An enzyme-modified electrochemical electrode and its preparation method and ultra-thin enzyme plating device

Abstract

The present invention provides an enzyme-modified electrochemical electrode and a preparation method thereof and an ultra-thin enzyme plating device, wherein the enzyme-modified electrochemical electrode comprises a substrate electrode and an enzyme plating layer on the surface of the substrate electrode; the thickness of the enzyme plating layer is 10nm to 300nm, and the detection range of the enzyme-modified electrochemical electrode for inert substrates is 0.01μmol / L to 50mmol / L. The enzyme-modified electrochemical electrode of the present invention has low enzyme consumption and low production cost, which is conducive to large-scale promotion and use; the enzyme plating layer of the enzyme-modified electrochemical electrode has a nanometer-level thickness, fast response to the substrate, and a response delay time of microseconds; the enzyme plating layer of the enzyme-modified electrochemical electrode has little effect on the size of the substrate electrode, which is conducive to the use of the enzyme-modified electrochemical electrode in micrometer-level and nanometer-level scenarios; the enzyme-modified electrochemical electrode has a long life and is easy to preserve.

Description

Technical Field

[0001] The invention belongs to the technical field and relates to an enzyme modified electrochemical electrode, in particular to an enzyme modified electrochemical electrode and a preparation method thereof and an ultra-thin enzyme plating device. Background Art

[0002] Enzyme-based electrochemical electrodes are important tools for the detection and analysis of electrochemically inert substances in current life science research. With the help of a specific enzyme coating modified on the electrode surface, inert substrates can be catalyzed to produce electrochemically active products. The electrochemically active products can act as reporter molecules to undergo electrochemical reactions on the surface of enzyme-based electrochemical electrodes and generate signals, thereby obtaining concentration change information related to the inert substrate.

[0003] The enzyme coating modification method of the traditional enzyme-based electrochemical electrode is to evenly mix the biological enzyme with the cross-linking agent and then apply it to the electrode surface by infiltration or the like. First of all, this method has low requirements on the user's operating ability, but the large consumption of the enzyme leads to a high production cost of the electrode, especially for enzymes such as glutamate oxidase, which have a high unit price. Secondly, the commonly used cross-linking agent materials (such as glutaraldehyde, etc.) are toxic to biological enzymes, and often affect the activity of enzyme molecules during the modification process, affecting the sensitivity of the electrode. In addition, the traditional enzyme coating modification method is time-consuming. In order to stably modify the biological enzyme on the electrode surface, it is necessary to repeatedly immerse the electrode in the enzyme-containing droplets until the visible enzyme coating on the electrode surface will not dissolve when immersed in the enzyme-containing droplets. It usually needs to be repeated more than 20 times, and the modification of an electrode usually takes 5 to 15 minutes. In addition, in the actual operation of traditional enzyme coating modification, as the operation time becomes longer, the viscosity of the droplets containing biological enzymes and cross-linking agents continues to increase, resulting in uncontrollable amount of enzyme coated on the electrode surface, making it difficult to achieve uniformity of electrochemical electrode substrate sensitization, and affecting the quality control of the electrode. Finally, based on the traditional enzyme coating modification method, the thickness of the enzyme coating formed on the surface of the electrochemical electrode is usually above 2μm. On the one hand, the thickness above 2μm limits the application of such enzyme-based electrodes in microscopic to nanoscopic recording scenarios. On the other hand, the thicker enzyme coating also causes the electrode to respond slowly to substrate detection.

[0004] CN101196487A discloses a method for preparing a modified electrode by electrodepositing a chitosan-ionic liquid-enzyme composite film. During the preparation and modification, the electrode is placed as a cathode in an acidic solution containing chitosan, ionic liquid and enzyme, and an electric current is applied for electrodeposition. Chitosan, ionic liquid and enzyme are attached to the surface of the cathode electrode to obtain a modified electrode. However, the thickness of the modified electrode prepared by the method for electrodepositing a chitosan-ionic liquid-enzyme composite film is relatively large, which limits the application of the modified electrode in microscopic to nanoscopic level recording scenarios, and the detection response to inert substrates is slow.

[0005] CN111172571A discloses a method for preparing organic-inorganic hybrid nanoflowers by electrodeposition, which relates to the technical field of enzyme immobilization. The present invention mixes a rare earth nitrate aqueous solution with a biological enzyme and a nitrate to obtain a mixed solution; the rare earth ions in the rare earth nitrate are one or more of La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Yb and Y ions; the biological enzyme is α-amylase, horseradish peroxidase or laccase; then a three-electrode system is used to electrodeposit the obtained mixed solution, and an electrodeposited film is deposited on the surface of the working electrode; the electrodeposited film is then washed and dried in sequence to obtain an organic-inorganic hybrid nanoflower. However, the method for preparing organic-inorganic hybrid nanoflowers by electrodeposition adopts a relatively complex three-electrode system, and the operation process is relatively complicated.

[0006] CN102175736A discloses an enzyme electrode for detecting sterigmatocystin and a preparation method thereof, as well as a biosensor for detecting sterigmatocystin assembled with the enzyme electrode as a substrate electrode to fix aflatoxin oxidase. A functional L-cysteine ​​film is first self-assembled on the surface of a gold electrode, and then inserted into a hybrid Prussian blue electrodeposition working solution, and a Prussian blue hybrid electrode can be prepared by a constant potential method. Carbon nanotubes are used as electron transfer bodies, which are uniformly dispersed in a chitosan solution as a carrier for embedding aflatoxin oxidase, and then chitosan-aflatoxin oxidase-carbon nanotube mixed films are assembled on the surface of a hybrid Prussian blue modified electrode, and a biosensor for detecting sterigmatocystin is prepared. However, the preparation method of the enzyme electrode for detecting sterigmatocystin is complicated and has a high manufacturing cost, and is not suitable for large-scale promotion and use.

[0007] The currently disclosed enzyme-modified electrochemical electrodes and their preparation methods have certain defects, such as high preparation cost, long operation time, uncontrollable enzyme content on the surface of the substrate electrode, slow response to inert substrates, and difficulty in application in micron and nanometer scenarios. Therefore, it is very important to develop and design a new type of enzyme-modified electrochemical electrode and its preparation method and ultra-thin enzyme plating device. Summary of the invention

[0008] In view of the deficiencies in the prior art, the purpose of the present invention is to provide an enzyme-modified electrochemical electrode, a preparation method thereof, and an ultra-thin enzyme plating device. The enzyme-modified electrochemical electrode of the present invention has low enzyme consumption and low production cost, which is conducive to large-scale promotion and use; the enzyme plating thickness of the enzyme-modified electrochemical electrode is nanometer-level, and the response to the substrate is fast, and the response delay time is microsecond-level; the enzyme plating of the enzyme-modified electrochemical electrode has little effect on the size of the substrate electrode, which is conducive to the use of the enzyme-modified electrochemical electrode in micrometer-level and nanometer-level scenarios; the enzyme-modified electrochemical electrode has a long life and is easy to preserve.

[0009] To achieve this object, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides an enzyme-modified electrochemical electrode, wherein the enzyme-modified electrochemical electrode comprises a substrate electrode and an enzyme coating on the surface of the substrate electrode;

[0011] The thickness of the enzyme plating layer is 10nm-300nm, and the detection range of the enzyme-modified electrochemical electrode for inert substrates is 0.01μmol / L-50mmol / L.

[0012] The present invention limits the thickness of the enzyme coating to 10 nm to 300 nm, for example, it can be 10 nm, 20 nm, 30 nm, 50 nm, 80 nm, 100 nm, 120 nm, 150 nm, 200 nm or 300 nm, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0013] The present invention limits the detection range of the enzyme-modified electrochemical electrode for inert substrates to 0.01 μmol / L to 50 mmol / L, for example, it can be 0.1 μmol / L, 0.2 μmol / L, 0.5 μmol / L, 1 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, 50 μmol / L, 80 μmol / L, 0.1 mmol / L, 0.2 mmol / L, 0.3 mmol / L, 0.4 mmol / L, 0.5 mmol / L, 0.6 mmol / L, 0.7 mmol / L, 0.8 mmol / L, 0.9 mmol / L mol / L, 1mmol / L, 1.2mmol / L, 1.5mmol / L, 1.7mmol / L, 2mmol / L, 3mmol / L, 5mmol / L, 7mmol / L, 10mmol / L, 12mmol / L, 15mmol / L, 17mmol / L, 20mmol / L, 22mmol / L, 25mmol / L, 30mmol / L, 35mmol / L, 40mmol / L, 45mmol / L or 50mmol / L, but is not limited to the listed values, and other unlisted values ​​within the numerical range are equally applicable.

[0014] The inert substrate of the present invention includes any one of glutamate, glucose, ethanol, aspartic acid, pyruvic acid, lysine, lactic acid or acetylcholine, or a combination of at least two thereof. Typical but non-limiting combinations include a combination of glutamate and glucose, a combination of ethanol and aspartic acid, a combination of aspartic acid and pyruvic acid, a combination of pyruvic acid and lysine, a combination of lactic acid and acetylcholine, a combination of glucose and lactic acid, a combination of lactic acid and acetylcholine, a combination of glutamate, glucose and lactic acid, or a combination of glutamate, glucose, lactic acid and acetylcholine.

[0015] The enzyme modified electrochemical electrode of the present invention has low enzyme consumption and low production cost, which is conducive to large-scale promotion and use; the enzyme coating thickness of the enzyme modified electrochemical electrode is nanometer-level, the response to the substrate is fast, and the response delay time is microsecond-level; the enzyme coating of the enzyme modified electrochemical electrode has little effect on the size of the substrate electrode, which is conducive to the use of the enzyme modified electrochemical electrode in micrometer-level and nanometer-level scenarios; the enzyme modified electrochemical electrode has a long life and is easy to preserve.

[0016] As a preferred technical solution of the present invention, the thickness of the enzyme coating is 100nm to 300nm, for example, it can be 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 220nm, 240nm, 260nm, 280nm or 300nm, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0017] Preferably, the detection range of the enzyme-modified electrochemical electrode for inert substrates is 0.01 μmol / L to 100 μmol / L, for example, it can be 0.01 μmol / L, 0.02 μmol / L, 0.05 μmol / L, 0.1 μmol / L, 0.5 μmol / L, 0.1 μmol / L, 0.5 μmol / L, 10 μmol / L, 15 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, 60 μmol / L, 70 μmol / L, 80 μmol / L, 90 μmol / L or 100 μmol / L, but is not limited to the listed values, and other unlisted values ​​within the numerical range are equally applicable.

[0018] In a second aspect, the present invention provides a method for preparing the enzyme-modified electrochemical electrode as described in the first aspect, the preparation method comprising:

[0019] The substrate electrode is used as the working electrode and constitutes a two-electrode system with the auxiliary electrode, and electrodeposition is performed in an enzyme-containing solution to obtain an enzyme-modified electrochemical electrode.

[0020] The present invention adopts an electroplating method to replace the coating operation. The preparation method of the enzyme-modified electrochemical electrode is simple to operate, does not need to perform repeated coating operations, reduces the operation time, the amount of enzyme modified on the electrode surface is controllable, and the enzyme consumption in the preparation process is low.

[0021] As a preferred technical solution of the present invention, the substrate electrode includes any one of a platinum electrode, a gold electrode, a platinum alloy electrode, a gold alloy electrode or a glassy carbon electrode, or a combination of at least two thereof. Typical but non-limiting combinations include a combination of a platinum electrode and a gold electrode, a combination of a gold electrode and a platinum alloy electrode, a combination of a platinum alloy electrode and a gold alloy electrode, a combination of a gold electrode and a glassy carbon electrode, a combination of a platinum electrode and a glassy carbon electrode, or a combination of a platinum electrode, a gold electrode and a glassy carbon electrode.

[0022] Preferably, the material of the auxiliary electrode includes any one of platinum, gold or silver, or a combination of at least two of them. Typical but non-limiting combinations include a combination of platinum and gold, a combination of gold and silver, and a combination of platinum and silver.

[0023] As a preferred technical solution of the present invention, the enzyme-containing solution is obtained by mixing an enzyme solution with a chitosan solution.

[0024] The chitosan solution in the present application affects the response time of the enzyme-modified electrochemical electrode to the inert substrate and the binding force between the enzyme coating and the substrate electrode. This is because chitosan is an alkaline polysaccharide with a large number of free amino groups in its structure, which makes it have good biocompatibility and biosafety, and thus has a good protective effect on the catalytic performance of the enzyme molecules; the polymer product formed by chitosan and enzyme molecules on the electrode surface is thin, but has a uniform texture, a smooth surface and no obvious gaps, which is suitable for the rapid and stable enzymatic reaction.

[0025] Preferably, the solute of the enzyme solution includes any one of glutamate oxidase, glucose oxidase, lactate oxidase or acetylcholinesterase, or a combination of at least two of them. Typical but non-limiting combinations include a combination of glutamate oxidase and glucose oxidase, a combination of glucose oxidase and lactate oxidase, a combination of lactate oxidase and acetylcholinesterase, a combination of glutamate oxidase, glucose oxidase and lactate oxidase, or a combination of glutamate oxidase, glucose oxidase, lactate oxidase and acetylcholinesterase.

[0026] Preferably, the volume ratio of the enzyme solution to the chitosan solution is (0.8-1.2):1, for example, it can be 0.8:1, 0.85:1, 0.9:1, 0.95:1, 1:1, 1.05:1, 1.1:1, 1.15:1 or 1.2:1, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0027] In the present application, the volume ratio of the enzyme solution to the chitosan solution will affect the response time of the enzyme-modified electrochemical electrode to the inert substrate and the binding force between the enzyme coating and the substrate electrode; when the volume ratio of the enzyme solution to the chitosan solution is low, the response time will be longer and the binding force will be enhanced, because the lower proportion of enzyme molecules weakens the enzyme reaction efficiency, while the higher proportion of chitosan enhances the binding between the modified layer and the electrode surface; when the volume ratio of the enzyme solution to the chitosan solution is high, the response time will be shorter and the binding force will be weakened, because the higher proportion of enzyme molecules enhances the enzyme reaction efficiency, while the lower proportion of chitosan weakens the binding between the modified layer and the electrode surface.

[0028] Preferably, the concentration of the enzyme solution is 10 unit / mL to 500 unit / mL, for example, it can be 10 unit / mL, 20 unit / mL, 30 unit / mL, 50 unit / mL, 70 unit / mL, 100 unit / mL, 120 unit / mL, 150 unit / mL, 170 unit / mL, 200 unit / mL, 250 unit / mL, 300 unit / mL, 350 unit / mL, 400 unit / mL, 450 unit / mL or 500 unit / mL, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0029] Preferably, the mass concentration of the chitosan solution is 0.5% w / v to 2.0% w / v, for example, it can be 0.5% w / v, 0.6% w / v, 0.7% w / v, 0.8% w / v, 0.9% w / v, 1% w / v, 1.1% w / v, 1.2% w / v, 1.3% w / v, 1.4% w / v, 1.5% w / v, 1.6% w / v, 1.7% w / v, 1.8% w / v, 1.9% w / v or 2% w / v, but is not limited to the listed values, and other unlisted values ​​within the numerical range are equally applicable.

[0030] Preferably, the pH of the chitosan solution is 5.0-6.0, for example, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0, but is not limited to the listed values, and other values ​​not listed within the range are also applicable.

[0031] As a preferred technical solution of the present invention, the electrodeposition is constant potential electrodeposition, and the potential of the constant potential electrodeposition is -0.18V to -0.13V, for example, it can be -0.18V, -0.17V, -0.16V, -0.15V, -0.14V or -0.13V, but it is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0032] Preferably, the area of ​​the working electrode immersed in the enzyme solution is 40 μm to 800,000 μm 2 , for example, it can be 40 μm 2 , 50μm 2 、60μm 2 、70μm 2 、80μm 2 , 100μm 2 , 200μm 2 , 500μm 2 , 1000μm 2 , 2000μm 2 , 5000μm 2 、7000μm 2 , 10000μm 2 , 20000μm 2 、30000μm 2 , 40000μm 2 , 50000μm 2 、60000μm 2 、70000μm 2 、80000μm 2 , 100000μm 2 、300000μm 2 or 800000μm 2 , but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0033] Preferably, the electrodeposition time is 10s to 500s, for example, it can be 10s, 11s, 12s, 15s, 18s, 20s, 25s, 28s, 30s, 35s, 40s, 50s, 80s, 100s, 150s, 200s, 250s, 300s, 350s, 400s, 450s or 500s, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0034] As a preferred technical solution of the present invention, illustratively, the preparation method comprises:

[0035] The substrate electrode is used as a working electrode and forms a two-electrode system with an auxiliary electrode, and constant potential electrodeposition is performed in a mixed solution of an enzyme solution with a concentration of 10 unit / mL to 500 unit / mL and a chitosan solution with a concentration of 0.5% w / v to 2.0% w / v and a pH of 5.0 to 6.0 to obtain an enzyme-modified electrochemical electrode;

[0036] The volume ratio of enzyme solution to chitosan solution is (0.8-1.2):1, and the area of ​​the working electrode immersed in the mixed solution is 40 μm 2 ~80000μm 2 The potential of constant potential electrodeposition is -0.18V to -0.13V, and the time is 10s to 500s.

[0037] In a third aspect, the present invention provides an ultra-thin enzyme plating device based on the preparation method described in the second aspect, wherein the ultra-thin enzyme plating device comprises an enzyme plating pool and an enzyme plating pool control member movably connected to the enzyme plating pool;

[0038] The enzyme plating pool control component controls the enzyme plating pool to move in three-dimensional directions.

[0039] The enzyme plating pool control component of the present invention controls the movement of the enzyme plating pool with an accuracy of 3μm to 10μm, for example, it can be 3μm, 4μm, 5μm, 6μm, 7μm, 8μm, 9μm or 10μm, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0040] As a preferred technical solution of the present invention, the enzyme plating pool includes a protective shell, an auxiliary electrode slot is arranged in the protective shell, an enzyme-containing solution is arranged in the auxiliary electrode slot, and an auxiliary electrode connecting line is connected to the auxiliary electrode slot.

[0041] The material of the protective shell of the present invention includes insulating materials, such as polypropylene plastic, but is not limited to polypropylene plastic, and other insulating materials not listed are also applicable.

[0042] The auxiliary electrode connection wire of the present invention includes a metal wire with an insulating layer on its surface, for example, a copper single wire covered with an insulating layer, but is not limited to a copper wire, and other metal wires not listed are also applicable.

[0043] Preferably, the material of the auxiliary electrode groove includes any one of platinum, gold or silver, or a combination of at least two of them, for example, a combination of platinum and gold, a combination of gold and silver, or a combination of platinum, gold and silver.

[0044] As a preferred technical solution of the present invention, the ultra-thin enzyme plating device further comprises a bottom plate, and the enzyme plating pool control component is fixedly arranged on the bottom plate.

[0045] Preferably, the bottom plate comprises a magnetic metal plate.

[0046] The magnetic metal plate of the present invention can be adsorbed on the base, which is convenient for the electrodeposition and the disassembly of the ultra-thin enzyme plating device after the electrodeposition.

[0047] As a preferred technical solution of the present invention, a working electrode control component is also provided on the bottom plate, one end of the working electrode is fixedly provided on the working electrode control component, and the other end is placed in the enzyme plating pool; the working electrode control component controls the movement of the working electrode in three-dimensional directions.

[0048] The working electrode control component of the present invention comprises a working electrode fixing groove, in which one end of the working electrode is fixed.

[0049] The working electrode control component of the present invention controls 1 to 10 working electrodes simultaneously, for example, it can be 1, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0050] Preferably, the material of the working electrode control element includes any one of sponge, rubber or foam plastic or a combination of at least two of them, typical but non-limiting combinations are a combination of sponge and rubber, a combination of rubber and foam plastic, or a combination of sponge, rubber and foam plastic.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] (1) The enzyme-modified electrochemical electrode of the present invention has low enzyme consumption and low production cost, which is conducive to large-scale promotion and use;

[0053] (2) The enzyme coating thickness of the enzyme-modified electrochemical electrode of the present invention is in the nanometer range, and the response to the substrate is fast, with a response delay time in the microsecond range;

[0054] (3) The enzyme coating of the enzyme-modified electrochemical electrode of the present invention has little effect on the size of the substrate electrode, which is conducive to the use of the enzyme-modified electrochemical electrode in micrometer- and nanometer-scale scenarios;

[0055] (4) The enzyme-modified electrochemical electrode of the present invention has a long life and is easy to store;

[0056] (5) The present invention adopts an electroplating method to replace the coating operation. The preparation method of the enzyme-modified electrochemical electrode is simple to operate, does not require repeated coating operations, reduces the operation time, and the amount of enzyme modified on the electrode surface is controllable. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 A schematic structural diagram of an ultrathin enzyme plating device provided for a specific embodiment of the present invention.

[0058] Among them, 1-enzyme plating pool control part; 2-protective shell; 3-auxiliary electrode slot; 4-enzyme solution; 5-auxiliary electrode connecting line; 6-bottom plate; 7-working electrode fixing slot; 8-working electrode. DETAILED DESCRIPTION

[0059] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0060] In a specific embodiment, Figure 1 As shown, the present invention provides an ultra-thin enzyme plating device, which includes an enzyme plating pool and an enzyme plating pool control member 1 movably connected to the enzyme plating pool;

[0061] The enzyme plating pool control component 1 controls the enzyme plating pool to move in three-dimensional directions.

[0062] The enzyme plating pool control component 1 of the present invention controls the movement of the enzyme plating pool with an accuracy of 3 μm to 10 μm.

[0063] Furthermore, the enzyme plating cell comprises a protective shell 2 , an auxiliary electrode slot 3 is arranged in the protective shell 2 , an enzyme-containing solution 4 is arranged in the auxiliary electrode slot 3 , and an auxiliary electrode connecting line 5 is connected to the auxiliary electrode slot 3 .

[0064] The material of the protective shell 2 of the present invention includes insulating materials, such as polypropylene plastic, but is not limited to polypropylene plastic, and other insulating materials not listed are also applicable.

[0065] The auxiliary electrode connection line 5 of the present invention includes a metal wire with an insulating layer on its surface, for example, a copper single wire covered with an insulating layer, but is not limited to a copper wire, and other metal wires not listed are also applicable.

[0066] Furthermore, the material of the auxiliary electrode groove 3 includes any one of platinum, gold or silver, or a combination of at least two of them.

[0067] Furthermore, the ultra-thin enzyme plating device also includes a bottom plate 6 , and the enzyme plating pool control component 1 is fixedly disposed on the bottom plate 6 .

[0068] Furthermore, the bottom plate 6 comprises a magnetic metal plate.

[0069] The magnetic metal plate of the present invention can be adsorbed on the base, which is convenient for the electrodeposition and the disassembly of the ultra-thin enzyme plating device after the electrodeposition.

[0070] Furthermore, a working electrode control component is also provided on the bottom plate 6, one end of the working electrode 8 is fixedly provided on the working electrode control component, and the other end is placed in the enzyme plating pool; the working electrode control component controls the movement of the working electrode 8 in three-dimensional directions.

[0071] The working electrode control component of the present invention comprises a working electrode fixing groove 7 , in which one end of a working electrode 8 is fixed.

[0072] The working electrode control component of the present invention controls 1 to 10 working electrodes 8 simultaneously.

[0073] Furthermore, the material of the working electrode control element includes any one of sponge, rubber or foam plastic, or a combination of at least two of them.

[0074] Example 1

[0075] This embodiment provides a glutamate oxidase modified electrochemical electrode, wherein the glutamate oxidase modified electrochemical electrode comprises a platinum electrode and a glutamate oxidase coating on the surface of the platinum electrode;

[0076] The thickness of the glutamate oxidase coating is 300 nm, and the detection range of the glutamate oxidase modified electrochemical electrode to glutamate is 0.01 μmol / L to 100 μmol / L.

[0077] This embodiment provides a method for preparing the glutamate oxidase modified electrochemical electrode, the preparation method comprising:

[0078] A platinum electrode was used as a working electrode and a platinum auxiliary electrode formed a two-electrode system, and constant potential electrodeposition was performed in a mixed solution of a glutamate oxidase solution with a concentration of 33 unit / mL and a chitosan solution with a mass concentration of 1% w / v and a pH of 5.5 to obtain a glutamate oxidase modified electrochemical electrode;

[0079] The volume ratio of glutamate oxidase solution to chitosan solution was 0.9:1, and the area of ​​the platinum electrode immersed in the mixed solution was 40 μm 2 The potential of constant potential electrodeposition was -0.15 V and the time was 500 s.

[0080] Example 2

[0081] This embodiment provides a glucose oxidase modified electrochemical electrode, wherein the glucose oxidase modified electrochemical electrode comprises a platinum electrode and a glucose oxidase coating on the surface of the platinum electrode;

[0082] The thickness of the glucose oxidase coating is 80 nm, and the detection range of the glucose oxidase modified electrochemical electrode for inert substrates is 1 mmol / L to 25 mmol / L.

[0083] This embodiment provides a method for preparing the glucose oxidase modified electrochemical electrode, the preparation method comprising:

[0084] A platinum electrode was used as a working electrode and a platinum auxiliary electrode formed a two-electrode system, and constant potential electrodeposition was performed in a mixed solution of a glucose oxidase solution with a concentration of 100 unit / mL and a chitosan solution with a mass concentration of 0.8% w / v and a pH of 5.2 to obtain a glucose oxidase modified electrochemical electrode;

[0085] The volume ratio of glucose oxidase solution to chitosan solution is 1.1:1, and the area of ​​the platinum electrode immersed in the mixed solution is 200 μm 2 The potential of constant potential electrodeposition was -0.16 V and the time was 50 s.

[0086] Example 3

[0087] This embodiment provides a lactate oxidase modified electrochemical electrode, wherein the lactate oxidase modified electrochemical electrode comprises a glassy carbon electrode and a lactate oxidase coating on the surface of the glassy carbon electrode;

[0088] The thickness of the lactate oxidase coating is 100 nm, and the detection range of the lactate oxidase modified electrochemical electrode for inert substrates is 10 μmol / L to 10 mmol / L.

[0089] This embodiment provides a method for preparing the lactate oxidase modified electrochemical electrode, the preparation method comprising:

[0090] A glassy carbon electrode was used as the working electrode and a gold auxiliary electrode formed a two-electrode system. Constant potential electrodeposition was performed in a mixed solution of a lactate oxidase solution with a concentration of 100 unit / mL and a chitosan solution with a mass concentration of 1.2% w / v and a pH of 5.7 to obtain an enzyme-modified electrochemical electrode.

[0091] The volume ratio of lactate oxidase solution to chitosan solution is 1:1, and the area of ​​the glassy carbon electrode immersed in the mixed solution is 800000 μm 2 The potential of constant potential electrodeposition was -0.13 V and the time was 100 s.

[0092] Example 4

[0093] This embodiment provides a glutamate oxidase modified electrochemical electrode, wherein the glutamate oxidase modified electrochemical electrode comprises a gold electrode and a glutamate oxidase coating on the surface of the gold electrode;

[0094] The thickness of the glutamate oxidase coating is 10 nm, and the detection range of the glutamate oxidase modified electrochemical electrode for inert substrates is 1 mmol / L to 50 mmol / L.

[0095] This embodiment provides a method for preparing the glutamate oxidase modified electrochemical electrode, the preparation method comprising:

[0096] A gold electrode was used as the working electrode and a platinum auxiliary electrode formed a two-electrode system, and constant potential electrodeposition was performed in a mixed solution of a glutamate oxidase solution with a concentration of 10 unit / mL and a chitosan solution with a concentration of 2% w / v and a pH of 6.0 to obtain an enzyme-modified electrochemical electrode;

[0097] The volume ratio of glutamate oxidase solution to chitosan solution is 1.2:1, and the area of ​​the gold electrode immersed in the mixed solution is 200 μm 2 The potential of constant potential electrodeposition was -0.18 V and the time was 10 s.

[0098] Example 5

[0099] This example provides a method for preparing a glutamate oxidase modified electrochemical electrode, which is the same as Example 1 except that the chitosan solution is replaced with an equal volume of glutamate oxidase solution.

[0100] Example 6

[0101] This example provides a method for preparing a glutamate oxidase modified electrochemical electrode, which is the same as Example 1 except that the volume ratio of the glutamate oxidase solution to the chitosan solution is 0.6:1.

[0102] Example 7

[0103] This example provides a glutamate oxidase modified electrochemical electrode, which is the same as Example 1 except that the volume ratio of the glutamate oxidase solution to the chitosan solution is 1.4:1.

[0104] The response time of the glutamate oxidase modified electrochemical electrode in Examples 1 and Examples 4 to 7 to 2 μmol / L glutamate was tested, the response time of the glucose oxidase modified electrochemical electrode in Example 2 to 1 mmol / L glucose was tested, and the response time of the lactate oxidase modified electrochemical electrode in Example 3 to 1 mmol / L lactic acid was tested. The test method of the response time is: electrochemical amperometry, that is, a three-electrode system is used, the working electrode is the electrochemical electrode to be tested, and the Ag / AgCl electrode is the reference electrode. The electrode is a platinum electrode as a counter electrode; the working voltage of the electrochemical electrode to be tested is 0.6-0.8V; the test is carried out at 24-26°C, the three electrodes are inserted into a reaction cell containing 20mL of a phosphate buffer solution with a pH of 7.2-7.4, the volume of the reaction cell is 100mL, the solution is magnetically stirred at a speed of 300-500rpm, an appropriate amount of a test mother solution is added dropwise to the solution, and the electrochemical current response is observed, and the time interval from the addition of the test mother solution to the current response reaching 90% of its maximum value is calculated as the value of the response time.

[0105] The shelf life of the enzyme-modified electrochemical electrodes in Examples 1 to 7 was tested, and the obtained shelf life is shown in Table 1. The test method for the shelf life is as follows: the electrochemical electrode is stored in a 4°C refrigerator in the dark on a daily basis; the test method is the same as the test method for the response time, and an appropriate amount of test stock solution is added to the solution to observe the current response of the electrochemical electrode, thereby comparing the responses of the electrochemical electrodes with different shelf lives (with a pass standard of not less than 80% of the initial response).

[0106] Table 1

[0107]

[0108]

[0109] From Table 1, we can see that:

[0110] (1) The enzyme-modified electrochemical electrode obtained in Examples 1 to 4 has a short response time to an inert substrate and a long shelf life. The enzyme-modified electrochemical electrode of the present invention has a low enzyme consumption and a low production cost, which is conducive to large-scale promotion and use; the enzyme coating thickness of the enzyme-modified electrochemical electrode is nanometer-level, and the response to the substrate is fast, and the response delay time is microsecond-level; the enzyme coating of the enzyme-modified electrochemical electrode has little effect on the size of the substrate electrode, which is conducive to the use of the enzyme-modified electrochemical electrode in micrometer-level and nanometer-level scenarios; the enzyme-modified electrochemical electrode has a long life and is easy to preserve;

[0111] (2) By comparing Example 1 with Example 5, it can be seen that the chitosan solution affects the response time of the enzyme-modified electrochemical electrode to the inert substrate and the binding force between the enzyme coating and the substrate electrode. This is because chitosan is an alkaline polysaccharide with a large number of free amino groups in its structure, which makes it have good biocompatibility and biosafety, and thus has a good protective effect on the catalytic performance of the enzyme molecules; the polymer product formed by chitosan and enzyme molecules on the electrode surface is thin, but has a uniform texture, a smooth surface and no obvious gaps, which is suitable for the rapid and stable enzymatic reaction;

[0112] (3) By comparing Example 1 with Examples 6 and 7, it can be seen that the volume ratio of the enzyme solution to the chitosan solution affects the response time of the enzyme-modified electrochemical electrode to the inert substrate and the binding force between the enzyme coating and the substrate electrode; when the volume ratio of the enzyme solution to the chitosan solution is low, the response time will be longer and the binding force will be enhanced. This is because the low proportion of enzyme molecules weakens the enzyme reaction efficiency, while the high proportion of chitosan enhances the binding between the modified layer and the electrode surface; when the volume ratio of the enzyme solution to the chitosan solution is high, the response time will be shorter and the binding force will be weakened. This is because the high proportion of enzyme molecules enhances the enzyme reaction efficiency, while the low proportion of chitosan weakens the binding between the modified layer and the electrode surface.

[0113] In summary, the enzyme modified electrochemical electrode of the present invention has low enzyme consumption and low production cost, which is conducive to large-scale promotion and use; the enzyme coating thickness of the enzyme modified electrochemical electrode is nanometer-level, the response to the substrate is fast, and the response delay time is microsecond-level; the enzyme coating of the enzyme modified electrochemical electrode has little effect on the size of the substrate electrode, which is conducive to the use of the enzyme modified electrochemical electrode in micrometer-level and nanometer-level scenarios; the binding force between the enzyme coating of the enzyme modified electrochemical electrode and the substrate electrode is stronger and not easy to fall off, and the enzyme modified electrochemical electrode has a long life and is easy to preserve; the present invention adopts an electroplating method to replace the coating operation, and the preparation method of the enzyme modified electrochemical electrode is simple to operate, does not need to perform repeated coating operations, reduces the operation time, and the amount of enzyme modified on the electrode surface is controllable.

[0114] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention are within the protection scope and disclosure scope of the present invention.

Claims

1. An ultra-thin enzyme plating device, characterized in that: The ultra-thin enzyme plating device comprises an enzyme plating pool and an enzyme plating pool control component movably connected to the enzyme plating pool; The enzyme plating pool control component controls the enzyme plating pool to move in three-dimensional directions; The enzyme plating pool comprises a protective shell, an auxiliary electrode slot is arranged in the protective shell, an enzyme-containing solution is arranged in the auxiliary electrode slot, and an auxiliary electrode connecting line is connected to the auxiliary electrode slot; The ultra-thin enzyme plating device also includes a bottom plate, and the enzyme plating pool control component is fixedly arranged on the bottom plate; The bottom plate is also provided with a working electrode control component, one end of the working electrode is fixedly provided on the working electrode control component, and the other end is placed in the enzyme plating pool; the working electrode control component controls the movement of the working electrode in three-dimensional directions; The ultra-thin enzyme plating device is prepared based on the following method: The substrate electrode is used as the working electrode and forms a two-electrode system with the auxiliary electrode, and electrodeposition is performed in an enzyme-containing solution to obtain an enzyme-modified electrochemical electrode; The enzyme-modified electrochemical electrode comprises a substrate electrode and an enzyme coating on the surface of the substrate electrode; The thickness of the enzyme plating layer is 10nm-300nm, and the detection range of the enzyme-modified electrochemical electrode for inert substrates is 0.01μmol / L-50mmol / L.

2. The ultra-thin enzyme plating device according to claim 1, characterized in that: The thickness of the enzyme coating is 100nm to 300nm.

3. The ultra-thin enzyme plating device according to claim 1, characterized in that: The detection range of the enzyme-modified electrochemical electrode for inert substrates is 0.01 μmol / L to 100 μmol / L.

4. The ultra-thin enzyme plating device according to claim 1, characterized in that: The substrate electrode includes any one of a platinum electrode, a gold electrode, a platinum alloy electrode, a gold alloy electrode or a glassy carbon electrode, or a combination of at least two thereof.

5. The ultra-thin enzyme plating device according to claim 1, characterized in that: The material of the auxiliary electrode includes any one of platinum, gold or silver, or a combination of at least two of them.

6. The ultra-thin enzyme plating device according to claim 1, characterized in that: The enzyme-containing solution is obtained by mixing an enzyme solution with a chitosan solution.

7. The ultra-thin enzyme plating device according to claim 6, characterized in that: The solute of the enzyme solution includes any one of glutamate oxidase, glucose oxidase, lactate oxidase or acetylcholinesterase, or a combination of at least two thereof.

8. The ultra-thin enzyme plating device according to claim 6, characterized in that: The volume ratio of the enzyme solution to the chitosan solution is (0.8-1.2):

1.

9. The ultra-thin enzyme plating device according to claim 6, characterized in that: The concentration of the enzyme solution is 10 unit / mL to 500 unit / mL.

10. The ultra-thin enzyme plating device according to claim 6, characterized in that: The mass concentration of the chitosan solution is 0.5% w / v to 2.0% w / v.

11. The ultra-thin enzyme plating device according to claim 6, characterized in that: The pH of the chitosan solution is 5.0-6.

0.

12. The ultra-thin enzyme plating device according to claim 1, characterized in that: The electrodeposition is constant potential electrodeposition, and the potential of the constant potential electrodeposition is -0.18V to -0.13V.

13. The ultra-thin enzyme plating device according to claim 1, characterized in that: The area of ​​the working electrode immersed in the enzyme solution is 40 μm 2 ~800000μm 2 .

14. The ultra-thin enzyme plating device according to claim 1, characterized in that: The electrodeposition time is 10s to 500s.

15. The ultra-thin enzyme plating device according to claim 1, characterized in that: The material of the auxiliary electrode groove includes any one of platinum, gold or silver, or a combination of at least two of them.

16. The ultra-thin enzyme plating device according to claim 1, characterized in that: The bottom plate includes a magnetic metal plate.

17. The ultra-thin enzyme plating device according to claim 1, characterized in that: The material of the working electrode control element includes any one of sponge, rubber or foam plastic or a combination of at least two of them.

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