www.rsc.org/analyst | The Analyst
i-SECTION: CRITICAL REVIEW
Microcantilever biosensors based on
conformational change of proteins
Hai-Feng Ji,*a Hongyan Gao,a Koutilya R. Buchapudi,a Xin Yang,a Xiaohe Xua and
Marvin K. Schulteb
DOI: 10.1039/b713330h
Microcantilevers (MCLs) hold a position as a cost-effective and highly sensitive sensor
platform for medical diagnostics, environmental analysis and fast throughput analysis.
MCLs are unique in that adsorption of analytes on the microcantilever (MCL) surface
changes the surface characteristics of the MCL and results in bending of the MCL. Surface
stress due to conformation change of proteins and other polymers has been a recent
focus of MCL research. Since conformational changes in proteins can be produced
through binding of anylates at specific receptor sites, MCLs that respond to
conformational change induced surface stress are promising as transducers of chemical
information and are ideal for developing microcantilever-based biosensors. The MCL can
also potentially be used to investigate conformational change of proteins induced by
non-binding events such as post-translational modification and changes in temperature
or pH. This review will provide an overview of MCL biosensors based on conformational
change of proteins bound to the MCL surface. The models include conformational change
of proteins, proteins on membranes, enzymes, DNA and other polymers.
1. Introduction to
microcantilever technology and
conformational changes of
proteins
Advances in the field of micro-electromechanical systems (MEMS) and their
a
Institute for Micromanufacturing, Louisiana
Tech University, Ruston, LA, 71272, USA.
E-mail: hji@chem.latech.edu;
Fax: 01-318-257-5104; Tel: 01-318-257-5125
b
Department of Chemistry & Biochemistry,
University of Alaska Fairbanks, Fairbanks, AK,
99775-6160, USA
uses offer unique opportunities in the design of small-size and cost-effective analytical methods. In 1994, it was realized that
microcantilevers (MCLs) could be made
extremely sensitive to chemical and physical changes.1–3 To date, physical sensing
has been demonstrated by detecting thermal energy, strain, magnetic field, electric charge, viscosity, density and infrared
radiation. Extremely sensitive chemical
and biological sensors based on microcantilevers, for analytes including DNA,
alcohol, mercury, antigens, metal ions,
organophosphates, bacteria, pathogens,
etc., have been reviewed.4–8
Electron micrographs of MCLs are
shown in Fig. 1. The low cost and disposable characters of MCL chips make
the MCL sensing platform appropriate for
developing biological based sensor arrays.
Two characteristics of an MCL can be
used to detect chemicals.
a. Resonance frequency: the resonance
frequency of a MCL can be used to detect
chemical species in air due to changes in
mass as well as changes in spring constants
due to adsorption.
b. Bending: MCLs undergo bending
due to molecular adsorption by confining the adsorption to one side of the
Dr Hai-Feng Ji (second from right) received his PhD degree of chemistry from
the Chinese Academy of Science, China, in 1996. In 2000, he joined the faculty
in the Institute for Micromanufacturing at Louisiana Tech University. His
research interests focus on MEMS devices, surface modification for sensors,
and nanoassembly of organic molecules. He is currently a co-author of 80 peerviewed journal articles and book chapters. Dr Xin Yang (far left) is currently
a postdoctoral associate in Dr Ji’s lab. Dr Xiaohe Xu (second from left) is a
research engineer at Louisiana Tech University. Mr Koutilya R. Buchapudi (far
right) is pursuing his master degree in Dr Ji’s lab. Ms Hongyan Gao (not in the
picture) is currently a professor at Yili Normal University, Xin Jiang, China.
Dr Marvin Schulte (not in the picture) received his PhD at the University of
Minnesota in 1992 and is currently an associate professor of Biochemistry at
the University of Alaska Fairbanks.
434 | Analyst, 2008, 133, 434–443
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Fig. 1 Electron micrographs of several MCLs. The sizes of MCLs on the right vary from 5 lm to 200 lm in extent from the support. The far
left micrograph shows a piezoresistive cantilever.
cantilever. Adsorption or intercalation of
the analyte will change the surface characteristics of the MCL or the film volume on
the cantilever, and results in bending of the
MCL. Using Stoney’s formula,9 the radius
of curvature of bending of the cantilever
due to adsorption can be written as:
3(1 − m)L2
ds
(1)
DZ =
ET 2
where Dz is the observed deflection at the
end of the cantilever, m and E are Poisson’s
ratio (0.2152) and Young’s modulus (156
GPa for silicon) for the substrate, respectively, T and L are the thickness and length
of the cantilevers, respectively, and ds is the
differential stress on the cantilever.
MCLs can bend down10 or up11 on binding of specific species in the environment
depending on different effects, as shown
in Fig. 2. From a molecular point of view,
binding results in electrostatic repulsion,12
attraction,10 steric effects,13 intermolecular
interactions, or a combination of these
that alter the surface stresses on the cantilever.
Both frequency and bending approaches have been demonstrated to de-
tect chemicals with sensitivity as high as
parts-per-trillion to parts-per-quadrillion
range.13–15 Each approach has its own
advantages and disadvantages and each
will be used for specific applications. The
frequency method is limited to measuring mass increase on the sensor surface
and can fail if the potential ligand has
a low molecular weight. The bending
approach based on adsorption-induced
surface stress is of particular interest in
the study of conformational change in
proteins that is the subject of this review.
Overall, MCLs are cost-effective, sensitive
sensors that could be used for numerous biomedical, industrial, diagnostic and
homeland security applications.
The surface stress resulting from conformation change of proteins has been a
recent focus of MCL research. Conformational changes are capable of altering
orientation of molecules on the surface,
distances between molecules, and surface
interactions, thus it is reasonable that
conformational changes will alter MCL
bending. Surface stress changes due to
protein conformational change on interaction with analytes could act as transducers
of chemical information. MCLs respond-
Fig. 2 Two mechanisms of binding-induced surface stress in different types of responsive
coatings. (Top) Expansive stresses for antigen–antibody interaction. (Bottom) Compressive
stress due to neutralization of excessive charge on the surface.
This journal is © The Royal Society of Chemistry 2008
ing to these stresses would be ideal for
high sensitivity detection of the small dimensional changes expected. These MCLs
could also be used to investigate conformational changes that do not involve
analyte interactions.
Configuration and conformational
changes in biomolecules, including phase
transitions, play a critical role in many
biological processes. Traditional methods
employed for studying conformational
changes in proteins have included optical
rotation, immunoassays, SDS-PAGE,
Western blotting, spectrophotometry,
X-ray crystallography, viscometry, fluorescence, circular dichroism (CD), hydrogen
exchange, thermodynamics and nuclear
magnetic resonance (NMR).16
More novel methods include electrospray ionization mass spectrometry,17 hot
tritium bombardment technique,18 surface
plasmon resonance (SPR),19 atomic force
microscopy (AFM),20 Fourier transform
infrared (FTIR) difference spectroscopy,21
fast photochemical oxidation of proteins
(FPOP),22 quartz crystal microbalance
(QCM),23 molecular simulation, molecular simulation combined with experimental measurements,24 small angle X-ray
scattering25 and synchrotron radiation Xray resolution scattering.26
While these techniques are useful in
the detailed characterization of protein
conformation and the protein folding and
unfolding process, all require instrumentation that is expensive and complex. If
conformation changes in proteins can be
detected with MCL sensors, they could
be used to study differences in conformational effects of ligands, receptor
mechanism or conformational stability of
proteins. This would provide unique opportunities for protein structure/function
studies in a small, inexpensive instrument.
The cost of MCLs in comparison to other
available techniques would place the MCL
Analyst, 2008, 133, 434–443 | 435
technology on the benchtop of a much
larger pool of investigators, increasing
the rate of discovery. The MCL technique would permit rapid investigations
of protein conformation, compounds that
alter conformation and conformational
stability.
2. Surface stress changes
induced by conformational
changes in proteins
The surface stress induced by conformational changes in proteins was first
reported by Welland’s group in 1999.10
In this work, the surface stress induced
by protein adsorption onto a gold surface was measured. Two proteins, immunoglobulin G (IgG) and albumin
(BSA), were studied. Each protein produced a different surface stress change on
adsorption. IgG produced a compressive
stress, which may be due to expansion of
the proteins on the gold surface, whereas
BSA produced tensile stress, which might
be due to contraction of protein molecules
(Fig. 3). In this case, the MCL technique
provided a sensitive tool to probe the
adsorption of proteins onto solid surfaces,
particularly over long time scales. This
method could provide important information in biotechnology applications involving protein-functionalized surfaces.
Several other examples of surfacerelated conformational changes of proteins on MCLs have been demonstrated
and the molecular interaction kinetics was
recently studied.27
Although the paper cited above on
conformational change of proteins on
MCL surfaces was published in 1999,
the use of conformational change in proteins on MCLs did not receive much
attention as a sensing concept until
2004.28 A MCL modified with a layer of
acetylcholinesterase (AChE) underwent
bending due to the inhibition of AChE
by paraoxon; a compound that slightly
changes the conformation of AChE. The
AChE was immobilized on the MCL
through a cross-linker to a monolayer
on the gold surface of the MCL. Numerous reports and the X-ray crystallographic structure of AChE and conjugates show that AChE changes its structure on binding of organophosphorus
compounds (OPs) at the catalytic serine site.29,30 Studies with mouse AChE
revealed that the inhibitor produces a
conformational change at the omega loop
69–96, far away from the active site. The
CD spectra of stabilized AChE displayed
two parameters which can be influenced
independently from each other: the magnitude of the ellipticity bands and the accentuation of the bands at 220 and 208 nm.
The inhibition by many OPs, including
paraoxon, of AChE is irreversible. Since
OPs are electrically neutral molecules and
the bound OPs do not have interactions
each other, the bending of the MCL was
most probably generated from the slight
Fig. 3 Left: (a) the compressive change in surface stress induced by a 10 ll injection of IgG on a gold surface. The inset shows the response
immediately after IgG injection. The experimental drift was subtracted (5 × 10−5 N m−1 min−1 ) from the response shown in the inset in order
to observe the initial change of slope due to the adsorption of proteins. (b) Schematic diagram showing how protein–surface interactions
may cause the proteins to unfold. The proteins try to “spread” or expand on the surface, and since they are confined within the monolayer
a compressive surface stress occurs. (c) Schematic diagram showing how attractive (hydrophobic) protein–protein interactions may cause the
proteins to rearrange. Since the proteins are relatively immobile within the monolayer surface, rearrangement results in deformation of the
proteins (i.e. the biomolecules tend to “flatten”) and a compressive surface stress results. Right: (a) the tensile change in surface stress induced
by a 10 ll injection of BSA on a gold surface. The inset shows the response immediately after BSA injection. (b) Schematic diagram showing
that if the proteins are relatively mobile on the surface, attractive (hydrophobic) protein–protein forces could cause the biomolecules to pack
together. Since the proteins are trying to contract, a tensile surface stress results. Reprinted from ref. 10 with permission from the American
Chemical Society.
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conformational change in AChE. Because
AChE only slightly changes its conformation on complexation with organophosphates, the change in surface stress on
the MCL was small (0.014 N m−1 surface
stress). This work demonstrated that the
slight conformational change in AChE
produced by OP inhibition could be used
to detect OPs by confining AChE on an
MCL. The same concept can be expanded
through the use of other proteins to
develop new MCL biosensors. Since the
bending amplitude of a MCL generated by
inhibition of AChE was relatively small,
this MCL sensor would not be useful as
a real time sensor for field detection of
organophosphates.
The role of conformational change in
MCL bending was addressed again in
later studies using calmodulin (CaM),
haemoglobin and myoglobin.31 Calmodulin (CaM) is a small (17kD), heat stable acidic protein with approximately 148
amino acids residues and is an abundant
and ubiquitous calcium binding protein
that serves as an activator of numerous cellular enzymes. In the absence of
Ca2+ , CaM consists of a short dumbbell
structure with two globular domains connected by a helical linker. Each of the
globular domains contains two EF hands
(helix–12-residue-loop–helix motif) which
bind to a Ca2+ ion at the loop region
with intermediate affinity (K D from 1 to
10 lM).32 Upon binding of four Ca2+
per molecule of calmodulin, CaM undergoes large conformational changes, and
becomes more elongated.32 This altered
conformation of the Ca2+ –CaM complex
enables it to interact with other proteins,
and this action is critical to various aspects of cell metabolism. If conformational change contributes to MCL bending, then an analyte–protein interaction
such as Ca2+ /calmodulin that generates
a large conformational change in the
protein would be expected to generate
larger bending of an MCL compared to
proteins that do not show large conformational changes. On the other hand,
if conformational changes play little or
no role in bending then the assay will
not be able to distinguish between two
analytes via response amplitude. Experimental results showed that the modified
MCL bent when calmodulins change their
conformation on binding of Ca2+ . Different degrees of conformational change
induced by altering the ionic strength
Fig. 4 Bending responses of a (PEI/CaM)3 multilayer modified MCL to a 10−5 M
concentration of Ca2+ in 0.01 M, 0.001 M, and 1 M of NaCl solutions. Reprinted from
ref. 31 with permission from the American Chemical Society.
of the solution produced different deflection amplitudes (Fig. 4). The maximum surface stress generated was 0.098 N
m−1 . No surface stress induced cantilever
bending was observed for proteins that
do not show significant conformational
changes (such as haemoglobin or myoglobin with oxygen). While not conclusive,
these data suggest that conformational
changes in proteins contribute to MCL
bending.
Another report in 2005 used a different
approach to detect specific protein
conformations.33 In this work, oestradiol
(E2) was pre-bound on a human oestrogen
receptor (EPa-LBD) protein, which
changes the conformation of the protein.
The EPa-LBD and EPa-LBD-E2 complex
were distinguished by MCLs modified by
conformation-specific peptides a/bI (SerSer-Asn-His-Gln-Ser-Ser-Arg-Leu-Ile-GluLeu-Leu-Ser-Arg), which recognizes EPaLBD-E2 complex, and a/bII (Ser-AlaPro-Arg-Ala-Thr-Ile-Ser-His-Tyr-Leu-MetGly-Gly), which recognizes EPa-LBD
(Fig. 5). This is an indirect and complementary approach for studying conformational change in proteins. It could be
used when the conformational change
of the protein is too small and does
not produce detectable bending of the
MCL.
The number of publications reporting the detection of conformational
change in proteins using microcanti-
This journal is © The Royal Society of Chemistry 2008
levers has significantly increased since
2006, and more analysis and calculations have been discussed. Promising proteins suitable for MCL-based sensors are
stimulus-responsive, elastin-like polypeptides (ELPs).34 Conformational changes
of grafted ELPs, included by a phase
transition or changes in osmotic pressure, lead to significant changes in surface stress in the ELP graft layer and
translate into changes in MCL deflection.
The conformational mechanics of ELPs
in response to changes in solution pH
and ionic strength have been investigated.
In an ionic strength study, the ELPs
modified MCLs produced a downward
deflection in response to changes in salt
concentrations (Fig. 6), suggesting that
the grafted ELPs on the top surface of
a MCL cause lateral steric interactions
(driven by the osmotic swelling pressure
of the grafted ELP layer). These interactions led to increases in the cantilever
surface and increased cantilever deflection
compared to that of unmodified control
cantilevers. The pH effect showed that the
cantilever deflection decreases (becomes
less negative) with increasing solution pH
in the range from 6 to 11, a result of
ELP collapse with increasing solvent pH
caused by decreasing ionization of the
NH3 + group on the lysine side chain.
The ELP surface conformation thus depends on a balance between restoring
elastic forces in the hydrated graft layer
Analyst, 2008, 133, 434–443 | 437
Fig. 5 (a) Schematic drawing showing a two-cantilever configuration, the a/bI attached on
one cantilever and the a/bII on the other, and the preferential binding of ERa-LBD, E2-bound
or free, onto the a/bI and a/bII, respectively. (b) A cartoon showing the sensor layer on top of
the surface and the blocking layer at the bottom surface of a cantilever and its bending upon
target binding onto the top sensing surface. Reprinted from ref. 34 with permission from the
American Chemical Society.
Fig. 6 Net microcantilever deflection plotted as a function of time for two different ionic
strengths (PBS and PBS + 1.5 M NaCl). Net deflection is determined as the difference between
the deflection of a microcantilever with end-grafted ELP1-180 in PBS or in PBS + 1.5 M NaCl
and deflections of a bare reference microcantilever under the same solution conditions. Dd
indicates the effective difference in cantilever deflection at steady state. Reprinted from ref. 35
with permission from the American Chemical Society.
and repulsive electrostatic forces (ionic
osmotic pressure). The change in integral
surface stress associated with cycling the
solution pH between 5.9 and 11.9 was
438 | Analyst, 2008, 133, 434–443
Dd ∼ 0.055 N m−1 . These results showed
that reversible switching of ELP conformation on a cantilever surface leads
to reversible cantilever bending, and the
magnitude of cantilever actuation can be
modulated by the level of the applied
external stimulus.
In an MCL sensing assay for detecting activated cyclic adenosine monophosphate (cyclic AMP)-dependent protein kinase (PKA) using a peptide derived from
the heat-stable protein kinase inhibitor
(PKI), Kown et al. compared the interaction of adenosine triphosphate (ATP) and
the peptide inhibitor with the kinase by
a solution phase capillary electrophoretic
assay, by surface plasmon resonance technology, and by the MCL method.35 The
MCL method exhibited much higher sensitivity and wider dynamic range than
the conventional activity assay. The resonant frequency shifted more dramatically
than expected from theoretical calculations, likely resulting from the compressive
stress exerted by repulsive electrostatic
intermolecular interactions or changes in
the hydrophobicity on the functionalized
side of the cantilever.
3. Surface stress resulting
from conformational change of
proteins on membranes
Recent work on membrane modified cantilevers shows that conformational change
of proteins in membranes can also produce microcantilever bending and could
be used for drug screening and other applications. A membrane preparation containing serotonin 5-HT3AS receptors was
used to modify an MCL.36 The 5-HT3AS
receptor is a membrane bound, centrally
and peripherally localized, ligand-gated
ion channel mediating membrane depolarization and neuronal excitation. The
modified MCL was found to bend on application of the naturally occurring 5-HT3
receptor agonist (5-hydroxytryptamine,
which is also called serotonin) or the
antagonist MDL-72222, due to the conformational change of the proteins in the
membrane. Application of other similar,
but non-interacting molecules, such as
tryptophan produced no bending of the
MCL. K d values obtained for serotonin
and MDL-72222 are identical to those
obtained from radio-ligand binding assays. These results suggest that the MCL
system has potential for use in labelfree, drug screening applications involving
membrane bound receptors.
In another study,37 bacteriorhodopsin
(BR) proteoliposomes were used as a
This journal is © The Royal Society of Chemistry 2008
model system to explore the applicability of micromechanical MCL arrays to
detect conformational changes in membrane protein patches. BR assembles in
its native form as a two-dimensional (2D)
crystal leading to the highest possible
density at the cell surface. The hydrolysis
of the retinal of BR can be emulated by
the addition of hydroxylamine to form
retinaloxime. This chemical removal of
photoactivated retinal is accompanied by
structural changes in the BR protein and
to the loss of the crystallinity of the BR
2D crystals. Visualization of the BR proteoliposomes on the MCLs was performed
using a tapping mode AFM (Fig. 7).
Based on these, the authors demonstrated
that the MCL can quantitatively detect
retinal removal from BR. The data analysis showed that the MCL bending is
caused from the conformational change of
the protein. The authors concluded that
the cantilever-based technique would be
able to detect structural changes of these
membrane proteins on ligand binding or
unbinding. These results show this technique to be a potential tool to measure
membrane protein-based receptor–ligand
interactions and conformational changes
of proteins on membranes.
4. Photon-induced
conformational change of
proteins
Another two interesting examples demonstrated the photon-induced conformational change of the proteins on MCL surfaces. In one example,38 purple membranes
from Halobacterium salinarum were deposited electrophoretically on platinumcoated MCLs. By illuminating the bacteriorhodopsin (BR)-containing purple membranes, the protein undergoes its photochemical reaction cycle, during which
a conformational change occurs in the
protein, changing its shape and size. The
on–off change occurs in millisecond. The
shape of the signal, the action spectrum
of the deflection amplitude, and the blue
light inhibition of the deflection all prove
that the origin of the signal is the conformational change arising in the bacteriorhodopsin during the photocycle. From
the size of the signal, the magnitude of
the protein motion could be established.
Using polarized light, the orientation of
the motion was detected, relative to the
transition moment of the retinal. In air,
Fig. 7 Functionalization of the upper cantilever surface with BR membrane patches
visualized by tapping mode AFM. The scale bar corresponds to 1 mm. The dashed line, also
indicated by two arrowheads in panel A, corresponds to the position of the captured height
profile (B). (C) Nonlabeled BR membrane patches immobilized on ultraflat gold (in air, tapping
mode). (D) Immunoassayed BR patches. Antibodies are specific against the extracellular side
of BR, indicating a preferential orientation of BR with the cytoplasmatic side facing the
cantilever. Scale bar, 500 nm. Reprinted from ref. 38 with permission from the Biophysical
Society.
the smaller dilatation of the protein could
be explained as a smaller conformational
change than that in water because the
dried protein is more rigid. The average
energy per BR molecule contributing to
MCL bending was estimated to be 195kT
(in terms of the Boltzmann energy at 295
K). This calculated energy provides an
estimate of the order of magnitude and
compares to the energy of a photon of
84kT with a wavelength of 580 nm, which
triggers the photocycle of BR. In another
paper,39 the same BR protein model was
used for the photocycle and the author
This journal is © The Royal Society of Chemistry 2008
attributed the MCL bending to proton release caused by the conformational change
in BR.
5. Surface stress change
resulted from conformational
change of enzymes
In addition to conformation changes of
stimulus responsive proteins and membranes, it has been shown that the conformational changes of enzymes also contribute to the surface stress change and
Analyst, 2008, 133, 434–443 | 439
subsequent MCL bending. In 2005, Ji and
Yan reported a glucose oxidase (GOx)
functionalized MCL sensor for glucose
measurement.40 The results showed that
the MCL underwent bending when it
was exposed to glucose. The possible
contributions to MCL bending include
heat release, pH change, H2 O2 production,
and the conformational change of the
enzymes. The basis these effects is the oxidation of glucose, as shown in the following equation:
D-glucose + O2 + H2 O
GOx
−→ gluconic acid + H2 O2
(2)
The reaction results in a decrease in
pH and O2 , and increase in H2 O2 , which
has been monitored as an indirect measurement of glucose concentration. The
calculations show that the heat release
would produce only a very small amount
of bending (∼7.45 × 10−3 nm). This is
only about 0.03% of the observed 20 nm
deflection. pH change only contributes a
10 nm cantilever bending. Furthermore,
the MCL bending vs. pH change does not
match with MCL bending on exposure to
glucose. Calculations also show that the
H2 O2 production does not contribute to
cantilever bending. These analyses suggested that the conformational change of
the GOx enzyme may contribute half of
the MCL deflection.
Similar phenomena have been observed on other enzymes, such as
organophosphorus hydrolase (OPH)41
and horseradish peroxidase (HRP).42 In
both cases, calculations showed that the
conformational changes of the enzymes
contribute greatly to cantilever bending.
6. Surface stress change due to
conformational change of DNA
and other polymers
It should be noted that the concept of
conformational change induced surface
stress also applies to DNA and other
polymers. Shu et al. reported the direct
integration between an ensemble of DNA
motors and an array of microfabricated
silicon cantilevers.43 The forces exerted by
the precise duplex to nonclassical i-motif
conformational change were probed via
differential measurements using an in-situ
reference cantilever coated with a nonspecific sequence of DNA (Fig. 8). The open
to close stroke of the motor by photons
induced a 0.032 ± 0.003 N m−1 compressive surface stress, which corresponds to a
single motor force of approximately 11 pN
m−1 . Furthermore, the surface-tethered
conformational change was highly reversible, in contrast to classical DNA
motors which typically suffer rapid system
poisoning. The direction and amplitude
of motor-induced cantilever motion was
tunable via control of buffer pH and
ionic strength, indicating that electrostatic
forces play an important role in stress
generation. Hybrid devices which directly
harness the multiple accessible conformational states of dynamic oligonucleotides
and aptamers, translating biochemical energy into micromechanical work, present a
radical new approach to the construction
of “smart” nanoscale machinery.
Surface stress changes in response
to thermal dehybridization of doublestranded DNA (dsDNA) oligonucleotides
that are grafted on one side of a MCL
have been observed.44 Changes in surface stress occur when one complementary DNA strand melts and diffuses
away from the other, resulting in alterations of the electrostatic, counterionic,
Fig. 8 Harnessing duplex to i-motif conformation changes on a micromechanical cantilever array. (a) Chemical structure of a C+ :C base pair
on strand X at pH 5.0 to show the three hydrogen bonds formed between a single pair of hemiprotonated cytosine bases and a schematic
diagram to show the intramolecular interdigitation of strand X to form the i-motif. (b) Scanning electron microscope image of an array of eight
rectangular silicon cantilevers. (c) Schematic diagram to show a cantilever functionalized on one-side with a thin film of gold and a monolayer of
thiolated X. At pH > 6.7 hybridization of surface-tethered X to strand Y in solution (1 lM) forms the duplex structure. (d) At pH 5.0, X forms
the self-folded i-motif and induces repulsive in-plane surface forces (compressive surface stress) which cause the cantilever to bend downward,
Dz. Strand Y is shown to be present in free solution in a random coil conformation. Reprinted from ref. 44 with permission from the American
Chemical Society.
440 | Analyst, 2008, 133, 434–443
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and hydration interaction forces between the remaining neighboring surfacegrafted DNA molecules. As the temperature of the cantilever is raised, there is
a conformational change of the DNA
molecules as it undergoes a transition
from double- to single-stranded, resulting
in a change in the surface stress of the
layer. When single strands are immobilized, the strands are in a more coiled
and compact conformation. Upon introduction and subsequent hybridization of
complementary target DNA strands, the
double-stranded DNA takes on a rodlike
conformation, which results in an increase
in the DNA film thickness. The transduction of phase transitions into a mechanical signal is ubiquitous for DNA, making cantilever-based detection a widely
useful and complementary alternative to
calorimetric and fluorescence measurements.
Poly(N-isopropylacrylamide) (pNIPAAM) alone or as a copolymer is a stimulusresponsive polymer that undergoes an
inverse phase transition triggered by
changes in the solvent quality, such as temperature, ionic strength, pH, or co-solvent
concentration. Associated with this phase
transition is a significant conformational
change. Studies showed that pNIPAAM
brush or poly(N-isopropylacrylamide-coN-vinylimidazole) (pNIPAAM-VI) (7 :
3) brush grafted MCLs can be used to
detect and transduce this phase transition behavior. Changes in the conformational state of the brush, induced by
the phase transition or changes in osmotic pressure, cause significant changes
in the surface stress in the brush that
leads to detectable changes in cantilever
deflection.45,46 The large stress values for
pNIPAAM-VI are mainly due to much
thicker brushes and much higher grafting
densities.
7. Perception of
conformational change of
protein based MCL biosensors
It is noteworthy that the bending mode
of MCL sensors requires two different
MCL surfaces in order to differentiate
the surface stress on the two surfaces.
Typically, one side of a MCL had a thin
film of gold coating and the other side of
the MCL was made of silicon with a thin
naturally grown oxide layer. In general,
one of these two sides of MCLs would
be modified by special coatings, such as a
perfluorocarbons monolayer31,40 or BSA,33
to inhibit the protein adsorption, and
thus the protein immobilization could be
realized on a single side of the MCLs. The
receptors have also been applied directly
onto the top side of MCLs by an ink-jetspotting dispensing system (such as MDP-705-L system, Microdrop, Norderstedt,
Germany).37
These results demonstrate that conformational change of proteins and other
biopolymers can be used to develop surface stress change-based biosensors. Surface stress change phenomenon due to
the conformational change of proteins not
only offers unique opportunities in the
design of small and sensitive analytical
methods, but may also provides an alternative, label-free bioassay to study the
protein–ligand interaction under varying
conditions of ionic strength or electrolyte
identity. The simplicity of such label-free
methods also significantly increases the
likelihood this technology will be utilized
and reduces the costs. The fast throughput
characteristics of these technique will facilitate studies of protein conformational
change in the future.
MCLs provide an unique platform for
conformational change based biosensing,
and conformational change-based MCL
bending would dramatically broaden the
usefulness of MCL sensors. We can foresee
the emergence of more MCL biosensors
in this field; however, it is a challenge
to directly prove that the surface stress
change results from the conformational
change of proteins. Gaining a fundamental understanding of this particular
phenomenon requires correlation of conformational change to the degree of the
MCL bending. This is difficult due to
the complexity involved in quantification
of conformational change. So far, no
other instruments, except for MCL systems, can be used for studying conformational change-induced surface stress.
The conformational change of proteins
on the cantilever surface may be characterized by field emission scanning electron microscopy (FESEM), surface plasmon resonance (SPR), atomic force microscopy (AFM), and infrared reflectionabsorption spectroscopy (IRAS), etc. for
limited information. These instruments
may provide information on how the
proteins change their conformation when
compared with the MCL deflection re-
This journal is © The Royal Society of Chemistry 2008
sults. Alternatively, molecular models may
be extremely helpful to quantitatively correlate the conformational change in the
proteins with MCL bending response.
It is generally recognized that many
proteins undergo conformational changes
upon complexation with analytes. Understanding how the conformational change
in proteins correlated with surface stress
under varying conditions is very important, since it should give new insight into
improving these protein-based biosensors.
Most of the conclusions in reviewed papers, however, were drawn based on calculation or indirect evidences. Due to the
lack of other tools to verify this concept,
these conclusions may not be precise and
may conflict with each other. For instance,
several researchers have observed that the
MCLs have a reduced deflection in higher
concentration buffer solutions. Several researchers attribute this phenomenon to
less conformational change of proteins
under higher ionic strength, i.e. the steric
effect of the proteins plays the major
role in change of surface stress,31,38 while
others suggested that the surface stress
change was due to the proton release or
electrostatic forces that resulted from the
conformational change of the proteins.39,43
Further study will be needed to further
investigate the origin of surface stress, including the entropic, hydrophobic, hydration forces, orientation and the grafting
density of the proteins.
As discussed in the Introduction, all of
the current techniques for characterization of protein conformation and protein
folding are expensive and complex. MCL
techniques would provide unique opportunities for protein structure/function
studies in a small, inexpensive instrument.
Compared to existing technologies for
protein conformation studies, the MCL
sensor technology has three key advantages:
Low cost: electronics for operation and
control for static mode (deflection) are
relatively simple and inexpensive when a
piezoresistive approach is used. A Wheatstone bridge electronic circuit can be used
to conveniently measure the resistance
change.
Low-power consumption: for static
mode, since the MCL bending signal is
driven by molecular recognition, the only
power needed is for detection and display,
allowing use of light-weight battery power
or photovoltaic cells.
Analyst, 2008, 133, 434–443 | 441
Small size: the entire sensor could fit
in an area with sides less than a few
millimetres. The device can be readily
integrated into a robot system without
much increase of weight in order to protect
the operator and to guarantee uniform
detection strategies.
However, there is still a long way to go
to convert the proof-of-concept results to
robust, commercially-available products.
Efforts in the following areas are critical
for commercialization:
(1) Development of robust MCL modification procedures. In general, the critical step in developing a bioassay is the
immobilization of the biological reagents
to the surface of the transducer without
a significant change in the nature of
the reagents. The modification procedure
should be easy to use and the resulting
sensor should be reproducible, reusable,
and reliable. The lifetime of the sensor
will also be extended. For static mode,
the MCL sensor procedure should be
optimized to ensure significant bindinginduced surfaces stresses.
(2) Optimization of the sample analysis
system and MCL materials, dimensions,
shapes and array for best performance.
The flow conditions and the geometric
variation of the MCL supporting system
can affect the cantilever measurement accuracy. A specifically designed microfluidic supporting system is needed to avoid
error and noise in the measurements. A
reference MCL will be needed in the
final product to account for non-specific
medium effects. An MCL array for averaging the raw experimental data would be
of great benefit to improve the accuracy
and sensitivity.
(3) Deflection detection system. The
optical method is the most widely utilized
and most sensitive method of qualifying MCL deflection.5 One limitation of
this method is the complexity inherent
in optical instruments, which needs laser
adjustment. The advantage of piezoresistive and piezoelectric approaches47,48 is
that the electronic device can be made
extremely simple and cost effective, but
the sensitivity is much less than that of
the optical method. The electronics of
the capacitive method is also simple but
the parallel plates may stick together and
terminate the data collection. Recently,
interferometric6 and MOSFET49 methods
have show certain advantages over other
methods, but they suffer from light scatter442 | Analyst, 2008, 133, 434–443
ing or not enough sensitivity, respectively.
Either novel or improvement of the existing methods is required for commercialization of the MCL technology for study
of protein conformation change.
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