CN105301086A - Preparation method and application of glucose oxidase biosensor - Google Patents
Preparation method and application of glucose oxidase biosensor Download PDFInfo
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- CN105301086A CN105301086A CN201510880316.9A CN201510880316A CN105301086A CN 105301086 A CN105301086 A CN 105301086A CN 201510880316 A CN201510880316 A CN 201510880316A CN 105301086 A CN105301086 A CN 105301086A
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- glucose oxidase
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Abstract
The invention discloses a preparation method and application of a glucose oxidase biosensor. The biosensor comprises a matrix electrode, wherein a malachite green membrane coats the sensing end of the electrode; and a glucose modification enzyme is modified on the membrane. The preparation method comprises the following steps: forming the malachite green membrane on a glassy carbon electrode by virtue of an electric polymerization method, and modifying glucose oxidase on the membrane by utilizing a dispensing method. The composite membrane biosensor prepared by the method can be used for measuring the glucose concentration in solution, and the composite membrane enzyme electrode has good response on glucose oxidation. The method disclosed by the invention is simple in preparation steps, and the prepared biosensor is high in conductivity, high in selectivity, high in catalytic activity and high in sensitivity and has excellent prospects in the aspects of glucose catalysis and detection.
Description
Technical field
The invention belongs to enzyme biologic sensor technical field, particularly a kind of preparation method of highly sensitive glucose oxidase enzyme biologic sensor and application thereof.
Background technology
Biology sensor is widely used in the Electrochemical Detection of living matter due to its fast sharp detection perform and the advantage such as required instrument price is cheap.The highly sensitive glucolase biology sensor that exploitation is used for the subtle change of concentration of glucose in detection bodies has important scientific meaning and use value.
Malachite green belongs to triphenylmethane dye, is a kind of material of modified electrode of novelty, and its excellent electric conductivity can promote the electron transmission of bio-electrical activity molecule well, is applied to the responsiveness that electrochemical sensor can significantly improve to detection material.Compared with the biology sensor of the glucose oxidase modified with other, the film modified glucose oxidase enzyme biologic sensor of malachite green makes simple, highly sensitive, reaction velocity is fast, by force anti-interference, sensing range is wide, will bring larger progress for the development of biology sensor.
Glucose oxidase is widely studied because having extremely important catalytic activity.And it is cheap, stable in properties, practical, often by people as the desirable molecule preparing biology sensor.
Summary of the invention
The object of this invention is to provide a kind of preparation method and application thereof of glucose oxidase enzyme biologic sensor.
Principle of the present invention and thinking: utilize the method for electropolymerization that malachite green solution is modified at electrode surface, form a kind of carrier thin film, glucose oxidase solution is dripped at film surface, in enzyme solutions, mix glutaraldehyde, that Nafion (perfluorinate high molecular polymer sulfonate cation exchanger) makes glucose oxidase more firm is crosslinked together with malachite green film, form a kind of film glucose being had to specific response.Utilize the surperficial glucose oxidase of this modified electrode to carry out catalytic oxidation to glucose, generate grape acid, hydrogen peroxide, quantitative test can be carried out to glucose accurately by cyclic voltammetry.
Concrete steps are:
(1) by base electrode on gold sand printing paper with the burnishing powder of 0.5 μm corase grind, after clean with ultrapure water on tSuede with the Al of 0.35 μm
2o
3burnishing powder fine grinding, be more totally placed on ultrapure water the HNO that volume ratio is 1:3
3each ultrasonic cleaning 2min in/absolute ethyl alcohol mixed solution and intermediate water, cleaner with ultrapure water, dry, i.e. obtained pretreated base electrode.
(2) with step (1) pretreated base electrode for working electrode, at the H of 0.1mol/L
2sO
4in carry out cyclic voltammetry scanning, scanning potential range is-1.0 ~ 1.4V; Sweep speed is 0.1V/s; Scanning hop count is 20 sections, stablizes, namely complete the activation of base electrode to cyclic voltammetry curve.
(3) using the base electrode after step (2) activation as working electrode, in 15ml beaker, 2mL3 × 10 are added successively
-3the NaNO of the malachite green solution of mol/L, the phosphate buffer solution of 5mL0.2mol/L and 3mL1mol/L
3solution, scans by cyclic voltammetry, and scanning potential range is-1.4 ~ 1.8V; Sweep speed is 0.1V/s; Scanning hop count is 30 sections, after having scanned, and with the clean base electrode of ultrapure water, dries, on base electrode, has just prepared the malachite green film of electropolymerization.
(4) centrifuge tube getting 4mL add successively 10 μ L mass percent concentrations be 2% glutaraldehyde water solution, 50 μ LpH values be 6.0 PBS solution and 5mg glucose oxidase, then 500 μ L are settled to Nafion solution, mix, shake up, be i.e. obtained glucose oxidase enzyme mixation.
(5) the glucose oxidase enzyme mixation that painting 20 μ L step (4) is obtained is dripped on the surface of the base electrode processed through step (3), on the malachite green film of electropolymerization, just define glucose oxidase layer after drying, namely complete the preparation of the film modified glucose oxidase enzyme biologic sensor of electropolymerization malachite green.
Described base electrode is glass-carbon electrode or indium-tin oxide electrode.
Described PBS solution is the NaH of 0.2mol/L
2pO
4-Na
2hPO
4buffer solution.
Glucose oxidase biosensor application prepared by the present invention is in carrying out quantitative test to glucose.
The inventive method preparation process is simple, obtained biology sensor good conductivity, and selectivity is good, and catalytic activity is high, highly sensitive, has fine prospect in the catalysis of glucose and context of detection.
Accompanying drawing explanation
Fig. 1 is naked glass-carbon electrode in the embodiment of the present invention to the response diagram of blank solution and glucose solution.
Fig. 2 is the response diagram of glass-carbon electrode to blank solution and glucose solution of electropolymerization malachite green in the embodiment of the present invention.
Fig. 3 is malachite green/glucose oxidase enzyme modified electrode in the embodiment of the present invention to the response diagram of blank solution and glucose solution.
Fig. 4 is malachite green/glucose oxidase/nafion modified electrode in the embodiment of the present invention to the response diagram of blank solution and glucose solution.
Fig. 5 is the uv absorption figure of malachite green modified electrode under the different number of turns in the embodiment of the present invention.
Fig. 6 is through the absorption peak of electropolymerization at electrode face finish malachite green and the ultra-violet absorption spectrum of malachite green solution in the embodiment of the present invention.
Fig. 7 be in the embodiment of the present invention the film modified glucose oxidase enzyme biologic sensor of electropolymerization malachite green under different scanning rates to the effect diagram of peak shape.
Fig. 8 be the film modified glucose oxidase enzyme biologic sensor of electropolymerization malachite green that the embodiment of the present invention obtains under different scanning rates with the linear relationship chart of peak current.
Fig. 9 is the cyclic voltammogram of the film modified glucose oxidase enzyme biologic sensor of electropolymerization malachite green under different glucose system that the embodiment of the present invention obtains.
Figure 10 be the film modified glucose oxidase enzyme biologic sensor of electropolymerization malachite green that the embodiment of the present invention obtains under different glucose system with the function relation figure of peak current.
Embodiment
Below with reference to Figure of description and specific embodiment, the present invention is described in further details.
embodiment:
(1) by glass-carbon electrode on gold sand printing paper with the burnishing powder of 0.5 μm corase grind, after clean with ultrapure water on tSuede with the Al of 0.35 μm
2o
3burnishing powder fine grinding, be more totally placed on ultrapure water the HNO that volume ratio is 1:3
3each ultrasonic cleaning 2min in/absolute ethyl alcohol mixed solution and intermediate water, cleaner with ultrapure water, dry, i.e. obtained pretreated base electrode.
(2) with step (1) pretreated base electrode for working electrode, at the H of 0.1mol/L
2sO
4in carry out cyclic voltammetry scanning, scanning potential range is-1.0 ~ 1.4V; Sweep speed is 0.1V/s; Scanning hop count is 20 sections, stablizes, namely complete the activation of base electrode to cyclic voltammetry curve.
(3) using the base electrode after step (2) activation as working electrode, in 15ml beaker, 2mL3 × 10 are added successively
-3the NaNO of the malachite green solution of mol/L, the phosphate buffer solution of 5mL0.2mol/L and 3mL1mol/L
3solution, using Ag/AgCl electrode as contrast electrode, platinum electrode, as to electrode, scans by cyclic voltammetry, and scanning potential range is-1.4 ~ 1.8V; Sweep speed is 0.1V/s; Scanning hop count is 30 sections, after having scanned, and with the clean base electrode of ultrapure water, dries, in the induction end of base electrode, has just prepared the malachite green film of one deck electropolymerization.
(4) get 4mL centrifuge tube add successively 10 μ L mass percent concentrations be 2% glutaraldehyde water solution, 50 μ LpH values be 6.0 PBS solution and 5mg glucose oxidase, then 500 μ L are settled to Nafion solution, mix, shake up, be i.e. obtained glucose oxidase enzyme mixation.
(5) the glucose oxidase enzyme mixation that painting 20 μ L step (4) is obtained is dripped on the surface of the base electrode processed through step (3), on the malachite green film of electropolymerization, just define glucose oxidase layer after drying, namely complete the preparation of the film modified glucose oxidase enzyme biologic sensor of electropolymerization malachite green.
Characterized different phase in the biology sensor preparation process of the present embodiment by cyclic voltammetry, result as shown in drawings.Naked glass-carbon electrode is to glucose solution almost no response as shown in Figure 1; The glass-carbon electrode only having malachite green to modify as shown in Figure 2 does not almost respond glucose solution; The electric current electrode that malachite green is modified as seen from Figure 3 dripping the reduction peak after being coated with glucose oxidase near-0.4V just reduces; As shown in Figure 4 drip be coated with glucose oxidase after modify again after Nafion film, peak shape is more a lot of than not having fashion.Hydrogen peroxide inhibits reduction peak as shown in Figure 4, and reduction peak current is reduced.
The sign of electropolymerization malachite green film modified electrod in ultraviolet absorption spectroscopy of the present embodiment, Fig. 5 is with under the different number of turns, the absorption peak of electropolymerization malachite green film modified electrod, can see, along with the continuous increase of the electropolymerization number of turns, the malachite green film thickness of electrode surface increases, uv absorption strengthens, meanwhile ultraviolet absorption peak also there occurs red shift, this demonstrate the process that malachite green modified glassy carbon electrode is electropolymerization, be not absorption, because the change that can not cause absorption peak of absorption, and when there is electropolymerization, polymkeric substance is due to the increase of double bond conjugated degree, absorbing wavelength is caused to increase.Fig. 6 is through electropolymerization and there occurs red shift at the absorption peak of electrode face finish malachite green compared with the ultraviolet absorption peak of malachite green solution.These facts can show that malachite green defines polymkeric substance in glassy carbon electrode surface, and have been fixed on electrode through electropolymerization.
In the present embodiment the film modified glucose oxidase enzyme biologic sensor of electropolymerization malachite green under different scanning rates on the impact of peak shape, as in Fig. 7, along with the continuous increase of sweep speed, peak point current also constantly increases, and still can obtain the good cyclic voltammogram of symmetry, the malachite green showing to be fixed on electrode surface presents good reversibility.As in Fig. 8, peak current with sweep rapid-result linear relationship, linear equation is y=-7.5-185*x.This shows that the electrochemical process of modified electrode in PBS is by granule surface contral.In experiment, sweep speed controls at 100mV/s.
The glucose solution of the film modified glucose oxidase enzyme biologic sensor of the electropolymerization malachite green of the present embodiment to variable concentrations is utilized to measure.As in Fig. 9 and Figure 10, be 2.4mg/mL, 0.02mol/L and PBS buffer solution, 0.1mol/LNaCl, time of repose that pH value is 7 are that in the system of 250 seconds, concentration of glucose is 2 × 10 in the glucose oxidase enzyme concentration of modifying malachite green electrode
-6~ 6 × 10
-5during mol/L, the logarithm value of reduction peak current becomes good linear relation with the logarithm value of concentration, and equation of linear regression and linearly dependent coefficient are respectively C=-0.2988X+1.253; R=0.9996.
The biology sensor obtained with the present embodiment is to the glucose solution of different volumes concentration known in identical 4 parts of blood serum samples, system when 10 times of ascorbic acid and 10 times of uric acid exist carries out the calculating of sample test and recovery of standard addition, and result is as shown in table 1 below.
Table 1 measures blood serum sample and recovery of standard addition
From upper table 1, the mensuration of the biology sensor that the present embodiment obtains to glucose sugar concentration is accurately and effectively.Electropolymerization malachite green film modified glucose oxidase biology sensor prepared by the present embodiment is a kind of ideal examination of glucose concentration device, is expected to for life science, clinical medical on-line monitoring.
Investigate the sensitivity of this biology sensor, specificity and stability to the detection of serum sample.
Below be only the preferred embodiment of the present invention, protection scope of the present invention be not only confined to above-described embodiment, conceives various process programs without substantial differences all in protection scope of the present invention with the present invention.
Claims (2)
1. a preparation method for glucose oxidase enzyme biologic sensor, is characterized in that concrete steps are:
(1) by base electrode on gold sand printing paper with the burnishing powder of 0.5 μm corase grind, after clean with ultrapure water on tSuede with the Al of 0.35 μm
2o
3burnishing powder fine grinding, be more totally placed on ultrapure water the HNO that volume ratio is 1:3
3each ultrasonic cleaning 2min in/absolute ethyl alcohol mixed solution and intermediate water, cleaner with ultrapure water, dry, i.e. obtained pretreated base electrode;
(2) with step (1) pretreated base electrode for working electrode, at the H of 0.1mol/L
2sO
4in carry out cyclic voltammetry scanning, scanning potential range is-1.0 ~ 1.4V; Sweep speed is 0.1V/s; Scanning hop count is 20 sections, stablizes, namely complete the activation of base electrode to cyclic voltammetry curve;
(3) using the base electrode after step (2) activation as working electrode, in 15ml beaker, 2mL3 × 10 are added successively
-3the NaNO of the malachite green solution of mol/L, the phosphate buffer solution of 5mL0.2mol/L and 3mL1mol/L
3solution, scans by cyclic voltammetry, and scanning potential range is-1.4 ~ 1.8V; Sweep speed is 0.1V/s; Scanning hop count is 30 sections, after having scanned, and with the clean base electrode of ultrapure water, dries, on base electrode, has just prepared the malachite green film of electropolymerization;
(4) centrifuge tube getting 4mL add successively 10 μ L mass percent concentrations be 2% glutaraldehyde water solution, 50 μ LpH values be 6.0 PBS solution and 5mg glucose oxidase, then 500 μ L are settled to Nafion solution, mix, shake up, be i.e. obtained glucose oxidase enzyme mixation;
(5) the glucose oxidase enzyme mixation that painting 20 μ L step (4) is obtained is dripped on the surface of the base electrode processed through step (3), on the malachite green film of electropolymerization, just define glucose oxidase layer after drying, namely complete the preparation of the film modified glucose oxidase enzyme biologic sensor of electropolymerization malachite green;
Described base electrode is glass-carbon electrode or indium-tin oxide electrode;
Described PBS solution is the NaH of 0.2mol/L
2pO
4-Na
2hPO
4buffer solution.
2. the application of glucose oxidase enzyme biologic sensor prepared of preparation method according to claim 1, is characterized in that this glucose oxidase biosensor application is in carrying out quantitative test to glucose.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109164154A (en) * | 2018-10-28 | 2019-01-08 | 桂林理工大学 | The preparation method and applications of poly- acridine orange modification ITO electrode |
| CN111326743A (en) * | 2019-12-09 | 2020-06-23 | 中国人民解放军军事科学院军事医学研究院 | Application of porous carbon derived from bamboo as electrode material for glucose biosensing and glucose biofuel cell |
| CN114894865A (en) * | 2022-05-24 | 2022-08-12 | 四川大学 | High-precision glucose sensor and preparation method thereof |
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| CN111326743A (en) * | 2019-12-09 | 2020-06-23 | 中国人民解放军军事科学院军事医学研究院 | Application of porous carbon derived from bamboo as electrode material for glucose biosensing and glucose biofuel cell |
| CN114894865A (en) * | 2022-05-24 | 2022-08-12 | 四川大学 | High-precision glucose sensor and preparation method thereof |
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Application publication date: 20160203 |