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Diagnostics
microcantilever sensors are physical sen-
sors that respond to surface stress changes
due to chemical or biological processes.1
When fabricated with very small force
constants, they can measure forces and
Ram Datar, Seonghwan Kim, Sangmin Jeon, stresses with extremely high sensitivity.
Peter Hesketh, Scott Manalis, Anja Boisen, The very small force constant (less than 0.1
N/m) of a cantilever allows detection of
and Thomas Thundat surface stress variation due to the adsorp-
tion (or specific surface-receptor interac-
tion) of molecules. Adsorption of
molecules on one of the surfaces of the
Abstract
typically bimaterial cantilevers (silicon or
Cantilever sensors have attracted considerable attention over the last decade
silicon nitride cantilevers with a thin gold
because of their potential as a highly sensitive sensor platform for high throughput and
layer on one side) results in a differential
multiplexed detection of proteins and nucleic acids. A micromachined cantilever platform
surface stress due to adsorption-induced
integrates nanoscale science and microfabrication technology for the label-free detec-
forces, which manifests as a deflection. In
tion of biological molecules, allowing miniaturization. Molecular adsorption, when addition to cantilever bending, the reso-
restricted to a single side of a deformable cantilever beam, results in measurable bend- nance frequency of the cantilever can vary
ing of the cantilever. This nanoscale deflection is caused by a variation in the cantilever due to mass loading. These two signals,
surface stress due to biomolecular interactions and can be measured by optical or elec- adsorption-induced cantilever bending
trical means, thereby reporting on the presence of biomolecules. Biological specificity in when adsorption is confined to one side of
detection is typically achieved by immobilizing selective receptors or probe molecules the cantilever and adsorption-induced fre-
on one side of the cantilever using surface functionalization processes. When target quency change due to mass loading, can
molecules are injected into the fluid bathing the cantilever, the cantilever bends as a be monitored simultaneously.2
function of the number of molecules bound to the probe molecules on its surface. Mass-
produced, miniature silicon and silicon nitride microcantilever arrays offer a clear path to Mass Detection Using Variation in
the development of miniature sensors with unprecedented sensitivity for biodetection Resonance Frequency
applications, such as toxin detection, DNA hybridization, and selective detection of As described earlier, the resonance fre-
pathogens through immunological techniques. This article discusses applications of quency, f, of a cantilever varies sensitively
cantilever sensors in cancer diagnosis. as a function of mass loading (m),
according to:
1 k
f= . (1)
Introduction 2 m * + m
The detection of multiple target mole- disease process. Detection of a single bio-
cules in a small volume of sample has marker has only limited specificity and The spring constant of the cantilever is
immediate relevance in the early detec- therefore cannot be sufficiently informa- k, m* is the effective mass of the can-
tion of diseases, such as cancer. It is well tive. Hence, cancer diagnostics have been tilever, and is a numerical constant.
known that many cancers can be cura- shifting from traditional monitoring of Increasing the surface area of a cantilever
tively treated if diagnosed early when the single biomarkers to the detection of mul- by nanopatterning can lead to higher
tumors are still small and localized. tiple markers. Detection of multiple bio- adsorbed mass and higher sensitivity of
However, the unfortunate reality is that a markers is particularly important in detection. Lee et al. has demonstrated a
significant proportion of cancers are diag- screening for cancers of low prevalence, cantilever with nanofabricated holes for
nosed only after the tumors have spread such as ovarian cancer. Ideally, these increasing the adsorbed mass.3 Although
distally through blood or lymphatic fluid measurements would be done in a single mass detection using the cantilever reso-
(metastasized) to multiple locations. Since readout with samples that are readily nance frequency is well suited for measur-
cancer is a complex disease, its diagnosis available and minimally invasive, such as ing mass in vacuum and air, its mass
will require monitoring for alterations in blood serum. Therefore, a technique that resolution is very poor when operated
multiple parameters at molecular, cellu- can detect multiple biomarkers simulta- under solution.4 Resonance frequency
lar, and tissue levels to provide a compre- neously using a single sensor platform variations, therefore, are generally not
hensive picture of the extent of the and minimal sample volume would be used for the highly sensitive detection of
adsorbed mass in liquid environments. piezoelectricity,11,12 embedded MOSFET tilever beam has been developed where the
Detecting surface stress variations is (metal oxide semiconductor field-effect doped silicon layer is only on one side of
therefore a method of choice when bio- transistor),13 capacitance,14 and electron the neutral axis of the cantilever beam and
markers have to be detected in body flu- tunneling.15 In optical beam deflection, the shows variation in its resistance as a func-
ids, such as serum. However, Burg et al. cantilever motion is detected by reflecting tion of the extent of the deflection.17 These
recently demonstrated a hollow cantilever a focused beam of light from the tip of a cantilevers are composed of several layers
concept called the suspended microchan- cantilever beam into a position-sensitive of materials at different regions where sin-
nel resonator (SMR) that is capable of detector. In the piezoresistive technique, gle crystal silicon serves as an active func-
detecting biological interactions in liquids the resistance of an asymmetrically doped tional element. The active silicon region is
with unprecedented sensitivity.5 (See the cantilever varies sensitively as a function sandwiched between silicon nitride and
section on Suspended Microchannel of bending. The piezoelectric technique silicon dioxide layers for insulation and
Resonators later in this article.) involves coating the cantilevers with protection, Figure 1a. These piezoresistive
piezoelectric materials, which develop a cantilevers are fabricated using predoped
Mechanics of Cantilever measurable charge due to cantilever silicon with insulating layers of silicon
Deflection bending. In the embedded MOSFET read- nitride on both sides. The thickness of the
Adsorption of molecules on a surface out method, a field-effect transistor is insulating layers (silicon nitride and silicon
results in a decrease in surface free energy. embedded at the base of the cantilever. oxide) is adjusted in such a way that the
If the adsorption of molecules on a surface The stress from the bending of the can- neutral axis of the bending cantilever is
is restricted mostly to one side, for exam- tilever changes the carrier mobility and outside of the doped silicon. The can-
ple, by making the opposite surface inert, drain current. In the capacitance method, tilevers fabricated using this method have
a differential surface stress is generated the capacitance between a bending can- a higher signal-to-noise ratio, less can-
between the two surfaces of a cantilever tilever and a fixed substrate varies as a tilever drift, and increased sensitivity, as
beam. Surface stress, g, and surface free function of cantilever bending. The elec- compared to piezoresistive cantilevers fab-
energy, , can be related using the tron tunneling method is extremely sensi- ricated, where the neutral axis lies at
Shuttleworth equation: tive, and it is based on the tunneling of the boundary between the doped and
electrons between a cantilever and a fixed undoped regions of the silicon. Since the
dg , electrode.16 silicon nitride/silicon oxide layer is insulat-
g=g+ (2)
de ing, these piezoresistive cantilevers can be
Piezoresistive Cantilever Array used in liquid environments.
where the surface stress, epsilon, is defined Fabrication For the cantilevers fabricated, there are
as the ratio of the change in surface area to Piezoresistive materials, such as doped two parallel silicon stripes (20 m wide)
the total area. Since the bending of the silicon, show piezoresistivity where the in each cantilever (see Figure 1). Two
cantilever is very small compared to the resistance of the material varies as a func- gold/titanium metal leads connect silicon
length of the cantilever, the strain contri- tion of applied stress. A multilayer can- stripes at their base and end at the contact
bution is often neglected. However, this is
a subject under active discussion in the lit-
a
erature.68
The differential surface stress created by
molecular adsorption results in cantilever
bending. Stoneys equation relates the dif-
ference in surface stress g between the L
chemically modified surface and the
untreated surface with the cantilever
deflection, h:
LR
Eh t 2,
g = (3)
4(1n) L
w
h
where, is the Poissons ratio of the mate-
rial, E is Youngs (elastic) modulus of the
b
cantilever material, and t and L are the
thickness and the length of the cantilever, c
respectively. The surface stress also can be Sensing Layer
thought of as a change in surface energy
density or a change in surface tension. It is Si3N4
clear that the longer the cantilever, the Si
more sensitive the cantilever to measure
surface stresses. SiO2
Si3N4
Modalities of Cantilever Deflection-
Based Sensing
The motion of the cantilever response
can be sensitively monitored using a vari- Figure 1. (a) Schematic diagram of a microcantilever indicating dimensions length, L,
ety of techniques, such as variations in resistor length, LR, width, w, and thickness, h. (b) An array of piezoresistive cantilevers.
optical beam deflection,9 piezoresistivity,10 (c) Cross-sectional diagram through the layers of the microcantilever sensors.
coating chemistry employed, typically all Figure 4 shows the surface stress variation PSA. PSA is a serine protease secreted by
experimental surfaces are freshly pre- of a piezoresistive cantilever as a function prostatic luminal epithelial cells. When
pared no more than 48 hours prior to time due to ssDNA (thiol link) immobiliza- used in population screening for the
assay. Stability studies to determine the tion and subsequent hybridization with detection of elevated serum, PSA is cred-
effects of aging on the prepared surfaces complementary ssDNA. The specific bind- ited with dramatic advances in the early
remain to be done. ing between the complementary DNA diagnosis and management of men with
strands on the cantilever results in a sur- prostatic carcinoma. The majority of the
Biomolecular Detection Assays face stress variation of 3040 mN/m. Wu recently marketed assays are based on the
Antigen-antibody interactions are a et al. investigated the origins of cantilever commonly used reference range (<4 ng of
class of highly specific protein-protein deflection due to biomolecular interactions PSA in a ml of blood), and almost all of
binding that play a critical role in molecu- and found that the deflection resulted from them employ some variation of the tech-
lar biology. Since cantilever bending origi- a change in free energy of one cantilever nique of enzyme-linked immunosorbent
nates from the free energy change induced surface.22 The interplay between the ener- assay (ELISA).
by specific biomolecular binding, this bio- getic and entropic contributions deter- Figure 5 shows the potential of micro-
molecular detection assay offers a com- mined the direction of cantilever motion. cantilevers as a platform for developing a
mon platform for high-throughput, Although both DNA hybridization and sensitive and specific assay for PSA using
multiplexed label-free analysis of biomol- protein-protein (antigen-antibody) bind- the optical beam deflection method for
ecules, such as protein-protein binding, ing can be detected using cantilever cantilever bending. The cantilevers were
DNA hybridization, and DNA-protein deflections, what remained unclear for a immobilized with polyclonal anti-PSA
interactions.1927 When antibody mole- while was whether this technique had suf- antibodies. Binding of PSA on the immo-
cules are immobilized on one surface of a ficient specificity and sensitivity to be bilized polyclonal antibody resulted in a
cantilever, specific binding between anti- used for the detection of disease-related deflection of the cantilever. Furthermore,
bodies and antigens produces cantilever proteins at clinically relevant conditions changes in surface stress were related
deflection. Similarly, the cantilever under- and concentrations. To address this tech- quantitatively to the concentration of PSA.
goes bending when single-stranded DNA nologically critical issue, sensitive and Results in Figure 5 are from a model sol-
(ssDNA) probes are immobilized on the specific detection of a prostate cancer vent system prepared with phosphate-
cantilever hybridized with complemen- marker, prostate specific antigen (PSA), buffered saline and shows steady-state
tary ssDNA (target) molecules in the solu- was conducted as an example of both cantilever deflection as a function of PSA
tion. Such specific deflection was not seen protein-protein binding in general and of concentration against a much higher back-
when the incoming DNA strands were a cancer diagnostic tumor marker detec- ground of bovine serum albumin. Similar
noncomplementary due to the absence of tion in particular.21 Prostate cancer has tests were performed against high back-
hybridization. Therefore, it is possible to emerged as the most common nonskin grounds of human serum albumin and
design ssDNA probes specifically to cancer and the second leading cause of human plasminogen, both of which are
detect mutations in the DNA sequence of cancer death in men in North America and found abundantly in human sera. Of note
target DNA responsible for many cancers. Europe (www.cancer.gov). While transrec- was the finding that PSA concentrations
The following section briefly describes tal ultrasonography and digital rectal can be detected below 4 ng/ml, the clini-
development of this assay. examination are common clinical exami- cal threshold for prostate cancer. In fact,
nations, the most widely used biochemical concentrations down to 0.2 ng/ml were
Detection of Specific DNA test involves analyzing the presence of detected. Since for the same PSA concen-
Sequences trations cantilever deflections varied with
Several groups have shown can- their geometry, it is important to stan-
60
tilever deflections due to adsorption of dardize these measurements in terms of
ssDNA on virgin cantilevers as well surface stress rather than cantilever
as hybridization of complementary 30 deflections using Stoneys formula. The
Stress (mN/m)
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