WO2020048245A1

WO2020048245A1 - Oxygen-enriched nano bio-enzyme electrode, sensor device, preparation method, and application

Info

Publication number: WO2020048245A1
Application number: PCT/CN2019/096766
Authority: WO
Inventors: 封心建
Original assignee: 赛特世纪（苏州）生物科技有限公司
Priority date: 2018-09-05
Filing date: 2019-07-19
Publication date: 2020-03-12
Also published as: CN108872344B; CN108872344A

Abstract

An oxygen-enriched nano bio-enzyme electrode, a sensor device, a preparation method, and application. According to the oxygen-enriched nano bio-enzyme electrode, an electrode substrate is modified with a hollow nano material having an oxygen enriching function, hydrogen peroxide catalyst particles, and an oxidase corresponding to an object under test. The oxygen-enriched nano bio-enzyme electrode and device have a wide detection linear range, high sensitivity, and good interference resistance performance, and can be mass produced. These advantages mainly derive from the hollow material having an oxygen storage function.

Description

Oxygen-enriched nano biological enzyme electrode, sensor device, preparation method and application thereof

Technical field

The invention relates to the technical field of electrode material preparation and electrochemical detection, and in particular, to an oxygen-enriched nano-biological enzyme electrode material, a sensor device, and a preparation method and application thereof.

Background technique

Glucose is a worldwide public health problem and has become a dangerous disease next to cardiovascular disease, that is, cancer. Today, there are as many as 300 million person-times of diabetes in the world. Among them, as many as 100 million people with severe diabetes need to control blood glucose concentration in real time. Therefore, in daily life, it is of great significance to achieve accurate and rapid detection of blood glucose in patients with diabetes. Since 1962, Leland Clark first proposed a theoretical model for measuring glucose with enzyme electrodes, and research on glucose sensors has never stopped. At present, current-type glucose sensors are mainly divided into three generations, namely the first type of glucose sensors that use oxygen as an electronic mediator; the second type of glucose sensors that use artificial electronic mediators instead of oxygen as the electronic receptors for enzyme reactions; and A third type of glucose sensor that has no mediator and allows direct electron transfer between the enzyme and the electrode. The first type of glucose sensor uses oxygen as its natural receptor, which is environmentally friendly, non-toxic and non-polluting. However, due to the following disadvantages, it has not been widely used:

1) The response signal of the glucose sensor is affected by the solubility of oxygen in the solution to be measured. Insufficient oxygen concentration limits the linear range of glucose detection;

2) The oxygen content in the test solution fluctuates, which limits the accuracy of the sensor detection;

3) The detection of hydrogen peroxide usually needs to be performed at a high potential of> 0.6V, but at this potential most endogenous interfering substances (ascorbic acid, uric acid, etc.) and reducing drugs (acetaminophen, acetylsalicylic acid) Acid, etc.) can also be oxidized on the electrode, which will interfere with the glucose response signal and affect the accuracy of glucose detection.

In order to avoid the above-mentioned problems of the first type of glucose sensor in practical applications, in the prior art, most glucose sensors use a second type of glucose sensor based on a synthetic electronic medium. These sensors avoid many of the following problems:

1) The current electronic mediators mainly include conductive organic salts, quinones and their derivatives, dyes, ferrocene and their derivatives. These electronic mediators are toxic to the human body, and are easily detached from the electrodes, resulting in poor stability.

2) The electronic medium will compete with oxygen for electrons, which will cause oxygen interference, which will affect the accuracy of the glucose sensor detection, especially for low concentration glucose solutions.

In the prior art, a three-phase oxygen-rich electrode was used to solve the above problem, and one end was in contact with the liquid to be measured and the other end was in contact with an oxygen gas system. However, although this two-electrode or three-electrode system can continuously measure, the electrode structure is difficult to device It is not easy to operate and carry, and cannot meet people's daily needs and mass production.

In view of this, the present invention is proposed.

Summary of the Invention

A first object of the present invention is to provide an oxygen-enriched nanobioenzyme electrode to solve the above-mentioned problem. The oxygen-enriched nanobioenzyme electrode is modified with a hollow structure on an electrode substrate, and the hollow structure has an internal structure. Or multiple air or oxygen-enriched cavities, this electrode adds an oxygen-enriched hollow structure. The hollow structure is rich in oxygen. During the measurement process, the oxygen diffuses to the electrode surface to achieve a constant and sufficient oxygen supply at the moment of measurement. In addition, the electrode structure does not need to be in contact with an oxygen-containing gas phase such as the atmosphere to achieve a constant and sufficient oxygen supply. It is more convenient and flexible in use, has the advantages of easy deviceization, and can be produced in batches.

A second object of the present invention is to provide a preparation method of the preparation method of the oxygen-enriched nano-biological enzyme electrode, which is convenient, simple, and easy to mass-produce and prepare.

A third object of the present invention is to provide an oxygen-enriched nano-biological enzyme sensor device. The device is an electrochemical detection device using a two-electrode or three-electrode system, wherein the oxygen-enriched nano-biological enzyme electrode is used as a working electrode and at the same time as a cathode. Use, has a wider detection limit, higher sensitivity, good selectivity and accuracy.

A fourth object of the present invention is to provide an oxygen-enriched nano-biological enzyme sensor system. The system further includes a display system, which can effectively read the measured current signal in real time, and is convenient for carrying around.

A fifth object of the present invention is to provide an electrochemical detection method for an oxygen-enriched nano-biological enzyme. The concentration of a sample to be measured is obtained from a measured cathodic reduction current signal. The cathodic reduction current is measured by cyclic voltammetry and linear scanning voltammetry Method, current-time test and other methods, and compared with the standard curve to obtain the concentration of the test object.

The sixth object of the present invention is to provide a method for reducing interference and increasing the upper measurement limit in the electrochemical detection of glucose. The glucose sample concentration is obtained from the measured cathodic reduction current signal, and the test of the cathodic reduction current is performed by cyclic voltammetry and linear scanning. Obtained by voltammetry, current-time test, and compared with the glucose standard curve to obtain the glucose concentration. This method is accurate, fast, and efficient.

A seventh object of the present invention is to provide an application of the aforementioned material or device.

In order to achieve the above object of the present invention, the following technical solutions are particularly adopted:

An oxygen-enriched nano-bioenzyme electrode, a hollow structure is modified on an electrode substrate, and the hollow structure has one or more air or oxygen-rich cavities inside; preferably, hydrogen peroxide is also modified on the electrode substrate. Catalyst particles and oxidase corresponding to the analyte.

Preferably, the hollow structure is selected from one of a cube, a cuboid, a cylinder, a vertebra, a sphere structure and an irregular three-dimensional structure.

Preferably, the material of the hollow structure is selected from: metal materials, inorganic materials, polymer materials, or composite materials of any combination thereof; more preferably, the metal material is selected from nickel, copper, titanium, aluminum, and gold One or an alloy thereof; more preferably, the inorganic material includes a metal oxide, a carbon material, or a combination thereof; even more preferably, the carbon material is selected from one of carbon, graphene, and reduced graphene Or a combination of several; more preferably, the metal oxide is selected from one or a combination of nickel oxide, silicon oxide, zirconia, aluminum oxide, copper oxide, and titanium oxide; more preferably, the high The molecular material is selected from one or a combination of polystyrene, polyaniline, polypyridine, and polypyrrole.

Preferably, the hydrogen peroxide reduction catalyst is selected from one or a combination of carbon, metal, alloy, metal oxide, metal salt, and organic material reduction catalyst; more preferably, the carbon is selected from graphene One or a combination of reducing graphene and carbon nanotubes; more preferably, the metal is selected from the group consisting of platinum, rhodium, iron, nickel, cobalt, gold, or an alloy thereof; More preferably, the organic material reduction catalyst is selected from biomaterials and / or metal organic complexes; even more preferably, the biomaterial is selected from cytochrome C, catalase, horseradish peroxidase, Prussia One or a combination of several types of blue.

Preferably, the material of the electrode substrate is selected from one or a combination of metal materials, inorganic materials, polymer materials, and more; more preferably, the metal material is selected from nickel, copper, titanium, aluminum, One of gold or an alloy thereof; more preferably, the inorganic material is selected from metal oxides, carbon materials, or a combination thereof; even more preferably, the carbon material is selected from carbon fiber materials, carbon nanotubes, graphene, One or more combinations of reduced graphene; more preferably, the metal oxide is selected from one or more of nickel oxide, silicon oxide, zirconia, alumina, copper oxide, and titanium oxide Combination; more preferably, the polymer material is selected from one or a combination of a polyaniline film, a polypyridine film, and a polypyrrole film.

Preferably, the electrode substrate is selected from a hydrophobic material or a hydrophilic material.

Preferably, the surface morphology of the electrode material is smooth or rough; more preferably, the rough morphology includes: linear, rod-like, sheet-like, porous, or irregular bodies.

Preferably, the test substance is selected from one or a combination of uric acid, urea, glucose, lactic acid, acetylcholine, and alcohol; more preferably, the alcohol is cholesterol.

Preferably, the oxidase is selected from the group consisting of glucose oxidase, α-phosphoglycerol oxidase, cholesterol esterase, cholesterol dehydrogenase, cholesterol oxidase, glucose oxidase, glucose dehydrogenase, lactate dehydrogenase, and malate dehydrogenase. One or a combination of catalase, bilirubin oxidase, ascorbate oxidase, peroxidase, urase, collagenase, protease, protease or proteolytic enzyme.

Preferably, the hollow structure is a hollow sphere;

More preferably, the surface morphology of the hollow sphere is uniform or uneven;

More preferably, there are several through holes on the surface of the hollow sphere, and the through holes pass into the cavity from the surface of the hollow sphere; even more preferably, the diameter of the through holes is 0.1nm-20nm;

More preferably, the diameter of the hollow sphere is 0.03-2 μm;

More preferably, the wall thickness of the hollow sphere is 1-500 nm.

The preparation method of the oxygen-enriched nano-biological enzyme electrode includes the following steps:

The film-forming substance is used to spread and fix the hollow structure on the surface of the electrode substrate, and the one or more hollow structures contain air or oxygen-rich gas.

Preferably, the catalyst particles and oxidase of hydrogen peroxide are also spread and fixed on the surface of the substrate;

Preferably, the hollow structure, the catalyst particles of hydrogen peroxide and the oxidase are mixed to form a film on the surface of the electrode substrate;

Preferably, the hollow structure, the catalyst particles of hydrogen peroxide, and the oxidase are respectively formed on the surface of the electrode substrate, and the film formation is not sequential.

Preferably, the film-forming material includes chitosan and a perfluorosulfonic acid proton membrane;

Preferably, a surfactant is added during the film forming process; more preferably, the surfactant is selected from the group consisting of sodium dodecyl benzoate, cetyltrimethylammonium bromide, and lauryl sulfonated amber One or a combination of disodium acid monoester, disodium cocomonoethanolamide sulfosuccinate monoester, monolauryl phosphate, and potassium monododecyl phosphate.

Preferably, the method for fixing the oxidase is performed by covalent crosslinking and / or embedding;

More preferably, the covalent cross-linking method specifically includes the following steps: mixing the chitosan acetic acid solution, the hollow structure dispersion solution, the oxidase solution, the glutaraldehyde aqueous solution, and the solvent, and dropping the solution onto the surface of the electrode substrate after being left to stand , The electrode is obtained after drying;

More preferably, the mass concentration of the hollow structure dispersion is 0.1% -30%; even more preferably, the solvent of the hollow structure dispersion is water or ethanol;

More preferably, the drying is performed by natural drying and / or drying, the drying temperature is 30-200 ° C, and the drying is 0.5-12 hours;

More preferably, the placing time is 5-60 minutes;

More preferably, the concentration of the chitosan acetic acid is 0.5-5 mg / mL,

More preferably, the concentration of the oxidase solution is 10-200 mg / L;

More preferably, the concentration of the glutaraldehyde aqueous solution is 1% -10%.

Preferably, the embedding method specifically includes the following steps: after the perfluorosulfonic acid proton membrane solution, the oxidase solution, and the hollow structure dispersion are uniformly mixed, the solution is dropped on the electrode substrate immediately and dried to obtain the electrode;

More preferably, the solvent of the perfluorosulfonic acid proton membrane solution is water or ethanol;

More preferably, in the perfluorosulfonic acid proton membrane solution, the mass ratio of the perfluorosulfonic acid proton membrane to the solvent is 1 / 1000-1 / 2;

More preferably, the mass concentration of the hollow structure dispersion is 0.1% -30%; even more preferably, the solvent of the hollow structure dispersion is water or ethanol.

An oxygen-enriched nano-biological enzyme sensor device includes an electrode system. The electrode system includes the oxygen-enriched nano-biological enzyme electrode according to

claim

1 or 2. The oxygen-enriched nano-biological enzyme electrode is a working electrode, at least in part. The working electrode is in contact with the solution to be measured;

Preferably, the oxygen-enriched nano-biological enzyme electrode is a cathode;

Preferably, the electrode system further includes a counter electrode selected from one of a carbon rod electrode, a Pt electrode, a titanium electrode, or a platinum black electrode;

Preferably, the device further comprises a counter electrode and a reference electrode connected to the counter electrode; more preferably, the reference electrode is selected from the group consisting of a calomel electrode, a silver / silver chloride electrode, a mercury / mercury oxide electrode, and mercury / Mercury sulfate electrode.

An oxygen-enriched nano-biological enzyme test system includes the oxygen-enriched nano-biological enzyme sensor device and a power supply system and a display system connected to the sensor device;

Preferably, the power supply system includes an electrochemical workstation.

An oxygen-enriched nano-bioenzyme electrochemical detection method uses the oxygen-enriched nano-bioenzyme sensor device or the oxygen-enriched nano-bioenzyme test system to detect an object to be tested, and reduces a current signal or an anode through a measured cathode. The signal obtains the concentration of the sample to be measured;

Preferably, the electrolyte used in the test is a buffer solution of pH = 5-8; more preferably, the concentration of the buffer solution is 0.1-2M; more preferably, the buffer solution includes: PBS buffer solution, sodium sulfate Buffer solution, disodium hydrogen phosphate-citrate buffer solution, acetic acid-sodium acetate buffer solution;

Preferably, the test object is selected from one or a combination of uric acid, urea, lactic acid, acetylcholine, and alcohol.

Preferably, in the electrochemical detection method for oxygen-enriched nano-biological enzymes, the method for testing the cathode reduction current signal includes cyclic voltammetry, linear scanning voltammetry, and current-time testing.

A method for reducing interference and increasing the upper detection limit in the electrochemical detection of glucose, characterized in that, using the oxygen-enriched nano-biological enzyme sensor device or the oxygen-enriched nano-biological enzyme test system to detect a substance to be tested, Compare the measured cathodic reduction current signal with the standard curve of glucose, thereby reducing the interference caused by fluctuation or deficiency of oxygen content;

Preferably, the standard curve of glucose is prepared by adding standard samples of different known concentrations to the electrolyte for testing, and recording the relationship between the current signal and the glucose concentration to obtain a standard curve;

Preferably, the method for testing the cathode reduction current signal includes cyclic voltammetry, linear scanning voltammetry, and current-time testing;

More preferably, in the cyclic voltammetry test, the highest potential is scanned at 0.4-0.6V, the lowest point is -0.3V, the sweep speed is 0.01-0.1V / s, and the output signal is obtained at -0.1V or -0.2V. Reduction current

More preferably, in the linear scanning voltammetry, the highest potential is scanned at 0.4-0.6V, the lowest point is -0.3V, the sweep speed is 0.01-0.1V / s, and the output signal is obtained at -0.1V or -0.2V Cathode reduction current

More preferably, in the current-time test, -0.1V or -0.2V is used as the constant potential, the scanning time is 10-30s, and the current value of the 5s is taken as the cathode reduction current.

The oxygen-enriched nano-bioenzyme electrode, the oxygen-enriched nano-bioenzyme sensor device, or the oxygen-enriched nano-bioenzyme test system is in a device for detecting the concentration of uric acid, urea, glucose, lactic acid, acetylcholine or cholesterol. Applications.

Compared with the prior art, the beneficial effects of the present invention are:

(1) The oxygen-enriched nano-bioenzyme electrode and device provided in this application have a wider detection limit, more sensitive sensitivity, good stability, good biocompatibility, non-toxic and pollution-free, anti-interference, and accuracy High degree and can be mass produced.

(2) The oxygen-enriched nano-bioenzyme electrode provided in this application has a hollow structure modified on the electrode substrate, and the hollow structure has one or more air or oxygen-enriched cavities inside the electrode. The structure body and the hollow structure body are rich in oxygen. During the measurement process, the oxygen diffuses to the electrode surface to realize a constant and sufficient oxygen supply at the moment of measurement. In addition, the electrode structure does not need to be in contact with an oxygen-containing gas phase such as the atmosphere to achieve a constant and sufficient oxygen supply. It is more convenient and flexible in use, has the advantages of easy deviceization, and can be produced in batches.

(3) The hollow sphere material used in the oxygen-enriched nano-bioenzyme electrode and device in the present invention has low cost, simple preparation and easy mass production.

(4) The preparation technology adopted for the oxygen-enriched nano-bioenzyme electrode and device in the present invention requires simple conditions and no severe temperature and pressure requirements.

(5) The oxygen-enriched nano-biological enzyme electrode and device of the present invention are convenient to apply, can be used for a long time, and have good stability.

(6) The oxygen-enriched nano-bioenzyme electrode and device in the present invention are made of biocompatible materials, are harmless to the organism, and can be used for human body detection and continuous detection.

(7) The method for glucose detection in the present invention can detect up to 60 mM on-line, realize the accurate detection of hydrogen peroxide by the cathodic reduction method, and avoid the interference of substances in the human body that are easy to be electrochemically oxidized.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the specific embodiments or the description of the prior art are briefly introduced below. Obviously, the appendixes in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to these drawings without paying creative labor.

1 is a transmission electron microscope image of the hollow mesoporous silica nanospheres prepared in Example 1;

2 is a transmission electron microscope image of the hollow mesoporous silica nanospheres prepared in Example 2;

3 is a transmission electron microscope image of the hollow mesoporous silica nanospheres prepared in Example 3;

4 is a current-time test curve of the oxygen-enriched nano-biological enzyme electrode prepared in Example 2;

FIG. 5 is a linear curve of a glucose concentration test current of an oxygen-enriched nanobiological enzyme electrode prepared in Example 1, Example 2, and Example 3; FIG.

FIG. 6 is a characterization of the interference resistance of the glucose-enriched nano-bioenzyme sensor device prepared in Example 2; FIG.

7 is a transmission electron microscope image of the hollow alumina prepared in Example 5;

FIG. 8 is a current linear curve of the glucose concentration measured by the hollow alumina electrode prepared in Example 5; FIG.

9 is a transmission electron microscope image of the hollow alumina prepared in Example 6;

FIG. 10 is a linear curve of glucose concentration current measured by an oxygen-enriched nano-bioenzyme sensor device prepared by hollow alumina prepared in Example 6; FIG.

11 is a transmission electron microscope image of the hollow titanium oxide prepared in Example 7;

FIG. 12 is a linear curve of glucose concentration current measured by an oxygen-enriched nano-biological enzyme sensor device prepared by hollow titanium oxide prepared in Example 7; FIG.

13 is a transmission electron microscope image of a solid silica prepared in a comparative example;

FIG. 14 is a linear curve of glucose concentration current measured by a glucose sensor prepared from a solid silica prepared in a comparative example.

detailed description

The technical solution of the present invention will be clearly and completely described below with reference to the drawings and specific embodiments, but those skilled in the art will understand that the embodiments described below are part of the embodiments of the present invention, but not all of them. It is only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. If the specific conditions are not indicated in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

The following methods were used to prepare hollow mesoporous silica spheres:

(1) 25ml aqueous solution includes 100nm polystyrene microspheres 0.2wt%, cetyltrimethylammonium bromide 0.2wt%, sodium hydroxide 0.056wt% and 1,2-bis (triethoxysilyl) Ethane was 1.152 wt%, and the reaction was stirred at 80 ° C for 2 hours, and then washed with water and ethanol 3 times to obtain a sample.

(2) Drop the polystyrene microspheres @mesoporous silicon balls washed in (1) on the cleaned glass slides, put them in a tube furnace, pass oxygen at 550 ° C, and calcine for 6 hours.

(3) Scrape the hollow mesoporous silica ball prepared in (2) from the glass slide with a clean blade, add a certain amount of ethanol or deionized water, and prepare a certain amount of hollow mesoporous silica ball. Ethanol or water solution.

The above method for cleaning the slide glass uses an organic solvent of ethanol: acetone: water 1: 1 as a cleaning agent, ultrasonication for 15 minutes, and an oven at 80 ° C.

The following methods are used to prepare and detect the oxygen-enriched nano-biological enzyme sensor device:

(1) 1.6 microliters of platinum nanoparticle dispersion with a volume ratio of 2: 1: 1: 14: 2, a 1/100 perfluorosulfonic acid solution, 1% -2% A mixed solution of sodium dodecyl benzoate solution, ethanol and deionized water.

(2) 1.6 microliters of a 12% by weight hollow mesoporous silica ball ethanol solution with a volume ratio of 30: 12: 43: 85: 30 were added dropwise to the electrode in (1), 1/2 of perfluorosulfonic acid Solution, 100mg / ml glucose oxidase aqueous solution, mixed solution of deionized water and ethanol.

(3) After the electrode is air-dried, it is dried in an oven at 60 ° C for 2 hours.

(4) Cover the dried electrode in (3) with a polymer protective film, and cut the electrode for electrochemical test.

(5) The present invention adopts a three-electrode system for testing. The working electrode is connected to one side and the counter electrode and the reference electrode are connected to one side. First, the cyclic voltammetry test is used to scan the highest potential 0.6, the lowest point -0.3V, and the sweep speed 0.05V. / s, then select -0.2V as the constant potential for IT (current-time) detection, the scan time is 10s, and take the 5s current value as the output signal. Record the current signals corresponding to different concentrations of glucose and different interferences.

Example results are shown in Figures 1 and 5. FIG. 1 is a transmission electron microscope image of a hollow sphere, and FIG. 5 is a glucose concentration-current curve of Example 1. It can be seen that the present invention can effectively improve the detection linear range.

Example 2

The following method is used to prepare hollow mesoporous silica spheres:

(1) 25ml aqueous solution includes 100nm polystyrene microspheres 0.2wt%, cetyltrimethylammonium bromide 0.2wt%, sodium hydroxide 0.056wt% and 1,2-bis (triethoxysilyl) 0.768 wt% of ethane was stirred at 80 ° C. for 2 hours and centrifuged and washed 3 times with water and ethanol to obtain a sample.

(2) Drop the polystyrene @ mesoporous silica ball washed in (1) on the cleaned glass slide, put it in a tube furnace, and pass oxygen at 550 ° C for 6 hours.

(1) 1.6 microliters of platinum nanoparticles with a volume ratio of 2: 1: 1: 14: 2, 1/100 of perfluorosulfonic acid, and 1% -2% dodecane were added dropwise to the disposable electrode substrate. A mixed solution of sodium phenylbenzoate solution, ethanol and deionized water.

(2) 1.6 microliters of a 12% by weight hollow mesoporous silica ball ethanol solution with a volume ratio of 40: 12: 43: 85: 30, and 1/2 of perfluorosulfonic acid were dropped on the electrode in (1). Solution, 100 mg / ml glucose oxidase aqueous solution, a mixture of deionized water and ethanol.

(5) The present invention adopts a three-electrode system for testing. The working electrode is connected to one side, and the counter electrode and the reference electrode are connected to one side. First, the cyclic voltammetry test was used to scan the highest potential of 0.6, the lowest point of -0.3V, and the sweep speed of 0.05V / s. Then select -0.2V as the constant potential for I-T detection, the scan time is 10s, and take the 5s current value as the output signal. Record the current signals corresponding to different concentrations of glucose and different interferences.

Example results are shown in Figures 2, 5, and 6. FIG. 1 is a transmission electron microscope image of a hollow sphere, and FIG. 5 is a linear curve of glucose concentration-current in Example 2. It can be seen that the present invention can effectively improve the detection linear range. Figure 6 shows that the hydrophilic oxygen-enriched glucose sensor can effectively prevent interference from common interferences.

Example 3

The following methods were used to prepare hollow mesoporous silica spheres:

(1) 25ml aqueous solution includes 100nm polystyrene microspheres 0.2wt%, cetyltrimethylammonium bromide 0.2wt%, sodium hydroxide 0.056wt% and 1,2-bis (triethoxysilyl) 0.576 wt% of ethane was stirred at 80 ° C for 2 hours, and then washed with water and ethanol for 3 times to obtain a sample.

(2) On the electrode in (1), 1.6 microliters of a 12 wt% hollow mesoporous silica ball ethanol solution with a volume ratio of 60: 12: 43: 85: 30, and 1/2 of perfluorosulfonic acid were added dropwise. Solution, 100 mg / ml glucose oxidase aqueous solution, a mixture of deionized water and ethanol.

Example results are shown in Figures 3 and 5. FIG. 3 is a transmission electron microscope image of a hollow sphere, and FIG. 5 is a linear curve of glucose concentration-current in Example 3. It can be seen that the present invention can effectively improve the detection linear range.

Example 4

The following methods were used to prepare hollow alumina spheres:

(1) 0.6303 g of ammonium formate was dissolved in 50 ml of deionized water, and the pH was adjusted to 4.4 with formic acid. An aqueous solution of 3 ml (2.5 wt%) of 100 nm polystyrene microspheres and 0.351 g of aluminum sulfate were added for 15 minutes. After reacting at 70 ° C for two hours, it was washed by centrifugation with ethanol.

(2) Drop the washed polystyrene microspheres @ alumina ball ball in (1) on the cleaned glass slide, put it in a tube furnace, and calcinate it at 550 ° C for 2 hours.

(3) The hollow alumina ball prepared in (2) is scraped off the glass slide with a clean blade, and a certain amount of ethanol or deionized water is added to prepare a certain amount of ethanol or aqueous solution of the hollow alumina ball.

(1) 1.6 microliters of platinum nanoparticles with a volume ratio of 2: 1: 1: 14: 2, a 1/100 perfluorosulfonic acid aqueous solution, and 1% -2% twelve are added dropwise to a disposable electrode substrate. A mixture of sodium alkyl benzoate solution, ethanol and deionized water.

(2) On the electrode in (1), 1.6 microliters of a 12 wt% hollow alumina sphere ethanol solution with a volume ratio of 30: 12: 43: 85: 30 respectively, a 1/2 perfluorosulfonic acid solution, 100 mg / ml glucose oxidase aqueous solution, a mixture of deionized water and ethanol.

Example 5

The following methods were used to prepare hollow alumina spheres:

(1) 0.6303 g of ammonium formate was dissolved in 50 ml of deionized water, and the pH was adjusted to 4.4 with formic acid. An aqueous solution of 3 ml (2.5 wt%) of 200 nm polystyrene microspheres and 0.351 g of aluminum sulfate were added for 15 minutes. After reacting at 70 ° C for two hours, it was washed by centrifugation with ethanol.

(2) Put the washed polystyrene microspheres @ alumina balls dripped in (1) on the cleaned glass slide, put them in a tube furnace, and calcinate them at 550 ° C for 2 hours.

Example results are shown in Figures 7 and 8. FIG. 7 is a transmission electron microscope image of a hollow sphere, and FIG. 8 is a linear curve of glucose concentration-current in Example 5. It can be seen that the present invention can effectively improve the detection linear range.

Example 6

The following methods were used to prepare hollow alumina spheres:

(1) 0.6303 g of ammonium formate was dissolved in 50 ml of deionized water, and the pH was adjusted to 4.4 with formic acid. An aqueous solution of 3 ml (2.5 wt%) of 300 nm polystyrene microspheres and 0.351 g of aluminum sulfate were added for 15 minutes. After reacting at 70 ° C for two hours, it was washed by centrifugation with ethanol.

Example results are shown in Figures 9 and 10. FIG. 9 is a transmission electron microscope image of a hollow sphere, and FIG. 10 is a glucose concentration-current linear curve of Example 6. It can be seen that the present invention can effectively improve the detection linear range.

Example 7

The following methods were used to prepare hollow titanium dioxide spheres:

(1) 6 ml (2.5 wt%) of an aqueous solution of 100 nm polystyrene microspheres are added with deionized water having a mass ratio of 10: 34.716: 2: 0.88, ethanol, tetrabutyl titanate, and acetylacetone. The temperature was raised to 40 ° C, and the reaction was performed at a constant temperature for 10 hours. The sample was then centrifuged and washed three times with ethanol.

(2) Drop the polystyrene @ titanium dioxide ball washed in (1) on the cleaned glass slide, put it in a tube furnace, and calcinate it at 550 ° C for 2 hours.

(3) The hollow titanium dioxide ball prepared in (2) is scraped off the glass slide with a clean blade, and a certain amount of ethanol or deionized water is added to prepare a certain amount of ethanol or aqueous solution of the hollow titanium dioxide ball.

(2) On the electrode in (1), 1.6 microliters of a 12 wt% hollow alumina sphere ethanol solution with a volume ratio of 30: 12: 43: 85: 30, a 1/2 Nafion solution, and 100 mg / ml glucose were dropped. A mixture of an oxidase aqueous solution, deionized water, and ethanol.

Example results are shown in Figure 11 and Figure 12. FIG. 11 is a transmission electron microscope image of a hollow sphere, and FIG. 12 is a linear curve of glucose concentration-current in Example 7. It can be seen that the present invention can effectively improve the detection linear range.

Example 8

The following methods were used to prepare hollow TiO ₂ spheres:

(1) 6 ml (2.5 wt%) of an aqueous 200 nm polystyrene microsphere solution is added deionized water with a mass ratio of 10: 34.716: 2: 0.88, ethanol, tetrabutyl titanate, and acetylacetone. The temperature was raised to 40 ° C, and the reaction was performed at a constant temperature for 10 hours. The sample was then centrifuged and washed three times with ethanol.

(5) The present invention adopts a three-electrode system for testing. The working electrode is connected to one side, and the counter electrode and the reference electrode are connected to one side. First use cyclic voltammetry to scan the highest potential of 0.6, the lowest point of -0.3V, the sweep speed of 0.05V / s. Then select -0.2V as the constant potential for I-T detection, the scan time is 10s, and take the 5s current value as the output signal. Record the current signals corresponding to different concentrations of glucose and different interferences.

Example 9

(1) 6 ml (2.5 wt%) of an aqueous 300 nm polystyrene microsphere solution was added deionized water with a mass ratio of 10: 34.716: 2: 0.88, ethanol, tetrabutyl titanate, and acetylacetone. The temperature was raised to 40 ° C, and the reaction was performed at a constant temperature for 10 hours. The sample was then centrifuged and washed three times with ethanol.

(4) Cover the baked electrode in (3) with a polymer protective film, and cut the electrode for electrochemical test.

Comparative example

The following methods are used to prepare solid silica spheres:

(1) A mixed solution of 75: 10: 3.15: 2 volume ratio of ethanol, water, ammonia and tetraethyl orthosilicate was stirred and reacted at room temperature for 1 hour, and then washed with water and ethanol for 3 times to obtain a solid silica. ball.

(2) Drop the prepared silica solid ball on a clean glass slide, and then scrape it off the glass slide with a clean blade, add a certain amount of ethanol or deionized water, and prepare a certain amount of ethanol as a solid ball. Or aqueous solution. The above method for cleaning the slide glass uses an organic solvent of ethanol: acetone: water 1: 1 as a cleaning agent, ultrasonication for 15 minutes, and an oven at 80 ° C.

Example results are shown in Figures 13 and 14. FIG. 13 is a transmission electron microscope image of a solid SiO ₂ sphere, and FIG. 14 is a linear curve of glucose concentration-current of a comparative example. It can be seen that the linear range of the two-phase glucose sensor with a non-hollow structure is significantly lower than that of the three-phase oxygen-enriched glucose sensor described above.

Although specific embodiments have been used to illustrate and describe the present invention, it should be appreciated that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit the present invention; those skilled in the art should understand that: Without departing from the spirit and scope of the present invention, the technical solutions described in the foregoing embodiments may be modified, or some or all of the technical features may be equivalently replaced; and these modifications or replacements do not make the corresponding technical solutions Essentially deviate from the scope of the technical solutions of the embodiments of the present invention; therefore, this means that all such substitutions and modifications that fall within the scope of the present invention are included in the appended claims.

Claims

An oxygen-enriched nano-bioenzyme electrode, characterized in that a hollow structure is modified on an electrode substrate, and the hollow structure has one or more air or oxygen-rich cavities inside;

Preferably, the electrode substrate is further modified with hydrogen peroxide catalyst particles and an oxidase corresponding to the test object;

Preferably, the hollow structure is selected from one of a cube, a cuboid, a cylinder, a vertebra, a sphere structure and an irregular solid structure;

Preferably, the material of the hollow structure is selected from: metal materials, inorganic materials, polymer materials, or composite materials of any combination thereof; more preferably, the metal material is selected from nickel, copper, titanium, aluminum, and gold One or an alloy thereof; more preferably, the inorganic material includes a metal oxide, a carbon material, or a combination thereof; even more preferably, the carbon material is selected from one of carbon, graphene, and reduced graphene Or a combination of several types; more preferably, the metal oxide is selected from one or a combination of nickel oxide, silicon oxide, zirconia, aluminum oxide, copper oxide, titanium oxide, and cerium oxide; more preferably, The polymer material is selected from one or a combination of polystyrene, polyaniline, polypyridine, and polypyrrole;

Preferably, the hydrogen peroxide reduction catalyst is selected from one or a combination of carbon, metal, alloy, metal oxide, metal salt, and organic material reduction catalyst; more preferably, the carbon is selected from graphene One or a combination of reducing graphene and carbon nanotubes; more preferably, the metal is selected from the group consisting of platinum, rhodium, iron, nickel, cobalt, gold, or an alloy thereof; More preferably, the organic material reduction catalyst is selected from biomaterials and / or metal organic complexes; even more preferably, the biomaterial is selected from cytochrome C, catalase, horseradish peroxidase, Prussia One or a combination of several types of blue;

Preferably, the material of the electrode substrate is selected from one or a combination of metal materials, inorganic materials, polymer materials, and more; more preferably, the metal material is selected from nickel, copper, titanium, aluminum, One of gold or an alloy thereof; more preferably, the inorganic material is selected from metal oxides, carbon materials, or a combination thereof; even more preferably, the carbon material is selected from carbon fiber materials, carbon nanotubes, graphene, One or more combinations of reduced graphene; more preferably, the metal oxide is selected from one or more of nickel oxide, silicon oxide, zirconia, alumina, copper oxide, and titanium oxide Combination; more preferably, the polymer material is selected from one or a combination of polyaniline film, polypyridine film, and polypyrrole film;

Preferably, the electrode substrate is selected from a hydrophobic material or a hydrophilic material;

Preferably, the surface morphology of the electrode material is smooth or rough; more preferably, the rough morphology includes: linear, rod-like, sheet-like, porous, or irregular bodies;

Preferably, the test substance is selected from one or a combination of uric acid, creatinine, urea, glucose, lactic acid, acetylcholine, triglycerides, and alcohol; more preferably, the alcohol is cholesterol;

Preferably, the oxidase is selected from the group consisting of glucose oxidase, α-phosphoglycerol oxidase, cholesterol esterase, cholesterol dehydrogenase, cholesterol oxidase, glucose oxidase, glucose dehydrogenase, lactate dehydrogenase, and malate dehydrogenase. One or a combination of catalase, bilirubin oxidase, ascorbate oxidase, peroxidase, urase, collagenase, protease, protease or proteolytic enzyme.
The oxygen-enriched nanobiological enzyme electrode according to claim 1, wherein the hollow structure is a hollow sphere;

Preferably, the surface morphology of the hollow sphere is uniform or uneven;

Preferably, there are several through holes on the surface of the hollow sphere, and the through holes pass into the cavity from the surface of the hollow sphere; more preferably, the diameter of the through holes is 0.1nm-20nm;

Preferably, the diameter of the hollow sphere is 0.03-2 μm;

Preferably, the wall thickness of the hollow sphere is 1-500 nm.
The method for preparing an oxygen-enriched nanobiological enzyme electrode according to claim 1 or 2, further comprising the following steps:

A film-forming substance is used to fix the hollow structure on the surface of the electrode substrate, and the one or more hollow structures include air or oxygen-enriched gas;

Preferably, the catalyst particles and oxidase of hydrogen peroxide are also fixed on the surface of the substrate;

Preferably, the hollow structure, the catalyst particles of hydrogen peroxide and the oxidase are mixed to form a film on the surface of the electrode substrate;

Preferably, the hollow structure, the catalyst particles of hydrogen peroxide, and the oxidase are respectively formed on the surface of the electrode substrate, and the film formation is not sequential.

Preferably, the film-forming material includes chitosan, bovine serum albumin and perfluorosulfonic acid proton membrane;

Preferably, a surfactant is added during the film forming process; more preferably, the surfactant is selected from the group consisting of sodium dodecyl benzoate, cetyltrimethylammonium bromide, and lauryl sulfonated amber One or a combination of disodium acid monoester, disodium cocomonoethanolamide sulfosuccinate monoester, monolauryl phosphate, and potassium monododecyl phosphate.
The method for preparing an oxygen-enriched nanobiological enzyme electrode according to claim 3, wherein the method for fixing the oxidase is performed by covalent crosslinking and / or embedding;

Preferably, the covalent cross-linking method specifically includes the following steps: mixing the chitosan acetic acid solution, the hollow structure dispersion liquid, the oxidase solution, the glutaraldehyde aqueous solution and the solvent, and adding the solution dropwise to the surface of the electrode substrate, The electrode is obtained after drying;

More preferably, the mass concentration of the hollow structure dispersion is 0.1% -30%; even more preferably, the solvent of the hollow structure dispersion is water or ethanol;

More preferably, the drying is performed by natural drying and / or drying, the drying temperature is 30-200 ° C, and the drying is 0.5-12 hours;

More preferably, the placing time is 5-60 minutes;

More preferably, the concentration of the chitosan acetic acid is 0.5-5 mg / mL,

More preferably, the concentration of the oxidase solution is 10-200 mg / L;

More preferably, the concentration of the glutaraldehyde aqueous solution is 1% -10%;

Preferably, the embedding method specifically includes the following steps: after the perfluorosulfonic acid proton membrane solution, the oxidase solution, and the hollow structure dispersion are uniformly mixed, the solution is dropped on the electrode substrate immediately and dried to obtain the electrode;

More preferably, the solvent of the perfluorosulfonic acid proton membrane solution is water or ethanol;

More preferably, in the perfluorosulfonic acid proton membrane solution, the mass ratio of the perfluorosulfonic acid proton membrane to the solvent is 1 / 1000-1 / 2;

More preferably, the mass concentration of the hollow structure dispersion liquid is 0.1% -30%; even more preferably, the solvent of the hollow structure dispersion liquid is water or ethanol.
An oxygen-enriched nano-biological enzyme sensor device includes an electrode system, wherein the electrode system comprises the oxygen-enriched nano-biological enzyme electrode according to claim 1 or 2, characterized in that the oxygen-enriched nano-biological enzyme electrode is a working electrode, At least part of the working electrode is in contact with the solution of the object to be measured;

Preferably, the oxygen-enriched nano-biological enzyme electrode is a cathode;

Preferably, the electrode system further includes a counter electrode selected from one of a carbon rod electrode, a Pt electrode, a titanium electrode, or a platinum black electrode;

Preferably, the device further comprises a counter electrode and a reference electrode connected to the counter electrode; more preferably, the reference electrode is selected from the group consisting of a calomel electrode, a silver / silver chloride electrode, a mercury / mercury oxide electrode, and mercury / Mercury sulfate electrode.
An oxygen-enriched nano-biological enzyme test system, comprising the oxygen-enriched nano-biological enzyme sensor device according to claim 5 and a power supply system and a display system connected thereto.

Preferably, the power supply system includes an electrochemical workstation.
An electrochemical detection method for an oxygen-enriched nano-biological enzyme, using the oxygen-enriched nano-biological enzyme sensor device according to claim 5 or the oxygen-enriched nano-biological enzyme test system according to claim 6 to detect an object to be tested, which is characterized by The reason is that the concentration of the sample to be measured is obtained through the measured cathode reduction current signal or anode signal;

Preferably, the concentration of the sample to be measured is obtained through the measured cathode reduction current signal;

Preferably, the electrolyte used in the test is a buffer solution with pH = 5-8; more preferably, the concentration of the buffer solution is 0.1-2M; more preferably, the buffer solution includes: PBS buffer solution, sodium sulfate Buffer solution, disodium hydrogen phosphate-citrate buffer solution, acetic acid-sodium acetate buffer solution;

Preferably, the test object is selected from one or a combination of uric acid, glucose, urea, lactic acid, acetylcholine, and alcohol.
The electrochemical detection method for an oxygen-enriched nano-biological enzyme according to claim 7, wherein the test method of the cathode reduction current signal comprises cyclic voltammetry, linear scanning voltammetry, and current-time test.
A method for reducing interference and increasing the upper limit of detection in electrochemical detection of glucose, characterized in that the oxygen-enriched nano-biological enzyme sensor device according to claim 5 or the oxygen-enriched nano-biological enzyme test according to claim 6 is used. The system detects the object to be measured, which is characterized by comparing the measured cathodic reduction current signal with a standard curve of glucose, thereby reducing interference caused by fluctuations or shortages of oxygen content;

Preferably, the standard curve of glucose is prepared by adding standard samples of different known concentrations to the electrolyte for testing, and recording the relationship between the current signal and the glucose concentration to obtain a standard curve;

Preferably, the method for testing the cathode reduction current signal includes cyclic voltammetry, linear scanning voltammetry, and current-time testing;

More preferably, in the cyclic voltammetry test, the highest potential is 0.6V, the lowest point is -0.5V, the sweep speed is 0.01-1V / s, and the output signal is between -0.5-0.6V to obtain an anodizing current or a cathode. Reduction current

More preferably, in the linear scanning voltammetry, the highest potential is 0.6V, the lowest point is -0.3V, and the sweep speed is 0.01-0.1V / s, and the output signal is obtained at -0.1V or -0.2V to obtain cathode reduction. Current

More preferably, in the current-time test, a potential between 0-0.6V is used as a constant potential, the scanning time is 10-30s, and the current value between 5-10s is taken as the cathode reduction current.
The oxygen-enriched nanobiological enzyme electrode according to claim 1-2, or the oxygen-enriched nanobiological enzyme sensor device according to claim 5, or the oxygen-enriched nanobiological enzyme test system according to claim 6 detecting uric acid. , Creatinine, urea, glucose, lactic acid, acetylcholine, triglycerides, or cholesterol concentration equipment.