Preparation method of nano-enzyme microelectrode and nano-enzyme microelectrode

Abstract

The invention discloses a preparation method of a nano-enzyme microelectrode and the nano-enzyme microelectrode. The method comprises the following steps: providing a substrate, and removing impurities on the surface of the substrate; sufficiently mixing solid powder of nano-enzyme with deionized water according to a preset proportion to obtain a nano-enzyme solution; the nanometer enzyme comprises an Au24Cd1 nanometer enzyme, an Au24Er1 nanometer enzyme or an Au25 nanometer enzyme; by taking an Ag / AgCl electrode as a reference electrode, a platinum sheet electrode as a counter electrode and the substrate as a working electrode, carrying out nano-enzyme deposition treatment in the nano-enzyme solution through an electrochemical workstation based on a multi-potential step method; and depositing the nano-enzyme on the surface of the substrate to obtain the nano-enzyme microelectrode. According to the embodiment of the invention, the preparation of various nano-enzyme microelectrodes can be realized, and the forest sensitivity and biocompatibility of the microelectrodes can be improved. The method can be widely applied to the technical field of electrochemical detection.

Description

Technical Field

[0001] The invention relates to the technical field of electrochemical detection, in particular to a preparation method of a nanozyme microelectrode and the nanozyme microelectrode. Background Art

[0002] Traditional metal neural electrodes have high stability when recording neural signals. However, due to their inherent high impedance characteristics, weak signals cannot be detected during long-term implantation. At the same time, due to the large difference between the surface properties of traditional metal electrodes and the biological system, the biocompatibility of the material is poor, which may cause the peripheral nerve tissue to reject foreign bodies, thereby inducing chronic neuroinflammation. In related technologies, the most advanced nanotechnology is applied to improve sensitivity and biocompatibility. However, existing nanotechnology products are limited and cannot achieve multi-type signal detection. Summary of the invention

[0003] The purpose of the present invention is to solve one of the technical problems existing in the prior art to at least a certain extent.

[0004] To this end, the object of the present invention is to provide a method for preparing a highly sensitive nanozyme microelectrode and a nanozyme microelectrode.

[0005] In order to achieve the above technical objectives, one aspect of the embodiments of the present invention provides a method for preparing a nanozyme microelectrode, comprising the following steps: providing a substrate, removing surface impurities of the substrate; wherein the substrate comprises silver wire, gold wire or pure EcoGME; taking a solid powder of nanozyme and mixing it with deionized water in a preset ratio to obtain a nanozyme solution; the nanozyme comprises Au 24 Cd 1 Nanozymes, Au 24 Er 1 Nanozymes or Au 25 Nanozyme; using the Ag / AgCl electrode as the reference electrode, the platinum electrode as the counter electrode, and the substrate as the working electrode, through an electrochemical workstation, in the nanozyme solution, based on the multi-potential step method, the nanozyme deposition treatment is performed; the nanozyme is deposited on the surface of the substrate to obtain a nanozyme microelectrode; the nanozyme microelectrode is insulated to obtain a nanozyme microelectrode with a partial area exposed; wherein the exposed part is used as the sensing area of ​​the electrode. The embodiment of the present application uses an electrochemical workstation, in the nanozyme solution, based on the multi-potential step method, to achieve the modification of multiple electrodes with multiple artificial nanozymes, and the catalytic activity of the nanozyme itself is used to reduce the inherent high electrochemical impedance of the electrode. At the same time, this gold-based clustered nanozyme also has a high antioxidant activity, thereby effectively reducing the chance of inducing neuroinflammation. The embodiment of the present application can realize the preparation of multiple nanozyme microelectrodes, which is beneficial to improving the forest sensitivity and biocompatibility of the microelectrode.

[0006] In some embodiments, the method for preparing the nanozyme microelectrode of the embodiment of the present invention, the nanozyme deposition treatment based on the multi-potential step method, comprises:

[0007] If the substrate is a silver wire, the electrodeposition parameters are set to set a plurality of potentials in the voltage range of 0.2 to 2 V, with a step amplitude between the potentials of 0.1 to 0.2 V; and the duration of each potential is set;

[0008] Set the deposition current to 0.3 to 0.5 mA; start the electrodeposition process;

[0009] Alternatively, if the substrate is a gold wire or pure EcoGME, the electrodeposition parameters are set to set a number of potentials in the voltage range of 1.7 to 3.4 V, with a step amplitude of 0.1 to 0.2 V between the potentials; and the duration of each potential is set;

[0010] Set the deposition current to 0.3 to 0.5 mA; start the electrodeposition process.

[0011] In some embodiments, in one embodiment of the present invention, providing a substrate and removing surface impurities of the substrate comprises:

[0012] A silver wire is provided, and after being polished with sandpaper, the silver wire is placed in distilled water for ultrasonic impurity removal treatment;

[0013] Alternatively, a gold wire is provided, and after being polished with sandpaper, the gold wire is placed in distilled water for ultrasonic impurity removal treatment;

[0014] Alternatively, a substrate is provided, and patterned magnetron sputtering is performed on the substrate through a preset mask, and Cr and Au are deposited in sequence to obtain pure EcoGME.

[0015] In some embodiments, in one embodiment of the present invention, the Au 24 Cd 1 Nanozymes are prepared by the following steps:

[0016] Add HauCl to the first volume of ultrapure water 4 Aqueous solution, Cd(NO 3 ) 2 aqueous solution and MPA aqueous solution, and stirred continuously; wherein, the added HauCl 4 Aqueous solution, Cd(NO 3 ) 2 The aqueous solution and the MPA aqueous solution correspond to the preset density and the preset volume respectively;

[0017] Add NaOH aqueous solution dropwise until the pH value of the reaction solution is 12; add a second volume of NaBH solution;

[0018] Saturated CO is introduced and closed treatment is performed; the duration of the closed treatment is the first duration;

[0019] After stirring for a second time, the brown solution was freeze-dried to obtain pure Au 24 Cd 1 Nanozymes should be stored at -18 to -22°C.

[0020] In some embodiments, in one embodiment of the present invention, the microelectrode is characterized by the following steps:

[0021] The Ag / AgCl was used as the reference electrode, the platinum electrode as the counter electrode, and the test electrode as the working electrode for testing;

[0022] During the test, the test solution is replaced to change the solution concentration; the test electrode includes a microelectrode and a nanozyme microelectrode, the microelectrode includes an AgME microelectrode, an AuME microelectrode and an EcoGME microelectrode; the nanozyme microelectrode includes a nanozyme-modified AgME microelectrode, a nanozyme-modified AuME microelectrode, and a nanozyme-modified EcoGME microelectrode;

[0023] If the solution is changed, the test is suspended; and the test solution is allowed to stand for a third period of time before continuing the test.

[0024] In some embodiments, in one embodiment of the present invention, the method comprises:

[0025] The microelectrode was tested based on the CV and EIS methods. Phosphate buffer solution was used as the working solution. The test environment was built and the test parameters were set before starting the test.

[0026] Alternatively, DA solutions of different concentrations are tested by DPV, SWV, NPV, and LSV methods; the concentration range of the DA solution includes 1 μM to 200 uM;

[0027] Alternatively, AA solutions of different concentrations are tested by DPV, SWV, NPV, and LSV methods; the concentration range of the AA solution is 200 μM to 1200 uM.

[0028] In some embodiments, in one embodiment of the present invention, the test parameters are set by the following steps:

[0029] For CV testing, set the test voltage to -0.4 to 0.6 V and the scan rate to 0.05 to 0.15 V / s.

[0030] For EIS testing, set the initial voltage to 0V and the frequency range to 1 to 105Hz.

[0031] In some embodiments, in one embodiment of the present invention, the test parameters are set by the following steps:

[0032] For DPV test, set the test voltage to -0.3 to 0.6V, the pulse width to 0.03 to 0.06s, the sampling width to 0.01 to 0.03s, and the pulse period to 0.4 to 0.6s;

[0033] For SWV test, set the test voltage to -0.3 to 0.6V and the frequency to 8 to 12Hz;

[0034] For LSV testing, set the scan rate to 0.08 to 0.12 V / s and the sampling interval to 0.001 V;

[0035] For NPV testing, set the pulse width to 0.04 to 0.06 s, the sampling width to 0.01 to 0.03 s, and the pulse period to 0.4 to 0.6 s.

[0036] In some embodiments, in one embodiment of the present invention, the AA solution is prepared by the following steps:

[0037] A first weight of DA powder is fully dissolved in a third volume of PBS solution to obtain a 50 mM AA solution; the ratio of the first weight to the third volume is a first preset value;

[0038] The fourth volume of 50 mM DA solution is dissolved in the fifth volume of PBS solution, and the mixture is fully mixed to obtain a 10 mM DA solution; the ratio of the fourth volume to the fifth volume is the second preset value;

[0039] The sixth volume of 10 mM DA solution is dissolved in the seventh volume of PBS solution to obtain a 1 mM DA solution; the ratio of the sixth volume to the seventh volume is a third preset value;

[0040] dissolving the eighth volume of 1 mM DA solution in the ninth volume of PBS solution to obtain a 100 nM DA solution; the ratio of the eighth volume to the ninth volume is a fourth preset value;

[0041] The second weight of AA powder is taken and fully dissolved in the tenth volume of PBS solution to obtain a 10 mM AA solution; the ratio of the second weight to the tenth volume is the fifth preset value.

[0042] On the other hand, an embodiment of the present invention provides a nanozyme microelectrode, which is prepared by the above-mentioned method for preparing the nanozyme microelectrode.

[0043] The embodiments of the present application at least have the following beneficial effects: the method provided by the embodiments of the present invention comprises: providing a substrate, removing surface impurities of the substrate; wherein the substrate comprises silver wire, gold wire or pure EcoGME; taking the solid powder of nanozyme and fully mixing it with deionized water in a preset ratio to obtain a nanozyme solution; the nanozyme comprises Au 24 Cd 1 Nanozymes, Au 24 Er 1 Nanozymes or Au 25 Nanozyme; using the Ag / AgCl electrode as the reference electrode, the platinum electrode as the counter electrode, and the substrate as the working electrode, through an electrochemical workstation, in the nanozyme solution, based on the multi-potential step method, the nanozyme deposition treatment is performed; the nanozyme is deposited on the surface of the substrate to obtain a nanozyme microelectrode; the nanozyme microelectrode is insulated to obtain a nanozyme microelectrode with a partial area exposed; wherein the exposed part is used as the sensing area of ​​the electrode. The embodiment of the present application uses an electrochemical workstation, in the nanozyme solution, based on the multi-potential step method, to achieve the modification of multiple electrodes with multiple artificial nanozymes, and the catalytic activity of the nanozyme itself is used to reduce the inherent high electrochemical impedance of the electrode. At the same time, this gold-based clustered nanozyme also has a high antioxidant activity, thereby effectively reducing the chance of inducing neuroinflammation. The embodiment of the present application can realize the preparation of multiple nanozyme microelectrodes, which is beneficial to improving the forest sensitivity and biocompatibility of the microelectrode. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the embodiments of the present invention or the drawings of related technical solutions in the prior art are introduced below. It should be understood that the drawings introduced below are only for the convenience of clearly describing some embodiments of the technical solutions of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0045] Figure 1 A schematic diagram of surface features of an embodiment of a substrate provided by the present invention;

[0046] Figure 2 A schematic structural diagram of an embodiment of three nanozyme materials provided by the present invention;

[0047] Figure 3 A schematic diagram of the detection result of an embodiment of the current signal on the microelectrode provided by the present invention;

[0048] Figure 4 Surface SEM images of embodiments of AgME microelectrodes modified with different nanozyme materials provided by the present invention;

[0049] Figure 5 Surface SEM images of embodiments of AuME microelectrodes modified with different nanozyme materials provided by the present invention;

[0050] Figure 6 Surface SEM images of examples of EcoGME microelectrodes modified with different nanozyme materials provided by the present invention;

[0051] Figure 7 A schematic diagram showing the comparison of the basic electrochemical characterization test results of the silver wire microelectrode provided by the present invention;

[0052] Figure 8 A schematic diagram showing the comparison of the basic electrochemical characterization test results of the gold wire microelectrode provided by the present invention;

[0053] Fig. 9 A schematic diagram showing the comparison of the basic electrochemical characterization test results of the EcoG microelectrode provided by the present invention;

[0054] Fig.10 A schematic diagram showing a comparison of the CV curve of the silver wire microelectrode and the cathode charge storage capacity provided by the present invention;

[0055] Fig.11 A schematic diagram showing a comparison of the CV curve of the gold wire microelectrode and the cathode charge storage capacity provided by the present invention;

[0056] Fig.12 A schematic diagram showing a comparison of the CV curve and cathode charge storage capacity of the EcoG microelectrode provided by the present invention;

[0057] Fig.13 Schematic diagram of the response current of nanozyme deposited by the multi-potential step method provided by the present invention. DETAILED DESCRIPTION

[0058] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and are not to be construed as limitations of the present invention. For the step numbers in the following embodiments, they are only provided for the convenience of explanation, and the order between the steps is not limited in any way, and the execution order of each step in the embodiment can be adaptively adjusted according to the understanding of those skilled in the art.

[0059] Implantable neural microelectrodes are important tools for monitoring brain diseases. According to the depth of signal acquisition, implantable neural microelectrodes can be divided into microelectrodes for recording cerebral cortex signals and wire electrodes for deep brain recording. EcoGME usually consists of multiple electrode recording sites. These recording sites are arranged in an array, covering a large area of ​​the cerebral cortex. Therefore, EcoGME has the advantage of high spatial resolution and is often used in brain function localization and brain-computer interface research before epilepsy surgery. Wire electrodes are usually made of metal materials such as Au, Ag, Pt / Ir. ​​Metal materials have the advantages of high conductivity and high stability. At the same time, the wire electrode has the characteristics of small size and flexibility, and is minimally invasive during implantation. Metal wire electrodes are usually used for deep brain stimulation and EEG signal recording. However, the clinical application of traditional metal wire electrodes is limited by their inherent high impedance and potential chronic neuroinflammatory response. Therefore, it is crucial to develop new implantable neural microelectrodes and improve their sensing performance and biocompatibility.

[0060] In recent years, the application of nanotechnology in the field of neural electrodes has made significant progress. Artificial nanozymes have unique advantages such as catalyzing inflammatory factors and enhancing biocompatibility, and show great potential in the field of biosensing. Gold nanoparticles have various types of enzyme activities, such as nucleases, glucose oxidases, peroxidases, catalases, and superoxide dismutases. Among them, the Michaelis constant of gold nanoparticles for hydrogen peroxide substrates in the enzymatic reaction of peroxidase is less than 1mM, but the catalytic activity is weak. In recent years, artificial nanozymes based on gold clusters have attracted much attention due to their inherent high catalytic activity and selectivity. This type of artificial nanozyme achieves enhanced catalytic activity and reprogramming of substrate selectivity by adjusting bond length. Gold cluster nanozymes have enzyme-like preferential activity and can catalyze the reduction of peroxides in injured brain tissue. This process greatly reduces inflammatory factors, highlighting their key role in alleviating neuroinflammation. In previous studies, nanozyme-modified electrodes have been studied to reduce the impedance of the electrodes. At the same time, nanozymes can inhibit the growth of glial cells and reduce the risk of inflammation during electrode implantation. However, previous studies mainly selected representative silver wire electrodes and preferred Au 24 Cd nanozyme is shown as a representative. Further exploration is needed for other similar implantable electrodes and nanozymes.

[0061] In the study of brain diseases, the monitoring of electrochemical signals is as important as the monitoring of electrophysiological action potentials. In particular, the monitoring of neurotransmitters such as dopamine (DA) and ascorbic acid (AA) is important. DA plays a key role in neurotransmission, mood regulation, and motor control. By monitoring DA levels, the pathological mechanisms of neuropsychiatric diseases such as Parkinson's disease, depression, and schizophrenia can be better understood and diagnosed. Ascorbic acid acts as a powerful antioxidant in the central nervous system, protecting nerve cells from oxidative stress and free radical damage. Changes in its levels are associated with the onset of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. By monitoring ascorbic acid levels, we can gain a deeper understanding of the pathological mechanisms of these diseases. Electrochemical methods such as differential pulse voltammetry (DPV), square wave voltammetry (SWV), linear sweep voltammetry (LSV) and normal pulse voltammetry (NPV) are widely used to monitor DA and AA. The principle of monitoring DA and AA by electrochemical methods is to detect the current signal generated by the redox reaction of the two at a specific potential through the working electrode to determine their presence and concentration. Its advantages include high sensitivity, fast response, high selectivity, simple operation, low cost, and miniaturization and portability. DPV and SWV reduce the influence of charging current by superimposing a series of forward and reverse pulse signals, thereby improving the sensitivity and resolution of detection. LSV focuses on studying the trend of current change with potential by applying a linearly changing unidirectional scanning voltage. NPV applies a series of short and constant voltage pulses on the working electrode to reduce the influence of capacitive current and improve the detection sensitivity of Faraday current. These voltammetric electrochemical methods provide powerful tools for accurately detecting DA and gaining in-depth understanding of its electrochemical properties.

[0062] Traditional metal neural electrodes have high stability when recording neural signals. However, due to their inherent high impedance characteristics, weak signals cannot be detected during long-term implantation. At the same time, due to the large difference between the surface properties of traditional metal electrodes and the biological system, the biocompatibility of the materials is poor, which may cause the peripheral nerve tissue to reject foreign bodies, thereby inducing chronic neuroinflammation.

[0063] In order to solve the high electrochemical impedance caused by slow electron transfer of traditional metal neural electrodes and the biocompatibility limitations caused by chronic neuroinflammation and implant trauma, many people are working hard to apply the most advanced nanotechnology to improve sensitivity and biocompatibility. In this regard, the embodiments of the present application explore the modification of various electrodes with a variety of artificial nanozymes, using the catalytic activity of the nanozymes themselves to reduce the inherent high electrochemical impedance of the electrodes. At the same time, this gold-based clustered nanozyme also has a high antioxidant activity, thereby effectively reducing the chance of inducing neuroinflammation. The present invention can improve the recording sensitivity and biocompatibility of the electrode, so that it can be better used to monitor brain diseases that require high sensitivity and long-term biocompatibility.

[0064] The following is a detailed description of the preparation method and system of the nanozyme microelectrode proposed in accordance with the embodiment of the present invention with reference to the accompanying drawings. First, the preparation method of the nanozyme microelectrode proposed in accordance with the embodiment of the present invention will be described with reference to the accompanying drawings. The preparation method of the nanozyme microelectrode in the embodiment of the present invention mainly comprises the following steps:

[0065] Providing a substrate, and removing surface impurities of the substrate; wherein the substrate comprises silver wire, gold wire or pure EcoGME;

[0066] The solid powder of nanozyme is mixed with deionized water in a preset ratio to obtain a nanozyme solution; the nanozyme includes Au 24 Cd 1 Nanozymes, Au 24 Er 1 Nanozymes or Au 25 Nanozymes;

[0067] Using an Ag / AgCl electrode as a reference electrode, a platinum electrode as a counter electrode, and the substrate as a working electrode, an electrochemical workstation is used to perform nanozyme deposition in the nanozyme solution based on a multi-potential step method; the nanozyme is deposited on the surface of the substrate to obtain a nanozyme microelectrode;

[0068] The nanozyme microelectrode is insulated to obtain a nanozyme microelectrode with a partial area exposed; wherein the exposed part serves as the sensing area of ​​the electrode.

[0069] In some possible embodiments, the present application provides three different methods for modifying microelectrodes with nanozymes. Figure 1 The surface morphology characteristics of EcoGME, AuME, and AgME shown in FIG. Among them, EcoGME is patterned with gold deposition process (indicated by the yellow part) on PI substrate (indicated by the orange part). The present application example provides three different nanozymes, referring to Figure 2 As shown, three nanozyme materials (including Au 24 Cd1 Nanozymes, Au 24 Er 1 Nanozymes, Au 25 Schematic diagram of the molecular structure of nanozyme. Electrodeposition is performed on the electrode sensing site using the multi-step potential chromoamperometry method, so that the nanozyme material is attached to the surface of the electrode sensing site. The current response diagram of the multi-step potential chromoamperometry (STEP) method is also shown in the figure. 3 )n represents the nitrate corresponding to different nano metal elements (Cd / Er / Au). Figure 3 As shown in the figure, after the microelectrode is prepared, different electrochemical methods are used to detect the current signal on the implanted microelectrode, including differential pulse voltammetry (DPV), square wave voltammetry (SWV), linear sweep voltammetry (LSV) and normal pulse voltammetry (NPV). The results are shown in the figure. Figure 3 As shown. Figure 4 Figure 1 is a SEM image of the surface of pure AgME (bare AgME). Figure 4 The second picture shows the modified Au 24 Cd 1 Nanozyme material AgME (Au 24 Cd1-AgME) surface SEM image. Figure 4 The third picture shows the modified Au 24 Er 1 Nanozyme material AgME (Au 24 Er1-AgME) surface SEM image. Figure 4 Figure 4 shows the modified Au 25 Nanozyme material AgME (Au 25 -AgME) surface SEM image. Figure 5 Figure 1 is a SEM image of the surface of pure AuME (bare AuME). Figure 5 The second picture shows the modified Au 24 Cd 1 Nanozyme material AuME (Au 24 Cd 1 -AuME) surface SEM image. Figure 5 The third picture shows the modified Au 24 Er 1 Nanozyme material AuME (Au24 Er 1 -AuME) surface SEM image. Figure 5 Figure 4 shows the modified Au 25 The AuME surface of nanozyme material (Au 25 -AuME) SEM image. Figure 6 Figure 1 is a SEM image of the surface of pure EcoGME (bareEcoGME). Figure 6 The second picture shows the modified Au 24 Cd 1 EcoGME(Au 24 Cd 1 -EcoGME) surface SEM image. Figure 6 The third picture shows the modified Au 24 Er 1 EcoGME(Au 24 Er 1 -EcoGME) surface SEM image. Figure 6 Figure 4 shows the modified Au 25 EcoGME(Au 25 -EcoGME) surface SEM image.

[0070] This application explores three different nanometal cluster materials and different nanozyme materials (including Au 24 Cd 1 Nanozymes, Au 24 Er1 nanozyme, Au 25The modification and detection methods of silver wire microelectrodes (AgME), gold wire microelectrodes (AuME) and electrocortical microelectrodes (EcoGME) by nanozymes were studied. A series of detection methods were used to evaluate their performance. We used electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) to characterize the electrodes before and after modification with nanozymes. The results showed that the modification of different nanozymes can reduce the low-frequency impedance of the electrode and increase the electron transfer rate to varying degrees. Among them, the improvement effect of AgME is better than that of AuME and EcoGME. Specifically, after modification of nanozymes, the AC impedance of AgME at a frequency of 100Hz was reduced by up to 18 times, the AC impedance of AuME at a frequency of 100Hz was reduced by up to 9 times, and the AC impedance of each channel of EcoGME at a frequency of 100Hz was reduced by up to 13 times. In addition, we characterized the current curves of AuME and EcoGME modified with three nanozyme materials in solutions of DA and AA by four methods: DPV, SWV, NPV, and LSV. The results show that after modification with nanozymes, the electrochemical detection performance of AuME and EcoGME for DA and AA has been improved, which is specifically manifested in the improvement of current response intensity and detection sensitivity during electrochemical reactions. In addition, compared with LSV and NPV, experiments using DPV and SWV have more stable oxidation peak waveforms and sensitivity. Implantable neural electrodes modified with nanozymes are expected to become an effective tool for clinical monitoring of DA and AA, which is of great significance for the study of various brain diseases.

[0071] Optionally, in one embodiment of the present invention, the nanozyme deposition treatment based on a multi-potential step method comprises:

[0072] If the substrate is a silver wire, the electrodeposition parameters are set to set a plurality of potentials in the voltage range of 0.2 to 2 V, with a step amplitude between the potentials of 0.1 to 0.2 V; and the duration of each potential is set;

[0073] Set the deposition current to 0.3 to 0.5 mA; start the electrodeposition process;

[0074] Alternatively, if the substrate is a gold wire or pure EcoGME, the electrodeposition parameters are set to set a number of potentials in the voltage range of 1.7 to 3.4 V, with a step amplitude of 0.1 to 0.2 V between the potentials; and the duration of each potential is set;

[0075] Set the deposition current to 0.3 to 0.5 mA; start the electrodeposition process.

[0076] Optionally, in one embodiment of the present invention, providing a substrate and removing surface impurities of the substrate comprises:

[0077] A silver wire is provided, and after being polished with sandpaper, the silver wire is placed in distilled water for ultrasonic impurity removal treatment;

[0078] Alternatively, a gold wire is provided, and after being polished with sandpaper, the gold wire is placed in distilled water for ultrasonic impurity removal treatment;

[0079] Alternatively, a substrate is provided, and patterned magnetron sputtering is performed on the substrate through a preset mask, and Cr and Au are deposited in sequence to obtain pure EcoGME.

[0080] In some possible embodiments, the preparation process of the nanozyme electrode is as follows:

[0081] For AgME with silver wire as the substrate, the silver wire with a diameter of 0.15 mm was sandpapered and then placed in distilled water for 10 min of ultrasonic treatment to remove surface impurities. Then, 0.5 g of nanozyme (three nanozymes were used in this embodiment, including Au 24 Cd 1 Nanozymes, Au 24 Er1 nanozyme, Au 25 Nanozyme) solid powder was thoroughly mixed with 10ml deionized water to obtain a 5mg / ml nanozyme solution. Next, a three-electrode electrochemical system was used, with a commercial Ag / AgCl electrode as the reference electrode, a platinum electrode as the counter electrode, and a silver wire as the working electrode. Nanozyme deposition was performed by multi-step potential chronoamperometry (STEP) in a CHI760E electrochemical workstation. Next, the electrodeposition parameters were set, and 12 potentials were set between 0.2 and 2V, with a step amplitude of 0.1 to 0.2V between the potentials, and each potential lasting for 2s. To ensure the uniformity of the nanozymes deposited on the AgME surface, the deposition current was controlled at about 0.4mA, and then the electrodeposition was started. Finally, the deposited nanozyme electrode was insulated with polyimide (PI), and the top 1mm portion was exposed as the electrode sensing area. It can be understood that the above process is an example of an embodiment, and those skilled in the art can adjust the parameters therein according to actual needs to achieve the same effect; this application does not limit the parameters in the preparation process.

[0082] For AuME based on gold wire, the gold wire with a diameter of 0.1 mm was sandpapered and placed in distilled water for 10 min to remove surface impurities. Then, 0.5 g of solid powder of nanozyme material was mixed with 10 ml of deionized water to obtain a 5 mg / ml nanozyme solution. Next, a three-electrode electrochemical system was used, with a commercial Ag / AgCl electrode as the reference electrode, a platinum electrode as the counter electrode, and a gold wire as the working electrode. Nanozyme deposition was performed by the STEP method in a CHI760E electrochemical workstation. Next, the electrodeposition parameters were set, and 12 potentials were set between 1.7 and 3.4 V, with a step amplitude of 0.1 to 0.2 V between the potentials, and each potential lasted for 2 s. To ensure the uniformity of the nanozymes deposited on the surface of AuME, the deposition current was controlled at about 0.4 mA, and then the electrodeposition was started. Finally, the deposited nanozyme electrode was insulated with PI, and the top 1 mm portion was exposed as the electrode sensing area.

[0083] For EcoGME, a customized mask was used to perform patterned magnetron sputtering on a PI substrate, and 10nm Cr and 100nm Au were deposited in sequence to obtain pure EcoGME. Then, 0.5g of solid powder of nanozyme material was mixed with 10ml of deionized water to obtain a 5mg / ml nanozyme solution. Next, a three-electrode electrochemical system was used, with a commercial Ag / AgCl electrode as a reference electrode, a platinum sheet electrode as a counter electrode, and EcoGME as a working electrode, and nanozyme deposition was performed by the STEP method in a CHI760E electrochemical workstation. Next, the electrodeposition parameters were set, and 12 potentials were set between 1.7 and 3.4V, with a step amplitude of 0.1 to 0.2V between the potentials, and a duration of 2s for each potential. To ensure the uniformity of the nanozymes deposited on the surface of EcoGME, the deposition current was controlled at about 0.4mA, and then the electrodeposition was started. Finally, the deposited nanozyme electrode was insulated with PI, and the top disk-shaped electrode site and the bottom gold finger interface were exposed.

[0084] Optionally, in one embodiment of the present invention, the Au 24 Cd 1 Nanozymes are prepared by the following steps:

[0085] Add HauCl to the first volume of ultrapure water 4 Aqueous solution, Cd(NO 3 ) 2 aqueous solution and MPA aqueous solution, and stirred continuously; wherein, the added HauCl 4 Aqueous solution, Cd(NO 3 ) 2The aqueous solution and the MPA aqueous solution correspond to the preset density and the preset volume respectively;

[0086] Add NaOH aqueous solution dropwise until the pH value of the reaction solution is 12; add a second volume of NaBH solution;

[0087] Saturated CO is introduced and closed treatment is performed; the duration of the closed treatment is the first duration;

[0088] After stirring for a second time, the brown solution was freeze-dried to obtain pure Au 24 Cd 1 Nanozymes should be stored at -18 to -22°C.

[0089] In some possible embodiments, the preparation process of nanozyme is as follows:

[0090] The nanozymes used in this work use two thiolates with opposite charges as ligands, and the reaction is carried out at room temperature and in the dark. 24 Cd 1 Nanozyme preparation is taken as an example: HauCl is added to ultrapure water (2.35 ml, i.e., the first volume in the embodiment of the present application) 4 Aqueous solution (20 mM, 0.25 mL), Cd(NO 3 ) 2 aqueous solution (20 mM, 10 ul) and MPA aqueous solution (5 mM, 2 mL) were stirred for 5 min. The solution was yellow-green. During the stirring process, the Au atoms in HauCl were replaced by Cd(NO 3 ) 2 The Cd2+ atoms in the Au were substituted with a molar ratio of 24:1. 24 Cd 1 Nanozyme clusters. Continue to add NaOH aqueous solution (1M, 0.3ml) until the pH of the reaction solution is 12, and then add 0.1ml (i.e., the second volume in the embodiment of the present application) NaBH solution. At this time, 10ml, 0.2M NaOH solution reacts and fuses with 43mg of NaBH powder, so the solution becomes clear and transparent. Next, pass saturated CO2 and seal for 2min (i.e., the first time in the embodiment of the present application). After stirring for 3h, the brown solution was freeze-dried to obtain pure Au. 24 Cd 1 The nanozyme product was stored at -20°C. 24 Er 1 Nanozyme clusters and Au 25 The synthesis of nanozyme clusters only requires the addition of Cd(NO 3 ) 2The aqueous solution is replaced with a nitrate aqueous solution of the corresponding element and the subsequent operations are repeated.

[0091] Optionally, in one embodiment of the present invention, the microelectrode is characterized by the following steps:

[0092] The Ag / AgCl was used as the reference electrode, the platinum electrode as the counter electrode, and the test electrode as the working electrode for testing;

[0093] During the test, the test solution is replaced to change the solution concentration; the test electrode includes a microelectrode and a nanozyme microelectrode, the microelectrode includes an AgME microelectrode, an AuME microelectrode and an EcoGME microelectrode; the nanozyme microelectrode includes a nanozyme-modified AgME microelectrode, a nanozyme-modified AuME microelectrode, and a nanozyme-modified EcoGME microelectrode;

[0094] If the solution is changed, the test is suspended; and the test solution is allowed to stand for a third period of time before continuing the test.

[0095] Optionally, in one embodiment of the present invention, the method further comprises:

[0096] The microelectrode was tested based on the CV and EIS methods. Phosphate buffer solution was used as the working solution. The test environment was built and the test parameters were set before starting the test.

[0097] Alternatively, DA solutions of different concentrations are tested by DPV, SWV, NPV, and LSV methods; the concentration range of the DA solution includes 1 μM to 200 uM;

[0098] Alternatively, AA solutions of different concentrations are tested by DPV, SWV, NPV, and LSV methods; the concentration range of the AA solution is 200 μM to 1200 uM.

[0099] Optionally, in one embodiment of the present invention, the test parameters are set by the following steps:

[0100] For CV testing, set the test voltage to -0.4 to 0.6 V and the scan rate to 0.05 to 0.15 V / s.

[0101] For EIS testing, set the initial voltage to 0V and the frequency range to 1 to 105Hz.

[0102] Optionally, in one embodiment of the present invention, the test parameters are set by the following steps:

[0103] For DPV test, set the test voltage to -0.3 to 0.6V, the pulse width to 0.03 to 0.06s, the sampling width to 0.01 to 0.03s, and the pulse period to 0.4 to 0.6s;

[0104] For SWV test, set the test voltage to -0.3 to 0.6V and the frequency to 8 to 12Hz;

[0105] For LSV testing, set the scan rate to 0.08 to 0.12 V / s and the sampling interval to 0.001 V;

[0106] For NPV testing, set the pulse width to 0.04 to 0.06 s, the sampling width to 0.01 to 0.03 s, and the pulse period to 0.4 to 0.6 s.

[0107] In the present application embodiment, the electrochemical characterization method of the electrode, wherein, all electrochemical tests in the present application embodiment are completed at room temperature using CHI760E electrochemical workstation. The experiment uses a three-electrode system for electrochemical characterization, with Ag / AgCl as the reference electrode, a platinum electrode as the counter electrode, and a test electrode (AgME, nanozyme-modified AgME, AuME, nanozyme-modified AuME, EcoGME, nanozyme-modified EcoGME) as the working electrode. During the test, we change the solution concentration by changing the test solution, suspend the test when the solution is changed, and place it for 30s (i.e., the third duration in the present application embodiment) before continuing the test to stabilize the solution. We use six test methods of CV, EIS, DPV, SWV, NPV, and LSV. Test parameter settings need to be performed in the electrochemical workstation before testing. For CV test, the test voltage range is set to -0.4~0.6V, and the scan rate is set to 0.1V / s. For EIS test, the initial voltage is set to 0V, and the frequency range is set to 1~105Hz. For DPV test, the test voltage range is set to -0.3~0.6V, the pulse width is set to 0.05s, the sampling width is set to 0.02s, and the pulse period is set to 0.5s. For SWV test, the test voltage range is set to -0.3~0.6V, and the frequency is set to 10Hz. For LSV test, the scan rate is set to 0.1V / s, and the sampling interval is set to 0.001V. For NPV test, the pulse width is set to 0.05s, the sampling width is set to 0.02s, and the pulse period is set to 0.5s. When testing the electrode by CV and EIS method, use PBS solution as the working solution, set up the test environment and adjust the test parameters, and then test directly. When testing DA solutions of different concentrations by DPV, SWV, NPV, and LSV methods, DA solutions with a concentration range of 1μM to 200uM were tested respectively. DA solutions with a concentration range of 1μM to 200uM were prepared by dropping 10mM, 1mM, and 100μM DA solutions into 10mL of PBS solution, respectively, so that the concentration of the DA solution varied within the range of 5uM to 50uM. Similarly, when testing AA solutions of different concentrations by DPV, SWV, NPV, and LSV methods, AA solutions with a concentration range of 200μM to 1200uM were tested respectively.

[0108] For example, refer to Figure 7 As shown, Figure 1 and Figure 2 are the AC impedance and Bode diagrams of the pure silver wire microelectrode and the silver wire nanozyme electrode modified with three nanozymes; Figure 3 and Figure 4 are the impedance value comparison diagram and phase margin comparison diagram of the pure silver wire microelectrode and the silver wire nanozyme electrode modified with three nanozymes at 100Hz. Figure 8As shown, Figure 1 and Figure 2 are the AC impedance and Bode diagrams of the pure gold wire microelectrode and the gold wire nanozyme electrode modified with three nanozymes; Figure 3 and Figure 4 are the impedance value comparison diagram and phase margin comparison diagram of the pure gold wire microelectrode and the gold wire nanozyme electrode modified with three nanozymes at 100Hz. Fig. 9 As shown, Figure 1 and Figure 2 are the AC impedance and Bode diagrams of the pure EcoG microelectrode and the three nanozyme-modified EcoG microelectrodes; Figure 3 and Figure 4 are the impedance value comparison diagram and phase margin comparison diagram of the pure EcoG microelectrode and the three nanozyme-modified EcoG microelectrodes at 100Hz. Fig.10 As shown in the figure, the two figures are the CV curves of the pure silver wire microelectrode and the silver wire nanozyme electrode modified with three nanozymes and the cathode charge storage capacity comparison. Fig.11 As shown in the figure, the two figures are the CV curves of the pure gold wire microelectrode and the gold wire nanozyme electrode modified with three nanozymes and the cathode charge storage capacity comparison. Fig.12 As shown, Figure 1, Figure 2 and Figure 3 are CV curves of pure EcoG microelectrode and EcoG microelectrode modified with three nanozymes, respectively; Figure 4 is a comparison of cathode charge storage capacity of pure EcoG microelectrode and EcoG microelectrode modified with three nanozymes.

[0109] Optionally, in one embodiment of the present invention, the AA solution is prepared by the following steps:

[0110] A first weight of DA powder is fully dissolved in a third volume of PBS solution to obtain a 50 mM AA solution; the ratio of the first weight to the third volume is a first preset value;

[0111] The fourth volume of 50 mM DA solution is dissolved in the fifth volume of PBS solution, and the mixture is fully mixed to obtain a 10 mM DA solution; the ratio of the fourth volume to the fifth volume is the second preset value;

[0112] The sixth volume of 10 mM DA solution is dissolved in the seventh volume of PBS solution to obtain a 1 mM DA solution; the ratio of the sixth volume to the seventh volume is a third preset value;

[0113] dissolving the eighth volume of 1 mM DA solution in the ninth volume of PBS solution to obtain a 100 nM DA solution; the ratio of the eighth volume to the ninth volume is a fourth preset value;

[0114] The second weight of AA powder is taken and fully dissolved in the tenth volume of PBS solution to obtain a 10 mM AA solution; the ratio of the second weight to the tenth volume is the fifth preset value.

[0115] The preparation process of DA and AA solutions in the embodiment of the present application can be:

[0116] Phosphate buffered saline (PBS) was used as solvent for both DA and AA solutions, and the pH of PBS was 7.4. In this experiment, DA and AA powders were purchased from Aladdin, and PBS reagent was purchased from Biosharp. In detail, for the DA solution, 0.984 g (i.e., the first weight in the embodiment of the present application) of DA powder was fully dissolved in 10 mL (i.e., the third volume in the embodiment of the present application) of PBS solution to obtain a 50 mM DA solution; 2 mL (i.e., the fourth volume in the embodiment of the present application) of 50 mM DA solution was added to 8 mL (i.e., the fifth volume in the embodiment of the present application) of PBS solution, and the mixture was fully mixed to obtain a 10 mM DA solution; similarly, 1 mL (i.e., the sixth volume in the embodiment of the present application) of 10 mM DA solution was placed in 9 mL (i.e., the seventh volume in the embodiment of the present application) of PBS solution to obtain a 1 mM DA solution; finally, 1 mL (i.e., the eighth volume in the embodiment of the present application) of 1 mM DA solution was dissolved in 9 mL (i.e., the ninth volume in the embodiment of the present application) of PBS solution to obtain 100 nM DA. For the AA solution, 0.1761 g (i.e., the second exemplary weight in the embodiment of the present application) of AA powder was weighed and fully dissolved in 10 mL (i.e., the tenth exemplary volume in the embodiment of the present application) of PBS solution to obtain a 10 mM AA solution, which was used for subsequent dropwise addition tests. Considering that the chemical properties of the above solutions are unstable, all of the above solutions must be prepared and used immediately and stored at room temperature and away from light. It will be understood that the weights and volumes in the above embodiments are exemplary examples, and those skilled in the art can adjust the weights and volumes according to the ease and density of the required solution; this application does not impose specific limitations.

[0117] The preparation method and the corresponding testing process provided by the present application are described in detail below with a specific embodiment:

[0118] Embodiment 1:

[0119] Preparation and SEM characterization of nanozyme electrodes.

[0120] In this study, we used three different nanozyme materials (including Au 24 Cd 1 Nanozymes, Au 24 Er 1 Nanozymes, Au 25Three different types of microelectrodes (including AgME, AuME, and EcoGME) were modified with artificial nanozymes to explore the effect of modification of artificial nanozymes on the electrochemical properties of implantable microelectrodes. In order to compare the effects of different metal atomic layer interfaces on the performance of nanozyme electrodes, this study used AgME, AuME, and EcoGME as carrier electrodes. We modified the nanozyme material onto the electrode surface by electroplating using the multi-potential step method. For AgME, we scanned 4 circles at a potential of 0.2V to 1.3V to obtain AgME modified with nanozymes. For AuME, AuME modified with nanozymes was obtained by scanning 10 circles at a potential of 1.7V to 3.4V. For EcoGME, we used an adapter circuit board to electroplated 8 channels at the same time, and obtained EcoGME modified with nanozymes by scanning 8 circles at a potential of 0.7V to .4.4V (the current response diagram of electroplating is shown in the figure). Fig.13 ).

[0121] In this study, scanning electron microscopy was used to analyze the surface of each electrode. Figure 4 , Figure 5 or Figure 6 As shown in the figure, the surface of the unmodified bare electrode is relatively smooth, while the surface of the modified nanozyme electrode is distributed with atomic cluster layers. This shows that after the artificial nanozyme material is modified, the roughness of the electrode surface increases. Previous studies have shown that the increase in the roughness of the electrode surface is conducive to increasing the reaction sites on the electrode surface, thereby accelerating its interfacial electron transfer rate.

[0122] Embodiment 2:

[0123] Basic electrochemical characterization tests of nanozyme electrodes.

[0124] In order to explore the effect of nanozyme modification on the electrochemical sensing performance of implantable microelectrodes, we first performed basic electrochemical characterization of AgME, AuME and EcoGME before and after modification of the three nanozyme materials by EIS and CV. The entire electrochemical detection experiment was completed using an electrochemical workstation. In order to verify the inherent characteristics of each electrode, we first performed EIS tests on each electrode, and the impedance detection frequency was set to 1Hz~Hz ( Figure 7 , Figure 8 , Fig. 9 ). The results showed that in the range of 1Hz to Hz, the impedance of the electrode modified with nanozymes was significantly reduced, with AgME reducing by up to 18 times, AuME reducing by up to 9 times, and EcoGME reducing by up to 13 times. Then we performed CV tests on each electrode in PBS solution ( Fig.10 , Fig.11 , Fig.12). For AgME, CV tests were performed at a voltage of -0.8 to 0.1 V. The comparison curves show that the modified Ag nanozyme electrode has a higher oxidation current and a larger response current area than pure AgME. The results of obtaining its cathode charge storage capacity show that the cathode charge storage capacity of nanozyme-modified AgME is up to 21 times higher than that of unmodified AgME. For AuME, CV tests were performed at a voltage of -0.4 to 0.6 V. The comparison curves show that the modified Au nanozyme electrode has a higher oxidation current and a larger response current area than that of pure AuME. Similarly, the cathode charge storage capacity of nanozyme-modified AuME is up to 16 times higher than that of unmodified AgME. For EcoGME, we performed CV tests on 8 channels at a voltage of -0.4 to 0.6 V. The comparison curves show that the modified nanozyme EcoGME has a higher oxidation current and a larger response current area than pure EcoGME. The data results show that the cathode charge storage capacity of nanozyme-modified EcoGME is increased by up to 23 times compared with unmodified EcoGME.

[0125] In summary, the method provided in the embodiment of the present application includes: providing a substrate, removing surface impurities of the substrate; wherein the substrate includes silver wire, gold wire or pure EcoGME; taking the solid powder of nanozyme and deionized water and mixing them in a preset ratio to obtain a nanozyme solution; the nanozyme includes Au 24 Cd 1 Nanozymes, Au 24 Er 1 Nanozymes or Au 25 Nanozyme; using the Ag / AgCl electrode as the reference electrode, the platinum electrode as the counter electrode, and the substrate as the working electrode, through an electrochemical workstation, in the nanozyme solution, based on the multi-potential step method, the nanozyme deposition treatment is performed; the nanozyme is deposited on the surface of the substrate to obtain a nanozyme microelectrode; the nanozyme microelectrode is insulated to obtain a nanozyme microelectrode with a partial area exposed; wherein the exposed part is used as the sensing area of ​​the electrode. The embodiment of the present application uses an electrochemical workstation, in the nanozyme solution, based on the multi-potential step method, to achieve the modification of multiple electrodes with multiple artificial nanozymes, and the catalytic activity of the nanozyme itself is used to reduce the inherent high electrochemical impedance of the electrode. At the same time, this gold-based clustered nanozyme also has a high antioxidant activity, thereby effectively reducing the chance of inducing neuroinflammation. The embodiment of the present application can realize the preparation of multiple nanozyme microelectrodes, which is beneficial to improving the forest sensitivity and biocompatibility of the microelectrode.

[0126] The embodiment of the present invention also provides a nanozyme microelectrode, which is prepared by the above-mentioned method for preparing the nanozyme microelectrode.

[0127] Similarly, the contents of the above method embodiments are all applicable to the present nanozyme microelectrode embodiment. The functions specifically implemented by the present nanozyme microelectrode embodiment are the same as those of the above method embodiments, and the beneficial effects achieved are also the same as those achieved by the above method embodiments.

[0128] In some selectable embodiments, the function / operation mentioned in the block diagram may not occur in the order mentioned in the operation diagram. For example, depending on the function / operation involved, the two boxes shown in succession can actually be executed substantially simultaneously or the boxes can sometimes be executed in reverse order. In addition, the embodiment presented and described in the flow chart of the present invention is provided by way of example, for the purpose of providing a more comprehensive understanding of technology. The disclosed method is not limited to the operation and logic flow presented herein. Selectable embodiments are expected, wherein the order of various operations is changed and the sub-operation of a part for which is described as a larger operation is performed independently.

[0129] In addition, although the present invention is described in the context of functional modules, it should be understood that, unless otherwise specified, one or more of the functions and / or features can be integrated into a single physical device and / or software module, or one or more functions and / or features can be implemented in a separate physical device or software module. It is also understood that a detailed discussion of the actual implementation of each module is unnecessary for understanding the present invention. More specifically, in view of the properties, functions and internal relationships of the various functional modules in the device disclosed herein, the actual implementation of the module will be understood within the conventional skills of the engineer. Therefore, those skilled in the art can implement the present invention set forth in the claims without excessive experimentation using ordinary techniques. It is also understood that the specific concepts disclosed are merely illustrative and are not intended to limit the scope of the present invention, which is determined by the full scope of the appended claims and their equivalents.

[0130] If the functions are implemented in the form of software functional units and sold or used as independent products, they can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present invention, or the part that contributes to the prior art or the part of the technical solution, can be embodied in the form of a software product. The computer software product is stored in a storage medium, including several programs to enable a computer device (which can be a personal computer, a server, or a network device, etc.) to perform all or part of the steps of the methods described in each embodiment of the present invention. The aforementioned storage medium includes: U disk, mobile hard disk, read-only memory (ROM, Read-Only Memory), random access memory (RAM, Random Access Memory), disk or optical disk, etc., which can store program codes.

[0131] The logic and / or steps represented in the flowchart or otherwise described herein, for example, may be considered as an ordered list of executable programs for implementing the logical functions, and may be embodied in any computer-readable medium for use by a program execution system, device or apparatus (such as a computer-based system, a system including a processor, or other system that can fetch and execute a program from a program execution system, device or apparatus), or in conjunction with such program execution systems, devices or apparatuses. For purposes of this specification, a "computer-readable medium" may be any device that can contain, store, communicate, propagate or transmit a program for use by a program execution system, device or apparatus, or in conjunction with such program execution systems, devices or apparatuses.

[0132] More specific examples of computer-readable media (a non-exhaustive list) include the following: an electrical connection with one or more wires (electronic device), a portable computer disk case (magnetic device), a random access memory (RAM), a read-only memory (ROM), an erasable and programmable read-only memory (EPROM or flash memory), an optical fiber device, and a portable compact disk read-only memory (CDROM). In addition, the computer-readable medium may even be a paper or other suitable medium on which the program is printed, since the program may be obtained electronically, for example, by optically scanning the paper or other medium, followed by editing, deciphering or, if necessary, processing in another suitable manner, and then stored in a computer memory.

[0133] It should be understood that the various parts of the present invention can be implemented by hardware, software, firmware or a combination thereof. In the above-mentioned embodiments, a plurality of steps or methods can be implemented by software or firmware stored in a memory and executed by a suitable program execution system. For example, if implemented by hardware, as in another embodiment, it can be implemented by any one of the following technologies known in the art or their combination: a discrete logic circuit having a logic gate circuit for implementing a logic function for a data signal, a dedicated integrated circuit having a suitable combination of logic gate circuits, a programmable gate array (PGA), a field programmable gate array (FPGA), etc.

[0134] In the above description of this specification, the description with reference to the terms "one embodiment / example", "another embodiment / example" or "certain embodiments / examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0135] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

[0136] The above is a specific description of the preferred implementation of the present invention, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of the present invention.

Claims

1. A method for preparing a nanozyme microelectrode, characterized in that: The following steps are involved: Providing a substrate, and removing surface impurities of the substrate; wherein the substrate comprises silver wire, gold wire or pure EcoGME; The solid powder of nanozyme is mixed with deionized water in a preset ratio to obtain a nanozyme solution; the nanozyme includes Au 24 Cd1 nanozyme, Au 24 Er1 nanozyme or Au 25 Nanozymes; Using an Ag / AgCl electrode as a reference electrode, a platinum electrode as a counter electrode, and the substrate as a working electrode, an electrochemical workstation is used to perform nanozyme deposition in the nanozyme solution based on a multi-potential step method; the nanozyme is deposited on the surface of the substrate to obtain a nanozyme microelectrode; The nanozyme microelectrode is insulated to obtain a nanozyme microelectrode with a partial area exposed; wherein the exposed part serves as the sensing area of ​​the electrode.

2. The method for preparing the nanozyme microelectrode according to claim 1, characterized in that: The nanozyme deposition process is based on a multi-potential step method, comprising: If the substrate is a silver wire, the electrodeposition parameters are set to set a plurality of potentials in the voltage range of 0.2 to 2 V, with a step amplitude between the potentials of 0.1 to 0.2 V; and the duration of each potential is set; Set the deposition current to 0.3 to 0.5 mA; start the electrodeposition process; Alternatively, if the substrate is a gold wire or pure EcoGME, the electrodeposition parameters are set to set a number of potentials in the voltage range of 1.7 to 3.4 V, with a step amplitude of 0.1 to 0.2 V between the potentials; and the duration of each potential is set; Set the deposition current to 0.3 to 0.5 mA; start the electrodeposition process.

3. The method for preparing the nanozyme microelectrode according to claim 1, characterized in that: The method of providing a substrate and removing surface impurities of the substrate comprises: A silver wire is provided, and after being polished with sandpaper, the silver wire is placed in distilled water for ultrasonic impurity removal treatment; Alternatively, a gold wire is provided, and after being polished with sandpaper, the gold wire is placed in distilled water for ultrasonic impurity removal treatment; Alternatively, a substrate is provided, and patterned magnetron sputtering is performed on the substrate through a preset mask, and Cr and Au are deposited in sequence to obtain pure EcoGME.

4. The method for preparing the nanozyme microelectrode according to claim 1, characterized in that: The Au 24 Cd1 nanozyme was prepared by the following steps: Adding HauCl4 aqueous solution, Cd(NO3)2 aqueous solution and MPA aqueous solution to the first volume of ultrapure water, and stirring continuously; wherein the added HauCl4 aqueous solution, Cd(NO3)2 aqueous solution and MPA aqueous solution correspond to preset densities and preset volumes, respectively; Add NaOH aqueous solution dropwise until the pH value of the reaction solution is 12; add a second volume of NaBH solution; Saturated CO is introduced and closed treatment is performed; the duration of the closed treatment is the first duration; After stirring for a second time, the brown solution was freeze-dried to obtain pure Au 24 Cd1 nanozyme is stored at -18 to -22°C.

5. The method for preparing the nanozyme microelectrode according to claim 1, characterized in that: The microelectrodes were characterized by the following steps: The Ag / AgCl was used as the reference electrode, the platinum electrode as the counter electrode, and the test electrode as the working electrode for testing; During the test, the test solution is replaced to change the solution concentration; the test electrode includes a microelectrode and a nanozyme microelectrode, the microelectrode includes an AgME microelectrode, an AuME microelectrode and an EcoGME microelectrode; the nanozyme microelectrode includes a nanozyme-modified AgME microelectrode, a nanozyme-modified AuME microelectrode, and a nanozyme-modified EcoGME microelectrode; If the solution is changed, the test is suspended; and the test solution is allowed to stand for a third period of time before continuing the test.

6. The method for preparing the nanozyme microelectrode according to claim 1, characterized in that: The method comprises: The microelectrode was tested based on the CV and EIS methods. Phosphate buffer solution was used as the working solution. The test environment was built and the test parameters were set before starting the test. Alternatively, DA solutions of different concentrations are tested by DPV, SWV, NPV, and LSV methods; the concentration range of the DA solution includes 1 μM to 200 uM; Alternatively, AA solutions of different concentrations are tested by DPV, SWV, NPV, and LSV methods; the concentration range of the AA solution is 200 μM to 1200 uM.

7. The method for preparing the nanozyme microelectrode according to claim 6, characterized in that: The test parameters are set by following the steps below: For CV testing, set the test voltage to -0.4 to 0.6 V and the scan rate to 0.05 to 0.15 V / s. For EIS testing, set the initial voltage to 0V and the frequency range to 1 to 105Hz.

8. The method for preparing the nanozyme microelectrode according to claim 6, characterized in that: The test parameters are set by following the steps below: For DPV testing, set the test voltage to -0.3 to 0.6V, the pulse width to 0.03 to 0.06s, the sampling width to 0.01 to 0.03s, and the pulse period to 0.4 to 0.6s; For SWV test, set the test voltage to -0.3 to 0.6V and the frequency to 8 to 12Hz; For LSV testing, set the scan rate to 0.08 to 0.12 V / s and the sampling interval to 0.001 V; For NPV testing, set the pulse width to 0.04 to 0.06 s, the sampling width to 0.01 to 0.03 s, and the pulse period to 0.4 to 0.6 s.

9. The method for preparing the nanozyme microelectrode according to claim 6, characterized in that: The AA solution was prepared by the following steps: A first weight of DA powder is fully dissolved in a third volume of PBS solution to obtain a 50 mM AA solution; the ratio of the first weight to the third volume is a first preset value; The fourth volume of 50 mM DA solution is dissolved in the fifth volume of PBS solution, and the mixture is fully mixed to obtain a 10 mM DA solution; the ratio of the fourth volume to the fifth volume is the second preset value; The sixth volume of 10 mM DA solution is dissolved in the seventh volume of PBS solution to obtain a 1 mM DA solution; the ratio of the sixth volume to the seventh volume is a third preset value; dissolving the eighth volume of 1 mM DA solution in the ninth volume of PBS solution to obtain a 100 nM DA solution; the ratio of the eighth volume to the ninth volume is a fourth preset value; The second weight of AA powder is taken and fully dissolved in the tenth volume of PBS solution to obtain a 10 mM AA solution; the ratio of the second weight to the tenth volume is the fifth preset value.

10. A nanozyme microelectrode, characterized in that: The nanozyme microelectrode is prepared by the preparation method of the nanozyme microelectrode according to any one of claims 1 to 9.

Images