CN105380668A - Method for utilizing magneto-acoustic resonance to continuously monitor blood glucose change - Google Patents

Abstract

The invention provides a method for utilizing magneto-acoustic resonance to continuously monitor blood glucose change. The method includes the following steps that 1, a resonator capable of continuously monitoring the blood glucose change and a sensor are led or implanted into a body of a subject, wherein the sensor and the resonator are mechanically connected, the resonator comprises a magnet capable of guiding a magnetic field to the sensor and an exciter mechanically connected with the sensor, the sensor includes a sensor material in the state changing with surrounding environment change when sensing the surrounding environment change and is driven by the resonator, and the sensor material is in an acoustic thick-layer form; 2, a signal responding to the state change of the sensor material is detected outside the body of the subject through a detector, and the detector is connected with an adjustable electromagnetic field generator for guiding an electromagnetic field to the sensor; 3, the signal detected by the detector is continuously processed so as to continuously monitor the blood glucose change in the body of the subject.

Description

A kind of method utilizing the continuous Monitoring Blood Glucose change of magnetoacoustic resonant

The application is Chinese invention patent application (denomination of invention: magnetic acoustic resonator sensor; Application number: 201180026587.8; The applying date: on December 9th, 2011) divisional application.

Technical field

The present invention relates to sensing device, preferably adopt the chemokinesis type electromechanical transducer device of hydrogel.

Background technology

The hydrogel that can change shape or volume when contacting different chemical environments is used as pH sensor, ion transducer, chemical sensor, gas sensor and temperature sensor, and is used as mechanical drive element.For example, see: Bashir, R. etc., MicromechanicalcantileverasanultrasensitivepHmicrosensor, AppliedPhysicsLetters, 2002,81 (16): 3091 pages ~ 3093 pages; Zhang, L. and W.R.Seitz, ApHsensorbasedonforcegeneratedbypH-dependentpolymerswell ing, AnalyticalandBioanalyticalChemistry, 2002,373 (7): 555 pages ~ 559 pages; VanderLinden, H. etc., Developmentofstimulus-sensitivehydrogelssuitableforactua torsandsensorsinmicroanalyticaldevices.SensorsandMateria ls, 2002,14 (3): 129 pages ~ 139 pages; Mayes, A.G. etc., Metalion-sensitiveholographicsensors.AnalyticalChemistry, 2002,74 (15): 3649 pages ~ 3657 pages; Mayes, A.G. etc., Aholographicalcoholsensor.AnalyticalChemistry, 1999,71 (16): 3390 pages ~ 3396 pages; Mayes, A.G. etc., Aholographicsensorbasedonarationallydesignedsyntheticpol ymer.JournalofMolecularRecognition, 1998,11 (1-6): 168 pages ~ 174 pages; Millington, R.B. etc., Ahologrambiosensorforproteases.SensorsandActuatorsB-Chem ical, 1996,33 (1-3): 55 pages ~ 59 pages; Blyth, J. etc., Holographicsensorforwaterinsolvents.AnalyticalChemistry, 1996,68 (7): 1089 pages ~ 1094 pages; Herber, S., W.Olthuis and P.Bergveld, Aswellinghydrogel-basedP-C02sensor.SensorsandActuatorsB-Chemical, 2003,91 (1-3): 378 pages ~ 382 pages; Kuckling, D. etc., Photocross-linkablepoly (N-isopropylacrylamide) copolymersIII:micro-fabricatedtemperatureresponsivehydro gels.Polymer, 2003,44 (16): 4455 pages ~ 4462 pages; Hilt, J.Z. etc., Ultrasensitivebiomemssensorsbasedonmicrocantileverspatte rnedwithenvironmentallyresponsivehydrogels.BiomedicalMic rodevices, 2003,5 (3): 177 pages ~ 184 pages.

Based on the complexion changed of modified water gel, the change in size of hydrogel crystal or the optical detection of diffraction grating are provided and has sensed metering (sensorimetric) signal easily, latter provided more general detection platform.For example, see: Asher, S.A. Photoniccrystalcarbohydratesensors:Lowionicstrengthsugar sensing.JournaloftheAmericanChemicalSociety is waited, 2003,125 (11): 3322 pages ~ 3329 pages and Marshall, A.J. PH-sensitiveholographicsensors.AnalyticalChemistry is waited, 2003,75 (17): 4423 pages ~ 4431 pages.

But, it is believed that hydrogel has several aspect to have embody rule.For example, see Hoffman, A.S., Hydrogelsforbiomedicalapplications.AdvancedDrugDeliveryR eviews, 2002,43:1 page ~ 12 page.An important consideration for optics hydrogel system maintains the position limiting the granule of complexion changed subsequently.This stability rigidity needing hydrogel must keep enough, but low cohesiveness is provided simultaneously, make it possible to the change in volume occurring surveying.These mechanical aspects of hydrogel by the amount of stretch number of the rectangular element of poly-HEMA (methacrylate) centimetre is studied, to measure its elasticity at different pH value.For example, see Johnson, B. etc., MechanicalpropertiesofapHsensitivehydrogel, SEM nd Annual Meeting in 2002, Milwaukee, WI.

Another kind of method for sensing uses the sonic sensor in response to viscoelastic solution.Sonic sensor is included in the resonator of excited vibration in viscoelastic solution.Solution viscosity change (such as, due to chemical reaction) causes the frequency of vibration/amplitude variations of detectable resonator.

The adaptation of sensor as aforementioned comprises provides monomolecular film on the resonator, and described monomolecular film is used as the probe of surrounding chemical/physical environment.Monomolecular film can with the molecular reaction in surrounding medium, such as, cause the mass/volume of resonator to change, thus changes the frequency/amplitude of resonator vibrates.Such setting can use monolayer hydrogel to detect nucleotide.For example, see Kanekiyo, Y. etc., Novelnucleotide-responsivehydrogelsdesignedfromcopolymer sofboronicacidandcationicunitsandtheirapplicationsasaQCM resonatorsystemtonucleotidesensing.JournalofPolymerScien ceParta-PolymerChemistry, 2000,38 (8): 1302 pages ~ 1310 pages; Tang, A.X.J. etc., Immunosensorforokadaicacidusingquartzcrystalmicrobalance, AnalyticaChimicaActa, 2002.471 (1): 33 pages ~ 40 pages; Nakano, Y., Y.Seida and K.Kawabe, Detectionofmultiplephasesinecosensitivepolymerhydrogel, KobunshiRonbunshu, 1998.55 (12): 791 pages ~ 795 pages; And Serizawa, T. etc., Thermoresponsiveultrathinhydrogelspreparedbysequentialch emicalreactions.Macromolecules, 2002.35 (6): 2184 pages ~ 2189 pages.

Acoustic sensor therefore can be used as surrounding probe and without the need to combining diffraction grating.But, in above-mentioned setting, in order to change detected, need carefully to follow the tracks of the disturbance of the usual <0.01% occurred in audio frequency.Namely, existing sonic sensor has following problems: the effect produced in the sensor because of physical/chemical environmental change is less, in order to obtain the useful information about change, needs to carry out the very high measurement of sensitivity.

Summary of the invention

The present invention attempts to utilize the change by the physics and/or chemical property that drive sensor to carry out the change of surrounding being monitored.Particularly, the present invention attempts the muting sensitivity problem solving above-mentioned existing sonic sensor.

According to a first aspect of the invention, a kind of sensing device is provided, described sensing device comprises resonator, sensor and detector, described sensor and described resonator are mechanically connected, the sensor material that described sensor changes when being included in contact surrounding environment change between a first state and a second state, wherein said sensor is driven by described resonator, and described detector response changes in the state of described sensor material, and wherein said sensor material is the form of acoustics thick-layer.

The main advantage of this acoustic method is the information of the interiorization educational level can passed back caused by crosslinked and copolymerization, and these information are obvious for hydrogel elastic, but are all unconspicuous for any change in size of optical collection.The advantage of embodiments of the present invention is that the sound phase change across aquagel membrane (acoustics thick film) thickness is comparatively large and close to Pi/2, thus substantially resonance is changed to elimination point (pointofextinction), and thus provide the useful mechanical switch effect between vibration with non-vibrational state.

Therefore, embodiments of the present invention relative to the Advantageous techniques effect of prior art are, when chemical/physical environment changes, the amplitude of the acoustical signal of resonator moves larger, thus only have with wherein audio frequency compared with the prior art of small size movement arranges, more easily detect.Sensor layer (can be bio-identification layer) self becomes the resonance structure of energy storage or power consumption.Thus sensor response is controlled by the chemical composition of sensor layer or upper layer.Aquagel membrane is a kind of examples of many successful of this kind of upper layer.

This Advantageous techniques effect realizes by providing the sensor material of acoustics thick-layer form, and it is different from the thin layer disclosed in prior art.Namely, acoustics thick film is provided directly to cause acoustical signal greatly to offset.

For viscoelastic material, the conversion between acoustics thin layer performance and acoustics thick-layer performance is most conveniently limited by phase factor.Therefore, the thickness of acoustics thick film roughly meets following equation:

$\mathrm{\&omega;}\sqrt{\frac{\mathrm{\&rho;}}{G}}t>\&ap;\mathrm{\&pi;}/2$

Wherein, ω is angular frequency, and ρ is density, and G is modulus of shearing, and t is film thickness.

Acoustics thick-layer is when sound wave is perpendicular to the layer can supporting sound wave remarkable phase shift during this Es-region propagations.This restriction does not comprise monolayer disclosed in arranging of prior art.This type of monolayer can not support the sound phase shift perpendicular to described layer.Further, if this type of monolayer does not use high frequency according to above-mentioned equation, the sound phase shift being parallel to described layer can not be supported.

In the context of this application, " sonic vibration " refers to the standing sound wave reflected between the up-and-down boundary of sensor.

Preferably, detector comprises the electromagnetic field generator that can arrange for by electromagnetic field orientation sensor.This is favourable, because sensor can the long-range use without the need to connecting line.Preferably, electromagnetic field generator and detector comprise for generation of electromagnetic field and the conventional structure element for detection of electromagnetic fields.This provide compact setting.Advantageously, electromagnetic field generator is adjustable.This makes device have larger variability and susceptiveness in use.Electromagnetic field generator can be spiral winding, and sensing device can comprise the signal generator and lock-in amplifier that are connected with electromagnetic field generator and detector to provide larger susceptiveness.Detector can comprise the difference diode detecting circuit deducting the signal detected in the signal for producing from signal generator.In one is arranged, resonator comprises the magnet that can arrange for by magnetic field orientation sensor.This arrangement provides magnetic acoustic resonator sensor.Resonator can comprise the exciter be connected with sensor mechanism, and it can be as piezoelectrics such as quartz layers.The thickness of piezoelectric layer is preferably 50 μm ~ 1000 μm.

Other sensing devices can be used for the electrical impedance of detector for measuring, thus measure acoustic response.Such as, the impedance measuring circuit of commercially available electric impedance analyzer or other customizations can be added.

Exciter can derive from magnetostriction materials, and wherein the magnetic component of resonator electromagnetic field is used for excite sound waves.Exciter can comprise metal material.It can be the layer form that thickness is preferably 50 μm ~ 1000 μm.

It is thick that the sensor layer comprising acoustics thick-layer sensor material is preferably 0.1 μm ~ 1mm, be more preferably 0.1 μm ~ 100 μm thick, and then be more preferably 0.1 μm ~ 10 μm thick, most preferably be 0.5 μm ~ 5 μm thick.Advantageously, to be greater than a part thick for sensor layer.

Sensor material is preferably hydrogel, such as methacrylate-methacrylate copolymer, poly-(acrylamide-co-3-acrylamido phenylboric acid) or poly-(acrylamide-co-2-acrylamido phenylboric acid).

Preferably, described sensing device is chemical flooding ejector half electromechanical transducer device.Described sensing device can be used in method for sensing.Further, described sensing device can be used as switch or in the method for monitoring continuously.In addition, described sensing device can be used for change based on surrounding and in the method for the hierarchy of control.

According to a further aspect in the invention, provide a kind of method preparing combined probe, described combined probe comprises the hydrogel acoustics thick film on piezoelectricity resonator element and described resonator element, and described method comprises: prepare described hydrogel monomer mixture; Described monomer mixture is applied to as on the adherent layers such as aluminum polyester sheet; Resonator element is applied on described monomer mixture; Make described polymerize monomer mixtures to form the described hydrogel acoustics thick film adhering to described resonator element, thus form combined probe; And described combined probe and described adherent layer are peeled off.Applying described monomer mixture before can process with binding agent (as methacrylic acid trimethoxy-silylpropyl ester) as described in resonator element thus promote bond.In one embodiment, hydrogel is prepared by the mixture of HEMA (HEMA), Ethylene glycol dimethacrylate (EDMA), methacrylate (MAA) and light trigger (as dimethoxy-phenyl acetone).In another kind is arranged, hydrogel is prepared by the mixture of 3-acrylamido phenylboric acid, acrylamide and N,N methylene bis acrylamide.In another is arranged, hydrogel is prepared by the mixture of 2-acrylamido phenylboric acid, acrylamide and N,N methylene bis acrylamide.

Embodiments of the present invention attempt to utilize material acoustics qualitative change caused by the physics of material and/or chemical environment change to provide chemokinesis type electromechanical transducer device.

In one embodiment, hydrogel changes in response to chemical environment, and preferably wherein chemical environment change is pH change.In the present embodiment, hydrogel normally by the change of its viscoelastic properties in response to the polymer of pH.The volume of hydrogel also can change in response to pH change.The hydrogel material being suitable for this object is methacrylate-methacrylate copolymer, it is the copolymer of HEMA and methacrylate, is preferably cross-linked with a certain amount of Ethylene glycol dimethacrylate (being also referred to as ethylene glycol dimethacrylate).Amount for the formation of the methacrylate of hydrogel is generally about 6 % by mole, and the amount of Ethylene glycol dimethacrylate is generally 1.5 % by mole ~ 7.5 % by mole.Usually, the hydrogel had more than 3.5 % by mole of Ethylene glycol dimethacrylate tends to become harder and lacks flexible.The methacrylate of surplus is 86.5 % by mole ~ 92.5 % by mole.

Different from the quartz crystal microbalance of routine, device of the present invention can radio operation thus eliminate non-dish type (non-disk) electrode.Compared with the 5MHz operating frequency of routine, this makes operating frequency extend to the scope of 6MHz ~ 1.1GHz.In addition, radio operation makes the resonator of sensing device and sensor element can implant such as human or animal subject.Now, detector can away from resonator and sensor manipulation in vitro.

In another embodiment, hydrogel changes in response to chemical environment, and wherein chemical environment change is the change of analyte concentration.Analyzing thing can be as physiological analytes such as sugar.This type of sugar can comprise glucose, fructose, galactose and mannose.Glucose is the physiological analytes of particular importance, plays an important role in such as diabetes and other diseases and sweat and living cells metabolism.

In one is arranged, hydrogel is the polymer containing phenylboronic acid group, and this phenylboronic acid group is generally the side base on main polymer chain.Preferably, described polymer is poly-(acrylamide-co-3-acrylamido phenylboric acid).This polymer in conjunction with glucose and other sugar, and can think that the boric acid moieties of this polymer is combined with the c/s-diol of sugar.

In one is arranged, sensing device of the present invention is the form of the implanted equipment implanting human or animal subject, and it comprises resonator and sensor.In the present arrangement, detector, in vitro in response to sensor, thus makes it possible to the remote monitoring of simple non-intruding mode as physiological analytes such as glucoses.Therefore, implanted equipment provides for implanting human or animal subject, and comprises resonator of the present invention and sensor, and wherein sensor and resonator are mechanically connected.In the method for remote sense study subject physiological status, detector can use together with implanted equipment, and wherein detector response is in the sensing material of sensor.The sensing of the physiological status of study subject can comprise the sensing to analyzing thing, and such as, described sensing can be continuous monitoring analysis substrate concentration.

From less stable during life-time service and the commercially available enzyme glucose sensor that carries out applying in body of being difficult to sterilize is different with monitor, sensing equipment of the present invention is durable and be easy to implant suitable study subject.

In the implementation method of the apparatus and method be mentioned before, the signal of amplitude moves greatly, can be more than 95%.Therefore be easy to detect, and significantly simplify sensor apparatus.Namely, observing very large signal intensity makes instrument can be simpler and cost is lower.Such as, can remove phase-locked and adopt the impedance bioelectrical measurement method that conventional susceptiveness is lower.Further, to compare physical and chemical changes sensitiveer in the setting of embodiments of the present invention and prior art.

For a better understanding of the present invention and how display implements the present invention, existing will exemplarily with reference to accompanying drawing, wherein:

Fig. 1 (a) demonstrates to be in and is produced and the hydrogel switch of the elect magnetic field of monitoring by measuring system, wherein the detection system LabVIEW that comprises signal generator, detector and run with PC (not shown) ^tMthe lock-in amplifier connected---be retained in the upper surface of hydrogel switch on plastic components (not shown) by contacting solution with o ring;

Fig. 1 (b) demonstrates the fundamental resonance peak of quartz disk compared with the identical quartz disk after the poly-HEMA aquagel membrane of coating 10 μm---and the top surface of quartz disk and hydrogel complex is all in the solution of pH3.5;

Fig. 2 demonstrates the comparison of cutting between wave resonance spectrum of three kinds of different hydrogels-quartzy complex, shows there is significant decay at 100MHz;

Fig. 3 (a) demonstrates the phase contrast image at aquagel membrane edge, shows that its thickness is about 3 μm;

Fig. 3 (b) demonstrates the frequency displacement with primary harmonic and triple-frequency harmonics after hydrogel layer coating quartz sense device, and hydrogel layer is originally with 5 μ l monomer mixtures (dish 1), 8 μ l monomer mixtures (dish 2), 12 μ l monomer mixtures (dish 3) and 15 μ l monomer mixtures (dish 4) preparation;

The acoustic resonance harmonic wave of the same hydrogel-quartzy complex after Fig. 4 demonstrates contact pH3.5 and pH8---film expands and causes the frequency at 6.7MHz place to subtract, and the resonance at 33MHz place stops and the frequency at 60MHz place increases;

Fig. 5 (a) demonstrate there is thickness evenly aquagel membrane hydrogel-quartzy complex on-off action---pH3.5 makes switch in "On" state, and pH8 makes switch in "Off" state;

Fig. 5 (b) demonstrates the switching characteristic being expressed as a series of continuous peak resonance flyback with 15 seconds intervals;

Fig. 6 (a) demonstrates pH calibration curve, and its increment resonance loci described between the open state of pH3.5 and the off status of pH8 changes;

Fig. 6 (b) demonstrates the change relative to pH value of solution of the amplitude of resonance and the Q-factor;

Fig. 7 demonstrates the Reversible binding occurred between aqueous medium mesoboric acid and c/s-diol;

Fig. 8 demonstrates the chemical constitution of poly-(acrylamide-co-3APB) copolymer;

Fig. 9 demonstrates the pH curve of poly-(acrylamide-co-3APB);

Figure 10 demonstrate be coated with poly-(acrylamide-co-3APB) film MARS relative to the response amplitude of concentration of glucose;

Figure 11 (a) demonstrate there is poly-(acrylamide-co-3APB) film MARS to the response of glucose solution;

Figure 11 (b) demonstrates the response of MARS relative to concentration of glucose;

Figure 12 demonstrate there is poly-(acrylamide-co-3APB) film MARS to the amplitude variations difference of 15mM glucose;

Figure 13 demonstrates the change for pH4 ~ 10, has the amplitude variations curve of MARS relative to different 3-APB concentration of poly-(acrylamide-co-3APB) film;

Figure 14 demonstrates poly-(acrylamide-co-3APB) sensor relative to the Δ Q between % by mole response in the pure buffer of pH7.4 of 3-APB and the response in the 10mM dextrose buffer liquid of pH7.4;

Figure 15 demonstrates when glucose incremental increase from the difference of vibration of the signal of MARS relative to cross-linking agent MBA concentration;

Figure 16 (a) demonstrate there is poly-(acrylamide-co-3APB) film MARS relative to the response amplitude of concentration of glucose;

Figure 16 (b) demonstrates the response amplitude when concentration of glucose is not more than 7.5mM and is directly proportional to concentration of glucose;

Figure 17 demonstrates when pH7.4, for the continuous change 56. of concentration of glucose from 0 ~ 100mM

Figure 18 demonstrates MARS based on 3-APB for the response of 5mM fructose, glucose, galactose and mannose solution; With

Figure 19 demonstrates the curve of the amplitude variations of MARS sensor relative to 3-APB % by mole and (a) apparent pKa, (b) hydrophobicity (being measured as Δ Α 4) and (c) viscoelasticity (being measured as Δ Q).

Detailed description of the invention

Embodiment 1

This document describes the new chemical activated form mechanical switch based on compound acoustic resonator.

Equipment drawing is shown in Figure 1.Planar spiral winding is provided thus excites by quartz disk and the harmonic wave of compound resonator made based on the hydrogel acoustics thick film coating of pH sensitivity methacrylate-methacrylate copolymer by electromagnetic method.Spiral winding and signal generator, coaxially to connect between AM detector and lock-in amplifier.Detector adopts difference diode detecting circuit to deduct the radiofrequency signal returned by coil in the larger excitation signal produced from signal generator.In order to pass the frequency of composite resonant equipment, amplitude and Q factor back, adopt LabView ^tMthe original amplitude that software processes is collected by spectrogram and frequency number strong point.

Carry out the measurement of the acoustic resonance characteristic up to 100MHz with the above-mentioned activating system based on coil, described activating system can keep strong signal amplitude in multi-harmonic frequency.Acoustics track from signal generator front panel selection operating frequency and for passing each harmonic resonance back is poor, and this acoustics track difference is relevant to the chemical property of hydrogel again.

250 μm of thick AT quartz disks in the 2.1 μm of thick aquagel membranes deposited observe on-off action, excites at fundamental frequency (6.6MHz) in pH3.5, and stop in pH8, open/close amplitude ratio is 500:1.Find to limit switching point well by the independent variable (argument) of the hyperbolic tangent function (Tanh [kt]) of wave vector (k)-thickness (t) result, therefore just can record film elasticity when not using multi-parameter fitting system is 5.8 × 10 ⁵n/m ²and cut wave-wave speed for 24ms ^-1.The method makes the expansion of hydrogel respond significantly amplification and can determine the engineering properties of film.Therefore, the acoustics provided based on pH sensitive aqueous gel cuts ripple switch.

Maximize to make signal response and significantly improve the resonance in described method, advantageously the resonant frequency of probe being controlled.Equally, in order to make aquagel membrane be easy to operation and adhere to, advantageously glass instead of usually can through chemically treated surface of metal electrode is adopted.Therefore, for technical reason, the above-mentioned adjustable equipment based on simple coil and exposed quartz substrate is favourable.Use this common-mode, multiple hydrogel material and thickness can be adopted.

In embodiment of the present invention, when contiguous multi-frequency quartz-crystal resonator is placed, ionizable poly-HEMA hydrogel can be utilized relative to the mechanical property of pH.Electrical characteristic and the examine repair thereof of much frequency resonance device can be assessed.Understand aquagel membrane formation and and sound wave between interaction be another importance of architectural characteristic.Therefore, in the method that the present invention is used, obtain the phase contrast image of the hydrogel of deposition and estimate its thickness.In addition, explain the characteristic of system according to selected operating frequency, and estimate the acoustic impedance of hydrogel resonator device relative to pH value.

Be difficult to determine the thickness of aquagel membrane, density, elasticity and viscosity, because these parameters are mostly unknown.Such as, because of the potential change of thickness in the soft character of hydrogel and dish thereof, be difficult to determine the thickness thickness of measurement film as described below (but, can).On the contrary, determine that whether elasticity be that the determiner of thickness possible range is even more important, more specifically, determine whether to set up the physical-chemical conditions causing sound wave to open or close.It should be noted that described system is more more complicated than those systems containing molecular film in some aspects, because thickness is the pith of wave length of sound.Varied in thickness is generation sound difference in film, the overall characteristic of this appreciable impact system.The wideband acoustic measurement of the water-setting lacquer disk(-sc) complex manufactured according to the present embodiment adopts the signal generator and lock-in amplifier that easily obtain, and these parts are with previously identical with the parts of protein adsorption research for viscous fluid.Spiral winding is winding by hand formula, and diode detector is in internal build.

Once sound field is produced by electrostriction in crystal, then acoustic characteristic is relevant to the hydration status of polymer, and hydration depends on the pH of bath medium.Measured the acoustical coupling of crystal and thick aquagel membrane (elasticity is " rigidity "), and chemical relevant elasticity is relevant to the switching characteristic of self.

Example

Methacrylic acid 2-hydroxyethyl ester (HEMA; 97%), Ethylene glycol dimethacrylate (EDMA; also be referred to as ethylene glycol dimethacrylate), methacrylic acid (MAA), dimethoxyphenylacetophenone (DMAP), 2-propanol and (methyclyloxypropyl) triethoxysilane provide by AldrichChemicalCo. (Gillingham, Britain).Every other chemicals is AG and is provided by Sigma or Aldrich.100 μm of thick polyester films (MET401 level) of aluminizing are purchased from HiFiIndustrialFilmLtd. (Stevenage, Britain).

Prepared by compound resonator

Monomer mixture is prepared by HEMA (89 % by mole), EDMA (cross-linking agent, 5 % by mole), MAA (6 % by mole) and isopyknic 2-propanol.Then adding light trigger dimethoxyphenylacetophenone (DMAP) is 1% (weight/volume) to ultimate density.Spend the night with the acetone soln process crystal of the methacrylic acid trimethoxy-silylpropyl ester of 1% (weight/volume).Aluminum polyester sheet is placed on smooth glass plate, and the monomer mixture pipetting about 5 μ l with pipet is to produce fluid " pearl ".Treated quartz disk is placed on monomer mixture, and pressing is to make its underpart evenly be coated with polymer.The radical polymerization of UV initiation within 15 minutes, is carried out by the middle exposure of UV radiation (about 350nm) produced at UV exposing unit.After polymerization, peeled off the side of aluminizing of dish from polyester sheet, the effect of hydrogel-quartzy complex adherent layer is played in the side of aluminizing of polyester sheet.Last step thoroughly cleans complex in methanol.

Spiral winding

Prepare that DC resistance is 1 ohm, inductance is 0.5mH by the 0.085mm enamel covered wire deriving from RSElectronics (Britain) and overall diameter is the planar spiral winding of 5mm, and this coil is connected to the thick epoxy laminate of the 0.25mm with cyanoacrylate adhesive thin layer.

In order to design effective hydrogel sensor, it must be understood that relation that its acoustic impedance change of deriving from the signal of telecommunication detected and the hydrogel action of chemical induction change how.

The acoustic impedance of hydrogel-dish

The compound resonator that the system studied is made up of the resonant panel being attached to additional hydrogel resonator (thickness, density and viscoelasticity are unknown).In addition, there is the 3rd liquid level of contact complex upper surface.The acoustic impedance of complex system describes by (1-4) usually:

$Z_q=\sqrt{{\mathrm{\&rho;}}_f{G}_f}Tanh\&lsqb;kt\&rsqb;---\left(1\right)$

$Z_f=\sqrt{G_f{\mathrm{\&rho;}}_f}Taah\&lsqb;k_f{t}_f\&rsqb;---\left(2\right)$

G _w＝2πfπω(3)

$Z_c=Z_q+\frac{Z_f+Z_w}{1+(Z_w{Z}_f/G_f{\mathrm{\&rho;}}_f)}---\left(4\right)$

Wherein k and t is wave vector and the thickness of layer respectively, and Zq, Zf and Gw are that quartzy impedance, hydrogel impedance and water resistance are anti-.The combination Zc of these impedances characterizes compound resonator, different from the independent resonator represented by the equation 2 of second item.This analysis display is when the independent variable kt of hyperbolic tangent function is pi/2, and namely when the chemical disturbance in hydrogel matrix causes sound amplitude and audio frequency very big mobile, hydrogel impedance Z f can sharply change.Because hydrogel size can define well unlike quartz plate, and show larger size and metataxis, thus can estimate that film resonance occurs over just wherein acoustic wave front and keeps in parallel plane comparatively low harmony wave.

Hydrogel chemistry

The acoustic impedance of hydrogel comes from total (aggregate) internal force produced by the hydrophilic cross-linking copolymer layer absorbed water from solution.The existence of faintly acid carboxyl makes gel carry out the change [Montheard of hydration according to the pH of bath medium, J.P., M.Chatzopoulos and D.Chappard, 2-EydroxythylMethacrylate (Hema)-Chemical-PropertiesandApplicationsinBiomedicalFields.Jou rnalofMacromolecularScience-ReviewsinMacromolecularChemi stryandPhysics, 1992, C32 (l): 1 page ~ 34 pages].What is interesting is, thickness and elasticity are with pH simultaneously and consecutive variations.In this case, pH changes thickness t because of hydration, and because known elasticity changes k [Johnson according to k=ω √ (pc) with pH change, B. etc., MechanicalpropertiesofapHsensitivehydrogel, SEM nd Annual Meeting in 2002, Milwaukee, WI].This changes the independent variable kt of film acoustic impedance (1), and from being acoustically significantly exaggerated pH change in whole tanh.

The detection of water-setting lacquer disk(-sc) complex

Specific features [the Stevenson of the acoustics configuration adopted in this research, and C.R.Lowe A.C., NoncontactexcitationofhighQacousticresonancesinglassplat es.AppliedPhysicsLetters, 1998.73 (4): 447 pages ~ 449 pages] [Sindi, H.S., A.C.Stevenson and C.R.Lowe, Astrategyforchemicalsensingbasedonfrequencytunableacoust icdevices.AnalyticalChemistry, 2001.73 (7): 1577 pages ~ 1586 pages] be connected with dish through air gap, make exciting/testing process in can not apply machinery or electric disturbance.Different from the hard-wired acoustic equipment of routine, equipment of the present invention is that field drives and detects with spiral winding.Can think excite/testing process comprises two synchronous behaviors.By generating process, the radio-frequency current coming from signal generator circulates with the rotation of coil.These processes create contiguous radio-frequency (RF) magnetic field, and himself supports rf electric field.Depend on the dielectric boundaries generation effect of the field distribution of coil geometric layout (geometry) at dielectric substance, thus cause surface current [Stevenson, and C.R.Lowe A.C., Magnetic-acoustic-resonatorsensors (MARS): anewsensingmethodology.SensorsandActuatorsa-Physical, 1999.72 (1): 32 pages ~ 37 pages] or electric charge [Stevenson, A.C etc., Theacousticspectrophonometer:Anovelbioanalyticaltechniqu ebasedonmultifrequencyacousticdevices.Analyst, 2003.128 (10): 1222 pages ~ 1227 pages].Different electric charges is concentrated on different borders (such as contacting upper surface and the lower surface of the piezoelectricity crystal of water and air respectively), crystal action [Stevenson is driven as applied electric charge by electrode, A.C. etc., Hypersonicevanescentwavesgeneratedwithaplanarspiralcoil. Analyst, 2003.128 (9): 1175 pages ~ 1180 pages], [Thompson, M. etc., Electromagneticexcitationofhighfrequencyacousticwavesand detectionintheliquidphase.Analyst, 2003.128 (8): 1048 pages ~ 1055 pages].Also carry out contrary process, this action is reversed, cause caused electric current to be got back in coil.Final result is that the change of hydrogel chemistry is converted to metataxis immediately, and this can affect sound scissoring vibration, and for above-mentioned reasons, the signal of telecommunication measured by impact.

Electrical impedance

The another kind of method of observe system be the amplification of the hypothesis signal of telecommunication depend on magnetic distribution (with coil and surrounding dielectric about) relevant translation function.The change of frequency displacement and acoustics Q factor can directly be determined by equation (1), and relevant with frequency displacement to the electricity Q factor recorded.

Assuming that thick film condition of resonance can be realized, then model estimate when the thickness of film, frequency and elasticity reach in kt pi/2 move time, will the significant change of the acoustic impedance of film be observed.To can obtain between zero resonance and thick film resonance chemistry initiation conversion condition and scheme interested especially.

The detection of dish-hydrogel resonance

The fundamental resonance of inspection plate in water, then attached water gel, make the frequency of resonator and Q reduce (Fig. 1).All pH3.5 is selected in two groups of tests, because the tightr and stable [Marshall of hydrogel with this understanding, A.J. etc., PH-sensitiveholographicsensors.AnalyticalChemistry, 2003.75 (17): 4423 pages ~ 4431 pages].Attached water gel makes the energy loss process in hydrogel outside amount, and therefore Q factor declines.The system with more open moving structure of " swing " from fluid around, ahead of estimate, the acoustical material of hydrogel matrix seemingly more " power consumption ".The decay produced makes resonant frequency lower and reduces Q factor.As selection, possible film thickness change (may than much bigger in quartz disk) avoids phase coherence, thus reduces resonant Q factor.

The determination of thickness

Adopt phase microscope independent measurement film thickness, because film thickness is for determining that the resonance characteristic of complex is extremely important.Microscopical non-intruding feature avoids any breaking-up of aquagel membrane, then may damage with the such as contact contourograph such as Dektac.But, importantly measured film is transparent, thus utilizes the microscope that matches to carry out profile mensuration.This mensuration operation must carry out the step of the upper surface imaging from the naked surface of quartz crystal to hydrogel.Effectively, describe the isogram of membrane boundary, wherein each contour is separated by the fixed interval cycle that (Fig. 3 a).The dielectric constant of known poly-HEMA copolymer is 1.5, and the wavelength of light source is 600nm, can when by isogram estimation, to pipette volume be 10 μ L, and film thickness is 2.1 μm.

Carry out with ultraviolet crosslinked before whether four that contain different volumes monomer mixture independent equipment are pipetted on crystal, to be acoustics thick film according to definition [Anal.Chem.2000,72,141 ~ 149] evaluated for film of Martin.The frequency displacement that Fig. 3 b shows between resonance plate that naked resonance plate and hydrogel apply is roughly linearly, and has nothing to do with the volume applied.Find that the frequency displacement recorded is greater than the Sauerbrey class frequency displacement calculated by optical thickness, show that cross-film acoustic phase shift is remarkable.These measurements confirm that applied aquagel membrane is " thick " film.

Hydrogel-dish spectrogram

Do not change chemical or equipment geometric layout to cause different acoustic phase shift, regulating frequency makes it in certain frequency scope, pass series of harmonic (Fig. 2) back when pH3.5.In a word, find that resonance amplitude increases with frequency and sharply declines, be difficult to differentiate the harmonic wave higher than 100MHz.This characteristic is attributable to the change of material damping and film thickness.

By record sound spectrogram change (comprising its frequency, amplitude and Q factor), have studied pH and become 8 for the impact of initial quintuple harmonics from 3.5.During fundamental frequency (the first width figure in Fig. 4), observe thickness increase film specific to frequency move down.This response is due at higher ph values, the carboxylic ions that hydrogel matrix combines because of polymer, attracts gegenion and water and hydration occurs, and therefore polymer phase expands and thickness increases.

At 33MHz harmonic wave, there is no obvious frequency displacement, because resonance peak disappears completely (the second width figure in Fig. 4).But, in higher frequency, frequency displacement pattern reversion (the 3rd width figure in Fig. 4).The frequency upper shift that the aquagel membrane thicker to pH8 is relevant.It is believed that this characteristic is an example of the thick film characteristic when cross-film acoustic phase shift is enough to the resonance in supporting film self.This characteristic is attributable to film disengaging dish resonance, makes to support that the key reflections border of standing wave resonance is between the lower surface and dish-hydrogel interface of dish, instead of between dish and hydrogel-water termination.

Above-described embodiment illustrates harmonic wave (particularly 33MHz harmonic wave) can switch transition.But for suitable acoustic sensor thick-layer, any frequency (fundamental frequency or harmonic wave) all can be used for realizing switching characteristic.

On-off action

For the compound resonator with thick film, on-off action is feature of greatest concern.Now, the transformation between open state and off status has high contrast.The instance graph of the good switching characteristic of fundamental frequency is shown in Fig. 5 a, and wherein when pH becomes 8 from 3.5, peak disappears completely.It is reversible that Fig. 5 b illustrates switch.Resonance peak in this example with continuous print by time mode repeated collection.The left side of curve represents the peak that passing repeated collection is in time same.After adding pH8 buffer, in about 10 seconds, peak disappears completely, and is again in pH3.5, has passed original peak back, has shown that the elastic characteristic of hydrogel-dish complex is reversible.The reason that originally can obtain on-off action is because produce extremely short wave length of sound in film under low sheraing velocity of wave, is about 24ms-1 according to estimates.This value is suitable with the value 15ms-1 of the poly-HEMA hydrogel recorded separately.In addition, because the wave length of sound in switching point place hydrogel is through well defining, therefore by c=ρ .V ²the elasticity that can calculate poly-HEMA during pH8 is 5.8 × 10 ⁵n/m ², conform to the engineering properties of " soft " polymeric material.

Calibration curve

In order to each stage of clear and definite switch transition, compound resonator is carried out to the increment change of pH.Resonator carries out reversible switch transition, stops completely at pH8 resonance.These changes in acoustic resonance figure and the change of amplitude pH are shown in Fig. 6 a.The respective change of resonance amplitude and Q factor is shown in Fig. 6 b.Data show significant resonance damping, show that energy is regained from quartz element by the absorbed inside in hydrogel.In Q factor and amplitude acoustics figure, the position of turning point is by replacing different ionogens to estimate, as the diffraction grating previously reported prove.

Chemical flooding ejector half mechanical switch can be made up of the composite resonant body comprising quartz disk and the aquagel membrane on it.When using appropriate frequency and multi-frequency acoustic equipment, this on-off action occurs when film works under thick film condition of resonance, and wherein sound is 90 degree mutually, hydrogel thickness 2.1 μm.The susceptiveness of the Flexible change of hydrogel is also significantly strengthened under Switching Condition.In addition, this technology not only contributes to selecting suitable monomer, cross-linking agent and copolymer ratio to provide mechanical response acoustics or optics hydrogel, if film is thinner and detect in high frequency, it can also play the effect for studying the dynamic (dynamical) chemical classes switch module of protein in field of acoustics (proteinacious) film.

Embodiment 2

The following describes can the glucose sensor of remote monitoring concentration of glucose.

N,N methylene bis acrylamide (MBA) is share as cross-linking agent synthesis 3-acrylamido phenylboric acid (3-APB) and the glucose susceptiveness copolymer of acrylamide by the radical polymerization that UV causes in room temperature.The pKa of poly-(acrylamide-co-3-APB) is about 8.6, but glucose knot

Main method described herein is incorporated by poly-for glucose responding type (acrylamide-co-3-APB) film in magnetic acoustic resonator sensor (MARS) and studies the response of sensing device to glucose in solutions concentration change.

The complexation of boric acid and c/s-diol

Reversible binding between boric acid and c/s-diol is shown in Figure 7, and this combination is pH dependent form.The chemical constitution of poly-(acrylamide-co-3-APB) is shown in Fig. 8.At a low ph, boric acid is uncharged three-legged structure state, changes tetrahedron state into when it is charged and pH increases.In tetrahedral geometry, boric acid is for the affinity of c/s-diol higher than triangular structure form, and under three-legged structure, the complex of itself and c/s-diol is more easily hydrolyzed.Glucose comprises syn diol structure.Glucose and polymer with two kinds of potential mechanism being combined of phenylboronic acid group comprise: 1:1 monomer combines and 1:2 is crosslinked combines.Equimolar 1:1 combines the expansion causing polymeric film, and 1:2 combination causes shrinking.

The pH response of poly-(acrylamide-co-3-APB) film

Because poly-(acrylamide-co-3-APB) is pH sensitivity, and glucose is more stable than three-legged structure with the complex of tetrahedral 3-APB, therefore have studied the characteristic of polymeric film at different pH.Determine and measure the pH titration curve of polymer, and calculating pKa value by pH curve.The process of synthetic copolymer is almost identical with the synthesis of poly-(HEMA-co-MAA), and difference is to expose 30 minutes to UV.Pre-polymer solution is made up of the 5mol/l monomer in 500 μ l solvents (DMSO containing 2% (weight/volume) light trigger DMPA).Buffer system is acetic acid (pH4,4.5,5,5.25 and 5.5), MES (pH5.75,6,6.5), phosphate (pH7,7.5,8), Tris (Tris, pH8.5), glycine (pH9), CHES (pH10) and phosphate (pH12).Buffer concentration is 10mM, and ionic strength sodium chloride is fixed as 154mM.

Along with the pH value of buffer increases, poly-(acrylamide-co-3-APB) is charged gradually.There is Donna (Donnan) current potential between polymer and the aqueous medium around it, order about water and enter hydrogel and hydrogel is expanded.Meanwhile, osmotic effect also causes the hydration of polymer.Fig. 9 shows the pH curve collected at 6MHz, and wherein poly-(acrylamide-co-3-APB) film is containing 78.5 % by mole of acrylamides, 1.5 % by mole of MBA and 20 % by mole 3-APB.The apparent pKa value calculated is 8.47.

There is the MARS of poly-(acrylamide-co-3-APB) to the response of glucose

Glucose combines the apparent pKa caused to be reduced

Fig. 9 contains the pH curve gathering (acrylamide-co-3-APB) of 78.5 % by mole of acrylamides, 1.5 % by mole of MBA and 20 % by mole MAA when showing 6MHz; The apparent pKa value calculated by this opisometer is 8.47.In Fig. 9, poly-(acrylamide-co-3-APB) polymer is almost neutrality in physiological pH 7.4, and wherein the 3-APB side base of about 91.5% is triangle configuration and is combined with glucose more weak.5mMD-(+)-glucose solution is prepared with assessment through applying resonator in the response of different pH value to glucose solution in buffer listed above.The pH curve negative sense of 6MHz moves, and apparent pKa value is reduced to 8.02 by 8.47 (Fig. 9).Now, under pH7.4, the boric acid base group of 24% ratio has become anion and has been tetrahedral.

As shown in Figure 7, because the combination of glucose and three-legged structure 3-APB is more weak, therefore dissociating of neutral 3-APB is determined by the proton concentration in solution and the combination between glucose and tetrahedral 3-APB.

The calibration of the glucose responding of MARS

Poly-(acrylamide-co-3-APB) MARS of glucose responding type is calibrated to the response of concentration of glucose in buffer solution with the polymer identical with shown in Fig. 9.Glucose solution is prepared in the 10mMPBS buffer of pH7.4.Concentration of glucose is 0 ~ 15mM, and amplification is 2.5mM.Figure 10 show be coated with poly-(acrylamide-co-3-APB) film (78.5 % by mole of acrylamides, 1.5 % by mole of MBA and 20 % by mole 3-APB) MARS relative to the response amplitude of concentration of glucose; At 20 mhz three tests are carried out to the glucose solution of 0 ~ 15mM and obtain response data.Figure 10 display is from the almost routine relation of linear proportional of the concentration of glucose within the scope of signal amplitude and the 0 ~ 15mM of MARS.When concentration of glucose is comparatively obvious higher than 7.5mM time error.The sensitivity of glucose meter is about 87.07mV/mM.It is believed that under high glucose concentration to be due to the conversion between the 1:1 binding pattern between boric acid and glucose molecule and 2:1 cross-linking patterns compared with big error.

Collect the data shown in Figure 10: with pure PBS buffer, the polymer be coated on quartz disk is cleaned three times, and again test under similarity condition.

Figure 11 (a) has the MARS of poly-(acrylamide-co-3-APB) film (78.5 % by mole of acrylamides, 1.5 % by mole of MBA and 20 % by mole 3-APB) to the cubic response of glucose solution (the PBS buffer of 0mM ~ 15mM, pH7.4) when showing 20MHz; Figure 11 (b) depicts the response results of three tests relative to concentration of glucose respectively.In Figure 11 (b), noticing that poly-(acrylamide-co-3-APB) MARS tests slightly larger than first time in rear twice test containing the response higher than the glucose solution of 10mM glucose, is then similar by the response of second time test and third time test acquisition.This may be because the aperture of polymer network is expanded by glucose molecule when first time tests, and glucose obtains the approach more easily entering polymer also makes hydrogel expand subsequently.

The optimization of 3-APB concentration in glucose sensor

The susceptiveness of MARS is enough to the minor variations detecting concentration of glucose in buffer solution.Relation between response (amplitude of signal) and concentration of glucose is roughly linearly (Figure 10); But, in order to strengthen response, the composition of polymeric film is optimized.

Different 3-APB concentration is to the response of glucose

Have studied there is different % by mole 3-APB polymer to the response of glucose.Glucose solution system is same as described above, and amplitude variations (Δ Α) is defined as follows:

Δ A ₀ ¹⁵=amplitude (15mM glucose)-amplitude (0mM glucose) (20MHz)

Figure 12 is presented at 20MHz relative to different 3-APB concentration, has the MARS of poly-(acrylamide-co-3-APB) film to the Δ A of 15mM glucose ₀ ¹⁵; Three samples are tested to each % by mole.Amplitude variations (Δ A ₀ ¹⁵) curve relative to 3-APB % by mole is bell (Figure 12).Peak response is observed for the 3-APB of about 20 % by mole, and response declines along with more 3-APB adds in polymer.The ionizing of the result in table 12 and poly-(acrylamide-co-3-APB) (with 3-APB % by mole about) contribution and the glucose accessibility (relevant with hydrophilic/hydrophobic with the aperture of polymer network) that enters polymer network be correlated with.When there is different concentration of glucose, the apparent pKa of polymeric film or the apparent pKa change caused by glucose are depended in the ionizing of poly-(acrylamide-co-3-APB).Poly-(acrylamide-co-3-APB) apparent pKa (20MHz) relative to % by mole 3-APB is calculated in table 1; As the concentration <20 % by mole of 3-APB, apparent pKa change is remarkable, but at higher % by mole, apparent pKa increases.Containing the apparent pKa of height of poly-(acrylamide-co-3-APB) of the 3-APB of >20 % by mole, namely the ionizing of low degree can explain Δ A in Figure 12 ₀ ¹⁵the decline of response.

There is during table 1:46MHz the apparent pKa value of poly-(acrylamide-co-3-APB) (three samples) of different 3-APB concentration

^*sTD: standard deviation

PK caused by glucose combines _avalue change

Owing to having lower apparent pK _apoly-(acrylamide-co-3-APB) comprise more tetrahedral boric acid and more glucose can be caught at physiological ph, therefore have studied poly-(acrylamide-co-3-APB) because of glucose combine caused by apparent pK _athe 3-APB concentration dependent of change.Prepare the polymer with 15 % by mole, 20 % by mole, 25 % by mole and 30 % by mole 3-APB respectively, and collect the pH curve in response to the pure buffer of pH4 ~ 10.Again record pH curve existing under 5mM glucose.For four kinds of polymeric films, pKa change is respectively-0.52 ,-0.32 ,-0.66 and-0.46.

The impact % by mole on the hydrophilic/hydrophobic of poly-(acrylamide-co-3-APB) of 3-APB

The hydrophilic/hydrophobic of 3-APB polymer is analyzed by the amplitude variations of observing caused by water suction when pH changes to 4 from 10.Amplitude (Δ A ₄ ¹⁰) be defined as:

Δ A ₄ ¹⁰=amplitude (pH10)-amplitude (pH4) (20MHz)

Figure 13 is presented at the MARS of 20MHz containing poly-(acrylamide-co-3-APB) film to the Δ A of pH change (pH4 ~ 10) relative to different 3-APB concentration ₄ ¹⁰; Three samples are analyzed to each % by mole.Collection has the pH response of the polymer of different 3-APB concentration (5 ~ 30 % by mole, amplification is 5 % by mole).Figure 13 illustrates the amplitude variations relative to 3-APB percentage ratio in copolymer.When polymer is maximum containing amplitude variations during 20 % by mole of 3-APB; As % by mole >20 % by mole of 3-APB, polymeric film expands less.In theory, the density increase of 3-APB can improve Donna current potential and osmotic effect, and thus more water can enter polymer network.But the hydrophobicity of 3-APB hinders water and glucose enters hydrogel simultaneously.So 20 % by mole time, osmotic effect and hydrophobic effect reach balance.Therefore, the hydrophilic/hydrophobic proposing poly-(acrylamide-co-3-APB) is the principal element of the susceptiveness determining acoustics glucose sensor.

The impact % by mole on polymer viscoelastic of 3-APB

The swelliong power of poly-(acrylamide-co-3-APB) copolymer also affecting glucose molecule is infiltrated polymer and is limited qualitatively by the Q factor of the sensor being coated with polymer.As mentioned above, poly-(acrylamide-co-3-APB) copolymer shows as elastomeric material when the high operate frequency of MARS; The elasticity of polymer can be strengthened with the hydrogen bonding of water or glucose and improve Q factor.

The Q factor of poly-(acrylamide-co-3-APB) sensor when < is about 140MHz with operating frequency increase in direct ratio, subsequently higher than saturated during 140MHz; Such as, the Q factor of polymer under saturated level with 15 % by mole of 3-APB is about 3250, and the Q factor with the polymer of 20 % by mole of 3-APB is about 2000, and the swelling polymer with 30 % by mole of 3-APB is less and Q factor is about 900.Q factor may be that the elasticity of polymer increases simultaneously is also possible cause owing to penetrate and the acoustic energy dissipated in the polymer is less with the increase of operating frequency.During high frequency, acoustic energy concentrates on polymer-silica interfaces, and the viscoelasticity of polymer reaches balance, and therefore, Q factor is saturated when frequency >140MHz.

Also observe the expansion characteristics of same polymer in the 10mM glucose phosphate salt buffer of pH7.4.In the case, the saturated level containing the phase emergencing copolymer Q factor of 15 % by mole of 3-APB is about 4000, and the polymer containing 20 % by mole of 3-APB is about 3000, and the swelling polymer containing 30 % by mole of 3-APB is less and Q factor is about 1500.The Q factor difference of polymer in pure buffer and 10mM glucose solution (Δ Q) is: 15 % by mole of 3-APB are 750,20 % by mole of 3-APB be 1000,30 % by mole of 3-APB is 600.The Δ Q of the saturated level relative to 3-APB % by mole is shown in Figure 14, which show poly-(acrylamide-co-3-APB) sensor relative to 3-APB the response % by mole in the 10mM glucose solution of pure pH7.4 buffer and pH7.4 between Δ Q.

The result of Figure 14 shows: glucose combines the elasticity enhancing polymer, between 20 % by mole of 3-APBA and 25 % by mole 3-APBA, observe optimum efficiency.Except the hydrophilic/hydrophobic of polymer, the susceptiveness of the swelliong power of polymer network and aperture also affecting glucose sensor.In Figure 12, the first half of curve reflects that the concentration of the sensitivity of glucose polymer and phenylboronic acid group is in direct ratio, and the susceptiveness that latter half shows polymer is also determined by the hydrophilic/hydrophobic sexual balance of network, viscoelasticity and aperture.

The optimization of crosslinked % by mole in 3-APB polymer

It is believed that the performance of the aperture of hydrogel network and viscoelasticity also affecting glucose sensor, wherein steric hindrance may play an important role.

According to the above results, when 3-APB concentration is 20 % by mole, reach peak response (Δ A ₀ ¹⁵).By the percentage ratio of 3-APB is remained 20 % by mole and respectively monitoring polymer containing 0.5 % by mole, 1.5 % by mole and 2.5 % by mole of MBA time response study crosslinked impact (Figure 15).Figure 15 show with reference to solution (concentration of glucose is 0mM) and concentration of glucose is 15mM when from the signal of MARS relative to the difference of vibration of cross-linking agent MBA concentration.Dextrose buffer solution is injected continuously with the amplification of 2.5mM in the concentration range that Figure 15 is presented at 0 ~ 15mM.Result in Figure 15 show to reduce cross-linking agent % by mole can't to contribute to strengthen poly-(acrylamide-co-3-APB) sensor to the sensitivity of glucose.Also adopt Δ Q research when contacting 10mM glucose solution, crosslinked scope is the expansion characteristics of poly-(acrylamide-co-3-APB) copolymer of 0.5 ~ 2.5 % by mole.

The detection of 5.7 hyperglycemias and hypoglycemia

In diabetics, blood sugar concentration rises to the level (hyperglycemia) far above normal concentration (about 4.4mM), and when patient has taken insulin inadequately, blood sugar level can be brought down below normal level (hypoglycemia).Therefore, the efficient working range of glucose sensor should be measured.In the PBS buffer of pH7.4 compound concentration be 0, the glucose solution of 2.5mM, 5mM, 7.5mM, 10mM, 12.5mM, 15mM, 20mM, 40mM, 60mM, 80mM and 100mM.Polymeric film is made up of 83.5 % by mole of acrylamides, 1.5 % by mole of MBA and 15 % by mole 3-APB.

Figure 16 shows: (a) has the MARS of poly-(acrylamide-co-3APB) film (83.5 % by mole of acrylamides, 1.5 % by mole of MBA and 15 % by mole 3-APB) relative to the response amplitude of concentration of glucose (0 ~ 100mM), utilizes SigmaPlot to fit to exponential equation; B () response amplitude as concentration of glucose≤7.5mM is directly proportional to concentration of glucose.

As shown in figure 16, MARS becomes saturated relative to the response of concentration of glucose at more than 20mM.Therefore, the efficient working range based on the glucose sensor of MARS is 0 ~ 20mM, and is substantially directly proportional (Figure 16) to concentration of glucose lower than response amplitude during 7.5mM.Shown in Figure 16 (a), data fitting is exponential equation, and it shows that MARS sensor may follow index ascending fashion relative to the response of concentration of glucose.Figure 17 shows the response time for 0 ~ 100mM concentration of glucose, higher than the glucose of 20mM, and response time <10 minute.Figure 17 has poly-(acrylamide-co-3APB) film (83.5 % by mole of acrylamides, 1.5 % by mole of MBA and 15 % by mole 3-APB) MARS when being presented at pH7.4 for concentration of glucose from 0 ~ 100mM continually varying response time.

The response time when saturated level during concentration of glucose >20mM observed in Figure 16 and 3-APB content >20 % by mole reduces (Figure 17) may be relevant with the expansion of polymer network.Hydrogel expands more in containing the solution of high glucose concentration, and this is conducive to the infiltration of glucose solution on the contrary.But, because poly-(acrylamide-co-3-APB) copolymer is crosslinked, the swelliong power of network is limited, therefore, the saturated level reached in Figure 16 may correspond to expansion limit, and the steric effect of the complex of glucose and aromatic boric acid.

Electric field is on the impact of glucose responding

According to above-mentioned research, the apparent pKa value measured by operating frequency influences glucose sensor.In order to understand the performance of glucose sensor within the scope of wide sound spectrum, introduce the concept of polymer brush theoretical draw portion pKa.In this study, by determining that the valid analysing range of sensor have studied the performance of glucose sensor how by the impact of operating frequency.

Consisting of of polymer: acrylamide 78.5 % by mole, cross-linking agent MBA1.5 % by mole and 3-APB20 % by mole, finds that MARS is rather similar each other relative to the response of concentration of glucose in the frequency range of 6MHz ~ 73MHz.Therefore, the impact of operating frequency on the MARS response with poly-(acrylamide-co-3-APB) film is negligible.

Sugar detects

Show that glucose molecule is combined with phenylboronic acid group with two kinds of approach based on holographic sensor in front research: 1:1 monomer combines and the crosslinked combination of 1:2.Stoichiometry 1:1 combines the expansion causing polymeric film, and the stoichiometry of 1:2 causes shrinking.Glucose has five kinds of chemical constitutions:

The isomer of research glucose is to understand the mechanism combined between phenylboric acid and c/s-diol.In addition, except glucose, the sugar of maximum contained in blood is fructose and galactose.In 10mMPBS buffer, prepare fructose, galactose and mannose solution (5mM), ionic strength is 154mM at physiological ph.The quartz disk with poly-(acrylamide-co-3-APB) co-polymer membrane reaches balance in three kinds of sugar juices, and between measurements, carries out cleaning return to reference level (phosphate buffer) to response with phosphate buffer to film

It is sweet that Figure 18 shows beta-D-fructofuranose, α-D-galactopyranose and β-D-pyrans

Polymer is significantly greater than the response to other sugar to the response of fructose, and the amplitude variations compared with reference is 362.95mV (Figure 18).To the response of glucose, galactose and mannose, its three less is similar toward each other.The Δ Α of glucose is 52.25mV, and galactose is 30.15mV, and mannose is 15.2mV.Can the attribution ratio that is c/s-diol to the affinity of boric acid and effectively isomer to the strong response of fructose.Report compared with fructose and other three kinds of sugar, to boric acid, there is higher affinity.

Discuss and conclusion

The phenylboric acid of synthesis, 3-acrylamido phenylboric acid (3-APB) are added MARS thus produce glucose selective sensor, because MARS is by RF electric field wireless excitation, so this sensor constitutes the promising alternative for developing the continuous glucose detector of implanted.3-APB is phenyl boronic acid derivative, and the copolymer of itself and acrylamide can at physiological pH response glucose.Degeneration due to enzyme reduces stability and the susceptiveness of sensor, and therefore by traditional enzymatic glucose oxidase being replaced with the phenyl boronate part of synthesis, the performance of glucose sensor can be more durable and reversible.Also think that the glucose responding type hydrogel of synthesis can realize glucose-insulin closed loop system in body.Susceptiveness, repeatability, selectivity and response time are the important parameters of glucose monitoring system.Have studied the mechanism that the MARS with poly-(acrylamide-co-3-APB) co-polymer membrane combines in polymeric film the response of glucose and glucose.

The MARS being coated with poly-(acrylamide-co-3-APB) adsorption layer (adlayer) determines (Figure 19) mainly through the concentration of 3-APB, the hydrophobicity of polymer and viscoelasticity the sensitivity of glucose.Figure 19 (a) display relative to 3-APB % by mole, the Δ A of MARS sensor ₀ ¹⁵) and the apparent pKa value of poly-(acrylamide-co-3-APB); (b) relative to 3-APB % by mole, the Δ A of MARS sensor ₀ ¹⁵) and the Δ A of poly-(acrylamide-co-3-APB) ₄ ¹²; (c) relative to 3-APB % by mole, the Δ A of MARS sensor ₀ ¹⁵) and the Δ Q of poly-(acrylamide-co-3-APB).

In a word, preliminary study has been carried out to the novel glucose sensing system based on MARS.Because the viscoelastic change gathered in (acrylamide-co-3-APB) network caused in conjunction with glucose is measured by MARS, and the corresponding glucose level of recoverable.Glucose monitoring system has good reversibility and repeatability.The sensitivity average out to of sensor is about 87.07mV/mM.Result shows that MARS can be the general-purpose platform of physical property for studying very thin polymeric film and developing intellectual resource type polymer class biosensor.

Although reference the preferred embodiment of the present invention is to invention has been concrete displaying and description, but it will be appreciated by those skilled in the art that can carry out various change to form of the present invention and details and do not deviate from claims limit scope of the present invention.

Claims (10)

1. utilize a method for the continuous Monitoring Blood Glucose change of magnetoacoustic resonant, described method comprises the steps:

1) in subject import or implant can continuously Monitoring Blood Glucose change resonator and sensor, described sensor and described resonator are mechanically connected; Described resonator comprises and can arrange for the magnet of the described sensor that led in magnetic field and the exciter that is connected with described sensor mechanism; Described sensor is included in the sensor material that when contacting surrounding environment change, state changes thereupon, and wherein said sensor is driven by described resonator, and wherein said sensor material is the form of acoustics thick-layer; The thickness of described acoustics thick film roughly meets following equation:

$\mathrm{\&omega;}\sqrt{\frac{\mathrm{\&rho;}}{G}}t>\&ap;\mathrm{\&pi;}/2$

Wherein, ω is angular frequency, and ρ is density, and G is modulus of shearing, and t is film thickness; Described acoustics thick-layer is when sound wave is perpendicular to the layer can supporting sound wave remarkable phase shift during this Es-region propagations;

2) detect by detector the signal that the state in response to described sensor material changes subject is outer, described detector is connected with the adjustable electromagnetic field generator that can arrange for the described sensor that led by electromagnetic field;

3) signal that described detector detects is processed, to monitor the change of blood sugar in subject.

2. the method for claim 1, described method also comprises and processing the signal detected with the signal generator be connected with described electromagnetic field generator and described detector and lock-in amplifier.

3. the method for claim 1, described method also comprises use blood sugar test standard curve and calibrates.

4. the method for claim 1, wherein said exciter comprises the piezoelectric being preferably quartz, and described piezoelectric is the form of layer, and its thickness is 50 μm ~ 1000 μm.

5. as aforementioned method according to claim 1, wherein, the thickness of described acoustics thick-layer be 0.1 μm ~ 1mm, be preferably 0.1 μm ~ 100 μm, be more preferably 0.1 μm ~ 10 μm, be most preferably 0.5 μm ~ 5 μm.

6. as the method in aforementioned claim as described in 4 or 5, wherein, to be greater than a molecule thick for described acoustics thick-layer.

7. as the method in aforementioned claim as described in 1, wherein, described sensor material is hydrogel.

8. method as described in any of claims 7, wherein, described hydrogel is the polymer containing phenylboric acid side base.

9. method as claimed in claim 8, wherein, described polymer is poly-(acrylamide-co-3-acrylamido phenylboric acid) or poly-(acrylamide-co-2-acrylamido phenylboric acid).

10. the method as claimed in any one of the preceding claims, wherein described resonator and sensor are the forms of the implanted equipment for implanting human or animal subject; And wherein said detector is in vitro in response to described sensor.