Resonator for the detection of a mass analyte and method for operation of the resonator

Abstract

A resonator is disclosed for the detection of a mass analyte, such as a biological analyte. The resonator has: a piezoelectric layer formed of a piezoelectric material; a first resonator region and a second resonator region each occupying a corresponding region of the piezoelectric layer; electrodes disposed to apply a driving signal to the piezoelectric layer to generate bulk acoustic waves, the electrodes being common to the first resonator region and the second resonator region. In operation, the first resonator region has a first resonant frequency and the second resonator region has a second resonant frequency. The first resonator region and the second resonator region differ from each other in that the first resonator region is adapted to receive a mass analyte for the mass analyte selectively to attach to a surface of the first resonator region. In operation, attachment of the mass analyte selectively at the first resonator region causes a greater frequency shift in the first resonant frequency than in the second resonant frequency. Also disclosed is a corresponding method for the detection of a mass analyte.

Claims

1. A resonator for the detection of a mass analyte, the resonator comprising: a piezoelectric layer formed of a piezoelectric material; a first resonator region and a second resonator region each occupying a corresponding region of the piezoelectric layer; electrodes disposed to apply a driving signal to the piezoelectric layer to generate bulk acoustic waves, the electrodes being common to the first resonator region and the second resonator region; in operation, the first resonator region having a first resonant frequency and the second resonator region having a second resonant frequency; wherein the first resonator region and the second resonator region differ from each other in that the first resonator region is adapted to receive a mass analyte for the mass analyte selectively to attach to a surface of the first resonator region, wherein, in operation, attachment of the mass analyte selectively at the first resonator region causes a greater frequency shift in the first resonant frequency than in the second resonant frequency, wherein the first and second resonator regions are formed as parts of the same resonator, wherein the first resonator region comprises a mass analyte receiving layer, and wherein the first resonant frequency (f.sub.1) and the second resonant frequency (f.sub.2) are different to each other, even before the mass analyte is received at the first resonator region (R1).

2. The resonator according to claim 1 wherein the first and second regions are contiguous parts of the same resonator.

3. The resonator according to claim 1 wherein the electrodes sandwich the piezoelectric layer.

4. The resonator according to claim 1 wherein the first and second resonant frequencies are each greater than 0.5 GHz.

5. The resonator according to claim 1 wherein the resonator is a solidly mounted resonator (SMR) with an acoustic wave reflector structure arranged under the piezoelectric layer and electrodes.

6. The resonator according to claim 1 wherein the mass analyte receiving layer comprises binding sites at the first resonator region for selectively attaching to the mass analyte.

7. A sensor device comprising an array of resonators, each resonator in said array comprising: a piezoelectric layer formed of a piezoelectric material; a first resonator region and a second resonator region each occupying a corresponding region of the piezoelectric layer; electrodes disposed to apply a driving signal to the piezoelectric layer to generate bulk acoustic waves, the electrodes being common to the first resonator region and the second resonator region; in operation, the first resonator region having a first resonant frequency and the second resonator region having a second resonant frequency; wherein the first resonator region and the second resonator region differ from each other in that the first resonator region is adapted to receive a mass analyte for the mass analyte selectively to attach to a surface of the first resonator region, wherein, in operation, attachment of the mass analyte selectively at the first resonator region causes a greater frequency shift in the first resonant frequency than in the second resonant frequency, wherein the first and second resonator regions are formed as parts of the same resonator, wherein the first resonator region comprises a mass analyte receiving layer, and wherein the first resonant frequency (f.sub.1) and the second resonant frequency (f.sub.2) are different to each other, even before the mass analyte is received at the first resonator region (R1).

8. The sensor device according to claim 7 wherein the array of resonators is formed on a single chip.

9. A method for detecting a mass analyte, the method comprising providing a resonator comprising: a piezoelectric layer formed of a piezoelectric material; a first resonator region and a second resonator region each occupying a corresponding region of the piezoelectric layer; electrodes disposed to apply a driving signal to the piezoelectric layer to generate bulk acoustic waves, the electrodes being common to the first resonator region and the second resonator region, the method further comprising: operating the resonator to detect a first resonant frequency, corresponding to the first resonator region, and a second resonator frequency, corresponding to the second resonator region; selectively receiving a mass analyte at the first resonator region, attachment of the mass analyte selectively at the first resonator region causing a greater frequency shift in the first resonant frequency than in the second resonant frequency, wherein the first and second resonator regions are formed as parts of the same resonator, wherein the first resonator region comprises a mass analyte receiving layer, and wherein the first resonant frequency (f.sub.1) and the second resonant frequency (f.sub.2) are different to each other, even before the mass analyte is received at the first resonator region (R1).

10. The method according to claim 9 wherein the method further comprises: detecting a shift in one or both of the first and second resonant frequencies and carrying out one or both of (i) and (ii): (i) identifying a portion of the shift caused by an environmental change at the sensor other than attachment of the mass analyte selectively at the first resonator region; and (ii) identifying another portion of the shift caused by attachment of the mass analyte selectively at the first resonator region.

11. The method according to claim 10 wherein the environmental change is a temperature change at the resonator.

12. The resonator according to claim 1 wherein the mass analyte receiving layer comprises a metal layer.

13. The sensor device according to claim 7 wherein the mass analyte receiving layer comprises a metal layer.

14. The method according to claim 9 wherein the mass analyte receiving layer comprises a metal layer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Embodiments of the invention will now be described by way of example with reference to the accompanying drawings in which:

(2) FIG. 1 shows a schematic perspective view of a ZnO based SMR with a functional Au layer only on a pentagonal section of the Mo top electrode corresponding to the first resonator region (R1), the remaining part of the Mo top electrode corresponding to the second resonator region (R2).

(3) FIG. 2 shows a schematic view of the layout of a one chip device array (5×5) in which 25 resonators are arrayed. FIG. 2 also shows an optical microscope image of one resonator in the array.

(4) FIG. 3 shows a typical frequency split (f.sub.1/f.sub.2) due to the two resonator regions (R1 and R2) observed in both the thickness shear mode (TSM) and thickness longitudinal mode (TLM) of the same device.

(5) FIG. 4 shows a schematic plot of the resonant behaviour of a resonator prior to deposition of gold to form the first resonator region (R1).

(6) FIG. 5 shows a schematic plot of the resonant behaviour of the resonator of FIG. 4 after deposition of gold to form the first resonator region (R1).

(7) FIG. 6 shows a schematic cross sectional view through the first and second resonator regions of a resonator according to an embodiment of the invention.

(8) FIGS. 7-10 show displacement maps in the x and the z directions from FEM simulation demonstrating the resonator regions (R1 and R2) vibrating at different frequencies.

(9) FIG. 11 shows FEM simulated spectra of the TSM split resonances for different Au thicknesses at the first resonator region (R1) showing the spectral shift observed for R1.

(10) FIG. 12 shows corresponding FEM simulated spectra to FIG. 11 but of the TLM split resonances for different Au thickness.

(11) FIG. 13 shows a plot of the difference between the FEM simulated resonant frequencies of R2 and R1 for the TSM and TLM depending on the Au thickness at the first resonator region (R1).

(12) FIG. 14 indicates the increasing frequency difference between split resonances (f.sub.2 and f.sub.1) measured in a representative SMR device due to gradual Au deposition at the pentagonal section of the Mo top electrode, corresponding to the first resonator region (R1). The left hand plot shows TSM and the right hand plot shows TLM. Each of these shows a single mode for 0 nm of Au and split modes at different frequencies for 5 and 10 nm of Au.

(13) FIG. 15 shows the measured difference between the resonant frequencies (f.sub.2 and f.sub.1) for the TSM and TLM depending on the Au thickness.

(14) FIG. 16 shows a schematic of the device architecture of a 2R FBAR biosensor with the functional Au layer only on a pentagonal section of the Mo top electrode, similar to the arrangement shown in FIG. 1. The regions corresponding to the first (R1/f.sub.1) and second (R2/f.sub.2) resonances are shown.

(15) FIG. 16 also shows a schematic of the sensing surface (thiol+blocking/anti-PSA) and target (PSA antigen) detection.

(16) FIG. 17 shows the performance of a device according to an embodiment of the invention, in which there is an increase in the difference between the two resonances f.sub.2-f.sub.1 observed in the 2R FBAR sensor due to the attachment of subsequent molecules ((1) thiols; (2) anti-PSA; (3) PSA antigen) onto the Au active surface.

(17) FIG. 18 shows a linear fit for the gravimetric detection of PSA antigen from EDTA whole blood samples of known concentrations (determined by ELISA); point zero represents control measurements where no capture antibody (anti-PSA) was attached to the sensor surface.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS, AND FURTHER OPTIONAL FEATURES OF THE INVENTION

(18) In summary, the preferred embodiments of the present disclosure provide a dual resonator acoustic device which is self-compensating and can be used as an advanced tool in gravimetric sensing. Bio-sensing electro-acoustic devices use frequency shifts due to mass accretion after successful target-receptor interactions to track specific biomolecules. However, the device response to target molecules and other environmental factors (i.e. temperature, humidity, pressure) can show considerable variability across devices, leading to poor reliability. The dual resonator device according to the preferred embodiments of the invention has two regions. The first region is sensitive to target molecules by depositing a functional layer (Au). The second region is mass insensitive. Resonators with both a shear mode (about 1 GHz) and a longitudinal mode (about 2 GHz) are fabricated with different Au thicknesses and modelled to illustrate this method. The separation of the two frequencies of the two regions increases by about 50 MHz and about 120 MHz for the shear and longitudinal modes respectively as the Au thickness increases from 0 to 30 nm.

(19) We now set out a discussion of the preferred embodiments of the invention in more detail.

(20) Gravimetric biosensors based on thin film bulk acoustic wave (BAW) resonators have attracted significant interest in recent years mainly due to their high sensitivities. These devices can operate in the GHz frequency range; their sensing principle is based on the resonant frequency shift, Δf.sub.r, due to mass attachment and this mass sensitivity is proportional to f.sub.r.sup.2 according to Sauerbrey..sup.[1] However, these sensors cannot distinguish among different binding molecules, therefore they need a functionalized layer on the surface of the resonator in order to provide a signal representative of selective binding to specific binding molecules. Indeed, such functionalization layers have been reported in DNA sequencing,.sup.[2] detection of human fibrinogen,.sup.[3] pesticides,.sup.[4,5] potassium ions.sup.[6] and cancer biomarkers..sup.[7,8] Typical functional layers with biological affinity are known, such as gold, carbon nanotubes, graphene oxide, ZnO nanowires, supramolecular monolayers or titanium dioxide and have been shown to provide suitable selectivity with acoustic wave devices..sup.[6,9-13] Despite this, shifts in frequency are still observed due to changes in the surroundings (i.e. temperature, pressure, humidity) of these devices and cannot be differentiated from “real” mass attachment..sup.[14-16] Such effects pose a major challenge in the reliable detection of low quantities of biological compounds or other deposited material.

(21) Several methods have been proposed to compensate or measure the surrounding conditions of gravimetric BAW resonators. Active compensation techniques using external circuits or a reference device close to resonator are complex and are costly..sup.[17,18]

(22) A passive compensation technique reported by García-Gancedo et al. was based on using a thicker silicon dioxide as the first acoustic reflector layer, which produced two resonances that can be tracked simultaneously to monitor temperature variations and mass attachment..sup.[19] The disclosure in reference 19 corresponds to WO 2013/088163. The SiO.sub.2 layer, which is part of the reflector, does not add selectivity to the device and its thermal properties are strongly influenced by the deposition conditions.

(23) In this work a simpler mechanism to split the fundamental resonance mode of the gravimetric sensor into two closely separated distinct frequencies using a functional layer thickness is proposed as shown in FIG. 1. In FIG. 1, the first resonator region (R1) has a functional layer (Au is used as example due to its larger mass density to observe the shifts) that can be functionalized to become sensitive and selective to biological species. The second resonator region (R2) without the functional layer has negligible frequency shifts since it is not selective to the biological species. Therefore, the second resonator region can be used for calibrating Au thickness and other surface effects. In this manner a self-referenced device is achieved, which obviates the need for prior knowledge of the initial resonant frequency as the separation of the resonances can be used as the sensing mechanism for mass attachment. In this work the effect of Au thickness on the resonance spectral separation is modelled and characterized by fabricating ZnO based solidly mounted resonators (SMRs).

(24) FIG. 1 shows in general the construction of the SMR device. Substrate 10 (formed of single crystal Si) has a series of alternating reflector layers 12, 14, 15, 18, 20. Bottom electrode 22 is formed over the reflector layers. Bottom electrode 22 may be formed of Ir, for example. Piezoelectric layer 24 is formed over the bottom electrode 22. A suitable material for the piezoelectric layer is ZnO. On top of the piezoelectric layer is formed top electrode 26, defining the first resonator region R1 and the second resonator region R2. A suitable material for the top electrode 26 is Mo. At the first resonator region R1, a layer 28 of Au or Au/Cr is formed. There is no corresponding layer formed at the second resonator region R2.

(25) FIG. 2 shows a schematic view of the layout of a one chip device array (5×5) in which 25 resonators are arrayed. FIG. 2 also shows an optical microscope image of one resonator in the array. As can be seen in FIG. 1 and FIG. 2, the first resonator region has a generally pentagonal shape in plan view. This low symmetry shape is of use to suppress unwanted resonances or harmonics that may arise due to reflections at the edges of the electrode.

(26) FIG. 3 shows a typical frequency split (f.sub.1/f.sub.2) due to the two resonance regions (R1 and R2) observed in both the thickness shear mode (TSM) and thickness longitudinal mode (TLM) of the same device. FIGS. 4 and 5 show the effect of the deposition of gold to form the first resonator region (R1) on the resonance. No scales are indicated in FIGS. 4 and 5 but it can be seen that the data of FIG. 5 corresponds to that of FIG. 3.

(27) FIG. 6 shows a schematic cross sectional view through the first and second resonator regions of a resonator according to an embodiment of the invention. Note that the substrate and reflectors shown in FIG. 1 are omitted. Au layer 28 is formed only at first resonator region R1 and not at second resonator region R2. There is shown a layer of mass analyte 30 attached to Au layer 28.

(28) Finite element method (FEM) simulations shown in FIGS. 7-10 demonstrate the resonator region that is activated at different frequencies. Specifically, FIGS. 7-10 show displacement maps in the x and the z directions from FEM simulation demonstrating the resonator regions (R1 and R2) vibrating at different frequencies.

(29) FIG. 11 shows FEM simulated spectra of the TSM split resonances for different Au thicknesses at the first resonator region (R1) showing the spectral shift observed for R1. FIG. 12 shows corresponding FEM simulated spectra to FIG. 11 but of the TLM split resonances for different Au thickness. FIG. 13 shows a plot of the difference between the FEM simulated resonant frequencies of R2 and R1 for the TSM and TLM depending on the Au thickness at the first resonator region (R1).

(30) The lower frequency (f.sub.1) corresponds to the first resonator region (R1) whereas the higher frequency (f.sub.2) corresponds to the second resonator region (R2) only, for both the thickness shear mode (TSM) and the thickness longitudinal mode (TLM). The separation of the split resonances depends on the Au thickness, and increases with a thicker Au layer, as shown in FIGS. 11-13. Without the Au/Cr layer 28, the SMR exhibits a single resonance as shown in FIGS. 11 and 12 and the admittance amplitude is higher than when a layer of Au/Cr is attached because of the reduced size of the uncoated region. The FEM simulations in FIGS. 11-13 illustrate the frequency separation, which is approximately linear within the Au thickness range investigated.

(31) The electro-acoustic responses of fabricated split mode resonators according to preferred embodiments of the invention are shown in FIGS. 14 and 15. FIG. 14 indicates the increasing frequency difference between split resonances (f.sub.2 and f.sub.1) measured in a representative SMR device due to gradual Au deposition at the pentagonal section of the Mo top electrode, corresponding to the first resonator region (R1). The left hand plot shows TSM and the right hand plot shows TLM.

(32) Each of these shows a single mode for 0 nm of Au and split modes at different frequencies for 5 and 10 nm of Au. FIG. 15 shows the measured difference between the resonant frequencies (f.sub.2 and f.sub.1) for the TSM and TLM depending on the Au thickness.

(33) After the deposition of the Au/Cr layer 28 on the pentagonal section of the Mo top electrode of the SMR device, a clear split of the TSM and TLM in FIG. 14 into two distinct frequencies is observed. Similar to the FEM simulations, f.sub.1 that appears after Au deposition shifts towards lower frequencies, whilst the original resonance (f.sub.2) stays at the same frequency region at all times. This is because the added Au/Cr layer acts as a mass load on the first resonator region (R1). The experimental (f.sub.2-f.sub.1) plotted in FIG. 15 is slightly different to the simulated (f.sub.2-f.sub.1) probably due to uncertainty in the thickness or the assumption of ideal material properties. In addition the acoustic losses of thicker Au layers cause significant damping of the resonance in the experiments.

(34) Accordingly, it is shown here that the two fundamental modes of resonance thickness shear mode (TSM) and thickness longitudinal mode (TLM) are present at a low frequency region (about 0.95-1.2 GHz) and a high frequency region (about 1.9-2.2 GHz), respectively. They split into two closely separated distinct resonances after thermal evaporation of Au on the pentagonal section of the Mo top electrode of the SMR device, corresponding to the first resonator region (R1).

(35) It is worthwhile noting that the devices presented here are based on both TSM and TLM, rendering these devices suitable for sensing in both liquid and gas environments..sup.[20] The advantage of having mass sensitive and mass insensitive modes in a single device results in much more controllable and reliable tracking tool for various molecule detection, whilst eliminating other surface effects (e.g. temperature, humidity, pressure). This therefore helps to avoid undesirable false responses without the need for additional reference devices or complicated electronics. Another benefit of having the split resonances is the fact that significant deviations of the resonant frequencies across devices are due to thickness variation and orientation of the piezoelectric layer in BAW resonators. In particular this affect devices operating in the shear resonance that need off-axis piezoelectric layers, which cannot be grown uniformly over large substrates..sup.[16,20] Having two frequencies—one that is mass sensitive and one that is mass insensitive—in this case provides each sensor with its own reference resonant frequency to extract the frequency shift, and eventually the amount of target molecules detected.

(36) The resonators disclosed here have applications in the sensing of cancer biomarkers for example.

Experimental

(37) FEM Simulation

(38) FEM simulations were carried out using COMSOL® Multiphysics 5.3 (solid mechanics and electrostatics module) in 3D with the dimensions of the SMR fabricated. Due to the symmetry of the device, only half of the structure was simulated to reduce computation time. For each resonance region, 500 frequency points were simulated using a free triangular mesh with a 2 μm minimum element size to generate 106074 degrees of freedom to solve. Material properties from the default COMSOL® Multiphysics library are used.

(39) Device Fabrication

(40) The fabrication process of the shear and longitudinal mode ZnO based SMRs is described in our previous works..sup.[11,20] After the SMR fabrication, the pentagonal region for the functional layer is defined by standard ultra-violet photolithography using AZ2020 nLoF photoresist. By means of thermal evaporation (E306, Edwards, West Sussex, UK), 5 nm of Cr is deposited as adhesion layer for the subsequent Au layer, the thickness of which is varied from 5 to 30 nm. Lift-off is carried out in n-methyl-2-pyrrolidone, acetone, iso-propanol and de-ionized water to leave a pentagonal shape region (area of 1.42×10.sup.−4 cm.sup.2) on the resonator with the added Au/Cr layer.

(41) Electro-Acoustic Characterization

(42) Fabricated SMRs are then characterized on a coplanar probe station by measuring the impedance (Z) in the frequency range from 0.5 GHz to 3.0 GHz using 150 μm pitch ground-signal-ground radio-frequency (RF) probes (Picoprobes, GGB industries Inc., Naples, Fla., USA), connected to a vector network analyzer (Model E5062A, Keysight Technologies, Santa Rosa, Calif., USA).

(43) There follows a description of a dual resonator (2R) FBAR biosensor for PSA antigen detection from EDTA whole blood sample, according to an embodiment of the invention.

(44) Materials

(45) Thiol molecule, 11-mercaptoundecanoic acid (11-MUA) was purchased from ChemCruz; 6-mercapto-1-hexanol, ethanolamine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (Merck); Anti-PSA polyclonal capture antibody was purchased from GeneTech; EDTA whole blood samples were used as received from Addenbrookes hospital, Cambridge UK.

(46) Methods

(47) Self-assembled monolayers (SAMs) of thiolate compound were formed by the spontaneous chemisorption of thiolate groups on Au surface. For the SAM formation on gold, the devices were immersed in the freshly prepared 5 mM 11-mercaptoundecanoic acid (11-MUA) in ethanolic solution for 24 hours at 4° C. This formed a SAM of the alkanethiol on the gold active area on the FBAR. Next, the modified surface was rinsed with ethanol to remove any unbound molecules and dried with nitrogen. Formation of SAM was followed by additional surface-blocking step with ethanolic solution of 6-mercapto-1-hexanol (0.1 M) for 1 hour at RT and followed by ethanol rinsing/nitrogen drying steps.

(48) For antibody coupling, treatment of water-soluble carbodiimide and succinimide compounds was performed immediately by immersing the devices in EDC (0.2M) and NHS (50 mM) for 30 min followed by rinsing with MiliQ water. This activates the carboxylic acid terminated-group on the modified gold surfaces. Capture antibodies (anti-PSA, polyclonal) were covalently immobilized on the NHS-activated SAM by spotting with 10 μL of a 0.5 mg/mL solution for 1 h at RT. For control measurements, no capture antibody was attached in this step. The remaining NHS-active ester sites were blocked with 1.0 M ethanolamine pH 8.5 for 30 min at RT. The prepared devices were subsequently used for the PSA antigen detection from the whole blood samples.

(49) EDTA whole blood samples containing various concentrations of PSA antigen were used as received. The PSA concentrations for each sample were previously determined by ELISA. A small volume (10 μL) of blood samples from 7 patients were applied on the antibody-modified Au active surface and left to incubate for 15 min to allow the recognition of the target protein by the immobilized receptors. After extensive rinsing with MiliQ water and drying with nitrogen, devices were measured on a network analyzer (NA-E5062A).

(50) Results

(51) FIG. 16 shows a schematic of 2R FBAR biosensor, where the resonator (R1) with Au layer is functionalized with thiol molecules and capture antibody (anti-PSA). That prepared surface is selective to the PSA antigen detection.

(52) The sensor response to the attachment of the (1) thiol (11-MUA)+blocking (6-mercapto-1-hexanol), (2) anti-PSA, (3) PSA antigen onto Au active surface was monitored by measuring the real part of the electrical admittance (Y) in the frequency range from 0.5 GHz to 3.0 GHz and it is shown in FIG. 17.

(53) In this experiment, the developed sensor was applied to the detection of PSA from EDTA whole blood samples from cancer patients. As can be seen from FIG. 18, we obtained a linear trend in a PSA detection from a complex matrix, such as whole blood. Direct PSA detection from the whole blood significantly reduces time of the test as there is no need for sample pre-treatment and therefore it can simplify the whole process of PSA testing. To measure specificity of the assay, we performed control measurements, where no anti-PSA capture antibody was attached to the sensing surface of the FBAR sensor. For this, no significant response was obtained after incubation with EDTA whole blood samples. This indicates low non-specific interactions with FBAR sensor's surface and shows good selectivity for PSA antigen detection.

(54) While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

(55) All references referred to above and/or listed below are hereby incorporated by reference.

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