Microelectrode array and surface modification method and application thereof

Abstract

The invention relates to a microelectrode array as well as a surface modification method and application thereof, belongs to the technical field of biosensors, and aims at solving one of the problems that a traditional electrode in the prior art is complicated in preparation and low in sensitivity and cannot detect glutamic acid and dopamine at the same time. According to the method disclosed by the invention, the polypyrrole films are deposited on the dopamine detection microelectrode and the glutamic acid detection microelectrode, so that interference substances can be isolated. Meanwhile, the interference of dopamine with positive charges also needs to be eliminated in addition to the elimination of AA on the glutamic acid detection site; according to the invention, the selected Nafion membrane is a cation selective permeable membrane, and cations can pass through the membrane to isolate anions; the peroxide polypyrrole thin film is also a cation exchange membrane, polypyrrole is formed through electropolymerization of pyrrole, then high-potential electrooxidation is carried out, and in the electrooxidation process, electronegative carbon groups formed along the main chain of the peroxide polypyrrole can effectively attract cations and repel anions.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and in particular to a microelectrode array and a surface modification method and application thereof. Background Art

[0002] Glutamate and dopamine are two key neurotransmitters, and changes in neurotransmitter concentrations directly reflect abnormalities in the nervous system. Implantable analytical probes that can synchronously monitor glutamate and dopamine in real time with high spatial resolution are very important for neuroscience research. However, neurotransmitter monitoring probes face many challenges, including low detection sensitivity (because the concentration of neurotransmitters in the brain is low, generally in the nanomolar to micro-nanomolar range) and interference signals from electroactive stems in the extracellular fluid.

[0003] Dopamine is an electroactive substance that contains special groups that can directly undergo redox reactions. It reacts at a certain voltage to form the gain and loss and transfer of electrons. Glutamate is a non-electroactive substance. Its detection uses glutamate oxidase to catalyze glutamate in the presence of oxygen and water to produce α-ketoglutaric acid, ammonia and hydrogen peroxide. Neutral and electroactive small molecules of hydrogen peroxide can be oxidized at an appropriate potential on the electrode surface to obtain a current signal related to the glutamate concentration in the detection area of ​​the brain. In addition, the electrode where hydrogen peroxide is oxidized is usually a platinum electrode, which can undergo redox reactions.

[0004] The traditional way to fix glutamate enzyme on the electrode surface is to use a mixture of stabilizer glutaraldehyde and cross-linker bovine serum albumin (BSA) under a microscope to fix the enzyme on a specific electrode site. First, it is difficult to manually apply the enzyme to the target site, and second, the thickness and consistency of the enzyme deposition on the site are difficult to control. In addition, the microelectrode of the existing technology cannot detect dopamine and glutamate at the same time. Summary of the invention

[0005] In view of the above analysis, the present invention aims to provide a microelectrode array and a surface modification method and application thereof, so as to solve one of the problems in the prior art, such as the complex preparation of traditional electrodes, low sensitivity, and inability to simultaneously detect glutamate and dopamine.

[0006] In a first aspect, a surface modification method for a microelectrode array for synchronously detecting glutamate and dopamine is provided, wherein the microelectrode array comprises a glutamate detection microelectrode and a dopamine detection microelectrode, and the method comprises the following steps:

[0007] (1) depositing a layer of polypyrrole film on a glutamate detection microelectrode and a dopamine detection microelectrode respectively, subjecting the polypyrrole film to peroxidation treatment to form a peroxidized polypyrrole film, and then dipping in a Nafion solution, drying, and forming a layer of Nafion membrane outside the oxidized polypyrrole film;

[0008] (2) depositing a layer of chitosan film on the glutamate detection microelectrode treated in step (1), then immersing it in a glutamate oxidase solution and refrigerating it.

[0009] Furthermore, in step (1), the thickness of the polypyrrole peroxide film on the glutamate detection microelectrode is greater than the thickness of the polypyrrole peroxide film on the dopamine detection microelectrode.

[0010] Furthermore, in step (1), a layer of polypyrrole film is deposited on the glutamate detection microelectrode. The specific method is as follows: the microelectrode array is added to a 150-250 mM pyrrole PBS solution, and the glutamate detection microelectrode is electroplated at a voltage of 0.8-1 V for 5-10 minutes.

[0011] Furthermore, in step (1), a layer of polypyrrole film is deposited on the dopamine detection microelectrode. The specific method is as follows: the microelectrode array is added to a 2-5mM pyrrole PBS solution, and the dopamine detection microelectrode is scanned at a voltage of 0.2-1.2V and a scanning speed of 20mv / s for 2-5 cycles.

[0012] Furthermore, the polypyrrole film is electro-oxidized at a voltage of 0.9-1V to form a peroxidized polypyrrole film.

[0013] Furthermore, in step (1), the mass concentration of the Nafion solution is 5%, and the solution is baked at 180° C. for 3 minutes.

[0014] Furthermore, step (2) further comprises dissolving chitosan in deionized water, adding acid to adjust the pH value to 3, then adding alkali to adjust the pH value to 5, and then depositing a chitosan film. Preferably, the mass volume ratio of chitosan to deionized water is 0.3-0.5 mg / mL.

[0015] Furthermore, in step (2), the treated glutamate detection microelectrode is electroplated at a voltage of -0.8 to -1.2 V for 2 minutes to form a chitosan film.

[0016] Furthermore, in step (2), the glutamate oxidase solution is a PBS solution of glutamate peroxidase, the enzyme activity of glutamate oxidase in the PBS solution is 125 units / mL, and the refrigerated temperature is 4°C.

[0017] In a second aspect, the present invention provides a microelectrode array modified by the method for synchronously detecting glutamate and dopamine.

[0018] In a third aspect, the present invention provides an application of a microelectrode array in the synchronous detection of glutamate and dopamine, wherein the microelectrode array is modified by the method described above.

[0019] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects:

[0020] (1) The method of the present invention deposits a layer of polypyrrole film on the dopamine detection microelectrode and the glutamate detection microelectrode, which can achieve the isolation of interfering substances. The interfering substances in the extracellular fluid are mainly negatively charged ascorbic acid (AA). At the same time, in addition to excluding AA, the interference of positively charged dopamine (DA) needs to be excluded from the glutamate detection site; the Nafion membrane selected in the present invention is a cation selective permeable membrane, which can pass cations and isolate anions; the peroxidized polypyrrole film is also a cation exchange membrane, which is first formed into polypyrrole (PPy) by electropolymerization of pyrrole (Py), and then subjected to high potential electrooxidation. During the electrooxidation process, the electronegative carbon group formed along the main chain of the peroxidized polypyrrole can effectively attract cations and repel anions;

[0021] (2) The method of the present invention uses chitosan as an adsorption substrate for glutamate oxidase fixation, and selectively deposits chitosan on a glutamate detection microelectrode. Specifically, the pH value of the chitosan solution is changed by applying different potentials to the electrode. When a high negative potential is applied, the local pH value around the electrode increases, and chitosan is deposited on the electrode, thereby achieving selective deposition of chitosan to form a chitosan film, thereby achieving selective glutamate oxidase fixation.

[0022] (3) The electrodeposition on the surface of the microelectrode for detecting dopamine and glutamate of the present invention adopts different deposition conditions so that the thickness of the peroxide polypyrrole (OPPy) film deposited on the two electrodes is different, and the thickness on the dopamine detection microelectrode is less than the thickness on the glutamate detection microelectrode; the thin peroxide polypyrrole film can enhance the adsorption of dopamine on the electrode surface to improve the sensitivity of detecting dopamine, and the relatively thick peroxide polypyrrole film can simultaneously repel negatively charged ascorbic acid and positively charged dopamine, allowing neutral small molecule hydrogen peroxide to penetrate. The present invention can achieve selective detection of dopamine by electrodepositing thin / thick peroxide polypyrrole films, and exclude dopamine from the glutamate detection site to achieve simultaneous detection of dopamine and glutamate;

[0023] (4) The microelectrode array modified by the method of the present invention is an implantable electrode that can realize implantable analysis of real-time synchronous monitoring of glutamate and dopamine with high spatial resolution, which is crucial for research in the field of brain-computer interface.

[0024] In the present invention, the above-mentioned technical solutions can also be combined with each other to achieve more preferred combination solutions. Other features and advantages of the present invention will be described in the subsequent description, and some advantages can become obvious from the description, or can be understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained through the contents particularly pointed out in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The drawings are only for the purpose of illustrating particular embodiments and are not to be considered limiting of the present invention. Like reference symbols denote like components throughout the drawings.

[0026] Figure 1 It is a schematic diagram of the local structure of the dopamine detection microelectrode modified by the method of the present invention;

[0027] Figure 2 is a schematic diagram of the local structure of the glutamate detection microelectrode modified by the method of the present invention;

[0028] Figure 3 This is a current response diagram of the microelectrode array prepared in Example 1 of the present invention for detecting dopamine;

[0029] Figure 4 This is a current response diagram of glutamate detected by the microelectrode array prepared in Example 1 of the present invention.

[0030] Reference numerals:

[0031] 1- platinum electrode, 2- polypyrrole peroxide film, 3- Nafion membrane, 4- insulating substrate, 5- chitosan film, 6- glutamate oxidase. DETAILED DESCRIPTION

[0032] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings, wherein the accompanying drawings constitute a part of the present invention and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not used to limit the scope of the present invention.

[0033] A specific embodiment of the present invention discloses a surface modification method for a microelectrode array for synchronously detecting glutamate and dopamine, wherein the microelectrode array comprises a glutamate detection microelectrode and a dopamine detection microelectrode, and the method comprises the following steps:

[0034] (1) depositing a layer of polypyrrole film on the glutamate detection microelectrode and the dopamine detection microelectrode respectively, subjecting the polypyrrole film to peroxidation treatment to form a peroxidized polypyrrole film 2, and then dipping in Nafion solution, drying, and forming a layer of Nafion membrane 3 outside the oxidized polypyrrole film;

[0035] (2) A layer of chitosan film 5 is deposited on the glutamate detection microelectrode treated in step (1), and then immersed in a glutamate oxidase 6 solution and refrigerated.

[0036] The schematic diagram of the local structure of the dopamine detection microelectrode and the glutamate detection microelectrode modified by the method of the present invention is shown in FIG. Figure 1 and 2 As shown, the microelectrode array is arranged on an insulating substrate 4 , and both the dopamine detection microelectrode and the glutamate detection microelectrode are platinum electrodes 1 .

[0037] The positions of the glutamate detection microelectrode and the dopamine detection microelectrode in the present invention can be selected according to actual detection needs, and the positional relationship of the two electrodes is not specifically limited in the present invention. Specifically, when the glutamate detection microelectrode is electro-deposited, only the glutamate detection microelectrode is energized. When the dopamine detection microelectrode is electro-deposited, only the dopamine detection microelectrode is energized.

[0038] Compared with the prior art, the microelectrode array modified by the method of the present invention can detect glutamate and dopamine simultaneously. The method of the present invention deposits a layer of polypyrrole film on both the dopamine detection microelectrode and the glutamate detection microelectrode, which can achieve the isolation of interfering substances. The interfering substances in the extracellular fluid are mainly negatively charged ascorbic acid (AA). At the same time, in addition to excluding AA, the interference of positively charged dopamine (DA) needs to be excluded from the glutamate detection site; the Nafion membrane 3 selected in the present invention is a cation selective permeable membrane, which can pass cations and isolate anions; the peroxidized polypyrrole film 2 is also a cation exchange membrane, which is first formed by electropolymerizing pyrrole to form polypyrrole, and then subjected to high potential electrooxidation. During the electrooxidation process, the electronegative carbon group formed along the main chain of the peroxidized polypyrrole can effectively attract cations and repel anions.

[0039] It should be noted that the surface modification of the microelectrode array in the present invention follows the following principles:

[0040] (a) Nafion membrane 3 is a cation-selective permeable membrane that excludes negatively charged substances AA;

[0041] (b) The thin peroxidized polypyrrole film 2 and Nafion membrane 3 modification separated the negatively charged AA and the positively charged DA, allowing only DA to enter and excluding AA;

[0042] (c) Thick peroxide polypyrrole film 2 and Nafion membrane 3 modification separates the neutral hydrogen peroxide small molecules, anionic AA and cationic electroactive substances DA, allowing only hydrogen peroxide (H2O2) to enter and excluding AA and DA;

[0043] (d) Glutamic acid is catalyzed by magnesium oxide of glutamate to produce hydrogen peroxide.

[0044] In a specific embodiment, in step (1), the thickness of the polypyrrole peroxide film 2 on the glutamate detection microelectrode is greater than the thickness of the polypyrrole peroxide film 2 on the dopamine detection microelectrode.

[0045] In a specific embodiment, the thickness of the peroxidized polypyrrole film on the glutamate detection microelectrode is 80-120 nm, and the thickness of the peroxidized polypyrrole film on the dopamine detection microelectrode is 0.5-1.5 nm.

[0046] It should be noted that the thin polypyrrole peroxide film 2 can enhance the adsorption of dopamine on the electrode surface to improve the sensitivity of detecting dopamine, and the relatively thick polypyrrole peroxide film 2 can simultaneously repel negatively charged ascorbic acid and positively charged dopamine, allowing neutral small molecule hydrogen peroxide to penetrate. The present invention can achieve selective detection of dopamine by electrodepositing thin / thick polypyrrole peroxide films 2, and exclude dopamine from glutamate detection sites to achieve simultaneous detection of dopamine and glutamate.

[0047] In a specific embodiment, in step (1), a layer of polypyrrole film is deposited on the glutamate detection microelectrode. The specific method is as follows: the microelectrode array is added to a 150-250mM pyrrole PBS solution, and the glutamate detection microelectrode is electrodeposited at a voltage of 0.8-1V for 5-10min.

[0048] In a specific embodiment, in step (1), a layer of polypyrrole film is deposited on the dopamine detection microelectrode. The specific method is as follows: the microelectrode array is added to a 2-5mM pyrrole PBS solution, and the dopamine detection microelectrode is scanned at a voltage of 0.2-1.2V and a scanning speed of 20mv / s for 2-5 cycles.

[0049] It should be noted that the polypyrrole films are deposited on the glutamate detection microelectrode and the dopamine detection microelectrode, and the concentrations of the selected pyrrole PBS solutions are different, so that the thicknesses of the deposited polypyrrole films are different. The thickness of the peroxidized polypyrrole film 2 on the glutamate detection microelectrode is greater than the thickness of the peroxidized polypyrrole film 2 on the dopamine detection microelectrode.

[0050] In a specific embodiment, the polypyrrole film is electro-oxidized at a voltage of 0.9-1 mV to form a peroxidized polypyrrole film 2 .

[0051] It should be noted that the thickness of the peroxidized polypyrrole film 2 electrodeposited on the glutamate detection microelectrode and the dopamine detection microelectrode in the present invention is controlled by controlling the electrodeposition condition parameters. The above-mentioned electrodeposition condition parameters of the present invention can make the polypyrrole electrodeposited with different thicknesses on the glutamate and dopamine detection microelectrodes, so that the thickness of the peroxidized polypyrrole film on the glutamate detection microelectrode is greater than the thickness of the peroxidized polypyrrole film on the dopamine detection microelectrode.

[0052] In the present invention, there is no specific requirement for the order of electrodepositing the polypyrrole film on the surface of the glutamate detection microelectrode and the dopamine detection microelectrode. The glutamate detection microelectrode can be electrodeposited, or the dopamine detection microelectrode can be electrodeposited first. In the specific embodiment of the present invention, the polypyrrole film is first deposited on the dopamine detection microelectrode as an example for explanation.

[0053] In a specific embodiment, in step (1), the mass concentration of the Nafion solution is 5%, and the solution is baked at 180° C. for 3 minutes.

[0054] In a specific embodiment, step (2) further comprises dissolving chitosan in deionized water, adding acid to adjust the pH value to 3, then adding alkali to adjust the pH value to 5, and then depositing chitosan film 5.

[0055] Specifically, the acid is hydrochloric acid, and the alkali is sodium hydroxide.

[0056] In a specific embodiment, the mass volume ratio of chitosan to deionized water is 0.3-0.5 mg / mL.

[0057] It should be noted that the change in the pH value of the chitosan solution can change the charge state of chitosan. At a lower pH value (such as pH = 3), the amine groups of chitosan are protonated and soluble in aqueous solution. At a higher pH value (such as pH = 5), these amine groups are deprotonated and become insoluble. The pH value of the solution is changed by applying different potentials to the electrodes. When a high negative voltage is applied, the local pH value around the electrode increases, and chitosan is deposited on the electrode, achieving selective deposition of chitosan, and then the enzyme is adsorbed by the chitosan membrane to achieve selective fixation of glutamate oxidase 6.

[0058] In a specific embodiment, in step (2), the treated glutamate detection microelectrode is electroplated at a voltage of -0.8 to -1.2 V for 2 minutes to form a chitosan film 5 .

[0059] In a specific embodiment, in step (2), the glutamate oxidase solution is a PBS solution of glutamate peroxidase, the enzyme activity of glutamate oxidase in the PBS solution is 125 units / mL, and the refrigerated temperature is 4°C.

[0060] In a second aspect, the present invention provides a microelectrode array modified by the method for synchronously detecting glutamate and dopamine.

[0061] In a third aspect, the present invention provides an application of a microelectrode array in the synchronous detection of glutamate and dopamine, wherein the microelectrode array is modified by the method described above.

[0062] The microelectrode array modified by the method of the present invention can synchronously detect glutamate and dopamine with high sensitivity, and can realize implantable analysis of real-time synchronous monitoring of glutamate and dopamine with high spatial resolution, which is crucial for research in the field of brain-computer interaction.

[0063] The technical solution of the present invention is further explained below in conjunction with specific embodiments.

[0064] Example 1

[0065] This embodiment discloses a surface modification method for a microelectrode array for synchronously detecting glutamate and dopamine, wherein the microelectrode array comprises a glutamate detection microelectrode and a dopamine detection microelectrode, and the method comprises the following steps:

[0066] (1) Electrodepositing a polypyrrole film on a dopamine detection microelectrode, the specific method is as follows: adding the microelectrode array to a 2 mM pyrrole PBS solution, scanning the dopamine detection microelectrode at a scanning voltage of 0.2-1.2 V and a scanning speed of 20 mv / s for 2 cycles;

[0067] Electrodepositing polypyrrole film on glutamate detection microelectrode, the specific method is as follows: adding the microelectrode array into 200mM pyrrole PBS solution, electrodepositing the glutamate detection microelectrode at a voltage of 0.85V for 5min;

[0068] The microelectrode array of the electro-deposited polypyrrole film is subjected to PPy peroxidation treatment at a voltage of 989 mV until a stable current response is achieved to form a peroxidized polypyrrole film 2, wherein the thickness of the peroxidized polypyrrole film on the glutamate detection microelectrode is 80-120 nm, and the thickness of the peroxidized polypyrrole film on the dopamine detection microelectrode is 0.5-1.5 nm, and then the electrodes are dip-coated with a 5% Nafion solution, and baked at 180° C. for 3 min, and repeated twice to form a Nafion film 3 with a thickness of 50-200 μm;

[0069] (2) Preparation of chitosan solution: 20 mg of chitosan was dissolved in 50 mL of deionized water, and hydrochloric acid was added to adjust the pH value to 3. Ultrasonic stirring was performed until all the chitosan was dissolved, and the solution was filtered. Then, sodium hydroxide was added to adjust the pH value of the solution to 5 to obtain a chitosan solution.

[0070] The microelectrode array treated in step (1) was added to the chitosan solution, and the treated glutamate detection microelectrode was electroplated at a voltage of -1.0 V for 2 minutes, and repeated twice to form a chitosan film 5 on the glutamate detection microelectrode, and then immersed in a glutamate oxidase PBS solution, the enzyme activity of glutamate oxidase in the PBS solution was 125unit / mL, and stored in a refrigerator at 4°C.

[0071] Example 2

[0072] This embodiment discloses a surface modification method for a microelectrode array for synchronously detecting glutamate and dopamine, wherein the microelectrode array comprises a glutamate detection microelectrode and a dopamine detection microelectrode, and the method comprises the following steps:

[0073] (1) Electrodepositing a polypyrrole film on a dopamine detection microelectrode, the specific method is as follows: adding the microelectrode array to a 3.5 mM pyrrole PBS solution, scanning the dopamine detection microelectrode at a scanning voltage of 0.2-1.2 V and a scanning speed of 20 mv / s for 5 cycles;

[0074] Electrodepositing polypyrrole film on glutamate detection microelectrode, the specific method is as follows: adding the microelectrode array into 150mM pyrrole PBS solution, electrodepositing the glutamate detection microelectrode at a voltage of 0.8V for 7.5min;

[0075] The microelectrode array of the electro-deposited polypyrrole film is subjected to PPy peroxidation treatment at a voltage of 0.9 V until a stable current response is achieved to form a peroxidized polypyrrole film 2, wherein the thickness of the peroxidized polypyrrole film on the glutamate detection microelectrode is 80-120 nm, and the thickness of the peroxidized polypyrrole film on the dopamine detection microelectrode is 0.5-1.5 nm, and then the electrodes are dip-coated with a 5% Nafion solution, and baked at 180° C. for 3 min, and repeated twice to form a Nafion film 3 with a thickness of 50-200 μm;

[0076] (2) Preparation of chitosan solution: 15 mg of chitosan was dissolved in 50 mL of deionized water, and hydrochloric acid was added to adjust the pH value to 3. Ultrasonic stirring was performed until all the chitosan was dissolved, and the solution was filtered. Then, sodium hydroxide was added to adjust the pH value of the solution to 5 to obtain a chitosan solution.

[0077] The microelectrode array treated in step (1) was added to the chitosan solution, and the treated glutamate detection microelectrode was electroplated at a voltage of -0.8 V for 2 minutes, and repeated twice to form a chitosan film 5 on the glutamate detection microelectrode, and then immersed in a glutamate oxidase PBS solution, the enzyme activity of glutamate oxidase in the PBS solution was 125unit / mL, and stored in a refrigerator at 4°C.

[0078] Example 3

[0079] This embodiment discloses a surface modification method for a microelectrode array for synchronously detecting glutamate and dopamine, wherein the microelectrode array comprises a glutamate detection microelectrode and a dopamine detection microelectrode, and the method comprises the following steps:

[0080] (1) Electrodepositing a polypyrrole film on a dopamine detection microelectrode, the specific method is as follows: adding the microelectrode array to a 5 mM pyrrole PBS solution, scanning the dopamine detection microelectrode at a scanning voltage of 0.2-1.2 V and a scanning speed of 20 mv / s for 4 cycles;

[0081] Electrodepositing polypyrrole film on glutamate detection microelectrode, the specific method is as follows: adding the microelectrode array into 250mM pyrrole PBS solution, electrodepositing the glutamate detection microelectrode at a voltage of 1V for 10min;

[0082] The microelectrode array of the electro-deposited polypyrrole film is subjected to PPy peroxidation treatment at a voltage of 1 V until a stable current response is achieved to form a peroxidized polypyrrole film 2, wherein the thickness of the peroxidized polypyrrole film on the glutamate detection microelectrode is 80-120 nm, and the thickness of the peroxidized polypyrrole film on the dopamine detection microelectrode is 0.5-1.5 nm, and then the electrodes are dip-coated with a 5% Nafion solution, and baked at 180° C. for 3 min, and repeated twice to form a Nafion film 3 with a thickness of 50-200 μm;

[0083] (2) Preparation of chitosan solution: 25 mg of chitosan was dissolved in 50 mL of deionized water, and hydrochloric acid was added to adjust the pH value to 3. Ultrasonic stirring was performed until all the chitosan was dissolved, and the solution was filtered. Then, sodium hydroxide was added to adjust the pH value of the solution to 5 to obtain a chitosan solution.

[0084] The microelectrode array treated in step (1) was added to the chitosan solution, and the treated glutamate detection microelectrode was electroplated at a voltage of -1.2 V for 2 minutes, and repeated twice to form a chitosan film 5 on the glutamate detection microelectrode, and then immersed in a glutamate oxidase PBS solution, the enzyme activity of glutamate oxidase in the PBS solution was 125 unit / mL, and stored in a refrigerator at 4°C.

[0085] Test Example 1

[0086] Detection of dopamine and glutamate by the microelectrode array prepared in Test Example 1

[0087] 1. Place 1×PBS buffer solution into the sample pool and the microelectrode array prepared in Example 1 into the electrochemical workstation. Figure 3 As shown, when 250 μM ascorbic acid (AA) was added to 1×PBS buffer solution, the dopamine detection microelectrode had no current response, but after dopamine was added, the dopamine detection microelectrode had a current response. Figure 3 ①②③④ in the figure indicate adding dopamine solution of the same concentration, specifically, the dopamine concentration is 5μM. Figure 3 It can be seen that the microelectrode array prepared by the present invention is selective for dopamine.

[0088] Among them, the principle of dopamine detection is: dopamine is an electroactive substance, which can directly undergo redox reaction under a certain voltage, resulting in the gain and loss and transfer of electrons, and generate a current signal.

[0089] 2. Place 1×PBS buffer solution into the sample pool and the microelectrode array prepared in Example 1 into the electrochemical workstation. Figure 4 As shown, when 250 μM ascorbic acid (AA) was added to 1×PBS buffer solution, the glutamate detection microelectrode had no current change. After adding glutamate, the glutamate detection microelectrode showed a current change. Figure 4 ①②③ indicate that glutamate of the same concentration is added, specifically, the glutamate concentration is 20 μM. When dopamine of 5 μM concentration is added, the glutamate detection microelectrode has no current response, indicating that the glutamate detection microelectrode of the present invention is selective for glutamate.

[0090] Among them, the principle of glutamate detection is: glutamate is a non-electroactive substance, and the detection uses glutamate oxidase as a recognition component. Glutamate oxidase undergoes an enzymatic reaction on the electrode surface. Glutamate oxidase catalyzes glutamate to produce α-ketoglutaric acid, ammonia and hydrogen peroxide in the presence of oxygen and water. Electroactive hydrogen peroxide can be oxidized on the electrode surface to generate a current signal.

[0091] The above experiments show that the dopamine detection microelectrodes in the microelectrode array of the present invention detect dopamine and the glutamate detection microelectrodes detect glutamate without affecting each other. Therefore, the microelectrode array prepared by the present invention can detect dopamine and glutamate at the same time.

[0092] The inventors also conducted the above test on the microelectrode arrays prepared in other embodiments, and the results were basically the same. Due to limited space, they are not listed one by one.

[0093] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A surface modification method for a microelectrode array for synchronous detection of glutamate and dopamine, characterized in that: The microelectrode array comprises a glutamate detection microelectrode and a dopamine detection microelectrode, and the method comprises the following steps: (1) depositing a layer of polypyrrole film on the glutamate detection microelectrode and the dopamine detection microelectrode respectively, subjecting the polypyrrole film to peroxidation treatment to form a peroxidized polypyrrole film, and then dipping in Nafion solution, drying, and forming a layer of Nafion membrane 3 outside the oxidized polypyrrole film; (2) depositing a layer of chitosan film on the glutamate detection microelectrode treated in step (1), then immersing it in a glutamate oxidase solution and refrigerating it.

2. The surface modification method of a microelectrode array for synchronously detecting glutamate and dopamine according to claim 1, characterized in that: In step (1), the thickness of the polypyrrole peroxide film on the glutamate detection microelectrode is greater than the thickness of the polypyrrole peroxide film on the dopamine detection microelectrode.

3. The surface modification method of a microelectrode array for synchronously detecting glutamate and dopamine according to claim 2, characterized in that: In step (1), a layer of polypyrrole film is deposited on the glutamate detection microelectrode. The specific method is as follows: add the microelectrode array into a 150-250 mM pyrrole PBS solution, and electro-deposit the glutamate detection microelectrode at a voltage of 0.8-1 V for 5-10 minutes.

4. The surface modification method of a microelectrode array for synchronously detecting glutamate and dopamine according to claim 2, characterized in that: In step (1), a layer of polypyrrole film is deposited on the dopamine detection microelectrode. The specific method is as follows: add the microelectrode array into a 2-5mM pyrrole PBS solution, and scan the dopamine detection microelectrode at a scanning voltage of 0.2-1.2V and a scanning speed of 20mv / s for 2-5 cycles.

5. A surface modification method for a microelectrode array for synchronously detecting glutamate and dopamine according to any one of claims 1 to 4, characterized in that: The polypyrrole film is electro-oxidized at a voltage of 0.9-1V to form a peroxidized polypyrrole film.

6. A surface modification method for a microelectrode array for synchronously detecting glutamate and dopamine according to any one of claims 1 to 4, characterized in that: In step (1), the mass concentration of the Nafion solution is 5%, and the solution is baked at 180° C. for 3 minutes.

7. The surface modification method of a microelectrode array for synchronous detection of glutamate and dopamine according to claim 1, characterized in that: Step (2) further comprises dissolving chitosan in deionized water, adding acid to adjust the pH value to 3, then adding alkali to adjust the pH value to 5, and then depositing a chitosan film. Preferably, the mass volume ratio of chitosan to deionized water is 0.3-0.5 mg / mL. Preferably, the treated glutamate detection microelectrode is electroplated at a voltage of -0.8 to -1.2 V for 2 minutes to form a chitosan film.

8. The surface modification method of a microelectrode array for synchronous detection of glutamate and dopamine according to claim 1, characterized in that: In step (2), the glutamate oxidase solution is a PBS solution of glutamate peroxidase, the enzyme activity of glutamate oxidase in the PBS solution is 125 units / mL, and the refrigerated temperature is 4°C.

9. A microelectrode array for synchronously detecting glutamate and dopamine modified by the method according to any one of claims 1 to 8.

10. An application of a microelectrode array in the simultaneous detection of glutamate and dopamine, characterized in that: The microelectrode array is modified by any one of the methods of claims 1-8.

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