WO2012073011A2

WO2012073011A2 - Method and apparatus for characterising molecules

Info

Publication number: WO2012073011A2
Application number: PCT/GB2011/052349
Authority: WO
Inventors: Sourav Ghosh; Victor Ostanin; Ashwin Seshia
Original assignee: Cambridge Enterprise Ltd
Current assignee: Cambridge Enterprise Ltd
Priority date: 2010-11-30
Filing date: 2011-11-29
Publication date: 2012-06-07
Anticipated expiration: 2013-05-30
Also published as: WO2012073011A3; GB201020237D0

Abstract

The present invention relates to a method and apparatus for characterising molecules. In particular, the present invention relates to a method and apparatus of determining force-extension characteristics of a molecule, a method and apparatus of determining an activation energy of a molecule or bound molecular pair, and a method and apparatus of determining a rates of binding and of unbinding of a molecule. In each of the methods, a transduced signal from an oscillating sensor is used to determine the molecular characteristics of interest. The output of the sensor is dependent upon interactions of the molecules of interest attached to microbeads, which are bound to the surface of the sensor through molecular bindings such as ligand- receptor bindings or covalent bonds.

Description

Method and Apparatus for Characterising Molecules

FIELD OF THE INVENTION The present invention relates to a method and apparatus for characterising molecules. In particular, the present invention relates to a method and apparatus of determining force-extension characteristics of a molecule, a method and apparatus of determining an activation energy of a molecule or bound molecular pair, and a method and apparatus of determining a rates of binding and of unbinding of a molecule.

BACKGROUND OF THE INVENTION

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 interaction affinity. Receptor-based detection is often challenged by non-specific interactions, which necessitates that the detection technique is able to differentiate between specific and non-specific interactions.

However, most existing techniques, labelled or non-labelled, are unable to detect true affinity of interaction and are often plagued with the issue of false positive response. Current force spectroscopic techniques, such as optical tweezers, biomembrane force probe and atomic force microscopy, can measure the activation energy of interaction, but 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.

We have therefore appreciated the need for an alternative method and apparatus of characterising molecules, for example to determine a measure of the affinity of interaction, quantified by the activation energy, force-extension characteristics, and rates of binding and of unbinding. Background prior art can be found in WO2010/096439, US2010/08701 1 , EP1573329 and EP0295965.

SUMMARY OF THE INVENTION

The invention is set out in the independent claims.

In embodiments the present invention therefore provides a method of determining force-extension characteristics of a molecule, the method comprising: exposing a prepared surface of a sensor to a plurality of microbeads for a binding period, each microbead being functionalised with a molecule of interest, said sensor comprising a transducer that oscillates in response to a drive signal applied to said sensor, said prepared surface of said sensor comprising a binding partner for binding with said molecule of interest, and said binding period being sufficient to allow binding between said molecule of interest and said binding partner; applying a driving signal to said sensor for a first period, said driving signal comprising an alternating voltage having a frequency and amplitude sufficient to cause said sensor to oscillate; recording a transduced signal from said sensor over said first period, said transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said driving signal, said transduced signal resulting from oscillation of said sensor; comparing said transduced signal with a model defining force-extension characteristics of a molecule; adjusting parameters of said force-extension characteristics in said model to substantially fit said model with said transduced signal; and determining force- extension characteristics of said molecule of interest from said adjusted parameters that result in said fit.

The method described herein 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 true affinity- based screening and provides an additional level of selectivity in biosensing applications. Also unlike existing force spectroscopic techniques, the method 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. The technique is also applicable to a small biological entity and a cell, virus, bacteria, spore (or part thereof) and the like.

In embodiments, said applying a driving signal comprises ramping an amplitude of said driving signal over said first period between a minimum amplitude sufficient to cause said sensor to oscillate, and a maximum amplitude sufficient to cause detachment of said molecules bound with said binding partner, and wherein said frequency of said driving signal is substantially a resonant frequency of said sensor. Preferably, said amplitude of said driving signal is ramped from said minimum amplitude to said maximum amplitude and/or from said maximum amplitude to said minimum amplitude.

Alternatively, said applying a driving signal comprises ramping said frequency of said driving signal over said first period between a minimum frequency and a maximum frequency (changing the driving frequency effectively changing the amplitude of the sensor response). The minimum and maximum frequencies are sufficient to cause said sensor to oscillate. The amplitude of said driving signal may be less than or equal to a maximum amplitude sufficient to cause detachment of said molecules bound with said binding partner, so that the molecules do not become detached during this process, or greater than or equal to this maximum amplitude, for example to probe the force- extension curve beyond a point of a conformational change or unbinding event of the molecule. Preferably the frequency of the driving signal is ramped from said minimum frequency to said maximum frequency and/or from said maximum frequency to said minimum frequency. In embodiments, said transduced signal is recorded against a magnitude of oscillation of said sensor. Preferably, said magnitude of oscillation is determined by using a voltage and a current of said transduced signal at said driving frequency to calculate said magnitude of oscillation of said sensor. In some embodiments, said transduced signal is recorded against a measured current of said transduced signal at said driving frequency.

In preferred embodiments of the inventions, the method comprises generating an Anharmonic Detection (ADT) signal from said transduced signal, said ADT signal representing a deviation of said recorded transduced signal from a baseline transduced signal, and said baseline transduced signal representing a phase and/or amplitude response at an odd harmonic frequency of a driving signal for a sensor absent said microbeads functionalised with said molecule of interest, said generating said ADT signal comprising: comparing said baseline signal with said transduced signal to identify a deviation between said baseline signal and said transduced signal, and wherein said comparing said transduced signal with a model comprises comparing said ADT signal with said model defining a force-extension characteristics of a molecule.

Preferably in this embodiment, said baseline transduced signal is obtained prior to said exposing step and wherein said baseline signal is obtained by: applying a driving signal to said sensor for a first period, said driving signal comprising an alternating voltage having a frequency and amplitude sufficient to cause said sensor to oscillate, and said sensor being devoid of microbeads functionalised with said molecules of interest; recording a baseline transduced signal from said sensor over said first period, said baseline transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said driving signal, said baseline transduced signal resulting from oscillation of said sensor. Preferably, said applying a driving signal comprises ramping an amplitude of said driving signal over said first period between a minimum amplitude sufficient to cause said sensor to oscillate, and a baseline maximum amplitude, wherein said frequency of said driving signal is substantially a resonant frequency of said sensor and wherein said baseline maximum amplitude is greater than an amplitude sufficient to cause detachment of said molecules when bound with said binding partner. In one embodiment, said amplitude of said driving signal is ramped from said minimum amplitude to said maximum amplitude and/or from said maximum amplitude to said minimum amplitude. Alternatively, said applying a driving signal comprises ramping said frequency of said driving signal over said first period between a minimum frequency and a maximum frequency. Again the minimum and maximum frequencies are sufficient to cause said sensor to oscillate, and the amplitude of the driving signal may be less than or greater than an amplitude sufficient to cause detachment of said molecules when bound with said binding partner. In the alternative embodiment, said frequency of said driving signal is ramped from said minimum frequency to said maximum frequency and/or from said maximum frequency to said minimum frequency. In embodiments using the baseline transduced signal, said baseline transduced signal is recorded against a magnitude of oscillation of said sensor. Preferably, said magnitude of oscillation is determined by using a voltage and a current of said baseline transduced signal at said driving frequency to calculate said magnitude of oscillation of said sensor.

In other embodiments using the baseline transduced signal, said baseline transduced signal is recorded against a measured current of said baseline transduced signal at said driving frequency. In some embodiments, the method comprises performing a cleaning step prior to said applying a drive signal to remove molecules of interest not bound to said binding partners and unwanted molecules, said cleaning step comprising washing said sensor with a buffer solution. Furthermore, the above methods method may comprise performing a cleaning step prior to said applying a drive signal to remove molecules of interest not bound to said binding partners and unwanted molecules, said cleaning step comprising: applying said driving signal to said sensor; recording said amplitude and/or phase of said transduced signal at substantially an odd harmonic frequency of said frequency of said driving signal; and comparing data derived from said recorded transducer signal from each applying step with data derived from said recorded transducer signal from a previous applying step until said data is substantially reproduced from one applying step to the next applying step, thereby indicating that molecules of interest not bound to said binding partners and unwanted molecules are no longer on said surface.

In some embodiments, the method comprises counting said microbeads prior to said applying said driving signal. In further embodiments, said odd harmonic is a third or fifth harmonic of said driving frequency. In yet further embodiments, said binding between said molecule of interest and said binding partner is a ligand-receptor binding, or a covalent bond. In some embodiments, said sensor is in a liquid, gas, air or a vacuum. In embodiments the present invention also provides apparatus for determining force- extension characteristics of a molecule, comprising: a sensor comprising a transducer that oscillates in response to a drive signal applied to said sensor, said sensor comprising a prepared surface comprising a binding partner for binding with molecules of interest, said molecules of interest being supported on a plurality of microbeads; a driving signal generator for generating a driving signal comprising an alternating voltage having a frequency and amplitude sufficient to cause said sensor to oscillate; and a controller operatively coupled to said sensor and said driving signal generator, said controller being configured to: controllably apply said driving signal from said driving signal generator to said sensor; receive and record an amplitude and/or phase of a transduced signal at substantially an odd harmonic frequency of said frequency of said driving signal, said transduced signal resulting from an oscillation of said transducer; compare said transduced signal with a model defining force-extension characteristics of a molecule; adjust parameters of said force-extension characteristics in said model to substantially fit said model with said transduced signal; and determine force-extension characteristics of said molecule of interest from said adjusted parameters that result in said fit. In embodiments of the apparatus, said controller is configured to: generate an Anharmonic Detection (ADT) signal from said transduced signal, said ADT signal representing a deviation of said recorded transduced signal from a baseline transduced signal, and said baseline transduced signal representing a phase and/or amplitude response at an odd harmonic frequency of a driving signal for a sensor absent said microbeads functionalised with said molecule of interest, said controller generating said ADT signal by: comparing said baseline signal with said transduced signal to identify a deviation between said baseline signal and said transduced signal, wherein said compare said transduced signal with a model comprises comparing said ADT signal with said model defining force-extension characteristics of a molecule.

Preferably, the baseline transduced signal is obtained prior to said exposing step and wherein said controller is adapted to obtain said baseline signal by: controllably apply a driving signal to said sensor for a first period, said driving signal comprising an alternating voltage having a frequency and amplitude sufficient to cause said sensor to oscillate, and said sensor being devoid of microbeads functionalised with said molecules of interest; receive and record a baseline transduced signal from said sensor over said first period, said baseline transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said driving signal, said baseline transduced signal resulting from oscillation of said sensor.

In embodiments the present invention also provides a method of determining an activation energy of a molecule or bound molecular pair, the method comprising: exposing a prepared surface of a sensor to a plurality of microbeads for a binding period, each microbead being functionalised with a molecule of interest, said sensor comprising a transducer that oscillates in response to a drive signal applied to said sensor, said prepared surface of said sensor comprising a binding partner for binding with said molecule of interest, and said binding period being sufficient to allow binding between said molecule of interest and said binding partner; determining a first rate of unbinding of (a conformational change of) a molecule or bound molecular pair of interest over a first period; determining a second rate of unbinding of (a conformational change of) a molecule or bound molecular pair of interest over a second period; optionally determining a log of a first slope of said first rate of unbinding against time and a log of a second slope of said second rate of unbinding against time; and determining an activation energy of said molecule or bound molecular pair using (optionally said determined log of) said first and second slopes, wherein said second rate of unbinding is greater than said first rate of unbinding.

The skilled person will appreciate that in this method, and also in the corresponding apparatus described below, when referring to determining a log of a slope, what is meant is determining the log (value) represented by the slope: In embodiments a log is taken on the concentration equation, which is an exponential equation. The resulting equation is a straight line and activation energy is evaluated using the slope of this line.

The activation energy of the molecule may be, for example, an activation energy of a conformational change of the molecule such as an uncoiling of the molecule. The activation energy of the bound molecular pair may be, for example, the activation energy for unbinding a pair of molecules, one of which may be attached to a functionalised surface. The skilled person will be aware of many useful bound molecular pairs to which the technique may be applied including, but not limited to: an antibody-antigen pair, a DNA-cDNA pair, a covalently bound pair, an aptamer-antigen pair, and the like. Optionally a biological unity may be interposed between a microbead and the surface; the molecule of interest may be on a surface of the biological entity itself. In embodiments of this method, determining a first rate of unbinding of (a conformational change of) a molecule or bound molecular pair comprises: applying a first driving signal to said sensor for said first period, said first driving signal comprising an alternating voltage having a frequency and a first amplitude sufficient to cause said sensor to oscillate at a first mechanical oscillation amplitude, said first mechanical oscillation amplitude being sufficient to cause detachment of said bound molecules from said binding partner; recording a first transduced signal from said sensor over said first period, said first transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said driving signal, said first transduced signal resulting from oscillation of said sensor in response to said first driving signal; determining said first rate of unbinding of a molecule or bound molecular pair from said recorded first transduced signal.

In further embodiments of this method, determining a second rate of unbinding of (a conformational change of) a molecule or bound molecular pair comprises: applying a second driving signal to said sensor for said second period, said second driving signal comprising an alternating voltage having a frequency and a second amplitude sufficient to cause said sensor to oscillate at a second mechanical oscillation amplitude, said second mechanical oscillation amplitude being sufficient to cause detachment of said bound molecules from said binding partner; recording a second transduced signal from said sensor over said second period, said second transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said driving signal, said second transduced signal resulting from oscillation of said sensor in response to said second driving signal; determining said second rate of unbinding of a molecule or bound molecular pair from said recorded second transduced signal.

In preferred embodiments of this method, this method comprise generating a first Anharmonic Detection (ADT) signal from said first transduced signal, said first ADT signal representing a deviation of said recorded first transduced signal from a first baseline transduced signal, and said first baseline transduced signal representing a phase and/or amplitude response at an odd harmonic frequency of said first driving signal for a sensor absent said microbeads functionalised with said molecule of interest, said generating said first ADT signal comprising: comparing said first baseline signal with said first transduced signal to identify a deviation between said first baseline signal and said first transduced signal, and wherein said determining said first rate of unbinding of a molecule or bound molecular pair from said recorded first transduced signal comprises determining said first rate of unbinding of a molecule or bound molecular pair from said first ADT signal.

Preferably, the method further comprise generating a second Anharmonic Detection (ADT) signal from said second transduced signal, said second ADT signal representing a deviation of said recorded second transduced signal from a second baseline transduced signal, and said second baseline transduced signal representing a phase and/or amplitude response at an odd harmonic frequency of said second driving signal for a sensor absent said microbeads functionalised with said molecule of interest, said generating said second ADT signal comprising: comparing said second baseline signal with said second transduced signal to identify a deviation between said second baseline signal and said second transduced signal, and wherein said determining said second rate of unbinding of a molecule or bound molecular pair from said recorded second transduced signal comprises determining said second rate of unbinding of a molecule or bound molecular pair from said second ADT signal.

In embodiments of the above method using the ADT signal, said first or second baseline transduced signal is obtained prior to said exposing step and wherein said first or second baseline signal is obtained by: applying said first or second driving signals to said sensor, said sensor being devoid of microbeads functionalised with said molecules of interest; recording a respective first or second baseline transduced signal from said sensor, said first or second baseline transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said first or second driving signal, said first or second baseline transduced signal resulting from oscillation of said sensor.

In embodiments of this method, the method comprises performing a cleaning step prior to said determining a first and second rate of unbinding to remove molecules of interest not bound to said binding partners and unwanted molecules, comprising washing said sensor with a buffer solution. In other embodiments of this method, the method comprises performing a cleaning step prior to determining a first and second rate of unbinding to remove molecules of interest not bound to said binding partners and unwanted molecules, said cleaning step comprising: applying a driving signal to said sensor; recording said amplitude and/or phase of said transduced signal at substantially an odd harmonic frequency of said frequency of said driving signal; and comparing data derived from said recorded transducer signal from each applying step with data derived from said recorded transducer signal from a previous applying step until said data is substantially reproduced from one applying step to the next applying step, thereby indicating that molecules of interest not bound to said binding partners and unwanted molecules are no longer on said surface.

In other embodiments of this method, the method further comprises: determining a third rate of unbinding of a molecule or bound molecular pair of interest over a third period; determining a log of a third slope of said third rate of unbinding against time, wherein said determining an activation energy of said molecule or bound molecular pair comprises using said determined log of said first, second and third slopes, wherein said third rate of unbinding is greater than said second rate of unbinding.

In embodiments three activation energies are derived from a combination of three equations defined by respective said first, second and third slopes, and averaged. In embodiments, said odd harmonic is a third or fifth harmonic of said driving frequency. In some embodiments, said binding is a ligand-receptor binding or a covalent bond. In embodiments, said sensor is in a liquid, gas, air or a vacuum.

In embodiments the present invention also provides apparatus for determining an activation energy of (a conformational change of) a molecule or bound molecular pair, comprising: a sensor comprising a transducer that oscillates in response to a drive signal applied to said sensor, said sensor comprising a prepared surface comprising a binding partner for binding with molecules of interest, said molecules of interest being supported on a plurality of microbeads; a driving signal generator for generating a driving signal comprising an alternating voltage; and a controller operatively coupled to said sensor and said driving signal generator, said controller being configured to: controllably apply said driving signal to said sensor to determine a first rate of unbinding of a molecule or bound molecular pair of interest over a first period; controllably apply said driving signal to said sensor to determine a second rate of unbinding of a molecule or bound molecular pair of interest over a second period; optionally determine a log of a first slope of said first rate of unbinding against time and a log of a second slope of said second rate of unbinding against time; and determine an activation energy of said molecule or bound molecular pair using (optionally said determined log of) said first and second slopes, wherein said second rate of unbinding is greater than said first rate of unbinding. In embodiments of this apparatus, said controller determines said first rate of unbinding of a molecule or bound molecular pair by: controllably applying a first driving signal to said sensor for said first period, said first driving signal comprising an alternating voltage having a frequency and a first amplitude sufficient to cause said sensor to oscillate at a first mechanical oscillation amplitude, said first mechanical oscillation amplitude being sufficient to cause detachment of said bound molecules from said binding partner; receiving and recording a first transduced signal from said sensor over said first period, said first transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said driving signal, said first transduced signal resulting from oscillation of said sensor in response to said first driving signal; and determining said first rate of unbinding of a molecule or bound molecular pair from said recorded first transduced signal.

In embodiments of this apparatus, said controller determines said second rate of unbinding of a molecule or bound molecular pair by: controllably applying a second driving signal to said sensor for said second period, said second driving signal comprising an alternating voltage having a frequency and a second amplitude sufficient to cause said sensor to oscillate at a second mechanical oscillation amplitude, said second mechanical oscillation amplitude being sufficient to cause detachment of said bound molecules from said binding partner; receiving and recording a second transduced signal from said sensor over said second period, said second transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said driving signal, said second transduced signal resulting from oscillation of said sensor in response to said second driving signal; and determining said second rate of unbinding of a molecule or bound molecular pair from said recorded second transduced signal. In some embodiments of this apparatus, the controller is configured to generate a first Anharmonic Detection (ADT) signal from said first transduced signal, said first ADT signal representing a deviation of said recorded first transduced signal from a first baseline transduced signal, and said first baseline transduced signal representing a phase and/or amplitude response at an odd harmonic frequency of said first driving signal for a sensor absent said microbeads functionalised with said molecule of interest, said controller being configured to generate said first ADT signal by: comparing said first baseline signal with said first transduced signal to identify a deviation between said first baseline signal and said first transduced signal, wherein said determining said first rate of unbinding of a molecule or bound molecular pair from said recorded first transduced signal comprises determining said first rate of unbinding of a molecule or bound molecular pair from said first ADT signal. Preferably, the controller is configured to generate a second Anharmonic Detection (ADT) signal from said second transduced signal, said second ADT signal representing a deviation of said recorded second transduced signal from a second baseline transduced signal, and said second baseline transduced signal representing a phase and/or amplitude response at an odd harmonic frequency of said second driving signal for a sensor absent said microbeads functionalised with said molecule of interest, said controller being configured to generate said second ADT signal by: comparing said second baseline signal with said second transduced signal to identify a deviation between said second baseline signal and said second transduced signal, wherein said determining said second rate of unbinding of a molecule or bound molecular pair from said recorded second transduced signal comprises determining said second rate of unbinding of a molecule or bound molecular pair from said second ADT signal.

In embodiments of the apparatus using the ADT signal, said first or second baseline transduced signal is obtained prior to exposing of said molecules to said sensor and wherein said controller is configured to obtain said first or second baseline signals by: controllably applying said first or second driving signals to said sensor, said sensor being devoid of microbeads functionalised with said molecules of interest; receiving and recording a respective first or second baseline transduced signal from said sensor, said first or second baseline transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said first or second driving signal, said first or second baseline transduced signal resulting from oscillation of said sensor.

In embodiments the present invention also provides a method of determining a rate of binding of a molecule, said method comprising: exposing a prepared surface of a sensor to a substantially constant flow of a plurality of microbeads for a binding period, each microbead being functionalised with a molecule of interest, said sensor comprising a transducer that oscillates in response to a drive signal applied to said sensor, said prepared surface of said sensor comprising a binding partner for binding with said molecule of interest, and said binding period being sufficient to allow binding between said molecule of interest and said binding partner; applying a driving signal to said sensor for a first period, said driving signal comprising an alternating voltage having a frequency and amplitude sufficient to cause said sensor to oscillate at a first mechanical oscillation amplitude, said first mechanical oscillation amplitude being below a mechanical oscillation amplitude sufficient to cause unbinding of the binding molecules; recording a transduced signal from said sensor over said first period, said transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said driving signal, said transduced signal resulting from oscillation of said sensor; changing an amplitude of said driving signal such that for a second period said driving signal has an adjusted amplitude, in particular removing said driving signal for a second period, wherein said second period is longer than said first period; repeating said applying said driving signal to said sensor for said first period, said recording said transduced signal from said sensor over said first period and said removing said driving signal for said second period over said binding period; comparing said recorded transduced signals from said repeated recording steps to determine a deviation between the recorded transduced signals over said binding period, said deviation defining a rate of binding of said molecule. In embodiments the comparing to determine the deviation comprises (optionally subtracting a baseline and then) fitting a curve, in embodiments an exponential curve, to the recorded signals to determine the rate of binding.

In embodiments it is not necessary to change the amplitude of the driving signal - repeated measurements monitoring the progress of the binding over time are sufficient. Conveniently but not essentially successive measurements may be performed over substantially the same periods (intervals) of time. In embodiments the driving is continuous and the, for example 3fo, signal may also be recorded continuously.

In the second period the driving signal amplitude may be removed, or decreased, or increased. Removing the driving allows the binding to happen without hindrance, but in some situations the oscillation may assist the binding.

In embodiments of this invention, the method comprises: applying said driving signal for said first period before said exposing step; and recording said transduced signal from said sensor over said first period to define a baseline transduced signal, said baseline transduced signal representing a phase and/or amplitude response at an odd harmonic frequency of a driving signal for a sensor absent said microbeads functionalised with said molecule of interest. In some embodiments, said comparing step comprises determining a gradient logarithm of said deviation against time over said binding period.

In other embodiments, said magnitude of mechanical oscillation of said sensor is determined by using a voltage and a current of said transduced signal at said driving frequency to calculate said magnitude of oscillation of said sensor. In embodiments, said odd harmonic is a third or fifth harmonic of said driving frequency. In embodiments, said binding between said molecule of interest and said binding partner is a ligand-receptor binding, or a covalent bond. In further embodiments, said sensor is in a liquid, gas, air or a vacuum.

In embodiments the present invention also provides apparatus for determining a rate of binding of a molecule, comprising: a sensor comprising a transducer that oscillates in response to a drive signal applied to said sensor, said sensor comprising a prepared surface comprising a binding partner for binding with molecules of interest; a driving signal generator for generating a driving signal comprising an alternating voltage having a frequency and an amplitude sufficient to cause said sensor to oscillate; and a controller operatively coupled to said sensor and said driving signal generator, said controller being configured to: controllably expose said prepared surface of said sensor to a substantially constant flow of a plurality of microbeads for a binding period, each microbead being functionalised with a molecule of interest, said binding period being sufficient to allow binding between said molecule of interest and said binding partner; controllably apply a driving signal to said sensor for a first period, said driving signal comprising an alternating voltage having a frequency and amplitude sufficient to cause said sensor to oscillate at a first mechanical oscillation amplitude, said first mechanical oscillation amplitude being below an oscillation amplitude sufficient to cause unbinding of the binding molecules; receive and record a transduced signal from said sensor over said first period, said transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said driving signal, said transduced signal resulting from oscillation of said sensor; change an amplitude of said driving signal such that for a second period said driving signal has an adjusted amplitude, in particular remove said driving signal for a second period; wherein said second is longer than said first period; repeating said controllably applying said driving signal to said sensor for said first period, said receive and record said transduced signal from said sensor over said first period and said remove said driving signal for said second period over said binding period; compare said recorded transduced signals from said repeated recording to determine a deviation between the recorded transduced signals over said binding period, said deviation defining a rate of binding of said molecule.

In embodiments of this apparatus, the controller is configures to: controllably apply said driving signal for said first period before said sensor is controllably exposed to said flow of said plurality of microbeads; and receive and record said transduced signal from said sensor over said first period to define a baseline transduced signal, said baseline transduced signal representing a phase and/or amplitude response at an odd harmonic frequency of a driving signal for a sensor absent said microbeads functionalised with said molecule of interest.

In embodiments, said controller compares said recorded transduced signals to determine a deviation between the recorded transduced signals comprises determining a gradient logarithm of said deviation against time over said binding period.

In some embodiments, said magnitude of mechanical oscillation of said sensor is determined by using a voltage and a current of said transduced signal at said driving frequency to calculate said magnitude of oscillation of said sensor. In embodiments, said odd harmonic is a third or fifth harmonic of said driving frequency. In embodiments, said binding between said molecule of interest and said binding partner is a ligand-receptor binding, or a covalent bond.

In the above aspects and embodiments of the invention the sensor may operate in a liquid, gas, air or in a vacuum. In the above aspects and embodiments of the invention the molecule/entity of interest may either be on the functionalized surface or on the microbead.

In the above aspects and embodiments of the invention the microbeads may be replaced by biological entities such as cells, spores or viruses, or parts thereof, and the like. The technique may be used (with or without microbeads) to classify biological entities, for example for further analysis and or treatment.

LIST OF FIGURES

Embodiments of the present invention will now be described, by way of example only, and with reference to the accompanying figures, in which:

Figure 1 shows an example system used for characterising molecules of interest;

Figure 2 shows a model of a biological entity attached to a sensor via molecular tether; and

Figures 3 to 5 show experimental data.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The acoustic response of conventional mechanical oscillators, such as a piezoelectric crystal, is predominantly harmonic at modest amplitudes. However, we have observed from the electrical response that significant motional anharmonicity (non-linearity) is introduced in presence of an attached analyte. In brief, the present invention uses changes in the electrical response of an oscillator, caused by the presence of molecules of interest, to determine the molecular properties of the molecules of interest. The molecules are supported on microbeads and are attached to the surface of the oscillator via molecular tethers comprised of specific receptors for the molecules. 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 label-free screening of biomolecules based on affinity and provides an additional level of selectivity in receptor-based detection that is often associated with non-specific interactions.

The Anharmonic Detection Technique (ADT) described below 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 known that nonlinearity in mechanical stresses can be primarily dominated by interactions at the contact interface. Contact acoustic nonlinearity is thus widely used to characterize material defects. This fundamental principle is the basis of ADT.

We observed that the response of a TSM AT-cut quartz crystal resonator, oscillating in- plane, becomes significantly nonlinear (anharmonic) in presence of surface-bound streptavidin-coated polystyrene microbeads (SCPM) even at modest oscillation amplitudes, both in air and 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 (/) 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.

Initial experiments indicate a detection capability of more than one order of magnitude better than existing Quartz Crystal Microbalance (QCM) and Surface Plasmon Resonance (SPR), which are notable non-labelled detection techniques. Most remarkably, for a given drive frequency (/), 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 characteristics 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. Figure 1 shows an example system used for determining the characteristics of molecules.

Generator 1 with controllable frequency and amplitude (e.g. Agilent 33220A) produces a sinusoidal signal at its output (e.g. around 1 5 MHz, typically 14.3MHz, in this embodiment). The signal is amplified by power amplifier 2 to increase the voltage amplitude to several volts (e.g. 1 2 volts). The signal is then filtered to remove higher harmonics by Low Pass Filter LPF 3 with cut-off frequency above the driving frequency, but well below the 3rd harmonic of 45MHz (e.g. cut-off 20 MHz). The filtered signal is fed to one electrode of a sensor 4 (a transducer e.g. a quartz crystal with fundamental resonance 1 5 MHz). A second electrode of transducer 4 produces electric current at all frequencies (including harmonics) that is fed into the input of a High Pass Filter 5 that has a pass frequency band starting close to the third harmonic (e.g. 35 MHz). The filtered signal is fed into the input of a general purpose receiver 6 (e.g. Stanford Research Systems SR844 Lock-In Amplifier), which is tuned to the desired harmonic frequencies (e.g. 3f or third harmonic of the driving frequency, or 5f or the fifth harmonic of the driving frequency f) and records the in-phase and quadrature (vector) components at the desired harmonic frequency. A passive frequency tripler was used to generate the reference signal for the lock-in (not shown in the figure). Additionally, two quadrature receivers were employed to detect the 1 / (fundamental) voltage and the quartz output current (not shown). The reference clock output from generator 1 synchronous with the main output also optionally drives the Clock input of receiver 6 to assist synchronisation of the generator and receiver.

After preparation of sensor surface (discussed below) the generator 1 is then controlled (e.g. by computer 7) to make varying drive amplitude via control interface (e.g. Ethernet). The amplitude (and preferably phase) of the harmonic content of the transducer current is digitised by receiver 6 and transferred to computer via control interface (e.g. GPIB - IEEE488 for receiver). The obtained data are further processed to deliver measurement results.

The sensor 4, comprises a disc or platform or surface of suitable dimension and shape, which can be actuated by an electrical input to oscillate at the desired frequency and amplitude, and whose oscillatory response can be transduced into electrical output. An example transducer is AT-cut TSM quartz.

Microbeads of suitable material and dimension are functionalized with molecules of the compound of interest. The choice of material and dimension is to ensure that the microbeads are visible under an optical or fluorescent microscope and that suitable signal to noise ratio is obtained.

The sensor surface is functionalized with a suitable receptor or binding partner for the molecule. This binding can be a ligand-receptor binding (e.g. antigen-antibody) or can be a covalent binding (e.g. through EDC-NHS chemistry) depending on the molecule in question. Thus the molecule is sandwiched between the microbead and the sensor surface. The chain of molecules between the microbead and the surface is referred to as the linker.

In case the binding in the linker is a ligand-receptor one, the microbeads can be functionalized with either the ligand or the receptor (where the molecule is a ligand- receptor complex) or either the molecule or the receptor (e.g. where the molecule is a protein and the receptor is an antibody). The sensor surface is then functionalized with the complementary binding partner. The sensor surface comprising the receptor is designed such that any diffusion of the microbead-linker complex on the surface at high sensor oscillation amplitude is prevented. Hence, the only unbinding that can occur at high oscillation amplitudes of the sensor is the irreversible unfolding of the molecule (in case of a covalently bonded attachment) or unbinding of the ligand-receptor bond.

The concentration of the molecules on the microbead and the concentration of the receptor on the surface are maintained such that the microbead is attached to the surface via a single molecular linker. The number of microbeads to be employed is determined by optimizing between the decrease in quality factor (and hence the decrease in resolution or signal-to-noise ratio (SNR)) and the increase in signal with increase in number of beads. This number does not vary with the molecule being characterized but with the material and size of the microbeads.

Determination of Baseline Signal

Whilst the various molecular characteristics may be determined using the transduced harmonic (e.g. 3F or 5F etc) output of the sensor 4 for high quality factor oscillatory systems, it has been found that for low quality factor systems the measurement is more accurate when the characteristics are determined using data from an Anharmonic Detection (ADT) signal, which is calculated from the sensor output.

In order to calculate the ADT signal, a baseline signal, representing the sensor's output absent any microbeads, first needs to be established. Once the baseline signal has been achieved, the ADT signal may be calculated as the difference between the transduced signal in the presence of microbeads, and the baseline signal. The difference in the two signals arises due to the presence of the microbeads (and molecular interactions).

The baseline signal is determined as follows:

1 . A drive signal is applied to the sensor without microbeads and the electrical response is measured at f (at the drive frequency) and at 3f, i.e. three times the drive frequency (or any higher odd multiple). The 3f response against the λ ί response is recorded, and/or the 3f response against the oscillation amplitude is recorded.

The oscillation of amplitude can be determined by one of the following two methods: a. Multiplying the calibrated voltage across the sensor by a factor 1 .4pm/volt and by the quality factor of the sensor (when driven at resonance); here the quality factor is given by the ratio of the wave resistance and the motion resistance of the sensor, measured by fitting the frequency characteristics (voltage versus frequency) of the sensor with the electrical model of the network.

b. Taking an X-ray measurement of the sensor surface (direct measurement).

Two schemes for driving the sensor are envisaged:

a. keeping the driving frequency f constant close to one of the resonant frequencies of the sensor and varying the driving voltage thereby varying the oscillation amplitude of the sensor; or

b. keeping the driving voltage constant and sweeping the frequency about one of the resonant frequencies of the sensor thereby varying the oscillation amplitude of the sensor

2. This 3f versus ^"I f or oscillation amplitude response is taken over a wide range of λ ί or oscillation amplitude in the absence of microbeads. These responses form the baseline signal.

Once the baseline signal has been determined, the sensor is scanned in a similar way in the presence of the microbeads. Any deviation in the 3f (or higher odd f) response from the baseline signal against ^"I f or oscillation amplitude represents the Anharmonic Detection (ADT) signal.

In alternative embodiments, it is sometimes possible to obtain the baseline signal differently to the method described above. In these alternative embodiments, the baseline is obtained as follows.

A second transducer (a reference transducer of the same specifications as the first transducer, but without the functionalised surface and/or microbeads) is provided. The molecules may be introduced simultaneously on both the sensor and the reference and the 3f (or higher odd f) response against oscillation amplitude or 1 / current is measured from both the sensor. The deviation in the 3f (or higher odd f) response against oscillation amplitude or 1 f current is the signal of interest and referred to as the Anharmonic Detection (ADT) signal. The deviation denotes the presence of the microbeads, and the nature of variation of deviation with the sensor oscillation amplitude or 1 / current, or with time is used to extract the characteristics of the linker - its force-extension characteristics, activation energy and rates of binding and unbinding. Determination of Maximum Drive Voltage

A useful parameter to know is the 'maximum drive voltage', applied to the transducer, which corresponds to the maximum amplitude of oscillation at which unbinding of the ligand-receptor bond or unfolding of a covalently-bonded molecule occurs. This maximum drive voltage is a parameter unique to the molecular linker being used for the same microbead (material and size).

This parameter is determined as follows: 1 . Again, a drive signal is applied to the sensor (in the presence of microbeads) and the electrical response is measured at f (at the drive frequency) and at 3f, i.e. three times the drive frequency (or any higher odd multiple). The 3f response against the λ ί response is recorded, and/or the 3f response against the oscillation amplitude is recorded. Again, two schemes for driving the sensor are envisaged:

a. keeping the driving frequency f constant close to one of the resonant frequencies of the sensor and varying the voltage of drive to vary the oscillation amplitude of the sensor; or

b. keeping the drive voltage constant and sweeping the frequency about one of the resonant frequencies of the sensor to vary the oscillation amplitude of the sensor

2. The maximum oscillation amplitude is gradually increased in successive scans following one of the schemes above. The deviation in 3f versus 1 f or oscillation amplitude is compared with the same from the previous scan.

3. If the ADT signal does not change between successive scans, (i.e. the graph from the successive scan is reproducible), the maximum oscillation amplitude is further increased.

4. If a decrease in the 3f deviation from the baseline against 1 f or oscillation amplitude relative to the previous scan occurs, this indicates unbinding and the corresponding drive voltage is noted as the 'maximum drive voltage'.

Alternatively, the maximum drive voltage may be determined as follows:

The sensor could be driven over a wide oscillation amplitude range in the first scan. This can be done by setting the final voltage for a constant frequency scan to a high value or by setting the voltage for constant voltage scan to a high value. The deviation in 3f from the baseline against 1 f or oscillation amplitude is recorded.

If there is a 'maxima' in the graph, i.e. the 3f deviation decreases after increasing up till a certain λ ί or oscillation amplitude, and the graph from the successive scan with the same drive parameters is not reproducible, this indicates unbinding. The drive voltage corresponding to the 'maxima' is noted as the 'maximum drive voltage'.

Cleaning of Prepared Sensor Surface After the binding step and prior to driving the sensor and taking any measurements, it is advisable to perform a cleaning step to remove beads with molecules of interest not bound to said binding partners. Two schemes are envisaged: 1 . Washing the sensor with a suitable buffer solution (e.g. Phosphate Buffer Saline (PBS) of 7.4 pH); or

2. Driving the sensor at a constant voltage close to but below the 'maximum drive voltage' at one of its resonant frequencies for suitable time duration.

Either both or one of the cleaning schemes above can be carried out. The cleaning process is repeated until the 3f response against ~\ f or oscillation amplitude from each subsequent scan is reproducible from one scan to the next. This indicates that beads with molecules of interest not bound to the binding partners are no longer on the surface of the sensor.

Determination of Force-Extension Characteristics

The method for determining the force-extension characteristics of the molecules of interest is as follows:

1 . The sensor is driven as described above (using one of the two alternative driving schemes) in the presence of microbeads and the ADT signal is recorded. The final or the constant drive voltage (depending on the driving scheme) is set to greater than or equal to the determined 'maximum drive voltage' for the sensor.

2. The measured ADT signal is compared with a model defining force-extension characteristics of a molecule (described below). 3. Parameters of the force-extension characteristics in the model are adjusted to fit the ADT signal computed from the model with the experimental ADT signal.

4. The force-extension characteristics may then be determined from the adjusted parameters that resulted in the abovementioned fit. We will now discuss the quantitative model used in the determination of the force- extension characteristics.

A biological entity attached to the sensor via molecular tether is modelled as a spring- mass system.

Figure 2 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 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. So only the horizontal component {Ft,) of the interaction force is considered for estimating the deviation in electrical response. Any Fourier series harmonic component n in F^ is given by

AF(n) = + F_a sin(q>)] exp( jnax)dt

T

ω Ν,,

- j^" (F_s (s) + k_a ) cos(^) exp(jnox)dt

where time period 7=1 // and angular frequency ω = 2i f. The higher odd harmonic components are non-zero and the third harmonic (3/) component is a significant proportion (- 1 /3) of the first harmonic (1 /) even for small amplitudes (i.e. φ~ττ/2). Also, the symmetricity of the expression of indicates that only odd harmonics are generated (i.e. even harmonic components are absent or zero). The odd harmonic components of F_tx are transduced into electrical charge, the time derivative of which is current, and this modifies the overall electrical response of the quartz. This explains the relative deviation in the overall 3f response observed in experiments due to attached microbeads.

The biological entity is modelled as a sphere and the molecular tether as an equivalent nonlinear spring of length l₀ comprised of multiple (Ni_inks) ligand-receptor linker-springs connected in parallel. The force along the tether is given by F_j = -N_UnksF_s (s) , where F_s(s) represents the force-extension characteristics of a linker-spring. The force perpendicular to tether due to its angular stiffness k_a is modelled as F_a = -k_aN_Unks cot(<p) where cot(^) models the impenetrable surface barrier.

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 given a and f, AF(3) depends on F_s(s) and k_a (which are mechanical characteristics of the tether) and extension s and inclination φ (which depend on mass and size of the particle and nature and length of the tether). Hence, for a given f, the deviation in 3f response, AF(3), expressed as a function of a, i.e. F_3F(a), provides a signature for the particle-tether system.

F_3F(a) is obtained by solving the describing differential equations for the particle (Eq. 2) for F^f), deriving its third Fourier harmonic component using Eq. 1 and plotting versus a(t). m_xX(t) = F_tx -≠(t) - A(X(t) - V_s (t)) = F_tx - (_r+ A)X(t) + AV_s (t)

m_yY(t) = F_ty - (r+ fi)Y(t) Here, m_x and m_y are the effective masses of the particle; V_s(t) is the velocity of the surface; γ is the coefficient of Stokes' viscous resistance from bulk liquid; A and β are coefficients of viscous drag in the horizontal and vertical directions respectively at the particle-surface interface due to the liquid. F_3F(a) can then be compared with the experimentally observed ADT signal to extract the force-extension characteristics F_s(s). It needs to be noted that F_s(s) is temperature dependent and hence the temperature needs to be controlled in the experiments.

The force-extension characteristics for a linker-spring is assumed as follows.

F_s (s) = (<¾ sin (s / + a₂ sinh³ (s /b₂ ))exp[-(cs + d)ⁿ ] (3) The form of this function is based on force versus tip-displacement results previously reported in AFM experiments.

Experimental Results

Experiments were carried out with surface-bound streptavidin-coated polystyrene microbeads (SCPM) of various sizes (5.61 μηι, 3.09 μπι and 0.39 μηπ) in 7.4 pH 100 mM phosphate buffer saline (PBS) solution. Gold-electrode-plated AT-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)n-(EG)₆-Biotin]. For non-specific or physisorbed interaction, a hydroxyl-terminated-thiol [HS-(CH₂)n-OH, FT 00.1 1 ] was used. 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.

In each scan the voltage was raised linearly from 0.07 l/ rms to 12 l/ rms in 2 minutes for specific interaction and to 4 l rms for the physisorbed case. To carry out the tests in liquid, a 8 μΖ. 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 that the same volume of liquid was on the sensor for each scan. This replacement is not required where an enclosed flow cell is used.

Figure 3a presents the 3f electrical response against oscillation amplitude of three successive scans from a sensor with 5.61 μιη 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 . Also, the response rises steadily at first and later drops and becomes unsteady; this characteristics 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 can be explained by the phenomenon of beads diffusing on and desorbing from surface at higher amplitudes. The increase in quality factor of the sensor observed after the scans confirms decrease in losses at the interface due to 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 l/ rms (instead of to 4 V rms reported here) is sufficient to acoustically clean the surface of most non-specifically bound particles.

Figure 3b presents the 3f response of successive scans from a sensor, initially without beads and then with specifically captured 0.39 μιη beads. Here again the relative increase in the 3f response due to introduction of beads is significant, given that the number of beads is only -42,000 (-1 .3 ng). The striking difference with the response from sensor with physisorbed beads is noteworthy. The response with specifically captured beads is reproducible over successive scans in contrast to the case with physisorbed beads; also, the response here is steadily increasing, without any drop. Both 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 4a presents the deviation in 3f response from the baseline response versus the oscillation amplitude due to introduction of different numbers of 0.39 μιη beads. This is referred to as the anharmonic detection 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 3a. It is this difference in signal shape that differentiates between specific and non-specific interactions. Also, the magnitude of the signal is nearly proportional to the number of beads on the surface.

This is also observed in experiments with 3.09 μιη beads (Figure 4b). Hence, the ADT signal recorded after the screening out of non-specifically bound species, confirmed from the reproducibility of successive scans, can potentially quantify specifically bound biological entities on the sensor, by comparision with the signal from a known concentration of the same recorded in library.

Interestingly however, the shape of the signal with 3.09 μιη is different from that with 0.39 μιη beads although the same molecular linker is used in both the cases. This demonstrates selectivity of ADT toward particles with similar affinity to the receptor but of different sizes and mass. The quantitative model explains the basis of selectivity of ADT toward 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 Figures 4a and 4b.

Referring back to Eq 3 above, 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 μιη. F_tx is then evaluated using Eq. 2 as F_tx = m_xX (t) + (Y+ A)X (t) - AV_s (t) .

The 3f component is then computed by numerically integrating Eq. 1 , multiplied by the appropriate force-to-charge conversion factor for an AT-cut quartz and by a factor for proximity to third overtone resonance, and then differentiated with respect to time to compute the deviation in the 3f current. This deviation 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 F_s(s) in Eq. 3.

The final fit, which match closely with the experiment (Figure 4b), is for the following values of the coefficients of F_s(s) in Eq. 3: a1 = 840; b1 = 20; a2 = 1200; b2 = 20; c = 18; d = 0.275. F_s(s) with these coefficients define the force-extension characteristics of the streptavidin-biotin-thiol linker used in the reported experiments.

Figure 5 shows the resulting force-extension function F_s(s) of the linker-spring (dotted line) and the force-extension encountered for 3.09 μιη (red line) employing the model. The dip in the red line can be explained by possible initiation of unbinding in the ligand- receptor bond. The magnitude of the peak force (-469 pN) is around three times that reported in AFM experiments. We attribute this to inaccurate estimation of the number of ligand-receptor linker-springs (Ni_inks = 18, using estimations reported before), 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.

The final fit is obtained from model after fitting the signal estimated from model with that observed in the experiment. The highlighted parts in Figure 5 show the actual force-extensions encountered by the linkers for 3.09μιη (red solid line) and 0.39μιη (green solid line) beads.

The signal for 42000 beads of 0.39 μιη computed with this F_s(s) fitted satisfactorily with that observed from the corresponding experiment (Figure 4a) for number of linkers Nnnks = 1 , which closely matches with our estimation of Ni_inks = 2. Thus apart from extracting the force-extension characteristics of the molecular linker, the model also enables quantitative estimation of the ADT signal for a linker with known force- extension characteristics.

Determination of Activation Energy

The activation energy for unfolding of the molecule used as a covalently bonded linker or the activation energy for unbinding of a ligand-receptor linker can be determined using the following steps.

1 . The molecule under investigation is attached in a sandwich structure between the microbead and the surface as explained above.

2. The sensor is driven at constant oscillation amplitude at the 'maximum driving voltage', determined as stated earlier, at the fundamental resonant frequency in the absence of the microbeads and the 3f response (baseline) is recorded. This oscillation amplitude is just sufficient to cause unbinding of the ligand-receptor complex or unfolding of a covalent linker. At this drive voltage, the sensor experiences oscillations at a 'first mechanical oscillation amplitude'.

3. Another baseline is recorded at slightly elevated drive voltage (e.g. 5% higher), to slightly increase the constant oscillation amplitude above the aforementioned 'first mechanical oscillation amplitude'. At this elevated drive voltage, the sensor experiences oscillations at a 'second mechanical oscillation amplitude'.

4. After attaching the microbeads via the linker to the surface, the sensor is again driven to achieve the first mechanical oscillation amplitude in a similar way for a time necessary to drop the 3f response (the deviation from the first baseline) by an amount sufficient to determine the rate of unbinding of the molecules. 5. Since this 3f (deviation) response is proportional to the number of particles, so the 3f deviation versus time provides a snapshot of the number of attached particles over time.

The gradient (m,) of the logarithm of 3f deviation versus time is obtained numerically by linear regression fitting.

6. The sensor with microbeads is then driven at the elevated (second) mechanical oscillation amplitude (as in step 3) again for a time necessary to drop the 3f response by an amount sufficient to determine a rate of unbinding of the molecules. Of course, it would be understood that the second rate of unbinding will be greater than the first rate of unbinding, since a higher force is applied in the second case. The corresponding gradient (m₂) of the logarithm of 3f deviation versus time is obtained numerically by linear regression fitting.

7. The activation energy E_a and the parameter δ^' (the decrease in depth of potential well relative to k_BT caused by application of force) can then be obtained by solving the following set of two equations. m₁ = -{k_BTI h) e^ ^Bessel O, δ ) + Struvel_(0, δ )]

m₂ = -{k_BTI h) e^ ^Bessel O, δβ) + Struvel_(0, δβ)]

Here k_B is the Boltzmann constant, T is the absolute temperature, h is the Planck's constant and ?' is the ratio of force applied (determined experimentally). Bessell is the modified Bessel function of the first kind and StruveL is the modified Struve function Conveniently, but not essentially, these simultaneous equations may be solved for Ea (and optionally δ) by taking the log of the gradients and m₂ .

8. Since δ^' = F a /(k_BT), where F is the force applied along the linker (which is determined from the force-extension characteristics) and a is the inter-nuclear distance between the ground and transition states projected on the force vector, it is also possible to estimate the value of a . 9. E_a and a provide important information on the potential energy profile of the interaction.

Optionally, a third rate of unbinding and a third logarithm (m₃) of the slope may be calculated. In such embodiments, the activation energy can be determined from all possible pair combinations from the first, second and third rates (first and second; first and third; second and third etc), and the activation energy obtained from each combination can be averaged to obtain a final result. Determination of Unbinding Rate

Rate of unbinding of the ligand-receptor complex can be directly determined from the graph of 3f (deviation) versus time as obtained above. E.g. the gradient of the logarithm of 3f (deviation) against time (m₍) may represent the rate of unbinding of the ligand- receptor complex at the given force or oscillation of amplitude. However, it needs to be noted that since the molecular complex is tagged to microbeads and sandwiched between the microbead and a surface, this unbinding rate is different from in-solution unbinding kinetics. Also since the surface is oscillating, the ligand and receptor moves away from the binding zone after unbinding thus preventing any possible reversible reaction. Like unbinding of the complex due to forces from large oscillation amplitude, unbinding due to rise in temperature can also be studied. Here, it is possible to remove the hindrance to reversible reaction due to the oscillation of the surface by making the measurement time short-spanned (around 100 ms) and repeating periodically with long time period (5 sees).

Determination of Binding Rate

As mentioned above, the 3f signal (deviation) is proportional to the number of microbeads on the sensor. This can also be used to measure the binding rate of the molecules tagged to the microbeads similar to the unbinding rate discussed above. The steps are as follows.

1 . Drive the sensor in the absence of the microbeads at a constant oscillation amplitude, which is less than an amplitude sufficient to cause unbinding of the molecular complex, but sufficient enough to give a sufficient signal to noise ratio. The resultant transduced signal is recorded as a 'baseline' signal.

2. Without driving the sensor, introduce the microbeads tagged with the molecule of interest, whose binding with the receptor on the sensor is going to be studied, at a constant flow rate.

3. Drive the sensor intermittently and periodically at the oscillation amplitude same as the one used for the baseline to measure the 3f response and record the deviation. The measurement time is around 100 ms. The time period of repeating of the measurement is around 5 seconds.

4. The 3f (deviation) gives the rate of increase of bound microbeads on the surface and the gradient logarithm of the 3f (deviation) curve against time gives a measure of the rate of binding of the molecules tagged to the microbeads with the receptor on the sensor surface.

In the above embodiments, the sensor is described as being in a liquid or having one surface of the sensor exposed to liquid (or having a liquid deposited on the surface of the sensor). In alternatives, the sensor may operate in air, a gas or a vacuum.

The ability to measure the true affinity of molecular interactions rapidly on a large scale and at an affordable cost is one of the pivotal needs in life sciences and pharmacology. The methods presented here are new biophysical characterization and screening techniques that can potentially enable a wide range of applications in clinical diagnostics, drug discovery, food and environment safety and bio-defence as well as the fundamental understanding of biological events and processes.

1 . The methods allow measurement of activation energy, which gives a measure of affinity of molecular interactions, and the resulting data can be used to infer the mechanical force-extension characteristics of molecular tethers. Thus, it presents a potential method for affinity-based screening and provides an additional level of selectivity in biosensing applications. The knowledge of the activation energy and mechanical force-extension characteristics also helps in the analytical studies, such as design and choice of specific receptor/antibodies. 2. The ability to determine force-extension characteristics and activation energy also presents the method as a novel force-spectroscopic technique. The conventional force spectroscopic techniques such as the Atomic Force Microscope (AFM) is expensive, time consuming and demands expertise. They also allow investigation of only one molecule at a time, requiring time consuming multiple measurements to obtain statistically useful data. However, since the above method is entirely electronic it is integrable and scalable thus enabling cost-effective, rapid and easy-to-use force-spectroscopic technique.

3. Some potential applications for these methods are in the pharmaceutical industry for drug discovery (in vitro trials and companion diagnostics), in point-of-care diagnostics, food and environmental safety, biosecurity and in the chemical and biochemical research laboratories for the study of fundamental biological events and processes, identification of biomolecular interaction networks, reaction kinetics and pathways.

4. Information on activation energy and force-extension characteristics of the bond between the drug molecules being tested and the binding partner simultaneously for all drug molecules provides extra criteria, that allow reduction of number of potential candidate drugs dramatically. Hence, with a few candidate drugs to be tested by the end of "in vitro" trial stage, this potentially makes "in vivo" testing feasible.

No doubt many other effective alternatives will occur to the skilled person. It will be understood that the invention is not limited to the described embodiments and encompasses modifications apparent to those skilled in the art lying within the scope of the claims appended hereto.

Claims

CLAIMS:

1 . A method of determining an activation energy of a molecule or bound molecular pair, the method comprising:

exposing a prepared surface of a sensor to a plurality of microbeads for a binding period, each microbead being functionalised with a molecule of interest, said sensor comprising a transducer that oscillates in response to a drive signal applied to said sensor, said prepared surface of said sensor comprising a binding partner for binding with said molecule of interest, and said binding period being sufficient to allow binding between said molecule of interest and said binding partner;

determining a first rate of unbinding of a molecule or bound molecular pair of interest over a first period; and

determining a second rate of unbinding of a molecule or bound molecular pair of interest over a second period; and

determining an activation energy of said molecule or bound molecular pair using said first and second rates;

wherein said second rate of unbinding is greater than said first rate of unbinding.

2. A method according to claim 1 further comprising determining a log of a first slope of said first rate of unbinding against time and a log of a second slope of said second rate of unbinding against time; and wherein said determining said activation energy uses said determined log of said first and second slopes.

3. A method according to claim 1 or 2, wherein determining a first rate of unbinding of a molecule or bound molecular pair comprises:

applying a first driving signal to said sensor for said first period, said first driving signal comprising an alternating voltage having a frequency and a first amplitude sufficient to cause said sensor to oscillate at a first mechanical oscillation amplitude, said first mechanical oscillation amplitude being sufficient to cause detachment of said bound molecules from said binding partner;

recording a first transduced signal from said sensor over said first period, said first transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said driving signal, said first transduced signal resulting from oscillation of said sensor in response to said first driving signal;

determining said first rate of unbinding of a molecule or bound molecular pair from said recorded first transduced signal.

4. A method according to claim 1 , 2, or 3 wherein determining a second rate of unbinding of a molecule or bound molecular pair comprises:

applying a second driving signal to said sensor for said second period, said second driving signal comprising an alternating voltage having a frequency and a second amplitude sufficient to cause said sensor to oscillate at a second mechanical oscillation amplitude, said second mechanical oscillation amplitude being sufficient to cause detachment of said bound molecules from said binding partner;

recording a second transduced signal from said sensor over said second period, said second transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said driving signal, said second transduced signal resulting from oscillation of said sensor in response to said second driving signal;

determining said second rate of unbinding of a molecule or bound molecular pair from said recorded second transduced signal.

5. A method according to claim 3, comprising generating a first Anharmonic Detection (ADT) signal from said first transduced signal, said first ADT signal representing a deviation of said recorded first transduced signal from a first baseline transduced signal, and said first baseline transduced signal representing a phase and/or amplitude response at an odd harmonic frequency of said first driving signal for a sensor absent said microbeads functionalised with said molecule of interest, said generating said first ADT signal comprising:

comparing said first baseline signal with said first transduced signal to identify a deviation between said first baseline signal and said first transduced signal, and wherein said determining said first rate of unbinding of a molecule or bound molecular pair from said recorded first transduced signal comprises determining said first rate of unbinding of a molecule or bound molecular pair from said first ADT signal.

6. A method according to claim 4, or claim 5 when dependent on claim 4, comprising generating a second Anharmonic Detection (ADT) signal from said second transduced signal, said second ADT signal representing a deviation of said recorded second transduced signal from a second baseline transduced signal, and said second baseline transduced signal representing a phase and/or amplitude response at an odd harmonic frequency of said second driving signal for a sensor absent said microbeads functionalised with said molecule of interest, said generating said second ADT signal comprising:

comparing said second baseline signal with said second transduced signal to identify a deviation between said second baseline signal and said second transduced signal, and

wherein said determining said second rate of unbinding of a molecule or bound molecular pair from said recorded second transduced signal comprises determining said second rate of unbinding of a molecule or bound molecular pair from said second ADT signal.

7. A method according to claim 5 or 6, wherein said first or second baseline transduced signal is obtained prior to said exposing step and wherein said first or second baseline signal is obtained by:

applying said first or second driving signals to said sensor, said sensor being devoid of microbeads functionalised with said molecules of interest;

recording a respective first or second baseline transduced signal from said sensor, said first or second baseline transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said first or second driving signal, said first or second baseline transduced signal resulting from oscillation of said sensor.

8. A method according to any one of claims 1 to 7, comprising performing a cleaning step prior to said determining a first and second rate of unbinding to remove molecules of interest not bound to said binding partners and unwanted molecules, comprising washing said sensor with a buffer solution.

9. A method according to any one of claims 1 to 8, comprising performing a cleaning step prior to determining a first and second rate of unbinding to remove molecules of interest not bound to said binding partners and unwanted molecules, said cleaning step comprising:

applying a driving signal to said sensor; recording said amplitude and/or phase of said transduced signal at substantially an odd harmonic frequency of said frequency of said driving signal; and

comparing data derived from said recorded transducer signal from each applying step with data derived from said recorded transducer signal from a previous applying step until said data is substantially reproduced from one applying step to the next applying step, thereby indicating that molecules of interest not bound to said binding partners and unwanted molecules are no longer on said surface.

10. A method according to any one of claims 2 to 8 when dependent on claim 2, comprising:

determining a third rate of unbinding of a molecule or bound molecular pair of interest over a third period;

determining a log of a third slope of said third rate of unbinding against time, wherein said determining an activation energy of said molecule or bound molecular pair comprises using said determined log of said first, second and third slopes,

wherein said third rate of unbinding is greater than said second rate of unbinding.

1 1 . A method according to claim 10, comprising determining said activation energy from a combination of activation energies in equations defined by said first, second and third slopes.

12. A method according to any one of claims 1 to 1 1 , wherein said odd harmonic is a third or fifth harmonic of said driving frequency.

13. A method according to any one of claims 1 to 12, wherein said binding is a ligand-receptor binding or a covalent bond.

14. Apparatus for determining an activation energy of a molecule or bound molecular pair, comprising:

a sensor comprising a transducer that oscillates in response to a drive signal applied to said sensor, said sensor comprising a prepared surface comprising a binding partner for binding with molecules of interest, said molecules of interest being supported on a plurality of microbeads;

a driving signal generator for generating a driving signal comprising an alternating voltage; and a controller operatively coupled to said sensor and said driving signal generator, said controller being configured to:

controllably apply said driving signal to said sensor to determine a first rate of unbinding of a molecule or bound molecular pair of interest over a first period;

controllably apply said driving signal to said sensor to determine a second rate of unbinding of a molecule or bound molecular pair of interest over a second period; and

determine an activation energy of said molecule or bound molecular pair using said first and second slopes;

15. Apparatus according to claim 14 wherein said controller is configured to determine a log of a first slope of said first rate of unbinding against time and a log of a second slope of said second rate of unbinding against time, and to determine said activation energy using said determined log of said first and second slopes.

16. Apparatus according to claim 14 or 15, wherein said controller determines said first rate of unbinding of a molecule or bound molecular pair by:

controllably applying a first driving signal to said sensor for said first period, said first driving signal comprising an alternating voltage having a frequency and a first amplitude sufficient to cause said sensor to oscillate at a first mechanical oscillation amplitude, said first mechanical oscillation amplitude being sufficient to cause detachment of said bound molecules from said binding partner;

receiving and recording a first transduced signal from said sensor over said first period, said first transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said driving signal, said first transduced signal resulting from oscillation of said sensor in response to said first driving signal; and

17. Apparatus according to claim 14, 15 or 16, wherein said controller determines said second rate of unbinding of a molecule or bound molecular pair by:

controllably applying a second driving signal to said sensor for said second period, said second driving signal comprising an alternating voltage having a frequency and a second amplitude sufficient to cause said sensor to oscillate at a second mechanical oscillation amplitude, said second mechanical oscillation amplitude being sufficient to cause detachment of said bound molecules from said binding partner; receiving and recording a second transduced signal from said sensor over said second period, said second transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said driving signal, said second transduced signal resulting from oscillation of said sensor in response to said second driving signal; and

18. Apparatus according to claim 16, wherein the controller is configured to generate a first Anharmonic Detection (ADT) signal from said first transduced signal, said first ADT signal representing a deviation of said recorded first transduced signal from a first baseline transduced signal, and said first baseline transduced signal representing a phase and/or amplitude response at an odd harmonic frequency of said first driving signal for a sensor absent said microbeads functionalised with said molecule of interest, said controller being configured to generate said first ADT signal by:

comparing said first baseline signal with said first transduced signal to identify a deviation between said first baseline signal and said first transduced signal,

wherein said determining said first rate of unbinding of a molecule or bound molecular pair from said recorded first transduced signal comprises determining said first rate of unbinding of a molecule or bound molecular pair from said first ADT signal.

19. Apparatus according to claim 17, wherein the controller is configured to generate a second Anharmonic Detection (ADT) signal from said second transduced signal, said second ADT signal representing a deviation of said recorded second transduced signal from a second baseline transduced signal, and said second baseline transduced signal representing a phase and/or amplitude response at an odd harmonic frequency of said second driving signal for a sensor absent said microbeads functionalised with said molecule of interest, said controller being configured to generate said second ADT signal by: comparing said second baseline signal with said second transduced signal to identify a deviation between said second baseline signal and said second transduced signal,

20. Apparatus according to claim 18 or 19, wherein said first or second baseline transduced signal is obtained prior to exposing of said molecules to said sensor and wherein said controller is configured to obtain said first or second baseline signals by: controllably applying said first or second driving signals to said sensor, said sensor being devoid of microbeads functionalised with said molecules of interest;

receiving and recording a respective first or second baseline transduced signal from said sensor, said first or second baseline transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said first or second driving signal, said first or second baseline transduced signal resulting from oscillation of said sensor.

21 . A method of determining a rate of binding of a molecule, said method comprising:

exposing a prepared surface of a sensor to a substantially constant flow of a plurality of microbeads for a binding period, each microbead being functionalised with a molecule of interest, said sensor comprising a transducer that oscillates in response to a drive signal applied to said sensor, said prepared surface of said sensor comprising a binding partner for binding with said molecule of interest, and said binding period being sufficient to allow binding between said molecule of interest and said binding partner; applying a driving signal to said sensor for a period, said driving signal comprising an alternating voltage having a frequency and amplitude sufficient to cause said sensor to oscillate at a first mechanical oscillation amplitude, said first mechanical oscillation amplitude being below a mechanical oscillation amplitude sufficient to cause unbinding of the binding molecules;

recording a transduced signal from said sensor over said period, said transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said driving signal, said transduced signal resulting from oscillation of said sensor; and

processing said recorded transduced signals from said recording to determine a rate of binding of said molecule.

22. A method as claimed in claim 21 further comprising:

changing an amplitude of said driving signal such that for a second period said driving signal has an adjusted amplitude, in particular removing said driving signal for a second period; wherein said second period is longer than said first period; and

repeating said applying said driving signal to said sensor for a first said period, said recording said transduced signal from said sensor over said first period and said removing said driving signal for said second period over said binding period; and

wherein said processing comprises comparing said recorded transduced signals from said repeated recording steps to determine a deviation between the recorded transduced signals over said binding period, said deviation defining said rate of binding of said molecule.

23. A method according to claim 21 or 22, comprising:

applying said driving signal for said first period before said exposing step; and recording said transduced signal from said sensor over said first period to define a baseline transduced signal, said baseline transduced signal representing a phase and/or amplitude response at an odd harmonic frequency of a driving signal for a sensor absent said microbeads functionalised with said molecule of interest.

24. A method according to claim 21 , 22 or 23, wherein said comparing step comprises determining a gradient logarithm of said deviation against time over said binding period.

25. A method according to claim 21 , 22, 23, or 24 wherein said magnitude of mechanical oscillation of said sensor is determined by using a voltage and a current of said transduced signal at said driving frequency to calculate said magnitude of oscillation of said sensor.

26. A method according to any one of claims 21 to 25, wherein said odd harmonic is a third or fifth harmonic of said driving frequency.

27. A method according to any one of claims 21 to 26, wherein said binding between said molecule of interest and said binding partner is a ligand-receptor binding, or a covalent bond.

28. Apparatus for determining a rate of binding of a molecule, comprising:

a sensor comprising a transducer that oscillates in response to a drive signal applied to said sensor, said sensor comprising a prepared surface comprising a binding partner for binding with molecules of interest;

a driving signal generator for generating a driving signal comprising an alternating voltage having a frequency and an amplitude sufficient to cause said sensor to oscillate; and

a controller operatively coupled to said sensor and said driving signal generator, said controller being configured to:

controllably expose said prepared surface of said sensor to a substantially constant flow of a plurality of microbeads for a binding period, each microbead being functionalised with a molecule of interest, said binding period being sufficient to allow binding between said molecule of interest and said binding partner;

controllably apply a driving signal to said sensor for a first period, said driving signal comprising an alternating voltage having a frequency and amplitude sufficient to cause said sensor to oscillate at a first mechanical oscillation amplitude, said first mechanical oscillation amplitude being below an oscillation amplitude sufficient to cause unbinding of the binding molecules;

receive and record a transduced signal from said sensor over said first period, said transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said driving signal, said transduced signal resulting from oscillation of said sensor; and

process said recorded transduced signals to determine a rate of binding of said molecule.

29. Apparatus according to claim 28, wherein the controller is configured to: change an amplitude of said driving signal such that for a second period said driving signal has an adjusted amplitude, and in particular remove said driving signal for a second period, wherein said second period is longer than said first period; and repeat said controllable applying of said driving signal to said sensor for a first said period, said receiving and recording of said transduced signal from said sensor over said first period and said removing of said driving signal for said second period over said binding period.

30. Apparatus according to claim 28 or 29, wherein the controller is configures to: controllably apply said driving signal for said first period before said sensor is controllably exposed to said flow of said plurality of microbeads; and

receive and record said transduced signal from said sensor over said first period to define a baseline transduced signal, said baseline transduced signal representing a phase and/or amplitude response at an odd harmonic frequency of a driving signal for a sensor absent said microbeads functionalised with said molecule of interest.

31 . Apparatus according to claim 28, 29 or 30, wherein said controller compares said recorded transduced signals to determine a deviation between the recorded transduced signals comprises determining a gradient logarithm of said deviation against time over said binding period.

32. A method according to claim 28, 29, 30, or 31 , wherein said magnitude of mechanical oscillation of said sensor is determined by using a voltage and a current of said transduced signal at said driving frequency to calculate said magnitude of oscillation of said sensor.

33. A method according to any one of claims 28 to 32, wherein said odd harmonic is a third or fifth harmonic of said driving frequency.

34. A method according to any one of claims 28 to 33, wherein said binding between said molecule of interest and said binding partner is a ligand-receptor binding, or a covalent bond.

35. A method of determining force-extension characteristics of a molecule or biological entity, the method comprising:

applying a driving signal to said sensor for a first period, said driving signal comprising an alternating voltage having a frequency and amplitude sufficient to cause said sensor to oscillate;

recording a transduced signal from said sensor over said first period, said transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said driving signal, said transduced signal resulting from oscillation of said sensor;

comparing said transduced signal with a signal estimated from a model defining force-extension characteristics of a molecule;

adjusting parameters of said force-extension characteristics in said model to substantially fit said signal estimated from said model with said transduced signal; and determining force-extension characteristics of said molecule of interest from said adjusted parameters that result in said fit.

36. A method according to claim 35, wherein said applying a driving signal comprises ramping an amplitude of said driving signal over said first period between a minimum amplitude sufficient to cause said sensor to oscillate, and a maximum amplitude sufficient to cause detachment of said molecules bound with said binding partner, and wherein said frequency of said driving signal is substantially a resonant frequency of said sensor.

37. A method according to claim 36, wherein said amplitude of said driving signal is ramped from said minimum amplitude to said maximum amplitude and/or from said maximum amplitude to said minimum amplitude.

38. A method according to claim 35, wherein said applying a driving signal comprises ramping said frequency of said driving signal over said first period between a minimum frequency and a maximum frequency, said minimum and maximum frequencies being sufficient to cause said sensor to oscillate, and wherein said amplitude of said driving signal is greater than or equal to a maximum amplitude sufficient to cause detachment of said molecules bound with said binding partner.

39. A method according to claim 38, wherein said frequency of said driving signal is ramped from said minimum frequency to said maximum frequency and/or from said maximum frequency to said minimum frequency.

40. A method according to any one of claims 35 to 39, wherein said transduced signal is recorded against a magnitude of oscillation of said sensor.

41 . A method according to claim 40, wherein said magnitude of oscillation is determined by using a voltage and a current of said transduced signal at said driving frequency to calculate said magnitude of oscillation of said sensor.

42. A method according to any one of claims 35 to 39, wherein said transduced signal is recorded against a measured current of said transduced signal at said driving frequency.

43. A method according to any one of claims 35 to 41 , comprising generating an Anharmonic Detection (ADT) signal from said transduced signal, said ADT signal representing a deviation of said recorded transduced signal from a baseline transduced signal, and said baseline transduced signal representing a phase and/or amplitude response at an odd harmonic frequency of a driving signal for a sensor absent said microbeads functionalised with said molecule of interest, said generating said ADT signal comprising:

comparing said baseline signal with said transduced signal to identify a deviation between said baseline signal and said transduced signal, and

wherein said comparing said transduced signal with a model comprises comparing said ADT signal with said model defining a force-extension characteristics of a molecule.

44. A method according to claim 43, wherein said baseline transduced signal is obtained prior to said exposing step and wherein said baseline signal is obtained by: applying a driving signal to said sensor for a first period, said driving signal comprising an alternating voltage having a frequency and amplitude sufficient to cause said sensor to oscillate, and said sensor being devoid of microbeads functionalised with said molecules of interest;

recording a baseline transduced signal from said sensor over said first period, said baseline transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said driving signal, said baseline transduced signal resulting from oscillation of said sensor.

45. A method according to claim 44, wherein said applying a driving signal comprises ramping an amplitude of said driving signal over said first period between a minimum amplitude sufficient to cause said sensor to oscillate, and a baseline maximum amplitude, wherein said frequency of said driving signal is substantially a resonant frequency of said sensor and wherein said baseline maximum amplitude is greater than an amplitude sufficient to cause detachment of said molecules when bound with said binding partner.

46. A method according to claim 45, wherein said amplitude of said driving signal is ramped from said minimum amplitude to said maximum amplitude and/or from said maximum amplitude to said minimum amplitude.

47. A method according to claim 44, wherein said applying a driving signal comprises ramping said frequency of said driving signal over said first period between a minimum frequency and a maximum frequency, said minimum and maximum frequencies being sufficient to cause said sensor to oscillate, and wherein said amplitude of said driving signal is greater than an amplitude sufficient to cause detachment of said molecules when bound with said binding partner.

48. A method according to claim 47, wherein said frequency of said driving signal is ramped from said minimum frequency to said maximum frequency and/or from said maximum frequency to said minimum frequency.

49. A method according to any one of claims 43 to 48, wherein said baseline transduced signal is recorded against a magnitude of oscillation of said sensor.

50. A method according to claim 49, wherein said magnitude of oscillation is determined by using a voltage and a current of said baseline transduced signal at said driving frequency to calculate said magnitude of oscillation of said sensor.

51 . A method according to any one of claims 43 to 48, wherein said baseline transduced signal is recorded against a measured current of said baseline transduced signal at said driving frequency.

52. A method according to any one of claims 43 to 51 , comprising performing a cleaning step prior to said applying a drive signal to remove molecules of interest not bound to said binding partners and unwanted molecules, said cleaning step comprising washing said sensor with a buffer solution.

53. A method according to any one of claims 43 to 52, comprising performing a cleaning step prior to said applying a drive signal to remove molecules of interest not bound to said binding partners and unwanted molecules, said cleaning step comprising:

applying said driving signal to said sensor;

recording said amplitude and/or phase of said transduced signal at substantially an odd harmonic frequency of said frequency of said driving signal; and

54. A method according to any one of claims 43 to 53, comprising counting said microbeads prior to said applying said driving signal.

55. A method according to any one of claims 43 to 54 wherein said odd harmonic is a third or fifth harmonic of said driving frequency.

56. A method according to any one of claims 43 to 55, wherein said binding between said molecule of interest and said binding partner is a ligand-receptor binding, or a covalent bond.

57. Apparatus for determining force-extension characteristics of a molecule or biological entity, comprising: a sensor comprising a transducer that oscillates in response to a drive signal applied to said sensor, said sensor comprising a prepared surface comprising a binding partner for binding with molecules of interest, said molecules of interest being supported on a plurality of microbeads;

a driving signal generator for generating a driving signal comprising an alternating voltage having a frequency and amplitude sufficient to cause said sensor to oscillate; and

controllably apply said driving signal from said driving signal generator to said sensor; receive and record an amplitude and/or phase of a transduced signal at substantially an odd harmonic frequency of said frequency of said driving signal, said transduced signal resulting from an oscillation of said transducer;

compare said transduced signal with a signal estimated from a model defining force-extension characteristics of a molecule;

adjust parameters of said force-extension characteristics in said model to substantially fit said signal estimated from said model with said transduced signal; and determine force-extension characteristics of said molecule of interest from said adjusted parameters that result in said fit.

58. Apparatus according to claim 57, wherein said controller is configured to:

generate an Anharmonic Detection (ADT) signal from said transduced signal, said ADT signal representing a deviation of said recorded transduced signal from a baseline transduced signal, and said baseline transduced signal representing a phase and/or amplitude response at an odd harmonic frequency of a driving signal for a sensor absent said microbeads functionalised with said molecule of interest, said controller generating said ADT signal by:

comparing said baseline signal with said transduced signal to identify a deviation between said baseline signal and said transduced signal,

wherein said compare said transduced signal with a model comprises comparing said

ADT signal with said model defining force-extension characteristics of a molecule.

59. Apparatus according to claim 58, wherein said baseline transduced signal is obtained prior to said exposing step and wherein said controller is adapted to obtain said baseline signal by: controllably apply a driving signal to said sensor for a first period, said driving signal comprising an alternating voltage having a frequency and amplitude sufficient to cause said sensor to oscillate, and said sensor being devoid of microbeads functionalised with said molecules of interest;

receive and record a baseline transduced signal from said sensor over said first period, said baseline transduced signal comprising a phase and/or amplitude substantially at an odd harmonic frequency of said driving signal, said baseline transduced signal resulting from oscillation of said sensor.

60. A method or apparatus according to any one of claims 1 to 34 wherein said microbeads are replaced by biological entities.