CN105380668B

CN105380668B - Method for continuously monitoring blood glucose change by using magnetoacoustic resonance

Info

Publication number: CN105380668B
Application number: CN201510669909.0A
Authority: CN
Inventors: C·B·洛维; A·史蒂文森; B·阿莱雅雷斯特贝尔
Original assignee: Zhongkekang Magnetic Medical Technology Suzhou Co ltd
Current assignee: Zhongkekang Magnetic Medical Technology Suzhou Co ltd
Priority date: 2011-12-09
Filing date: 2011-12-09
Publication date: 2020-10-16
Anticipated expiration: 2031-12-09
Also published as: CN105380668A

Abstract

The invention provides a method for continuously monitoring blood glucose change by using magnetoacoustic resonance, which comprises the following steps: 1) introducing or implanting into a subject a resonator and a sensor that can continuously monitor changes in blood glucose, the sensor being mechanically coupled to the resonator; the resonator comprises a magnet positionable to direct a magnetic field toward the sensor and an exciter in mechanical communication with the sensor; the sensor comprises a sensor material which changes state when exposed to a change in the surrounding environment, wherein the sensor is driven by the resonator, and wherein the sensor material is in the form of an acoustically thick layer; 2) detecting a signal in response to a change in state of the sensor material outside the subject's body by a detector to which is connected an adjustable electromagnetic field generator configured to direct an electromagnetic field towards the sensor; 3) the signals detected by the detector are continuously processed to continuously monitor changes in blood glucose in the subject.

Description

Method for continuously monitoring blood glucose change by using magnetoacoustic resonance

The application is a divisional application of Chinese invention patent application (title of the invention: magnetoacoustic resonance sensor; application number: 201180026587.8; application date: 2011, 12 months and 9 days).

Technical Field

The present invention relates to sensing devices, preferably chemically activated electromechanical sensing devices employing hydrogels.

Background

Hydrogels that change shape or volume when exposed to different chemical environments have been used as pH, ion, chemical, gas, and temperature sensors, and as mechanical drive elements. See, for example: bashir, R.et al, micromechanic cantilers as an ultra-sensitive pH microsensor, applied Physics Letters,2002,81(16): pages 3091-3093; zhang, L, and W.R.Seitz, A pH sensitive on force generated by pH-dependent polymer spinning, Analytical and Analytical Chemistry, 2002,373(7) pp.555-559; van der Linden, H. et al, Development of pharmaceutical-sensitive hydrogels available for activators and microorganisms in microbiological devices, 2002,14(3): pages 129-139; mayes, A.G., et al, Metal ion-sensitive pharmacological sensors, analytical chemistry, 2002,74(15): pages 3649-3657; mayes, A.G., et al, A. pharmacological analytical Chemistry, 1999,71(16): pages 3390-3396; eyes, A.G., et al, ecological sensor based on a defined synthesized polymer, journal Molecular Recognition,1998,11(1-6): pages 168-174; millington, R.B. et al, Ahologram biosensiser for proteins, Sensors and actors B-Chemical,1996,33(1-3): pages 55 to 59; blyth, J.et al, Hologrphic sensor for water in solvents, analytical chemistry, 1996,68(7): pages 1089-1094; herber, S., W.Olthuis and P.Bergveld, assembling hydrogel-based P-C02sensor.Sensors and Actuators B-Chemical,2003,91(1-3): pages 378-382; kuckling, D.et al, Photo cross-linkable poly (N-isopyracrylamide) copolymers III: micro-fabricated responsive polymers, 2003,44(16), pages 4455 to 4462; hilt, J.Z., et al, ultrasensitive biological sensors based on microcantilever patterns with environmental responses, 2003,5(3), pp.177-184.

Based on the color change of the modified hydrogel, optical detection of the dimensional change of the hydrogel crystals or diffraction gratings has provided a convenient sensorometric (sensorometric) signal, which provides a more versatile detection platform. See, for example: photonic crystal carbonate sensors, such as Asher, S.A. et al, Low ionic structural h super sensitive. journal of the American Chemical Society, 2003,125(11), pages 3322-3329 and Marshall, pH-sensitive pharmacological sensors, analytical Chemistry, such as A.J. 2003,75(17): pages 4423-4431.

However, hydrogels are believed to have several specific applications. See, for example, Hoffman, A.S., hydrogels for biological applications, advanced Drug Delivery Reviews,2002, pages 43: 1-12. An important consideration for optical hydrogel systems is to maintain the position of the particles that define the subsequent color change. This requires that the hydrogel must retain sufficient stiffness for stability, but at the same time provide low cohesion, so that a measurable volume change can occur. These mechanical aspects of hydrogels have been studied by stretching the size of the rectangular units of poly-HEMA (hydroxyethyl methacrylate) by several centimeters to measure their elasticity at different pH values. See, for example, Johnson, B.et al, Mechanical properties of a pH sensitive hydrogel,2002 annual SEM treatise, Milwaukee, Wis.

Another sensing method uses an acoustic wave sensor responsive to a viscoelastic solution. An acoustic wave sensor includes a resonator that is excited to vibration in a viscoelastic solution. A change in the viscosity of the solution (e.g., due to a chemical reaction) results in a detectable change in the vibration frequency/amplitude of the resonator.

Adaptation of the aforementioned sensor involves providing a monomolecular film on the resonator which acts as a probe for the surrounding chemical/physical environment. The monomolecular film may react with molecules in the surrounding solution, for example, causing a change in the mass/volume of the resonator, thereby changing the frequency/amplitude of the resonator vibration. Such an arrangement has enabled the detection of nucleotides using a single layer of hydrogel. See, for example, Kanekiyo, Y., et al, Novel nucleotide-reactive hydrogels designed from polymers of boron acid and reactive units and the first applications as a QCM Resononator system to nucleotide sensing. journal of Polymer Science Part a-Polymer Chemistry, 2000,38(8): pages 1302-1310; tang, A.X.J., et al, Immunosensor for dietary acid using quartz crystal microbalance, analytical Chimica Acta,2002.471(1): pages 33-40; nakano, y. seida and k. kawabe, Detection of multiple phases in an ecosensitive polymer hydrogel, Kobunshi Ronbunshu,1998.55(12): pages 791-795; and Serizawa, T.et al, thermal emulsion chemical reactions, macromolecules,2002.35(6): pages 2184-2189.

The acoustic sensor can thus be used as a probe of the surrounding environment without the need to incorporate a diffraction grating. However, in the above arrangement, to detect changes, careful tracking of disturbances occurring in the audio, typically < 0.01%, is required. That is, the existing acoustic wave sensor has the following problems: the effect in the sensor due to the physical/chemical environmental change is small and very sensitive measurements are needed to obtain useful information about the change.

Disclosure of Invention

The present invention seeks to monitor changes in the surrounding environment using changes in the physical and/or chemical properties of the driven sensor. In particular, the present invention seeks to solve the problem of low sensitivity of the prior art acoustic wave sensors described above.

According to a first aspect of the present invention there is provided a sensing device comprising a resonator, a sensor and a detector, the sensor being in mechanical communication with the resonator, the sensor comprising a sensor material which changes between a first state and a second state upon change in contact with the surrounding environment, wherein the sensor is driven by the resonator and the detector is responsive to changes in the state of the sensor material, wherein the sensor material is in the form of an acoustically thick layer.

The main advantage of the present acoustic method is the ability to return information on the internal chemical forces due to cross-linking and co-polymerization, which is evident for hydrogel elasticity but not for any dimensional changes of the optical collection. An advantage of embodiments of the present invention is that the acoustic phase variation across the thickness of the hydrogel membrane (acoustically thick membrane) is large and close to Pi/2, essentially changing the resonance to the point of cancellation (point of extinction) and thus providing a useful mechanical switching action between the vibrating and non-vibrating states.

An advantageous technical effect of embodiments of the invention over the prior art is therefore that the amplitude of the acoustic signal of the resonator shifts more when a change in the chemical/physical environment occurs, and is therefore easier to detect than in prior art arrangements where the acoustic frequency shifts only slightly. The sensor layer (which may be a biological recognition layer) itself becomes a resonant structure that stores or consumes energy. The sensor response is thus controlled by the chemical composition of the sensor layer or upper layer. Hydrogel films are a successful example of such a top layer.

This advantageous technical effect is achieved by providing the sensor material in the form of an acoustically thick layer, which is different from the thin layers disclosed in the prior art. That is, providing an acoustically thick film directly results in a large excursion of the acoustic signal.

For viscoelastic materials, the transition between acoustically thin layer properties and acoustically thick layer properties is most conveniently defined by a phase factor. Thus, the thickness of the acoustically thick film substantially satisfies the following equation:

where ω is angular frequency, ρ is density, G is shear modulus, and t is film thickness.

An acoustically thick layer is a layer that can support a significant phase shift of an acoustic wave when the acoustic wave propagates perpendicular to the layer. This definition does not include the single layer disclosed in the prior art arrangement. Such a monolayer cannot support an acoustic phase shift perpendicular to the layer. Also, such a single layer cannot support an acoustic phase shift parallel to the layer if high frequencies are not used according to the above equation.

In the context of this application, "acoustic vibration" refers to standing sound waves that are reflected between the upper and lower boundaries of the sensor.

Preferably, the detector comprises an electromagnetic field generator positionable to direct an electromagnetic field towards the sensor. This is advantageous because the sensor can be used remotely without a connecting wire. Preferably, the electromagnetic field generator and detector comprise conventional structural elements for generating an electromagnetic field and for detecting an electromagnetic field. This provides a compact arrangement. Advantageously, the electromagnetic field generator is adjustable. This allows for greater variability and sensitivity of the device in use. The electromagnetic field generator may be a helical coil and the sensing means may comprise a signal generator and a lock-in amplifier connected to the electromagnetic field generator and the detector to provide greater sensitivity. The detector may comprise a differential diode detector circuit for subtracting the detected signal from the signal generated by the signal generator. In one arrangement, the resonator comprises a magnet which may be arranged to direct a magnetic field towards the sensor. This arrangement provides a magnetoacoustic resonant sensor. The resonator may include an exciter mechanically coupled to the sensor, which may be a piezoelectric material such as a quartz layer. The thickness of the piezoelectric layer is preferably 50 μm to 1000 μm.

Other sensing devices may be used to measure the electrical impedance of the detector, and thus the acoustic response. For example, a commercially available impedance analyzer or other custom impedance measurement circuitry may be incorporated.

The exciter may be derived from magnetostrictive materials in which the magnetic component of the resonator electromagnetic field is used to excite the acoustic wave. The exciter may comprise a metallic material. It may be in the form of a layer having a thickness preferably of from 50 μm to 1000. mu.m.

The sensor layer comprising the acoustically thick layer sensor material is preferably 0.1 μm to 1mm thick, more preferably 0.1 μm to 100 μm thick, even more preferably 0.1 μm to 10 μm thick, most preferably 0.5 μm to 5 μm thick. Advantageously, the sensor layer is more than one molecule thick.

The sensor material is preferably a hydrogel, such as hydroxyethyl methacrylate-methacrylate copolymer, poly (acrylamide-co-3-acrylamidophenylboronic acid), or poly (acrylamide-co-2-acrylamidophenylboronic acid).

Preferably, the sensing device is a chemically actuated electromechanical sensing device. The sensing device can be used in a sensing method. Also, the sensing device may be used as a switch or in a method of continuous monitoring. Furthermore, the sensing device may be used in a method of controlling a system based on changes in the surrounding environment.

According to another aspect of the present invention there is provided a method of making a composite probe comprising a piezoelectric resonator element and an acoustically thick hydrogel film on the resonator element, the method comprising: preparing the monomer mixture for the hydrogel; applying the monomer mixture to a release layer such as an aluminized polyester sheet; applying a resonator element to the monomer mixture; polymerizing the monomer mixture to form the acoustic thick hydrogel film adhered to the resonator element, thereby forming a composite probe; and peeling the composite probe from the anti-adhesion layer. The resonator element may be treated with a binder (such as trimethoxysilylpropyl methacrylate) prior to application of the monomer mixture to promote adhesion. In one embodiment, the hydrogel is prepared from a mixture of 2-hydroxyethyl methacrylate (HEMA), ethylene glycol dimethacrylate (EDMA), Methacrylate (MAA), and a photoinitiator (e.g., dimethoxyphenylacetone). In another arrangement, the hydrogel is prepared from a mixture of 3-acrylamidophenylboronic acid, acrylamide, and N, N-methylenebisacrylamide. In yet another arrangement, the hydrogel is prepared from a mixture of 2-acrylamidophenylboronic acid, acrylamide, and N, N-methylenebisacrylamide.

Embodiments of the present invention seek to provide a chemically activated electromechanical sensing device using changes in the acoustic properties of a material caused by changes in the physical and/or chemical environment of the material.

In one embodiment, the hydrogel is responsive to a change in chemical environment, preferably wherein the change in chemical environment is a change in pH. In this embodiment, the hydrogel is generally a polymer that responds to pH by a change in its viscoelastic properties. The volume of the hydrogel will also change in response to a change in pH. Hydrogel materials suitable for this purpose are hydroxyethyl methacrylate-methacrylate copolymers, which are copolymers of 2-hydroxyethyl methacrylate and methacrylate, preferably crosslinked with a certain amount of ethylene glycol dimethacrylate (also known as ethylene glycol dimethacrylate). The amount of methacrylate used to form the hydrogel is typically about 6 mole percent and the amount of ethylene glycol dimethacrylate is typically 1.5 to 7.5 mole percent. Generally, hydrogels with more than 3.5 mole% ethylene glycol dimethacrylate tend to become stiffer and lack flexibility. The rest is 86.5 to 92.5 mol percent of hydroxyethyl methacrylate.

Unlike conventional quartz crystal microbalances, the device of the present invention is wireless operable to eliminate non-disk (non-disk) electrodes. This extends the operating frequency to the range of 6MHz to 1.1GHz compared to the conventional 5MHz operating frequency. Furthermore, wireless operation allows the resonator and the sensor component of the sensing device to be implanted in, for example, a human or animal subject. At this point, the detector may be operated outside the body, away from the resonator and sensor.

In another embodiment, the hydrogel is responsive to a change in chemical environment, wherein the change in chemical environment is a change in analyte concentration. The analyte may be a physiological analyte such as a sugar. Such sugars may include glucose, fructose, galactose and mannose. Glucose is a particularly important physiological analyte and plays an important role in conditions such as diabetes, as well as in fermentation processes and in the metabolism of living cells.

In one arrangement, the hydrogel is a polymer containing phenylboronic acid groups, which are typically pendant from the polymer backbone. Preferably, the polymer is poly (acrylamide-co-3-acrylamidophenylboronic acid). The polymer is capable of binding glucose and other sugars, and it is believed that the boronic acid moiety of the polymer binds to the cis diol of the sugar.

In one arrangement, the sensing device of the invention is in the form of an implantable device implanted in a human or animal subject, which includes a resonator and a sensor. In this arrangement, the detector is responsive to the sensor externally of the body, thus enabling remote monitoring of physiological analytes such as glucose in a simple non-invasive manner. Thus, an implantable device is provided for implantation in a human or animal subject and includes a resonator and a sensor according to the present invention, wherein the sensor is mechanically coupled to the resonator. In a method of remotely sensing a physiological state of a subject, a detector may be used with an implantable device, where the detector is responsive to a sensing material of a sensor. Sensing of the physiological state of the subject may include sensing of an analyte, which may be, for example, continuous monitoring of analyte concentration.

Unlike commercially available enzyme-based glucose sensors and monitors, which are less stable over long-term use and difficult to disinfect for in vivo applications, the sensing device of the present invention is durable and easy to implant in a suitable subject.

In the above-mentioned method and device, the signal shift of the amplitude is very large, and can be more than 95%. Thus facilitating detection and significantly simplifying the sensor apparatus. That is, the observation of very large signal changes allows for simpler and less costly instrumentation. For example, the phase lock may be removed and conventional less sensitive electrical impedance measurements employed. Also, embodiments of the present invention are more sensitive to physical and chemical changes than prior art arrangements.

For a better understanding of the present invention and to show how the same may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, in which:

FIG. 1(a) shows a hydrogel switch in an electromagnetic field generated and monitored by a measurement system comprising a signal generator, detector and LabVIEW operating with a PC (not shown)^TMConnected lock-in amplifier-with o-ring and plastic element (not shown) to keep the contact solution on the upper surface of hydrogel switch;

FIG. 1(b) shows the fundamental resonance peak of a quartz disk compared to the same quartz disk after coating with a 10 μm poly-HEMA hydrogel film-the top surfaces of both the quartz disk and the hydrogel composite were in solution at pH 3.5;

FIG. 2 shows a comparison between the shear resonance spectra of three different hydrogel-quartz composites, showing significant attenuation at 100 MHz;

FIG. 3(a) shows a phase contrast image of the edge of a hydrogel film, indicating that it is about 3 μm thick;

FIG. 3(b) shows the frequency shifts of the fundamental and third harmonics after coating a quartz sensor with a hydrogel layer initially prepared with 5. mu.l monomer mix (plate 1), 8. mu.l monomer mix (plate 2), 12. mu.l monomer mix (plate 3) and 15. mu.l monomer mix (plate 4);

FIG. 4 shows the acoustic resonance harmonics of the same hydrogel-quartz composite after exposure to pH3.5 and pH 8-membrane swelling results in a frequency decrease at 6.7MHz, a resonance cessation at 33MHz, and a frequency increase at 60 MHz;

FIG. 5(a) shows the switching action of a hydrogel-quartz composite with a hydrogel film of more uniform thickness-pH 3.5 places the switch in the "on" state, while pH8 places the switch in the "off" state;

FIG. 5(b) shows the switching characteristics expressed as a series of consecutive peak resonances retraced at 15 second intervals;

fig. 6(a) shows a pH calibration curve depicting incremental resonant trace changes between the on state at pH3.5 and the off state at pH 8;

FIG. 6(b) shows the amplitude of resonance and the change in Q-factor versus solution pH;

FIG. 7 shows the reversible binding that occurs between boric acid and cis diol in an aqueous medium;

FIG. 8 shows the chemical structure of poly (acrylamide-co-3 APB) copolymer;

FIG. 9 shows the pH profile of poly (acrylamide-co-3 APB);

FIG. 10 shows the response amplitude of a MARS coated with a poly (acrylamide-co-3 APB) membrane with respect to glucose concentration;

FIG. 11(a) shows the response of MARS with poly (acrylamide-co-3 APB) membrane to glucose solution;

FIG. 11(b) shows the response of MARS versus glucose concentration;

FIG. 12 shows the difference in amplitude change of MARS with poly (acrylamide-co-3 APB) membrane to 15mM glucose;

FIG. 13 shows the plot of MARS with poly (acrylamide-co-3 APB) membrane versus the amplitude of different 3-APB concentrations for variations in pH 4-10;

FIG. 14 shows the Δ Q between the response of a poly (acrylamide-co-3 APB) sensor in pure buffer at pH7.4 and the response in 10mM glucose buffer at pH7.4 in mole% relative to 3-APB;

FIG. 15 shows the difference in amplitude of the signal from the MARS versus the crosslinker MBA concentration as the glucose increment was increased;

FIG. 16(a) shows the response amplitude of MARS with poly (acrylamide-co-3 APB) membrane versus glucose concentration;

FIG. 16(b) shows that the response amplitude is proportional to the glucose concentration when the glucose concentration is not more than 7.5 mM;

FIG. 17 shows a continuous change 56 from 0 to 100mM for glucose concentration at pH 7.4.

FIG. 18 shows the response of 3-APB-based MARS to 5mM fructose, glucose, galactose and mannose solutions; and

FIG. 19 shows the plot of the change in amplitude of the MARS sensor relative to 3-APB mole% and (a) apparent pKa, (b) hydrophobicity (measured as Δ Α 4) and (c) viscoelasticity (measured as Δ Q).

Detailed Description

Example 1

Described herein are novel chemically activated mechanical switches based on composite acoustic resonators.

The apparatus is illustrated in figure 1. A planar spiral coil is provided to electromagnetically excite harmonics of a composite resonator made of a quartz disk and a hydrogel acoustic thick film coating based on a pH sensitive hydroxyethyl methacrylate-methacrylate copolymer. The spiral coil is coaxially connected with the signal generator, the AM detector and the phase-locked amplifier. The detector employs a differential diode detector circuit to subtract the radio frequency signal returned by the coil from the larger excitation signal generated by the signal generator. For transmitting back the frequency, amplitude and Q factor of the composite resonance device, LabView is used^TMThe software processes raw amplitude and frequency data points collected from the spectrogram.

Measurements of acoustic resonance characteristics up to 100MHz were made with the above-described coil-based excitation system, which was able to maintain strong signal amplitudes in multiple harmonic frequencies. The operating frequency is selected from the signal generator front panel and used to transmit back the acoustic trace difference for each harmonic resonance, which in turn is related to the hydrogel chemistry.

The switching action was observed in a 2.1 μm thick hydrogel film deposited on a 250 μm thick AT quartz disk, excited AT pH3.5 AT fundamental frequency (6.6MHz) and terminated AT pH8 with an amplitude ratio of 500:1 on/off. The hyperbolic tangent function (Tanh [ kt ]) as a result of the wave vector (k) and the film thickness (t) was found]) The independent variable (alignment) of (a) can well define the switching point, so that a membrane elasticity of 5.8 × 10 can be measured without using a multiparameter fitting system⁵N/m²And the shear wave velocity is 24ms^-1. This method allows the swelling response of the hydrogel to be greatly amplified and enables the mechanical properties of the membrane to be determined. Thus, acoustic shear wave switches based on pH-sensitive hydrogels are provided.

In order to maximize the signal response and greatly improve resonance in the method, it is advantageous to control the resonance frequency of the probe. Also, in order to make the hydrogel film easy to handle and adhere, it is advantageous to use glass instead of the metal electrode surface which can usually be chemically treated. The above-described adjustable device based on a simple coil and a bare quartz substrate is therefore advantageous for technical reasons. Using this general model, a variety of hydrogel materials and thicknesses can be employed.

In the described embodiments of the invention, the mechanical properties of ionizable poly-HEMA hydrogels with respect to pH can be exploited when placed adjacent to a multifrequency quartz crystal resonator. The electrical characteristics of the multi-frequency resonator and its detection characteristics can be evaluated. Understanding the formation of hydrogel films and their interaction with acoustic waves is another important aspect of system performance. Thus, in the method used in the present invention, a phase-contrast image of the deposited hydrogel is obtained and its thickness is estimated. In addition, the characteristics of the system were interpreted according to the selected operating frequency and the acoustic impedance of the hydrogel resonator device was estimated with respect to pH.

It is difficult to determine the thickness, density, elasticity and viscosity of hydrogel films, since these parameters are largely unknown. For example, due to the soft nature of the hydrogel and its potential variation in thickness in the disc, it is difficult to determine the thickness (although the thickness of the film can be measured as described below). Rather, it is more important to determine whether elasticity is the determining factor for the possible range of thicknesses, and more specifically, whether a physico-chemical condition can be established that causes the sound waves to turn on or off. It should be noted that the systems are in some respects more complex than those containing molecular films, since thickness is a significant fraction of the wavelength of the acoustic wave. The thickness variation creates an acoustic phase difference in the film, which significantly affects the overall characteristics of the system. Broadband acoustic measurements of hydrogel disc composites made according to this embodiment employ readily available signal generators and lock-in amplifiers, which are identical to those previously used for viscous fluid and protein adsorption studies. The spiral coil is hand wound and the diode detector is built in.

Once the acoustic field is generated by electrostriction in the crystal, the acoustic properties are related to the hydration state of the polymer, which in turn depends on the pH of the bath medium. The acoustic coupling of the crystal to a thick hydrogel film (elastic is "rigid") has been measured, and the chemically-related elasticity has been correlated with its switching characteristics.

Examples of the invention

2-hydroxyethyl methacrylate (HEMA, 97%), ethylene glycol dimethacrylate (EDMA, also known as ethylene glycol dimethacrylate), methacrylic acid (MAA), Dimethoxyphenylacetophenone (DMAP), 2-propanol and (methacryloxypropyl) triethoxysilane were supplied by Aldrich Chemical Co. All other chemicals were of analytical grade and were supplied by Sigma or Aldrich. Aluminized 100 μm thick polyester Film (MET401 grade) was purchased from hifi industrial Film Ltd (Stevenage, uk).

Composite resonator preparation

A monomer mixture was prepared from HEMA (89 mol%), EDMA (crosslinker, 5 mol%), MAA (6 mol%) and an equal volume of 2-propanol. The photoinitiator Dimethoxyphenylacetophenone (DMAP) was then added to a final concentration of 1% (w/v). The crystals were treated overnight with 1% (w/v) trimethoxysilylpropyl methacrylate in acetone. An aluminized polyester sheet was placed on a flat glass plate and approximately 5 μ l of the monomer mixture was pipetted to create a fluid "bead". The treated quartz disk was placed on top of the monomer mixture and pressed to uniformly coat its lower portion with polymer. The UV-initiated free radical polymerization was carried out by exposure to UV radiation (about 350nm) generated by a UV exposure unit for 15 minutes. After polymerization, the disc was peeled off the aluminized side of the polyester sheet, which served as an anti-adhesive layer for the hydrogel-quartz composite. The final step was to wash the complex thoroughly in methanol.

Spiral coil

A planar spiral coil with a DC resistance of 1 ohm, an inductance of 0.5mH, and an overall diameter of 5mm was prepared from 0.085mm enameled copper wire from RS Electronics (uk) and connected to a 0.25mm thick epoxy laminate with a thin layer of cyanoacrylate adhesive.

In order to design an effective hydrogel sensor, it must be understood how the change in its acoustic impedance, derived from the detected electrical signal, relates to the chemically induced change in hydrogel behavior.

Hydrogel-disk acoustic impedance

The system studied was a composite resonator consisting of a resonator plate attached to an additional hydrogel resonator (thickness, density and viscoelasticity unknown). In addition, there is a third liquid layer contacting the upper surface of the composite. The acoustic impedance of a composite system can generally be described by (1-4):

G_w＝2πfπω (3)

where k and t are the wave vector and thickness of the layer, respectively, and Zq, Zf, and Gw are the quartz impedance, the hydrogel impedance, and the water impedance. The combination of these impedances Zc characterizes a composite resonator, unlike the individual resonators represented by equation 2 in the second clause. The analysis shows that the hydrogel impedance Zf changes dramatically when the argument kt of the hyperbolic tangent function is pi/2, i.e. when chemical perturbations in the hydrogel matrix cause a large shift in acoustic amplitude and acoustic frequency. Since the hydrogel dimensions are not as well defined as quartz plates and exhibit larger dimensional and mechanical changes, it can be expected that the film resonance will only occur in the lower harmonics where the acoustic wavefronts remain plane-parallel.

Hydrogel chemistry

The acoustic impedance of the hydrogel is due to the total (aggregate) internal force generated by the hydrophilic cross-linked copolymer layer that absorbs water from the solution. The presence of weakly acidic carboxyl groups allows the gel to undergo a change in hydration depending on the pH of the bath medium [ Montheoard, J.P., M.Chatzopoulos and D.Chappard, 2-epoxy methyl Methacrylate (Hema) -Chemical-Properties and Applications in biological fields of journal of Macromolecular Science-Reviews in Macromolecular Chemistry and Physics,1992, C32(l): pages 1-34 ]. Interestingly, the thickness and elasticity vary with pH simultaneously and continuously. In this case, the pH changes the thickness t due to hydration and k according to the known elasticity as a function of pH [ Johnson, b. et al, Mechanical properties of a pH sensitive hydrogel,2002 annual SEM treatise, Milwaukee, WI ], according to k ═ ω √ (pc). This changes the independent variable kt of the membrane acoustic impedance (1) and acoustically amplifies the pH change significantly across the hyperbolic tangent.

Detection of hydrogel disc complexes

The specific features of the acoustic configurations employed in this study [ Stevenson, A.C. and C.R.Lowe, non-detail excitation of high Q-acidic resonances in glass plates applied physics Letters,1998.73(4): pages 447-449 ] [ Sindi, H.S., A.C.Stevenson and C.R.Lowe, analysis for chemical sensing based on systematic mechanical Chemistry, 2001.73(7): pages 1577-1586 ] were connected across the air gap to the disk so that no mechanical or electrical disturbance was applied during excitation/detection. Unlike conventional hardwired acoustic devices, the device of the present invention is field driven and detects with a helical coil. The excitation/detection process can be considered to comprise two simultaneous actions. By the generation process, radio frequency current from the signal generator circulates as the coil rotates. These processes generate a nearby radio frequency magnetic field that itself supports the radio frequency electric field. The field distribution, which depends on The coil geometry (geometry), acts at The dielectric boundaries of The dielectric material, thus inducing surface currents [ Stevenson, A.C. and C.R.Lowe, Magnetic-acidic-resistive sensors (MARS): a new sensing method. sensors and Actuators a-Physical,1999.72(1): pages 32 to 37 ] or charges [ Stevenson, A.C. et al, The acidic semiconducting meter: A. non-biological and electrolytic conductive base on multiferroic-catalytic device. analysis, 2003.128(10): pages 1222 to 1227 ]. Different boundaries (e.g., the upper and lower surfaces of a piezoelectric crystal that contact water and air, respectively) concentrate different charges as the crystal is driven by the application of charges through electrodes Stevenson, A.C., et al, Hypersonic electronic waves generated with an anodic crystalline phase, analysis, 2003.128(9) pages 1175-1180, [ Thompson, M. et al, electrochemical excitation of high electronic environmental waves and detection in-situ phase analysis, 2003.128(8) pages 1048-1055 ]. The reverse process is also performed, reversing the action, resulting in the induced current returning to the coil. The end result is that the change in hydrogel chemistry is immediately converted to a mechanical change, which affects the acoustic shear vibration and, for the reasons mentioned above, the measured electrical signal.

Electrical impedance

Another way of observing the regime is to assume that the amplification of the electrical signal depends on the transduction function associated with the electromagnetic field distribution (related to the coil and the surrounding dielectric). The frequency shift and the change in acoustic Q-factor can be determined directly from equation (1) and related to the measured electrical and frequency shifts.

Assuming that a thick film resonance condition can be achieved, the model predicts that a significant change in the acoustic impedance of the film will be observed when the thickness, frequency and elasticity of the film reach π/2 shift in kt. Conditions and protocols that enable chemically induced transitions between zero resonance and thick film resonance are of particular interest.

Detection of disc-hydrogel resonance

The plate was examined for fundamental resonance in water and then hydrogel was attached, lowering the frequency and Q of the resonator (fig. 1). In both tests, pH3.5 was chosen because the hydrogel is more compact and stable under these conditions [ Marshall, A.J., et al, pH-sensitive therapeutic sensors. analytical Chemistry, 2003.75(17): pages 4423-4431 ]. The attachment of the hydrogel causes additional energy loss processes to occur in the hydrogel, and thus the Q factor decreases. Starting from a system with a more open mobile structure that "oscillates" in the surrounding fluid, it is unexpected that the hydrogel matrix appears to be a more "energy-consuming" acoustic material. The resulting attenuation shifts the resonant frequency down and lowers the Q factor. Alternatively, possible film thickness variations (possibly much larger than in a quartz disk) avoid phase coherence, thus lowering the resonant Q-factor.

Determination of thickness

The film thickness was measured separately using a phase contrast microscope, since the film thickness is very important for determining the resonance characteristics of the complex. The non-invasive nature of the microscope avoids any damage to the hydrogel film that may occur with a contact profiler such as Dektac. However, it is important that the film being measured is transparent so that the profilometry can be performed using a phase-contrast microscope. The measurement procedure must be performed with the step of imaging from the bare surface of the quartz crystal to the upper surface of the hydrogel. Effectively, contour plots of the membrane boundaries are depicted, where the contours are separated by a fixed interval period (fig. 3 a). It is known that the poly-HEMA copolymer has a dielectric constant of 1.5 and a light source wavelength of 600nm, and the film thickness is 2.1 μm when the migrated volume is 10. mu.L as estimated from the contour plot.

Four separate devices containing different volumes of monomer mixture were removed from the crystal before crosslinking with uv light to evaluate whether the film was acoustically thick according to Martin's definition [ anal. chem.2000,72,141-149 ]. Fig. 3b shows that the frequency shift between the bare and hydrogel-coated resonant disks is approximately linear, regardless of the applied volume. The measured frequency shift was found to be greater than the Sauerbrey-like frequency shift calculated from the optical thickness, indicating a significant transmembrane acoustic phase shift. These measurements confirmed that the applied hydrogel film was a "thick" film.

Hydrogel-disc spectrogram

In order to induce different acoustic phase shifts without changing the chemistry or the device geometry, the frequency is adjusted to transmit back a series of harmonics in a certain frequency range at ph3.5 (fig. 2). In summary, it was found that the resonance amplitude decreases sharply with increasing frequency, and harmonics above 100MHz are difficult to resolve. This characteristic can be attributed to material damping and film thickness variations.

The effect of pH change from 3.5 to 8 on the first fifth harmonic was investigated by recording sonogram changes, including its frequency, amplitude and Q factor. At fundamental frequency (first plot in fig. 4), a shift in frequency characteristic of films of increasing thickness is observed. This response is due to the hydrogel matrix becoming hydrated at higher pH values due to ionization of the polymer-bound carboxyl groups, attraction of counter ions and water, and the polymer phase swelling and increasing in thickness.

At the harmonic of 33MHz, there is no significant frequency shift because the resonance peak disappears completely (second graph in fig. 4). However, at higher frequencies, the frequency shift pattern is reversed (third diagram in fig. 4). The frequency associated with the hydrogel film with a thicker pH8 was shifted upward. This property is believed to be an example of a thick film property when the acoustic phase shift across the membrane is sufficient to support resonance in the membrane itself. This characteristic is attributable to the membrane being out of resonance with the disk, so that the primary reflection boundary supporting the standing wave resonance is located between the lower surface of the disk and the disk-hydrogel interface, rather than between the disk and the hydrogel-water interface.

The above embodiments illustrate that harmonics, particularly 33MHz harmonics, can be switched. However, for a suitable acoustic sensor thick layer, any frequency (fundamental or harmonic) can be used to achieve the switching characteristics.

Switching action

For composite resonators with thick films, switching is the most interesting feature. At this time, the transition between the on state and the off state has an extremely high contrast. An example of a good switching characteristic of the fundamental frequency is illustrated in fig. 5a, where the peak completely disappears when the pH changes from 3.5 to 8. Fig. 5b illustrates that the switch is reversible. The resonance peaks in this example were collected repeatedly in a continuous time-wise manner. The left side of the curve shows that the same peak was collected repeatedly over time. Buffer with pH8 additionAfter the liquid, the peak completely disappeared within about 10 seconds, and was again at pH3.5, returning the original peak, indicating that the elastic properties of the hydrogel-disc complex were reversible. The reason for the switching action initially available is because of the extremely short acoustic wave wavelength generated in the film at low shear wave speeds, estimated to be about 24 ms-1. This value is comparable to the value of 15ms-1 for poly-HEMA hydrogel measured alone. Furthermore, since the wavelength of the acoustic wave in the hydrogel at the switching point is well defined, it is defined by c ═ ρ²The elasticity of poly-HEMA at pH8 can be calculated to be 5.8 × 10⁵N/m²Consistent with the mechanical properties of "soft" polymeric materials.

Calibration curve

To clarify the stages of the switching transition, incremental changes in pH are made to the composite resonator. The resonator performs a reversible switching transition with the resonance completely suspended at pH8. These changes in the acoustic resonance plot are shown in figure 6a, as well as the change in amplitude with pH. The corresponding changes in resonance amplitude and Q factor are shown in fig. 6 b. The data showed significant resonance damping, indicating that energy was withdrawn from the quartz element by internal absorption in the hydrogel. The Q-factor and the position of the inflection point in the amplitude acoustics plot can be predicted by substituting different ionizing groups, as demonstrated by previously reported diffraction gratings.

A chemically driven mechanical switch may be constructed from a composite resonator body comprising a quartz disk and a hydrogel membrane thereon. This switching occurs when the membrane is operated under thick film resonance conditions, where the acoustic phase is 90 degrees and the hydrogel film is 2.1 μm thick, when the appropriate frequency and multi-frequency acoustic device is used. The sensitivity to elastic changes of the hydrogel is also significantly enhanced under switching conditions. Furthermore, this technique not only facilitates the selection of suitable monomer, crosslinker and copolymer ratios to provide mechanically responsive acoustic or optical hydrogels, but it can also serve as a chemical-based switching component for studying protein (proteinaceus) membrane dynamics in the acoustic field if the membrane is thin and probed at high frequencies.

Example 2

A glucose sensor capable of remotely monitoring glucose concentration is described below.

Synthesis of a glucose-sensitive copolymer of 3-acrylamidophenylboronic acid (3-APB) and acrylamide by UV-initiated free radical polymerization at room temperature with N, N-Methylenebisacrylamide (MBA) as crosslinker. Poly (acrylamide-co-3-APB) has a pKa of about 8.6, but is glucose nodule

The main approach described here was to incorporate glucose responsive poly (acrylamide-co-3-APB) membranes into magneto-acoustic resonant sensors (MARS) and study the response of the sensing device to changes in glucose concentration in solution.

Complexation of boronic acids with cis-diols

The reversible binding between boronic acid and cis diol, which is pH dependent, is shown in figure 7. The chemical structure of poly (acrylamide-co-3-APB) is shown in FIG. 8. At low pH, boric acid is an uncharged trigonal state that transitions to a tetrahedral state when it is charged and pH is increased. In the tetrahedral form, the boronic acid has a higher affinity for the cis diol than in the trigonal form, where its complex with the cis diol is more easily hydrolyzed. Glucose comprises a cis-diol structure. Two potential mechanisms by which glucose binds to the phenylboronic acid groups carried by the polymer include: 1:1 monomer bonding and 1:2 cross-linking bonding. Equimolar 1:1 bonding results in expansion of the polymer film, while 1:2 bonding results in shrinkage.

pH response of Poly (acrylamide-co-3-APB) membranes

Since poly (acrylamide-co-3-APB) is pH sensitive and the complex of glucose with tetrahedral 3-APB is more stable than the trigonal structure, the characteristics of the polymer membrane at different pH were investigated. A pH titration curve of the polymer is determined and measured, and the pKa value is calculated from the pH curve. The procedure for synthesizing the copolymer was almost the same as that for the poly (HEMA-co-MAA) except that UV exposure was performed for 30 minutes. The prepolymer solution consisted of 5mol/l monomer in 500. mu.l solvent (DMSO containing 2% (w/v) photoinitiator DMPA). The buffer system was acetic acid (pH4, 4.5, 5, 5.25 and 5.5), MES (pH5.75, 6, 6.5), phosphate (pH7, 7.5, 8), Tris (pH 8.5), glycine (pH9), CHES (pH10) and phosphate (pH 12). The buffer concentration was 10mM, and the ionic strength was fixed at 154mM with sodium chloride.

As the pH of the buffer increased, the poly (acrylamide-co-3-APB) became increasingly charged. A Donnan potential exists between the polymer and the aqueous medium surrounding it, driving water into the hydrogel and causing it to swell. At the same time, the osmotic effect also leads to hydration of the polymer. FIG. 9 shows the pH profile collected at 6MHz where the poly (acrylamide-co-3-APB) membrane contained 78.5 mol% acrylamide, 1.5 mol% MBA, and 20 mol% 3-APB. The calculated apparent pKa value was 8.47.

Response of MARS with poly (acrylamide-co-3-APB) to glucose

Apparent pKa reduction due to glucose binding

FIG. 9 shows the pH profile of poly (acrylamide-co-3-APB) containing 78.5 mol% acrylamide, 1.5 mol% MBA and 20 mol% MAA at 6 MHz; the apparent pKa value calculated from this curve was 8.47. In FIG. 9, the poly (acrylamide-co-3-APB) polymer is nearly neutral at physiological pH7.4, with about 91.5% of the 3-APB pendant groups in a triangular configuration and weakly bound to glucose. A 5mM D- (+) -glucose solution was prepared in the above listed buffers to evaluate the response of the coated resonators to the glucose solution at different pH values. The pH curve at 6MHz shifted negatively and the apparent pKa value was reduced from 8.47 (FIG. 9) to 8.02. At this time, at ph7.4, a proportion of 24% of the borate groups had become anionic and tetrahedral.

As shown in FIG. 7, since the binding of glucose to the trigonal 3-APB is weak, the dissociation of neutral 3-APB was determined by the proton concentration in the solution and the binding between glucose and the tetrahedral 3-APB.

Calibration of glucose response of MARS

The response of glucose-responsive poly (acrylamide-co-3-APB) MARS to glucose concentration in the buffer solution was calibrated with the same polymer as shown in FIG. 9. A glucose solution was prepared in 10mM PBS buffer, pH 7.4. The glucose concentration is 0-15 mM, and the amplification is 2.5 mM. FIG. 10 shows the response amplitude of MARS coated with poly (acrylamide-co-3-APB) membrane (78.5 mol% acrylamide, 1.5 mol% MBA and 20 mol% 3-APB) versus glucose concentration; response data were obtained by performing three tests at 20MHz on 0-15 mM glucose solution. FIG. 10 shows that the signal amplitude from MARS is almost linearly proportional to the glucose concentration in the range of 0-15 mM. Errors were evident when the glucose concentration was higher than 7.5 mM. The sensitivity of the glucometer was about 87.07 mV/mM. It is believed that the large error at high glucose concentrations is due to the transition between the 1:1 binding mode and the 2:1 cross-linking mode between the boronic acid and glucose molecules.

The data shown in FIG. 10 is collected: the polymer coated on the quartz plate was washed three times with pure PBS buffer and tested again under the same conditions.

FIG. 11(a) shows the three responses of MARS with poly (acrylamide-co-3-APB) membrane (78.5 mol% acrylamide, 1.5 mol% MBA and 20 mol% 3-APB) to glucose solution (0 mM-15 mM, pH7.4 PBS buffer) at 20 MHz; fig. 11(b) plots the response results of three tests against glucose concentration, respectively. In FIG. 11(b), it is noted that the response of poly (acrylamide-co-3-APB) MARS to glucose solutions containing more than 10mM glucose was slightly greater in the two latter tests than in the first, while the responses obtained from the second and third tests were similar. This is probably due to the fact that the pore size of the polymer network is enlarged by the glucose molecules at the first test, glucose gaining access to the polymer more easily and subsequently swelling the hydrogel.

Optimization of 3-APB concentration in glucose sensors

MARS sensitivity is sufficient to detect small changes in glucose concentration in buffer solution. The relationship between response (amplitude of signal) and glucose concentration is approximately linear (fig. 10); however, in order to enhance the response, the composition of the polymer film is optimized.

Response of different 3-APB concentrations to glucose

The response of polymers with different mole% 3-APB to glucose was investigated. The glucose solution system was the same as above, and the amplitude change (Δ Α) was defined as follows:

ΔA₀ ¹⁵amplitude (15mM glucose) -amplitude (0mM glucose) (20MHz)

FIG. 12 shows poly (C) at 20MHz versus different 3-APB concentrationsDelta A of MARS of enamide-co-3-APB) membrane to 15mM glucose₀ ¹⁵(ii) a Three samples were tested for each mole%. Amplitude variation (Δ A)₀ ¹⁵) The curve relative to 3-APB mol% is bell shaped (FIG. 12). The maximum response was observed for about 20 mole% of 3-APB and the response decreased as more 3-APB was added to the polymer. The results in Table 12 correlate with the contribution of ionization of poly (acrylamide-co-3-APB) (related to mole% of 3-APB) and the accessibility of glucose into the polymer network (related to pore size and hydrophilicity/hydrophobicity of the polymer network). Ionization of poly (acrylamide-co-3-APB) in the presence of different glucose concentrations depends on the apparent pKa of the polymer membrane or the change in apparent pKa due to glucose. The apparent pKa (20MHz) of poly (acrylamide-co-3-APB) relative to mole% 3-APB is calculated in Table 1; when the concentration of 3-APB is<At 20 mole%, the apparent pKa did not change significantly, but at higher mole%, the apparent pKa increased. Comprises>The high apparent pKa, i.e., low degree of ionization, of 20 mole% of poly (acrylamide-co-3-APB) of 3-APB can be explained by the Δ A in FIG. 12₀ ¹⁵A drop in response.

Table 1: apparent pKa values of poly (acrylamide-co-3-APB) with different 3-APB concentrations at 46MHz (three samples)

^*STD: standard deviation of

pK due to glucose binding_aChange in value

Due to lower apparent pK_aThe poly (acrylamide-co-3-APB) contains more tetrahedral boric acid and can capture more glucose at physiological pH value, so the apparent pK of the poly (acrylamide-co-3-APB) caused by glucose combination is researched_aVarying 3-APB concentration dependence. Polymers with 15 mole%, 20 mole%, 25 mole% and 30 mole% 3-APB were prepared separately and the pH curves of the pure buffers were collected in response to pH 4-10. The pH curve was again recorded in the presence of 5mM glucose. pKa changes were-0.52, -0.32 for the four polymer films, respectively-0.66 and-0.46.

Effect of mole% 3-APB on hydrophilicity/hydrophobicity of Poly (acrylamide-co-3-APB)

The hydrophilicity/hydrophobicity of the 3-APB polymer can be analyzed by observing the change in amplitude due to water absorption when the pH is changed from 10 to 4. Amplitude (Δ A)₄ ¹⁰) Is defined as:

ΔA₄ ¹⁰amplitude (pH10) -amplitude (pH4) (20MHz)

FIG. 13 shows the Δ A of MARS versus pH change (pH 4-10) for poly (acrylamide-co-3-APB) -containing membranes at 20MHz versus different 3-APB concentrations₄ ¹⁰(ii) a Three samples were analyzed for each mole%. The pH response of polymers with different 3-APB concentrations (5-30 mol%, 5 mol% amplification) was collected. FIG. 13 graphically depicts the change in amplitude relative to the percentage of 3-APB in the copolymer. The amplitude change was greatest when the polymer contained 20 mole% 3-APB; when mole% of 3-APB>At 20 mole%, the polymer film swells less. Theoretically, an increase in the density of the 3-APB can increase the polynorbic potential and osmotic effect, so that more water will enter the polymer network. However, the hydrophobicity of 3-APB prevents water and glucose from entering the hydrogel simultaneously. So at 20 mole%, the osmotic effect and the hydrophobic effect reach equilibrium. Therefore, it was proposed that the hydrophilicity/hydrophobicity of poly (acrylamide-co-3-APB) is a major factor in determining the sensitivity of acoustic glucose sensors.

Effect of mol% 3-APB on Polymer viscoelasticity

The swelling capacity of poly (acrylamide-co-3-APB) copolymers also affects the penetration of glucose molecules into the polymer and is qualitatively defined by the Q-factor of the polymer coated sensor. As described above, poly (acrylamide-co-3-APB) copolymers behave as elastic materials at the high operating frequencies of MARS; hydrogen bonding with water or glucose enhances the elasticity of the polymer and increases the Q factor.

The Q-factor of the poly (acrylamide-co-3-APB) sensor increases proportionally with operating frequency at < about 140MHz, and then saturates above 140 MHz; for example, a polymer with 15 mole% 3-APB has a Q factor at saturation level of about 3250, while a polymer with 20 mole% 3-APB has a Q factor of about 2000, a polymer with 30 mole% 3-APB swells less and a Q factor of about 900. The increase in Q factor with operating frequency may be due to less acoustic energy penetrating and dissipating in the polymer, while the increase in elasticity of the polymer may also be due. At high frequencies, the acoustic energy is concentrated at the polymer-quartz interface and the viscoelasticity of the polymer reaches equilibrium, so the Q factor saturates at frequencies >140 MHz.

The swelling behaviour of the same polymer in 10mM glucose phosphate buffer pH7.4 was also observed. In this case, the saturation level of Q-factor for the corresponding polymer containing 15 mol% 3-APB is about 4000, the polymer containing 20 mol% 3-APB is about 3000, the polymer containing 30 mol% 3-APB swells less and the Q-factor is about 1500. The difference in Q factors (Δ Q) of the polymers in pure buffer and 10mM glucose solution was: 15 mol% 3-APB was 750, 20 mol% 3-APB was 1000 and 30 mol% 3-APB was 600. The Δ Q relative to the saturation level of 3-APB mol% is shown in FIG. 14, which shows the Δ Q between the response of a poly (acrylamide-co-3-APB) sensor relative to the mol% of 3-APB in pure pH7.4 buffer and 10mM glucose solution at pH 7.4.

The results of fig. 14 show that: glucose incorporation enhanced the elasticity of the polymer, with the best effect observed between 20 and 25 mole% 3-APBA. In addition to the hydrophilicity/hydrophobicity of the polymer, the swelling capacity and pore size of the polymer network also affect the sensitivity of the glucose sensor. The first half of the curve in fig. 12 reflects that the sensitivity of the glucose polymer is directly proportional to the concentration of phenylboronic acid groups, and the second half indicates that the sensitivity of the polymer is also determined by the hydrophilic/hydrophobic balance of the network, the viscoelasticity, and the pore size.

Optimization of crosslink mole% in 3-APB polymers

It is believed that the pore size and viscoelasticity of the hydrogel network also affect the performance of the glucose sensor, where steric hindrance may play an important role.

According to the above results, the maximum response (. DELTA.A) was achieved at a 3-APB concentration of 20 mol%₀ ¹⁵). The polymer content was monitored separately by maintaining the percentage of 3-APB at 20 mol% and monitoring the content of 0.5 molThe effect of crosslinking was investigated in% response, 1.5 mol% response and 2.5 mol% MBA (fig. 15). FIG. 15 shows the difference in amplitude of the signal from MARS versus the concentration of crosslinker MBA for the reference solution (glucose concentration 0mM) and for a glucose concentration 15 mM. FIG. 15 shows continuous infusion of glucose buffer solution at an increase of 2.5mM over a concentration range of 0-15 mM. The results in FIG. 15 show that reducing the mole% of the crosslinker does not help to enhance the sensitivity of the poly (acrylamide-co-3-APB) sensor to glucose. Delta Q was also used to study the swelling characteristics of poly (acrylamide-co-3-APB) copolymers crosslinked in the range of 0.5 to 2.5 mole% when exposed to 10mM glucose solution.

5.7 detection of hyperglycemia and hypoglycemia

In diabetic patients, blood glucose levels rise to levels far above normal (about 4.4mM) (hyperglycemia), and when the patient takes insulin inappropriately, blood glucose levels fall below normal (hypoglycemia). Therefore, the effective operating range of the glucose sensor should be measured. Glucose solutions were prepared at concentrations of 0, 2.5mM, 5mM, 7.5mM, 10mM, 12.5mM, 15mM, 20mM, 40mM, 60mM, 80mM, and 100mM in PBS buffer at pH 7.4. The polymer film consisted of 83.5 mol% acrylamide, 1.5 mol% MBA and 15 mol% 3-APB.

FIG. 16 shows: (a) the response amplitude of MARS with poly (acrylamide-co-3 APB) membrane (83.5 mol% acrylamide, 1.5 mol% MBA and 15 mol% 3-APB) versus glucose concentration (0-100 mM) was fitted as an exponential equation using SigmaPlut; (b) when the glucose concentration is less than or equal to 7.5mM, the response amplitude is proportional to the glucose concentration.

As shown in FIG. 16, the response of MARS with respect to glucose concentration became saturated above 20 mM. Thus, the MARS-based glucose sensor works effectively in the range of 0-20 mM, and below 7.5mM the amplitude of the response is substantially proportional to the glucose concentration (FIG. 16). The data shown in FIG. 16(a) is fit to an exponential equation, which indicates that the response of the MARS sensor with respect to glucose concentration may follow an exponential rise pattern. FIG. 17 shows response times for glucose concentrations from 0 to 100mM, above 20mM, response times <10 minutes. FIG. 17 shows the response time of MARS with poly (acrylamide-co-3 APB) membrane (83.5 mol% acrylamide, 1.5 mol% MBA and 15 mol% 3-APB) for continuous variation of glucose concentration from 0 to 100mM at pH 7.4.

The saturation levels observed in FIG. 16 for glucose concentrations >20mM and the decrease in response time (FIG. 17) for 3-APB levels >20 mol% may be related to swelling of the polymer network. The hydrogel swells more in solutions containing high glucose concentrations, which in turn facilitates the penetration of the glucose solution. However, since the poly (acrylamide-co-3-APB) copolymer is crosslinked, the network has limited swelling capacity, and thus the saturation level achieved in fig. 16 may correspond to the swelling limit, as well as the steric effect of the complex of glucose with the aromatic boronic acid.

Effect of electric field on glucose response

According to the above study, the operating frequency affects the apparent pKa value measured by the glucose sensor. To understand the performance of glucose sensors over a broad spectral range, the polymer brush theory and the concept of local pKa was introduced. In this study, it was investigated how the performance of the glucose sensor is affected by the operating frequency by determining the effective detection range of the sensor.

The composition of the polymer is: acrylamide 78.5 mol%, crosslinker MBA1.5 mol% and 3-APB20 mol%, the MARS responses with respect to glucose concentration were found to be rather similar to each other in the frequency range from 6MHz to 73 MHz. Thus, the effect of operating frequency on the MARS response with poly (acrylamide-co-3-APB) membranes was negligible.

Sugar detection

Previous studies based on holographic sensors showed that glucose molecules bind to phenylboronic acid groups in two ways: 1:1 monomer bonding and 1:2 cross-linking bonding. The stoichiometric 1:1 binding results in expansion of the polymer film, while the 1:2 stoichiometry results in contraction. Glucose has five chemical structures:

isomers of glucose were studied to understand the mechanism of binding between phenylboronic acid and cis-diol. In addition, the largest amount of sugar contained in blood is fructose and galactose, in addition to glucose. Fructose, galactose and mannose solutions (5mM) were prepared in 10mM PBS buffer, with an ionic strength of 154mM at physiological pH. The quartz plate with poly (acrylamide-co-3-APB) copolymer membrane was equilibrated in three sugar solutions and between measurements the membrane was washed with phosphate buffer until the response returned to the reference level (phosphate buffer)

FIG. 18 shows β -D-fructofuranose, α -D-galactopyranose and β -D-mannopyranose

The response of the polymer to fructose was significantly greater than to the other sugars, with an amplitude change of 362.95mV from the reference (fig. 18). The responses to glucose, galactose and mannose are smaller and the three are relatively similar to each other. Δ Α for glucose was 52.25mV, galactose was 30.15mV and mannose was 15.2 mV. The strong response to fructose can be attributed to the affinity of the cis-diol for boronic acid and the proportion of the effective isomer. Fructose has been reported to have a higher affinity for boronic acids than the other three sugars.

Discussion and conclusions

Synthetic phenylboronic acid, 3-acrylamidophenylboronic acid (3-APB), has been added to MARS to produce a glucose selective sensor, which, because MARS is wirelessly excited by an RF electric field, constitutes a promising alternative for the development of implantable continuous glucose meters. 3-APB is a phenyl boronic acid derivative, the copolymer of which with acrylamide can respond to glucose at physiological pH. By replacing the traditional enzyme glucose oxidase with a synthetic phenyl boronate ligand, the performance of the glucose sensor is more durable and reversible, since denaturation of the enzyme reduces the stability and sensitivity of the sensor. It is also believed that the synthetic glucose responsive hydrogels enable an in vivo glucose-insulin closed loop system. Sensitivity, reproducibility, selectivity and response time are important parameters of glucose monitoring systems. The response of MARS with poly (acrylamide-co-3-APB) copolymer membranes to glucose and the mechanism of glucose incorporation in the polymer membrane have been studied.

Sensitivity of MARS coated with poly (acrylamide-co-3-APB) adsorption layer (adlayer) to glucose was determined mainly by the concentration of 3-APB, hydrophobicity and viscoelasticity of the polymer (FIG. 19)). FIG. 19(a) shows Δ A for the MARS sensor relative to mole% of 3-APB₀ ¹⁵) And the apparent pKa value of poly (acrylamide-co-3-APB); (b) delta A of MARS sensor relative to mole% of 3-APB₀ ¹⁵) And Delta A of poly (acrylamide-co-3-APB)₄ ¹²(ii) a (c) Delta A of MARS sensor relative to mole% of 3-APB₀ ¹⁵) And Δ Q of poly (acrylamide-co-3-APB).

In summary, preliminary studies were conducted on a novel MARS-based glucose sensor system. Changes in viscoelasticity within the poly (acrylamide-co-3-APB) network due to bound glucose can be measured by MARS and the corresponding glucose levels can be corrected. The glucose monitoring system has good reversibility and repeatability. The sensitivity of the sensor averaged about 87.07 mV/mM. The results indicate that MARS can be a versatile platform for studying the physical properties of very thin polymer films and developing intelligent polymer-based biosensors.

While the present invention has been particularly shown and described with reference to the preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

1. A method of monitoring sugar concentration using magnetoacoustic resonance, the method comprising the steps of:

1) placing a resonator and a sensor in a physiological analyte of a subject, the sensor being mechanically coupled to the resonator, wherein the resonator is capable of monitoring a concentration of a sugar; the resonator comprises a magnet positionable to direct a magnetic field toward the sensor and an exciter in mechanical communication with the sensor; the sensor comprises a sensor material that changes between a first state and a second state upon contact with a change in the surrounding environment, wherein the sensor is driven by the resonator, and wherein the sensor material is in the form of an acoustically thick layer; the thickness of the acoustically thick layer substantially satisfies the following equation:

where ω is angular frequency, ρ is density, G is shear modulus, and t is film thickness; the acoustically thick layer is a layer capable of supporting a significant phase shift of an acoustic wave when the acoustic wave propagates perpendicular to the layer;

wherein the first state is a state in which the sensor material resonates with the resonator, and the second state is a state in which the sensor material does not resonate with the resonator due to an increase in film thickness,

wherein the environmental change is a pH change, the sensor material is a hydrogel, and the hydrogel is a hydroxyethyl methacrylate-methacrylate copolymer;

2) detecting a signal in response to a change in state of the sensor material outside the subject's body by a detector to which is connected an adjustable electromagnetic field generator configured to direct an electromagnetic field towards the sensor;

3) the signal detected by the detector is processed to monitor the sugar concentration in the sample.

2. The method of claim 1, further comprising processing the detected signals with a signal generator and a lock-in amplifier connected to the electromagnetic field generator and the detector.

3. The method of claim 1, wherein the exciter comprises a piezoelectric material of quartz, and the piezoelectric material is in the form of a layer having a thickness of 50 μm to 1000 μm.

4. The method of claim 1, wherein the acoustically thick layer has a thickness of 0.5 μm to 5 μm.

5. The method of claim 4, wherein the acoustically thick layer is greater than one molecule thick.

6. The method of claim 1, wherein the sensor material is a hydrogel.

7. The method of any one of claims 1-6, wherein the hydrogel is a polymer comprising pendant phenyl boronic acid groups.

8. The method of claim 7, wherein the polymer is poly (acrylamide-co-3-acrylamidophenylboronic acid) or poly (acrylamide-co-2-acrylamidophenylboronic acid).