Frontiers in Drug Design and Discovery - Rahman Et Al. (Vol.2)

Frontiers in Drug Design & Discovery
Bentham Science Publishers Ltd.

http://www.bentham.org/fddd
Volume 2, 2006
Contents
Editorial: Biomakers to Biosensors: Technology and i
Applications to Improve the Drug Discovery Process
G.W. Caldwell, Atta-ur-Rahman and M.R. D’Andrea and
M.I. Choudhary
Strategies of Biomarker Discovery for Drug Development 5

X.J. Lou, S.M. Belkowski, J.M. Dixon, B. Hertzoh, D. Horwitz,
S.I. Ilyin, D. Lawrence, D. Polkovitch, M. Towers and
M.R. D’Andrea
Protein and Antibody Microarrays: Clues Towards 23

Biomarker Discovery
K.Usni-Aoki, M. Koy, M. Kawai, M. Murakami,
K. Imai, K. Shimada and H. Koga
The use of Biomarkers to Detect Cervical Neoplasia and 35

to Diagnose High-Grade Cervical Disease
D.P. Malinowski
New Developments in the Field of Protein and Metabolism 55

Assays Aimed at Drug Discovery Processes
K. Narasimhan, P. Sukumar and M. Choolani
Proteomic Screening for Novel Therapeutic Targets 87
in Kidney Diseases
V. Thongboonkerd
Aptamer-Based Technologies as New Tools for Proteomics 103

in Diagnosis and Therapy
V. de Franciscis and L. Cerchia
Recent Developments in Proteomics: Mass Spectroscopy 121

and Protein Arrays
V. Kulkarni and M. Rao
NMR Spectroscopy Based Metabonomics: Current 151

Technology and Applications
C.A. Daykin and F. Wulfert
NMR-Based Metabonomics of Urine from an Exploratory 175

Study of Ciprofibrate in Healthy Volunteers and Patients
with Type 2 Diabetes Mellitus
G.C. Leo, E.J. van Hoogdalem and M.B.A. van Doorn
Chromatography Mass Spectrometry Based 193

Metabonomic Analysis Method
G.W. Caldwell, W. Lang, G.C. Leo, J.A. Masucci, W. Jones
and A. Mahan
OWLS—A Versatile Technique for Drug Discovery 211

S.E. Ramsden
Cell-Based Biosensors in Proteomic Analysis 225

S.E. Kintzios
Current Approaches in Natural Biopolymer-Nanoparticle 241
Hybird Functional Materials: From Drug Delivery to
Bio-Detection Application
R. Brayner
Spectroscopic Analysis of Cell Physiology and Function 259

M. Riley, I.M. Fernandez and P. Lucas
Encapsulates Biomolecules Using Sol-Gel Reaction for 273

High-Throughput Screening
K. Sakai-Kato, M. Kato, N. Utsunomiya-Tate and T. Toyo’oka
Modeling of Environmentally Sensitive Hydrogels for 295

Drug Delivery: An Overview and Recent Developments
H. Li, R. Lou and K.Y. Lam
Polyelectrolyte Nanocapeules – Promising Progress 333

in Development of New Drugs and Therapies
S. Krol, A. Gliozzi and A. Diaspro
Contributors 349
Subject Index 353

Editorial Frontiers in Drug Design & Discovery, 2006, Vol. 2 i
Editorial: “Biomarkers to Biosensors – Technologies and Applications

to Improve the Drug Discovery Process”
Moving forward in the 21st century, the discovery and development of ethical
therapeutics “drugs” remains rich with opportunities but hampered by challenging
technological and financial obstacles. The Frontier in Drug Design and Discovery series
is dedicated to pharmaceutical scientists around the world who seek to bring affordable,
effective and safe drugs to patients. The first volume brought together experts to discuss
the advantages and limitations of screening techniques used in the drug discovery
process to identify potential drug candidates. While screening techniques have advanced
steadily in the last decade, a large number of these drug candidates fail in clinical studies
because we cannot accurately predict how effective or how safe these candidates will
behave in humans based solely on animal studies. In the second volume, experts discuss
new technological and conceptual approaches to accelerate and to improve the
predictability of the discoveries made in the laboratory into clinical testing.
Twenty years ago, it was common to have a wealth of background information available
on potential drug targets thanks to years of academic and pharmaceutical basic research.
In today’s drug discovery world, therapeutic targets are typically poorly understood at
the conception of projects. Moreover, the understanding of the concordance of efficacy
and toxicity of pharmaceuticals observed in animals with that observed in humans is
usually lacking. The debate in the pharmaceutical industry on how to proceed ahead on
projects without a wealth of background information has focused on the use of
innovative biomarker strategies, establishing proof of mechanism in human subjects,
market differentiation, and efficiently terminating the development of unsuccessful
projects. The areas of bioinformatics, genomics, proteomics, and metabolomics are
leading the way to identify the details of the machinery that make up a living cell and
thus, establish a solid scientific platform for biomarker and biosensor discoveries.
Scientists are now embarking on an endeavor to discover how these biomarkers and
biosensors can be used to understand the complex behavior that underlies the
development and the progression of diseases. It is our firm belief that the investigation
of the effects of drugs and the nature of disease will become ever more feasible because
of advances in biomarker and biosensor research.
We have selected authors to contribute their expertise to assemble a one-stop reference
book to discuss the full range of biomarker and biosensor programs. X.J. Lou and
colleagues have contributed a chapter outlining practical guidelines for the discovery,
validation and use of biomarkers to accelerate the drug discovery process. These
guidelines have the potential to shorten timelines and costs for developing drugs. The
chapter by K. Usui-Aoki and colleagues give a well-balanced review of the status of
protein and antibody microarray technologies. The surface plasmon resonance-based
antibody microarray approach looks very promising for clinical applications. The
discussion of combining protein/antibody and cDNA microarray data clearly illustrates
the difficulties of validating large data sets. D.P. Malinowski introduces the reader to the
use of biomarkers for the detection of cervical carcinoma and for the diagnoses of high-
ii Frontiers in Drug Design & Discovery, 2006, Vol. 2 Editorial
grade cervical disease. These biomarkers appear promising in molecular diagnostic

applications to detect malignant cells in both histology and cervical cytology specimens.
K. Narasimhan and colleagues have written an excellent chapter describing the use of
high-throughput proteomic and metabolic assays to accelerate the drug discovery
process. Novel applications of chromatographic, electrophoretic, immunologic and mass
spectrometry technologies are used to analyze the proteome and the metabolome for
clinical benefit. The chapter by V. Thongboonkerd gives an overview of gel-based,
surface-enhanced laser desorption ionization, and liquid chromatography coupled to
tandem mass spectrometry proteomic methods. The chapter focuses mainly on applying
these techniques to renal and urinary proteomics to define novel therapeutic targets in
kidney diseases. V. De Franciscis and L Cerchia have written a chapter reviewing
the use of specific nucleic acid-based compounds “aptamers” as biosensors for protein
detection. The aptamers protein detection approach looks very promising for a large
number of applications in all parts of the drug discovery process. A. Kulkarni and M
Rao have prepared a chapter reviewing different mass spectrometry approaches being
used to identify and characterize proteins from diverse sources and recent developments
in protein array technology.
C. Daykin and F. Wülfert introduce the reader to nuclear magnetic resonance (NMR)
spectroscopy data handling, data analysis strategies and metabonomics as a tool for
understanding the development and the progression of diseases at the metabolic level.
NMR based-metabonomics contains a wealth of information on the endogenous
biochemical processes in living cells. G.C Leo and colleagues present the results from a
urinary NMR based metabonomic study of ciprofibrate in healthy volunteers and
patients with type 2 diabetes mellitus. The study showed that supervised statistical
analysis is able to separate ciprofibrate treated versus placebo treated male and female
subjects. G.W. Caldwell and colleagues have prepared a chapter reviewing
chromatography mass spectrometry based metabonomic strategies applied to diverse
areas such as fermentation, bacteria, plants and animals.
J.J. Ramsden presents an interesting chapter on the use of optical waveguide lightmode
spectroscopy to measure binding and dissociation between biomolecules, protein
conformational changes, and interaction of drugs with lipid membranes. The potential
uses of this technique for drug design and discovery are discussed. S.E. Kintzios
introduces the reader to cell based biosensors in proteomic analysis. The chapter by R.
Brayner review the current approaches for hybrid functional materials based on natural
biopolymers such as proteins and polysaccharides and nanoparticles consisting of
metals, oxides, quantum dots for drug delivery and biosensors applications. M. Riley and
colleagues present an interesting chapter on the use of infrared and Raman spectroscopy
to evaluate biochemical pathways of mammalian cells. A critical review of equipment,
experimental approaches, and analytical methods is provided for drug screening
applications. K. Sakai-Kato and colleagues have written an excellent chapter describing
how biomolecules can be immobilized on capillary-, microchip-, and microarray-based
analytical systems using the sol-gel reactions. This technology has potential applications
as biosensors and screening systems. H. Li and colleagues give an overview of the
Editorial Frontiers in Drug Design & Discovery, 2006, Vol. 2 iii
mathematical modeling for simulation of environmentally sensitive hydrogels. The

chapter discusses the properties and performance of the hydrogels as drug delivery
systems. S. Krol and colleagues review the use of encapsulation techniques for living
cells and artificial tissues.
In total, we hope that these chapters will provide a well-rounded overview of the
application of today's Frontier technologies and how they can be applied to do Drug
Design and Discovery.
Gary W. Caldwell
Atta-ur-Rahman
Michael R. D’Andrea
M. Iqbal Choudhary
Frontiers in Drug Design & Discovery, 2006, 2, 5-22 5
Strategies of Biomarker Discovery for Drug

Development
Xiang Jian Lou, S.M. Belkowski, James M. Dixon, Brenda Hertzog,

Dan Horowitz, Sergey I. Ilyin, Danielle Lawrence, D. Polkovitch,
Meghan Towers, Michael R.D’Andrea*
Drug Discovery, Target Validation, Johnson & Johnson Pharmaceutical Research and
Development, LLC, Welsh and McKean Roads, Spring House, PA 19477-0776, USA
Abstract: Drug development is a long and costly process. Although the length
of drug development may vary depending on the target class, attrition is the
main contributor to the financial burden. Therefore, various biological markers
(biomarkers) are needed to determine compound efficacy and dosing.
Biomarkers provide comprehensive information about the molecular network
of the target and lead compound in vivo. Such information has the potential of
shortening time of development and reducing costs by facilitating the decision
of what lead compound should move forward at early development stages.
This review article focuses on the strategies for biomarker discovery by giving
readers a practical guideline to approaches that are used for the discovery,
validation and use of biomarkers to accelerate the process of drug discovery.
INTRODUCTION
Latest calculations estimate that the costs for bringing new medication to the market
is approximately $600-800 million, which is largely due to high attrition rates [1].
Hence, it is essential to improve success rates in order to maintain the long-term
economic viability necessary to provide novel health care solutions to unmet medical
needs [1]. New and more accurate, knowledge-based decisions are required to advance
or stop a lead candidate compound as early as possible in the discovery process. Many
types of biomarker measurements help support key decisions throughout the drug
discovery process, from pre-clinical evaluations to regulatory approval. This, of course,
is a huge commitment of time and resources. It is thought that pre-clinical animal model
data should help steer or direct biomarker efforts as the candidate compound is
scheduled for first in human studies. Therefore, it is key to assess drug exposure-
response relationships such as specificity and potency toward the molecular target,
potential toxic side effects and therapeutic applications. This is an impressive challenge
for biomarkers and can consume as much time validating potential (translatable) pre-
clinical biomarkers for the human condition, as it would take to validate targeted-based
compounds.
*Corresponding author: Tel: 215-628-5619; Fax: 215-540-4887; E-mail: MDANDREA@PRDUS.JNJ.COM
Garry W. Caldwell / Atta-ur-Rahman / Michael R. D'Andrea / M. Iqbal Choudhary (Eds.)

All rights reserved – © 2006 Bentham Science Publishers.
6 Frontiers in Drug Design & Discovery, 2006, Vol. 2 Lou et al.
Biomarker discovery and validation has been extensively discussed in the literature
for both disease diagnosis and prognosis, but no guideline of this process has been
provided for drug development. Fundamentally there are three types of biomarkers
important in drug development: disease-related, target-related and toxicity biomarkers.
However, since drug development is a long process involving multiple stages of in vitro,
in vivo animal and human testing, various forms of the above three types of biomarkers
can be used in each stage. Biomarker names can be quite confusing. To facilitate
understanding, we wish to define our use of biomarker terminology. We define disease-
related biomarkers used in the preclinical stage (in vivo animal testing) as animal
efficacy biomarkers, while target-related biomarkers are referred to as pharmacodynamic
(PD) biomarkers. When target-related biomarkers are also disease related, the term of
efficacy or PD biomarkers are often interchangeable. In the clinical stage (clinical trials
phase I, II, III), both target-related and disease-related biomarkers are referred to as
human efficacy biomarkers and toxicity biomarkers are called human toxicity
biomarkers.
The biomarker discovery process can be envisioned as a rolling wheel without an
actual start or end (Fig. 1). In this wheel, a drug target can be selected from disease-
related biomarkers discovered either for diagnosis or for target selection. Once a target is
selected, target-related biomarkers can be discovered by altering the amount or activity
of the target. Target-related biomarkers can often be used to screen lead compounds and
to elucidate the mechanism of action (MOA) of a compound in model systems at the
stage of lead generation and optimization. When either target-related biomarkers are
specific for the target and are present in the animal model or disease-related biomarkers
are present in the animal model, then they can serve as PD and/or animal efficacy
biomarkers for validating compounds at the preclinical stage. In the best-case scenario,
these animal efficacy and PD biomarkers might also translate to potential clinical
biomarkers to accelerate phase II and III clinical trials. In the case of toxicity,
biomarkers for the preclinical stage (animal toxicity biomarkers) also have the potential
to serve as human toxicity biomarkers. On the other hand, human efficacy and toxicity
biomarkers can also translate back to early stage in vitro or animal models for the
measurements of efficacy and toxicity.
Before a biomarker strategy is formulated, information obtained from the literature
coupled with any pre-clinical data will help to determine what technologies are best
suited for biomarker discovery. We prefer to initiate a biomarker development plan
when the target is proposed. In addition to traditional molecular biology and
biochemistry technologies such as polymerase chain reaction (PCR), mutagenesis and
Western blotting, recently developed high-throughput technologies provide essential
tools for large-scale differential analysis. These novel techniques are categorized as
genomics, proteomics and metabolomics.
Genomic technologies allow genome-wide analysis of disease-unique gene expres-
sion profiles or polymorphisms. The most commonly used gene expression profiling
technologies include DNA microarrays [2-5], quantitative PCR [6-8], serial analysis of
gene expression [9,10] and representative difference analysis (RDA) [11,12]. The
polymorphism analysis technologies mainly involve genome-wide linkage mapping
using either sequencing [13] or high-density single nucleotide polymorphism (SNP)
microarrays [14].
Strategies of Biomarker Discovery Frontiers in Drug Design & Discovery, 2006, Vol. 2 7
Proteomic and metabolomic approaches screen for the disease-specific expression

patterns for proteins and metabolites, respectively. The most commonly used differential
protein analysis are two-dimensional electrophoresis (2D-GE), surface enhanced laser
desorption time-of-flight mass spectrometry (SELDI-TOF), liquid chromatography/mass
spectrometry (LC-MS/MS), multidimensional protein identification technology
(MudPIT, which couples 2D-LC to MS/MS), isotope-coated affinity tag (ICAT) and
laser capture microdissection (LCM) coupled with reverse phase protein arrays (for a
review see [15]). As opposed to genomics, the advantage of proteomics is that an
identified protein biomarker is itself a functional end point and thus a true signature of a
disease state.
Fig. (1). The Biomarker discovery wheel shows that many aspects of biomarker research originate
from previous studies to impact future studies and decisions. Although the wheel presentation may
appear to be an oversimplification, biomarkers support or validate four fundamental areas: target
selection, lead validation and optimization, and pre-clinical and clinical situations. All together,
the information obtained from each of these areas should help advance drug discovery compounds
to the clinic.
There are a multitude of differences between genomic and proteomic techniques. For
example, unlike PCR and reverse transcriptase related methods in genomic analysis, no
proteomic technology for amplification detection exists. Also, genomic methods are
deemed more quantitative than their proteomic counterparts. While proteomic analysis
has been considered the more valuable technique, it is also the most challenging
biomarker discovery technology. The range between the most and least expressed
proteins in a proteome may exceed ten orders of magnitude [16]. Proteins exhibit a wide
range of post-translational modifications and various truncated or proteolytically
degraded forms. Finally, proteins, unlike DNA and RNA, do not have complementary
binding partners with high-specificity. Most of the protein-protein or protein-DNA
interactions are highly sensitive to experimental conditions. Regardless of these
challenges, proteomic analysis has delivered many potential disease-specific biomarkers
[17,18]. Differential metabolite measurements are usually performed using NMR and
mass spectrometry. Details of metabolomics are discussed in several sections of this
book.
Although designing a strategy for biomarker discovery depends on the drug
development stage the biomarker is meant for and the biological properties of the target,
ultimately, it is the question at hand that determines the most suitable technology to
address the need. The remainder of the chapter will discuss such strategies for each stage
of drug development.
TARGET SELECTION STAGE

Traditionally, most new drug candidates are launched through one or more of the
following four approaches: (1) to discover or select a new drug target, (2) to design a
drug based on the increased knowledge for a known target, for example a new drug
targeting a receptor with increased specificity and affinity, (3) to modify the chemical
structure of a known compound (4) to search for biological activity of large numbers of
natural products, banks of previously discovered chemical entities, large libraries of
peptides, proteins, nucleic acids and other organic molecules. Major attention is now
being given to the discovery and selection of entirely new targets for drug therapy since
a good target is fundamental to the success of drug development [19]. A good target
should be specific to disease conditions and can be enhanced or disrupted by either small
molecules or antibodies. The interruption of the target should not do harm to other
physiological conditions, specifically it will not cause significant side effects. Target
selection cannot start from the clinical endpoints in human subjects because some
disease conditions take more than 10 years to reach significant clinical endpoints. More
importantly, the test of target interruption cannot be performed directly on human
subjects. Therefore, target discovery and selection usually rely on the measurement of
disease-related and target-related biomarkers in model systems. Furthermore, if a
disease-related biomarker is proven to cause the disease (under certain conditions) and
this disease-related biomarker is “drugable”, namely it can be specifically enhanced or
interrupted, this disease-related biomarker can be a drug target.
Disease-related biomarkers are generally discovered through two approaches:
unbiased differential analysis of disease vs. normal samples and pathway-based
candidate searching. Recently developed high-throughput technologies for genomics
(vide supra ), proteomics (vide supra ) and metabolomics studies provide essential tools
for the differential analysis approach. Each technology can convey different information
and have different technical capabilities. These technologies by themselves or in
combination with other methods have generated many successful examples of disease-
related biomarker discoveries [20-22]. For example, using oligonucleotide-based DNA
microarrays, Le Page et al. identified two differentially expressed genes encoding
cytokine IL-8 and FGF-2 as potential biomarkers by comparing primary cultures of 39

ovarian cancer specimens with 11 normal ovarian epithelia. The increased expression
levels of these two epithelial ovarian cancer biomarkers were confirmed by quantitative
PCR and immunohistochemistry (IHC) using an independent tissue array representing
different grades and pathologies of ovarian disease. To date, they have shown by ELISA
that the levels of these two cytokines are elevated in epithelial ovarian cancer patients
and a combination of these two biomarkers with CA125 can detect epithelial ovarian
cancer with a higher specificity than CA125 alone [23].
Pathway-based approaches usually design pathway-biased tools according to existing
knowledge. These pathway-biased tools are then used to search for biomarkers [22,24-
26]. For example, it has been hypothesized that abnormal DNA methylation is one of the
major mechanisms of imbalanced gene expression in cancer. Therefore, methylation-
detection arrays may be used for cancer biomarker discovery [27,28]. More often,
pathway-based approaches are combined with unbiased “omics” differential analysis.
The recent discovery of a type II collagen peptide as a biomarker for osteoarthritis (OA)
is one example of such a combination [29]. In order to determine OA biomarkers, the
authors first generated antibodies against the collagen degradation product in the
presence of MMP-13 (collagenase-3). Collagen degradation is reported to be elevated in
OA. Transgenic over-expression of MMP-13 in hyaline cartilages and joints induced
cartilage changes in mice characteristic of human OA [30]. By using these antibodies as
bait, they discovered peptides in urine that are elevated in OA patients. These peptides
have been applied, in a small clinical setting, as biomarkers for OA recognition and
MMP inhibitor treatment and have provided favorable results [29]. Many biological and
pharmacological pathway examples are available on the websites of various consortiums
and databases [31-33] (www.BioCarta.com).
Target-related biomarkers are usually discovered through functional enhancement
and interruption of the target in in vitro or animal models. The most commonly used
functional enhancement and interruption methods include: (1) over-expressing
(transgenic) and knocking out target genes in animals, (2) virus-mediated knocking in
(transfection), (3) small interfering RNA (siRNA) mediated knocking down of the target
genes in cell culture systems and (4) modification by tool compound or bioactive
molecule induced stimulatory or inhibitory effects [34]. Target-related biomarkers are
then discovered in these biologically manipulated model systems using the similar
unbiased differential analysis and/or pathway-specific approaches (vide supra). Once
discovered, a causal relationship can be confirmed by another round of functional
enhancement and interruption. A causal relationship is not necessary for the validation of
biomarker, but will enhance the merit of the biomarker. For example, GREB 1 is a
potential down-stream biomarker for estrogen receptor (ER) in breast cancer. This is
because GREB1 was induced by beta-estradiol in the ER-positive endometrial cell line
ECC-1 and MCF-7 cells respectively [35,36] and the suppression of GREB 1 using
siRNA blocked estrogen induced growth in MCF-7 [35]. If the target is an enzyme, the
down stream biomarkers can be first searched for among its substrates and/or products,
such as phosphorylated products (in the case of kinases) and digested products (in the
case of proteases).
In addition to facilitating target selection, many disease-related biomarkers such as
low-density lipoprotein (LDL), high-density lipoprotein (HDL), C-reactive protein
(CRP) and CD40 also become drug targets (for review papers, see [37,38]). Due to the
heterogeneity of many diseases such as atherosclerosis, cancer and Alzheimer’s disease

it is important to first examine whether a putative target from the literature or a potential
disease-specific biomarker obtained using approaches discussed above is abnormally
expressed in a representative sampling of disease tissues. A causal relationship between
the target and disease-related biomarkers also needs to be confirmed by any of the
knocking in or knocking down model systems. More importantly, correcting the
abnormally functioning targets should only bring disease-related biomarkers back to
normal levels instead of causing damage to other parts of the body. Knowledge
integration through correlation of data obtained using different technologies and systems
approach of understanding the biological networks should have synergistic effects on
target selection [39] in addition to biomarker discovery [40]. The “omics” technologies
provide the means to comprehensively monitor the molecular network of biomarker,
target and disease processes. Drug discovery through a validated target with biomarkers
and model systems indicating normal and perturbed networks has shown the potential of
providing improved therapy [41] and/or accelerating the process of drug development
[13,42,43]. In the latter case, through a population-based, genome-wide study involving
hundreds of heart attack patients and their family members in Iceland, deCODE genetics
discovered variations in the gene encoding 5-lipoxygenase activating protein (FLAP)
that doubles the risk of the disease [13]. The same genetic variation was also found to
confer a significantly increased risk of stroke in the Scottish population [43]. It has been
known for many years that important down stream biomarkers of 5-lipoxygenase are
leukotrienes (for a review, see [44]). These are potent drivers of inflammation and may
contribute to the instability and rupture of atherosclerotic plaques. Since plaque rupture
is the event immediately preceding most heart attacks, a drug that can down-regulate the
activity of the leukotriene pathway may offer a highly targeted and effective means of
reducing risk of heart attack. Well-tolerated anti-leukotriene reagents have also been
discovered for the treatment of asthma (for reviews see [45]). One of the reagents is
DG031 developed by Bayer AG. DG031 inhibits leukotriene synthesis through its
binding to FLAP. deCODE genetics therefore licensed DG031 from Bayer AG. Because
of the extensive safety and clinical data already gathered on the DG031, it only took
deCODE genetics a little over one year to bring DG031 to phase II clinical trial from
target discovery. In the phase II trial, DG031 showed the effect of decreasing the levels
of target leukotriene B4 and heart attack related biomarkers such as CRP and
myeloperoxidase [42]. Regardless of the limitations of this trial, the deCODE genetics
FLAP example provides an exciting example of translating genomic findings and
biomarker measurements to target selection and clinical application.
LEAD VALIDATION AND OPTIMIZATION STAGE

Once lead compounds are developed or selected, biomarkers are used to validate (or
invalidate) the efficacy of these early lead compounds in animal models. Although many
of the disease- and target-related biomarkers discussed previously could be directly used
in this stage, this is a critical stage where a biomarker strategy must be in place to
provide decision tree analyses as the compound progresses. A myriad of issues should be
considered in the development of a biomarker plan for this stage, such as (1) is the MOA
determined for the target and/or compound, (2) are there downstream profiles that can
confirm receptor inhibition or activation, (3) can these effectors be assayed in fluids or
biopsies and (4) are these pre-clinical endpoints translatable to the clinic? The biomarker
strategy is dependent upon answering these questions. As an example, oncological
targets affect cell proliferation, differentiation and death; however, in an effort to

improve specificity beyond the less selective chemotherapeutical and radiological
approaches, new therapeutic approaches should be implemented to characterize the
MOA for these anticancer agents in preclinical and in clinical trails.
Although, in theory, it is required to understand a compound’s MOA, if there are
selective assays that support the progression of a compound through the drug discovery
process map and there is ample in vivo evidence, the compound will continue to
progress. In fact, it may take years and numerous studies to fully validate a compounds
MOA. Such is the case for the application of bisphosphonates (BPPs) and a newer-
generation BPP, zoledronic acid, in the management of advanced, metastatic bone
disease. Skeletal metastases are a common form of cancer metastasis, ranking in
frequency behind only that of liver and lung [46]. Further complications include pain,
hypercalcemia, pathologic fractures and spinal cord compression, which negatively
impact the patient’s quality of life and function [46]. BPPs have been recently used to
effectively manage and prevent bone pain and skeletal complications in a number of
malignancies, such as pamidronate in multiple myeloma [47] and clondronate in breast
cancer [48]. Although BPPs provided a novel therapeutic intervention back in 1996, it is
the subsequent work by a myriad of reports that continue to elucidate the MOA of the
BPPs, which inhibit osteoclast function and bone resorption [49]. After binding to
mineralized bone, BPPs are subsequently released and osteoclasts internalize the
compounds leading to apoptosis and decreases resorption [50,51]. Continuing research
embellish these earlier findings by expanding on the understanding of the MOA for the
BPPs as it is reported that TGF-β and Il-6 may play roles in the mechanisms underlying
bone metastasis and resorption, and the BPPs may also have a direct anti-angiogenic
effect [51,52]. This example, which is nicely reviewed in Saba and Khuir [46], presents
a situation that precludes the need to clearly elucidate the MOA of a particular
compound to advance the compound to a clinical setting.
Understanding the pathway of the target activation can also help generate a
predictable biomarker plan in the event that off-target effects are adverse and toxic. For
example, our lab demonstrated the cellular impact of a compound on the intended
biomolecule in vivo [53]. We developed an in vivo assay to validate the
pharmacodynamic activity of JNJ-10198409. This compound is a relatively selective
inhibitor of platelet-derived growth factor receptor tyrosine kinase (PDGF-RTK) in
tumor tissues after administering the compound orally in a nude mouse xenograft model
of human LoVo colon cancer. Since we were aware of the signaling pathway of PDGF-
RTK, we developed a novel assay to quantify the in vivo anti-PDGF-RTK activity of the
inhibitor in tumor tissue by determining the phosphorylation status of phospholipase Cγ1
(PLCγ1), a key downstream cellular molecule in the PDGF-RTK signaling cascade. We
used two antibodies, one specific for the total (phosphorylated and unphosphorylated
forms) PLCγ1 (pan-PLCγ1) and the other, specific for phosphorylated form of PLCγ1
(ph-PLCγ1) to immunohistochemically detect their expression in tumor tissues.
Computer-assisted image analysis was then used to directly compare the ratio of ph-
PLCγ1 to pan-PLCγ1 immunolabeling intensities in serial sections (5 µms) of tumors
obtained from vehicle- and compound-treated tumor bearing mice. Our data
demonstrated statistically significant, dose-dependent differences in the ph-PLC/pan-
PLC ratio among the four treatment groups (vehicle, 25, 50, and 100 mg/kg b.i.d.).
These results confirmed the ability of this compound to suppress PDGF-RTK

downstream signaling in tumor tissues in vivo.
Not only are drug developing companies assessing the relationship between the
compound and its target in the animal models, but they also have to extrapolate the
human experience from these preclinical in vivo models and to determine if any of the
preclinical biomarkers are translatable to the human condition. A recent example
describes the application of biomarkers of the anticancer activity of R115777 (Zarnestra)
in an in vitro model of human breast cancer [54]. In this study, they illustrated the
inhibition of HDJ-2 farnesylation, up-regulation of RhoB, inhibition of VEGF and
MMP-1 secretion in addition to other markers in cultured cells. These markers were
consistent with the MOA and they demonstrated that the compound did interfere with
tumor growth, survival and angiogenesis pathways in breast cancer models with low or
over expressed HER2/neu receptor [54].
These studies highlight the need to elucidate the MOA of a particular compound for
biomarker discovery efforts. MOA studies support the selection of therapeutic agents,
appropriate models of efficacy and experimental design, as well as rational
characterization and prediction of non tumor (host) effects [55]. For example, in the field
of cancer research, about six physiological changes that lead to malignant growth are
represented among targeted therapies such as those pathways involved in growth,
angiogenesis and metastasis [56]. Elucidating the MOA of these targeted pathways will
not only enable the stratification of patients and cancer subtypes, but will lead to the
identification and characterization of a variety of new targets and biomarkers for anti-
cancer therapies [55].
PRECLINICAL STAGE
Preclinical biomarkers can be discovered and employed in the PD/PK (pharmaco-
kinetics) model often used in drug discovery. PD describes the intensity of a drug effect
in relation to its concentration in the body fluid [57] resulting in response-time profiles
[58]. PK describes the time course of the concentration of a drug in a body fluid
(preferably plasma or blood) that results from the administration of a certain dose [57]
resulting in drug concentration-time profiles [58]. Simply put, PK is “what the body does
to the drug” and PD is “what the drug does to the body” [59]. Many target- and disease-
related biomarkers discussed before can be used at this stage to evaluate the PD effect in
animal. PK/PD models are a vital part of drug development. They play an important role
in the selection of drug candidates and provides useful information to investigators in the
early “go/no-go” decision making process. In these animal models, a simplified version
of a true biological process is performed in a non-clinical environment and the data are
then used to predict the possible drug effect in humans.
Animal species, dose and dosing intervals, as well as mode and duration of
administration, are all important factors of a successful PK/PD model [60]. A study
performed by Barrett et al. [61] using agonist-induced platelet aggregation as a
biomarker to predict the efficacy of GP IIb/IIIa receptor antagonists, selected the guinea
pig over the rat and dog because of their platelet characteristics. Guinea pig platelet
functions resemble those of human platelets and their platelet membrane glycoproteins
exhibit similar homology to human GP IIb/IIIa and GP Ib/IX. This species had a
considerable advantage over the others and was the optimum choice for this type of
study.
Once the animal species has been selected, a mechanism-based modeling, currently
the preferred methodology for PK/PD correlations [62], can be setup. The biomarkers in
these model systems should mimic the biochemical and molecular changes seen in the
disease process [63]. An example is the suppression of Factor IX activity and the
prolonged response of activated partial thromboplastin time (aPTT) in Cynomolgus
monkeys [64]. This modeling system was useful in describing the dose-dependent
effects of a novel humanized anti-Factor IX monoclonal antibody for anti-coagulant
therapy [64]. This model approach is generally applicable in predicting the effect of
similar therapeutic monoclonal antibodies. In another example, Van der Graaf et al.
applied a mechanism-based operational model of agonism to evaluate the adenosine A1
receptor-mediated in vivo effect of N6-cyclopentyladenosine analogs in rats [65]. Using
heart rate as the biomarker, measures of agonist affinity and efficacy were determined.
Once established, PK/PD relationships can also be used to evaluate and optimize various
dosage forms and drug delivery systems [62].
Based on animal studies, the next step is to predict and evaluate in vivo potency and
intrinsic activity of the compound, as well as any other drug interactions, in humans
[62]. A well-chosen biomarker can translate between species and allow this deter-
mination to be made. The use of EEG effect intensity as an endpoint for determining
EC50 values for various opioids in rats was employed by Cox et al. [66]. Others have
shown that concentration-EEG effect relationships of synthetic opioids obtained in this
study have also been found in humans [67,68]. This PD characterization of synthetic
opioids may be useful in the design of new compounds at the preclinical stage.
Another important effort of biomarker discovery in the preclinical phase is the
determination of toxicity biomarkers. The attrition rate of compounds due to toxicity is
very high and if this can be determined earlier in preclinical drug discovery then money
and time can be saved. Determining toxicity biomarkers in preclinical studies can be
quite difficult. These issues may not be apparent in initial drug discovery experiments
because short-term dosing may not reveal or produce toxicity signatures. In many cases
it is not until later development when chronic dosing is performed that these effects are
apparent. Many of the toxic effects of compounds occur unexpectedly and are
unpredictable in nature. The first sign of these toxic effects are often determined by
observing a change in serum chemistry, hematological parameters or microscopic
morphology [69]. Liver toxicity is one of the most common forms of drug-induced
system damage. The liver is exposed to a wide variety of metabolic, toxic, microbial and
neoplastic insults that are potentially hepatotoxic. Therefore, compounds that have a
distinct type of toxicity due to their chemistry should be suspected for their capability to
induce these toxicities. Knowledge of specific toxicities led to the development of free
radical toxicity based models such as the one developed for pyrogallol [70].
Unfortunately, not all toxicities are predictive based on the chemistry of the compound.
A toxicity marker must be developed for a class of drug that may act in a particular way.
Due to the high number of different toxicities that can arise, Steiner et al. developed an
algorithm that can outline the type of toxicity being induced by a compound based on
hepatic gene expression in rats [71]. Using this algorithm, investigators were able to
accurately predict the hepatic toxicity of hydrazine and coumarin while showing the lack
of hepatic toxicity after administration of gentamicin. This readout can identify the
potential toxicity that an uncharacterized compound may exhibit.
Some compounds may have expected potential organ specific toxicities. One
approach is to look at toxicity biomarkers defined for a predicted target organ. In the
case of drug induced heart toxicities, markers described in humans could also be used in
preclinical studies. Serum troponins have been described as reliable markers for acute
myocardial infarction in humans [72]. A high degree of similarity between the levels of
cardiac troponins found in animal studies and humans provides strong evidence that they
may play a role in bridging biomarkers for both preclinical and clinical studies in
monitoring drug-induced cardiac injury [73].
Unfortunately, there are also many other types of toxicity that may not be as
straightforward making a standard marker quite elusive because the damage may not
occur in organs that are expected to undergo toxicity. Therefore an alternate approach is
to look for biomarkers associated with physiological and pathological changes in a toxic
condition in areas of potential damage based on MOA of the compound. A description
of this approach is the study performed by Searfoss et al. In this study a target specific
compound, functional gamma secretase inhibitor, was tested in rats at high doses.
Adipisin was found to be expressed in the intestine, an organ known to be a potential site
of injury based on the MOA of the compound, and was directly correlated with the
damage to the tissue [74]. The adipisin was excreted by the rats making this an easily
assessable and tissue specific toxicology biomarker.
Discovery of both efficacy and toxicity biomarkers can be performed with the use of
computer aided drug design (CADD). Studies have shown the ability to use PK/PD data
from one species and apply this predicted outcome to another species [75]. One such
example was rat PK/PD data that was modeled to a rhesus monkey. Comparison of the
positron emission tomography (PET) experiment in the rhesus monkey confirmed the
model to be relevant. Modeling of this nature could extrapolate data and biomarker use
for both efficacy and toxicity from preclinical to clinical setting with far better accuracy.
CLINICAL STAGE
One of the ultimate goals of biomarker discovery is to identify a biomarker that is
practical for use in clinical studies. These markers would have to acquire samples by
minimally invasive or non-invasive procedures and give correct information about the
state of a disease (disease-related biomarkers) or action of a compound (human efficacy
biomarkers).
General approaches for disease-related biomarker discovery has been discussed in
the target selection stage. One of the potential problems in disease-related biomarker
development is the accuracy of the biomarker. An example of disease-related biomarkers
can be illustrated by the screening of men for prostate-specific antigen for early prostate
cancer. Unfortunately, it is not usually wise to depend on the information of a single
biomarker because of the possibility of obtaining false positive and false negative data.
To that end, e-cadherin, an important cell-cell adhesion protein, has been linked to the
malignant progression of adenocarcinomas including prostate cancer [76-78]. Most
excitingly, a soluble 80-kDa fragment of e-cadherin detected in the serum was
significantly increased during prostate cancer progression [79]. As with many
biomarkers, e-cadherin is not specific to prostate cancer as decreased e-cadherin

expression is associated with poor survival in bladder cancer patients [80,81]. In spite of
these contrary findings, increased serum levels of soluble e-cadherin was also detected in
patients with early relapse of superficial bladder tumors [82].
Although some diseases provide the patient symptoms, other diseases may provide
the primary health care physician insight into the patient’s well being. Conditions such
as ovarian cancer do not provide overt clues until they are presented with a late stage
disease. Once the disease has developed into a late stage disease, the survival decreases
from 95% at stage I (surgical and chemotherapeutic intervention is not needed) to
approximately 20-25% five-year survival despite appropriate treatment [83]. Therefore,
there is a need to diagnose a disease such as ovarian cancer much earlier. Unfortunately,
the pathology is confined to the ovary and the patient rarely becomes symptomatic, in
contrast to the cancers of the breast, prostate and colon. Furthermore, the ovary is
anatomically more difficult to assess during routine examinations. Even imaging
frequently cannot distinguish between a benign and a malignant condition. The need for,
and the development of, reliable biomarkers, especially in the serum, is a priority. Since
it may not be realistic to envision a discovery of a single ovarian biomarker, perhaps a
combination of biomarkers may hold accuracy to diagnose ovarian cancer earlier. One
such study by Rapkiewicz et al. [83] was able to correctly identify all cases of early-
stage ovarian cancer (n=116) based on their proteomic signature. These experiments
were based on the premise that pathological changes in tissue such as the ovary, will be
presented in a particular and potentially diagnostic protein biosignature pattern in the
serum [84-86]. Three of those biosignatures were identified as glyoxaolase I, RhoGD1a
and FK506 binding protein, all of which have been implicated in oncogenesis pathways
that include cell apoptosis, DNA synthesis and mutagenesis [87,88]. In concert, Ye and
colleagues [89] reported the identification of the haptoglobin-α subunit in sera of
patients with ovarian cancer for the use as a potential serum biomarker for the diagnosis
of the disease [89]. Although a specific early stage ovarian cancer biomarker has yet to
be identified and validated, it may be that a panel of many biomarkers, such as those
described above, may provide enough diagnostic potential to alert the physician to
perform additional testing.
Although, as mentioned above, various technologies can be used to assess
biomarkers in tissue and bodily fluid samples in clinical settings, relevant biopsies may
not always be feasible. Noninvasive imaging approaches offer significant advantages in
monitoring the action of a compound in both preclinical and clinical settings.
Noninvasive imaging techniques include light imaging, computed tomography (CT),
magnetic resonance imaging (MRI), PET as well as different combinations of these
modalities. Even though, all of these approaches can facilitate preclinical and clinical
activities, in this chapter we focus on PET. PET provides an opportunity to non-
invasively evaluate in vivo PK, PD and PK/PD as well as the response to therapy if
relevant clinical paradigm is established. Applications of PET for PK/PD analysis are
particularly important for drugs with central MOA. Previously published studies
reported on the application of PET to establish relationship between drug plasma
concentration, central nervous system (CNS) receptor occupancy and efficacy to
facilitate dose selection [90, 91]. This information helps to de-risk clinical development
by providing better prediction of dosing to be used in the phase II and/or III. It also aids
in reducing the cost and duration of phase II clinical trials. A significant number of
CNS-related PET tracers has been developed and validated. A list of validated PET
tracers (albeit not complete) is available at http://www.snidd.org/. Co-development of
new CNS drug and related PET tracer(s) offers an efficient biomarker strategy, but
requires substantial investment of time and effort if previously validated tracers are not
available for the target of interest. It should be noted that, even though PET-enabled
studies are fairly easy to understand from a conceptual point, logistics of PET
applications in the clinic are fairly challenging and subject to FDA regulation in US.
Among many other requirements, clinical deployment of a novel PET tracer requires
submission of an IND (Investigational New Drug) to the FDA. An IND needs to be
supported by appropriate toxicity and safety data. PET tracer precursors have to be
manufactured under GMP (Good Manufacturing Practices) compliant conditions.
Monitoring of patients’ radioactive exposure and ensuring overall compliance with GCP
(Good Clinical Practice) are also very important. PET tracers have fairly short half-lives
and consequently require PET studies to have very effective coordination between
radiochemistry lab and personnel administering the tracer and performing the scan. Post-
scan data processing and interpretation is also very important [92-96]. Quite a few PET
tracers are routinely used in current medical practices and include: FDG ([18F]fluoro-
deoxyglucose), FLT (3'-Deoxy-3'-[18F]fluorothymidine) and FDOPA (6-[18F]fluoro-L-
3,4-dihydroxyphenylalanine). These and other already validated tracers are available for
both preclinical and clinical studies from local cyclotron facilities as well as from
Siemens cyclotron network (http://www.ctimi.com/cti_petnet) and can be used with
relative ease for preclinical and clinical applications. For example, Alzheimer’s and
other dementias significantly alter brain metabolism early, even before manifestations of
mild cognitive symptoms [97-99]. Clinical FDG-PET detects this altered metabolism
and provides noninvasive diagnostic assessment. In Oncology, tumor glucose utilization
can be assessed by FDG-PET as a measure of tumor metabolism. FDG-PET can provide
valuable diagnosis, staging and prognosis information for many types of tumors. In the
study by Weber et al. 40 patients (3 female, 37 male, age 55 ± 11 years) with locally
advanced adenocarcinomas of the esophagogastric junction and preoperative chemo-
therapy (cis-Platin, 5FU, Paclitaxel) had FDG-PET prior to and 14 days after initiation
of therapy. This study demonstrated a strong correlation of changes in FDG-uptake with
histopathological tumor regression and patient survival [100]. Whole Body FDG-PET
imaging could also predict the outcome for patients with lymphoma following
chemotherapy treatment [101]. In fact, predictive values generated by FDG-PET are
superior to those of conventional imaging.
Pharmacogenetic and pharmacogenomic biomarkers may also be very important at
the clinical stage. Using pharmacogenetic and pharmacogenomic biomarkers to select
the appropriate patient group may significantly decrease the cost of drug development as
clinical trials focused on the right population (responders without adverse reaction) may
decrease the trial size and increase the success rate [102]. However, there is still debate
about the cost-effectiveness of genetic screening before drug trials. Nevertheless, details
on pharmacogenetic and pharmacogenomic biomarkers are beyond the scope of this
review article.
BIOMARKER VALIDATION
Validation of key biomarkers is critical for their use in preclinical studies or to
promote their advancement into clinical studies. The goals of validation, among others,
are to determine the fitness of a marker in distinguishing normal from treated samples or
normal from diseased samples in a specific manner. In addition, validation aims to
determine the sample availability of the marker (i.e. located in blood or secretions) and
translatability of the marker into other models or into the clinic. Specificity of a
biomarker is key in allowing its use for assessing treatment efficacy and understanding
the potential mechanism of action of the compounds being used. Validation of
accessibly of the biomarker is important to assure the user of a highly reproducible and
stable result. It is also essential to validate the translation of a marker from preclinical to
clinical studies to give the user confidence that the biomarker is suitable for the model
system/clinical study.
An excellent example of each of these validation steps is the validation of adipocyte
complement-related protein of 30 kDa (Acrp30) expression for PPARγ activity. PPARγ
agonists such as rosiglitizone were found to induced Acrp30 mRNA expression in 3T3-
L1 preadipocytes [103]. This observation in cell cultures was then extended into a
preclinical mouse model. The distribution of Acrp30 was found to be primarily in
adipose tissue and qPCR analysis of white adipose tissue of db/db mice showed an
increase in Acrp30 message expression after rosiglitizone treatment. This was further
validated by examination of the protein level in plasma and a correlative increase in
circulating levels of this protein was found in treated animals [103]. The increase was
found to be specific to PPARγ as PPARα agonists did not increase message or protein
levels in the mouse model. Therefore, this marker had been defined as an adipose
specific marker that could be used as a read out of metabolic activity. Having shown
specificity and an easily accessible source as well as a cause and effect relationship to
the biologic endpoint, the biomarker was assessed for its usefulness in clinical studies.
The increase in circulating Acrp30 after rosiglitizone treatment was confirmed in human
studies and it was found that PPARα agonists did not affect the expression of Acrp30
showing its specificity as a clinical biomarker. These data together validate the use of
this biomarker, PPARγ agonists, in both pre-clinical and clinical studies [103].
In the case just outlined, the protein biomarker was determined to be available in
plasma. Validation of biomarker availability is quite easily performed if working with
plasma or urine. However, not all relevant biomarkers are present in an easily accessible
source. If the site of action of a compound is limited to the analysis of biopsies it is
important to validate the availability and reproducibility of the biopsy material. Boyle et
al. hypothesized that message levels of key cytokine mRNAs in the synovium were a
good indicator of rheumatoid arthritis (RA) [104]. However, the levels of these
cytokines in synovium were not easily standardized or reproduced. They found that IL-6
message levels measured from biopsied synovial tissue of RA patients using qPCR were
increased over osteoarthritis patients and could be standardized using peripheral blood
mononuclear cells (PBMC) [104]. This allowed a high reproducibility in the patient
samples removing drift from sample storage and handling. Prior to this methodology,
synovial tissue could not accurately depict the state of RA in patients even though it was
the immediate site of action of the disease. Validation of the assay conditions led to the
use of synovium as a convenient source of patient samples to monitor RA.
As the previous examples show, the value of a biomarker is increased with each
validation step. This leads to use of the biomarker in more levels of drug development
with increased confidence of the outcome leading to better go/no-go decisions.
SUMMARY
The value of biomarkers cannot be over emphasized. Although the mainstream
definition of a biomarker includes those biological markers that will have value in the
clinic, it is clear to us that the term, biomarker, represents a conglomerate of markers
obtained to address questions and validations that arise throughout the entire drug
discovery process (Fig. 1). Each will answer a particular question, and may not carry
through to the next process, while others may translate from animal models to human
trials. It is also possible that particular biomarker(s) in a Phase I study will not support
subsequent Phase II studies. In closing, it is our approach to first define the question or
hypothesis, assemble the technological platform, obtain and analyze the data to
ultimately help advance the lead compound to the clinical setting.
ABBREVIATIONS
PD = Pharmacodynamic
MOA = Mechanism of action
PCR = Polymerase chain reaction
RDA = Representative difference analysis
SNP = Single nucleotide polymorphism
2D-GE = Two-dimensional electrophoresis
SELDI-TOF = Surface enhanced laser desorption time-of-flight mass spectrometry
LC-MS/MS = Liquid chromatography/mass spectrometry
MudPIT = Multidimensional protein identification technology
ICAT = Isotope-coated affinity tag
LCM = Laser capture microdissection
IHC = Immunohistochemistry
OA = Osteoarthritis
siRNA = Small interfering RNA
ER = Estrogen receptor
LDL = Low-density lipoprotein
HDL = High-density lipoprotein
CRP = C-reactive protein
FLAP = 5-lipoxygenase activating protein
BPPs = Bisphosphonates
PDGF-RTK = Platelet-derived growth factor receptor tyrosine kinase
PLCγ1 = Phospholipase Cγ1
PK = Pharmacokinetics
aPTT = Activated partial thromboplastin time
CADD = Computer aided drug design
PET = Positron emission tomography
CT = Computed tomography
MRI = Magnetic resonance imaging
IND = Investigational New Drug
GMP = Good Manufacturing Practices
GCP = Good Clinical Practice
FDG = [18F]fluorodeoxyglucose
FLT = 3'-Deoxy-3'-[18F]fluorothymidine
FDOPA = 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine
Acrp30 = Adipocyte complement-related protein of 30 kDa
RA = Rheumatoid arthritis
PBMC = Peripheral blood mononuclear cells
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Protein and Antibody Microarrays:

Clues Towards Biomarker Discovery
Kazue Usui-Aoki1,3,*, Motoki Kyo2, Makoto Kawai1, Masatoshi

Murakami1, Kazuhide Imai1, Kiyo Shimada1, Hisashi Koga1, 3
1
Chiba Industry Advancement Center, 2-6 Nakase, Mihama-ku, Chiba 261-7126, Japan;
2
Biotechnology Frontier Project, Toyobo Co., Ltd., 10-24 Toyo-cho, Tsuruga, Fukui
914-0047, Japan and 3Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari,
Kisarazu, Chiba 292-0818, Japan
Abstract: The recent advancement of proteomics technologies has provided us
a variety of approaches for protein-expression profiling. Among these
approaches, protein and antibody microarrays are promising new ones for
biomarker discovery. Although at present they have several limitations with
respect to sample preparation, sensitivity, specificity, and so on, protein and
antibody microarrays will no doubt become a standard adjunctive method in
the actual clinical scene. With this in mind, we have been establishing a novel
system for antibody microarray in which surface plasmon resonance (SPR)
technology is utilized for the signal detection. Up to 400 real-time antibody-
target bindings could be measured simultaneously within a single hour.
Although SPR is assumed to be an expedient technology for protein and
antibody microarrays, here we describe its advantages and disadvantaged
compared to other detection technologies. This review focuses on the
technological aspects of these two methods and a discussion of their clinical
usefulness. We further emphasize the interpretation of the protein and antibody
microarray results in combination with the results of DNA microarray and
intracellular pathways mainly constructed from data on protein-protein
interaction.
INTRODUCTION
Such as western blot and immunohistochemical analyses are robust but low-
throughput methods for investigation of protein expression. A high-throughput method
for rapid screening of expression against hundreds of proteins in complicated biofluids is
necessary for deep understanding of disease processes on a proteomic level.
As is known from comparative expression studies, mRNA and protein expression
levels do not always correlate [1-3]. A protein expression level is often modulated
depending on several kinds of post-transcriptional and -translational processes.
Therefore, detailed expression information on multiple proteins is required to appreciate
a complicated biological phenomenon. High-throughput methodologies, which allow
*Corresponding author: Tel: +81-438-52-3919; Fax: +81-438-52-3918; E-mail: ukazue@kazusa.or.jp

24 Frontiers in Drug Design & Discovery, 2006, Vol. 2 Usui-Aoki et al.
fast, direct and quantitative detection of hundreds of proteins, are also required. The
current state of protein profiling technologies, such as 2D-PAGE combined with mass
spectrometry, must still cope with significant drawbacks before they are able to fulfill all
the needs for high-throughput proteomic analysis.
Antibody microarrays are a solid-phase technology that can be used to screen
expression of multiple proteins concurrently [4]. Several antibody-based techniques have
been developed and used to profile protein expression [5-9]. The present platform for
antibody microarrays utilizes the technologies developed for either DNA microarrays [5,
9] or sandwich immunoassay techniques [10]. The use of two differentially labeled
extracts is similar to conventional DNA microarray analysis and allows for pair-wise
comparisons. This kind of dual label system can be surveyed in the literature and is now
commercially available [5,9]. Micro-sandwich immunoassay technique, another
representative approach to antibody microarrays, requires two antibodies to capture and
detect a target protein, but its sensitivity and specificity are superior to those of other
techniques.
We attempted to develop a high-throughput antibody based protein detection system
that could be used to screen protein expression patterns in several biological fluids. For
this purpose, we utilized surface plasmon resonance (SPR) technology and established
an SPR-based antibody microarray system [11]. For SPR biosensors, target molecules
are immobilized on a gold-coated chip, unlabeled biological samples are loaded, and the
angle change of reflected light is measured [12-15]. The angle change indicates the
change of the target protein in different biological samples. Two key advantages of SPR
biosensors are the ability to use unlabeled samples, in which native protein conformation
is preserved, and the ability to continuously monitor binding kinetics. On the other hand,
the SPR biosensors have insufficient sensitivity (especially compared with sandwich
immunoassay), and consequently require more protein for loading samples. In addition,
there has been no available detector for multiple SPR-signals. To exploit the advantages
and overcome the disadvantages of this method, we developed a novel platform for the
antibody microarrays based on SPR technology. In this system, up to 400 real-time
antibody-target bindings could be measured simultaneously within a single hour. We
first provide an overview of protein and antibody microarray technologies and then
introduce our system and most recent improvements to the system; finally, we discuss
how to validate the antibody microarray results.
OVERVIEW OF HIGH-THROUGHPUT PROTEIN EXPRESSION ANALYSIS

Currently, array formats for protein analysis fall into two major classes, protein
arrays, Fig. (1A) and antibody arrays, Fig. (1B-D). In the protein array format, numerous
recombinant or purified proteins (reverse-phase protein microarrays) are immobilized on
the solid phase. Patient samples such as serum from autoimmune disease are incubated
onto the microarray, and the bound ligands (e.g., autoantibody) are detected by direct or
indirect labeling. In the antibody format, numerous antibodies are immobilized onto the
substratum as specific capture molecules, and endogenous protein levels related to
patient’s pathophysiological status are comprehensively monitored.
Although technologies established for DNA microarrays have been adapted to
protein and antibody microarrays, protein and antibody microarrays are faced with
significant challenges that have yet to be solved by the establishment of DNA
Protein and Antibody Microarrays Frontiers in Drug Design & Discovery, 2006, Vol. 2 25
microarray technologies. The most serious obstacle is the vast range of protein
concentrations in different biological fluids. For example, the range of protein
concentration in any cell is more than 1010; this range can not be covered by preexisting
methodologies. Furthermore, PCR-like direct amplification methods do not exist for
proteins. Consequently, chemical enhancement of the detected signals is an obligatory
step for protein and antibody microarrays [16-19]. Expected sensitivity is at least at the
femtomolar level within acceptable background. Moreover, the labeling and chemical
enhancement methods must have linear reactions and be reproducible to insure reliable
quantitative analysis. The elimination of natural contamination of reagents for chemical
enhancement (biotin, peroxidases, alkaline phophatases, fluorescent proteins, and so on),
should also be attempted so as to minimize background activity.
Fig. (1). Classification of protein/antibody microarray platforms. (A) Schematic illustration of

protein microarray format. Numerous recombinant or purified proteins (reverse-phase protein
microarrays) are immobilized on the solid phase (red). An analyte containing a specific ligand
(e.g., antibody; black) is incubated onto the microarray. The bound ligands are detected by direct
or indirect labeling (yellow stars). (B-D) Schematic illustration of protein microarray formats. In a
sandwich immunoassay format (B), unlabeled targets (red) are captured by immobilized antibodies
(black) and then detected by labeled detection antibodies (green). In a direct labeling format (C),
fluorescently labeled targets (red with yellow stars) are captured by immobilized antibodies
(black) and the signals correlate with the amount of captured targets that are directly detected. In
our SPR-based format (D), unlabeled targets (red) are captured by immobilized antibodies (black),
and then SPR arises when light is reflected from the thin layer of gold at the surface. When targets
in the sample bind and concentrate at the surface, the incident angle of the reflected light changes
and an SPR signal is detected.
Protein Microarray
Protein microarrays should be useful in screening ligands (e.g., antibodies) in sample
fluids as mentioned before, Fig. (1A). Protein microarrays can also analyze other
interactions with peptides, small molecules, oligosacchaloides or DNA as well as
proteins. Several different platforms for protein microarray are already available for
clinical use; we only introduce “reverse-phase” protein microarrays in this short review
article.
Because most potential molecular markers and targets are proteins, proteomic
profiling is expected to yield more direct answers to functional and pharmacological
questions than does transcriptional profiling. Reverse-phase protein microarrays

represent a technology uniquely suited to screening a broad range of molecular markers
or pathway targets in large numbers of samples simultaneously in a high-throughput
manner [20-22]. An advantage of reverse-phase protein microarrays is their high-
throughput capabilities using low sample volumes. Typical reverse-phase protein
microarrays for patient biopsy materials are printed with nL-µL of whole cell lysate onto
immobilized support materials to allow for probing with specific antibodies. This
advantage is perfectly matched for molecular profiling of clinical patient samples.
Frequently only a small amount of patient material is available for molecular analysis.
Nishizuka et al., developed a reverse-phase protein lysate microarray system with the
60 human cancer cell lines used by National Cancer Institute to screen for new
anticancer agents (NCI60) [23]. To identify molecular markers, they also developed a
multistep protocol starting with NCI60. The first step of this protocol is the selection of
candidate markers based on differential transcript expression levels with cDNA
microarrays [24]. To confirm and quantify the protein expression level of these
candidates, reverse-phase protein microarrays were utilized. Finally, they identified two
best candidates, villin for colon cancer cells and moesin for ovarian cancer cells [24].
Villin appears at last as useful as the currently used colon marker cytokeratin 20 [25].
Reverse-phase protein microarrays, prepared using simple procedures and standard
microarray equipment, represent a powerful tool for the discovery of new biomarkers.
Antibody Microarray
Antibody microarrays are a high-throughput technology concurrently used to screen
for protein expression and allow the identification and the quantification of a large
number of target proteins from a minute amount of a sample within a single experiment.
Antibody microarrays based on traditional sandwich immunoassay, Fig. (1B) [4, 26], do
not require direct labeling of proteins contained in sample fluids. Instead of protein
labeling, detection antibodies recognizing an epitope opposite to immobilized antibody-
recognizing sites are labeled with a fluorescent dye. The obvious limitation of this assay
is the requirement of having two non-overlapping accessible epitopes and two validated
antibodies for a target molecule. Nevertheless, antibody microarrays based on sandwich
immunoassay are the most sensitive and specific assay. For example, the IL-6 assay
demonstrated sensitivity at 2pg/mL [27]. Especially in the cytokine field, this type of
assay is rapidly expanding.
On the other hand, direct labeling of proteins contained in sample fluids is a less
sensitive but more convenient method for comprehensive analysis of protein expression
without complete sets of paired antibodies, Fig. (1C). All of the proteins in the sample
are labeled with either a fluorophore or a hapten tag such as biotin, Fig. (1C). Direct
labeling has several advantages. One is that the paired antibodies essential for antibody
microarrays based on sandwich immunoassay are not required. Thus, it is easier to
expand array-contents as soon as a single antibody to a target becomes available.
Another advantage is in the adaptation of the differential display technique, a commonly
used technique in DNA microarrays. More specifically, a mixture of two samples
labeled with different tags is incubated on the same microarray. Co-incubation of the
sample in question with a control sample makes it possible to provide internal
normalization of the target proteins. A disadvantage of the direct labeling method is the
potential interruption of antibody-antigen interactions depending on excess labeling at
the epitope and/or its surrounding area. The background signal arising from direct
labeling is another disadvantage of this method. Because all proteins in a sample are
labeled, including highly sticky proteins, strong nonspecific binding of these proteins to
antibodies or to the microarray substrate may cause measurable interference.
Nonspecific binding could also reduce detection sensitivity to target proteins and thus
deteriorate the accuracy of the data. The background in the direct labeling method can be
reduced by manipulation of surface chemistries [28] or through further improvement in
blocking and/or washing protocols. Although much effort has been exerted to improve
the direct labeling method, the protocol’s optimization has not been accomplished
SPR-BASED ANTIBODY MICROARRAY

The availability of high quality, specific antibodies is the limiting factor, and starting
point, for successful utilization of antibody microarray technology [4]. Unfortunately,
high-quality antibodies are currently available for only a small percentage of the known
proteins involved in signal networks and gene regulation. The generation of large
comprehensive libraries of fully characterized specific antibodies is a significant
challenge for the new generation of antibody microarrays.
Taking into consideration this difficulty, we have been conducting a project
comprehensively generating antibodies against mouse KIAA proteins and performing
their validation [29, 30]. Using our libraries of antibodies, we established a novel
antibody microarray system in which surface plasmon resonance (SPR) technology is
utilized for signal detection, Fig. (1D), [11]. Up to 400 real-time antibody-target
bindings could be measured simultaneously within a single hour. This rapid detection
was achieved by a direct readout of the bindings using SPR technology. Protein A-
purified polyclonal antibodies were spotted onto a Bare Gold Affinity Chip in 20 X 20
formats as shown in Fig. (2A). All antibodies used here had their titers checked by
ELISA and their specificities checked by several immunological techniques, including
western blot and immunohistochemistry. Some of these data are freely available through
our InGaP database, a comprehensive database of gene/protein expression profiles of
mouse KIAA (http://www.kazusa.or.jp/create).
Anti-tubulin antibody was used for a positive control and spotted at each corner of
the matrix. For the reference SPR angle changes, reference ROIs (regions of interests)
were drawn in close proximity to each target and simultaneously analyzed with the target
angle changes, Fig. (2B). The reference data were subtracted from each target datum,
and the subtracted data were used for subsequent validation and analysis. Each curve in
Fig. (2C) consists of chronologically detected SPR angle changes (Resonance change
units: RCU), and each color indicates a different target. To assess the reproducibility of
this SPR-based antibody microarray system, we performed regression analysis using the
results from independent experiments. Fig. (2D) is a scatter plot of the RCU of the
corresponding targets on differently spotted Affinity Chips. The results using brain
sample showed high reproducibility between replicate experiments, with correlation
coefficients of 0.94.
Several technical and practical problems remain unresolved, however. A central
unresolved issue in SPR-based antibody microarray concerns the detection of low-
abundance proteins from small tissue samples. Clinical samples, such as patient biopsies,
are often limited in the amount and concentration of the targeted proteins. Although a
cubic centimeter of tissue contains approximately 109 cells, tissue samples from needle
biopsy or cell aspiration contain less than 105 cells. It is therefore necessary to improve
Fig. (2). SPR-based antibody microarray system on which approximately 400 different
antibodies are spotted. (A) Overview of the Affinity Chip. 400 spots are located within 1 cm2
grid area, and an adhesive gasket forms a flow cell with an approximate volume of 47 µl between
the Affinity Chip and the gasket. (B) Immobilized antibodies were visualized through a CCD
camera and defined as regions of interest (ROIs). Reference ROIs were also drawn in close
proximity to each target ROI by the system software. SPR signals of these ROIs were measured by
a FLEXCHIP™ Kinetic Analysis System (HTS Biosystems). (C) We prepared a sample derived
from an adult mouse brain and performed an analysis using the SPR-based antibody microarray
system. Each curve consists of chronologically detected RCU, and each color indicates a different
target. (D) RCUs for replicated experiments are plotted against each other. The plot shows a high
correlation between two independent experiments with correlation coefficients of 0.94 (brain
sample).
the sensitivity for an antibody microarray system. In our SPR-based antibody microarray
system, this can be satisfied by avoidance of the electrostatic non-specific adsorption.
We reduced the density of the carboxyl group on the gold surface by using a mixture of
HS-PEG-COOH and HS-PEG-OH, Fig. (3A, Scheme C). Fig. (3B(c)) shows that the
SPR signal change on the blank spot (which indicates the non-specific adsorption was
completely abolished by preparation of the mixture solution with 0.1 mM HS-PEG-
COOH and 0.9 mM HS-PEG-OH. Using clued protein samples, satisfactory sensitivity
(approximately 30 ng/ml) was achieved by surface chemistries for antibody immobiliza-
tion. Further improvement has been achieved by refinement of the detection instrument
MultiSPRinter® (Toyobo Co., Ltd.), Fig. (3C), [31]. Especially, the refinement of the
flow cell and flow pass enabled high-throughput SPR analysis with minimal sample
volume (~ 200 µl).
Fig. (3). Surface chemistries and a novel instrument for improvements to the SPR-based
antibody microarray system. (A) Carboxyl groups were introduced on the gold surface via three
procedures. SAMs of 11-CDT (Scheme A), HS-PEG-COOH (Scheme B), a HS-PEG-COOH and
HS-PEG-OH mixture (Scheme C) were formed on the UV-irradiated surface. The introduced
carboxyl groups were activated by EDC and NHS, and then reacted with antibodies. (B) The
unreacted NHS ester groups were blocked by H2N-PEG-OH. SPR signal changes on the antibody
microarray by exposure of the crude lysate (mouse brain) using 1.5 % BSA containing HEPES
buffer as the running buffer. The antibody arrays were prepared via (a) Scheme A, (b) Scheme B
and (c) Scheme C. The data shown in panels ( A) and (B) are reproduced from Kyo et al., [31]. (C)
An overview of the detection unit for MultiSPRinter® (Toyobo).
THE INTERPRETATION OF THE PROTEIN AND ANTIBODY

MICROARRAY RESULTS
Measurement of gene/protein-expression profiles using microarray technology is
becoming increasingly popular among the biomedical research community. Although
there has been great progress in this field, we are still confronted with a difficult
question after completing their experiment: how to validate the large data sets regarding
gene/protein-expression? Experimental verification of microarray results is extremely
time-consuming. In this context, the browser system shown in Fig. (4) was developed to
enable us to readily integrate microarray results on an intracellular pathway related to
spotted targets molecules.
Fig. (4). The interpretation of protein and antibody microarray results. (A) Schematic
representation of the pathway including mKIAA1027. The proteins are essentially represented by
ellipses. A modification of the shape refers to a protein’s functions (e.g., an eclipsed shape
represents protein kinase). The proteins are also displayed with several different colors. Dark blue
indicates the targeted mKIAA protein. Light blue indicates identified mKIAA-interactors. Red
indicates other components of the cellular pathway selected by Ingenuity Pathway Analysis. The
interactions and regulations among the proteins are illustrated by the different lines of connection.
A detailed explanation of these differences is recorded on the Search Results of InCeP
(IntraCellular Pathway, based on mKIAA protein-protein interactions) database (http://www.
kazusa.or.jp/create/). These data will be freely available through our InCeP database. Each
component of the pathway is represented by the gene symbol (e.g., Promyelocytic leukemia, PML,
etc.). (B) Confirmation of microarray results by MAKOT. Temporal gene expression data from
GEO (Gene Expression Omnibus: http://www.ncbi.nlm.nih.gov/geo/) including mKIAA1027
genes were incorporated into MAKOT, and then the data were visualized on reconstructed
pathway views. Each gene is represented by a circular symbol; each time a point is represented by
a different diagram. The color of each circular symbol represents the expression level of an
individual gene at each time point (red = upregulated, black = unchanged, green = downregulated).
Gene names are also indicated at the lower right of each symbol (only the diagram at 0 hour is
represented).
Since the spotted targets of our antibody microarray system are mKIAA proteins,
which are large proteins with an average length of 4.6 kb and deduced gene products of
830 amino acid residues [32], we decided to generate intracellular pathways related to
the mKIAA proteins. For the first step, we developed an intracellular pathway database
based on mKIAA protein-protein interactions. mKIAA protein interactions were
identified by MS/MS analysis following immunoprecipitation with anti-mKIAA

antibodies. Most mKIAA proteins are still functionally unresolved, although the
interactions with biologically known molecules should enable us to extend the pathway
using pathway-assist softwares such as Ingenuity Pathway Analysis software (Ingenuity
Systems, Mountain View, CA) and PathwayAssist (Ariadne Genomics, Inc., Rockville,
MD). The generated pathways are now distributed through InCeP (IntraCellular
Pathway, based on the mKIAA protein-protein interactions) database (http://www.
kazusa.or.jp/create). Users can freely access InCeP through the Internet and download
the graphical display as well as the curated information, Fig. (4A). For the next step, we
generated MAKOT (Mutualistic Associations of Knowledge obtained by Computational
Comparative Technology), in which researchers are able to superimpose the information
across gene/protein-expression datum on a related pathway. Temporal changes in the
gene/protein-expression profile could be visualized on the pathway and subsequently
validated from the detectable expression-linkage among proximate edges (represented by
a circular symbol in Fig. (4B); proteins, protein complexes, and small molecules).
Furthermore, the reliability between two proximate edges of the pathway could be
statistically evaluated by Pearson’s correlation coefficient. The statistical evaluation on
MAKOT could also consider delayed expression-linkage of downstream target proteins
on the same signal cascade. We are now preparing to distribute MAKOT through our
website (http://www.kazusa.or.jp/create).
Fig. (4A) represents the pathway related to mKIAA1027, also known as talin 1,
which plays a significant role in the cell-cell and cell-extracellular matrix adhesions
through interactions with the intracellular domain of integrin β. In addition to the
previously identified function of mKIAA1027/talin 1 in the transduction of the integrin
signal, we have identified a novel functional aspect of mKIAA1027/talin 1 mediated by
Promyelocytic leukemia (Gene Symbol in Fig. (4A) is PML), a novel binding partner for
mKIAA1027/talin 1. PML was originally identified as a fusion protein with retinoic acid
receptor α, but this novel interaction was experimentally verified by us. To demonstrate
the usefulness of MAKOT in the validation of gene/protein-expression profiles, we
superimposed temporal gene expression changes of the pathway members such as
mKIAA1027/talin 1 and PML on this pathway. For instance, the application of MAKOT
to this expression profile revealed statistical significance in many proximate edges such
as mKIAA1027/talin 1 and PML, Fig. (4B). Therefore, superimposition of gene/protein-
expression profiles on the intracellular pathway would be helpful in validating the
profiles. In this review article, we used gene-expression datum transferred from GEO
(Gene Expression Omnibus: http://www.ncbi.nlm.nih.gov/geo/). MAKOT is similarly
applicable to protein-expression data.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge all staff at Kazusa DNA Research Institute. This
study was supported by the CREATE Program from JST , a grant from Kazusa DNA
Research Institute to H.K. and a Grant-in-Aid for Young Scientists (B) by MEXT
KAKENHI (17710173) to K.U.
ABBREVIATIONS
NCI60 = The 60 human cancer cell lines used by National Cancer Institute to screen
for new anticancer agents
KIAA = “KI” stands for “Kazusa DNA Research Institute” and AA are reference
characters.
SAMs = Self-assembled monolayers
11-CDT = 11-carboxy-1-decanethiol
EDC = 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride
NHS = N-hydroxysuccinimide
SPR = Surface plasmon resonance
RCU = Resonance change unit
InGaP = Integrative Gene and Protein expression database
InCeP = IntraCellular Pathway based on mKIAA protein-protein interactions
MAKOT = Mutualistic Associations of Knowledge obtained by Computational
Comparative Technology
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The Use of Biomarkers to Detect Cervical Neoplasia

and to Diagnose High-Grade Cervical Disease
Douglas P. Malinowski*
TriPath Oncology, 4025 Stirrup Creek Drive – Suite 400, Durham,
North Carolina 27703, USA
Abstract: The detection of cervical carcinoma and its malignant precursors is

currently accomplished using the Pap smear in conjunction with testing for the
presence of human papillomavirus (HPV). This screening approach is
successful at identifying patients with cervical disease with a very high
sensitivity, but with limited specificity. In order to improve the accuracy of
cervical disease detection and diagnosis, a number of approaches have been
employed to incorporate molecular diagnostics into the testing procedure. Two
investigational approaches to identify biomarkers have been employed: (1) the
use of specific biomarkers based upon known keratinocyte response to HPV
infection; and (2) the use of genome-wide expression profiles to identify new
genes whose expression is altered in response to HPV infection and the
transformation process. Both approaches have identified biomarkers that
appear suitable for the detection of the mild dysplasia precursors of disease,
the malignant precursors of moderate-severe dysplasia and cervical carcinoma.
Some biomarkers are suitable for the detection of HPV infected cells
displaying mild dysplasia while others are more specific to moderate-severe
dysplasia and carcinoma (disease-specific markers). The disease-specific
markers appear to be over-expressed in high-grade cervical disease and
represent aberrant entry of the infected cells into the S-phase of the cell cycle.
These markers appear promising in molecular diagnostic applications to detect
malignant cells in both histology and cervical cytology specimens. An
emerging diagnostic paradigm will be discussed where HPV DNA analysis
represents measurements of transient infection and risk for future disease;
analysis of HPV oncogene transcripts distinguishes active versus transient
infection; and detection of aberrant S-phase induction represents a measure of
active disease.
INTRODUCTION
Cancer of the uterine-cervix represents a major gynecologic cancer that affects
women on a worldwide basis. Each year, there are approximately 517,000 new cervical
cancer cases diagnosed on a global basis. In the US and Europe, the incidence of cervical
cancer has diminished during the last five decades due to the introduction of the Pap
*Corresponding author: Tel: (919) 206-7102; E-mail: dmalinowski@tripathimaging.com

36 Frontiers in Drug Design & Discovery, 2006, Vol. 2 Douglas P. Malinowski
smear for the routine detection of cervical cancer and its malignant precursors. In
comparison to the global incidence of cervical cancer, there are approximately 10,370
new cases of cervical carcinoma diagnosed in the USA during 2005.
Cervical neoplasia is a slowly progressing disease that is initiated through the
infection of cervical keratinocytes by oncogenic strains of Human Papillomavirus
(HPV). The development of cervical carcinoma is preceded by the malignant precursors
of moderate-severe dysplasia, which constitute Cervical Intraepithelial Neoplasia 2, 3
(CIN2, 3) in histology specimens and High-Grade Squamous Intraepithelial Lesion
(HSIL) in cytology specimens. Mild dysplasia, which can harbor underlying cervical
disease, is referred to as CIN1 in histology and Low-Grade Squamous Intraepithelial
Lesion (LSIL) in cytology. In this review, high-grade cervical disease is defined as
moderate-severe dysplasia in a cervical biopsy specimen (CIN2+ lesion). Finally,
suspicious appearing cells can be categorized as either Atypical Squamous Cells –
cannot rule out HSIL (ASC-H) or Atypical Squamous Cells of Uncertain Significance
(ASC-US). Underlying cervical disease can still be present within the LSIL (CIN1),
ASC-H and ASC-US specimens. Appropriate clinical management guidelines exist to
manage patients across this spectrum of morphological classifications of cervical
abnormalities [1-3].
Current diagnostic methods used to screen for the presence of cervical neoplasia
include the use of testing for the presence of HPV DNA in a cervical sample and the use
of the Pap smear for the detection of morphologically abnormal cells within the cervical
specimen. These current methods provide high sensitivity for the detection of cervical
neoplasia, but with a low specificity for disease detection. Furthermore the reliance on
morphology to detect cervical neoplasia if often confounded by the appearance of
cellular abnormalities related to benign physiological conditions that often appear as
malignant. This can result in a large number of false positive cases being referred to
follow up examination and unnecessary procedures. As a result of these diagnostic
limitations, considerable research has been conducted to identify additional molecular
markers that could be used to improve the specificity for disease detection and to
eliminate the subjectivity inherent in morphology-based disease classification methods.
Investigations into the genomic alterations of the HPV infected cervical keratinocytes,
primary human cervical lesions and known host genes affected by HPV have been
investigated to identify suitable markers for use in cervical disease screening. Of
particular note, alterations in cell-cycle control, DNA replication, RNA transcription and
extracellular matrix interactions have been consistently identified using a number of
different microarray-based transcriptional profiling approaches. In this review, we will
review the current status of translational research related to the characterization of these
changes and in particular, the characterization of markers suitable for the detection of
HPV induced dysplasia and the specific identification of malignant transformation with
subsequent diagnosis of high-grade cervical disease. Markers suitable for the detection
of cervical dysplasia and the detection of high-grade cervical disease (CIN2+) will be
discussed. Markers specific for high-grade cervical disease represent aberrant entry of
the cervical cells into the S-phase of the cell cycle and are distinct alterations associated
with cervical disease. Thus, the detection of aberrant S-phase entry appears to be a
specific characteristic of high-grade cervical disease that can be used to detect malignant
cells in both histology and cervical cytology specimens. An emerging diagnostic
paradigm will be discussed where HPV DNA analysis represents measurements of
The Use of Biomarkers Frontiers in Drug Design & Discovery, 2006, Vol. 2 37
transient infection and risk for future disease, analysis of HPV oncogene transcripts
represents active infection versus transient infection and detection of aberrant S-phase
induction represents a measure of active disease.
MOLECULAR BIOLOGY OF CERVICAL NEOPLASIA:

Human papillomavirus (HPV) is recognized as the pathogenic agent responsible for
the development of cervical cancer and the presence of HPV has been associated with
>99% of all cervical cancers. HPV is a circular DNA virus, consisting of approximately
8 kilobases of double-stranded DNA and encodes a small number of viral proteins.
Currently, there are over 100 HPV viral subtypes identified, yet all these viruses share a
tropism for infection and replication within epithelial cells. Within the HPV family of
viruses, there are both non-oncogenic forms and oncogenic forms of the virus. The non-
oncogenic forms of the virus (including HPV types 6 and 11) are associated with
common warts and condyloma. The oncogenic forms of HPV (including types 16 and
18) are associated with cervical carcinoma. The oncogenic forms of HPV can be
classified into high-risk and intermediate-risk viral subtypes. The high-risk HPV viral
subtypes include: HPV types 16, 18, 45, and 58; and the intermediate-risk HPV viral
subtypes include: HPV types 31, 33, 35, 39, 51, 52 and 69 [4]. Of particular interest are
the viral subtypes associated with cervical cancer [5, 6]. These high-risk HPV viral
subtypes are DNA tumor viruses that encode two viral specific oncogene proteins (E6
and E7) responsible for the transformation of infected cervical keratinocytes [7, 8]. The
pathogenesis of cervical cancer involves changes in cell cycle control, alterations in
HPV structure and expression patterns and finally changes in the chromosome integrity
of the infected keratinocytes. These changes are summarized in Fig. 1.
Some of the molecular changes within HPV infected keratinocytes include the
inactivation of the key tumor suppressor pathway functions of p53, Rb [5, 9] and TGF-
Beta [10]; the immortalization of cells [11]; abrogation of DNA damage response
[12,13]; and the generation of centrosome abnormalities leading to genomic instability
[14,15]. The HPV oncogenes E6 and E7 are known to produce alterations in the cell
cycle including over-expression of cyclin dependent kinases (CDK) [9], cyclin proteins
[16-18] and CDK inhibitors such as p16INK4A [19-21]. Likewise, an increase in
proliferation is observed with HPV infected cells including activation of telomerase [22-
24]. Cervical neoplasia is also characterized by re-entry of the differentiated
keratinocytes into the cell cycle. This is evidenced by detectable expression of
Minichromosome Maintenance (MCM) Proteins within the stratified epithelial layers of
the uterine-cervix based upon detection of MCM5 and MCM2 [25, 26]. These effects are
summarized in Fig. 2 and in Table 1.
HPV TESTING AND DNA DETECTION IN CERVICAL SCREENING

Most HPV infections are transient in nature, with the viral infection resolving itself
within a 12-month period. For those individuals who develop persistent infections with
one or more oncogenic subtypes of HPV, there is a risk for the development of neoplasia
in comparison to patients without an HPV infection. Given the importance of HPV in the
development of cervical neoplasia, the clinical detection of HPV has become an
important diagnostic tool in the identification of patients at risk for cervical neoplasia
development. Both the high-risk and the intermediate-risk HPV viral subtypes are
Fig. (1). The molecular characteristics of cervical neoplasia. The alterations that occur within
cervical neoplasia are initiated through infection of the undifferentiated cervical keratinocyte by
oncogenic viral subtypes of HPV. HPV viral replication occurs concomitantly with the
differentiation of the cervical keratinocyte. In order to maintain DNA replication, the HPV
oncogenes E6 and E7 re-initiate entry into the cell cycle. The activities of the HPV also result in
additional abnormalities that include: (i) alteration in the structure of the HPV virus from an
episome to viral integration within the keratinocyte genome; (ii) genetic instability of the infected
keratinocyte leading to tetraploidy, aneuploidy, and chromosomal amplification (3q+); (iii) loss of
HPV E2 expression; and (iv) increased expression of the HPV oncogenes E6 and E7.
detectable through the use of the DiGene Hybrid Capture II assay (DiGene Corporation,
Gaithersburg, MD.) The NCI ALTS clinical trial (ASCUS LSIL Triage Study) helped
define the clinical utility of HPV testing in combination with annual Pap screening.
Within the ASCUS patient population, 50-60% of these patients harbored HPV
infections with 7% of patients presenting high-grade disease upon colposcopy and
biopsy confirmation. The ALTS trial concluded that HPV triage of ASCUS patients was
more effective for disease detection upon referral to colposcopy than repeat cytology or
direct referral to colposcopy. However, HPV testing of LSIL patients was not useful as a
triage for colposcopy because of the high prevalence of HPV infection in this patient
population [27-31]. The use of HPV testing has been approved by the FDA in
combination with Pap screening as both a reflex test within the ASCUS patient
population and as a primary screening for cervical disease detection [32]. The utility for
HPV testing in a primary diagnostic setting has been recommended in recent guidelines
from the American College of Obstetrics and Gynecology (ACOG) [33]. In addition to
the Hybrid Capture II assay, a number of PCR-based methods have been shown to
Fig. (2). Molecular pathogenesis of cervical cancer. A variety of cellular processes are altered
through HPV induced cervical neoplasia and collectively contributes to neoplastic transformation
and the onset of cervical cancer.
support HPV detection and viral genotyping using liquid-based cervical cytology
specimens [34-38]. The presence of HPV positive patients has been shown to predict the
future occurrence of CIN3+ disease [39]. This study monitored patients with a normal
history of Pap smears and followed the natural history of disease development over a 10-
year period in relation to the presence or absence of HPV. The presence of HPV was
shown to correlate with a higher incidence of CIN3+ disease over the 10 year period
than HPV negative patients as shown in Fig. 3.
A similar study evaluated disease development in women with a normal Pap history
over a 10 year period. This study demonstrated that the presence of HPV types 16 and
18 correlated with a higher likelihood of disease recurrence than the other high-risk HPV
types within the HPV positive, cytology normal patients [40].
The clinical utility of HPV-based screening for cervical disease is in its negative
predictive value. An HPV negative result in combination with a history of normal Pap
smears is an excellent indicator of a disease-free condition and a low risk of cervical
neoplasia development during the subsequent 1-3 years. However a positive HPV result
is not diagnostic of cervical disease; rather it is an indication of infection. Although the
majority of HPV infections is transient and will spontaneously clear within a 12-month
period, a persistent infection with a high-risk HPV viral subtype indicates a higher risk
for the development of cervical neoplasia. To supplement HPV testing, a number of
molecular markers associated with cervical neoplasia have been evaluated in order to
improve the clinical specificity for cervical disease diagnosis.
Table 1. Human Papillomavirus Open Reading Frame Genes
HPV Gene Viral Function Keratinocyte Interactions
LCR Regulatory control of transcription, None reported

replication and host interactions
L1 Major capsid protein None reported
L2 Minor capsid protein None reported
E1 Viral replication
Maintenance of viral episome
E2 Transcriptional regulation Repression of telomerase expression

Cofactor for DNA replication
E4 Keratin interactions Actin-cytoskeleton interactions

Viral shedding
E5 Growth factor receptor interactions EGFR and MAPK signaling pathways

and signal transduction
E6 and E7 Prolongs division phase of the - MAPK Signaling pathway activation

cell-cycle to promote replication. - p53 and Rb inactivation
Responsible for malignant - Abrogation of G1/S and G2/M cell-cycle
transformation of the cervical checkpoints
keratinocyte following infection with
- Activation of E2F transcription pathways
oncogenic subtypes of HPV
- Activation of centrosome duplication
- TGF B pathway inativation
- c-myc interaction
- Telomerase activation
HPV E6 AND E7 mRNA DETECTION AND ACTIVE HPV INFECTIONS

The ability to detect HPV E6 and E7 mRNA has been reported using the NASBA
nucleic acid amplification technology for potential clinical applications. The detection of
the E6 and E7 mRNA from cervical samples infected with high-risk viral subtypes has
been reported in several publications [41-46]. The utility of detecting mRNA for E6 and
E7 in comparison to the detection of HPV DNA, based upon the L1 major capsid protein
gene, was compared for the ability to detect disease within the ASCUS and LSIL patient
population in a 2-year follow-up study. The results of this study showed that the
detection of E6 and E7 mRNA was more specific for the detection of patient with a high
likelihood of future cervical disease when compared to the detection of HPV L1 DNA
[44]. These results suggest that mRNA detection reflects active viral transcription
Fig. (3). Predictive value of HPV testing in cervical disease screening. The presence of HPV
positive specimens is associated with an increase in the development of CIN3+ disease. The
absence of HPV is associated with a significantly lower incidence of cervical disease over the
same time period.
associated with ongoing disease whereas the detect of HPV DNA is more likely to detect
transient infections in patients who will clear the viral infection within a 12-24 month
period and thus will not progress on to cervical disease. These results are summarized in
Table 2.
Table 2. Prediction of CIN2+ Cervical Disease During 2-Year Follow-up of ASCUS and
LSIL Patients: Comparison of Detection Methods for High-Risk HPV E6/E7
mRNA and L1 DNA*
Measurement NASBA Detection of E6/E7 mRNA PCR Detection of L1 DNA
HPV Subtypes Detected 16,18,31,33,45 13 HPV Viral subtypes
HPV Prevalence 23.4% 54.6%
Sensitivity 85.7% 85.7%
Specificity 84.9% 50.0%
PPV 37.5% 15.4%
NPV 98.3% 97.1%
Odds Ratio 69.8 5.7

* Data summarized from reference [44].
BIOMARKERS ASSOCIATED WITH HPV INDUCED CERVICAL

NEOPLASIA
Microarray Analysis of Cervical Carcinoma and HPV Cell Line Models
The application of DNA microarray transcriptional profiling of primary cervical
carcinoma lesions, cultured primary cervical carcinoma cell lines and HPV infected cell
line model systems has yielded information related to both the over-expression and the
reduction of gene expression associated with HPV-induced cervical neoplasia [47-59].
Among these different experimental approaches, a common set of genes with altered
expression patterns have been detected including: (i) activation of a proliferation
signature (including cell cycle regulation, signal transduction, DNA replication, and
cellular proliferation); and (ii) increase in gene expression associated with extracellular
matrix (ECM) interactions and proteolysis (ECM remodeling, tissue invasion and
metastasis) [60-72]. The common over-expressed genes that have been identified in
cervical neoplasia using these transcriptional profiling approaches are shown in Table 3.
A number of these genes have been examined for expression patterns of the encoded
proteins within cervical neoplasia. The most interesting biomarkers have also been
characterized for potential utility in molecular diagnostic applications. A number of
genes associated with regulation of the cell cycle and DNA replication have been shown
to undergo an increase in expression level in cervical neoplasia by microarray analysis.
These include various cyclins, the cyclin dependent kinase inhibitor, p16INK4A and
proteins associated with DNA replication including PCNA, CDC6, members of the
MCM family of DNA licensing factors (MCM2-7) and Topoisomerase II alpha
(TOP2A) [51,53,58,59].
Cell Cycle Regulatory Proteins and Cervical Neoplasia

Cyclins and Altered Expression in Cervical Neoplasia
Results from microarray analyses have consistently identified cyclins as over-
expressed genes in cervical neoplasia and HPV induced cellular transformation. Cyclin
E, A and B have all been shown to be over-expressed in squamous cell cervical
neoplasia as well as in LSIL and HSIL lesions [9,15-18, 60-62]. Likewise, Cyclins A
and B have been shown to be over-expressed in cervical adenocarcinoma and its
malignant precursors [18]. Over-expression of these various cyclins are detected in
HPV-induced neoplasia and are summarized in Table 4.
Cyclin Dependent Kinase Inhibitors
p16INK4A (p16) is an inhibitor of cyclin dependent kinases (CDK 4 and CDK6) and
functions in the progression from G1 to S phase of the cell cycle. In response to
infection by high-risk HPV infectious, p16 is over-expressed in cervical neoplasia
including HSIL lesions and carcinoma [63]. Over-expression of p16 has been shown to
correlate with HPV type 16 and 18 infections and can be detected in both squamous cell
carcinoma and adenocarcinoma [9, 63-66]. The specificity of p16 over-expression has
been examined and is associated with carcinoma, biopsy confirmed CIN 2+ lesions and a
significant number of LSIL – CIN 1 lesions [67-70]. The p16 protein can be detected in
both histology specimens as well as liquid-based cytology specimens [9, 64, 71, 72].
Immunohistochemical detection of p16 has been shown in both the cytoplasm and the
nucleus of mild-severe dysplasia (CIN1-CIN3), squamous cell carcinoma and adeno-

carcinoma [73].
Table 3. Microarray Analysis of Cervical Neoplasia and HPV Infected Cell Lines.
Identification of Common Gene Categories and Specific Over-Expressed Genes*
Extracellular
Investigational Signal Cell cycle DNA replication Cellular
Matrix Ref.
Approach Transduction regulation and transcription proliferation
Interactions
Primary genes IGFBP3 Cyclin A TOP2A BST2 Claudin 1 [50]

identified by Cyclin B1 MCM2 IFI27 Claudin7 [51]
transcriptional Cyclin B2 MCM4 Co-actosin Aquaporin 5 [54]
profiling of Cyclin E MCM6 MDK Mesothelin [55]
human cervical CDK2 B-Myb NP25 ITGB6 [58]
carcinoma FOSL2 CRIP1 ITGA3
BTEB1 Laminin V
PCNA MMP-2
MMP-9
UPA
Additional Pleitrophin p16INK4A MCM3 Bcl-2 ITGA9 [22]

genes p21 Waf-1 MCM5 Survivin [47]
identified using MCM7 Ubiquitin [48]
HPV induced Telomerase E2-C [49]
expression in TOP2B [52]
cell line model c-myc [59]
systems CDC6
* Gene identities are listed using the currently accepted gene ID nomenclature available at the National Center for
Biotechnology Information (http://www.ncbi.nlm.nig.gov).
DNA Replication and Cellular Proliferation

Cell division cell protein 6 (CDC 6) and minichromosome maintenance (MCM)
proteins participate in the early stages of eukaryotic DNA replication through the
regulated assembly of the pre-replication complex onto DNA during the G1 phase of the
cell cycle. CDC6 participates through recruiting additional proteins into the pre-
replication complex consisting of the origin recognition complex (ORC), CDC6, Cdt1
and the MCM hexamer (MCM2, MCM3, MCM4, MCM5, MCM6, MCM7). This multi-
protein complex is responsible for the initiation of DNA replication during the S-phase
of the cell cycle. The MCM proteins consist of 6 distinct protein members, MCM2-7,
which form a hexamer and participate in both the formation of the pre-replication
complex and function as a helicase to unwind DNA during replication [74]. CDC6 has
been shown to be elevated in cervical neoplasia at both the mRNA and the protein level
[73, 75]. Furthermore, CDC6 expression has been reported to show some specificity for
high-grade cervical dysplasia [75]. MCM5 was the first member of the MCM family that
was demonstrated as a potential biomarker in cervical neoplasia [25]. Subsequently, the
proteins for MCM2 and MCM7 were also shown to be promising biomarkers in cervical
neoplasia; detection was consistent with re-entry of the differentiated keratinocytes into
the proliferative phase of the cell cycle [26, 76]. More recently, MCM5 has been
characterized in cervical neoplasia and shown to be elevated at both the mRNA and the
protein levels [73, 75]. Likewise, characterization of MCM2, MCM6 and MCM7
expression in cervical neoplasia has been reported and shown to be elevated at both the
mRNA and the protein levels for moderate-severe dysplasia and carcinoma [77, 78].
Table 4. Alterations of Cyclin Expression in Cervical Neoplasia
Cyclin Function Expression Status in References

Protein Cervical Neoplasia
Cyclin D1 Activation of G1 Phase of - Cyclin D1 decreased in 97% [9, 17, 61, 62]
and D3 Cell Cycle of HSIL and 72% of invasive
Regulatory subunits of cervical cancer
CDK4 and CDK6 - Cyclin D3 decreased in 51%
Links mitogenic stimuli to of squamous cell carcinoma
cell-cycle progression - Increased expression within
cervical carcinoma associated
with poor disease-free survival
Cyclin E Activation of G1 Phase Increased in 97% of LSIL, 92% [9, 16, 160]
of the Cell Cycle of HSIL and 82% of squamous
CDK2 regulatory subunit cell carcinoma
Cyclin A Activation of S and M Increased in 35% of squamous [9, 18]

Phases of the Cell Cycle cell carcinoma and
Regulatory subunit for adenocarcinoma
CDK2 (S-phase) and
CDK1 (M-phase)
Links anchorage dependent
cell growth to cell cycle
progression
Cyclin B Activation of the G2 and M Increased in cervical neoplasia, [9, 18]

Phases of the Cell Cycle squamous cell carcinoma and
CDK1 regulatory subunit adenocarcinoma
The application of these markers has been reported to detect atypical cervical cells
that are consistent with aberrant S-phase entry and display a high likelihood of CIN2+
disease upon biopsy [77-79]. A summary of the expression patterns for these
proliferation signature markers is shown in Table 5.
The MCM genes are known to be under the control of the E2F-1 transcription factor.
As a result of HPV E7 disruption of the Rb tumor suppressor pathway, the constitutive
activation of the E2F-1 transcription factor results in constitutive transcription of S-
phase genes and permits the aberrant entry of the transfected cells into the S-phase of the
Table 5. Analysis of Cell Cycle Regulatory and S-Phase Genes in Cervical Neoplasia
Marker Function Increase in mRNA Protein Over Reference

Between Cervical Expression in
Carcinoma and Normal Cervical Disease
Cervical Epithelium by IHC Analysis
MCM2 DNA Licensing Factor 5-fold CIN2+ >> CIN1 [26, 77, 78]
MCM6 DNA Licensing Factor 3-fold CIN2+ >> CIN1 [77, 78]
MCM7 DNA Licensing Factors 3-fold CIN2+ >> CIN1 [76-78]
TOP2A DNA unwinding 4-fold CIN3+ > CIN2 > CIN1 [77, 78]
INK4A
p16 CDK inhibitor 6-fold CIN1+ [73, 75]
MCM5 DNA Licensing Factor Cancer, CIN3 >>> Normal CIN1+ [73, 75]
CDC6 Pre-initiation complex Cancer, CIN3 > Normal CIN3+ > CIN2, CIN1 [73, 75]
formation
cell cycle [9, 79]. The aberrant over-expression of the S-phase genes, including MCM2-
7 and TOP2A, appears similar to the same set of genes implicated in the proliferation
signature of tumorigenic cell lines [80]. This aberrant expression of the S-phase genes
also appears to be a characteristic of cervical neoplasia with potential diagnostic
applications [79] (Fig. 4).
Extracellular Matrix Proteins and Cervical Neoplasia

Several categories of genes associated with extracellular matrix proteins have been
identified by DNA microarray profiling of cervical carcinoma including Claudin 1 and 7,
Laminin V and various members of the integrin family. Claudins are a group of
membrane proteins that form tight junctions in epithelial tissues and appear to participate
in the formation of watertight barriers in tissues. Claudins have been shown to be
elevated in cervical neoplasia, with tissue expression increasing with the severity of
cervical disease [81-83]. Similar observations have been made for several proteases
including cathepsins, and matrix metalloproteinases [84], Laminin V [85-86] and
integrin beta 3 [87-89]. Although useful in histology applications, none of these markers
has demonstrated utility in cervical cytology applications. These results are summarized
in Table 6.
Signal Transduction Pathways and Biomarker Characterization
MAP Kinase and ERK
Investigations into the function of the HPV oncoproteins E5, E6 and E7 have
revealed that these HPV encoded proteins interact with a number of key signal
transduction and proliferation pathways within infected cervical keratinocytes. The E5
protein has been shown to interact with EGFR [90, 91], and both E5 and E6 have been
shown to activate the MAP kinase pathway [92-94]. Both EGFR and MAP kinase link
external growth signals to cellular proliferation through signal transduction pathways.
The activation of the MAP kinase pathway, through the phosphorylation of ERK1 and
ERK2, has been shown to increase with increasing severity of cervical disease.
However, there was no correlation with the phosphorylation status of ERK1 and ERK2
and the clinical outcome of cervical neoplasia or the detectable presence of high-risk
HPV [94].
Fig. (4). Molecular pathogenesis of cervical carcinoma. The persistent infection of cervical
keratinocytes with oncogenic subtypes of HPV ultimately results in the aberrant entry of the
infected cells into the S-phase of the cell cycle. The resulting constitutive over-expression of S-
phase genes occurs through abrogation of the normal cell-cycle control of S-phase entry through
the combined action of E6 and E7. This aberrant S-phase induction appears to be a molecular
characteristic of HPV induced neoplastic transformation.
Beta-Catenin and Signal Transduction Pathways

HPV has been shown to interact with the Notch signal transduction pathway. The
Notch signaling pathway is an evolutionarily conserved pathway in metazoan organisms
that is responsible for the determination of cell fate based upon interaction with adjacent
cells an as such, participates in the fundamental processes of embryogenesis, cell fate
determination and cellular differentiation. Alterations in the Notch pathway have been
described in cervical neoplasia [95-99].
The Wnt signal transduction pathway has been implicated in cervical neoplasia and
one of the key components of the Wnt pathway, beta-catenin, has been demonstrated to
be over-expressed in cervical neoplasia [100]. Beta-catenin is known to interact with E-
cadherin in the generation of functional complexes that also participate in the linkage of
extracellular matrix interactions though the Focal Adhesion Kinase (FAK) pathway
[101,102]. Activation of the Wnt signaling pathway in cervical neoplasia results in
changes to beta-catenin that include increased expression levels, re-localization of the
protein from the nucleus to the cytoplasm and phosphorylation. In the majority of
cervical neoplasia, beta-catenin was shown to be over-expressed and localized in the
cytoplasm. However, these alterations have not been observed in all carcinomas and thus
represent one of the heterogeneous alterations that are observed within cervical
carcinoma. These same studies have also demonstrated alterations in E-cadherin in
cervical neoplasia, most notably the loss of detectable E-cadherin in cervical neoplasia.
Like Beta-catenin, there are no consistent alterations in the expression levels of E-
cadherin that can be demonstrated consistently in cervical neoplasias [100,101,103-106].
Table 6. Extracellular Matrix Gene Over-expression in Cervical Neoplasia
Marker Function Cervical Disease Status Reference
Claudin 1 Tight junctions in Differentially expressed in cervical [81-83]

epithelial tissues neoplasia with expression increasing
with severity of lesion
Claudin 7 Tight junctions in Differentially expressed in cervical [81-83]

epithelial tissues neoplasia with expression increasing
with severity of lesion
Cathepsin F Protease Increased in cervical carcinoma [84]
MMP-9,10,11,12 Protease Increased in cervical carcinoma [84]
Laminin V Extracellular matrix Increased in cervical carcinoma and [85,86]

component CIN3 lesions. Cytoplasmic localization
of the protein observed in cervical
carcinoma
Integrin Beta 3 Extracellular matrix Increased expression in cervical [87-89]

component carcinoma
Alterations in the TGF-beta signal transduction pathway have been described in

cervical neoplasia that include inhibitory effects on Smad binding to DNA and the
abrogation of G1/S boundary checkpoint of the cell cycle [10].
Proliferation Signatures in Cervical Neoplasia
Activation of proliferation within keratinocytes includes E6 interaction with c-myc
and telomerase activation [22-24] and the activation of the E2F1 transcription pathway
leading to over-expression of genes related to entry into the S-phase of the cell cycle and
DNA replication [9, 107]. A summary of the signal transduction pathways affected in
cervical carcinoma and the malignant precursors are summarized in Table 7.
The results from these investigations clearly demonstrate that alterations in key
signal transduction pathways occur within cervical neoplasia and most are produced as a
Table 7. Oncogenic HPV and Key Molecular Pathway Interactions
HPV Key Pathway Clinical

Pathways Diagnostic Utility Reference
Protein Component Observation
Rb Tumor E7 pRb Rb protein Not routinely utilized [5, 9]

Suppressor degraded in in diagnostic
cervical neoplasia applications
Loss of Rb
detected by IHC
E2F-1 E7 E2F-1 target genes Induction of S- Increased expression [9, 79,

Transcription phase genes of S-phase genes at 107]
including MCM mRNA and protein
proteins level in both dysplasia
and carcinoma
c-myc E6 Telomerase Increased Telomerase activation [22-24]

Transcription expression telomerase activity is observed as an early
event in cervical
neoplasia
No diagnostic utility
EGFR E5 EGFR EGFR required for No consistent [90]

Signaling HPV induced alterations in EGFR
hyperplasia observed in cervical
neoplasia
MAPK E5, E6 ERK1 Increased MAPK Levels of [91-94]

Signaling ERK2 activity reported in phosphorylated Erk1
response of E6 and Erk2 correlated
sequence variants with stage of cervical
disease.
No correlation
observed with HPV
presence of clinical
outcome
Wnt Signaling Beta-catenin - Increased beta- Beta-catenin over- [100-106]

E-Cadherin catenin expression expression not
and cytoplasmic observed in all
localization as an cervical carcinomas
early event in Decreased expression
cervical neoplasia of E-cadherin not
- Decreased consistent in all
expression of E- cervical neoplasia
cadherin in
cervical neoplasia
result of interactions with oncogenic HPV viral subtypes. Based upon our current
understanding of cancer biology, it is tempting to speculate that the observed alterations
in these signal transduction pathways are likely key events in the early stages of cervical
neoplasia. However, none of these alterations have demonstrated diagnostic utility to
detect cervical neoplasia in a majority of lesions or to yield prognostic information about
disease progression and clinical outcome.
BIOMARKERS AND CLINICAL UTILITY

The investigations summarized in this chapter have revealed a number of molecular
alterations in cervical neoplasia at both the gene and protein expression level. Some of
the alterations, such as those described for the MAPK and Wnt signaling pathways have
demonstrable alterations in component proteins within these signal transduction
pathways. However, the variability observed in many of these markers renders them
insufficient for use in routine clinical settings. However, a number of other alterations
have been described with consistent correlations in cervical neoplasia. These
investigations have revealed that a number of biomarkers, such as p16INK4A, MCM5 and
Claudin 1 show protein expression levels that increase with the severity of the cervical
dysplasia. These markers do not show a preference for any particular stage of cervical
disease, yet all show a strong correlation with cervical dysplasia. These markers appear
suitable for use in the detection of cervical dysplasia with the ability to detect all stages
of cervical dysplasia in both histology and cytology samples. As such, these markers
would appear suitable to help resolve suspicious lesions and cells where the morphology
is inadequate to permit a clear diagnosis of cervical dysplasia versus more benign
metaplasia or reactive repair processes within the uterine-cervix. There are a second
group of biomarkers that appear to demonstrate a more quantitative distinction between
moat-severe dysplasia and cervical carcinoma with minimal detectable expression in
mild dysplasia or normal proliferating cervical cells. Examples of the biomarkers
specific for high-grade cervical disease (CIN2+) are MCM2, MCM6, MCM7 and
TOP2A. It is anticipated that future investigations into these markers will permit the
development of specific molecular diagnostic tests to detect high-grade cervical disease
within suspicious ASCUS and ASC-H cases or within indeterminate SIL cases. Finally,
a small group of biomarkers associated with the extracellular matrix have been shown to
correlate with invasive cervical cancers. Markers such as Laminin V appear suitable to
detect the invasive potential of cervical carcinoma. These applications have been
summarized in Table 8.
BIOMARKERS AND THE EMERGING DIAGNOSTIC PARADIGM FOR

CERVICAL DISEASE TESTING
Given the causal affect of oncogenic HPV on the development of cervical carcinoma,
testing for HPV is an important component of cervical disease testing. However, as we
have reviewed in this chapter, multiple investigations into HPV have shown a strong
correlation with future risk for cervical disease. However, the measurement of HPV (as
practiced with the HC2 test) is not specific for cervical disease, and thus is not
diagnostic for patients who have current infection versus those patients with transient
infections. Measurements of the E6 and E7 mRNA transcripts within cervical samples
have demonstrated a higher specificity and PPV for the detection of patients with active
HPV infections and are more accurate to identify patients with an active disease process
versus those patients with a transient HPV infection. These dilemmas in HPV testing
have been recently summarized in an article by Elizabeth Unger et al. [108]:
“While viral load, transcript copies and transcript pattern were statistically associated
with CIN III, none of these measures effectively discriminated between HPV-16 women
with disease requiring treatment and those who could be followed. Cellular proliferation
and differentiation pathways affected by HPV should be investigated as biomarkers for
cervical cancer screening”.
Table 8. Markers of Cervical Neoplasia
Marker Set Marker Expression Clinical Utility
Markers to detect cervical dysplasia

INK4A
p16 Biomarker protein expression linearly Useful to distinguish dysplasia from
Claudin 1 correlated with severity of disease benign lesions and to resolve
Claudin 7 indeterminate lesions
MCM5
Markers to detect high-grade cervical disease (CIN2+)
CDC6 Biomarker protein preferentially Promising markers to aid in the

MCM2 expressed in CIN2+ lesions diagnosis of CIN2+ disease in cytology
MCM6 and suspicious histology SIL cases
MCM7
TOP2A
Markers to detect invasive cancer
Laminin V Biomarker expression correlated with Promising markers to aid in the

MMP-9 invasive carcinoma prognosis of cervical cancers
It is interesting to note that many of the proliferation biomarkers characterized in this

review appear to mirror the advice of Dr. Unger and reflect a series of critical
keratinocyte response to active HPV infections. More importantly, a number of these
biomarkers appear to be very specific for the detection of high-grade cervical. As such, it
is tempting to speculate that future diagnostic paradigm for cervical disease screening
and diagnosis could employ two or three testing methodologies. HPV testing for DNA
would indicate those patients at risk for future disease – applicable for general screening.
The detection of HPV E6 and E7 transcripts would further identify those patients with
active HPV infections that represent a higher risk for disease. The use of biomarkers
would permit the identification of those patients within the HPV positive population that
have active cervical disease and require immediate clinical follow-up. Thus molecular
diagnostic tests to identify patients at risk and patients with disease would constitute a
future diagnostic paradigm in cervical disease screening and diagnosis. This diagnostic
paradigm is shown in Fig. 5. As suggested in this paradigm, the detection of oncogenic
HPV DNA in a cervical specimen would identify patients at high-risk for disease. The
application of molecular diagnostic tests to these HPV positive patients would identify
the subset of HPV positive patients that have active disease and require immediate
referral to colposcopy and treatment. An HPV positive patient with a negative molecular
diagnostic assay result could be referred to the HPV mRNA assay to identify patients
with active viral infection and an increased likelihood for cervical disease development
within 2 years. These high-risk patients would be monitored routinely with a molecular
diagnostic test for the detection of cervical disease.
Fig. (5). Emerging diagnostic paradigm for cervical disease detection. The presence of HPV DNA
in cervical specimens is a useful measurement of transient HPV infections since the majority of
these viral infections are normally cleared within 12 months. HPV DNA testing is thus a measure
of risk for future cervical disease development. The detection of the E6 and E7 transcripts is
associated with a higher incidence of cervical disease and appears to be a useful measurement of
active viral replication and a higher likelihood of disease development within 12-24 months.
Finally the detection of aberrant S-phase induction is a characteristic of neoplastic transformation
and is a measurement of current cervical disease.
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New Developments in the Field of Protein

and Metabolism Assays Aimed at
Drug Discovery Processes
Kothandaraman Narasimhan, Ponnusamy Sukumar and

Mahesh Choolani*
Diagnostic Biomarker Discovery Laboratory, Department of Obstetrics
and Gynaecology, National University Hospital, 5 Lower Kent Ridge Road,
Singapore 119074
Abstract: The advent of new high-throughput proteomic and metabolic assays
using mass spectrometry (MS) has significantly benefited drug discovery
process. This process is accelerated with the miniaturization of detection
devises (nanotechnology and biosensors) to carry out rapid and effective
screening. Using proteomics (protein arrays) it is possible to globally
investigate the molecular basis of disease, drug action leading to drug
development. Similarly metabolic assays for nutritional status, expanded
neonatal screening through tandem MS could swift through several thousand
data points associated with a particular disease (either proteins/peptides or
metabolites) in a short span of few minutes and come up with highly sensitive
and accurate diagnosis. Nanobiotechnology raises fascinating possibilities for
new analytical array based assays (receptor-ligand binding, DNA-DNA
hybridization, or both) and microanalytic separations, each of which will be
mentioned here with respect to their ability to affect the drug discovery
processes. Molecular profiling by DNA microarray technology has made
significant contributions to the understanding of molecular targets for diseases
such as cancer. One of the challenges is how to efficiently utilize the
accumulated research data to develop new diagnostic and/or prognostic
markers and therapeutic targets. Proteomics based disease therapeutic research
involves high-throughput protein structure determination (e.g. structural
biology of protein tyrosine kinases (PTKs). These processes involve
conventional antibody based arrays as well as targets identified using structural
biology for narrowing down targets for drug delivery. Drug discovery
processes aimed at generating inhibitors for the treatment of malignancies are
believed to be dependent on the gain of function of specific PTKs. Current
research include Src as a target for pharmaceutical intervention, JAK kinases
in leukemias/lymphomas, and phosphoproteomics. The following areas
mentioned above will form the key focus of this chapter.
*Corresponding author: Tel: +65-68741625; Fax: +65-68723056; E-mail: obgmac@nus.edu.sg

56 Frontiers in Drug Design & Discovery, 2006, Vol. 2 Narasimhan et al.
INTRODUCTION
Impact of Proteomics Technologies on Drug Discovery
The early 2000 saw the advent of highly sophisticated technologies with the merger
of chemistry and biology resulting in “Chembiology”. One of the major benefactors of
this amalgamation is for the field of clinical diagnostics, which has seen tremendous
opportunity to develop new and effective screening approaches using modern
technologies such as protein array and mass spectrometry. Over the last five years we
have seen advances both in instrumentation as well as introduction of new techniques,
which has resulted in higher sensitivity as well as miniaturization to accommodate more
reaction surface and also considerable reduction in time required to carry out metabolic
assays. The major advances were observed for tagging bio-molecules such as proteins
and nuclei acids on different surfaces to enhance the assay capabilities.
This chapter on the “New developments in the field of protein and metabolism assays
aimed at drug discovery processes” focus on the recent developments associated with
metabolic assays using (i) proteins arrays (ii) mass spectrometry (iii) recent advances in
miniaturization technologies and specific assays with nanobiology and nanotechnology
and (iv) finally different strategies employed for designing better drugs against kinases
involved in different diseases especially for cancer. This comprehensive review covers
all the recent advances as well as the most recent scientific literature, which has
mentioned these technologies for better development of more sophisticated metabolic
assays.
Advances in proteomics have resulted in a bigger role for proteomics associated
interventions in clinical screening for different diseases [1]. This technology offers
researchers new approaches to investigate the molecular basis of disease, drug action,
and development [2]. A proteomics approach, identifying protein targets associated with
various diseases, can ultimately provide a basis for early disease detection and
eventually guide to a rational design for pharmacological intervention [3]. The following
sections will especially explore novel applications of chromatographic, electrophoretic,
immunologic, and mass spectrometric technologies to analyze proteomes for clinical
benefit. Modern proteomic technologies will have an important role in drug discovery
especially for molecular diagnostics and practice of medicine in the post-genomic era.
The commonly used technologies are 1-D and 2-D gel electrophoresis (1D and 2D-GE)
for protein separation and followed by analysis of proteins by mass spectrometry (MS).
Microanalytical protein characterization with multidimensional liquid chromatography/
mass spectrometry (MD LC-MS) improves the throughput and reliability of peptide
mapping [4]. Matrix-Assisted Laser Desorption Mass Spectrometry (MALDI-MS) has
become a widely used method for determination of biomolecules including peptides,
proteins [5].
Functional proteomics technologies include yeast two-hybrid system for studying
protein- protein interactions [6-7]. Establishing a proteomics platform in the industrial
setting initially requires implementation of a series of robotic systems to allow a high-
throughput approach for analysis and identification of differences observed on 2-D
electrophoresis gels. Protein chips are also proving to be useful for several applications
associated with disease diagnosis and high-throughput assays. Proteomic technologies
are now being integrated into the drug discovery process as complimentary to genomic
approaches. Toxicoproteomics involves the evaluation of protein expression for
New Developments in the Field of Protein Frontiers in Drug Design & Discovery, 2006, Vol. 2 57
understanding of toxic events, is an important application of proteomics in preclincial

drug safety [8-9]. Use of bioinformatics is essential for analyzing the massive amount of
data generated from both genomics and proteomics [10].
Proteomics is providing a better understanding of pathomechanisms of human
diseases. Analysis of different levels of gene expression in healthy and diseased tissues
by proteomic approaches is as important as the detection of mutations and
polymorphisms at the genomic level and may be of more value in designing a rational
therapy. Protein distribution / characterization in body tissues and fluids, in healthy as
well as in diseased, is the basis of the use of proteomic technologies for molecular
diagnostics. Proteomics will play an important role in medicine of the future which will
be personalized and will combine diagnostics with therapeutics [11].
Clinical Proteomics Market

Clinical proteomics market is comprised of innovators and futurists whose work will
help shape the product expectations of future researchers. For suppliers hoping to
dominate this technology, understanding the current experiences of today’s visionaries
and monitoring how their needs change over time is critical. The effectiveness of clinical
proteomics will depend on two technological components: rapid, multiplex protein
detection assays and data analysis systems to assimilate vast amounts of protein
expression data from healthy and diseased individuals into clinically relevant data sets.
The number of companies involved in proteomics has increased remarkably during
the past few years. More than 300 companies have been identified to be involved in
proteomics and 202 of these are profiled in the report with 450 collaborations. The
markets for proteomic technologies are difficult to estimate as they are not distinct but
overlap with those of genomics, gene expression, high throughput screening, drug
discovery and molecular diagnostics [12]. Collectively based on a recent estimate
available in the public domain, the value of markets for proteomic technologies in the
year 2005 is about $6 billion and is expected to increase to $10 billion by the year 2010
and $18 billion by the year 2015, with a projected annual growth of 30%. The largest
expansion will be in bioinformatics and protein biochip technologies. Important areas of
application are cancer and neurological disorders.
Introduction to Protein Arrays

Microarrays were first developed as a tool for high-throughput genotyping and gene
expression analysis [13]. By combining small sample volumes and the ability to generate
massive amounts of information in a single experiment, microarrays vastly accelerated
the search for functional effects of single nucleotide polymorphisms (SNPs) and
modified gene expression (increases and decreases in mRNA production) in normal and
diseased physiological states. Gene expression arrays operate under the pretence that
changes in mRNA levels ultimately correlate to changes in encoded protein levels; often
this assumption does not hold true. Additionally, gene expression arrays provide no
information on protein post-translational modifications (phosphorylation, glycosylation,
etc.) that affect cell function. To examine expression at the protein level and otherwise
acquire quantitative and qualitative information on proteins of interest, this has led to the
development of protein microarrays.
A protein microarray consists of antibodies, proteins, protein fragments, peptides,

aptamers or carbohydrate elements that are coated or immobilized in a grid-like pattern
on small surfaces. The arrayed molecules are then used to screen and assess patterns of
interaction with samples containing distinct proteins or classes of proteins [14-15]. A
brief overview of each type of protein array techniques is mentioned in the following
sections. Protein array technology allows high-throughput screening for gene expression
and molecular interactions. Protein arrays appear as new and versatile tools in functional
genomics, enabling the translation of gene expression patterns of normal and diseased
tissues into protein product catalogue. Protein function, such as enzyme activity,
antibody specificity, and other ligand-receptor interactions and binding of nucleic acids
or small molecules can be analyzed on a whole-genome level by using this new
technology.
Currently development in proteomics associated with diagnostics is associated with
(1) detection of antigens and antibodies in blood samples; (2) profiling of sera to
discover new disease markers; and (3) in environment and food monitoring. One of the
chief formats is the capture array, in which ligand-binding reagents, which are usually
antibodies but may also be alternative protein scaffolds, peptides or nucleic acid
aptamers, are used to detect target molecules in mixtures such as plasma or tissue
extracts [15]. In diagnostics, capture arrays can be used to carry out multiple
immunoassays in parallel, both testing for several analytes in individual sera for example
and testing many serum samples simultaneously [16].
As the array technology develops, an ever-increasing variety of formats become
available (e.g. nanoplates, patterned arrays, three-dimensional pads, flat-surface spot
arrays, microfluidic chips), and proteins can be arrayed onto different surfaces (e.g.
membrane filters, polystyrene film, glass, silane, gold). Various techniques are being
developed for producing arrays, and robot-controlled, pin-based, or ink-jet printing
heads are the preferred tools for manufacturing protein arrays. CCD cameras or laser
scanners are used for signal detection; atomic force microscopy and mass spectrometry
are upcoming options. The emerging future array systems will be used for high-
throughput functional annotation of gene products. In addition, their involvements in
molecular pathways and their response to medical treatment will become the doctor's
indispensable diagnostic tools [17].
Of all the applications for protein microarrays, molecular diagnostics is most
clinically relevant, and would fit in with the emerging trend in individualized treatment,
or personalized medicine [18]. Different proteins such as antibodies, antigens, and
enzymes can be immobilized within protein microchips. Miniaturized and highly parallel
immunoassays will greatly improve efficiency by increasing the amount of information
acquired with single examination and will reduce cost by decreasing reagent
consumption. Application of protein biochips and microarrays in molecular diagnostics
has good commercial prospects.
Types of Protein Arrays

Antibody Arrays in Cancer Research
The most common type of protein arrays are antibody arrays. Antibody arrays have
valuable applications in cancer research [19]. Many different antibody array technol-
ogies have been developed, each with particular advantages, disadvantages, and optimal
applications. The methods have been demonstrated on various sample types, such as
serum, plasma, and other body fluids; cell culture supernatants; tissue culture lysates;
and resected tumor specimens. The applications to cancer research have included
profiling proteins to identify candidate biomarkers, characterizing signalling pathways,
and the measurement of changes in modification or expression level of cancer-related
proteins [20]. The different other format and applications of antibody arrays include
arrays to measure whole cells, arrays to measure enzyme activities, reverse phase arrays,
and bead-based arrays. Further innovations in the methods and experimental strategies
are broadening the scope of the applications and the type of information that can be
gathered.
Antibody microarrays fall into one of two subtypes: those using matched antibody
pairs for sandwich-type assays and those utilizing single antibodies and a sample
labelling methodology. Several published manuscripts demonstrate the utility and
effectiveness of the sandwich immunoassay microarray [14]. This type of microarray
consists of arrayed capture antibodies and appropriate control and orientation elements.
Assays are performed by adding an antigen standard or test sample, followed by a
detector antibody. The detector antibody is either modified with a directly detectable
label (enzyme, fluorescent molecule, isotope, etc.), or it is biotinylated for detection after
subsequent probing with labelled streptavidin.
Antibody-pair microarrays essentially are multiplexed ELISAs [20]. In fact, standard
commercially available ELISA pairs are readily adapted for microarray use once the
pairs have been screened for cross-reactivity when multiplexed. Sandwich-style antibody
pair microarrays can be used for qualitative or comparative (i.e. treated versus non-
treated) detection of protein analytes or for protein quantification when appropriate
standards are used to assemble calibration curves. When matched antibody pairs are not
available, single-antibody protein microarray protocols involving labelled samples can
be used. As in sandwich antibody pair arrays, the array platform consists of arrayed
antibody (or antibodies). In this assay format, however, the captured protein analytes are
themselves labelled for direct detection, obviating use of a detector antibody. The
method requires that protein samples be labelled beforehand (e.g. with fluorescent
molecule, isotope, or biotin). The label enables detection of any proteins in the sample
that interact with the microarrayed antibody and associated elements. This technique is
useful for examining protein targets, such as poorly characterized cell signalling
proteins, for which paired antibodies do not yet exist. The main drawback to this method
is its lack of antibody redundancy, which helps to ensure specific antigen recognition.
Additionally, since all sample constituents are labelled (i.e. the target as well as other
proteins in the sample), non-specific background signal increases. This technique is
primarily used for comparative and qualitative studies.
Reverse Phase Protein Microarrays

Protein arrays are described for screening of molecular markers and pathway targets
in patient matched human tissue during disease progression. Reverse-phase protein
microarrays represent a new technology that can generate a multiplex readout of dozens
of phosphorylated events simultaneously to profile the state of a signalling pathway
target even after the cell is lyzed and the contents denatured [21]. This technology may
offer a new opportunity to measure and profile these signalling pathways, providing data
on post-translational phosphorylation events not obtainable by gene microarray analysis.

In contrast to previous protein arrays that immobilize the probe, reverse phase protein
array immobilizes the whole repertoire of patient proteins that represent the state of
individual tissue cell populations undergoing disease transitions [22]. A high degree of
sensitivity, precision and linearity was achieved, making it possible to quantify the
phosphorylated status of signal proteins in human tissue cell subpopulations. Using this
novel protein microarray longitudinal analysis of the state of pro-survival checkpoint
proteins at the microscopic transition stage from patient matched histologically normal
prostate epithelium to prostate intraepithelial neoplasia (PIN) and then to invasive
prostate cancer has been carried out [22]. Reverse phase protein microarrays have been
widely used for the development of reference standard development for molecular
network analysis of metastatic ovarian carcinoma [23]. In addition to elucidation of the
molecular network within a tumor specimen, critical questions are to what extent
signalling changes occur upon metastasis and are there common pathway elements that
arise in the metastatic microenvironment. For individualized combinatorial therapy, ideal
therapeutic selection based on proteomic mapping of phosphorylation end points may
require evaluation of the patient's metastatic tissue. Extending these findings to the
bedside will require the development of optimized protocols and reference standards. At
present a reference standard based on a mixture of phosphorylated peptides is available
to begin to address this challenge [23].
3-Dimensional MALDI-MS Protein Array

Protein profiling and characterization of protein interactions in biological samples
ultimately require indicator-free methods of signal detection, which likewise offer an
opportunity to distinguish specific interactions from non-specific protein binding. A
more recent development in the area of protein array technology is the analysis of
protein interaction and function with a 3-dimensional MALDI-MS protein array [24]. A
new 3-dimensional protein microchip for detecting biomolecular interactions with
MALDI-MS; the microchip comprises a high-density array of methacrylate polymer
elements containing immobilized proteins as capture molecules and directly interfaces
with a commercially available mass spectrometer. The chip was tested in three types of
experiments by detecting antibody-antigen interactions, enzymatic activity, and enzyme-
inhibitor interactions. MALDI-MS biochip-based tumor necrosis factor alpha (TNF-
alpha) immunoassays demonstrated the feasibility of detecting antigens in complex
biological samples by identifying molecular masses of bound proteins even at high
nonspecific protein binding. By detecting model interactions of trypsin with trypsin
inhibitors, the protein binding capacity of methacrylate polymer elements and the
sensitivity of MALDI-MS detection of proteins bound to these elements surpassed that
of other 2- and 3-dimensional substrates tested Immobilized trypsin retained functional
(enzymatic) activity within the protein microchip and the specificity of macromolecular
interactions even in complex biological samples. The underlying technology should
therefore be extensible to whole-proteome protein expression profiling and interaction
mapping.
Protein and Peptide Arrays for Quantifying Kinase Activity in Cell Extracts
High-throughput quantitation of kinase activity in cell extracts has applications in
biomedical research and medical diagnostics. Kinetic analysis of signal transduction
pathways can provide insight into cellular processes such as mitosis and differentiation.
In addition, many disease states are associated with upregulated levels of cellular
signaling molecules. However, accurate measurements of kinase activity directly from
cell extracts are complicated by the complex nature of the extracts, as they contain
multiple kinases as well as phosphatases. Protein arrays have been developed to assay
(to quantify) the activity of multiple tyrosine kinases in cell extracts [25]. Substrates for
different kinases are spotted onto a surface and covalently linked into a polyacrylamide
hydrogel. This hydrogel provides a porous three-dimensional support for the kinase
substrates and maintains the substrates in a hydrated environment [26]. Cell extract is
prepared and incubated over the array. Phosphorylation of the substrates can be detected
using fluorescently labeled antiphosphotyrosine antibodies and scanning the array for
fluorescence. Glutathione-S-Transferase Green Fluorescent Protein (GST-GFP) fusion
protein arrayed on a glass microscope slide.
The other option is to use peptide arrays. Peptide microarray uses protein fragments
on an array [27]. An example of a peptide microarray and its uses can be found in work
performed by Pellois, et al. (2002) [28] and Houseman, et al. (2002) [29], these papers
demonstrate the use of peptide chips for profiling p53 and kinase activities, respectively.
This diverse set of applications clearly demonstrates that the protein microarray is a
powerful tool. Unfortunately, costs for equipment such as arraying robots and slide
scanners limit the number of researchers able to take advantage of this technology.
Future developments in this field will focus on direct methods to detect
phosphorylation of the immobilized substrates and applying this assay to high-
throughput analysis of signal transduction networks and to screening of chemical kinase
inhibitors.
Cellular Lysate Protein Arrays to Study Kinase Activity

Microarrays of cellular proteins are either arrayed as complex protein mixtures, or
arrayed as purified or overexpressed proteins. Complex protein mixture arrays are
essentially dot blots of cellular lysates. Investigations performed by Paweletz, et al. 2001
[22] demonstrate the utility of arraying cellular lysates and probing these arrayed
elements with a variety of antibodies recognizing various intracellular proteins. Creating
microarrays consisting of libraries of purified or overexpressed proteins allows for
screens for protein:protein interactions and kinase activities. A representation of this
type of array is the yeast kinase microarray produced by Zhu, et al. (2000) [30], this
array consisted of 119 of the 122 known yeast kinases. Using this novel protein chip
technology high-throughput analysis of biochemical activities, and analysis of nearly all
of the protein kinases from Saccharomyces cerevisiae is possible. The protein chips were
made from disposable arrays of microwells in silicone elastomer sheets placed on top of
microscope slides. The high density and small size of the wells allows for high-
throughput batch processing and simultaneous analysis of many individual samples.
Only small amounts of protein are required. Of 122 known and predicted yeast protein
kinases, 119 were overexpressed and analysed using 17 different substrates and protein
chips. The results of this study showed many novel activities and that a large number of
protein kinases are capable of phosphorylating tyrosine. This study also showed that,
tyrosine phosphorylating enzymes often share common amino acid residues that lie near
the catalytic region [30].
Surface Plasmon Resonance Imaging-Based Protein Arrays

The most recent technique in the use of protein arrays is the development of surface
plasmon resonance imaging-based protein arrays for high-throughput screening of
protein-protein interaction inhibitors [31]. This technique was first applied for the high-
throughput screening of inhibitor molecules targeting RB-E7 interaction. The E7 protein
produced by high-risk human papillomavirus (HPV) induces a degradation of the
retinoblastoma tumor suppressor RB through direct interaction, which suggests that an
inhibitor for the interaction can be a potential anticancer drug. The glutathione S-
transferase-fused E7 protein (GST-E7) was first layered onto a glutathionylated gold
chip surface that had been designed to specifically bind to GST-fused proteins.
Subsequently, a microarrayer was used to spot the hexa-histidine-tagged RB proteins
(His(6)-RB) onto the GST-E7-layered gold chip surface, and the resulting SPR image
could be analyzed. Upon increased His(6)-RB concentration in the spotting solution, the
SPR signal intensity increased proportionally, indicating that His(6)-RB bound to GST-
E7 in a concentration-dependent manner. The His(6)-RB/GST-E7 interaction was
challenged by spotting the His(6)-RB solution in the presence of a RB binding peptide
(PepC) derived from a motif on E7. The SPR imaging data showed that PepC inhibited
the His(6)-RB/GST-E7 interaction in a concentration-dependent manner. This report
show that the SPR imaging-based protein array chip can be applied to screen small
molecule inhibitors that target protein-protein interaction.
Arrays for Detection of Lipid-Protein Interactions

Protein domains that bind phosphoinositides specifically have emerged as major
determinants in localizing proteins to their site of function. PIP Array and PIP Strips
membranes were used for identifying proteins that have phosphoinositide recognition
domains and for analyzing the lipid-binding specificities of proteins. PIP Array
membranes provide eight different phosphoinositides arrayed in amounts from 100 to
1.6 picomoles, allowing assessment of the strength of protein binding, in addition to the
lipid specificity of protein to lipids. Phosphoinositide-mediated binding of proteins to
PIP Array and PIP Strips membranes is typically analyzed by protein-lipid overlay
assays [32]. Proteins may be detected using standard Western blot procedures in
conjunction with our high-performance, fluorescence-based alkaline phosphatase- and
horseradish peroxidase (HRP)-mediated signal generation systems.
Protein Arrays Based on SELDI-TOF-MS

Another area of research is the development of protein arrays is the use of surface
enhanced laser desorption ionisation-time of flight-mass spectrometry (SELDI-TOF-
MS). This technology could be used for the identification and quantification of specific
protein profiles in crude biological samples as well as to characterize bioactive peptides,
Fig. (1). A typical example of SELDI-TOF-MS application is in the identification of
apolipoprotein variants in plasma [33]. Expression levels of apoA-I and apoA-II and
their glycosylated products were carried out using 1 microL plasma samples.
Strong anionic and weak cationic exchanger ProteinChips (SAX2 and WCX2 chip
surfaces) and WCX2 chip was found to be selective for specific apolipoproteins. Using
the WCX2 chip and analysis via SELDI-MS, apoA-I and apoA-II were separated as
sharp peaks at 28 and 17 kD and did not overlap with other serum protein peaks. Since
these assays can be completed on a large number of clinical samples in approximately 1

h, further development of this technique will facilitate both epidemiological studies and
therapeutic trials in assessing the role of the apolipoproteins and their glycosylated
products in atherosclerosis [33]. Similarly a further modification of SELDI-TOF-MS by
coupling the chip surface with antibodies could facilitate to carry out immunoassays. In
one such study microheterogeneity of transthyretin (TTR) was identified in plasma and
urine using SELDI-TOF-MS immunoassay [34]. TTR was extracted from plasma or
urine onto an antibody-coated (via protein A) affinity chip surface (PS20) using the
SELDI technique. Subsequently samples were subjected to SELDI analysis. In healthy
individuals, TTR in plasma occurred rather consistently in two variants of 13732 +/- 12
and 13851 +/- 9 Da for the native and S-cysteinylated forms and at a smaller signal of
14043 +/- 17 Da for the S-glutathionylated form. In urine of pregnant women, various
signals were observed with a dominant signal at 13736 +/- 10 Da and a varying number
of smaller immunoreactive fragments. These fragments are possibly the consequence of
metabolism in plasma or kidney.
Fig. (1). Identification and characterisation of bioactive peptides using a combination of

combinatorial approach coupled with SELDI-TOF-MS approach.
Other novel applications were also developed for protein arrays based on binding
antigens to array surface. In one such study employing protein arrays, with 266 target
antigens spotted on a protein chip investigators were able to predict if the present
autoantibody repertoire be consulted to predict resistance or susceptibility to the future
development of an autoimmune disease [35]. A selected panel of 27 different antigens
(10% of the array) revealed a pattern of IgG antibody reactivity in the pre-CAD sera that
discriminated between the mice resistant or susceptible to CAD with 100% sensitivity
and 82% specificity (P = 0.017). Surprisingly, the set of IgG antibodies that was
informative before CAD induction did not separate the resistant and susceptible groups
after the onset of CAD; new antigens became critical for post-CAD repertoire
discrimination. Thus, at least for a model disease, present antibody repertoires can
predict future disease, predictive and diagnostic repertoires can differ, and decisive
information about immune system behaviour can be mined by bioinformatic technology
[35].
Metabolomics of Small Molecules Aiding Biomarker Discovery

Metabolomics is the "systematic study of the unique chemical fingerprints that
specific cellular processes leave behind" - specifically, the study of their small-molecule
metabolite profiles [36]. The metabolome represents the collection of all metabolites in a
biological organism, which are the end products of its gene expression. Gene expression
data based on mRNA turnover and proteome analyses do not tell the whole story of what
might be happening in a cell, while metabolic profiling can give an instant 'snapshot' of
the physiology of that cell.
Metabolomics as a Tool for Biomarker Discovery

Metabolomics is a new field of research were high-throughput techniques for
metabolic profiling is carried out using a combination of different chromatography and
MS techniques. Techniques such as gas chromatography (GC), high-pressure liquid
chromatography (HPLC), nuclear magnetic resonance spectroscopy (NMR) and
capillary electrophoresis (CE) are used to separate metabolites according to their various
chemical and physical properties. The molecules are then identified using methods such
as MS. The benefit of metabolomics analysis is the ability to monitor immediate
biochemical consequences of consequences of mutations, changes in the environment
and the effect of treatment with drugs as well as monitor drug metabolism in the body
and their interaction with proteins. The outcome of this new application will help in the
development of new and better drugs for specific type of diseases.
In the following sections a combination of advanced analytical techniques for global
metabolic profiling and chemometrics will be described to illustrate the use of
metabonomics for biomarker detection and identification in different types of metabolic
diseases as well as cancer.
Tandem Mass Spectrometry (MS/MS) an Ideal Tool for Newborn Screening

Tandem mass spectrometry (MS/MS) was introduced in the 1990s for population-
based newborn screening has enabled health-care providers to detect an increased
number of metabolic disorders in a single process by using dried blood-spot specimens
routinely collected from newborns [37-38]. One of the initial applications were MS was
used is in the development of high-throughput screening approaches to detect inherent

metabolic errors in new born babies. The advantages of using tandem mass spectrometry
(MS/MS) for newborn screening substantially increases the number of metabolic
disorders that can be detected from dried blood-spot specimens. Metabolic assays based
on this technique could also be used to monitor the nutritional status of newborn child
for inborn metabolic disorders.
Currently during each year, approximately 4 million babies in the United States have
dried blood spots analyzed through newborn screening programs. This screening is
intended to detect inborn disorders that can result in early mortality or for life
threatening diseases later in life. Detectable metabolic disorders include e.g. (a)
phenylketonuria (PKU), (b) endocrinopathies e.g. congenital hypothyroidism and (c)
hematologic disorders e.g. sickle cell disease. These three groups of disorders account
for approximately 3,000 new cases of potentially fatal or debilitating disease each year
for which outcomes are improved with early identification and treatment through
newborn screening systems.
At present electrospray tandem mass spectrometry (ESI- tandem-MS/MS) is used as
an alternative automated high throughput and broad-spectrum approach to screen for
relatively large number of disorders using minimal amount of patient blood samples in a
very short period of time and was also found to be cost effective. This highly sensitive
technique could result in the compilation of metabolic screening methods to detect
amino acid disorders such PKU, maple syrup urine disease, and homocystinuria among
newborns, and does so with a low false-positive rate [39]. By using specific scan
functions, a large number of amino acids and acylcarnitines in blood spots are quantified
in 2 minutes analytical time. This approach is successfully used to quantify and screen
for argininosuccinic acid in blood spots, which is a key metabolite in the diagnosis of
argininosuccinase deficiency [39].
Tandem Mass Spectrometry enables health-care providers to detect an increased
number of metabolic disorders in a single process by using dried blood spot specimens
routinely collected for newborn screening. MS/MS allows screening of 31 metabolic
disorders using a single analytical run and can detect these disorders within 1 to 2
minutes (Table 1). The advantages of this new platform is to incorporate a number of
target candidate markers for several diseases in one single panel i.e. the number of
disorders that can be detected, by incorporating an acylcarnitine profile, which enables
detection of fatty acid oxidation disorders (e.g. medium-chain acyl-CoA dehydrogenase
[MCAD] deficiency) [40-41] and other organic acid disorders.
Frontiers in Metabolomics for Cancer Research

The term ‘tumour metabolome’ was coined by Mazurek and Eigenbrodt (2003) [42]
to indicate the metabolic characteristics of tumour cells. The tumor metabolome
primarily constitute high levels of glycolytic and glutaminolytic capacities, high
phosphometabolite levels, and a high channelling of glucose carbons to synthetic
processes. Among the different enzymes associate with the tumor metabolism, tumor
M2-pyruvate kinase (M2-PK) has been investigated in detail [43]. Tumor M2-PK is a
key regulator of the tumor metabolome. The Tumor M2-PK test is unique in the sense it
measures the tumor metabolic activity, which in turn acts as a measure of tumor
aggressiveness. Consequently, tumor M2-PK can also be very effective for monitoring
the effects of therapy, detecting possible relapses or metastases, and providing useful
supportive information in the diagnosis and detection of various new secondary tumours.
Table 1. Common metabolic disorders studied in newborns using tandem MS. For a more
comprehensive description please refer to reference [49]. The disorders have
been detected from analyses of dried blood-spot specimens collected during the
newborn period. Certain disorders require complex metabolic profiles and
intermetabolic relation to detect disease with low false-positive and no false-
negative rates. The list of primary metabolic indicators serves only as a guideline
Disorder Primary metabolic indicator
Amino Acids
Phenylketonuria Phe
Maple syrup urine disease Leu/Ile, Val
Homocystinuria (cystathione synthase deficiency) Met
Hypermethioninemia Met
Citrullinemia Cit
Argininosuccinic aciduria Cit
Tyrosinemia, type I Tyr
Fatty Acids
Medium-chain acyl-CoA dehydrogenase deficiency C8, C10, C10:1, C6
Very-long–chain acyl-CoA dehydrogenase deficiency C14:1, C14, C16
Short-chain acyl-CoA dehydrogenase deficiency C4
Multiple acyl-CoA dehydrogenase deficiency C4, C5, C8:1, C8, C12, C14, C16, C5DC
Carnitine palmitoyl transferase deficiency C16, C18:1, C18
Carnitine/acylcarnitine translocase defect C16, C18:1, C18
Long-chain hydroxy acyl-CoA dehydrogenase C16OH, C18:1OH,
deficiency C18OH
Trifunctional protein deficiency C16OH, C18:1OH, C18OH
Organic Acids
Glutaric acidemia, type I C5DC
Propionic acidemia C3
Methylmalonic acidemia C3
Isovaleric acidemia C5
3-hydroxy-3-methylglutaryl CoA lyase deficiency C5OH
3-methylcrotonyl CoA carboxylase deficiency C5OH
Similarly using metabolomics approach it is possible to monitor the dynamics of

glucose metabolism in cancer tissues and body fluids. Another area of research is the
lipidomic profiling changes in carcinogenesis. All the above approaches could lead to a
next wave of metabolomics preceding proteomics techniques to understand the
metabolic phenotype of the tumor. This could facilitate in the deciphering the metabolic
phenotype changes in response to different types of therapy during a particular type of
cancer.
Lipidomics in Cancer Research

Lipids like other metabolites play an important role in cell, tissue and organ
physiology. This fact is demonstrated by a large number of genetic studies as well as
human diseases that involve the disruption of lipid metabolic enzymes and pathways.
Examples of such diseases include cancer, diabetes, as well as neurodegenerative and
infectious diseases. The field of lipids is still in its early stages, and is largely due to the
complexity of lipids and the lack of powerful tools for their analysis [44]. With advances
in analytical techniques such as LC and MS for systems-level analysis of lipids and their
interacting partners, lipidomics could become a promising area of clinical research, and
drug discovery.
One of the established studies using metabolomics based biomarker discovery was in
the field of ovarian cancer research. The phospholipid, lysophosphatidic acid (LPA) was
identified as potential diagnostic and prognostic biomarker for ovarian and other
gynaecologic cancers [45]. Initial studies to detect LPA were dependent on using GS-
MS approaches [46]. To further improve the accuracy and potentially increase the
sensitivity and specificity of the assay, ESI-MS-based method to analyze LPA and
related lysophospholipids [44, 47]. LPA, lysophosphatidylinositol (LPI),
lysophosphatidylserine (LPS), and lysophosphatidylcholine (LPC) could be detected
with high sensitivity (in low pmol range) using this method. LPA and closely related
lysophospholipids isolated from thin-layer chromatography (TLC) plates were analyzed
directly by ESI-MS. This ESI-MS-based assay allows simultaneous detection and
quantitation of all different species of LPAs and LPIs in a sample over a range of at least
5-300 pmol. Moreover, this test was at least 50 times more sensitive when a multiple
reaction-monitoring (MRM) mode was used. Using these protocols in a limited set of
analysis, both LPA and LPI were found to be much elevated in patients with ovarian
cancer.
Other key issue metabolome could address are those were proteomics and genomics
has failed to address. Obviously the focus of this technology would be to carry out
metabolic profiling, identify individuals who will respond from treatments with either
drugs or dietary components? Answers might be associated with a much higher platform
with integration of the data with systems biology approaches. Some of the key questions
to be addressed for metabolomics would be to understand the role of metabolites in
understanding the tumor behaviour. The identification of key metabolites, development
of metabolic biomarkers and type of tissue and body fluids to be looked into will the
future of metabolomics in tumor biology. Similarly one the challenges would be to use
this technology to differentiate metabolic biomarkers that are distinct in tumor cell
environment or in pre-cancerous tissues.
Cost-Benefit Analysis of Universal Tandem MS for Newborn Screening

A study was carried out to estimate potential costs and benefits of routinely using
tandem mass spectrometry (MS/MS) to screen newborns for inborn errors of metabolism
[48]. The analysis of costs and benefits resulting from use of MS/MS in screening of 32,
000 newborn infants using data from the Kaiser Permanente Medical Care Program of
Northern California in addition to other published data showed in the base scenario, the
cost per quality-adjusted life year saved by MS/MS screening was $5827; in the least
favourable scenario, this cost was $11,419, and in the most favourable scenario, $736.
This clearly indicate, the costs per quality-adjusted life year saved by MS/MS screening
for inborn errors of metabolism compare favourably with other mass screening
programs, including those for breast and prostate cancer [48].
The Role of Metabonomics in Understanding Global Systems Biology and Disease

Processes
Metabolic profiles give deeper insights into interactions between the gene and
surrounding environment. This could give a better knowledge about the development of
many diseases. Metabonomics and metabolomic approaches provide important
phenotypic benchmarks in systems biology studies and novel chemometric methods
allow direct integration of multi-omics data with real-world metabolic and physiological
end-points. With developments in both hardware and software fronts this new
technology could be successfully applied to problems in clinical scenarios, toxicology,
functional genomics, personalized healthcare, patient stratification scenarios and for
large scale molecular epidemiology studies in human populations and drug
administration.
Nanoscience and Nanotechnology: an Emerging Opportunity in Biomedical Science

Nanotechnology is an enabling technology, with high potential impact on industrial,
health-related, biomedical, environmental, economy. This is an emerging technology
with bright future in a clinical perspective. This technology has been termed as
“BioNanoMedicine” and has been envisaged as a platform for the 21st century [50]. The
prospects include tailor made therapy for different diseases, diagnosis and drug
targeting. The following sections describe the latest developments and possible impact
of this new technology on the biomedical field.
Nanobiotechnology
Nanotechnology is currently evolving in two ways, one in the development of
nanodevises to screen different diseases conditions [51], drug discovery [52] and the
other in the delivery of specific types of drugs to specific organs affected with specific
diseases [53-54]. The advantages are many as this technology is very flexible and
requires very little amount of target molecules to be coated on the microspheres required
for the disease detection. The ability to study processes at the single-cell level promises
to provide a host of information with benefits in the area of therapeutics and drug
discovery. The terms “microchip”, “microdevice” and “lab-on-a-chip” all refer to
minute, devices that are engineered for biomedical applications. They are rapidly being
developed for use on a single-test basis and show promise as tools for clinical research
and at home self-testing.
Areas of intense research in nanotechnology currently focus on (a) cellular analysis
in microdevices (b) development and fabrication of innovative devices (c) novel biofluid
separators; (d) advances in microanalytical systems; (e) electrokinetics;
dielectrophoresis; (f) advances in chemical, electrochemical, and optical in-line sensor
technology; and (g) novel low concentration detection in capillary electrophoresis
systems. Microfluidic technology to probe chemical and biochemical responses at the
cellular and sub-cellular levels. Subsequent techniques which require novel technology
associated with nanotechnology is in simulation and modelling studies, materials
modification to improve system performance, novel sample preparation protocols, and

analytical techniques.
Nanobiotechnology raises fascinating possibilities for new analytical assays in
various fields such as bioelectronic assembly, biomechanics and sampling techniques, as
well as in chips or micromachined devices. Recently, nanotechnology has greatly
impacted biotechnological research with its potential applications in smart devices that
can operate at the level of molecular manipulation. Micro total analysis system (micro-
TAS) offers the potential for highly efficient, simultaneous analysis of a large number of
biologically important molecules in genomic, proteomic and metabolic studies. This
review aims to describe the present state-of-the-art of microsystems for use in
biotechnological research, medicine and diagnostics.
This technology has been envisioned as an innovative strategy for identifying
biological markers for cancer detection. Development of Flow-thru Chip Proteomics
[55] as well as chemical amplification using continuous–Flow PCR on Chip are
significant advances, which has taken place in the field of nanotechnology and micro-
fluidics, which could significant effects on the future drug delivery, patient screening
and therapeutics. Similarly development in the field of micro analytical chemistry has
opened up a macro-opportunity for clinical nutrition.
Chemotherapy on its own or in combination with other treatments is very effective
for anticancer therapy. Introduced in the middle of the last century, chemotherapy today
still faces the problem of determining which specific agent or agents are able to yield the
desired clinical therapeutical effect for a particular tumor and patient. Numerous tests in
vitro have been developed to detect chemosensitivity and chemoresistance and also for
screening new drugs. Three groups of tests can be defined: (i) cell viability tests (ii)
measurements of cell metabolism and (iii) clonogenic assays. Test time, tissue
preparation, complexity of test performance, and correlation with the clinical progress of
the disease are criteria used to judge how successful the tests are. The introduction of Si-
sensor chips [56], which are able to detect metabolic changes in living cells, has opened
up new possibilities in this field. Basically two sensor principles or types can be
considered: (i) the light-addressed potentiometric sensor (LAPS) and (ii) the multisensor
array (MSA). Whereas LAPS measures one, MSA registers online many parameters (for
instance, impedance, pH, O2, temperature).
Targeted Drug Delivery Achieved with Nanoparticle-Aptamer Bioconjugates

For the first time that targeted drug delivery is possible using nanoparticle-apatamer
conjugates is becoming a reality [57]. Nucleic acid ligands (referred to as aptamers) are
short DNA or RNA fragments that can bind to target antigens with high specificity and
affinity; analogous to monoclonal antibodies. In the field of cancer nanotechnology,
aptamers have the potential to act as targeting molecules – directing the delivery of
nanoparticles to tumour-antigens, present on the surface of cancer cells. In general terms,
therapeutic nanoparticles (~50 – ~250 nanometer) are specially designed delivery
vehicles that can encapsulate a drug within them and release the drug in a pre-
determined and regulated manner which can vary from a sudden release to a slow release
over a period of several years.
Using prostate cancer as a model disease, proof of concept nanoscale targeted drug
delivery vehicles were developed (1 nanometer = 0.000000001 meter), which can target
prostate cancer cells with high specificity and efficiency [57]. Once bound to prostate
cancer cells, the nanoparticle/aptamer bioconjugates were internalised making it possible
for their cytotoxic payload to get released directly inside the cancer cells. The
combination of targeted delivery and controlled release of drugs at the site of cancer will
likely result in "smart therapeutics" that are more effective, yet safer than what is
available today. It as been successfully shown that bioconjugates can efficiently target
and get taken up by the prostate LNCaP epithelial cells, which express the prostate-
specific membrane antigen protein (77-fold increase in binding versus control, n = 150
cells per group). In contrast to LNCaP cells, the uptake of these particles is not enhanced
in cells that do not express the prostate-specific membrane antigen protein [57].
As the initial step, researchers synthesised nanoparticles for controlled drug release
made from a biocompatible and biodegradable PLA polymer system and encapsulated a
fluorescently labelled model drug within them, in order to visualise nanoparticle uptake
into target cells. The nanoparticles in question were designed for attachment to aptamers
so that the binding properties of aptamers for targeting could be preserved. Additional
design criteria consisted of the development of nanoparticles that demonstrated a long
circulating half-life (meaning that they are not readily cleared by the body's immune
system) and nanoparticles that exhibited a strong preferential binding to targeted cancer
cells.
In another study using prostate cancer targeting was modelled using a microfluidic
device and shown to occur under physiological fluid flow conditions that are present in
systemic microvasculature, making their use after intravenous administration
therapeutically relevant [58]. Experimental results show that these bioconjugates
successfully and selectively adhered to PSMA-positive prostate cancer cells, while
PSMA-negative cells were not targeted. The investigators also used high magnification
microscopy and 3-D image reconstruction to study the localisation of the bioconjugates
after incubation with the prostate cancer cells and confirmed that the particles were
rapidly internalised into the targeted cells – an important fact since the payload of
nanoparticles may be released inside the cancer cells in a regulated manner over an
extended period of time. Further studies are going in different laboratories were
chemotherapeutic agent such as docetaxel are encapsulated within the nanoparticles and
the preliminary results were found to be promising.
Bioconjugates represent an exciting prospect in the advancing field of cancer
nanotechnology and hold significant promise for future cancer treatment. With further
modification of the controlled-release polymer system or tweaks to the aptamer targeting
group it may be possible to produce a diverse range of specific and selective
bioconjugates. Drug delivery 'vehicles' can be made to target a many of important human
cancers. Nanoscale drug delivery vehicles are getting closer to clinical realization."
Application of Nanotechnology Based Assays: Nanotechnology Enables New

Approaches for Detecting Proteins and DNA
Controlling the architecture of materials at the scale of 1 to 100 nanometers is a
grand challenge for chemists and engineers and will take another fifty years or more to
accomplish. However the results from this accomplishments will change the way we
diagnose disease and, perhaps more importantly, to give researchers the ability to ask
entirely new questions about the etiology of cancer and other diseases. Most approaches
use (1) antibody-labeled magnetic microparticle (2) DNA barcodes (3) conjugating a
second antibody to the DNA-conjugated gold nanoparticle (4) ELISA-type assay for
detecting proteins and all these techniques often achieve PCR-like sensitivity. In one
example this approach was used to detect a potential marker of Alzheimer’s disease
present in attomolar concentrations in spinal fluid [59]. Doctors currently don’t have any
means for diagnosing Alzheimer's disease in their patients. The disease can only be
confirmed after death, by studying brain tissue. Nanotechnology-based technique could
lead to a test for diagnosing the early signs of Alzheimer’s disease [60]. The Bio-
Barcode-Assay can recognize ADDL, a protein that accumulates in the brains of
sufferers [61]. The first set of experiments that quantify the number of ADDLs in
cerebrospinal fluid was reported in early 2005 [59]. It is several times more sensitive
than conventional tests and could revolutionize disease detection. In future, it might
form the basis not only of a test for Alzheimer’s but also for types of cancer, the human
form of mad cow disease and HIV. The next exciting step would be to move to blood.
Detection of these diseases in blood opens up a vast sea of new opportunities in clinical
diagnostics based on nanotechnology. To perform a Bio-Barcode-Assay, researchers
select antibodies on the basis of the biomarker they need to detect in a solution. Some
antibodies are fixed to magnetic particles while others are attached to spherical gold
particles just 30 nanometres in diameter. Strands of DNA are fixed to the gold
nanoparticle. When antibodies bind to a target biomarker, it becomes sandwiched
between a magnetic particle on one side, and a gold particle and its strands of DNA on
the other. Applying a magnetic field brings this entire "complex" out of solution.
Researchers then release the DNA strands and use a DNA detection device to recognise
their signature sequences. Attempts were also pursued to determine if a protein known as
inhibin can be used to detect ovarian cancer in its earliest stages using the same
technology.
Future Avenues for Nanobiotechnology Research

Utilizing nanoscale atomic force microscope tips of various shapes, dip-pen
lithography is capable of creating gene and protein chips containing millions of spots. In
just a short time, this technology has become the standard method for creating materials
one atomic layer at a time, making it an important tool for developing new nanoscale
materials and characterizing their unique chemical and physical properties. The private
sector is already commercializing dip-pen lithography equipment and working to
develop additional applications.
Development of new probe techniques such as single molecule FRET (fluorescence
energy transfer) at the tip-cell/macromolecule interface, near-IR scanning near-field
optical microscopy using microfabricated devices and magnetic property imaging could
be used for probing electron transport in cells. Large-scale sensor arrays for diagnostics
of biological fluids. The arrays in question can consist of a wide variety of devices,
ranging from micromechanical cantilevers, each with a slightly different chemical
binding property, to microcalorimeters to measure energy released upon chemical
reactions with different molecules resident within each calorimeter.
Electronic Properties and Exploitation of Biomolecules
Though the electrical characteristics of biomolecules may not generally be relevant
to their biological function, they do represent a fascinating class of new materials for
possible use in electronics. Typical examples for these include use of scanning
tunnelling microscopy (STM) as well as special-purpose substrates prepared in the
nanofabrication facility to measure and eventually to make use of the electrical
characteristics of biomolecules.
Disease Specific Assays

Miniaturized Assays in Biotechnology
Molecular profiling by DNA microarray technology has made significant
contributions to the understanding of cancer. One of the challenges is how to efficiently
utilize the accumulated research data to develop new standards for cancer classification
and to develop new diagnostic and/or prognostic markers and therapeutic targets. The
ultimate value of these molecular markers will be determined by testing in large-scale,
multi-centre clinical trials that require an integrated, easy-to-use and robust assay
system. The rationale for miniaturization is several-fold: (i) reduction of reagent
and—more importantly—(ii) sample consumption, (iii) improving analytical speed by
shortening analysis time or by running several analyses in parallel.
Microarray Technology
The introduction of DNA microarray and SAGE techniques has had dramatic
implications on clinical research especially for cancer research, Fig. (2), allowing
researchers to analyze expression of multiple genes in concert and relate the findings to
clinical parameters [62]. The main discoveries in breast cancer, as well as in other
malignancies, have so far been with respect to two key issues [63]. First, individual
tumours arising from the same organ may be grouped into distinct classes based on their
gene expression profiles, independent of stage and grade. Second, the biologic relevance
of such classification is corroborated by significant prognostic impact. Microarray
technologies can provide unique possibilities to explore the mechanisms of tumor
behaviour in vivo that will allow evaluation of prognosis and, potentially, drug
resistance. These techniques have the capabilities to be implemented for routine clinical
use, whether to define prognostic factors or to predict sensitivity to therapy. A typical
example is the use of microarray for breast cancer prognosis [64].
Microarray technology has been widely used for studying the prognosis predictor for
stage II and III colon cancer patients [65] as well as to discriminate low malignant
potential and invasive epithelial ovarian tumours [66]. Molecular profiling showed
estimated accuracies of 78 and 83%, respectively for prognosis prediction [65]. The
other use of microarray technology is to study the cancer outcome and subtype
classification by gene expression as carried out for bladder cancer [67]. Using a total of
10,368 human gene elements, genes driving the separation between tumor subsets were
shown to be important biomarkers for bladder cancer development and progression and
eventually candidates for therapeutic targeting [67].
A more recent use of this high-throughput technology is to profile cytochrome P450
profile of ovarian cancer and identify novel therapeutic targets and establish the
prognostic significance of expression of individual cytochrome P450s in this type of
cancer [68-69]. The cytochromes P450 are a multigene family of enzymes with a central
role in the oxidative metabolism of a wide range of xenobiotics, including anticancer
drugs and biologically active endogenous compounds. The study showed several P450s
show increased expression in ovarian cancer and this provides the basis for developing
P450-based therapeutics in ovarian cancer.
Fig. (2). Flowchart representing the use of SAGE and microarray based approach for drug
discovery in cancer research.
Enzymatic Assays in a Microsphere Environment

The study of enzymatic reactivity is an important part of current proteomics research.
Enzyme assays include identification and localization of enzymes within the cellular
network, screening of drug candidates, development of diagnostic assays as well as
optimization of enzymatic function through gain or loss of functions. Since many of
these applications require high-throughput analysis with minimal consumption of the
precious samples, the transfer of such assays to microarrays is desirable. Several
investigations regarding the transfer of enzymatic assays to nanowells and microarrays
have been reported. Majority of the reported studies are related to the enzymatic
phosphorylation of peptides and proteins by kinases [70-71]. Similarly for drug
discovery process, cell-free expression and analysis of green fluorescent protein and
bgalactosidase (b-gal) in nanowells has been performed [70]. One of the major
challenges is to retain the unbound reaction products on the spot of reaction. This could
be performed by chemical compounds in nanoliter droplets on flat surfaces and enzyme
solution on top by aerosol deposition [72]. Similarly for signal amplification in
microarray a new technique named Rolling Circle Amplification was introduced recently
[73]. This technique relies on the enzymatic extension of a primer-antibody conjugate
followed by hybridization of labeled probes to the generated DNA strand.
Cemiluminescence for the sensitive detection of multiple cytokines [74] has also bee
used as to an alternative to fluorescent detection. Using ‘‘classical’’ methods, a common
measurement principle can be employed, both for measuring enzyme levels as well as
for measuring metabolite levels, i.e. methods that are based on the formation or
consumption of NAD(P)H. This allows the use of fluorescence as a common detection
method, which can be adapted for a wide range of analyses. Currently the technology
evolving at a rapid pace and more challenges includes the use of multiplex screening of
analytes against enzymes on single platform. To perform this more sophisticated liquid
handling, surface modifications and additional equipment will be required in the near
future.
Targeting Kinase for Cancer Therapy

Overview of Protein Tyrosine Kinases
Protein tyrosine kinases (PTKs) are enzymes which catalyze the phosphorylation of
tyrosine residues. It is expected that the total number of PTKs does not exceed 1000,
based on the genome project analysis. There are two main classes of PTKs: receptor
PTKs and cellular, or non-receptor, PTKs. Of the 91 protein tyrosine kinases identified
thus far, 59 are receptor tyrosine kinases and 32 are non-receptor tyrosine kinases. These
enzymes are involved in cellular signaling pathways and regulate key cell functions such
as proliferation, differentiation, anti-apoptotic signaling and neurite outgrowth.
Unregulated activation of these enzymes, through mechanisms such as point mutations
or over-expression, can lead to various forms of cancer as well as benign proliferative
conditions. Indeed, more than 70% of the known oncogenes and proto-oncogenes
involved in cancer code for PTKs. The importance of PTKs in health and disease is
further underscored by the existence of aberrations in PTK signalling occurring in
inflammatory diseases and diabetes.
Increasing knowledge of the structure and activation mechanism of RTKs and the
signalling pathways controlled by tyrosine kinases provided the possibility for the
development of target-specific drugs and new anti-cancer therapies [75]. Approaches
towards the prevention or interception of deregulated RTK signalling include the
development of selective components that target either the extracellular ligand-binding
domain or the intracellular tyrosine kinase or substrate-binding region.
Receptor PTKs possess an extracellular ligand binding domain, a transmembrane
domain and an intracellular catalytic domain. The transmembrane domain anchors the
receptor in the plasma membrane, while the extracellular domains bind growth factors.
Characteristically, the extracellular domains are comprised of one or more identifiable
structural motifs, including cysteine-rich regions, fibronectin III-like domains,
immunoglobulin-like domains, EGF-like domains, cadherin-like domains, kringle-like
domains, Factor VIII-like domains, glycine-rich regions, leucine-rich regions, acidic

regions and discoidin-like domains.
The intracellular kinase domains of receptor PTKs can be divided into two classes:
those containing a stretch of amino acids separating the kinase domain and those in
which the kinase domain is continuous. Activation of the kinase is achieved by ligand
binding to the extracellular domain, which induces dimerization of the receptors.
Receptors thus activated are able to autophosphorylate tyrosine residues outside the
catalytic domain via cross-phosphorylation. The results of this auto-phosphorylation are
stabilization of the active receptor conformation and the creation of phosphotyrosine
docking sites for proteins, which transduce signals within the cell. Signaling proteins,
which bind to the intracellular domain of receptor tyrosine kinases in a phosphotyrosine-
dependent manner include RasGAP, PI3-kinase, phospholipase C, phosphotyrosine
phosphatase SHP and adaptor proteins such as Shc, Grb2 and Crk.
In contrast to receptor PTKs, cellular PTKs are located in the cytoplasm, nucleus or
anchored to the inner leaflet of the plasma membrane. They are grouped into eight
families: SRC, JAK, ABL, FAK, FPS, CSK, SYK and BTK. Each family consists of
several members. With the exception of homologous kinase domains (Src Homology 1,
or SH1 domains), and some protein- protein interaction domains (SH2 and SH3
domains), they have little in common, structurally. Of those cellular PTKs whose
functions are known, many, such as SRC, are involved in cell growth. In contrast, FPS
PTKs are involved in differentiation, ABL PTKs are involved in growth inhibition, and
FAK activity is associated with cell adhesion. Some members of the cytokine receptor
pathway interact with JAKs, which phosphorylate the transcription factors, STATs. Still
other PTKs activate pathways whose components and functions remain to be
determined.
Tyrosine kinases are important mediators of the signalling cascade, determining key
roles in diverse biological processes like growth, differentiation, metabolism and
apoptosis in response to external and internal stimuli. Recent advances have implicated
the role of tyrosine kinases in the pathophysiology of cancer. Though their activity is
tightly regulated in normal cells, they may acquire transforming functions due to
mutation(s), overexpression and autocrine paracrine stimulation, leading to malignancy.
Constitutive oncogenic activation in cancer cells can be blocked by selective tyrosine
kinase inhibitors and thus considered as a promising approach for innovative genome
based therapeutics. The modes of oncogenic activation and the different approaches for
tyrosine kinase inhibition, like small molecule inhibitors, monoclonal antibodies, heat
shock proteins, immunoconjugates, antisense and peptide drugs are reviewed in light of
the important molecules. As angiogenesis is a major event in cancer growth and
proliferation, tyrosine kinase inhibitors as a target for anti-angiogenesis can be aptly
applied as a new mode of cancer therapy [76].
Classification of Tyrosine Kinases

1. Receptor tyrosine kinase (RTK) e.g. EGFR, PDGFR,FGFR and the IR
Note: receptor tyrosine kinases are not only cell surface transmembrane receptors,
but are also enzymes having kinase activity.
2. Non-receptor tyrosine kinase (NRTK) e.g. SRC, ABL, FAK and Janus kinase.
Structural Biology of Protein Tyrosine Kinases

Crystallography for Protein Kinase Drug Design
Proteomics based disease therapeutic research and drug discovery involves high-
throughput protein structure determination i.e., elucidation of potential targets for
docking target drugs by studying the structural biology of PTKs. Strategies to produce
crystallisable protein kinase constructs include (i) truncation to the catalytic domain (ii)
co-crystallization with rigidifying ligands (iii) crystallization of known rigid forms and
(iv) point mutation to improve homogeneity or mimic less crystallisable proteins. In an
ideal case especially for protein kinases, a broad range of crystal structures should be
obtained to characterize target flexibility, structure modulation via co-factor binding or
posttranslational modification, ligand induced conformational changes, and off-target
complex structures for selectivity optimization. PKA, the prototypical serine/threonine
protein kinase, and SRC, a tyrosine kinase and the first identified oncoprotein, provide
multiple examples of these various approaches to protein kinase crystallography for drug
design. Protein crystallography can be used throughout the drug discovery process to
obtain diverse information critical for structure based drug design [77]. Src structures of
a major inactive form have shown how the protein kinase is rigidified by several
interdomain interactions. Hence relatively little structural information available
regarding the presumably more flexible active forms.
Site and Means of Targeting

Inhibiting the activity of tyrosine kinases by low molecular weight compounds
capable of interfering with either ligand binding (in the case of receptor tyrosine kinases)
or with protein substrate (in case of non receptor tyrosine kinase) has proved to be
difficult. Although the bisubstrate inhibitor approach offered promise, but with very
little practical progress. Approaches to generate non-competitive or allosteric inhibitors
have also failed. The ATP competitive inhibitors appear to be the target of choice. The
major strategies currently employed for tyrosine kinase inhibitions are described in, Fig.
(3). The five different categories could broadly classified based on the type of receptor
used (1) ligand (2) monoclonal antibody (3) immunotoxins (4) Hsp inhibitor and by the
use of (5) antisense technology.
The ATP Binding Site the Most Studied Target for Kinase Inhibition
The characterisation of the human kinome [78-79] has resulted in the emergence of
numerous kinase drug targets in a variety of therapeutic areas [80]. Through the
elucidation of the sequence and structural composition of kinase active sites, coupled
with the solution of numerous ATP competitive ligand complex structures, significant
advances have been made in developing inhibitors that are highly selective. ATP binds
within a deep cleft formed between the two lobes of the tyrosine kinase domain. Though
the ATP binding site is highly conserved the architecture in the regions proximal to the
ATP binding site does afford some key diversity for designing new drug and has
potential application in drug discovery [81]. This has shown to be the case not only for
kinases that are divergent in primary structure, but also for isoforms with highly
conserved structure and ATP binding sites. Hence selection of selective inhibitors and
design of highly potent and selective kinase ATP competitive ligands are key to specific
kinase inhibition. These include the use of small molecules to sequester kinases in
inactive conformations, and to block phospho-transferase activity by preventing

substrate docking and recruitment.
Fig. (3). Common strategies used for inhibition of tyrosine kinase activity. Broadly they could be
classified into five different categories based on the type of receptor used (1) ligand (2)
monoclonal antibody (3) immunotoxins (4) Hsp inhibitor and by the use of (5) antisense
technology [Modified and adapted from reference 99].
Substrate recruitment sites are promising from a structure based design perspective
as they contain features unique to individual protein kinases [81]. A recent report by
Breitenlechner et al. (2005) [82] used three crystal structures of a dimeric active c-Src
kinase domain, in an apo and two ligand complexed forms, with resolutions ranging
from 2.9A to 1.95A. The structures show how the kinase domain, in the absence of the
rigidifying interdomain interactions of the inactivation state, adopts a more open and
flexible conformation. The ATP site inhibitor CGP77675 binds to the protein kinase
with canonical hinge hydrogen bonds and also to the c-Src specific threonine 340. In
contrast to purvalanol B binding in CDK2, purvalanol A binds in c-Src with a
conformational change in a more open ATP pocket.
A similar approach was also used to study the structural basis of Janus kinase 2
inhibition by a potent and specific pan-Janus kinase inhibitor [83]. The development of
JAK2 specific inhibitors has tremendous clinical relevance. JAK2, a member of the
Janus kinase (JAK) family of PTKs, is an important intracellular mediator of cytokine
signalling. Mutations of the JAK2 gene are associated with hematological cancers and
aberrant JAK activity is also associated with a number of immune diseases including
rheumatoid arthritis. Accordingly, critical to the function of JAK2 is its protein tyrosine
kinase (PTK) domain. A crystal structure of the active conformation of the JAK2 PTK
domain in complex with a high affinity, pan-JAK inhibitor that appears to bind via an
induced fit mechanism. This inhibitor, the tetracyclic pyridone 2-tert-butyl-9-fluoro-3,6-
dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-one, was buried deep within a
constricted ATP-binding site, in which extensive interactions, including residues that are
unique to JAK2 and the JAK family, are made with the inhibitor [83].
Antibody Arrays and Protein Kinase Assays

Profiling Receptor Tyrosine Kinase Activation by Using Ab Microarrays
A protein-based microarray allows the global observation of biochemical activities,
where hundreds or thousands of proteins can be concurrently screened for protein-
protein, protein-nucleic acid, and small molecule interactions. This technology holds
great potential for basic molecular biology research, disease marker identification, and
toxicological response profiling and pharmaceutical target screening [14]. The
development of methods to analyze intracellular signaling molecules on microarrays
would make Ab arrays widely useful in systems biology. Multiplex Ab arrays are ideal
and sensitive to the amounts and modification states of signal transduction proteins in
crude cell lysates and the integration of these arrays with 96-well microtiter plate
technology to create microarrays in microplates. Ab arrays were used to monitor the
activation, uptake, and signalling of ErbB receptor tyrosine kinases in human tumor cell
lines Nielsen et al. (2003) [84]. Data obtained from multicolor ratiometric microarrays
correlate well with data obtained by using traditional approaches, but the arrays are
faster and simpler to use. The integration of microplate and microarray methods for
crude cell lysates should make it possible to identify and analyze small molecule
inhibitors of signal transduction processes with exceptional speed and precision. Direct
scale-up to array-based screening in 96- and 384-well plates should allow small
molecules to be identified with specific inhibitory profiles against a signaling network.
Ab microarrays are mainly used for (i) profiling protein abundance, (ii) profiling the
functional state of a signaling system, (iii) analyzing the kinetics of ligand-activated
signaling, (iv) measuring the in vivo inhibitory constant of a small molecule EGFR
inhibitor, and (v) array-based profiling in 96-well plates for screening [84].
Microtiter Plate Array System for Protein Kinase Assays
The microtiter plate array system is well suited to the study of protein kinase
substrates, antigens, binding molecules, and inhibitors since these all can be
quantitatively studied at a single uniform, reproducible interface. Synthetic peptides
have played a useful role in studies of protein kinase substrates and interaction domains.
Fully automated synthesis of (phospho) peptide arrays in microtiter plate wells provides
efficient access to protein tyrosine kinase characterization [85]. Synthesis of peptides
and phosphopeptides on microtiter plate wells overcomes previous limitations and
demonstrates utility in determination of the epitope of an autophosphorylation site,
phospho-motif antibody and utility in substrate utilization assays of the protein tyrosine
kinase, p60c-src [85].
The most recent type of assay is to use protein-acrylamide copolymer hydrogels for
array-based detection of tyrosine kinase activity from cell lysates and extracts [26]. For
this purpose glutathione S-transferase-Crkl (GST-Crkl) fusion proteins are covalently

immobilized into polyacrylamide gel pads via copolymerization of acrylic monomer and
acrylic-functionalized GST-Crkl protein constructs on a polyacrylamide surface. The
resulting hydrogels resist non-specific protein adsorption, permitting quantitative and
reproducible determination of Abl tyrosine kinase activity and inhibition, even in the
presence of a complex cell lysate mixture. This approach will have a direct application to
the detection and treatment of cancers resulting from upregulated tyrosine kinase
activity, such as chronic myeloid leukemia (CML). These findings also establish a basis
for screening tyrosine kinase inhibitors and provide a framework on which protein-
protein interactions in other complex systems can be studied.
Monoclonal Antibody as Inhibitors for PTKs

The extracellular domain of the receptor tyrosine kinase provides an excellent target
for monoclonal antibodies. With the advancement of genomics, design, selection and
production of therapeutic monoclonal antibodies have become much easier. The
revolution in antibody technology now allows us to produce humanized, human chimeric
or bispecific antibody for targeted cancer therapy [86]. Monoclonal antibodies (mAbs)
against growth factors or their receptors have been revealed to be effective therapeutic
agents for solid tumours. Blocking angiogenesis is now considered to be a promising
approach for anticancer therapy and the use of anti-VEGF MAb has demonstrated tumor
suppression [87]. Similarly certain types of antigens are over expressed in cancers. P12
antigen over expression is associated with a wide range of cancer cell lines and tissues
and antibody directed towards these antigens may serve as important contributors to
cancer therapeutics as exemplified by the results of preliminary trials. Mab P12 reacts
with the carbohydrate sequence present on high molecular weight glycoproteins.
Oncologists are now interested in newer MAbs as promising agents for the treatment of
cancer.
The EGFR family of receptor tyrosine kinase comprises of four members – the
EGFR/ Erb B1, HER-2/ Erb B2, HER-3/ Erb B3 and HER-4/ Erb B4. Members of the
EGFR family are involved in some complex biological signal transduction network.
EGFR and HER-2/ neu are amplified in tumor samples of breast, lung and colorectal
tissues. Two targets in particular-the process of new blood vessel development, or
angiogenesis, and the epidermal growth factor receptor and its signalling pathway-are
exploited by the newest monoclonal antibodies that are available for use in colorectal
cancer patients [88]. The orphan receptor tyrosine kinase HER2 gene is amplified in
breast cancer and acts as a major signalling partner for other EGFR family members
leading to proliferation, differentiation, antiapoptosis and tumor progression [89].
Herceptin was the first genome based targeted anticancer therapeutic, approved by FDA
in 1998. Binding of Herceptin to HER receptor leads to receptor internalization,
inhibition of cell cycle progression and antibody dependent cellular toxicity or eliciting
the immune response. Herceptin induces normalization and regression of angiogenesis in
HER-2 overexpressing breast cancer [90] and even blocks cleavage of HER2, which
generates a membrane-bound constitutively active truncated receptor.
Hsp 90 and Other Novel Strategies

Heat shock protein 90 is a molecular chaperone whose association is required for
stability and function of multiple mutated, chimeric, and over-expressed signalling
proteins that promote cancer cell growth and/or survival. The accumulation of Hsp-s is
seen in pathological conditions and tumours [91]. Heat shock proteins (Hsp-s) are
ubiquitous proteins known for the maintenance of cellular homeostasis and are inducible
under variety of stresses. Hsp-s are mainly involved in the proper folding of other
proteins and hence referred to as molecular chaperons. Most kinases require molecular
chaperons to maintain their activation competent conformation. Hsp-s interacts with and
stabilizes various kinases [92]. Chaperon based inhibitors other than interacting with
protein kinases, prevent the associated chaperon(s) from maintaining the activation
competent conformation of the kinase. The result being the proteosomal degradation of
the misfolded kinases, thus diminishing the level of many kinases. The Hsp-s has an
unique ATP binding site, including a Bergerat fold characteristic of bacterial gyrase,
topoisomerases and histidine kinases. Thus the ATP binding site serves as a robust
antitumor target for kinase related chaperone machinery. Important examples are
Geldanamycin, Cisplatin, Novobiocin, Radicol and other purine based inhibitors.
Geldanamycin affects ErbB2, EGF, v-Src, Raf-1 etc. Hsp90 small molecule inhibitors,
by interacting specifically with a single molecular target, thus promote the
destabilization and eventual degradation of multiple cancer cell survival and growth
promoting proteins, and these inhibitors have shown promising anti-tumor activity in
preclinical breast cancer model systems and it has also the unique ability to inhibit
multiple survival pathways utilized by cancer cells. This property of Hsp 90 has been
used as a target for breast cancer [93].
Immunotoxins Based Therapeutics

One of the ways to increase the efficacy of the antibodies that targets specific
molecules expressed by tumor cells can be increased by attaching toxins to them. Few
common immunotoxins are (i) bacterial toxins like pseudomonas exotoxin, (ii) plant
exotoxin like ricin or (iii) radio-nucleotides. The toxins are chemically conjugated to a
specific ligand such as the variable domain of the heavy or light chain of the monoclonal
antibody. The advantage of this technology is that normal cells lacking the cancer
specific antigens are not targeted by the targeted antibody. The most promising
immunotoxin is the EGF fusion protein DAB389EGF [94], which is a fused EGF
specific sequence and diphtheria toxin and have been found to be effective in EGFR
over expressing breast tumor and non small cell lung cancer. The use of therapeutic
antibody for the development of antibody-drug conjugate has been tried to improve the
therapeutic potential e.g. Tositumomab by GlaxoSmithcline, Anti-Tac(Fv)-PE38(LMB-
2) against CD25, in B, T cell lymphoma and anti-B4-bR against CD19 in B-Non
Hodgkins lymphoma. As molecular studies of cancer have started revealing an increased
epitope repertoire due to great strides in genomics and proteomics, the search for more
effective Antibody-drug conjugate has got an impetus recently.
Antisense Strategies and Peptide Drugs

Antisense are small pieces of synthetic oligonucleotides that are designed to interact
with the mRNA by Watson-Crick base pairing to block the transcription and thus
translation of target proteins. Antisense oligodeoxynucleotides (ODN) targeting IGF-1R
induces apoptosis in malignant melanoma and is also effective in breast cancer [95].
Peptides are other potential mediators for inhibition of specific types of kinase
activities. Peptide and peptidomimetics that interfere with the interaction of protein-
protein interaction has a significant impact on the cellular processes. A typical examples
is provided by Grb2 which is an essential component in the Ras signaling pathway and
it’s interaction with Sos is responsible for the down stream signaling process. Proline
rich “Peptidimer” having two VPPVPPRRR sequence specifically recognizes Grb2 and
selectively blocks Grb2-Sos interaction an important step in EGFR over expressing
cancers [96]. Small peptides mAZpTyr-(_-Me)pTyr-Asn-NH2 [97] and peptidomimetics
like AHNP anti erbB2 are even known to inhibit unwanted tyrosine kinase dimerisation
by competing with target proteins thus the peptides or peptidomimetics acts as
antagonist of RTK [98].
Mining the Kinome

With the completion of Genome project, mining the kinome is an effective strategy
to meet the future challenges to identify suitable target for cancer therapy. Cancer arises
by clonal proliferation from a cell, which builds up a series of mutations leading to
abnormal signaling were kinases play a major role during each event from cancer onset
to metastasis. With over 500 kinases in the Extracellular Domain Disease relevant
signaling of RTK in human kinome and as many as 200-300 protein kinases mediating a
large number of pathways in a cell at a particular time, selectivity becomes very
important considering the cost of drug development. Hence strategies, Fig. (4) to move
kinase drug discovery in a more rapid, comprehensive and efficient manner, including
tyrosine kinase target validation, selectivity and durability are key to the success of drug
discovery [100]. For a more comprehensive review on methods for deciphering the
kinome please refer to the review article by Johnson and Hunter, (2005) [101].
Fig. (4). A unique strategy to identify unknown kinase based on known substrate candidate or
unknown substrate candidate. (Modified based on reference [100]).
Biochemical, cell based assay and screening methods for the through profiling of the
kinase inhibitors using monoclonal antibody based multi-immunoblotting, fluorescent
polarization assays, non-radioactive high throughput assays, 2-D NMR approaches
should be exploited. Structure based drug design (SBDD) strategies depending on
computational approaches, mathematical models of tumor and normal tissue response,

high-throughput X-ray crystallography and chemogenomic approaches can be used to
advance molecules through the routine drug discovery process. The use of recent
advances in RNA interference (RNAi) appears to be a promising approach for silencing
gene expression, thus elucidating genetics of human disease with emphasis into the
biological role of the kinase signalling pathways [102]. The promising progress made by
the drugs Gleevec, Iressa and Herceptin has brought to light the potential of new
innovative genome based molecular therapeutics.
CONCLUSION
The future looks very bright for clinical diagnostics. The advent of newer
technologies will revolutionize the way many diseases are currently treated. Protein
arrays will evolve both in sensitivity and specificity with the discovery of more markers
for various diseases. Miniaturization will have a significant impact on disease diagnosis
and targeted therapy. Kinome based drug discovery will be one of the major areas of
research in the years to come and will have a major role in the discovering better drugs
for cancer therapy. Drug delivery will hold key for better and efficient treatment
strategies. Hence a combination of nanotechnology coupled with efficient delivery
process will determine the effectiveness of these new advances in the field of newer
metabolic assays and disease screening as well as therapeutic applications.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge National Medical Research Council (NMRC,
Singapore) and Biomedical Research Council (BMRC, Singapore) for providing funds
(Grant numbers- NMRC: R174-000-071-213 and BMRC: R174-000-091-305) for
research.
ABBREVIATIONS
1-D and 2-D GE = 1 dimensional and 2 dimensional gel electrophoresis
Ab = Antibody
ATP = Adenosine tri phosphate
CE = Capillary electrophoresis
CML = Chronic myeloid leukaemia
Da = Dalton
DNA = Deoxyribonucleic Acid
EGFR = Epithelial growth factor receptor (another name - Erb B)
ELISA = Enzyme Linked ImmunoSorbent Assay
ESI- tandem-MS/MS = Electrospray tandem mass spectrometry
FRET = Fluorescence energy transfer
GC = Gas chromatography
GST = Glutathione S-transferase

GFP = Green Fluorescent Protein
HER-2 = Human epidermal growth factor receptor 2
HIV = Human Immunodeficiency virus
HPLC = High-pressure liquid chromatography
HPV = High-risk human papillomavirus
Hsp 90 = Heat shock protein 90
LAPS = Light-addressed potentiometric sensor
LPA = Lysophosphatidic acid
mAbs = Monoclonal antibodies
MALDI-MS = Matrix-Assisted Laser Desorption Mass spectrometry
MCAD = Medium-chain acyl-CoA dehydrogenase
MD LC/MS = Multidimensional liquid chromatography/mass spectrometry
micro-TAS = Micro total analysis system
MRM = Multiple reaction-monitoring
mRNA = Messenger ribonucleic acid
MSA = Multisensor array
NMR = Nuclear magnetic resonance spectroscopy
NRTK = Non-receptor tyrosine kinase
ODN = Oligodeoxynucleotides
PIN = Prostate intraepithelial neoplasia
PKU = Phenylketonuria
PS20 = protein pull down Ciphergen ProteinChip™
[www.ciphergen.com]
PTK = Protein Tyrosine Kinase
RNAi = RNA interference
RTK = Receptor tyrosine kinase
RTK = Receptor Tyrosine Kinase
SAGE = Serial Analysis of Gene Expression
SBDD = Structure based drug design
SELDI-TOF-MS = Surface Enhanced Laser Desorption-time of flight mass
spectrometry
SNPs = Single nucleotide polymorphisms

SPR = Surface Plasmon Resonance
STM = Scanning tunnelling microscopy
TLC = Thin-layer chromatography
TNF-alpha = Tumor necrosis factor alpha
TTR = Transthyretin
VEGF = Vascular endothelial growth factor
WCX2 = Weak Cation Exchange
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Proteomic Screening for Novel Therapeutic

Targets in Kidney Diseases
Visith Thongboonkerd*
Siriraj Proteomics Facility, Medical Molecular Biology Unit, Office for Research and
Development, Faculty of Medicine Siriraj Hospital, Mahidol University, 12th Fl.
Adulyadej Vikrom Bldg., 2 Prannok Rd., Bangkoknoi, Bangkok 10700, Thailand
Abstract: The high-throughput capability of proteomics allows simultaneous
examination of numerous proteins and makes a global analysis of proteins in
cell, tissue, organ or biofluid possible. This strength of proteomics has been
extensively applied to examine altered proteins caused by various diseases.
Some of these altered proteins may particularly be important for the disease
progression and/or complications. Thus, information on these altered proteins
is valuable for better understanding of the pathogenesis and pathophysiology
of medical diseases. Additionally, functional analysis of such proteins may
lead to identification of novel therapeutic targets and development of new
drugs for improving therapeutic outcome as well as for preventing serious
complications. This review focuses mainly on applications of renal and urinary
proteomics to define novel therapeutic targets in kidney diseases. Several
recent studies on various kidney diseases have successfully identified altered
renal and urinary proteins, some of which may potentially be the novel
therapeutic targets. Urinary proteome profiling has also been applied to
biomarker discovery that will be useful for clinical diagnostics, prognosis,
prediction of treatment response, and development of personalized medicine.
Finally, potential roles of proteomics for drug design and discovery are
discussed.
INTRODUCTION
Traditional approach to define therapeutic targets for drug design and discovery has
applied conventional biochemical methods to hypothesis-driven research focusing on a
specific disease pathway and on involving protein(s) as well as metabolite(s). Although
successful, it is time-consuming and the targets to be studied require a priori
assumption. Currently known therapeutic targets are underestimated and, thus, there is a
need of other methods to screen for a large number of potential therapeutic targets
simultaneously. In the post-genomic era, especially after the Human Genome Project
was completed, several biotechnologies have been developed to utilize the genomic
information to examine other cellular elements (proteins, transcripts, metabolites, lipids,
etc.) on the genomic scale. Since then, respective ‘omics’ fields (proteomics,
transcriptomics, metabolomics, lipomics, etc.) have been defined. The successfulness of
*Corresponding author: Tel/Fax: +66-2-4184793; E-mail: thongboonkerd@dr.com (or) vthongbo@yahoo.com

88 Frontiers in Drug Design & Discovery, 2006, Vol. 2 Visith Thongboonkerd
these omics fields is due mainly to advances in separation sciences (using either gel-
based or gel-free methods) and mass spectrometry (MS). The great contribution of MS
technology to life science has been confirmed as the 2002 Nobel Prize in Chemistry
went to John B. Fenn (who developed electrospray ionization; ESI) and Koichi Tanaka
(who developed matrix-assisted laser desorption/ionization; MALDI) [1]. Because the
main sites of drug action mainly compose of proteins and many of the
pharmacologically regulating systems operate through proteins it is most likely,
therefore, that extensive analysis of proteins in disease states will lead to the successful
identification of novel therapeutic targets. This chapter focuses mainly on the potential
role of proteomics for the identification of novel therapeutic targets as well as for drug
design and discovery, particularly in kidney diseases.
RENAL AND URINARY PROTEOMICS: A BRIEF OVERVIEW

The kidney is one of the major organs responsible for (i) eliminating waste products
(particularly urea and metabolites) from the plasma; (ii) maintaining normal homeostasis
of water, acid-base, minerals and solutes; and (iii) producing hormones (i.e. renin,
erythropoietin and 1,25 dihydroxy vitamin D3) [2]. It contains several microstructures
including glomerulus, proximal tubule, the loop of Henle, distal tubule, collecting duct,
renal pelvis, vessels, etc. The kidney size is only 0.5% of total body mass but generates
150-180 L/day ultrafiltrate from plasma (renal plasma flow is approximately 350-400
mL/100 g of tissue per min) [2]. The kidney then selectively reabsorbs water, nutrients
and necessary constituents in the ultrafiltrate until less than 1% of ultrafiltrating volume
is excreted with waste products as the urine [2]. Because the kidney and urine are related
to several physiological functions, data on their protein compositions during normal and
disease states would be expected to provide a wealth of information to better understand
normal renal physiology and pathophysiology of kidney diseases.
To study proteins, two different but complementary fields need to be clarified.
Protein chemistry is to examine structure, physical and chemical properties as well as
function of each protein in details, whereas proteomics is to simultaneously examine a
large number of proteins or the entire proteome in a complex mixture of biological
samples [3]. Both of them are aimed to better understand the cellular biology and
physiology, and to determine functional significance of proteins in normal and disease
states. Therefore, these two fields are different but complementary. Proteomics can be
divided into two broad categories: ‘expression proteomics’ and ‘functional proteomics’.
Expression proteomics is to define normal proteome profile and differential protein
expression among different sets of biological samples, and to determine alterations in
protein expression caused by experimental interventions, physiological stimuli, or
pathogenic conditions. Functional proteomics is to examine function of the protein of
interest; e.g., its complexes, protein-protein interactions, and post-translational
modifications [4].
Proteomics has been extensively applied to several fields of biomedical research with
four main objectives: (i) to better understand normal biology and physiology of cells,
microorganisms, tissues, and organs; (ii) to explore the pathogenic mechanisms and to
better understand the pathophysiology of medical diseases; (iii) to identify novel
biomarkers for early disease detection, prediction, and prognosis; and (iv) to identify
novel therapeutic targets for drug and vaccine discovery. In renal research, proteomics
has also been applied with similar objectives as those applied to other research areas.
Proteomics for Drug Design & Discovery Frontiers in Drug Design & Discovery, 2006, Vol. 2 89
Kidney diseases are caused by several etiologies including congenital defects (abnormal
genes), metabolic derangement, physical injury, neoplasm, infection, inflammation,
autoimmunity, hemodynamic dysregulation, toxicity from drugs or chemicals, and renal
involvement of systemic diseases. These various pathogenic mechanisms affect kidney
microstructures differently. Expression and function of proteins in affected intrarenal
microstructures are expected to be altered. Therefore, extensive analyses of the kidney
proteome and/or subproteome(s) using expression and functional proteomics during
disease states are crucial to better understand the complexity of the pathogenesis and
pathophysiology of kidney diseases.
As the urine is a body fluid excreted from the kidney, proteomic analysis of the urine
also provides a wealth of information. While renal proteomics may lead to identification
of novel therapeutic targets, urinary proteomics is suitable for biomarker discovery
because of the availability of urine samples in almost all of patients and the simplicity of
specimen collection. To date, renal and urinary proteomics have been applied to a wide
variety of kidney diseases including glomerular diseases (e.g., IgA nephropathy, focal
segmental glomerulosclerosis or FSGS, and diabetic nephropathy); tubulointerstitial
disorders (e.g., Dent’s disease and nephrotoxicity induced by cyclosporine, gentamicin,
radiocontrast medium and lead); renal vascular disease (i.e. renovascular hypertension);
renal cancers; renal transplant allograft rejection; acute renal failure; and end-stage renal
disease [5-7].
PROTEOMIC TECHNOLOGIES FOR ANALYSIS OF KIDNEY AND URINE

Gel-Based Methods
Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is the most
commonly used method in current proteomics studies. The first dimension of 2-D PAGE
separates proteins on the basis of differential pH or isoelectric point (pI), whereas the
second dimensional separation resolves proteins on the basis of differential molecular
size (M r) [8]. Separated proteins in 2-D gel are visualized by various stains (Coomassie
Brilliant Blue, silver, fluorescence, etc.). Visualized protein spots can then be excised,
in-gel digested with proteolytic enzymes (trypsin, chymotrypsin, Arg-C, Asp-N, Lys-C,
PepsinA, V8-E, V8-DE, etc.), and identified by MALDI-MS followed by peptide mass
fingerprinting (PMF). The most common type of mass analyzer employed in MALDI
analysis is time-of-flight (TOF). MALDI-TOF MS provides a high-throughput manner
of protein identification – hundreds of proteins can be identified within a day [9-11].
Consequently, MALDI-TOF MS has become an integral part of today’s modus operandi
in proteome analysis. Even with lots of advantages, gel-based approach has some
limitations. 2-D PAGE procedures are time-consuming, particularly when a large
number of clinical samples are analyzed. Additionally, low abundant, transmembrane
and highly hydrophobic proteins may not be detectable in a 2-D gel. Note that SDS-
PAGE or 1-D PAGE can also be applied to proteomic analysis, particularly when
membrane or highly hydrophobic proteins are analyzed.
Gel-Free Methods
Liquid Chromatography (LC) Coupled to Tandem MS (MS/MS)
High-performance (HP) LC coupled to electrospray ionization-tandem MS (ESI-
MS/MS) has gained a wide acceptance for gel-free proteomic analysis and become a
method-of-choice for analysis of membrane and low abundant proteins [12-14]. ESI is
the process of ionization from electrospray source, whereas tandem MS refers to the
strategy of multi-step mass analyses. When compared to 2-D PAGE approach, LC-based
methods are more effective for analysis of small proteins and peptides, as well as
membrane or highly hydrophobic proteins. Recently, the high-throughput LC approach
has been developed namely ‘multidimensional protein identification technology
(MudPIT)’ or 2D-LC-MS/MS [15]. This approach involves proteolytic digestion of the
total protein mixture to obtain a set of protein-derived peptides that are then separated by
strong cation-exchange (SCX) chromatography (‘Bottom-Up’ approach). Peptides
present in fractions from this SCX step are separated further by reversed-phase (RP) LC
and then sequenced by MS/MS. Several thousands of peptides can be sequenced by this
way in a relatively short time. In contrast to the Bottom-Up approach, the undigested
protein mixture can also be separated by HPLC and resolved proteins in each fraction
are then digested with a proteolytic enzyme prior to MS/MS sequencing (‘Top-Down’
approach) [16, 17].
Surface-Enhanced Laser Desorption/Ionization (SELDI) or Protein Chip Technology
SELDI is a suitable method for proteome profiling of human body fluids. SELDI-
TOF MS combines MALDI-TOF MS with surface retentate chromatography.
Specifically, a protein sample is applied onto a chip surface carrying a functional group
(e.g., normal phase, hydrophobic, cation- or anion-exchange). After incubation, proteins
that do not bind to the surface are removed by a simple washing step and bound
peptides/proteins are analyzed by TOF mass spectrometer. The detection of a protein by
SELDI-TOF MS is critically determined by its concentration in the sample, its binding
to the chromatographic surface and its ionization process within the mass spectrometer.
This approach reduces the complexity of the sample being analyzed by selecting only a
subset of proteins. SELDI-TOF MS offers some advantages for urinary proteome
analysis. Only 5 to 10 µL of urine is needed for a single analysis and this method can be
readily automated, making it particularly useful for the high-throughput study. For
comparative analysis of urine samples, it is important to determine factors that may
affect the composition and relative abundance of urinary proteins, but are not related to
the investigated disease. Additionally, operating parameters must be strictly the same for
each run to reduce the inter-assay variability.
Capillary Electrophoresis Coupled to MS (CE-MS)
CE-MS is another method suitable for proteome profiling of human body fluids. It
offers some advantages as it is fairly robust, uses inexpensive capillaries and is
compatible with essentially all buffers and analytes [18-21]. In contrast to LC, CE
generally has no flow rate but requires a closed electric circuit. Various MS coupling
techniques can be applied to CE [22, 23]. The predominant ionization method for CE-
MS is ESI, while MALDI has also been used extensively [24, 25]. The main advantages
of MALDI appear to be the enhanced stability as well as an easier handling in
comparison to ESI. Additionally, once the analytes are deposited on the target, they can
be re-analyzed several times without the need of a new CE run. Moreover, the deposited
analytes can be subsequently manipulated. However, the disadvantages of MALDI are
the decreased dynamic range in comparison to ESI and the higher sensitivity towards
signal suppression. For detection of the narrow CE-separated analyte zones, a fast and
sensitive mass spectrometer is required. Thus, both ion trap and TOF systems appear
adequate. While ion trap MS acquires data over a suitable mass range with the rate of
several spectra per second, the resolution is generally too low to resolve the single
isotope peaks of >3-fold charged molecules. Consequently, assignment of charge to
these spectra is hampered. Modern ESI-TOF mass analyzers record up to 20 spectra per
second and provide the resolution of more than 10,000 and a mass accuracy better than 5
ppm. Thus, the most suitable MS method to date for this type of analysis is ESI-TOF
MS.
Mass Spectrometric Immunoassay (MSIA)
This method combines immunoassays with MALDI-TOF MS [26-39]. Proteins are
first captured by micro-scale affinity techniques and are subsequently examined
qualitatively and quantitatively using MALDI-TOF MS [40]. This approach has a
potential of greatly extending the range, utility and speed of biological research and
clinical assays. In the initial stage of development, agarose beads (derivatized with an
affinity ligand) were used to create a microliter-volume column inside a micropipettor
tip (thus, creating an affinity pipette) [26, 28]. More recently, tailored, high-flow rate,
high binding-capacity affinity micropipettes have been manufactured and used in
combination with robotic platforms for the preparation of up to 96-samples in parallel
[30, 34, 38]. Using this approach, the proteins of interest can be selectively retained and
concentrated by repeat flowing of a biofluid through the affinity pipette. After washing
to remove unspecified compounds, the retained proteins are eluted, mixed with a
MLADI matrix (i.e. α-cyano-4-hydroxycinnamic acid), and targeted onto the MALDI
plate. The eluted proteins are then analyzed with a mass spectrometer (TOF-MS). For
quantification, a known variant of the analyte (chemically modified to shift the
molecular mass without significantly altering the affinity for the immobilized ligand) is
introduced into the biofluid at a constant concentration. The protein variant is then
retained, eluted and analyzed simultaneously with the investigated protein. The protein
of interest can then be quantified by normalizing its signal with that of the internal
reference [26-39].
URINARY PROTEOME PROFILING FOR BIOMARKER DISCOVERY AND

CLINICAL DIAGNOSTICS
Proteomics of human urine can be performed using a ‘classical approach’ or an
‘alternative approach’ [7]. The ‘classical approach’ is to extensively and systematically
examine protein expression and function to better understand the normal physiology and
pathophysiology of medical diseases [7]. The analytical techniques involve in this
approach include expression proteomics, bioinformatics, quantitative analysis and
functional proteomics. The ‘alternative approach’ is to examine proteome profiles or
patterns of overall protein expression in biological samples to differentiate types or
groups of the samples (i.e. normal versus diseases; a specific disease versus others),
rather than focusing on a specific protein or a particular disease pathway [7]. Analytical
techniques commonly used in this approach are microarrays [41], SELDI [42], and CE-
MS [43]. The advantage of the latter approach is that detailed characterizations (identity,
protein-protein interactions, post-translational modifications, etc.) of proteins are
unnecessary. This approach is, therefore, suitable for clinical diagnostics and biomarker
discovery, especially in multi-factorial diseases for which one marker may not be
sufficient for effective detection or diagnosis.
The alternative approach or proteome profiling also offers an opportunity of its use
as a complementary diagnostic tool for some medical diseases, in which clinical
diagnosis relies only on invasive procedures that may be limited in some occasions. For
example, renal biopsy, which remains the gold standard for the diagnosis of glomerular
diseases, may not be possible in patients whose indications for this invasive procedure
are not fulfilled and those with bleeding tendency or flank skin infection, thereby
delayed diagnosis. In these cases, urinary proteome profiling may potentially be useful
as a complementary diagnostic tool. Moreover, information about dynamic changes of
the urinary proteome profile during or after treatment may be useful to predict
therapeutic outcome and/or prognosis of the disease. Evaluation of treatment response
by various regimens of therapy, looking at urinary proteome profiles, may also lead to
the development of personalized medicine for each individual.
SCREENING FOR NOVEL THERAPEUTIC TARGETS OF KIDNEY

DISEASES USING PROTEOMIC TECHNOLOGIES
Proteomics is probably the best screening tool, to date, to screen for the novel
therapeutic targets that have not previously been defined by conventional methods. One
of the major objectives in recent renal and urinary proteomics studies is to identify
altered proteins modified by kidney diseases. Major findings from recent renal and
urinary proteomics studies applied to kidney diseases are summarized in Table 1.
Table 1. Partial List of Altered Proteins Identified in Kidney Diseases Using Proteomic
Technologies
A. Glomerular diseases
IgA nephropathy
Plasma proteins
- IgA1
Focal segmental glomerulosclerosis
Proteases
- MBL-associated serine protease
Plasma proteins
- Albumin
- Apolipoprotein J
- γ-Fibrinogen
- Vitronectin
Matrix proteins
- Fibulin
Diabetic nephropathy
Cytoskeletal proteins
- Myosin
- Tropomyosin
(Table 1) contd….
- Vimentin
Proteases
- Contraception-associated protein
- Elastase
Protease inhibitors
- Anti-thrombin III
- Elastase inhibitor
- Phosphatidylethanolamine-binding protein
- T-kininogen
Apoptosis-related proteins
- Deoxyribonuclease I
- Histone H3.2
Redox-associated proteins
- Ferritin
Calcium-binding proteins
- Annexin V
- Calbindin-D28k
- Calmodulin
- Crocalbin-like protein
Transport regulators
- Na-H exchanger regulator
- Retinol-binding protein
- Syntaxin 11
- Transthyretin
Signaling proteins
- C1q-binding protein
Plasma proteins
- Apolipoprotein A-IV
Matrix proteins
- Elastin
Miscellaneous
- HSCP207
B. Tubulointerstitial diseases
Cyclosporin A nephrotoxicity
- Calbindin-D28k
Lead nephrotoxicity
Metabolic enzymes
- Aflatoxin B1 aldehyde reductase
(Table 1) contd….
- Aldose reductase
- Argininosuccinate synthase
- Glutathione S-transferase P
- Sorbitol dehydrogenase
- Transketolase
- Calbindin-D28k
- Calcineurin
Stress regulatory proteins
- Heat shock protein 70 (HSP70)
- Heat shock protein 90 (HSP90)
Plasma proteins
- α2-Microglobulin
- Transferrin
Radiocontrast nephrotoxicity
Plasma proteins
- α2-Microglobulin
Gentamicin nephrotoxicity
- Actin
- α-Tubulin
Protease inhibitors
- Serine protease inhibitor (SPI1)
Metabolic enzymes
- Acetyl-CoA carboxylase
- ATP synthase α subunit
- ATP synthase β subunit
- ATP-specific succinyl-CoA synthetase
- cGMP-specific 3'5'cyclic phosphodiesterase
- Cytosolic malate dehydrogenase
- DNA polymerase α subunit IV
- α-Enolase
- F1-ATPase
- Fructose 1,6 bisphosphatase
- Glutamate decarboxylase
- Glycine amidinotransferase
- GTP-specific succinyl-CoA synthetase
- Methylacyl-CoA racemase α
- NG,NG-dimethylarginine dimethylaminohydrolase 1
- Phospholipase B
(Table 1) contd….
- Regucalcin
- Fatty acid transport protein
Signaling proteins
- Moesin
- DnaK-type molecular chaperone
Plasma proteins
- Albumin
- α2-Microglobulin
Dent's disease
- Profilin
Protease inhibitors
- Cystatin C
- Cystatin M
- Phosphatidylethanolamine-binding protein
Metabolic enzymes
- Carbonic anhydrase
- Lysozyme
- Cubilin
- Transthyretin
Signaling proteins
- Epidermal growth factor precursor
- Insulin-like growth factor-binding protein
Plasma proteins
- Angiotenogen
- Apolipoprotein A-I
- Apolipoprotein A-IV
- Complement factor H-related protein
- β2-Glycoprotein I
- Hemopexin
- Megalin
- β2-Microglobulin
- Pigment epithelium-derived factor
- Uromodulin
- Vitamin D-binding protein
(Table 1) contd….
Miscellaneous
- Lipocalin
C. Vascular disease
Renovascular hypertension
- Troponin T
Metabolic enzymes
- 3-Mercaptopyruvate sulfurtransferase
- Aldehyde dehydrogenase family 7
- Pyruvate kinase M1 isozyme
- Vacuolar ATP synthase subunit B
- Nucleolin-related protein
Signaling proteins
- Phosphotyrosyl protein phosphatase
- Serine/threonine protein kinase 10
Matrix proteins
- Lamin A
Miscellaneous
- 3010027A04Rik
- Type A/B hnRNP p40
D. Renal cancers
- Cytokeratin 8
- Keratin 10
- Smooth muscle protein 22-α
- Stathmin
- α-Tubulin
- β-Tubulin
- Vimentin
Metabolic enzymes
- Aconitase
- Agmatinase
- Aldehyde dehydrogenase 1
- Aldolase A
- Aldose reductase
- Aminoacylase-I
- Carbonic anhydrase I
(Table 1) contd….
- α-Enolase
- γ-Enolase
- Enoyl-CoA hydratase
- FK506-binding protein 4
- Glutathione peroxidase
- Glutathione-S-transferase P
- Glyceraldehyde-3-phosphate dehydrogenase
- α-Glycerol-3-phosphate dehydrogenase
- Inorganic pyrophosphatase
- Lactate dehydrogenase
- Mn-superoxide dismutase
- N-acetylglucosamine phosphate mutase
- NADH-ubiquinone oxidoreductase complex I
- Nicotinamide N-methyl transferase
- Phosphoglucomutase
- Phosphoglycerate kinase 1
- Phosphoglycerate mutase
- Triosephosphate isomerase
- Ubiquinol cytochrome C reductase
- Ubiquitin carboxyl terminal hydrolase 1
- Ubiquitin-activating enzyme E1
- Valosin-containing protein
Redox-associated proteins
- Thioredoxin
- Annexin I
- Annexin II
- Annexin IV
- Annexin V
Transporters
- CLIC-4
- α-S1 Casein
Signaling proteins
- Cofilin 1
- G protein
- Moesin
- Platelet-derived endothelial cell growth factor
(Table 1) contd….

- Glucose regulatory protein (GRP78)
- HSP27
- HSP90
- Protein disulfide isomerase
Plasma proteins
- β-Fibrinogen
Matrix proteins
- Lamin B1
Miscellneous
- Elongation factor 2
- Major vault protein
E. Renal transplant allograft rejection

Plasma proteins
- β2-Microglobulin
F. Acute renal failure

Proteases
- MBL-associated serine protease
- Transthyretin
Plasma proteins
- Albumin
- Apolioprotein A-IV
- β2-Microglobulin
- Transferrin
- Vitamin D-binding protein
- Zn α-2 glycoprotein
G. End-stage renal disease

Protease inhibitors
- α1-Antitrypsin
- Cystatin C
Plasma proteins
- Albumin
- Complement factor D
(Table 1) contd….
- α-Fibrinogen
- β2-Microglobulin
- Salivary proline-rich protein
The altered proteins that have been identified in each disease from different studies
are integrated and classified into various functional groups, based on their major
functions appeared in the Swiss-Prot/TrEMBL database and/or literatures. Some of the
listed proteins have multiple functions (e.g., annexin, phosphatidylethanolamine-binding
protein, and aldose reductase) but are designated only in one functional class in the table.
Note that these altered proteins can be one of the following possibilities:
(a) Altered proteins that are the causes of kidney diseases and definitely are the
therapeutic targets.
(b) Altered proteins that are not the direct causes but play important roles in the
pathogenic mechanisms of kidney diseases (i.e. those involve in the cascade of
disease pathways) and may be the novel therapeutic targets.
(c) Proteins that are altered as a result of kidney diseases and their changes are to
compensate for the disease mechanisms or to maintain the normal homeostasis.
(d) Proteins that are altered as a result of kidney diseases but their changes have no
functional significance or have only minor effects on renal physiology.
(e) Proteins that are not actually altered but their changes are observed in a
proteomic analysis as a spurious result of quantitative analysis (as equal amount
of total protein is normally used for quantitative analysis, especially in gel-
based methods).
(f) Proteins that are altered only in a specific kidney disease and may be the novel
biomarkers (regardless of their functional significance in the disease
mechanisms).
Although some of the altered proteins showed in Table 1 may potentially be the
novel therapeutic targets of kidney diseases, none of them has been verified. Extensive
functional evaluation and bioinformatic analysis of these altered proteins, which have
been identified by expression proteomics, are required to define the validated therapeutic
targets.
PHARMACOPROTEOMICS FOR DRUG DESIGN AND DISCOVERY

After defining the validated novel therapeutic targets for kidney diseases, the next
step is to discover novel drug compounds by designing their molecular structures to fit
into the functional parts of protein molecules that are the therapeutic targets.
Bioinformatics plays a critical role for such design. Proteomics applied to
pharmaceutical purposes or ‘pharmacoproteomics’ involves almost all of basic
methodologies described above, particularly MS techniques. Indeed, various mass
spectrometric methods; including electrospray and nanospray ionization, atmospheric
pressure chemical ionization, photoionization, and their interface with LC have been
utilized to measure levels of drugs and their metabolites in the plasma and urine for quite
some times [44]. Recent advances in HPLC coupled to tandem MS (HPLC-MS/MS)
make the identification of drug compounds more sensitive with a better resolution. A
high-throughput capability of HPLC-MS/MS, with or without stable isotope labeling,
facilitates the studies of in vitro and in vivo drug metabolisms, examination of metabolite
activities, identification and characterization of impurities in the pharmaceuticals,
analysis of chiral impurities in drug substances, and drug discovery [45-48].
Pharmacoproteomics can also be applied for prediction of therapeutic response to a
specific drug. However, response to a particular drug may not be predicted easily
because of the inter-individual variability [49], which is partly due to genetic factors
[50]. Therefore, combination of pharmacoproteomics and pharmacogenomics is
important for prediction of therapeutic outcome as well as for evaluation of genetically
and biochemically dynamic processes during medications [51]. Thus, proteomic
technologies are not used alone for drug design and discovery or for other
pharmaceutical purposes, but rather they are integrated with genomic and chemical
approaches.
INTEGRATIVE ‘OMICS’ FOR PERSONALIZED MEDICINE

Currently, the suffix ‘-omics’ is used frequently for the nomenclature of several
fields in biomedical research (i.e., genomics, transcriptomics, proteomics, lipomics,
metabolomics, interactomics, and several others). Most of recent studies have applied
each of these omics sciences separately for individual study project. It is unlikely that the
complexity of disease mechanisms will be completely understood by a single omics
approach. Integrating all of them is required for future biomedical research. With the
capability of bioinformatics to link all these omics fields together, each omics field is
complementary to the others and serves as an individual piece of the jigsaw to fulfill the
dynamic images of the pathogenesis and pathophysiology of medical diseases, as well as
their complications. Recently, the concept of ‘systems biology’ has been emerging for
the global evaluation of biological systems and has included ‘integrative omics’ as one
of the analytical processes [52, 53]. Systems biology has been defined by Weston and
Hood [54] as “the analysis of the relationships among the elements in a system in
response to genetic or environmental perturbations, with the goal of understanding the
system or the emergent properties of the system”. A system may be a few protein
molecules carrying out a particular task such as galactose metabolism (termed a
biomodule), a complex set of proteins and other molecules working together as a
molecular machine such as the ribosome, a network of proteins operating together to
carry out an important cellular function such as giving the cell shape (protein network),
or a cell or group of cells carrying out particular phenotypic functions. Thus, a biological
system may encompass molecules, cells, organs, individuals, or even ecosystems [54].
The major keys that make systems biology being the ideal approach for future
biomedical research are: (i) the successfulness of the Genome Projects and the
availability of databases; (ii) the emergence of cross-disciplinary biology that allows
biologists, chemists, physiologists, computer scientists, engineers, mathematicians,
statisticians, physicians and healthcare professionals work closely together; (iii) the
availability of bioinformatics that serves as a link for a cross-talk among different fields;
and (iv) advances in the high-throughput platforms of biotechnologies that permit
simultaneous study of a large complement of genes, transcripts, proteins, lipids or other
elements. Systems biology or integrative omics is, therefore, the ideal approach for
future biomedical research and should result to: (i) better understanding of the
pathogenesis and pathophysiology of medical diseases and their complications; (ii)
identification of new therapeutic targets; (iii) discovery of novel drugs and biomarkers;
(iv) better therapeutic outcomes; and (v) successful prevention of the diseases and their
complications. Because of the wide spectrum of data to be obtained from integrative
omics or systems biology, this ideal approach can also be utilized to develop and
optimize personalized medicine.
CONCLUSIONS
Proteomics has lots of advantages and strengths to be used in renal research. The data
obtained from recent renal and urinary proteomics studies have demonstrated the
potential of using proteomics as a tool to screen for novel therapeutic targets of kidney
diseases. Additionally, pharmacoproteomics in combination with pharmacogenomics,
chemoproteomics, and bioinformatics is very important for drug design and discovery in
the post-genomic era. Finally, integrative omics or systems biology holds the greatest
promise for future biomedical research. This ideal approach may lead to the ultimate
goals of improved therapeutic outcome and successful prevention of diseases, as well as
development of personalized medicine to gain the best therapeutic response for
individual patients.
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Aptamer-Based Technologies as New Tools for

Proteomics in Diagnosis and Therapy
Vittorio de Franciscis* and Laura Cerchia*

Istituto di Endocrinologia ed Oncologia Sperimentale del CNR
“G. Salvatore”, via S. Pansini 5, 80131 Naples, Italy
Abstract: Proteomics has provided a tool to define protein profile of a specific

cell or tissue and to associate protein expression levels and post-translational
modifications with disease states therefore developing innovative technologies
for measurement of protein levels has become a major challenge of the last few
years. Specific nucleic acid-based compounds, named aptamers, have been
shown as high-affinity ligands and potential antagonists of disease-associated
proteins. Aptamers, isolated from combinatorial libraries by an iterative in
vitro selection process, discriminate between closely related targets thus
representing a valid alternative to antibodies or other bio-mimetic receptors,
for the development of biosensors and other bio-analytical methods. Moreover
they can be easily stabilized by chemical modifications for in vivo applications
and numerous examples have shown that stabilized aptamers against
extracellular targets such as growth factors, receptors, hormones or coagulation
factors are very effective inhibitors of the corresponding protein function. By
integrating the aptamer-based biosensor development with the maturing
technology for in vitro selection of anti-protein aptamers results in the high-
throughput production of proteome chips. Furthermore, aptamer arrays and
biosensors will reveal the most effective tools for the detection of biomolecular
interactions and the identification of protein targets, particularly with regard to
those not detectable by known receptors like enzymes or antibodies. We will
review here the main and innovative methods based on the use of aptamers as
biosensors for protein detection that, in alternative or combined to the classical
proteomic approaches, could reveal suitable for both diagnostic and therapeutic
purposes.
INTRODUCTION
The deciphering of the human genome and the enormous amount of data collected on
disease mechanisms has lead to the discovery of an increasing number of potential
specific targets for new therapeutics or diagnostics. Indeed, through the use of different
technologies, including mass spectrometry and bioinformatics, proteomic has recently
provided the possibility to define the protein expression profile for a given tissue or
*Corresponding authors: Tel: +390817462036; Fax: +390817701016; E-mails: defranci@unina.it and

cerchia@unina.it

104 Frontiers in Drug Design & Discovery, 2006, Vol. 2 de Franciscis and Cerchia
organ and its alterations caused by a disease or infection [1]. The detailed knowledge of
potential disease-associated targets is an extremely useful tool for the rational design of
novel therapeutic molecules or diagnostics. Therefore, the selective targeting of these
signatures has become a main goal to develop patient-oriented new therapeutic agents
especially for devastating disease as cancer and neurodegenerative diseases.
In cancer an increasing number of proteins involved in cell growth, including growth
factors, receptors, intracellular mediators and transcription factors have been found to be
altered through multiple mechanisms of activation. These include the altered
expression/activity of cell surface proteins as receptor-type tyrosine kinases, including
the epidermal growth factor receptor (EGFR) that is frequently found over-expressed in
non-small cell lung carcinoma, bladder, cervical, ovarian, kidney and pancreatic
carcinoma, and the HER-2/neu receptor that is over-expressed in various types of
cancers, including breast (were it occurs in 30% of early stage cases), ovarian, gastric,
lung, bladder, and kidney carcinomas [2,3]. Likewise, the altered activation or
expression of intracellular proteins as the serine/threonine kinases Akt and Erk or
nuclear effectors as p53 and pRB is needed to uncontrolled cell survival and
proliferation. Therefore, these proteins are suggested as possible therapeutic or
prognostic biomarkers, however, no single biomarker was able to identify those patients
with the best (or worst) prognosis or those which would be responsive to a given
therapy.
On the other hand, the anomalous protein misfolding and aggregation, with an
accompanying "toxic gain of function" occurs in several neurodegenerative conditions
including Prion diseases, Alzheimer’s disease (AD), Parkinson’s disease (PD), and
Huntington’s disease and is central to their pathogenesis. For example, in AD, misfolded
amyloid beta peptide 1-42 (Abeta), a proteolytic product of amyloid precursor protein
metabolism, accumulates in the neuronal endoplasmic reticulum and extracellularly as
plaques. In contrast, in PD cases there is abnormal accumulation of alpha-synuclein in
neuronal cell bodies, axons, and synapses [4].
Hence, finding specific ligands capable to detect and measure the altered pattern of
gene expression is a strategic and plausible objective for the diagnosis and therapy of
important diseases.
To be effective as tools to detect disease-associated biomarkers these ligands should
have the following characteristics: (i) ability to discriminate between different
conformations of the same target proteins; (ii) capability to quantify the level of
expression of the altered versus the physiologic forms; (iii) ideally, be usable both for in
vitro and in vivo purposes; and finally, for therapeutic applications they should (iv) have
the power to interfere with the altered product. To this goal, different types of molecules
have been shown to be of potential utility for diagnosis and therapy, including small
chemical compounds, peptides, antibodies and nucleic acid ligands.
Here we will discuss the properties of nucleic acid-based compounds (named
aptamers) isolated from combinatorial libraries by a selection procedure, the Systematic
Evolution of Ligands by EXponential enrichment (SELEX) technology [5-7], which, in
the last few years, has yielded several high-affinity ligands and potential antagonists of
disease-associated proteins [8-15]. Aptamers are single-stranded nucleic acids that
unlike ribozymes and antisense oligonucleotides, assume three-dimensional shapes that
dictates high-affinity binding to a variety of targets. In particular, in this review we will
Aptamer-Based Technologies Frontiers in Drug Design & Discovery, 2006, Vol. 2 105
focus on the main methods based on the use of aptamers as biosensors for protein
detection.
OVERVIEW OF THE SELEX TECHNOLOGY

The SELEX technology is an evolutionary, in vitro combinatorial chemistry process
used to identify aptamers, as specific ligands of a given target, from large pools of
diverse oligonucleotides, Fig. (1). The starting point for the generation of an aptamer is
the synthesis of a nucleic acid library (RNA, DNA or modified RNA) of large sequence
complexity followed by the selection for oligonucleotides able to bind with high affinity
Fig. (1). Schematic representation of the SELEX process. The single-stranded (ss) DNA library is
amplified by Polimerase Chain Reaction (PCR) in order to generate the double-stranded DNA
pool that will be transcribed by T7 RNA Polymerase. The pool of RNA molecules with different
conformations will be used for the selection process (see text for details).
and specificity to a target molecule. Randomisation of a synthetic sequence stretch from

22 up to 100 nucleotides in length have been used to create an enormous diversity of
possible sequences (4N different molecules) which in consequence generate a vast array
of different conformations with different binding properties. The SELEX method
includes steps of: (i) incubating the library with the target molecule under conditions
favourable for binding; (ii) partitioning: molecules that, under the conditions employed,
adopt conformations that permit binding to a specific target are then partitioned from
other sequences; (iii) dissociating the nucleic acid-protein complexes and (iv) amplifying
of the nucleic acids pool to generate a library of reduced complexity enriched in
sequences that bind to the target. This library will be then used as starting pool for the
next round of selection. After reiterating these steps (the number of rounds of selection
necessary is determined by both the type of library used as well as by the specific
enrichment achieved per selection cycle), the resulting oligonucleotides are subjected to
DNA sequencing. The sequences corresponding to the initially variable region of the
library are screened for conserved sequences and structural elements indicative of
potential binding sites and subsequently tested for their ability to bind specifically to the
target molecule.
Affinities of aptamers for the targeted proteins are typically very high, with
dissociation constants ranging from low picomolar (1 x 10-12 M) to low nanomolar (1 x
10-9 M) that are better than those obtained for peptides derived from phage display
selection and comparable to antibody-antigen interactions. Furthermore, due to the fact
that the specific, three-dimensional arrangements of a small number of contact points of
the aptamer mediates the protein-aptamer interaction, rather than a general affinity for
the sugar-phosphate backbone of the nucleic acid, aptamers can achieve high target
selectivity. In addition, the binding characteristics of aptamers can be influenced by the
type of the experimental system used for the selection and counter-selection (depletion
of aptamers that bind to non-target molecules).
IN VIVO APPLICATIONS OF APTAMERS

During the last few years an increasing interest has emerged for aptamers designed
against cellular or viral targets of biomedical interest in vivo. Several aptamers are
actually in clinical trials [16] and the Food and Drug Administration has recently
approved one aptamer developed by Eyetech (Macugen™) that inhibits the human
Vascular Endothelial Growth Factor 165 (VEGF165), for the treatment of age-related
macular degeneration [17].
Since the first description of SELEX in 1990, aptamers have been generated toward a
variety of different targets, including proteins, peptides, small molecules, organic dyes,
viruses etc. that are potential targets for therapeutic or diagnostic intervention. In several
cases it has been shown that the SELEX procedure permits to generate molecules that
display high selectivity for the target thus allowing to easily discriminate between even
very close molecules. Indeed, it has been demonstrated that anti-theophylline aptamer
can discriminate between caffeine and theophylline, two molecules that differ only by a
methyl group [18]. For instance, RNA aptamers with high selectivity have been
generated that bind with nanomolar affinities the protein kinase C, a potential target in
cancer medicine, and are capable of discriminating between the beta II from the highly
related beta I isoenzymes [19]. DNA aptamers have been obtained that recognise both
the native and the denatured state of ERK-2, a member of the family of mitogen-
activated protein kinases, which are central transducers of extracellular signals [20].
RNA ligands with high affinity for the Ras binding domain (RBD) of Raf-1 have been
isolated and shown to inhibit either Ras binding to Raf-1 and Ras-induced Raf-1
activation, but they did not affect the interaction of Ras with B-Raf, a Raf-1 related
protein [21]. Furthermore, highly specific aptamer has been generated against platelet-
derived growth factor (PDGF) that suppress PDGF B-chain (PDGF-BB) but not the
epidermal- or fibroblast-growth-factor-2-induced proliferation [22]. Other proteins for
aptamer targeting are: tenascin-C, an extracellular matrix protein that is over-expressed
during tissue remodelling processes, including tumour growth [23]; human epidermal
growth factor receptor-3 (ErbB3/HER3; 24) and the IFN-γ-inducible CXCL10
chemokine [25]. In the latter case, Marro et al. identified a series of nuclease-resistant
RNA aptamers with high binding affinity for human and/or mouse CXCL10. CXCL10 is
a chemokine involved in a variety of inflammatory diseases. Since some of the aptamers
are highly selective for CXCL10, they represent a powerful tool to further elucidate the
complex cross-talk between the CXCL10/CXCR3 receptor and other chemokine/
receptor system.
Even though a large number of aptamers have been selected for preferential targeting
of extracellular proteins or protein epitopes the use of living cells as complex target has
been recently described to develop a differential whole-cell SELEX protocol to target
cell surface bound proteins in their natural physiological environment. The aptamers
produced bind specifically to the Ret receptor tyrosine kinase and inhibit its downstream
signalling effects [26].
Despite the increasing number of aptamers isolated of potential medical importance
their use in therapy is still lagging behind because of the lack of an efficient and safe
delivery system to target specific cells with adequate amounts of aptamer.
Indeed, to be successfully applied in therapy aptamers must possess defined
molecular properties needed to cross the collagen microfibrillar network of the
extracellular matrix, and reach the target tissue or cells and, most importantly, also
penetrate the cell membrane. Coupling aptamers to inert large molecules, as cholesterol
or polyethylene glycol, have been used to keep them in circulation anchored to liposome
bilayers [27]. Furthermore, since aptamers, especially RNA-based aptamers, are rapidly
degraded by nucleases in whole organisms major efforts have therefore been addressed
to improve their stability by a variety of approaches [28]. RNA aptamers with 2'-fluoro
(amino) pyrimidine modifications, 2'-O-alkyl nucleotides, 3'end cap and locked nucleic
acids, LNA [29] modifications that significantly enhanced their stability, may survive
for several hours in vivo against degradation by nucleases [30]. Hence, the development
of a safe, efficient, specific, and non-pathogenic system for the delivery of therapeutic
RNA is highly desirable.
Protocols have been also developed that allow the targeting of intracellular proteins
with inhibitory aptamers (named intramers) that are delivered into intra-cellular
compartments either by direct transfection or through the use of expression systems for
the aptamer sequences [31-33]. The cytoplasmic expression of intramers demonstrates
the aptamers striking potential as rapidly generated intracellular inhibitors of
biomolecules.
Nevertheless and despite the very short time from the development of the SELEX
process several aptamers are actually enrolled in phase 2 or phase 3 clinical trials as
promising therapeutics. In this respect, the application for in vivo imaging is especially
promising due to the very wide range of possibilities available to introduce changes in
their structure that will enhance the bioavailability and tune the pharmacokinetics
properties. Indeed, apart those mentioned above, there are very few drawbacks for the
use of aptamers in vivo. In fact, there is no experimental evidence so far for aptamers
being immunogenic, a very useful property for reagents that need to be administered
repeatedly to the same individual for therapy or diagnostic when studying disease
progression.
DEVELOPMENT OF BIOSENSORS WITH APTAMERS AS BIO-

RECOGNITION ELEMENT
Proteins are critical for the normal functioning of the human body. They provide
structure, transmit and receive information, and carry out important chemical reactions.
In persons with a disease, many proteins may be affected. Compared to normal health,
some proteins may become overly abundant, while others may become scarce. By
measuring these proteins, associated with a clinical state of interest, such as the presence
of disease or response to a therapy, it becomes possible to discover new insights into
disease and health. For these reasons there is an obvious need to develop new
technologies that could speed both diagnosis and therapy of diseases by enabling direct
detection of the expression and modification of proteins closely correlated with disease
and by accelerating the process of drug discovery and development.
Proteomics is defined as the study of the structure, function, expression, cellular
localisation, interacting partners, and regulation of every protein produced from a
complete genome [1]. In the last few years a big effort has been devoted to design
innovative approaches alternative or combined to the classic proteomic methods,
represented by two-dimensional gel separation followed by mass spectrometric analysis,
to define protein profile of a specific cell, tissue, or organism and to associate protein
expression levels and post-translational modifications with disease states. The aim of the
improvement of the proteomic technology is to overcome the evident limitations in
speed and sensitivity in samples processing by allowing the quantification of even the
most weakly expressed proteins. Instead of identifying proteins on the basis of their
charge and size a useful approach is to identify them by specific recognition. In this way
it is possible to discriminate proteins that have similar physical properties but different
conformations.
As described above, nucleic acid aptamers like antibodies recognize and bind a
specific protein target on the basis of their three-dimensional structure. Labelled
aptamers have been used in the same way as antibodies, for example in enzyme-linked
immunosorbent assay (ELISA)-like assays, chromatographic and electrophoretic
techniques, Western blotting analyses and more generally as biosensors, including in
arrays [34,35].
Several features render aptamers a promising alternative to antibodies as tools for the
specific detection of proteins. Differently from antibodies they are synthetic molecules
and are generated entirely in vitro; the synthetic nature of aptamers will facilitate
manufacture and arraying, while the in vitro SELEX process, has facilitated automation
for high throughput probe generation [36,37].
Moreover, the minimal cross-reactivity reached by the addition of negative counter-

selection steps during aptamers generation and the high affinity for the target, provide
the potential for producing multiplex chips. The use of the aptamers, in fact, allows to
overcome the most critical obstacle to the development of antibody microarrays for
protein detection which is the lack of specificity of many available antibodies that cross-
react with multiple proteins [38]. In addition, while many antibodies are temperature-
sensitive and can denature upon contact with surfaces, aptamers are stable to long-term
storage, can be transported at room temperature, and undergo reversible denaturation.
Furthermore, since the chemistry for the production and the modification of oligo-
nucleotides is well developed, once aptamers are selected, they can be functionalised
using a wide variety of fluorophores, as well as cobalt or iron paramagnetic particles,
gold, radio-isotopes and biotin. These characteristics render the aptamers suitable as
ligands for protein detection in a great number of different methodologies.
In addition to report in detail the most innovative methods using the aptamers as
biosensors in chip arrays (see “Aptamers as probes in microarray format” paragraph), we
have summarised in Table 1 some of the recent applications for protein detection and
quantisation that use immobilised aptamers in chromatographic, electrophoretic and
Mass Spectrometry techniques. As revealed by the reported examples, the aptamers
employed as ligands in separation methods, are all raised against proteins of biomedical
importance.
Table 1. Different Aptamer-Based Separation Methods for Protein Detection
Aptamer-based
Aptamer Application Ref.
separation method
Affinity Chromatography DNA-aptamer for L-selectin Purification of human [39]

L-selectin receptor globulin
Affinity Chromatography Aptamer for the thyroid Purification of TTF1 from [40]
transcription factor 1 (TTF1) bacterial lysates
Affinity capillary DNA-aptamer for HIV-1 Detection of HIV-1 reverse [41, 42]
electrophoresis reverse transcriptase transcriptase
Non-equilibrium capillary Aptamer against human Quantitative analysis of [43]

electrophoresis of equilibrium thrombin thrombin
mixtures (NEC-EEM)
MALDI-Mass Spectrometry DNA aptamer for human Detection of thrombin from [44]
thrombin human plasma
Aptamers as Probes in Microarray Format

The functional features of the aptamers allows their use as biosensors and their
adaptation to chip arrays. The nature of the platform most suitable for the generation of
aptamer arrays is open to question. Simply printing aptamers on polylysine-coated slides
is not a good approach since the aptamers will denature upon electrostatic capture. Walt
and co-workers have adapted an anti-thrombin DNA aptamer to high-density fiber-optic

arrays [45]. The aptamer was immobilised on the surface of silica microsheres and the
resulted beads were loaded in microwells on the distal tip of an imaging fiber. Beads
prepared by using a different oligonucleotide were included to detect the non-specific
binding. The imaging fiber was coupled to a modified epifluorescence microscope
system, and the distal end of the fiber was incubated with a fluorescein-labelled
thrombin solution. The system showed a good selectivity towards thrombin and could be
reused without any sensitivity change. It presented an apparent dissociation constant of
300 nM for thrombin binding and a detection limit of 10 nM.
Another use of DNA and RNA aptamers as bio-recognition element in optical
sensors is represented by a chip-based biosensor for multiplex analysis of proteins
related to cancer in complex biological mixtures [46]. In this study, carried out at the
company Archemix, four fluorescently labelled aptamers (RNA-based aptamers against
basic fibroblast growth factor (bFGF), inosine monophosphate dehydrogenase, and
VEGF, and an anti-thrombin DNA-based aptamer) were immobilized onto a glass
surface within a flow cell and fluorescence polarization anisotropy was used for solid-
phase measurements of target protein binding. It has been demonstrated specific
detection and quantification of cancer-associated proteins (inosine monophosphate
dehydrogenase II, VEGF and bFGF) in the context of human serum as well as in cellular
extracts. The anti-thrombin aptamer yielded a 10-fold increase in signal above
background, while RNA aptamers against bFGF, inosine monophosphate dehydrogenase
and VEGF, showed 2-, 1.4-, and 25-fold increases, respectively, in signal compared to
background controls. Furthermore the dissociation constant values for the complex
aptamer-target in the array were comparable to those from solution-phase experiments,
thus suggesting that the immobilization of the aptamers did not interfere with their
functionality.
Furthermore, the aptamer arrays technology has been integrated with a device that
could also deliver samples to perform complex assay procedures [47]. In more detail, the
“electronic tongue”, a chip-based microsphere array developed for the digital analysis of
complex fluid by using chemical sensors and antibodies [48], has been adapted to the
use of aptamers as biosensors. The aptamer-based electronic tongue setup contained a
fluid delivery system, a fluorescence microscope, a digital camera, a flow cell in which
the aptamer chips were loaded, and a computer for data analysis. The aptamer chips
were constituted by silicon chips with multiple wells in which were deposited the beads
modified with the sensor. To this aim, streptavidin agarose beads were incubated with
5’ends-biotinylated aptamers to produce aptamer-bead sensor elements. The aptamer
biosensor array was tested to functionally screen aptamers anti-lysozyme, previously
selected. Furthermore, it was used to quantify labelled proteins by means of aptamers as
capture reagents, and to quantify unlabelled proteins, in a sandwich assay format with
antibodies. In the first case, an aptamer originally selected to bind to biothreat agent ricin
was biotinylated, immobilised and probed with a solution contained Alexa-Fluor488-
labelled ricin, once introduced into the chip wells. In the sandwich assay, the anti-ricin
aptamer bound to unlabelled ricin, while a fluorescent anti-ricin antibody served as a
reporter. The limits of detection (320 ng/ml) in sandwich assay format were comparable
to those observed with antibody anti-ricin assay. The biosensors were reusable by
heating or by washing with a buffer containing 7 M urea.
Integrating aptamer microarray production with the maturing technology for

automated in vitro selection of antiprotein aptamers result in the high-throughput
production of proteome chips. Recently, high-throughput methods for generating
aptamer microarrays have been described [49]. As a proof-of-principle, the microarrays
have been used to screen the affinity and specificity of a pool of robotically selected
anti-lysozyme RNA aptamers. 5’-biotinylated RNA aptamers were spotted on
streptavidin-coated microarray slides and the resultant arrays were validated by showing
the specific, dose-dependent detection of lysozyme target protein. The lower limit of
detection was in the low-pg/ml range, and the protein could be detected even against a
background of labelled cellular lysate.
Recently, an attractive method represented by the direct site-specific biotinylation at
the desired positions by transcription using unnatural base pairs, has been used to
immobilise a previously selected RNA-based aptamer on sensor chips [50]. The same
research group developed unnatural base pairs between 2-amino-6-(2-thienyl)purine
(denoted by s) and 2-oxo(1H)-pyridine (denoted by y) or 2-amino-6-(2-thiazolyl)-purine
(denoted as v) and y. These unnatural base pairs function in transcription and can be
used in the direct site-specific biotinylation of RNA molecules. In fact, the position 5 of
y can be modified to introduce various functional groups via the nucleoside of 5-iodo-2-
oxo-(1H)pyridine (5-iodo-y). In this study, the procedure for the synthesis of the
substrate of biotinylated-y (Bio-yTP) and the incorporation of Bio-y into RNA by T7
RNA polymerase transcription, was used to transcribe a biotinylated anti-(Raf-1) RNA
aptamer [21]. The biotinylated RNA aptamer was efficiently immobilised on
streptavidin-coated sensor chips and accurately recognised its target protein. The
aptamer interaction with Raf-1 was detected by 27 MHz quartz-crystal microbalance
(QCM) or surface plasmon resonance (SPR) technology. The unmodified RNA aptamer,
in which Bio-y was not incorporated, did not bind to either sensor chip. The protein
bound on the sensor chips could be removed by urea treatment, to regenerate the sensor
chip surface. Furthermore, the binding of another RBD, GST-RBD of RGL, to the
sensor chip-bound aptamer was not detected, indicating that the immobilised RNA
aptamer retains its specificity for Raf-1.
By using the QCM technology it has been recently developed an aptamer-based
biosensor to detect the protein trans-activator of transcription (Tat) of human
immunodeficiency virus type 1 (HIV-1) [51]. Tat protein controls the early phase of the
HIV-1 replication cycle [52]. With the aim to generate a detection method for the full-
length Tat protein, it has been successfully used the aptamer selected against Tat [53],
which displays an efficient binding specificity against Tat, but not for other cellular
factors. This aptamer is a very promising bio-recognition element for the detection of
Tat, since it combines unique characteristics such as high affinity for Tat and possibility
of altering its design to enhance sensitivity and stabilisation against nucleases, allowing
monitoring of viral protein levels in vivo. For these reasons, it has been used as a model
also in aptamer beacon generation (54, see “Protein detection by molecular aptamer
beacons” paragraph).
For the production of the biosensor, the RNA aptamer was biotinylated and
immobilised on streptavidin deposited on a gold electrode of piezoelectric quartz-
crystals. The interaction with Tat protein in solution has been studied following the
changes in the oscillation frequency of the crystal. Furthermore, the aptamer-based
biosensor has been compared with the corresponding immuno-sensor, based on the
specific monoclonal anti-Tat antibody. The antibody was immobilised on a layer of

carboxylated dextran previously deposited on the gold electrode. Both receptors,
aptamer and antibody, detected Tat at a minimum concentration of 0.25 ppm with a
comparable reproducibility in terms of coefficient of variation. The functional properties
of the developed piezoelectric aptamer-biosensor showed that the approach can be
applied to detect with specificity and reproducibility other different protein targets with
the advantage to regenerate and reuse the biosensor.
As demonstrated by all the above studies, in addition to strengthen the basic research
in proteomics such functional aptamer-based microarrays could accelerate the analysis
of combinatorial libraries of already selected aptamers. Furthermore, the aptamer array
technology combined to bioinformatics allows to discover disease-specific biomarkers
and protein signatures and to verify drug compounds efficacy. The measurement, using
an aptamer array, of the concentrations of a large number of proteins in a patient’s
clinical sample could be obtained. A patient’s protein profile is likely to change in the
presence of disease, and different profiles may be associated with varying responses to
therapeutics or other clinically relevant parameters.
PhotoSELEX and Photoaptamer-Based Chips as Highly Sensitive and Specific

Capture Agents
Aptamers with modified DNA bases that photo-crosslink to captured proteins
(photoaptamers) have been investigated for use as arrayed probes for proteomic analysis.
They are highly sensitive and specific capture agents developed using the photochemical
SELEX (PhotoSELEX) process. This new in vitro selection methodology was used by
Golden et al. [55] to identify ssDNA aptamers capable of photo-crosslinking the human
bFGF, representing the target molecule. The bFGF photoaptamers could cross-link
picomolar concentrations of target in the presence of serum with very little non-specific
cross-linking. They displayed functional properties comparable with commercially
available ELISA monoclonal antibodies and perfectly distinguished bFGF from
consanguine proteins, VEGF and PDGF.
The PhotoSELEX method is based on the incorporation of at least one
bromodeoxyuridine (BrdU) (in place of a T nucleotide) in the oligonucleotide libraries
that, as a consequence of the modification, can form a specific covalent crosslink with
the target proteins when exposed to ultraviolet light. Because crosslinking requires both
affinity-based binding and close proximity between a BrdU (at a specific location in the
photoaptamer) and an amino acid (at a specific location in the target protein),
photoaptamers can offer extraordinary specificity.
Photoaptamers can be assembled into aptamer arrays in order to measure
simultaneously large numbers of proteins, eventually thousands [56,57]. Since
photoaptamers covalently bind to their target analytes before fluorescent signal
detection, the photoaptamer arrays can be vigorously washed to remove background
proteins, providing the potential for superior signal-to-noise ratios and lower limits of
quantification in biological matrices.
The sensitivity and specificity of photoaptamers, combined with the ability to
automate and scale up their selection and the ability to use them on solid surfaces,
indicate that they could become an important factor in the development of proteomic
technology.
The application of photoaptamer technology to proteomics was investigated in

microarray format by using the above bFGF photoaptamer [55] and a new photoaptamer
raised against the HIV coat protein gp120MN [58]. To this aim, the photoaptamers were
synthesised with a 5’ C6-amino linker and immobilised by spotting on N-hydroxy-
succinimide -activated slides. The aptamers were then assayed for photocross-linking
activity by dose response to their targets. The obtained results showed that the behaviour
of the aptamers was the same on surface as well in solution, in fact they displayed
subnanomolar sensitivity with little or no cross-reactivity.
Furthermore, a mathematical model for the kinetic analysis of photoaptamer-protein
photocross-linking reactions has been reported by using two photoaptamers that cross-
linked human bFGF and one against HIV MN envelope glycoprotein [59].
Multiplexed photoaptamer-based arrays that allows for the simultaneous measure-
ment of multiple proteins of interest in serum samples have been recently described [60].
Microarrays spotted with photoaptamers specific for 17 extracellular proteins were
probed with target proteins, illuminated with 308-nm light to covalently crosslink the
photoaptamers to their affinity-bound target proteins, subjected to harsh denaturing
washes to remove non-specific binders, treated with an amine-reactive fluouorescent
dye, and then scanned. The array exhibited limits of detection below 10 fM for several
analytes including interleukin-16,VEGF, bFGF and endostatin and able to measure
proteins in 10% serum samples.
To date some photoaptamer-based chips are commercially available as highly
sensitive and specific capture agents to discover disease-specific biomarkers and protein
signatures (Somalogic Inc.). Furthermore, a continuous and crescent consideration is
given to the challenges involved in producing multiplex aptamer chips composed of
aptamers taken from disparate literature sources, and to the development of standardised
methods for characterising the performance of capture reagents used in biosensors.
PROTEIN DETECTION BY APTAMER-BASED PROXIMITY LIGATION

ASSAYS
In the field of the development of innovative specific and sensitive procedures to
evaluate proteomes, worthy of note is the literature by Landegren and co-workers. They
have dedicated a lot of work to the study of the ligation of oligonucleotide probes as a
mechanism for protein detection [as a review see 61]. The mechanism of proximity
ligation is depicted in Fig. (2).
The act of DNA ligation creates sequences that can be used for amplified detection of
macromolecules with excellent specificity and sensitivity. The mechanism has been used
for analyses of nucleic acid, but it can also be applied to detect proteins if affinity probes
are coupled to DNA sequence extensions. In fact, in the proximity ligation technique,
target proteins are analysed using two or more “proximity probes” each composed of one
ligand-binding component able to bind the target protein, and one attached
oligonucleotide strand. Binding of the probes to the same target molecule brings the
oligonucleotides into close proximity. Thereby the DNA strands can be joined through
ligation, generating a sequence with elements required for amplification of the ligation
product to detectable levels. Probes that fail to bind the target molecule do not come in
proximity and give rise to low non-specific signal.
Fig. (2). The mechanism of proximity ligation.
Antibodies such as aptamers can be used as protein binders, even if DNA aptamers
are ideal reagents in such assays, since the attachment of DNA sequence extensions is
trivial, and since reagents can be selected, recorded as DNA sequences, and shared in the
scientific community just like PCR primers. The method, based on the use of a DNA
aptamer for the PDFG-BB protein [62], has been successfully applied to detect the
homodimer of PDGF-BB [63]. It has been demonstrated that homogeneous proximity
ligation assays can be established that exceed the detection sensitivity of standard
ELISAs by a factor of a thousand. The technique can also be combined with
amplification via the rolling-circle replication mechanism by which ligated probes can
be induced to form circles of DNA that represent an ideal tool for localised analysis of
individual protein molecules and of interactions between proteins in situ.
PROTEIN DETECTION BY MOLECULAR APTAMER BEACONS

Large impulse is given to the development of molecular aptamer beacons for protein
recognition and quantitative analysis. The design of molecular beacons by using nucleic
acid aptamers employs the high versatility of target recognition of the aptamers so it will
be possible to develop beacons to detect a variety of target proteins of biomedical
importance. The unique properties of the molecular beacons, which combines the signal
transduction mechanism of molecular beacons and the high specificity of aptamers, will
enable the development of a class of protein probes for real time protein tracing in living
specimen and for efficient biomedical diagnosis in homogeneous solutions.
Molecular beacons are oligonucleotide probes that assume a hairpin structure in
which the single-stranded loop can pair with complementary sequences and the paired
stem contains fluorescent reporters (or a fluorophore and a quencher) that interact with
one another [64]. Hybridisation of a complementary target sequence leads to the
formation of a long duplex region, destabilisation of the hairpin, and a spatial separation
between the two dyes. Ultimately, interaction with target oligonucleotides leads to either
the loss of fluorescence resonance energy transfer (FRET) or to dequenching of a

fluorophore, optical signals that can be readily detected. The utility of this kind of
biosensors relies in their capability to report the presence of specific nucleic acids in
homogeneous solutions and are used in real time PCR.
Molecular beacons have been modified by using nucleic acid aptamers in order to
detect non-nucleic acid ligands, including proteins. A lot of examples have recently been
reported in which the aptamer dictates the specificity of target recognition. Similar to
molecular beacons, aptamer beacons can adopt two or more conformations, one of which
allows ligand binding. Some uses of molecular beacon aptamers have been reported that
employ the thrombin as model ligand. In the study of Hamaguchi et al. [65], an anti-
thrombin aptamer has been engineered by adding nucleotides to the 5'-end, which are
complementary to nucleotides at the 3'-end of the aptamer. In the absence of thrombin,
the aptamer beacon is forced into a stem-loop structure. In the presence of thrombin, the
aptamer beacon undergoes a conformational change that in turn causes a change in the
distance between a fluorophore attached to the 5'-end and a quencher attached to the 3_-
end. The fluorescence-quenching pair is used to report changes in conformation induced
by ligand binding.
An alternative strategy has been reported by Li et al. [66]. They constructed two
aptamer beacons, one labelled with a fluorophores-quencher pair and the other with two
fluorophores. The binding of thrombin to the beacon causes significant fluorescent
signal change attributed to a significant conformational change from a loose random coil
to a compact unimolecular quadruplex. The aptamer beacon recognises its target at 112
picomolar thrombin concentration in homogeneous solutions and allows protein
quantisation in living specimen.
A special class of signalling aptamers named "structure-switching signalling
aptamers" has been reported [67,68]. Signalling aptamers refer to aptamers or modified
aptamers able to generate a recordable signal. This new class of signalling aptamers are
designed to function by switching structures from a pre-formed, lowly fluorescent
duplex assembly to a ligand-aptamer complex having a higher level of fluorescence. In
the assay having the thrombin as a model target, in the absence of the protein the
fluorophore-labelled DNA aptamer forms a duplex with a complementary
oligonucleotide modified with a quencher. In the presence of the target, it is favourite the
formation of the complex aptamer-protein with a consequent increase in fluorescence
intensity.
A recent recent approach has been described [69] involving protein-induced co-
association of two aptamers recognising two distinct epitopes of the same protein.
Two fluorophore-labelled “signalling” oligonucleotides are attached to both the
aptamers by non-DNA linker. When the aptamers bind the protein, the signalling
oligonucleotides are brought in proximity causing a change of FRET between the
fluorophores. This assay has been developed by using thrombin as a model system but it
could detect different disease-related proteins in biological samples.
Furthermore, a novel electrochemical detection method for aptamer biosensors has
been recently reported for detection of thrombin [70]. Methylene blue (MB), intercalated
into the beacon sequence, represents the electrochemical marker. When the beacon
aptamer, immobilised on a gold surface, binds thrombin, the hairpin forming beacon
aptamer undergoes a conformational change leading to the release of the intercalated

MB. As a consequence, a decrease in electrical current intensity is registered in
voltamogram. The peak signal of the MB is clearly decreased by the binding of thrombin
onto the aptamer beacon. This method was able to linearly and selectively detect
thrombin with a detection limit of 11nM.
Moreover, even quaternary structural changes have been exploited to create aptamer
biosensors [54]. Two RNA oligomers derived from an anti-Tat aptamer were constructed
to analyse the Tat protein of HIV-1. One of the oligomer forms a hairpin structure that
contains fluorophore and quencher at both ends of the RNA and another was a non-
structured oligomer. Specifically in the presence of Tat or its peptides, but not in the
presence of other RNA binding proteins, the two oligomers undergo a conformational
change to form a duplex that leads to relieving of fluorophore from the quencher, and
thus to the generation of a fluorescent signal.
In addition, to overcome the step of aptamer engineering to obtain a functional
aptamer beacon, it has been reported the possibility to directly select aptamer beacons
starting from a pool of random sequence DNA molecules [71]. This method couples in
vitro selection for ligand binding to a nucleic acid conformational change that in turn
leads to the production of a fluorescent signal.
A very recent study [72], reports the development of an aptamer beacon as a
synthetic high-affinity DNA probe that exhibits FRET in response to a specific protein
biomarker, the PDGF. This approach could be applicable to protein biomarkers relevant
in cancer and other important diseases. It consists in a novel assay method that is based
on specific conformational features of synthetic, high-affinity DNA aptamers in the
presence of the specific protein targets to which these aptamers bind. This assay can
detect as little as 2.5 ng of PDGF in the presence of a nearly 1000-fold excess of serum-
derived proteins commonly present in cell culture media. It has been observed that, with
a DNA aptamer carrying multiple binding sites for a multimeric protein target, the FRET
bioassay can be accomplished by using a mixture of two individually labelled DNAs,
one carrying the fluorophore and the other with the matching quencher. The incubation
of the aptamers and the target protein results in FRET because of the specific closed
conformation of both aptamer molecules. This finding could be useful for the future
design of more selective DNA-based FRET bioassays that use more than one ligand for
the same protein target.
CONCLUDING REMARKS
One of the most pressing problems facing those attempting to understand the
regulation of gene expression and translation is the necessity to monitor protein
production in a variety of metabolic states. Many of the available methods for
characterising and quantitating protein biomarkers are time consuming, labour intensive
and require multiple steps, such as immobilisation, repetitive incubations and washings,
as well as additional reagents to amplify the signal. The use of aptamers as bio-
recognition element for the development of biosensors to detect protein targets offers
over classical methods mainly based on antibodies, a lot of advantages, such as the
possibility of easily regenerate the immobilised aptamers, their homogeneous
preparation and the possibility of using different detection methods due to easy labelling.
Moreover, the enormous diversity of random oligonucleotide libraries can exceed the
diversity of antibodies in the mammalian genome by several orders of magnitude. Since

aptamers are nucleic acids, experience with DNA, as in the production of DNA-arrays,
should be applicable to the development of aptamer-based biosensors. On the other side,
the aptamer arrays can potentially expand the scope of DNA microarrays to recognise
expressed proteins as well expressed mRNAs. In this regard, numerous aptamers have
already been selected against a wide array of proteins, and the possibility of acquiring
aptamers against proteomes has been advanced by automation of the in vitro selection
procedure. These considerations, explain why now the aptamer-based technology for
protein detection is in advanced stages of development as useful tools in clinical
diagnosis and therapy.
ACKNOWLEDGEMENTS
This work was supported by the European Union FP6 EMIL LSHC-CT-2004-
503569. We wish to thank members of our laboratory C. L. Esposito and I. Amelio for
scientific advice and fruitful discussions.
ABBREVIATIONS
AD = Alzheimer’s disease
bFGF = Basic fibroblast growth factor
EGFR = Epidermal growth factor receptor
ELISA = Enzyme-linked immunosorbent assay
FRET = Fluorescence resonance energy transfer
HIV-1 = Human immunodeficiency virus type 1
MB = Methylene blue
PD = Parkinson's disease
PDFG-BB = Platelet-derived growth factor B-chain
PCR = Polymerase chain reaction
QCM = Quartz-crystal microbalance
RBD = Ras binding domain
SELEX = Systematic Evolution of Ligands by EXponential enrichment
SPR = Surface plasmon resonance
TAT = Trans-activator of transcription
VEGF = Vascular endothelial growth factor
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Recent Developments in Proteomics:

Mass Spectroscopy and Protein Arrays
Aarohi Kulkarni and Mala Rao*

Division of Biochemical Sciences, National Chemical Laboratory,
Pune 411008, Maharashtra, India
Abstract: Proteomics has been gaining increasing attention in order to

understand the relevance of the genomic information available through high
throughput DNA sequencing and microarray techniques. Proteomics can be
viewed as an experimental approach to explain the information contained in
the genomic sequences in terms of the structure, function and control of
biological processes and pathways. It thus systematically analyses the proteins
expressed in the cell. It is well known that several modifications of proteins
like posttranslational modifications and splicing are not visible at the DNA
level but alter the functions of the proteins involved greatly. These can be
envisaged at the protein level wherein the functions can be assigned. Coherent
strategies and technologies are required to elucidate protein expression,
interactions and functions. The most well established approach to the
identification of proteins and their separation is the 2-D PAGE. It allows the
resolution and identification of proteins from a wide variety of sources without
understanding their functions. There are limitations to this approach, which
lacks sensitivity and its inability to separate effectively hydrophobic membrane
proteins. The technique however remains the leader methodology for delivery
of protein expression and for recording change in protein structure. Recently
mass spectrometry has become the method of choice for the identification and
characterization of proteins after their purification. In the current methods of
mass spectroscopy the protein is identified from fragments and as such does
not yield information about posttranslational modifications. Its application is
still at a developing stage and in future it will be one of the leader techniques
for depicting protein structure. Proteomics has recently seen advancement in
the form of microarray technique. Microarray based analysis is a high
throughput technology, which can be performed at a dynamic range and also
has the added advantage of performing functional proteomics. Its ease of
operation and ability to control key parameters such as temperature, pH and
cofactor concentrations makes it suitable to automation. This review will
essentially focus on the different mass spectroscopy approaches being used to
identify and characterize proteins from diverse sources and the developments
in protein array technology.
*Corresponding author: Tel: +91-20-25902228; Fax: +91-20-2588 4032; E-mail: mb.rao@ncl.res.in

122 Frontiers in Drug Design & Discovery, 2006, Vol. 2 Kulkarni and Rao
INTRODUCTION
Proteomics is defined as the study of the proteome, but is more commonly viewed as
a collection of technologies and tools. The technology has seen enormous progress over
the past few years and significant optimism for continued progress. Proteomics has
recently received a lot of attention as the focus for gaining a wider and comprehensive
understanding of the relevance of genomic information generated. This information is
now being generated at an accelerating rate. Ideally it is possible to study the proteins at
both expression and functional levels but for their diverse physicochemical properties. In
addition to their physical properties, expression levels of proteins also vary widely
within a cell, often showing poor correlation to mRNA levels [1]. Proteomics aims to
supplement analytical techniques designed to study proteins by ‘‘one species-at-a-time’’
with methodologies that enable thousands of proteins to be studied concomitantly.
Rather than being hypothesis-driven where subsequent studies are directed based on
previous findings and specific results are anticipated, proteomics is largely discovery-
driven where newly acquired data provide details about the system under study, mostly
without inclination of predictable results. A generalized approach to analyzing protein
expression having common applicability is required [2]. Recently analysis of protein-
protein interactions for pathway elucidation has been undertaken at a meaningful scale.
Despite the complete sequencing of several genomes, the functions of most of the
putatively expressed proteins remain to be established. A very challenging task is the
development of computational methods which will be able to assign predicted function
to proteins on the basis of the genetic information [3]. It is known that several
modifications of proteins, not immediately apparent at the DNA level, such as
posttranslational modification and differential splicing can significantly alter protein
function. It will therefore only be through practical studies at the protein level that
functions will be assigned. Rational strategies and technologies are required to expound
protein expression, interactions and function. Despite the challenges, some studies have
attempted to analyse the proteome at a near genomic scale. The subcellular localization
of expressed proteins and their mapping has been made possible by the tagging
experiments with yeast proteins [4], whilst protein-protein interaction data for
Helicobacter pylori and other organisms is emerging [5-8]. At the functional level, 6144
yeast open reading frames (ORFs) were expressed as glutathione S-transferase (GST)
fusions and screened for activity, allowing ORFs of previously unassigned function to be
assigned.
Mass spectrometry (MS) plays a central role in proteomics research, not only since
the Nobel Prize in Chemistry has been awarded to John B. Fenn and Koichi Tanaka in
2002 [9,10], for their role in the development of electrospray ionization (ESI) and laser
desorption/ionization (LDI) but because of its potential application in understanding the
structure and function of proteins. In the 1980s, these so called “soft ionization”
techniques have laid the groundwork for the modern MS analysis of proteins and
peptides. Mass spectrometers have become more powerful, easy to use and affordable in
recent years. For the analysis of proteins from complex biological mixtures, a number of
limitations still remain that cannot be overcome simply by improvements of MS systems
alone. A common strategy applied in performing proteomics is outlined in Fig. (1).
Recent Developments in Proteomics Frontiers in Drug Design & Discovery, 2006, Vol. 2 123
Fig. (1). A common strategy to perform proteomics.
TWO DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS

The need to analyze proteins at a genomic scale has led to a number of parallel
approaches being developed. A well known technique for protein separation is two
dimensional polyacrylamide gel electrophoresis (2-D PAGE). 2-D PAGE allows the
separation and detection of proteins from a wide variety of sources without the need for
any prior knowledge of function. Presently, numerous hyphenated front-end protein and
peptide separation methods are utilized to increase the depth and breadth of proteomic
measurements. In the earliest applications of proteomics, 2D-PAGE was utilized to
resolve, visualize and compare protein abundances between two samples. The principle
procedure is based on isoelectric focusing (IEF), which separates the proteins based on
their isoelectric points (pI), followed by electrophoresis in the presence of sodium
dodecyl sulphate (SDS) to separate the proteins based on their molecular weight [11].
These separation parameters allow for the resolution of proteins differing by a single
charge, thereby allowing such in vivo modifications such as phosphorylation and certain
mutations to be detected. The high resolving power of 2D-PAGE and the development
of various staining procedures to visualize these protein ‘‘spots’’ have resulted in a very
robust methodology for identifying protein abundance changes between two proteome
samples. In conducting such studies, proteome samples from a control and treated (or
generically different) cell (or organism or tissue) are extracted and separated on distinct
gels. The proteins can be visualized by colorimetric staining and the spots resolved on
both gels are aligned to enable the relative staining intensities of each protein on the
distinct gels to be compared. Protein spots that show a difference in staining intensity
can be excised from the gel and digested within the gel plug [12, 13]. The resulting
peptides can be extracted from the gel and analyzed by either MS or tandem MS. The
MS or tandem MS data are bioinformatically compared with an appropriate database to
identify their protein of origin.
Minor modifications in the techniques have led to increased specificity. These
include the introduction of immobilized pH gradients (IPGs), which provide increased
reproducibility and resolution [14-17]. As a result of such advances, 2-D PAGE is
capable of profiling many thousands of proteins on a single matrix with intense
resolution, separating even isoforms differing in post-translational modifications as
minor as a single deamidation event. Other post-translational modifications detectable by
2-D PAGE include phosphorylation and glycosylation. In conjunction with major
advances in image analysis and improved statistical tools, it is possible to define those
proteins whose expression alters under defined conditions. As a result, skilled scientists
are able to minimise the experimental variance and produce reliably quantitative data.
Mass spectrometry (MS) has become the method of choice for protein identification
and characterization following separation by 2-D PAGE. This is achieved by either
protein mass fingerprinting using MALDI-TOF mass spectrometry or de novo
sequencing using electrospray MS. MS is a rapidly evolving discipline and both of these
methodologies together have recently seen major improvements in terms of sensitivity
and automation. However, although a powerful technique, there are limitations to 2-D
PAGE based approaches. A typical two dimensional gel electrophoresis pattern is shown
in Fig. (2).
The most important drawback of 2D-PAGE-based proteomics is its inability to
analyze the entire proteome. Proteins displayed in a single 2D gel represent only a
portion of all the proteins that are present in a sample. The low-abundance proteins are
mostly missed out in 2-D PAGE. The limit for the detection of proteins with silver
staining is approximately 1 ng, (i.e., 20 fmol for a 50 kDa protein). Proteins, which are
very small and very large proteins, alkaline proteins, and hydrophobic proteins, are also
not open to identification and analysis by 2D-PAGE. One particular class of proteins that
is not readily amenable to 2D-PAGE is membrane proteins.
2D-PAGE is labor-intensive and has a relatively low throughput. The throughput of
2D-PAGE is adequate for many basic research studies, but it may present a serious
obstacle for projects that involve screening of a large number of clinical samples, or for
any other research where the analysis is critical.
The narrow range of silver stain is a limiting factor in the accuracy of the method.
Another troublesome aspect is that different proteins have different staining
characteristics. Coomassie Blue stain is more suitable for protein quantification, but the
sensitivity is significantly lower than the sensitivity of silver staining. MS analysis of
proteins from silver-stained 2D gels is not routine. Silver stain detects proteins down to
the fmol level and, consequently, the quantity of protein in a silver-stained spot is
usually low. The amount of sample available for analysis is further reduced through
losses that occur during the preparation of peptide digests. MS data from digests of
silver-stained proteins may contain only a few peptide signals, which may not be enough
for unambiguous protein identification. Because of the low amounts of the analyte,
analysis of proteins from silver-stained gels is also more susceptible to interferences
from sample contamination [18]. One particularly bothersome class of contaminating
proteins is keratins from human skin and hair that can create a serious problem for
MALDI-ToF-MS. Despite these disadvantages the technique remains the major
established methodology for delivery of protein separation data.
Fig. (2). A typical two-dimensional gel pattern of liver proteins as adapted from Cutler, P (Cutler,
P., 2003). The liver proteins are exquisitely separated on the two dimensional electrophoresis gel
yielding a pattern easy for identification of individual proteins.
SOLUTION-BASED PROTEOMICS
Due to the recognized limitations of 2D-PAGE, a significant amount of effort has
been focused on the development of alternative, non-gel methods of resolving proteome
samples prior to MS analysis. Yates and co-workers [19-22] developed an early example
showing the utility of solution-based fractionation techniques. Their separation method,
called MudPit, is a hyphenated strong cation exchange/reversed-phase liquid chromato-
graphy (i.e., SCX/RPLC) separation method coupled online with MS detection. In the
MudPIT experiment, the entire proteome sample is digested with trypsin and the tryptic
peptides were systematically resolved based on charge in the first dimension (i.e., SCX)
and hydrophobicity (i.e., RPLC) in the second. The peptide mixture is loaded onto a
SCX column and discrete fractions are displaced directly onto the RPLC column, and
are then separated and eluted from the RP column and directly analyzed by MS/MS to
identify the eluting peptides. To maximize sample transfer from the stationary phases,
the SCX and RP stationary phases are sequentially packed into a single capillary
column.
MASS SPECTROMETRY (MS): A POWERFUL TOOL IN PROTEOMICS

Sequencing of an entire genome (genomics) or analysis of multitudes of gene
transcripts using mRNA array technologies (transcriptomics) are efficient tools in
studying the genetic make up of diverse organisms including the human genome. Like
genomics and transcriptomics, the current state-of-the-art proteomic techniques relate
strongly to the development of specific new methodologies and instrumentation that
have come up in the past two decades. This includes mass spectrometry (MS),
protein/peptide fractionation techniques, and bioinformatics to name a few. The current
chapter will be focusing on mass spectrometry and its applications in understanding the
structure of proteins and peptides. Mass spectrometry is an analytical technique which
determines the mass-to-charge (m/z) ratio of ions [23]. It is generally used to find the
composition of a physical sample by generating a spectrum representing the individual
masses of the components of a sample. It has several broad applications exemplified by
the following
• Identifying unknown compounds by the mass of the compound and/or
fragments thereof.
• Determining the isotopic composition of one or more elements in a compound.
• Determining the structure of compounds by observing the fragmentation of the
compound.
• Quantitating the amount of a compound in a sample using carefully designed
methods (mass spectrometry is not inherently quantitative).
• Studying the fundamentals of gas phase ion chemistry (the chemistry of ions
and neutrals in vacuum).
• Determining other physical, chemical or even biological properties of
compounds with a variety of other approaches.
In the analysis of complex proteomes, intact proteins are rarely used for
identification; rather, they are more typically digested by chemical or enzymatic means
into their constituent peptides. These are then identified in the MS mode to produce a
‘‘peptide map’’ or a ‘‘peptide fingerprint’’. These measured masses can be used to
compare with theoretical peptide maps to identify the protein. Mass spectrometers
measure the mass/ charge ratio of analytes. For protein studies, this can include intact
proteins and protein complexes, fragment ions produced by gas-phase activation of
protein ions (top-down sequencing), peptides produced by enzymatic or chemical
digestion of proteins (mass mapping), and fragment ions produced by gas-phase
activation of mass-selected peptide ions (bottom-up sequencing) [24]. The application of
mass spectrometry and MS/MS to proteomics takes advantage of the vast and growing
array of genome and protein data stored in databases. The information produced by the
mass spectrometer, lists of peak intensities and mass-to-charge (m/z) values, can be
manipulated and compared with lists generated from ‘‘theoretical’’ digestion of a protein
or ‘‘theoretical’’ fragmentation of a peptide. Applications to analyze piezomolar
quantities of sample are driving the development of more sensitive mass spectrometers,
as well as low flow, high-resolution separation technologies, to provide structural
information on individual components in complex mixtures of thousands of proteins
derived from biological samples. Protein identification by mass spectrometry requires

interplay between mass spectroscopy instrumentation and gas phase peptide chemistry.
Mass spectroscopy instrumentation deciphers the ionization, activation and detection of
molecules while gas phase peptide chemistry identifies the bonds broken and the rate of
cleavage. The bond cleavage depends on factors such as peptide/protein charge state,
size, composition and sequence.
MASS SPECTROSCOPY
Mass Spectrometers
A mass spectrometer is a device that produces the mass spectrum of a sample to find
its composition. The first mass spectrography technique was described in an 1899 article
by English scientist J.J. Thomson. Arthur Jeffrey Dempster and F.W. Aston in 1918 and
1919 [23] respectively devised the processes that more directly gave rise to the modern
versions. The schematic view of a mass spectrophotometer is outlined in Fig. (3).
Fig. (3). Schematic representation of a mass spectrophotometer; the basic instrument.
The basic principle used in a mass spectrometer is that different molecules have
different masses. For example, table salt (NaCl), is vaporized (turned into gas) and
broken down (ionized) into electrically charged particles, called ions, in the first part of
the mass spectometer. The sodium ions and chloride ions have specific molecular
weights. They also have a charge, which means that they will be moved under the
influence of an electric field. These ions are then sent into an ion acceleration chamber
and passed through a slit in a metal sheet. A magnetic field applied to the chamber pulls
on each ion equally and deflects them onto a detector. The lighter ions deflect further
than the heavy ions because the force on each ion is equal but their masses are not (this
is derived from the equation F = ma which states that if the force remains the same, the
mass and acceleration are inversely proportional). The detector measures exactly how far
each ion has been deflected, and from this measurement, the ion's 'mass to charge ratio'
can be worked out [25, 26]. From this information it is possible to determine with a high
level of certainty what the chemical composition of the original sample was.
Ion Source
The ion source is the part of the mass spectrometer that ionizes the material under
analysis (the analyte). Magnetic or electrical fields then transport the ions to the mass
analyzer. Techniques for ionization have been instrumental in determining what types of
samples can be analyzed by mass spectrometry. Electron ionization and chemical
ionization are used for gases and vapors. In chemical ionization sources the analyte is
ionized by chemical ion-molecule reactions during collisions in the source. Two
techniques often used with liquid and solid biological samples include electrospray
ionization [9, 27] and matrix-assisted laser desorption/ionization [28, 29]. Inductively
coupled plasma sources are used primarily for metal analysis on a wide array of samples
types. Others include fast atom bombardment (FAB), thermo spray, atmospheric
pressure chemical ionization (APCI), secondary ion mass spectrometry (SIMS) and
thermal ionization.
Mass Analyzer
Mass analyzers separate the ions according to their mass per charge (m/z). There are
many types of mass analyzers usually categorized on the basis of the principles of
operation.
Sector MS: It uses an electric and/or magnetic field to affect the path and/or velocity
of the charged particles in some way. The force exerted by electric and magnetic fields is
defined by the Lorentz force law:
F=q (E + v x B)
where E is the electric field strength, B is the magnetic field induction, q is the charge of
the particle, v is its current velocity (expressed as a vector). All mass analyzers use the
Lorentz forces in some way either statically or dynamically in mass-to-charge
determination. Sector instruments change the direction in which ions are flying through
the mass analyzer. The ions enter a magnetic or electric field which bends the ion paths
depending on their mass-to-charge ratios (m/z), deflecting the more charged and faster-
moving, lighter ions more. The ions eventually reach the detector and their relative
abundances are measured. The analyzer can used to select a narrow range of m/z's or to
scan through a range of m/z's to catalog the ions present.
TOFMS: Perhaps the easiest to understand is the Time-of-flight (TOF) analyzer. It
increases the kinetic energy of ions by passage through an electric field and measures the
time they take to reach the detector [30, 31]. Although the kinetic energy is the same, the
velocity is different so the lighter more highly charged ion would reach the detector first.
QMS: Quadrupole mass analyzers use oscillating electrical fields to selectively stabilize
or destabilize ions passing through a RF quadrupole field.
QIT: The quadrupole ion trap works on the same physical principles as the QMS, but the
ions are trapped and sequentially ejected. Ions are created and trapped in a mainly
quadrupole RF potential and separated by m/z, non-destructively or destructively. There

are many mass/charge separation and isolation methods but most commonly used is the
mass instability mode. The cylindrical ion trap mass spectrometer is a derivative of the
quadrupole ion trap mass spectrometer.
Linear QIT: In the linear quadrupole ion trap the ions are trapped in a 2D quadrupole
filed instead of the 3D quadrupole field of the QIT.
FTMS: Fourier transform mass spectrometry measures mass by detecting the image
current produced by ions cyclotroning in the presence of a magnetic field. Instead of
measuring the deflection of ions with a detector such as an electron multiplier, the ions
are injected into a Penning trap (a static electric/magnetic ion trap) where they
effectively form part of a circuit. Detectors at fixed positions in space measure the
electrical signal of ions that pass near them over time producing cyclical signal. Since
the frequency of the ions' cycling is determined by its mass to charge ratio, performing a
Fourier transform on the signal can deconvolute this effect.
Each analyzer type has its strengths and weaknesses. In addition, there are many
more or less common mass analyzers. Many mass spectrometers use two or more mass
analyzers for tandem mass spectrometry (MS/MS).
Detector
The final element of the mass spectrometer is the detector. The detector records the
charge induced or current produced when an ion passes by or hits a surface. In a
scanning instrument the signal produced in the detector during the course of the scan
versus where the instrument is in the scan (at what m/z) will produce a mass spectrum, a
record of how many ions of each m/z are present. Typically, some types of electron
multiplier are used, though other detectors (such as Faraday cups) have been used.
Because the number of ions leaving the mass analyzer at a particular instant is typically
quite small, significant amplification is often necessary to get a signal. Microchannel
Plate Detectors are commonly used in modern commercial instruments. In FTMS, the
detector consists of a pair of metal plates within the mass analyzer region that the ions
only pass near. No DC current is produced; only a weak AC image current is produced
in a circuit between the plates.
Types and Applications

Mass spectroscopy was earlier limited to volatile compounds and therefore was
largely a technique for organic chemists. The development of methods of ionization
which could be applied to non-volatile compounds were developed in the last two
decades. This widened the spectrum of application of mass spectroscopy which could
then be applied to biological molecules which were not studied till then due to their non
volatile nature. The major development in this field was the development of ‘soft
ionization techniques’. These award winning developments were the development of
electrospray ionization (ESI) and of soft laser desorption (SLD) by John Fenn and
Koichi Tanaka (2002, Nobel Prize in Chemistry) [9, 10]. An improved SLD method,
matrix-assisted laser desorption/ionization (MALDI), was developed by Franz
Hillenkamp and Michael Karas in 1988. Improved versions of these techniques have
come up in the recent past which will be described further in the chapter.
ELECTROSPRAY IONIZATION (ESI)

Electrospray Ionisation (ESI) is one of the Atmospheric Pressure Ionisation (API)
techniques and is adapted suitably to the analysis of polar molecules ranging from less
than 100 Da to more than 1,000,000 Da in molecular weight. An ESI module is
simplified in Fig. (4).
Fig. (4). Schematic representation of the principle of ESI.
Electrospray ionization (ESI) occurs during the electrostatic nebulization of a

solution of charged analyte ions by a large electrostatic field gradient. The advantage of
ESI is that highly charged droplets are formed at near atmospheric pressure. Although
the exact mechanism awaits full experimental verification, it is understood that
molecular ions are produced from liquid solution under mild conditions. ESI was
originated by Dole et al. in 1968 [32]. The experiments were extended by Fenn et al.
after a gap of almost 10 years. The work of Fenn outlined the fundamental aspects of
ESI demonstrating its application in the analysis of biomolecules of modest molecular
weights. Access to higher molecular weights by the production of ions bearing multiple
charges was demonstrated later. ESI is especially useful in producing ions from
macromolecules because it overcomes the propensity of these molecules to fragment
when ionized.
In electrospray ionization a liquid is pushed through a very small charged, usually
metal, capillary. The liquid contains the substance which is to be studied, the analyte, as
well as a large amount of solvent, which is usually much more volatile than the analyte.
The charge contained in the capillary is transfered to the liquid and the analyte molecule
becomes charged. As like charges repel, the liquid pushes itself out of the capillary and
forms a mist or an aerosol of small droplets about 10µm across. A neutral carrier gas,
such as nitrogen gas, is sometimes used to help nebulize the liquid and to help evaporate
the neutral solvent in the small droplets. As the small droplets, now suspended in air,
evaporate the charged analyte molecules are forced closer together. The proximity of the
molecules becomes unstable as the similarly charged molecules come closer together
and the droplets once again explode. This is referred to as Coulombic fission because it
is the repulsive Coulombic forces between charged analyte molecules that drive it. This
process repeats itself until the analyte is free of solvent and is a lone ion [33]. There
remains debate as to the exact mechanisms of electrospray processes particularly in the
later part of the process as the lone ion is formed. The lone ion then continues along to
the mass analyzer of a mass spectrometer. There are two major competing theories about
the final production of lone ions, the charged residue model (CRM) and the ion
evaporation model (IEM). The CRM states that Coulombic fission (explosions)
continues until a lone ion is formed. The IEM, however, suggests that ions are
evaporated (usually surrounded by a layer of solvent) from the surface of the small
droplets produced later in the cascade of Coulombic fissions. It has been suggested that
both models probably occur for different analytes/solvents and in the limit of both
models they have a tendency to converge. That is to say that the distinction between a
droplet containing an analyte molecule and an analyte molecule surrounded by a layer of
solvent eventually disappears and columbic fission looks a lot like ion evaporation. The
real question is scale and timing and the techniques to definitively determine this are not
available.
The use of the word "ionization" in "electrospray ionization" is criticized by some
due to that many of the ions observed are thought to be preformed in solution before the
electrospray process or created by simple changes in concentrations as the aerosolized
droplets shrink. It is argued that electrospray is simply an interface for transferring ions
from the solution phase to the gas phase.
Some of the common immonium ions used as reference ions are listed in Table 1
obtained from [23].
In electrospray processes the ions observed are quasimolecular ions that are ionized
by the addition of a proton (hydrogen ion) to give [M+H] (M=analyte molecule,
H=hydrogen ion), or other cation such as sodium ion [M+Na], or the removal of a proton
[M-H] for example. In electrospray multiply charged ions such as [M+2H] are often
observed. For large macromolecules there will often be a distribution of many charge
states and the charge on the ions can be great such as [M+25H]. Note that these are all
even-electron species. Electrons themselves (alone) have neither been added or removed
as with some other ionizations. The formation of ions in electrospray is somewhat
homologous to acid-base reactions. Redox reactions do occur and a circuit with
measurable current flow exists but atomic and molecular ions are the primary carriers of
charge in the solution and gas phases. Recent variant, called electrosonic spray
ionization (ESSI) produces ions with one or only a few charge states [34]. In ESI, higher
voltages favor lower charged forms, and if a peptide is large, the lower charged forms
might not be within the mass range of the mass analyzer. However, lower voltage is
better for smaller analytes. Instrument and ionization parameters are a compromise;
when analyzing complex samples such as peptide digests that have widely varying
chemical properties. Also in ESI there is competition between analytes for charge as
they are extruded from the spray droplets. If a protein digest is analyzed by infusion,
with no separation of the peptides, only a small number of the most easily ionized
peptides are observed. To detect more ions, peptides are separated by a chromatographic
method directly coupled to the MS. Thus, only a few peptides elute at the same time, and
nearly complete coverage of a protein can be achieved, although the chromatography has
its limitations. For instance, the most commonly used method is reverse phase
chromatography, where peptides bind to beads packed into a column and binding is via
hydrophobic interactions with alkyl-terminating chains covalently bound to the beads.
When carbon loading is high, smaller or more hydrophilic peptides are recovered in high
yield, but the larger or more hydrophobic peptides are poorly recovered; when carbon
loading is lower, the larger or more hydrophobic peptides give higher yield, but the
smaller, hydrophilic peptides do not bind. To get around these issues, the easiest
approach is to produce different types of digests, to achieve complete coverage of a
protein. Although it is not yet widely used in practical proteomics studies, ion mobility,
in which ions of different cross sections, charge states, and m/z are separated by
Table 1. m/z Values of Common Immonium Ions
Immonium ion (m/z) Amino acid residue Major (M) or minor (m) peak
60.04 S M
70.07 R or P M
72.08 V M
73.00 R m
74.06 T M
84.08 K or Q M
86.1 I or L M
87.09 N or R M
88.04 D M
100.09 R m
101.11 K or Q M
102.06 E M
104.05 M M
110.07 H M
112.09 R M
120.08 F M
126.06 P M
129.1 K or Q m
136.08 Y M
138.07 H m
159.09 W M
collisions with a bath gas in a uniform electric field, is being explored as an additional
separation that may improve the numbers of peptides that can be successfully identified
from a digest of a protein mixture. ESI interface is combined in various ways with
different mass analyzers. In ESI quadrupole mass analyzers resolve m/z by applying
radio frequency (RF) and DC voltages, allowing only a narrow mass/charge range to
reach the detector. Quadrupoles however have low resolution. Commercially available
instruments usually have mass/charge limits ranging from 0 to 4000 m/z and at best are
normally set to resolve the various 13C isotope peaks for a singly charged ion (which
differ by one Da), although the resolution may be intentionally degraded to improve
sensitivity. In ESI, multiple charging enables quadrupole mass measurement of
molecules >100,000 Da, if the molecule can be charged sufficiently.
Variants
There exist many variations on the basic electrospray technique. Two important ones
are microspray (µ-spray) and nanospray. The primary difference is in the reduced flow
rate of the analyte containing liquid; however many other differences, such as the
reduced internal diameter of the tubing or lack of nebulization gas, exist because of the
difference in flow rate. These variants are important because they generally offer better
sensitivity over traditional electrospray. The µ and nano designations refer to the flow
rate of the analyte containing liquid; µLiters/minute and nanoLiters/minute respectively.
Applications
ESI has been used for the analysis of small proteins and large oligopeptides. They all
show a distribution of multiply charged molecular ions arising by either proton or alkali
ion attachment. The largest protein examined so far is the native covalent dimer of
bovine serum albumin with a molecular weight of more than 130kDa. Electrospray
ionization is the primary ion source used in liquid chromatography-mass spectrometry
and many other advanced forms of spectrometry. This is due to being a liquid-gas
interface that is capable of coupling liquid chomatography with mass spectrometry.
Electrospray ionization is the method of choice in studying noncovalent gas phase
interactions. The electrospray process is capable of transferring liquid-phase noncovalent
complexes into the gas phase without disrupting the noncovalent interaction. This means
that a cluster of two molecules can be studied in the gas phase by other mass
spectrometry techniques.
Although ESI is a powerful technique applied to analysis of bioorganic molecules, it
has some distinct shortcomings. The flowing nature of ESI demands a large amount of
sample. The continuous flow cannot be analysed by the available mass analyzers and
therefore it results in sample loss. ESI is also susceptible to ion suppression ion effects.
All samples thereofre have to be desalted rpior to application onto an ESI analyser. If
complex mixtures are present the highter-concnetration analytes can suppress ion
formation by lower concentration analytes. This shortcoming has been addresses in the
development of MALDI.
MATRIX ASSISTED LASER DESORPTION IONIZATION (MALDI)

Koichi Tanaka received 1/4 of the 2002 Nobel Prize in Chemistry for the
development of soft laser desorption (SLD), however MALDI itself was developed by
Franz Hillenkamp and Michael Karas previously to SLD. SLD is not used currently for
biomolecules analysis, meanwhile MALDI is widely used in mass spectrometry research
laboratories. MALDI deals well with thermolabile, non-volatile organic compounds
especially those of high molecular weight and is used successfully in biochemical areas
for the analysis of proteins, peptides, glycoproteins, oligosaccharides, and oligo-
nucleotides. It is relatively straightforward to use and reasonably tolerant to buffers and
other additives. The mass accuracy depends on the type and performance of the analyser
of the mass spectrometer, but most modern instruments should be capable of measuring
masses to within 0.01% of the molecular weight of the sample, at least up to 40,000 Da.
It is most similar in character to electrospray ionization both in relative softness and
ions produced. However unlike ESI the ions in MALDI are produced in pulses, the
sample is cocrystallized with a solid matrix that can absorb the wavelength of light
emitted by the laser [35]. The ionization is triggered by a laser beam (normally a
nitrogen-laser). A matrix is used to protect the biomolecule from being destroyed by
direct laser beam. MALDI is based on the bombardment of sample molecules with a
laser light to bring about sample ionisation. The sample is pre-mixed with a highly
absorbing matrix compound for the most consistent and reliable results. The matrix
converts the laser energy into excitation energy for the sample, which leads to sputtering
of analyte and matrix ions from the surface of the mixture. In this way energy transfer is
efficient and also the analyte molecules are spared excessive direct energy that may
otherwise cause decomposition. Most commercially available MALDI mass
spectrometers now have a pulsed nitrogen laser of wavelength 337 nm [10, 29, 36,
37]. This basic principle is outlined in Fig. (5).
Fig. (5). The basic principle of MALDI: A diagrammatic representation.
The choice of the matrix is critical in MALDI. The matrix consists of crystallized
molecules, of which the three most commonly used are 3,5-dimethoxy-4-hydroxy-
cinnamic acid (sinapinic acid), α-cyano-4-hydroxycinnamic acid (alpha-cyano or alpha-
matrix) and 2,5-dihydroxybenzoic acid (DHB). A solution of one of these molecules is

made, in a mixture of highly purified water and another organic compound (normally
acetonitrile (ACN) or ethanol). Normally some trifluoroacetate (TFA) is also added. The
matrix-solution is then mixed with the analyte molecule (e.g. protein-sample) under
investigation. The organic compound ACN allows the hydrophobic proteins in the
sample to dissolve into the solution, while the water allows the water-soluble
(hydrophilic) proteins to do the same. This solution is spotted onto a MALDI plate
(usually a metal plate designed for this purpose). The solvents vaporize, leaving only the
recrystallized matrix, but now with proteins spread throughout the crystals. The matrix
and the analyte are said to be co-crystalized in a MALDI spot. Although multiply
charged ions can be produced, more typically, only singly charged ions are observed in
MALDI [38]. Charging can be induced by addition or loss of protons (at acidic or basic
pH values for the delivering solvent, respectively), by loss or gain of electrons or by
adduction of small ions.
It is important to remember the limitations of these methods when interpreting results
of analysis of a sample. For example, after digesting a protein with trypsin, the analysis
of the digest by MALDI will produce only a limited subset of the expected peptide ions.
Peptides must be able to cocrystallize efficiently with the matrix.
MALDI predominantly generates singly charged molecular-related ions regardless of
the molecular weight; hence the spectra are relatively easy to interpret. Fragmentation of
the sample ions does not usually occur. In positive ionisation mode the protonated
molecular ions (M+H+) are usually the dominant species, although they can be
accompanied by salt adducts, a trace of the doubly charged molecular ion at
approximately half the m/z value, and/or a trace of a dimeric species at approximately
twice the m/z value. Positive ionisation is used in general for protein and peptide
analyses. In negative ionisation mode the deprotonated molecular ions (M-H-) are
usually the most abundant species, accompanied by some salt adducts and possibly
traces of dimeric or doubly charged materials. Negative ionisation can be used for the
analysis of oligonucleotides and oligosaccharides.
The laser is fired, the energy arriving at the sample/matrix surface optimised, and
data accumulated until an m/z spectrum of reasonable intensity is amassed. The time-of-
flight analyser separates ions according to their mass (m)-to-charge (z) (m/z) ratios by
measuring the time it takes for ions to travel through a field free region known as the
flight or drift tube. The heavier ions are slower than the lighter ones. The m/z scale of
the mass spectrometer is calibrated with a known sample that can either be analysed
independently (external calibration) or pre-mixed with the sample and matrix (internal
calibration). The laser is fired at the crystals in the MALDI spot. The spot absorbs the
laser energy and it is thought that primarily the matrix is ionized by this event. The
matrix then transfers a part of its charge to the analyte (e.g. a protein), thus ionizing it.
Ions observed after this process are quasimolecular ions that are ionized by the addition
of a proton to [M+H]+, or other cation such as sodium ion [M+Na]+, or the removal of a
proton [M-H]- for example. MALDI generally produces singly-charged ions, but
doubly-charged ions such as [M+2H]2+ have been observed as well. Note that these are
all even-electron species. Ion signals of radical cations can be observed eg. in case of
matrix molecules and other stable molecules.
The proteins are then ready to be extracted into a mass spectrometer. The type of a
mass spectrometer most widely used with MALDI is the TOF (time-of-flight mass
spectrometer), mainly due to its large mass range [39]. In Time-of-flight mass
spectrometry the mass accuracy can be enhanced by increasing the length of the tube for
a given detection system. Common TOF-MALDI instruments are equipped with a
"reflectron" which acts as an "ion mirror", deflecting molecular ions within an electric
field at the end of the tube, thus nearly doubling the traveling distance and increasing
precision.
MALDI spectrum and the identification scheme of a protein are outlined in Fig. (6).
Fig. (6). The MALDI spectrum of a protein leading to database search and protein identification.
Attributes
MALDI has several favorable attributes. Due to the pulsed nature of most lasers, ions
are formed in discrete events. If mass analysis is then synchronised with ion formation
very little sample is wasted. High level of sensitivity is achieved is obtained especially
when coupled to time-of-flight analysers. It can be used to provide analysis of one or
more analytes. Singly charged analytes are generated and therefore analysis is rapid and
high-throughput analysis can be achieved. A practical advantage is its resistance to salts
and buffers. Even then MALDI has some distinct disadvantages. MALDI has problems
in being coupled to some mass analysers. The presence of a matrix which facilitates
ionization causes a large degree of chemical noise and the ratios below 500 daltons have
a lot of background interference.
LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY (LC-MS)

LC-MS is an analytical chemistry technique that combines the physical separation
capabilities of various chromatographic techniques (Gas/ liquid) with the mass analysis
capabilities of mass spectrometry [40]. LC-MS is a powerful technique with high
sensitivity and specificity. Generally its applications are oriented towards the specific
detection and potential identification of chemicals in the presence of other chemicals in a
complex mixture.
Various parameters like the size of the column, flow splitting, mass analyzer used
and the interface have to be considered for judging the efficacy of the instrument. A
major difference between traditional HPLC and the chromatography used in LC-MS is
that the scale is smaller with respect to the internal diameter of the column and even
more so with respect to flow rate since it scales as the square of the diameter.
Initially1mm columns were standard for LC-MS (as opposed to 4.6mm for HPLC).
Recently 300µm and 75µm capillary columns have become more prevalent. At the low
end of these column diameters the flow rates approach 100nL/min and are generally
used with nanospray sources [22, 31, 41-43].When standard bore (4.6mm) columns are
used the flow is often split ~10:1. This can be beneficial by allowing the use of other
techniques in tandem however at the cost of MS sensitivity. Not splitting the flow
generally does not increase sensitivity since the interface is usually concentration
dependent and can actually decrease sensitivity. Scaling the chromatography
appropriately is the answer for increased sensitivity. Some ion sources/interfaces have
been made to be more flow dependent and can handle higher flow rates. Most mass
spectrometers can be used in LC-MS however quadrupole and quadrupole ion traps are
most common. An interface between a liquid phase technique which continuously flows
liquid and a gas phase technique that must remove all but the gas phase ions seems
initially to be difficult and was for a long time. The advent of electrospray ionization
changed this. The interface is most often an electrospray ion source or variant such as a
nanospray source; however fast atom bombardment, thermospray and atmospheric
pressure chemical ionization interfaces are also used.
Applications
Pharmacokinetics
LC-MS is commonly used in pharmacokinetic studies (how quickly the drug clears
the body) in animals and humans. It is well suited for this application because of its
sensitivity and specificity in detecting and quantitating the drug in the complex matricies
such as blood and urine of these applications.
Proteomics
LC-MS is also used in the study of proteomics where the components of a complex
mixture are to be detected and identified. The bottom-up LC-MS approach to proteomics
generally involves protease digestion (usually Trypsin) followed by LC-MS with peptide
mass fingerprinting or LC-MS/MS (tandem MS) to derive sequence of indiviual peptides
Fig. (7).
Fig. (7). A typical peptide mass mapping using LC-MS/MS.
TANDEM MASS SPECTROMETRY (MS-MS)

Tandem mass spectrometry (MS-MS) is used to produce structural information about
a compound by fragmenting specific sample ions inside the mass spectrometer and
identifying the resulting fragment ions. This information can then be pieced together to
generate structural information regarding the intact molecule. Tandem mass
spectrometry also enables specific compounds to be detected in complex mixtures on
account of their specific and characteristic fragmentation patterns.
A tandem mass spectrometer is a mass spectrometer that has more than one analyser,
in practice usually two. The two analysers are separated by a collision cell into which an
inert gas (e.g. argon, xenon) is admitted to collide with the selected sample ions and
bring about their fragmentation. The analysers can be of the same or of different types,
the most common combinations being:
quadrupole - quadrupole
magnetic sector – quadrupole
magnetic sector – magnetic sector
quadrupole – time-of-flight.
Fragmentation experiments can also be performed on certain single analyser mass

spectrometers such as ion trap and time-of-flight instruments, the latter type using a
post-source decay experiment to effect the fragmentation of sample ions.
The basic modes of data acquisition for tandem mass spectrometry experiments are
as follows:
Product or daughter ion scanning
Precursor of parent ion scanning
Constant neutral loss scanning
Selected/multiple reaction monitoring.
Product or daughter ion scanning: the first analyser is used to select user-specified
sample ions arising from a particular component; usually the molecular-related (i.e.
(M+H)+ or (M-H) -) ions. These chosen ions pass into the collision cell, are bombarded
by the gas molecules which cause fragment ions to be formed, and these fragment ions
are analysed i.e. separated according to their mass to charge ratios, by the second
analyser. All the fragment ions arise directly from the precursor ions specified in the
experiment, and thus produce a fingerprint pattern specific to the compound under
investigation.
This type of experiment is particularly useful for providing structural information
concerning small organic molecules and for generating peptide sequence information.
Precursor or parent ion scanning: the first analyser allows the transmission of all
sample ions, whilst the second analyser is set to monitor specific fragment ions, which
are generated by bombardment of the sample ions with the collision gas in the collision
cell. This type of experiment is particularly useful for monitoring groups of compounds
contained within a mixture, which fragment to produce common fragment ions.
Constant neutral loss scanning: this involves both analysers scanning, or collecting
data, across the whole m/z range, but the two are off-set so that the second analyser
allows only those ions which differ by a certain number of mass units (equivalent to a
neutral fragment) from the ions transmitted through the first analyser .
Selected/multiple reaction monitoring: both of the analysers are static in this case as
user-selected specific ions are transmitted through the first analyser and user-selected
specific fragments arising from these ions are measured by the second analyser. The
compound under scrutiny must be known and have been well-characterised previously
before this type of experiment is undertaken. This methodology is used to confirm
unambiguously the presence of a compound in a matrix. This method combines not only
high specificity but also high sensitivity [44].
Applications
Peptide Sequencing by Tandem Mass Spectrometry
The most common use of MS-MS in biochemical areas is the product or daughter ion
scanning experiment that is particularly successful for peptide and nucleotide
sequencing.
Peptides fragment in a reasonably well-documented manner. The protonated

molecules fragment along the peptide backbone and also show some side-chain
fragmentation. There are three different types of bonds that can fragment along the
amino acid backbone: the NH-CH, CH-CO, and CO-NH bonds. Each bond breakage
gives rise to two species, one neutral and the other one charged, and only the charged
species is monitored by the mass spectrometer. The charge can stay on either of the two
fragments depending on the chemistry and relative proton affinity of the two species.
Hence there are six possible fragment ions for each amino acid residue and these are
labelled as in the diagram, with the a, b, and c” ions having the charge retained on the N-
terminal fragment, and the x, y”, and z ions having the charge retained on the C-terminal
fragment. The most common cleavage sites are at the CO-NH bonds which give rise to
the b and/or the y” ions.
The extent of side-chain fragmentation detected depends on the type of analysers
used in the mass spectrometer. A magnetic sector – magnetic sector instrument will give
rise to high-energy collisions resulting in many different types of side-chain cleavages.
Quadrupole – quadrupole and quadrupole – time-of-flight mass spectrometers generate
low energy fragmentations with fewer types of side-chain fragmentations.
Immonium ions appear in the very low m/z range of the MS-MS spectrum. Each
amino acid residue leads to a diagnostic immonium ion, with the exception of the two
pairs leucine (L) and iso-leucine (I), and lysine (K) and glutamine (Q), which produce
immonium ions with the same m/z ratio, i.e. m/z 86 for L, m/z 101 for K and Q and I.
The table of immonium ions has already been mentioned in ESI.
An example of an MS-MS daughter or product ion spectrum is illustrated below. The
molecular weight of the peptide was measured using standard mass spectrometric
techniques and found to be 680.4 Da, the dominant ions in the MS spectrum being the
protonated molecular ions (M+H+) at m/z 681.4. These ions were selected for
transmission through the first analyser, then fragmented in the collision cell and their
fragments analysed by the second analyser to produce the following MS-MS spectrum.
The sequence (amino acid backbone) ions have been identified, and in this example the
peptide fragmented predominantly at the CO-NH bonds and gave both “b” and “y””
ions. (Often either the b series or the y” series predominates, sometimes to the exclusion
of the other). The b series ions have been labelled with blue vertical lines and the y”
series ions have been labelled with red vertical lines. The mass difference between
adjacent members of a series can be calculated e.g. b3-b2 = 391.21 – 262.16 = 129.05
Da which is equivalent to a glutamine (E) amino acid residue; and similarly y4 – y3 =
567.37 – 420.27 = 147.10 Da which is equivalent to a phenylalanine (F) residue. In this
way, using either the b series or the y” series, the amino acid sequence of the peptide can
be determined and was found to be NFESEGK (n.b. the y” series reads from right to
left!). The immonium ions at m/z 102 merely confirm the presence of the glutamine (E)
residue in the peptide. The protein identification by the MS/MS daughter ion scanning is
represented in Fig. (8).
Generally a protein identification study would proceed as follows:
The protein under investigation would be analysed by mass spectrometry to generate
a molecular mass to within accuracy of 0.01%. The protein would then be digested with
a suitable enzyme. Trypsin is useful for mass spectrometric studies because each
proteolytic fragment contains a basic arginine (R) or lysine (K) amino acid residue, and
thus is eminently suitable for positive ionisation mass spectrometric analysis. The digest
mixture is analysed by mass spectrometry to produce a rather complex spectrum from
Fig. (8). MS-MS Daughter or Product Ion Spectrum adapted from Johnson, R.S; Biemann, K.
(1989).
which the molecular weights of all of the proteolytic fragments can be read. This
spectrum, with its molecular weight information, is called a peptide map. For these
experiments the Q-Tof mass spectrometer is operated in the “MS” mode. With the digest
mixture still spraying into the mass spectrometer, the Q-Tof mass spectrometer is
switched into “MS-MS” mode. The protonated molecular ions of each of the digest
fragments can be independently selected and transmitted through the quadrupole
analyser, which is now used as an analyser to transmit solely the ions of interest into the
collision cell. An inert gas is introduced into the collision cell and the sample ions are
bombarded by the collision gas molecules causing them to fragment. The optimum
collision cell conditions vary from peptide to peptide and must be optimised for each
one. The fragment (or daughter or product) ions are then analysed by the second (time-
of-flight) analyser. In this way an MS-MS spectrum is produced showing all the
fragment ions that arise directly from the chosen parent or precursor ions for a given
peptide component.
An MS-MS daughter ion spectrum is produced for each of the components identified
in the proteolytic digest. Each fragmentation spectrum provides different sequence
information and the spectra need to be interpreted carefully (Fig. (9)).
Fig. (9). Q-TOF Operating in MS-MS Mode (courtesy of Micromass UK Ltd., UK).
The amount of sequence information generated will vary from one peptide to
another, Some peptide sequences will be confirmed totally, other may produce a partial
sequence of, say, 4 or 5 amino acid residues.
Table 2 lists the mass values of amino acid residues in peptides and has been adapted
from [23].
Limitations of Mass Spectroscopy

The complex nature of cellular proteomes has time and again proved to be a
challenge for proteomic technology development. Unlike a genome, a proteome is a
highly dynamic entity. Protein expression in a biological system changes with the state
of development, in response to environmental stimuli, with the progression of a disease,
etc. In addition, different cells within a multi-cellular organism have different pro-
teomes. The number of proteins in a proteome is very large. Although no precise
calculations can be made, it is estimated that up to 50,000 protein species may be
simultaneously present in a eukaryotic cell. Furthermore, proteins within a proteome are
structurally diverse and have various physicochemical characteristics. Because of these
considerations, comprehensive characterization of cellular proteomes is an enormous
undertaking. 2D-PAGE in combination with MS plays a central role in proteomics.
CONCLUDING REMARKS
During the last two decades, mass spectrometry applications have revolutionized
analysis of proteins, moving from simple studies of purified proteins, blocked N-termini,
Table 2. Mass Values of Amino Acid Residues in Peptides
Residue Mass
Amino acid Single letter code
Monoisotopic Average
Glycine G 57.02147 57.052
Alanine A 71.03712 71.079
Serine S 87.03203 87.078
Proline P 97.05227 97.117
Valine V 99.06842 99.133
Threonine T 101.04768 101.105
Cysteine C 103.00919 103.144
Isoleucine I 113.08407 113.160
Leucine L 113.08407 113.160
Asparagine N 114.04293 114.104
Aspartic Acid D 115.02695 115.089
Glutamine Q 128.05858 128.131
Lysine K 128.09497 128.174
Glutamic Acid E 129.04260 129.116
Methionine M 131.04049 131.198
Histidine H 137.05891 137.142
Phenylalanine F 147.06842 147.177
Arginine R 156.10112 156.188
Tyrosine Y 163.06333 163.17
Tryptophan W 186.07932 186.213
Homoserine lactone 83.03712 83.090
Homoserine 101.04768 101.105
Pyroglutamic Acid 111.03203 111.100
Carbamidomethylcysteine 160.03065 160.197
Carboxymethylcysteine 161.01466 161.181
Pyridylethylcysteine 208.06703 208.284

modified peptides, and analysis of peptide synthesis reactions, to the current array of
new methods and instruments, as well as inspiration for the new field of systems
biology. Proteomics is a significant and upcoming area of research. In the same time, we
have shifted from an era where our understanding of protein and peptide gas-phase
chemistry was built up slowly, to an era where large datasets can now be mined for rules
that rapidly increase our understanding of the fundamental chemical processes. This will
enhance identification of components of complex samples and yield significant advances
in medicine and biology. The great reliance of the field on mass spectrometry for protein
characterization has spurred many advances in mass spectrometry instrumentation,
separations technology, and software, and data management capabilities. Yet, this
amazing growth has only whetted our appetites, and we can look forward to even more
powerful instrumentation and algorithms used in creative ways to yield a rich
information source in the future.
Protein Arrays
The coming to age of miniaturized technology for the interactive studies of
molecules has changed the way in which cell biology is studied. Microarray based
analysis is a relatively high-throughput technology, which is suited to automation and
has the potential for genome and proteome wide analysis. A key advantage of the
microarray format is the use of nonporous solid surface such as glass, which permits
precise deposition of capture molecules (probes) in a high density and ordered fashion.
Various methods exist for creating arrays. Photolithography employs light, directed
spatially via a photo mask, to specifically modify surfaces for derivatization.
Piezoelectric approaches use accurate dispensing techniques to deliver reagents onto the
surface at subnanolitre levels. Microspotting makes use of direct contact with the surface
by solid or split pins. In addition, there are means of in situ synthesis of probes onto
solid phases. Following creation of the chip, a fluidic system delivers the reagents, a
laser scanner reads the chips and sophisticated software analyses and interprets the data.
Microarray technology is still at a developing stage. This is mainly because the
properties of proteins are changed markedly by the ambient conditions. Proteins do not
behave as predictably as DNA when immobilized on solid surfaces, and certain classes
may not be expected to be stable under such conditions. One must be aware of the
possible changes in protein conformation and hence activity or affinity induced by
common operations such as immobilization to a solid surface, derivatization with a
fluorescent probe, heating or drying. However, the concept of protein arrays has the
potential advantage of being scalable, flexible, ease of automation and performance [45].
Arrays may also enable the control of key parameters such as temperature; pH and
cofactor concentration not easily afforded by cell based systems [46].
All proteins from higher eukaryotic genomes may not be recognized by the first
generation of arrays. Bearing in mind the aforementioned need to discriminate different
isoforms generated by post-translational modification, in conjunction with the need to
validate the selectivity and affinity for each analyte, highlights the magnitude of the task
required to produce a fully optimized array.
Antibodies are relatively stable to immobilization and are attractive ligands in arrays
technology. However, the key disadvantage of this technology is the inherent lack of
stability of protein reagents. A number of workers have therefore investigated aptamers,
which have affinity for individual protein molecules. In addition, they offer the
capability to directly detect interacting proteins using generic protein stains without
interference from the probe. The sensitivity of such an approach may not match that
achievable by more sophisticated detection approaches. Extensive selection procedure is
essential in selecting a particular aptamer. Briefly, this includes binding of proteins to a
solid phase for exposure to aptamer libraries and aptamer selection. The importance of
aptamers in protein arrays has yet to be demonstrated, and the ability to select them
without having the target protein readily available may prove problematic. The need for
rapid parallel expression and purification of proteins has led Cahill and co-workers from
the Max Planck Institute in Berlin to develop protein expression arrays of tagged
proteins in Saccharomyces cerivisiae and Pichia pastoris under the control of inducible
promoters. Antibody screening, protein-protein interactions, epitope mapping and
protein expression profiling are some of the key areas where arrays seem to have
application. Other groups have created arrays using GST fusion proteins. This tag can be
used for either purification [47], immobilization [48], or in combination with another
tag, for both purification and immobilization [49].
The combination of surface plasmon resonance (SPR) and MS has created a novel
approach in protein investigations. SPR quantifies interactions between proteins and
ligands while MS deciphers the structural features of the bound proteins. Proteins are
captured by affinity tags from solutions using ligands which are covalently attached to
the SPR-sensor surface. The technique is non-destructive and therefore retains the active
structure of proteins which are directly analysed by MS from the SPR surface or
separately after elution and recovery from the surface [48]. The technique was first
demonstrated in 1996 and immediately was followed by advancements. Although
offering a potential insight into ligand binding, this method cannot be seen as a test of
receptor function due to the inability to demonstrate downstream signaling in response to
ligand binding. Similarly, protein arrays have been formed in lipid monolayers, using
specific lipids to form ordered arrays of both hydrophobic and hydrophilic proteins [51].
As shown in Fig. (10), specific protein capture on microarrays can be performed
using affibodies (a), aptamers (b) or antibodies (c, d) as immobilized specific capture
reagents. Detection of bound analytes is usually performed by direct labelling of the
analyte molecules (a–c) or by antibody sandwich formation (d). Such assays can be used
for protein identification and quantification and therefore will be useful tools in
multiparametric diagnostics in the near future. The reverse screening approach uses
immobilized cell lysates, which represent the whole repertoire of proteins in cells at a
distinct state. Unspecific adsorption of protein mixtures can be governed by electrostatic,
hydrophobic van der Waals, or metal–chelate interactions. Captured proteins can be
identified using mass spectrometry (e) or specific antibodies (f). Different antibodies or
patient sera can be used to screen these microarrays for the presence of absence of
distinct target proteins (f). Immobilized tissue samples (g) or cells (h) can be used for
reverse screening approaches with antibody detection of specific markers as well.
Reversed screening assays have a high potential as tools for biomarker detection in
proteome analyses. Specific interaction microarrays have been described for
receptor–ligand (e.g. small molecule drug candidates, phospholipids) interactions (i),
enzyme–substrate interactions (j), protein–protein interactions (k), protein-oligosaccha-
ride interactions (l) and protein–DNA interactions (m) and can be used to identify
interaction partners of proteins in a highly parallelized manner.
Fig. (10). Types of protein interaction and capture microarrays (adapted from Stoll et al., 2004).
Expression Arrays
One of the simplest formats of protein array consists of a large number of defined
spots on a planar support comprised of reagents capable of recognizing individual
proteins. The 96-well microtitre plate format of the standard ELISA can be used in
setting up a protein microarray [1,52,53]. New developments may help in reaching the
potential of moving from current standard arraying throughput of 96-wells/30s to a more
appropriate scale of 5000–10 000 assays. Robotic picking and high-density gridding of
phage display libraries provided filter based ELISA format assays. The dream
development in protein arrays is the demonstration of methodologies capable of assaying
proteins at physiologically relevant concentrations. Applications to date have mainly
restricted themselves to relatively low-density arrays. Huang et al. [54,55] recently
described an ECL based immunoassay array for the simultaneous assay of 24 cytokines
from either cultured media or patient sera. The system was based on the standard
sandwich ELISA technology but the initial capture antibodies raised to the various
cytokines were transferred in an ordered format onto a membrane. Cytokines were
detected at levels as low as 60 pg. Specific analytes in clinical samples have been
determined fluorimetrically at physiologically relevant levels by an antibody array using
a CCD camera [56]. The scope for increased levels of sensitivity may be realised by use
of minaturised ligand binding assays employing affinity capture to “harvest” the analyte
[57].
A recent review discussed the relevance of spot density to sensitivity and the
limitations of such approaches [58]. Madoz-Gurpide and coworkers [59] have proposed
a method of prefractionation prior to arraying proteins. Preferring a sandwich
immunoassay format they were able to demonstrate subpicomole sensitivity in lung
adrenocarcinoma lysates. Using a similar direct labeling approach, protein expression
changes have been described for colon carcinoma following exposure to ionizing
radiation [60]. In an array of almost 2000 proteins probed with 146 antibodies, a limit of
detection of approximately 6 pg was obtained. A number of protein expression changes
were observed including proteins implicated in apoptosis, signal transduction and
activation of transcription. In the suspension array probe molecules are bound to the
surface of colour coded microspheres in a microfluidics based instrument using
sophisticated optics. The colour coded microspheres identify the reaction. A second
molecule is used as a reporter of the magnitude of the interaction. The two colour coding
of the sphere and the reporter define the assay and the magnitude of the result
respectively. Although currently limited to 100 analytes, recent developments in the area
of quantum dots, a technology being developed independently of the Luminex system,
may ultimately offer an increased capacity for multiplexing bead based assays [61].
Although microspheres are currently limited to approximately one hundred, quantum
dots are nanocrystals of semiconducting material, which emit light at very precise
wavelengths, dependent on the spot size. Defined beads may be prepared by the quantum
dot technique. Different types of planar and bead-based arrays are shown in Fig. (11).
Either planar microarrays or bead-based arrays can be employed for multiplexed
ligand-binding assays. Planar microarrays can be generated with multiples of different
capture spots whereas multiplexing in bead based arrays is limited to the number of
distinguishable beads. In planar arrays analyte spots are easily distinguishable by their
xy-coordinates in the array. Detection on planar arrays is performed using chemo-
luminescence, radioactivity, mass spectrometry or fluorescence. The latter is the
preferred detection method for bound analytes in bead-based microarray assays.
Interaction Arrays
Interaction studies involving different proteins have wide reaching importance in
understanding biological and pharmacological processes. Immunoprecipitation is a
classical technique used in these interactive studies. This relatively simple technique has
found wide applicability but can be complicated by nonspecific interactions which can
confound the interpretation of data. Tandem affinity purification (TAP) has been
described as a method to overcome this [62], although this is a relatively low-throughput
technique. Interaction methodologies in a microarray format would be extremely
advantagous. A universal protein array for quantitative detection of protein interactions
with a range of proteins, nucleic acid and small molecules has been reported by Ge [63].
A key aim of the study was to produce low-density arrays of purified, native proteins as
a sensitive screen for proteins drug targets in disease. Probably the first example of a
protein array applied to an entire genome was demonstrated by Zhu et al. [64,65] who,
overexpressed and purified 5800 ORFs from yeast, representing in excess of 90% of the
yeast genome as double N-terminal GST, His6-fusion proteins in yeast. A 96-well
format glutathione affinity purification was used for rapid parallel isolation. The isolated
protein was immobilized via the His6- tag, on nickel coated glass slides and they were
able to screen for protein and phospholipid interaction at a near genomic scale. A new
interaction motif was described for binding with calmodulin. Notwithstanding the
limitations of detecting protein interactions in vitro on solid surfaces, the studies were
able to classify phospholipid binding in terms of both specificity and strength of binding.
This approach demonstrates the possibility of not only high-density applications but the
potential for genomic coverage on a functional chip.
Fig. (11). Different types of planar and bead based microarrays.
A biosensor technology was applied for soluble recombinant insulin-like growth

factor receptor domains isolated on a chip to define protein-protein interactions [66].
This enabled protein domains to be characterized in terms of binding of ligand, binding
of other proteins and provided further information about the function of the nonbinding
domains.
Functional Arrays
A key goal of proteomics is to assign function to proteins. The determination of
protein function in an array format offers a number of advantages. Kodadek [67]
prophesied that functional protein arrays composing of native proteins will precede the
development of protein expression arrays. This is in part due to the need for the
requirement for high throughput isolation of high selectivity ligands and the requirement
for sensitive methods of detection without derivatisation [68-70] discusses the use of
common hosts (e.g. E. coli) and their ability to express proteins with correct folding
which is highly significant in the field of protein expression arrays where various claims
have been made for expressing functional arrays. Zhu and coworkers [64,65] have
developed a method for expression of yeast genes and have expressed 119 of a predicted
122 yeast kinases, as GST-fusion proteins via a high copy expression vector under the
control of a galactose-inducible promoter.
Future Aspects
Some of the challenges that will be faced by researchers attempting to implement
high-throughput microarray technologies for proteomics include maintaining diverse
protein activities on chips, especially membrane proteins, creating sufficiently diverse
antibody or ligand libraries and finding attachment strategies that allow three-
dimensional access for binding. Although these hurdles may be difficult to surmount, it
is likely that protein micro- and macroarrays will bear fruit, especially when comple-
mented by other ‘‘classic’’ proteomics technologies. In summary, protein analytical
applications in the future will benefit from the explosion of new DNA- based
technologies in array and detection [47]. The rapid growth of the proteomics market and
the climate for new technology is driving the new generation of companies and academic
efforts that are developing novel protein microarray techniques for the future.
ACKNOWLEDGEMENTS
Aarohi Kulkarni acknowledges the Council of Scientific and Industrial Research for
Research Fellowship.
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NMR Spectroscopy Based Metabonomics: Current

Technology and Applications
Clare A. Daykin1,* and Florian Wülfert2

1
School of Pharmacy, University of Nottingham, University Park, Nottingham,
UK, NG7 2RD and 2School of Biosciences, University of Nottingham, Sutton
Bonington, Loughborough, Leicestershire, UK, LE12 5RD
Abstract: Metabonomics has, in the past decade demonstrated enormous
potential in furthering the understanding of disease processes, toxicological
processes, phenotypic outcome of gene expression and biomarker discovery.
However, implementation of metabonomic methodology requires the
development of generic, rapid, advanced analytical tools to comprehensively
profile biosample (biofluids and tissues) metabolites. 1H NMR spectroscopy is
arguably the most powerful tool for profiling of biosamples with inherent
advantages over other analytical techniques e.g.: 1) lack of requirement for
extensive sample preparation; 2) non-destructive; 3) non-equilibrium
perturbing; 4) small (down to 1 µl) sample volumes and 5) quantitative and
qualitative information are available from the same data set. Whilst
conventional ‘target analysis’ approaches may apply more sensitive analytical
methods than 1H NMR spectroscopy for detection of low metabolite levels in
biological materials, the ‘target analysis’ approach involves consideration of
critical biochemical pathways and pre-selection of the metabolites of interest.
The subjective selection of a battery of biochemical methods is then required
which is a necessary, but complex and time-consuming process and if an
inappropriate or restricted range of biochemical methods or parameters are
chosen, important metabolic disturbances may be over-looked. On the other
hand, the use of 1H NMR spectroscopy to follow biochemical responses does
not require such a pre-selection of metabolites and allows subsequent multi-
component analysis, without bias imposed by the experimenters’ expectations,
hence it has been demonstrated in a wealth of publications that NMR spectra
of biosamples are extraordinarily rich in information on endogenous
biochemical processes in both good health and disease.
This review will aim to discuss technical developments relevant to the field,
commonly used data handling and data analysis strategies and the use of
metabonomics as a tool to facilitate our understanding of health status at the
metabolic level.
*Corresponding author: Tel: (+)44 115 9515052; Fax: (+)44 115 9515102;
E-mail: clare.daykin@nottingham.ac.uk

152 Frontiers in Drug Design & Discovery, 2006, Vol. 2 Daykin and Wülfert
INTRODUCTION
Systems Biology
Whilst the definition of systems biology, also previously referred to as ‘bionomics’
[1] or ‘systeomics’ (a trademark name used by the Californian Separation Society) [2], is
highly variable in the literature, our working definition for systems biology is ‘the study
of biology as an integrated system of genetic (genomics), protein (proteomics),
metabolite (metabonomics or metabolomics), cellular and pathway events that are in
flux and interdependent’ [3, 4]. In more general terms, systems biology has come to
mean an integrated approach to e.g. drug discovery where the effects of compounds on
whole pathways and networks of pathways are considered, as opposed to their effects on
only isolated endogenous molecules.
To give a brief overview of each of the major *omics technologies, which together
make up ‘systems biology’ in turn, genomics involves the observation of differential
gene expression as a result of external influence such as toxicity, disease, xenobiotic
exposure, or environmental factors. The technology involves new generations of
proprietary “gene chips”, which are small, disposable, but expensive devices, encoded
with an array of genes that respond to extracted cellular mRNAs produced as a result of
external influence, the effect of which is the switching on or off of various genes [e.g. 5-
7].
However, although many genes can be placed on a chip array and patterns of gene
switching can be monitored rapidly, the technology is not cheap (although the
technology is cheaper than it was several years ago). Furthermore, relationships between
gene regulation/expression and the integrated function and control of cellular systems
(so-called functional genomics) are still far from clear and are likely to remain so for
many years. The main reason for this is that the vast majority of DNA is non-coding, yet
genes i.e. DNA sequences which code for proteins, can not function as isolated units
without the presence of neighboring genes and non-coding DNA.
This lack in the understanding of the biological consequences of altered gene
expression led to the development of proteomics, a term first suggested by Australians
Marc Wilkins and Keith Williams in 1995 and which is defined as, “the study of
proteins, how they're modified, when and where they're expressed, how they're involved
in the metabolic pathways and how they interact with each other” [8, 9]. However,
amongst other problems, the 2D gels used in the early days of proteomics were
notoriously irreproducible and labor-intensive. Since this time, several significant
advances have begun to overcome some of the formidable obstacles associated with
proteomic analysis. For example, Washburn and Yates [10, 11] led the way from 2D
gels to multidimensional liquid chromatography (LC) coupled with mass spectrometry
(MS), which has resulted in higher throughput, greater proteome coverage and improved
dynamic range and sensitivity. Wilm and Mann [12] developed nano-electrospray, which
further enhanced sensitivity limits. However, despite these advances, and the fact that
proteomics is potentially less expensive than genomics; it is still very slow and labor
intensive at present. More importantly, although these measurements may ultimately
provide new biomarkers of disease, lead to new drug discovery targets and provide
insights into toxicological mechanisms, it is currently very difficult to relate genomic
and proteomic findings to classical indices of toxicity. Explanations for this include
limitations in terms of both speed and sensitivity of the current technology, which
NMR Spectroscopy Based Metabonomics Frontiers in Drug Design & Discovery, 2006, Vol. 2 153
precludes the measurement of a detailed time-course of the proteomic response to

disease or drug exposure and the measurement of responses is usually focused on one
organ such as liver and hence effects in other important tissues such as kidney, lung or
heart muscle could be overlooked.
The most recent of the *omics technologies is metabo*omics. However, whilst
metabonomics or metabolomics are considered relatively new technologies, with the
terms and their definitions not coming into existence until late last century/ early this
century [13, 14], a large number of publications reporting methods for the metabolic
profiling of biological fluids date back to the 1970’s [e.g. 15-17]. Since this time, Jeremy
Nicholson and his group based at Imperial College, London in the UK has carried out
much of the pioneering work in NMR spectroscopy-based metabonomics whilst the
group of Jan van der Greef from Leiden University in the Netherlands has lead the way
in the parallel LC-MS based arena. In both cases, their published work in this field dates
back to the early 1980’s [e.g. 18-23]. Now, the field is sufficiently advanced that
numerous companies offer metabo*omic analysis as a contract research service.
Although metabo*omics is complementary to genomics and proteomics, it also has
certain advantages. In particular, the metabonome (or metabolome) is further down the
line from gene to function and so reflects more closely the activities of the cell or
organism at a functional level. Furthermore, metabolic fluxes are not regulated by gene
expression alone, but all physiological perturbations result in disturbances in the ratios
and concentrations, binding or fluxes of endogenous biochemicals, either by direct
chemical reaction or by binding to key macromolecules that control metabolism. If these
disturbances are of sufficient magnitude, they will affect the efficient functioning of the
whole organism. In bodyfluids, metabolites are in dynamic equilibrium with those inside
cells and tissues and, consequently, abnormal cellular processes in tissues of the whole
organism following a toxic or metabolic insult will be reflected in altered biofluid
compositions. Thus whilst genomics and proteomics characterize the potential for
relevant effects, metabo*omics actually catalogues the direct effects of pathological
processes.
Nomenclature: Are Metabonomics and Metabolomics the Same?

As the most recent addition to the ‘systems biology’ toolbox, the definition and even
in fact, the name, metabo*omics, is less well defined than e.g. genomics or proteomics,
as reflected by the fact that different authors use different definitions for similar terms or
conversely, the same approach is given different names, e.g. metabonomics [13],
metabolomics [24] and metabolic profiling [25].
Whilst some research groups use these terms interchangeably, others argue that the
terms refer to philosophically different aims of analysis. For example, Nicholson et al.
previously defined metabonomics as: “quantitative measurement of multiparametric
response of living organisms to (patho)physiological stimuli or genetic modification”
[13] and later extended this definition with: “an approach to understanding global
metabolic regulation of an organism and its commensal and symbiotic partners” [26].
Nicholson argues that the name ‘metabonomics’ incorporates the analysis of products of
non-enzymatic reactions, otherwise known as metabonates, which can interact with and
influence metabolite formation [26]. Groups adhering to this definition use the term
metabolomics to discuss only work dealing with simple cell systems and mainly
intracellular metabolite concentrations.
Aims of Analysis
A change in environmental, genetic or chemical (e.g. xenobiotic, nutriceutical,
functional food and cosmaceutical) factors to a cell, tissue or whole, complex organism
will cause changes in the ratios, concentrations and fluxes of endogenous biochemicals
in or through key intermediary metabolic pathways. In cases of mild change, cells will
attempt to maintain homeostasis and metabolic control by varying the composition of the
body fluids that either perfuse through them or are secreted by them and consequently,
following metabolic perturbation there are characteristic alterations in biofluid
composition, which are organ- and mechanism-specific.
To investigate these complex metabolic consequences to metabolic stresses, such as
disease processes, toxic reactions or genetic manipulation, non-selective, but specific
“informanation-rich” analytical approaches are required. Whilst this review is limited to
the recent advances in NMR spectroscopy based metabonomics, several analytical
methods in addition to NMR spectroscopy can and do serve as powerful means of
generating multivariate metabolic data [27, 28] including; direct injection mass
spectrometry (MS) [29], GC/MS [25, 30], HPLC/MS [31], HPLC-DAD [32] and optical
spectroscopic techniques [33, 34]. It is also recognized that no single analytical
technique is, at the present time, capable of covering the entire metabonome. Therefore,
a range of techniques and approaches will be required in order to gain as comprehensive
coverage of the metabonome as possible.
NMR TECHNOLOGY AND METABONOMICS

Background to the Role of NMR Spectroscopy in Metabonomics
A key point of metabonomics studies is to have in disposition one or more generic
analytical methods for rapid biological sample profiling. NMR spectroscopy is a
relatively mature technique in this field and it is well established that high resolution
NMR spectroscopy can provide useful qualitative and quantitative biochemical
information relating to the metabolic status of a person, animal or cell system [32].
Various studies have demonstrated the application of 1H NMR spectroscopic analysis
to almost every imaginable biofluid or tissue type, including amongst others; urine [35],
blood plasma [36-38] or serum [39, 40], bile [41-43], seminal fluid [44, 45],
cerebrospinal fluid [46-48], kidney [49-51], liver [51] and heart [52]. Furthermore, the
spectral assignment of many endogenous metabolite peaks has been made previously
through the concerted use of 1- and 2-dimensional NMR methods [53-56].
Proton NMR has traditionally dominated the biofluid NMR field, due to a number of
advantages, such as the experimental simplicity, the speed at which data can be
generated and the large amount of data which can be generated in a relatively short time-
frame. Whilst other nuclei, in particular 13C and 31P can prove useful for metabonomics,
traditionally their use has been limited in large studies due to the long experiment times
involved. As NMR spectroscopy becomes more sensitive and technology such as
cryoprobes become more widely available it is expected that the exploitation of 13C
NMR and 31P NMR will become more widespread.
The features of NMR spectroscopy that prove useful in this type of study are
summarized in Table 1.
Table 1. Summary of Useful Features of 1H NMR Spectroscopy in Biofluid Studies

(Reproduced From [37] with Modifications)
Feature Comment
Selectivity There is no need for the pre-selection of analytical conditions based on the chemical
properties of the analyte, or postulation of the metabolites affected by a disease or
toxicological process. Thus, a wide range of low MW metabolites and macromolecules
can be simultaneously monitored in a short space of time, without prior knowledge or
expectation of the results.
Non-Invasive NMR spectroscopy is non-invasive, non-destructive and non-equilibrium perturbing.

It thus allows subsequent analysis of a sample with other techniques.
Speed A typical single pulse 1H NMR biofluid spectrum is obtainable in <10 minutes.
Sample With a standard NMR probe only 500 µl of sample is required for routine
Volumes spectroscopy, or 1-2 µl with a micro-volume NMR probe.
Sample Only the addition of deuterium oxide is required for analysis. Although, where
Preparation physical removal of proteins is required, e.g. in order to study protein-bound low MW
metabolites in blood plasma, care should be exercised in the deproteinization method
selected. Different deproteinization methods will lead to different degrees of protein
unfolding and hence, methods can vary widely in terms of the subsequent NMR-
observable metabolites [38, 57].
Dynamic Information can be obtained on dynamic biochemical processes in complex matrices

Information and molecular interactions such as protein-ligand binding [58, 59].
Structural Data provides qualitative structural information that can also be quantitative with
Information addition of internal standards at known concentrations.
Reproducibility NMR spectroscopy is an inherently reproducible analytical method [60, 61].
Automation
High quality, reliable automated sample analysis is critical for metabonomic studies
because large numbers of samples must be handled quickly, efficiently and with a high
degree of reproducibility on a routine basis. A single study may involve analysis of well
over a thousand samples. A traditional 60- or 120-place autosampler may be useful
where retention of samples is important, or where the instrument may be required for
handling mixtures of both biofluid and non-aqueous samples at short notice. However,
this approach can be costly due to the requirement for large numbers of expensive NMR
tubes.
An alternative to the traditional autosampler is a flow-injection (FI-) NMR
accessory. These have become widely available as a result of the need for high
throughput in combinatorial chemistry studies [62] and the technique is also well suited
to biofluid studies [63]. When compared to using conventional autosampler racks, the
use of FI-NMR results in faster throughput of samples and minimal spectrometer
optimization whilst neglecting the requirement for expensive NMR tubes. In some cases,
FI-NMR has even been extended to include other analytical techniques such as MS and
infra red (IR) on-line in order to fully exploit the total amount of analytical information
available on a sample in a single run [64].
Spectral Editing Methods

Spectral editing is an essential part of the successful application of NMR
spectroscopy to biological analysis, due to the complexity of 1D spectra. Spectral editing
methods may be applied for peak assignment, spectral simplification or studies on the
molecular properties of the compound(s) of interest. Some of the most commonly
applied spectral editing methods are summarized in Table 2, together with brief
explanations of their purpose.
Table 2. Summary of Commonly Applied Methods for the Simplification of Biofluid

NMR Spectra
Method Application
Spectroscopic: 1D NMR with water Good starting point for biofluid analysis. Allows
1D presaturation e.g. endogenous metabolite profiles to be monitored and
NOESYPRESAT related to abnormal effects. Gives an indication of
spectral complexity and concentration of components.
Carr-Purcell-Meiboom-Gill Spectral editing is based on molecular mobility. This

(CPMG) Spin Echo experiment allows attenuation of macromolecules and
therefore low MW metabolites can be studied.
Diffusion-Edited Originally introduced to measure diffusion constants,

this experiment has found use in the removal of peaks
from low MW metabolites in highly heterogeneous
biofluid samples e.g. blood plasma [37, 52,65].
RECUR-NMR This method allows detection of small molecule NMR

resonances which appear underneath the large, dominant
water peak typical of biofluid NMR spectra [66].
Spectroscopic: Correlation Spectroscopy Usually applied to metabolite identification problems by

2D (COSY) establishing connectivity between protons in a molecule,
it has also been applied to the screening phase of some
metabonomic studies [e.g.52]
J-Resolved Spectroscopy As for COSY, whilst usually applied to metabolite

(J-RES) identification problems where it is used to establish the
multiplicity of peaks in regions of heavy spectral
overlap, it has also been applied to the screening phase
of some metabonomic studies.
(Table 2) contd….
Method Application
Physicochemical Sample Extraction Extraction can be used to simplify spectra by physical

separation of the compounds under study (e.g. low MW
metabolites or lipids) from interfering compounds.
Whilst this approach is used by many groups, it is often
applied with little consideration of the effects of the
extraction methods applied. It has previously been
shown that e.g. different deproteinization methods can
vary widely in their ability to extract different low MW
metabolites [38]. The same holds true for lipid
extraction methods.
Freeze Drying Freeze drying of urine samples can help to reduce the
large, dominant water peak typical of biofluid spectra
and remove the large urea peak, thus enabling
visualization of resonances from low MW metabolites
which may be obscured by these peaks. Reconstitution
of the freeze-dried sample in deuterated buffer will also
help control sample pH. However, freeze-drying of
samples is also labor intensive and unsuitable for use
with whole blood plasma or serum because integrity of
lipoproteins will be lost.
HPLC-NMR Whilst HPLC-NMR and HPLC-NMR-MS have proved

invaluable additions to the metabolite identification
toolboxes of many labs, personal experience has shown
that its usefulness during the profiling stages of a
metabonomic study is limited. This is largely due to the
sensitivity loss encountered in running samples in high-
throughput on-flow mode or extra experiment time
required for ‘time-slicing’ through chromatographic
peaks [67]. For profiling stages of a study, NMR (on
either whole biosamples or extracts) and parallel HPLC-
DAD yields much more useful data [32].
Micro-Volume and Cryogenic Probes

Whilst NMR spectroscopy possesses a number of inherent advantages for
metabonomic studies, two disadvantages have traditionally limited the field; low
sensitivity and the requirement for sample volumes of at least 500 µl. However, two
recent welcome developments have been the introduction of micro-volume NMR probes,
which allow the study of as little as 1-2 µl of sample [68, 69] and cryogenically cooled
NMR probes, or cryoprobes, which improve the sensitivity of experiments by a factor or
3.5-4.0 [70]. An example of a typical 750 MHz blood plasma spectrum acquired using
the Bruker 1mm NMR probe with 5 µl of mouse plasma is shown in Fig. (1). The use of
cryoprobes has not only enhanced the sensitivity of 1H NMR spectroscopy for biofluids,
but also makes the use of heteronuclear experiments, such as 13C NMR more assessable
[71], where experiment times have previously been prohibitively long.
Fig. (1). Typical 750 MHz 1H CPMG NMR Spectrum of Mouse Plasma (5µl) acquired using the
Bruker 1mm NMR probe.
High Resolution Magic Angle Spinning (HR-MAS)

Whilst ‘standard’ NMR methods can be used for samples which are in the liquid
state, the same methodology is not applicable to semi-solid material due to line-
broadening artifacts. High-resolution magic angle spinning (HR-MAS) on the other hand
cancels out these undesired line-broadening effects and improves resolution. This
technique was originally successfully applied in combinatorial chemistry to study
compound libraries covalently linked to solid supports [72]. Later the technique also
found employment in biochemistry [e.g.73], polymer [e.g.74] and colloid chemistry
[e.g.75]. More recently, HR-MAS has also been explored to map metabolite patterns in
tissues for diagnostic purposes [e.g. 76, 77]. As such, it is a powerful analytical tool,
potentially bridging the divide between 1H NMR spectroscopy of tissue extracts and
magnetic resonance spectroscopy (MRS) in vivo. Typical standard 1D and CPMG-edited
600 MHz 1H NMR spectrum of ca. 10 mg of unextracted liver tissue, acquired using
HR-MAS are illustrated in Fig. (2). These spectra demonstrate the wealth of metabolic
information that can be acquired without the requirement for tissue extraction.
On the other hand, the study of tissue using extraction procedures, followed by
conventional liquid state NMR requires more tissue sample, in order to reproducibly
extract the tissue (typically 50 mg for extraction vs. 10 mg for HR-MAS). Extraction is
also complicated by the need to study both lipophobic and lipophilic phases and does not
address the composition of cell membrane constituents. Furthermore, sample extraction
is principally destructive in nature. However, as for spectroscopic analysis of blood
plasma, information can be lost if spectral-editing is used alone [38]. Therefore, a
combined strategy of HR-MAS with spectral editing and sample extraction is
recommended.
Fig. (2). Typical 600 MHz 1H High Resolution Magic Angle Spinning NMR Spectra of Liver
Tissue where (A) standard 1D NMR spectrum acquired with water presaturation and (B) Carr-
Purcell-Meiboom-Gill (CPMG) Spin-Echo spectrum, which allows attenuation of resonances from
compounds with short T2 relaxation times, such as proteins and lipids. These spectra were
acquired using approximately 10 mg of tissue. Some typically observed metabolites are labeled.
BIOFLUID OR TISSUE NMR SPECTROSCOPIC DATA AS INPUT FOR

MULTIVARIATE ANALYSIS (MVA)
By definition, metabonomics projects generate large amounts of complex data. The
datasets consisting of e.g. NMR spectra and/or clinical data contain biological
information spread over a large number of correlated variables, i.e. peaks. In the case of
NMR spectra, the correlation between the peaks is given by the fact that one metabolite
will typically have more than one peak in a spectrum and metabolites will have
biological relationships with each other. Multivariate analysis uses these correlations and
trends in order to summarize the data by reducing the dimensionality from e.g. several
hundreds of peaks to a few coefficients, allowing visualization of the data and

subsequent interpretation to be carried out in a much more comprehensible and natural
manner than spectral comparison by eye.
Principal Component Analysis (PCA)

If a theoretical sample were characterized by three metabolites, each expressed as a
single peak in its NMR spectrum, the area or height of the peak can be plotted on its own
axis and each spectrum can be expressed as a point in the three-dimensional space as
depicted in Fig. (3).
Fig. (3). Representation of spectra with metabolite peaks A, B and C in multivariate space.
Applying PCA to the data in the original 3-dimensional data space will reveal the
lower (1- or 2-) dimensional projection which still conserves as much as possible the
distances between the data points, as shown in Fig. (4). In a typical metabonomic study,
spectra will consist of hundreds or thousands of peaks. Therefore, concentrating the bulk
of the information in only a few dimensions is an invaluable tool.
In statistical terms the PCA algorithm will find the new dimensions or “Principal
Components” as the linear combinations of the original measurements that maximize the
variance. The variances of the original variables and their correlation will lead to a
unique solution, greatly reducing the complexity of the dataset while preserving as much
as possible information.
The linear combinations of the original variables (called loading vectors) summarize
the measurements along their characteristic correlations, revealing therefore the “data
trends”. Furthermore, each sample has a score value for each loading vector,
representing the strength of the particular trend present the sample in question. Plotting
the scores will therefore position each sample according to how prominent the data
trends are represented in the respective sample.
Fig. (4). PCA as a manner to rotate the original axes in such a way that the distances between the
sample points are maximized when projected on the plane given by the paper.
Interpretation of the scores of the samples (e.g. clustering due to disease) and
inspection of the data trends responsible for these clusters greatly enhances the
possibility to explain complex metabolomic changes in simple terms of clusters and
systematic data trends, as illustrated in Fig. (5).
Principal Component Discriminant Analysis (PCDA)

PCDA is a PCA-based method where the difference between pre-defined groups of
samples is maximized rather than the overall variance. When applying PCA, clustering
will only be observed when the data contains a strong clustering effect and hence, inter-
cluster variation must be greater than intra-cluster variation, i.e. subject-specific
variation must be smaller than the metabolic impact of treatment or disease under study.
However, particularly in clinical studies this is often not the case.
The PCDA method finds the linear combination of original measurements that
maximizes the inter-class distances. Therefore, projection of the original data on these
Fig. (5). The difference between healthy and diseased subjects visible on the scores plot can be
translated back as pattern of metabolites
discriminant axes will yield a scores plot, separating the a priori assigned classes as
much as possible. The difference between the projection planes found by PCA and
PCDA can best be demonstrated by the example illustrated in Fig. (6).
Partial Least Squares regression (PLS)

Where PCA is used for exploratory data analysis and PCDA for clustering according
to predefined classes, PLS is used for regression. PLS uses a lower dimensional
projection of the predictors (e.g. NMR spectra), similar to the earlier mentioned
multivariate techniques. However, the projection is not only optimized to express as
much of variance for the predictors but also simultaneously maximizes the correlation
with the variable(s) to be predicted.
This results in a regression vector where a regression coefficient is calculated for
each predicting variable (e.g. each peak). Each coefficient can be compared to the slope
of a calibration line and these individual contributions add up to the final estimate. The
regression coefficients can be interpreted directly as a positive or negative contribution
of the respective peak to the variable to be predicted.
Fig. (6) . (A) Raw Data consisting of 120 samples with 3 variables plotted on it’s original basis;
(B) Raw data colored according to classes, showing it actually consists of three tightly layered
clusters; (C) PCA scores finding the widest spread for all samples on projection plane, projecting
roughly “from above”, even coloring according to class does not reveal clusters; (D) PCDA scores
finding the biggest distance between clusters; projecting roughly “from the side”, using a priori
class information.
It should be noted however, that there is a fundamental difference between

constructing individual calibration lines for each predictor (and e.g. averaging their
results) or using PLS regression. Constructing individual univariate lines would use all
variance, including that which is uncorrelated and hence, would introduce more
prediction error than aid in prediction. PLS will find the projection dimensions (or latent
variables) where the scores are best correlated to the predicted variable and uncorrelated
variance will remain in dimensions unused for prediction. Furthermore, by summarizing
the correlated predictors into the latent variables, significant predictive power can be
generated where the single predictors alone may have too low signal to noise ratio to
contribute to the estimate.
Partial Least Squares Discriminant Analysis (PLS-DA)

Whilst PLS regression is normally used to predict continuous variables, such as
concentrations, it is possible to use the same algorithm to predict discrete class-
assignments, resulting in the PLS-DA method. PLS-DA is straightforward for a two-

classes-study, where e.g. healthy and diseased subjects will be assigned values of +1 or
–1 respectively. When more classes are to be modeled, the assignment of different
discrete values for each class in one variable would imply an ordering between classes
which is almost never the case. Therefore, each class must be represented by its own
predicted binary variable, leading to a PLS2 model that will simultaneously predict the
different class assignments. Generally this will only lead to stable models if the
predicted variables are correlated, which is rarely the case in class assignments. PLS-DA
is therefore a complementary tool to PC-DA: where PC-DA suffers from a lack of
degrees of freedom in a two-class situation PLS-DA will perform optimally.
Unsupervised Clustering
Many metabonomic studies are carried out without experimenters’ access to subject
group assignments in the first instance. In these cases, unsupervised clustering is
required in order to study the inherent groupings within the data. Although PCA can
provide a good overview of inherent similarities and differences between spectra it is
often difficult to determine discreet clusters in the data by this method. Distance-based
clustering methods, on the other hand, are more focused on this task. The methods, i.e.
hierarchical or maximum likelihood based methods (typical examples are K-Nearest-
Neighbor or K-Means algorithms) will use the distance between the samples and/or
cluster centers to visualize the similarities and differences in dendrograms. Dendrograms
are binary trees where neighboring branches are a representation of similar samples and
the length of the branches a measure of the difference between them. Different measures
for distance (Euclidian, Mahalanobis, City Block etc) and eventual data reduction
methods lead to a wide variety of possibilities for unsupervised exploratory clustering
with these techniques. Although the interpretation of the underlying mechanisms for
clustering might be difficult, these techniques are valuable tools to formulate hypotheses
for clusters that can be confirmed in follow up studies.
Neural Networks
Within metabonomics, Artificial Neural Networks (ANN’s) are largely applied to the
prediction of metabolite concentration or classification of biosamples. ANN’s are non-
linear learning algorithms which need to be trained before being able to predict either
continuous numerical or discrete class values. By their nature, the training of the
algorithms is far from simple and decisions taken during the training stage will greatly
influence the quality of the trained model. Aspects to be considered are the characteristic
parameters for the algorithm itself, i.e. type of network, number of nodes and layers,
types of transfer functions etc, as well as the very crucial choice of training, validation
and test sets. The latter aspect is important to all types of data analysis but due to the
powerful learning ability of ANN’s, these are more susceptible to unrepresentative or
unevenly distributed training data sets. Very characteristic to ANN’s are their empirical
and ‘black box’ nature, leading to models that, although high in predictive power, are
difficult to interpret. This almost enigmatic character makes the use of ANN’s for
exploratory modeling, where the why is more important than the how much, unattractive.
If on the other hand, the metabolic mechanism is understood but highly non-linear,
ANN’s can be a very powerful tool for prediction and classification using metabonomic
data.
Examples of the application of ANN’s in the prediction of metabolites are given by

Hiltunen et al. [78] and Ala-Korpela et al. [79, 80], where plasma lipoprotein
concentrations were predicted from the 1H-NMR spectra of whole blood plasma.
Kaartinen et al. [81] predict human brain metabolites from in vivo NMR spectra,
interestingly training the ANN using simulated 1H-NMR spectra and obtaining accurate
predictions of experimental ‘real’ samples. The use of ANN’s for classification purposes
is exemplified by Holmes et al. [82], where a probabilistic neural network was used for
classifying different types of toxicity using a large set of rat urine 1H-NMR spectra.
Another example is the classification of plant extracts into different classes of herbicides
and their modes of action using NMR and ANN by Ott et al. [83]. Although it is not
strictly an ANN but uses a model topology similar to probabilistic neural networks, the
CLOUDS approach proposed by Ebbels et al. [84], is also a classification tool.
Data Pre-Processing
Because all data analysis methods presented are data driven models, the outcome and
quality of each model will depend solely on the quality of data presented to the model.
Therefore data pre-processing is arguably the most important stage of data analysis and
different labs express preferences for a wide variety of different approaches. Generally,
the pre-processing will contain different additive and/or multiplicative operations on the
raw data. It is crucial that these steps will neither introduce artifacts nor remove too
much variation and therefore the data pre-processing must be included in the necessary
validation steps such as internal cross-validation or prediction of an external test set.
Methods of Removing Undesired Variation in the Data Set

The most common source of undesired variation in a data set is ‘peak shifts’, where
small changes in pH or ionic strength between samples result in chemical shift changes
for various compounds. Some metabolites, e.g. citrate are particularly sensitive to these
changes and whilst these shifts pose little problem to a spectroscopist examining the data
by eye, from a data analysis perspective, if a certain peak is not always expressed in the
same position, the model will interpret it as different entities (e.g. different metabolites),
making the model at best unnecessarily complex or even useless. Different tactics have
been proposed to tackle this problem. Probably the most commonly used and robust
method is simple resolution reduction, commonly referred to as ‘bucketing’ or ‘binning’
[85] where each spectrum is segmented into equally sized regions of e.g. 0.04 ppm wide.
However, whilst this technique is adequate in most cases, it can be rather crude and
subtle metabolic differences between groups of data may be lost. For this reason, many
authors prefer to align peaks and a number of approaches have been proposed for this
purpose [86-90].
All metabonomics data sets will contain some degree of both biological and
analytical variation the source of which is not the biochemical state under study, e.g.
day-to-day differences in spectrometer operation, diurnal variation, subject-to-subject
variation (particularly when studies involve two or more groups of healthy individuals).
A number of different methods can be applied to help reduce this problem. The most
suitable method for removal of this variation is dependent on the design of the study.
In many cases, the application of a predicting or ‘supervised’ data analysis method
such as PLS, PC-DA or PLS-DA is enough to exclude any interfering factors from the
data analysis model. However when dealing with very diverse groups of subjects e.g.
healthy human volunteers, this is usually insufficient. In these cases, some groups have
reported good results with the application of orthogonal signal correction (OSC) [91, 92]
prior to discriminant analysis. OSC can be considered as a ‘backwards’ PLS, i.e. dummy
‘y’ variables are assigned to the data, dependent on the known groups and any
information in the data which is orthogonal i.e. uncorrelated to this group information is
removed. The usefulness of this kind of approach is however, disputed. Whilst some
groups appear to have used it with a large degree of success, others have failed to
improve on results obtained with standard discriminant analysis [93]. It should also be
remembered that OSC is technically a supervised method of data analysis and hence, the
use of appropriate validation steps is essential if meaningful results are to be obtained.
Another, milder approach, which is useful in longitudinal (time-course) studies, is to
mean-centre the data per subject. In this case, the ‘average’ state of each individual is set
to the centre of the plot, which will then show each individual’s variation around their
own average state [94]. A similar approach can also be applied where day-to-day
variation in the spectrometer operation causes confounding factors, provided that
spectrometer variation can be separated from group or individual variation. Therefore,
the importance of acquiring data in a random order or considering sample order in the
study design can not be overstated.
Variable-selection or -elimination methods can be used to improve the robustness
and power of models when the interfering variation, such as instrumental, environmental
or individual effects, is concentrated on a subset of the measured variables. The PLS-
based Uninformative Variable Elimination (PLS-UVE) is an example of this type of
method, where an estimation of the stability of the regression coefficients serves as a
measure to eliminate those variables that are not robust. Some methods use the
regression vector or the loading vectors to select variables that dominate the model,
whilst others use optimizing algorithms such as genetic algorithms, neural networks or
simulated annealing to find a stable subset of variables. The most useful algorithm will
depend on the selection and elimination criteria whether the subsets can lead to models
with enhanced stability, predictive power or interpretability.
METABOLITE IDENTIFICATION
Although some may argue that identification of unknown metabolites is not strictly
part of a metabonomic study, for biological interpretation of the data generated, the
chemical identification of metabolites of interest obviously plays a critical role.
When studying commonly analyzed biosamples, such as urine and blood plasma, the
majority of metabolite identification problems can be solved with straight forward
strategies such as comparison with spectral databases, literature, standard addition and
two-dimensional NMR spectroscopy. However, occasionally, these avenues of
investigation will not result in positive metabolite identification. In these cases, the
usefulness of directly coupled LC-NMR-MS is unrivalled [95]. LC-NMR-MS, can allow
unequivocal identification of metabolites where neither LC-NMR nor LC-MS alone
could generate all the necessary information. An example of this situation, illustrated in
Fig. (7), was observed during the study published in reference [94]. In this example, the
molecule contains 3 groups with exchangeable (‘NMR-blind’) protons, whereas the
application of LC-MS alone could not have distinguished the symmetry of the molecule.
Hence, only acquisition of both NMR and MS data allowed identification of the
compound.
Fig. (7). An example of metabolite identification by directly coupled HPLC-NMR-MS, where

neither NMR spectroscopy nor mass spectrometry alone provided sufficient information for
unequivocal metabolite identification.
However, an important point to note with respect to metabolite identification is that

the complexity of the task and the time required should not be underestimated.
BIOLOGICAL CHALLENGES AND METABONOMICS

Toxicological Studies
The majority of the earliest published metabonomic studies studied the effects of
well-known toxins [96], e.g. alpha-naphthylisothiocyanate (ANIT), carbon tetrachloride
(CCl(4)), 2-bromoethylamine (BEA), 4-aminophenol (PAP), hydrazine and many others.
Whilst metabonomics has expanded into other application areas, as outlined below,
toxicology remains a dominant field for its application [97-100].
Probably the largest toxicology-based studies concentrating on development of
metabonomics methodology, technology and knowledge are the COMET and
subsequent COMET II consortia, led by Imperial College, London, UK and involving
six major pharmaceutical companies [101]. The COMET project has applied
metabonomic techniques to generate a database of approximately ten thousand 600 MHz
1
H NMR spectra of urine and blood serum from animals treated with approximately one
hundred and fifty model toxins. This information will be used to build ‘expert systems’
with the aim of predicting the most likely toxicological effect for new compounds.
However, the methods available for constructing such expert systems are still under
development.
The Study of Disease in Man

The area of clinical metabonomics is still relatively immature in comparison with
toxicological metabonomics and brings with it a host of additional challenges.
In animal studies, whether using either metabolomics or conventional methods, the
exact biological history of the experimental animals i.e. diet, age, gender and compound
dose are known and sample timing and environment are well controlled. In the case of
clinical studies, there are many more variables in the human biological history and
experimental conditions can not be met, only described and recorded as
comprehensively as possible. This intrinsic variability is demonstrated in some recent
normality studies which demonstrate the importance of rigorous study design and
statistical analysis of metabonomic information obtained from man [102, 103].
However, despite the increased variability of data in clinical studies, NMR-based
metabonomics has been applied to the study of numerous diseases with success, for
example: coronary heart disease [40], cancer [104, 105], meningitis [48] and a number
of inborn errors of metabolism [106-109], to name a few.
Normality Studies
Following on from the study of disease, a number of published studies have recently
focused on the sources of inherent variation in control animals [110-113] and the
‘normal, healthy’ human population [114, 115]. The knowledge gained from these
studies will both allow studies to be better designed by ensuring that biological variation
in the ‘control’ animals or subjects is either minimized or at least explainable and may
eventually pave the way to studies whose aim is prediction rather than diagnosis of
disease.
Nutritional Studies
The application of metabonomics to nutritional studies is the least mature of all the
application areas because from both the analytical and chemometric perspectives it is
probably the most challenging field. This can be attributed to a number of complexities
which are either less applicable or not applicable in other areas: 1) food products are
aimed at the ‘normal, healthy’ population; 2) the metabolic effects of foods are much
more subtle than the effects of drugs; 3) response of individuals to bioactive ingredients
can vary dramatically depending upon other lifestyle factors such as age, habitual diet,
exercise regime etc; 4) a food product is frequently largely uncharacterized. However,
despite these difficulties, metabonomics has been applied with success to nutritional
studies in both animals [116, 117] and more recently, in humans [94,118, 119] and is
well placed to become of major importance in nutritional research.
CONCLUSIONS
In recent years, there has been a shift in approach to the study of biochemical
metabolism from the traditional hypothesis-driven ‘target analysis’ approaches, to the
hypothesis-generating *omics approaches. The *omics approaches present a number of
inherent advantages over the targeted approaches, not least that pre-assumption and
selection of metabolites of interest are unnecessary. Hence, observations regarding the
effects of environmental, genetic or chemical influences on cells, tissues or whole
complex organisms are not biased by scientists’ expectations. The combination of NMR
spectroscopy and multivariate data analysis has been shown via numerous publications
on the subject, to be well suited to the profiling of high abundance (≥ low µM)
metabolites in biosamples and technical developments in the field such as the
introduction of cryoprobes and microvolume probes will further strengthen the technique
by increasing sensitivity, reducing sample volume requirements and increasing the
accessibility of heteronuclear experiments e.g. 31P and 13C NMR spectroscopy.
However, with increasing ability to measure more metabolites in ‘single shot’
experiments, new problems are encountered from a data handling and information
extraction perspective which were not an issue in the days of metabolic ‘target analysis’.
Probably the largest problem which scientists encounter when handling *omics data is
the presence (and subsequent strategies for removal) of confounding variation in the
metabolic profiles. Inherently all sources of variation from sample collection, sample
handling, instrumental variation and biological variation will be present in *omics data.
The enforcement of rigorous sample handling protocols and thorough consideration of
study design is essential. Implementation of the *omics disciplines therefore involves
more than simply adopting extra analytical methodology. New working philosophies,
where clinicians, analysts and chemometricians are all involved at each stage of a study
from design to conclusion are critical.
Despite the complexities of setting up *omics methodologies and project teams, the
developments and results already accomplished in this young discipline demonstrate that
the investment is clearly worthwhile. The field has already demonstrated such significant
advances to medicine as increased ability to diagnose disease, discovery of new
biomarkers and increased understanding of how the environment which we live in
affects our health and well-being. These developments are set to continue as the
methodology matures and becomes more wide-spread and the scientific community in
general becomes better able to cope with the astronomical amounts of data we are likely
to see being generated from these studies.
ABBREVIATIONS
ANN = Artificial neural network
CSF = Cerebrospinal fluid
DAD = Diode array detector
FI = Flow injection
GC = Gas chromatography
HPLC = High performance liquid chromatography
HRMAS = High resolution magic angle spinning
KNN = K-nearest neighbor
MS = Mass spectrometry
MVA = Multivariate analysis
NMR = Nuclear magnetic resonance
OSC = Orthogonal signal correction

PCA = Principal components analysis
PCDA = Principal component discriminant analysis
PLF = Partial linear fit
PLS = Partial least squares
TIC = Total ion chromatogram
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NMR-Based Metabonomics of Urine from

An Exploratory Study of Ciprofibrate in
Healthy Volunteers and Patients with
Type 2 Diabetes Mellitus
Gregory C. Leo1,*, Ewoud J. van Hoogdalem2, and

Martijn B.A. van Doorn3
1
Johnson & Johnson Pharmaceutical Research and Development, LLC, Welsh and
McKean Roads, Springhouse, PA 19477-0776, USA; 2Johnson & Johnson
Pharmaceutical Research and Development, LLC, Turnhoutseweg 30,
B-2340 Beerse, Belgium and 3 Centre for Human Drug Research,
Zernikedreef 10, 2333 CL Leiden, The Netherlands
Abstract: This report presents the results from the NMR-based metabonomics
of urine collected as part of a multiple dose, double-blind, randomized, placebo
controlled parallel group exploratory study of the pharmacodynamics of
ciprofibrate in healthy volunteers and patients with type 2 Diabetes Mellitus.
Using supervised statistical analysis it was possible to distinguish the various
treatment groups (healthy and diabetic: placebo-treated or drug-treated) when
males and females were analyzed separately.
INTRODUCTION
Pharmaceutical companies strive to discover and to develop drugs that are safe and
efficacious, but the process is scientifically complex and financially risky [1]. Much
financial cost is incurred by high attrition of new compound entities in pre-clinical and
clinical development. The reasons for failure of these compounds generally fall into one
or more of the following categories: poor efficacy, safety deficiencies or economic
reasons [2]. The current pre-clinical paradigm for drug development is based on the
premise that efficacy and safety issues reveal themselves in preclinical efficacy and
safety data in relation to drug exposure, and in pharmacokinetics, toxico-kinetics, drug-
drug interactions and drug metabolism studies. Detecting defects in these areas at an
early stage of drug discovery and preclinical development would be highly valuable
[2,3] and one approach currently utilized by the pharmaceutical industry is systems
biology. Systems biology is the combined application of “-omics” technologies (geno-
mics, proteomics, transcriptomics, metabonomics). From a size perspective metabono-
mics has a simpler task set before it as there are only a few thousand metabolites
whereas the number of human genes is greater than 30,000 and the number of proteins
greater than 100,000 [4]. The focus of the present work will be metabonomics,
*Corresponding author: Tel: 215-628-5884; Fax: 215-628-7064; E-mail: GLEO@PRDUS.JNJ.COM

176 Frontiers in Drug Design & Discovery, 2006, Vol. 2 Leo et al.
an area that we have invested [5,6] an effort to understand and apply within our drug
discovery and development environment.
Metabonomics has progressed as a field because of many analytical and compu-
tational advances. Initial metabonomic studies were predominantly NMR-based but MS-
based applications are rapidly appearing as well as other analytical techniques (HPLC
and IR). A recent discussion and review of metabonomics as applied to pharmaceutical
research and development has been published by some of the pioneers of the field [7].
As noted in the literature many of the pharmaceutical applications have involved
biofluids (primarily urine and plasma) from animals. NMR-based metabonomic profiling
has been applied towards human disease diagnosis [8] and recently a report was
published using NMR to analyze human blood serum samples as a diagnostic tool for
coronary heart disease [9]. Applications of metabonomics in a clinical trial setting to aid
in the evaluation of a drug or drug candidate have not appeared in the literature. It is in
this context that we investigated the utility of NMR-based metabonomics as part of an
investigational trial to study the pharmacological effects of ciprofibrate and to search for
biomarkers in healthy volunteers and in patients with type 2 diabetes.
Ciprofibrate is an established and effective treatment for three main types of
atherogenic hyperlipoproteinaemia: type 2a hypercholesterolaemia, type 2b combined
hyperlipidaemia, and type IV hyper-triglyceridaemia [10]. Its therapeutic effects are
mediated by binding and activating the peroxisome proliferator-activated receptor alpha
(PPARα). The PPARs are members of the nuclear hormone receptor family of ligand-
activated transcription factors and control the expression of many target genes in various
tissues [11,12]. The pharmacokinetic profile of ciprofibrate has been accurately
determined in several studies [13-15]. Ciprofibrate exhibits high bioavailability, which is
attributable to almost complete intestinal absorption of the dose administered and
absence of significant first-pass metabolism. Maximum plasma levels after a single oral
dose of 100 mg/day are 85 µg/ml, with a peak level 2 hours after administration,
whereas continuous therapy of ciprofibrate 100 mg/day results in a steady state plasma
concentration of 80-86 µg/ml. The half-life of ciprofibrate is 80 hours in subjects with
normal renal function. In patients with renal failure, the half life of ciprofibrate is
prolonged, and can reach up to 170 hours. As a consequence, there is a risk of drug
accumulation; this phenomenon likely contributes to the muscle toxicity observed with
ciprofibrate in such conditions and explains why it is contraindicated in patients with
severe renal failure. Thus, precautions must be taken when treating patients with
moderate renal failure with ciprofibrate. Ciprofibrate is highly bound to plasma proteins,
with 95% being bound to albumin. Approximately 75% of the administered dose of
ciprofibrate is metabolized in the liver by UDP-glucuronosyltransferase and 80%-97%
of the dose is eliminated via the kidneys, with only approximately 3% being excreted in
the feces.
Measuring plasma levels of biochemical markers (i.e. triglycerides and cholesterol)
in plasma samples is currently the method of choice to evaluate the efficacy and safety
of novel PPARα agonists in early clinical development ‘proof of concept’ studies.
However, because it is expected that activation of PPARα nuclear receptors regulates
the transcription of several known, but also many as yet unknown genes, it is likely that
significant downstream metabolic effects are overlooked when measuring a limited
panel of conventional markers. Thus, metabonomics could provide a more compre-
hensive picture of the metabolic changes induced by the study drug. Therefore, the main
NMR-Based Metabonomics of Urine Frontiers in Drug Design & Discovery, 2006, Vol. 2 177
objective of this study was to explore the usefulness of NMR-based metabonomic

profiling (system response profiling or ‘Systems Pharmacology’ [16]) for the pharma-
cological effects of ciprofibrate (as prototype for the PPARα class) in urine samples of
type 2 diabetes mellitus patients (T2DM) and healthy volunteers (HV). As a secondary
objective, we sought to explore disease and gender related differences in urine
metabolite profiles (‘Systems Pathology’ [16]) between T2DM patients and HV.
EXPERIMENTAL
Clinical Trial Protocol and Urine Collection
This was a single center, randomized, double blind, placebo-controlled, parallel
groups multiple oral dose study. After a one-week placebo run-in period, subjects were
randomly assigned to a three-week treatment with 100 mg ciprofibrate q.d. or placebo
(Table 1). Treatment allocation took place according to randomly permuted blocks and
was stratified by gender and subject type (diabetic patient or HV). Study medication (an
encapsulated tablet) was taken prior to breakfast.
Table 1. Study Design Showing Different Populations, Treatment Periods (V) Double
Blind Placebo Controlled Randomized Design
1 wk run-in period Treatment period Follow-up
V1 V2 V3 V4 V5 V6
Baseline 1 wk 2 wk 3wk
• Male T2DM :8 Placebo

randomization
• Female T2DM :8 Placebo 50% ciprofibrate

• Male Healthy :8 Placebo 50% placebo
• Female Healthy :8 Placebo
Subjects entered the study 7 to 21 days after screening. They visited the study center
weekly for five weeks (Visits 1 to 5 and a post-study visit). These study visits were
scheduled on the same day of the week as Visit 1, but a deviation of one day either way
was allowed.
Eight male and eight female patients with an established diagnosis type 2 diabetes
mellitus uncontrolled by diet alone, aged between 18 and 75 years, and a fasting plasma
triglyceride (TG) concentration > 1.5 mM (>133 mg/dl), were included. Patients were
excluded if they had a significant medical history or current symptoms of clinically
relevant conditions, or had used any non-sterioidal anti-inflammatory drug, PPAR
agonist (α,β/δ or γ) or lipid lowering medication within two weeks prior to the expected
study start date.
In addition, eight male and eight female healthy subjects (as determined by medical
history, physical examination and routine laboratory tests), aged between 18 and 45
years, were included. The demographics and baseline characteristics of both study
populations are presented in Table 2. Fasting urine was collected during the morning
hours in PE containers and 12 mL (2 x 6mL) was stored in PET tubes (Vacutainer) at
minimally -20 ºC until analysis.
Table 2. Demographics for T2DM and HV Groups Shown as the Average and in the
Parentheses, the Standard Deviation
Parameter T2DM HV
Age (years) 59 (7.8) 27 (7.8)

2
Body Mass Index (kg/m ) 30.4 (5.22) 24.0 (2.62)
Waist/Hip ratio 1.0 (0.07) 0.8 (0.05)
Disease duration (yrs) 6 (4.2) N.A.
Current treatment: No medication N=1 N = 18
OAD Monotherapy N=8 N.A.
OAD Combination N=3 N.A.
Insulin N=2 N.A.
OAD and Insulin N=2 N.A.
Current Antihypertensive N=6 N.A.
Heart rate (bpm) 74 (14.5) 71 (10.2)
Systolic blood pressure (mmHg) 145 (19.0) 116 (12.9)
Diastolic blood pressure (mmHg) 82 (8.5) 70 (10.0)
OAD – oral antidiabetic drug.
Proton NMR Analysis

The preparation of the urine for NMR analysis was based upon a protocol previously
reported in the literature [5,17]. To minimize chemical shift variation due to differences
in the urine pH, 300 µl of 0.2 M, pH 7.4 phosphate buffer was added to 600 µl aliquots
of the urine. After 10 minutes the samples were centrifuged at 10,000 rpm for 10
minutes and 800 µl of the supernatant was removed and placed in 1.5 ml sample vials.
Before capping the vials with septum caps 50 µl of a 1 mg/ml solution TSP (3-
trimethylsilylpropionic-(2,2,3,3-d4)-acid) in D 2O was added to each sample. The NMR
data were acquired on a Bruker Biospin DMX-600 spectrometer (600.03 MHz 1H
frequency) interfaced with Bruker Biospin’s BEST flow injection accessory (a Gilson
215 liquids sample handler) [18]. The probe used was an inverse configuration, single
cell, 4 mm, proton-carbon LC probe. The injection volume was 400 ul. The pulse
sequence used was a 1D-NOESY using water pre-saturation (47 Hz field strength)
during the recycle delay (3.3 s) and the mixing delay (0.08 s) [19]. This experiment is
robust and easy to implement in automation giving superior water suppression compared
to a single pulse experiment using presaturation. Since urine normally lacks the amount
of protein as found in plasma, the CPMG edited 1D experiment is not commonly used
for urine samples. The acquisition time was 1.7 seconds using 32K complex points for a
16 ppm sweep width. The number of scans collected for each urine sample was 256 and
the data were processed using 0.3 Hz exponential line broadening. Residual water signal
was eliminated using the method of Marion, Ikura and Bax [20]. Spectra were manually
phased and baseline corrected before reducing the data using the AMIX software by
Bruker Biospin. Data reduction involved dividing the spectra into 228 segments, each
0.04 ppm wide for the spectral window ranging from 0.5 to 9.6 ppm. The sum of the
area for each segment was used to define each variable. Each variable was normalized
by the total area and a scale multiplier of 100 was used.
Chemometric Analysis
The reduced data described above were imported into Simca-P+ version 10.5.0.0 by
Umetrics AB (Umeå, Sweden) for principal component analysis (PCA) and partial least-
squares – discriminant analysis (PLS-DA). Multivariate analysis is well documented
[21-23] and has been applied in the areas of analytical science and specifically, meta-
bonomics [24]. Briefly, principal component analysis is a mathematical manipulation of
the data whereby linear combinations of the reduced data are optimized with respect to
the variance and are plotted using axes redefined by the factors or principal components
instead of the original measurement variables (scores plot). The corresponding loadings
from the scores plot indicates which variables (spectral regions) are most important for
the separation seen along each factor. Partial least-squares (PLS) is a generalization of
PCA where a projection model is developed predicting Y (e.g., an outcome) from X (the
variables used in PCA as just described) via the scores of X. When PLS is combined
with discriminant analysis (DA) the Y’s are defined by the user and the algorithm
allocates observations (a set of urine samples) to one class of a given set of classes. In
the present work, after unblinding the study the urine sample was classified, for
example, as being from a placebo-treated male diabetic subject. In analyzing PLS-DA
results one will make use of contribution plots to highlight those variables that
distinguish the members in the different classes. The variables found in the contribution
plots (or the loadings plots) can guide closer scrutiny of the proton spectra and in
conjunction with metabolite chemical shift tables [8,25] lead to possible metabolite
identification. The computations were carried out on a desktop personal computer. There
were 205 variables that were used for the statistical analyses after the variables from
4.72 to 5.08 (water) and 5.52 to 6.08 ppm (urea) were eliminated from the workset.
Univariate scaling was used in the PCA and PLS-DA calculations. There were four
spectra, each from different subjects, excluded from the statistical analyses because three
had low metabolite concentrations and the other had poor water suppression. After the
initial unsupervised analysis, samples having elevated glucose were excluded resulting
in the absence of all six time points for two placebo-treated, diabetic subjects (one male
and one female) and half the time points for two ciprofibrate-treated, diabetic subjects
(one male and one female).
The PLS-DA models for the males and females were not tested using another set of
samples since there were none available and the number of patients for each group was
too small to subdivide the group so that a portion could be used for validation of the
model. The method of validation used was response permutation [23] that permutes the
sample’s class identity and then re-calculates the R2Y and Q2 values. The R2Y and Q2
represent the model’s ability to fit the data and the predictive ability of the model. This
permutation procedure was repeated 50 times to generate reference distributions of R2Y
and Q 2 for appraising the significance of the parent model values. A regression plot of
the permutations should give intercept values less than 0.05 for Q2 and should not
exceed 0.3 – 0.4 for R2Y [23]. For the four classes generated for the female subjects
these two criteria were met – the R2Y values hovered around 0.4 and Q2 was less than
0.05. The models generated for the male subjects were weaker – Q2 was less than 0.05
but the R2Y was about 0.5. The number of PLS components used to create a model is
informative. For G well conditioned and well separated classes, there are G-1 significant
PLS components expected [26]. For the models generated for the male and female
subjects in the present study four PLS components were calculated. Since four classes
were used in the PLS-DA calculation, three PLS components would have been ideal. An
extra component indicated non-linearities, such as sub-clusters, are present in the data
[26]. The models are useful as indicators that the methodology has value but lack the
strength to be of real predictive value. This suggests a more careful consideration is
needed with respect to the number of samples for future trials.
RESULTS AND DISCUSSION

A typical proton spectrum of human urine from a healthy male volunteer is shown in
Fig. (1). The bottom spectrum shows all the peaks to scale and the top spectrum is the
vertical expansion of the same data to better illustrate some of the smaller peaks and the
amount of noise generally observed. The two dominant peaks (the sharp singlets to the
left of 3 and 4 ppm) are from creatinine. Residual water after suppression is seen at the
center of the spectrum and urea is the broad peak to its left. The peak to the far right is a
reference (TSP) added to the sample. The regions of the spectrum containing the urea,
residual water and TSP are not used in the statistical treatment of the data. Ciprofibrate
or its major metabolite, the beta-O-acylglucuronide [27] were not observed in the
spectra.
Fig. (2) shows the PCA scores plot of 197 urine samples excluding the four samples
that were outliers as mentioned in the experimental section. The urine samples in Fig. (2)
were collected during visits 1-6 but only during visits 3-5 were the actively treated
individuals exposed to ciprofibrate. The most notable feature of the scatter plot is the
group of data points projecting along the negative axis of factor 1 (t1). These points
(urine samples) were from two placebo-treated diabetic patients (one male and one
female) and two ciprofibrate-treated diabetic patients (one male and one female) with
high levels of glucose in the urine (glucosuria). In the main group of samples no
clustering was observed. Other combinations of factors in two- and three-dimensions
were checked but clustering was not observed in the larger group of points. If the points
with the high glucose were excluded from the data set and the calculation repeated some
discrimination of healthy subjects from diabetic subjects was observed if certain
combinations of the higher factors were viewed (data not shown).
If we focused only on visits 3-5 (the active treatment period); excluded the patients
with elevated levels of glucose in the urine; excluded the obvious outliers and selected
only male or female subjects it was possible to separate the different treatment groups
using supervised statistical analysis, PLS-DA, as shown in Fig. (3) and (4). Of the
original 201 urine samples 42 samples were used to generate Fig. (3) and 45 samples
Fig. (1). Typical 600 MHz urine spectrum of urine from a healthy male volunteer at the first office
visit. The bottom spectrum shows all the peaks to scale and the top spectrum is an eight-fold
vertical expansion of the bottom spectrum. Abbreviations: ac - acetate; cr – creatinine; dma -
dimethylamine; h - hippurate; sw - suppressed water; T - trimethylamine-N-oxide; TSP - 3-
trimethylsilylpropionic-(2,2,3,3-d4)-acid.
were used to generate Fig. (4). Figures (3) and (4) demonstrate the power of discriminant
analysis over the unsupervised analysis with the caveat that the study be unblinded. In
PLS-DA the healthy volunteers are separated from diabetic patients and within each of
these groups, separation is observed based on treatment. It is worth cautioning the reader
that for the diabetic groups there are less than four individuals, because of the exclusions
of the patients with high urinary glucose and even though the statistical modeling
separated the classes, greater confidence would be gained by a significantly higher
number of participants in each of the categories. In Fig. (4) it should be noted that the
ciprofibrate-treated females separated into two groups. The difference between the two
groups was that the group of four urine samples removed from the all the other samples
(cubes in the upper left region of the chart) had in general reduced metabolite
concentrations such that many of the smaller peaks seen in the other spectra were buried
in the baseline noise in these spectra. This group of four samples was excluded when
making the comparisons in the contribution plots that follow.
Fig. (2). Scores plot of urinary samples from visits 1-6.
To find those regions of the spectra responsible for differences between the various
groups contribution plots were generated. In a contribution plot the values along the x-
axis are the spectral regions (i.e., buckets) used for the calculations and the y-axis shows
in units of standard deviations the difference between the two groups. For example,
those variables that are found along the negative y-axis are for metabolites found in
greater amounts in the bottom group than for the top group. The first set of contribution
plots show the effects of treatment upon the healthy volunteers or the diabetic
individuals. Fig. (5) shows the contribution plot for the placebo-treated healthy male
subjects and the ciprofibrate-treated healthy males. Looking at variables that were
elevated greater than 2 sd in either group it was not possible to identify the metabolites
using published chemical shift tables for metabolites[8,25] with the exception of the
buckets at 3.58 and 2.86 that are consistent with glycine and trimethylamine,
respectively. Fig. (6) shows the comparable plot for healthy female volunteers given
either placebo or ciprofibrate. The only identifiable metabolite found at greater than 2 sd
Fig. (3). PLS-DA three-dimensional score plot of urinary samples from male volunteers. The
symbols represent the different classes: plus sign – healthy/ciprofibrate-treated; wedge –
healthy/placebo-treated; cube – diabetic/ciprofibrate-treated and sphere – diabetic/placebo-treated.
was acetate (1.94). The variables that exceeded the 2 sd limit in the males were not
found in the females but similarities do exist. Acetate and the 1.62 bucket were found
elevated in ciprofibrate-treated, healthy females but less so in males (greater than 1 sd
but less than 2 sd). For the ciprofibrate-treated, healthy males the bucket labeled as
glycine was seen elevated in the females but below the 2 sd threshold and for the
placebo-treated males trimethylamine was significantly elevated but less so for the
comparable female group (slightly greater than 1 sd). The fact that there are not many
differences between the two treatment groups for both the males and females might
suggest that the compound is well tolerated but this view would ignore ciprofibrate’s
known impact on lipid metabolism. Alternatively, as will be seen in other contribution
plots where there are not many variables that are dramatically different even between the
healthy and diabetic groups, it could be that in the current study design and methods of
analysis the patient to patient variation is sufficient to overwhelm the differences
between the various classes. The inherent lack of sensitivity to monitor more metabolites
or the data reduction method (bucketing) could also be limitations. With some of the
current NMR instrumentation a significant increase in the number of measurable
resonances can be obtained (see for example [28]). Also, it could be that serum would
provide additional information to increase the class distinctions. For example, serum
fatty acid profiles in conjunction with supervised chemometric methods have been used
to discriminate type 2 diabetes mellitus patients from healthy controls [29].
Fig. (4). PLS-DA three-dimensional score plot of urinary samples from female volunteers. The
symbols represent the different classes: plus sign – healthy/ciprofibrate-treated; wedge –
healthy/placebo-treated; cube – diabetic/ciprofibrate-treated and sphere – diabetic/placebo-treated.
The following figures compare placebo-treatment versus ciprofibrate-treatment for

diabetic male volunteers (Fig. (7)) and diabetic female volunteers (Fig. (8)). Identifying
the metabolites at >|2| sd for both groups compared in Figures (7) and (8) was not
possible for many of the peaks. In the diabetic males the ciprofibrate-treated group had
elevated acetoacetate (2.34) and the metabolite represented by the bucket at 1.62.
Acetoacetate could result from keto-acidosis. The variables (4.62, 5.46, 5.5) seen around
the water region appear to be more due to the unit variance scaling blowing up the noisy
patterns in the data. This is related to baseline perturbations and is seen in some of the
Fig. (5). Contribution plot showing the buckets that differentiate urinary samples of placebo-
treated, healthy males (top) with those from ciprofibrate-treated, healthy males. Some bucket
identifications are: glycine (3.58) and trimethylamine (2.86).
Fig. (6). Contribution plot showing the buckets that differentiate urinary samples of placebo-
treated, healthy females (top) with those from ciprofibrate-treated, healthy females. Bucket 1.94 is
acetate.
Fig. (7). Contribution plot showing the buckets that differentiate urinary samples from placebo-
treated, diabetic males (top) with those from ciprofibrate-treated, diabetic males. Bucket 2.34 is
acetoacetate.
treated, diabetic females (top) with those from ciprofibrate-treated, diabetic females. Bucket 2.86
is trimethylamine.
other contribution plots. Using a less harsh scaling method such as pareto scaling gave
similar results. The bucket at 1.62 was also elevated for ciprofibrate-treated diabetic
females (Fig. (8)) and in the ciprofibrate-treated healthy females (Fig. (6)). This peak
was always broad, suggesting elevated protein levels in the urine. In Fig. (7) there are
some interesting buckets found elevated for the ciprofibrate-treated group in the
aromatic region that are suggestive of a substituted indole (7.34, 7.7, 7.22 and 7.26).
Diabetes related ketoacidosis can induce changes in indolamine levels in the rodent brain
(rat [30] and mouse [31]) but whether these changes would be measurable in human
urine by NMR is unclear. Keto-acidosis is a severe complication found in subjects
exhibiting high levels of gluocose in diabetes mellitus and in the present case, keto-
acidosis seems unlikely given that the glucose levels of the subjects studied remained
fairly stable throughout the study (data not shown). More analytical work is required to
assign the resonance in the 2.34 ppm bucket. In Fig. (8) trimethylamine (2.86) appears
elevated in the ciprofibrate-treated diabetic females and less so in the males (Fig. (7)).
The last two figures compare placebo-treated diabetic and healthy individuals (men,
Fig. ( 9)) and women, Fig. (10)). In Fig. (9) metabolites elevated (greater than 2 sd) in
diabetic, placebo-treated males were N-methylnicotinate (8.86, 8.82, 4.46) and citrate
(2.7, 2.66, 2.54). The metabolites that were diminished for the diabetic males were not
identified (i.e. those buckets elevated for the control group – the placebo-treated, healthy
males). The same comparison for the female subjects is shown in Fig. (10). For the
females, the elevated N-methylnicotinate and citrate were not found and instead there
was a diminution of several buckets for the diabetic females. The biggest change was the
bucket at 4.06 which is consistent with creatinine even though the bucket for the
corresponding N-methyl resonance of creatinine (3.06) is not seen. The spectra from the
placebo-treated, diabetic females had an unidentified shoulder on the upfield side of the
creatinine peak at 3.05 ppm that was not present in the spectra from the healthy females
that could have offset the decrease in creatinine. The same phenomenon appears in the
males but to a lesser extent (the 4.06 bucket is between 1 and 2 standard deviations).
This is opposite the previous findings in serum which showed that fibrates, in general,
increased serum creatinine [32]. The lack of similarities between the males and the
females when making the comparison between healthy and diabetic could be due to
several factors. One of the factors that could be very prominent is that of diet which was
not controlled as part of the study. This would cause variability in the metabolite
concentrations that could mask more subtle changes.
Previously reported was a proton NMR study that profiled the urine of type 2
diabetic patients (n=33) versus the urine from a control group (n=20) [33]. Treatment
status was not indicated. In this report 17 metabolites were quantitated and a subset of
six metabolites were found to differ between the healthy controls and the diabetic group.
Lactate, alanine, citrate, dimethylamine, trimethylamine-N-oxide (TMAO) and hippurate
were found elevated in diabetic patients relative to controls. Resonances from these and
the set of 17 metabolites were observed in our urine samples but they were not found to
be discriminating factors with the exception of citrate (placebo-treated diabetic males,
Fig. 9). The paper by Messana et al. normalized the metabolites to the urinary creatinine
whereas in the present work the buckets were normalized to the total integrated area.
Creatinine was found to be relatively constant (99.8 ± 18.5 umol/L) for the study group
used in the aforementioned report and thus the authors were justified to normalize to
treated, healthy males (top) with those from placebo-treated, diabetic males. Some bucket
identifications are: N-methylnicotinate (8.86, 8.82, 4.46) and citrate (2.7, 2.66, 2.54).
treated, healthy females (top) with those from placebo-treated, diabetic females. Bucket 4.06 is
creatinine.
creatinine. The patient ages were similar between the two reports; in the present report
the average age of the diabetic group was 59 ± 7.8 years (Table 2) and in the other report
the patient age was 65 ± 13 years. Other patient and healthy volunteer criteria are shown
in Table 2. With respect to creatinine, the critical piece was that for the ciprofibrate-
treated diabetic females the group size was, after the exclusions mentioned, rather small
(n=3) and this was more likely the cause for the discrepancy observed.
Type 2 diabetes has been shown to be an appropriate disease to study the effect of
pharmacological interventions using metabonomics approaches. A mouse model of type
2 diabetes using obese (NZO x NON)F1 male mice treated with rosiglitazone, a PPARγ
agonist, was studied using a focused metabonomic approach whereby the authors
analyzed the lipid metabolome in plasma and myocardial, liver and adipose tissues [34].
In this report the authors used capillary gas chromatography as the analytical platform to
evaluate the lipid metabonome. A couple of the findings made were that rosiglitazone
induced de novo fatty acid synthesis and the effects on lipid metabolism were tissue
specific. The information gleaned in this report could not be expected to be obtained by
profiling urine using NMR methods. The approach by Watkins et al. was very targeted
and in the present study a global approach was pursued using a body fluid that was very
easy to collect and to prepare for analysis.
In conclusion, this study has shown that supervised statistical analysis (PLS-DA) of
urine samples collected as part of an exploratory trial of ciprofibrate in human volunteers
is able to separate the various study groups – ciprofibrate-treated versus placebo-treated
for the male and female subjects. The contribution plots have shown those regions of the
urine spectra that are responsible for the differences between the various treatment
groups. In addition, the diabetic patients and healthy subjects were easily separated but
the variation in the urine profiles limited the number of metabolites observed that
distinguished one group from another. This could possibly be improved by increasing
the study size and the application of some filtering method such as orthogonal signal
correction to enhance the separation of the groups. Also, moderate dietary limitations
could reduce some variation in the urine profiles. In comparison with another NMR
study that profiled a panel of urinary metabolites for type 2 diabetic patients and healthy
individuals, the other study showed a greater number of metabolites to discern the one
group from the other [33]. Furthermore, choosing a targeted metabonomic approach for
the related drug, rosiglitazone, several lipid metabolites were identified in plasma and
tissue to characterize the different treatment groups in a diabetic mouse model [34].
Here, NMR-based metabonomics of easily collected urine samples with minimal sample
preparation has discerned healthy from type 2 diabetic individuals for both treated and
untreated categories. This work adds to the growing body of literature showing that
global and targeted approaches using “omics” technologies will bring about a new era in
clinical analytical approaches for evaluating metabolic profiles in disease and for
studying mechanisms of drug response in drug development within the pharmaceutical
industry.
ABBREVIATIONS
CPMG = Carr-Purcell-Meiboom-Gill
HPLC = High performance liquid chromatography
IR = Infra-red
LC = Liquid chromatography
MS = Mass spectroscopy
NMR = Nuclear magnetic resonance
NSAID = Non-steroidal anti-inflammatory drug
PCA = Principal component analysis
PE = Polyethylene
PET = Polyethyleneterephthalate
PLS-DA = Partial least squares-discriminant analysis
PPAR = Peroxisome proliferator-activated receptor
ppm = Parts per million
TMAO = Trimethylamine-N-oxide
TSP = 3-trimethylsilylpropionic-(2,2,3,3-d4)-acid
UDP = Uridine-diphosphate
1D-NOESY = One-dimensional nuclear Overhauser spectroscopy
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Chromatography Mass Spectrometry Based

Metabonomic Analytical Methods
Gary W. Caldwell*, Wensheng Lang, Gregory C. Leo, John A. Masucci,

William Jones and Andrew Mahan
Johnson & Johnson Pharmaceutical Research and Development, L.L.C., Welsh &
McKean Roads, P.O. Box 776, Spring House, PA 19477, USA
Abstract: The concept of a system biology approach to understanding
biological functions such as the development and the progression of chronic
diseases and drug-induced toxicity in humans has generated widespread
interest in the pharmaceutical community. Techniques including genomics,
transcriptomics, proteomics, peptidomics, lipidomics and metabolomics are
being integrated not only to understand the entire biological system but also to
discover novel biomarkers. Analytical “Xomics” assays are playing a major
role in the development of these methods. We will review some of these
analytical techniques and specifically focus on chromatography mass
spectrometry based metabonomic strategies applied to diverse areas such as
fermentation, bacteria, plants and animals
INTRODUCTION
System biology can be defined as a global systems-level insight into complex
biological chemical events in order to understand the whole organism or biological
functions such as, metabolism, cell signaling, cell cycle, apoptosis, differentiation, and
transformation [1]. While the goal of system biology is to study whole cells or
organisms, it is extremely difficult to measure every biological chemical event since they
are spatial, temporal, and typically interdependent to each other. A focused system
biology approach around a subset of biological chemical events is considered a more
practical approach. Another way of stating the above definition is that the heterogeneous
parts of a biological system (e.g. genome, transcriptome, proteome, peptidome, and
metabolome) are studied independently and eventually combined to model the whole
organism through integration of experimental and computational techniques (Fig. 1).
Thus, a system biology approach requires the integration of interdisciplinary teams such
as, analytical sciences to collect the “Xomics” data (e.g. genomics, proteomics and
metabolomics), biological sciences to integrate genetic, protein, metabolite, cellular and
pathway events, and mathematical sciences to process the information with techniques
such as biostatistics and bioinformatics to useful knowledge [2]. The system biology
approach for the understanding of complex behaviors that underlies the development and
the progression of chronic diseases and drug safety in humans has generated widespread
*Corresponding author: Tel: 215-628-5537; E-mail: GCALDWEL@PRDUS.JNJ.COM

194 Frontiers in Drug Design & Discovery, 2006, Vol. 2 Caldwell et al.
insight into biological functions should permit better diagnoses of human diseases and
stimulate the discovery of novel therapeutic drugs.
Fig. (1). Illustration of a system biology approach and analytical “Xomics” methods. The numbers
of DNA (genes), mRNA, proteins and metabolites refer to humans.
Significant analytical progress in the last few years has been made in the design of
instrumentation for the collection of genomic [4], proteomic [5] and metabolomic [6, 7]
data. Once deoxyribonucleic acids (DNA), messenger ribonucleic acids (mRNA),
proteins and metabolites are qualitatively and quantitatively known, this information can
be theoretically pieced together into a system-level view of how a biological system
functions. There are several ways to fingerprint or profile “Xomics” data. In many
reviews these areas are broadly classified as:
• Global Fingerprinting
• Target Profiling
• Functional Profiling
• Structural Profiling
Global fingerprinting refers to the large-scale measurement of all compounds (DNA,
mRNA, proteins, or metabolites) within a biological system. This approach is typically
used as a screening tool to discriminate between samples (e.g., plasma, urine, cells,
tissues, etc.) of different biological status (control versus stressed). As shown in Table
(1), global fingerprinting of the genome, transcriptome and metabolome is possible for
Chromatography Mass Spectrometry Frontiers in Drug Design & Discovery, 2006, Vol. 2 195
many organisms using present analytical technology. Target profiling refers to the
qualitative and quantitative analysis of pre-defined sets of compounds. Analytical
targeting profile assays can be established for all “Xomes”. In addition, global and
targeting approaches can be used to establish validated biomarkers [8]. Here we are
defining biomarkers as specific or combinations of genes, proteins or metabolites in the
organism that have a particular molecular feature that makes them useful for measuring
physiological status such as drug-induced toxicity, the progress of a disease or the
effects of a drug treatment. Functional profiling is defined based on genes, proteins and
metabolites interactions within the cellular process. Other than the metabolome, the
functions of most genes and proteins are unknown in many organisms. Structural
profiling refers to the three-dimensional (3-D) structures of DNA (mRNA), proteins and
metabolites. Other than the genome and metabolome, most 3-D structures of mRNAs
and proteins are unknown in many organisms.
Table 1. Summary of Analytical Technologies Capable to Analyze “Xomes”
Genome Transcriptome Proteome Metabolome
Global Fingerprinting Yes Yes Incomplete Yes
Target Profiling Yes Yes Yes Yes
Functional Profiling Incomplete Incomplete Incomplete Yes
Structural Profiling Yes Incomplete Incomplete Yes
In this chapter, we will review analytical “Xomics” assays associated with global
fingerprinting or target profiling. Due to technological limitations, a single analytical
technique cannot provide sufficient visualization of the genome, the proteome and the
metabolome (Fig. 1). Multiple analytical “Xomics” assays are required for a
comprehensive view. A range of analytical techniques have been developed including
DNA sequencing, gene and protein chips, DNA and protein microassays, gas
chromatography (GC), high pressure liquid chromatography (HPLC), capillary
electrophoresis (CE), mass spectrometry (MS) and nuclear magnetic resonance (NMR).
We will specifically focus on chromatography-MS based metabolomic methods applied
to diverse areas such as fermentation, bacteria, plants and animals.
GENOME
The genome is the complete set of all DNA in an organism. Genomics refers to the
study of the entire set of DNA sequences both coding (genes) and noncoding (i.e., global
fingerprinting). For example, the Human Genome Project (HGP) has estimated that the
complete human nucleotide sequence is approximately 3.2 billion base pairs with
approximately 28,000 to 35,000 genes [9]. For comparison purposes, the complete
nucleotide sequence of the human pathogen Mycoplasma genitalium genome is 580,070
base pairs with 470 genes [10]. These prokaryotes are one of the smallest known
genomes of a self-replicating organism. Analytical methods such as automated capillary-
array electrophoresis (CAE) have played a major role in determining DNA sequences
[11]. Because of HGP, research interests have shifted from DNA sequencing to DNA
sequent variations in a population. A DNA sequence variant may provide diagnostic

markers for predisposition to specific diseases, thus permitting better diagnoses of
human diseases. Recently, MS techniques such as matrix-assisted laser desorption/
ionization (MALDI) and electrospray ionization (ESI) are being utilized in genome
variation studies [12].
The DNA sequencing of organisms has revealed many genes that are encoded in the
genome (i.e. functional profiling). Some of these genes have known functionality;
however, many of these newly discovered genes have no known or predictable
functions. A powerful method to study gene function involves qualitative and
quantitative measurements of the steady-state levels of cellular mRNA (i.e., gene
expression or transcriptomic). There may be as many as 100,000 mRNAs in a human.
The most common functional profiling experiment is the determination of patterns of
differential gene expression, that is, comparing differences in mRNA expression levels
between different cellular phenotypes or developmental stages or between identical cells
subjected to different stimuli. Today it is possible to perform massively parallel analysis
of gene expression on tens of thousands of genes from a given sample. From studies of
multiple samples, a statistical analysis of gene expression over a large number of
experimental conditions can be obtained.
Serial analyses of gene expression (SAGE) [13] and microarray hybridization
(termed cDNA microarray) [14] technologies have emerged as the dominant analytical
methods used to study gene expression. The analytical approach most often used is
cDNA microarray. Briefly, DNA segments representing the collection of genes to be
studied are amplified by polymerase chain reaction (PCR) techniques and mechanically
spotted at high density on glass microscope slides. Co-hybridization assays using two or
more fluorescently labeled probes prepared from mRNA from the cellular phenotypes of
interest are used to query the microarrays. The rate of hybridization allows relative
mRNA expression levels to be determined using a confocal laser scanner to
simultaneously measure all fluorescence intensities. cDNA microarray techniques have
been used to identify relevant disease genes, aberrant cellular signaling pathways and
expression signatures correlated with disease or drug-induced toxicity. For example, a
genomic expression signature assay for drug-induced oxidative stress/reactive
metabolites toxicity in rat liver has been recently published [15]. In this study, cDNA
technology was used to compare transcriptional profiles elicited for a series of drug
candidates that were known to form free radicals or reactive metabolites in rat liver. A
69-gene set was selected, which was able to separate drug candidates into control,
macrophage activator, peroxisome proliferators and oxidative stress/reactive metabolites
classes using multivariate analysis. The results clearly showed that gene-signature
profiling has the potential to predict many other types of hepatotoxicity.
PROTEOME
Proteins are the products of mRNA with the majority of reactions in a cell being
carried out by them. The proteome is the complete set of all post-transcriptionally
regulated (i.e. protein expression) and post-translationally modified proteins in an
organism. Since the splicing of an mRNA transcript can yield many different protein
isoforms from one DNA sequence and post-translational modifications controlling
protein interactions are present, there may be as many as one million proteins in a human
with a dynamic concentration range as large as 7 to 12 orders of magnitude [16]. In
contrast to genomics, it is not possible to simultaneously determine the complete

proteome (i.e., global fingerprinting) of an organism using a single analytical technique.
In addition, there is no strict linear relationship between mRNA expression levels and
the proteome of a cell. With this in mind, proteomics can be defined as a system-level
study of protein expression, post-translational modifications, 3-D conformational
changes and protein-protein interactions to obtain a global system view of cellular
processes. We can broadly classify proteomics as:
• Target Profiling proteomics: identifying proteins in biological samples or
proteins that are differentially expressed between different samples.
• Functional proteomics: identifying post-translational modifications and
protein-protein (or -ligand) interactions.
• Structural proteomics: determining the tertiary structure of proteins.
Thus, the study of proteomics is more complex than the determination of the primary
sequence as is done in genomic projects. It is clear that proteomics is a complementary
approach to genomics.
Profiling proteomics (or protein mapping) is used to compare the spectrum of
proteins in cells subjected to different stimuli (i.e. diseased versus normal tissues). A
number of analytical technologies are available for proteomic mapping including two-
dimensional (2-D) gel electrophoresis [17, 18] and chromatographic-based mass
spectrometry methods [19, 20] but among them the 2-D gel electrophoresis is widely
utilized. Briefly, 2-D gel electrophoresis involves first separating proteins based on their
isoelectric points (pI) using an isoelectric focusing (IEF) technique. Typically, an
electric field is applied to a pH gradient and proteins migrate to the position in the
gradient equivalent to their pI. The second step involves the separation of proteins based
on their molecular weight using sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). Individual proteins can be detected using techniques such
as, Coomassie staining, silver staining, autoradiography, MALDI time-of-flight (TOF)
or ESI tandem MS. Using these detection techniques, 2D-gel electrophoresis can
separate up to 10,000 proteins and identify proteins at the femtomole level. For example,
a recent proteomics analysis of human uveal melanoma tumor cells using a combination
of 2-D gel electrophoresis and LC-MS/MS analyzed 270 gel spots and identified 683
proteins, which were derived from 393 different genes [21]. It was determined that 96%
of the proteins identified had never been reported in uveal melanoma tumor cells.
Despite 2-D gels’ excellent resolving power, the majority of proteins expressed at lower
concentration levels could not be detected or identified even using mass spectrometry.
Thus, many proteins were missing from the proteomic expression map. It is very often
the case that proteins at these low expression levels have regulatory functions and are of
great biological importance. In addition, membrane bound proteins are under-
represented in 2-D gel systems which is a serious problem considering these proteins can
account for approximately 30% of total proteins in cells.
Multidimensional HPLC/MS/MS technologies are also used in proteomics [22].
After a mixture of proteins is extracted from a biological sample, the mixture is digested
in solution to produce a more complex mixture of peptides. The mixture of peptides is
separated online either by 2-D HPLC or capillary-HPLC and continuously analyzed by
ESI MS/MS. The MS/MS peptide spectra are searched against genomic and proteomic
databases to identify the various proteins from the biological sample. While bottom up
measurements (i.e. peptide to proteins) of proteomes are frequently applied, there are
still problems with sensitivity and quantitative information obtained from this method.
Functional and structural proteomics are as important as profiling proteomics. These
topics have been reviewed in several excellent papers and the reader is referred to these
authors [23, 24].
METABOLOME
The metabolome is the complete set of all intracellular and extracellular metabolites that
can be expressed in an organism. Here we are defining metabolites as the intermediates
of biochemical reactions within the organism. Hence, metabolites are small molecules
that can be analyzed using standard small molecule chemical analyses (i.e. NMR and
chromatographic-based MS techniques). The concentration of these metabolites
represents the integrative information of cellular functions such as, the phenotype of an
organism in response to genetic or environmental changes. Thus, metabolomics refers to
the detection and quantitation of endogenous metabolites that are involved in key
intermediary cellular pathways. Using this information, metabolomic global
fingerprinting or target profiling gives an indication of an organism’s physiological
status, for example, stress, disease, chemical or other insults [25].
In contrast to genomics and proteomics, a direct link between genomics and
metabolomics is extremely difficult to establish. It has been observed that for complex
organisms, such as plants, there are more metabolites than genes, while in
microorganisms, such as yeast, there are fewer metabolites than genes [26] (Table 2). In
addition, it is also well known that the same metabolite generated in one biochemical
pathway can participate in many other biochemical pathways [27]. While this situation
will complicate the interpretation of metabolomic data, the biochemical relationships
(i.e. functional profiling) of almost all of the endogenous metabolites in the metabolome
have been extensively investigated [27].
Table 2. Xomics of Selected Species from http://BioCyc.org/
Microorganisms Genes Proteins Metabolites
Anabaena sp 6070 6070 651
Caulobacter crescentus 3818 3780 674
Esherichia coli K12 4479 5224 1048
Drosophila melanogaster 16,904 16,924 382
Mycoplasma genitalium G37 480 496 324
Neurospora crassa 10,065 10,088 395
Vibrio cholerae N16961 3950 3911 652
For the purposes of this review, we will consider only endogenous metabolites under
1000 daltons to be part of the metabolome. With this definition in mind, it is estimated
that humans have approximately 2,500 metabolites. In contrast to proteomics, the

structures of many of these metabolites are known including complex natural products,
hydrophilic carbohydrates, hydrophobic lipids, amino and non-amino organic acids,
alcohols, ketones, and inorganic species [27]. Due to the diverse ionic and non-ionic
character of these metabolites, it is not possible to simultaneously determine the
complete metabolome of an organism using a single analytical technique. A combination
of analytical techniques will need to be used to generate metabolomic data including
NMR [28], GC/MS [29], LC/MS [30], CE/MS [31] and near-infrared spectroscopy
(NIRS) [32].
NMR-BASED METABOLOMICS
NMR-based metabolomic assays for global fingerprinting or target profiling have
several advantages including limited sample pretreatment, non-destructive analysis, non-
biased method to metabolite classes, ease of quantification, lack of any need to pre-select
the experimental condition employed for the analysis, experimental times on the order of
minutes per sample and moderate sample throughput. The technique is very useful for
structural characterization of unknown metabolites using 1- and 2-D NMR methods [33].
NMR spectroscopy functions due to the presence of a strong magnetic field and radio
frequency pulses. The energy levels of nuclei, such as protons, in the presence of a
strong magnet field split into a two-spin energy state. Absorption of radio frequency
energy causes the nuclei to be promoted from a low-energy to a high-energy spin state.
This change in energy states is subsequently detected. The major disadvantage of NMR
is that the limit of detection (or the sensitivity) of proton nuclei is in the micromolar
(µM) range. Major improvements in sensitivity have included the introduction of 900
and 800 MHz magnets, cryoprobe and nanoproble sampling technology.
Various NMR studies have demonstrated the usefulness of this technology to
examine the metabolome. For example, drug-induced toxicity causes subtle to dramatic
changes in the biochemical composition of cells trying to maintain homeostasis. These
changes in endogenous metabolites in urine and plasma composition are reflected by
changes in the pattern of the NMR spectra. Pattern recognition and chemometric
methods are used to discern these pattern modulations such that information on the
mechanism of toxicity can be determined. Each drug candidate will give rise to a unique
NMR-metabonomics profile; however, if a toxic event occurs, then the pattern will also
contain endogenous molecules reflective for this type of toxicity. Compounds that target
the same tissue will have similar profiles. For example, an increase in urinary acetate
and glucose together with bile aciduria generally indicates cholestasis toxicity.
Metabolic markers that are associated with other target organs and toxicity types have
been recently published [34].
NMR methods also have been used to study the metabolome to evaluate behavioral
characteristics of rats [35]. The dominant-submissive relationship (DSR) between pairs
of rats based on a food competition paradigm was studied. Briefly, DSRs were
established in rat pairs that were competing for access to a feeder filled with sweetened
milk. The amount of time spent in the feeder was measured for each rat. Dominant rats
spent significantly longer amounts of time at the feeder than their submissive partners.
This competition paradigm was maintained for 2 weeks. At the end of 2 weeks,
dominant and submissive rats were selected and urine was collected. Principal
component analysis (PCA) revealed a metabolite from milk sugar, galactose, as a
discriminating factor between rats classified as dominant and those classified as

submissive.
GC/MS -BASED METABOLOMICS

GC/MS is a combined technology whereby volatile and thermally stable metabolites
are first chromatographically separated by GC and then eluting metabolites are detected
by MS [36]. Briefly, a small aliquot (1 µL) of a pre-prepared sample is injected by split
or splitless techniques onto a capillary GC column. These columns provide both high
chromatographic resolution and high sensitivity. Ions are generated by passing a beam of
electrons through a gas sample or a volatilized liquid sample in the gas phase (i.e.,
electron ionization (EI)). In many cases, eluting metabolites are mass separated on
single-quadrupole (Q) mass spectrometers. Since the EI technique is a high-energy
process, excess fragmentation of the molecular ion (M+) occurs, which gives rise to
many ions in the MS spectrum. Detection limits are typically in the nanomolar (nM) or
picomolar (pM) concentration range. Greater sensitivity can be obtained if the GC/MS is
configured to detect only those masses characteristic of the metabolites being monitored
(i.e., single ion monitoring). Higher scan speeds have been introduced using GC-TOF
mass spectrometers to improve cycle times [37] and multistage MS/MS and ion trap MS
systems have been added to increase fragmentation information [38].
While volatile metabolites can be analyzed directly, analysis of semi-volatile
metabolites must be derivatized. Volatile metabolites of biological samples are typically
compounds with boiling points less than 300 °C including aldehydes, alcohols, esters,
furans, small hydrocarbons, isocyanates, isothiocyanates, ketones, small lipids, pyrroles
and sulfide derivatives. These metabolites are either extracted from the sample with an
organic solvent or directly collected from the sample headspace. Semi-volatile
metabolites include long chain alcohols, amines, amides, alkaloids, amino acids, sugars,
sugar-phosphates and organic acids. GC/MS is used to analyze a wide range of volatile
non-polar metabolites and through chemical derivatization, the range can be extended to
semi-polar metabolites. A comparison of GC/MS with other chromatographic-MS
techniques is shown in Fig. (2).
Fig. (2). Illustration of the relationship between molecular weight and polarity of metabolites for
various chromatographic-MS techniques.
In any metabolome analysis, the methods used to prepare samples for GC/MS
analysis need to be fully understood in order to measure the concentration of
metabolites. For example, the metabolite concentrations in an organism are in flux,
therefore, rapid quenching of all cellular activity is extremely important to obtain
reproducible results. This is typically accomplished by rapidly changing the temperature
or the pH of the sample. Freezing samples in liquid nitrogen or acidic treatments with
perchloric or nitric acid are used for plant and animal tissues. Since raw samples are too
complex to be directly injected onto the capillary GC column, samples are usually
extracted with organic solvents. Solvents such as methanol and ethanol or combinations
of these alcohols with water are used to extract polar metabolites, while chloroform,
ethyl acetate, and hexane are used to extract the lipophilic components. Extractions at
elevated temperatures can also be used to extract metabolites. In addition, microwave
and ultrasound assisted extraction methods have been shown to be effective in extracting
metabolites from biological samples [39]. The extraction volumes are typically reduced
by evaporation to partially or totally remove organic solvents from the sample and to
concentrate the metabolites further. Freeze-drying (or lyophilization) can be used to
remove water from aqueous samples. Extraction procedures using supercritical fluid
chromatography (SFC) have been used for plants [40] and animal tissues [41]. Since this
technique uses liquid carbon dioxide (CO2) as the extraction medium, the removal of
CO2 to concentrate the metabolite sample is very easy.
The most widely used derivatization procedure involves the use of silylation reagents
in which a silyl group (i.e. -Si(CH3)3) replaces the hydrogen of a hydroxyl, thiol, amine
or carboxylic groups. Recently, a study was completed that comprehensively examined
the effects of quenching, solvent extraction and derivatization procedures on the
metabolome of the leaves of Arabidopsis thaliana [42]. Using a design-of-experiment
procedure, conditions were systematically varied and the effect on 66 endogenous
metabolites was analyzed. While a set of experimental conditions was found to allow for
a reliable metabolomic analysis of Arabidopsis, the results clearly demonstrated that it is
impossible to obtain both high accuracy and precision for all metabolites. Thus, when
using GC/MS for global fingerprinting experiments, the goal is to obtain the highest
accuracy and precision for as many classes of metabolites as possible. It should always
be remembered that GC/MS-based metabolomic assays could be biased toward many
classes of metabolites due to sample preparation procedures.
Another source of variability in these types of experiments is the stability of the
metabolites and the derivatized metabolites. Samples must be stable since they reside in
the autosampler for long periods of time before being analyzed. Evaporation of solvents
or metabolites from sample tubes is another source of variability. One way to verify
stability is to measure the inter-day and intra-day variability of the GC/MS protocol.
For accurate quantification of metabolites in complex biological samples, it is
absolutely necessary to have stable isotope-labeled internal standards for all metabolites
of interests. Using a GC/MS target profiling approach, it may be possible to fulfill this
requirement since there are a limited number of metabolites under investigation.
However, for global fingerprinting metabolome analysis, this situation is not possible.
The approach used to circumvent this problem is to discriminate between samples of
different biological status. In other words, it is not necessary to measure absolute
concentrations if one is only interested in a comparison between two samples. However,
the reproducibility (i.e. the relative responses) of the GC/MS peaks from experiment to
experiment is crucial for metabolome analysis. Data processing software is used to

distinguish and integrate the areas under peaks. If the GC/MS spectrum contains
hundreds of peaks, many will be overlapping with each other and thus, the software must
deconvolute these peaks. Problems in the deconvolution of peaks from sample to sample
could be wrongly interpreted as a true difference between samples (i.e., the metabolomes
between the samples are different). Recently, a novel semi-automated strategy for peak
deconvolution using a hierarchical multivariate curve resolution procedure was
introduced [43]. While this procedure does not fully eliminate this bottleneck, the results
appear very promising.
GC/MS has extensively been used in metabolome analysis as a target profiling
technique due to its high separation efficiency of complex biological samples, reliable
identification of metabolites based on retention times and mass-to-charge ratios (m/z),
ease of operation, automation capabilities and low cost for analysis of metabolites. One
of the first applications of GC/MS was a urinary target profiling assay to predict diseases
related to organic acidemias [44]. It has also been used to analyze the human breath [45],
volatile metabolites from plants [46], potatoes [47], tomatoes [48], Arabidopsis thaliana
[42, 49] and the discrimination of Type 2 diabetic patients from healthy controls [50].
Major improvements in GC/MS-based metabolomic analysis may arise from the
application of multi-dimensional separation and rapid scanning strategies, such as the
use of GC/GC-TOF MS systems [51]. In GC/GC, separated peak clusters are transferred
from the first column to a second column for further separation, thus providing
orthogonal resolution on a comprehensive basis. TOF MS is preferred to fulfill the fast
detection rate requirements. This combination of technologies should have greater
advantages in peak specificity and sample sensitivity. Recently, the potential of a
GC/GC-TOF MS system was demonstrated studying the metabolome of tissues from
New Zealand obese (NZO) mice and lean BL/6 mice [52]. The results from this study
clearly demonstrated the advantages of GC/GC-TOF MS.
LC/MS -BASED METABOLOMICS

LC/MS is a combined technology whereby semi-polar and polar metabolites are first
chromatographically separated by LC, eluting metabolites are ionized by either ESI or
atmospheric pressure chemical ionization (APCI) and finally detected by MS [53]. A
comparison of this technique with other chromatographic systems is shown in Fig. (2).
LC separation is based on the selective distribution of metabolites between a liquid
mobile phase and a stationary phase. Many LC applications are performed using a non-
polar stationary phase and a polar mobile phase (i.e. reverse phase (RP)
chromatography). RP HPLC offers flexibility in stationary phases, mobile phases and
solvent gradient separation technology. The technique is ideally matched for the analysis
of polar metabolites, which are not amenable to GC analysis. For example, in a typical
experiment, a 10 µL aliquot of human urine was injected onto an Agilent 1100 LC
system equipped with a Zorbax SB C18 column (5µm, 2.1x50 mm). The column was
maintained at 25 °C and the elution was performed with a linear gradient with 0 - 80% in
0.1% aqueous formic acid/acetonitrile in 40 min at a flow rate of 0.3 ml/min. Data were
collected over the mass range 50 – 1000 daltons in ESI positive mode on a Q-TOF mass
spectrometer. In Fig. (3) are shown the total ion current (TIC), and the mass ranges 250
– 300 and 300 – 350 daltons.
Fig. (3). HPLC/MS analysis of human urine on a Zorbax SB C18 column (5µm, 2.1x50 mm) using
a gradient elution with 0-80% in a 0.1% aqueous formic acid/acetonitrile in 40 min at a flow rate
of 0.3 ml/min.
In general, an MS system is comprised of three parts: an ion source to generate ions,

a mass analyzer to differentiate ions based on their m/z ratio, and a detector. The three
types of ion sources used for metabolomic analysis are ESI, APCI and MALDI. ESI and
MALDI techniques are more widely used than APCI in metabolomic analysis. Briefly,
the ESI method involves both a desolvation and ionization process. When a liquid
sample is passed through a capillary, whose tip is connected to a high voltage potential,
charged aerosol droplets are formed. These droplets are desolvated using both heated
inlets and a coaxial flow of gas to produce singly charged and multi-charged molecular
ions. The multi-charge ionization, mechanism of ESI can provide a mass range of greater
than 100,000 Daltons. In MALDI, samples are prepared as dried spots mixed with a co-
crystallizing matrix (dihydroxy benzoic acid or α-cyano-4-hydroxycinnamic acids). By
exposing these spots to a laser at the absorption wavelength of the matrix, desorption of
metabolites occurs producing primarily singly charged molecular ions. These ionization
techniques are considered “soft’, since they produce little or no fragmentation. Mass
analyzers are usually one or a combination of three types; namely, Q, TOF, and Fourier
transform ion cyclotron resonance (FT-ICR). The detectors used to amplify the signals
coming from the mass analyzer are typically electron multipliers.
All the ion sources and mass analyzers can be operated in both a positive and
negative ion mode. In the positive mode, a proton binds to the metabolite to produce an
M+1 cation, while in the negative mode, the metabolite gives up a proton to produce an
M-1 anion. These positive/negative ion mode experiments can either be done separately
or combined using a polarity-switching scheme. Many metabolites are detected in one
but not both ion modes and therefore, using both modes will expand the metabolomic
range.
Since high injection port temperatures, metabolite volatility, and sample
derivatization are not required for many LC/MS applications, sample preparation is
simplified. However, rapid quenching of all cellular activity is still extremely important
to obtain reproducible results. In many cases, samples can be directly analyzed after
minimum sample preparation such as, protein precipitation, liquid phase extraction or
solid phase extraction. When global fingerprinting of the metabolome is required,
sample pre-treatments steps must be carefully optimized in order to not eliminate
important low concentration metabolites.
The performance of HPLC/ESI-MS can be compromised by sample preparation
processes, which induce ESI ion suppression effects [54]. Ion suppression effects render
a decreased signal response to metabolites in the metabolome due to the presence of
increasing levels of chemical background that completes for ionization with the
metabolite. The complete evaluation of ion suppression effects encompasses isotope
dilution techniques whereby stable isotope analogs of metabolites are used as internal
standards in the MS analysis. However, the availability of isotopically labeled
metabolites is scarce. For microbial metabolome analysis, isotopic metabolites are
obtained by biosynthesis using isotopic labeled feedstocks. For example, the quantitative
analysis of the metabolome of Saccharomyces cerevisiae was completed using
metabolite from uniformly 13C labeled cell extract as internal standards [55]. For plant
and animal metabolome analysis, isotopic dilution experiments are not straightforward
since labeled metabolites cannot be easily synthesized.
Recently, a new ultra high pressure liquid chromatography (UPLC) technology was
introduced that uses small particle sizes (i.e. 1.7 µm) combined with very high mobile
phase operating pressures [56]. As shown in Fig. (4), the UPLC technique has higher
peak capacity, greater resolution and increased sensitivity when compared to the 5 µm
column in Fig. (3). The UPLC/Q-TOF MS data in Fig. (4) were collected using the same
UPLC experimental conditions as the HPLC/MS data shown in Fig. (3). Due to the
extremely high pressure used in UPLC/MS, the chromatographic analysis time also can
be shortened, in some cases, to one tenth of the time used in conventional LC/MS
applications. The shortening of run times can be used to develop higher throughput
screening assays for metabolomics.
LC/MS/MS with ESI and MALDI are frequently used for metabolome analysis. A
novel column-switching RP HPLC/MS/MS was developed for multiplex quantitation of
eicosanoids and platelet-activating factor [57]. The assay was optimized for 14 lipid
mediators with a sample throughput of 96 samples/24 hours. The lower limit of detection
was 5 pg with a linear range from 2000 – 5000 pg. A HPLC/TOF MS assay was
developed to investigate rat urinary metabolic perturbations associated with D-serine
induced nephrotoxicity [58]. Changes in the rat metabolome were observed including
increases in tryptophan, phenylalanine, and lactate. Concentration decreases were
observed for methylsuccinic and sebacic acid. Recently, the metabolomic analysis of
islets of Langerhans and Escherichia coli strain DH5-α were analyzed using a MALDI-
TOF MS in negative ionization mode [59]. The islet extracts contained 44 metabolites of
which 29 were known compounds, while the Escherichia coli contained 60 metabolites
of which 39 were known compounds. Approximately 27% overlap in metabolites was
discovered between islets of Langerhans and Escherichia coli strain DH5-α. The results
clearly showed that MALDI can be used for metabolomic analysis.
Fig. (4). UPLC/QTOF-MS analysis of human urine on an Acquity BEH C18 column (1.7µm,
2.1x50 mm) using a gradient elution with 0-80% acetonitrile in 40 min at 0.3 ml/min.
CE/MS -BASED METABOLOMIC

CE/MS is a combined technology whereby polar metabolites are first
chromatographically separated by CE and then eluting metabolites are detected by MS
[60]. CE uses electroosmotic flow to separate charged molecules in a capillary column
filled with buffer or buffer containing gel. A strong electrical voltage potential is applied
across the ends of the capillary column whereby charged metabolites move toward the
electrode with the opposite charge. The rate at which these charged metabolites move
depends on their size and charge strength. These CE systems are designed to operate
with very small amounts of samples and to deliver a very concentrated sample to the MS
system.
CE/MS has been applied to many target profiling metabolomic analyses. For
example, CE/MS has been successfully applied for the diagnosis of various human
metabolic disorders [61]. The CE/MS analysis time for this assay was approximately
fifteen minutes per sample and potentially could replace standard clinical GC/MS
assays, which take in some cases, several hours. A CE-ESI/MS method for the
determination of 19 amino acids was developed [62]. This assay could be used as a
replacement assay for GC/MS since the amino acids did not have to be derivatized.
CE/MS has been used for the separation and detection of phosphorylated and acid
metabolites in extracts of prokaryotes [63]. In this study, 119 metabolites were detected
from extracts of Escherichia coli strain DH5-α. Nineteen of these metabolites were
identified and their limits of detection in full scan mode ranged from 20 nM to 2.5 µM.
CONCLUSION
It is essential to use analytical strategies that provide the widest coverage of
metabolites for a metabolomic analysis. Combinations of NMR, GC/MS, LC/MS and
CE/MS analytical techniques will have to be used for global fingerprinting analysis.
However, many potential problems will have to be solved. For example, combining
these metabolomic data sets into a single data set is not straightforward. Pattern
recognition algorithms allow identification of significant metabolic differences. Many of
the pattern recognition strategies currently used in metabolome analysis are based on
supervised PCA techniques instead of unsupervised techniques. Sample preparation
plays a major role in metabolomic analysis and understanding sample clean up and pre-
fractionation steps are crucial, since these procedures determine the minor changes in the
metabolomic analysis. It should also be remembered that sample origins and handling
procedures outlined in the literature are not always comprehensive and therefore, it is not
clear if only a portion of the metabolome is being analyzed and modeled.
Conventional techniques and data analysis involving NMR, GC/MS, LC/MS and
CE/MS can be used for target profiling of the metabolome. Metabolite classes including
lipids, steroids, eicosanoids and bile acids have been analyzed. An impressive amount of
metabolomic data has been generated in the literature; however, these publications have
served more to highlight the complexity of cellular processes than to elucidate
mechanisms. In the future, metabolomic analysis of plasma or urine may replace
classical clinical chemistry or histopathology approaches.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the management at Johnson & Johnson
Pharmaceutical Research and Development.
ABBREVIATIONS
APCI = Atmospheric Pressure Chemical Ionization
CAE = Automated Capillary-Array Electrophoresis
2D = Two-dimensional
3D = Three-dimensional
cDNA = Complementary DNA
DNA = Deoxyribonucleic Acid
DSR = Dominant-Submissive Relationship
EI = Electron Ionization
ESI = Electrospray Ionization
FT-ICR = Fourier transform ion cyclotron resonance
GC = Gas Chromatography
HPLC = High Pressure Liquid Chromatography
HGP = Human Genome Project
pI = Isoelectric Point
IEF = Isoelectric Focusing
MALDI = Matrix-Assisted Laser Desorption/Ionization
MS = Mass Spectrometer
+
M = molecular ion
mRNA = Messenger RNA
m/z = Mass-to-charge ratio
µL = microliter
µM = micromolar
NIRS = Near-Infrared Spectroscopy
NZO-mice = New Zealand obese mice
NMR = Nuclear Magnetic Resonance
nM = nanomolar
PCA = Principal Component Analysis
PCR = Polymerase Chain Reaction
pM = picomolar
Q = quadrupole mass filters
RP = Reverse phase chromatography
RNA = Ribonucleic Acid
SAGE = Serial Analysis of Gene Expression
SDS-PAGE = Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
SFC = Supercritical fluid chromatography
TOF = Time-of-Flight
TIC = Total Ion Current
UPLC = Ultra high pressure liquid chromatography
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OWLS—A Versatile Technique for Drug Discovery
Jeremy J. Ramsden*
School of Industrial and Manufacturing Science, Cranfield University, MK43 0AL, UK
Abstract: Optical waveguide lightmode spectroscopy (OWLS) is forecast to

become a key measurement technology in drug design and discovery. This
review focuses on what the technology is, why it is superior to existing
technologies and how it may be expected to develop for drug discovery
applications.
1. INTRODUCTION
The quantification of molecular interactions is an essential part of the drug discovery
process. By the time it starts, the target has usually already been selected—e.g. an
enzyme, a transcription factor, or a promoter site. If the structure is known, or, if not, if
at least that of the active site can be predicted with confidence, molecular modelling is
used to assist the search for a small molecules that bind to the active site. Typically a
large number of candidate molecules is generated at that stage, of which some fraction
will be synthesised and their interaction with the target measured in the laboratory. It is a
measure of the still primitive state of this procedure that dozens of molecules may
emerge from that process, not one of which may ultimately prove to be suitable. In vitro
tests and preclinical trials follow, a major goal of which is to assess all the other
elements of performance necessary for a successful drug. These elements are often
grouped under the ADME umbrella—adsorption, distribution, metabolism and
excretion. There is at present no satisfactory way of quantifying performance other than
globally. Clearly, if molecular methods can also be used to characterise all these
interaction-based processes, very significant savings in both time and cost will be
realised. Furthermore, there are many examples of drugs successfully passing all stages
of the testing procedures, including clinical trials, only to founder because of
unacceptably harmful side effects. These side effects are also manifestations of
molecular interactions, in this case ones that are stronger than desired. There is a very
urgent need to include screening for undesirable interactions long before preclinical
trials begin, let alone the clinical ones.
Present approaches are typically so far removed from the natural conditions under
which the interactions will take place that they can only offer very limited guidance. For
example, the adsorption stage, which, it is recognised, usually involves passage across a
lipid bilayer membrane as a first step, is typically quantified by measuring the partition
coefficient of the drug between oil and water. Unsurprisingly, the correlation between
this partition coefficient and membrane permeability is poor.
*Corresponding author: Tel: +44 1234 754100; Fax: +44 1234 751346; E-mail: J.Ramsden@Cranfield.ac.uk

212 Frontiers in Drug Design & Discovery, 2006, Vol. 2 Jeremy J. Ramsden
Optical waveguide lightmode spectroscopy (OWLS) belongs to the family of

evanescent wave methods, the more recent members of which are based on integrated
optics (nanophotonics). Changes of the spatial distribution of electronic polarisation
within the evanescent optical field generated by a guided light wave can be used to make
a plethora of inferences concerning the behaviour of molecules at the solid/liquid
interface—at which most biologically relevant interactions take place. Hence OWLS is
also a “molecular microscope” or, more precisely, a “molecular nanoscope”, since
changes may be determined with nanometre or subnanometre resolution.
In recent years, optical biosensors have become objects of intense interest as the next
major field of application of integrated optics (based on planar waveguides) and fibre
optics. The first widely-used evanescent field technique, ellipsometry, is however
unsuitable as a practical drug design and discovery tool, because of its relatively low
sensitivity and cumbersome instrumentation. It was followed by surface plasmon
resonance (SPR), which, while more sensitive, requires a noble metal surface that is
incompatible with most biological systems, and compatibilisation has only been
achieved (e.g. by coating with a polysaccharide such as dextran) at the cost of making all
kinetics transport-limited. OWLS is the most recent arrival on the scene: it is
intrinsically an order of magnitude more sensitive than SPR (and that sensitivity can be
further increased by many orders of magnitude using interferometric detection schemes);
it is highly versatile, since waveguides can be made from virtually any transparent
material, including polymers; it can be miniaturised and incorporated into lab-on-a-chip
devices; and the raw data generated by an OWLS device can be processed in highly
informative ways, yielding a wealth of structural detail on the biomolecular interactions
under investigation.
Some of the types of measurements relevant to drug discovery that will be described
include: binding and dissociation between biomolecules; conformational changes of
biomacromolecules due to small molecule binding; absorption by and release from novel
drug delivery materials; interactions of drugs with lipid membranes; and the response of
living cells to drugs.
Evanescent fields are also used to excite fluorescently labelled molecules bound to
receptors. Confining the excitation in this way to the vicinity of the receptor layer helps
to reduce background emission from unbound ligands in solution. The fluorescence from
the bound ligands is observed through a microscope. This technique has, of course, the
great disadvantage of requiring the ligands to be modified, except for intrinsically
fluorescent ligand molecules, among which not so many are emitting at wavelengths
useful for microscopy.
The structure of this article is as follows: firstly the technology will be summarised
and briefly compared with other technologies; and then the applications to drug
discovery will be explained.
2. THE TECHNOLOGY OF OWLS

The fundamental physical principle behind all the evanescent field-based techniques
is that changes in the electronic polarisability within the evanescent field generated at the
interface between two transparent dielectrics by light totally internally reflected at that
interface change the phase shift undergone by the light upon reflexion [1]. The change in
A Versatile Technique For Drug Discovery Frontiers in Drug Design & Discovery, 2006, Vol. 2 213
electronic polarisability arises through a change in the atomic composition of the matter
within the evanescent field. Replacement of the water molecules in the immediate
vicinity of the reflecting interface by a drug or a protein, for example, will usually
increase the polarisability, since the atomic constituents such as carbon or nitrogen are
more polarisable than hydrogen. Depending on how the phase shift is measured, the
number of drug or protein molecules within the evanescent field can be calculated with
high precision [2].
The signal-to-noise ratio of the measurement strongly depends on the number of
reflexions. Techniques such as scanning angle reflectometry or ellipsometry rely on a
single reflexion [3]. In an optical waveguide, on the other hand, there are thousands of
reflexions per centimetre. That is why waveguide techniques have unprecedented
sensitivity. An optical waveguide consists of a thin slab of higher refractive index
material surrounded by materials of lower refractive index.1 Once introduced into such a
structure, light propagates along it—following a zigzag path, and generating the
evanescent field at each reflexion. There is a certain minimum thickness (the cut-off
thickness) of high refractive index material below which this phenomenon cannot occur.
The phase shift occurring upon each reflexion determines the propagation constant of the
guided light, for which a convenient parameter is the so-called effective refractive index,
usually denoted by N. Just like a normal refractive index, it is defined as the ratio of the
phase velocity of the waveguided light to the velocity of light in vacuo. The difference
between the customary and effective refractive indices is that the latter are valid for the
waveguide as a whole, even though it consists of slabs of materials of very different
refractive indices; in other words the light travels at a uniform velocity through the
multilayer structure. The extent of penetration of the evanescent field beyond the
confines of the high refractive index layer (and perpendicular to the plane of the layer)
depends strongly on its thickness. At the cut-off thickness, the evanescent field is
formally infinite in extent. As the waveguide thickness increases the evanescent field
shrinks rapidly, and the response of the propagation constants to changes in
polarisability within the evanescent field rapidly increases, reaching a maximum, and
slowly declining thereafter [4].
One interesting consequence of this structure is that the light can only propagate at
discrete values of the propagation constant (or N). To see this, consider the round-trip
made by the beam starting from some point within the F layer, reflected at the F,C
interface, travelling back to the F,S interface, being reflected, and returning to its starting
point. If at that point the phase of the light differs from its value when it started by any
value other than an integral multiple of 2π (i.e. 2πm, m = 0,1,2,...), it will destructively
interfere with itself and be quenched. According to the value of m the discrete modes are
called zeroth, first, second order and so on. The sensitivity to polarisability changes
decreases with increasing m, hence the most sensitive waveguides are the thinnest ones
in which only the zeroth order modes can propagate (the cut-off thickness increases with
increasing m).
___________________________
1
Notation. The high refractive index slab is denoted by F. Its support—required for mechanical strength as well as for isolation
from the environment—is denoted by S. Its cover—the medium in which samples are introduced—is denoted by C and is
typically a liquid. Adlayers interposed at the F,C interface are denoted by A, B, etc. They might consist of the monolayer of
receptors, or a lipid membrane, etc.
Each mode exists in two orthogonal polarisations called transverse electric (TE) and
transverse magnetic (TM). Each interacts differently with the cover medium, and their
simultaneous measurement enormously increases the amount of information obtainable.
The Mode Equations. The condition of non-interference described above can be used to
construct the mode equations, one for each value of m and for each polarisation (usually
indicated by the parameter ρ = 0 and 1 for the TE and TM polarisations respectively).
One simply sums the phase changes Φ due to the reflexions at each interface together
with the change βF due to crossing the F layer and equates it to 2πm:
ΦF,S + ΦF,A,C + 2βF = 2πm , (1)
where
βF = k(n2F – N2)1/2dF . (2)
The expressions for the Φ can be derived from the (complex) Fresnel reflexion
coefficients R, noting that they can be written as
R= |R| exp(iΦ) . (3)
For example,
RF,A + RA,C exp(2ikz,AdA)
RF,A,C = , (4)
1 + RF,A RA,C exp(2ikz,AdA)
with
kz,A = k(n2A – N 2)1/2 (5)
and others are given in the literature [2 - 4].
Waveguide Fabrication: The F layer is typically very thin, 100–200 nm, and can be
made by physical vapour deposition or sol-gel technology [5].
3. THE TECHNOLOGY OF MEASURING THE LIGHTMODE SPECTRUM

The two main techniques available are grating coupling and interferometry. In the
former case, a diffraction grating, typically 1–2 mm long,2 is incorporated into the
waveguide. Light from an external beam impinges on the grating at a certain angle α, at
which it is coupled in to the waveguide, according to whether it fulfils the coupling
condition [2, 4]:
N = nair sin α + 2πl/Λ (6)
where l = 0,±1,±2, . . . is the diffraction order and Λ is the grating constant. Since Λ is
typically small (a few hundred nanometres) its fabrication is non-trivial. Either
semiconductor processing technology can be used, i.e. the usual sequence of
exposing—it must be done holographically—photoresist through a mask and developing
___________________________
2
Here and elsewhere in this article we tacitly assume that we are using visible light, e.g. the red light from a He-Ne laser,
propagating in the waveguide with refractive index nF ~ 1.8–2, supported by glass (nS ~ 1.5), and covered by an aqueous
solution (nC ~ 1.3).
and etching, or by embossing a master grating into a sol-gel film before final hardening.
In the future, self-assembly techniques might become available. The grating can be
created at either the S,F or F,C interface. The mode spectrum, i.e. the different values of
N, can be measured by scanning α. This is called incoupling. Exactly the same equation
(6) applies to outcoupling, in which the guided modes already introduced into the
waveguide are coupled out at the grating. In this case it is convenient to measure α using
a position-sensitive detector on which the outcoupled light falls. This configuration is
potentially more convenient for compact low-cost devices than incoupling, for which
expensive mechanical goniometry needs to be used. Incoupling however is still the
preferred configuration for high-precision work (a resolution of 10-6 can be reached
without undue difficulty) in the laboratory.
If interferometry is used, either two modes propagating in the same waveguide must
be made to interfere with each other (with the advantage that no structuring of the
waveguide is required), or the propagating beam must be divided into two waveguides,
one of which interacts with the sample before they are recombined [6]. Examples of the
latter are the Mach-Zehnder interferometer and the dual-slab waveguide interferometer.
The output power Pout is a measure of the phase difference ∆Φ.
Pout = Pin(1 + cos Φ)/2 (7)
where Pin is optical power input [7]. The phase change in the interaction zone (i.e. along
the sensing channel) is related to the effective refractive index N by
∆Φ = (2π/λ)L∆N (8)
where ∆N is the change of effective refractive index due to adsorption of matter in the
sensing zone (of length L). In multimode operation, the modes must be separated in time
and measured separately.
Since the difference between the sample and reference beams is proportional to their
length, extremely high sensitivities can in principle be reached by this approach
(although the requirements for a uniform temperature of the device are very demanding).
On the other hand, compared with grating coupling, it is difficult to obtain absolute
values of the N.
Other Technologies: These can be divided into reflexion-based ones, including
ellipsometry, scanning angle reflectometry (SAR) and the evanescent field-based surface
plasmon resonance (SPR) [3, 6], and the rest. Both ellipsometry and SAR benefit from
an extensive literature and much theoretical work has been carried out on these
techniques. On the other hand, they are less sensitive than OWLS, and SAR is very
slow. SPR is also less sensitive, and requires a noble metal substrate (in which the
surface plasmons are excited optically). Attempts have been made to simultaneously
overcome these two disadvantages3 by coating the metal with a dextran hydrogel,
typically a few hundred nanometres thick. If carboxydextran is used, convenient
chemistry for immobilising proteinaceous receptors is available. Unfortunately, the use
of the hydrogel introduces a high density of hydroxyl groups into the entire volume in
___________________________
3
The requirement for a metal is not a disadvantage if it is desired to carry out electrochemical work at the same time. This is an
important niche application for SPR.
which receptor-ligand binding takes place, which has a profound effect on the aqueous
chemistry [8, 9], and hence may drastically influence association and dissociation,
thereby severely distorting the binding affinity. To compound the difficulty, transport,
especially of macromolecules such as proteins, is strongly retarded within the hydrogel,
which tends to make the measured binding kinetics transport-limited [10], and which
strongly favours rebinding in the dissociation regime.
All these methods suffer from the disadvantage that a laborious fitting procedure
must be applied to the data in order to extract parameters of interest, with all the
attendant ambiguities. In fact, in many applications this is not even attempted, but a
single arbitrarily chosen experimentally measured signal is used to give an indication of
the state of binding via a calibration procedure.
Most of the other (non-optical) techniques available either require labelling (with a
radioactive atom) or the path leading from the measurement to deduction of the number
of bound molecules is tortuous and full of assumptions. The latter well-applies to the
family of electrochemical methods [3]. Hence, while they may be useful for calibrated
biosensing, they are less useful as research tools. Other methods [3] tend to be slow and
cumbersome, and hence unsuitable for the all-important high resolution kinetic
determinations.
4. APPLICATIONS TO DRUG DISCOVERY

The quantification of molecular interactions is fundamental to drug discovery. The
most basic parameter is the binding of the drug to its target. Using OWLS, the target (or
the drug) can be immobilised to the waveguide, and the binding of the drug (or the
target) introduced into the cover medium can be measured in real time. The partner on
the waveguide will be referred to as the “receptor”, and the partner introduced into the
liquid cover medium will be referred to as the “ligand”. Another very important
interaction is that between the drug and the lipid bilayer membrane. In this case, the
waveguide is coated with a lipid bilayer membrane, either using the Langmuir-Blodgett
(LB) technique [11] or by disrupting vesicles [12], and the drug is introduced into the
liquid cover medium. Many variants of these basic experiments are possible. The
interaction between living cells and drugs can be studied by placing one or more cells on
the waveguide surface and measuring their response to drugs introduced into the
medium bathing the cells.
Formally, the receptors constitute an adlayer A on the waveguide surface. The
adlayer has a thickness dA and a refractive index nA. Two mode equations (1) must be
solved simultaneously to yield these two parameters. The number of molecules v per unit
area of adlayer can then be determined using
v = dA(nA – nC)/dn/dc (9)
where the coefficient dn/dc, called the refractive index increment, and specific to the
particular system of molecule and solvent under study [13], must be determined or
estimated separately.
Only the simplest adlayers are characterised by only one refractive index. Layered or
columnar structures may be uniaxial and therefore characterised by two refractive
indices, ordinary and extraordinary [11, 14].
Binding and Dissociation: In this case, it is essential to measure the kinetics of the
ligand-receptor interaction. The typical measurement procedure consists of
1. waveguide preconditioning
2. deposition of receptors
3. establishment of baseline with pure (ligand-free) solvent
4. ligand association using ligand solution at concentration c1
5. ligand dissociation using pure (ligand-free) solvent
6. (if baseline restored) ligand association using ligand solution at concentration c2 etc.
It is definitely advantageous if the baseline and ligand association and dissociation steps
are carried out using continuous laminar flow of the liquid. This is particularly important
for the ligand association step, for under such conditions the number of association
attempts per receptor per unit time is constant.
The deposition of receptors may be carried out online, in which case the exact
number per unit area can be determined from the mode spectrum. The only ambiguity
concerns their orientation. Unless the receptor molecule has some particular asymmetry,
random deposition of the receptor is likely to lead to random orientation on the
waveguide surface, in which case the active site (i.e. the ligand binding site) may be
inaccessible in a certain fraction of the receptor molecules. In order to overcome this
problem, the receptor molecule should be immobilised in a definite orientation, by
modifying either it or the waveguide surface so that it is attached by a single point. A
good way of achieving this is to coat the waveguide with a bilayer lipid membrane and
attach a membrane anchor to the receptor molecule (if it is not naturally so endowed)
[15]. The problem then is to ensure that the ligand affinity of the receptor is not affected
by the modification.
If lipid membrane-based immobilisation is not used, the experimenter has a choice
between physisorption and covalently bonding the receptors to the waveguide surface.
There is a vast literature available concerning protein immobilisation. A very useful and
widely applicable method is to link the carboxy or amine groups on the protein with
respectively the amine or carboxy groups on the substrate [16]. The only real drawback
of this technique is that most proteins contain several carboxy or amine groups, and there
is thus ambiguity in which ones are linked covalently to the substrate. In order to create a
carpet of carboxy or amine groups on the substrate, if the substrate contains silica then
carboxylated or aminated silane derivatives may be bonded to it using very well known
procedures, or a poly-carboxy or poly-amino molecule may be allowed to adsorb
spontaneously [14], or a carboxylated or aminated Langmuir-Blodgett film may be
deposited on the substrate [16].
An indication of the receptor orientation can be obtained by examining the adlayer
thickness dA, and comparing it with the dimensions of the receptor molecule, if they are
known e.g. from X-ray crystallography.
The determination of the association and dissociation parameters is carried out with
the help of the canonical association and dissociation equation. We consider a monolayer
of receptors (antigens) of area aR and surface density (i.e. number per unit area) vR. The
two dimensional projected area of each ligand (i.e. the antibody) is aL, and its dissolved
bulk concentration is cb.
When the receptor layer is exposed to flowing ligand solution, the ligand is
transported to the layer via convective diffusion and the binding rate is:
dvL/dt = kacvφ (10)
where ka is the ligand-receptor association rate constant, cv is the vicinal ligand
concentration (i.e. just above the receptor layer) and φ is a function capturing the
probability that an arriving ligand will find a vacant receptor. Unsuccessful molecules
(i.e. those not finding a vacant receptor) will remain for some time in the vicinity of the
surface, which of course reduces the flux from the bulk, and this is captured in the ka
term. However, this has been extensively discussed elsewhere (e.g. [17]), and without
loss of generality we shall forthwith assume ka = 1.
If aL < aR then φ is simply 1 – θ, where θ is the fraction of the surface occupied by
ligands, i.e.
θ = aLvL , (11)
and has a maximum possible value of θ∞ = σ, where σ is a dimensionless receptor site
density, defined by
σ = a Lv R ; (12)
under this condition vL(∞) = vR. The same results apply if aL > aR but vR is very small
and hence the receptors are mostly isolated from each other.
If on the other hand the ligand is much larger than the receptor (and in order for the
response to be measurable, vR should be large), both vL(t) and vL(∞) will depend on both
vR and aL, and on vL, i.e. the fraction of occupiable sites occupied. Defining
θ* = θ/θ∞ (13)
where
1 + 0.3136σ2 + 0.45σ3
θ∞(σ) = θJ 1– , (14)
1 + 1.8285σ + 0.5075σ3 + σ7/2
one has [18, 19]
φ(θ*, σ) = (1 – θ*)(1 – B1θ* – B2θ*2) (15)
for substitution into eqn (10), where the constants are:
0.7126 + 1.404σ1/2
B1 = (16)
1/σ + 3.4363 + 2.4653σ1/2
and
0.07362 + 0.1204σ1/2
B2 = . (17)
1/σ + 0.5443 + 0.2725σ1/2
The experimentally available parameters are vR (measured at the close of receptor

deposition) and vL(t), i.e. the kinetic binding curve. Eqn. (10) with the appropriate
substitutions can then be fitted to the vL(t) data in order to determine as many of the
unknown parameters (including ka) as the experimental resolution—usually extremely
high using OWLS—permits. Dissociation is measured by flooding the system with
ligand-free solution, in which case we have
dvL/dt = –kd(t)vL ; (18)
good results may often be obtained by combining eqns (10) and (18) and carrying out a
global fit with a change of boundary conditions (the ligand concentration in solution
goes to zero) at the initiation of flooding. Time-dependent dissociation coefficients kd(t)
are discussed fully elsewhere (e.g. [17]).
Conformational Changes: Several waveguide parameters are useful in obtaining
indications of receptor conformation. If the dimensions of a molecule are known, and if
it is significantly aspherical, the average orientation of a layer of molecules deposited on
the waveguide can be directly obtained from the adlayer thickness dA. The molecular
orientation can also be derived from measurements of the birefringence of the adlayer,
i.e. the ordinary (no) and extraordinary (ne) refractive indices. The form factor u for a
layer constituted from substances 1 and 2 of differing refractive indices n1 and n2
respectively is given by [11].
nA2 nB2 nm2 - no2
u= (19)
no2 ne2 - no2
where nm is the mean refractive index of the layer,
nm = 2no2 /3 + ne2 /3 . (20)
If the predominant molecular orientation is perfectly columnar, i.e. perpendicular to

the plane of the layer, then u = 0, and if it is perfectly lamellar, then u = ∞.
Changes in these parameters, whether mean adlayer thickness, or form factor, or
indeed any other parameter characteristic of the polarisability profile perpendicular to
the plane of the receptor layer, upon addition of a drug may be used to deduce
conformational changes. Typically an experiment will begin with the receptor in
equilibrium with a drug-free solution flowing over it. The solution is then switched to
one containing the drug (if the concentration is sufficiently high the cover refractive
index may then also change, but the new value of nC is included in the mode equations
used to determine ne and no, hence this change does not affect the final results).
Release of Drugs from Novel Delivery Materials: OWLS is extremely well suited to
characterising influx into and efflux from microporous materials [20]. The technique is
particularly effective if the delivery material can itself become the waveguide, i.e. the F
layer. A typical experiment begins with the F layer in equilibrium with drug-free liquid.
The drug is then introduced into the solution, and flows into the pores. If the exact
functional form of the kinetics is not of importance, a simple model suffices to relate the
measured refractive index of the F layer to the refractive indices of the F layer matrix
and the drug (respectively nf and nd), and the volume fraction θ of the nanopores [21]:
nF = θnd + (1 – θ)nf . (21)
Here, nd would be the refractive index of the drug solution inside the pores. If the
concentration is cd, then
nd = cd dn/dc (22)
(this equation can also be used to ascertain whether the presence of the drug makes a
significant difference to the refractive index of the cover medium). The refractive index
increment dn/dc is best found by measuring the refractive indices of a range of solutions.
The technique is entirely reversible: a drug-loaded nanoporous layer exposed to a
drug-free solvent will release its contents into the solvent, and is directly measurable.
Should it be impracticable to create the entire waveguiding layer from the drug-
releasing material under investigation, the waveguide may be coated with a thin (tens of
nanometres) layer of the material of interest [24].
Interactions of Drugs with Lipid Membranes: Not least due to the necessity for nearly
every drug to cross at least one bilayer lipid membrane to reach its target, the precise and
accurate measurement of drug-lipid interactions is of tremendous importance. As already
mentioned, the traditional approach is to measure the oil/water partition coefficient.
Octanol is typically used as the oil phase. A significant increase in sophistication was
represented by the measurement of the uptake of drugs by lipid membrane vesicles, but
the method is cumbersome, possibly unrealistic because of the high curvature of the
vesicles compared with natural membranes of interest, and does not allow measurement
of dissociation of the drug from the membrane, which is of equal importance to the
association. Hence the introduction of OWLS for the determination of drug-lipid
membrane interactions was a further very significant increase in sophistication.
The first application of OWLS to this domain was for the determination of partition
coefficients of drugs between the aqueous solutions and bilayer lipid membranes [22].
Owing to the extremely high sensitivity of OWLS, the drug uptake of a single lipid
bilayer coating the waveguide can be accurately measured. As already mentioned, the
optical waveguides can be coated with the lipid bilayer either using the Langmuir-
Blodgett technique [11] or from vesicles [12]. Careful measurements on these so-called
supported bilayers has shown that the fluidity of both the upper and lower leaflets is
comparable to that of natural cell membranes [25]. The reason for this is that the lower
leaflet is actually resting on a cushion of a few molecular layers of water more or less
tightly bound to the metal oxide waveguide material.
Other experiments have focused on determining the partial molar volume (v̄2) of a
drug in a bilayer lipid membrane [23]. The mean molar volume Vm is given by
Vm = [x(RM,2 – RM,1) + RM,1](nA2 + 2)/(nA2 – 1) (23)
where x is the mole fraction of the drug (component 2) in the membrane (component 1),
and the RM are the molar refractivities, and nA is the mean membrane refractive index
(eqn 20). Partial molar volumes can be easily found from a plot of mean molar volume
versus mole fraction of the drug by means of the Gibbs-Duhem equation, for two and
components we have
Vm = x(dVm/dx) + v–1; (24)
–
the intercepts at x = 0 and 1 of tangents to this curve yield v and v respectively.
1 2
The partial molar volumes of drugs in membranes at different mole fractions,

especially when compared with the molecular volume, permits a wealth of mechanistic
interference concerning the drug-membrane interactions to be made [23]. A further
advantage of OWLS is that the kinetics of uptake and release of the drug by the
membrane can be measured in real time.
Most drugs are somewhat amphiphilic, and like any detergent will therefore destroy
the membrane if present at sufficient concentration. This may only happen for mole
fractions in excess of about one third. For more unusual molecules such as oligopeptides
however, this destruction may take place at far lower concentrations, as has been shown
to happen with the oligopeptide mellitin [26].
In principle, the permeability of a membrane with respect to a drug can be calculated
from the partition coefficient and the uptake and release kinetics. There is also interest in
directly measuring this parameter. One possible approach is to coat a porous silicon
Fabry-Perot cavity sensor [27] with the membrane. In this way, the rate of accumulation
of the drug on the trans side of the membrane can be directly measured.
The Response of Living Cells to Drugs: One of the most fascinating recent
developments of OWLS has been its application to the quantification, in situ and in real
time, of the optogeometric parameters of living cells [28]. This has been shown to work
very effectively even though the cell may be one or two orders of magnitude bigger than
the penetration depth of the evanescent field. The reason for the success is that the most
significant and relevant changes in cell morphology following environmental stimuli
tend to be at the interface with the substrate. To carry out such investigations, cells are
admitted into a cuvette whose floor is the optical waveguide, upon which they become
attached to the waveguide. Very weakly bound cells are removed by a gentle flow of
medium, and those remaining may then spread. A single cell is sufficient for
measurements to be carried out. Spreading—the transformation from a sphere to a
segment—involves extremely significant redistribution of the cell matter, especially at
the cell-substrate interface, engendering significant changes in the propagation constants
of the guided lightmodes. Thanks to the precision and accuracy of OWLS, the cell-
substrate contact area can be determined from these propagation constants with
subnanometre resolution.
From the mode spectrum, the cover refractive index nC is determined by solving a
mode equation (1). This refractive index depends both on that of the medium bathing the
cells and that of the cells themselves, i.e. nM and nK respectively, and the precise
functional form of the dependence is determined by the shape of the cells, i.e. [28]
nC = s[nKcv' + nM(1/scv')] (25)
where s is the inverse penetration length of the exponentially decaying evanescent field
into the cell-containing medium, c is the number of cells per unit area, and v' is the
effective volume of the cells, which is obtained by taking the Laplace transform of the
cross-section of the cell parallel to the waveguide surface (here use is made of the
exponential decay of the evanescent field). For example, for spherical cells of radius r0,
the effective volume is
v' = 2π(r0 - 1/s)/s2 (26)
and for a spread cell having the form of a segment of radius r and height h
v' = π[h(2r - h)/s + 2(h – r)/s2 - 2/s3] . (27)
If the volume V of the cells is known, as it will be if they were deposited as spheres
from culture, then either r or h can be eliminated since the volume of a segment is
πh2(r – h/3), assuming that they have neither grown nor shrunk. We have here an
excellent method for the rapid non-invasive determination of cell shape and size. The
area a in contact with the surface is simply given by πh(2r – h).
The most interesting applications of this approach are in investigating how the
attachment and spreading responses vary in the presence of drugs, which can very easily
be introduced into the medium bathing the cells. Again in contrast to most other
techniques, it is very easy to determine the reverse effect, i.e. the drug withdrawal,
simply by switching the flow to that of medium not containing the drug, or containing
some agent that complexes the drug.
New developments in waveguide design (the so-called ‘reverse symmetry’
waveguides [29] with significantly greater penetration depths of the evanescent field)
further add to the potential applications of this technique.
5. CONCLUSIONS
OWLS is as yet a relatively unknown technique, which has tremendous potential in
the field of drug discovery. Hitherto, it has been applied in the biological field mainly for
the study of protein adsorption problems. Its particular strengths are the versatility of
experimental set-ups possible, and the readiness with which pertinent information can be
extracted from the raw data by simple and transparent means.
6. ABBREVIATION
OWLS = Optical waveguide lightmode spectroscopy
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Cell-Based Biosensors in Proteomic Analysis
Spiridon E. Kintzios*
EMBIO/Laboratory of Plant Physiology, Faculty of Biotechnology, Agricultural
University of Athens, Iera Odos 75, 11855 Athens, Greece
Abstract: In recent years there has been a rapid increase in the number of
diagnostic applications based on biosensors, including live, intact cells, tissues,
organs or whole organisms. Whole cells provide multipurpose catalysts,
particularly in processes that require the participation of a number of enzymes
in sequence. However, the sensitivity and reliability of these sensors is often
limited by the signal transduction mechanisms and by non-specific
interferences, due both to analyte and environmental variations.
In similar fashion to DNA and protein microarrays, which deliver multiplex
detection via the high-density spatial arrangement of molecular recognition
elements, arrays of cells at high-density can form the basis of cell-based
sensors with extremely high-throughput capability. The expression of receptors
of interest within these arrays could yield cell-based sensors with defined
specificities. In addition, transfected cell microarrays composed of high-
density arrays of mammalian cells expressing defined genes, could be the basis
for future high-throughput cell-based protein sensing platforms. Such cellular
arrays could be used for the detection of molecular interactions in functional
proteomics in vitro, to the testing of proteins in functional studies in living
cells. Microarrays with ordered cell arrangements of GFP-producing or
luminescent bacteria may be used as an integral part of future biosensors.
Recent and representative applications in this direction include (i) the profiling
of antibody specificities and protein interactions with genetically engineered
human immune cells, (ii) cells containing surface antibodies, specific to
antigens of different pathogens and (iii) cell proliferation/metabolism sensors
dedicated to screening for drug candidates and drug kinetic analysis.
INTRODUCTION
According to the definition provided by Panisko et al. [1] “Proteomics seek to
identify proteins and their posttranslational modifications, elucidate protein-protein
interactions, and quantify relative protein abundance on a global scale. Proteomic studies
are designed to analyze hundreds or thousands of proteins in single analyses and provide
a global view of changes in protein expression that occur when cells are treated. The
primary advantage of studying cells at the proteome level is the amount of information
that can potentially be derived from a single experiment”. Proteome analysis methods
are technically very challenging due to the high complexity and diversity of proteins,
*Corresponding author: Tel: +3210 5294292; Fax: +3210 5294286; E-mail: skin@aua.gr

226 Frontiers in Drug Design & Discovery, 2006, Vol. 2 Spiridon E. Kintzios
especially those proteins present at very low concentrations in cells. Favourite methods
for the characterization of proteomes include mass spectrometry [2-5], two-dimensional
polyacrylamide gel electrophoresis (2-D PAGE) [6, 7] and stable isotope labelling [8].
Cell-based sensors are a particular class of biosensors. Having made their debut more
than fifteen years ago, they are likely to gain a dominant position among analytical
technologies of the 21st century. Indeed, research activities in the field of cell-based
sensors are rapidly increasing, with an approximate increase of 70% of the number of
published reports on cell biosensors between 2002 and 2004, which represents one
quarter of all biosensor-related publications and conference presentations [9].
A cell-based sensor design employs the physiological responses of whole living cells
as the sensing component, such as oxygen consumption, surface chemical or electrical
potential, mobility or genetic activity. Thus, whole cells provide multipurpose catalysts,
particularly in processes that require the participation of a number of enzymes in
sequence [10, 11]. Therefore, they are able to provide physiologically relevant data in
response to an analyte and to measure the bioavailability of the analyte [12]. In other
words, cell-based sensors that carry out functional assays as cells, not only possess the
ability to detect the presence of an agent, but also are capable of responding in a manner
that can offer insight into the physiological effect of an analyte [13, 14]. Cell-based
biosensors are also likely to have improved stability, higher biocatalytic activity, adding
low cost in their favour. However, very few of the constructed sensors have been
commercialised. The few that have become commercial products are generally used for
the detection of a range of substrates and are based on BOD measurement. Most of the
biosensors reported, use bacterial cells as the sensing element. Breakthrough advances in
animal cell storage capacity are expected to increase the commercial applicability of
cell-based sensors for high throughput pharmaceutical and disease screening.
In following, the emerging application of biosensors in microarray format is briefly
reviewed, i.e. biosensors based on arrays of cells at high-density, which can form the
basis of cell-based sensors with extremely high-throughput capability. Emphasis is given
on cell-based protein sensing platforms used for the detection of molecular interactions
in functional proteomics in vitro. Representative examples of applications include the
profiling of antibody specificities, detection of pathogens and screening for drug
candidates and drug kinetic analysis.
CLASSIFICATION OF CELL-BASED BIOSENSORS

A cell-based assay system can be classified using one or more of the following
criteria:
1. The cell type used for constructing the sensor (e.g. mammalian, bacterial, etc):
Although the overwhelming majority of reports refer to the use of micro-
organisms such as bacteria or yeast for constructing cell-based sensors,
mammalian cells are used predominantly in cellular microarrays-based proteome
assays. This is due to the fact that, contrary to bacteria and plants, animal cells do
not have a cell wall that would impede the direct interaction of the cell surface
with a protein molecule under investigation. Especially immortalized cell lines are
easily handled and propagated. A typical researchers’ favourite is the Vero
(African Green Monkey Kidney) fibroblast cell line [15-17]. In addition, cells of
Cell-Based Biosensors in Proteomic Analysis Frontiers in Drug Design & Discovery, 2006, Vol. 2 227
neural origin that can be extracted from primary sources and maintained in culture
are available [18]. These neural cell lines can be grown into nerve cell networks
on substrate-integrated, thin-film microelectrode arrays in which the spontaneous
electrical activity can be monitored by a large number of electrodes for several
months [19, 20]. These systems are accessible by pharmacological assays and
have shown a highly sensitive and reproducible, tissue-specific response to
neuroactive compounds [18-28]. Human glioblastoma and/or neuroblastoma cells
have also been used for monitoring different physiological parameters (such as
superoxide accumulation) after a certain chemical stimulus had been provided
[29-31], although the elicitation of cellular resting potential and cell
differentiation was suppressed in some cases.
2. The method used for achieving a desired level of specific response to a particular
molecule (e.g. cell selection, genetic engineering): a limitation to the generation
of a cell-based sensor system is the non-availability of intact signal transduction
pathways for the desired stimulus. Ideally, the cell line used for constructing a
sensor must have endogenous genes (usually by means of genetic engineering)
that are tightly regulated by a chosen stimulus: subsequently, these genes can be
tagged by a reporter gene encoding a quantifiable protein (e.g. by means of
fluorescence spectrometry or microscopy) [12, 32]. Many reporter genes have
been incorporated in microbial sensors [33-39]. A reporter molecule should be
highly specific and sensitive, demonstrate minimal cytotoxicity and maximum
stability, as well as minimal interference with endogenous cell components other
than the target analyte. In addition, it should be autofluorescent or generally not
requiring the addition of a substrate for signal generation [12]. Among various
types of reporters, bioluminescent proteins are widely used, such as reporter genes
encoding luciferases that yield luminescence as the reporter signal [40]. Two
other proteins, aequorin and green fluorescent protein (GFP) (both derived from
the jellyfish Aequorea victoria) are increasingly used as reporter molecules in
many applications, since their ability and spectral properties change through
structural alterations of the native protein [33, 34].
In another approach, orphan receptors are matched with regulatory ligands, thus
resulting to elevated Ca+2 concentrations in transfected cells that interact with
receptor-specific peptides. This method is analyzed in more detail below.
3. The assay method (optical, electrochemical, etc.): the two most common methods
of transducing cellular responses are optical and electrical. The instrumentation
used in order to detect visible, fluorescent, or luminescent signals from cells or
tissues, includes microscopes, fiber optics, CCD cameras and other optical
equipment. Due to its high sensitivity and the advantages in the measurement of
high-density microtiter plates, bio- and chemiluminescence imaging is quite
suitable for the development of high throughput screening (HTS) systems
[41].However, quantification of the results is limited by the lack of appropriate
calibration systems, the invasive nature of intracellular recording and the
influence of the sample properties on the emission spectrum and intensity. On the
other hand, microphysiometry is based on the principle that electrically active
cells or tissues can be interfaced with microelectrodes which allow the capture of
extracellular spikes or impedance changes associated with cellular or tissue
responses. Various potentiometric electrodes have been used to detect
extracellular metabolites, e.g. potentiometric pH electrodes measured the

acidification of the external environment caused by the production of acidic
catabolites such as organic acids and CO2 [42, 43]. Ion sensitive field effect
transistors (ISFETs), can also be used to detect the acidification of the
extracellular environment [44-46]. A significant advance in the field of
microphysiometry was contributed by Hafeman et al. [47] who developed the
light-addressable potentiometric sensor (LAPS): this system, which is very similar
to a pH ISFET, allows for detecting the sensor surface potential by illuminating a
small spot at any desired position with a focused pulsed light-pointer. In this way,
surface potential measurements are not limited by the ability to microfabricate
discretely insulated gates.
4. The degree of organization of the cell community within the sensor (e.g. single
cell vs. cell cluster vs. tissue): the overwhelming majority of cell biosensors are
still single-cell based systems, although in recent years a steady shift towards
using cell clusters in suspension or in an immobilized state is being observed.
Single cell sensors are usually combined with amperometric sensors for
measuring physiological parameters, e.g. in cultured human neutrophils [48],
fibroblasts [49], glioblastoma [50] and monocytic cell lines (THP-1) [51]. Cluster-
based sensors reflect an attempt to reconstruct the original three-dimensional
configuration of a tissue segment or an organ within the body of the biosensor. In
the same context, a further development is the use of whole tissues rather than
cultured cells. This alternative has evolved into the development of the Tissue
Microarray technology which will be discussed in detail in following.
CELLULAR MICROARRAYS – BASIC CONCEPTS

Microarray technologies represent a considerable advance in genomics and
proteomics, since they satisfy the need to evaluate large numbers of molecular targets for
their bioactive properties. Contrary to conventional analytical and molecular techniques,
they are characterized by minimal user input, speed and requirement of low sample
volumes, thereby significantly increasing both the number of tissues and the number of
targets that can be evaluated [52]. For example, in a single cDNA microarray
experiment, one is able to determine the expression status of 50,000 human genes.
Conceptually, microarrays are formed after the local immobilization of test
molecules on the surface of a substrate, by covalent or non-covalent interactions and a
signal is detected when the probe molecules interact with the test molecules at a specific
position of the array. DNA and protein microarrays deliver multiplex detection via the
high-density spatial arrangement of molecular recognition elements [53, 54]. Microarray
format applications include the detection of gene expression by cDNA and
oligonucleotide arrays [55], profiling of antibody specificities and protein interactions by
peptide arrays [56-58] and screening for drug candidates with arrays of small molecules
[59]. Protein microarrays have been developed for the detection of antibodies [60] and
cytokines [61] by spotting specific antibodies onto membranes in an array format.
In similar fashion, arrays of cells at high-density can form the basis of cell-based
sensors with extremely high-throughput capability. Cellular microarrays are still an
emerging technology; as a result, they share the weaknesses and inadequacies of any
cell-based analytical system. The most important problem is the lack of absolute
specificity against a particular target molecule. Secondly, the viability of cells

incorporated into the sensor system often declines rapidly with time, thus reducing
dramatically the storability of the biosensor. As it will be analysed below, considerable
progress has been made in ameliorating both the specificity and the viability of cell
microarrays.
TISSUE MICROARRAYS AND THEIR APPLICATIONS

Tissue microarrays (or TMAs) are a platform for high-throughput analysis of tissue
specimens in research. In principle, TMAs are an ordered array of tissue cores on a glass
slide, i.e. a simple assay system that can be constructed from archival paraffin-embedded
tissue for immunohistochemical staining, in situ hybridization, and other methodologies
[62]. Various techniques for the construction of TMAs have been have been described.
Generally, very small (< 6mm) tissue sections are embedded in paraffin, usually with the
aid of a common microscope bearing a special holing needle, a sampling needle, and a
proper box to fix paraffin blocks on the microscope carrier [63]. With the precise
mechanical control of the microscope, the holing procedure on the recipient paraffin
blocks and sampling procedure of core tissue biopsies and observation and localization
of sampling regions are performed. It is generally feasible to fabricate TMAs in a simple
and relatively cost-effective way. In addition, major attempts have been made to
automate the process of TMA construction and of data analysis [64]. For example,
commercial instrumentation for TMA fabrication is available, such as the Tissue Arrayer
series from Beecher Instruments (Silver Spring, MD).
TMAs represent the currently most favourite version of cellular microarrays. They
offer a compromise between in vitro systems and whole organisms. In a sense, TMAs
are sensor systems that identify themselves with the samples under investigation: they
are (almost exclusively) built on clinical tissue samples and therefore are inherently
related to a specific genotype. The TMA is then used in order to investigate the
interactions between a molecule and the tissue array; consequently, the nature of these
interactions is closely dependent on the origin of the tissue samples.
The number of published research studies on TMAs increased 100-fold in the last
eight years (from just four publications in 1998 to more than 450 in 2005). This is due to
the fact that, compared to regular tissue sections, TMA technology offers a number of
distinct advantages, including high speed, and throughput, easy handling of specimens
and minimal sample tissue requirement, a critical issue in clinical research.
In principle, all the histochemical and molecular detection techniques that can be
used with regular sections can also be used with tissue microarrays. The most common
application is the detection of protein expression in clinical tissue specimens by means
of immunohistochemical analysis, fluorescence in situ hybridisation (FISH), or RNA in
situ hybridisation (ISH) [65]. TMAs are also used to accelerate studies seeking for
associations between molecular changes and clinical endpoints, a topic that land marked
the era of TMA-based assays [66]. The ability to study archival tissue specimens is an
important advantage as such specimens are usually not applicable in other high-
throughput genomic and proteomic surveys. Construction and analysis of TMAs can be
automated, increasing the throughput even further [67]. A major obstacle to broad
acceptance of microarrays is that they reduce the amount of tissue analyzed from a
whole tissue section to a disk, a few mm in diameter that may not be representative of
the protein expression patterns of the entire tumor [68]. Although they have a potentially
very broad range of applications, TMAs have been used so far in two distinct fields,
namely cancer research and assaying inflammation-related signals.
TMAs and Cancer Research

Quantitative proteomics can be used as a screening tool for identification of
differentially expressed proteins as potential biomarkers for cancers. Candidate
biomarkers from such studies can be subsequently tested using other techniques for use
in early detection of cancers [8]. Applications of TMAs in cancer research include the
biochemical profiling of different tumor types and the evaluation of potential diagnostic
markers. In a standard procedure, tissue cylinders with a diameter less than one
millimetre can be dissected from hundreds of different primary tumor blocks and
subsequently brought into a recipient tissue microarray block. Sections from such array
blocks can then be used for simultaneous in situ analysis of hundreds or thousands of
primary tumors on DNA, RNA, and protein level. The possibility to miniaturize tissue
analyses will substantially facilitate translational and clinical cancer research in a
number of ways. In a single experiment, up to 500 to 1000 tissues can be evaluated on
the same microscope slide [64]. Tens of thousands of TMA sections can be generated
from one paraffin block containing 10x10 mm of tissue area with a depth of 3 m. This
increases the speed of analysis of very large clinical datasets, and will also facilitate the
standardization and interpretation of the results. In terms of practical application, the
staining of a single TMA slide provides a much greater degree of consistency and
standardization than the immunostaining of hundreds of individual slides, while the
quantitation of immunostainings is markedly easier on arrayed samples than on large
sections.
In a number of comparative studies on tumour tissues, results derived from TMA-
based assays correlated very well with investigations conducted on mount sections [69].
Torhorst et al. [52] applied a TMA-based immunohistochemical approach in analyzing
prognostic markers in a series of 553 breast carcinomas. Four independent TMAs were
constructed by acquiring 0.6 mm biopsies from one central and from three peripheral
regions of each of the formalin-fixed paraffin embedded tumors. Immunostaining of
TMA sections and conventional “large” sections were performed for two well
established prognostic markers, estrogen receptor (ER) and progesterone receptor (PR),
as well as for p53, another frequently examined protein for which the data on prognostic
utility in breast cancer are less unequivocal. Compared with conventional large section
analysis, a single sample from each tumor identified about 95% of the information for
ER, 75 to 81% for PR, and 70 to 74% for p53. However, all twelve TMA analyses (three
antibodies on four different arrays) yielded as significant or more significant associations
with tumor-specific survival than large section analyses (p < 0.0015 for each of the 12
comparisons). A single sample from each tumor was sufficient to identify associations
between molecular alterations and clinical outcome. It was concluded that, contrary to
expectations, tissue heterogeneity did not negatively influence the predictive power of
the TMA results.
Camp et al. [68] investigated the number to disks required to adequately represent
the expression of three common antigens in invasive breast carcinoma-estrogen receptor,
progesterone receptor, and the Her2/neu oncogene in 38 cases of invasive breast
carcinoma since 1932 by creating a breast cancer microarray and evaluating the
antigenicity of these markers and others. They demonstrated that many proteins retained
their antigenicity for more than 60 years, thus validating their study on archival tissues.
Using microarrays of complementary DNA, Dhanasekaran et al. [70] examined
gene-expression profiles of more than 50 normal and neoplastic prostate specimens and
three common prostate-cancer cell lines. Signature expression profiles of normal
adjacent prostate (NAP), BPH, localized prostate cancer, and metastatic, hormone-
refractory prostate cancer were determined. Many associations were established between
genes and prostate cancer. They assessed two genes - hepsin, a transmembrane serine
protease, and pim-1, a serine/threonine kinase at the protein level - using TMAs
consisting of over 700 clinically stratified prostate-cancer specimens. Expression of
hepsin and pim-1 proteins was significantly correlated with measures of clinical
outcome. Thus, the integration of cDNA microarray, high-density tissue microarray, and
linked clinical and pathology data was proven to be a powerful approach to molecular
profiling of human cancer. Another method combinatory approach was adopted by
Nishizuka et al. [71], who used cDNA microarrays, oligonucleotide chips, protein
microarrays and TMAs derived from the NIH panel of sixty human cancer cell lines in
order to identify molecular markers for the differential diagnosis between colon and
ovarian cancer. In this way they were able to identify villin as a promising candidate
marker for colon cancer cells and moesin for ovarian cancer cells. Finally, Turashvilli et
al. [72] used TMAs in order to investigate the genetic heterogenity of invasive breast
cancer (as reflected by the wide spectrum of histological types and differentiation
grades), in particular the differential expression of eight genes in lobular and ductal
cancers.
Cytokine Detection/Inflammation Sensors

Another major application of TMA-assisted assays is the detection of inflammatory
signals, such as interleukin-1 (IL-1) [73]. Certain immortalized endothelial cell lines,
including ECV304 have been used for the selective detection of pg concentrations of IL-
1, the cytokine tumor necrosis factor-α (TNF-α) an the phorbol ester phorbol myristate
acetate (PMA) [74]. In these assays, cellular response to inflammatory signals was
recorder by transfecting cells with hybrid DNA constructs between the coding sequence
of β-lactamase and the promoter and regulatory regions of the gene encoding for
inflammation-associated genes, such as the endothelial-leukocyte adhesion molecule
(ELAM) [75] and RelB [76]. Automated fluorescence analyzers further facilitated
sorting out of responding cells at throughputs near 100 million cells per hours.
TRANSFECTED CELL MICROARRAYS

The expression of receptors of interest within cellular arrays could yield cell-based
sensors with defined specificities [77]. Transfected cell microarrays composed of high-
density arrays of mammalian cells expressing defined genes could be the basis for future
high-throughput cell-based sensing platforms. The signals produced from these
microarrays are dependent upon the efficiency of DNA uptake by the cells within each
spot. Microarrays with ordered cell arrangements of GFP-producing or otherwise
luminescent bacteria may be used as an integral part of future biosensors. For example,
Andrews et al. [78] studied the movement of mobile genetic elements and chemical
signals (such as the pheromones acylhomoserine lactones) between bacterial cells by
guiding bacteria cells to specific areas of a microelectrode array by dielectrophoresis,

where they were immobilised on electrodes.
Expression screens have been also created by incubation of the whole microarray
with antigen-specific antibodies and immunofluorescence [79]. In a significant
development, Ziauddin and Sabatini [80] transferred plasmid DNA incorporated into
agarose gel pads soaked with plasmid DNA into cells seeded onto microarrays. For the
successful development of transfected cell microarrays for use in high-throughput cell-
based sensor applications, careful cconsideration must be given to the factors that
influence the signals generated from the spots of transfected cells. Perhaps the most
important factor affecting the resulting signal emanating from the spots is the
transfection efficiency, or the percentage of cells on each spot that take up and express
the deposited DNA [77]. However, investigation into the factors that influence the
transfection efficiency on cellular microarrays has been limited.
In yet another approach, Rider et al. [81] developed the CANARY (Cellular Analysis
and Notification of Antigen Risks and Yields) cellular sensor by genetically engineering
human immune cells (cultured B cells) in order for them to express antibodies, specific
to antigens of different pathogens. In addition, cells are engineered to produce aequorin,
which emits light after interaction with an antigen, through a calcium-mediated pathway.
In a particular application, B cell-based CANARY sensors were used for the
identification of Yersinia pestis. A similar technique was used by Whelan and Zare [82],
who constructed a single-cell detector that combined the natural signal amplification of
whole-cell biosensors with the flexibility and specificity of immunological recognition.
Their system was based on an immune cell expressing receptors for the constant region
of immunoglobulin G (IgG); the cell was loaded with a Ca2+-indicating dye and with
antibodies directed against the protein of interest. Introduction of a multivalent protein
antigen caused cross-linking of the receptors, which resulted in a detectable increase in
the concentration of cytosolic Ca2+. Finally, Thach et al. [83] investigated the response
of primary rat neural precursor cells (NPC) and human peripheral blood mononuclear
cells (PBMC) against Sinbis virus (SV) carrying GFP, suggesting their potential use as
detectors of SV in a cell-based biosensor paradigm.
The use of transfected cells in a microarray format is neither a flawless process nor
always applicable. For example, transient transfections result in the over-expression of
individual genes, with the probability of producing a biased cellular phenotype, whereas
not every cell line is amenable to transfection.
Electroinsertion of enzymes, antibodies and/or receptor-like molecules in the cell
membrane could become an attractive alternative to cell transformation with genes
expressing membrane-bound antibodies. Using this method, Moschopoulou and Kintzios
[84] recently reported the development of a new, hybrid type of ultra-sensitive
electrophysiological superoxide anion (O2•_) sensor. The membrane-engineering process
involved the electroinsertion of superoxide dismutase (SOD) molecules in the
membranes of Vero fibroblast cells, which acted as catalytic units able to convert O2•_ to
H2O2. Superoxide dismutation triggered changes to the cell membrane potential that
were measured by appropriate microelectrodes, according to the principle of the
Bioelectric Recognition Assay (BERA) (see below). The sensor instantly responded to
picomole concentrations of O2•_ with a detection limit (S/N=3) of 100 pM. This
technology was further modified by the authors’ research group for the detection of
human viruses, such as Hepatitis B, D and C viruses (HBV, HDV and HCV,
respectively) in whole blood samples (unpublished results).
CELL CLUSTER AND IMMOBILIZED CELL ARRAYS

Although the majority of cellular microarrays are based on single cell systems,
sensors including cell clusters have been frequently reported. The most basic form is a
suspension of genetically engineered cells, usually incorporated into a self-contained
instrument that allows various types of cell-based modules to be maintained at a preset
temperature and ambient or not CO2 level [13]. A number of microfabricated cellular
sensors has emerged, essentially based on assaying parameters of a cell suspension
culture [85-87], predominantly extracellular acidification [88, 89] as well as electric
impedance [90, 91]. Real-time detection in vivo contributes to better understanding
cellular physiology by monitoring the extra- and intracellular microenvironments in a
quantifiable manner.
Immobilized cell biosensors represent an even more advanced approach than cell
clusters, since immobilization considerably facilitates the required proximity between
the biomaterial and the transducer as well as the stability of the sensor for storage and
reuse. Viable cells have been immobilized by means of entrapment and adsorption
techniques, using various polymers. Natural polymers used for the entrapment of the
cells include alginate [92], carrageenan [93], low-melting agarose [15, 16, 94] and
chitosan [95]. Biopolymers such as calcium alginate are permeable and non-toxic but
have poor mechanical stability and may be degraded by some organisms [96]. Agarose
and related gelling agents form rigid matrixes with variable pore diameter may inhibit
diffusion of large molecules, such as viral coat proteins. On the other hand, they are
appropriate for long-term (e.g. several months) preservation of cell viability [15-17]. A
typical immobilized cell sensor system based on agarose or alginate is the Bioelectric
Recognition Assay (BERA) [15]. It is a biosensory method based on a unique
combination of a group of cells, whose immobilization in the matrix preserves their
physiological functions and measures the expression of the cell interaction with viruses,
through the change in electrical properties. Cells are selected to specifically interact with
the virus under detection. In this way, when a positive sample is added to the probe, a
characteristic, ‘signature-like’ change in electrical potential occurs upon contact between
the virus and the gel matrix. BERA has been used for the detection of viruses in humans
(Hepatitis B and C viruses, herpes viruses) and plants (tobacco and cucumber viruses) in
a remarkably specific, rapid (1-2 minutes), reproducible and cost-efficient fashion. The
sensitivity of the virus detection with BERA is equal or even better than with advanced
immunological, cytological and molecular techniques, such as the reverse transcription
polymerase chain reaction (RT-PCR). More recently, absolute cell specification has been
achieved by means of membrane engineering (see above).
For mammalian cells, collagen matrixes represent the material that is closest to the
natural tissue environment. Mao and Kisaalita [97] reported on the biocompatibility
properties of collagen, as a first step towards establishing a functional 3-D cell-based
biosensor platform, while Yang et al. [98] used a three-dimensional model for studying
lung tumour/collagen matrix interaction. Armbruster et al. [99] also described a method
to attach cultures of collagen lattices on a silicon microdevice.
A subgroup of immobilized cell systems are spheroid cultures, created by various

techniques, such as culturing on polymer-coated culture surfaces [100] or in a porous
substratum [101, 102]. Spheroid cultures have attracted attention as three-dimensional
organoid culture methods, in particular hepatocyte culture systems; isolated primary
cells in this culture spontaneously form spherical multicellular aggregates and maintain
their morphological and functional characteristics in vitro [103, 104]. Although the
spheroid culture technique is such a useful method, several obstacles hinder their
widespread use for biological application, such as the inability to immobilize spheroids
at a defined location and cell necrosis occurring within the core of large and coalesced
spheroids because of oxygen depletion [105, 106]. Quite recently, Fukuda et al. [107]
described a novel hepatocyte culture system – in actuality a spherical organoid
(spheroid) microarray culture system – using a combination of microfabrication and
microcontact printing. The system consisted of a chip that had cylindrical cavities of 300
mm diameter at a density of 700 cavities/cm2. Primary hepatocytes spontaneously
formed spheroids with a uniform diameter at the center of each cavity on the chip.
Hepatocytes forming spheroids had a cuboidal cell shape, similar to hepatocytes in vivo,
and stably maintained liver-specific phenotypes, such as liver-enriched transcriptional
factors, albumin secretion, urea cycle enzymes, and intercellular adhesion molecules.
PERSPECTIVES
In essence, cellular and tissue microarrays are newcomers in the field of proteomics.
As a logical result, there is presently no clear indication for an increasing or decreasing
probability of these methods and related instrumentation to replacing or substituting
more conventional HTS proteomic assays, in particular automated HPLC-MS based
systems, on a mass-scale, routine analysis level. Nevertheless, there is adequate evidence
for the rapid expansion of research on cell-based proteomic sensors. This is not only
apparent from the increasing number of related publications; a shift of research focus to
integrated sensor arrays with whole cells as sensing components is being fuelled by
advances in other areas, such as:
1. Bio- and chemiluminescence imaging techniques allowing for the simultaneous
measurement of multiple analytes in the same sample or even in vivo imaging at a
whole organism level [41, 108].
2. Advances in three-dimensional microfabrication technology opening new
possibilities for miniaturising cell culture and analysis devices. For example,
Kintzios et al. [17] recently developed a miniaturized biosensor system by
combining the electrophysiological response of immobilized cells with
superoxide-sensing technology, optical and fluorescence microscopy. This system
enables the correlation of seven different cell physiological parameters to each
other, as well as the prediction of cell proliferation or death by comparing the
relative response of the electrophysiological and superoxide sensor during a one-
week long culture period. A commercial prototype of the sensor (the EMBIO®
sensor, Fig. 1) is currently being disseminated in large-scale clinical trials. Further
advances are resulting by improving our understanding of the performance of
cell-based assays in three-dimensional substrates as more representative of an “in
vivo” environment than two-dimensional culture systems. For example, Alp et al.
[109] developed a cellular microarray that mimics a TMA: skin tissue constructs
were made from human fibroblast cells in a fibrin gel (at a density of 100.000
human fibroblast cells/ml). By measuring the cells dielectric properties, a linear

relation between the capacitance (at 0.4 MHz) and the cell number was shown.
Fig. (1). The EMBIO® sensor.
3. Novel approaches in microphysiometry that may boost the high throughput

efficiency of existing cell monitoring systems. An example is provided by the
work of Xu et al. [110], who improved LAPS chips in order to facilitate
monitoring the response of single neuronal cells to a pharmacological agent
(acetylcholine). This will be particularly important for measuring cellular
dynamics, e.g. neuronal communication between cells at distances of 10-100 µm
[111].
It is finally anticipated that cell-based systems will make a difference in proteomic
analysis since they reduce the complexity of the testing environment and therefore
enhance the sensitivity of the measurements [112, 113]. This progress will be further
sustained by parallel advances in microfabrication technology and the miniaturization of
cell culture systems.
ABBREVIATIONS
BERA = Bioelectric Recognition Assay
BOD = Biological Oxygen Demand
CCD = Charge-Coupled Device
FISH = Fluorescence In Situ Hybridisation
GFP = Green Fluorescent Protein
HTS = High Throughput Screening

ISFET = Ion Sensitive Field Effect Transistor
ISH = In Situ Hybridization
LAPS = Light-Addressable Potentiometric Sensor
ROS = Reactive Oxygen Species
RT-PCR = Reverse Transcription Polymerase Chain Reaction
TMA = Tissue Microarray
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Current Approaches in Natural Biopolymer-

Nanoparticle Hybrid Functional Materials:
From Drug Delivery to Bio-Detection Applications
Roberta Brayner*
Interfaces, Traitements, Organisation et Dynamique des Systèmes
(ITODYS) –UMR-CNRS 7086 and Université Paris 7 Denis Diderot,
case 7090; 2 Place Jussieu 75251 Paris Cedex 05 France
Abstract: This review is focused on current approaches emerging at the

intersection of hybrid nanomaterials research and biotechnology. This
interdisciplinary field of chemistry and biology is also associated with physical
and chemical properties of organic and inorganic nanoparticles, as well as to
DNA, proteins and polysaccharide studies.
INTRODUCTION
Astonishing advances have been made in biological sciences after the discovery of
the double helix structure of DNA. Biology has evaluated from descriptive and
phenomenological discipline to a molecular science. To improve and to create new
revolutionary materials, it seems crucial to combine biotechnology with materials
science. Fuse these important disciplines we can generate new advanced materials to
solve biological problems. These hybrid functional materials exhibit specific and strong
complementary recognition interactions e.g. nucleic acid-DNA, antigen-antibody.
Proteins may be genetically modified with specific anchoring groups such as thiols. This
facilitates the aligned binding to nanoparticles, or the site-specific linkage of the
biomaterial to surfaces. This review describes the utilization of hybrid functional
materials based on natural biopolymers such as proteins and polysaccharides and
nanoparticles consisting of metals (e.g. Au, Ag, Ni, Co), oxides (e.g. ZnO, Fe3O4) and
quantum dots (e.g. CdS, CdSe, CdSe@ZnS…) for drug delivery and bio-detection
applications.
1. Biosensors for the Recognition and Monitoring of Molecule Interactions

DNA sensors and gene chips are particularly relevant for directly applying the
information gathered from genome projects and polymorphism databases. Polymor-
phism identification, sequence recognition, pathogen identification, expression profiling
and mutation detection are examples of current DNA sensors applications [1-5]. We can
mention electrochemical techniques used to develop methodologies to detect DNA in
human genes that are potentially involved in the modulation of individual cancer
susceptibility as well as therapeutic efficacy [1]. These techniques are based on the
*Corresponding author: Tel: 33 1 44 27 95 41; Fax: 33 1 44 27 61 37; E-mail: brayner@ccr.jussieu.fr

242 Frontiers in Drug Design & Discovery, 2006, Vol. 2 Roberta Brayner
utilization of DNA-modified gold electrodes. In this case, the intensities and peak
potentials change according to the modification of the gold electrode surface by
anchoring of thiol-terminated double-stranded oligonucleotide. Surface-modified
colloidal gold and silver can be employed as biosensors [6-7] and dielectric nano-
particles enclosed with gold shell have been applied also in tumor therapy utilizing
absorption of near-infrared (NIR) light [8]. Although many biomedical applications of
Au nanoparticles their toxicological effects have been ignored. Recent studies have
indicated that colloidal Au does not cause acute cytotoxicity, it was demonstrated that
these nanoparticles could be rapidly modified by surrounding cellular environment [9].
Matrix-assisted laser desorption-ionization (MALDI) mass spectroscopy has become a
very powerful tool for biochemical analysis [10-11]. In this case, negatively charged Au
nanoparticles were employed as selective probes to trap oppositely charged proteins
from aqueous solutions [12]. To recover these probes from the solution, Au
nanoparticles were bound covalently to the surface of Fe3O4 magnetic nanoparticles to
generate Au@magnetic materials [12]. In this work, cytochrome C and myoglobin were
used as target species. The trapping capacity of Au@magnetic particles, as a function of
pH, was determinated by MALDI analysis (Fig. 1).
Fig. (1). MALDI mass spectra obtained from a mixture (0.1 mL) of cytochrome C (10- 6M) and
myoglobin (10 -6M) when using Au@magnetic particles (1 mg) as probes to trap the target species
for 1 h from buffer solutions of differing pH: (a) pH 6, (b) pH 8 and (c) pH 12. SA was used as
MALDI matrix (Reprinted from ref. [12] with permission. Copyright 2004 American Chemical
Society).
The most interesting and practical application is to employ this technique in the
analysis of enzymatic digest product of proteins. Another example of Au nanoparticles
Drug Delivery to Bio-Detection Applications Frontiers in Drug Design & Discovery, 2006, Vol. 2 243
used, as biological nanoprobes is a colorimetric DNA detection based on the sequence-

specific hybridization properties of DNA [13]. In this case, Au nanoparticles smaller
than 60 nm have extremely high extinction coefficients at ~520 nm [13a]. Moreover,
different Au agglomeration states can result in distinctive color changes that make these
nanoparticles an ideal color-reporting group form signaling molecular recognition events
and render nanomolar concentration detection available [14]. An example of this
detection assay is based on the use of concanavalin (conA) and mannopyranoside-
encapsulated Au nanoparticles (man-GNPs) to identify the binding partners for conA
[15]. Here, the binding constants are determined from wavelength shifts. A detection
scheme based on Chen at al work [15] is illustrated in Fig. 2.
Fig. (2). Schematic illustration for the colorimetric detection of protein-protein interactions based
on ref [15].
A protein named A binds the ligands protruding from the GNP surface and promotes
particle agglomerations via multivalent ligand-protein interactions giving rise to a blue
colored solution. However, the addition of a putative protein, named B, capable of
interacting with protein A, could influence the binding A and Au nanoparticles allowing
to Au nanoparticles re-dispersion. Consequently, the solution color can change from
blue to original red color. It was also studied the possibility of using the performed man-
GNPs/conA complex for a competitive colorimetric assay. Among ten proteins
considered herein, four proteins, i.e. thyroglobulin, BS-I, SBA and MAL were found to
have very drastic effects on the absorption spectrum of man-GNPs/conA. For these
proteins, the wavelength was blue-shifted and the absorption intensity increased. The
color changed from blue to red indicating that these proteins were able to compete with
man-GNPs/conA complex and disrupt particle agglomeration. The new technology
developed in this work provides not only qualitative but also quantitative evaluation of
protein-protein interactions by using Au nanoparticles-based competitive assays. This
method may also be straightforward to screen molecular libraries containing potential
drugs to disrupt cell-cell adhesion mediated by lectins.
Recently, hybrid structures of microorganisms with inorganic nanoscale moieties
have received great interest owing to their potential in fabricating electronic systems.
The electronic properties of metal nanoparticles, as a result of the single electron
transport of current [17] make them ideal materials for nanodevices. Concomitantly, the
nanostructure of microorganisms such as bacteria [18] and viruses [19-20] are attractive
scaffolds for the templating of metal nanoparticles through the interactions of the former
with surface charges and the affinity of certain metals for specific biological molecules
[18-23]. Berry and Saraf [24] present a simple method to build hybrid devices that use
the biological response of a microorganism to control the electrical properties of the
system. In our design, a monolayer of gold nanoparticles is deposited on the
peptidoglycan membrane of a live Gram-positive Bacillus cereus bacterium. The
hydrophilic peptidoglycan membrane is then actuated by humidity to modulate the width
of the electron-tunneling barrier between metal nanoparticles. In this work, Bacillus
cereus was deposited on a silicon substrate with a layer of 500 nm of thermally grown
silica and gold electrode lines spaced 7 ± 0.2 microns apart and coated with poly-L-
lysine. The bacteria-deposited ship was then immersed in a solution of poly-L-lysine
coated gold nanoparticles (diameter d = 30 nm) [18]. The deposition is highly selective,
with formation of a monolayer only on the negatively charged bacteria surface because
Au nanoparticles and the substrate are both positively charged (Fig. 3). The insets of Fig.
4 show a typical bacterial bridge, coated with a monolayer of Au nanoparticles
connected to Au electrodes.
Fig. (3). Scanning electron microscopy (SEM) images reveal the highly controlled and selective
deposition on bacteria of poly(L-lysine)-coated-30 nm Au nanoparticles from a solution at pH 7
over (a) 30 min; (b) 1 h; (c) 2 h; (d) 4 h; (e) 8 h; (f) Positively charged Au nanoparticles are
deposited on a negatively charged PSS-coated lysine/SiO2/Si substrate over 16 h. (Reprinted from
ref [24] with permission. Copyright 2005 Angewandte Chemie International Edition).
Here, one bridge constitutes a device. The electrical properties of Au nanoparticle

monolayer are controlled by actuating the bacterium peptidoglycan membrane. An
actuation of less than 8% in the peptidoglycan membrane, induced by change in
humidity from 20 to 0%, leads to more than 40-fold increase in the tunneling current.
These results open up new routes to obtain active coupling between microorganisms and
electrical, optical and/or magnetic devices. Another example of hybrid structures based
on microorganisms with inorganic nanomaterials is the utilization of cyanobacteria as
bioreactors to synthesize metallic nanoparticles [25a]. In this work, we showed that the
common Anabaena, Calothrix and Leptolyngbya cyanobacteria were able to form gold,
Fig. (4). Typical device current (I, normalized per bridge) as a function of relative humidity (Hrel)
for “up” (i.e. decreasing humidity; ∆) and “down” cycles (i.e. increasing humidity; ≤) at a bias
voltage of 10 V. The inset shows SEM images of two typical bacteria bridges which span the
electrodes. The peripheral strip is a (percolating) monolayer of deposited gold nanoparticles.
(Reproduced from ref [24] with permission. Copyright 2005 Angewandte Chemie International
Edition).
silver, platinum and palladium metallic nanoparticles with well-controlled size. These
nanoparticles were synthesized intracellulary (Fig. 5) and naturally released in the
culture medium where they are stabilized by the algal-polisaccharides, allowing their
easy recovery. In addition, the size of recovered particles as well as the synthesis yield
was shown to depend on the cyanobacteria genus, demonstrating the flexibility of this
approach [25a].
The advantages of this new process are: (i) the nanoparticles show a narrow size
distribution that depend on cyanobacteria genus employed as bioreactor (6 to 10 nm) in
contrast to similar reports in the literature; (ii) these particules are naturally released and
stabilized in the culture medium. This effect, associated to the proliferation capacity of
these algae could open the route to natural bioreactors for nanoparticle production in a
continuous way; (iii) this process is fully green both from reagents (without organic
solvents or reducing agents) and cost/energy (atmospheric pressure, ambient temperature
and water medium); (iv) a lot of seasoning cyanobacterial blooms was observed in the
world that open new horizons to improve this new original and low cost process. In
summary, Au and Ag nanoparticles have many attractive properties. They are nanometer
in size and may have various functional ligands on the surface. These special properties
provide many possible modes of interaction with biological cells, such as specific
binding to the cell membrane and penetration through the membrane to interact with
biological molecules inside a cell or a virus. For example, carbohydrate-functionalized

Au nanoparticles have been developed to explore their interactions with cell membrane
surfaces, such as lectins [25b]. These studies could have potential applications in lectin
detection, control of cell fertilization, proliferation, viral infection and also inflammatory
response.
Fig. (5). (a) TEM micrograph of Calothrix cyanobacteria thin section (presence of gold metallic
nanoparticles); (b) TEM mimicrograph of Anabaena cyanobacteria thin section (presence of silver
metallic nanoparticles).
The optical and electrical properties of colloidal gold nanoparticles are know to be
dramatically affected by their size, shape and surrounding surface environments [26].
Since size-dependent photoluminescence of nanosized semiconductor materials reported
by Mooradian [27a], the visible photoluminescence of small gold nanoclusters (< 25 nm)
has also been observed [27b]. The unique properties of nanoscale colloidal particles are
studied for their potential in various biosensor developments [28]. The optical properties
of 3D aggregations of gold nanoparticles have been used to detect hybridization of
specific DNA sequences in solution and or surfaces [13c, 29] as an alternative to
fluorescent labeling of DNA. It was demonstrated that fluorescent labels used in DNA
microarray analysis, such as cy3 and cy5 are very expensive, photosensive and special
care must be taken to avoid their exposure to light during labeling [30]. On the other
hand, gold nanorods are not photobleached and can interact with thiol-DNA targets
through S-Au bonds. Consequently, gold nanorods may be considered as an alternative
fluorescent label for heterogeneous DNA analysis. Sequence-specific DNA detection
techniques have been developed which rely upon target hybridization with radioactive,
fluorescent, chemiluminescent and other types of labeled probes [30]. Since the first
reports by Alivisatos and Mirkin [13c, 29a-b], gold nanoparticle labeling of DNA
molecules has been considered as an alternative marker for DNA hybridization moni-
toring events and even for the detection of a single base mismatch in the oligonucleotide
sequence. Following the design concept of DNA-nanoparticles, Luong et al. [31]
developed a fluorescence-based method for the determination of DNA sequences and
monitoring reversible DNA hybridization events. This method was attempted by using
DNA functionalized gold nanorods [31]. These authors discovered that sufficiently long
gold nanorods (aspect ratio > 13) exhibit novel optical properties by means of relatively
intense fluorescence emission at 743 nm and one weaker band at 793 nm. After
functionalization by DNA probes, the DNA hybridization event could be effectively
monitored by measuring the fluorescence intensity. The results suggest that the unique
fluorescent properties of gold nanorods could potentially be exploited as a sensitive
probe in fluorescence-based microarray assay and optical biosensor development. Work
is in progress using DNA sequences of interest towards the detection of important
pathogenic bacteria. Another example of biomarker is silver-dendrimer nanocomposites
[32]. Poly(amidoamine) (PAMAM) dendrimers hold great promise as templates for
metal composite nanoparticles because of their low toxicity and highly regular, branched
3D structure allowing them to host inorganic nanoclusters and form stable dendrimer
complexes and nanocomposites [33-36]. Dendrimer nanocomposites (DNC) are
nanometer-size inorganic/organic hybrid composite particles containing topologically
trapped guest atoms/molecules/nanodomains immobilized by dendritic polymer hosts of
well-defined size, charge and terminal functionality [33-34]. Fabrication of metal-
dendrimer nanocomposites by reactive encapsulation requires two steps: (i) binding of
metal ions to template dendrimer molecule to form complexes and (ii) immobilization of
the preorganized metal ions to form nanoclusters with dendrimer templates. It is
important to know that the composition and morphology of dendrimer nanocomposites
depend on many factors such as chemical structure, uniformity and concentration of the
template molecules, metal/template molar ratio, pH and temperature [37-38]. Balogh et
al. [32] have synthesized water-soluble, biocompatible, fluorescent and stable silver-
dendrimer nanocomposites that exhibit a potential for in vivo cell labeling. Amino-,
hydroxyl- and carbonyl-terminated ethylenediamine core generation 5poly(amidoamine)
dendrimers were used to prepare aqueous silver (I)-dendrimer complexes at the biologic
pH of 7.4. These hybrid nanocomposites are fluorescent and their surface charge,
cellular internalization, toxicity, and cell labeling capacities were determined by surface
functionalities of dendrimer templates. These materials exhibit potential applications as
cell biomarkers. It is important to note that while significant advances in biological
labeling have been made, few therapeutic applications of metal nanoparticles have been
reported in the literature. A great example is the anti-microbial properties of silver
nanoparticles, which have been used for wound healing [39]. Silver nanoparticles
fabricated in Hepes buffer exhibit potent cytoprotective and post-infected anti-HIV-1
activities toward Hut/CCR5 cells [40]. These nanoparticles inhibited HIV-1 replication
via an unknown mechanism [40]. In summary, it was demonstrated that gold
nanoparticles are biocompatible, nontoxic [41], bind compatible with a large range of
biomolecules such as amino acids [42], proteins [43] and DNA [13c, 44], and expose
large surface areas for biomolecule immobilization. These nanoparticles could also serve
as excellent delivery vehicles for a variety of molecules such as drugs and proteins. We
present here some examples of molecule delivery based on gold nanoparticles as
carriers. Sastry et al. [45] studied the binding of the hormone insulin to gold
nanoparticles and its application in transmucosal delivery for the therapeutic treatment of
diabetes mellitus. In this work, insulin was loaded onto bare gold nanoparticles and
aspartic acid-capped gold nanoparticles and delivered in diabetic Wistar rats by both oral
and intranasal (transmucosal) routes. It was observed a significant reduction of blood
glucose levels (postprandial hyperglycemia) when insulin is delivered using gold
nanoparticles as carriers by the transmucosal route in diabetic rats. Gu et al. [46] have
shown that gold nanoparticles in toluene react with bis(vancomycine)cystamide in water

to form vancomycin-capped gold nanoparticle. This hybrid nanomaterial enhanced
antibacterial activity against E. coli strains. More recently, SiO2@Au nanoparticles,
designed to absorb in the near infrared, have been used in cancer hyperthermia [47].
2. Plasmon Resonance Measurements, Surface-Enhanced Resonance Raman

Scattering (SERRS) and Resonance Light Scattering (RLS) of Biopolymer Onto Au
and Ag Nanoparticles
The adsorption of biopolymers onto metallic nanocolloids has industrial relevance in
such areas as pharmaceuticals, wastewater treatment as well as in various biological
systems. Optical, electronic, magnetic and catalytic properties of these metallic
nanocolloids strongly depend on their shapes as much as their sizes [48a]. This is
particularly important for Au and Ag, which have strong surface plasmon resonance
oscillations. Controlling the shape of these nanoparticles is crucial in scheming their
surface-related properties such as absorption and emission [48b], SERRS [48c] and other
nonlinear optical properties [49]. A shift in plasmon resonance surface was measured for
Au colloidal solutions upon adsorption of polyampholyte (gelatin) [50-51]. It was
demonstrated that the shift in wavelength of the absorption maximum can quantitatively
yield measurements of the adsorbed amount as well as information about the structure of
the adsorbed biopolymer layer [51]. SERRS applications in the biophysical, biochemical
and biomedical domains in the last decade are summarized in several review papers [52-
56]. These reviews present SERRS experiments performed on amino acids and peptides,
purine and pyrimidine bases and also on macromolecules such as proteins, DNA and
RNA. Other more medical applications include SERRS detection of stimulating drugs
[57] and selective analysis of anti-tumor drug interactions with DNA [58-62]. Detection,
identification and quantification of neurotransmitters in brain fluid are developing in
neurochemistry. For example, Fig. 6 shows SERRS spectra of dopamine and
norepinephrine anchoring on silver nanoparticle surfaces in aqueous solution. SERRS
spectra of these two neurotransmitters were measured at concentrations between 10 -6 and
10-9M with accumulation times as short as 0.025s [63].
In these experiments, albumin was added as a protein component to silver colloidal
solutions to make them close to a real biological environment. The low concentrations
and fast data acquisition are on the order of physiologically relevant concentrations and
very close to the time scale of neuronal events, respectively. The use of nanoparticles-
biopolymers systems for in situ detection of specific reactions has significant potential to
probe living cells and tissues. For example, the detection in a cell of DNA or
protein/receptor interactions will provide information of specific functionality. One form
of Raman spectroscopy, the SERRS, has much important properties for labeling
nanoparticles. Raman and SERRS techniques have similar sensitivities [64] but with
SERRS many more codes can be written onto the surface of the nanoparticles. SERRS
can be used for labeling both fluorophores and non fluorophores. To test the sensitivity
of the essay, 3,5-dimethoxy-4-(6’-azobenzotriazolyl)-phenylamine anchoring on Ag
nanoparticle surface was employed to discover the minimum amount that could be
detected by this method (about 10-10M) [65]. A surprising feature of this experiment,
which is not fully understood, is that the chromophore on the hybrid DNA-Ag
nanoparticles is at least one hundred times more sensitive than that of the other dyes
Fig. (6). SERRS spectra of neurotransmitters in silver colloidal solution. Spectra were collected in
50 ms using 100 mV NIR excitation. (Reproduced from ref [63b]. Copyright 2002 Institut of
Physics Publishing).
[65]. Ag nanoparticles have considerable potential for biochemical analysis and Au

colloids could be similarly developed. The advantage of Ag nanoparticles is that the
range of dyes which remain effective in biological media is much more extensive. On
the other hand, in some biological systems such as cell suspensions, silver can react
positively with the cell and it is well known as a bactericide. Natural polysaccharides
such as chitosan, alginate and carragheenan have some important properties such as
mucoadhesivity, biocompatibility and nontoxicity, which render them interesting
biomaterials. From a physicochemical point of view, these polysaccharides have the
special quality of gelling upon contact with cations (alginate and carragheenan) and
anions (chitosan) under very mild conditions [66-67]. Nevertheless, due to their large
size (1-3 mm), these beads are only appropriate for drugs delivery in the gastric cavity,
for bio-labelling [68] and heat-triggered drug release [69]. For example, polysaccharidic
alginate biopolymers have been used as templates for the controlled growth of gold
metallic nanoparticles [70]. Au3+ was used to form alginate gels as spherical capsules.
After reduction in mild conditions, Au metallic nanoparticles were obtained inside

alginate microcapsules (Fig. 7). These microcapsules were then immersed in a
methylene blue (MB) aqueous solution (used as a probe molecule) at a concentration of
10-5M. It was observed a wavelength plasmon band shift probably due to an electronic
coupling between Au nanoparticles and the MB molecule inside alginate network (Fig.
7). It was also observed an important SERRS effect for MB molecule adsorbed on these
Au nanoparticles compared to the same molecule adsorbed on a lithographically
designed 2D Au arrays film [71] (Fig. 7).
Fig. (7). (a) UV-visible spectrum and alginate microcapsules of Au3+; (b) UV-visible spectrum of
Au metallic nanoparticles (Au plasmon resonance) and Au metallic nanoparticles inside alginate
microcapsules; (c) UV-visible spectra of MB-Au-alginate compared with Au-alginate (red shift
observed in the presence of MB); (d) Raman spectra of methylene blue excited under Au
nanoparticles (SERRS effect) [70].
This polyfunctional hybrid material may be applied in drug delivery (because

alginates have a great protein loading capacity), magnetic targeting or magnetic
resonance imaging. A new method for the determination of proteins in aqueous solutions
has been developed based on RLS of Ag nanoparticles in the presence of proteins [72]. It
was showed that interaction between Ag and proteins such as bovine serum albumin
(BSA), human serum albumin (HSA) and human immunoglobulins (γ-IgG) results in
strong enhancement of the RLS intensity. This method has been applied to the synthetic
samples and also to the analysis of human serum samples and the results obtained were
very close with those reported by the hospital, indicating that the RLS method can be
used for practical applications [72].
3. Biopolymer-functionalized Magnetic nanoparticles

Magnetic nanoparticles are widely studied and applied in various fields of biology
and medicine such as magnetic targeting, magnetic resonance imaging, diagnostics,
DNA purification, etc [73]. Generally, we have advanced hybrid functional materials
based on biological species such as cells, nucleic acids and proteins connected to the
magnetic nanoparticles. Several synthetic approaches have been applied to anchoring
biomolecules to magnetic nanoparticles, which were then used in various bioanalytical
systems. For example, antibody biomolecules were adsorbed on Fe3O4 magnetic
nanoparticles to use for specific binding to cells and posteriori separation of these cells
in an external magnetic field [74-75]. Another very interesting example is the natural
fabrication of iron oxide by some bacteria [76-83]. The unexpected and unusual features
of these biogenic magnetite crystals are not only a narrow size distribution, but also a
diameter range of 40-120 nm, which thus allocates them the highest magnetic moment
(Fig. 8).
Fig. (8). Crystal morphologies and intracellular organization of magnetosomes from magnetotatic
bacteria: (a) cubooctahedral; (b) bullet-shaped; (c-d) pseudohexagonal. The magnetosomes are
arranged in one (c) or more (d) chains. The lengths of the bars represents 100 nm). (Reprinted
from ref. [78a]. Copyright 2003 Angewandte Chemie International Edition).
Magnetospirillum sp. MGT-1 [76], Magnetospirillum sp. AMB-1 [77] and

Magnetospirillum gryphiswaldense [78] produce natural Fe3O4 magnetic nanoparticles
aligned in chains and enveloped by a lipid membrane [79] (Fig. 9).
Fig. (9). TEM image of (A) Magnetospirillum gryphiwaldense with a chain of cubooctahedral
magnetite crystals and one flagellum at each pole (the length of the bar represents 500 nm) and (B)
it is isolated and purified magnetosomes enveloped by a membrane (the length of the bar
represents 20 nm). (Reprinted from ref. [78a]. Copyright 2003 Angewandte Chemie International
Edition).
These natural hybrid materials can be isolated from the parent bacteria [80] and
modified with biomolecules using bifunctional coupling reagents such as glutaric
dialdehyde. They have been applied for fluoroimmunoassay [81], chemiluminescence
immunoassay [82] and DNA carriers [83]. Another approach is the fabrication of
polyfunctional hybrid materials. The development of hydrophilic nanoparticles as drug
carriers has represented over the last few years an important challenge. For example,
polysaccharidic alginate biopolymers have been used as templates for the controlled
growth of magnetic nanoparticles [84]. Ni2+ and Co 2+ were used to form alginate gels as
spherical capsules (Fig. 10). After reduction under flowing H2/N2 at 350°C, SQUID
measurements indicated that Ni presents a superparamagnetic behavior with a blocking
temperature TB = 290 K [84].
Contrast agents (CAs) play an important role in magnetic resonance imaging (MRI)
in medicine [85]. MRI CAs are primarily used to improve disease detection by
increasing sensitivity and diagnostic confidence. There are several types of MR contrast
agents being used in clinical practice today. The lanthanide ion Gd3+ is usually chosen
for MRI CAs because it has a very large magnetic moment (µ = 63 µB) and a symmetric
Fig. (10). (a) alginate 50%Co50%Ni (after reduction); (b) TEM micrograph of 50%Co50%Ni
metallic nanoparticles inside alginate matrix.
8 
electronic ground state  S 7  . The Gd 3+ ion is toxic and in order to reduce its toxicity,
 2
it is always sequestered by chelation [86] or encapsulation [87-88]. Wilson et al. [89]
have employed ultra-short nanotubes or US-tubes (20-100 nm) [90-91] as
“nanocapsules” for MRI-active Gd3+ ions. US-tubes are probably best suited for cellular
uptake biocompatibility and eventual elimination from the body. In this work, the
authors presented the internal loading of US-tubes with aqueous GdCl3 to form
Gdn3@US-tube species. This hybrid material is formed by superparamagnetic metal-ion
clusters that present proton relaxation centers with relaxivities 40 to 90 times larger than
current clinical agents [89]. Several iron oxide-based magnetic labeling systems have
been developed for monitoring stem cell migration [92-95] and tracking lymphocytes
[96-97]. However, probes based on these materials have some difficulties for the
successful MR cellular imaging due to their relatively low cell transport efficiencies or
the use of macromolecules transport facilitating agents which can often cause unwanted
side effects such as nanocrystal aggregation and cytotoxicity at a high dose level [98].
Recently, Fe3O4 nanocrystals were also employed as a probe for efficient intracellular
labeling and their MRI applications [99]. In this case, by simple modulation of
nanocrystal surface charge properties, the authors were able to prepare magnetic
nanocrystals that efficiently label a variety of cell types. Since cell membranes are know
to be weakly negatively charged [100], it is expected that only cationic water soluble
iron oxide (WSIO) nanocrystals easily anchor to cell membranes through electrostatic
interactions and are internalized into cells by way of a charge-mediated endocytosis
process. The excellent labeling capacity of these cationic WSIO has led to a system for a
preliminary but successful MRI monitoring of neural stem cells in vivo in rat spinal cord.
Weissleber et al. [93] have developed a cell labeling approach using short HIV-Tat
peptides to derivatize superparamagnetic nanoparticles. The nanoparticles are efficiently
internalized into hematopoietic and neural progenitor cells in quantities up to 10-30 pg
of superparamagnetic iron per cell. Iron incorporation did not affect cell viability,
differentiation or proliferation of CD34+ cells. It was observed after intravenous
injection that 4% of magnetically CD34 + cells homed to bone marrow per gram of tissue
and single cells could be detected by MRI in tissues samples. Localization and retrieval
of cell populations in vivo enable detailed analysis of specific stem cell and organ
interactions critical for advancing the therapeutic use of stem cells.
4. Quantum Dots: Fluorescent Biosensing and Fluorescent Encoding

Colloidal semiconductor quantum dots (QDs) are extensively used in various
practical medical applications. For example, they have been used as immunofluorescent
probes to detect the Her2 breast cancer marker [101], as signal transduction components
in immunoassays for microbial toxins [102] and as labels for dynamic studies of cancer
cell motility and correlation of metastatic potential [103]. The QDs have several
advantages over conventional fluorescent dyes. Their fluorescence absorption and
emission are conveniently tunable by their size and material composition, and the
emission peaks have a narrow spectral linewidth. Typically, emission widths are 20-30
nm, which is only one third of the emission linewidth of a conventional organic dye
[104]. The high quantum yields often range from 35-50% for CdSe/ZnS nanoparticles
[104]. Moreover, QDs are about 100 times as stable against photobleaching as organic
dyes and they often present a long fluorescence lifetime of several hundred nanoseconds.
This allows time-delayed fluorescence measurements, which can be used to eliminate
the autofluorescence of biological matrices. Another QDs application is the development
of multifunctional nanoparticle probes based on QDs for cancer targeting and imaging in
living animals [105]. The preparation of these hybrid materials involves encapsulating
luminescent QDs with a triblock copolymer and linking this amphiphilic polymer to
tumor-targeting ligands and drug-delivery functionalities. Gao et al. [105] present in this
work, in vivo imaging results from three different QD surface modification: COOH
groups, PEG groups, and PEG plus prostate-specific membrane antigen ab (PSMA). No
tumors signals were detected with COOH probe, only weak tumor signals were observed
with PEG probe (passive targeting) and intense signals were detected in the PEG-PSMA
ab conjugated probe (active targeting). These results provides further evidence for the
conclusion that active targeting by using tumor-specific ligand is much faster and more
efficient than passive targeting based on tumor permeation, uptake and retention [105].
These new multifunctional QDs might be use in diagnosis and treatment of cancer,
cardiovascular plaques and neurodegenerative disease.
ABBREVIATIONS
MALDI = Matrix-assisted laser desorption-ionization
ConA = Concanavalin
Man-GNPs = Mannopyranoside-encapsulated Au nanoparticles
GNP = Gold nanoparticles
PAMAM = Poly(amidoamine)
DNC = Dendrimer nanocomposites
SERRS = Surface-enhanced resonance Raman scattering

RLS = Resonance light scattering
BSA = Bovine serum albumin
HSA = Human serum albumin
CAs = Contrast agents
MRI = Magnetic resonance imaging
WSIO = Water soluble iron oxide
QDs = Quantum dots
PSMA = Prostate-specific membrane antigen
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Spectroscopic Analysis of Cell

Physiology and Function
Mark Riley1,*, Iram Mondaca Fernandez1, and Pierre Lucas2

1
Agricultural and Biosystems Engineering, 2Materials Science and Engineering,
The University of Arizona, 1177 E. 4th Street, Shantz Building, Room 403,
Tucson, Arizona, 85721, USA
Abstract: Spectroscopic methods including infrared and Raman techniques
have tremendous promise for providing rapid, non-invasive information on the
impact of pharmaceuticals and toxicants on cells and tissues. Spectroscopy is
not a new field; however these methods have only recently been applied to
study the physiology and function of cells and tissue. The infrared spectrum
(2,500-25,000 nm) of a cell or tissue can supply information about the
fundamental vibrational modes of functional groups existing in biological
molecules thus permitting quantification of material composition and in some
cases can be connected to cell function. Spectroscopy permits observation of
changes within the cell occurring at the molecular level and is a rapid, non-
destructive, and reagent-free measurement.
Infrared and Raman spectroscopy have been applied to evaluate the
biochemical composition of mammalian cells or to discriminate between
cancerous and healthy cells. While discriminations can be made between
stages of the cell cycle due to alterations in spectral signatures of nucleic acids,
only very small differences are apparent between distinct subcellular
structures. Analyses of separate cell fractions, isolated by sucrose density
gradient centrifugation, present the most significant spectral differences in the
C-H (carbon and hydrogen) stretching region (2800–3000 cm-1), at the ester
carbonyl stretching band (1737 cm-1), and in the PO2- stretching region (1089
and 1242 cm-1).
This manuscript provides a critical review of these methods, including their
potential use and pitfalls for drug screening applications. Significant
innovations have been made over recent years in equipment, experimental
approaches, and in analysis methods.
INTRODUCTION
Spectroscopic methods including infrared and Raman techniques have promise for
providing rapid, non-invasive information for rapid screening of the impact of
pharmaceuticals and toxicants on cells and tissues. Measurements require only minutes
and can be performed without damaging the biological sample. Typically, no reagents
*Corresponding author: Tel: (520) 626-9120; Fax: (520) 621-3963; E-mail: riley@ag.arizona.edu

260 Frontiers in Drug Design & Discovery, 2006, Vol. 2 Riley et al.
are required and so a native sampling process can be performed repeatedly on the same
cell or tissue sample.
Infrared and Raman spectra yield molecular information about the chemistry of a
sample. This information is related not only to the chemical composition, but also to the
environment (pH, redox state, interaction between compounds, etc.). This can be used to
identify and quantify compounds in volumes of femtoliters. Recent research in this area
has shown that such methods can discriminate between cell types (including between
bacterial species and between cancerous and non-cancerous cells), can provide a means
to evaluate cell function (response to stimulating and inhibiting agents, induction of
apoptosis), and can present information on alterations in cellular physiology (cell
adhesion, cytoarchitecture, etc.). This review provides a background on the
spectroscopic methods, their prior applications, and future use in drug discovery.
APPLICATIONS OF SPECTROSCOPIC METHODS

Much of the early work in applying spectroscopic methods to biological samples
involved measurements of bacteria. Naumann and coworkers [1] provide one of the first
uses of infrared spectroscopy to cellular systems and this was used to analyze pathogenic
bacteria. They were successful at classifying bacteria using standard microbiological
methods and in correlating this classification to certain features of the infrared spectra.
Dehydrated samples were employed and this might explain a comparatively small degree
of differentiation between species. IR measurements are highly sensitive to water
content and so much of the literature in this area focuses on reducing the impact of water
or on dehydrating samples prior to analyses. New sampling tools (utilizing fibers or
crystals that employ an evanescent wave that samples only a small depth) can minimize
the effect of water, but not eliminate it. Irudayaraj and coworkers [2] have used
photoacoustic infrared spectroscopy to characterize microorganisms. The photoacoustic
method is ideal for characterizing bacteria on surfaces.
More recent bacterial studies have demonstrated very sensitive measurements of
nucleic acids of bacteria using microcalorimetric spectroscopy with detection limits of 1
ng obtained [3]. Filip and Hermann [4] used infrared spectroscopy for discriminating
Pseudomonas species (aeruginosa, fluorescens, putida, and stutzeri). They found distinct
features for many cellular components, but the species could not be differentiated. The
authors deduced and presented a result which has significance for all cellular
spectroscopy. They determined that alterations in the composition of the nutrient broth
used to grow the cells resulted in changes of the absorption ratios of multiple spectral
features between fatty acids: proteins and between proteins: peptidoglycans /
polysaccharides. Differences were observed between freshly harvested and starved cell
biomass. It appears that richer broths lead to a greater proportion of peptidoglycan to
protein than do less rich broths. Better spectra were obtained using the attenuated total
reflectance (ATR) method than with transmission measurements.
The difficulties presented in spectroscopic measurements of bacteria actually suggest
several directions for non-invasive monitoring of cells and tissue of mammalian origin.
Since cells (bacteria and otherwise) grown in a nutrient rich media have different cell
spectra than do cells grown in a nutrient poor environment, this suggests that the method
is highly sensitive to small variations in cellular physiology and perhaps in cell function.
Spectroscopic Analysis Frontiers in Drug Design & Discovery, 2006, Vol. 2 261
Infrared spectroscopy has been applied to evaluate the biochemical composition of

mammalian cells or to discriminate between cancerous and healthy cells [5-7]. Many of
the difficulties encountered with bacterial measurements have been circumvented with
mammalian cell work. While discriminations can be made between stages of the cell
cycle due to alterations in spectral signatures of nucleic acids [8], only very small
differences are apparent between distinct sub-cellular structures. Analyses of separate
cell fractions presented the most significant spectral differences in the lipids, carbonyls
and phosphates [5]. To best explain these results, we shall next discuss the fundamentals
of these methods.
FOUNDATIONS OF SPECTROSCOPIC METHODS

Cell physiology and activity have been characterized using the methods of Fourier
Transform infrared (FTIR) spectroscopy which is as a non-destructive technique [9-10].
A beam of infrared light is introduced to the sample and either the transmitted or
reflected light is collected using a highly sensitive detector. Light absorbance at specific
wavelengths usually follows a Beer’s law behavior in which light absorbance is
proportional to optical pathlength, absorbtivity, and concentration:
Absorbance = εbC (1)
Light in the infrared (750 nm – 1000 µm in wavelength) is low energy and so does
not disrupt proteins or nucleic acids as do high energy photons in the ultraviolet.
Molecules have a set of resonance vibrations produced by thermal energy. When a
molecule is exposed to radiation from a thermal source, the radiation is absorbed only at
frequencies that correspond to the molecular modes of vibration in the infrared region
(IR) of the electromagnetic spectrum. Absorbance features that arise from fundamental
molecular vibrations (rather than the complex combinations and overtones observed in
the visible and ultraviolet) can be used to identify and quantify a molecule (a protein, for
example) and can also provide information on that molecule’s environment. For
example, phosphates of nucleic acids absorb photons in the wavelength region of 9174 –
9216 nm. Throughout this text we will use the wavenumber (cm-1) convention. To
convert from wavenumbers to wavelength, apply the following.
Wavelength = 107 / Wavenumber (2)
Spectral features from the fingerprint region between 4,000-400 cm-1 (2,500-25,000
nm) can supply information about the fundamental vibrational modes of functional
groups existing in biological molecules thus permitting quantification of material
composition.
Fig. 1 shows an infrared spectrum of E. coli bacteria grown under nutrient rich
conditions. Table 1 provides a summary of the location of notable absorbance features of
biological molecules. Within this spectral region, there arise a number of features
specific for biological compounds. CH 2 (that is, one carbon bonded to two hydrogens) is
found predominantly in long chain hydrocarbons, such as in the lipids that comprise the
cell membrane.
Additional features include the so-called amide (I, II, and III) vibrations which are
indicative of the vibrations of the peptide bond of proteins. Not only do the amide
features characterize the amount of protein, but each feature is comprised of multiple
smaller features, detailed in Table 1. For example, portions of the amide I usually are
ascribed to α−helical protein structures, β-sheet structures, and random coils. This
information is particularly useful when characterizing the response of a cell to various
stresses which may alter protein production or ultimately lead to protein unfolding. For
example, protein unfolding due to elevated temperatures often leads to a loss of α-helical
content with an increase in β-sheet and random coils. Protein alterations are apparent in
the spectra of bacteria exposed to elevated temperatures (Fig. 2). This Fourier self-
deconvolution (FSD) analysis permits identification of peak locations to simplify
classification of the biochemical source of absorbance. In this case, a number of protein
features are altered due to the application of heat. Specific peak locations can move
several cm-1 based on hydration and temperature of the sample.
Fig. (1). Infrared absorbance spectrum of a healthy E. coli culture.
RAMAN SPECTROSCOPY
Raman spectroscopy is a vibrational technique which is complementary to infrared
spectroscopy. Raman spectroscopy involves the measurement of the wavelength and
intensity of inelastically scattered light from molecules. Raman features are significantly
less intense than are infrared absorbances as only approximately 1 out of every 106
photons is inelastically scattered.
Raman spectroscopy has a number of similarities to infrared spectroscopy; however,
Raman measurements are not as greatly impacted by the presence of water as are
infrared methods. Raman spectroscopy has potential for use as a means to monitor for
compounds or organisms in aqueous materials particularly as water does not present a
substantial Raman signal. The application of Raman spectroscopy to monitor biological
systems has received less attention previously, for a number of reasons, the primary one
being the low signal intensities and comparatively high limits of detection compared
with traditional biochemical analyses. There are a number of means to improve this
signal intensity substantially.
Table 1. Infrared Peak Assignments for Common Biochemicals Within Cells and Tissue
Assignment Wavenumber (cm-1) Reference
Asymmetric CH2 band (lipids) 2924 [11]
Symmetric CH2 band (lipids) 2854 [11]
C=O of Nucleic acids 1716 [12]
Amide I of β sheet protein structure 1695-1660 [13]
Amide I of α helical protein structure 1660-1648 [13]
Amide I of random coil structure 1637 [13]
Amide II of protein structure 1541 [13]
Tyrosine 1514 [13]
Methyl deformation in proteins 1458 [13]
Sugars 1419 [14]
Amide III 1350-1180 [13]
ADP 1230 [15]
ATP 1211 [15]
Carbohydrates 1150 [5]
ATP 1118 [15]
ADP 1112 [15]
Glucose 1103 [14]
P=O and P-O-P of nucleic acids and phospholipids 1090-1085 [16]
Glucose 1080 [17]
CO-O-C symmetric stretching of sugars 1058-1060 [17]
Sucrose, trehalose 1047 [14]
Osidic C-O stretching of glucose 1030 [17]
DNA, phospholipids 964 [5]

Raman microspectroscopy combines molecular specificity with diffraction-limited

resolution [18]. Biological samples can be investigated under physiological conditions
and without extensive sample preparation. Several years ago Schrader [19] provided a
critical review of Raman methods for application in medicine. Raman spectra have been
published for biological tissue [20], nucleic acids [18]; myeloperoxidase and eosinophil
peroxidase [18] and for specific locations within cells [18,22]. The approach has been
used to determine the oxidation state of myeloperoxidase within activated granulocytes
using resonance Raman spectroscopy [23]. Raman spectroscopy has been used to
characterize and compare related types of cells. Tumorigenic and non-tumorigenic cells
can be differentiated based on variations in protein and nucleic acid content [24-25].
Fig. (2). IR spectra of E. coli cells exposed to elevated temperature (57.5oC) for the prescribed
times. Solid arrow highlights the decrease in the 1631 cm-1 while the dotted arrow highlights the
increase in the 1619 cm-1 feature.
Raman spectroscopy has previously been used to identify microbial species of

medical interest [26-27]. Freeze dried cells were found to yield good spectra, but with a
comparatively low information content compared with wet cells particularly on nucleic
acid content. Maquelin and coworkers [26] applied a 6 hour incubation period to
increase cell numbers but had to employ a mathematical treatment to remove conflicting
information from the underlying and changing culture media. They did observe features
that could be used to classify bacterial species.
In biological samples, Raman signals are generally weak and often differences in
peak intensities are difficult to quantify, especially in single cells or when a single
molecular species is of interest, such as specific proteins, organelles, or components of
the cytoskeleton or DNA. In addition, the excitation intensity must be kept low and the
exposure time short in order to avoid having the light affect the cell under measurement
(photo-induced damage). Photodestruction can be reduced by selecting longer excitation
wavelengths, such as in the near IR (which also reduces the fluorescence background
issue). Puppels [28] noted variations in sample degradation with the wavelength of
excitation light. A wavelength of 525 nm caused chromosomal damage whereas 660 nm
did not. Notingher and coworkers [29-31], observed that human epithelial cells are quite
transparent in the range 785-800 nm. Exposure up to 20 minutes can be conducted at 785
nm with 20 mW without observable cell damage. Excitation with longer wavelength
light generates weaker signals proportionally to the frequency to the 4th power.
While differences in Raman spectra of healthy and non-viable cells can be subtle,
changes in spectral features may be enhanced through methods to increase the Raman
signal intensity. When using near infrared (NIR) excitation, the best possibility of
increasing the intensity of the Raman signal is to use the Surface Enhanced Raman
Scattering (SERS) effect. The SERS effect can increase Raman signals by as much as 12
orders of magnitude. The first applications of SERS were reported for the adsorption of
species on electrochemically roughened silver, gold, and copper electrode surfaces and
with gold and copper colloids [32]. The SERS enhancement is attributed to a metal
electron mediated resonance Raman effect via a charge transfer intermediate state.
Colloidal silver and gold clusters used in SERS can provide extremely high
enhancement factors. This permits quantification of molecular concentrations down to
10-12 M [33] and can provide single molecule detection in femtoliter volumes [34].
Krafft et al. [35] applied Raman microspectroscopy to evaluate single cells. They
found that freeze dried cells gave good spectra, but the freeze drying process apparently
caused changes in the DNA conformation. Their measurements were performed with an
integration time of 1 min with a power of 25 mW using 785 nm excitation. This
approach has significant promise particularly due to the low effect of water on signal
intensities. With a small degree of development, and interaction with other means to
reduce the number of cells to be quantified at any one time, this approach has substantial
promise for providing a rapid and near-continual measurement of cell physiology and
function.
Fig. 3 below shows Raman spectra of E. coli cells before or after application of an
elevated temperature procedure designed to induce protein unfolding. These spectra
were collected using a confocal micro Raman spectrometer. There are substantial
alterations in spectral features particularly at 1450 and 1220 cm -1, features assigned to C-
H deformations predominantly of proteins.
DATA ANALYSIS AND SPECTRAL INTERPRETATION

For the analysis of data, mathematical approaches applied to the chemical analysis,
called chemometric techniques are applied. Methods for the analysis of spectra are
divided in two types, unsupervised and supervised methods [26]. The unsupervised
methods are used without previous knowledge of the microorganisms used. Each
spectrum is compared with others in order to make homogeneous groups or hierarchical
clusters. These methods are used to show similarity between spectra representing
organisms: same genus, species, and susceptibility to antibiotics. In supervised methods,
each spectrum is formerly assigned to a definite class a relationship is sought with
calibration data that provides information on the quantity of the organisms.
Fig. (3). Raman spectra of E. coli – healthy cells (top spectrum) and after application of an
elevated temperature procedure (bottom spectrum).
Using supervised and unsupervised techniques of analysis it is possible to determine

if the “unknown” spectra are included in the spectral database. This requires
standardization of culturing conditions (such as medium composition, temperature and
time of culture) and instrument parameters for the creation of reference libraries of
microorganism spectra. These libraries are the basis of microorganism identification
algorithms and permit the identification of an unknown microorganism on the basis of
its mass, FTIR or Raman spectrum [26].
METHODS FOR PROTEIN AND METABOLIC PROFILING

We have recently applied a unique fiber-based spectroscopic measurement scheme to
investigate alterations in the physiology and function of lung epithelial cells exposed to
inhalation hazards [36-40]. The principle of measurement is based on the concept of
fiber evanescent wave spectroscopy (FEWS) [10, 41-42, 34]. When a light wave is
guided through an optical fiber, part of its E field extends outside the surface and
constitutes the evanescent wave. The detection principle of these optical fiber sensors is
based on the absorption of the evanescent wave by substances in contact with the fiber
surface. This technique yields IR spectral information very similar to that obtained with
ATR spectroscopy. A challenge for biological monitoring is avoiding the effects of high
water content in samples. This may be reduced by employing a pre-screening step such
as provided by an immunopurification or other means to increase cell numbers while
decreasing sample water content. Utilization of evanescent wave spectroscopy reduces
the water impact.
Anchorage dependent cells, such as epithelial type II pneumocytes (A549 cells) can
be grown attached to the surface of the fibers. Baseline spectra are collected with healthy
cultures. The cells are then exposed to various chemical hazards and their response
tracked through monitoring the spectral absorptions located between 3000-900 cm-1 due
to hydrocarbon vibrations of methyl and methylene groups in membrane lipids, due to
proteins (amide I and amide II vibrations), and other components. The cells are
maintained at near to complete confluence (full coverage of their attachment surface)
which inhibits cell replication thus reducing variations from cell to cell in their stage of
the growth cycle.
Absorption bands in the regions of 2800-3000 cm-1 (corresponding to membrane
lipid features) and within 1700-1400 cm-1 (corresponding primarily to protein features)
change rapidly upon exposure of the cells to each toxin. These alterations provide
mechanistic information on the cell damage and response which correlates with
information from standard biochemical analyses [34]. Some compounds cause initial
damage to the cell membrane which is displayed in rapid alterations to the lipid features,
followed by later changes in proteins. Compounds that damage nucleic acids alter the
features corresponding to phosphates of nucleic acids. Similarly, membrane damaging
agents primarily alter the CH2 features of lipids.
A comparison of the peak intensities of A549 cells exposed to differing concen-
trations of inhalation hazards demonstrates a dose response with specificity in the
cellular component affected [38]. For example, the alkylating agent methylmethane-
sulfonate (MMS) damages the cell membrane and hence lipid specific features at 2854
and 2924 cm-1, decrease by 0.7 and 0.8 absorbance units, respectively for cells exposed
to 5 mM MMS; these features decrease by 0.5 and 0.6 absorbance units, respectively for
cells exposed to 2 mM MMS. These changes correspond to the biochemical mechanisms
of MMS toxicity.
Exposure to the fungal metabolite gliotoxin leads to a complex absorbance profile
over time at a number of features (Fig. 4). 5 µM gliotoxin produces a small decline in
A549 cell absorbance features for lipids at 2852 cm-1 and 2922 cm-1. These changes
occur consistently over 20 hours of continual exposure to gliotoxin. α-helical protein
content and sugars show an increase for the first 4 hours of exposure, followed by a
decline from 12 - 20 hours. Many biochemical features are fairly constant for the first 4
hours, have a step change decrease at 12 hours, and then remain constant thereafter.
Some, such as ADP and glucose show a slow increase over time. Fig. 4 below shows
alterations in sugar features, specifically the C-O stretch, C-O-H bend, of carbohydrates
(1150cm-1).
This spectral analysis provides information on both mechanisms by which cells are
damaged by toxins and the approaches by which the cell responds. For example,
apoptosis inducing compounds, which lead to condensation of DNA, produce significant
changes in the spectral features of nucleic acids. Alterations in protein features
reasonably follow the time course of induction of a number of cell response genes.
Fig. (4). Alterations in sugar composition within A549 cells after application of 10 µM gliotoxin, a
fungal metabolite.
The infrared spectroscopic measurement approach has several advantages over more
traditional methods of evaluating cellular response to toxic or pharmacologically active
compounds. The measurement can be performed very quickly requiring only minutes
whereas cell responses can begin from minutes to hours after exposure to active
compounds. The cells are sensitive to a wide range of environmental toxins and their
response through initiation of a number of pathways to recover from the stressor can be
identified through spectral analysis.
A limitation of this spectroscopic analysis lies in the difficulty of identifying the
specific identity of a compound which has caused an alteration in spectral features. Most
environmental monitoring methods focus on quantifying a limited number of potential
toxins such as metals, pesticides, or polyaromatic hydrocarbons. As is typical of cell-
based sensing methods, the target for quantification is not specific identification of the
agent but rather biological activity. Hence specific concentrations of a toxin cannot be
determined, although a dose response relationship can be demonstrated. The mechanism
of action can also be identified through analysis of cell components which display
damage. Additional cell types can be employed in parallel so as to modulate sensitivity
to certain toxins.
The spectroscopic technique has the potential to be applied outside of laboratory
settings and permit continual monitoring of the potential impact of toxins on human
health. In a regular infrared spectroscopy experiment, the sample would have to be
extracted and placed across the beam path inside the analytical instrument. This requires
extremely thin samples and/or applied on an infrared transparent substrate. Similarly, an
ATR experiment would require that the sample be extracted and introduced into the
instrument. However the development of high quality infrared optical fibers now allows
for in situ measurement where the signal is brought to the sample and collected back to
the detector using a single optical fiber. This type of remote analysis has many
applications in medicine and drug discovery. It has been applied to the detection and
monitoring of a wide range of biomolecules [34]. This technique is especially suited for
in vivo analysis such as monitoring of biological processes [43], and biomedical sensing
including non-invasive glucose measurements [44] and cancer diagnosis [45].
Additionally, fiber analysis permits one to collect signals remotely from cell culture held
in controlled conditions in an environmental chamber.
Remote spectroscopic sensing can only be performed with fibers that have large
optical windows in the infrared, low optical losses and sufficient chemical durability.
Chalcogenide fibers are based on S, Se and Te containing glasses combined with nearby
elements on the periodic table, particularly, As, Ge, Sb. Owing to higher atomic mass,
these glasses exhibit low phonon energies and possess a wide transparency window in
the infrared region, from visible up to 18 µm depending on the composition. This optical
window encompasses the vibrational modes of virtually all organic molecules and is
therefore ideal for biochemical sensing.
In this fiber sensing technique, the evanescent wave that interacts with the sample
decreases in intensity exponentially with increasing distance from the fiber surface. The
technique therefore only collects signal from within about 0.5 µm of the fiber surface
where the evanescent wave is most intense. It then allows very localized probing that
could prove useful for bacterial strain identification (or for analysis of membrane acting
compounds) since many structural features providing the possibility of differentiation
are presented on the cell surface [46]. It is also beneficial to collect strong signals from
the cell itself while minimizing signal from the surrounding fluid.
Another benefit of utilizing a chalcogenide fiber is the possibility of shaping the fiber
into a reduced-diameter sensing zone. The fiber diameter is typically reduced from 400
µm to 100 µm along a distance of a few centimeters or less. This geometry considerably
increases the evanescent wave intensity along the reduced diameter zone. This provides
two major advantages to the technique. First, the sensitivity is greatly increased in
comparison to the equivalent ATR due to the much larger number of internal reflections
that result in many absorption events at the interface between the glass and the sample.
Second, the signal is entirely collected along the reduced diameter zone and therefore
prevents collecting noise signal from the surrounding environment. Finally, the
chalcogenide fiber surface exhibits a hydrophobic behavior that is beneficial in
minimizing interference of water during signal collection. Water has a strong infrared
signal that overlaps with many organic molecules. Since many biochemical processes
take place in an aqueous environment it is therefore beneficial to minimize this
interference. The hydrophobic nature of the glass surface results in selective interaction
and detection of organic species at the fiber surface relative to water molecules.
Hydrophobic organic species with a low dielectric constant can therefore be detected
more efficiently in aqueous media using chalcogenide fibers.
There are several significant challenges which need to be addressed before this
approach reaches field use capabilities. Cell adhesion to the fiber surface can be
inconsistent thus changing the number and mass of cellular matter in contact with the
evanescent wave. This leads to the requirement of performing relative measurements of
cell composition before and after exposure. The poor adhesion is potentially due to
compositional effects of the TAS material. Some arsenic does leach out of the fibers;
however a washing step appears to remove this pool of mobile arsenic and so direct
arsenic toxicity is likely not a significant factor. During the measurement process
variations in water content may impact measurements and could distort the trends
observed here. This can potentially be addressed through increased consistency in the
length and thickness of the drawn sensing zone of the fiber.
Interpretation of the complex spectral changes resulting from cell exposure to toxic
compounds presents a significant challenge at least in part due to the interference of
water vapor signals in the 900-1700cm-1 region of the cell spectrum. While the water
signal overlap complicates the interpretation, some consistent and reproducible changes
can be observed in some specific spectral features of the cellular material exposed to
toxicants.
Higher level analysis techniques such as partial least squares regression and principle
component analysis could be used to extract patterns in the evolving spectral features so
as to increase the predictive capability of the technique. For instance, the pattern of
changing spectral features indicative of cell response to gliotoxin is different from that of
MMS. Development of a substantial database of cell spectra along with such complex
analysis tools could assist in discrimination of a cell response pattern indicative of
gliotoxin exposure rather than that due to a genotoxin. Such a database will require
spectra of cell responses to each toxin at varying concentrations.
SUMMARY
Spectroscopic methods including use of infrared and Raman techniques can provide
information on cellular physiology and function. Alterations in cellular lipids, proteins,
carbohydrates, and nucleic acids are the most readily identified. These approaches have
significant potential for high throughput screening of pharmaceuticals, for testing
toxicity, and for quantification of environmental hazards.
ACKNOWLEDGEMENTS
This work was supported by DARPA contract # N66001-C-8041, by the NIEHS
sponsored Southwest Environmental Health Sciences Center # P30 ES06694, and by the
Arizona Board of Regents Technology and Research Initiative Fund.
ABBREVIATIONS AND SYMBOLS

ATR = Attenuated total reflectance
B = Optical pathlength
C = Concentration of an analyte
FEWS = Fiber evanescent wave spectroscopy
FSD = Fourier self-deconvolution
FTIR = Fourier transform infrared spectroscopy
IR = Infrared
MMS = Methylmethanesulfonate
SERS = Surface enhanced Raman scattering
TAS = Telurium arsenic sulfide
ε = Molar absorbtivity
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Encapsulated Biomolecules Using Sol-Gel Reaction

for High-Throughput Screening
Kumiko Sakai-Kato †, Masaru Kato*,‡,§,

Naoko Utsunomiya-Tate† and Toshimasa Toyo'oka‡
†
Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20
Shinmachi, Nishitokyo-Shi, Tokyo, 202-8585, Japan; ‡School of Pharmaceutical
Sciences and COE Program in the 21st Century, University of Shizuoka, 52-1 Yada
Suruga-ku, Shizuoka, 422-8526, Japan and §PRESTO, Japan Science and Technology
Agency (JST), Saitama, Japan
Abstract: Recently, the sol-gel encapsulation method has attracted much
attention for the development of desirable protein-doped matrices as
biosensors. Proteins are entrapped into a porous, silica matrix that is formed
via a low-temperature sol-gel reaction. The encapsulated proteins can retain
their structure and biological activity for a prolonged period. This sol-gel
encapsulation method allowed the reuse of expensive protein reagents multiple
times. Furthermore, the encapsulation method often improved the stability of
the immobilized proteins. Based on these reasons, this technology has been
used in various fields, and is expected to contribute to the effectiveness of
analytical systems and the application to high-throughput screening systems. In
this review, we introduce various studies in which biomolecules were
immobilized on capillary-, microchip-, and microarray-based analytical
systems using the sol-gel reaction. The interactions of the immobilized
biomolecules and analytes were detected using UV, fluorescence, mass
spectrometry, or electrochemical detection. On the other hand, many
researchers are studying the sol-gel processing to improve the biocompatibility
of the sol-gel derived materials using new biocompatible silane precursors and
processing methods. The microstructure of the silica matrix was also
investigated using various analytical systems. We also review some reports
that described the fundamental aspects of the sol-gel reaction.
1. INTRODUCTION
Recently, it became possible that many groups of compounds can be synthesized at
the same time due to the progress of combinatorial chemistry. Furthermore, due to the
development of bioinformatics, a large quantity of information can be accumulated
which is indispensable to research for drug discovery [1,2]. These situations calls for
flexible, fast and cost-effective strategies to meet the demands of evaluating the high
content lead series with improved aspects for clinical success [3,4]. Biomaterials
*Corresponding author: Tel: +81-54-264-5654; Fax: +81-54-264-5654; E-mail: daikato@u-shizuoka-ken.ac.jp

274 Frontiers in Drug Design & Discovery, 2006, Vol. 2 Sakai-Kato et al.
including proteins have attracted many researchers' interest due to their highly specific
recognition ability and effective catalytic ability. It was very natural that a bioreactor
was produced that made use of these biomaterials outside the living body for the food or
medical industries [5]. This was used in a variety of fields, and as a consequence, new
technologies for the immobilization of these biomaterials have been developed for their
more efficient use [6]. Various immobilization methods are reported as this way of
fixation as shown in Table 1, when the enzyme is being most widely used as a
biomaterial. These include the covalent attachment to supports [7-9], adsorption onto
solid supports [10], cross-linking with bifunctional reagents [11,12], entrapment in gels,
encapsulation in membranes with microcapsules, liposomes, hollow fibers, or dialysis
membranes [13, 14]. However, such techniques are not generic, and in most cases, can
be used only for a limited range of biomolecules or applications.
Table 1. Enzyme-Immobilization Technique
Method Characteristics Advantage Disadantage Ref.
Covalent The most popular supports Producing a very The potential for [7-9]
Attachment to are porous ceramics. rigid bond. inactivation of the
supports active site during the
immobilization process.
Adsorption on Physically adsorption on The simplest and The adsorbed product is [10]
solid supports various inorganic or cheapest approach. not very stable.
organic supports.
Cross-linking To form agglomerations Producing very The attachment can [11,12]

with bifunctional of enzymes or stable system. destroy catalytic
reagents incorporate enzymes into activity.
a polymer.
Entrapment in The enzymes were Inexpensive, The reagents may not [13]
gels entrapped in the gel performed under always reach enzymes.
during the polymerization. mild conditions.
Encapsulation in With microcapsules, Working well. Best Expensive. [14]

membranes liposomes, hollow fibers, suited to medical
or dialysis membranes. applications.
Recently, the sol-gel encapsulation method has attracted much attention for the
development of desirable protein-doped matrices for biosensors. The silicate matrix is
formed by the hydrolysis of alkoxide precursors followed by condensation to yield
polymeric oxo-bridged SiO2 networks. Conventional sol-gel methods involve the use of
a high concentration of methanol as a co-solvent for the precursors, often with high
acidity [15]. However, Elleby et al. developed a modified version of the process that
removes the need for the addition of methanol. This version does not expose the
biomolecules to the damaging effects of low pH [16]. These improvements enabled
encapsulated proteins to retain their structure [16] and biological activity for a prolonged
Encapsulated Biomolecules Frontiers in Drug Design & Discovery, 2006, Vol. 2 275
period [17] and to enhance its utility as a new type of matrix for biomolecules. Silica has
physical and chemical properties which are quite attractive from the viewpoint of joining
inorganic materials with biomolecules and living cells; the thermodynamic stability of
the Si-O bond, 452kJmol-1, indicates a very strong inertness that excludes any
interference with enzymes and functions typical of differentiated cells.
The entrapment of proteins and other biological species into a wide range of sol-gel
derived nanocomposite materials, and their use as biosensors and biocatalysis
alpplications, has been reviewed by some researchers [18-22]. In addition to the general
overview of the sol-gel encapsulation techniques for biomaterials, this review focused on
the utility of biocomposite materials in numerous applications to high-throughput
screening systems primarily for drug discovery, including the stationary phases for
affinity chromatography, capillary electrochromatography, microchip electrophoresis,
and microarrays. Finally, the additional possibility of sol-gel biocomposites and future
subjects are addressed.
2. THE SOL-GEL REACTION

The sol-gel reaction can be divided into the following steps, Fig. (1): (1) the
hydrolysis of an alkoxysilane; (2) the condensation of hydrated silica to form siloxane
bonding (≡ Si-O-Si ≡); and (3) the polycondensation that involves the linking of an
additional silanol group to form cyclic oligomers.
Fig. (1).Typical sol-gel reaction and monomer compounds.
The properties of each individual sol-gel matrix can be altered by a number of

factors. These include pH, temperature, reagent concentrations, reaction time, the rate of
hydrolysis and condensation, and the nature of the catalyst. The most common starting
reagents of the reaction are tetramethoxysilane (TMOS) or tetraethoxysilane (TEOS)

because they can be readily hydrolyzed and condensed under mild conditions. Many
alkoxysilanes and their derivatives are now commercially available. The hydrolysis is
mainly performed with a catalyst: acid or base catalysis. It is generally agreed that, under
acid catalysis, it is easy to form a linear network, because the velocity of the hydrolysis
reaction is faster than that of the condensation reaction. Whereas, under base catalysis,
entangled or randomly branched chains are formed, because the velocity of the
condensation reaction is faster than the hydrolysis reaction [15].
3. STRUCTURE OF THE MONOLITH

3.1. Porous Monolith
Many silane compounds are used as precursors of the silica monolith. These include
tetraalkoxysilanes, mono-, di- or tri-alkyl alkoxysilanes; many comprise functional
groups ranging from alkenyl, aryl, amino, carboxyl, thiol or other groups. The precursor
is hydrolyzed by water, either spontaneously or under acidic or base catalysis, to form
hydroxy derivatives (silicic acids, hydroxysilanes, etc.). A cascade of condensation
reactions gives rise to soluble, colloidal and ultimately phase-separated polymers
(polysilicates, polysiloxanes, etc.), which produce the final matrices (called monoliths).
The monolith has nano-sized pores, which are used for the encapsulation of
biomolecules. The initial gels are typically brittle or semiflexible, and contain 50-80%
interstitial water, with pore volumes of 0.4-3.4 mL/g, pore distributions of 4-200 nm,
and surface areas of 600-2100 m2/g [19, 20,23-25], Fig. (2).
Fig. (2). Schematic representation of the sol-gel process.
Aging of the wet silica network over a period of days to weeks promotes further
condensation and strengthens the network. Most of the monolith can been dried by
evaporation of the solvent. If only simple silica alkoxides are used as precursors, the wet
gels mostly carry Si-OH side groups which are hydrophilic and induce a considerable
capillary force (typically 70%) in the entire structure. That is to say, the walls of the
nano-sized pores shrink, and a monolith called an “xerogel” is produced.
There is another type of monolith which is termed an “aerogel”. The aerogel is made
by ambient pressure drying or by prevention of solvent evaporation during drying. A
supercritical drying is used for ambient pressure drying [26].
If the monolith is used for the encapsulation of some molecules, the monolith must
have a pore size that is sufficiently small to prevent leakage of the molecules, but large
enough to allow smaller analytes to enter the monolith with ease. These monoliths form
nano-capsules for the encapsulation of molecules or function as a glue for the immo-
bilization of the molecules. Examples of their applications are shown in the latter part of
this review. Although these types of monoliths can be prepared by a simple procedure,
they do not have µm-sized pores, which are termed “through-pores”, therefore, they
cannot be used for the analysis of large molecules that cannot enter the monolith. To
overcome the above-mentioned defect, another type of monolith, which has through-
pores, was developed.
3.2. Bimodal Porous Monolith

A bimodal porous monolith has two different pore sizes, which are µm-sized
through-pores and nm-sized mesopores, in the matrix. A porogen is often used for
creating a bimodal porous monolithic bed. Porogens play dual roles: they serve (a) as a
through-pore template, and (b) as a solubilizer of a silane reagent. A porogen is used to
create desired morphologies with intended permeabilities and surface areas for the
construction of a monolith. A water-soluble organic polymer, poly(ethylene oxide)
(PEO) or polyethylene glycol (PEG) has been used as a porogen by many researchers.
Toluene has also been used as a suitable porogen for a photopolymerized sol-gel (PSG)
monolith [27,28]. Using the proper porogen, spinodal decomposition occurs and a
porous monolith is produced, Fig. (3). Spinodal decomposition is a clustering reaction in
a homogeneous solution. The phase separation was solidified by gel formation, resulting
in a silica rod with a bimodal porous structure consisting of µm-sized through-pores and
meso- or nano-porous silica skeletons. The decomposition mechanism of the polycon-
densation of an alkoxysilane in the presence of PEO was extensively studied by
Nakanishi et al. [29-33].
Fig. (3). Schematics of spinodal decomposition.
Silica rods with a bimodal porous structure typically possess 0.3-5 µm silica
skeletons, 0.5-8 µm through-pores, and 2-20 nm mesopores in the skeleton [34]. The
sizes of the skeletons and through-pores were independently controllable by changing
the preparation conditions. A porogen has often been used for controlling the size, Fig.
(4). Alkali treatment of the monolith has been reported to form mesopores on the
surface, and the strength of the used alkali, treatment time, and temperature affect the
size of these meso-pores [35]. These monoliths were used for separation columns,
bioreactors, and so on. Examples of this application are shown below.
Fig. (4). Effect of porogen content on the bimodal porous structure.
4. SILANE PRECURSOR AND SOL-GEL PROCESSING FOR IMMOBILIZA-

TION OF BIOMOLECULES
The sol-gel reaction involves the formation of a colloidal sol solution by hydrolysis
of a precursor, such as TEOS or TMOS. A buffered solution containing the biomolecule
of interest is then added to the sol to initiate rapid polycondensation of the silane.
Following polycondensation, a hydrated gel is produced that immobilizes the biological
element without the need for a covalent tether.
Although the hydrolysis of the alkoxysilane, such as TMOS or TEOS, are widely
used for the immobilization of biomolecules due to the simple procedures, during the
hydrolysis of silane precursors, TMOS or TEOS, methanol or ethanol is produced. These
byproducts will readily dissolve or destabilize existing proteins or the bilayer membrane
structure. Therefore, another route using aqueous sodium silicate as a starting material
was developed in order to avoid the production of an alcohol [36,37]. In this route, SiO2
networks are produced by acidifying aqueous sodium silicate. By using colloidal silica
together, silicates condense on the surface of the colloidal particles that behave as
nucleation sites for condensation. Aqueous sol-gel processes were produced to reduce
the effect of the alcohol. This allowed the immobilization of the bacteria. Sakai-Kato et
al. also used this technique and showed the immobilization of cytochrome P-450 [38].
By using this method, the activity of the oxidation of testosterone drastically improved,
compared with that using TMOS-derived silica matrices. Ferrer et al. developed other
aqueous routes. The methods used vacuum elimination of the alcohol by a rotary
evaporation method. Biomolecues were added to the sol after removal of the alcohol and
then the condensation reaction occurred [39, 40].
To achieve entrapment of active biomolecules in the sol-gel derived silica, it is
necessary to maintain the active conformation of the biomolecules within the matrix, and
for the enzyme, ensure that the entrapped enzyme is accessible to substrates. A number
of sol-gel derived materials have been designed with the purpose of making the matrix
more biocompatible with the entrapped biological molecules. For example, new
biocompatible silane precursors and processing methods have recently been reported
based on glycerated silanes [20,41]. Brennan et al. developed the use of a silane
precursor bearing a covalently attached sugar for the formation of protein-doped sol-gel
derived silica. Silica materials containing covalently bound glucosamide moieties
provide a biocompatible environment for entrapped Src kinase, and a sufficient porosity
to allow polypeptide substrates to enter the glass and interact with the entrapped Src
kinase [42]. Another approach involves the use of protein-stabilizing additives to
increase the protein stability, including ligand-based stabilizers [43,44], organosilanes
[45,46], poly (ethylene glycol)[47], graft copolymers, such as polyvinylimidazole and
polyvinylpyridine [48-50], and charged polymers, such as poly(vinylimidazole) and
poly(ethyleneimine) [51]. Polymers such as polyvinyl alcohol, alginate, gelatin, and
chitosan were used as additives, directly mixed in the silica sol before gelation [52,53].
Entrapped proteins have also been shown to be stabilized by the addition of small
molecules such as sugars and amino acids (osmolytes) during sol-gel processing due to
changes in the excluded volume and protein hydration [54,55]. However, such species
could be easily removed from the matrix by washing, resulting in a significant loss in
protein stability and poor reusability of the entrapped protein [55]. As a further
improvement, it is possible to encapsulate biomolecules together with an additive that is
beneficial for the stability or bioactivity of the biomolecules. These additives can be
hydrophobic moieties brought about by alkoxysilane precursors, carrying, for instance,
alkyl R groups. In this regard, an important contribution was made by Reetz et al. [56]
who showed that such hydrophobic moieties could improve the activity of lipase beyond
that of the free enzymes.
5. INVESTIGATION OF SOL-GEL STRUCTURE

5.1. Structural Evolution
It is important to discuss the structural evolution during the process of hydrolysis or
condensation of a silica solution. Because the structural evolution determines the final
gel structures, it affects the mode of the biomolecules entrapment. The methods of
choice for determining structures are nonintrusive in situ methods such as nuclear
magnetic resonance (NMR) spectrometry, Raman and infrared spectroscopies, and X-
ray, neutron, and light scattering.
1
H and 29 Si NMR have been extensively employed to elucidate the extent and
kinetics of the hydrolysis and condensation reactions accompanying gelation, and the
speciation of silicate solutions during the early stage of the polymerization of TMOS,
TEOS and several oligomeric species, Si2O(OR)6, Si3(OR)8, and Si8O12(OR)12 [57-59].
In addition, 17O and 13C NMR have been used to monitor the water and solvent content
in several systems [60].
Raman and Infrared spectroscopies have been used both in combination with NMR
to identify specific oligomeric species in solution and to follow the evolution of
inorganic frameworks by comparison with model compounds of known structure [61].
Balfe et al. combined Raman and Fourier transform infrared (FT-IR) spectroscopies to
determine the hydrolysis behavior of model linear and cyclesilicate compounds. Both
methods provide information about the Si-O-Si bond strain with different vibration
modes [62-64]. Attenuated total reflectance (ATR)-FT-IR has many advantages over the
conventional method of using a potassium bromide disk. ATR enables one to measure
silanol groups without drying the gel. Furthermore, it allows a continuous measurement
during the time-course of gelation [65]. Small-angle-scattering investigations utilizing
neutron (SANS), X-rays (SAXS), or visible light have been employed to investigate the
growth and topology of macromolecular networks that precede gelation, the aggregation
of colloids, and the structures of porous gels and aerogels. Martin and Hurd [66],
Schaefer [67], and Schaefer and Keefer [68,69], have published excellent reviews of this
topic. During the process of gelation, the polycondensation of alkoxysilane monomers
leads to the formation and growth of soluble particles and then colloids. These clusters
coalesce and raise the solution viscosity for the sol-gel transition, at which bulk gelation
occurs (gelation point) [15]. Because dynamic light scattering (DLS) method is often
used for measuring the hydrodynamic radius of a macromolecule or colloid, this
technique was employed to evaluate the changes in the cluster diameter during the
gelation process and investigate how proteins influence the gelation [65, 70].
5.2. Gel Structure

The characteristics of the resulting gel were investigated using various methods. The
pore diameter of the xerogel is measured using nitrogen adsorption isotherms, and
specific surface areas were calculated from the Brunauer-Emmett-Teller (BET) analysis.
The microstructures of the xerogels were observed using scanning electron microscopy
(SEM) [71]. Compared with the xerogel, the structural investigation of the hydrogel is
more difficult due to its high water content. The pore size distribution of hydrogels was
measured using calorimetry [71, 72]. The porous materials had dynamic and thermo-
dynamic relationships applied for the freezing and melting curves of water in gels filled
with water [71, 73]. The microstructure of the hydrogel was observed using transmission
electron microscopy (TEM) [38].
5.3. The Characteristics of Immobilized Biomolecules

The structure of the gel is very important, because it regulates the activity or
conformation of the entrapped biomolecules. Because the porous glasses prepared by the
sol-gel technique are optically clear, it is possible to examine the spectroscopic
characteristics of the entrapped biomolecules for clarifying the physical environment of
the immobilized protein or the mechanism of entrapment [74]. The steady-state and
time-resolved fluorescence spectroscopy of the intrinsic Trp is used to probe the internal
microenvironment of the sol-gel-derived materials [75]. The nature of the interaction
between the sol-gel derived silica matrix and electron transfer proteins were explored
using the optical absorption spectra for the proteins for determining the synthesis
conditions of the materials [76]. Another approach is that the fluorescence probes are
used for investigation of the local structure and dynamics of encapsulated biomolecules
in the sol-gel materials. The small organic probes and fluorescent biomolecules provide
information about the pore-solvent composition and polarity, internal solvent and dopant
dynamics, environmental heterogeneity and phase segregation, and surface chemistry, as
determined by the molecule-matrix interaction [77,78].
Resonance Raman spectrometry is also used for the protein structure in the sol-gel
encapsulated state of the myoglobin [79]. IR also provides information about the
proteins entrapped in the gel [80,81]. Two representative bands that originated from
BSA were observed in the region between 1800 cm-1 and 1400 cm -1, Fig. (5). The signal
around 1657 cm-1 is the amide I band, which mainly consists of υC=O stretching
vibrations. The signal at 1549 cm-1 is the amide II band, which mainly consists of υN-H
bending vibrations. These two bands change the position depending on the structural
Fig. (5). IR spectra of BSA in gel and in free solution.
changes as well as the degree of hydrogen bonding [81]. It is also reported that the amide
I/ amide II intensity and area ratios may be associated with conformational changes in
the protein [81]. Table 2 shows a comparison of the peak positions and amide I/ amide II
area ratios obtained from BSA in the TMOS-derived gel and in free solution. The peak
positions of each peak show no differences in both formats. Furthermore, the peak area
ratios are very close to each other. These strongly suggest that BSA maintains its
conformation after encapsulation in the gel [65].
Table 2. Comparison of the Amide I and II Bands for BSA Encapsulated in Gel and in
Free Solution
Position Area ratio
amide I (cm-1) amide II (cm-1) (amide I/amide II)
encapsulated in gel 1657 1549 1.56
in solution 1657 1549 1.43
The image of the immobilized biomolecules were directly observed using the TEM
[38], Fig. (6).
Fig. (6). TEM image of immobilized microsome. Bar: 1 µm.
6. APPLICATION
Protein-doped silicate materials have been applied in a variety of forms. It is one of
the most important advantages of the sol-gel reaction. Therefore, we can optimize the
configuration of the sol-gel derived biomaterials to provide the utmost performance for a
given application. Typically, silicate biocomposite materials can be prepared as
monolithic blocks, powders, thin films, microarrays, or fibers, as shown in Fig. (7). The
choice of a particular configuration relies on factors such as required protein loading,
desired response time, sensitivity and detection limits, and the ability to interface the
material to common analytical devices.
Fig. (7). Configurations used to interface sol-gel derived materials.
6.1. Study for Protein Charactersitics

Bulk glasses (monoliths), which are prepared by adding the protein-doped sol to a
mold, such as a optical cuvette, and allowing gelation to occur, provide relatively high
total protein loadings and long pathlengths, making monoliths useful for the fundamental
studies of protein behavior in a glass (thermodynamic stability, dynamics, accessibility,
binding constants ) [75,77,82-85], and is also useful for biophysical studies, such as the
kinetics of the allosteric transition of proteins by restricting and slowing down protein
structural changes [86,87].
6.2. Sensor
The bulk gel entrapping proteins have been successfully used as platforms for optical
biosensors[16,43, 88]. The disadvantage of the bulk glass is the long response times for
sensing applications, due to the slow diffusion of reagents within the monolith [89].
One of the most technologically important aspects of the sol-gel process is that, prior
to gelation, the fluid sol or solution is ideal for preparing thin films by such common
processes as dipping, spinning, or spraying. Sol-gel film formation requires considerably
less equipment and is potentially less expensive. Sol-gel film was formed on one optical
face of a 1-cm pathlength polystyrene cuvette. This metalloprotein–entrapped gel was
used for nitrogen monoxide or carbon monoxide sensing [90,91].
Optical methods can be used to quantify reactions that generate a color change,
whereas electrochemical detection is suitable for redox reactions. Biomaterials that can
be used in electrochemical biosensors include enzymes, antibodies, antigens, oligo-
nucleotides and DNA fragments. Biomaterials can either be immobilized on the surface
of electrodes or trapped within conductive materials. Composite ceramic carbon
electrodes in which an enzyme-loaded carbon powder is mixed in the sol-gel solution
have been extensively developed [92,93]. These electrodes can be prepared in virtually
any desired shape; as thick supported films, as discs or rods, and even in the form of
microchips [94]. Sandwich configurations in which enzymes are deposited between two
sol-gel silica layers of controlled porosity have also been developed. They offer rapid
diffusion of the substrate through the upper porous layer and a shorter enzymatic
reaction along with high enzyme loading [95]. Glucose oxidase (GOD) is one of the
most widely used enzymes for the biosensing of glucose, due to its significance in
clinical analysis for the diagnosis and treatment of diabetes, and in biotechnology and
the food industry. GOD catalyzes the oxidation of glucose by molecular oxygen into
hydrogen peroxide and gluconic acid. Oxygen depletion can be electrochemically
determined by an oxygen-sensitive electrode [96,97].
6.3. Chromatographic Column

Although the bulk form provides relatively high total protein loadings and long path-
lengths, bulk glasses tend to have a long response time due to slow diffusion of the
reagents within the monolith. This is especially problematic when it comes to enzymes,
because its causes a decrease in the reaction efficiency within the monolith compared
with that in solution. To overcome this problem, the integration of the bio-doped gel into
a flow-through system is quite ideal. The on-line method is also expected to facilitate the
development of the high-throughput analytical system of multiple analytes for drug
discovery research.
6.3.1. Packed Column
A packed column was prepared by loading the ground glass containing proteins into
a column. An organic or non-organic molecule-doped sol-gel was prepared as a bulk gel.
The following typical preparatory procedures are as follows: after drying, gels are
crushed using a mortar and pestle, then sieved. The bioaffinity columns are fabricated by
loading a slurry containing the particles into a column. This format leads to good flow
rates (due to interparticle spacings) along with a high surface area due to the nanometer
scale pores in the material. Such materials also allow for high protein loadings. Alkaline
phosphatase, and a number of other enzymes were effectively entrapped in the sol-gel
glass with a retention of approximately 30% of their enzymatic activities [17]. A protein
A-trapped column was prepared for the purification of sheep immunoglobulin G [98].
The sol-gel was derivatized by γ-aminopropyltriethoxysilane to provide a matrix that
eliminates the nonspecific absorption of proteins. On the other hand, immunoaffinity
chromatography columns can be prepared by the encapsulation of antibodies in sol-gel
glass networks for the removal of carcinogenic pyrene. Pyrene could be isolated and
enriched by a factor of 125 by this column. No change in the specific retention
properties could be observed after 10 absorption/desorption cycles. The column was
used for the analysis of PAHs [53,99,100]. Other groups also encapsulated monoclonal
anti-atrazine antibodies in sol-gel matrices [101]. Zusman’s group has developed
columns using glass fibers covered with antibody-doped sol-gel glass and used them for
the affinity separation of tumor-associated antigens from blood [102]. Although the
entrapped materials were not biomolecules, a biogel column was also prepared using this
method. Narang et al. prepared a synthetic ribonuclease inhibitor-doped sol-gel column
for the removal of ribonuclease from solution [103].
6.3.2. Monolithic Column
Kato et al. prepared biomolecule-encapsulated columns using the sol-gel method,
Fig. (8) A, in a single step for capillary electrophoresis (CE) and capillary electrochro-
matography(CEC) [52, 104-110]. In their method, grinding the silica matrices before
packing was not required because the polymerization reaction was performed within the
separation column. The sol-gel reaction proceeds within a capped capillary, which can
minimize drying of the gel. The resultant hydrogel immobilizes various biomaterials
with wide range of sizes, from proteins to microsomes. The electroosomotic flow (EOF)
is generated as a flow when the voltage is applied at both ends of a capillary and can
carry the analyte to the encapsulated biomolecules within gel matrix. The water content
of this hydrogel calculated from the weight reduction after drying is around 70 %, which
coincides with the water content in living organisms. This is expected to retain the
encapsulated biomolecule function for a long period. The hydrogel was prepared using
TMOS as the starting material, then hydrolyzed by HCl so that the hydrolysis proceeded
form a fully or partially hydrolyzed silane, SiOH4-n (OMe)n. A buffered solution
containing proteins was added to the hydrolyzed silica solution. The mixture was put
into a capillary, which had previously been filled with running buffer. Both ends of the
capillary were sealed and placed at 4 ºC for more than 4 days. Bovine serum albumin
(BSA) was encapsulated within the hydrogel, and the column was used to separate
tryptophan enantiomers. A mixed solution of TMOS and methyltrimethoxysilane
(MTMS) was used as a precursor for an ovomucoid-encapsulated column, and the
column was used to separate benzoin and basic drug enantiomers [104]. The effects of
various factors, such as pH and concentration of the eluent, on enantiomeric separation
were examined. The BSA-encapsulated column was also used for the evaluation of the
binding factors using the retention times of the analytes. The binding factors were
calculated as 181 M-1 for D-Trp and 371 M-1 for L-Trp. Although the binding constants
were smaller than those obtained by other techniques using BSA as a chiral selector, the
ratio of the binding constants of D- and L-Trp to BSA was similar. This shows that this
protein-encapsulated column can be used for the measurement of the ratio of the binding
constants to the analytes. The advantages of this protein-encapsulated column are that
the size of the analyte and the required proteins in this system decreased a few orders of
magnitude from conventional schemes, as well as the simplicity of the preparatory
procedures.
Fig. (8). Schematics of separation and enzyme reaction by the biomolecule-encapsulated column
(A) and FAC/MS system (B).
This biomolecule-encapsulated column could also be used as an enzymatic bioreac-

tor. At the inlet of the capillary, trypsin is encapsulated in the gel matrix. The substrates
are electrokinetically introduced from the inlet of the capillary, Fig. (8). A a). The sub-
strates are converted into products by the encapsulated enzyme, Fig. (8). A b). Finally,
the unreacted substrates and products are separated by electrophoresis, Fig. (8). A c).
Sakai-Kato et al. prepared trypsin- and the microsomes-encapsulated columns. The
trypsin-encapsulated column digested substrates (amino acid derivative and peptides)
and separated the substrates and products within a single capillary [107]. They encap-
sulated microsomes which contained drug-metabolizing enzymes. The encapsulated
column was used for the production of metabolites and their separation from the
substrates [109]. It could also be used for metabolic inhibition screening by injection of
a mixed sample into the column [110]. In both cases, the resultant monolithic reactor
showed an excellent enzymatic activity with prolonged stability, and the separation of
the unreacted substrates and products in the same capillary also showed a high
selectivity. The sample size in this system decreased 3 orders of magnitude from con-
ventional reaction schemes. Because the analytical procedures, i.e., reaction, separation,
and detection, were integrated into a simple system, the systematic automation and the
reduction of the analytical times and required solvents were realized. Although the
preparation of the biomolecule-encapsulated column using a hydrogel is easy and
simple, the EOF was a major driving force for pumping the eluent, as hydrodynamic
flow does not work well in the column due to a lack of micro-sized pores (through-
pores) [111]. Therefore, this column was used only for CE or CEC. Another shortcom-
ing of this column was that large molecules could not be analyzed by the column,
because these molecules could not penetrate the nanopores of the hydrogel network.
To overcome this problem, the developmet of biocomposite stationary phases with
micro-sized pores for the penetration of the pressurized flow and the large molecules are
required. Until now, two techniques were reported for the solutions. Brennan et al.
developed the protein-doped monolithic silica columns. The spinodal decomposition of
the PEO-doped sol into two distinct phases prior to the gelation of the silica results in a
bimodal pore distribution that produces large macropores to allow the flow of the eluent
[112-114]. Kato et al. prepared a bio-doped column by coating a protein-containing gel
on a photopolymerized porous silica monolith [111]. They coated a macroporous PSG
monolith with a protein-containing film using the sol-gel reaction. Although Brennan’s
technique prepared the column in one step, the existence of biomolecules during the
spinodal decomposition limits the preparatory procedure resulting in a restriction in the
size of the skeleton or through-pore of the resulting monolith. In fact, the through-pore
of the resulting monolith is about 0.1um, while Kato’s techniques of the latter method
enabled the fabrication of the monolith with the size of more than 1 µm, although their
technique required a two-step reaction.
Brennan et al. entrapped the clinically relevant enzyme, dihydrofolate reductase,
within bioaffinity columns which were used to screen mixtures of small molecules using
frontal affinity chromatography [112], Fig. (8). B. The protein-doped monolith had a
bimodal pore distribution that produced large macropores (>0.1 µm) with minimal back
pressure and mesopores (~3-5-nm diameter) that retain a significant fraction of the
entrapped protein. Inhibitors present in compound mixtures were retained via bioaffinity
interactions, with retention times being dependent on both the ligand concentration and
the affinity of the ligand for the proteins. The results suggest that this column can
provide a useful platform for the high-throughput screening of lead compounds.
Furthermore, by using mass spectrometric detection, the number of compounds that can
be simultaneously analyzed was significantly increased [113].
Kato et al. prepared a macroporous monolithic column using the spinodal decom-
position of the sol, followed by covering the monolith with the protein-containing
silicate film. The large through-pores enabled the good pressurized flow of eluents, and
the analysis of large molecules. A porous monolith was used as a supporting matrix for
the enzyme immobilization. This technique is advantageous for the preparation of
biomolecule-immobilized reactors because (1) its micron-sized pores allow for the
penetration of large molecules, and (2) its large surface area allows for an increase in the
number of immobilized biomolecules. They prepared a pepsin-coated column [111]. The
sol-gel reaction was optimized so that the sol solution containing pepsin forms a thin
film on the photopolymerized sol-gel (PSG) monolith that was initially fabricated at the
inlet of the capillary. Pepsin was encapsulated into the gel matrix without losing its
activity. The large surface area of the PSG monolith enabled the immobilized pepsin to
achieve a high catalytic turnover rate, and the porous nature of the PSG promotes
penetration of large molecular proteins into the column. The immobilized pepsin
digested peptides and proteins, and the resulting mixture of peptide fragments could be
directly separated in the portion of the capillary where no PSG monolith existed. By
combination of in-capillary digestion, separation and mass spectrometry analysis, they
could identify proteins [111]. The on-line digestion of insulin chain β and lysozyme
provides identification of the proteolytic peptides. Therefore, this column is very
promising for use in proteomic research. Dulay et al. used the PSG for the trypsin-
immobilized column. Trypsin is covalently linked to a photopolymerized sol-gel
monolith modified by incorporating poly(ethylene glycol) (PSG-PEG) for the on-column
digestion of the Nα-benzoyl-L-arginine ethyl ester (BAEE) and two peptides,
neurotensin and insulin chain β. The coupling of the enzyme to the monolith is via
room-temperature Schiff chemistry in which an alkoxysilane reagent (linker) with an
aldehyde functional group links to an inactive amine on trypsin to form an imine bond
[115].
6.4. Biomolecule-Encapsulated Microfluidic and Microarray Chips, and Microtiter

Plates
The area of integrated chemistry is rapidly growing, and many analytical systems
have now been integrated into microfabricated devices. These integrated analytical
systems have been used in many fields because the integration provided small volumes,
faster responses, highly parallel analyses, and minimal cross contamination. The
technique was thought to be suitable for high-throughput screening. The sol-gel reaction
was used to immobilize biomolecules on microfluidic and microarray chips, and
microtiter plates. Clark et al. developed a microchip with an immobilized IgG antibody
and trypsin. They used a mixed solution of TMOS and MTMS for the immobilization of
these materials on a polydimethylsiloxane (PDMS) microfluidic chip [116]. To
minimize cracks in the gel microstructure, polyvinylalcohol was added in the sample
solution. They also developed arrays of sol-gel encapsulated enzymes, comprising 147
microwells, suitable for the repeated assays of bioactivity or enzyme inhibition, Fig. (9).
A. Liposome, glucose oxidase, and horseradish peroxidase (HRP) were encapsulated. A
hydrolase array was also prepared containing twenty different lipases and proteases
[117]. They recently developed a P450s-immobilized microchip for a toxicology assay
(they called it a “MetaChip”) [118], Fig. (9). B. The MetaChip used two arrayed plates
for the assessment of ADME/Tox (absorption, distribution, metabolism, excretion/
toxicology). P450s were microarrayed on the chip, and a prodrug was spotted on the
P450-immobilized spots. An MCF7 breast cancer cell was cultured as a monolayer on
another plate, and the effects of drugs and their metabolites on the cultured cell were
evaluated by a combination of these two microplates.
Sakai-Kato et al. used a trypsin-encapsulated hydrogel, which was previously
mentioned [107], for immobilization of trypsin on a microfluidic chip. By using the
microfluidic chip, large molecules, such as proteins, were hydrolyzed, and peptide
fragments were separated on the microchip [108], Fig. (9). C. They also prepared an
enzyme-doped bioreactor for a 96-well microtiter plate. Two approaches were evaluated;
1) a silica thin film containing enzymes was fabricated on the bottom of the microtiter
plate, and 2) a miniaturized enzyme reactor was prepared by coating a enzyme-
containing gel on a porous silica monolith which was fabricated so as to fit into a 96-
well microtiter plate well, and could then be easily removed. The first approach was
Fig. (9). Schematics of microarray chip (A), MetaChip platform cited from [118] (B), and Images
of a microchip used in [108] (C).
applied to the development of the P450 array [38]. The microsomes containing
expressed human P450 enzymes were immobilized on the microassay plate using sol-gel
chemistry. A thin-film hydrogel containing microsomes was fabricated using aqueous
silicate as the starting material. The different P450 isozymes were immobilized on the
microassay plate, and the metabolites by each isozyme were visualized as fluorescent
images, which creates opportunity for the inhibitor assays. Because this methodology
enabled the development of an assay system using P450 that is unstable and involves
other enzymes for its function, it can be applicable to various screening assays that
require complicated reactions involving many biological components, and paved the way
for the immobilization methods of protein-chips. The second method was applied to the
trypsin reactor in the microassay plate. The trypsin-containing gel was used for coating
the surface of a bimodal porous silica monolith, which was fabricated using TMOS and
MTMS along with PEG as a porogen [119]. The through-pores were important for fast
mass transfer and the nanopores were important for increasing the quantity of the
immobilized trypsin. The large surface area of the monolith enabled the immobilized
trypsin to achieve a high catalytic turnover rate. Furthermore, the kinetic parameter of
the immobilized trypsin indicates the absence of diffusional limitations. The
immobilized trypsin effectively hydrolyzed an amino acid derivative (Nα-benzoyl-
arginine-p-nitroanilide) and a protein (BODIPY-casein) [119].
Brennan et al. immobilized biomolecules on the bottom surface of a 96-well
microtiter plate. They compared the catalytic constant, Michaelis constant, and inhibition
constant of Factor X, dihydrofolate reductase, cyclooxygenase-2, and γ-glutamyl

transpeptidase before and after immobilization [120]. These biomolecules were
encapsulated by diglyceryl silane (DGS). They also encapsulated two membrane-
associated proteins, a nicotinic acetylcholine receptor, a ligand-gated ion channel, and a
dopamine D 2Short receptor, a G-protein coupled receptor, using the sol-gel method. The
protein-doped monolith was fabricated on the surface of the 96-well microtiter plate. The
two receptors showed 40-80% solution activity over periods of at least 1 month [121].
They also used sugar-modified silica N-(3-triethoxysilylpropyl) gluconamide (GLTES)
for encapsulation of an Src protein trypsine kinase, a cellular membrane protein which
catalyzes the transfer of phosphate from ATP to a tyrosine residue within a protein
[122]. Using this immobilized technique, an unstable protein, firefly luciferase, was
immobilized and used for ultrasensitive ATP detection (1 pM) [123]. The ATP sensor
could be repeatedly used. They also immobilized Factor Xa and urease using a mixture
of GLTES and DGS.
Luo et al. immobilized liposome containing two transmembrane proteins,
bacteriorhodopsin and F 0F1-ATP synthase, in a TMOS-derived gel containing PEG on a
glass slide [40]. Liposome provided membrane structure and protein orientation to two
proteins. This transmembrane-doped gel could couple the photo-induced proton gradient
to the production of ATP.
Sol-gel encapsulation technique is also used for the enzymatic reaction and detection
system for the determination of uric acid in human serum in microchip-based analysis.
HRP and luminol was co-immobilized at the detection area, and urinase was immo-
bilized in an enzymatic reactor. The uric acid was monitored by a chemiluminescence
reaction between the hydrogen peroxide produced from the enzymatic reactor and
luminol under the catalysis of HRP in the microreactor. The linear range of the uric acid
concentration was 1 to 100 mg/L and the detection limit was 0.1 mg/L [124].
Tsai and Doong developed an array-based optical biosensor. Urease and
acetylcholinesterase were used as model enzymes and were co-entrapped with the
sensing probe, FITC-dextran, in the sol-gel matrix to measure pH, urea, acetylcholine
and heavy metals (enzyme inhibitors) [125]. The biosensor exhibited a high specificity
in identifying multiple analytes. No obvious cross-interference was observed when a 50-
spot array biosensor was used for the simultaneous analysis of multiple samples in the
presence of multiple analytes.
6.5. Whole Cells in Sol-Gel Glasses

6.5.1. Chemical Applications
Many intracellular microbial enzymes are produced in quantities large enough to be
used in industry processes. However, the cost for their isolation and purification can be
quite high. Therefore, it would be of interest to be able to encapsulate whole cells, such
as yeast or bacteria, in order to avoid tedious separation and purification procedures. The
first paper was published in the late 1980’s by Carturan et al. reporting the
immobilization of yeast spores into thin SiO2 layers [126]. Encapsulated yeast has been
used for the conversion of sugar and carbohydrates into ethyl alcohol and CO2, or
environmental protection and metal recovery [127]. The sol-gel encapsulation of the
bacterium esherichia coli was reported by Livage et al. Using the aqueous precursors of
the sol-gel reaction, the cellular organization and the enzymatic activity was improved
and 50% of the bacteria was still able to form metabolites after one month of aging
[36,128,129].
6.5.2. Medical Application
An emerging approach to treating disease makes use of living cells encapsulated
within a porous matrix that shield the cells from immune attack. Recent studies have
shown that sol-gel silica could be used for cell plantation. The first experiments were
made with the pancreatic islets of Langerhans, which are known to produce insulin in
response of glucose. The silica spheres containing the inlets allowed the passage insulin
and cytokines but not the passage of antibodies. In addition to the in vitro experiments,
in vivo experiments have been performed via the transplantation of encapsulated islets
into a diabetic mouse [130,131]. Another process, called ‘biosil’ was developed for the
encapsulation of swine hepatocytes and rat liver. In this process, living cells are
deposited onto a substrate and then partially covered by a silica film via the gas phase
[24]. These cell encapsulation techniques were very promising not only for in vivo
applications for cell transplantation, but also for the development of an assay system
using cells for more precisely evaluating the drug candidate.
7. CONCLUSIONS
As described above, the sol-gel derived biocomposites provide significant advan-
tages for the immobilization methods used in the development of analytical instruments.
Sol-gel derived silica materials can be produced using a wide variety of compositions
and can immobilize a wide variety of biomolecules with a wide range of sizes, from
small chemical compounds or proteins to cells. This is ascribed to the fact that the gel
formation proceeds using the encapsulated biomolecules as templates. Another
advantage is that because the gel reaction was started from the liquid sol, the resultant
gel enabled a wide range of configurations, such as bulk, thin film, fibers, and
microarray.
While sol-gel derived biocomposites have been shown to be useful in many
analytical applications, some problems remain to be solved. For, example, some
properties of silica materials still need to be improved to reduce shrinkage, cracking,
pore collapse and phase separation. The changes in the material’s properties by the gel
aging should be also controlled for improving the reproducibility of the analytical
systems. Another disadvantage is that the entrapped biomolecules are leaked more or
less from the gel matirix during the repeated use. Non-specific interaction of the analyte
and residual silanol is also problematic in evaluating the affinity between analytes and
entrapped biomolecules. These would be resolved by thoroughly investigations of the
new biocompatible silane precursors. For that purpose, the analytical methods of the
structure and characteristics of the sol-gel matrials and entrapped biomolecules would be
also required. Based on these studies, useful encapsulated biomolecules for drug
discovery will be developed.
Recent improvement in the sol-gel technology for the immobilization of biomaterials
is very drastic. This shows that many researchers recognize that this technology is
perhaps the most facile and generic immobilization technology available today. This
recognition facilitates the application of the sol-gel technology to the medical or food
industries. For example, the analytical format using the sol-gel technology has been
shifted to the bulk form to the high-throughput format, such as high flow- rate
chromatography columns, microassay plates, or microfabricated devices. The type of
encapsulated biomaterials has been also extended from single molecules to the protein
complex or cells. Based on the basic studies mentioned above, useful assay systems
using encapsulated biomolecules will be further developed for drug design and drug
discovery.
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Modeling of Environmentally Sensitive Hydrogels

for Drug Delivery: An Overview and Recent
Developments
Hua Li*, Rongmo Luo and K.Y. Lam

Institute of High Performance Computing, National University of Singapore, 1 Science
Park Road, #01-01 The Capricorn, Singapore Science Park II, Singapore 117528
Abstract: A critical review of mathematical modeling for simulation of

environmentally sensitive hydrogels is presented for application of drug
delivery. The review demonstrates that there have been large numbers of
published studies on the model development, although the majority of
investigations in the research area of the drug delivery are experimental-based.
Therefore, a systematical review of mathematical modeling of environmentally
sensitive hydrogels is necessarily provided through comprehensive assessment
on several critical developments of mathematical models to simulate the
hydrogels for mechanisms of drug release. The present review classifies the
developed models into the fundamental and empirical/semi-empirical groups
and also discusses the properties and performance of the hydrogels as drug
delivery system in kinetics and equilibrium.
INTRODUCTION
Hydrogel is generally defined as a hydrophilic mixture being a form of the materials
that possess both the properties of solid and liquid [1, 2]. Its structure is formed from the
networks of randomly cross-linked macromolecules and it includes three phases,
polymeric-network matrix solid phase, interstitial fluid phase and ionic phase. The solid
phase of the hydrogels is a network of cross-linked polymeric chains where their three-
dimensional polymeric structure is usually described as a mesh, with the interstitial
space filled up with fluid. The network meshes hold the fluid in place and also create
rubber-like elastic force for expansion or contraction of the hydrogels, thus providing the
solidity to the hydrogels [3]. The cross-linked network can be formed physico-
chemically, for instance, by hydrogen bonding, van der Waals interactions between
chains, covalent bond, crystalline, electrostatic interactions or physical entanglements
[4]. The fluid phase of the hydrogels fills in the interstitial pores of the network and
gives the hydrogel wet and soft properties, which resembles, in some respects, to
biological tissues [5]. The ionic phase of the hydrogels is generally composed of the
ionizable groups bound to the polymer chains and the mobile ions (counter ions and co-
ions) due to the presence of electrolytic solvent. The ionizable groups dissociate in
*Corresponding author: Tel: +65-64191249; Fax: +65-64191280; E-mail: lihua@ihpc.a-star.edu.sg

296 Frontiers in Drug Design & Discovery, 2006, Vol. 2 Li et al.
solution completely for strong electrolyte or partially for weak polyelectrolyte groups,
while the network is left with same charged groups fixed to the chains. The fixed charge
groups produce an electrostatic repulsion force each other and play a role in the swelling
of the hydrogel.
In fact, there are various natural or synthetic materials being examples of the
hydrogels. The natural ones include the guar gum and collagens that are modified to
produce hydrogels, and the synthetic those poly(acrylic acid)− PAA, poly(hydroxyethyl
methacrylate)−PHEMA, poly(acrylamide)−PAM, poly(vinyl alcohol) poly(acrylic
acid)−PVA/PAA, poly(acrylonitrile)−PAN, poly(acrylonitrile) poly(pyrrole)−PAN/PPY,
poly(N-isopropylacrylamide)− PNIPA, etc. Depending on the physical and chemical
characteristics of the polymeric networks and fixed charge groups, the hydrogels may be
categorized in other ways. For example, the hydrogels can be synthesized to be either
neutral or ionic, determined by the characteristic of the pendant groups fixed onto the
polymeric matrix.
One of the important reasons that the responsive hydrogels attract considerable
research interest is their unique property of undergoing discrete or continuous volume
phase transition in response to the very small change of surrounding environment
conditions, such as solution pH [6-9], solvent composition [10], salt concentration [11],
temperature [12-14], light/photon [15, 16], electric field [17], electromagnetic field [18]
and so on. In fact, there are some environmental variables found in the body, such as low
pH and elevated temperature. Thus the environmentally sensitive hydrogels have
enormous potential in various applications of drug delivery. For example, either pH-
sensitive and/or temperature-sensitive hydrogels can be used for site-specific controlled
drug delivery. The hydrogels responsive to specific molecules such as glucose or
antigens can be used as biosensors and drug delivery systems. Light-, pressure- and
electro-sensitive hydrogels also have the potential in applications of drug delivery and
bioseparation. The significant features make the stimuli-responsive hydrogel better
known as smart/intelligent materials for wide range of applications, as they are able to
sense and eventually respond to the environmental changes without need of external
power source. Sensors and/or actuators [19], artificial muscle [20, 21], microfluidic
control [22], ultrafiltration [23], separation [24-26], and chromatographic packing [27]
are some examples of successful applications of the hydrogels. A more exceptional
promise of the hydrogels is their biocompatibility and biostability potential [28] for
possible substitution of the human body tissues and biomimetic applications, such as
articular cartilage [29], wound dressing [30], corneal replacement [31] and tissue
engineering [32, 33], especially in the medicine and pharmaceutical applications such as
the drug delivery system [34-39].
The drug delivery system is such a research area that people from almost every
scientific discipline can make a significant contribution. Due to the highly
interdisciplinary nature of the area, the researchers play a unique role in new strategy
development for drug delivery system, who emerge from the training programs that
integrate the biological science such as quantitative physiology and pathophysiology,
cell and molecular biology, the engineering technology such as polymer engineering and
microfabrication technology, and the mathematical modeling such as pharmaco-kinetics
and transport phenomena.
Modeling of Environmentally Sensitive HydrogelsFrontiers in Drug Design & Discovery, 2006, Vol. 2 297
In addition, the sciences of mathematical modeling and numerical simulation are

generally accepted as the third mode of scientific discovery, with the other two modes
being experiment and analysis, which makes this field an integral component of cutting
edge scientific and industrial research in most domains. This is especially so for
understanding and manipulating the fate of drug releases in humans as a classical
engineering endeavor, where basic science and mathematical analysis can be used to
achieve an important practical end, and the multi-physics and multi-phases are common
requirements.
Extensive search of literature has thus far revealed that there are numerous
researchers making efforts to develop the mathematical models for simulation of
environmentally sensitive hydrogels for drug delivery applications. A systematical
review is necessarily presented here through comprehensive assessment on the several
critical developments of mathematical models to simulate the hydrogels for applications
of the drug release.
FUNDAMENTAL MATHEMATICAL MODELS

Fick’s Law
Usually the drug delivery system may be modeled simply as one-dimensional
transient mass transfer in a hydrogel disk. The disk has thickness L and an initial drug
concentration profile in contact with a solvent maintained at sink conditions. The
common case is low drug concentration, which doesn’t have significant effect on the
drug diffusivity. In addition, the drug diffusion is often assumed to be the rate-
controlling step rather than swelling or drug dissolution. This is suitable assumption if
the kinetics of swelling can be neglected for the hydrogel, for example, the systems with
little swelling, or when the diffusive release occurs from a pre-swollen matrix. With the
assumptions mentioned, Fick’s second law is often employed for simulation of drug
diffusion, and generally expressed by
∂c ∂ ∂c
= (D ) (1)
∂t ∂x ∂x
with the boundary conditions
∂c
=0 at x = 0, t > 0 (2)
∂x
c(t , L) = 0 at x = L, t > 0 (3)
and the initial condition
c(0, x) = v( x) at t = 0, 0 < x < L (4)
where c = c(t , x) is the drug concentration, t is the release time, v(x) is the initial
drug concentration profile, x is the position normal to the effective area of diffusion in
one-dimension diffusion, and D is the drug diffusivity.
When the drug diffusivity D depends on spatial position in the hydrogel matrix, Eq.
(1) becomes the partial differential equation with variable coefficient, for which no
analytical solution is available, and numerical techniques such as the Crank-Nicholson
method are then employed to solve the problem [40]. The time-dependent drug flux and
cumulative fractional release may be obtained by solving a set of linear algebraic
equations at each time step.
If the polymeric network of the hydrogels is homogeneous and the drug diffusivity D
is independent of drug concentration, D can be assumed to be a constant. Then Eq. (1) is
simplified to
∂c ∂ 2c
=D 2 (5)
∂t ∂x
This is a partial differential equation with a constant coefficient D, and can be solved
analytically with the method of separation of variables.
Varshosaz et al. [41] suggested that the diffusion coefficient D may be calculated
according to Fick’s first law. This is the diffusion law, and in one dimension it describes
the flux of particles through a point x by
∂c( x, t )
(Jn )x = −D (6)
∂x
Varshosaz et al. [41] then derived
dQ ADK d (c0 − c)
= (7)
dt h
where dQ / dt is the rate of mass transfer, A is the surface area of the hydrogel film,
Kd is the partition coefficient, h is the hydrogel thickness. c0 and c are the drug
concentrations in both the sides of the hydrogel, respectively.
For the mass transfer in a three-dimensional cylindrical domain, Fu et al. [42] gave
an analytical solution of Fick’s second law as,
∞ ∞
Mt 8
M∞
= 1− 2 2
h r
∑α
m =1
−2
m exp(− Dα m2 t ) × ∑ β n− 2 exp(− Dβ n2 t )
n =1
(8)
with
(2n + 1)π
J 0 ( rα ) = 0 and βn = (9)
2h
where M t and M ∞ are the amounts of drug released at time t and infinite time,
respectively. h denotes the half-length and r the cylinder radius. D is a constant diffusion
coefficient. α and β are defined by Eq. (9) with a zero-order Bessel function J 0 , m
and n are integers. This model is applicable to tablets that range from the shape of a flat
disk with the radius much larger than the thickness to that of a cylindrical rod with the
radius much less than the thickness, where the drug is homogeneously distributed within
the system [43], but the model doesn’t consider the volume expansion of the hydrogels.
For the drug release from micro-hydrogel particle with consideration of drug
dissolution and diffusion in the continuous matrices of microgels as well as the limited
solubility of drug in the release medium, the present authors [44, 45] and Hombreiro-
Perez et al. [46] presented a mathematical model for simulation of the kinetics of drug
release from the microgel particles. In general, the initially loading drug concentration
c0 in spherical microgels is greater than the drug saturation concentration cs . This may
be achieved either by preparation of a solution and total evaporation of the solvent, or by
partial evaporation or phase inversion. When the polymeric microgels are put into a well
stirred release medium, the following four steps of mass transfer take place
consequently: (a) drug dissolution within the microgels; (b) drug diffusion within the
matrices of microgels; (c) drug diffusion through the unstirred liquid boundary layers on
the surfaces of the microgels; and (d) drug diffusion and convection within the release
medium. Since the convective transport within the medium is usually much faster when
compared with that of the diffusive mass, the convective transport can be neglected
when the overall rate of drug release from the polymeric microgels is calculated.
Therefore, it is reasonably assumed here that the drug dissolution and diffusion in the
continuous matrices of spherical non-swellable microgels control the drug release in a
well-stirred release medium. The kinetics of drug release from the microgels with radius
R can be simulated mathematically for the drug release process in a well stirred release
medium, by the following partial differential governing equation,
∂c(r , t ) ∂ 2c(r , t ) 2 ∂c(r , t )

= D( + ) + k (εcs − c(r , t )) (10)
∂t ∂r 2 r ∂r
and the following initial and boundary conditions,
c(r , t ) = εcs at t = 0, 0<r <R (11)
∂c(r , t )
=0 at t > 0, r=0 (12)
∂r
c(r , t ) = 0 at t > 0, r=R (13)
where c(r , t ) (g/cm3) is the drug concentration at the radial position r (cm) of the
microgel system at the release time t (s). D (cm2/s) is the drug diffusion coefficient, k
(s-1) is the first-order rate constant of drug dissolution, ε is a parameter for the
polymeric network meshes of the microgels and it is directly related to the cross-linking
density of the polymeric microspheres. If cs (g/cm3) is defined as the drug saturation
concentration in the system, εcs (g/cm3) refers to the equivalent drug saturation
concentration in microgels with the network mesh parameter ε .
The first term of the right-hand side in Eq. (10) is the well-known Fick’s second law
of diffusion for a spherical system, which describes the diffusive drug release process in
the microgels due to the continuous dissolution of the drug. The second term of the
right-hand side in the equation corresponds to the potential rate-limiting drug dissolution
process. It is observed that, when the initially loading drug concentration c0 is smaller
than the drug saturation concentration cs , Eq. (10) is reduced to the classic Fick’s
diffusion equation.
Although the drug diffusion coefficient D in the polymeric microgels may be
solvent-concentration dependent, usually it is reasonably assumed that D is
approximately a constant for simplicity. It is also assumed that the drug is uniformly
distributed throughout the microgels with equivalent drug saturation concentration εcs
initially. Under perfect sink conditions, the release medium may be considered to be well
stirred, thus the drug concentration outside of the microgels is further assumed to be
constant and equal to zero.
In addition, Gao et al. [47, 48] developed a model to predict the relative change in
drug release rate as a function of formulating composition for HPMC tablets of
adinazolam mesylate and alprazolam. This model is based on the steady-state
approximation of Fick’s law for the drugs release from solid matrices and it is modified
by Lapidus and Lordi [49, 50] as follows:
S D' t 0.5
Mt = M0 ( ) (14)
V π
in which M t denotes the amount of drug released at time t, M 0 is the initial amount of
drug within the tablet, S represents the surface area and V the volume available for
release, D ' is the effective diffusion coefficient and defined as:
D
D' = (15)
τ
where D is the true self-diffusion coefficient of the drug in the pure release medium
and τ is the tortuosity of the diffusing matrix. However, the swelling of the system is
not taken into account and the mathematical analysis may be reduced to one-dimension
problem.
Coiviello et al. [51] considered the drug release from non-eroding/swelling
cylindrical gel through the different experimental setups that may be designed to
calculate the diffusion coefficient [52]. By assuming a constant drug diffusion
coefficient D and neglecting the gel density variation due to diffusion, Fick’s second law
in two-dimensional cylindrical coordinates system is written as:
∂c D ∂ ∂c ∂ 2c
= (R ) + D 2 (16)
∂t R ∂R ∂R ∂Z
where t is time, c is the drug concentration in the cylinder. R and Z are the radial and
longitudinal coordinates, respectively. The corresponding initial and boundary
conditions are given as:
c( Z , R) = c0 , − Z c ≤ Z ≤ Z c , 0 ≤ R ≤ Rc at t=0 (17)
crel = 0 at t=0 (18)
c( Z , Rc , t ) = c(± Z c , R, t ) = k p crel (t ) (19)
Zc Rc
Vrel crel (t ) = πRc2 2 Z c c0 − ∫ ∫ c( Z , R, t )2πRdRdZ (20)
−Zc 0
where 2 Z c and Rc are the cylinder height and the radius respectively. c0 is the initial
drug concentration in the cylinder, crel is the drug concentration in the release medium,
Vrel is the volume of the releasemedium and k p is the drug partition coefficient
between the cylindrical gel and the environmental release medium. The set of the
equations (16)-(20) can be numerically solved by the control volume method [53].
Several studies reveal that the kinetics of the controlled release from scleroglucan
hydrogel matrices exhibit evidently the non-Fickian macroscopic features, because of the
instantaneous formation of a stagnant layer of thickness h between the gel matrix and
reservoir fluid. Such phenomenon is attributed to both the limited erosion of the gel
which is caused by the mechanical agitation of the fluid reservoir and the presence of the
gel sustaining net. In the conditions mentioned, the kinetic profile of relevant release
may be simulated well by solving the second Fick law in both gel and stagnant layer, by
assuming an equal diffusion coefficient for the theophylline in the two phases. It is also
assumed here that the distribution of the initial drug concentration within the gel follows
a step profile, in which it is equal to its nominal value c0 for 0 < x < L − h and it is
zero for L − h < x < L , where L is the sum of the thicknesses of the gel and stagnant
layer. Accordingly, the experimental release is described by solving the one-dimensional
second Fick law [54]:
∂c ∂ 2c
= Dg 2 (21)
∂t ∂x
with the initial and boundary conditions as
c=0 L−h< x< L at t=0 (22)
c = c0 0< x< L−h at t=0 (23)

∂c
=0 x=0 at t>0 (24)
∂x
c=0 x=L at t>0 (25)
Fick’s second law can also be used for simulation of diffusion of solute in PEG for a
planar sheet as
∂c ∂ 2c
=D 2 (26)
∂t ∂z
with the initial condition of uniform concentration ( c = c0 , − l < z < l at t = 0 ), and
the boundary condition of surface concentrations equal to zero by assuming sink
conditions ( c = 0 , at z = −1 and z = l for t > 0 ). The corresponding cumulative
fractional release or the amount of solute released relatively to total solute loaded in a
disk is obtained by [55]:
2 π t
∞ 2
Mt 8
= 1− ∑ exp{− D ( 2 n + 1) } (27)
n = 0 ( 2n + 1) π
2 2
M∞ 4l 2
in which the Fickian profiles are validated to fit the experimentally measured
concentration profiles to estimate the diffusion coefficients at each time point.
Gu et al. [56] used Fick’s second law for investigation of the diffusion of water in the
hydrogel film for the drying process. They believed that the water vapor pressure in
contact with the polymer film may be considered as a constant since there is a large
water vapor source, i.e. large amount of saturated aqueous salt solutions, in the sealed
container where the hydrogel film is kept for drying. The rate of water exchange
between the water vapor and the hydrogel film depends on the relative humidity of the
air and the water concentration at the film surface. It is assumed here that the rate of
water concentration change on the film surface is directly proportional at anytime to the
difference between the actual surface concentration Cs and the equilibrium surface
concentration C ∞ with the constant water vapor pressure in the air, namely
∂C
−D = α (C ∞ − C s ) (28)
∂x
where C is the concentration of water in the hydrogel (g/m3), x is the water diffusion
direction. D is the diffusion coefficient of water in the film, which is generally a function
of the water concentration. In the system with internal order and/or anisotropy, the
diffusion coefficient should be a function of the microstructure. α is a constant of
proportionality and it is related to the coefficient of mass transfer between the film
surface and the adjacent air. Eq. (28) may be used as a boundary condition to solve
Fick’s second equation. It is noted that Eq. (28) describes diffusion in one-dimensional
geometry. From the point of experimental view, this means that the finite diameter of the
cylindrical film specimens is neglected and the lateral homogeneity is assumed in the
film. This assumption is relatively reasonable for the small ratio of film thickness to film
diameter.
Kikkinides et al . [57] considered the combination of transport mechanisms that are
responsible for the sustained release of the drug encapsulated within the micro-domains
of the porous matrix, which is in analogy with the work of Varelas et al. [58, 59]. The
present hydrogel is initially impregnated with a drug or another solute, thus the drug
partitions preferentially into the micro-domains. By exposing the hydrogel into the drug-
free surrounding environment, the drug exits the gel from the bulk phase in accordance
with Fick’s law.
∂cb
(1 − φ ) = (1 − φ ) D b ∇ 2 cb + J s (29)
∂t
with the following boundary and initial conditions,
∇cb (0, t ) = 0 , c b ( Rb , t ) = 0 (30)
c b (r ,0) = c* (31)
where cb = cb (r , t ) is the concentration of drug in bulk phase, φ is the volume

fraction of micro-domains, r is the radial direction in the bulk phase and Rb is the
radius of the polymer matrix that is here assumed a cylindrical shape. Db is the
diffusion coefficient of the solute in the polymer matrix, J s is the flux term through the
micro-domains, and c* is the solute saturation concentration in the bulk phase.
In general, Fick’s law of diffusion for one-dimensional swelling of films assumes
that the diffusion coefficient D of the penetrating agent (solvent or solution) and film
thickness remain constant during the entire swelling process. However, Vazquez et al.
[60] considered that the film thickness obviously does not remain constant for extensive
swelling. They proposed the solutions of the differential forms of Fick’s law for thin
sheets, by neglecting diffusion through the edges,
Mt 8 ∞
1 (2n + 1) 2 π 2 Dt
M∞
= 1− 2
π
∑
n = 0 ( 2n + 1)
2
exp[
4l
] (32)
which may be approximately simplified to
Mt Dt
= 2[ 2 ]1 / 2 (33)
M∞ πl
in which M t and M ∞ are the masses of penetrant at time t and infinity, respectively. l
is the thickness of the sheet. D is the diffusion coefficient and usually is independent of
copolymer composition for all pHs, and the lowest D for swelling is found
experimentally in pure water [60].
Higuchi Equation
One of the most renowned mathematical equations for simulation of the release rate
of drugs from matrix system is the Higuchi model [49], and its basic formulation is
written as,
Mt
= D(2c0 − c s )c s t for c0 > c s (34)
A
where M t is the cumulative absolute amount of drug released at time t. A is the
surface area of the controlled release device, such as the hydrogel, immersed in the
release medium. D is the drug diffusivity in the polymer carrier. c 0 and c s are the
initial drug concentration and the solubility of the drug in the polymer, respectively.
Obviously Eq. (34) can be simplified to:
Mt
=K t (35)
M∞
where M ∞ is the absolute cumulative amount of drug release at infinite time, which
should be equal to the absolute amount of drug incorporated within the system at time
t=0. K is a constant reflecting the design variables of the system. Therefore, the
Higuchi model briefly reveals that the fraction of drug release is proportional to the
square root of time.
Higuchi initially examined the model only for planar system. The model was later
modified and extended to consider different geometries and matrix characteristics
including hydrogels. The classical Higuchi equation was developed by the pseudo-steady
state assumptions. Thus it is difficult to directly simulate the really controlled release
systems. Several assumptions of the Higuchi derivation should carefully be kept in mind
[43]: (a) the initial drug concentration in the release system is much higher than the
solubility of the drug. This assumption provides a platform for justification of applied
pseudo-steady state approach; (b) the model is for one-dimensional diffusion and thus
the edge effects are neglected; (c) the suspended drug is in a fine state such that the
particles are much smaller in diameter than the thickness of the release system; (d)
swelling or dissolution of the polymeric carrier is negligible; (e) the diffusivity of the
drug is constant; and (f) perfect sink conditions are maintained. It is apparent that the
assumptions mentioned above are far away from most really controlled drug delivery
systems. As result, the Higuchi equation is often used to examine the experimental data
of drug release to obtain a rough idea of the underlying release mechanism.
Siepmann et al. [43] derived a proportionality between the fractional amount of the
released drug and the square root of the time, by an exact solution of Fick’s second law
of diffusion for thin films with thickness δ , and based on the assumptions of the perfect
sink conditions, constant diffusivity and uniform initial drug concentration with
c0 < c s , i.e., monolithic solutions. The derived proportionality is given in the form as
Mt Dt ∞
nδ
= 4( 2 )1 / 2 {π −1 / 2 + 2∑ (−1) n ierfc } (36)
M∞ δ n =1 2 Dt
It should be noted that the second term in the second bracket will vanish after short
time. However, a sufficiently accurate approximation of Eq. (36) for M t / M ∞ < 0.6
can be written as follows:
Mt Dt
= 4( 2 )1 / 2 = k ' t (37)
M∞ πδ
where k ' is a constant. Therefore, the proportionality between the fraction of drug
release and the square root of time may also be based on the physical phenomena which
are totally different from those studied by Higuchi for his classical equation of
monolithic solutions versus monolithic dispersions.
Anyway, the diffusion always is dominant mechanism and thus the proportionality is
commonly regarded as an indicator for diffusion-controlled drug release. Despite the
approximation of the model, the Higuchi treatment for a rigid matrix under sink
conditions still is a widely used model for prediction of release phenomena. It is also
approximately available for swellable system [61].
Conaghey et al. [62] further simplified the Higuchi model of matrix diffusion
control, which is validated after less than 30% of the drug is released. This model is able
to simulate the kinetics, in which after release from the resin particles, the drug ions have
to pass through the hydrogel before they diffuse across the membrane. In the simplified
Higuchi model of matrix diffusion control, the quantity of drug M t released at time t is
given by
1
M t = 2c0 ( Dt / π ) 2
(38)
in which c0 is the initial concentration of the drug in the reservoir, and D the diffusion
coefficient through the matrix.
When a biodegradable polymer matrix is considered, the situation becomes more
complicated. However, Heller et al. [63] still assumed the first-order kinetics of
permeability coefficient and employed the modified Higuchi equation as [64]:
dM t A 2c0 P0 (exp(kt )) 1 / 2
= [ ] (39)
dt 2 t
where A is the surface area of the hydrogel system, c0 is the initial concentration of the
drug, P0 is the permeability of the drug in the absence of degradation of the polymer,
and k is the first-order rate constant of permeability coefficient.
In order to investigate the approximation of the Higuchi model, Kasasulu et al. [65]
took the dissolution rates of the theophylline as an example. The dissolution rates are
determined by the linear regression of the Higuchi square root of time. The theophylline
1/ 2
profiles are linear for the time period 4-22 min , but thereafter demonstrate positive
deviations. It is understood that the positive deviations from the Higuchi equation may
result from the air entrapped in the matrix. For hydrophilic matrices the deviations may
result from the erosion of gel layer. Moreover, the work of Cobby et al. [66]
demonstrates that the differences in rates of release are also related to the shape factors.
Power Law
Relatively compared with the Higuchi equation, a much simpler semi-empirical
equation is the power law for simulation of drug release from polymeric systems [67,
68]. The power law is written as
Mt
= kt n (40)
M∞
in which M t and M ∞ are the absolute cumulative amounts of drug released at time t
and infinite time, respectively. k is a constant incorporating the structural and geometric
characteristics of the drug device. n is defined as the release exponent indicating the
kinetics mechanism of drug release.
Obviously, it is seen that the Higuchi equations (34) and (35) and the short-time
approximation (37) of the exact solution of Fick’s second law for thin films are the
special case of the power law at the release exponent n = 0.5 . When the release
exponent n = 1 , it is known from Eq. (40) that the drug release rate is independent of
time, which corresponds to the zero-order release kinetics. For the case of slabs, the
mechanism creating the zero-release is known the case-II transport. In fact, one can
consider the power law as a generalization of the drug release, in which the
superposition of two apparently independent mechanisms of drug transport, a Fickian
diffusion and a case-II transport, predicts the most general case of kinetic swelling of
drug system and drug release from glassy polymers, regardless of the form of the
constitutive equation and the type of coupling of relaxation and diffusion [43].
Therefore, Eq. (40) has two distinct physical meanings in the two special cases,
n = 0.5 for the Fickian diffusion-controlled drug release and n = 1.0 for the constant
zero-order/case-II swelling-controlled drug release. When 0.5 < n < 1.0 , it is generally
regarded as an non-Fickian transport indicator through the superposition of both
phenomena for anomalous transport. For example, in the work done by Bajpai et al.
[67], the release exponent n of all the samples is larger than 0.5. This means that all the
samples demonstrate the non-Fickian swelling behavior, which may be attributed to the
fact that in water the carboxylic groups presenting along the macromolecular chains
undergo the ionization to yield –COO - groups. This results in the relaxation of polymeric
segments due to repulsion among similar charges, and then makes the swelling process
chain relaxation controlled [67]. In addition, it should noted that the two special values
mentioned, n = 0.5 and 1.0, are validated only for slab geometry. The release exponent
n will be different values for other geometries such as spheres and cylinders [69].
Furthermore, Peppas and Sahlin [70] developed another interesting model,
Mt
= k1 t m + k 2 t 2 m (41)
M∞
where k1 , k 2 and m are constants. The first term k1t m on the right-hand side represents
2m
the Fickian diffusion contribution F, and the second term k 2t the case-II relaxation
contribution R. The ratio of the two contributions can be calculated by [43],
R k 2t m
= (42)
F k1
Darcy’s Law
As well known, in a homogeneous matrix the pressure drop ∆P and the induced
fluid flow are related linearly through Darcy’s law, namely the mean velocity V of the
fluid flowing through the membrane can be written as,
k
V = ∆P (43)
µL
where k is the Darcy permeability of the membrane, µ is the viscosity of the solution,
and L is the membrane thickness. The Darcy permeability k is measured in Darcy (1
Darcy= 1µm ) and it is a quantitative measure of the ability of the fluid to convect
2
through the membrane and it depends only on the microstructure of the matrix.
Coluccio et al. [71] considered an enzymatically controlled system. An assumption
made is that the decrease of the driving force for drug diffusion is compensated by
increasing the Darcy permeability and the porosity of the polymeric matrix, due to
enzymatic erosion. For examination of their Darcy measurement, they measured the
mass flux J c of a solute diffusing and convecting through the membrane. This means
that the fluxJ c is driven by the difference of solute concentration ∆c = c s and by the
pressure drop ∆P , which are imposed at the two sides of the membrane. Thus,
dc
J c = cV − D (44)
dz
where z is the distance from the membrane surface, c = c(z ) is the local solute
concentration, and D is the effective diffusivity. V is the uniform mean velocity of the
carrier fluid and is driven by the pressure drop ∆P according to Eq. (43).
In order to study the motion of the hydrogel, Wolgemuth et al. [72] constructed a
force balance on each phase, with the constraint that the viscous drags between the solid
and fluid phases are equal and opposite. A Stokes-type of the equation for the fluid is
thus written as,
∇ xV + ∇ xV T
ζ (V − ut ) = µ∇ x ⋅ ( ) − ∇xP (45)
2
where ζ is the drag coefficient between the polymer and the fluid, V is the fluid
velocity, u t = ∂u / ∂t is the polymer velocity, µ is the fluid viscosity, ∇ x is the
gradient operator with respect to the position x , and P is the fluid pressure. This
equation presents the balance and the drag force acting between the fluid and polymer.
The first term on the right-hand side is the fluid shear, and the second term is the
hydrostatic pressure which is related to the osmotic pressure of the hydrogel. When the
fluid shear is negligible, Eq. (45) is equivalent to Darcy’s law.
Based on the biphasic theory, Netti et al. [73] developed a model which is applicable
to both macroscopically porous gels and highly entangled polymer solutions or hydrogel.
In the former case, the fluid phase is present within the macroscopically porous gels,
such as fibrilar gels or tissues, as a distinct phase within the porosity [74]. In these
systems, external mechanical stimuli may lead to a convective fluid transport due to a
non-balanced hydrostatic pressure gradient. On the other hand, if an external stress field
is applied to an entangled polymer solution or hydrogel, an osmotic pressure gradient
will arise to balance the solvation and the elastic forces of the polymeric network.
Therefore, there will be convective fluid flow associated with osmotic pressure gradients
in a similar profile to those occurring in macroscopically porous gels related to
hydrostatic pressure gradients. In both cases, the mathematical formulation of the
coupling of mechanics and transport can be given by a generalized Darcy’s Law.
Langmuir Theory − Absorption Isotherm Model

When the drug diffusion is considered in the microphase and in the bulk phase, the
solute diffuses from the interior of the microdomain to its surface where it rapidly
exchanges. This process may be simulated by a Langmuir absorption isotherm model
[57], in which each bonding site can only absorb one molecule,
[ MS ]
M +S←
→ MS ; K= (46)
[ M ][ S ]
where M represents the molecule of loading drug, and S and MS are the unoccupied and
absorbed sites, respectively. The absorption equilibrium constant K depends on the
functional groups in the hydrogels.
Several researchers used the Langmuir absorption isotherm theory for simulation of
the drug release from the hydrogels. For example, Chern et al. [75] studied the
absorption isotherm of caffeine in the hydrogels at 25 oC aqueous solution by the batch
tests. Their experimental results agree well with those predicted by the derived Langmuir
isotherm model as,
KQc
q= (47)
1 + Kc
where K is the absorption equilibrium constant, Q is the matrix absorption capacity, and
q and c are the equilibrium caffeine concentrations in the gel and solution phases,
respectively.
In addition, Lorenzo et al. [76] investigated the 2,6-naphthalenedisulfonic acid (NS-
2) loaded into the gel, based on the Langmuir absorption model. The NS-2 loaded is
higher at acidic pH with the low ionic strength of the medium and hydrogels in the
collapsed state. The absorption isotherms may be examined in terms of the Langmuir
equation:
SKceq
A= (48)
1 + Kceq
where A is the amount of NS-2 loaded per unit volume of gel, ceq is the final
equilibrium concentration in the solvent, S is the number of absorbing sites per unit
volume of gel or the amount of NS-2 that can be maximally bound per volume of gel,
and K is the affinity of one absorption site for a NS-2 molecule.
EMPIRICAL AND SEMI-EMPIRICAL MODELS

Peppas Model
Peppas and his co-workers are a noted group in the field of drug release. As
mentioned before, the classical power law expressed by Eq. (40) is valid for the slab
geometry only, and it is rewritten here as
Mt
= kt n (49)
M∞
where M t / M ∞ is the fractional release, k is a kinetic constant, and n is the release
exponent. When the drug systems have other geometries such as spheres and cylinders,
the release exponent n should be identified individually.
For a drug device with cylindrically geometrical shape, a simple semi-empirical

formula is proposed by Peppas et al. for prediction of the release mechanism in
cylinders. That is, n = 0.45 for the Fickian diffusion, 0.45 < n < 0.89 for non-
Fickian anomalous transport, and n = 0.89 for the case-II transport, respectively [77-
79].
Furthermore, Peppas and Sahlin [71] proposed a model for simulation of drug release
driven by both the Fickian and polymer relaxation:
Mt
= k1 t 1 / 2 + k 2 t (50)
M∞
where the first term on the right-hand side represents the Fickian contribution, and the
second term the case-II relaxational contribution. k1 and k 2 correspond to the release
rates of the case-I and case-II mechanisms respectively.
Based on the equation (50), Varshosaz et al. [80] gave a semi-empirical model for
ephedrine HCl, a water-soluble model drug, release from a hydrogel disk,
Mt
= k1t 0.78 + k2t 0.2 (51)
M∞
This is a biexponential pattern with two different mechanisms of the drug release. In
the first stage, there is an anomalous mechanism up to the first hour of drug release when
the cylinder radius r = 0.991, in which the swelling is the rate limiting step for drug
release. After the swelling, there is a Fickian release behavior up to 7hr when r = 0.996,
in which the drug Fickian diffusion is the rate controlling step. However, when pH=1.2,
the drug in the hydrogel demonstrates the case-II diffusion or a swelling-controlled
mechanism. The reason may be the acidic pHs restrict the water uptake of the hydrogel
and the swelling becomes much slower than diffusion rate.
Zero-Order Release
In general, a hydrogel device providing the zero-order drug release is necessarily
required to be designed for overcoming Fick’s law. So far many creative systems have
been developed for the zero-order drug release. Usually the drug release from swollen
hydrogels follows Fickian diffusion, in which the rate of diffusion is proportional to the
square root of time. However, the zero-order release is an often desired property of a
controlled release device. The zero-order drug release from the swollen hydrogels may
be achieved by the phasing-separated hydrogels or rate-controlling barriers [81].
Fell and Rowe [82, 83] discussed the zero-order kinetics of drug release from the
spheroids, which could be attributed to the high viscosity and swelling index of the
polysaccharide. The present high viscosity means the formation of a three-dimensional
gel-like network that may retard the drug release. This is supported by highly swelling
index of the polysaccharide, which is an indication of absorption of a large quantity of
water and swelling of the polysaccharide [84]. In addition, Lu et al. [85] proposed an
initially non-uniform drug loading to overcome the disadvantage of the first-order

release associated with conventional diffusion-controlled hydrogel matrix devices,
through a photolaminating method to control intimately the initially non-uniform drug
loading. The drug release pattern with a conventional uniform drug loading usually
shows a typical first-order release behavior, in which an initially high release rate with
the burst effect is followed by a rapidly declining the rate of drug release. Actually it is
desirable to eliminate the burst effect because it may cause negative side effects. The
concept of photolamination is to provide the initially non-uniform concentration profiles
to control the release pattern. The experimental results also demonstrate that, with
increasing the gradient of the drug distribution, the corresponding release profiles
approach more closely to the desired zero-order release, especially in the early release
period, and the burst effect is nearly eliminated.
Varelas et al. [59] studied a biphasic hydrogel as efficacious platform for zero-order
drug release. For the controlled release from the biphasic hydrogels, they developed two
mathematical models of mass transfer through dispersed-phase networks to identify the
mechanism of mass transfer within the hydrogels and to correlate the characteristics of
the release profiles with structural properties of the network. The two models are based
on different assumptions of the mechanism of solute release into the bulk. The first
model consists of two continuity equations, one for each phase of the network, as
follows:
∂cb ∂c
(1 − φ ) = (1 − φ ) Deff ∇ 2 cb − φ m (52)
∂t ∂t
∂c m
φ = f (c m , c b ) (53)
∂t
with the boundary conditions
∂cb (r , t )
cb (r , t ) r = R = 0 ; =0 (54)
∂r r = 0
and the initial conditions
cb (r , t ) t = 0 = c * ; cm (r , t ) t = 0 = c * / α (55)
where φ is the volume fraction in microdomains (cm3 microdomains/cm 3 bulk), cb is

the drug concentration in the bulk phase (g/cm3), t is time (s), Deff is the effective
diffusivity through the gel (cm2/s),c m is the drug concentration in the microdomains
3
(g/cm ), r is radial position within a cylindrical hydrogel device (cm), c * is the
concentration in the bulk phase when saturated (g/cm3), and α is the equilibrium
partition coefficient (cm3 microdomains/cm3 bulk). The second term on the right-hand
side of Eq. (52) is the source term giving the rate of introducing the drug into the bulk
phase from the microdomains. Eq. (53) generally expresses the source term. The first
model consisting of Eqs. (52) and (53) is able to simulate the mechanism of the zero-
order drug release by different forms of the source terms in Eq. (53), depending on the
mechanism of interfacial mass transfer.
In the first model, it is assumed that the drug is encapsulated in the dispersed phase
and the microdomain-bulk interface behaves as a barrier of constant mass transfer
resistance through which the drug must pass. It is also assumed that diffusion through
the interior of the domains is very rapid and thus the domains are well-stirred. Then the
source term in Eqs. (52) and (53) becomes
∂c m hA
φ = − i (αc m − cb ) (56)
∂t V
where h is the mass transfer coefficient across the interface between the microdomain
and bulk (cm/s), Ai is the total interfacial area (cm2), and V is the total gel volume
(cm3).
Typical release profiles predicted by the first model mentioned above demonstrate
the mechanism that produces a slow but steady decrease over time in the flux of drug to
the surrounding environment due to the fact that the driving force for interfacial mass
transfer, (αc m − cb ) , varies with time. However, the present model does not predict a
plateau followed by a sharp drop-off in the flux, for example, as observed in the
experiments with tryptophan and theophylline.
The second model Varelas et al. [59] developed is for the case where the
microdomains act as a perfect source, namely their composition remains unchanged for
some period of operation. It is similar to a salt crystal that dissolves without any
accompanying change in its surface area. The solute available for interfacial mass
transfer resides in a saturated phase with constant concentration c s at the microdomain
surface. The nature of the surface phase remains saturated for some period of time
because it is replenished rapidly from the source. At the end of this period all the
microdomains are depleted and the interfacial mass transfer ceases.
Therefore, the second model is a variation of the first model, has only different forms
of the source term as the second continuity equation by
 hA
∂cm − i (cs − cb ) cm > cmf
φ = V (57)
∂t  0 cm = cmf

where c mf is the final concentration of solute in the microdomains, corresponding to
any solute which is bound and thus unavailable for transport to the bulk. In brief, the
second model consists of Eqs. (52) and (57), with the same boundary and initial
conditions as Eqs. (54) and (55). Varelas et al. [59] employed the model to predict the
profile with a true zero-order plateau for the biphasic hydrogels, which is qualitatively in
agreement with the experiments.
Bezemer et al. [86] conducted another interesting study on the effect of polymer
degradation on the diffusion of lysozyme in the polymer matrix. A relation is thus
required between the diffusion coefficient and the molecular weight of the polymers.
The degraded polymers are subsequently used as matrix for protein loaded films. It is
observed that the release rate increases with decreasing molecular weight for the drug
release from 1000PEG70PBT30 matrices with a molecular weight of 35 000 g/mol. It is
also found that the drug release less follows the first-order kinetics, whereas near zero-
order release is found for the same polymer with a higher molecular weight. The
diffusion coefficients obtained from the first part of the release curves are given as
function of the molecular weight of the matrices. The data can be fitted by the following
linear relationship between D and M n−2 :
k2
D= + k3 (58)
(M n ) 2
the units of k2 and k3 are cm 2g 2s −1mol−2 and cm 2s −1 , respectively. The decrease of

the molecular weight of the polymer due to hydrolytic degradation during incubation in
Phosphate-buffered saline may be described by
1 1
= + k1t (59)
Mn Mn
where M n (g/mol) is the number average molecular weight at time t, M n (g/mol) is the
initial number average molecular weight. k1 (g −1s −1mol) is a degradation rate
constant.
Since the polymer is degraded hydrolytically during release, M n is a function of

time. The diffusion coefficient can thus be written as a function of time by combining
Eqs. (58) and (59) and substituting D = Dinitial at t=0:
D(t ) = Dinitial (1 + at + bt 2 ) (60)
This is an empirical equation for a time-dependent diffusion coefficient. For

diffusion of the lysozyne from PEG/PBT matrices, for example, Eq. (60) needs to be
incorporated into the well known equations for diffusion. By integration of Eq. (60), one
has
t
1 2 1 3
T = ∫ D(t )dt = Dinitial (t + at + bt ) (61)
0 2 3
Thus the lysozyne release from films with the thickness l is described by [87, 88]
Mt 8 π 2T
= 1 − 2 exp(− 2 ) for M t / M ∞ > 0.4 (62)
M∞ π l
Mt T
=4 for M t / M ∞ < 0.6 (63)
M∞ πl 2
and the lysozyne release from microspheres with average radius r is given by
Mt 6 π 2T
= 1 − 2 exp(− 2 ) for M t / M ∞ > 0.4 (64)
M∞ π r
Mt T 3T
=6 − 2 for M t / M ∞ < 0.6 (65)
M∞ πr 2
r
The experimental release profile of the lysozyne from the polymer matrices strongly
depends on the ratio of the degradation rate and diffusion one. For the highly swollen
matrices where the diffusion is fast as relatively compared with the degradation, no
effect of polymer degradation on the release is expected and a first-order profile is
observed. For less swollen matrices, the decline in release rate caused by the reduced
drug concentration in the matrix may be compensated by increasing the diffusion
coefficient due to polymer degradation for an almost constant release rate. The same
conclusion is valid for the effect of the molecular weight on the release profile. For the
polymeric matrix with a low molecular weight, diffusion is fast as compared with
degradation, resulting in a first-order release. For the release from high molecular weight
polymers near zero-order kinetics may be found.
Maxwell Model
Usually one can use the Maxwell-type models [89] for analysis of hydrogels
relaxation behavior and understanding of the mechanical properties of hydrogels and
polymeric network characteristics.
Michailova et al. [90] used the two types of Maxwell models to study the mechanical
relaxation spectra of the mixed HPPMC/NaCMC gels obtained by the experimentally
small deformation oscillatory. The two Maxwell models are the generalized Maxwell
model and the adapted Maxwell model, relating the discrete spectrum of relaxation times
with the material viscoelastic functions. The generalized Maxwell model is written as
G ' (ω ) = ∑ Gi ω 2τ i2 /(1 + ω 2τ i2 ) (66)

n
G ' ' (ω ) = ∑ Gi ωτ i /(1 + ω 2τ i2 ) (67)

n
η ' (ω ) = ∑ Giτ i /(1 + ω 2τ i2 ) (68)

n
The adapted Maxwell model is given as
G ' (ω ) = G '0 + ∑ Giω 2τ i2 /(1 + ω 2τ i2 ) (69)

n
G ' ' (ω ) = G ' '0 + ∑ Giωτ i /(1 + ω 2τ i2 ) (70)

n
where the material viscoelastic functions G ' and G ' ' are the storage and the loss
moduli, respectively, and they are functions of the angular frequency ω . As the
parameters of the relaxation spectrum, Gi and τ i are the modulus and relaxation time
of the ith-member of n-Maxwell elements. G '0 and G ' '0 are the second plateau moduli
at the frequencies of the G ' and G ' ' curves. Based on the discrete characteristics of the
relaxation spectrum, the zero-relaxation time τ 0 and the mean relaxation time θ are
computed by the following equations:
τ 0 = limη i / Gi = limτ i (71)

ω →0 ω →0
θ = ∑ Giτ i2 / ∑ Giτ i (72)

n n
Usually the generalized Maxwell model is suitable for the homogeneous gel systems
where the thermodynamic compatibility of the solvent and polymer is observed. The
model can be extrapolated towards very low frequencies, showing the flow curve in the
terminal region, based on which one can compute the coefficients characterizing the
entangled gel network. They include the zero shear viscosity η 0 , plateau modulus G N0
and zero-relaxation time τ 0 . The comparatively longer zero-relaxation time observed in
both media suggests a decreased mobility of the hydrated macromolecules, which is
related to the low quantity of the solvent in the swollen matrix system. The type of
system may demonstrate slower rates of both water penetration and hydrogel
erosion/dissolution. The generalized Maxwell model is not suitable for the
inhomogeneous gel systems such as the mixed HPMC/NaCMC gels. They may be
simulated by the adapted Maxwell model. Usually the hydrophilic matrices are highly
inhomogeneous during the swelling due to the presence of particles with different
degrees of hydration. The mixing of polymers with different chemical compositions and
viscoelastic behaviors increases the gel heterogeneity. With highly swelling and quickly
relaxing cellulose derivatives, probably an interpenetrating network forms as a third gel-
phase with its own rheological properties. This kind of hydrogels demonstrates a second
plateau in the loss modulus curves G ' ' , inhomogeneity in the process of polymer chain
oscillation, low coefficient of relaxation and residual stress.
Flory−Rehner and Flory−Huggins Models

For the most commonly cross-linked gels that are cross-linked randomly along the
backbone of a polymer and formed by a vulcanization process, the model developed by
Flory and Rehner is a suitable tool for numerical simulation. In the Flory-Rehner model,
it is assumed that the molecular weight is uniform between cross-links and the cross-
linking occurs in the solid state, in which state the polymer chains are in the most
probable conformation and elastic stress only develops upon addition of solvent to the
network. The swelling of a polymeric network is thus referenced to the volume of
polymer in the solid state.
Ebert et al. [91] employed the Flory-Rehner model for study of the gels formed by
the condensation of terminally modified and multi-armed polymers, termed the
condensation gels. Then the number v e of moles of elastically active chains in the
network may be calculated by the volume fraction v 2, s of polymer in the gel,
(V0 / V1 )[ln(1 − v2, s ) + v2, s + χ1v22, s ]

ve = − (73)
(v12,/s3 − 2v2, s / f )
where χ is the Flory interaction parameter, V1 is the molar volume of solvent, V0 is

the volume of polymer in the absence of solvent, and f is the functionality of the cross-
linking.
Actually the swelling characteristics and drug release behavior of a polymeric
network hydrogel depend principally upon the extent of cross-linking. One of the
important structural parameters is the average molecular weight M e between two
consecutive entanglements, which is generally calculated by the Flory-Rehner model as
1 2 (v / V1 )[ln(1 − v2, s ) + v2, s + χ1v22, s ]

= − (74)
Me Mn (v12,/s3 − v2, s / 2)
where M n is the number average molecular weight of the cellulose ether tested, v is
the corresponding volume. V1 is the molar volume of water, v 2, s is the polymer fraction
of the swollen hydrogel in equilibrium, and χ 1 is the Flory parameter of polymer-water
interaction.
This model is able to describe the volume swelling ratio of crosslinked polymers in
equilibrium state, which is based on the assumption that the elastic retractive forces of
the polymer chains balance with the thermodynamic compatibility of the polymer with
the solvent molecules during swelling [92]. This assumption is validated when the
hydrogels are neutral, the swelling is isotropic, there are tetra-functional crosslinks at
zero volume, four chains are connected at one point, and the polymer chains with
Gaussian distribution are crosslinked in the solid state.
Another important structural parameter characterizing the cross-linked polymer is the

average molar mass M c between cross-links, which is directly related to the cross-link
density. According to the data of experimentally swelling, the average molar mass Mc
between the cross links of hydrogel can be computed by the Flory-Rehner model in the
simplified form as follows [67],
M c = −d pVsφ 1 / 3 [ln(1 − φ ) + φ + χφ 2 ] −1 (75)
where d p is the density of polymer, Vs is the molar volume of solvent, and χ is the
Flory-Huggins interaction parameter between the solvent and polymer.
The osmotic pressure in equilibrium state approaches to zero with complex
dependence on the properties of hydrogel and solution. In the Flory-Rehner model, the
osmotic pressure results generally from three terms for the charged polymeric network
[93]:
π = π mixing + π elastic + π ion (76)
where the first term on the right-hand side accounts for the polymer-solvent mixing, the
second term is the elastic contribution due to the polymeric network deformation, and
the third term describes the electrostatic and ion-solvent interaction. The Flory-Huggins
theory provides a pattern for the mixing contribution as [94]
RT
π mixing = − (ln(1 − φ ) + φ + χφ 2 ) (77)
V1
where R is the gas constant, T is the temperature, V1 is the solvent volume, φ is the
volume fraction of polymer in the gel and χ is the Flory parameter.
One can use the Flory-Huggins theory to describe the chemical potential µ mix due to
the mixing by,
µ mix = kT (ln(1 − φ ) + φ + χφ 2 ) (78)
in which φ is defined as the polymer volume fraction in the hydrogel and χ is the
Flory interaction parameter. In the classic Flory theory, for a given polymer composition
at constant temperature, χ is treated as a constant. However, for the anionic hydrogels
with the pH-induced condensation, the parameter χ cannot be treated as a constant. The
balance of the hydrogel matrix between the hydrophilicity and hydrophobicity changes
with pH when protons bind to the polymeric backbone. Thus the hydrogel changes from
one polymer matrix for which water is a good solvent ( χ <0.5) to the other for which
water is a poor solvent ( χ >0.5). As a result, the hydrogel matrix at low pH collapses
and squeezes out water [95].
For modeling of phase transitions and swelling behaviors of polymeric hydrogels,

Bae et al. [96] proposed a thermodynamic model by combining the extended Flory-
Hugghins model in the free energy due to the mixing contribution with the modified
Flory-Rehner theory for elastic networks. In the model, the change in Gibbs free energy
of the network, ∆G net , due to isotropic swelling deformation with free of ions may be
written as,
∆Gnet = ∆Gel + ∆Gmix (79)
where ∆Gmix and ∆Gel are the changes of the Gibbs free energy due to the mixing and
elasticity contributions, respectively. The latter is written as
3Φ 02 / 3 A 1 / 3 B
∆Gel = NRT [( )(φ − φ ) + ( )φ ln φ ] (80)
2mc mc
Φ 0 and φ are the polymer volume fractions in the network

where R is the gas constant.
formation and swollen network states, respectively. mc is the number of lattice sites
occupied by an average network chain, and N is the number of moles of lattice sites. The
factors A and B are given as,
f − 2 2φ 2φ
A= + and B= (81)
f f f
where f is the functionality of the crosslinks for a perfect polymer network.
It is suggested here that the interaction parameter χ is computed by,
∆Gmix φ1 φ 1
= ln φ1 + 2 ln φ 2 + φ 2 ∫ χ (T , φ )dφ (82)
NRT r1 r2 φ2
whereφ1 , φ 2 , r1 and r2 are the volume fractions and relative molar volumes of the
components 1 and 2, respectively. The interaction parameter χ (T , φ ) is defined as the
product of two functions, depending on concentration and temperature, respectively,
χ (T , φ ) = D(T ) B(φ ) (83)
d1
D(T ) = d 0 + and B(φ ) = (1 − bφ ) −1 (84)
T
where d 0 , d1 and b are adjustable parameters. When the combinational term of the
crosslink hydrogels is negligible and the relative molar volume of solvent is
approximately unity, Eq. (82) may be rewritten as [97],
∆Gmix 1
= φ 0 ln φ 0 + φ ∫ χ (T , φ )dφ (85)
NRT φ
Where φ 0 and φ are the volume fractions of the solvent and polymer in the swollen
hydrogels, respectively.
Camera-Roda-Sarti Model
For chemically crosslinked swellable polymeric hydrogel in kinetic state, the drug
release process is strongly influenced by the diffusion of the water solvent inside the
matrix which, in turn, undergoes substantial structural or morphological modifications.
The kinetics of the concentrations of the species is also influenced by the kinetics of the
solvent sorption-desorption in hydrogels. The kinetics of these physical changes may be
modulated by the characteristic times of the corresponding molecular or internal stress
rearrangements. The model proposed by Camera-Roda and Sarti [98] may be applicable
for simulation of the diffusion process from and within a swollen hydrogel polymeric
matrix and prediction of the drug release during the swelling or deswelling phenomena
induced by temperature modification [94]. Despite the complexity of solvent penetration
in the hydrogel polymeric matrix due to the coupling of stress and concentration as well
as chemical potential fields, the formulation of the Camera-Roda-Sarti model may be
developed by the solvent conservation law and a viscoelastic constitutive equation for
the diffusive flux with concentration-dependent relaxation time and diffusivity. The
constitutive equation for the diffusive flux is composed of two terms, the Fickian term
J f and the relaxing term J r with a finite relaxation time τ , namely
∂J r
J = J f + J r = − D f gradc − ( Dr gradc + τ ) (86)
∂t
which should be combined with the conservation law,
∂c
= −divJ (87)
∂t
where c is drug concentration in the hydrogel polymeric matrix, and the convective
contribution to J is neglected. In order to accommodate a relaxation from an initial
Fickian behavior to a final one with different diffusivities, the expressions of D f and
Dr are considered as,
D f = Din and Dr = D∞ (c) − Din (88)
where Din is the diffusivity at the beginning of diffusion or when a change occurs from
a steady-state situation, and it is assumed to be equal to diffusion coefficient of the
unpenetrated polymer. The dependences of D∞ and τ on c are assumed in the
following exponential forms,
D∞ (c) = Deq exp( g (c − ceq )) (89)
τ (c) = τ eq exp( K (ceq − c)) (90)
where Deq and τ eq are the diffusivity and relaxation time when c = ceq , and here ceq
is the penetrant concentration at equilibrium with pure penetrant in external
environment. The exponential equation (89) for D∞ (c) is consistent with free volume
theory.
The above formulation can be rewritten in nondimensional terms as follows:
D∞+ (c + ) = Rd exp( gceq (c + − 1)) (91)
τ + (c + ) = De(exp( K (1 − c + ))) / Rd (92)
where c + = c / ceq , De = τ eq Deq / δ 2 is the Deborah number, and Rd = Deq / D i in

which Di is the diffusivity in the unpenetrated polymer. g and K are given according
to the assumption made by Camera-Roda and Sarti [98], De and Rd are adjustable
parameters. τ eq is the characteristic time for comparison between the experimentally
measured and theoretically computed data, and t = t +τ eq Rd / De , where t and t + are

the dimensional and nondimensional time, respectively.
Tanaka-Fillmore Model
Thickness of the hydrogel disk is one of important parameters to control the rate of
the drug release from a cylindrical disk hydrogel device. Tanaka and Fillmore proposed
a very simple model to estimate the effect of geometrical size on the drug release
behavior [99], that is
R2
t= (93)
D
where t, R and D are the characteristic time, the hydrogel size, and the co-operative
diffusion coefficient, respectively.
The experiments indicate that, as the diameter of the hydrogel disk decreases, the
amount of the drug release at various time-intervals increases [100]. This may simply be
attributed to the fact that, with increasing the diameter of the hydrogel disk, the surface
area available per gram of polymer decreases. As the flux is directly proportional to the
surface area for given values of other dependent variables, the rate of drug release also
decreases. Therefore, it may be concluded that the desired release rate can be achieved
by making the hydrogel device with a suitable geometrical size, such as the diameter of
cylindrical disk hydrogel device.
Hopfenberg Model
As mentioned above, the geometric effects play an important role in altering the
dissolution rate. Hopfenberg [101] developed a model for theoretical simulation of the
drug release from surface-eroding devices with various geometries including slabs,
spheres and infinite cylinders. In the Hopfenberg model for the slab, spherical and
cylindrical matrices displaying heterogeneous erosion, the governing equation is given
as,
Mt kt
= 1 − [1 − 0 ]n (94)
M∞ c0 a0
where M t is the amount of drug release from the device in time t, M ∞ is total amount
of drug release when the device is exhausted. k 0 is the erosion-rate constant, c0 is the
initially uniform concentration of drug in the matrix, a 0 is the initial radius of a sphere
or cylinder, or the half-thickness of a slab. n is the geometric shape factor, where n = 1
for a slab, n = 2 for a cylinder and n = 3 for a sphere. In the Hopfenberg model, it is
assumed that the release kinetics is not affected by time-dependent diffusional
resistances internally or externally acting on the eroding matrix, namely the actual
erosion process is the rate-limiting step. The contribution of the secondary surface area
is also neglected in release process.
Hopfenberg and Katzhendler et al. [101, 102] also developed another model to
describe the drug release from an erodible tablet matrix, in which the matrix swelling is
assumed to be slower, relatively compared with the erosion process. The matrix swelling
thus occurs prior to the release of drug from the matrix. The model may be applicable for
the erosion rates of the tablets with different erosion rates in the radial and axial
directions. For kinetics of drug release from the erodible tablets matrix, the model with
two coordinates, a in radial direction and b in axial one, is given as,
Mt kt 2k t
= 1 − (1 − a ) 2 (1 − b ) (95)
M∞ c0 a0 c0b0
where k a is the radial erosion-rate constant, k b is the axial erosion-rate constant, and
a 0 and b0 are the initial radius and thickness of the tablets, respectively. This model is
able to provide the fractional amount of drug release from the surface erodible tablets
[103]. In the special case when k a ≅ kb = k0 , Eq. (95) can be rewritten as
Mt kt 2k t
= 1 − (1 − 0 ) 2 (1 − 0 ) (96)
M∞ c0 a0 c0b0
Kim et al. [104] investigated the kinetic swelling of pH-sensitive anionic hydrogels
for oral protein delivery. It is assumed here that the penetrant sorption for long periods is
mainly controlled by relaxation of the polymeric network, and that the sorption process
by polymeric relaxation is first-order. The model for the relaxation may be written as,
dM t
= k 2 (M ∞ − M t ) (97)
dt
where k 2 is the relaxation rate constant. By integrating Eq. (97) one has
Mt
= 1 − A exp(−k 2 t ) (98)
M∞
where A is a constant. Usually the constants A and k 2 are obtained from the slopes and
intercepts of the experimental plot of ln(1 − M t / M ∞ ) versus time t at time later than
those at a given M t / M ∞ .
The model developed by Hopfenberg and Frisch [105], including both the effects of
Fickian diffusion and polymeric relaxation, may predict the solvent uptake in swelling-
controlled release systems, that is,
∂c ∂ ∂c
= ( D − vc) (99)
∂t ∂x ∂x
where c is the concentration of water within the polymeric network, D is the diffusion
coefficient of water through the hydrogel, x is distance, t is time, and v is the velocity of
the glassy/rubbery front during the swelling [106]. Lugo et al. [107] employed the above
governing equation to analyze the transport behavior of the salmon calcitonin in the
hydrogels, by defining c as the concentration of solute and v is the velocity of the
swelling front. They used the analytical solution of Eq. (99), known as the Berens-
Hopfenberg model,
Mt ∞
8 − D(2n + 1) 2 t
= φ F [1 − ∑ exp( )] + φ R (1 − exp(−kt )) (100)
n =1 ( 2n + 1)π
2
M∞ 4l 2
where k is the first-order relaxation constant, D is the diffusion coefficient, φ F and φ R
are the fractions of sorption contributed by Fickian diffusion and the chain relaxation,
respectively. l is the half-thickness of the slab. This model can estimate the overall
release in terms of Fickian and non-Fickian contributions. The analysis results in the
determination of both the diffusion coefficient and a characteristic relaxation time τ
defined as the reciprocal of the term k .
Crank [87] demonstrated a model similar to the above Berens-Hopfenberg model for
a plane film, that is
Mt  8 ∞ exp(−(2n + 1) 2 k F t 
= f F 1 − 2 ∑  + ∑ f R ,i [1 − exp(−k R ,it )] (101)
M∞  π n=0 (2n + 1) 2 
where
4π 2 D
kF = and f F = 1 − ∑ f R ,i (102)
τ 02 i
where f F and f R ,i are the fractions of contributions from the Fickian diffusion and
relaxation processes, and k R ,i is the respective relaxation rate constant. The semi-
empirical analytical equation (101) is applicable for the swelling of coupled film [108].
This model in fact makes the assumption that the water sorption into glassy polymer is a
linear superposition of Fickian diffusion and first-order relaxation.
Scott’s Second-Order Diffusion Model

Scott et al. [109] developed a second-order diffusion model for the kinetics of
swelling of the hydrogels as follows,
dH
= K (H ∞ − H ) 2 (103)
dt
where H is the maximum or equilibrium water uptake at time t. K is the rate constant. If
K = 1 / AH 2 , and replacing H ∞ by 1 / B and integrating Eq. (103), one can have
t
= A + Bt (104)
H
where A and B are the coefficients with the physical meanings [110]: after a long
treatment time Bt >> A and by Eq. (104), B = 1 / H ∞ , i.e. it is the reciprocal of the
maximum or equilibrium water uptake; at a very short treatment time A >> Bt and in
the limit, Eq. (104) becomes to:
dH 1
lim( )= (105)
t →0 dt A
The intercept A thus is the reciprocal of initial swelling rate. By rearranging and
differentiating Eq. (104), one can have
dH A
= (106)
dt ( A + Bt ) 2
Actually the above equation (106) is identical with Eq. (103), which indicates the
swelling rate being a function of the treatment time.
Nernst-Planck Equations
For simulation of ion transport within the hydrogels, the total flux of the ions may be
modeled by the Nernst-Planck equations with consideration of the fluxes due to the
concentration gradient, electrical migration and convection [111, 112],
∂ck ∂ψ
Γk = φ[− Dk − µ k zk ck ] + ckU (107)
∂x ∂x
∂ 2ck ∂ck ∂ψ ∂ 2ψ
Dk 2 + µ k zk + µ k zk ck 2 = 0 (k=1, 2, …, N) (108)
∂x ∂x ∂x ∂x
where Γk , Dk , c k , µ k and z k are the flux, effective diffusivity, concentration,
effective ionic mobility and valence of the kth ion species within the hydrogel. φ is the
gel porosity, ψ is the electric potential, U is the area-averaged fluid velocity relative
to the polymeric network, x is coordinate associated with the deformed hydrogel and N is
total number of ionic species. The electrostatic potential ψ is determined by
∂ 2ψ F N
= − (∑ z k c k ) (109)
∂x 2 εε 0 k =1
where ε 0 is the dielectric constant of vacuum, and ε is the relative dielectric constant
of the solvent.
Arrhenius Model
In the work of Kwak et al. [113], the self-diffusion is regarded as a result of
Brownian motions of the solute, consequently it is sensitive to temperature. An
increasing diffusion is observed as the temperature increases for the solute in curdlan
gels. Thus the temperature dependence scales in fact as an Arrhenius-type equation,
Ea
D = A exp(− ) (110)
RT
where D is the diffusion coefficient, A is a pre-exponential factor, E a is the
activation energy, R is the gas constant, and T is the absolute temperature. The present
activation energy E a associated with the diffusion is defined as the energy barrier that
should be overcome when the solute moves from one surrounding environment to the
other. From the slope of the linear least-square fits, E a of the self-diffusion may be
computed for different gel concentrations. The E a values increase as a function of the
molecular size of the diffusion species. The solute-solvent interactions should thus be
regarded as the major factor for the energy barrier of the diffusion, and the polymer
chains are considered as inert obstacles to the diffusion.
Noyes-Whitney Model
The rate of drug release from a polymeric matrix such as a tablet may also be
controlled by the solubility of the drug in water. Taking a hydrogel-coated polymeric
stent as example, the drug release from the stent requires the diffusion through a
nonerodable hydrogel layer to become available in the bulk solution. The rate of the drug
release through an unstirred liquid film in steady-state can be described by the Noyes-
Whitney model as follows [114],
dm DA(cs − c)
= (111)
dt h
where dm / dt is the rate of drug release, D is the diffusion coefficient, A is the total
surface area of drug in the bulk solution, cs is the drug solubility, c is the concentration
of drug in bulk solution, and h is the thickness of the diffusion layer.
Due to high degree of swelling in most hydrogel coating, the diffusion layer consists
primarily of water. The diffusion constant D for a given drug through the hydrogel
layer may be assumed essentially to be equivalent to its diffusion constant in water. It is
also reasonably assumed that the hydrogel coating swells quickly in water and it retains
the geometric dimension after a steady state is attained. Therefore, both A and h should
remain fairly constant for a given hydrogel coating. Based on the assumptions
mentioned, the Noyes-Whitney equation can be simplified into the following form for
the release of a sparingly-water-soluble drug from a hydrogel-coated polymeric matrix,
dm
= K (c H − c ) (112)
dt
where K is a characteristic constant of a specified drug in the system. For a water-
insoluble drug, it is known that the concentration of the dissolved drug in the diffusion
layer may not reach the saturated solubility of the drug. Then the cs in Eq. (111) should
be replaced by cH , which is the actual concentration of the drug in the diffusion layer.
In a practical environment such as the human body, usually the concentration c in the
bulk solution is much smaller than the drug concentration cH in the diffusion layer.
Thus c in Eq. (112) may be ignored reasonably. Then Eq. (112) can be further
simplified into the form as
dm
= KcH (113)
dt
For a given drug uniformly distributed in the coating matrix, it may be assumed that
the concentration of the dissolved drug in the diffusion layer is directly proportional to
the loading of the drug in the matrix, which should be a reasonable assumption when
cH < cs . Then Eq. (113) may be rewritten as follows,
dm
= K ' cL (114)
dt
where K ' is a characteristic constant of the drug and cL is the loading of the drug in the
hydrogel-coated polymeric matrix. In the Noyes-Whitney model therefore, the rate of the
sparingly-water-soluble drug release from the polymeric matrix is proportional to the
drug loading in the polymeric matrix. By Eq. (114), the rate of drug release from the
hydrogel-coated polymeric matrix is proportional to the drug loading in the polymeric
matrix [115].
Beltzmann Superposition Model

Bell et al. [116] developed a model for analysis of the kinetic response of hydrogel to
the pH changes of surrounding environment, including the expanding and contracting
phenomena. In the model, the strain ε of isotropic swelling of the hydrogel in a solution
is defined as
l − l 0 ∆l
ε= = (115)
l0 l0
where l and l 0 are the deformed and original lengths, respectively. The hydrogels
subjected to various pH conditions over time exhibit the following characteristics: (i) the
strain response is additive to the input of pH changes, and (ii) the response is
independent of the specific time at which the input is imposed. Then the Boltzmann
superposition principle is applicable and one can have
t
∂[ H + ]
ε (t ) = ∫ L(t − τ ) dτ (116)
0 ∂t
where ε (t ) is time-dependent strain, [ H + ] is the ionic concentration of hydrogen, τ

is a dummy variable. L(t − τ ) is the mechano-chemical compliance and it describes the
mechanical response to chemical stimulus or the conversion of chemical energy to
mechanical work. The model can be extended to the isotropic swelling of the hydrogel in
three-dimension domain by,
Vs (t ) l 3 (l 0 + ∆l ) 3
Q(t ) = = 3 = = [1 + ε (t )]3 (117)
Vd l0 l 03
in which Q (t ) is the degree of volume swelling. Vs and V d are the polymer volumes
in the swollen and dry states, respectively.
Guggenheim-Anderson-deBoer and Young-Nelson models

In order to understand the mechanisms of water uptake and the effect of the
hydrophobic component and the drying method used in the water-copolymer interaction,
Osuna et al. [117] used Guggenheim-Anderson-deBoer (GAB) and Young-Nelson

models, which are broadly applied in the characterization of water-amorphous products
systems. The GAB model is given as,
ηkawYm
Y= (118)
(1 − kaw )[1 + (η − 1)kaw ]
where Y is the moisture content of the solid on a dry basis, Ym is the moisture content of
the monolayer, and a w is the water activity. η and k are energetic constants related to
the heat of sorption. The parameters of the GAB model may be estimated by multi-linear
regression of Eq. (118). It is noted that the European Project Group COST 90 on
physical properties of foods has recommended the present GAB model as the
fundamental equation for characterization of water sorption by food materials.
In addition, the Young-Nelson model fits the experimental data of sorption and
desorption to equations in the following form
M s = A( β + θ ) + BθRH (119)
M d = A( β + θ ) + BθRH max (120)
where M s and M d are the amounts of water sorbed and desorbed at each relative
humidity, respectively, and they are expressed as a fraction of the dry mass of the
polymer. A and B are characteristic constants of each material,
ρ wVol M ρ wVol A
A= and B= (121)
Wm Wm
where ρ w is the water density, and W m is the weight of dry material. Vol M and
Vol A are the adsorbed and absorbed water volumes, respectively. θ is the fraction of
the material surface covered by at least one layer of water molecules, and Aθ is the
mass of water in a complete adsorbed monolayer, expressed as a fraction of the dry mass
of the polymer.
CONCLUSIONS
A comprehensive and systematical review has been presented on the recent
development of theoretically modeling for numerical simulation of environmentally
sensitive hydrogels for various applications of drug delivery. The review approximately
categorizes the developed models into two groups, the fundamental models and the
empirical/semi-empirical models, respectively. The models coming from the former
group are generally derived through conventionally fundamental theories or laws, and
the models from the latter one sometimes include the coefficients that are essentially
determined by experiment. The review reveals that the model development of the
responsive hydrogels for applications of drug delivery still is in preliminary stages. Most
of the developed models are continuous- and/or empirical-based. Actually they
difficultly provide precisely physical and chemical bases for description of drug delivery
phenomenon. As well known, the sciences of mathematical modeling and numerical
simulation have been accepted generally as the third mode of scientific discovery, and
other two modes are experiment and analysis, respectively. Therefore, the present
reviewers believe that the more accurately mathematical models and numerical
techniques, such as molecular simulating techniques in micro- and nano-scales, are
critically required and they emerge soon for simulation of advanced drug, protein and
gene delivery.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the financial support from the Agency for
Science, Technology and Research (A*STAR) of Singapore through A*STAR SERC
Grant – SRP on MEMS Phase II under the project number: 022 107 0009.
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Polyelectrolyte Nanocapsules – Promising Progress

in Development of New Drugs and Therapies
Silke Krol*, Alessandra Gliozzi, Alberto Diaspro

CNR-INFM, Department of Physics, University of Genoa, via
Dodecaneso 33, 16146 Genoa, Italy
Abstract: The most promising tool for future applications in the field of
science as well as in medicine is the use of nanobiotechnologies. Especially
self-assembly systems with tailored properties on a nanometer level fulfill the
requirements to nano-organized systems in a satisfactorily manner. Hence the
development of so-called nanocapsules prepared by means of Layer-by-Layer
technique was a great progress on the way to individual drug delivery systems
or nano-sized bioreactors. The preparation of hollow shells for drug delivery
use requires polyelectrolytes as well as a charged core that are not cytotoxic.
According to this purpose CaCO3 crystals with different shapes were
introduced as removable template for capsules with changeable permeability as
a result of pH variations. Due to the low toxic potential of the core it could be
valuable for applications in human body.
Furthermore the nano-organized shells are suitable as coating of living cells or
artificial tissue. With this “second” cell wall it is possible to target the
encapsulated material to predefined organs, and to prevent immune response.
Moreover one can choose between the breakage of the coverage using the
capsule only as targeted carrier or the production of proteins inside the
remaining shell. The requirements for this application are polyelectrolytes that
are not toxic to the tissue of the transplantation site as well as to the coated
cells.
INTRODUCTION
Since long it is known that the cause for many grave diseases can be found in the
partly or complete failure of organs as well as glands producing necessary hormones or
proteins. Still then it was a dream of researchers to replace them by healthy tissue. But as
easy as the idea sounds the conversion was hindered by a lot of nearly insolvable
problems.
The idea to cure diseases by substituting the damaged organ with donor tissue that
should adopt its function has arisen in the year 1883 when for the first time thyroid
tissue was transplanted. In the early 20th Century first experiments with the
transplantation of kidneys started and the surgeons tried to use of organs of foreign
origins like pigs, the so-called xenotransplants (donor and recipient are of different
*Corresponding author: Tel: +39-(0)10-3536309; Fax: +39-(0)10-311066; E-mail: krol@fisica.unige.it

334 Frontiers in Drug Design & Discovery, 2006, Vol. 2 Krol et al.
species). But within a short time period graft rejection occurred leading to inflammation
and destruction of the transplant. As a crucial problem in the replacement of organs or
tissue, the immune response of the recipient organism against the donor tissue was
determined. However, only autotransplantation (donor and recipient are the same
person) or in haploid twins succeeded while allotransplantation (donor and recipient are
human but different persons) mostly failed. Due to these grave problems and the need
for a permanent immunosuppressive drug therapy in order to avoid rejection, a broader
clinical use of organ replacement as cure is hampered.
One of the most serious problems in organ replacement is the mismatch between
available donor tissues and patients who urgently need an organ. Until now worldwide
470.000 kidneys, 74.000 livers and 54.000 hearts were replaced. But the major problem
in the transplantation of organs or tissue is the mismatch between needed and available
organs, for example in the case of hearts in 1998 in Germany around 1000 hearts are
needed but only 542 can be transplanted [1]. This is due to the fact that the crucial
requirement to the donor tissue is that it must fit to most of the recipient’s tissue
parameters [2, 3]. Hence, in many cases only relatives are eligible as donors.
Microencapsulation of tissues from different sources (allo- or xenografts) shall help to
overcome these problems by creating an immunoprotected transplant. The idea to protect
cells by means of a shell of biocompatible material has arisen in the early 70th of the
20th Century [4].
Coated Cells or Artificial Tissue – Hope for the Treatment of Grave Diseases
In the following three main applications for polymer capsule, microencapsulated
living cells or tissue, empty multilayer capsules as drug delivery system and the
application of the multilayer as immune protection for living cells were discussed. First
of all, whole or partial organs are coated to replace the damaged or restrictedly working
tissue without immune suppression. In this case the capsule should remain permanently
or at least for a long time on the enveloped artificial tissue to separate it from the
recipient’s immune system. The second task is to use coated cells as an alternative
approach to somatic gene therapy. For this, implanted recombinant cells with a
protective coating deliver therapeutic substances, e.g. hormones, messengers or proteins
directly to the target tissue. Also here only a stabile and permanent coating is useful. A
third trail leads to capsules as a delivery system for specific materials, e.g. stem cells,
DNA or drugs. For this application, it is absolutely necessary that the trapped material be
released from the capsule.
1. Alginate Microencapsulation
Since the concept of semipermeable microcapsules for transplantation without
immune suppression was introduced [4] this principle has been actively investigated as
therapy for different serious diseases. Encapsulated artificial tissues have been studied
for the treatment of diabetes, liver, or kidney failure. Moreover genetically engineered
cells producing proteins or factors raise hope as targeted drug delivery system or more
important as good alternative for viral induced gene therapy. Artificial cells containing
enzymes have been developed for example as a possible cure in hereditary enzyme
deficiency diseases. Besides, these types of cells are also useful in biotechnology, and
chemical engineering. Even modified hemoglobin as blood substitutes are now under
investigation and are already in Phase III clinical trials in patients [5].
Polyelectrolyte Nanocapsules Frontiers in Drug Design & Discovery, 2006, Vol. 2 335
In 2003 Yoshioka [6] called this strategy of implanted and immunoprotected living
cells as an in vivo drug delivery system ‘‘cytomedicine’’. In the past, the successful
transplantation of different types of living cells or tissues such as islets of Langerhans
[7-12], hepatocytes [13, 14], parathyroid cells [15-17], pituitary cells [18], and thymic
epithelial cells [19] with alginate microencapsulation or more advanced techniques was
reported.
But apart from technical problems with encapsulation of artificial tissue or cells also
ethic arguments especially in the case of xenotransplants have to be taken into account.
Due to the explosive development in this field, in the following, the introduced
applications for the method highlights only exemplarily some interesting progresses.
The most prominent example for microencapsulated artificial tissue is the pancreatic
islet or β-cells as possible therapy for insulin-dependent diabetes. The aim of this
therapy is a transplantation of the islets with a minimum or no immune suppression. Due
to the fact that there are several good reviews [5-8] summarizing the research in this
field, briefly the most important obstacles and advantages in the use of the different
coatings should be stated. But also for numerous other cell types or artificial organs the
alginate microencapsulation was used in the past (Table. 1).
Table 1. Overview of Cell Types Used with Alginate Microencapsulation
Cells Therapy against References
Encapsulated Artificial Tissue
Langerhans’ islets diabetes [5-8, 20-25]
insulin-secreting beta-cell line (BRIN-BD11) diabetes [26]
Liver Liver replacement [13, 14, 27]
parathyroid tissue Thyroid replacement [15-17, 28]
mesenchymal stem cells Cartilage replacement [29, 30]
Encapsulated Producer Cells as Bioreactor
Endostatin releasing producer cells Brain cancer [31-35]
NO producing cells cancer [36]
Interleukin-2 cancer [37]
Monoclonal antibodies Malignant brain cancer [38]
glial cell line-derived neurotrophic factor (GDNF) Parkinson’ disease [39, 40]
alpha-iduronidase producer cells mucopolysaccharidosis VII (MPS) [41]
iduronate-2-sulphatase producer cells Hunter syndrome (MPS II) [42]

But also for other serious diseases the microencapsulation in alginate beads was used
with satisfying success in some documented studies. A new cancer therapy basing on the
implantation of so-called producer cells is under investigation. As can be seen from
Tabble (1) the list of products from these cells span from anti-angionesis factors to
antibodies or transmitters. In all case neat but ultra-pure alginate beads or coated alginate
particles were implanted and the functionality of the enveloped cells was followed for up
to 12 month [32].
But for the therapeutic use of coated pancreatic islets there is still the problem with
the shortage of donor organs. In the last few years, significant progress was made for
xenotransplants from pigs [22]. Especially after the most serious constriction, a possible
infection with porcine endogenous retroviruses, was proved to be without cause [43, 44].
Another strategy is to transplant insulin-secreting beta-cell lines as substitute for the
native pancreas. But with immortal genetically engineered cell line there remain the risk
of cancer from the transplant [25].
Talking about microencapsulation in this context means, entrapment of cells or cell
clusters in high-viscose alginate, a marine polysaccharide, droplets stabilized with
divalent positively charged ions like e.g. barium or calcium are used (Fig. 1). An
exception is the work of Storrs et al. [45] using Langerhans’ islet sheets as a thin planar
bioartificial endocrine pancreas.
Fig. (1). Concept for microencapsulation of cells or cells clusters. 1 indicates the major compound
of the microbead, usually alginate; 2 is an additional coating e.g. to increase the tolerance of the
recipient or induce angiogenesis; 3 sketch the cells; and 4 one of the major problem e.g.
incomplete coating.
Analyzing the existing literature about alginate beads for microencapsulation, several
problems can be identified. The material must always be investigated under
consideration of biocompatibility of the building blocks. An important parameter for
biocompatibility is the fibrotic overgrowth of the implanted material. Overgrowth of the
capsule is on one side linked to attraction and adhesion of fibroblast by the polymer. On
the other side the release of factors like e.g. cytokines, nitroxide (NO) or antibodies by
the enveloped cells of allo- or xenografts can attract macrophages, lead to antibody-
mediated cytotoxicity or again fibrosis. Fibrosis can lead to necrosis of the enveloped
cells due to malnutrition or hypoxia. Apart from direct response of the immune system
another factor for long-term survival of the incorporated tissue is the angiogenesis of the
capsule surface which allows a good connection to the blood circuit. Only recently, e.g.
it was shown for a glucose sensor that the functionality is increased significantly if
revascularization occurs [46].
For alginate the researchers identified as major problem the ratio between the both
building blocks of alginate, L-guluronic (G) and D-mannuronic (M) acid. So the new
generation of capsules was usually prepared of alginates with an intermediate (G) and a
high (M) content because in this way biocompatibility could be gained [25]. Besides
neat alginate beads polyelectrolyte coatings were applied on top of the beads to improve
there biocompatibility, e.g. alginate/L-lysine capsules were constructed as a scaffold for
hepatocytes [27] or polyacrylic acid (PAA)/polyethylenimine (PEI) multilayers on
barium-alginate beads with parathyroid tissue or single parathyroid cells [28]. In case of
enveloped single cells a thinner fibrotic capsule was observable in case of the synthetic
polyion (PAA) as outermost layer.
In order to reduce the necrosis due to hypoxia and/or malnutrition, microcapsules of
barium cross-linked alginate with a high D-mannuronic acid to L-guluronic ratio
(reduced fibrosis) with incorporated perfluorocarbon (material with high oxygen storage
capacity, prevent hypoxia) were developed [25]. These hybrid capsules showed a good
functionality of the enveloped cells over a period of more than two years.
Anyhow, some important drawback for the alginate droplets must be mentioned. The
unfavorable ratio between encapsulated cell volume and overall capsule volume that
allow for a limited possible transplantation site like the peritoneal cavity. As well as that
the random trapping of the islets sometimes can lead to incomplete coverage, undefined
number of cells and prolonged response times to external stimulation.
2. Other Encapsulation Systems
Apart from the alginate microbeads other encapsulation systems or even complete
devices were investigated for their utility as barrier against the immune system. The
microbeads are a direct entrapment of the tissue but also hollow microporous fibers or
entrapment in synthetic polymer aggregates show big advantages. But in some specific
cases the requirements to the material are more demanding (Fig. 2).
One approach was the use of a TheraCyte® device plus a single dose of anti-CD4
antibodies to prevent rejection of different types of encapsulated xenogeneic cells [47].
They tested the protection ability in immunodeficient mice, normal animals and in
culture. Furthermore the foreign body reaction of the empty device in normal mice. But
the results were poor. The device elicited an immune response and only in immunodefi-
cient mice a survival of the implanted cells was observed. The device system seems to
be not flexible enough to solve the problem with infiltrating cells and induced
cytotoxicity of antibodies.
In case of cartilage reconstruction a multilayer photopolymerized hydrogel was
doped with chondrocytes taken from three different zones of native cartilage order to
take into consideration the complex structure of articular cartilage [48]. Another work
describes the encapsulation of human septal cartilage with polyelectrolyte complexes
Fig. (2). A. Concept for microencapsulation of cells or cells clusters in a device e.g. TheraCyte®.
B. Concept for the more demanding cartilage replacement approach. Here, the physical properties
of the material play a crucial role for the functionality.
consisting of sodium cellulose sulfate and poly-(diallyldimethylammoniumchloride)

(PDADMAC) [49]. Comparison of neat cartilage with a polyelectrolyte-protected one
showed that the coated tissue does not degrade and the level of chronic inflammation is
significantly lower. Encapsulation procedures basing only on the use of synthetic
polyelectrolytes were also tested in a new Parkinson’ disease therapy. Here cells were
genetically engineered for a continuous production of the neurotransmitter L-dopamine.
As a good example the investigations of Vallbacka et al. [50] can be mentioned. They
implanted hydroxyethyl methacrylate–methyl methacrylate (HEMA–MMA) coated
PC12 cells in rats while Yoshida et al. [51] used the same system in Japanese monkeys
as preclinical study. They checked for graft rejection as well as for continuous release of
dopamine and levodopa. The results proved a release even 8 weeks after implantation
and only a weak immune response.
Polymer fibers with incorporated producer cells for in vivo expression of
neurotrophic factor like ciliary neurotrophic factor (CNTF) or glial cell line-derived
neurotrophic factor (GDNF) were implanted. The experiments described by Zurn et al.
[52] in her review based on BHK cells encapsulated in hollow polymer fibers. These
cells are designed to release GDNF. They stated a satisfactorily protection of nigral
dopaminergic neurons against lesion-induced cell death in rodent as well as monkey
models of Parkinson’s disease by the released GDNF. But a comparative study by
Bensadoun et al. [53] revealed that 4 weeks post-surgery the GDNF level, released in the
striatum of rats, decreased significantly with polymer rods, whereas they remained stable
with encapsulated cells or lentiviral vectors as protective shell. Tao et al. [54] implantred
the same cell/fiber hybrid system into the eye of a canine model for retinitis pigmentosa.
The experiments indicate that the surgically transplanted, cell containing capsules were
well tolerated, and the cells inside remained viable for at least 7 weeks. The obtained
protection by the secreted CNTF was proved in all animals.
How widespread the possible impact in medical therapy for the hollow microporous
fiber/cell tool is, show studies of Schwenter et al. [55] or Boison and Huber et al . [56,
57]. In their approaches genetically engineered encapsulated cells were transplanted to
secrete the hormone human erythropoietin as a treatment of Epo-responsive anemia or
fibroblasts engineered to release adenosine by inactivating the activity of the adenosine-
metabolizing enzymes adenosine deaminase and adenosine kinase as epilepsy treatment.
After optimization of the infection conditions they found that in vivo erythropoietin
secretion leads to an increase in the hematocrit during the first 2 weeks and elevated
levels over a 6-week period. In case of epilepsy the implantation of the coated cells leads
to a complete protection from clonic seizures, and a nearly complete one from focal
seizures for at least 2 weeks.
Also as protection against tissue damage in case of cerebral infarcts encapsulated
grafts of basic fibroblast growth factor producer cells were tested [58]. In comparison
with non-transfected BHK cells and rats without coated cells implanted the producer
cells enveloped in polymer capsules with a semipermeable membrane revealed a
reduction of the infarct volume by approximately 30% and a significantly decreased
number of apoptotic cells were observed.
Direct interaction between polymer and DNA are well-described as new transfection
system in the gene therapy. But also encapsulated producer cells have an impact in gene
therapy. This became clear from the investigations by Saller et al. [59]. They trapped in
polymerized cellulose sulfate retroviral vector packaging amphotropic cells and tested
their in vitro function and in vivo release of virions in mice after implantation. For at
least 6 weeks survival of the coated cells was proved in culture as well as in two animal
models and also a gene transfer into lymphoid cells was achieved.
Summarizing the results for the different microencapsulation methods in which the
polymers not only cover the cells or cell clusters but also serves as a scaffold it became
clear that the problems are multifaceted. Especially if we memorize the requirements to
a cartilage-replacing scaffold like elasticity, low friction, good nutrition of the embedded
cells etc. These reflections together with the problems discussed in the first part of the
article lead to the conclusion that perhaps a single or double component system could be
too limited to fulfill all the demands to the polyelectrolyte matrix.
3. Polymer Capsule as Delivery System
In the first two parts of this review an overview was given of the impact of
microencapsulated living cells as “cytomedicine” as possible therapy for severe disease.
However, there are several drawbacks mainly related to the fact the used polymer system
is too simple. Basically the polymer capsules around the cells consist of only one or two
components. In the following part of the article polymers were introduced as transporter
and drug delivering unit. In this case, the crucial requirement for the polymer is
biodegradability and non-toxicity neither of the polymer itself nor of the breakdown
products, its metabolites. Furthermore, an ordered multilayer polyelectrolyte system will
be discussed which have the potential to overcome some of the drawbacks highlighted
earlier. Especially in the latter case only a very small section of the field could be
included in this review because the number of inventions is nearly exploded in the last
20 years.
3.1. Polymer-Based Drug Delivery Systems
Due to the importance of gene therapy as a promising strategy for the treatment of
many inheritable or acquired diseases that are currently considered incurable the
research focus on the delivery of DNA to damaged cells. There are two mayor routes,
which are followed: one is the transfection using a virus injection system as nano
syringe. The other one is a non-viral transfection of the cells by uptake through
endocytosis. One of the most interesting applications in this context is a polymer-based
DNA delivery exploits the interaction between a polycation and DNA as polyanion. In
this way the risk of infection or allergic reactions to the virus material can be excluded.
Two main tracks can be distinguished: First co-encapsulation of DNA and target cells in
a polymer matrix like showed by e.g. Quick and Anseth [60]; second, the condensation
of the DNA in a polycation matrix and uptake of the nanoparticles by the cells (Fig. 3).
Fig. (3). Concept for polymer condensation of DNA and gene delivery to the nucleus of the target
cell.
In the following the focus lays on the DNA/polymer system because here numerous
recently published works indicate the importance of the technique for a possible gene
therapy. In this context it becomes fast clear why polyelectrolytes are the perfect
transport for drugs into the human body. Apart from the polymer-based delivery also
cationic liposomes were developed as carrier for the DNA.
A large variety of polycations commercially available as well as exclusively
designed for the specific purpose are used to transfect cells with alien DNA.
Furthermore one can distinguish applications by means of more natural polymers like
poly-amino acids like poly-L-lysine (PLL) from synthetic one like poly-(ethylenimine)
(PEI) or poly-(methylacrylate) (PMA). Important for the entrance of the nanoparticles in

the cells is a slightly positive net charge.
Widely used in order to condense DNA into deliverable units is the polymer PEI like
e.g. described in the review article of by Lungwitz et al. [61]. In addition to linear PEI
also branched or better dendrimeric PEI serves as carrier for DNA They analyzed and
identified the intercellular obstacles preventing a successful transfection with the DNA
of the target tissue as well as potential disadvantages in the polyamine as low
transfection efficiency and cytotoxicity of the material. They suggested extending the
delivery system to endosomolytic agents or nuclear localization signals. Moreover they
refer to the requirement of strategies to prevent unspecific transfection to cells other than
the target tissue. In terms of a more complex polymer system to overcome obstacles the
work of Kim et al. [62] shall be mentioned. They investigated a bi-componental polymer
matrix composed of poly-(L-lactic acid) or the block copolymer poly-(D,L-lactide-co-
glycolide) mixed with PEI. In their preparation the plasmid DNA is attached on the
surface of polymer nanoparticles instead of a co-condensation incorporating the DNA in
the beads. Changing the amount of PEI hence increasing the positive net charge of the
beads can vary the amount of bound DNA. Unfortunately, the transfection efficiency of
bi-component beads is significantly lower in comparison to pure PEI beads. But the
cytotoxicity is diminished for bi-component beads. Both results support the conclusion
that first PEI is cytotoxic and second a positive charge is necessary to facilitate the
entrance of the particles into the cell.
In order to solve the problems with the quite high cytotoxicity another approach
deals with the design of co-polymers instead of constructing beads of two or more
components. Unfortunately in the past, modifications of PEI with dextran sulfate [63] or
human serum albumin [64] always led to a decrease in the transfection efficiency. But
the conjugation of a non-ionic hydrophilic polymer such as poly-(ethylene glycol) (PEG)
to PEI have showed a potential to overcome that drawback [65]. They build
nanoparticles with the co-polymer and DNA degrading at 37°C, non-cytotoxic and
depending on the molecular weight of PEG, the transfection rate for some of the
investigated cell types was augmented with respect to pure PEI/DNA polyplexes.
Furthermore, PEG can enhance the half-life of the particles in bloodstream by reducing
interactions with plasma proteins or other circulating cells [66-69]. But also chemical
modifications of the PEI can lead to reduced toxicity under preservation of DNA
condensing features like proved by Kim et al . [70] with their acid-labile PEI. The idea
that smaller breakdown-products of bigger PEI polymers are an efficient DNA carrier
but non-toxic was supported also the study of cross-linked PEI of different but low
molecular weights by Thomas et al. [71]. The results of their study were more than
promising because they found a higher DNA binding capacity; significantly lower
cytotoxicity and increased transfection rate by linkage of two PEIs. The mechanism for
the strong dependence of the toxicity on the molecular weight is clarified by Moghimi et
al. [72]. They have defined a two-step mechanism to describe the cell-polyion
interaction. In the first phase they observed necrotic-like changes resulting from
compromised membrane integrity, assessed by considerable lactate dehydrogenase
release and phosphatidylserine translocation from the inner plasma membrane to the
outer cell surface. At the second stage the polymer activated presumably a
“mitochondrially mediated apoptotic program”, due channel formation in the outer
mitochondrial membrane. This led to the release of proapoptotic cytochrome C,
subsequent activation of caspase 3, and alteration in mitochondrial membrane potential

as a result of caspase translocation into the mitochondria.
Another step in the direction of a tailored polymer fulfilling a couple of requirements
is the recently published work of Wang and Hsiue [73]. They combine the gene-loading
capability and high transfection efficiency provided by the polycation PEI with the
hydrophobicity of poly-(L-lactide) to enhance the ability of the particles to interact with
cells and better permeation of the tissue and folic acid in order to increase the selectivity
for tumors. The resulting block-co-polymer binds efficiently DNA, allows for
transfection of the cells with good yield and expresses a comparably low cytotoxicity.
A promising application for PEI-condensed DNA in conjunction with a second
polymer is the work of Huang et al. [74, 75]. They followed the bone regeneration in rats
with cranial defects after implantation of a scaffold of the co-polymer poly-(lactic-co-
glycolic acid) with embedded PEI-condensed DNA. The bone morphogenetic protein-4,
encoded by the plasmid, induced a significant increase in osteoid and mineralized tissue.
The example of PEI showed impressively the large diversity of tools to change and
improve the properties of a polyion or polymer with the potential to transport drugs but
some unfavorable features. Only for DNA delivery the list of studied polymers span
from natural glycoaminoglycans like hyaluronic acid [76], to synthetic block
copolymers, e.g. connecting poly-(2-dimethylaminoethyl)methacrylate, poly(ethylene
oxide), and poly(propylene oxide) [77] or permanently charged poly(amidoammonium)
salts [78].
In this context it must be mentioned the work of Cho et al. [79] because it presences
the first step towards multicomponent system combining the suitable functionality of the
single compound to a tailored construct able to fulfill the requirements in vivo. They
condensed plasmid DNA with PEI to nanoparticles to which they apply a block-co-
polymer of folate-poly-(ethylene glycol)-poly (L-lysine). This conjugate should allow
the folate receptor mediated uptake of the particles. The properties of PEG to prevent
cell binding with blood components were formerly mentioned. With this core-shell
construct they found an increased uptake in folate-receptor overexpressing cells in
comparison to deficient ones and a better cell viability and gene expression even in
comparison to PEI/DNA polyplexes.
From this part of the review we can summarize that only one or two component
systems can fulfill the demands of model cell systems but if the delivery or installation
of polymer-coated cells shall take place in a complex organism like the body the system
must be more flexible and consist of several compound with its own function. This is the
starting point of a new generation of coatings, more ordered and more tunable.
3.2. Polyelectrolyte Multilayer-Based Drug Delivery
Since the invention of the layer-by-layer (lbl) deposition of oppositely charged
polyelectrolytes [80, 81] as strategy to coat surfaces the field exploded. Apart from
developing the shells, empty or filled, as drug delivery system it was also used to tailor
the surface properties in order to serve a specific purpose. A good overview gives the
publication of Ai et al. [82]. Here, it was depicted how broad the spectrum of biomedical
applications for multilayered structures is.
Even a multilayered DNA delivery system was described [83]. Jewells and his
coworkers constructed layers of plasmid DNA that serves as polyanion and a
hydrolytically degradable synthetic polycation to transfect cells growing on the treated
surface as a possible transfection system from implants. But mainly the drug delivery
was carried out by colloidal systems, means hollow capsules afterwards filled with the
drug and sealed or capsules retarding the delivery of crystalline drugs used as core for
the layer self-assembly. The idea for ordered encapsulation of particles or entrapment in
multilayer shell is to protect valuable proteins, enzymes, DNA or hormones against
degradation. Moreover, with the shell it is possible to gain access to the delivery of
hydrophobic drugs without chemical modifications of the molecule. Usually the
chemical modification in order to increase the solubility of hydrophobic drugs leads to
decrease in activity. Furthermore encapsulation of drugs allows having a triggered or
sustained release [84]. This capability was investigated by Ai et al. for furosemide [85]
or by Qui et al . for ibuprofen [86]. Either for natural polyelectrolytes like polysaccha-
rides [86] or for synthetic polyions like PSS and PDADMAC (poly-(diallydimethyl-
ammonium chloride)) in addition to gelatin [85] a sustained drug release was found in
dependence of the number of layers. Notable in this context is the fact that furosemide is
practically insoluble in water.
But also the encapsulation of living cells (Fig. 4) is possible as shown by Diaspro,
Gliozzi and Krol et al. [87-90]. They were able to prove that encapsulation of yeast cells
as a model for living cells was firstly possible and second the cells maintain their
functionality and are even able to duplicate after the coating procedure. This hybrid
Fig. (4). Scheme of cell encapsulation by the Layer-by-Layer technique [81].

system of polyelectrolyte multilayer capsules with a specific property allows also for
non-invasive attachment of the cells to surfaces [91]. Furthermore they assume a
protection capability in aggressive environments. In order to study this feature in more
detail a model system was established. For this purpose yeast cells were encapsulated in
a multilayer under specific conditions under which the survival of the cells was
guaranteed [92]. In the first preliminary experiments it was shown that the system is
suitable as model system but the protection could only be gained under different ionic
strength or variations in the number of layers.
That the prevision of a multilayer capsule able to protect or even have a tuned
permeability based on experiments performed on empty capsules. The characterization
of the biophysical properties is mainly investigated by the group of Moehwald and
Sukhorukov. In their studies it could impressively be shown that the nanometer-sized
multilayer on colloids can be influenced by environmental factors like ionic strength,
nature of the polyion, number of layers or temperature [86, 93,94]. That the multilayers
can be functionalized to serve a specific purpose was shown by Diaspro et al. [95]. They
incorporated a pH-measuring unit in the capsule wall and were able to observe local pH
changes induced by an uncaging of protons under two-photon excitation.
Also calcium carbonate proved to be a good core because of its low toxicity and in
some cases its amorphous appearance allows for the controlled delivery and release of
attached drugs [96]. Due to the fact that ionic particles can be included in the capsule
walls [97] they can have a storage function for the embedded cells.
Moreover, experiments with uncoated cells onto polyelectrolyte multilayer surfaces
in order to trigger cells adhesion or repel them are very successful [98, 99]. The attention
was focused on the characterization of flat layers and the parameters leading to cell
adhesion or inhibition of the settlement.
But only a few works until now dealing with the tailoring of best-fitting multilayer
capsules for living single cells or cell clusters.
Future Vision
In this review we tried to figure out the general importance of native or genetically
engineered cells for medical therapies. The idea is that as long as physicians are not able
to repair damaged cells directly in vivo in a kind of nanosurgery the transplantation of
donor cells accomplishing the same function could be of help. But even now, the
donated organs do not satisfy the need and the prevision for the future is a rise in
patients with the urgent need of fitting tissue. Unfortunately, the use of tissue-engineered
material as well as organs from animals is problematic for several reasons. The new
form of encapsulated and in this way immune-protected cells are a useful and handy tool
against most of the severe diseases of our century like cancer, Alzheimer’s and diabetes.
Experiments in the past have showed that the microcapsules in use are too simple to
fulfil the numerous requirements in a complex system like the body. But in parallel in
the last 20 years new nanometer-sized multilayered capsules were developed. This new
generation of shells are actually investigated for a diversity of applications, e.g. drug
delivery, protection capability of proteins or enzymes with maintenance of their activity,
and immobilization of yeast cells without disturbance of the cell functionality. The
system seems to be more suitable for the requirements in the body. Due to the fact that
uncountable natural and synthetic polyelectrolytes are known the palette of instruments
to tailor shells best fitting for durability or degradation, biocompatibility or cell
repletion, incorporated drug release to enhance wound healing or suppress inflammation.
Imaginable is also that the outermost layer serves to transport coated cells to a target
tissue, then will be peeled off, giving way for the next layer which reduces the fibrosis in
that site for some weeks by degrading in subunits interacting with the macrophages or
cytokines and so on, so every layer has its specific function. Another advantage is the
small thickness of the shell because with that it is possible to reduce the volume of
implanted material (Fig. 5).
Fig. (5). Scheme of a multilayer polyelectrolyte capsule as tool to fulfill several functions to allow
long-term transplantation of immune protected cells or artificial tissue.
The diversity of materials and the possibility to arrange them in multilayers where
every layer can have its own feature rise hope that the shell can be as diverse as the
complex body demands for.
ACKNOWLEDGEMENT
S. Krol is grateful to the EU for the financial support by the EC contracts: BARP+:
NMP3-CT-2003-505614 and HPRN-CT- 2000-00159. Further the authors thank Prof. K.
Ulrichs for stimulating discussions and the careful correction of the manuscript.
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Contributors Frontiers in Drug Design & Discovery, 2006, Vol. 2 349
Contributors
Gary W. Caldwell Johnson & Johnson Pharmaceutical Research and

Development, LLC, Welsh and McKean Roads, P.O.
Box 776, Spring House, PA 19477-0776, USA.
Stanley M. Belkowski Johnson & Johnson Pharmaceutical Research and

Roberta Brayner Interfaces, Traitements, Organisation et Dynamique

des Systèmes (ITODYS) –UMR-CNRS 7086,
Université Paris 7 Denis Diderot, case 7090 ; 2 Place
Jussieu 75251 Paris Cedex 05 FRANCE.
Laura Cerchia Istituto di Endocrinologia ed Oncologia Sperimentale

del CNR “G. Salvatore”, via S. Pansini 5, 80131
Naples, ITALY.
Mahesh Choolani. Diagnostic Biomarker Discovery Laboratory,

Department of Obstetrics and Gynaecology, National
University Hospital, 5 Lower Kent Ridge Road,
119074, SINGAPORE.
Clare A. Daykin School of Pharmacy, University of Nottingham,

University Park, Nottingham, NG7 2RD, UK.
Michael R. D’Andrea Johnson & Johnson Pharmaceutical Research and

Alberto Diaspro CNR-INFM, Department of Physics, University of

Genoa, via Dodecaneso 33, 16146 Genoa, ITALY.
Vittorio De Franciscis Istituto di Endocrinologia ed Oncologia Sperimentale

del CNR “G. Salvatore”, via S. Pansini 5, 80131
Naples, ITALY.
James M. Dixon Johnson & Johnson Pharmaceutical Research and

Martijn van Doorn Centre for Human Drug Research, Zernikedreef 10,
2333 CL Leiden, THE NETHERLANDS.
Iram Mondaca Fernandez Agricultural and Biosystems Engineering, The

University of Arizona, 1177 E. 4th Street, Shantz
Building, Room 403, Tucson, Arizona, 85721, USA.
350 Frontiers in Drug Design & Discovery, 2006, Vol. 2 Contributors
Alessandra Gliozzi CNR-INFM, Department of Physics, University of

Brenda Hertzog Johnson & Johnson Pharmaceutical Research and

Ewoud J. van Hoogdalem Johnson & Johnson Pharmaceutical Research and

Development, LLC, Turnhoutseweg 30, B-2340
Beerse, BELGIUM.
Dan Horowitz Johnson & Johnson Pharmaceutical Research and

Sergey I. Ilyin Johnson & Johnson Pharmaceutical Research and

Williams Jones Johnson & Johnson Pharmaceutical Research and

Masaru Kato School of Pharmaceutical Sciences and COE

Program in the 21st Century, University of Shizuoka,
52-1 Yada Suruga-ku, Shizuoka, 422-8526, Japan.
PRESTO, JAPAN Science and Technology Agency
(JST), Saitama, JAPAN.
Spiridon E. Kintzios EMBIO/Laboratory of Plant Physiology, Faculty of

Biotechnology, Agricultural University of Athens,
Iera Odos 75, 11855 Athens, GREECE.
Silke Krol CNR-INFM, Department of Physics, University of

Aarohi Kulkarni Division of Biochemical Sciences, National

Chemical Laboratory, Pune, 411008, Maharashtra,
INDIA.
Wensheng Lang Johnson & Johnson Pharmaceutical Research and

K. Y. Lam Institute of High Performance Computing, National

University of Singapore, 1 Science Park Road, #01-
01 The Capricorn Singapore Science Park II, 117528
SINGAPORE.
Contributors Frontiers in Drug Design & Discovery, 2006, Vol. 2 351
Danielle Lawrence Johnson & Johnson Pharmaceutical Research and

Gregory C. Leo Johnson & Johnson Pharmaceutical Research and

Hua Li Institute of High Performance Computing, National

SINGAPORE.
Xing Jian Lou Johnson & Johnson Pharmaceutical Research and

Pierre Lucas Materials Science and Engineering, The University

of Arizona, 1177 E. 4th Street, Shantz Building,
Room 403, Tucson, Arizona, 85721, USA.
Rongmo Luo Institute of High Performance Computing, National

SINGAPORE.
Andrew Mahan Johnson & Johnson Pharmaceutical Research and

Douglas.P. Malinowski TriPath Oncology, 4025 Stirrup Creek Drive, Suite

400, Durham, North Carolina, 27702, USA.
John A Masucci Johnson & Johnson Pharmaceutical Research and

Kothandaraman Narasimhan Diagnostic Biomarker Discovery Laboratory,

119074, SINGAPORE.
Debbie Polkovitch Johnson & Johnson Pharmaceutical Research and

Jeremy J. Ramsden School of Industrial and Manufacturing Science,

Cranfield University, MK43 0AL, UK.
352 Frontiers in Drug Design & Discovery, 2006, Vol. 2 Contributors
Mala Rao Division of Biochemical Sciences, National

Chemical Laboratory, Pune, 411008, Maharashtra,
INDIA.
Mark Riley Agricultural and Biosystems Engineering, The

University of Arizona, 1177 E. 4th Street, Shantz
Building, Room 403, Tucson, Arizona, 85721, USA.
Kumiko Sakai-Kato Research Institute of Pharmaceutical Sciences,

Musashino University, 1-1-20 Shinmachi,
Nishitokyo-shi, Tokyo, 202-8585, JAPAN.
Ponnusamy Sukumar Diagnostic Biomarker Discovery Laboratory,

119074, SINGAPORE.
Visith Thongboonkerd Siriraj Proteomics Facility, Medical Molecular

Biology Unit, Office for Research and Development,
Faculty of Medicine Siriraj Hospital, Mahidol
University, 12th Fl. Adulyadej Vikrom Bldg., 2
Prannok Rd., Bangkoknoi, Bangkok 10700,
THAILAND.
Meghan Towers Johnson & Johnson Pharmaceutical Research and

Toshimasa Toyo'oka School of Pharmaceutical Sciences and COE

Program in the 21st Century, University of Shizuoka,
52-1 Yada Suruga-ku, Shizuoka, 422-8526, JAPAN.
Naoko Utsunomiya-Tate Research Institute of Pharmaceutical Sciences,

Musashino University, 1-1-20 Shinmachi,
Nishitokyo-shi, Tokyo, 202-8585, JAPAN.
Florian Wülfert School of Biosciences, University of Nottingham,

Sutton Bonington, Loughborough, Leicestershire,
LE12 5RD, UK.
Subject Index Frontiers in Drug Design & Discovery, 2006, Vol. 2 353
SUBJECT INDEX TO VOLUME 2
Absorption isotherm model 308 Aptamer 104, 106, 108,109

Acetonitrile 135 affinities of 106
as probes in microarray format
Activated partial thromboplastin time
109
(aPTT) 13 based separation methods for
Adenocarcinoma 42 protein deterection 109
Adenosine deaminase 339 in enzyme-linked immunosorbent
in epilepsy treatment 339 assay (ELISA)-like assays 108
in vivo applications of 106
Adenosine kinase 339
three-dimensional shapes of 104
in epilepsy treatment 339
Aptamer-based technologies 103
Age-related macular degeneration 106 as tools for proteomics 103
treatment of 106
Arrhenius model 324
Alginate microencapsulation 334
Artificial neural networks (ANNs) 164
Alkaline phophatases 25 for prediction of metabolite
Alpha-naphthylisothiocyanate (ANIT) concentration 164
167 Atherosclerosis 10
Alzheimer’s disease 10 heterogeneity of 10
heterogeneity of 10 Atmospheric pressure chemical
Aminophenol (PAP) 4-167 ionization (APC) 202
Anchorage dependent cells 267 ATP binding site 76
Antibody 58,144 as target for kinase inhibition 76
in cancer research 58 Atypical squamous cell 36
types of 58 Automated capillary electrophoresis
Antibody arrays 78 (CE) 195
profiling receptor tyrosine kinase
activation by 78
Beltzmann superposition model 326
Antibody-labeled magnetic microparticle
71 Beta I isoenzymes 106
Antibody microarray 26 Bimodal porous monolith 277
as high-throughput technology 26 Bioconjugates 70
Antibody-pair microarrays 59 in cancer nanotechnology 70
Antigens 217 Bioelectric recognition assays (BERA)
232
Anti-PDGF-RTK activity 11
Biofluid 159
Anti-ricin assay 110 for multivariate analysis (MVA)
Antisense oligodeoxynucleotides (ODN) 159
80 Biomarker 16,23,35
targeting IGF-1R induced associated with HPV induced
apoptosis in 80 cervical neoplasia 42
Antisense oligonucleotides 104 clinical utility of 49
354 Frontiers in Drug Design & Discovery, 2006, Vol. 2 Subject Index
diagnose high-grade cervical Brunauer-Emmett-Teller (BET) 280

disease 35 by immunohistochemistry 9
discovery of 23
for cervical disease testing 49
high-throughput methodologies Camera-Roda Sarti model 319
to 23 Cancer 10,35,104
pharmacogenomic type 16 heterogeneity of 10
protein/antibody microarrays in Cancer therapy 74
pharmacogenetic type 23
to detect cervical neoplasia 35 Capillary electrochromatography
validation of 16 (CEC) 284
Biomarker discovery 5 Capillary electrophoresis (CE) 195,284
for drug development 5 Carbon tetrachloride CCl4 167
strategies of 5 Cation exchange/reversed-phase liquid
Biomolecule 71,287 chromatography 125
electronic properties of 71 CE/MS based 205
exploitation of 71
sol-gel processing for Cell adhesion 269
immobilization of 278 to fiber surface 269
silane precursor of 278 Cell based assay system 226
Biopolymer 248,251 Cell based biosensors 225
plasmon resonance measurements classification of 226
of 248 in proteomic analysis 225
resonance light scattering (RLS) of Cell physiology function 259
248 spectroscopic analysis of 259
surface-enhanced resonance Cell proliferation 11
Raman scattering (SERRS)
of 248 Cell surface protein 104
expression/activity of 104
Biosensors 108,241
for recognition/monitoring of Cellular lysate protein arrays 61
molecule interactions 241 to study kinase activity 61
with aptamers as biorecognition Cellular microarrays 228
element 108 Cellular proliferation 42
Biotechnology 72 Central nervous system (CNS) receptor
miniaturiazed assays in 72
15
molecular profiling by 72
Cervical carcinoma 42,45
Biotin 25 claudin 1/7 in 45
Bisphosphonates 11 DNA microarray profiling of 45
anti-angiogenic effect of 11 laminin V in 45
Bovine serum albumin 250,284 microarray analysis of 42
Breast cencer 231 Cervical neoplasia 36
Bromoethylamine (BEA) 2-167 alterations of cyclin expression
in 44
Bronomics 152 altered expression in 42
analysis of cell cycle regulatory Congenital hypothyroidism 65

genes in 45 Cosmaceutical 154
beta-catenin in 46
Coumarin 13
by oncogenic stains of human
hepatic toxicity of 13
papillomavirus (HPV) 38
cell cycle regulatory proteins in C-reactive protein (CRP) 9
42 as disease-related biomarker 9
cellular proliferation in 43 Cross-linked macromolecules 295
cyclins in 42 Cryogenic probes 157
DNA replication in 43
ERK in 45 CXCL10/CXCR3 receptor 107
extracellular matrix proteins in 45 Cyclins 42
focal adhesion kinase (FAK) in HPV-induced neoplasia 42
pathway in 47 Cyclin dependent kinases (CDK) 37
MAP kinase in 45 over-expression of 37
markers of 50 CDK 4 as 42
microarray analysis of 43 CDK 6 as 42
molecular biology of 37 Cyclin dependent kinase inhibitors 42
origin recognition complex (ORC)
in 43 Cyclooxygenase-2 289
proliferation signatures in 47 Cytochrome P450 72
signal transduction pathways in and ovarian cancer 72
45,46 Cytokines 337
S-phase genes in 45 Cytomedicine 339
WNT signal transduction pathway
in 46
Charged residue model (CRM) 131 Darcy’s law 307
Chalcogenide fiber 269 Death 11
Chemiluminesence immunoassay 252 Delivery system 339
polymer capsule as 339
Chemotherapy 69
Dendrimer nanocomposites (DNC) 247
Chromatography mass spectrometry 193
based metabonomic analytical Deoxyribonucleic acids (DNA) 194
methods 193 Developed matrix-assisted laser
Ciliary neurotrophic factor (CNTF) desorption/ionization (MALDI)
338 88
glial cell line-derived 338 Differentiation 11
in vivo expression of 338 Dihydrofolate reductase 286
Ciprofibrate 175 Dihydroxybenzoic acid (DHB) 2,5-135
chemometric analysis of 179 Dihyrofolate reductase 289
PLS-DA models for 179 Disease specific assays 72
Collagenase-3 9 Disease-specific expression patterns 7
Collagens 296 proteomic/metabolomic
Condensation 275 approaches for 7
D-mannuronic (M) 337 E-cadherin 14,47

DNA barcodes 71 80-kDa fragment of 14
DNA hybridization 247 Electron microscopy (SEM) 280
DNA probes 247 Electroosomotic flow (EOF) 284
DNA replication 42 Electrosonic spray ionization (ESSI) 131
DNA RNA aptamers 110 Electrospray ionization (ESI)
bio-recognition element in optical 122,130,196
sensors 110 as atmospheric pressure ionization
DNA sequences 246 (API) techniques 130
application of 133
DNA tumor viruses 37
variants in 133
Drug 219,221
Encapsulated biomolecules 273
from novel delivery materials 219
combinational chemistry 273
response of living cells to 221
for high-throughput screening
Drug delivery 241,295 273
modeling of environmentally using sol-gel reaction 273
sensitive hydrogels for 295
Enzyme-immobilization technique 274
Drug development 5
Encapsulated microfluidic 287
biomarker in 6
DNA microarrays in 6 Encapsulation systems 337
high-dersity single nucleotide Endocrinopathies 65
polymorphism (SNP) in 6 Endothelial leukocyte adhesion molecule
microarrays in 6
(ELAM) 231
quantitative PCR in 6
Enzymatic assays 73
Drug discovery 55
in microsphere environment 73
based technique 71
clinical proteomics market in 57 Epithelial type II pneumocytes
dimensional MALDI-MS protein (A549 cells) 267
array in 60 Epidermal growth factor receptor
1-D/2-D gel electrophoresis in 56
(EGFR) 104
impact of proteomics technologies
on 56 Epithelial ovarian cancer biomarkers 9
in other novel strategies to 79 by quantitative PCR 9
metabolism assays in 55 Escherichia coli 261
protein assay in 55 Fourier self deconvolution (FSD) 262
reverse phase protein microarrays Estrogen receptor (ER) 230
in 59
Ethanol 135
Drug-induced system damage 13
Expression arrays 146
Dynamic light scattering (DLS) 280
Expression proteomics 88
in protein-protein interactions 88
E2F-1 transcription factor 44 in post-translational modifications
role of MCM genes 44 in 88
Factor IX 13
FGF-2 9 Genomics 8,126,193

as biomarker 9 high-throughput technologies
Fibrosis 337 for 8
Fick’s law 297 Genomic technologies 6
in genome-wide analysis of
Fiber evanescent wave spectroscopy
disease-unique gene expression
(FEWS) 266 profiles 6
Flow-injection (FI-) NMR accessory 155 Gentamicin 14
Fluorescence energy transfer 71 hepatic toxicity of 14
Fluorescence in situ hybridization Gibbs-Duhem equation 221
(FISH) 229 Global fingerprinting 194
Fluorescence resonance energy transfer Glucose oxidase (GOD) 283
(FRET) 115 Glutathione S-transferase (GST)
Fluorescent biosensing 254 fusion 122
Fluorescent encoding 254 Glutathione-S-transferase green
Fluorescent proteins 25 fluorescent protein (GST-GFP) 61
Fluoroimmunoassay 252 GP IIb/IIIa receptor antagonists 12
Fluorophore-labelled signaling efficacy of 12
oligonucleotides 115 Grave disease 334
coated cells artificial tissue in
Fourier transform infrared (FTIR)
334
spectroscopy 261,279 treatment of 334
Fourier transform ion cyclotron Guggenheim Anderson Deboer
resonance (FT-ICR) 203 Young models 326
Functional arrays 148
Functional profiling 194 H1NMR spectroscopy 151
Functionalized magnetic nanoparticles data pre-processing of 165
251 in metabolite identification 166
in metabonomics 154
Fundamental mathematical models in principal component analysis
297 (PCA) 160
role of automation 155
Gas chromatography (GC) 195 spectral editing methods of 156
Gene chips 152 Haptoglobin-α subunit 15
identification of 15
Gene/protein-expression profiles 29
using microarray technology 29 HDJ-2 farnesylation 12
inhibition of 12
Genetic heterogeneity 231
Hematologic disorders 65
Genome 195
HER2/neu receptor 12
Genome project 81
mining kinome in 81 High pressure liquid chromatography
(HPLC) 195
High-density lipoprotein (HDL) 9 IL-8 9

as disease-related biomarker 9 as biomarker 9
High-grade cervical disease 49 Immobilized biomolecules 280
MCM2 in 49 characteristics of 280
MCM6 in 49 Immobilized pH gradients (IPGs) 124
MCM7 in 49
Immunoprecipitation 31
TOP2A in 49
anti-mKIAA antibodies in 31
High-grade cervical dysplasia 43
Immunotoxins 80
CDC6 expression in 43
based therapeutics 80
High-grade squamous intraepithelial
In situ hybridization (ISH) 229
lesion 36
Inflammation 334
High resolution magic angle spinning
Inflammation-associated genes 231
(HR-MAS) 158
Inflammatory signals 231
High throughput 24
protein expression analysis of 24 Interaction arrays 147
High throughput assays 5 Interleukin-1 (IL-1) 231
High throughput screening (HTS) 2276 Intracellular pathway database 30
based on mKIAA protein-protein
Higuchi equation 304
interactions 30
HIV-1 replication 247
Ion evaporation model (IEM) 131
Hopfenberg model 321
Ion sensitive field effect transistors
Horse radish peroxidase (HRP) 287
(ISFETs) 228
HPV E6/E7/mRNA 40
Isoelectric focusing (IEF) 123
detection of 40
JAK2 gene 77
Human genome project (HPG) 195
JAK2 specific inhibitors 77
Human immunoglobulins 250
Janus kinase (JAK) family 77
Human papillomavirus (HPV) 35
DNA detection in cervical JNJ-10198409 11
screening of 37,39
induced cellular transformation 42 Keratins 125
infections by 40 from human skin/hair 125
open reading frame genes of 40
Kidney diseases 87
testing of 37
by congenital defects 89
Human serum albumin (HAS) 250 by hemodynamic dysregulation
HUT/CCR cells 247 89
Hydrazine 13,167 by infection 89
hepatic toxicity of 13 by inflammation 89
by metabolic derangement 89
Hydrogel 295
by neoplasm 89
Hydrolysis 275 by physical injury 89
Hypoxia 337 by toxicity from drugs 89
capillary electrophoresis coupled
to MS (CE-MS) in 90 Liver proteins 125

for 90 two-dimensional gel-pattern
gel-based methods to study 89 of 125
gel-free methods to study 89 Liver toxicity 13
integrative OMICS for 100
Low-density lipoprotein (LDL) 9
mass spectrometric immunoassay
as disease-related biomarker 9
(MSIA) in 91
pharmacoproteomics for drug
design/discovery in 99 Magnetic resonance imaging (MRI) 252
protein chip technology to 90 Contrast agents (Cas) in 252
proteome profiling in 92 Magnetic resonance spectroscopy (MRS)
proteomic screening for novel 158
therapeutic targets in 87
Magnetospirillum gryphiswaldense 251
role of liquid chromatography
(LC) coupled to tandem MS Magnetospirillum sp. AMB 251
(MS/MS) 89 Magnetospirillum sp. MGT-1 251
screening for novel therapeutic MALDI time of flight (TOF) 197
targets of 92
surface-enhanced laser desorption/ MALDI-ToF-MS 125
ionization (SELDI) 90 Mass mapping 126
Kinase activity 60 Mass spectrometers 127
protein peptide arrays for 60 Mass spectrometry (MS) 88,195
Mass spectroscopy 121
Langmuir theory 308 application of 129
Laser desorption/ionization (LDI) 122 developments in 121
in proteomics research 122
L-guluronic 337 ion source in 128
Light addressable potentiometric sensor limitations of 142
(LAPS) mass analyzer in 128
Lipid membranes `220 mass spectrometer in 127
interactions of 220 role of detector 129
tool in promteomics 126
Lipidomics 67
in cancer research 67 Matabolome 198
Lipid-protein interactions 62 Matabolomic analysis 204
arrays for detection of 62 Escherichia coli strain DH5-α
Lipoxygenase activating protein (FLAP) 204
of islets of langerhans 204
5-10
gene encoding of 10 Matabolomics 199
NMR-based 199
Liquid chromatography-mass
applications of 137 Matrix assisted laser desorption
in pharmacokinetics of 137 ionization (MALDI) 133,196,242
in proteomics 137 Matrix-assisted laser desorption mass
spectrometry (LC-MS) 137
spectrometry (MALDI-MS) 56
Matrix-assisted laser desorption/ Minichromosome maintenance (MCM)

ionization (MALDI) 129 43
Maxwell model 314 mKIAA proteins 30
MCM family 42 MMP-13 9
Messenger ribonucleic acids (mRNA) transgenic over-expression of 9
194 Molecular aptamer beacons 114
protein detection by 114
Metabolic disorders 65
Monoclonal antibody 79
Metabolic profiling 153,266
as inhibitors for PTKS 79
Methylmethane sulfonate (MMS) 267
Monolith 276
Metabolomic 8,64,65, 68,151,193, structure of 276
200,205 mRNA array technologies 126
application to nutritional studies
Multidiemensional liquid chromato-
168
applications of 151 graphy mass spectrometry (MD LC-
biological challenges in 167 MS) 56
for cancer research 65 Multidimensional 56
in biofluid studies 155 liquid chromatography 56
in disease processes 68
Multiplexed elisas 59
in normality studies 168
in toxicological studies 167 Mutualistic associations of knowledge
in understanding global system by computational comparative
biology 68 technology 31
metastatic bone disease 11
NMR spectroscopy based 151
NMR spectroscopy in 154 Nano technology 68
nomenclature of 153 in biomedical science 68
of small molecules aiding Nanobiotechnology research 68,71
biomarker discovery 64 Nanoparticle-aptamer bioconjugates 69
role in tumor metabolome 65
role of automation 155 Nanoscience 68
technology of 151 in biomedical science 68
to study disease in man 168 Nanotechnology based assays 70
tool for biomarker discovery 64 application of 70
Methylene blue (MB) 115,250 for detecting protein DNA 70
Methyltrimethoxysilane (MTMS) 284 Natural biopolymer nanoparticle hybride
approaches in 241
Microarray chips 287 bio-detection applications of 241
Microarray technology 72 functional materials 241
molecular profiling by 72 N-cyclopentyladenosine analogs 13
Microencapsulation 336 in vivo effects of 13
Microphysiometry 227 Nernst Planck equations 324
Microtiter plates 287
Neural networks 164 Partial least squares reguares regression

for prediction of metabolite (PLS) 162
concentration 164 for data analysis 162
Nitro oxide (NO) 337 Partial squares-discriminant analysis
N-methylnicotinate 188 (PLS-DA) 179
NMR-based metabonomics 175 PDGF B-chain (PDGF-BB) 107
of urine 175
Peptides 143
in healthy volunteers patients with
mass values of amino acid
type 2 diabetes mellitus 175
residues in 143
Nocogene proteins 37
Peptidoglycan 260
Non-receptor tyrosine kinase 75 proportion of 260
classification of 75
Peptidomimetics 80
Nosine mono phosphate dehydrogenase
Peripheral blood mononuclear cells
110
(PBMC) 17
Noyes Whitney model 325
Peroxidases 25
Nuclear magnetic resonance (NMR)
Pharmacokinetics mode 12
195,279 in drug discovery 12
Nucleic acid-based compounds 104 Phenylketonuria (PKU) 65
Nutriceutical 154 as metabolic disorder 65
Phorbol myristate acetate (PMA) 231
2’-O-alkyl nucleotides 107 Photo SELEX 112
Omics technologies 153 as capture agents 112
Optical waveguide lightmode Photoaptamer technology 113
applications to drug discovery application of 113
211,216 to proteomics 113
lightmode spectrum in 214 Photoaptamer-based chips 112
molecular interactions 211 Photoaptamer-protein photocross-linking
technology of 212
reactions 113
Orthogonal signal correction (OSC) 166
Photo-crosslink 112
application of 166
Photopolymerized sol-gel (PSG)
Osteoarthritis (OA) 9
type II collagen peptide as 277,287
biomarker for 9 Pichia pastor 145
PLA polymer system 70
p16INK4A(p16) 42 Platelet derived growth factor (PDGF)
Pappas model 309 107
Parkinson’s disease 338 Platelet-derived growth factor receptor
monkey models of 338 tyrosine kinase (PDGF-RTK) 11
Partial least squares discriminant analysis JNJ-10198409 as 11
(PLS-DA) 179 Platinum palladium metallic
nanoparticles 245 Protein detection 113

PLS-based uninformative variable by aptamer-based proximity
ligation assays 113
elimination (PLS-UVE) 166
Protein expression 23
Polu-l-lysine (PLL) 340
immunohistochemical analysis of
Poly amidoamine (PAMAM) 247 23
Poly ethylene oxide 277 role of western blot 23
Polyacrylic acid (PAA) 337 Protein ions 126
Polyanion 340 by gas-phase activation of 126
Polydimethylsiloxane (PDMS) 287 Protein kinase assays 78
microtiter plate array system for
Polyelectrolyte multiplayer-based
78
drug delivery 342
Protein kinase drug design 76
Polyelectrolyte nanocapsules 333
crystallography for 76
in development of new drug
therapies 333 Protein microarray 25
in interactions with peptides 25
Polyethlenimine (PEI) 337
role in oligosacchaloides 25
Polyethylene glycol (PED) 277 role in or DNA 25
Polymer-based drug delivery role in small molecules 25
Polymers chain reaction (PCR) 196 Protein misfolding 104
Porous monolith 276 in Alzheimer’s disease (AD) 104
in Huntington’s disease 104
Postprandial hyperglycemia 247 in Parkinson’s disease (PD) 104
Posttranslation modifications 225 in Prion disease 104
Post-translational modifications 196 Protein-protein interactions 56
Power law 306 proteomics technologies for 56
PPARα agonists 176 two-hybrid system for 56
Protein tyrosine kinase 74,76
Primary cervical carcinoma lesions 42
Protein/antibody microarray platforms
Principal component analysis (PCA) 179
25
Principal component discriminant
classification of 25
analysis (PCDA) 161
Protein-derived peptides 90
Progesterone receptor (PR) 230
role of strong cation-exchange
Protein 8,104,266 (SCX) chromatography 90
in cell growth 104
Proteolysis 42
in post-translational modifications
8 Proteome 196
methods for 266 Proteomic technologies 89
Protein analysis 7 for analysis of kidney/urine 89
Protein arrays 57,62,121,144, Proteomics 56,57,88,103,121,193
developments in 121 associated interventions in clinical
screening 56
Protein arrays based 62
developments in 121
on SELDI-TOF-MS 62
in diagnosis 103 model 323

in therapy 103 Serial analysis of gene expression
solution-based 125
(SAGE) 196
to study pathomechanisms of
human disease 57 Sickle cell disease 65
Proximity ligation 114 Single nucleotide polymorphisms
mechanism of 114 (SNPs) 57
PSMA-positive prostate cancer cells 70 functional effects of 57
Purvalanol A 77 Skeletal metastases 11
Sodium dodecyl sulfate polyacrylamide
Quantum. 254 gel electrophoresis (SDS-PAGE 197
Quartz-crystal microbalance (QCM) 111 Soft laser desorption (SLD) 129
Sol-gel glasses 289
chemical applications of 289
R115777 12
medical application of 290
anticancer activity of 12
whole cell in 289
Raman spectroscopy 262
Sol-gel reaction 275
Ras binding domain (RBD) 107 application of 282
of Raf-1 107 in study for protein characteristics
Reading frames (ORFs) 122 282
Receptor tyrosine kinases 75 Spectroscopic methods 260
classification of 75 applications of 260
Reverse-phase protein microarrays 24,59 foundations of 261
Reverse transcription polymerase chain S-phase genes 45
reaction (RT-PCR) 233 Spinodal decomposition 277
schematics of 277
Rheumatoid arthritis 17
Squamous cell carcinoma 42
RhoB 12
up-regulation of 12 Stable isotope labeling 226
RNA aptamers 107 Structural profiling 194
with 2’-fluoroaminopyrimidine Supercritical fluid chromatography
modifications 107 (SFC) 201
in extraction procedures 201
Saccharamyces cerivisae 61,145 Superoxide dismutase (SOD) 232
Sandwich assay 110 electroinsertion of 232
Sarface plasmon resonance (SPR) 145 Surface plasmon resonance (SPR)
combination of 145 24,111
Scanning angle reflectometry (SAR) 215 based antibody microarray system
24,27
Scanning electron microscopy (SEM)
Surface plasmon resonance imaging 62
244
System biology 193
Scott’s seconds order diffusion
Systematic evolution of ligands by Transmission electron microscopy 280

expontial entrichment (SELEX) Transplantation 334
technology 104 semipermeable microcapsules
as in vitro combinatorial chemistry for 334
process 105 Trifluroacetate (TFA) 135
used to identify aptamers 105
Triglyceride (TG) 176
Systems biology 152
Trimethylamine-n-oxide (TMAO) 187
Systeomics 152
Trypsin 287
on microfluidic chip 287
Tanaka-Fillmore model in 320 Tumor necrosis factor-α (TNF-α) 231
Tandem mass spectrometry (MS-MS) Two dimensional polyacrylamide gel
64,138 electrophoresis (2-PAGE) 123,226
applications of 139 in separation/detection of proteins
peptide sequencing by 139 123
newborn screening by 64 is membrane proteins 124
typical peptide mass mapping
using 138 Type 2 diabetes mellitus 178
demographics for 178
Target profiling 194
Tyrosine kinases 75
Target-related biomarkers 6 classification of 75
to elucidate mechanism of action
(MOA) 6
used to screen lead compounds 6 Urinary proteome profiling 91
Target-related biomarkers 9 for biomarker discovery 91
in clinical diagnostics 91
Thrombin 115
Urine 178
Tie-of flight (TOF) analyzer 128 role of NMR 175
Time-of-flight mass spectrometer 136 Uterine-cervix 35
Tissue microarrays 229
applications of 229
in cancer research 230 VEGF 12
inhibition of 12
Tissue NMR spectroscopic data 159
for multivariate analysis (MVA) Vide supra 8
159 Viral genotyping 39
Topoisomerase II alpha (TOP2A) 42 using liquid-based cervical
cytology specimens 39
Toxicoproteomics 56
Trans-activator of transcription (Tat) 111
of human immunodeficiency virus Watson-Crick base pairing 80
type 1 (HIV-1) 111
Transcriptional profiling 26 Xenobiotic 154
Transcriptomics 126 Xerogel 276
Transfected cell microarrays 231 X-ray crystallography 217
Zero-order release 310

Zoledronic acid 11
in management of

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Frontiers in Drug Design & Discovery

Bentham Science Publishers Ltd.

Strategies of Biomarker Discovery for Drug Development 5

Protein and Antibody Microarrays: Clues Towards 23

The use of Biomarkers to Detect Cervical Neoplasia and 35

New Developments in the Field of Protein and Metabolism 55

Aptamer-Based Technologies as New Tools for Proteomics 103

Recent Developments in Proteomics: Mass Spectroscopy 121

NMR Spectroscopy Based Metabonomics: Current 151

NMR-Based Metabonomics of Urine from an Exploratory 175

Chromatography Mass Spectrometry Based 193

OWLS—A Versatile Technique for Drug Discovery 211

Cell-Based Biosensors in Proteomic Analysis 225

Spectroscopic Analysis of Cell Physiology and Function 259

Encapsulates Biomolecules Using Sol-Gel Reaction for 273

Modeling of Environmentally Sensitive Hydrogels for 295

Polyelectrolyte Nanocapeules – Promising Progress 333

Subject Index 353

Editorial: “Biomarkers to Biosensors – Technologies and Applications

grade cervical disease. These biomarkers appear promising in molecular diagnostic

mathematical modeling for simulation of environmentally sensitive hydrogels. The

Strategies of Biomarker Discovery for Drug

Xiang Jian Lou, S.M. Belkowski, James M. Dixon, Brenda Hertzog,

*Corresponding author: Tel: 215-628-5619; Fax: 215-540-4887; E-mail: MDANDREA@PRDUS.JNJ.COM

Garry W. Caldwell / Atta-ur-Rahman / Michael R. D'Andrea / M. Iqbal Choudhary (Eds.)

Proteomic and metabolomic approaches screen for the disease-specific expression

TARGET SELECTION STAGE

cytokine IL-8 and FGF-2 as potential biomarkers by comparing primary cultures of 39

heterogeneity of many diseases such as atherosclerosis, cancer and Alzheimer’s disease

LEAD VALIDATION AND OPTIMIZATION STAGE

targets affect cell proliferation, differentiation and death; however, in an effort to

These results confirmed the ability of this compound to suppress PDGF-RTK

biomarkers, e-cadherin is not specific to prostate cancer as decreased e-cadherin

[46] Saba, N., Khuri, F. Oncology 2005, 68, 10-17.

Protein and Antibody Microarrays:

Kazue Usui-Aoki1,3,*, Motoki Kyo2, Makoto Kawai1, Masatoshi

*Corresponding author: Tel: +81-438-52-3919; Fax: +81-438-52-3918; E-mail: ukazue@kazusa.or.jp

Garry W. Caldwell / Atta-ur-Rahman / Michael R. D'Andrea / M. Iqbal Choudhary (Eds.)

OVERVIEW OF HIGH-THROUGHPUT PROTEIN EXPRESSION ANALYSIS

Fig. (1). Classification of protein/antibody microarray platforms. (A) Schematic illustration of

questions than does transcriptional profiling. Reverse-phase protein microarrays

SPR-BASED ANTIBODY MICROARRAY

THE INTERPRETATION OF THE PROTEIN AND ANTIBODY

identified by MS/MS analysis following immunoprecipitation with anti-mKIAA

The Use of Biomarkers to Detect Cervical Neoplasia

Abstract: The detection of cervical carcinoma and its malignant precursors is

*Corresponding author: Tel: (919) 206-7102; E-mail: dmalinowski@tripathimaging.com

Garry W. Caldwell / Atta-ur-Rahman / Michael R. D'Andrea / M. Iqbal Choudhary (Eds.)

MOLECULAR BIOLOGY OF CERVICAL NEOPLASIA:

HPV TESTING AND DNA DETECTION IN CERVICAL SCREENING

Table 1. Human Papillomavirus Open Reading Frame Genes

HPV Gene Viral Function Keratinocyte Interactions

LCR Regulatory control of transcription, None reported

L1 Major capsid protein None reported

L2 Minor capsid protein None reported

E2 Transcriptional regulation Repression of telomerase expression

E4 Keratin interactions Actin-cytoskeleton interactions

E5 Growth factor receptor interactions EGFR and MAPK signaling pathways

E6 and E7 Prolongs division phase of the - MAPK Signaling pathway activation

HPV E6 AND E7 mRNA DETECTION AND ACTIVE HPV INFECTIONS

Measurement NASBA Detection of E6/E7 mRNA PCR Detection of L1 DNA

HPV Subtypes Detected 16,18,31,33,45 13 HPV Viral subtypes

HPV Prevalence 23.4% 54.6%