Preparation method of an integrated nanopore electrode sensor and rapid interference-free detection for antibiotics

Abstract

The present invention discloses a preparation method of an integrated nanopore electrode sensor, which includes patterning an indium tin oxide substrate through a photolithography process and a wet etching process in sequence, depositing a silica nanochannel on the patterned indium tin oxide substrate, dropping a NaOH solution on a part of the silica nanochannel to expose a part of the indium tin oxide; respectively printing a reference electrode and a counter electrode onto the exposed part of the indium tin oxide through a screen printing process, so as to obtain an integrated nanopore planar three-electrode sensor constructed by an indium tin oxide / silica nanochannel working electrode, a reference electrode and a counter electrode. This preparation method can achieve rapid, stable and real-time detection of a target object. The present invention also provides an application of the integrated nanopore electrode sensor in detecting antibiotics in water.

Description

Technical Field

[0001] The invention belongs to the field of compound detection, and in particular relates to a preparation method and application of an integrated nanopore electrode sensor. Background Art

[0002] Antibiotics pose immeasurable risks to the environment, food safety, animals and human health. Antibiotic pollution in the environment can disrupt normal flora and lead to the emergence of drug-resistant bacteria; for products processed by microbial fermentation, antibiotic residues can inhibit the fermentation agent and interfere with the fermentation process, leading to production accidents and poor food quality; eating these contaminated products may cause allergies, destroy normal intestinal flora, and even have certain carcinogenic, mutagenic and teratogenic properties.

[0003] As a commonly used antibiotic in aquatic products, furazolidone has gradually attracted widespread attention from the industry and scientific research community. Furazolidone is unlikely to be metabolized in water and ordinary non-animal-derived foods, but it is unstable in animals and will be rapidly metabolized. Some metabolite molecules bind to cell membrane proteins and can remain stable for a long time. It is difficult to degrade by ordinary processing methods such as grilling, microwaves, and cooking. The side chain 3-amino-2-oxazolidinone (AOZ) in the protein-bound state dissociates in the gastric acid environment and is metabolized into the mutagenic and carcinogenic compound β-hydroxyethylhydrazine.

[0004] In the process of furazolidone national standard material calibration, the standard medium-performance liquid phase conditions of metronidazole furazolidone suppositories in the "National Drug Standard" (the sixth volume of local standards for chemical drugs upgraded to national standards) are often used for determination. The pretreatment process is complicated, the analysis cycle is long, the organic solvent consumption is large, and the professional ability of the operator is required to be high, which is not conducive to promotion and use.

[0005] In comparison, electrochemical methods have outstanding advantages such as fast detection speed, simple method, high sensitivity, low cost, and good real-time performance. However, the existing electrochemical sensors for detecting antibiotics such as furazolidone still have disadvantages such as the need to improve selectivity and stability and low sensitivity. In addition, the traditional electrochemical analysis experimental device includes multiple components such as electrodes, electrolytic cells, and electrochemical workstations, and electrolytes are required to connect the electrodes to form a circuit. Commercial electrochemical workstations are expensive and bulky, and are not suitable for rapid detection in various scenarios.

[0006] New electrodes have emerged. Screen-printed electrodes integrate electrodes and print them on an insulating substrate through screen printing technology, which greatly reduces the size of the experimental device. A small amount of sample can be used to complete the detection, making it easy to manufacture portable, batch-produced, and flexible sensors, which meets the needs of real-time detection devices. However, the reference current of the simple application of screen-printed electrodes for detection is often large, and the detection signal of the object to be tested is relatively not obvious, and the detection performance needs to be further improved. Therefore, it is necessary and significant to further study the real-time rapid detection sensor system with low background interference and high sensitivity.

[0007] The present invention is based on a self-made screen-printed electrode that is further modified into a local precise positioning nanopore planar three-electrode system, and based on the enrichment effect of the nanopore combined with low pH conditions, promotes low background, high sensitivity, direct and rapid detection of furazolidone. The modified nanopore electrode, combined with the optimized low pH conditions, can achieve low background, high sensitivity, direct and rapid detection of furazolidone. When used in conjunction with a micro-electrochemical workstation, the cost is low, the equipment size is small, the method of use is simple, and the operation is convenient. A higher degree of portability and miniaturization can be achieved, breaking through the bottleneck of rapid detection and real-time monitoring, and achieving rapid and real-time detection of target objects under various conditions. Summary of the invention

[0008] The present invention provides a method for preparing an integrated nanopore electrode sensor, which can realize rapid, stable and real-time detection of a target object.

[0009] The present invention provides a method for preparing an integrated nanopore electrode sensor, comprising:

[0010] (1) patterning an indium tin oxide substrate by photolithography and wet etching in sequence, depositing silicon dioxide nanochannels on the patterned indium tin oxide substrate, and dropping a NaOH solution on a portion of the silicon dioxide nanochannel to expose a portion of the indium tin oxide;

[0011] (2) The reference electrode and the counter electrode are respectively printed onto the exposed portion of the indium tin oxide by screen printing, thereby obtaining an integrated nanopore planar three-electrode sensor constructed by an indium tin oxide / silicon dioxide nanochannel working electrode, a reference electrode, and a counter electrode.

[0012] Preferably, the concentration of the NaOH solution is not less than 1 mol / L, and the reaction time is not less than 1 h. The present invention controls the solubility and reaction time of the NaOH solution to avoid the amount of silicon dioxide channels on the surface of the exposed indium tin oxide from affecting the firmness of the printed reference electrode and the counter electrode. In addition, compared with hydrochloric acid, hydrofluoric acid, etc., NaOH can specifically remove silicon dioxide, a nanochannel material, without damaging the glass substrate, because in a strong alkaline environment, the nanopores are more easily dissolved, resulting in peeling from the substrate.

[0013] Preferably, the screen printing process is as follows:

[0014] First, cover the areas corresponding to the working electrode and the reference electrode, print carbon on the uncovered area, and then keep it at 90 - 200 °C for 30 - 60 min to obtain the counter electrode. If the temperature is too high, it will damage the nanopores, resulting in a decrease in the selective blocking performance of the nanopores, as well as a weak bonding between the printed layer and the substrate, and a decrease in stability and conductivity; if the temperature is too low, it will lead to too long a curing time for the printed layer, which is not conducive to mass production.

[0015] Then, cover the areas corresponding to the working electrode and the counter electrode, print silver paste on the uncovered area corresponding to the reference electrode, and then keep it at 90 - 200 °C for 30 - 60 min. Preferably, the photolithography process includes: cleaning the indium tin oxide substrate deposited on the glass, coating with glue, pre-baking, exposure, development, and hard baking to obtain the lithographed indium tin oxide.

[0016] More preferably, the photolithography process includes:

[0017] (1) Substrate treatment

[0018] The conventional cleaning steps are cleaning with an ethanol solution of sodium hydroxide, acetone, isopropyl alcohol, and ethanol, and then drying to provide the adhesion of the substrate to the photoresist. In addition, it is necessary to heat on a hot plate at 150 - 200 °C for a few minutes (2 - 3 minutes) to remove the water vapor on the substrate surface.

[0019] (2) Photoresist coating

[0020] The photoresist is mainly coated by spin coating with a spin coater. For thin or relatively thick photoresist, the optimal spin coating speed is different, and the appropriate speed should be selected according to the photoresist for spin coating.

[0021] (3) Pre-baking

[0022] The purpose is to volatilize the organic solvents in the photoresist through temperature and cure the photoresist on the substrate surface. Pre-baking is generally carried out on a hot plate or in an oven, and each photoresist will have its specific pre-baking temperature and time.

[0023] (4) Exposure

[0024] It is completed through an exposure mask and an exposure system. For example, a step-and-repeat mask aligner or a contact exposure system exposes within their respective spectral working ranges, or direct laser maskless exposure can also be used. The exposure dose recommended in the product information description is obtained under standard processes and is for reference only. The specific dose needs to be verified according to the actual experimental conditions.

[0025] (5) Development

[0026] Develop using a specific developer. The developer concentration listed in the product information is the result under specific photoresist thickness and development process parameters, and is for reference only under other conditions. The accurate developer concentration should adapt to specific requirements. The optimal development time depends on the photoresist type, photoresist thickness, exposure wavelength, baking temperature, and development process.

[0027] (6) Hard baking

[0028] Hard baking is also known as hard bake. Hard baking is to make the photoresist film adhere more firmly to the substrate surface through heating and baking, and can increase the etching resistance of the photoresist layer.

[0029] Preferably, the wet etching includes: adding indium tin oxide after lithography to a hydrochloric acid solution to remove the exposed indium tin oxide, and then removing the remaining photoresist to obtain a patterned indium tin oxide substrate. According to its properties, a hydrochloric acid solution with a certain concentration is selected as the indium tin oxide etchant. Through the strong oxidation of hydrochloric acid, it reacts with indium tin oxide to accelerate the etching rate and obtain the electrode morphology.

[0030] Preferably, through solution growth method, deposit silica nanochannels on the patterned indium tin oxide substrate. Through The heating temperature of the solution growth method is 50 - 70 °C, and the heat preservation time is 12 - 48 h.

[0031] Preferably, the solution is prepared by adding ammonia solution and tetraethyl orthosilicate to a mixed solution of cetyltrimethylammonium bromide dissolved in water and ethanol. The mass ratio of cetyltrimethylammonium bromide, the mixed solution of water and ethanol, ammonia solution, and tetraethyl orthosilicate is 16 - 24:0.91 - 1.12:7.44.

[0032] Preferably, before depositing silica nanochannels on the patterned indium tin oxide substrate by solution growth method, ultrasonically clean the indium tin oxide conductive substrate successively with an ethanol solution of sodium hydroxide, acetone, ethanol, and deionized water.

[0033] On the other hand, the present invention also provides an application of the integrated nanopore electrode sensor prepared by the preparation method of the integrated nanopore electrode sensor in the rapid interference-free detection of antibiotics, including:

[0034] Drop an electrolyte solution containing antibiotics into the integrated nanopore planar three-electrode sensor, or add the integrated nanopore planar three-electrode sensor to an electrolyte solution containing antibiotics;

[0035] Use cyclic voltammetry and differential pulse voltammetry to detect furazolidone in the electrolyte solution.

[0036] Preferably, the antibiotic is furazolidone, and the pH value of the electrolyte solution containing furazolidone is 1 - 5.

[0037] Preferably, the working electrode is an indium tin oxide / silicon dioxide nanochannel working electrode, the reference electrode is Ag / AgCl, and the counter electrode is carbon.

[0038] Preferably, the electrolyte solution is an acetic acid - sodium acetate buffer salt solution or a hydrochloric acid solution.

[0039] Based on the selective effect of hydrophobic cetyltrimethylammonium bromide in the silicon dioxide nanochannel, the nanopores will promote the transport of hydrophobic substances and hinder the passage of hydrophilic substances. The lower the pH, the higher the hydrogen ion concentration, and the relatively more hydronium ions are formed in the solution. Under extremely acidic conditions, compared with other conditions, the silicon dioxide nanochannel will hinder the passage of hydrophilic hydronium ions to the electrode surface for reduction reaction to a greater extent; while greatly promoting the transport of hydrophobic furazolidone, making the detection signal of furazolidone further enhanced, so that the difference between the furazolidone detection signal and the background signal is greater, achieving an excellent anti-background interference effect, high sensitivity, and low detection limit.

[0040] The peak current signal is collected by using the integrated nanopore planar three - electrode sensor. The integrated nanopore electrode sensor prepared in the present invention can realize the detection of antibiotics in aquatic products under various conditions, and has prominent advantages such as good portability and strong adaptability compared with the traditional electrochemical test system.

[0041] Compared with the prior art, the beneficial effects of the present invention are:

[0042] In the present invention, NaOH is used to corrode part of the silicon dioxide channel to expose part of the indium tin oxide. Through the screen - printing process, the reference electrode and the counter electrode can be firmly printed on part of the indium tin oxide to achieve the purpose of obtaining a stable detector. Also, due to the enrichment effect of the nanopores in the silicon dioxide nanochannel, the detection baseline of the antibiotic is reduced, making the sensitivity higher, greatly reducing the background interference signal, and achieving low - background, high - sensitivity, strong anti - interference, and high - specificity detection. Description of the Drawings

[0043] Figure 1 Schematic diagram of the integrated nanopore electrode sensor prepared in Example 1;

[0044] Figure 2 Cyclic voltammetry curves of the electrochemical signals of the unmodified nanopore planar three - electrode sensor prepared in Example 1 in NaAc - HAc with pH = 5 (a), NaAc - HAc with pH = 3.6 (b), and HCl with pH = 1 (c);

[0045] Figure 3 Cyclic voltammetry curves of the electrochemical signals of the integrated nanopore planar three-electrode sensor prepared in Example 1 in NaAc-HAc (a) with pH = 5, NaAc-HAc (b) with pH = 3.6, and HCl (c) with pH = 1;

[0046] Figure 4 Differential pulse stripping voltammetry curve of the change in furazolidone concentration (0 - 100.0 μmol / L) recorded in a real sample by the integrated nanopore planar three-electrode sensor prepared in Example 1;

[0047] Figure 5 Calibration plot of the peak current of furazolidone recorded in a real sample by the integrated nanopore planar three-electrode sensor prepared in Example 1;

[0048] Figure 6 Cyclic voltammogram of the integrated nanopore planar three-electrode sensors prepared in Example 1, Example 2, Comparative Example 1, and Comparative Example 2 in the electrochemical probe hexaammine ruthenium;

[0049] Figure 7 Cyclic voltammogram of the integrated nanopore planar three-electrode sensors prepared in Example 1 and Comparative Example 3 in the electrochemical probe hexaammine ruthenium. Detailed implementation manners

[0050] The present invention will be further described below in conjunction with specific embodiments.

[0051] Example 1

[0052] (1) Cleaning the electrode: First, immerse the electrode in an ethanol solution containing 1 mol / L sodium hydroxide and ultrasonically treat it for 1 hour. Subsequently, immerse the electrode in acetone and ethanol successively, ultrasonically treat each for 15 minutes, then wash the electrode with deionized water for 15 minutes, repeat twice, and finally, dry the electrode with nitrogen for later use;

[0053] (2) Ultraviolet lithography steps:

[0054] 1. Coating photoresist and baking

[0055] According to requirements, aspirate a certain amount of AZ4620 photoresist and pour it onto indium tin oxide glass. According to the characteristics of AZ4620 positive photoresist, set the rotation speed of the spin coater in the first step to 500 r / min, spin coat for 10 - 20 s, and in the second step, set the rotation speed to 500 r / min and spin coat for 30 s to obtain a photoresist layer with a thickness of about 9 μm; after the spin coater evenly coats the indium tin oxide glass with the photoresist, take it out and place it on a heating plate for baking at 100 °C for 90 s in sequence, and use it after natural cooling.

[0056] 2. Exposure

[0057] Exposure is carried out using a mask, and various exposure parameters are optimized, such as the contact method, exposure duration, and other parameters. Finally, the exposure duration is determined to be 2 minutes.

[0058] 3. Development and hard baking

[0059] The indium tin oxide after natural cooling is placed in a special developer for development. After 60 seconds of development, it is taken out and rinsed with pure water. For the parts where the development is not uniform and there is photoresist residue, it is then rinsed clean with pure water. The rinsed indium tin oxide glass is placed on a hot plate and baked at a high temperature of 120 °C for 3 minutes.

[0060] (3) Wet etching step: In this experiment, 2 mol / L hydrochloric acid is configured to obtain an etching solution with good performance. This etching solution reacts with indium tin oxide through the strong oxidation of hydrochloric acid, which can accelerate the etching rate.

[0061] (4) Depositing silica nanochannels on the surface of the indium tin oxide electrode:

[0062] 1. Configuration Solution: Dissolve 0.16 g of cetyltrimethylammonium bromide in 100 mL of a water / ethanol mixed solution (70 mL / 30 mL). After the cetyltrimethylammonium bromide is completely dissolved, ammonia solution (10 μL) and tetraethyl orthosilicate (80 μL) are slowly added to the cetyltrimethylammonium bromide solution under stirring respectively;

[0063] 2. Modification of the nanoporous electrode: Immerse the self-made indium tin oxide electrode in the above solution, heat it in a water bath at 60 °C, and try to avoid vibration throughout the process. After 24 hours, take out the electrode, rinse it with a large amount of deionized water, and dry it with nitrogen. Subsequently, place the electrode in a dry box and place it at a temperature of 100 °C for 12 hours for aging. At this time, the nanopores of the obtained electrode are filled with the surfactant cetyltrimethylammonium bromide, and a SCNM / indium tin oxide composite material with a silica nanochannel array with pores perpendicular to the substrate is deposited on the surface of the indium tin oxide conductive substrate.

[0064] 3. Precise positioning of the nanopore modification layer: Using the chemical reagent removal method, configure 1 mol / L NaOH solution, precisely position the area where the template, that is, the silica nanochannels, needs to be removed, protect the working electrode area, and treat it at room temperature for 120 minutes to obtain an ideal nanopore electrode morphology, that is, there is exposed indium tin oxide and silica nanochannels on indium tin oxide existing simultaneously.

[0065] (5) Screen printing step: Cover the reference electrode and working electrode areas. Apply the ink onto the screen printing template on the exposed indium tin oxide, and use a squeegee to scrape the ink into the holes of the template to form an electrode pattern. First, print the carbon ink, and then cure the printed electrode sample in an oven at 90 °C for 1 h for drying and curing treatment, so that the ink forms a firm electrode layer on the substrate surface. Then, cover the counter electrode carbon and the working electrode and print the silver paste on the exposed indium tin oxide, and also cure it in the oven at 90 °C for 1 h for drying and curing treatment to obtain an integrated nanoporous electrode sensor, as Figure 1 shown.

[0066] Example 2

[0067] Different from Example 1, the concentration of the NaOH solution is 2 mol / L, and the reaction time is 2 h.

[0068] Comparative Example 1

[0069] Different from Example 1, the concentration of the NaOH solution is 0.5 mol / L, and the reaction time is 2 h.

[0070] Comparative Example 2

[0071] Different from Example 1, the concentration of the NaOH solution is 1 mol / L, and the reaction time is 20 min.

[0072] Comparative Example 3

[0073] Different from Example 1, the temperature of the oven is 250 °C.

[0074] Performance analysis:

[0075] As Figure 6 shown, the cyclic voltammogram of the planar three-electrode sensor obtained by precisely positioning and removing the nanopore region in Example 1 in the electrochemical probe hexammine ruthenium; the cyclic voltammogram of Example 2 with a NaOH solution concentration of 2 mol / L and a reaction time of 2 h for removing the nanopores in the electrochemical probe hexammine ruthenium; the cyclic voltammogram of Comparative Example 1 with a NaOH solution concentration of 0.5 mol / L and a reaction time of 2 h for removing the nanopores in the electrochemical probe hexammine ruthenium; the cyclic voltammogram of Comparative Example 2 with a NaOH solution concentration of 1 mol / L and a reaction time of 20 min for removing the nanopores in the electrochemical probe hexammine ruthenium. Among them, when the NaOH concentration and the reaction time are greater than the range described in the claims, such as in Example 2, the effect of removing NaOH is almost the same as that in Example 1, achieving the expected effect; if the NaOH concentration is too small or the reaction time is too short, such as in Comparative Example 1 and Comparative Example 2, the nanopore template is not completely removed, and there is a large hindrance effect on hexammine ruthenium.

[0076] AsFigure 7 As shown in the figure, it is the cyclic voltammetry curve of the planar three-electrode sensor obtained by curing the ink in an oven during the screen printing step in Example 1. Different from Example 1, the temperature of the oven provided in Comparative Example 3 is 250 °C, and the cyclic voltammetry curve of the prepared planar three-electrode sensor in the electrochemical probe hexammine ruthenium. Among them, since the nanopores will be damaged at this temperature and the curing of the ink will be affected, compared with the preferred conditions in Example 1, the detection performance decreases.

[0077] Application Example

[0078] Furazolidone with a concentration of 0 - 100 μmol / L was added to the HAc-NaAc (pH 3.6) solution. Under optimized conditions, a negative potential scan of 0 - -0.7 V was applied to the integrated nanopore planar three-electrode sensor. After enrichment for 60 s, the differential pulse stripping voltammetry curve of the solution was recorded, and the peak current signal was collected using a mini workstation. The peak current signal was linearly fitted with the furazolidone concentration data. According to the response current of furazolidone in the real sample and the linear relationship curve between current and concentration, the concentration of furazolidone in the test solution was calculated to achieve the determination of the concentration of furazolidone in the test solution.

[0079] The preparation steps of this micro electrochemical workstation: First, analyze the functional analysis requirements of the micro wearable electrochemical workstation, design the analog acquisition front-end and overall scheme, and then complete the implementation of the hardware circuit, as well as complete various program designs and functional tests.

[0080] In order to more accurately quantitatively detect furazolidone in actual samples, differential differential pulse stripping voltammetry was used, as Figure 4 shown. After optimizing the accumulation time, the electrochemistry of furazolidone with a concentration ranging from 0 to 100 μmol / L in the actual sample was recorded, and the peak current was linearly correlated with the increase in furazolidone concentration. The regression equation is: Ip (μA) = -0.3425C (μmol / L) + 1.17089e-7 (R 2 = 0.99136). According to the formula 3σ / S (σ is the standard deviation of 10 repeated blank signals), the detection limit of furazolidone in the actual sample was calculated to be 76 nmol / L (S / N = 3). The sensitivity calculated by this method is much lower than the requirement of less than 0.01 ug mL in NY / T 3410-2018 -1 , showing extremely high sensitivity in real samples.

[0081] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made according to the purpose of the invention of the present invention. Any changes, modifications, substitutions, combinations or simplifications made based on the spirit and principle of the technical solution of the present invention shall be equivalent replacement methods. As long as they meet the invention purpose of the present invention, they all fall within the protection scope of the present invention.

[0082] As Figure 2 shown, Figure 2 in (a), (b) and (c) of it are cyclic voltammograms of the electrochemical signals of the unmodified nanopore planar three-electrode sensor prepared in Example 1 in NaAc-HAc with pH = 5 (a), NaAc-HAc with pH = 3.6 (b), and HCl with pH = 1 (c). As the pH becomes lower, the difference between the test peak value of furazolidone and the background signal, i.e., the signal of water reduction, of the indium tin oxide planar three-electrode system becomes larger.

[0083] As Figure 3 shown, Figure 3 in (a), (b) and (c) of it are cyclic voltammograms of the electrochemical signals of the integrated nanopore planar three-electrode sensor prepared in Example 1 in NaAc-HAc with pH = 5 (a), NaAc-HAc with pH = 3.6 (b), and HCl with pH = 1 (c); with the modification of the nanopore, based on the selective effect of cetyltrimethylammonium bromide in the nanochannel, it has an excellent promoting effect on hydrophobic furazolidone and an obstructive effect on hydrophilic hydronium ions. As the pH decreases, the difference between the test peak value of furazolidone and the background signal of the integrated nanopore planar three-electrode system becomes larger. And compared with the planar electrode system with unmodified nanopores, the peak value is further increased. Combining the extremely acidic conditions and the selective effect of the nanopore, the sensitivity of furazolidone detection is further improved, the detection limit is further reduced, and the detection performance is better.

[0084] As Figure 5 shown, Figure 5 is the calibration graph of the peak current of furazolidone recorded in the real sample of the integrated nanopore planar three-electrode sensor prepared in Example 1.

Claims

1. Application of the integrated nanopore planar three-electrode sensor prepared by a preparation method of the integrated nanopore planar three-electrode sensor in detecting antibiotics in water, characterized in that, It includes: Dripping an electrolyte solution containing an antibiotic into the integrated nanopore planar three-electrode sensor, or adding the integrated nanopore planar three-electrode sensor into an electrolyte solution containing an antibiotic; The antibiotic is furazolidone, and the pH value of the electrolyte solution containing furazolidone is 1 - 5; Detecting furazolidone in the electrolyte solution by cyclic voltammetry and differential pulse voltammetry; The preparation method of the integrated nanopore planar three-electrode sensor includes: (1) Pattern the indium tin oxide substrate by photolithography and wet etching in sequence, deposit silicon dioxide nanochannels on the patterned indium tin oxide substrate, and drop NaOH solution on part of the silicon dioxide nanochannels to expose part of the indium tin oxide; The concentration of the NaOH solution is not less than 1 mol / L, and the reaction time is not less than 1 h; (2) Print the reference electrode and the counter electrode onto the exposed part of the indium tin oxide by screen printing process respectively, so as to obtain an integrated nanopore planar three-electrode sensor constructed by an indium tin oxide / silicon dioxide nanochannel working electrode, a reference electrode and a counter electrode; The screen printing process is: First cover the areas corresponding to the working electrode and the reference electrode, print carbon in the uncovered area, and then keep it at 90 - 200 °C for 30 - 60 min to obtain the counter electrode; Then cover the areas corresponding to the working electrode and the counter electrode, print silver paste in the area corresponding to the uncovered reference electrode, and then keep it at 90 - 200 °C for 30 - 60 min.

2. The application according to claim 1, characterized in that, The photolithography process includes: cleaning, coating, pre-baking, exposing, developing and hardening the indium tin oxide substrate deposited on the glass to obtain the photolithographed indium tin oxide.

3. The application according to claim 1, wherein The wet etching includes: adding the photolithographed indium tin oxide into hydrochloric acid solution to remove the exposed indium tin oxide, and then removing the remaining photoresist to obtain the patterned indium tin oxide substrate.

4. The application according to claim 1, wherein Deposit silicon dioxide nanochannels on the patterned indium tin oxide substrate by Stöber solution growth method, and the heating temperature of the Stöber solution growth method is 50 - 70 °C, and the heat preservation time is 12 - 48 h; The Stöber solution is prepared by adding ammonia solution and tetraethyl orthosilicate to a mixed solution of cetyltrimethylammonium bromide dissolved in water and ethanol, and the mass ratio of cetyltrimethylammonium bromide, the mixed solution of water and ethanol, ammonia solution and tetraethyl orthosilicate is 16 - 24: 0.91 - 1.12: 7.

44.

5. The application according to claim 1, characterized in that, Before depositing silicon dioxide nanochannels on the patterned indium tin oxide substrate, ultrasonically clean the indium tin oxide conductive substrate in sequence with an ethanol solution of sodium hydroxide, acetone, ethanol and deionized water.

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