Anharmonic Surface Interactions for Biomolecular Screening and

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Anal. Chem. 2011, 83, 549–554

Anharmonic Surface Interactions for Biomolecular Screening and Characterization Sourav K. Ghosh, Victor P. Ostanin, and Ashwin A. Seshia* University of Cambridge Nanoscience Centre, 11 J J Thomson Avenue, Cambridge, Cambridgeshire, United Kingdom, CB3 0FF The acoustic response of conventional mechanical oscillators, such as a piezoelectric crystal, is predominantly harmonic at modest amplitudes. However, here, we observe from the electrical response that significant motional anharmonicity is introduced in the presence of attached analyte. Experiments were conducted with streptavidin-coated polystyrene microbeads of various sizes attached to a quartz crystal resonator via specific and nonspecific molecular tethers in liquid. Quantitative analysis reveals that the deviation of odd Fourier harmonics of the response caused by introduction of microbeads as a function of oscillation amplitude presents a unique signature of the molecular tether. Hence, the described anharmonic detection technique (ADT) based on this function allows screening of biomolecules and provides an additional level of selectivity in receptor-based detection that is often associated with nonspecific interactions. We also propose methods to extract mechanical forceextension characteristics of the molecular tether and activation energy using this technique. 1. INTRODUCTION Knowledge of biomolecular interactions is of fundamental importance for the understanding of biological events and processes and applications in biosensing and drug discovery. In biosensing, this knowledge is crucial for choice of receptors or antibodies, which serve as specific recognition agents for target biomolecules or antigens. This requires tools for determination of affinity of interaction of the binding pair. Receptor-based detection is often challenged by nonspecific interactions that cannot be washed away easily; this necessitates that the detection technique is able to differentiate between specific and nonspecific interactions. However, most existing techniques, labeled or nonlabeled, are unable to detect affinity of interaction and are often plagued with the issue of false positive response.1-3 Current force spectroscopic techniques, such as optical tweezers,4 biomembrane force probe,5 and atomic force microscopy,6 can measure the * To whom correspondence should be addressed. E-mail: [email protected]. (1) Brewer, N. T.; Salz, T.; Lillie, S. E. Ann. Intern. Med. 2007, 146, 502–510. (2) Burman, W. J.; Stone, B. L.; Reves, R. R.; Wilson, M. L.; Yang, Z. H.; ElHajj, H.; Bates, J. H.; Cave, M. D. Am. J. Respir. Crit. Care Med. 1997, 155, 321–326. (3) Sayre, K. R.; Dodd, R. Y.; Tegtmeier, G.; Layug, L.; Alexander, S. S.; Busch, M. P. Transfusion 1996, 36, 45–52. (4) Grier, D. G. Nature 2003, 424, 810–816. (5) Merkel, R.; Nassoy, P.; Leung, A.; Ritchie, K.; Evans, E. Nature 1999, 397, 50–53. 10.1021/ac102261q  2011 American Chemical Society Published on Web 12/15/2010

activation energy of interaction, which gives a measure of the affinity of interaction, but can probe only one molecule at a time, requiring time-consuming multiple measurements to obtain statistically useful data. Besides, these techniques are associated with significant setup costs and are cumbersome. The anharmonic detection technique (ADT) described here allows measurement of activation energy of molecular interactions, and the resulting data can be used to infer the force-extension characteristics of molecular tethers. Thus, it presents a potential method for mapping molecular interaction networks and affinity-based molecular screening and provides a level of selectivity in addition to the efficacy of the receptor. Also, unlike existing force spectroscopic techniques, ADT has the advantage of simultaneously averaging over multiple molecular pairs of the same type. Moreover, being entirely electronic, it is integrable and scalable and enables cost-effective, rapid, and easy-to-use detection with minimal sample preparation. Alternative techniques reported in the literature aimed at fast and cost-effective detection of affinity of interaction include the bond rupture immunosensors,7 such as the rupture event scanning (REVS).8 However, a theoretical study of the physical mechanism responsible for generation of the said “rupture” signals, claimed to differentiate specific and nonspecific interactions, is largely missing in these works, and failure to reproduce these signals has also been reported.9 Moreover, unlike ADT, REVS does not report capability to measure activation energy or mechanical force-extension characteristic of a molecule. 2. TECHNIQUE DESCRIPTION ADT is an acoustic-based technique working on the basis of nonlinear molecular interactions with a surface. The electrical response of mechanical oscillators such as the thickness shear mode (TSM) quartz crystal is largely linear (harmonic) at low amplitudes, as the inherent material nonlinearity is insignificant. This implies that, if the resonator is driven by a pure sinusoidal voltage of frequency f, the electrical current (response) is also at the same frequency f. The electrical current flowing through the resonator is a time derivative of the charge directly transduced from internal mechanical stresses. It is well-known that nonlinearity in mechanical stresses can be primarily dominated by (6) Binnig, G.; Quate, C. F.; Gerber, C. Phys. Rev. Lett. 1986, 56, 930–933. (7) Hirst, E. R.; Yuan, Y. J.; Xu, W. L.; Bronlund, J. E. Biosens. Bioelectron. 2008, 23, 1759–1768. (8) Cooper, M. A.; Dultsev, F. N.; Minson, T.; Ostanin, V. P.; Abell, C.; Klenerman, D. Nat. Biotechnol. 2001, 19, 833–837. (9) Edvardsson, M.; Rodahl, M.; Kasemo, B.; Hook, F. Anal. Chem. 2005, 77, 4918–4926.

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interactions at the contact interface.10 Contact acoustic nonlinearity is, thus, widely used to characterize material defects.11-13 This fundamental principle is the basis of ADT. We observed that the response of the TSM AT-cut quartz crystal resonator, oscillating in-plane, becomes significantly nonlinear (anharmonic) in the presence of surface-bound streptavidin-coated polystyrene microbeads (SCPM) even at modest oscillation amplitudes, both in air and in liquid. This can be explained by anharmonicity in the interaction forces at the biological interface. The anharmonic interaction forces are added to the stresses (shear forces) in the bulk sensor, thus resulting in a modified electrical current (response) proportional to the shear forces. The modified anharmonic response is typically symmetric with surface displacement, and hence, only odd Fourier harmonics are primarily generated. The relative deviation of the first harmonic (f) response is negligible and cannot be measured in practice; however, the relative deviation of any higher harmonic response is significant with high signal-to-noise ratio (SNR). This provides a basis for sensitivity of the technique. Most remarkably, for a given drive frequency (f), the deviation of any higher odd harmonic response (3f, 5f, etc.) expressed as a function of the oscillation amplitude (referred to as anharmonic detection signal) is found to be uniquely dependent on the force-extension characteristic of the molecular tether, its length, and the size of microbeads. Hence, a variation in any of these parameters results in a different function (or shape of signal). This provides a basis for selectivity. Furthermore, since each particle contributes to the signal independently and synchronously, the magnitude of the signal is proportional (additive function) to the number of particles on the surface. This provides a basis for quantitative detection. Initial experiments with SCPM in air showed the capability to detect a single SCPM and to differentiate between specific and physisorbed interactions from the overall anharmonic response of the sensor.14 However, it was observed here that, for the low quality factors of the sensor operating in liquid, the anharmonicity from the bulk quartz or the liquid cannot be neglected and this response must be subtracted from the overall anharmonic response for accurate quantitative estimation of adsorbed analyte. In this paper, we present a detection and screening technique (ADT) based on the deviation in anharmonic response and demonstrate this principle experimentally with SCPM in liquid. Moreover, using ADT, we describe for the first time methods for molecular force spectroscopy and measurement of activation energy that is rapid and scalable. These methods are detailed in the Supporting Information, S1. The following sections present the experimental observations with SCPM and quantitative modeling. 3. EXPERIMENTS Materials. Experiments were carried out with SCPM of various sizes (5.61, 3.09, and 0.39 µm) in pH 7.4, 100 mM phosphate buffer saline (PBS) solution. Gold-electrode-plated AT(10) Fassbender, S. U.; Arnold, W. In Review of Progress in QNDE; Thompson, D.O., Chimenti, D.A., Eds.; Plenum Press: New York, 1996; Vol. 15, pp 1321-1328. (11) Solodov, I. Y.; Krohn, N.; Busse, G. In 1st Ultrasonics International Conference; Elsevier Science Bv: Delft, Netherlands, 2001; pp 621-625. (12) Zheng, Y. P.; Maev, R. G.; Solodov, I. Y. Can. J. Phys. 1999, 77, 927–967. (13) Krohn, N.; Stoessel, R.; Busse, G. Ultrasonics 2002, 40, 633–637. (14) Ghosh, S. K.; Ostanin, V. P.; Seshia, A. A. Anal. Chem. 2010, 82, 3929– 3935.

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cut TSM quartz crystals with a fundamental resonant frequency of 14.3 MHz were employed. For specific interaction with SCPM, one side of the crystals was functionalized with a self-assembled monolayer (SAM) of a biotinlylated-PEG-thiol [HS-(CH2)11(EG)6-biotin]. For nonspecific or physisorbed interaction, a hydroxyl-terminated-thiol [HS-(CH2)11-OH, FT 00.11] was used. The surface preparation process, along with the suppliers for the materials, is detailed in Supporting Information, S2. A quartz crystal resonator functionalized with SAM is referred to as a sensor. Separate sensors were prepared for different sized beads and types of interactions. Method. A sensor was driven close to its fundamental resonant frequency f by a sinusoidal AC signal using a 33220A Agilent function generator. Higher harmonics in the signal were substantially attenuated by a low pass filter. The transduced electrical signal (response) was received by an SR844 lock-in amplifier (Stanford Research Systems) that recorded the in-phase and quadrature (vector) components at third harmonic, 3f. A passive frequency tripler was used to generate the reference signal for the lock-in. Additionally, two quadrature receivers were employed to detect the 1f (fundamental) voltage and the quartz output current. In each scan, the voltage was raised linearly from 0.07 V rms to 12 V rms in 2 min for specific interaction and to 4 V rms for the physisorbed case. To carry out the tests in liquid, a 8 µL drop of PBS was maintained on the horizontally mounted sensor with attached SCPM or without. The sample compartment was closed and maintained under humid conditions to reduce evaporation of the drop at elevated amplitudes during the experiment. No significant evaporation was observed over one scan. A fresh drop was replaced before each new scan to ensure the same volume of liquid for all scans. 4. RESULTS AND DISCUSSION The results from physisorbed and specifically captured microbeads are presented in Figure 1. Figure 1a presents the 3f electrical response against oscillation amplitude of three successive scans from a sensor with 5.61 µm physisorbed beads and one scan from the same sensor but without beads. Clearly, the relative increase in the 3f response due to introduction of beads is significant in scan 1, indicating high sensitivity. Also, the response rises steadily at first and later drops and becomes unsteady; this characteristic is found to repeat over the next two scans with magnitude of the response decreasing in successive scans and becoming close to that from scan without beads toward the end of the third scan. This decrease can be explained by the phenomenon of beads diffusing on and desorbing from surface at higher amplitudes. This hypothesis is confirmed from the increase in quality factor of the sensor after the scans due to a decrease in losses at the interface from fewer beads in contact with the surface. This also demonstrates cleaning of particles bound via weaker interactions at high amplitudes of oscillation. One scan to 12 V rms (instead of to 4 V rms reported here) is sufficient to acoustically clean the surface of most nonspecifically bound particles. Figure 1b presents the 3f response of successive scans from a sensor, initially without beads and then with specifically captured 0.39 µm beads. Here, again, the relative increase in the 3f response

Figure 1. 3f response against oscillation amplitude from sensors in presence and absence of SCPM. (a) Successive scans with physisorbed beads and a scan without beads. (b) Successive scans with specifically captured beads compared to scans without beads.

due to introduction of beads is significant (given that the number of beads is only ∼42 000 or ∼1.3 ng), indicating high sensitivity. The striking difference with the characteristic response from sensor with physisorbed beads is noteworthy. The response with specifically captured beads is reproducible over successive scans unlike the case with physisorbed beads; also, the response here is steadily increasing, without any drop. Both these features indicate that no unbinding takes place at the surface in the range of scan. Also notable is the reproducibility of the response of scans without beads. This indicates that the contribution to anharmonicity from the crystal with the PBS drop alone is reproducible and insignificant compared to that from beads and can be used as the baseline to measure the deviation in 3f response caused by introduction of beads. Figure 2a presents the deviation in 3f response from the baseline response versus the oscillation amplitude due to introduction of different numbers of specifically bound 0.39 µm beads. This is referred to as the anharmonic detection (ADT) signal. The ADT signal rises steadily in all cases, and the shape of the signal is clearly different from the signal with physisorbed beads that can be extracted in a similar way from Figure 1a. It is this difference in signal shape that differentiates between a specific or high-affinity interaction and a nonspecific or low-affinity interaction. This provides selectivity in detection and can be used for affinity-based screening. The signal-to-noise (SNR) ratio observed for 1.3 ng of 0.39 µm beads (Figure 2a, red squares) indicates a detection capability of approximately 200 pg. The magnitude of the signal is found to be nearly proportional to the number of beads on the surface both for 0.39 µm (Figure 2a) and for 3.09 µm beads (Figure 2b). Hence, the ADT signal recorded after the screening out of nonspecifically bound species (confirmed from the reproducibility of successive scans) can potentially quantify specifically bound biological entities on the sensor, by

Figure 2. Shifts in 3f response from the baseline response due to specifically captured beads. Signals from experiments with a different number of beads are presented, and one of them is compared with a signal estimated from modeling for bead sizes of (a) 0.39 µm and (b) 3.09 µm. The magnitude of the signal is found to be proportional to the number of beads for both bead sizes.

comparison with the ADT signal from a known concentration of the same species, previously recorded in a library. Detailed methods for detection of surface-bound particles and the detection and screening of biomolecules are described in the Supporting Information, S1-I and -II. Interestingly, however, the shape of the signal with 3.09 µm is different from that with 0.39 µm beads although the same molecular linker is used in both the cases. This demonstrates selectivity of ADT toward particles with similar affinity of interaction to the receptor but of different sizes and mass. The quantitative model presented in Quantitative Modeling explains the basis of selectivity of ADT toward the interaction profile, mass and size of particles, and length of tether. The signals computed for the two sizes of beads from the model match quantitatively and qualitatively with those from experiments, as shown in Figure 2a,b. 5. QUANTITATIVE MODELING A biological entity attached to the sensor via molecular tether is modeled as a spring-mass system. Figure 3 shows this model along with the parameters involved. When the oscillator is driven by a pure sinusoidal signal of frequency f, which is typically its fundamental resonant frequency, forces act along the tether binding the particles to the oscillator surface. These modify the acoustic response of the oscillator, which is transduced into an electrical signal in the presence of a suitable transduction mechanism. For the TSM AT-cut quartz employed here, the transduction is piezoelectric and only the horizontal shear forces are transduced. Thus, only the horizontal component (Ftx) of the interaction force is considered for estimating the deviation in Analytical Chemistry, Vol. 83, No. 2, January 15, 2011

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¨ (t) ) Ftx - γX˙(t) - λ(X˙(t) - Vs(t)) ) mxX Ftx - (γ + λ)X˙(t) + λVs(t) ¨ myY(t) ) Fty - (γ + β)Y˙(t)

Figure 3. Spring-mass model of a biological entity bound to an oscillating sensor via molecular tether in liquid. The biological entity is modeled as a sphere, and the molecular tether as an equivalent nonlinear spring of length l0 composed of multiple (Nlinks) ligand-receptor linker-springs connected in parallel. The force along the tether is given byFl )-NlinksFs(s),whereFs(s)representsthemechanicalforce-extension characteristic of a linker-spring. The force perpendicular to tether due to its angular stiffness ka is modeled as Fa ) -kaNlinkscot (φ), where cot (φ) represents the impenetrable surface barrier.

electrical response. Any Fourier series harmonic component n in Ftx is given by

(2)

Here, mx and my are the effective masses of the particle (as derived in Supporting Information, S5); Vs(t) is the velocity of the surface; γ is the coefficient of Stokes’ viscous resistance from bulk liquid; λ and β are coefficients of viscous drag in the horizontal and vertical directions, respectively, at the particle-surface interface due to the liquid (as derived in Supporting Information, S6). F3F(a) can then be compared with the experimentally observed ADT signal to extract the mechanical force-extension characteristic Fs(s) (as detailed in Quantitative Modeling). It needs to be noted that Fs(s) is temperature dependent, and hence, the temperature needs to be controlled in the experiments for extraction of Fs(s). 6. EXTRACTION OF MECHANICAL FORCE-EXTENSION CHARACTERISTIC OF THE MOLECULAR LINKER The force-extension characteristic for a linker-spring is assumed as follows. Fs(s) ) [a1sinh(s/b1) + a2sinh3(s/b2)]exp[-(cs + d)n]

AF(n) )

ω π

-ωNlinks ) π

∫

T

0

∫

T

0

∫

ω T [Flcos(φ) + Fasin(φ)] × π 0 exp(jnωt)dt

Ftxexp(jnωt)dt )

(Fs(s) + ka)cos(φ)exp(jnωt)dt (1)

where time period T ) 1/f and angular frequency ω ) 2πf. The higher odd harmonic components are nonzero, and the third harmonic (3f) component is a significant proportion (∼1/3) of the first harmonic (1f) even for small amplitudes (i.e., φ ∼ π/2). Also, the symmetricity of the expression of Ftx indicates that only odd harmonics are generated (Supporting Information, S3). This explains the relative deviation in the 3f response observed in experiments due to attached microbeads. It is intuitive that the magnitude of AF(3) in eq 1, is influenced by the surface oscillation amplitude, a, which for the same drive parameters (voltage and frequency) depends on the mechanical transfer function of the resonator (including the quality factor) and the transduction factor and, hence, may vary from one sensor to the other. However, it is clear from eq 1 that for a given a and f, AF(3) depends on Fs(s) and ka (which are mechanical characteristics of the tether), extension s, and inclination φ (which depend on mass and size of the particle and nature and length of the tether). The dependence on these parameters is elaborated in Supporting Information, S4 with the aid of supplementary movies. Hence, for a given f, the deviation in 3f response, AF(3), expressed as a function of a, i.e., F3F(a), provides a signature for the particle-tether system. F3F(a) is obtained by solving the describing differential equations (eq 2) for the particle for Ftx(t), then deriving its third Fourier harmonic component using eq 1 and plotting versus a(t). 552

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(3) The form of this function is based on force versus tip-displacement results previously reported in AFM experiments.15,16 Making initial guesses for the coefficients of the above function, the differential equations in eq 2 are solved numerically using Wolfram Mathematica 7.0 for 1300 beads of 3.09 µm (values of other parameters reported in Supporting Information, S6). Ftx is then evaluated ¨ (t) + (γ + λ)X˙(t) - λVs(t). The 3f using eq 2 as Ftx ) mxX component is then computed by numerically integrating eq 1, multiplied by the appropriate force-to-charge conversion factor for an AT-cut quartz17 and by a factor for proximity to third overtone resonance, and then differentiated with respect to time to compute the change in the 3f current. This variation in 3f current plotted against oscillation amplitude is compared with that observed in the corresponding experiment and fitted to it by changing the coefficients of Fs(s) in eq 3 using trial and error. The final fit, which matches closely with the experiment (Figure 2b), is for the following values of the coefficients of Fs(s) in eq 3: a1 ) 840; b1 ) 20; a2 ) 1200; b2 ) 20; c ) 1/18; d ) 0.275; n ) 7. Figure 4 shows the resulting force-extension function Fs(s) of the linkerspring (dotted line) and the actual force-extension encountered for 3.09 µm (red line). The dip in the red line can be explained by possible initiation of unbinding in the ligandreceptor bond. The magnitude of the peak force (∼469 pN) is around three times that reported in AFM experiments by Lo et al.16 We attribute this to inaccurate estimation of the number (15) Kamruzzahan, A. S. M.; Ebner, A.; Wildling, L.; Kienberger, F.; Riener, C. K.; Hahn, C. D.; Pollheimer, P. D.; Winklehner, P.; Holzl, M.; Lackner, B.; Schorkl, D. M.; Hinterdorfer, P.; Gruber, H. J. Bioconjugate Chem. 2006, 17, 1473–1481. (16) Lo, Y. S.; Zhu, Y. J.; Beebe, T. P. Langmuir 2001, 17, 3741–3748. (17) Ward, R. W. The constants of alpha quartz. Proceedings of the 14th Piezoelectric Devices Conference and Exhibition; 1992; Vols. 2, pp 61-70.

Figure 4. Force-extension characteristic of a streptavidin-biotinPEG-thiol linker. This is obtained from the model after fitting the signal estimated from the model with that observed in the experiment. The highlighted parts show the actual force-extensions encountered by the linkers for 3.09 µm (red solid line) and 0.39 µm (green solid line) beads.

of ligand-receptor linker-springs (Nlinks ) 18, using estimations reported before14), due to insufficient knowledge on the concentration of receptor on the surface and the density of streptavidin on SCPM. However, appropriate design of the surface can eliminate these uncertainties. This method is described in detail in the Supporting Information, S1-III. The signal for 42 000 beads of 0.39 µm computed using this Fs(s) also fitted satisfactorily with that observed from the corresponding experiment (Figure 2a). The number of linkers that result in this fit is Nlinks ) 1, which closely matches with our theoretical estimation14 of Nlinks ) 2. Thus, apart from extracting the force-extension characteristic of the molecular linker, the model also enables quantitative estimation of the ADT signal. 7. METHOD FOR MEASURING ACTIVATION ENERGY Here, we present a method to extract the activation energy of a ligand-receptor interaction using ADT. Particles of appropriate size and density are attached to the sensor surface using the ligand-receptor linker, whose activation energy of interaction is to be determined. The receptor concentration on the surface is controlled such that each particle is attached via one ligand-receptor linker. By appropriate surface design, diffusion of the particle-linker complex on the sensor surface is prevented at high amplitudes. The surface design also ensures that the only place of unbinding is the ligand-receptor bond. An ADT experiment is then carried over a wide amplitude range, maintaining a constant known temperature in the physiological range, to determine the voltage at which unbinding initiates. The initiation of unbinding is confirmed by the dip in the ADT signal at high amplitude in a given scan followed by a decrease in magnitude of the signal in the next scan. The sensor is then driven at constant amplitude of oscillation at this voltage at the fundamental resonant frequency for a time necessary to drop the 3f response (the deviation) by a factor of around two. Since this is proportional to the number of particles, the 3f response (deviation) versus time provides a snapshot of the number of attached particles over time (Figure

Figure 5. Determination of activation energy. (a) 3f signals (deviation) against time from two fixed amplitude scan experiments at slightly different voltages. (b) Linear regression fits of the logarithm of the 3f signals (deviation) against time.

5a). Thus, this gives a measure of the rate of the unbinding reaction. The gradient (m1) of the logarithm of 3f response versus time is obtained numerically by linear regression fitting (Figure 5b). This is repeated at slightly elevated amplitude of oscillation (e.g., 5% higher), and the gradient (m2) is noted. The activation energy Ea and the parameter δ* (the decrease in depth of potential well relative to kBT caused by application of force) can then be obtained by solving the following set of two equations. m1 ) -(kBT/h)e-Ea/(kBT)[BesselI(0, δ*) + StruveL(0, δ*)] m2 ) -(kBT/h)e-Ea/(kBT)[BesselI(0, δ*β) + StruveL(0, δ*β)] Here, kB is the Boltzmann constant, T is the absolute temperature, h is the Planck’s constant, and β′ is the ratio of force applied (determined as explained in Supporting Information, S1-IV). BesselI is the modified Bessel function of the first kind, and StruveL is the modified Struve function. Since δ* ) Fa*/(kBT), where F is the force applied along the linker (which is determined from the force-extension characteristic) and a* is the internuclear distance between the ground and transition states projected on the force vector,18 it is also possible to estimate the value of a*. Ea and a* provide important information on the potential energy profile of the interaction. The derivation is presented in the Supporting Information, S1-IV. Besides, the gradients m1 and m2 also denote the rate of unbinding reaction of the molecular complex used as the linker, at the respective amplitudes of oscillation, at the temperature maintained in the experiment. However, it must be noted that the reaction kinetics of the complex near hard surface contacts could be different from near-solution kinetics. 8. CONCLUSION AND OUTLOOK Here, we present for the first time the anharmonic detection technique (ADT), which enables detection of surface-bound (18) Hyeon, C.; Thirumalai, D. J. Phys.: Condens. Matter 2007, 19, 12.

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particles and affinity-based screening of biomolecules, thus providing a feature of selectivity in the detection technique in addition to the efficacy of the receptor. With its ability to measure forces within and between molecules and extract activation energy through an entirely electronic method, ADT sets the stage for rapid, large-scale, and cost-effective identification of interaction networks and pathways, molecular force spectroscopy, and characterization. The initial results demonstrate high sensitivity, which could be further enhanced by studying the nature of dependence of the ADT signal on drive and particle parameters, building upon the model developed in this work (Supporting Information, S4). Thus, ADT as a platform technology can play an important role in understanding of fundamental biological events and find applications in pointof-care diagnostics, drug discovery, food and environment safety, and biosecurity.

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9. METHODS The methods for detection of biological entity, screening of biomolecules, and determination of molecular force-extension characteristics and activation energy are presented in detail in the Supporting Information. ACKNOWLEDGMENT This project was funded in part by the Engineering and Physical Sciences Research Council and the Cambridge Commonwealth Trust. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review August 27, 2010. Accepted November 19, 2010. AC102261Q