CN1277116C - Biological enzyme electrode for biosensor, and its prepn. method - Google Patents
Biological enzyme electrode for biosensor, and its prepn. method Download PDFInfo
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- CN1277116C CN1277116C CN 200410018772 CN200410018772A CN1277116C CN 1277116 C CN1277116 C CN 1277116C CN 200410018772 CN200410018772 CN 200410018772 CN 200410018772 A CN200410018772 A CN 200410018772A CN 1277116 C CN1277116 C CN 1277116C
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- KTWOOEGAPBSYNW-UHFFFAOYSA-N ferrocene Chemical compound [Fe+2].C=1C=C[CH-]C=1.C=1C=C[CH-]C=1 KTWOOEGAPBSYNW-UHFFFAOYSA-N 0.000 claims abstract description 39
- 229920000642 polymer Polymers 0.000 claims abstract description 31
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- 239000012212 insulator Substances 0.000 claims abstract description 10
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
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- NTDFJPCHHGBHCO-UHFFFAOYSA-N 7,9-dihydro-3H-purine-2,6,8-trione Chemical compound OC1=NC(O)=C2NC(O)=NC2=N1.N1C(=O)NC(=O)C2=C1NC(=O)N2 NTDFJPCHHGBHCO-UHFFFAOYSA-N 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The present invention relates to a biological enzyme electrode for biosensors and a preparation method thereof. The method comprises the following steps: firstly, an electric conducting material is made into the basal bodies of a work electrode and a counter electrode by printing through a spraying method on electrode substrates made of an organic insulating material; then, the surfaces of the middle parts of the basal bodies are coated with a layer of polycarbonate insulators; finally, the lower end part of the basal body of the work electrode is coated with a reaction layer formed by the integrated application of a high polymer built up film technology or a ferrocene overlay technology, an enzyme embedding technology, nanotechnology and a diffusion limitation film technology, and thus, the biological enzyme electrode is prepared. By the biologic enzyme electrode of the present invention, the properties and the practicality of the biosensor are integrally improved.
Description
Technical field
Technical scheme of the present invention relates to the used biochemical electrode of methods analyst material by the test electrochemical variable, is biologic enzyme electrode of using of a kind of biology sensor and preparation method thereof specifically.
Background technology
Biology sensor is a kind of new sensor technology that grows up in nearly decades, is widely used in clinical diagnosis, Industry Control, food and Pharmaceutical Analysis, bio-pharmaceutical exploitation, environmental analysis, military field, biotechnology and all many-sides of biochip.But the biologic enzyme electrode that biology sensors such as at present commercially available glucose, cholesterol are used all is a unimolecular film, exists that interference current thereby interference free performance are low, the enzyme layer easily comes off thereby serviceable life short, measurement sensitivity is low and the shortcoming of measure linear narrow range.In order to improve the interference free performance of biology sensor, US 5,229, and 282 have introduced a kind of preparation method of biology sensor, its principal character is to apply a responding layer on an end face of electrode, and this responding layer includes enzyme, electronics receptacle and a hydrophilic polymer; More method is to form the high molecular polymer built up film (also to claim the polyelectrolyte composite membrane on an end face of electrode, be called for short the PEM film), for example Decher G. once proposed the static self assembly and formed high molecular polymer built up film (Decher G.Makromol.Chem.Macromol.Symp.1991,46:321-327; Ber.Bunsen-Ges.Phys.Chem.1991,95 (11): 1403-1434; Thin Solid Films.1992,210-211:831-835), people such as HoshiT utilize polyallylamine (PAA), polyethyleneimine (PEI), diallyl dimethyl ammoniumchloride (PDDA), poly-sulfonated ethylene (PVS), poly-sulfonated phenylethylenes (PSS) etc. form PEM film (Hoshi T, Saiki H, Kuwazawa S, TsuchiyaC, Chen Q, Anzai J.ANALYTICAL CHEMISTRY, 73 (21): 5310-5315 NOV 1 2001), people such as Ram MK then utilize stack technology to prepare Cholesterol Biosensor, experimental result shows that some PEM film is to chaff interference, as uric acid, ascorbic acid has significant barrier effect, has overcome the existence of interference current.In order to improve the measurement sensitivity of biology sensor, the someone has designed nm of gold electrode (Gu HY.ANALYTICAL LETTERS.2002,35 (4): 647-661 that are used for electrochemica biological sensor; Kossek, Sebastian.Journal of the Electrochemical Society.1996,143 (12): 3890-3895).In order to prolong the serviceable life of biology sensor, it mainly is the method that adopts immobilized enzyme or embedding enzyme, for example, US 6,280,587 have disclosed catalyzing enzyme have been fixed on method on an organic polymer and one the infiltration limiting layer, 1211620 methods that disclose a kind of biological sensor produced by latex embedding enzyme of CN1186115, CN.And do not see relevant report as yet for enlarging improving one's methods of measure linear scope.Said method only limits to improve the performance of the single aspect of biology sensor, does not have the comprehensive effect of improving, poor practicability.Application number is that 03257848.2 Chinese patent is the utility model patent of application in early stage of the same applicant of the present invention and identical main inventor; it has disclosed the structure of the biologic enzyme electrode that the biology sensor of a kind of comprehensive PEM membrane technology, nanometer technology, embedding zymotechnic and protective seam technology uses; improved comprehensive effect; improved practicality, but still its structure of further improvement and preparation method's space has been arranged.
Summary of the invention
Technical matters to be solved by this invention is: biologic enzyme electrode that a kind of anti-interference, long service life, biology sensor highly sensitive and the measure linear wide ranges use and preparation method thereof is provided, and the biologic enzyme electrode that makes with this method has improved the every performance and the practicality of biology sensor on the whole.
The present invention solves this technical problem the technical scheme that is adopted:
The biologic enzyme electrode that biology sensor of the present invention is used is by working electrode, to electrode, carrier, the insulator on coated electrode surface and responding layer constitute, responding layer is by the anti-interference layer that covers in the working electrode lower end on the electrode matrix, nano particle, matrix embedding enzyme layer, oxidase and diffusion restriction film are formed, and anti-interference layer is to utilize cation high molecular polymer poly allylamine, polyethyleneimine, at least a and anionic polymer polymer poly sulfonated ethylene in the diallyl dimethyl ammoniumchloride, 2～15 strata electrolyte complex films of at least a common formation in the poly-sulfonated phenylethylene; Nano particle is selected one or more in nanogold particle, nano-Ag particles, nanometer silicon dioxide particle, the nano copper particle for use, its grain size scope is at 8～50nm, and nano particle is embedded in anti-interference layer by bag or is embedded in the matrix embedding enzyme layer by bag or is wrapped simultaneously and is embedded in anti-interference layer and the matrix embedding enzyme layer; Matrix embedding enzyme layer is selected 1～12 layer of 1～12 layer of shitosan or 1 layer of polyvinylpyrrolidone or 1 layer in gelatin or concanavalin A for use; Oxidase is selected glucose oxidase, Lactate Oxidase or cholesterol oxidase for use; Matrix embedding enzyme layer and oxidase are alternately absorption, and oxidase is embedded in the matrix embedding enzyme layer, when matrix embedding enzyme layer is selected shitosan or concanavalin A and multilayer embedding for use, is that one deck suffers one deck; The diffusion restriction film is the film made from the acetone soln that contains 1%～3% cellulose acetate, and the biologic enzyme electrode that the invention described above biology sensor is used is characterised in that: cover anti-interference layer or ferrocene electron mediator layer on the electrode matrix.
The responding layer of the working electrode lower end of the biologic enzyme electrode that biology sensor of the present invention is used is rectangle or circle.
The preparation method of the biologic enzyme electrode that biology sensor of the present invention is used is:
At the electrode base sheet of being made by organic insulation is on the carrier, conductive material print formed working electrode with spraying process and to the matrix of electrode, apply one deck polycarbonate insulator on their the center section surface, apply the responding layer of one deck integrated application high molecular polymer built up film technology or ferrocene soverlay technique, embedding zymotechnic, nanometer technology and the formation of diffusion restriction membrane technology at working electrode matrix end portion, promptly make biologic enzyme electrode, the concrete preparation method of its responding layer selects any one in following four kinds of methods for use:
A. the first step, with aluminium powder polishing working electrode end portion, use distilled water flushing, make electrode surface clean as minute surface, being immersed in the concentration that is mixed with 5% nano particle is the polyallylamine of 2mg/mL, polyethyleneimine, among at least a cation high molecular polymer solution 5mL in the diallyl dimethyl ammoniumchloride 20 minutes, take out and to immerse again in the damping fluid 5 minutes, take out again and be immersed in the poly-sulfonated ethylene that the concentration that is mixed with 5% nano particle is 2mg/mL, in the poly-sulfonated phenylethylene among at least a anionic polymer polymer solution 5mL 20 minutes, repeat this process until alternately plating 2～15 floor height Molecularly Imprinted Polymer built up films; Second step, the electrode that plates the high molecular polymer built up film is carried out the micro-embedded of enzyme with the embedding enzyme layer matrix that is mixed with 5% nano particle, the micro-embedded method that embedding enzyme layer matrix selects for use gelatin or polyvinylpyrrolidone to carry out enzyme is, gelatin or polyvinylpyrrolidone 1mL dissolving 2mg oxidase with the concentration 5% that is mixed with 5% nano particle, obtain the enzyme solutions of 2mg/mL, carry out gelatin or polyvinylpyrrolidone immobilized enzyme at electrode surface again, embedding enzyme layer matrix with the micro-embedded method that shitosan or concanavalin A carry out enzyme is, utilize the static self-assembling technique, accumulate with enzyme respectively with shitosan or concanavalin A, its operation steps is that electrode was immersed in chitosan solution that the concentration that is mixed with 5% nano particle is 2mg/mL or the concanavalin A solution embedding enzyme layer matrix 20 minutes, take out and to immerse again in the damping fluid 5 minutes, take out to immerse concentration again and be in the oxidase of 2mg/mL 20 minutes until alternately plating 1～12 layer of embedding enzyme layer for 1 time or repeatedly carry out this process; The 3rd step after the taking-up, applied with the acetone soln that contains 1%～3% cellulose acetate, dried naturally, formed the diffusion restriction film,
B. the first step, same a; Second step, the electrode polyvinylpyrrolidone of high molecular polymer built up film will be plated, gelatin, shitosan, or concanavalin A carries out the micro-embedded of enzyme, the micro-embedded method that embedding enzyme layer matrix selects for use gelatin or polyvinylpyrrolidone to carry out enzyme is, gelatin or polyvinylpyrrolidone 1mL dissolving 2mg oxidase with concentration 5%, obtain the enzyme solutions of 2mg/mL, carry out gelatin or polyvinylpyrrolidone immobilized enzyme at electrode surface again, embedding enzyme layer matrix with the micro-embedded method that shitosan or concanavalin A carry out enzyme is, utilize the static self-assembling technique, accumulate with enzyme respectively with shitosan or concanavalin A, its operation steps is that electrode was immersed in chitosan solution that concentration is 2mg/mL or the concanavalin A solution embedding enzyme layer matrix 20 minutes, take out and to immerse again in the damping fluid 5 minutes, take out to immerse concentration again and be in the oxidase of 2mg/mL 20 minutes until alternately plating 1～12 layer of embedding enzyme layer for 1 time or repeatedly carry out this process; The 3rd step, same a,
C. the first step, with aluminium powder polishing working electrode end portion, use distilled water flushing, make electrode surface clean as minute surface, being immersed in concentration is the polyallylamine that contains of 2mg/mL, polyethyleneimine, in at least a cation high molecular polymer solution in the diallyl dimethyl ammoniumchloride 20 minutes, take out and to immerse again in the damping fluid 5 minutes, take out again and be immersed in the poly-sulfonated ethylene that concentration is 2mg/mL, in the poly-sulfonated phenylethylene at least a anionic polymer polymer solution 20 minutes, repeat this process until alternately plating 2～15 floor height Molecularly Imprinted Polymer built up films; Second step, same a; The 3rd step, same a,
D. the first step is used 65%HNO successively
3: H
2O=1: 1 HNO
3With exposed part surface, acetone wiping working electrode lower end, spend dried up cleaning then, dry naturally; Second step, the alcoholic solution that adds 1 μ L concentration again on working electrode end portion surface and be 1% ferrocene forms ferrocene electron mediator layer, naturally add 5 μ L after drying again and contain 5% nano particle and oxidasic 5% gelatin solution of 2mg/mL, with 2% glutaraldehyde cross-linking 3 minutes, use deionized water rinsing, dry or 6 ℃ of preservations down in refrigerator; The 3rd step applied with the acetone soln that dropping 1 μ L～2 μ L contain 1%～3% cellulose acetate on working electrode end portion surface again, dried naturally, formed the diffusion restriction film,
The percent concentration of used solution is the weight/volume specific concentration in the said method, used nano particle is one or more in nanogold particle, nano-Ag particles, nanometer silicon dioxide particle, the nano copper particle, its grain size scope is at 8～50nm, used oxidase is glucose oxidase or Lactate Oxidase or cholesterol oxidase, and the prescription of used damping fluid is Na
2HPO
41.15 gram, KCl 0.2 gram, NaCl 8.0 grams, KH
2PO
40.2 gram, constant volume are in the volumetric flask of 1000mL, pH=7.4 is used as working electrode and is gold, carbon, platinum or silver to the conductive material of the matrix of electrode, and the organic insulation that is used as electrode base sheet is a polycarbonate.
When carrying out the micro-embedded processing of enzyme with embedding enzyme layer matrix in the said method, the embedding enzyme layer that is plated is 1～12 layer of 1 layer of polyvinylpyrrolidone, 1 layer in gelatin, 1～12 layer of concanavalin A or a shitosan.
Being preferably in working electrode end portion surface in the said method is to apply with the acetone soln that contains 2%～2.5% cellulose acetate, dries naturally, forms the diffusion restriction film.
The invention has the beneficial effects as follows:
(1) utilizes high molecular polymer accumulation multilayer PEM film or ferrocene electron mediator layer and cellulose acetate diffusion restriction film, chaff interference such as uric acid, ascorbic acid and trichloroacetic acid all there is significant barrier effect, overcome the existence of interference current, interference free performance obviously improves.
When detecting the glucose be present in the blood, cholesterol, lactic acid with biology sensor, these three kinds of materials have H entirely behind oxydasis separately
2O
2Produce, its chemical equation is as follows:
H
2O
2Produce electronics in the reaction of electrode surface generation oxidizing and electrochemical,
According to above-mentioned reaction, H
2O
2Produce electronics by the oxidizing and electrochemical reaction,, can measure the content of glucose, cholesterol and lactic acid according to the size of electric current when the electron transfer that produces promptly produces Ampere currents to electrode surface.Because a lot of enzyme sensors are to utilize common biochemical reaction secondary product H
2O
2The oxidation current that produces under 0.6V is worked indirectly, and when 0.6V, uric acid (uric acid), ascorbic acid (ascorbic acid) and trichloroacetic acid interfering materials such as (acetaminophen) also can be oxidized, produce the interference current influence and measure, promptly influence the electric current of enzymatic generation and the proportionate relationship between the substrate to be measured.Interfering material and H
2O
2The two class redox reactions that produce all occur in the surface of electrode.The biologic enzyme electrode that the biology sensor of the inventive method preparation is used utilizes polyallylamine (PAA), polyethyleneimine (PEI), diallyl dimethyl ammoniumchloride (PDDA), poly-sulfonated ethylene (PVS), poly-sulfonated phenylethylene (PSS) to wait formation PEM film or cellulose acetate diffusion restriction films, only allows product H
2O
2By and do not allow interfering material pass through, thereby anti-interference, or cover ferrocene electron mediator layer, reduce the response current of interfering material by utilizing the lower oxidation-reduction potential of ferrocene.The present inventor utilizes 283 electrochemical workstations that biologic enzyme electrode of the present invention and the naked biologic enzyme electrode that is attached with the PEM film carried out jamproof contrast test experiment, and the result is as shown in table 1 below, and the data in the table 1 are respectively 5mMH
2O
2, the current value that produces of 5mM ascorbic acid and 5mM uric acid, unit is a micromicroampere, the interference free performance of visible biologic enzyme electrode of the present invention obviously improves; The interference free performance that covers ferrocene electron mediator layer and cellulose acetate diffusion restriction film also is significantly improved, and measured result can be referring to embodiment 46.
(2) adding nano particle in the enzyme layer makes the sensitivity of biologic enzyme electrode improve 3～4 times (referring to embodiment 48).
(3) adopt the acetone soln that contains 1%～3% cellulose acetate to form the diffusion restriction film in the methods of the invention at electrode surface, the measure linear range widens of the biologic enzyme electrode that makes thus, the scope of can surveying is at 0-30mM (referring to embodiment 47).
(4) carry out micro-embedded with shitosan, polyvinylpyrrolidone, gelatin or concanavalin A and oxidase in the methods of the invention, increase the adsorbance of enzyme and prolonged the time of enzyme survival, thereby prolonged biologic enzyme electrode serviceable life, improved the stability of sensor.Wherein particularly directly adopt the static self-assembling method to prepare multilayer built up film immobilised enzymes with shitosan and oxidase, by increasing the amount that the assembling number of plies improves the enzyme that is fixed in the biologic enzyme electrode, prolonged the serviceable life of biologic enzyme electrode, enzyme membrane is put refrigerator 0-4 ℃ and should be preserved more than 1 year, and the stability of using in the air increased to 4～6 months from 1～2 month.
(5) the present invention has had further improvement, has improved and ripe preparation method than Chinese patent 03257848.2, anti-interference layer in the responding layer has also adopted ferrocene electron mediator layer except that adopting the macromolecule built up film, can simplify preparation technology, more choice is provided.
In a word, the biologic enzyme electrode that the biology sensor that uses the inventive method to make is used has improved the every performance and the practicality of biology sensor on the whole, thereby adopts biologic enzyme electrode of the present invention to make the biology sensor integral level obtain basic improvement.
The jamproof contrast test experimental result of table 1
| Electrode type | H 2O 2 | Ascorbic acid | Uric acid |
| Bare electrode | 4.9 | 2.5 | 3.2 |
| PAA/PVS | 4.0 | 0.2 | 0.9 |
| (PAA/PVS)PAA | 4.0 | 0.1 | 0.08 |
| (PAA/PVS) 2 | 3.7 | 0.02 | 0.02 |
| (PAA/PVS) 2PAA | 3.7 | 0.08 | 0.02 |
| PAA/PSS | 4.5 | 1.8 | 1.7 |
| (PAA/PSS)PAA | 4.5 | 1.0 | 0.7 |
| (PAA/PSS) 2 | 4.2 | 0.2 | 0.2 |
| (PAA/PSS) 2PAA | 4.0 | 0.1 | 0.05 |
Description of drawings
Fig. 1-I and Fig. 1-II are respectively that responding layer is the front elevation of the biologic enzyme electrode of the present invention of rectangle and circle.
Fig. 2 is the volume rendering synoptic diagram of the responding layer of biologic enzyme electrode of the present invention.
Fig. 3 is the square section synoptic diagram of the responding layer of biologic enzyme electrode of the present invention.
Fig. 4-I and Fig. 4-II are respectively that responding layer is the working electrode of rectangle and circular biologic enzyme electrode of the present invention and to the front elevation of electrode.
Fig. 5 is the three-electrode system figure that biologic enzyme electrode of the present invention adopts when measuring.
Fig. 6-I and Fig. 6-II are respectively that scanning voltage is the cyclic voltammogram of glucose biological sensor in damping fluid, uric acid, ascorbic acid, ferrocene solution of unmodified under 600mV and the 400mV condition.
Fig. 7-I and 7-II are respectively that scanning voltage is the cyclic voltammogram of glucose biological sensor in damping fluid, uric acid, ascorbic acid, ferrocene solution of modifying with cellulose acetate under 600mV and the 400mV condition.
Fig. 8 is a current curve of measuring glucose with the ferrocene for the glucose biological sensor of electron mediator layer preparation.
Fig. 9-I is respectively the I-t curve of the film modified glucose biological sensor of diffusion restriction of different cellulose acetate concentration with Fig. 9-II and Fig. 9-III.
Figure 10-I and Figure 10-II are respectively the work figure lines of the film modified glucose biological sensor of diffusion restriction of the cellulose acetate prepared at concentrations of variable concentrations.
Figure 11-I, 11-II and 11-III are respectively the cyclic voltammograms of the film modified glucose biology sensor of the cellulose acetate diffusion restriction of variable concentrations.
Figure 12 is the transmission electron microscope scanning spectra of institute's nano-golden particle-containing in the biologic enzyme electrode used of biology sensor of the present invention.
Figure 13-I is the glucose biological sensor modified with nanogold particle and not with the I-t curve of the glucose biological sensor of nanogold particle modification.
Figure 13-II is the current curve of cellulose acetate and the common glucose biological sensor of modifying of nanogold particle.
Figure 14 is the stability experiment test curve of biologic enzyme electrode of the present invention.
Among the figure, 1. working electrode, 2. pair electrode, 3. carrier, 4. the insulator on coated electrode surface, 5. responding layer, the 6. anti-interference layer of macromolecule built up film or the anti-interference layer of ferrocene electron mediator layer, 7. nano particle, 8. matrix embedding enzyme layer, 9. oxidase, 10. diffusion restriction film.
The present invention is further described below in conjunction with drawings and Examples.
Embodiment
Fig. 1 shows working electrode 1 and electrode 2 usefulness spraying processes is printed on the carrier of being made by polycarbonate 3, scribbles the polycarbonate insulator 4 on one deck coated electrode surface on their the center section face.Working electrode 1 and are a pair of anode and cathode that can change to electrode 2.Layer 5 responds in the lower end of working electrode 1.Fig. 1-I shows that responding layer 5 is rectangle, and Fig. 1-II shows that responding layer 5 is for circular.
Fig. 2 and Fig. 3 show the matrix of being close to working electrode 1 is 2～15 floor height molecule built up films or ferrocene electron mediator layer 6 in the responding layer 5, and then be the matrix embedding enzyme layer 8 and the enzyme 9 of alternately absorption, nano particle 7 is embedded in macromolecule built up film or ferrocene electron mediator layer 6 and the matrix embedding enzyme layer 8 by bag simultaneously.
Fig. 4 shows working electrode 1 and to the shape of electrode 2.Fig. 4-I shows that the responding layer 5 of working electrode 1 is rectangle, and Fig. 4-II shows that the responding layer 5 of working electrode 1 is for circular.
Fig. 5 shows when biologic enzyme electrode of the present invention is measured the three-electrode system that adopts, and wherein, A is that electrode is made in the metal working of adhering to the diameter 2.2mm of enzyme layer, B for exposed gold to electrode, C is the Ag/AgCl contrast electrode.
Fig. 6-I draws when scanning voltage is 600mV, and the electric current that uric acid and ascorbic acid produce is respectively 1495.2nA and 922.8nA; Fig. 6-II draws when scanning voltage is 400mV, and the electric current that uric acid and ascorbic acid produce is respectively 132.1nA and 163.5nA.Among Fig. 6, a. damping fluid, b.0.1mM ascorbic acid, c.0.1mM uric acid and 0.1mM ascorbic acid, d.0.1mM uric acid, 0.1mM ascorbic acid and 1mM ferrocene.
Fig. 7-I draws when scanning voltage is 600mV, and the electric current that uric acid and ascorbic acid produce is respectively 37.1nA and 100.2nA; Fig. 7-II draws when scanning voltage is 400mV, and the electric current that uric acid and ascorbic acid produce is respectively 9.4nA and 11.1nA.Among Fig. 7, a. damping fluid, b.0.1mM ascorbic acid, c.0.1mM uric acid and 0.1mM ascorbic acid, d.0.1mM uric acid, 0.1mM ascorbic acid and 1mM ferrocene.
Fig. 8 is a current curve of measuring glucose with the ferrocene for the glucose biological sensor of electron mediator layer preparation, arrow is represented the concentration of glucose respectively, arrow 1 is that the dropping final concentration is the uric acid of 0.3mM, and arrow 2 is to drip the ascorbic acid that final concentration is 0.1mM again.
The cellulose acetate solution concentration of each bar curve correspondence is a=0%, b=1%, c=1.5%, d=2%, e=2%+1%, f=2.5%, g=3% among Fig. 9, and their reflections have the response current of the film modified glucose biological sensor of the diffusion restriction of variable concentrations cellulose acetate solution preparation.
The cellulose acetate solution concentration of each bar curve correspondence is a=0%, b=1%, c=1.5%, d=2%, f=2.5%, g=3% among Figure 10.
Figure 11-I, 11-II and 11-III are respectively that concentration is the cyclic voltammogram of the film modified glucose biological sensor of 0%, 2% and 2.5% cellulose acetate diffusion restriction.
Contained diameter is the transmission electron microscope scanning spectra of the nanogold particle of 35nm in the biologic enzyme electrode that Figure 12 uses for biology sensor of the present invention.
Figure 13-I represents the contrast of the glucose biological sensor modified with nanogold particle and the I-t curve of the glucose biological sensor of not modifying with nanogold particle, wherein a is the current curve of the glucose biological sensor of nanogold particle modification, and b does not add the glucose biological sensor current curve of nanometer particle modification; Figure 13-II is the current curve of cellulose acetate and the common glucose biological sensor of modifying of nanogold particle.
Figure 14 shows that in the stability experiment test curve of biologic enzyme electrode of the present invention, first day current value is 100%, and current value is 80% during by 190 days.
Goldleaf is printed on spraying process on the carrier of being made by polycarbonate by shape shown in Figure 4, forms working electrode and the matrix of electrode.The size of carrier is long 40mm, wide 7mm, thick 0.2mm.Working electrode and to the outer length of side 35mm of electrode, interior length of side 32mm, upper end 1mm, the working electrode lower end is that rectangle or the diameter of wide 3.5mm, high 3mm is the circle of 1～3mm, to the wide 2mm of electrode lower end rectangle part, high 3mm.Working electrode is with mutually neat with a side broadside of carrier to the electrode upper end.With aluminium powder polishing working electrode end portion, use distilled water flushing, make electrode surface clean as minute surface, being immersed in the concentration that is mixed with 5% nanogold particle is the polyallylamine of 2mg/mL, polyethyleneimine, among at least a cation high molecular polymer solution 5mL in the diallyl dimethyl ammoniumchloride 20 minutes, immersed again after the taking-up in the damping fluid 5 minutes, take out again and be immersed in the poly-sulfonated ethylene that the concentration that is mixed with 5% nanogold particle is 2mg/mL, in the poly-sulfonated phenylethylene among at least a anionic polymer polymer solution 5mL 20 minutes, repeat this process until alternately plating 2～15 floor height Molecularly Imprinted Polymer built up films; The electrode that will plate the high molecular polymer built up film again carries out the micro-embedded of enzyme with the chitosan imbedded enzyme layer matrix that is mixed with 5% nanogold particle, method is to utilize the static self-assembling technique to carry out shitosan and enzyme is accumulated, concrete steps are that electrode was immersed in the chitosan solution embedding enzyme layer matrix that the concentration that is mixed with 5% nanogold particle is 2mg/mL 20 minutes, take out and to immerse again in the damping fluid 5 minutes, take out and to immerse again in the glucose oxidase that concentration is 2mg/mL 20 minutes, 1 time or repeatedly carry out this process until alternately plating 1～12 layer of embedding enzyme layer, after the taking-up, apply with the acetone soln that contains 1%～3% cellulose acetate, naturally dry, form the diffusion restriction film, form responding layer thus, at the distance working electrode with to electrode upper end 4mm and lower end rectangle or the covering of the center section more than circle one deck polycarbonate insulator, so far make the biologic enzyme electrode that a biology sensor is used.The prescription of used damping fluid is Na
2HPO
41.15 gram, KCl 0.2 gram, NaCl8.0 gram, KH
2PO
40.2 the gram, constant volume in the volumetric flask of 1000mL, pH=7.4.
Embodiment 2-4
Form working electrode with carbon, platinum or silver respectively and to the matrix of electrode, other is identical with embodiment 1.
Embodiment 5-10
Use nanogold particle and nanometer silicon dioxide particle and the nano copper particle and the nano-Ag particles of the nanogold particle of the nanogold particle of nano-Ag particles, nanometer silicon dioxide particle, nano copper particle, mixing and nanometer silicon dioxide particle, mixing and nanometer silicon dioxide particle and nano copper particle, mixing respectively, replace nanogold particle, other is identical with embodiment 1.
Remove with 5% the polyvinylpyrrolidone 1mL dissolving 2mg oxidase that is mixed with 5% nanogold particle, obtain the enzyme solutions of 2mg/mL, carry out immobilized enzyme at electrode surface again, form outside the 1 layers of polyethylene pyrrolidone matrix embedding enzyme layer, other with
Embodiment 12
Remove with 5% the gelatin 1mL dissolving 2mg oxidase that is mixed with 5% nanogold particle, obtain the enzyme solutions of 2mg/mL, carry out the gelatin immobilized enzyme at electrode surface again, form outside 1 layer of gelatin substrate embedding enzyme layer, other is identical with embodiment 1.
Embodiment 13
Except that matrix embedding enzyme layer was used 1～12 layer of concanavalin A instead, other was identical with embodiment 1.
Embodiment 14-15
Except that glucose oxidase being used instead into Lactate Oxidase or the cholesterol oxidase, other is identical with embodiment 1.
Embodiment 16
Goldleaf is printed on spraying process on the carrier of being made by polycarbonate by shape shown in Figure 4, forms working electrode and the matrix of electrode.The size of carrier is long 40mm, wide 7mm, thick 0.2mm.Working electrode and to the outer length of side 35mm of electrode, interior length of side 32mm, upper end 1mm, the working electrode lower end is that rectangle or the diameter of wide 3.5mm, high 3mm is the circle of 1～2mm, to the wide 2mm of electrode lower end rectangle part, high 3mm.Working electrode is with mutually neat with a side broadside of carrier to the electrode upper end.With aluminium powder polishing working electrode end portion, use distilled water flushing, make electrode surface clean as minute surface, being immersed in and being mixed with the concentration that contains 5% nanogold particle is the 2mg/mL polyallylamine, polyethyleneimine, among at least a cation high molecular polymer solution 5mL in the diallyl dimethyl ammoniumchloride 20 minutes, immersed again after the taking-up in the damping fluid 5 minutes, take out again and be immersed in the poly-sulfonated ethylene that the concentration that is mixed with 5% nanogold particle is 2mg/mL, in the poly-sulfonated phenylethylene among at least a anionic polymer polymer solution 5mL 20 minutes, repeat this process until alternately plating 2～15 floor height Molecularly Imprinted Polymer built up films; The electrode that will plate the high molecular polymer built up film again carries out the micro-embedded of enzyme with chitosan imbedded enzyme layer matrix, method is to utilize the static self-assembling technique to carry out shitosan and enzyme is accumulated, electrode was immersed in the chitosan solution embedding enzyme layer matrix that concentration is 2mg/mL 20 minutes, take out to immerse in the damping fluid 5 minutes, take out to immerse concentration again and be in the glucose oxidase of 2mg/mL 20 minutes, 1 time or repeatedly carry out this process until alternately plating 1～12 layer of embedding enzyme layer, after the taking-up, apply with the acetone soln that contains 1%～3% cellulose acetate, naturally dry, form the diffusion restriction film, form responding layer thus, at the distance working electrode with to electrode upper end 4mm and lower end rectangle or the covering of the center section more than circle one deck polycarbonate insulator, so far make the biologic enzyme electrode that a biology sensor is used.The prescription of used damping fluid is Na
2HPO
41.15 gram, KCl 0.2 gram, NaCl 8.0 grams, KH
2PO
40.2 the gram, constant volume in the volumetric flask of 1000mL, pH=7.4.
Embodiment 17-19
Form working electrode with carbon, platinum or silver respectively and to the matrix of electrode, other is identical with embodiment 16.
Embodiment 20-25
Use nanogold particle and nanometer silicon dioxide particle and the nano copper particle and the nano-Ag particles of the nanogold particle of the nanogold particle of nano-Ag particles, nanometer silicon dioxide particle, nano copper particle, mixing and nanometer silicon dioxide particle, mixing and nanometer silicon dioxide particle and nano copper particle, mixing respectively, replace nanogold particle, other is identical with embodiment 16.
Embodiment 26
Remove the polyvinylpyrrolidone 1mL dissolving 2mg oxidase with 5%, obtain the enzyme solutions of 2mg/mL, carry out immobilized enzyme at electrode surface again, form outside the 1 layers of polyethylene pyrrolidone matrix embedding enzyme layer, other is identical with embodiment 16.
Embodiment 27
Remove the gelatin 1mL dissolving 2mg oxidase with 5%, obtain the enzyme solutions of 2mg/mL, carry out the gelatin immobilized enzyme at electrode surface again, form outside 1 layer of gelatin substrate embedding enzyme layer, other is identical with embodiment 16.
Embodiment 28
Except that matrix embedding enzyme layer was used 1～12 layer of concanavalin A instead, other was identical with embodiment 16.
Embodiment 29-30
Except that glucose oxidase being used instead into Lactate Oxidase or the cholesterol oxidase, other is identical with embodiment 16.
Embodiment 31
Goldleaf is printed on spraying process on the carrier of being made by polycarbonate by shape shown in Figure 4, forms working electrode and the matrix of electrode.The size of carrier is long 40mm, wide 7mm, thick 0.2mm.Working electrode and to the outer length of side 35mm of electrode, interior length of side 32mm, upper end 1mm, the working electrode lower end is that rectangle or the diameter of wide 3.5mm, high 3mm is the circle of 1～2mm, to the wide 2mm of electrode lower end rectangle part, high 3mm.Working electrode is with mutually neat with a side broadside of carrier to the electrode upper end.With aluminium powder polishing working electrode end portion, use distilled water flushing, make electrode surface clean as minute surface, be immersed among at least a cation high molecular polymer solution 5mL in polyallylamine that concentration is 2mg/mL, polyethyleneimine, the diallyl dimethyl ammoniumchloride 20 minutes, take out and to immerse again in the damping fluid 5 minutes, take out again and be immersed among the anionic polymer polymer solution 5mL at least a in the poly-sulfonated ethylene that concentration is 2mg/mL, the poly-sulfonated phenylethylene 20 minutes, repeat this process until alternately plating 2～15 floor height Molecularly Imprinted Polymer built up films; The electrode that will plate the high molecular polymer built up film again carries out the micro-embedded of enzyme with the chitosan imbedded enzyme layer matrix that is mixed with 5% nanogold particle, method is to utilize the static self-assembling technique to carry out shitosan and enzyme is accumulated, electrode was immersed in the chitosan solution embedding enzyme layer matrix that the concentration that is mixed with 5% nanogold particle is 2mg/mL 20 minutes, take out and to immerse again in the damping fluid 5 minutes, take out to immerse concentration again and be in the glucose oxidase of 2mg/mL 20 minutes, 1 time or repeatedly carry out this process until alternately plating 1～12 layer of embedding enzyme layer, after the taking-up, apply with the acetone soln that contains 1%～3% cellulose acetate, naturally dry, form the diffusion restriction film, form responding layer thus, at the distance working electrode with to electrode upper end 4mm and lower end rectangle or the covering of the center section more than circle one deck polycarbonate insulator, so far make the biologic enzyme electrode that a biology sensor is used.The prescription of used damping fluid is Na
2HPO
41.15 gram, KCl 0.2 gram, NaCl 8.0 grams, KH
2PO
40.2 the gram, constant volume in the volumetric flask of 1000mL, pH=7.4.
Embodiment 32-34
Form working electrode with carbon, platinum or silver respectively and to the matrix of electrode, other is identical with embodiment 31.
Embodiment 35-40
Use nanogold particle and nanometer silicon dioxide particle and the nano copper particle and the nano-Ag particles of the nanogold particle of the nanogold particle of nano-Ag particles, nanometer silicon dioxide particle, nano copper particle, mixing and nanometer silicon dioxide particle, mixing and nanometer silicon dioxide particle and nano copper particle, mixing respectively, replace nanogold particle, other is identical with embodiment 31.
Embodiment 41
Remove with 5% the polyvinylpyrrolidone 1mL dissolving 2mg oxidase that is mixed with 5% nanogold particle, obtain the enzyme solutions of 2mg/mL, carry out immobilized enzyme at electrode surface again, form outside the 1 layers of polyethylene pyrrolidone matrix embedding enzyme layer, other with
Embodiment 31 is identical
Embodiment 42
Remove with 5% the gelatin 1mL dissolving 2mg oxidase that is mixed with 5% nanogold particle, obtain the enzyme solutions of 2mg/mL, carry out the gelatin immobilized enzyme at electrode surface again, form outside 1 layer of gelatin substrate embedding enzyme layer, other is identical with embodiment 31.
Embodiment 43
Except that matrix embedding enzyme layer was used 1～12 layer of concanavalin A instead, other was identical with embodiment 1.
Embodiment 44-45
Except that glucose oxidase being used instead into Lactate Oxidase or the cholesterol oxidase, other is identical with embodiment 31.
Embodiment 46
Anti-interference layer covers the antijamming capability contrast test of the glucose biological sensor of ferrocene electron mediator layer:
Use 65%HNO successively
3: H
2O=1: 1 HNO
3With exposed part surface, acetone wiping electrode lower end, spend dried up cleaning then, naturally dry, adding 1 μ L concentration at electrode surface then is that the alcoholic solution of 1% ferrocene forms ferrocene electron mediator layer, naturally add 5% gelatin solution that 5 μ L contain 5% nanogold particle and glucose oxidase 2mg/mL after drying again, with 2% glutaraldehyde cross-linking 3 minutes, use deionized water rinsing, dry, after drip the acetone soln that 2 μ L contain 2% cellulose acetate again, dry naturally, form the diffusion restriction film, make the biologic enzyme electrode that biology sensor is used, the overnight measurement.It is as follows to measure equipment therefor, condition and result: device is to measure on 283 type electrochemical workstations, the three-electrode system that adopts as shown in Figure 5, wherein, A is that electrode is made in the metal working of adhering to the diameter 2.2mm of enzyme layer, B for exposed gold to electrode, C is the Ag/AgCl contrast electrode; Measuring condition is the pH=6.8 of phosphate buffer, and sweep speed is 50mV/s, and voltage is for O.25V, 25 ℃ of experimental temperatures; The result as shown in Figure 6, Fig. 6 is the cyclic voltammogram of glucose biological sensor in damping fluid, uric acid, ascorbic acid, ferrocene solution that the electrode end portion of unmodified under the different scanning voltage conditions is coated with the ferrocene electron mediator, a. damping fluid among the figure, b.0.1mM ascorbic acid, c.0.1mM uric acid and 0.1mM ascorbic acid, d.0.1mM uric acid, 0.1mM ascorbic acid and 1mM ferrocene.Draw when scanning voltage is 600mV from Fig. 6-I, the electric current that uric acid and ascorbic acid produce is respectively 1495.2nA and 922.8nA; Draw when scanning voltage is 400mV from Fig. 6-II, the electric current that uric acid and ascorbic acid produce is respectively 132.1nA and 163.5nA.By reducing working electrode scanning voltage 200mV, then electric current reduces by 91.2% and 82.3% respectively, further confirms by reducing the interference that operating voltage can effectively reduce uric acid, ascorbic acid.Draw ferrocene under different scanning voltages from Fig. 6, oxidation peak and reduction peak potential difference (PD) present a pair of reversible redox peak less than 60mV, and its oxidation peak current potential is 250mV.As seen utilize ferrocene electron mediator layer to have anti-jamming capacity as the glucose biological sensor of anti-interference layer.Fig. 7 be under the different scanning voltage conditions with cellulose acetate is modified, the electrode end portion is coated with ferrocene electron mediator layer the glucose biological sensor cyclic voltammogram in damping fluid, uric acid, ascorbic acid and ferrocene solution respectively, a. damping fluid among the figure, b.0.1mM ascorbic acid, c.0.1mM uric acid and 0.1mM ascorbic acid.Draw when scanning voltage is 600mV from Fig. 7-I, the electric current that uric acid and ascorbic acid produce is respectively 37.1nA and 100.2nA.Draw when scanning voltage is 400mV from Fig. 7-II, the electric current that uric acid and ascorbic acid produce is respectively 9.4nA and 11.1nA.Relatively draw with Fig. 6-I respectively, the glucose biological sensor that cellulose acetate is modified is when scanning voltage is 600mV, and the uric acid electric current reduces by 97.5%, the ascorbic acid electric current reduces by 89.2%, when scanning voltage was 400mV, the uric acid electric current reduced by 99.4%, and the ascorbic acid electric current reduces by 98.8%.Therefore the glucose biological sensor antijamming capability that draws the cellulose acetate modification is strong.The cellulose acetate surface has a large amount of negative charges, and uric acid electronegative in the solution and ascorbic acid are had very strong repulsive interaction, makes them can not arrive electrode surface, thereby can not produce interference current.Fig. 8 is a current curve of measuring glucose with the ferrocene for the glucose biological sensor of electron mediator layer preparation, arrow is represented the concentration of glucose respectively, arrow 1 is that the dropping final concentration is the uric acid of 0.3mM, and arrow 2 is to drip the ascorbic acid that final concentration is 0.1mM again.The upper limit of detection that draws glucose from Fig. 8 reaches 30mM, the time that reaches 95% maximum effect electric current was less than 10 seconds, its sensitivity is 32.3nA/mM, uric acid and ascorbic acid do not produce tangible electric current, therefore the glucose biological sensor antijamming capability that with the ferrocene is the electron mediator layer is strong, and electrochemical properties is good.This shows, be the electron mediator layer with the ferrocene, be carrier with the gelatin, the glucose biological sensor anti-interference ability of utilizing the cellulose acetate of glutaraldehyde cross-linking preparation to modify is strong, upper limit of detection reaches 30mM, the time that reaches 95% maximum effect electric current is less than 10 seconds, its sensitivity 32.3nA/mM.Therefore with the inventive method preparation be that the glucose biological sensor of anti-interference layer can directly apply to clinical detection with ferrocene electron mediator layer, can satisfy requirements for clinical application.
Embodiment 47
The performance comparison test of the glucose biological sensor that variable concentrations cellulose acetate diffusion restriction is film modified.
Use 65%HNO successively
3: H
2O=1: 1 HNO
3With acetone wiping bare electrode end portion surface, spend dried up cleaning then, naturally dry, adding 1 μ L concentration at electrode surface then is that the alcoholic solution of 1% ferrocene forms ferrocene electron mediator layer, naturally add 5 μ L after drying again and contain 5% nano-Ag particles, nano copper particle, or 5% gelatin solution of Nano Silver and copper particle and glucose oxidase 2mg/mL, with 2% glutaraldehyde cross-linking 3 minutes, use deionized water rinsing, dry, after drip the acetone soln that 1 μ L contains 1～3% cellulose acetate again, dry naturally, form the diffusion restriction film, make the biologic enzyme electrode that biology sensor is used, the overnight measurement.It is as follows to measure equipment therefor, condition and result: device is to measure on 283 type electrochemical workstations, the three-electrode system that adopts as shown in Figure 5, wherein, A is that electrode is made in the metal working of adhering to the diameter 2.2mm of enzyme layer, B for exposed gold to electrode, C is the Ag/AgCl contrast electrode.Measuring condition is the pH=6.8 of phosphate buffer, and sweep speed is 50mV/s, and voltage is 0.25V, 25 ℃ of experimental temperatures.The result as shown in Figure 9, Fig. 9 is the I-t curve of the film modified glucose biological sensor of cellulose acetate diffusion restriction of variable concentrations, wherein the cellulose acetate solution concentration of each bar curve correspondence is a=0%, b=1%, c=1.5%, d=2%, e=2%+1%, f=2.5%, g=3%, their reflections have the response current of the film modified glucose biological sensor of the diffusion restriction of variable concentrations cellulose acetate solution preparation, can see that cellulose acetate diffusion restriction film has played certain barrier effect to the diffusion of glucose.A little less than the thin barrier effect of the film that forms when cellulose acetate concentration is low, so concentration of glucose just reached a among peak response electric current such as Fig. 9-I when low, and b is shown in the c curve.The thickness that forms when the cellulose acetate excessive concentration, the diffusion restriction effect is strong, has only the glucose of high concentration to spread and sees through, shown in the g curve among Fig. 9-II.Have only the effect of the suitably prepd cellulose acetate diffusion restriction of cellulose acetate concentration film diffusion restriction just moderate, the glucose of variable concentrations can spread in proportion and see through, shown in d curve and the f curve among Fig. 9-III among Fig. 9-II.The cellulose acetate concentration of the e curve correspondence among Fig. 9-II is 2%+1%, the cellulose acetate of 1 μ L 2% is dripped in expression earlier at the electrode surface of glucose oxidase enzyme modification, drip the cellulose acetate of 1 μ L 1% behind the natural air drying again, it does not meet The above results, this may be because the well combination of two-layer cellulose acetate diffusion restriction film is still needed and further studied.The response time that can also draw glucose biological sensor from Fig. 9 is respectively a=5 second, b=7 second, c=10 second, d=10 second, e=12 second, f=15 second, g=20 second, illustrate that cellulose acetate diffusion restriction film has certain influence to the response time, but it is just bigger to have only cellulose acetate concentration to reach influence in 2.5% o'clock, influence is less during low concentration.Figure 10-I is respectively the working curve of the film modified glucose biological sensor of diffusion restriction of different cellulose acetate prepared at concentrations with Figure 10-II.The cellulose acetate solution concentration of each bar curve correspondence is a=0%, b=1%, c=1.5%, d=2%, f=2.5%, g=3% among Figure 10, the sensing range that curve a, b from Figure 10, c, d, f, g can draw the film modified glucose biological sensor of the diffusion restriction of different cellulose acetate prepared at concentrations is respectively 0～4mM, 0～4mM, 0～13mM, 0～30mM, 0～30mM and 6～30mM.Along with the raising of cellulose acetate concentration, the sensing range of the film modified glucose biological sensor of cellulose acetate diffusion restriction increases, but after cellulose acetate was increased to finite concentration, the detection lower limit of the film modified glucose sensor of diffusion restriction began to improve.By relatively drawing concentration is that 2% and 2.5% cellulose acetate diffusion restriction film is more satisfactory, can satisfy the clinical detection needs, and sensitivity is respectively 32.3nA/mM and 13.7nA/mM, and related coefficient is respectively 0.99733 and 0.99781.Experimental result only shows can bring into play the diffusion restriction effect of cellulose acetate effectively in suitable cellulose acetate concentration range, widen the sensing range of glucose biological sensor effectively.Figure 11-I, 11-II and 11-III are respectively that concentration is the cyclic voltammogram of the film modified glucose biological sensor of 0%, 2% and 2.5% cellulose acetate diffusion restriction.The result shows, the glucose biological sensor of modifying with cellulose acetate is not at concentration of glucose during greater than 8mM, the oxidation peak peak value is overlapped to rise no longer in proportion, show that this moment, reaction showed as mixed order reaction earlier, gradually become 0 order reaction then, and with concentration be the sensing range of the glucose biological sensor modified of 2% and 2.5% cellulose acetate in glucose 0mM～30mM scope, the oxidation peak peak value rises in proportion, illustrate that react is 1 order reaction always.In a word, in the suitable cellulose acetate concentration range, along with the diffusion control effect of the increase cellulose acetate diffusion restriction film of concentration of glucose is strengthened, i.e. glucose diffusion sees through resistance to be increased thereupon, sensing range increases, and Michaelis constant increases but response current reduces.The sensing range 0-4mM of the glucose biological sensor of modifying with cellulose acetate not, and the sensing range of the glucose biological sensor of modifying with cellulose acetate can reach 0-30mM, and this explanation cellulose acetate diffusion restriction film can well be regulated and control the performance of glucose biological sensor.
Embodiment 48
Select known technology (Stober W.Controlled growth of monodisperse silica in the microsize rang.Journal of colloid and interface science, 1968,26:62-69 for use; Zhang Zhi plum etc. the preparation of nanoscale copper powder. fine chemistry industry .2002,17 (2): 69-71; Duan Chunying etc., the preparation of silver nano-grain and sign, chemical research, 2003,14 (3): 18-21; Tang Fangqiong etc.; hydrophilic gold and hydrophobic silicon dioxide nano particle are to the humidification of glucose biological sensor response sensitivity; SCI; 2001; 21 (1): 91-94) preparation nanogold particle, nano-Ag particles, nanometer silicon dioxide particle or nano copper particle, measure the effect that nano particle strengthens the glucose biological sensor electric current.Contained diameter is the transmission electron microscope scanning spectra of the nanogold particle of 35nm in the biologic enzyme electrode that Figure 12 uses for biology sensor of the present invention.Figure 13-I represent the glucose biological sensor modified with nanogold particle and the glucose biological sensor of not modifying with nanogold particle the I-t curve contrast and Figure 13--II is the I-t curve of nanogold particle and the common glucose biological sensor of modifying of cellulose acetate.A is that b is the glucose biological sensor current curve that does not add the nanometer gold grain with the current curve of the glucose biological sensor of nanogold particle modification among Figure 13-I.Both contrasts can draw, and the glucose sensor sensitivity that nanogold particle is modified is increased, and response current is big; Figure 13-II is the current curve of nanogold particle and the common glucose of modifying of cellulose acetate, and visible enzyme electrode prepared by this method can meet clinical needs.
% concentration in the foregoing description is weight/volume percent concentration.
Claims (5)
1. biologic enzyme electrode that biology sensor is used, be by working electrode, to electrode, carrier, the insulator on coated electrode surface and responding layer constitute, it is characterized in that: responding layer is by the anti-interference layer that covers in the working electrode lower end on the electrode matrix, nano particle, matrix embedding enzyme layer, oxidase and diffusion restriction film are formed, and anti-interference layer is to utilize cation high molecular polymer poly allylamine, polyethyleneimine, at least a and anionic polymer polymer poly sulfonated ethylene in the diallyl dimethyl ammoniumchloride, 2～15 strata electrolyte complex films or the ferrocene electron mediator layer of at least a common formation in the poly-sulfonated phenylethylene; Nano particle is selected one or more in nanogold particle, nano-Ag particles, nanometer silicon dioxide particle, the nano copper particle for use, its grain size scope is at 8～50nm, and nano particle is embedded in anti-interference layer by bag or is embedded in the matrix embedding enzyme layer by bag or is wrapped simultaneously and is embedded in anti-interference layer and the matrix embedding enzyme layer; Matrix embedding enzyme layer is selected 1～12 layer of 1～12 layer of shitosan or 1 layer of polyvinylpyrrolidone or 1 layer in gelatin or concanavalin A for use; Oxidase is selected glucose oxidase, Lactate Oxidase or cholesterol oxidase for use; Matrix embedding enzyme layer and oxidase are alternately absorption, and oxidase is embedded in the matrix embedding enzyme layer, when matrix embedding enzyme layer is selected shitosan or concanavalin A and multilayer embedding for use, is that one deck suffers one deck; The diffusion restriction film is the film made from the acetone soln that contains 1%～3% cellulose acetate.
2. the biologic enzyme electrode that biology sensor according to claim 1 is used is characterized in that: the responding layer of working electrode lower end is rectangle or circle.
3. the preparation method of the biologic enzyme electrode used of the described biology sensor of claim 1, it is characterized in that: at the electrode base sheet of being made by organic insulation is on the carrier, conductive material print formed working electrode with spraying process and to the matrix of electrode, apply one deck polycarbonate insulator on their the center section surface, apply one deck integrated application high molecular polymer built up film technology or ferrocene soverlay technique at working electrode matrix end portion, the embedding zymotechnic, the responding layer that nanometer technology and diffusion restriction membrane technology form, promptly make biologic enzyme electrode, the concrete preparation method of its responding layer selects any one in following four kinds of methods for use:
A. the first step, with aluminium powder polishing working electrode end portion, use distilled water flushing, make electrode surface clean as minute surface, being immersed in the concentration that is mixed with 5% nano particle is the polyallylamine of 2mg/mL, polyethyleneimine, among at least a cation high molecular polymer solution 5mL in the diallyl dimethyl ammoniumchloride 20 minutes, take out and to immerse again in the damping fluid 5 minutes, take out again and be immersed in the poly-sulfonated ethylene that the concentration that is mixed with 5% nano particle is 2mg/mL, in the poly-sulfonated phenylethylene among at least a anionic polymer polymer solution 5mL 20 minutes, repeat this process until alternately plating 2～15 floor height Molecularly Imprinted Polymer built up films; Second step, the electrode that plates the high molecular polymer built up film is carried out the micro-embedded of enzyme with the embedding enzyme layer matrix that is mixed with 5% nano particle, the micro-embedded method that embedding enzyme layer matrix selects for use gelatin or polyvinylpyrrolidone to carry out enzyme is, gelatin or polyvinylpyrrolidone 1mL dissolving 2mg oxidase with the concentration 5% that is mixed with 5% nano particle, obtain the enzyme solutions of 2mg/mL, carry out gelatin or polyvinylpyrrolidone immobilized enzyme at electrode surface again, embedding enzyme layer matrix with the micro-embedded method that shitosan or concanavalin A carry out enzyme is, utilize the static self-assembling technique, accumulate with enzyme respectively with shitosan or concanavalin A, its operation steps is that electrode was immersed in chitosan solution that the concentration that is mixed with 5% nano particle is 2mg/mL or the concanavalin A solution embedding enzyme layer matrix 20 minutes, take out and to immerse again in the damping fluid 5 minutes, take out to immerse concentration again and be in the oxidase of 2mg/mL 20 minutes until alternately plating 1～12 layer of embedding enzyme layer for 1 time or repeatedly carry out this process; The 3rd step after the taking-up, applied with the acetone soln that contains 1%～3% cellulose acetate, dried naturally, formed the diffusion restriction film,
B. the first step, same a; Second step, the electrode polyvinylpyrrolidone of high molecular polymer built up film will be plated, gelatin, shitosan, or concanavalin A carries out the micro-embedded of enzyme, the micro-embedded method that embedding enzyme layer matrix selects for use gelatin or polyvinylpyrrolidone to carry out enzyme is, gelatin or polyvinylpyrrolidone 1mL dissolving 2mg oxidase with concentration 5%, obtain the enzyme solutions of 2mg/mL, carry out gelatin or polyvinylpyrrolidone immobilized enzyme at electrode surface again, embedding enzyme layer matrix with the micro-embedded method that shitosan or concanavalin A carry out enzyme is, utilize the static self-assembling technique, accumulate with enzyme respectively with shitosan or concanavalin A, its operation steps is that electrode was immersed in chitosan solution that concentration is 2mg/mL or the concanavalin A solution embedding enzyme layer matrix 20 minutes, take out and to immerse again in the damping fluid 5 minutes, take out to immerse concentration again and be in the oxidase of 2mg/mL 20 minutes until alternately plating 1～12 layer of embedding enzyme layer for 1 time or repeatedly carry out this process; The 3rd step, same a,
C. the first step, with aluminium powder polishing working electrode end portion, use distilled water flushing, make electrode surface clean as minute surface, being immersed in concentration is the polyallylamine that contains of 2mg/mL, polyethyleneimine, in at least a cation high molecular polymer solution in the diallyl dimethyl ammoniumchloride 20 minutes, take out and to immerse again in the damping fluid 5 minutes, take out again and be immersed in the poly-sulfonated ethylene that concentration is 2mg/mL, in the poly-sulfonated phenylethylene at least a anionic polymer polymer solution 20 minutes, repeat this process until alternately plating 2～15 floor height Molecularly Imprinted Polymer built up films; Second step, same a; The 3rd step, same a,
D. the first step is used 65%HNO successively
3: H
2O=1: 1 HNO
3With exposed part surface, acetone wiping working electrode lower end, spend dried up cleaning then, dry naturally; Second step, the alcoholic solution that adds 1 μ L concentration again on working electrode end portion surface and be 1% ferrocene forms ferrocene electron mediator layer, naturally add 5 μ L after drying again and contain 5% nano particle and oxidasic 5% gelatin solution of 2mg/mL, with 2% glutaraldehyde cross-linking 3 minutes, use deionized water rinsing, dry or 6 ℃ of preservations down in refrigerator; The 3rd step applied with the acetone soln that dropping 1 μ L～2 μ L contain 1%～3% cellulose acetate on working electrode end portion surface again, dried naturally, formed the diffusion restriction film,
The percent concentration of used solution is the weight/volume specific concentration in the said method, used nano particle is one or more in nanogold particle, nano-Ag particles, nanometer silicon dioxide particle, the nano copper particle, its grain size scope is at 8～50nm, used oxidase is glucose oxidase or Lactate Oxidase or cholesterol oxidase, and the prescription of used damping fluid is Na
2HPO
41.15 gram, KCl 0.2 gram, NaCl 8.0 grams, KH
2PO
40.2 gram, constant volume are in the volumetric flask of 1000mL, pH=7.4 is used as working electrode and is gold, carbon, platinum or silver to the conductive material of the matrix of electrode, and the organic insulation that is used as electrode base sheet is a polycarbonate.
4. the preparation method of the biologic enzyme electrode that biology sensor according to claim 3 is used, it is characterized in that: when carrying out the micro-embedded processing of enzyme with embedding enzyme layer matrix, the embedding enzyme layer that is plated is 1～12 layer of 1 layer of polyvinylpyrrolidone, 1 layer in gelatin, 1～12 layer of concanavalin A or a shitosan.
5. the preparation method of the biologic enzyme electrode that biology sensor according to claim 3 is used is characterized in that: the acetone soln that working electrode end portion surface-coated is used is the acetone soln that contains 2%～2.5% cellulose acetate.
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