Sensors and Actuators B 114 (2006) 1071–1082

Review

Preparation of ormosil and its applications in the

immobilizing biomolecules
Vijay Shyam Tripathi, Vivek Babu Kandimalla, Huangxian Ju ∗
Key Laboratory of Analytical Chemistry for Life Science (Education Ministry of China), Department of Chemistry,
Nanjing University, Nanjing 210093, PR China

Received 8 April 2005; received in revised form 6 July 2005; accepted 6 July 2005
Available online 10 August 2005

Abstract

Biomolecules such as enzymes, antibodies, etc., are highly sensitive and specific in catalysis and recognition. These characteristics make
them as potential recognition and catalytic agents in different fields. Attempts have been made to utilize their harness by immobilizing them in
suitable matrices/supports. In recent years sol–gel technology has appeared as a greatly promising tool in entrapment of active biomolecules.
The introduction of various organic functional groups, such as amino, glycidoxy, epoxy, hydroxyl, etc., into alkoxide monomers leads to
organically modified sol–gel glasses (ormosil). The preparation of such organic/inorganic composites provides a means to produce silicate
materials with continuously tunable chemical and physical properties by simply changing the precursors employed, their molar ratio, or both.
Recently ormosils have been employed in multifarious applications in industrial and medical fields and show promising results in preserving
native activity of biomolecules. This review article discusses about the basic chemistry, characterization, advances and biosensor applications
of ormosil. The attractive features of ferrocene linked/entrapped ormosil are also incorporated.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Ormosil; Biosensors; Ferrocene; Encapsulation; Mediated biosensors; Non-mediated biosensors

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1071
2. Chemistry of sol–gel process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1072
3. Characterization of ormosil films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1073
4. Advances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1075
5. Ormosil based biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1075
5.1. Non-mediated biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1076
5.2. Mediated biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1076
6. Other applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1078
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1078
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1078
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1079

1. Introduction temperature and triggered several domestic and technological

applications [1,2]. The preparation of silica gel via hydroly-
The developments in sol–gel technology have permitted sis of tetraethyl orthosilicate, [Si(OC2 H5 )4 ] in acidic medium
the formation of ceramic materials in desired shapes at low resulted in the productions of glass-like materials in various
forms like fibers, monolithic optical lenses and composite
∗ Corresponding author. Tel.: +86 25 83593593; fax: +86 25 83593593. glass [3]. Major limitation in preparation of conventional
E-mail address: hxju@nju.edu.cn (H. Ju). ceramic materials is the need of high temperature during

0925-4005/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.snb.2005.07.037
1072 V.S. Tripathi et al. / Sensors and Actuators B 114 (2006) 1071–1082
processing and the difficulty in forming desired complex geo-
metrical configurations of the materials. During 1970s Roy
and his co-workers [4] recognized the potential of sol–gel
process for achieving very high level of chemical homo-
geneity. They synthesized a large number of ceramic oxide
compositions involving Al, Si, Ti, Zn, etc., which could not be
produced by traditional ceramic technology. The pioneering
work of Iler’s group [5] in silica chemistry led to commer-
cial development of colloidal silica powders [6]. This concept
has led to the production of a wide variety of composites with
controlled morphologies and particle size [6,7].
The most widely used starting precursors for the fabrica-
tion of silica-based materials (sol–gels) are tetramethoxysi-
lane (TMOS) and tetraethoxysilnae (TEOS). The introduc-
tion of various organic functional groups into inorganic
alkoxide has led to organically modified sol–gel glasses,
known as ormosils. Ormosils have several attractive features
compared to inorganic sol–gels. Firstly, they allow specific
binding of an enzyme to the silica network, for example on
silica grafted with aminosilane by Michael coupling through
glutaraldehyde to an amine bearing enzyme. Secondly, they
allow the encapsulation of catalysts with effective reten-
tion properties in case of strong interaction with the organic
branch, or better, the covalent bonding of a charge transfer
cofactor to the composite material via a chemical reaction
with the previously grafted groups. And they make it possible
to tune the wettability of composite material by a judicious
choice of the ratio of hydrophilic to hydrophobic monomers
[8,9]. With respect to analytical applications, ormosil derived
materials can be designed to a controlled active thickness
of the sensing device and controlled porosity and provide
a versatile way to prepare modified electrodes [10–12]. The
major advantages of the ormosil technology are that the mate-
rials can be prepared at a relatively low temperature and
their compositions can be easily changed according to the
applications. For example, it is possible to prepare the elec-
trodes using different conducting species, organosilanes or
polymer additives, redox mediators and several enzymes,
each of which can be used to fine tune the properties of
the electrode. The conducting species contain graphite and
palladium and the incorporation of gold colloids that was
reported in recent work [13]. Ormosils and polymers are
good to modulate enzyme activity. For example, some of the
researchers demonstrated that the use of polycationic poly-
mers into ormosil materials could improve the performance of
flavoproteins [14,15], while some studies have demonstrated
that the incorporation of copolymers into silica-based glasses
can improve the activity of entrapped glucose oxidase for
amperometric detection of glucose [16]. The biomolecules
such as atrazine chlorohydrolase [17], lipase [18], lipase
and human serum albumin [19] entrapped in ormosils show
improved performances including storage stability, excellent
activity retention, etc. By taking these advantages and utiliz-
ing the advances in ormosil technology in last two decades
several enzymes have been successfully encapsulated into
ormosil and employed in design of biosensors [9]. In recent
years several reviews were also appeared on sol–gel encapsu-
lated biomolecules [20–27], and few on ormosil [9,28]. This
review tries to describe all important aspects and reports of
the ormosil with emphasis on active biomolecules.
2. Chemistry of sol–gel process
The sol–gel chemistry paves a versatile path for the low
temperature synthesis of silica matrices. In typical sol–gel
process alkoxide monomers (TMOS or TEOS) undergo
hydrolysis to form silanols, silanols then link together to form
siloxanes, finally through condensation silanols react with
siloxanes to form porous sol–gel matrices after aging and
drying processes under ambient atmospheres [28]. The chem-
istry of such a process can be expressed in Fig. 1. Sol is the
dispersion of colloidal particles with diameters of 1–100 nm
in a liquid. Gel is an interconnected, rigid network with pores
of submicrometer dimensions and polymeric chains whose
average length is greater than a micrometer. The term “gel”
embraces a diversity of combinations of substances that can
be classified into four categories: (a) well-ordered lamel-
lar structures, (b) covalent polymeric networks, completely
disordered, (c) polymer networks formed through physical
aggregation, predominantly disordered, and (d) particular
disordered structures [28]. When the pore of the liquid is
removed at or near ambient pressure by thermal evapora-
tion, drying, shrinkage occurs and the monolith is termed as
xerogel. If the pore liquid is primarily alcohol, the monolith
is often termed as alcogel. The generic term of gel usually
applies to either xerogels or alcogels. A gel is defined as
dried when the physically adsorbed water is evacuated; dry-
ing process substantially reduces the pore size. As mentioned
above, the introduction of organic functional groups such as
amino-, glycidoxy- and epoxy-hydroxyl, etc. into the alkox-
ide monomers leads to the ormosil [28]. TEOS and some other
ormosil monomer structures and ormosil formation are given
in Fig. 2. The organic modification is mainly employed to
reduce the degree of cross-linking, improve film adhesion to
its support, reduce the concentration of surface silanol groups
and the ion exchange capacity, alter partition coefficients
or introduce reactive functional groups that can be subse-
quently used for anchoring molecular recognition species
on pre-prepared xerogels. While selecting ormosil matri-
ces for biosensor applications the characteristics, such as
hydrophobicity, hydrophilicity, porosity, optical properties,
film thickness, hard ness and cracking, should be taken into
consideration. When hydrophobic silica-forming monomers
are used such as Epoxy, the resulting matrices will reject
Fig. 1. Reaction scheme of sol–gel film formation.
 V.S. Tripathi et al. / Sensors and Actuators B 114 (2006) 1071–1082
Fig. 2. TEOS and commonly used ormosil monomers and reaction scheme
for ormosil formation.
water, leaving only segregated islands of carbon at the outer-
most surface in contact with electrolyte [1]. Hence, the ratio
of hydrophilic and hydrophobic monomers should be in opti-
mal ratio for sensor design or retaining the better activity
of the biomolecule. The variation of the number and type
of organic moieties included on the silicon monomer results
in a variety of pore sizes that can be created in the ormosil
network. The pore size corresponds to the physical size of
the organic subsistent (Fig. 2). The mechanical and opti-
cal properties of glass, prepared by the sol–gel process, can
be improved using modified alkoxide precursors RSi(OEt)3
to form organic–inorganic hybrid matrices. Where R is an
organic group such as methyl, vinyl or amyltriethoxysilane.
The covalently bound organic groups decrease the mechani-
cal tension during the drying process [29]. In order to avoid
phase separation, functionalized alkoxides F-R -Si(OEt)3 ,
where F is a functional group such as amino or isocyanate
and R is an alkyl spacer, are usually used to graft cova-
lently dopants onto the matrix. After drying, optically clear
and dense inorganic–organic hybrid xerogels (30 mm diam-
eter and 15 mm thick) can be obtained [30,31]. The reactive
area of ormosil can be determined by its wettability, which
can be increased by incorporating readily leachable/water-
soluble components such as polyethylene glycol in the matri-
ces and dissolving them out by immersing the electrodes
in an electrolyte solution [32]. This increases the porosity
for the penetrating electrolyte, thereby increasing the wetted
section inside the sol–gel matrices [1]. Changing the ratio
of tetralkoxysilane to organotrialkoxysilane can control the
cation exchange capacity and polarity of porous surface [1].
The homogeneous sized nano particles can also be obtained
via aqueous dispersion process, which is difficult thorough
normal sol–gel drying and grinding processes [33].
Processes such as ageing, drying, stabilization and densi-
fication influence the sol–gel structure since they are related
to the rates of hydrolysis and condensation that determine the
structure of the gel. It is essential to understand the kinetics
of the hydrolysis and condensation reactions. Additionally,
1073
many other factors including many species that present in the
solution influence the kinetics of hydrolysis and condensa-
tion [34]. The variables of major importance are temperature,
condensation electrolyte (acid/base), nature of the solvent
and the type of alkoxide precursor. In fact, many studies have
reported the variation of gelation time, viscosity or textural
characteristics (specific surface area) of the gel as a func-
tion of experimental conditions [35–39], without alternation
in hydrolysis and condensation. The influence of electrolyte
concentration on the hydrolysis of TEOS in different sol-
vents shows that the hydrolysis rate increases linearly with
the increasing concentration of H+ or H3 O+ in acidic media
or OH− in basic medium. The nature of alkoxy group on
the silicon atom also influences the rate constant. In general,
the long and bulky alkoxy group leads to low rate constant
of hydrolysis [36]. The nuclear magnetic resonance spectra
(NMR) of silicon were reported to investigate the conden-
sation of aqueous silicates at high pH [40]. Hydrolysis and
condensation reactions initiate numerous reactive sites when
mixing TMOS with water. The size of the sol particles and
the cross-linking within the particles (i.e. density) depend
upon the pH and the ratio of [H2 O]/[Si(OR)4 ]. The time of
gelation (tg ) also changes the chemistry of sol–gel signifi-
cantly [41]. Yamane et al. [42] reported that the curve of tg
versus pH was bell shape, in other words, gelation could be
nearly instantaneous for very acidic or basic solution of sili-
con alkoxides. This behavior is very different from the gels
prepared by destabilization of a silica sol where the curve
has a S shape, with maximum tg around the isoelectric point
of silica (pH ∼2) and a minimum tg near pH 5–6 [43]. The
anion and solvent also play an important role in the kinetics
of gelation, which can be either acidically or basically cat-
alyzed. The amount of water for hydrolysis of alkoxysilane
has a dramatic influence on gelation time [43]. For low water
content, generally an increase in the amount of hydrolysis
water decreases the gelation time, though there is a dilution
effect as well. It can be predicted that for higher water con-
tent the gelation time increases with the increasing quantity
of water. Furthermore viscosity of sol–gel precursors signif-
icantly influences the physical form, e.g. fiber coating and
monolith of casted gel [44–48].
3. Characterization of ormosil films
Although many reports indicated silica net works are
able to retain the structure and activity of a wide variety of
enzymes [49,50], some proteins may completely unfold upon
encapsulation as reported for apomyoglobin by Eggers and
Valentine [51]. Biomolecules are highly sensitive and fragile
in nature; hence their vicinity should be mild and closer to
the native environment (inside the cell) after immobilization.
Commonly the factors such as polarity, local microviscosity
and interactions with pore walls, and preferential partitioning
into a given phase have an impact on the dynamics, stability
and accessibility of the dopant. Hence there is a great need to
1074 V.S. Tripathi et al. / Sensors and Actuators B 114 (2006) 1071–1082
understand the nature of the local microenvironments within
nano composites. It requires suitable methods to characterize
the sol–gel materials. Following characterization procedures
of sol–gel structure have been adopted so far.
Firstly, the nature of the local microenvironment(s) within
a sol–gel-derived nanocomposite is an important factor in
designing materials for sensing applications. The widely
employed methods to characterize the sol–gel matrices
include small-angle X-ray scattering (SAXS), neutron scat-
tering (SANS) and light scattering (SALS), NMR [52], and
fluorescence spectroscopy [53–55]. SAXS allows the deter-
mination of a characteristic length of a particle (Guiniers
radius of gyration or electronic radius of gyration) and a frac-
tal dimension, which give some information on the structure
of the polymer (branched versus linear) and on the growth
mechanism. The application of SAXS to a number of gel
systems has been reported by various authors [53–55]. SANS
has been applied to the study of silica sols [56,57], as it yields
structural information on complex biological systems in real
time without damaging the structures involved [58]. More
recently lypase enzyme was entrapped into ormosil prepared
using tetramethylorthosilicate and methyltrimethoxysilane
using NaF as a catalyst. The relationship between gel struc-
ture and catalyst was evaluated using SANS. Scattering stud-
ies were conducted on both immobilized lipase and lipase
in free solution. Scattering studies on free enzyme provided
evidence for multiple populations of enzyme aggregates and
showed that choice of solvent affected the degree of aggre-
gation. The presence of the enzyme during the gel formation
conferred structural changes in the gel matrix [58]. SALS
have received very little attention in the sol–gel literature.
However, its characteristic dimension probed by visible light
scattering is 710 nm, and therefore, it cannot be used to
characterize the early stage of the gelation process. Recent
development of short wavelength UV lasers may make it pos-
sible to extend light-scattering studies to 3 nm, and thereby
to follow most of the gelation process.
Ormosil can be characterized by scanning electron
microscopy, BET adsorption experiments, differential ther-
mogravimetric analysis, and impedance spectroscopy to get
information on the surface characters (smoothness, cracks,
thick ness, etc.), porosity, structure and properties of the
materials [57]. X-ray photoelectron spectroscopy has been
employed to evaluate the chemical stability of sol–gel films
containing covalently-bonded ferrocene moieties, coated on
glassy carbon, and to monitor the chemical oxidation of tetra-
sulfur groups immobilized by covalent grafting to a silicate
film coated on indium–tin oxide electrode [59]. The distribu-
tion of ZrO2 particles within a sol–gel silica may be studied by
various techniques before incorporating the resulting material
into carbon paste, including gas adsorption, scanning elec-
tron microscopy, X-ray diffraction, and thermogravimetry
[60]. Composite films made of hybrids of nafion and sil-
ica have been analyzed by transmission electron microscopy,
energy-dispersive analysis of X-ray, modulated differential
scanning calorimetry, and electrochemical impedance spec-
troscopy using two stainless steel electrodes, which reveal
that the composite displays additional properties with respect
to those of the isolated Nafion and silica materials [61].
The thermogravimetry/mass spectra and Fourier transform
infrared (FTIR) spectra indicate the entrapment of urease in
a sol–gel silica film can form a silica network [62]. FTIR spec-
troscopy and UV–vis absorption spectrometry are extensively
used methods for characterizing organically modified or pure
SiO2 modified electrodes containing encapsulated organic or
organometallic species [59,63–68]. In fluorescence charac-
terization of the sol–gel-derived material the selection of a
fluorescent probe is the most critical factor. A wide variety of
fluorescent probes exist and typically fall into various classes
such as pH sensitive probes, solvatochromic probes, rigi-
dochromic probes, anisotropy probes, and so on [69]. These
(probes) tend to be incorporated into the materials during
the early stages of the sol–gel process when the hydrolyzed
precursor and entrapped molecule(s) are mixed and allowed
to gel. During the subsequent condensation reactions the
probe molecules are entrapped within the pores as the mate-
rial forms and may become part of the matrix network [69].
Bottini et al. [70] studied the conformation and stability of
myoglobin in organically net works through absorption and
fluorescence spectra. Marino et al. [71] used Raman spec-
troscopy to investigate the protonation of doped molecules
such as disperse red dye molecules in ormosil matrices.
Among the electrochemical methods cyclic voltamme-
try has been often used to characterize the electrochemical
behaviors of ormosil modified electrodes comprising elec-
troactive centers or encapsulating electroactive components.
The electrochemistry of CuII in ammonical medium at a silica
gel modified carbon paste electrode [72,73] shows that copper
species are electrochemically accessible only because they
retain enough mobility to diffuse out of the silica material on
the time scale of the voltammetric measurement. This agrees
with a subsequent work by Borgo et al. [67] who reported
a redox behavior for CuII initially loaded on aminopropyl-
grafted silica similar to that usually observed for solution
species. On the contrary, Bond et al. [74] suggested an intra-
silica charge transfer mechanism when studying the voltam-
metric reduction of several metal ions (HgII , AgI , CuII , PbII )
adsorbed onto a new thick-walled form of mesoporous sil-
ica. The electrochemical characterization of ceramic-carbon
composite electrodes was mainly performed by the Lev’s
group [75,76]. They studied the influence of the carbon source
(graphite, acetylene black or Ketjen black) and compared the
results to conventional carbon electrodes by using various
organic and inorganic redox couples.
Electrochemical techniques are also exploited to evaluate
the porosity of ormosil-based materials as well as the asso-
ciated mass transfer reactions. For example, Collinson et al.
[77] demonstrated that ultra microelectrodes could be effec-
tively used to probe molecular transport in microstructure gel
material and provided an effective mean to get accurate val-
ues of diffusion coefficients of target molecules encapsulated
in sol–gel-derived silica monoliths. Voltammetry can also be
 V.S. Tripathi et al. / Sensors and Actuators B 114 (2006) 1071–1082
directly used to characterize mass transport in SiO2 modified
electrodes, as well as to select the optimal experimental con-
ditions to get a sensor device displaying the highest sensitivity
as possible [78,79]. Besides the mass transfer limitation, the
overall electrochemical reaction might also be limited by
the heterogeneous rate of electron transfer, as exemplified
by Markovich and Mandler [80], via the electrochemistry of
hexacyanoferrate at an indium tin oxide electrode covered
with an octadecylsilane monolayer.
4. Advances
Inorganic sol–gel matrices are not highly biocompatible
and bristle in nature. Organic modification in sol–gel precur-
sor may provide better way of controlling nanoporous geome-
try of ormosil suitable for sensor design [81,82]. The addition
of synthetic or natural polymers such as poly(ethylene glycol)
(PEG) to TEOS and organosilane-derived sol–gel material
offers enhanced material properties such as optical clarity
and dehydration/rehydration stability, results in significant
improvement in the medium term stability of entrapped
lipase as compared to entrapment in absence of polymer
additives [83]. A graft copolymer of poly(vinylalcohol) with
4-vinylpyridine has been hybridized into sol–gel net, and the
formed organic–inorganic composite film displays an excel-
lent adhesion to electrodes and increases the sensitivity of the
enzyme electrode [16]. The ormosil methyltrimethoxysilane
(MTMOS) doped with chitosan shows good biocompati-
bility for immobilization of glucose oxidase (GOD) [84].
Pandey et al. [8,32] added an appropriate organic polymer
PEG in the starting sol solution to prevent the formation of
crack, thus increasing the film stability. The ormosil–carbon
electrodes modified by the incorporation of Meldola1s
blue into the MTMOS (hydrophobic) greatly reduced
the potential value to −0.2 in sensing the NADH [85].
The surface renewability of the electrodes by mechanical
polishing showed relative standard deviation less than
8% for successive surface renewals of mediator-modified
electrodes.
By taking the advantages of ormosils, Lev and Tsion-
sky [86] prepared macroporous chromatographic media
by incorporation of cluster-forming materials along with
the precursors of films. Nakanishi et al. [87] introduced
sol–gel derived monoliths for high-pressure liquid chro-
matography. Lev and co-workers [88] reported that the
incorporation of H2 O2 into sol–gel precursors of methyl-
ormosil favored in synthesis of macroporous monoliths,
which would be useful in chromatographic applications. Dur-
ing the sol–gel process the decomposition of the H2 O2 from
miniature bubbles, which formed templates for the poly-
condensation, yielded fractured or powdery materials. The
encapsulation of myoglobin in the organically modified sil-
ica bulks prepared using three organic functionalities (3-
aminopropyl)-trimethoxysilane, 3-(trimethoxysilyl)-propyl
methacrylate and (3-glycidyloxypropyl)-trimethoxysilane
1075
retained its activity and structure even after treated with
denaturing agent guanidinium hydrochloride (GdHCl). The
organic functionalities acted as surfactants that interacted
directly with the hydrophobic residues of myoglobin then
formed micelles around the protein to prevent the forma-
tion of unfavorable water structure promoted by GdHCl
[89].
5. Ormosil based biosensors
The use of sol–gel glass for the development of elec-
trochemical biosensors has received great attention because
of its sturdiness and possible commercial applications. A
number of publications are available on the applications of
sol–gel glass for the development of electrochemical sensors
[90]. Four approaches dependent on the convenience, stabil-
ity and response can be used for immobilizing the enzyme
on electrode surface: entrapment of enzyme in ormosil film,
attachment of enzyme on the surface of ormosil film, immo-
bilization of enzyme in a sandwich configuration and a
bilayer configuration (Fig. 3). Wang and Pamidi [91] devel-
oped biogel-based carbon inks that displayed compatibility
with the screen-printing device for microband electrodes. In
recent years Pandey’s group [92–94] successfully immobi-
lized glucose oxidase (GOD), horseradish peroxidase (HRP)
and acetylcholinesterase in ormosils. These immobilized
enzymes were evenly distributed in the sol–gel matrices and
gave good response when employed for biosensing. The addi-
tion of pore forming agents and conductive materials, PEG,
poly vinylalcohol (PVA) and graphite, palladium, ruthenium,
respectively can improve the electrochemical signals consid-
erably. Diffusional penetration of a mediator into the protein
yields a sufficiently short electron transfer distance for the
electrical activation of the biocatalyst. Penetration of the
mediator close to the enzyme active center inside the pro-
tein matrix can be controlled by the hydrophobic/hydrophilic
properties of the mediator and the enzyme, the size and
shape of the mediator and the electrostatic charge interac-
tion between the mediator and the enzyme. The mediators
can diffusionally shuttle electrons between the electrode and
Fig. 3. Different approaches for the immobilization of biomolecules and
mediator molecules in ormosil matrices.
1076 V.S. Tripathi et al. / Sensors and Actuators B 114 (2006) 1071–1082
enzymes in several configurations: soluble, immobilized as
monolayers (or multilayers), or incorporated into porous
matrices.
5.1. Non-mediated biosensors
The amperometric biosensor that does not require the
participation of redox molecules having reversible electro-
chemistry is referred to as non-mediated biosensor, whereas
the participation of redox mediator in signal transduction gen-
erates a category of mediated biosensors [95–97]. Usually the
charge transfer and sensitivity of non-mediated biosensors
must be improved because the most of redox enzyme active
sites are deep inside, which makes the electron shuttle become
slower or poor. The porosity and wettablility of the ormosil
film play key roles in biosensor performance. The ormosil
film developed using 3-aminopropyl-triethoxysilane (rela-
tively hydrophilic precursor) and 2-(3,4-epoxycyclohexyl)-
ethyltrimethoxysilane (relatively hydrophobic precursor) is
very smooth without cracking and has a good performance
when doping GOD [32]. Furthermore, the preparation of
this ormosil film is very simple with one-step gelation pro-
cess as compared to those prepared by following com-
plex protocol of gelation, which requires sonication for
the homogenization of monomers, and additive suspension
[75,98,99]. The porosity and wettability can also be modified
by employing the additives such as PEG along with bioac-
tive compounds [8,32]. To evaluate the effects of PEG and
graphite on GOD entrapped ormosil that were prepared using
3-aminopropyltriethoxysilane and 2-(3,4-epoxycyclohexyl)-
ethyl-trimethoxy silane as precursors, different compositions
such as GOD, GOD along with PEG, GOD and graphite pow-
der (1–2 (m), and GOD along with PEG and graphite pow-
der were designed. Composition 4 exhibited relatively good
response in the presence of glucose in 0.1 M pH 7.0 phos-
phate buffer at 25 ◦ C. Upon addition of glucose the anodic
current corresponding to the oxidation of hydrogen peroxide
increased greatly. The magnitude of anodic current in com-
position 4 was greater than those in other compositions. This
was mainly due to relatively less concentration of oxygen
at the site of enzymatic reaction required for the formation
of hydrogen peroxide. It was also been reported that neither
highly hydrophobic nor totally hydrophilic sol–gel matrices
were desirable for sensing application [1,100]. When chem-
ical modifiers such as metal dispersion, water-soluble poly-
mers and proteins were added to the materials, the resulting
electrodes became more hydrophilic. It has been reported [1]
that a blank sol–gel electrode without any hydrophilic mod-
ifier shows the highest water content angle (80◦ ) and in turn
the lowest wettability, whereas the sol–gel electrodes with
all hydrophilic modifiers (carbon, PEG and Pd-GOD) show
the lowest water contact angle (42◦ ) and the highest wetta-
bility (42 m2 /g). An increase in the wetted area increases the
wetted conductive surface accessible to the solution and also
the corresponding electrochemically active area and capac-
itive current. On the other hand, the non-wetted area does
not contribute to the capacitive or faradic currents. Ingersoll
and Bright [99] reported on the effect of addition time of
dopant and used oxygen as the analyte to study sensor per-
formance. The ormosil entrapped GOD also exhibited good
performance by the addition of hydorphilic modifiers such as
graphite and PEG into ormosils mainly due to the increase in
wettability, which increased the oxygen diffusion [92].
The non-mediated potentiometric biosensor has also
been reported. Gulcev et al. [101] co-entrapped carboxy-
seminaphtharhodafluor-1-dextran conjugate and hydrolytic
enzyme (urease/lipase) in ormosil to develop a reagentless
pH based biosensor. The doping of PVA improved enzyme
activity and enhanced the accessibility of different films.
The response was ∼25-fold higher than that obtained from
TEOS alone.
5.2. Mediated biosensors
Ferrocene derivatives, organic dyes, ferricyanide, Ru-
complexes and other electrochemically active substances
have been employed as mediators to improve the elec-
tron transfer of biomolecules with the conductive support
[102]. Among these ferrocene derivatives are particularly
well studied for electrochemical biosensing owing to their
good stability, high degree of characterization and their appli-
cation potential bioanalysis. Soluble redox-enzymes electri-
cally contacted by the use of diffusional electron transfer
mediators have been extensively reviewed [102]. The reac-
tion of GOD has been extensively studied with a number of
artificial electron acceptors including organic dyes such as
phenazine methosulfate, 2,6-dichlorophenolindophenol, and
N,N,N ,N -tetramethyl-4-phenylenediamine. However, these
mediators have a number of limitations such as poor stabil-
ity and the pH dependence of their redox potentials [102].
Other simple inorganic redox species such as hexacyanofer-
rate, hexacyanoruthenate and pentaamine pyridine ruthenium
[103] do not suffer from these problems. These inorganic
compounds have almost ideal electrochemistry and are more
stable than the organic dyes. The application of inorganic
mediators has been exemplified with other oxidases such
as sarcosine oxidase and lactate oxidase [104]. Inorganic
mediators are difficult to ‘tune’ for solubility and electro-
chemical properties, as they cannot be modified or derivatized
nearly as easily as their organic counterparts. The major-
ity of these problems have been overcome by the use of
ferrocene derivatives as electron acceptors for soluble oxi-
dases (e.g. GOD). The published values for the second-order
rate constant (ket ) for the reaction of the reduced active cen-
ter of GOD (FADH2 ) and an oxidized ferrocene derivative
range from 2.6 × 104 to 5.25 × 105 M−1 s−1 [102]. There
is no simple correlation between ket and formal potential
E0 , the positively charged ferrocene derivatives are favored
for the mediated electron transfer from GOD. This effect
originates from the electrostatic attraction of the positively
charged oxidized mediator and the negatively charged GOD.
Comparison of the mediating efficiency of charged electron
 V.S. Tripathi et al. / Sensors and Actuators B 114 (2006) 1071–1082
relays and charged enzymes always takes electrostatic inter-
actions into account [105]. The size of the mediator is also
an important factor. It has been shown that ferrocene deriva-
tives included in cyclodextrin cavities do not mediate electron
transport from enzyme [106]. To improve the contact between
electron relay molecules and redox enzymes, micellar sys-
tems composed of ferrocene-functionalized surfactants have
been applied [107]. The incorporation of graphite powder and
PEG into the ormosil as precursors exhibits excellent bio-
electrochemical properties [32]. The voltammograms of the
soluble ferrocene monocarboxylic acid shows reversible elec-
trochemistry of ferrocene, and the incorporation of graphite
particles increases the wetted surface area of the electrode
as well as facilitates the electron transfer within the sol–gel
matrix as a result of increases electronic conductivity of the
electrode. Chen et al. [84] reported a glucose biosensor using
ferrocene as a mediator, in which enzyme was immobilized
in ormosil–chitosan composite. The adsorbed ferrocene pro-
vided good shuttle of electrons between the enzyme and the
electrode and the presence of chitosan provided stabilizing
microenvironment around the enzyme. In some of the reports
ferrocene was firstly adsorbed on electrode surface, and then
the ormosil composite containing GOD was covered with
ormosil in construction of glucose biosensor [84].
In the absence of ferrocene an ormosil made using palla-
dium-linked glycidoxypropyltrimethoxysilane precursor,
trimethoxysilane, HCl and tetrathifulvalenetetracyanoquin-
odimethane (TTF-TCNQ) powder at 25 ◦ C was used to
prepare an electrocatalytic biosensor for glucose [108]. A
large electrocatalytic current to the order of 8000 ␮A/cm2
was observed on the addition of 300 mM glucose. Pyrorolo-
quinoline quinone (PQQ) is a redox cofactor present in a
number of dehydrogenases. It has been shown to catalyze
non-enzymatic reactions, including the oxidation of thiols to
disulfide, and successfully immobilized in ormosil prepared
from 3-aminopropyltrimethoxysilane (APTES) and 2-(3,4-
epoxycyclohexyl) ethyl-trimethoxysilane. The entrapped
PQQ exhibits good electrochemical performance against cys-
teine and glutathione oxidation and reproducibility [109].
The encapsulation/linking (with precursors) of redox
materials within sol–gel glass has gained a significant share
of attention in sensor designing because such a compos-
ite system provides very close contact of biomolecules
with transducer surface. Pankratov and Lev [110] reported
ferrocene-mediated carbon ceramic electrodes with lim-
ited storage and in-use stability. Several other reports on
ferrocene-encapsulated sol–gel glasses, including those of
Lev et al. [11,74,98], are available. Audebert et al. [111]
reported several electrodes modified with organic–inorganic
hybrid gels containing a ferrocene unit covalently linked
in silica network and organic–inorganic hybrid gels formed
by hydrolysis–polycondensation of some trimethoxysilyl-
ferrocenes [112]. Collinson et al. [113] reported the elec-
troactivity of redox probes encapsulated in sol–gel-derived
silicate film based on anionic and cationic gel-doped probes,
i.e., [Fe(CN)6 3−/4− ], [IrCl6 2−/3− ] and ferrocenemethanol
1077
Fig. 4. Ormosil monomers conjugated with ferrocene derivatives.
[FcCH2 OH0/+ ]. Fig. 4 shows typically mediator molecules
(ferrocene derivatives) incorporated into sol–gel matrices
by physically doping or by using organosilicon precursors.
Other reports on ferrocene-based sol–gel sensors are also
available [114]. A biocompatible Pd-linked ormosil mate-
rial with encapsulation of ferrocene has also been reported,
which shows redox electrochemistry similar to that of soluble
ferrocene in solution and good electrocatalytic behaviors to
redox proteins (alcohol dehydrogenase) and cofactors such
as NADH [115].
Two ormosils prepared using ferrocene carboxaldehyde
and a mixture of 3-aminopropyltrimethoxy silane with
2-(3,4-epoxycyclohexyl)-ethyltrimethoxy silane and ferro-
cene monocarboxylic acid and a mixture of 3-glycidoxy-
propyltrimethoxysilane with trimethoxysilane have been
reported [116]. The encapsulated ferrocene derivatives in
ormosils show quasi-reversible electrochemistry compared
to ferrocene-linked to ormosil [28,116]. Another ferrocene-
encapsulated palladium-linked ormosil shows a reversible
electrochemistry with a peak separation of 58 mV at the scan
rates less than 50 mV/s [117]. The good reversibility is due
to the presence of palladium, which plays a crucial role in
electron transfer [118]. This ferrocene-encapsulated ormosil
has been employed in development of glucose and dopamine
biosensors [118–120]. The silicate-based electrodes prepared
with ferrocene-linked ormosil precursors show good stability
without any leaching of the mediator and the enzyme [121].
Recently we conjugated the ferrocene monocarboxylic acid
with an inert protein bovine serum albumin (BSA–FMC)
through carbodiimide linkage. The conjugate was success-
fully entrapped in an ormosil prepared using APTES and 2-
(3,4 epoxycyclohexyl)-ethyltrimethoxy silane as monomers.
The entrapped BSA–FMC exhibited reversible redox peaks
without any leaching of mediator [122]. To evaluate the
applicability of the BSA–FMC doped ormosil in biosensors,
1078 V.S. Tripathi et al. / Sensors and Actuators B 114 (2006) 1071–1082
enzymes and multiwall carbon nanotubes were also doped
into the ormosil composite. The glassy carbon electrode mod-
ified with enzymes and nanoparticles doped ormosil com-
posite film exhibited good sensitivity, reproducibility and
stability towards the determination of glucose and H2 O2
[122,123].
6. Other applications
Ormosils have also been used in other different fields such
as manufacture of new contact lenses and fresnel lenses,
preparation of laser components for opticsm, second-order
non-linear optically active nanocomposites and bone repair-
ing materials, synthesis of photochromic coatings and porous
solvent absorbers, and so on [29,124–129]. The biomolecules
or dyes entrapped in ormosil nanoparticles can be efficiently
employed in drug delivery and other pharmaceutical and
medical applications [130]. The organically modified and
anticancer drug doped nanoparticles (diameter ∼30 nm)
obtained through aqueous dispersion have been used in
photodynamic cancer treatment [131]. Water-insoluble pho-
tosensitizing anticancer drug, 2-devinyl-2-(1-hexyloxyethyl)
pyropheophorbide, was entrapped in non-polar core of
micelles by hydrolysis of triethoxyvinylsilane, the resulted
nanoparticles were spherical, highly monodispersed, and
stable in aqueous system. Irradiation of the photosensitizing
drug entrapped in the nanoparticles with light of 650 nm
wavelength resulted in efficient generation of singlet oxygen,
which could inactivate the cells. The drug-doped nanoparti-
cles were up taken into the cytosol of tumor cells and caused
the damage to the impregnated tumor cells upon irradiation.
DNA molecules bound through electrostatic interaction
onto ormosil nanoparticles could be employed in non-viral
vector gene therapy [132]. The bound DNA molecules
were highly resistant from nucleases degradation. By
paying more affords, it would be possible to use efficiently
the manipulation of surface reactive groups on ormosil
nanoparticles in optical tracking, drug delivery and drug
interactions.
A fiber-optic microbial sensor for determination of
biochemical oxygen demand (BOD) was reported using
an oxygen-sensitive fluorescent material and two dif-
ferent kinds of seawater microorganisms immobilized
in ormosil. This report used Tris(4,7-diphenyl-1,10-
phenanthroline)ruthenium(II) perchlorate as the oxygen fluo-
rescent quenching indicator [133]. Nano-sized (20–100 nm)
photonic explorers for bioanalysis with biologically local-
ized embedding have been developed for specifically use
in biological environments to monitor small species like
H+ , Ca2+ , Na+ , Mg2+ and glucose, etc. [134,135]. Ana-
lytes can diffuse through the matrix and interact with the
sensing dye. These sensors typically contain a reference
dye. Koo et al. [136] reported an oxygen sensor based on
entrapment of platinum porphyrins in ormosil nanoparti-
cles. The ormosil nanoparticles (∼120 nm) were prepared
via a sol–gel-based process, which included the formation
of core particles with phenyltrimethoxysilane as a precur-
sor followed by the coating layer with methyltrimethoxysi-
lane as a precursor. The highly permeable structure and
the hydrophobic nature of the ormosil nanoparticles, as
well as their small size made the sensor show good sensi-
tivity for detection of dissolved oxygen. The hydrophobic
ormosil matrices such as methyltriethoxysilane (MTEOS)
and ethyltriethoxysilane (ETEOS) show enhanced perfor-
mance of dissolved oxygen sensor [137]. Chen et al. [138]
reported an oxygen sensor using a ruthenium complex as oxy-
gen sensitive indicator and TMOS, dimethyldimethoxysilane
(DiMe-DMOS) as ormosil precursors. The enhanced sen-
sitivity to dissolved oxygen was reported to be due to the
high hydrophobicity of the ormosil film. Another potential
application of ormosils is design of molecularly imprinted
polymer against diverse analytes [139]. Marx et al. [140,141]
reported molecularly imprinted polymers using ormosils
against to parathion and paroxon and employed them for
quantification.
7. Conclusions
This review article discussed the ormosil basic chem-
istry, characterization, advances and biosensor/biological
applications. Ormosil matrices offer tailorable hydrophilic,
hydrophobic, ionic, and H-bonding capacities as well as
electrochemical activities and controllable porosity and
are highly stable, inert and non-biodegradable. By doping
conductive materials and natural polymers into ormosil
matrices their conductivity and biocompatibility can be
further improved. By creating more reactive groups on
ormosil surfaces, these matrices can be exploited for
anchoring molecular-recognizing receptor and attaching
sensing elements on optrode, electrode and on several other
transduction surfaces especially for mimicking biological
processes. Another challenge is, despite widely utilized, the
sol–gel process is inherently complicated, and its mecha-
nism, the gel microstructure, the effects of different factors
on the stability and activity of immobilized biocatalysts
need to be understood. The applications of ormosil matrices
can further be extended and utilized in optical tracking
of cell metabolisms, nano medicine, gene therapy, chro-
matography and biosensors by paying more affords in near
future.
Acknowledgements
We gratefully acknowledge the financial support of
the Distinguished Young Scholar Fund to H.X. Ju
(20325518), the National Natural Science Foundation of
China (20275017). V.S. Tripathi and V.B. Kandimalla are
highly thankful to Nanjing University for providing Post-
doctoral fellowships.
 V.S. Tripathi et al. / Sensors and Actuators B 114 (2006) 1071–1082
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Biographies
Vijay Shyam Tripathi received his MSc degree in chemistry and PhD
degree in analytical chemistry from Banaras Hindu University, Varanasi,
India in 1997 and 2003, respectively. He is currently working as a
Post-doctoral fellow in the Department of Chemistry, Nanjing University,
China. His research interest includes electroanalytical chemistry, sol–gel
chemistry, biosensor and bioelectronics.
Vivek Babu Kandimalla received his BSc degree (1993) in biology and
MSc degree (1996) in biotechnology from Nagarjuna University, Nagar-
juna Nagar, India and PhD in biotechnology in 2002 form Andhra Univer-
sity, Visakhapatnam, India. He is a Post-doctoral fellow in the Department
of Chemistry, Nanjing University, Nanjing China. His research interests
include the development of electrochemical biosensors, antibodies pro-
duction, quantum dots (QDs) conjugation with antibodies and aptamers,
cellular imaging and in situ imaging tools design.
Huangxian Ju received his BSc (1986), MSc (1989) and PhD (1992)
degrees in chemistry from Nanjing University, Nanjing, China. He was
appointed as a Research Scientist at this University in 1992 and became
a Full Professor in 1999. During January 1996 to July 1997 he was a
Post-doctoral fellow in the Department of Chemistry, Montreal University,
Canada. His research interests include analytical biochemistry, biosensors,
electroanalysis and clinical chemistry.