CN114486860A - Hydrogen peroxide real-time in-situ quantitative analysis method based on bipolar nano electrode array - Google Patents
Hydrogen peroxide real-time in-situ quantitative analysis method based on bipolar nano electrode array Download PDFInfo
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- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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Abstract
The invention discloses a hydrogen peroxide real-time in-situ quantitative analysis method based on a bipolar nano electrode array, which comprises the following steps: preparing a gold nano-electrode array by using a template-assisted electrochemical deposition method; preparing a driving electrode Ag/AgCl; constructing a closed bipolar electrode by using the nano electrode array and the driving electrode; adding polylysine at the cathode end of the closed bipolar electrode, adding a cell suspension for in-situ incubation, and adding a stimulant to induce the release of hydrogen peroxide in cells; and adding an ECL luminescent system at the anode end of the closed bipolar electrode, and quantifying the content of hydrogen peroxide released by the living cells at the cathode end through the ECL intensity of the anode under an applied potential. The invention utilizes a label-free method to induce the release of hydrogen peroxide in cells, and converts sensitive electrochemical signals into visual optical signals by combining with the bipolar electrode, thereby realizing the imaging analysis of endogenous hydrogen peroxide in cells.
Description
Technical Field
The invention belongs to the technical field of biosensing, relates to an electrochemiluminescence method for imaging hydrogen peroxide, and particularly relates to a real-time in-situ quantitative analysis method for hydrogen peroxide based on a bipolar nano electrode array.
Background
Currently, the intracellular hydrogen peroxide is mainly analyzed by a chemiluminescence method, a fluorescence method and a chemical method. The chemiluminescence method is used for detecting hydrogen peroxide by taking hydrogen peroxide as a co-reactant and luminol as a luminophore, but the hydrogen peroxide and the luminophore are mixed together to easily generate interference, and the sensitivity of the luminol and hydrogen peroxide system is relatively low, so that some biological enzymes, enzyme-like catalysts or metal nanoparticles are needed for catalyzing reaction to amplify signals, and the signals are further used for analysis. However, the fluorescence reagent required for the fluorescence method has insufficient sensitivity due to the presence of autofluorescence and the phenomenon of fluorescence bleaching. The chemical method usually requires that the micro-nano electrode is inserted into cells to cause certain damage to the cells, and the preparation process of the micro-nano electrode is complicated and the required instruments are expensive.
Disclosure of Invention
The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide an electrochemiluminescence imaging analysis method for hydrogen peroxide in cells by using a bipolar nano electrode array which does not need to be modified.
The technical scheme is as follows: the invention provides a bipolar nano-electrode array-based real-time in-situ quantitative analysis method for hydrogen peroxide, which comprises the following steps:
1) preparing a gold nano-electrode array by using a template-assisted electrochemical deposition method;
2) preparing a driving electrode Ag/AgCl;
3) constructing a closed bipolar electrode by using the gold nano-electrode array obtained in the step 1) and the driving electrode prepared in the step 2);
4) adding polylysine at the cathode end of the closed bipolar electrode, adding the cell suspension for in-situ incubation, and adding a stimulating agent to induce the release of hydrogen peroxide in the cells;
5) and adding an ECL luminescent system at the anode end of the closed bipolar electrode, and quantifying the content of hydrogen peroxide released by the living cells at the cathode end through the ECL intensity of the anode under an applied potential.
The gold nano-electrode array in the step 1) is prepared by taking commercial bi-pass anodic aluminum oxide as a template, fixing the template on a support, taking the template as a working electrode, soaking the working electrode into a self-made gold electrolyte, taking mercury/mercurous sulfate as a reference electrode, taking a platinum sheet electrode as a counter electrode, performing electrodeposition by adopting a step constant current density method to prepare the gold nano-electrode array, then respectively performing grinding and polishing by using diamond millstones with different particle sizes, then performing polishing by using a diamond suspension, and finally performing ultrasonic cleaning by using deionized water to obtain the spare gold nano-electrode array.
The preparation method of the driving electrode Ag/AgCl in the step 2) comprises the specific steps of soaking silver wires in a mixed solution containing Cl ions, and then washing with distilled water.
The specific steps of constructing the closed bipolar electrode in the step 3) are as follows:
A. spin-coating a negative photoresist on a clean and flat glass mold, placing the clean and flat glass mold on a hot plate, heating and curing the glass mold, cooling the glass mold, cutting the glass mold into a circular patch with the diameter of about 1.5 +/-0.2 cm, and drilling a micro-cavity with the diameter of about 3 +/-0.1 mm on the circular patch for storing liquid;
B. assembling the polydimethylsiloxane chip and the polished gold nano electrode array into a sandwich-like structure, bonding the sandwich-like structure by an oxygen plasma cleaning machine, drying the sandwich-like structure, and then using Ag/AgCl as a driving electrode to construct a closed bipolar electrode.
Wherein, the stimulant in the step 4) is myristoyl phorbol ethyl ester, and the concentration of the stimulant is 1 +/-0.2 mu g/mL.
Wherein the driving voltage applied in the step 5) is 1.8V.
Wherein, the luminescent system added into the anode end in the step 5) comprises ruthenium bipyridyl and a coreactant, namely dibutylaminoethanol.
Wherein, the concentration of ruthenium bipyridyl of the luminophor added at the anode end in the step 5) is 1 plus or minus 0.3mmol/L, and the concentration of the coreactant dibutylaminoethanol is 20 plus or minus 0.5 mmol/L.
Has the advantages that: the invention is based on the photoelectric conversion effect of a closed bipolar electrode, a certain voltage is applied to two ends of the bipolar electrode, and when the voltage value reaches a critical value, the two poles of the bipolar electrode are induced to generate oxidation and reduction reactions. By incubating live cells in situ at the cathode of the bipolar electrode, adding electrochemiluminescence molecules and a co-reactant at the anode, under an external voltage, a luminescent system at the anode end is oxidized to emit electrochemiluminescence signals, and hydrogen peroxide in cells at the cathode end is reduced. The hydrogen peroxide content in the cells at the cathode end can be monitored through the intensity of the electrochemiluminescence signal at the anode end. The surface of the gold nano electrode array does not need any signal amplification biological enzyme, enzyme-like enzyme or nano particles, and the detection of the content of hydrogen peroxide in cells is effectively realized. Based on the structural advantages of the closed bipolar electrode, the detection end and the light-emitting end are effectively separated, mutual interference is avoided, and the sensitivity of imaging analysis is further improved. The combination of the bipolar electrode system and the electrochemiluminescence system effectively combines a high-sensitivity electrochemical method and a high-resolution imaging technology, and the system does not need complex and expensive instruments and equipment and is simple and convenient to operate. The combination of the bipolar electrode system and the electrochemiluminescence system effectively combines a high-sensitivity electrochemical method and a high-resolution imaging technology, and the system does not need complex and expensive instruments and equipment and is simple and convenient to operate. Because the hydrogen peroxide is highly expressed in cancer cells and is low in expression level in normal cells, the method can well distinguish the cancer cells from the normal cells, and provides certain guiding significance for the initial diagnosis and treatment of cancer diseases.
Drawings
FIG. 1 is a schematic diagram of imaging studies of intracellular endogenous hydrogen peroxide based on gold nanoelectrode arrays; the amplification part is the detailed process of the stimulators to stimulate the cells to produce hydrogen peroxide.
FIG. 2: FIG. 2A is an SEM image of a porous alumina template; FIG. 2B is an SEM image of a porous alumina template after silver plating; fig. 2C is an SEM image of gold nanowires grown in porous alumina pores.
FIG. 3: FIG. 3A is a graph of electrochemiluminescence signal-potential relationship based on gold nanoplates for (a)5mM potassium ferricyanide, (b) 5. mu.M hydrogen peroxide, and (c)0.01M PBS (pH 7.0). FIG. 3B is the electrogenerated chemiluminescence imaging map of the gold nanoelectrode array boundary. FIG. 3C is the electrochemiluminescence intensity curve values of the portion of FIG. 3B marked 7 yellow. Fig. 3D is a quantitative analysis of different hydrogen peroxide concentrations by an EMCCD imaging system. The inset in fig. 3D is a linear relationship of EMCCD image gray scale value versus hydrogen peroxide concentration.
FIG. 4: FIG. 4A is an image of electrochemiluminescence of cells in 5mM potassium ferricyanide medium at the cathode of an array of gold nanoelectrodes at different potentials; FIG. 4B is an imaging diagram of electrochemiluminescence of cells in 0.01M PBS (pH 7.0) medium at the cathode of the gold nano-electrode array under different potentials; fig. 4C is an imaging diagram of electrochemiluminescence with the cathode of the gold nano-electrode array as a stimulator stimulating cells to generate hydrogen peroxide under different potentials.
FIG. 5: FIG. 5A is an SEM image of cells fixed on a gold nanoelectrode array with 4% paraformaldehyde; FIG. 5B is an electrochemiluminescence diagram of HeLa cells in the absence of a stimulator stimulating the cells to produce hydrogen peroxide; FIG. 5C is an electrochemiluminescence diagram of HeLa cells stimulating the production of hydrogen peroxide by cells under the action of 1 + -0.2 μ g/mL of a stimulator.
FIG. 6: FIG. 6A is an electrochemiluminescence diagram of LO2 cells in the absence of stimulation of the cells with a stimulating agent to produce hydrogen peroxide; FIG. 6B is an electrochemiluminescence diagram of LO2 cells stimulating the production of hydrogen peroxide by 1. + -. 0.2. mu.g/mL of the stimulator.
Detailed Description
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments. It should be noted that variations and modifications can be made by those skilled in the art without departing from the principle of the present invention, and these should also be construed as falling within the scope of the present invention.
Reagents and instruments used in this experiment
Reagents used for the experiments: chloroauric acid trihydrate (HAuCl)4·3H2O) was purchased from Alfa Aesar (shanghai, china). Triammonium citrate, ethylenediaminetetraacetic acid (EDTA), phosphoric acid (H)3PO4) Hydrogen peroxide solution (30 wt% in H)2O) and anhydrous sodium sulfite (Na)2SO3) All purchased from alatin industries ltd (shanghai, china). Poly 1-lysine solution (0.1%, PLL) was purchased from bekken biotechnology (shanghai, china). Tris (2, 2' -bipyridyl) ruthenium (II) hexahydrate (Ru (bpy))3Cl2·6H2O), carnosic acid ester (PMA) and 2- (dibutylamino)) ethanol (DBAE) were supplied by Sigma Aldrich (shanghai, china). A commercially available Anodized Aluminum (AAO) membrane (diameter 13. + -.2 mm, pore diameter 200. + -.5 nm, thickness 50. + -.10 μm) was purchased from Shenzhen TopMembranes Inc (Shenzhen, Shanghai). Poly (dimethylsiloxane) (PDMS) monomers and curing agents were obtained from Dow core Inc. (Midland, Mich.). Human cervical cancer cells (HeLa) and human normal hepatocytes (LO2) were purchased from shanghai bioscience institute of china academy of sciences. All chemicals used in this study were of analytical grade and were not further purified unless otherwise indicated. Ultrapure water (. gtoreq.18.2 M.OMEGA.cm) was obtained from a Millipore system (Direct-Q3 UV, Millipore, USA) and used throughout the study.
Instruments used for the experiment: scanning Electron Microscopy (SEM) measurements were performed using a FEI Inspect F50 scanning electron microscope (FEI, USA). Electrochemiluminescence (ECL) experiments were performed using MPI-E electrochemistry and an ECL analyzer (siennamii ltd, china). Electrochemical measurements were performed at CHI660E electrochemical workstation (shanghai chen instruments ltd). An Electrochemiluminescence (ECL) imaging device consists of a Nikon Eclipse Ci upright microscope, an Andor iXon 888 electron charge multiplication CCD camera (EMCCD), an air objective lens (4x) and a water immersion objective lens (60x, Nikon). The electrochemiluminescence intensity-voltage curve was measured using Cyclic Voltammetry (CV) in CHI660E electrochemical workstation (shanghai chenhua instruments ltd, china). The distance between the microscope lens and the tested sample is adjusted by moving the electric translation table. The recorded electrochemiluminescence imaging data were analyzed using ImageJ software.
Example 1: preparation of gold nano-electrode array
And obtaining the gold nano-electrode array by using a template-assisted electrodeposition method. Briefly, Anodized Aluminum Oxide (AAO) is used as a template, which is mounted on a specific holder as a working electrode. Before electrodeposition, a silver thin film having a thickness of-1 μm was vapor-deposited on one side of the AAO film by an electron beam vapor deposition method (Kurt J. Lesker). The mixture was mixed with a solution containing 25g/L chloroauric acid, 80g/L triammonium citrate, and 150g/L anhydrous sodium sulfite (Na)2SO3) And 60g/L Ethylene Diamine Tetraacetic Acid (EDTA), and a repeated timing potential method is adopted to carry out the self-made electrolyte at 8.512mA/cm2Obtaining the gold nano-electrode array under constant current density. The electrodeposition experiments were carried out in a conventional three-electrode system, Hg/Hg2SO4 electrode and a platinum electrode were used as Reference electrodes, and Reference600 constant current potentiostat/ZRA (Gamry Instruments,usa) as a counter electrode. After electrodeposition, the resulting sample was immersed in a room temperature nitrite solution for 5s to remove the silver layer. And then washing the sample with ethanol and deionized water in sequence, drying and polishing to obtain the flat gold nano electrode array.
Example 2: cell culture
HeLa cells (human cervical cancer cells) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 1% penicillin, 1% streptomycin and 10% fetal bovine serum at 37 ℃ with 5% CO2. LO2 cells (normal hepatocytes) were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 1% penicillin, and 1% streptomycin.
The method comprises the steps of firstly carrying out preliminary polishing, cleaning and sterilization on a nano-electrode array, then adding 20 microliter of polylysine solution (PLL, 1mg/mL) to one side of the gold nano-electrode array to promote cell adhesion growth, carrying out incubation at 37 ℃ for 2 hours, then digesting cells from a culture dish by trypsin, carrying out centrifugal collection, and taking a certain amount of suspension in a fresh culture medium. Then adding a part of the cell suspension to one end of the gold nano-electrode array. Finally, the cells were incubated in an incubator for 8h for subsequent experiments.
Example 3: principle verification
1) Firstly, the bipolar electrode system constructed by the gold nano electrode array is verified to be used for realizing the photoelectric conversion function. To the cathode side of the bipolar electrode were added (a)5mM potassium ferricyanide, (b) 5. mu.M hydrogen peroxide, and (c) PBS (0.01M, pH 7.0) solution, respectively, and to the anode side was added a luminescent system (ruthenium bipyridine, 1mM Ru (bpy)3 2+) And a co-reactant (dibutylamine ethanol, 20mM DBAE), potassium ferricyanide is obtained through cyclic voltammetry, and the reduction potentials of hydrogen peroxide and water are 1.0V, 1.8V and 2.2V respectively, so that a stronger electrochemiluminescence signal is obtained at the potential. The result shows that the gold nano electrode array can effectively realize photoelectric conversion. See fig. 3A.
2) And secondly, the gold nano electrode array can be further used for experimental electrochemiluminescence imaging. And carrying out imaging analysis on the edge of the gold nano electrode array. The results show that the exposed gold nano-electrode array can effectively obtain electrochemiluminescence imaging, and electrochemiluminescence imaging can not be obtained without the exposed gold nano-electrode array, as shown in fig. 3B. The result shows that the gold nano electrode array can effectively realize electrochemiluminescence imaging. FIG. 3C is a graph of the gray scale values of the portion of FIG. 3B where the yellow line is drawn.
Example 4: imaging quantitative analysis of hydrogen peroxide solution on gold nano electrode array
To study the amount of endogenous hydrogen peroxide released in the cells, ECL of standard hydrogen peroxide solutions of different concentrations (1 μ M to 1mM) on gold nanoelectrode arrays was first imaged using EMCCD and read for grey values, see figure 3D. The curve for quantifying the hydrogen peroxide by the EMCCD is obtained by linear fitting the hydrogen peroxide concentration and the EMCCD image gray value, see fig. 3D inset, the linear relationship of which is that I is 31.3cH2O2+792.9, detection limit 0.3. mu.M.
Example 5: imaging of cells on gold nanoelectrode arrays
To study electrochemiluminescence imaging of cells on gold nanoelectrode arrays, cells were incubated in situ at the cathode end of gold nanoelectrodes. When 5mM potassium ferricyanide is added at the cell end, under the action of cyclic voltammetry, the potential window is 0-1V, as can be obtained from FIG. 4A, the imaging intensity is also continuously increased along with the increase of the potential, because potassium ferricyanide is a hydrophilic reagent and the cell membrane is composed of phospholipid bilayer, the potassium ferricyanide molecule can not enter the cell, the cell membrane acts as a physical barrier to block the electron transfer, and thus, a negative electrochemiluminescence imaging image is obtained. When PBS (0.01M, pH 7.0) is added at the cell end, under the action of cyclic voltammetry, the potential window is 0-2.2V, and the imaging intensity is continuously increased along with the increase of the potential, and the cell membrane is composed of phospholipid bilayers and acts as a physical barrier to block electron transfer, so that a negative electrochemiluminescence imaging image is also obtained. Hydrogen peroxide is produced intracellularly when a stimulator of 1. mu.g/mL myristoyl phorbol ethyl ester (PMA) is added to the cell microcavity of the cultured cells. Under the action of cyclic voltammetry, the potential window is 0-1.8V, and the image intensity is continuously increased along with the increase of the potential, so that a positive electrochemiluminescence image is obtained, wherein the image intensity is the same as the image intensity of the image. Therefore, the gold nano electrode array can effectively realize electrochemiluminescence imaging of intracellular endogenous hydrogen peroxide.
Example 6: in situ incubation of HeLa cells on gold nanoelectrode arrays
The bipolar electrode based on the gold nano-electrode array can effectively realize the imaging of intracellular endogenous hydrogen peroxide. Firstly, constructing a gold nano-electrode array into a bipolar electrode system, spraying 75% alcohol to the system for surface disinfection, then placing the system in a super clean bench, and performing further sterilization treatment for 40min by an ultraviolet lamp; subsequently, 200 microliters of 1mg/mL polylysine was added to the cathode microcavity of the bipolar electrode, placed at 37 ℃ for 2h, finally the polylysine was aspirated away, and the cell-containing medium suspension was added to the microcavity and placed in an incubator. After 8h, the suspension was aspirated and the residual medium was washed with 0.01M PBS until clean. And finally, adding 4% paraformaldehyde to fix the cells, drying, and then characterizing the gold nano electrode array with the cells by using a scanning electron microscope. FIG. 5A shows that cells grown on the gold nanoelectrode array are spindle-shaped, similar to the growth of cells in a culture dish; the inset in fig. 5A is an enlarged SEM image, which clearly shows the outline of the cell and has a better morphology, indicating that the gold nano-electrode array has good biocompatibility and is beneficial to the growth of the cell. FIG. 5B is an image of electrochemiluminescence images of HeLa cells cultured on the cathode side of a bipolar electrode in 0.01M PBS medium when a voltage of 2.2V is applied, and the cell area is in a black non-luminescence state due to the non-conductivity of the cell membrane and the blocking of electron transfer; while FIG. 5C shows that the addition of 1. + -. 0.2. mu.g/mL myristoyl phorbol ethyl ester (PMA) to the microcavity of the cell based on 5B cells stimulates the production of hydrogen peroxide by the cell, and the area of the cell is illuminated when a voltage of 1.8V is applied, indicating that the cell can produce hydrogen peroxide under the stimulation of PMA, and the system can effectively realize the imaging of endogenous hydrogen peroxide in the cell, and the endogenous hydrogen peroxide released by a single HeLa cell is measured to be about 45. + -. 3. mu.M.
Example 7: in situ incubation of LO2 cells on gold nanoelectrode arrays
This part of the experiment was identical to the specific procedure of example 6, except that the cultured cells were changed from the HeLa cancer cells of example 6 to the normal cells LO2 cells, and the other procedures were identical. The resulting phenomena are substantially uniform. Because the content of hydrogen peroxide in normal cells is less than that of cancer cells, the electrochemiluminescence intensity value obtained by the hydrogen peroxide generated by the normal cells LO2 under the stimulation of 1 +/-0.2 mu g/mL myristoyl phorbol ethyl ester (PMA) is lower, and the linear equation obtained in figure 3D is substituted to obtain the content of endogenous hydrogen peroxide released by a single normal cell LO2, which is about 7 +/-0.5 mu M.
Claims (8)
1. A real-time in-situ quantitative analysis method for hydrogen peroxide based on a bipolar nano electrode array is characterized by comprising the following steps:
1) preparing a gold nano-electrode array by using a template-assisted electrochemical deposition method;
2) preparing a driving electrode Ag/AgCl;
3) constructing a closed bipolar electrode by using the gold nano-electrode array obtained in the step 1) and the driving electrode prepared in the step 2);
4) adding polylysine at the cathode end of the closed bipolar electrode, adding the cell suspension for in-situ incubation, and adding a stimulating agent to induce the release of hydrogen peroxide in the cells;
5) and adding an ECL luminescent system at the anode end of the closed bipolar electrode, and quantifying the content of hydrogen peroxide released by the living cells at the cathode end through the ECL intensity of the anode under an applied potential.
2. The method for real-time in-situ quantitative analysis of hydrogen peroxide based on a bipolar nanoelectrode array according to claim 1, wherein the gold nanoelectrode array in step 1) is prepared by fixing commercial bi-pass anodic aluminum oxide as a template on a support, soaking the template as a working electrode into a self-made gold electrolyte, preparing the gold nanoelectrode array by using mercury/mercurous sulfate as a reference electrode and a platinum sheet electrode as a counter electrode through electrodeposition by using a step constant current density method, then respectively polishing by using diamond millstones with different particle sizes, then polishing by using a diamond suspension, and finally performing ultrasonic cleaning by using deionized water to obtain the spare gold nanoelectrode array.
3. The method for real-time in-situ quantitative analysis of hydrogen peroxide based on bipolar nanoelectrode array according to claim 1, wherein the driving electrode Ag/AgCl in step 2) is prepared by immersing silver wire in a mixed solution containing Cl ions, and then washing with distilled water.
4. The method for real-time in-situ quantitative analysis of hydrogen peroxide based on bipolar nanoelectrode array according to claim 1, wherein the steps of constructing the closed bipolar electrode in the step 3) are as follows:
A. spin-coating a negative photoresist on a clean and flat glass mold, placing the clean and flat glass mold on a hot plate, heating and curing the glass mold, cooling the glass mold, cutting the glass mold into a circular patch with the diameter of about 1.5 +/-0.2 cm, and drilling a micro-cavity with the diameter of about 3 +/-0.1 mm on the circular patch for storing liquid;
B. assembling the polydimethylsiloxane chip and the polished gold nano electrode array into a sandwich-like structure, bonding the sandwich-like structure by an oxygen plasma cleaning machine, drying the sandwich-like structure, and then using Ag/AgCl as a driving electrode to construct a closed bipolar electrode.
5. The method for real-time in-situ quantitative analysis of hydrogen peroxide based on bipolar nanoelectrode array according to claim 1, wherein the stimulant in step 4) is myristoyl phorbol ethyl ester, and the concentration of the myristoyl phorbol ethyl ester is 1 ± 0.2 μ g/mL.
6. The real-time in-situ quantitative analysis method for hydrogen peroxide based on bipolar nanoelectrode array as claimed in claim 1, wherein the driving voltage applied in step 5) is 1.8V.
7. The method for real-time in-situ quantitative analysis of hydrogen peroxide based on bipolar nanoelectrode array according to claim 1, wherein the luminescent system added at anode end in step 5) comprises ruthenium bipyridyl and a co-reactant dibutylaminoethanol.
8. The real-time in-situ quantitative analysis method for hydrogen peroxide based on bipolar nanoelectrode array according to claim 1, wherein the concentration of ruthenium bipyridyl of the luminophor added to the anode end in the step 5) is 1 ± 0.3mmol/L, and the concentration of the co-reactant dibutylaminoethanol is 20 ± 0.5 mmol/L.
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