CN108896634A

CN108896634A - A kind of oxygen-enriched jamproof glucose electrochemical detection method

Info

Publication number: CN108896634A
Application number: CN201811033174.2A
Authority: CN
Inventors: 封心建
Original assignee: Suzhou Yi Biological Sensor Technology Co Ltd
Current assignee: Suzhou Yi Biological Sensor Technology Co Ltd
Priority date: 2016-07-19
Filing date: 2016-12-13
Publication date: 2018-11-27
Also published as: CN107632050B; CN107632050A

Abstract

The present invention provides a kind of oxygen-enriched jamproof glucose electrochemical detection method, the method is detected using oxygen-enriched electrochemical detection device, and the device includes：Three-electrode system, the three-electrode system includes a reference electrode, a pair of electrodes and a working electrode, wherein the working electrode is the oxygen-enriched electrode of three-phase, and the first surface of the oxygen-enriched electrode of the three-phase is contacted with air or oxygen, second surface and electrolyte contacts；And the working electrode is cathode；And the oxygen-enriched electrode of three-phase is surface modification hydrogen-peroxide reduction catalyst, and is fixed with the carrier of the corresponding oxidizing ferment of test substance.The method strong antijamming capability, detection accuracy are good.

Description

A kind of oxygen-enriched jamproof glucose electrochemical detection method

Technical field

The present invention relates to oxygen-enriched jamproof glucose electrochemical detection methods, and specifically, the present invention is based on the first generation Glucose enzyme electrode uses oxygen as electron acceptor, passes through the redox for the hydrogen peroxide that detection enzymatic reaction generates Electric current to carry out glucose the improvement of the method for Electrochemical Detection.

Background technique

Diabetes are global public health problems, and global diabetic's quantity alreadys exceed 300,000,000.Therefore to blood glucose It is accurate detection have great importance in clinical treatment and daily monitoring.Since 1962, enzyme electrode is due to its side Just fast and effective feature has become the main method of detection blood glucose.Wherein first generation glucose enzyme electrode passes through detection enzymatic The concentration of hydrogen peroxide caused by reacting, and then monitor glucose content.The electron acceptor that this method uses is natural Oxygen, electron-transport speed obtain transmission speed faster than artificial electron's mediator used in second generation glucose enzyme electrode, And environmentally protective, without toxicity, cost is relatively low.But there is some disadvantages for this method：

Glucose sensor response signal first is affected by dissolved oxygen content, when the content amount deficiency of dissolved oxygen It is difficult to be measured the blood glucose of high concentration, and the fluctuation of oxygen concentration equally will affect response signal.

Secondly, the electrode of detection hydrogen peroxide generally requires the higher operating potential of selection, the method for anodic oxidation is used It is detected, however some endogenous reductase substances (ascorbic acid, uric acid etc.) and reproducibility drug (paracetamol, second Acyl salicylic acid etc.) etc. electroactive substances be equally easy to aoxidize in electrode surface, to current signal generate severe jamming, This will will affect the detection of concentration of glucose.

Have been reported that provide certain methods oxygen-deficient to solve the problems, such as at present.Such as increase by one layer of limitation in electrode surface Property diffusion barrier (polyurethane, polycarbonate) increases the detection of glucose sensor to improve oxygen/glucose diffusion ratio Range.But the sensitivity of detection can be reduced in this way, extend the response time.It also has been reported that and has used oxygen-enriched electrode, it is molten with oxygen The higher oxygen-rich material of Xie Du such as polymer mineral oil (Kel-F) provides additional oxygen for glucose oxidase.But it is this The upper limit of detection of electrode can only achieve about 10 mMs of glucose, still be unable to satisfy the demand to high concentration glucose test.Separately A kind of solution is using second generation glucose enzyme electrode, i.e., the small molecule compound manually synthesized replaces as mediator Oxygen transmits electronics in the activated centre of enzyme and electrode surface.But when in this way, there is higher electron-transport speed Oxygen can with artificial electron's mediator compete electronics, it is low that this will lead to accuracy in detection.And compared with natural oxygen, make Manually the cost of electron mediator is also higher.

It for the interference problem of reducing substances, also has been reported that and proposes solution, such as increase by one layer in enzyme electrode surface Selectivity through not diffusing to electrode surface to reduce interfering substance, but in this way when film thickness homogeneity not Easy to control, adhesion strength is weak, and the repeatability that these will lead to detection glucose is poor, and can reduce sensitivity.Also having been reported that makes With by modified electrode, (+0.2V~0VvsAg/AgCl) detects hydrogen peroxide under lower operating potential, can subtract in this way Oxidation reaction of few most of electroactive interfering substances in electrode surface.But be lowered in the oxidizing potential of hydrogen peroxide, Meanwhile the oxidizing potential of some other chaff interferent can also reduce, and can not avoid interfering so completely.If can pass through in negative potential Interference problem can be solved to detect glucose by restoring hydrogen peroxide.However, the method using cathodic reduction is difficult to detect Realize because oxygen can equally be reduced and generate stronger reduction current under reduction potential, with enzymatic reaction into It goes, the concentration of the dissolved oxygen in solution can change, and the fluctuation of the hydrogen reduction signal generated therewith can go back hydrogen peroxide Primary current generates severe jamming, causes output signal that can not form proportional relation with concentration of glucose.

Almost all of test substance can all generate hydrogen peroxide (H under the action of oxidizing ferment and oxygen₂O₂), therefore, pass The electrochemical sensor of system is by measuring H₂O₂Output determine the concentration of test substance.This method was since 1973 Since generation electrochemical sensor is by invention, it has been used always.However measurement H₂O₂Electrochemical method usually require it is one high Oxidizing potential (such as 0.3-0.6V is on Pt electrode), under this current potential, various substances in human body, such as ascorbic acid, uric acid are right Acetaminophen, lactic acid etc. can be also oxidized, and cause the selectivity of sensor.Relative to traditional electrochemical oxidation process, Negative potential measures H by the method for electrochemical reduction₂O₂It can be easy to avoid these by the interference of oxide material.But Based on existence conditions, which can not be implemented always on existing electrochemical sensor.

In conclusion there is an urgent need in the art to one kind can be avoided interference, the high examination of glucose concentration device of accuracy.

Summary of the invention

The present inventor allows oxygen quick by building solid-liquid-gas three-phase coexistence electrochemical sensor electrodes From gas phase diffusion to electrode surface, the content constant of the oxygen of electrode surface ensure that, and then realize in lower negative potential H is detected by the method (such as-0.1-- 0.3V) restored down₂O₂, influence of a variety of chaff interferents to measurement result is avoided, Substantially increase the selectivity of electrochemical sensor.Meanwhile the sensor also has wide linear detection range, high spirit simultaneously The advantages such as sensitivity and energy conservation.

The first aspect of the present invention provides a kind of oxygen-enriched electrochemical detection method, the method includes the steps：

(a) an oxygen-enriched electrochemical detection device is provided, the device includes：

Three-electrode system, the three-electrode system include a reference electrode, a pair of electrodes and a working electrode, wherein institute The working electrode stated is the oxygen-enriched electrode of three-phase, and the first surface of the oxygen-enriched electrode of the three-phase is contacted with air or oxygen, second Surface and electrolyte contacts；And the working electrode is cathode；

And the oxygen-enriched electrode of three-phase is surface modification hydrogen-peroxide reduction catalyst granules, and is fixed with test substance The substrate of corresponding oxidizing ferment；

(b) it contacts described device with the sample containing test substance, carries out Electrochemical Detection, obtain cathodic reduction electric current Signal；

(c) by the cathodic reduction current signal, the concentration of the sample containing test substance is obtained.

In another preferred example, the working electrode is the electrode for having solid-liquid-gas three phase interface.

In another preferred example, the cathodic reduction current signal is obtained at -1.0V~-0.05V；Preferably It is obtained at -0.6V~-0.1V.

In another preferred example, the step (c) further includes：It is dense with standard by the cathodic reduction current signal The standard curve that degree sample test obtains is compared, and obtains test substance concentration.

In another preferred example, the electrochemical detection device is biosensor.

In another preferred example, the Electrochemical Detection includes：Carry out cyclic voltammetry scanning, and test output signal Corresponding cathodic reduction current signal at current potential；Or potentiostatic scanning is carried out under negative voltage, and test cathodic reduction electric current letter Number.

In another preferred example, the hydrogen-peroxide reduction catalyst granules is selected from the group：Carbon, metal, metal salt, conjunction Gold, organic material reducing catalyst, or combinations thereof；Preferably, the metal is selected from the group：Platinum, rhodium, ruthenium, gold, cobalt, iron or nickel, But it is not limited to this；

The organic material reducing catalyst is selected from the group：Biomaterial and/or metal organic complex, it is preferable that institute State biomaterial be cromoci, hydrogen peroxide oxidation enzyme, Prussian blue, or combinations thereof.

In another preferred example, the catalysis material may include distribution metal and/or metal oxide nanoparticles, but It is without being limited thereto.

In another preferred example, the substrate is selected from the group：Metal material, carbon material, high-molecular porous material；It is preferred that Ground, the substrate are selected from the group：Carbon fiber paper, carbon nanotube, 3D graphene, foam copper.

In another preferred example, nickel foam, foam copper, titanium foam, foam can be selected but be not limited to the metal material Ferro-aluminum net, foam copper mesh, foam aluminium net, ferro-aluminum net, copper mesh or aluminium net.

In another preferred example, the carbon material may include graphene, carbon nanotube construction, carbon fiber, expansion stone Ink, photoetching graphite, porous carbon materials, or combinations thereof.

In another preferred example, the high-molecular porous material may include polyaniline film, polypyridine film or polypyrrole film, Or combinations thereof；As one of more preferred embodiment party, described substrate is porous, electrically conductive carbon material and carbon fibre material, or A combination thereof.

In another preferred example, the substrate is hydrophobized carbon fiber paper.

It is hydrophobic in another preferred example, the test substance is selected from the group：Glucose；And/or cholesterol, lactic acid, second Phatidylcholine, alcohols, or combinations thereof；And/or

The corresponding oxidizing ferment of the test substance is selected from the group：Glucose oxidase, alpha-phosphate glycerol oxidase, gallbladder are solid Alcohol esterase, cholesterin dehydrogenase, cholesterol oxidase, glucose oxidase, glucose dehydrogenase, lactic dehydrogenase, malic acid Dehydrogenase, bilirubin oxidase, ascorbic acid oxidase, peroxidase, uricase, clostridiopetidase A, matter acid enzyme, protease or egg White hydrolase, or combinations thereof.

In another preferred example, the oxygen-enriched electrochemical detection device is prepared by the following method：

(1) conductive base with hydrophobic structure is provided；

(2) the fixed catalysis material is loaded on the hydrophobic structure；

(3) apply the solution containing the corresponding oxidizing ferment of test substance, and drying at room temperature on the hydrophobic structure, thus The corresponding oxidizing ferment of the test substance is set to be attached to the hydrophobic structure surface.

In another preferred example, the conductive base with hydrophobic structure is prepared by the following method：To carbon fiber paper Hydrophobization modification is carried out, hydrophobized carbon fiber paper is obtained.

In another preferred example, the hydrophobization, which is modified, further includes：At above-mentioned hydrophobized carbon fiber paper plasma Reason obtains the hydrophobic carbon fiber paper of the hydrophilic another side of one side.

In another preferred example, in the step (1), the carbon fiber paper is 1.2cm ﹡ 1.2cm carbon fiber paper.

In another preferred example, the hydrophobization, which is modified, further includes：It will be formed after the dilution of polytetrafluoro lotion with water outstanding Turbid impregnates the carbon fiber paper.

In another preferred example, after the dilution, the concentration of the polytetrafluoro cream suspension is 0.5-5wt.%.

In another preferred example, carbon fiber paper is 0.5-5 hours in soaking time wherein.

In another preferred example, after the soaking step, the carbon fiber paper is dried at room temperature.

In another preferred example, it is described dry step after, the carbon fiber paper is put into Muffle furnace, (240 At DEG C) heating (60 minutes) after, by its natural cooling.

In another preferred example, the heating temperature in Muffle furnace is 200-550 DEG C.

In another preferred example, the heating time is 5-120 minutes.

In another preferred example, the power of the corona treatment is 100-300W.

In another preferred example, the air mass flow of the corona treatment is 0.1-0.8L/min.

In another preferred example, the nano-platinum particle, which is modified, includes：Use electrochemical deposition or physical vapour deposition (PVD) Method carries out the modification of nano-platinum particle to the carbon fiber paper.

In another preferred example, the electrochemical deposition method hydrophobization, which is modified, includes：

Above-mentioned carbon fiber paper is mounted on electrochemical cell side wall；

Configure certain density chloroplatinic acid containing nanocatalyst, the mixed solution of sulfuric acid pours into device as electrodeposit liquid Electrolytic cell in, be submerged the hydrophilic surface of above-mentioned carbon fiber paper；

Electro-deposition is carried out using three-electrode system：Using carbon fiber paper as working electrode, platinum filament is used as to electrode, Ag/ AgCl is as reference electrode, using current-vs-time method (I-t method) in certain potentials electro-deposition certain time；

Then electrode surface is washed with water to get the carbon fiber paper of top fiber surface finish nano platinum grain is arrived.

In another preferred example, when carrying out nano-platinum particle modification to carbon fiber paper using electrochemical deposition method, by carbon Fibrous paper is mounted on electrochemical cell side wall, to make inside of its hydrophilic surface towards electrochemical cell, makes hydrophobic surface towards electrochemistry The outside in pond.

In another preferred example, when carrying out nano-platinum particle modification to carbon fiber paper using electrochemical deposition method, electricity is heavy The concentration of chloroplatinic acid is 1-15mmol/L in hydrops.

In another preferred example, when carrying out nano-platinum particle modification to carbon fiber paper using electrochemical deposition method, electricity is heavy The concentration of sulfuric acid is 0.1-0.8mol/L in hydrops.

In another preferred example, when carrying out nano-platinum particle modification to carbon fiber paper using electrochemical deposition method, electricity is heavy Product is -0.3V using current potential.

In another preferred example, when carrying out nano-platinum particle modification to carbon fiber paper using electrochemical deposition method, electricity is heavy The product time is 20-500 seconds.

In another preferred example, the use physical gas-phase deposite method hydrophobization, which is modified, includes：Deposited by electron beam evaporation Mode modifies certain thickness nano-platinum particle in carbon fiber paper surface.

In another preferred example, it when carrying out nano-platinum particle modification to carbon fiber paper using physical gas-phase deposite method, repairs Adorn nano-platinum particle with a thickness of 5-20 nanometers.

In another preferred example, include in the step (3)：It is total using embedding fixation or covalent cross-linking and investment Method carries out the fixation of glucose oxidase to the carbon fiber paper of above-mentioned top fiber surface finish nano platinum grain.

In another preferred example, in the step (3), when the described embedding fixation, the method includes：

The carbon fiber paper of the top fiber surface finish nano platinum grain is fixed on electrochemistry according to certain method On the side mold of reaction unit, and exposed portion region (preferably diameter is 8 millimeters of border circular areas)；

Glucose oxidase aqueous solution, and naturally dry are added dropwise in above-mentioned zone；

When aqueous solution will dry, certain chitosan solution is added dropwise into above-mentioned zone；

It is put into and is dried in drier to get the hydrophobic oxygen-enriched electrode assembly of three-phase is arrived；

And

When the method shared using covalent cross-linking and investment, the method includes：

Certain mixed solution is added dropwise in above-mentioned zone, is put into drier and dries, so that it is hydrophobic oxygen-enriched to obtain three-phase Electrode assembly.

In another preferred example, when carrying out the fixation of glucose oxidase using embedding fixation, fixing means is to make to repair The side for being decorated with nano-platinum particle is upward.

In another preferred example, the concentration of the glucose oxidase aqueous solution of the dropwise addition is 2-30mg/mL.

In another preferred example, the volume that the glucose oxidase aqueous solution is added dropwise is 5-50 microlitres.

In another preferred example, the chitosan solution of dropwise addition is 5-50 μ L.

In another preferred example, the chitosan solution is the chitosan-acetic acid solution of 0.1-5mg/mL, wherein acetic acid Concentration be 0.5-2wt.%.

In another preferred example, the fixation of glucose oxidase is carried out using the method that covalent cross-linking and investment share When, fixing means is to keep the side for being modified with nano-platinum particle upward.

In another preferred example, the preparation method of the mixed solution of dropwise addition is to gather the shell of the 0.5-5mg/mL of 10-100 μ L Sweet and sour acid solution, the glucose oxidase solution of the 10mg/mL of 20-200 μ L, 1-10 μ L 1-10wt.% glutaraldehyde it is water-soluble Liquid and the blending of 10-100 μ L water are configured to uniform mixed liquor, and placement is added dropwise after 5-60 minutes again at room temperature.

In another preferred example, the volume that the mixed solution is added dropwise is 5-50 μ L.

In another preferred example, include in the step (4)：Under reduction potential, the glucose detection is carried out.

In another preferred example, which is characterized in that in the step (4), the glucose detection includes：To above-mentioned The hydrophobic oxygen-enriched electrode assembly of three-phase in inject electrolyte, and into solution be added various concentration glucose master sample is made It is tested, so that the relationship of record current signal and concentration of glucose, obtains standard curve.

In another preferred example, the glucose detection is carried out using three-electrode system.

In another preferred example, the glucose detection is tested using cyclic voltammetry or i-t method.

In another preferred example, the step (4) further includes：The glucose detection is carried out to sample to be tested, from And current signal is obtained, and be compared with the standard curve, obtain concentration of glucose.

In another preferred example, the electrolyte of the injection is the KCl/PBS buffer solution of pH=5-8.

In another preferred example, it when being tested using cyclic voltammetry, is stirred after glucose or chaff interferent is added with magnetic force It mixes device and stirs evenly and be scanned again.

In another preferred example, the scanning includes：The maximum potential of scanning range be 0.4V, potential minimum be- 0.3V, sweeping speed is 0.01-0.1V/s, and output signal takes corresponding cathodic reduction electric current at -0.1V current potential.

In another preferred example, when being tested using I-t method, use -0.1V as constant potential, sweep time 80 Second, take the 60th second current value as output signal.

The second aspect of the present invention, provides a kind of glucose electrochemical detection device, and the device includes：

And the oxygen-enriched electrode of three-phase is surface modification hydrogen-peroxide reduction catalyst granules, and is fixed with test substance The substrate of corresponding oxidizing ferment.

In another preferred example, the device further includes electrolyte.

In another preferred example, the electrolyte is KCl/PBS buffer solution.

In another preferred example, the oxygen-enriched electrode of the three-phase is the hydrophobic oxygen-enriched electrode of three-phase.

In another preferred example, the preparation method of the device includes step：By the carbon of surface finish nano platinum grain Fibrous paper is fixed on the side mold of electrochemical reaction appts, exposes the border circular areas that diameter is 8 millimeters；In above-mentioned zone Certain mixed solution is added dropwise, is put into drier and dries；

In another preferred example, the hydrophobic oxygen-enriched electrode of the three-phase is prepared via a method which：

(1) hydrophobization modification is carried out to carbon fiber paper, obtains hydrophobized carbon fiber paper；

(2) nano-platinum particle modification is carried out to the upper layer carbon fiber surface of the hydrophobized carbon fiber paper, obtains upper layer fibre Tie up the carbon fiber paper of surface modification nano-platinum particle；

(3) fixation of glucose oxidase is carried out to the carbon fiber paper of above-mentioned top fiber surface finish nano platinum grain, Obtain the hydrophobic oxygen-enriched electrode of three-phase.

In another preferred example, the hydrogen-peroxide reduction catalyst granules is selected from the group：Carbon, metal, metal salt, conjunction Gold, organic material reducing catalyst, or combinations thereof；Preferably, the metal is selected from the group：Platinum, rhodium, ruthenium, gold, cobalt, iron or nickel, Or combinations thereof；The organic material reducing catalyst is selected from the group：Biomaterial and/or metal organic complex；Preferably, institute State biomaterial be cromoci, hydrogen peroxide oxidation enzyme, Prussian blue, or combinations thereof.

In another preferred example, the substrate is carbon fiber paper.

In another preferred example, the substrate is hydrophobic carbon fiber paper.

In another preferred example, the substrate is the hydrophobic carbon fiber paper of the hydrophilic another side of one side.

In another preferred example, the corresponding oxidizing ferment of the test substance is selected from the group：Glucose oxidase, alpha-phosphate Glycerol oxidase, cholesterol esterase, cholesterin dehydrogenase, cholesterol oxidase, glucose oxidase, glucose dehydrogenase, cream Acidohydrogenase, malic dehydrogenase, bilirubin oxidase, ascorbic acid oxidase, peroxidase, uricase, clostridiopetidase A, matter Sour enzyme, protease, proteolytic enzyme, or combinations thereof.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Detailed description of the invention

Fig. 1 is obtained the schematic diagram of the hydrophobic oxygen-enriched electrode assembly of three-phase by the embodiment of the present invention 1；

The electric current-that Fig. 2 obtains the hydrophobic oxygen-enriched electrode cyclic voltammetry measurement glucose of three-phase by the embodiment of the present invention 1 is dense Spend working curve；

Fig. 3 is obtained electric current-concentration work of the hydrophobic oxygen-enriched electrode i-t method measurement glucose of three-phase by the embodiment of the present invention 2 Make curve；

The electric current-that Fig. 4 obtains the hydrophobic oxygen-enriched electrode cyclic voltammetry measurement glucose of three-phase by the embodiment of the present invention 3 is dense Spend working curve；

Fig. 5 is obtained electric current-concentration work of the hydrophobic oxygen-enriched electrode i-t method measurement glucose of three-phase by the embodiment of the present invention 4 Make curve；

Fig. 6 is obtained to be added when the hydrophobic oxygen-enriched electrode cyclic voltammetry of three-phase detects glucose by the embodiment of the present invention 1 and be done Disturb the influence of object；

Fig. 7 is obtained when the hydrophobic oxygen-enriched electrode i-t method of three-phase detects glucose by the embodiment of the present invention 2 and chaff interferent is added It influences；

Fig. 8 is electric current-concentration working curve of enzyme platinum carbon electrode cyclic voltammetry measurement glucose as a comparison；

The influence of chaff interferent is added in Fig. 9 when being enzyme platinum carbon electrode i-t method detection glucose as a comparison.

Obviously, it can see using the hydrophobic oxygen-enriched electrode of three-phase according to Fig. 1-Fig. 9 in the method for cathodic reduction hydrogen peroxide The detection of glucose is carried out, current value increases with the increase of the concentration of glucose, and the range of linearity upper limit can reach 140 mmoles You, when using cyclic voltammetry and i-t method, the chaff interferent ascorbic acid, the paracetamol that are easily electrochemically oxidized in human body It is negligible with interference of the uric acid to the signal of glucose, it has broad application prospects, has in field of medical device There is good Social benefit and economic benefit.

Figure 10 is hydrogen peroxide both electrochemical oxidation and electrochemical reduction detection method comparison diagram, but electrochemical oxidation side Method (Figure 10 a) will receive its interference for being oxidized easily substance.And if detected under reduction potential, it not will receive these objects The interference (such as Figure 10 b) of matter.

Figure 11 is that the result of glucose is detected by the principle of reduction hydrogen peroxide and oxidation hydrogen peroxide in serum Comparison restores hydrogen peroxide method not by the interference of other substances (such as Figure 11 a), and detects Portugal by traditional method for oxidation Grape sugar, then interfere very serious (such as Figure 11 b).

Specific embodiment

A kind of the present inventor in-depth study by long-term, it has unexpectedly been found that oxygen-enriched jamproof glucose electrochemistry The glucose detection upper limit is greatly improved in detection method, has obtained in reduction potential proportional with concentration of glucose Current signal realizes and carries out glucose detection in the method for cathodic reduction hydrogen peroxide, so as to avoid easily electric in human body It interferes caused by the substance of chemical oxidation, has broad application prospects in field of medical device, there is society's effect well Benefit and economic benefit.

Oxygen-enriched electrochemical detection method

For achieving the above object, present invention employs a kind of oxygen-enriched jamproof electrochemical detection method, the sides Method includes：

(1) carbon fiber paper (1.2cm ﹡ 1.2cm) is impregnated with the mixed solvent of organic solvent and water, one timing of ultrasonic cleaning Between, it is then placed in baking oven and is dried for standby using certain temperature；

(2) polytetrafluoro lotion is diluted to certain density suspension with water, the above-mentioned carbon fiber paper handled well is put into It impregnates certain time in above-mentioned suspension, then dries at room temperature, be finally putting into Muffle furnace using certain temperature heating one After fixing time natural cooling to get arrive hydrophobized carbon fiber paper；

(3) by above-mentioned hydrophobized carbon fiber paper corona treatment to get the carbon fiber hydrophobic to the hydrophilic another side of one side Paper；

(4) nano-platinum particle is carried out to above-mentioned carbon fiber paper using electrochemical deposition or physical gas-phase deposite method to repair Decorations：

When using electrochemical deposition method, above-mentioned carbon fiber paper is mounted on electrochemical cell side wall according to certain method；Match Set certain density chloroplatinic acid, the mixed solution of sulfuric acid pours into device in electrolytic cell as electrodeposit liquid, make above-mentioned carbon fiber The hydrophilic surface of paper is submerged；Electro-deposition is carried out using three-electrode system.Using carbon fiber paper as working electrode, platinum filament is used as to electricity Then pole, Ag/AgCl are used as reference electrode using current-vs-time method (I-t method) in certain potentials electro-deposition certain time Water cleaning electrode surface is to get the carbon fiber paper for arriving top fiber surface finish nano platinum grain；

When using physical gas-phase deposite method, deposited by electron beam evaporation mode modifies certain thickness receive in carbon fiber paper surface Miboplatin particle；

(5) above-mentioned top fiber surface modification is received using the method that embedding fixation or covalent cross-linking are shared with investment The carbon fiber paper of Miboplatin particle carries out the fixation of glucose oxidase：

When using embedding fixation, by the carbon fiber paper of above-mentioned top fiber surface finish nano platinum grain according to certain side Method is fixed on the side mold of electrochemical reaction appts, is exposed the border circular areas that diameter is 8 millimeters, is added dropwise in above-mentioned zone Certain glucose oxidase aqueous solution, when aqueous solution will dry, then that certain chitosan is added dropwise into above-mentioned zone is molten Liquid is finally putting into drier and dries to get the hydrophobic oxygen-enriched electrode assembly of three-phase is arrived；

When the method shared using covalent cross-linking and investment, by the carbon of above-mentioned top fiber surface finish nano platinum grain Fibrous paper is fixed on the side mold of electrochemical reaction appts according to certain method, exposes the border circular areas that diameter is 8 millimeters, Certain mixed solution is added dropwise in above-mentioned zone, is put into drier and dries to get the hydrophobic oxygen-enriched electrode assembly of three-phase is arrived；

(6) certain electrolyte is injected into the above-mentioned hydrophobic oxygen-enriched electrode assembly of three-phase, various concentration is added into solution Glucose tested using three-electrode system using cyclic voltammetry or i-t method, record different glucose and plus Enter corresponding current signal when disturbance object.

The organic solvent be ethyl alcohol, acetone, ethyl alcohol, acetone, water ratio be 1:1:1；The ultrasonic cleaning time is 1-30 minutes；Drying temperature is 60-150 DEG C.

The concentration of polytetrafluoro cream turbid liquid concentration is 0.5-5wt% after the dilution；The carbon fiber paper is in leaching wherein Steeping the time is 0.5-5 hours；The heating temperature in Muffle furnace is 200-550 DEG C, and heating time is 5-120 minutes.

When the progress corona treatment to hydrophobized carbon fiber paper, power 100-300W, air mass flow 0.1- 0.8L/min。

The method that carbon fiber paper is mounted on electrochemical cell side wall is to make inside of its hydrophilic surface towards electrochemical cell, Make outside of the hydrophobic surface towards electrochemical cell.

The concentration of chloroplatinic acid is 1-15mM in the electrodeposit liquid, and the concentration of sulfuric acid is 0.1-0.8M；Electro-deposition uses electricity Position is -0.3V, and electrodeposition time is 20-500 seconds.

When the use physical gas-phase deposite method carries out nano-platinum particle modification to carbon fiber paper, nano-platinum particle is modified With a thickness of 5-20 nanometers.

When the fixation for using embedding fixation progress glucose oxidase, fixing means is to make to be modified with Platinum Nanoparticles The side of grain is upward；The concentration of the glucose oxidase aqueous solution of dropwise addition is 2-30mg/mL, and the volume of dropwise addition is 5-50 microlitres； The chitosan solution of dropwise addition is 5-50 microlitres, the chitosan-acetic acid solution of 0.1-5mg/mL, and wherein the concentration of acetic acid is 0.5- 2wt%.

When the method shared using covalent cross-linking and investment carries out the fixation of glucose oxidase, fixing means is Keep the side for being modified with nano-platinum particle upward；The preparation method of the mixed solution of dropwise addition is by 10-100 microlitres of 0.5-5mg/ The chitosan-acetic acid solution of mL, the glucose oxidase solution of the 10mg/mL of 20-200 μ L, 1-10 microlitres of 1-10wt% penta 2 Aldehyde aqueous solution and 10-100 microlitres of water blending are configured to uniform mixed liquor, and placement is added dropwise after 5-60 minutes again at room temperature；Drop The volume added is 5-50 microlitres.

The electrolyte of the injection is the KCl/PBS buffer solution of pH=5-8.

When the cyclic voltammetry is tested, be added after glucose or chaff interferent with magnetic stirrer uniformly again into Row scanning；The maximum potential of scanning range is 0.4V, and potential minimum is -0.3V, and sweeping speed is 0.01-0.1V/s, output signal takes - Corresponding cathodic reduction electric current at 0.1V current potential.

When the I-t method is tested, use -0.1V as constant potential, sweep time is 80 seconds, takes the 60th second electricity Flow valuve is as output signal.

Compared with the conventional method, advantages of the present invention includes：Glucose detection range has been increased considerably, glucose is detected Upper limit of detection can reach 140 mMs, realize with the oxidase catalyzed substrate of the method for cathodic reduction hydrogen peroxide progress Accurate detection, avoid interference caused by the substance being easily electrochemically oxidized in human body.

Term

As used herein, term " biological enzyme sensor that hydrophobic solid-liquid-gas three phase coexists ", " inventive sensor ", " this The biological enzyme sensor of invention " is used interchangeably, and is referred to hydrophobic property, and there are solid-liquid-gas three in working sensor The biological enzyme sensor of the state (or phenomenon) mutually coexisted.

As used herein, term " hydrophobic ", " hydrophobic performance " are used interchangeably, and refer to object (such as biological enzyme sensor) or material Expect that (such as substrate) has excellent hydrophobic performance.Hydrophobic performance can be measured and be characterized with conventional method.In general, when normal Temperature, under normal pressure test condition, when contact angle >=120 degree (preferably >=150 °), it is believed that the substance has excellent hydrophobicity.

As used herein, room temperature refers to environment temperature, usually 0-50 DEG C, preferably 4-30 DEG C.

Biological enzyme sensor

One aspect of the present invention is intended to provide a kind of biological enzyme sensor that hydrophobic solid-liquid-gas three phase coexists, by Hydrophobic material surface forms the structure of solid gas liquid three-phase coexistence, so as to provide sufficient oxygen to supply enzymatic reaction, after And the working performance (such as detection range and detection limit) of biological enzyme sensor can be effectively improved.

As shown in fig.1, the biological enzyme sensor that the hydrophobic solid-liquid-gas three phase in the present invention coexists is in use, can be used as Working electrode, and combine reference electrode required for electrochemistry, or in combination with a reference electrode and one to electrode, And it is equipped with corresponding conducting medium, it can carry out analysis detection, such as the calibration or test of glucose.

The biological enzyme sensor that a kind of hydrophobic solid-liquid-gas three phase of the invention coexists may include：

Conductive base with hydrophobic structure,

And it can be reacted with test substance and generate the biological enzyme of hydrogen peroxide and urging with catalyzing hydrogen peroxide function Change material.

Further, as one of preferred embodiment, the sensor can also include：It is at least described to protect Biological enzyme, and the biological enzyme is fixed on to the protective film on the hydrophobic structure surface.

More specifically, the protective film is covered on the hydrophobic structure (hydrophobic electrode), be at least able to use with The solvent molecule and the test substance for forming the selected liquid-phase system penetrate, but can stop the biological enzyme.

Substrate with hydrophobic performance,

It distribution catalysis material with catalyzing hydrogen peroxide function on the substrate and can be reacted with test substance Generate the biological enzyme of hydrogen peroxide.

The biological enzyme sensor that the hydrophobic solid-liquid-gas three phase of another kind of the invention coexists may include：

Conductive base with hydrophobic performance, wherein the substrate includes inherently with catalyzing hydrogen peroxide function Catalysis material, and, the biological enzyme for generating hydrogen peroxide can be reacted with test substance.

The biological enzyme sensor that the hydrophobic solid-liquid-gas three phase of another of the invention coexists may include：

Conductive base with hydrophobic structure, and, can be reacted with test substance generate hydrogen peroxide biological enzyme and Catalysis material with catalyzing hydrogen peroxide function；Also, when the hydrophobic structure surface is applied selected liquid-phase system, until Less in the liquid-phase system for covering the hydrophobic structure and the oxygenous gas phase system being filled in the hydrophobic structure The biological enzyme and the catalysis material are distributed on interface, constitutes the form that solid-liquid-gas three phase coexists.

Conductive base with hydrophobic structure, wherein the material to form the hydrophobic structure includes to have to be catalyzed The catalysis material of hydrogen oxide function,

And the biological enzyme for generating hydrogen peroxide can be reacted with test substance；

Also, when the hydrophobic structure surface is applied selected liquid-phase system, at least the hydrophobic structure is being covered It the liquid-phase system and is filled on the interface of the oxygenous gas phase system in the hydrophobic structure biological enzyme is distributed with With the catalysis material, the form that solid-liquid-gas three phase coexists is constituted.

Substrate can select the rear surface by hydrophobic treatment to have hydrophobic performance the conductive base in the present invention Conductive material, also can be selected itself i.e. with hydrophobic surface conductive material, these materials can by it is commercially available or self-control etc. it is more Kind approach obtains.

For example, for needing by, just with the conductive material of hydrophobic performance, can use with low after hydrophobic treatment The substance of surface energy is modified it and is obtained, and such low-surface energy substance includes (but being not limited to)：Fluorocarbons, fluorine Silicon compound, silicone couplet or long chain alkyl compound etc. also can be the particle etc. with low-surface-energy, such as polytetrafluoroethylene (PTFE) Micro mist, perfluoroethylene-propylene (copolymer) micro mist etc., but not limited to this.And the method modified accordingly can refer to CN102815052A, CN The documents such as 102011153A.

Aforesaid conductive substrate can select conductive hydrophobic material, including (but being not limited to)：Metal material, carbon material or height Molecular porous material etc..For example, the metal material includes (but being not limited to) nickel foam, foam copper, titanium foam, foam ferro-aluminum Net, foam copper mesh, foam aluminium net, ferro-aluminum net, copper mesh or aluminium net, and carbon material includes (but being not limited to) graphene, carbon nanometer Pipe construction, carbon fiber, expanded graphite, photoetching graphite, porous carbon materials etc., high-molecular porous material include (but and unlimited In) polyaniline film, polypyridine film or polypyrrole film etc..

Hydrogen-peroxide reduction catalyst hydrogen-peroxide reduction catalyst above-mentioned is to refer to catalyzing hydrogen peroxide to be gone back The inorganic, biological of former electrochemical reaction, metal and metal oxide materials, for example, carbon nanotube, graphene can be selected in it, carefully Cytochrome C, hydrogen peroxide oxidation enzyme, Prussian blue, platinum (Pt), rhodium (Rh), ruthenium (Ru), golden (Au), cobalt (Co) oxide, iron (Fe) oxide, nickel (Ni) oxide etc., and its form is also unrestricted.

In the present invention, the catalysis material may include inorganic material and/or organic material；

Wherein, the inorganic material may include carbon and/or metal and/or the compound containing metallic element；

The organic material includes (but being not limited to) biomaterial and/or metal organic complex, for example, the biology Material can be selected but be not limited to cromoci, hydrogen peroxide oxidation enzyme or Prussian blue etc..

The metal includes (but being not limited to) platinum, rhodium, ruthenium, gold, cobalt, iron or nickel.

As more one of preferred embodiment, the catalysis material include (but being not limited to) distribution metal and/or Metal oxide nanoparticles.

Oxidizing ferment

As used herein, oxidizing ferment can be selected and any can aoxidize tested substance (for example, glucose) and generate The organized enzyme of hydrogen oxide, such as comprising (but being not limited to)：Glycerokinase, alpha-phosphate glycerol oxidase, cholesterol esterase, Cholesterin dehydrogenase, cholesterol oxidase, glucose oxidase, glucose dehydrogenase, lactic dehydrogenase, malic dehydrogenase, Bilirubin oxidase, ascorbic acid oxidase, peroxidase, uricase, clostridiopetidase A, matter acid enzyme, protease, proteolysis Enzyme, or combinations thereof.

Film forming matter is as used herein, and film forming matter, which refers to, can form a film on the surface of the material and play protection and fixed enzyme molecule Compound, include but are not limited to chitosan, cell nafion proton membrane (Nafion) also can be for can be to be configured to Biomaterial is prevented to pass through, the material of commonly seeing such as semi-permeable membrane or permeable membrane for being penetrated without limitation small molecule or ion, for example (,) it is common The material for preparing have cuprammonium process regenerated cellulose, cellulose acetate, polyacrylonitrile, ethylene-vinyl alcohol copolymer and poly- methyl Methyl acrylate, polysulfones, polyacrylamide etc..

As soon as in addition, in embodiment of the invention, for itself having the conductive base of Above-mentioned catalytic performance, especially It is the conductive base that its hydrophobic structure includes the catalysis material with catalyzing hydrogen peroxide function, then can also saves aforementioned hydrophobic The operation of catalysis material is loaded in structure.

The preparation method of biological enzyme sensor

Another aspect provides a kind of preparation method of biological enzyme sensor that hydrophobic solid-liquid-gas three phase coexists, It includes：The conductive base with hydrophobic structure is provided, and fixation is loaded with catalyzing hydrogen peroxide on the hydrophobic structure The catalysis material of function and the biological enzyme that generation hydrogen peroxide can be reacted with test substance.

" selected liquid-phase system " above-mentioned refers to water or at least contains the aqueous solution etc. of the test substance, also can be blood Liquid or other physiological fluids etc..

" test substance " above-mentioned is to refer to by the biological oxydasis and generate the substance of hydrogen peroxide, including Portugal Grape sugar etc., but not limited to this.

" oxygenous gas phase system " above-mentioned, including air or by oxygen with other auxiliary gases (for example, nitrogen etc. is non- The inert gases such as active gases and argon gas) formed gas.

In one embodiment, which can specifically include following steps：

(1) conductive base with hydrophobic structure is provided；

(2) the fixed catalysis material is loaded on the hydrophobic structure；

(3) apply the solution containing biological enzyme, and drying at room temperature on the hydrophobic structure, to keep the biological enzyme attached In the hydrophobic structure surface.

As more one of preferred embodiment, which can also include：

Apply film forming matter, and drying at room temperature on the hydrophobic structure for having been charged with the biological enzyme, to dredge described It is formed and is at least able to use to form the solvent molecule of the selected liquid-phase system and the test substance and penetrate on water-bound, but It can stop the protective film of the biological enzyme.

In one embodiment, the preparation method of sensor as aforementioned may include steps of, respectively：Hydrophobic material The preparation of material；Surface is handled using metal or metal oxide materials；Treated, and biological enzyme solutions are added in surface；It uses Filmogen film forming is protected.

A kind of preferred preparation process is as follows：

(1) preparation of hydrophobic material

There is the conductive base of hydrophobic function not need further hydrophobic treatment itself.And for hydrophilic material itself Material can be modified with the substance with low-surface-energy, impregnate 3-24h for example, can be put into the ethanol solution of silicon fluoride, then Take out use ethyl alcohol clean the surface residuals, finally the sample of processing heat in 100 DEG C of baking ovens polymerize 2h obtain it is hydrophobic Electrode material (also that is, aforesaid conductive substrate or abbreviation hydrophobic material).

(2) it is modified using metal or metal oxide materials

By above-mentioned steps 1) obtained in hydrophobic material be fixed in electrode cell, be added in electrode cell and need to be loaded into In the relevant solution of catalysis material.By taking Pt as an example, to deposit Pt metal on hydrophobic material surface, chloroplatinic acid is added in electrode cell Solution (10g/L H₂PtCl₆, concentration ratio is H₂PtCl₆:1M H₂SO₄:H₂O=13:25:12).Use processed hydrophobic material Material is used as working electrode, and platinum electrode is added and is used as to electrode, and Ag/AgCl electrode is as reference electrode, using instant current method, The electro-deposition 150-1500s under the potential of 0~-0.5V.

(3) loading of biological enzyme

Continue the above-mentioned metal prepared and/or the processed hydrophobic material of metal oxide materials to be fixed on electrode cell In, and the solution containing biological enzyme is added dropwise on hydrophobic material surface, such as the aqueous solution of glucose oxidase (wherein contains enzyme amount Can be 0.1-20U, U is enzyme activity unit), then, hydrophobic material is placed in natural drying at room temperature, the hydrophobic material after drying Material is loaded with biological enzyme, and biological enzyme possesses reactivity.

(4) film forming protection

It is protected on the above-mentioned hydrophobic material surface for being loaded with biological enzyme using protective film, hydrophobic material table is loaded in protection The biological enzyme molecule in face is unlikely to dissolve or fall off.Its operating method can be：Hydrophobic material in above-mentioned steps after drying Surface applies a certain amount of film forming matter, continues thereafter with hydrophobic material being placed in natural drying at room temperature, the hydrophobic material after drying Material surface has one layer of film forming matter to be formed by film, so that biological enzyme molecule will not be molten after so that hydrophobic material is applied aqueous solution Solution is in the solution.

By taking chitosan is film forming matter as an example, on the above-mentioned hydrophobic material surface for being loaded with biological enzyme, 10 μ L chitosans are added dropwise Solution, wherein chitosan content can be 1mg-250mg, then, hydrophobic material is placed in natural drying at room temperature, after dry Hydrophobic material surface can obviously observe the presence of chitosan film.

In addition, above-mentioned steps (3) and (4) can also concentrate on step completion, i.e., biological enzyme and Protective coatings are blended It forms a film together afterwards.

It in the present invention, can be processed hydrophobic by metal and/or metal oxide materials that above-mentioned steps (2) prepare Material, is fixed on electrode cell, and contains the mixed of biological enzyme and film forming protection materials in hydrophobic material surface addition (as being added dropwise) Close solution, such as glucose oxidase and the solution containing film forming matter (such as chitosan).In general, the water containing enzyme can be used in the solution Solution is mixed with the solution containing film forming matter, wherein the mixed proportion of the two is not particularly limited, as long as protection can be formed Film, usually 1:50 to 50:1, preferably 1:10 to 10:1, it is more preferably 2:1-1:2.In the solution, enzyme Content and the content of film forming matter can be determined according to situations such as the type of substance, as long as can be formed on surface has centainly The protective film of enzyme content.Typically for 1-4cm²Surface for, in the protective film containing enzyme amount be usually 0.1- 20U (U is enzyme activity unit), and the content of film forming matter (such as chitosan) is usually 1mg-250mg.

Then, hydrophobic material is dried (as spontaneously dried at room temperature).

In the present invention, the hydrophobic material surface biological enzyme formed after drying has reactivity.In addition, hydrophobic material quilt After being put into aqueous solution, biological enzyme molecule can formed film quality guarantee shield because without being dissolved in solution.

Main advantages of the present invention include：

The present invention provides a kind of oxygen-enriched jamproof glucose electrochemical detection methods, and using realization has in high detection The glucose Electrochemical Detection of limit, and interference caused by the substance being easily electrochemically oxidized in human body is avoided, it is set in medical treatment There is huge commercial value in standby field.

Embodiment 1

1) by carbon fiber paper (1.2cm ﹡ 1.2cm) with ethyl alcohol, acetone, water (ratio 1:1:1) mixed solvent impregnates, and surpasses Sound cleans 10 minutes, is then placed in baking oven and is dried for standby using 60 DEG C；

2) polytetrafluoro lotion is diluted to the suspension of 0.5wt% with water, the above-mentioned carbon fiber paper handled well is put into It states in suspension and impregnates 1 hour, then dry at room temperature, be finally putting into Muffle furnace after being heated 30 minutes using 200 DEG C certainly It so cools down to get hydrophobized carbon fiber paper is arrived；

3) by above-mentioned hydrophobized carbon fiber paper corona treatment, using power 100W, air mass flow 0.3L/min to get The carbon fiber paper hydrophobic to the hydrophilic another side of one side；

4) modification of nano-platinum particle is carried out to above-mentioned carbon fiber paper using physical gas-phase deposite method：Deposited by electron beam evaporation Mode modifies the nano-platinum particle with a thickness of 5 nanometers in carbon fiber paper surface；

5) using the shared method of covalent cross-linking and investment to the carbon of above-mentioned top fiber surface finish nano platinum grain The fixation of fibrous paper progress glucose oxidase：By the chitosan-acetic acid solution of 10 microlitres of 4mg/mL, the 10mg/mL of 20 μ L Glucose oxidase solution, the glutaraldehyde water solution of 2 microlitres of 10wt% and 100 microlitres of water blendings are configured to uniform mixed liquor, It places stand-by after twenty minutes at room temperature；The carbon fiber paper of above-mentioned top fiber surface finish nano platinum grain is fixed on electrification On the side mold for learning reaction unit, keep the side for being modified with nano-platinum particle upward, exposes the circle that diameter is 8 millimeters 10 microlitres of above-mentioned mixed solutions are added dropwise in domain in above-mentioned zone, are put into drier and dry to get the hydrophobic oxygen-enriched electrode of three-phase is arrived Device；Device is as shown in Fig. 1.

6) the KCl/PBS buffer solution of pH=7.2 is injected into the above-mentioned hydrophobic oxygen-enriched electrode assembly of three-phase；Delay to above-mentioned Rush in solution and the glucose of various concentration be added, with magnetic stirrer it is uniform after circulation volt is carried out using three-electrode system again Peace method scanning, embodiment result is as shown in 2 left side of attached drawing, wherein the maximum potential of scanning range be 0.4V, potential minimum be- 0.3V, sweeping speed is 0.05V/s, and output signal takes corresponding cathodic reduction electric current at -0.1V current potential, records different glucose Corresponding current signal obtains concentration-current curve, as shown in 2 right side of attached drawing.

7) above-mentioned cyclic voltammetric detection method is used, in scanning buffer solution and after 5mM glucose is added, is continued to molten 0.1mM ascorbic acid, 0.1mM paracetamol and 0.1mM uric acid are added in liquid, after each chaff interferent is added in record respectively Obtained cyclic voltammetric signal, embodiment result are as shown in Fig. 6.From fig. 6 it can be seen that when there are chaff interferents in system When, the cyclic voltammetric signal of detection does not change significantly, and it is common dry to illustrate that test method of the invention can be effectively prevented Disturb the interference of object.

Embodiment 2

1) by carbon fiber paper (1.2cm ﹡ 1.2cm) with ethyl alcohol, acetone, water (ratio 1:1:1) mixed solvent impregnates, and surpasses Sound cleans 30 minutes, is then placed in baking oven and is dried for standby using 140 DEG C；

2) polytetrafluoro lotion is diluted to the suspension of 3wt% with water, the above-mentioned carbon fiber paper handled well is put into above-mentioned It impregnates 2.5 hours in suspension, then dries at room temperature, be finally putting into Muffle furnace after being heated 90 minutes using 300 DEG C certainly It so cools down to get hydrophobized carbon fiber paper is arrived；

3) by above-mentioned hydrophobized carbon fiber paper corona treatment, using power 200W, air mass flow 0.8L/min to get The carbon fiber paper hydrophobic to the hydrophilic another side of one side；

4) modification of nano-platinum particle is carried out to above-mentioned carbon fiber paper using electrochemical deposition method：Above-mentioned carbon fiber paper is pacified Mounted in electrochemical cell side wall, make inside of its hydrophilic surface towards electrochemical cell, makes outside of the hydrophobic surface towards electrochemical cell；Configuration Chloroplatinic acid, sulfuric acid mixed solution (wherein the concentration of chloroplatinic acid is 3mM, and the concentration of sulfuric acid is 0.6M) poured into as electrodeposit liquid In device in electrolytic cell, it is submerged the hydrophilic surface of above-mentioned carbon fiber paper；Electro-deposition is carried out using three-electrode system.With carbon fiber As working electrode, platinum filament is used as to electrode paper, Ag/AgCl as reference electrode, using current-vs-time method (I-t method)- 0.3V electro-deposition 420 seconds, wash with water electrode surface then to get the carbon fiber of top fiber surface finish nano platinum grain is arrived Paper；

5) glucose is carried out using carbon fiber paper of the embedding fixation to above-mentioned top fiber surface finish nano platinum grain The fixation of oxidizing ferment：The carbon fiber paper of above-mentioned top fiber surface finish nano platinum grain is fixed on electrochemical reaction appts On the mold of side, keep the side for being modified with nano-platinum particle upward, exposes the border circular areas that diameter is 8 millimeters, in above-mentioned zone Middle to be added dropwise 45 microlitres, the glucose oxidase aqueous solution of 5mg/mL is added dropwise when aqueous solution will dry, then into above-mentioned zone 20 microlitres, the chitosan-acetic acid solution (wherein the concentration of acetic acid is 0.8wt%) of 2mg/mL is finally putting into drier and dries, Obtain the hydrophobic oxygen-enriched electrode assembly of three-phase；

6) the KCl/PBS buffer solution of pH=6.2 is injected into the above-mentioned hydrophobic oxygen-enriched electrode assembly of three-phase；Delay to above-mentioned Rush in solution be added various concentration glucose, with magnetic stirrer it is uniform after use again three-electrode system with -0.1V make I-t scanning is carried out for constant potential, embodiment result is as shown in Fig. 3, wherein sweep time is 80 seconds, is taken the 60th second Current value obtains concentration-current curve as output signal.

7) the KCl/PBS buffer solution that pH=6.2 is injected into the above-mentioned hydrophobic oxygen-enriched electrode assembly of three-phase, persistently uses magnetic The stirring of power blender；5mM glucose, 0.1mM ascorbic acid, 0.1mM acetparaminosalol are sequentially added into above-mentioned buffer solution Phenol, 0.1mM uric acid and 5mM glucose use three-electrode system to carry out i-t using -0.1V as constant potential in the process and sweep It retouches, embodiment result is as shown in Fig. 7, wherein the time interval that glucose and chaff interferent is added is 80 seconds.By what is be added The current signal increase that the current signal that three kinds of interfering substances obtain increases with glucose compares, embodiment result such as attached drawing 11 Shown in a left side.It can be seen from fig. 11 that when, there are when chaff interferent, the cyclic voltammetric signal of detection does not change significantly in system Become, illustrates that test method of the invention can be effectively prevented the interference of common interference object.

Embodiment 3

1) by carbon fiber paper (1.2cm ﹡ 1.2cm) with ethyl alcohol, acetone, water (ratio 1:1:1) mixed solvent impregnates, and surpasses Sound cleans 5 minutes, is then placed in baking oven and is dried for standby using 90 DEG C；

2) polytetrafluoro lotion is diluted to the suspension of 5wt% with water, the above-mentioned carbon fiber paper handled well is put into above-mentioned It impregnates 0.5 hour in suspension, then dries at room temperature, be finally putting into Muffle furnace and heated after twenty minutes certainly using 400 DEG C It so cools down to get hydrophobized carbon fiber paper is arrived；

3) by above-mentioned hydrophobized carbon fiber paper corona treatment, using power 300W, air mass flow 0.1L/min to get The carbon fiber paper hydrophobic to the hydrophilic another side of one side；

4) modification of nano-platinum particle is carried out to above-mentioned carbon fiber paper using electrochemical deposition method：Above-mentioned carbon fiber paper is pacified Mounted in electrochemical cell side wall, make inside of its hydrophilic surface towards electrochemical cell, makes outside of the hydrophobic surface towards electrochemical cell；Configuration Chloroplatinic acid, sulfuric acid mixed solution (wherein the concentration of chloroplatinic acid is 12mM, and the concentration of sulfuric acid is 0.2M) fallen as electrodeposit liquid Enter in device in electrolytic cell, is submerged the hydrophilic surface of above-mentioned carbon fiber paper；Electro-deposition is carried out using three-electrode system.With carbon fiber Tie up paper and be used as working electrode, platinum filament as to electrode, Ag/AgCl as reference electrode, using current-vs-time method (I-t method)- 0.3V electro-deposition 30 seconds, wash with water electrode surface then to get the carbon fiber of top fiber surface finish nano platinum grain is arrived Paper；

5) using the shared method of covalent cross-linking and investment to the carbon of above-mentioned top fiber surface finish nano platinum grain The fixation of fibrous paper progress glucose oxidase：By the chitosan-acetic acid solution of 100 microlitres of 0.5mg/mL, the 10mg/ of 200 μ L The glucose oxidase solution of mL, the glutaraldehyde water solution of 8 microlitres of 2wt% and 30 microlitres of water blendings are configured to uniformly mix Liquid is placed stand-by after sixty minutes at room temperature；The carbon fiber paper of above-mentioned top fiber surface finish nano platinum grain is fixed on On the side mold of electrochemical reaction appts, keep the side for being modified with nano-platinum particle upward, exposes the circle that diameter is 8 millimeters 40 microlitres of above-mentioned mixed solutions are added dropwise in region in above-mentioned zone, are put into drier and dry to get the hydrophobic oxygen-enriched electricity of three-phase is arrived Pole device；

6) the KCl/PBS buffer solution of pH=7.8 is injected into the above-mentioned hydrophobic oxygen-enriched electrode assembly of three-phase；Delay to above-mentioned Rush in solution and the glucose of various concentration be added, with magnetic stirrer it is uniform after circulation volt is carried out using three-electrode system again The scanning of peace method, embodiment result is as shown in Fig. 4, wherein and the maximum potential of scanning range is 0.4V, and potential minimum is- 0.3V, sweeping speed is 0.08V/s, and output signal takes corresponding cathodic reduction electric current at -0.1V current potential, records different glucose Corresponding current signal obtains concentration-current curve.

Embodiment 4

1) by carbon fiber paper (1.2cm ﹡ 1.2cm) with ethyl alcohol, acetone, water (ratio 1:1:1) mixed solvent impregnates, and surpasses Sound cleans 20 minutes, is then placed in baking oven and is dried for standby using 150 DEG C；

2) polytetrafluoro lotion is diluted to the suspension of 2.5wt% with water, the above-mentioned carbon fiber paper handled well is put into It states in suspension and impregnates 5 hours, then dry at room temperature, be finally putting into Muffle furnace after being heated 120 minutes using 100 DEG C certainly It so cools down to get hydrophobized carbon fiber paper is arrived；

3) by above-mentioned hydrophobized carbon fiber paper corona treatment, using power 150W, air mass flow 0.5L/min to get The carbon fiber paper hydrophobic to the hydrophilic another side of one side；

4) modification of nano-platinum particle is carried out to above-mentioned carbon fiber paper using physical gas-phase deposite method：Deposited by electron beam evaporation Mode modifies the nano-platinum particle with a thickness of 20 nanometers in carbon fiber paper surface；

5) glucose is carried out using carbon fiber paper of the embedding fixation to above-mentioned top fiber surface finish nano platinum grain The fixation of oxidizing ferment：The carbon fiber paper of above-mentioned top fiber surface finish nano platinum grain is fixed on electrochemical reaction appts On the mold of side, keep the side for being modified with nano-platinum particle upward, exposes the border circular areas that diameter is 8 millimeters, in above-mentioned zone Middle to be added dropwise 10 microlitres, the glucose oxidase aqueous solution of 30mg/mL drips when aqueous solution will dry, then into above-mentioned zone Add 40 microlitres, the chitosan-acetic acid solution (wherein the concentration of acetic acid is 1.5wt%) of 4mg/mL is finally putting into drier and dries in the air It does to get the hydrophobic oxygen-enriched electrode assembly of three-phase is arrived；

6) the KCl/PBS buffer solution of pH=5.8 is injected into the above-mentioned hydrophobic oxygen-enriched electrode assembly of three-phase；Delay to above-mentioned Rush in solution be added various concentration glucose, with magnetic stirrer it is uniform after use again three-electrode system with -0.1V make I-t scanning is carried out for constant potential, embodiment result is as shown in Fig. 5, wherein sweep time is 80 seconds, is taken the 60th second Current value obtains concentration-current curve as output signal.

Embodiment 5

1) with ethyl alcohol, acetone, water (ratio 1 after glass-carbon electrode (diameter 3mm) being polished:1:1) mixed solvent impregnates, Ultrasonic cleaning 3 minutes, is then dried with nitrogen spare；

2) modification of nano-platinum particle is carried out to above-mentioned glassy carbon electrode surface using physical gas-phase deposite method：Use electron beam Evaporation mode modifies the nano-platinum particle with a thickness of 5 nanometers in glassy carbon electrode surface；

3) method shared using covalent cross-linking and investment to the glass-carbon electrode of above-mentioned surface finish nano platinum grain into The fixation of row glucose oxidase：By the chitosan-acetic acid solution of 10 microlitres of 4mg/mL, the grape glycosyloxy of the 10mg/mL of 20 μ L Change enzyme solutions, the glutaraldehyde water solution of 2 microlitres of 10wt% and 100 microlitres of water blendings and be configured to uniform mixed liquor, is placed on room Under temperature after twenty minutes, it takes 3 microlitres to be added dropwise in above-mentioned glassy carbon electrode surface, is put into and is dried in drier to get arriving as a comparison Enzyme glass-carbon electrode.

4) glucose of various concentration is added into the KCl/PBS buffer solution of pH=7.2, it is equal with magnetic stirrer Cyclic voltammetry scanning is carried out using three-electrode system after even, wherein working electrode is above-mentioned enzyme glass-carbon electrode, embodiment result As shown in 8 left side of attached drawing, wherein the maximum potential of scanning range is 0.4V, and potential minimum is -0.3V, and sweeping speed is 0.05V/s, defeated Signal takes corresponding cathodic reduction electric current at -0.1V current potential out, and the corresponding current signal of record different glucose obtains dense Degree-current curve, as shown in 8 right side of attached drawing, preferable linear relationship is presented in concentration-electric current.

Comparative example 6

2) modification of nano-platinum particle is carried out to above-mentioned glass-carbon electrode using electrochemical deposition method：Above-mentioned glass-carbon electrode is soaked In the mixing electrodeposit liquid (wherein the concentration of chloroplatinic acid is 3mM, and the concentration of sulfuric acid is 0.6M) for entering chloroplatinic acid, sulfuric acid, using three Electrode system carries out electro-deposition.Using glass-carbon electrode as working electrode, platinum filament is used as to electrode, Ag/AgCl as reference electrode, Using current-vs-time method (I-t method) at -0.3V electro-deposition 420 seconds, electrode surface is washed with water then to get surface modification is arrived The glass-carbon electrode of nano-platinum particle；

3) glucose oxidase is carried out using glass-carbon electrode of the embedding fixation to above-mentioned surface finish nano platinum grain It is fixed：4.5 microlitres are added dropwise on the glass-carbon electrode of above-mentioned surface finish nano platinum grain, the glucose oxidase of 5mg/mL is water-soluble Liquid is added dropwise 2 microlitres when aqueous solution will dry, then to above-mentioned glassy carbon electrode surface, chitosan-acetic acid solution (its of 2mg/mL The concentration of middle acetic acid is 0.8wt%), it is finally putting into and is dried in drier to get enzyme glass-carbon electrode as a comparison is arrived；

4) 1mM glucose, 0.1mM ascorbic acid, 0.1mM pairs are sequentially added into the KCl/PBS buffer solution of pH=6.2 Paracetamol, 0.1mM uric acid and 1mM glucose, use in the process three-electrode system using+0.5V as constant potential into Row i-t scanning, wherein working electrode is above-mentioned enzyme glass-carbon electrode, and embodiment result is as shown in Fig. 9, wherein grape is added The time interval of sugar and chaff interferent is 40 seconds.The current signal that be added three kinds of interfering substances are obtained increases and glucose Current signal increase compares, and embodiment result is as shown in figure 1 lb.The results show that when being tested using electrochemical oxidation method, Chaff interferent present in system such as paracetamol, uric acid and ascorbic acid etc. can generate apparent influence to test result.

Comparative example 7

3) the KCl/PBS buffer solution of pH=7.2 is prepared respectively, and contains 10Mm H₂O₂With the pH of 10Mm ascorbic acid =7.2 KCl/PBS buffer solution；Linear voltammetry scanning is carried out to above-mentioned solution using three-electrode system, wherein the electricity that works The glass-carbon electrode of extremely above-mentioned surface finish nano platinum grain, embodiment result are as shown in Fig. 10, wherein scanning range is most High potential is 0.6V, and potential minimum is -0.6V, and sweeping speed is 0.05V/s, and scanning direction is from negative potential to positive potential.From Figure 10 In as can be seen that using electrochemical oxidation method test when, be highly susceptible to the influence (Figure 10 a) of the chaff interferent in system； However, when using being detected such as the restoring method in the present invention, when, there are when chaff interferent, the cyclic voltammetric of detection is believed in system Number without significantly change (Figure 10 b), illustrate that test method of the invention can be effectively prevented the interference of common interference object.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Claims

1. a kind of oxygen-enriched electrochemical detection device, including three-electrode system, the three-electrode system include reference electrode, to electrode And working electrode, wherein the working electrode is the oxygen-enriched electrode of three-phase, it is characterised in that：First table of the oxygen-enriched electrode of three-phase Face is contacted with air or oxygen, and second surface and electrolyte contacts, the working electrode is as cathodic reduction hydrogen peroxide；

Preferably, the oxygen-enriched electrode of the three-phase is surface modification hydrogen-peroxide reduction catalyst granules, and is fixed with test substance The substrate of corresponding oxidizing ferment.

2. oxygen-enriched electrochemical detection device as described in claim 1, it is characterised in that：The hydrogen-peroxide reduction catalyst particles Grain is selected from the group：Carbon, metal, metal salt, alloy, organic material reducing catalyst, or combinations thereof；Preferably, the metal choosing From the following group：Platinum, rhodium, ruthenium, gold, cobalt, iron or nickel, or combinations thereof；The organic material reducing catalyst is selected from the group：Biomaterial And/or metal organic complex；Preferably, the biomaterial be cromoci, hydrogen peroxide oxidation enzyme, Prussian blue, or A combination thereof.

3. oxygen-enriched electrochemical detection device as described in claim 1, which is characterized in that the substrate is selected from the group：Metal material Material, carbon material, high-molecular porous material；Preferably, the substrate is selected from the group：Carbon fiber paper, carbon nanotube, 3D graphene, Foam copper.

4. oxygen-enriched electrochemical detection device as described in claim 1, which is characterized in that the substrate is hydrophobic carbon fiber Paper.

5. oxygen-enriched electrochemical detection device as described in claim 1, which is characterized in that the test substance is selected from the group：Portugal Grape sugar, cholesterol, lactic acid, acetylcholine, alcohols, or combinations thereof；And/or the corresponding oxidizing ferment of test substance is selected from down Group：Glucose oxidase, alpha-phosphate glycerol oxidase, cholesterol esterase, cholesterin dehydrogenase, cholesterol oxidase, glucose Oxidizing ferment, glucose dehydrogenase, lactic dehydrogenase, malic dehydrogenase, bilirubin oxidase, ascorbic acid oxidase, peroxide Compound enzyme, uricase, clostridiopetidase A, matter acid enzyme, protease or proteolytic enzyme, or combinations thereof.

6. a kind of oxygen-enriched electrochemical detection method is filled using oxygen-enriched Electrochemical Detection according to any one of claims 1 to 5 Set the detection for carrying out test substance, it is characterised in that：

The first surface of the oxygen-enriched electrode of three-phase is contacted with air or oxygen, second surface and electrolyte contacts, the work Electrode is as cathodic reduction hydrogen peroxide；

Preferably, the oxygen-enriched electrode of the three-phase is surface modification hydrogen-peroxide reduction catalyst granules, and is fixed with test substance The substrate of corresponding oxidizing ferment；

It is highly preferred that the hydrogen-peroxide reduction catalyst granules is selected from the group：Carbon, metal, metal salt, alloy, organic material Reducing catalyst, or combinations thereof；It is further preferred that the metal is selected from the group：Platinum, rhodium, ruthenium, gold, cobalt, iron or nickel, or A combination thereof；The organic material reducing catalyst is selected from the group：Biomaterial and/or metal organic complex；It is further excellent Selection of land, the biomaterial be cromoci, hydrogen peroxide oxidation enzyme, Prussian blue, or combinations thereof；

It is highly preferred that the substrate is selected from the group：Metal material, carbon material, high-molecular porous material；Preferably, the substrate It is selected from the group：Carbon fiber paper, carbon nanotube, 3D graphene, foam copper；

It is highly preferred that the substrate is hydrophobic carbon fiber paper；

It is highly preferred that the test substance is selected from the group：Glucose, cholesterol, lactic acid, acetylcholine, alcohols, or combinations thereof； And/or the corresponding oxidizing ferment of test substance is selected from the group：Glucose oxidase, alpha-phosphate glycerol oxidase, cholesterol Esterase, cholesterin dehydrogenase, cholesterol oxidase, glucose oxidase, glucose dehydrogenase, lactic dehydrogenase, malic acid are de- Hydrogen enzyme, bilirubin oxidase, ascorbic acid oxidase, peroxidase, uricase, clostridiopetidase A, matter acid enzyme, protease or albumen Hydrolase, or combinations thereof.

7. oxygen-enriched electrochemical detection method as claimed in claim 6, which is characterized in that the substrate is to make by the following method Standby：(1) conductive base with hydrophobic structure is provided；(2) the fixed catalysis material is loaded on the hydrophobic structure； (3) apply the solution containing the corresponding oxidizing ferment of test substance, and drying at room temperature on the hydrophobic structure, thus make it is described to It surveys the corresponding oxidizing ferment of substance and is attached to the hydrophobic structure surface.

8. a kind of glucose electrochemical detection device, the device include three-electrode system, the three-electrode system includes ginseng Than electrode, to electrode and working electrode, wherein the working electrode is the oxygen-enriched electrode of three-phase, it is characterised in that：The three-phase is rich The first surface of oxygen electrode is contacted with air or oxygen, second surface and electrolyte contacts；And the working electrode is as cathode Restore hydrogen peroxide.

9. glucose electrochemical detection device as claimed in claim 8, it is characterised in that：The oxygen-enriched electrode of three-phase is surface Hydrogen peroxide reducing catalyst particle is modified, and is fixed with the substrate of the corresponding oxidizing ferment of test substance；

Preferably, the substrate is selected from the group：Metal material, carbon material, high-molecular porous material；

It is highly preferred that the substrate is selected from the group：Carbon fiber paper, carbon nanotube, 3D graphene, foam copper；

Preferably, the hydrogen-peroxide reduction catalyst granules is selected from the group：Carbon, metal, metal salt, alloy, organic material are also Raw catalyst, or combinations thereof；Preferably, the metal is selected from the group：Platinum, rhodium, ruthenium, gold, cobalt, iron or nickel, or combinations thereof；It is described Organic material reducing catalyst is selected from the group：Biomaterial and/or metal organic complex；Preferably, the biomaterial is Cromoci, hydrogen peroxide oxidation enzyme, Prussian blue, or combinations thereof；

Preferably, the corresponding oxidizing ferment of the test substance is selected from the group：Glucose oxidase, alpha-phosphate glycerol oxidase, gallbladder Sterol esterase, cholesterin dehydrogenase, cholesterol oxidase, glucose oxidase, glucose dehydrogenase, lactic dehydrogenase, apple Acidohydrogenase, bilirubin oxidase, ascorbic acid oxidase, peroxidase, uricase, clostridiopetidase A, matter acid enzyme, protease, Proteolytic enzyme, or combinations thereof.

10. a kind of reduce the method interfered and improve upper limit of detection in glucose Electrochemical Detection, such as claim 8-9 is used Any one of described in glucose electrochemical detection device carry out glucose detection, it is characterised in that：The oxygen-enriched electricity of three-phase The first surface of pole is contacted with air or oxygen, and second surface and electrolyte contacts, the working electrode is as cathodic reduction Hydrogen oxide；

Preferably, the hydrogen-peroxide reduction catalyst granules is selected from the group：Carbon, metal, metal salt, alloy, organic material are also Raw catalyst, or combinations thereof；It is highly preferred that the metal is selected from the group：Platinum, rhodium, ruthenium, gold, cobalt, iron or nickel, or combinations thereof；Institute Organic material reducing catalyst is stated to be selected from the group：Biomaterial and/or metal organic complex；It is highly preferred that the biology material Material be cromoci, hydrogen peroxide oxidation enzyme, Prussian blue, or combinations thereof；

Preferably, the substrate is selected from the group：Metal material, carbon material, high-molecular porous material；It is highly preferred that the base Material is selected from the group：Carbon fiber paper, carbon nanotube, 3D graphene, foam copper.