[go: up one dir, main page]

WO2011033285A1 - Appareil et procédé permettant de détecter des espèces cibles dans un analyte - Google Patents

Appareil et procédé permettant de détecter des espèces cibles dans un analyte Download PDF

Info

Publication number
WO2011033285A1
WO2011033285A1 PCT/GB2010/051511 GB2010051511W WO2011033285A1 WO 2011033285 A1 WO2011033285 A1 WO 2011033285A1 GB 2010051511 W GB2010051511 W GB 2010051511W WO 2011033285 A1 WO2011033285 A1 WO 2011033285A1
Authority
WO
WIPO (PCT)
Prior art keywords
sensor
target species
signal
transducer
frequency
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB2010/051511
Other languages
English (en)
Inventor
Sourav Ghosh
Victor Ostanin
Ashwin Seshia
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cambridge Enterprise Ltd
Original Assignee
Cambridge Enterprise Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cambridge Enterprise Ltd filed Critical Cambridge Enterprise Ltd
Publication of WO2011033285A1 publication Critical patent/WO2011033285A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01G—WEIGHING
    • G01G3/00—Weighing apparatus characterised by the use of elastically-deformable members, e.g. spring balances
    • G01G3/12—Weighing apparatus characterised by the use of elastically-deformable members, e.g. spring balances wherein the weighing element is in the form of a solid body stressed by pressure or tension during weighing
    • G01G3/13—Weighing apparatus characterised by the use of elastically-deformable members, e.g. spring balances wherein the weighing element is in the form of a solid body stressed by pressure or tension during weighing having piezoelectric or piezoresistive properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01G—WEIGHING
    • G01G3/00—Weighing apparatus characterised by the use of elastically-deformable members, e.g. spring balances
    • G01G3/12—Weighing apparatus characterised by the use of elastically-deformable members, e.g. spring balances wherein the weighing element is in the form of a solid body stressed by pressure or tension during weighing
    • G01G3/16—Weighing apparatus characterised by the use of elastically-deformable members, e.g. spring balances wherein the weighing element is in the form of a solid body stressed by pressure or tension during weighing measuring variations of frequency of oscillations of the body

Definitions

  • the present invention relates to apparatus and methods for detecting and determining the concentration of target species in an analyte.
  • the present invention relates to apparatus and methods for detecting and determining the concentration of target species in an analyte using oscillating transducers, and methods of generating a library of data from the oscillating transducers.
  • 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.
  • the present invention provides a method of detecting and 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
  • the library enables the concentration to be determined by comparing the recorded transduced signals with transduced signals for known concentrations of the target species.
  • 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 transduced signal data in said library further comprising oscillation magnitude data.
  • 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.
  • a plot of the transducer signal (for example magnitude and/or phase at the fifth harmonic - 5F, i.e. 5 times the drive frequency F) can be recorded as a function of amplitude of mechanical oscillation of the surface.
  • said transducer signal which is magnitude and/or phase at third of higher odd harmonic of the drive frequency F (3F or 5F or higher), can be recorded as a function of current at F i.e. drive frequency.
  • 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.
  • the transducer signal is recorded at odd harmonic, such as the third or fifth harmonics, of said driving frequency.
  • the drive signal is ramped from said minimum voltage to said maximum voltage and/or from said maximum voltage to said minimum voltage.
  • 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.
  • the method further comprises: obtaining baseline data from said sensor, said baseline data defining an amplitude and/or phase of a transduced signal at substantially an odd harmonic frequency of said driving frequency when there is substantially no bonding between said target species and said recognition agent; comparing said baseline data with said data derived from said recorded transducer signal when said target species is bonded to said recognition agent, to identify a deviation between said baseline data and said derived data, wherein said comparing data derived from said recorded transduced signal with a library comprises comparing said deviation with said library to determine a concentration of said target species.
  • obtaining said baseline data is performed prior to said exposing step and wherein said obtaining comprises: exposing said prepared surface of said sensor to an analyte substantially devoid of said target species; applying said driving signal to said sensor; ramping an amplitude of said driving signal over a second period between said minimum voltage and said maximum voltage; and 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, said transduced signal defining said baseline signal.
  • said obtaining said baseline data comprises: exposing a prepared surface of a second transducer comprising said sensor, to said analyte comprising said target species for said first period, said prepared surface of said second transducer being substantially devoid of said recognition agent; and performing said applying and said ramping to said sensor comprising said transducer and said second transducer, wherein said recording step further comprises recording an amplitude and/or phase of a second transduced signal from said second transducer at substantially an odd harmonic frequency of said frequency of said driving signal, said second transduced signal resulting from said oscillation of said sensor, said second transduced signal defining said baseline signal.
  • the method comprises performing a cleaning step prior to said applying of 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 reproducible between ramps of the driving signal and ensures than non-specific attachments do not contribute to false positives.
  • the prepared surface comprises a plurality of beads covalently bonded with said surface of said transducer, and wherein both the said surface and the said beads comprise a recognition agent.
  • the target species is a gas or moisture
  • the prepared surface provides an area creating pockets for gas or moisture underneath beads thus increasing the sensitivity of detection.
  • the prepared surface comprises regions of recognition agent.
  • the transducer comprises a quartz crystal having metallic electrodes and said recognition agent is bonded to one or both of said electrodes via metal-sulphur bond between said metal electrode and the sulphur of the thiol group of one terminal of the receptor, which is the terminal opposite to the one having said recognition agent, e.g. said recognition agent is bonded via Au-S bond between a gold electrode and the sulphur.
  • gold electrode can be replaced with harder metals like platinum to stop diffusion at high amplitudes.
  • said recognition agent is bonded covalently to electrodes of a quartz crystal by having layers of chromium, silicon and/or silicon oxide on said electrode (for example using silane chemistry).
  • the quartz can be electrodeless (driven using an electric field) and the recognition agent can be covalently bonded onto the quartz.
  • 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.
  • the transducer comprises a metal body.
  • 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
  • the library enables the concentration to be determined by comparing the recorded transduced signals with transduced signals for known concentrations of the target species.
  • the senor 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 transduced signal data in said library further comprising oscillation magnitude data.
  • the data processor calculates said magnitude of oscillation using a voltage and a current of said transduced signal at said driving frequency.
  • the senor 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.
  • the odd harmonic at which data recorder records data is a third or fifth (or any higher odd) harmonic of said driving frequency.
  • 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.
  • 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.
  • the prepared surface comprises a plurality of beads covalently bonded with said surface of said transducer, and wherein said beads comprise a recognition agent.
  • the target species is a gas or moisture
  • the prepared surface provides an area creating pockets for gas or moisture underneath beads thus increasing the sensitivity of detection.
  • the prepared surface comprises regions of recognition agent.
  • the transducer of the target species sensor comprises a quartz crystal having metallic electrodes.
  • 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.
  • the transducer comprises a metal body.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • said odd harmonic at which the transducer signal is recorded is a third or fifth harmonic of said driving frequency.
  • said drive signal is ramped from said minimum voltage to said maximum voltage and/or from said maximum voltage to said minimum voltage.
  • 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.
  • 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.
  • the present invention also 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 an amplitude below a voltage sufficient to cause detachment of said target species bonded with said prepared surface from said prepared surface, and said driving signal causing said sensor to oscillate; ramping a frequency of said driving signal over a second period between a minimum frequency sufficient to cause said sensor to oscillate, and a maximum frequency sufficient to cause said
  • the immobilized analyte or binding partner may be a metal, a polymer, a dendrimer, a self-assembled monolayer, a peptide, a protein, an antibody, an antigen, an enzyme, an enzyme inhibitor, a biologically active molecule, a drug, a polynucleotide or a peptide polynucleotide.
  • 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 20 show experimental results obtained from a target species sensor.
  • 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 (e.g. 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. 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).
  • a general purpose receiver 6 e.g. Stanford Research Systems SR844 Lock-In Amplifier
  • the desired harmonic frequencies e.g. 3F or third harmonic of the driving frequency, or 5F 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.
  • the generator 1 is then controlled (e.g. by computer 7) to make varying drive amplitude via control interface (e.g. Ethernet).
  • 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.
  • 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. cutoff 20 MHz).
  • the resulting signal is further amplified by power amplifier 3, which produces several volts amplitude (e.g. 8V).
  • the resulting drive signal 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).
  • 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 5F 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.
  • FIG. 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 (BP1 F), 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.
  • detector-regulator 4 e.g. Analog Devices AD8318
  • DAC Digital to Analog Amplifier
  • 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.
  • BP3F Band Pass Filter 7
  • 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.
  • ADC Analog To Digital Converter
  • Obtained data are further processed to deliver measurement results.
  • the senor comprises a quartz crystal sandwiched between gold electrodes coated with a self-assembled monolayer (SAM) of receptor molecules.
  • SAM self-assembled monolayer
  • 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.
  • the senor is instead driven at higher overtones.
  • a surface of the sensor is prepared with desired recognition properties to specifically interact with target species.
  • 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). After the target has been given sufficient time to selectively bond with the recognition agent, an optional washing step may be performed to remove non-specific species on the surface of the sensor.
  • 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 (or any higher overtone). 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).
  • 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 (less vibrating) area of the surface.
  • 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
  • the anharmonic signal is 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 (e.g. 3F or 5F etc).
  • the transduced signal is substantially simultaneously recorded at the driving frequency.
  • 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.
  • 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.
  • 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.
  • 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. This ensures that the non-specific attachments do not give rise to false positives.
  • 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.
  • 5F fifth harmonic
  • the concentration may additionally be determined by comparing a baseline signal with the transduced signal described above.
  • the baseline is the 3F (or higher odd F) response against oscillation amplitude or 1 F current in absence of the target-receptor binding.
  • a sample analyte without the target is introduced to the sensor before step 2 given above.
  • a cleaning step may be performed after the introduction of the sample.
  • the 3F (or higher odd F) response against oscillation amplitude or 1 F current is then measured. This is a one time step for the sensor.
  • the method then proceeds as described above in steps 2 onwards to provide the measured transduced signal of the 3F (or higher odd F) response against oscillation amplitude or 1 F current.
  • the deviation in the 3F (or higher odd F) response against oscillation amplitude or 1 F current between the situation where there was no target species in the analyte and the situation where the target species was present in the analyte is the signal of interest and referred to as the Anharmonic Detection (ADT) signal.
  • ADT Anharmonic Detection
  • the deviation denotes the presence of the target.
  • the concentration of the target is measured by comparing the ADT signal from this unknown target concentration with the library noted with known concentration of the target.
  • a second transducer (a reference transducer of the same specifications as the target transducer, but without the immobilized specific receptor for the target) is provided.
  • the analyte can be introduced simultaneously on both the sensor and the reference and the 3F (or higher odd F) response against oscillation amplitude or 1 F 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.
  • ADT Anharmonic Detection
  • the deviation denotes the presence of the target.
  • the concentration of the target is measured by comparing the ADT signal from this unknown target concentration with the library noted with known concentration of the target.
  • the device is calibrated and the library of known data (against which results are compared) generated as follows:
  • 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.
  • 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.
  • 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.
  • the senor may be calibrated and the library generated using the baseline steps described above.
  • 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 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.
  • 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.
  • 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. 1 1 ) from the experiments.
  • the ratio of slope350:slope100 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 1 1 %.
  • 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.
  • Fig. 14 demonstrates the reproducibility of the 3F signal in successive scans when the N2 flow is kept off.
  • Figure 15a presents the 3F electrical response against oscillation amplitude of three successive scans from a sensor with 5.61 ⁇ physisorbed beads and one scan from the same sensor but without beads.
  • the relative increase in the 3F response due to introduction of beads is significant in scan 1 .
  • the response rises steadily at first and later drops and becomes unsteady; this characteristic is found to repeat over the next two scans with magnitude of the response decreasing in successive scans and becoming close to that from scan without beads toward the end of the third scan. This can be explained by the phenomenon of beads diffusing on and desorbing from surface at higher amplitudes.
  • Figure 15b presents the 3F response of successive scans from a sensor, initially without beads and then with specifically captured 0.39 ⁇ beads.
  • 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.
  • 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 16a presents the deviation in 3F response from the baseline response versus the oscillation amplitude due to introduction of different numbers of 0.39 ⁇ 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 15a. 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 ⁇ beads ( Figure 16b).
  • 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.
  • Figure 17 demonstrates that the 3F response deviates with introduction of Bacillus subtilis spores (majority of which are specifically bound). Around 30,000 spores were introduced onto the sensor (30 ⁇ . of 10 6 cfu/mL). cfu stands for colony forming units. It is estimated that in absence of incubation enhancement a significant proportion of the spores did not bind with the sensor in 15 minutes of incubation time allowed. The SNR indicates that the sensitivity is more than one order better than existing QCM and SPR in similar absence of incubation enhancement.
  • Figure 18 presents the deviation in the 3F response with introduction of spores (30 ⁇ . of 10 6 cfu/mL) against oscillation amplitude, which is termed as the anharmonic detection technique (ADT) signal. Signals from three successive scans are found to be reproducible. In contrast ADT signals from non-specifically bound streptavidin-coated polystyrene microbeads (SCPM) are found to decrease on successive scans ( Figure 19 - successive scans with non-specifically bound SCPM).
  • SCPM streptavidin-coated polystyrene microbeads
  • Figure 20 presents the ADT signals from two different concentrations of spores.
  • the signals are approximately proportional to the number of spores estimated on the surface.
  • the oscillatory surface can be quartz with metallic electrodes coated with recognitions agents.
  • the metallic electrodes can be gold and the receptors can be attached to the gold electrodes via gold-thiol interaction.
  • the oscillatory surface can be a silicon or silicon oxide body with a covalently bonded recognition agent.
  • the oscillatory surface can be pure metal or any solid material body.
  • the sensor can perform its detection in vacuum, air and liquid medium. Mode of Oscillation
  • Oscillation can be made to occur in the plane of the surface.
  • Oscillation can be made to occur normal to the surface.
  • Oscillation can be made to occur in combination of the above like in Surface Acoustic Wave (SAW).
  • SAW Surface Acoustic Wave
  • the drive can be an electromagnetic force.
  • the drive can be an electrostatic force.
  • the drive can be piezoelectric.
  • the drive can be magnetostriction force.
  • the drive can be an acoustic wave.
  • the drive can be a combination of two or more of the above
  • the transduction can be electromagnetic.
  • the transduction can be electrostatic.
  • the transduction can be piezoelectric.
  • the transduction can be piezoresistive.
  • the transduction can be optical
  • the transduction can be a combination of two or more of the above.
  • Driving and receiving frequency :
  • the sensor can be driven at a constant voltage and the frequency can be swept about the resonant frequency (against its fundamental or any higher overtone). In either way of driving, it is ensured that the oscillation amplitude of the sensor is below the maximum that initiates any unbinding or diffusion at the surface.
  • 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).
  • the sensor is driven at two frequencies simultaneously and received at sum or difference of the frequencies.
  • 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 or moisture sensor with the specific recognition agent for the gas coated on the surface.
  • 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.

Landscapes

  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Investigating Or Analyzing Materials By The Use Of Ultrasonic Waves (AREA)

Abstract

La présente invention concerne un appareil et des procédés permettant de déterminer la concentration d'espèces cibles dans un analyte à l'aide de transducteurs oscillants, et des procédés de génération d'une bibliothèque de données à partir des transducteurs oscillants. La présente invention concerne un procédé de détermination d'une concentration d'espèces cibles dans un analyte. Le procédé consiste à exposer une surface préparée d'un capteur à un analyte comprenant une espèce cible pendant une première période, ledit capteur comprenant un transducteur qui oscille en réponse à un signal de commande appliqué audit capteur, ladite surface préparée dudit capteur comprenant un agent de reconnaissance qui se lie de façon sélective à ladite espèce cible, et ladite première période étant suffisante pour que ladite espèce cible se lie à ladite surface préparée ; à appliquer un signal de commande audit capteur, ledit signal de commande comprenant une tension alternative dont la fréquence est sensiblement égale à une fréquence de résonance dudit capteur, et ledit signal de commande amenant ledit capteur à osciller ; à augmenter une amplitude dudit signal de commande pendant une seconde période entre une tension minimale suffisante pour entraîner l'oscillation dudit capteur et une tension maximale, ladite tension maximale étant inférieure à une tension suffisante pour amener ladite espèce cible liée à ladite surface préparée à se séparer de ladite surface préparée ; à enregistrer une amplitude et/ou phase d'un signal converti par transduction sensiblement à une fréquence harmonique impaire de ladite fréquence dudit signal de commande, ledit signal converti par transduction résultant de ladite oscillation dudit capteur ; et à comparer des données dérivées dudit signal converti par transduction enregistré avec une bibliothèque afin de déterminer une concentration desdites espèces cibles, ladite bibliothèque représentant des données de signal converti par transduction provenant de concentrations connues desdites espèces cibles.
PCT/GB2010/051511 2009-09-18 2010-09-10 Appareil et procédé permettant de détecter des espèces cibles dans un analyte Ceased WO2011033285A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0916389.0 2009-09-18
GB0916389A GB2473635A (en) 2009-09-18 2009-09-18 Detecting concentration of target species

Publications (1)

Publication Number Publication Date
WO2011033285A1 true WO2011033285A1 (fr) 2011-03-24

Family

ID=41277923

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2010/051511 Ceased WO2011033285A1 (fr) 2009-09-18 2010-09-10 Appareil et procédé permettant de détecter des espèces cibles dans un analyte

Country Status (2)

Country Link
GB (1) GB2473635A (fr)
WO (1) WO2011033285A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013110023A1 (fr) * 2012-01-20 2013-07-25 Purdue Research Foundation Procédé et dispositif d'imagerie optique avec un ensemble amplificateur résonnant

Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998041820A1 (fr) 1997-03-17 1998-09-24 Q-Sense Ab Procede et appareil de mesure des proprietes et des processus des cellules au niveau de surfaces
WO1999030159A1 (fr) 1997-11-24 1999-06-17 Q-Sense Capteur pour detection de substance biologique
WO1999040397A1 (fr) 1998-01-23 1999-08-12 Q-Sense Ab Dispositif destine a un oscillateur piezoelectrique a quartz
WO2000055613A1 (fr) 1999-03-17 2000-09-21 Q-Sense Ab Procede d'examen des proprietes chimico-physiques d'un film mince
WO2001001121A1 (fr) * 1999-06-29 2001-01-04 Ut-Battelle, Llc Analyse plurimodale de structures micromecaniques destinee a la detection d'applications
WO2002095398A1 (fr) * 2001-05-24 2002-11-28 University Of Florida Procede et appareil de detection de l'exposition a un environnement de fumee
US6589727B1 (en) * 1999-04-22 2003-07-08 Akubio Ltd. Measurement and use of molecular interactions
WO2004095011A1 (fr) 2003-04-17 2004-11-04 Akubio Limited Detection de capteurs de phenomenes de rupture
US20050150778A1 (en) * 2002-11-18 2005-07-14 Lewis Nathan S. Use of basic polymers in carbon black composite vapor detectors to obtain enhanced sensitivity and classification performance for volatile fatty acids
WO2005121769A1 (fr) 2004-06-12 2005-12-22 Akubio Limited Appareil d'analyse comprenant une mosaique de capteurs et un element d'etalonnage
WO2006103439A2 (fr) 2005-04-01 2006-10-05 Akubio Limited Cartouche pour analyseur d'echantillons fluides
WO2009005452A1 (fr) 2007-07-04 2009-01-08 Q-Sense Ab Membrane pour microbalance à quartz
WO2009070108A1 (fr) 2007-11-28 2009-06-04 Q-Sense Ab Montage de système de réflectométrie optique

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6222366B1 (en) * 1999-05-10 2001-04-24 Fisher Controls International, Inc. High frequency measuring circuit with inherent noise reduction for resonating chemical sensors

Patent Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998041820A1 (fr) 1997-03-17 1998-09-24 Q-Sense Ab Procede et appareil de mesure des proprietes et des processus des cellules au niveau de surfaces
WO1999030159A1 (fr) 1997-11-24 1999-06-17 Q-Sense Capteur pour detection de substance biologique
WO1999040397A1 (fr) 1998-01-23 1999-08-12 Q-Sense Ab Dispositif destine a un oscillateur piezoelectrique a quartz
WO2000055613A1 (fr) 1999-03-17 2000-09-21 Q-Sense Ab Procede d'examen des proprietes chimico-physiques d'un film mince
US6589727B1 (en) * 1999-04-22 2003-07-08 Akubio Ltd. Measurement and use of molecular interactions
WO2001001121A1 (fr) * 1999-06-29 2001-01-04 Ut-Battelle, Llc Analyse plurimodale de structures micromecaniques destinee a la detection d'applications
WO2002095398A1 (fr) * 2001-05-24 2002-11-28 University Of Florida Procede et appareil de detection de l'exposition a un environnement de fumee
US20050150778A1 (en) * 2002-11-18 2005-07-14 Lewis Nathan S. Use of basic polymers in carbon black composite vapor detectors to obtain enhanced sensitivity and classification performance for volatile fatty acids
WO2004095012A1 (fr) 2003-04-17 2004-11-04 Akubio Limited Oscillateur a quartz
WO2004095011A1 (fr) 2003-04-17 2004-11-04 Akubio Limited Detection de capteurs de phenomenes de rupture
US20070062287A1 (en) * 2003-04-17 2007-03-22 Ostanin Victor P Rupture event sensors
US7570125B2 (en) 2003-04-17 2009-08-04 Akubio Limited Crystal oscillator
WO2005121769A1 (fr) 2004-06-12 2005-12-22 Akubio Limited Appareil d'analyse comprenant une mosaique de capteurs et un element d'etalonnage
WO2006103439A2 (fr) 2005-04-01 2006-10-05 Akubio Limited Cartouche pour analyseur d'echantillons fluides
US20090100914A1 (en) * 2005-04-01 2009-04-23 Daniel Peterson Godfrey Cartridge for a fluid sample analyser
WO2009005452A1 (fr) 2007-07-04 2009-01-08 Q-Sense Ab Membrane pour microbalance à quartz
WO2009070108A1 (fr) 2007-11-28 2009-06-04 Q-Sense Ab Montage de système de réflectométrie optique

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013110023A1 (fr) * 2012-01-20 2013-07-25 Purdue Research Foundation Procédé et dispositif d'imagerie optique avec un ensemble amplificateur résonnant
US9222878B2 (en) 2012-01-20 2015-12-29 Purdue Research Foundation Method and device for optical imaging with a resonant amplifier assembly

Also Published As

Publication number Publication date
GB2473635A (en) 2011-03-23
GB0916389D0 (en) 2009-10-28

Similar Documents

Publication Publication Date Title
Lucklum et al. Acoustic microsensors—the challenge behind microgravimetry
Lee et al. Label free novel electrical detection using micromachined PZT monolithic thin film cantilever for the detection of C-reactive protein
Lee et al. Immunoassay of prostate-specific antigen (PSA) using resonant frequency shift of piezoelectric nanomechanical microcantilever
US7458265B2 (en) Piezoelectric cantilever sensors
US8286486B2 (en) Molecular control of surface coverage
Yi et al. In situ cell detection using piezoelectric lead zirconate titanate-stainless steel cantilevers
Lee et al. Effect of mass and stress on resonant frequency shift of functionalized Pb (Zr 0.52 Ti 0.48) O 3 thin film microcantilever for the detection of C-reactive protein
Su et al. Quartz tuning fork biosensor
Park et al. Capacitive micromachined ultrasonic transducer (CMUT) as a chemical sensor for DMMP detection
US20050082944A1 (en) Electromagnetic piezoelectric acoustic sensor
US9151752B2 (en) Method for amplifying variation of frequency of signal in piezoelectrical biosensor
Bizet et al. Biosensors based on piezolectric transducers
Xu et al. Micro-machined piezoelectric membrane-based immunosensor array
Faegh et al. Ultrasensitive piezoelectric-based microcantilever biosensor: theory and experiment
Liu et al. A microfabricated thickness shear mode electroacoustic resonator for the label-free detection of cardiac troponin in serum
Wang et al. Label-free immunosensor based on micromachined bulk acoustic resonator for the detection of trace pesticide residues
De Simoni et al. A surface-acoustic-wave-based cantilever bio-sensor
US20080163689A1 (en) Electromagnetic piezoelectric acoustic sensor
Kao et al. A micromachined quartz resonator array for biosensing applications
Zhu Microcantilever sensors in biological and chemical detections
Hwang et al. Label-free detection of prostate specific antigen (PSA) using a bridge-shaped PZT resonator
WO2011033285A1 (fr) Appareil et procédé permettant de détecter des espèces cibles dans un analyte
Mandal et al. Surface acoustic waves (SAW) sensor for the active detection of Microcystin-LR (Cyanobacteria)
Kao et al. Characterization of viscoelastic properties of adsorbed biomolecules and biomolecular assemblies with high frequency micromachined quartz resonators
Lee et al. Capacitive micromachined ultrasonic transducer (CMUT)-based biosensor for detection of low concentration neuropeptide

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10763409

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10763409

Country of ref document: EP

Kind code of ref document: A1