GB2473635A

GB2473635A - Detecting concentration of target species

Info

Publication number: GB2473635A
Application number: GB0916389A
Authority: GB
Inventors: Souray Ghosh; Victor Ostanin; Ashwin Seshia
Original assignee: Cambridge Enterprise Ltd
Current assignee: Cambridge Enterprise Ltd
Priority date: 2009-09-18
Filing date: 2009-09-18
Publication date: 2011-03-23
Also published as: GB0916389D0; WO2011033285A1

Abstract

A method of determining target species concentration in an analyte comprises; exposing an oscillating sensor surface to an analyte comprising a target species, said surface comprising an agent that selectively bonds with said target species; applying a driving signal to said sensor to cause oscillation, comprising an alternating voltage having a frequency equal to a resonant frequency of said sensor; ramping an amplitude of said driving signal between a minimum voltage sufficient to cause said sensor to oscillate, and a maximum voltage below a voltage sufficient to cause detachment of bonded target species from said surface; recording an amplitude and/or phase of a transduced signal at an odd harmonic frequency of said driving signal, said transduced signal resulting from said sensor oscillation; and comparing data derived from said recorded transduced signal with a library to determine concentration of target species, said library representing transduced signal data from known concentrations of target species.

Description

Apparatus and Method for Detecting Target Species in an Analyte

FIELD OF INVENTION

The present invention relates to apparatus and methods for detecting and determining the concentration of target species in an analyte. In particular, the present invention relates to apparatus and methods for determining the concentration of target species in an analyte using oscillating transducers, and methods of generating a library of data from the oscillating transducers.

BACKGROUND OF INVENTION

The use of oscillating transducers to detect target species in analytes is known for example in US 7,570,125, W098/41820, W099/30159, W099/40397, W000/55613, W02009/005452, W02009/0701 08, W02006/1 03439, W005/1 21769, W004/09501 2 and W004/09501 1.

The applicant has appreciated the need for an improved method of detecting and determining the concentration of target species in an analyte using oscillating transducers.

SUMMARY OF INVENTION

The present invention provides a method of determining a concentration of a target species in an analyte, comprising: exposing a prepared surface of a sensor to an analyte comprising a target species for a first period, 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 recognition agent that selectively bonds with said target species, and wherein said first period is sufficient for said target species to bond with said prepared surface; applying a driving signal to said sensor, said driving signal comprising an alternating voltage having a frequency substantially equal to a resonant frequency of said sensor, and said driving signal causing said sensor to oscillate; ramping an amplitude of said driving signal over a second period between a minimum voltage sufficient to cause said sensor to oscillate, and a maximum voltage, said maximum voltage being below a voltage sufficient to cause detachment of said target species bonded with said prepared surface from said prepared surface; recording 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 said oscillation of said sensor; and comparing data derived from said recorded transduced signal with a library to determine a concentration of said target species, said library representing transduced signal data from known concentrations of said target species.

By recording the amplitude and/or phase of the signal produced by a transducer at odd harmonics of the driving frequency, qualitative and quantitative measurements of the concentration of the target species in an analyte can be made. Driving the transducer with a ramped amplitude up to a maximum voltage that is below the voltage at which the target species bonded with the prepared surface detaches from the prepared surface enables the method to be repeated on the same analyte using the same sensor.

The library enables the concentration to be determined by comparing the recorded transduced signals with transduced signals for known concentrations of the target species.

In embodiments of the method, the method comprises determining a magnitude of oscillation of said transducer to provide oscillation magnitude data representing a magnitude of oscillation of said transducer, and wherein said comparing comprises comparing data derived from said determined oscillation magnitude data with said library, said library further comprising oscillation magnitude data from known concentrations of said target species. Preferably, said determining of said magnitude of oscillation comprises using a voltage and a current of said transduced signal at said driving frequency to calculate said magnitude of oscillation of said transducer.

By determining the magnitude of oscillation of the transducer, a plot of the transducer signal (for example at the fifth harmonic -5F) can be recorded as functions of amplitude of mechanical oscillation of the surface.

In preferred embodiments, the method comprises repeating said applying, said ramping and said recording steps to produce a plurality of recorded amplitudes and/or phases of said transduced signals, and wherein said plurality of recorded amplitudes and/or phases of said transduced signals are averaged, and wherein said averaged signals are compared with said library.

Repeating the applying, ramping and recording steps enables multiple measurements to be taken and averaged. As discussed above, this is possible as a result of the transducer only being driven up to the point below which detachment of the target species bonded with the prepared surface from the prepared surface. Multiple averaged signals improve the signal to noise ratio of the received signals as noise is averaged out.

Preferably, the transducer signal is recorded at odd harmonic, such as the third or fifth harmonics, of said driving frequency.

In embodiments, the drive signal is ramped from said minimum voltage to said maximum voltage and/or from said maximum voltage to said minimum voltage.

In embodiments, the driving signal can be ramped from said minimum voltage to said maximum voltage and from said maximum to said minimum voltage to reveal a hysteresis loop in said recorded transducer signals, and wherein an area of said hysteresis loop is determined to determine a concentration of said target species.

In further preferable embodiments, the method comprises performing a cleaning step prior to said applying a drive signal to remove target and non-target species that are not bonded with said prepared surface from said prepared surface, said cleaning step comprising: applying said driving signal to said sensor; ramping an amplitude of said driving signal between said minimum voltage and said maximum voltage two or more times; 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 ramping step with data derived from said recorded transducer signal from a previous ramping step until said data is substantially reproduced from one ramping step to the next ramping step, thereby indicating that target and non-target species not bonded with said prepared surface are no longer on said surface. This ensures that the results are reproducable between ramps of the driving signal.

In embodiments, the prepared surface comprises a plurality of beads covalently bonded with said surface of said transducer, and wherein said beads comprise a recognition agent. This is particularly useful when the target species is a gas, as the prepared surface provides an area creating pockets for gas underneath beads thus increasing the sensitivity of detection.

In other embodiments, the prepared surface comprises regions of recognition agent.

In some embodiments, the transducer comprises a quartz crystal having metallic electrodes. Alternatively, the transducer comprises a silicon or silicon oxide body, and wherein said recognition agent is covalently bonded to a surface of said silicon or silicon oxide body. Alternatively, the transducer comprises a metal body (for example the metal body comprises a MEMS (Microelectromechanical System)).

The present invention also provides a target species sensor for determining a concentration of a target species in an analyte, comprising: a transducer that oscillates in response to a drive signal applied to said transducer, said transducer comprising a prepared surface comprising a recognition agent that selectively bonds with a target species; a driving signal generator for generating a driving signal comprising an alternating voltage having a frequency substantially equal to a resonant frequency of said transducer and wherein said driving signal generator controllably ramps an amplitude of said driving signal between a minimum voltage sufficient to cause said transducer to oscillate, and a maximum voltage, said maximum voltage being below a voltage sufficient to cause detachment of said target species bonded with said prepared surface from said prepared surface; a data recorder for recording 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; a comparator for comparing data derived from a recorded transduced signal with a library to determine a concentration of said target species, said library representing transduced signal data from known concentrations of said target species.

In embodiments, the sensor comprises a data processor for determining a magnitude of oscillation of said transducer to provide oscillation magnitude data representing a magnitude of oscillation of said transducer, and wherein said comparator compares data derived from said determined oscillation magnitude data with said library, said library further comprising oscillation magnitude data from known concentrations of said target species. Preferably, the data processor calculates said magnitude of oscillation using a voltage and a current of said transduced signal at said driving frequency.

By determining the magnitude of oscillation of the transducer, a plot of the transducer signal (for example at the fifth harmonic -SF) can be recorded as functions of amplitude of mechanical oscillation of the surface.

In preferred embodiments, the sensor comprises an averager for averaging a plurality of amplitudes and/or phases of a plurality of said transduced signals, and wherein said comparator compares said averaged amplitudes and/or phases of said plurality of transduced signals are compared with said library.

Multiple measurements are enabled with this sensor, as the transducer is driven up to a maximum voltage that is below the voltage at which the target species bonded with the prepared surface would detach from the prepared surface. Multiple averaged signals improve the signal to noise ratio of the received and recorded signals as noise is averaged out.

In embodiments, the odd harmonic at which data recorder records data is a third or fifth harmonic of said driving frequency.

Preferably, the driving signal generator generates a driving signal comprising an amplitude ramped from said minimum voltage to said maximum voltage and/or from said maximum voltage to said minimum voltage.

In embodiments, the driving signal generator generates a driving signal comprising an amplitude ramped from said minimum voltage to said maximum voltage and from said maximum to said minimum voltage to reveal a hysteresis loop in said recorded transducer signals, and wherein a data processor determines an area of said hysteresis loop to determine a concentration of said target species.

In embodiments, the transducer of the target species sensor comprises a quartz crystal having metallic electrodes. Alternatively, the transducer comprises a silicon or silicon oxide body, and wherein said recognition agent is covalently bonded to a surface of said silicon or silicon oxide body. Alternatively, the transducer comprises a metal body (for example a MEMS (Microelectromechanical System)).

The present invention further provides a method of generating a library comprising data representing transduced signal data from a sensor for determining a concentration of a target species in an analyte using said sensor, said sensor comprising a transducer that oscillates in response to a drive signal applied to said sensor, said method comprising: exposing a prepared surface of said sensor to an analyte comprising a known concentration of a target species for a first period, said prepared surface of said sensor comprising a recognition agent that selectively bonds with said target species, and wherein said first period is sufficient for said target species to bond with said prepared surface; applying a driving signal to said sensor, said driving signal comprising an alternating voltage having a frequency substantially equal to a resonant frequency of said sensor, and said driving signal causing said sensor to oscillate; ramping an amplitude of said driving signal over a second period between a minimum voltage sufficient to cause said sensor to oscillate, and a maximum voltage; recording 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 said oscillation of said sensor; and storing said recorded amplitude and/or phase of said transduced signal in a library.

A library of recorded amplitudes and/or phase of a transduced signal of known concentrations of target species in analytes enables a concentration of a target species to be calculated using the methods described above.

In embodiments, the maximum voltage is below a voltage sufficient to cause detachment of said target species bonded with said prepared surface from said prepared surface. This enables the measurements to be repeated.

In embodiments, the maximum voltage is a voltage sufficient to cause detachment of said target species bonded with said prepared surface from said prepared surface, and wherein a value representing said maximum voltage sufficient to cause detachment of said target species from said prepared surface is stored in said library. By storing this parameter, future measurements to determine the concentration of a target species in an analyte may be performed, as the point at which the target species bonded to the prepared surface detaches from the prepared surface is then a known parameter.

In preferred embodiments, the method comprises determining a magnitude of oscillation of said transducer to provide oscillation magnitude data representing a magnitude of oscillation of said transducer, and wherein said determining a magnitude of oscillation of said transducer comprises using a voltage and a current of said transduced signal at said driving frequency to calculate said magnitude of oscillation of said transducer, and wherein said oscillation magnitude data is stored in said library.

Preferably, the method comprises repeating said applying, said ramping and said recording steps to produce a plurality of recorded amplitudes and/or phases of said transduced signals, and wherein said plurality of recorded amplitudes and/or phases of said transduced signals are averaged, and wherein said averaged signals are stored said library.

Preferably, said odd harmonic at which the transducer signal is recorded is a third or fifth harmonic of said driving frequency.

Preferably, said drive signal is ramped from said minimum voltage to said maximum voltage and/or from said maximum voltage to said minimum voltage.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows an example system used for detecting the concentration of target species in an analyte; Figure 2 shows an alternative system of figure 1; and Figure 3 shows an alternative system of figures 1 and 2; and Figures 4 to 14 show experimental results obtained from a target species sensor.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Figure 1 shows an example system used for detecting the concentration of target species in an analyte.

Generator 1 with controllable frequency and amplitude (e.g. Agilent 33220A) produces sinusoidal signal at its output (e.g. 15 MHz in this embodiment). The signal is amplified by power amplifier 2 to increase the voltage amplitude to several volts (e.g. 8 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 (eg. cut-off 20 MHz).

The filtered signal is fed to one electrode of transducer 4 (e.g. a quartz crystal with fundamental resonance 15 MHz). A second electrode of transducer 4 produces electric current at all frequencies (including harmonics) that are fed into the input of a High Pass Filter 5 that has a pass frequency band starting well above driving frequency (e.g. 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 SF or the fifth harmonic of the driving frequency).

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 -1EEE488 for receiver). The obtained data are further processed to deliver measurement results.

Figure 2 shows an alternative system setup. The system is largely similar to figure 1, but shows more detail. Generator 1 with controllable frequency and amplitude (e.g. Analog Devices AD9912) produces sinusoidal signal at DAC_OUT output (e.g. 15 MHz in this embodiment).

The signal is filtered to remove higher harmonics by Low Pass Filter LPF 2 with cut-off frequency above the driving frequency but well below 3rd harmonic of 45MHz (e.g. cut-off 20 MHz). The resulting signal is further amplified by power amplifier 3, which produces several volts amplitude (e.g. 8V).

After final filtering to remove higher harmonics by Low Pass Filter 4 (e.g. similar to filter 2) the resulting drive signal, which is clean from harmonic content, is fed to one electrode of transducer 5 (e.g. quartz crystal with fundamental resonance 15 MHz). A second electrode on the transducer 5 produces electric current at all frequencies (including harmonics) and is fed to input of a High Pass Filter 6, which has a pass frequency band starting well above driving frequency (e.g. 35 MHz).

The resultant filtered signal is fed to IFIN input of a general purpose receiver (e.g. Analog Devices AD9874 low noise heterodyne receiver). The receiver is tuned to a desired harmonic frequency (e.g. 3F or third harmonic of driving, or SF or the fifth harmonic frequency).

The output sinusoid from filter 2 also drives input FDBK IN of clock comparator and driver producing digital clock synchronous with driving frequency on output OUT_CMOS, which then serves as a master clock for receiver 7 by feeding it to FREF input of receiver.

After preparation of sensor surface (discussed below) the generator is then controlled by computer 9 varying drive amplitude via control interface 8 while the amplitude (and preferably phase) of harmonic content of the transducer current is digitised by receiver 7 and transferred to computer via control interface 8. Obtained data are further processed to deliver measurement results.

Figure 3 shows a further alternative system setup.

The output signal from Variable Gain Amplifier 1 (VGA) with voltage controllable gain (e.g. Analog Devices AD8367) is filtered by Band Pass Filter 2 (BP1F), which feeds one electrode of quartz resonator 3. The second electrode of the resonator 3 carries electric current propagated through resonator fed back to input of amplifier 1. The central frequency of Band Pass Filter 2 substantially matches fundamental resonance (1 F) of quartz resonator 3 (e.g. 15 MHz).

The circuit self oscillates if the gain of the amplifier is sufficient high; the phase shift across the amplifier and filter 2 is provided by design. The Voltage on resonator 2 is detected by detector-regulator 4 (e.g. Analog Devices AD8318) and used by regulator 4 for control of amplifier 1 to obtain the desired amplitude set-up level provided by Digital to Analog Amplifier (DAC) 5 on Amp Set reference input of regulator.

The DAC 5 is controlled by computer to provide repeated ramps or saw-tooth wavewform.

The second electrode of resonator 3 carrying electric current at all frequencies (i.e. including harmonics) is fed to input of Band Pass Filter 7 (BP3F) with central frequency matching 3 times of driving frequency or third harmonic of it.

The filtered signal is fed to the input of a general purpose low noise RF detector 8 (e.g.Analog Devices AD606). Detected output of it carries 3F harmonic of current digitised by Analog To Digital Converter (ADC) and finally digital data from ADC recorded by computer 6.

Obtained data are further processed to deliver measurement results.

In a preferred embodiment of the invention, the sensor comprises a quartz crystal sandwiched between gold electrodes coated with a self-assembled monolayer (SAM) of receptor molecules. For the purposes of this application, we describe a mixed SAM of biotinylated thiol and hydroxyl-terminated thiol, although the skilled reader would appreciate that other SAMs with different receptor molecules could be used.

The analyte used is a solution of 5 micron streptavidin-coated polystyrene beads in Dl water. Biotin-streptavidin binding pair was chosen since their interaction closely represents the strength of typical antigen-antibody type bonds. The presence of hydroxyl terminated thiol reduces steric hindrance and improves yield of beads capture.

The percentage of biotinylated thiol used is 25%. This percentage was arrived at after multiple trial experiments to test maximum yield of beads on the surface from an analyte of given concentration.

The sensor is driven by a purely sinusoidal AC voltage at fundamental resonant frequency (F) of the combined system of sensor with beads at linearly increasing amplitude from 0.07 to 2 V RMS. The output signal is rich in harmonics. The harmonics arise primarily due to the nonlinearity in quartz motion at higher amplitudes of oscillation and nonlinear dynamics of the adsorbents of the analyte on the sensor surface. The nonlinearity from the latter is much greater in magnitude than that from the former. Nonlinear dynamics of adsorbed particles on the sensor surface include the nonlinear forces of interaction between the particles and the sensor due to the oscillation and diffusion of the former on the latter. Charge induced by even harmonics of nonlinear shear forces on quartz cancel out and so only odd harmonics of the drive frequency are present in the output electrical signal. Since quartz is a single-port device the output signal interferes with the drive signal. Hence if the sensor is driven at fundamental resonant frequency (1 F), the detection is made at third (3F) or higher odd harmonics. In this case the third harmonic (3F) signal was measured.

The method of determining a concentration of a target species in an analyte shall now be described.

1. A surface of the sensor is prepared with desired recognition properties to specifically interact with target species.

2. An analyte with a mixture of target and non-target species of unknown concentrations is brought into contact with the surface and allowed time to attach with the surface both specifically (i.e. the target species selectively bonds with the recognition agent) and non-specifically (i.e. merely resting on the surface of the sensor).

3. The surface is brought into controllable periodical mechanical oscillation by driving the quartz oscillator using a driving signal. The driving signal has a frequency substantially equal to the resonant frequency of the sensor.

4. The amplitude of oscillation is varied in time in a controlled manner in the operating range of interest (i.e. between a minimum value to cause the sensor to oscillate, and a maximum value that is below the value sufficient to cause detachment of the target species bonded with the prepared surface from the prepared surface).

The operating range of amplitude variation is dependent on the target species and the sensor and is determined from a library of known parameter for known target species and sensor surface combinations (the generation of the library is discussed below).

In this range of operation, the interaction between the non-specific species and the surface undergo irreversible change. The non-specific species may lose contact with surface or diffuse away to quieter area of the surface. Hence, the signal contribution from non-specifically interacting species is not reproducible and disappears in second or subsequent scans.

The amplitude of oscillation can be either monotonically increased or monotonically decreased or both. This variation in amplitude can be achieved by directly varying the amplitude of drive or by varying the frequency of drive, which in turns varies the amplitude of vibration, or by both.

5. The anharmonic periodical interaction forces (forces with frequencies that are multiples of driving frequency) between the attached target species and the surface are transduced into an electrical signal, sometimes called the anharmonic signal. The magnitude and/or phase of this transduced signal is recorded at an odd harmonic of the driving frequency (eg 3F or 5F etc).

6. Furthermore, a calculation of the magnitude of oscillation of the transducer can be made from the Voltage and Current of the transduced signal at the driving frequency. The magnitude and/or phase of the one or more frequency components in the anharmonic signal can then be recorded separately as functions of amplitude of mechanical oscillation of the surface. For example, the magnitude of the fifth harmonic (5F) of the drive frequency in the anharmonic signal is recorded as a function of the amplitude of oscillation.

7. To separate out the signal contribution from only the target species, the scans are repeated until the signal stabilizes and becomes reproducible. The driving signal is ramped a number of times and the recorded signals are compared with previously obtained signals from earlier ramps. When the signals are reproduced substantially the same between from one ramp to the next, this is an indication that target and non-target species merely resting on the surface of the sensor (i.e. not specifically bonded with the recognition agent) have been removed from the surface of the sensor. The sensor is now sufficiently clean' to proceed with measurements.

The functions from the reproducible scans are then averaged and recorded. This process of repetition also helps to improve the Signal to Noise ratio of the results as the noise is averaged out, hence the sensitivity and accuracy of quantification are improved.

8. If the shapes of the averaged functions are similar with the library functions, these averaged functions are then used for comparision in terms of both magnitude and shape with the stored library functions for a known concentration of the target species on the sensor surface recorded in the calibration stage. The results of comparision give qualitative and quantitative information on the concentration of target species in the analyte.

For example, a ratio is taken between the averaged function of magnitude of the fifth harmonic (5F) of the drive frequency in the anharmonic signal versus amplitude of oscillation and the corresponding library function. This ratio multiplied by the concentration of the target species in the library calibrating sample gives the concentration of the species in the analyte.

9. If the shapes do not resemble that of the library functions that implies there is more than one species whose interaction with the surface is close to specific and reversible in the range of operation. In such a scenario, library functions are prepared for all possible species that might be present in the analyte and whose interaction with the surface is close to that of the target species for the recognition agent concerned. A linear fitting of the experimental functions to the set of library functions for the different known species is performed. This gives the concentration of each of the specifically interacting species listed in library.

By ramping a driving signal up and down, it is possible to determine a hysteresis loop in the plot of the 3F response versus amplitude. One may determine the area of the hysteresis to allow determination of the concentration of the target species.

The device is calibrated and the library of known data (against which results are compared) is carried out as follows: 1. The sensor and sensor surface are prepared as described in steps 1 and 2 of the main method above. However, in this instance, the concentration of the target species is known.

2. For a given combination of a known concentration of target species and a surface with the desired specific recognition properties, anharmonic signals are generated as described in steps 3 to 7 given for the main method above.

3. The range of variation of the amplitude of oscillation is increased until the anharmonic signal becomes irreproducible indicating irreversible changes in the interaction forces between the target species and the surface (i.e. indicating that the target species bonded with the prepared surface has now detached from the prepared surface). The safe reproducible range (i.e. the range over which the target species remains bonded to the prepared surface) becomes a library parameter for the concerned target species and surface.

4. The functions obtained at this operating range of amplitude are averaged and recorded as library functions for the given combination of target species of the known concentration and the surface. These functions represent the nature of interaction of the species of interest with the surface carrying the specific recognition agent. For different profiles of interaction potential, the recorded anharmonic signal functions are different too.

5. In cases where the same sensors need to be calibrated for more than one species, the library functions are recorded for known concentrations of the different species in the same way as that of the target species described above. The operating amplitude range is however the same as that determined for the target species.

We shall now describe measured results obtained from such a system.

In the following results, a bare quartz, clean sensor and sensor with physisorbed beads was used. Physisorption takes place when the sensor surface has no receptor for the antigen. In this case the SAM constituted of 100% hydroxyl-terminated thiol and no biotin.

Fig. 4 shows the 3F signal measured from the three versus time.

Fig. 5 shows the 3F signals from the three sensors plotted against amplitude of quartz oscillation, calculated using the quality factor of the three systems. This demonstrates 1 5 that the 3F signal from the sensor with beads is significantly distinguishable from those from the other two. It may be noted that 3F signal from quartz is slightly higher than that from bare sensor. This may be attributed to the adsorbent on the bare gold electrode surface, since gold has higher affinity toward adsorbing gases and moisture compared to bare sensor, which is gold surface coated with SAM.

The magnitude of 3F signal normalised by amplitude of quartz motion (in nm) reveals interesting shape of the signal (Fig. 6). The hill' shape of the signal at the beginning is caused due to onset of diffusion of the physisorbed beads on the surface. This hill feature was absent for chemisorbed beads i.e. where specific binding took place between streptavidin on the beads and the biotin of the SAM on sensor surface.

Computation modelling done matches qualitatively with these experimental observations. A quantitative model is being developed with realistic parameters used in experiments.

More recent experiments, with chemisorbed beads of different concentration, reveal the potential for quantitative detection using this technique. The following figures show results obtained from three sensors with different concentrations of beads chemisorbed onto the surface. Fig. 7 plots the magnitude of 3F signal versus time.

Fig. 8 plots the magnitude of the 3F signal versus amplitude of quartz oscillation. The proportionality of 3F signals for the three sensors can be clearly noted.

Fig. 9 plots the magnitude of 3F signal per unit of quartz oscillation. Absence of the hill' shaped feature reveals that no diffusion of the chemisorbed beads initiated due to quartz oscillation.

Fig. 10 plots the magnitude of 3F signal observed from three successive scans of one particular sensor (with 20 beads) against time. This demonstrates reproducibility of the results.

By fitting the 3F signal obtained for the abovementioned three sensors, it was found that the 3F signal is a linear function of the cubic of amplitude of quartz oscillation (Fig. 11).

Qualitative measuring of number of beads: The slope of the fitting function (as shown in Fig.1 1) is also estimated by computational modelling to be linearly proportional to the density or number of particles on sensor surface. This is supported by observations (Fig. 11) from the experiments. The ratio of slope35O:slopelOO is 3.478, which is 0.6% away from ratio of numbers of beads counted under microscope, which is 350:100 i.e. 3.5. Standard statistical deviation error for the fit is estimated as 11%.

Similarly slope35O/slope2o = 22, which is 27% away from counted ratio of 17.5 with STDev=23% (well inside statistical error bars of +1-2*STDev).

Finally slopelOO/slope2o = 6.4, which is 28% away from counted ratio of 17.5 with STDev=24% (well inside statistical error bars of +1-2*STDev as well).

As discussed, at low oscillation amplitudes well inside onset of diffusion, 3F signal exists and is reproducible in second and further scans. We discovered in this region signal value is a cubic function of oscillation amplitude specifically bound particles (Fig. 10). This implies that tether of streptavidin-biotin thiol bond that holds the beads and is around 5 nm long, imparts lower force on the bead at smaller amplitudes of oscillation and the force rises with rising amplitude. The 3F signal from non-specifically bound or physisorbed beads is however a square function (graphs being prepared) of the amplitude of oscillation. Modelling of motion of beads in periodical potential (both chemisorbed and physisorbed) yield exactly the same conclusion. The fundamental reason behind this is the difference in the shape of the interaction potential at its bottom. So, the shape of 3F signal carries direct information on the profile of potential well which is fundamental to an adsorbate-surface pair. So it is claimed that functions of 3F signal truly represents the nature of interaction between adsorbate and surface and can be used to analyse a sample with a mixture of different kinds of adsorbates (specific and non-specific) from previously known interaction potential or 3F characterisation.

The following results demonstrate the potential of the technique to be used as a gas sensor. Fig. 12 and Fig. 13 demonstrate the difference in the 3F signal obtained with N2 flow on and off.

The primary difference in the cases with N2 flow on and N2 flow off is the presence of moisture and 02 in the latter as opposed to the former. The difference is the 3F signal is much more significant that the resonant frequency or even quality factor change in the two cases.

Fig. 14 demonstrates the reproducibility of the 3F signal in successive scans when the N2 flow is kept off.

We shall now describe alternative embodiments of the invention Oscillatory Surface 1. The oscillatory surface can be quartz with metallic electrodes coated with recognitions agents. For a biosensor application, the metallic electrodes can be gold and the receptors can be attached to the gold electrodes via gold-thiol interaction.

2. The oscillatory surface can be a silicon or silicon oxide body with a covalently bonded recognition agent.

3. The oscillatory surface can be pure metal or any solid material body. In embodiments, this metal body can comprise a MEMS (Microelectromechanical System).

Medium The sensor can perform its detection in vacuum, air and liquid medium.

Mode of Oscillation 1. Oscillation can be made to occur in the plane of the surface.

2. Oscillation can be made to occur normal to the surface.

3. Oscillation can be made to occur in combination of the above like in Surface Acoustic Wave (SAW).

Method of Drive (dependent on the transducer) 1. The drive can be an electromagnetic force.

2. The drive can be an electrostatic force.

3. The drive can be piezoelectric.

4. The drive can be magnetostriction force.

5. The drive can be an acoustic wave.

6. The drive can be a combination of two or more of the above.

Method of Transduction of anharmonic interaction forces (dependent on the transducer) 1. The transduction can be electromagnetic.

2. The transduction can be electrostatic.

3. The transduction can be piezoelectric.

4. The transduction can be piezoresistive.

5. The transduction can be optical 6. The transduction can be a combination of two or more of the above.

Driving and receiving frequency: The driving of the sensor can be performed at single frequency or two or more different frequencies. In the case of two or more driving frequencies the anharmonic forces may be received at combinatorial frequencies such as sum or difference of driving frequencies.

1. The sensor is driven at close to one of its overtone frequencies (fundamental resonant frequency or higher) and received at odd harmonic of the driving frequency (third or higher).

2. The sensor is driven at two frequencies simultaneously and received at sum or difference of the frequencies.

3. As in 2 where the sensor is driven at frequencies close to overtone frequencies (fundamental resonant frequency or higher) of the sensor-analyte system.

4. As in 3 where the sensor is driven at frequencies close to odd and even overtones and received at close to odd overtone.

5. As in 3 where the sensor is driven at frequencies close to odd overtones and received at close to even overtone.

6. As in 3 where the sensor is driven at frequencies close to even overtones and received at close to odd overtone.

7. As in 2 where one surface is in liquid and one of the excitations is applied acoustically via liquid.

8. As in 2 where at least one surface is in liquid and receiving is done acoustically via liquid.

9. As in 2 to 8, where the types of drive are different Possible Applications 1. The technique described above can be employed with any set of chosen surface, oscillation type, drive and transduction method as listed above and used as a biosensor with a specific recognition agent for the target biological entity immobilized on the surface.

2. The same as described in 1 can be used as a gas sensor with the specific recognition agent for the gas coated on the surface.

3. The technique described under Description of Main Claim can be employed with any set of chosen oscillatory surface, mode of oscillation, method of drive and transduction and driving and receiving frequencies as listed above along with introduction of microbeads, also functionalized with same recognition agent, and used as a biosensor. The microbeads can be magnetic and can be driven to come in contact with the surface or pulled away using an external magnetic field.

4. The same as described in 3 can be used as a gas sensor.

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 a concentration of a target species in an analyte, comprising: exposing a prepared surface of a sensor to an analyte comprising a target species for a first period, 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 recognition agent that selectively bonds with said target species, and wherein said first period is sufficient for said target species to bond with said prepared surface; applying a driving signal to said sensor, said driving signal comprising an alternating voltage having a frequency substantially equal to a resonant frequency of said sensor, and said driving signal causing said sensor to oscillate; ramping an amplitude of said driving signal over a second period between a minimum voltage sufficient to cause said sensor to oscillate, and a maximum voltage, said maximum voltage being below a voltage sufficient to cause detachment of said target species bonded with said prepared surface from said prepared surface; recording 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 said oscillation of said sensor; and comparing data derived from said recorded transduced signal with a library to determine a concentration of said target species, said library representing transduced signal data from known concentrations of said target species.
2. A method according to claim 1, comprising: determining a magnitude of oscillation of said transducer to provide oscillation magnitude data representing a magnitude of oscillation of said transducer, and wherein said comparing comprises comparing data derived from said determined oscillation magnitude data with said library, said library further comprising oscillation magnitude data from known concentrations of said target species.
3. A method according to claim 2, wherein said determining of said magnitude of oscillation comprises using a voltage and a current of said transduced signal at said driving frequency to calculate said magnitude of oscillation of said transducer.
4. A method according to claims 1, 2 or 3, comprising repeating said applying, said ramping and said recording steps to produce a plurality of recorded amplitudes and/or phases of said transduced signals, and wherein said plurality of recorded amplitudes and/or phases of said transduced signals are averaged, and wherein said averaged signals are compared with said library.
5. A method according to claims 1, 2, 3, or 4, wherein said odd harmonic is a third or fifth harmonic of said driving frequency.
6. A method according to any preceding claim, wherein said drive signal is ramped from said minimum voltage to said maximum voltage and/or from said maximum voltage to said minimum voltage.
7. A method according to any one of claims 1 to 6, wherein said driving signal is ramped from said minimum voltage to said maximum voltage and from said maximum to said minimum voltage to reveal a hysteresis ioop in said recorded transducer signals, and wherein an area of said hysteresis loop is determined to determine a concentration of said target species.
8. A method according to any preceding claim, comprising performing a cleaning step prior to said applying a drive signal to remove target and non-target species that are not bonded with said prepared surface from said prepared surface, said cleaning step comprising: applying said driving signal to said sensor; ramping an amplitude of said driving signal between said minimum voltage and said maximum voltage two or more times; 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 ramping step with data derived from said recorded transducer signal from a previous ramping step until said data is substantially reproduced from one ramping step to the next ramping step, thereby indicating that target and non-target species not bonded with said prepared surface are no longer on said surface.
9. A method according to any preceding claim, wherein said prepared surface comprises a plurality of beads covalently bonded with said surface of said transducer, and wherein said beads comprise a recognition agent.
10. A method according to any preceding claim, wherein said prepared surface comprises regions of recognition agent.
11. A method according to any preceding claim, wherein said target species is a gas.
12. A method according to any preceding claim, wherein said transducer comprises a quartz crystal having metallic electrodes.
13. A method according to any one of claims 1 to 12, wherein said transducer comprises a silicon or silicon oxide body, and wherein said recognition agent is covalently bonded to a surface of said silicon or silicon oxide body.
14. A method according to any one of claims 1 to 12, wherein said transducer comprises a metal body.
15. A method according to claim 14, wherein said metal body comprises a MEMS (Microelectromechanical System).
16. A target species sensor for determining a concentration of a target species in an analyte, comprising: a transducer that oscillates in response to a drive signal applied to said transducer, said transducer comprising a prepared surface comprising a recognition agent that selectively bonds with a target species; a driving signal generator for generating a driving signal comprising an alternating voltage having a frequency substantially equal to a resonant frequency of said transducer and wherein said driving signal generator controllably ramps an amplitude of said driving signal between a minimum voltage sufficient to cause said transducer to oscillate, and a maximum voltage, said maximum voltage being below a voltage sufficient to cause detachment of said target species bonded with said prepared surface from said prepared surface; a data recorder for recording 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; a comparator for comparing data derived from a recorded transduced signal with a library to determine a concentration of said target species, said library representing transduced signal data from known concentrations of said target species.
17. A target species sensor according to claim 16, comprising a data processor for determining a magnitude of oscillation of said transducer to provide oscillation magnitude data representing a magnitude of oscillation of said transducer, and wherein said comparator compares data derived from said determined oscillation magnitude data with said library, said library further comprising oscillation magnitude data from known concentrations of said target species
18. A target species sensor according to claim 17, wherein said data processor calculates said magnitude of oscillation using a voltage and a current of said transduced signal at said driving frequency.
19. A target species sensor according to claim 16, 17 or 18, wherein said sensor comprises an averager for averaging a plurality of amplitudes and/or phases of a plurality of said transduced signals, and wherein said comparator compares said averaged amplitudes and/or phases of said plurality of transduced signals are compared with said library.
20. A target species sensor according to any one of claims 16 to 19, wherein said odd harmonic is a third or fifth harmonic of said driving frequency.
21. A target species sensor according to any one of claims 16 to 21, wherein said driving signal generator generates a driving signal comprising an amplitude ramped from said minimum voltage to said maximum voltage and/or from said maximum voltage to said minimum voltage.
22. A target species sensor to any one of claims 16 to 21, wherein said driving signal generator generates a driving signal comprising an amplitude ramped from said minimum voltage to said maximum voltage and from said maximum to said minimum voltage to reveal a hysteresis loop in said recorded transducer signals, and wherein a data processor determines an area of said hysteresis ioop to determine a concentration of said target species.
23. A target species sensor according to any one of claims 16 to 22, wherein said prepared surface comprises a plurality of beads covalently bonded with said surface of said transducer, and wherein said beads comprise a recognition agent.
24. A target species sensor according to any one of claims 16 to 23, wherein said prepared surface comprises regions of recognition agent.
25. A target species sensor according to any one of claims 16 to 24, wherein said target species is a gas.
26. A target species sensor according to any one of claims 16 to 25, wherein said transducer comprises a quartz crystal having metallic electrodes.
27. A target species sensor according to any one of claims 16 to 25, wherein said transducer comprises a silicon or silicon oxide body, and wherein said recognition agent is covalently bonded to a surface of said silicon or silicon oxide body.
28. A target species sensor according to any one of claims 16 to 25, wherein said transducer comprises a metal body.
29. A target species sensor according to claim 28, wherein said metal body comprises a MEMS (Microelectromechanical System).
30. A method of generating a library comprising data representing transduced signal data from a sensor for determining a concentration of a target species in an analyte using said sensor, said sensor comprising a transducer that oscillates in response to a drive signal applied to said sensor, said method comprising: exposing a prepared surface of said sensor to an analyte comprising a known concentration of a target species for a first period, said prepared surface of said sensor comprising a recognition agent that selectively bonds with said target species, and wherein said first period is sufficient for said target species to bond with said prepared surface; applying a driving signal to said sensor, said driving signal comprising an alternating voltage having a frequency substantially equal to a resonant frequency of said sensor, and said driving signal causing said sensor to oscillate; ramping an amplitude of said driving signal over a second period between a minimum voltage sufficient to cause said sensor to oscillate, and a maximum voltage; recording 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 said oscillation of said sensor; and storing said recorded amplitude and/or phase of said transduced signal in a library.
31. A method according to claim 30, wherein said maximum voltage is below a voltage sufficient to cause detachment of said target species bonded with said prepared surface from said prepared surface.
32. A method according to claim 30, wherein said maximum voltage is a voltage sufficient to cause detachment of said target species bonded with said prepared surface from said prepared surface, and wherein a value representing said maximum voltage sufficient to cause detachment of said target species from said prepared surface is stored in said library.
33. A method according to claim 30, 31, or 32, comprising: determining a magnitude of oscillation of said transducer to provide oscillation magnitude data representing a magnitude of oscillation of said transducer, and wherein said determining a magnitude of oscillation of said transducer comprises using a voltage and a current of said transduced signal at said driving frequency to calculate said magnitude of oscillation of said transducer, and wherein said oscillation magnitude data is stored in said library.
34. A method according to any one of claims 30 to 33, comprising repeating said applying, said ramping and said recording steps to produce a plurality of recorded amplitudes and/or phases of said transduced signals, and wherein said plurality of recorded amplitudes and/or phases of said transduced signals are averaged, and wherein said averaged signals are stored said library.
35. A method according to any one of claims 30 to 34, wherein said odd harmonic is a third or fifth harmonic of said driving frequency.
36. A method according to any one of claims 30 to 35, wherein said drive signal is ramped from said minimum voltage to said maximum voltage and/or from said maximum voltage to said minimum voltage.
37. A method according to any one of claims 30 to 36, wherein said driving signal is ramped from said minimum voltage to said maximum voltage and from said maximum to said minimum voltage to reveal a hysteresis loop in said recorded transducer signals, and wherein an area of said hysteresis loop is determined to determine a concentration of said target species, and wherein said area is stored in said library.
38. A method according to any one of claims 30 to 37, comprising performing a cleaning step prior to said applying a drive signal to remove target and non-target species that are not bonded with said prepared surface from said prepared surface, said cleaning step comprising: applying said driving signal to said sensor; ramping an amplitude of said driving signal between said minimum voltage and said maximum voltage two or more times; 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 ramping step with data derived from said recorded transducer signal from a previous ramping step until said data is substantially reproduced from one ramping step to the next ramping step, thereby indicating that target and non-target species not bonded with said prepared surface are no longer on said surface.