Multi-layer modified ultra-micro array electrode and method for detecting selenium ions by using multi-layer modified ultra-micro array electrode

Abstract

The invention discloses a multi-layer modified ultra-micro array electrode and a method for detecting selenium ions by using the multi-layer modified ultra-micro array electrode, and relates to the technical field of electrochemical analysis. L-cysteine is adopted as a modifier, and a firm Au-S covalent bond can be formed by sulfydryl in the molecular structure of L-cysteine and the surface of the gold film, so that an orderly-arranged and stable modification layer is constructed; meanwhile, amino groups and carboxyl groups in molecules can generate specific recognition and coordination with selenium ions, especially Se (IV), through hydrogen bonds and electrostatic attraction, so that the selective adsorption capacity to selenium is remarkably enhanced. The enrichment efficiency of selenium on the surface of the electrode is improved by more than 10 times under the dual action of anchoring multilayer modification and specific recognition, and the detection limit is as low as 0.1 microgram / L by combining the high resolution of differential pulse voltammetry and is reduced by 1-2 orders of magnitude compared with a traditional unmodified gold electrode, a glassy carbon electrode or other modification systems. The precise capture and detection of the ultra-trace selenium in the complex matrix are successfully realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of electrochemical analysis, in particular to a multilayer modified ultramicroarray electrode and a method for detecting selenium ions. BACKGROUND

[0002] Selenium, as a key trace element for maintaining normal physiological activities of living organisms, performs multiple core functions in the body: as an active center component of glutathione peroxidase (GPx), it can efficiently catalyze the decomposition of peroxide, block the free radical chain reaction, and protect biological membranes and DNA from oxidative damage; at the same time, by participating in the synthesis and activation of iodine deiodinase (DI), it precisely regulates the transformation balance of triiodothyronine (T3) and thyroxine (T4), ensuring normal energy metabolism and growth and development; in addition, it also regulates immune cell differentiation, cytokine secretion, and tumor suppressor gene (such as p53) expression, and plays a unique role in enhancing immune defense and inhibiting malignant proliferation. When the body is deficient in selenium intake, it can lead to heart disease, liver cancer, Keshan disease, Kashin-Beck disease, autoimmune thyroiditis, and a series of diseases, while excessive intake can cause toxicity and oxidative stress, leading to hair loss, nail white spots, and nerve damage. The human body cannot synthesize selenium elements independently and needs to rely on dietary intake, mainly from selenium-rich cereals, animal offal, and seafood. However, selenium distribution shows significant regional differences, with a wide low-selenium zone from northeast to southwest of China, and combined with the loss of selenium during high-temperature processing and long-term storage of food, the problem of insufficient selenium intake in the population is prominent; while excessive intake (>400 μg / day) can cause acute and chronic poisoning symptoms such as hair loss, nail cracking, and nervous system damage. World Health Organization (WHO) data shows that about 1 billion people worldwide live in areas with selenium deficiency, and serum selenium concentration <120 μg / L has been listed as a core risk indicator for cardiovascular diseases, Keshan disease, and Kashin-Beck disease; the International Food Codex Commission (CAC) also stipulates that the safe daily intake of selenium for adults is 400 μg, and excessive intake will significantly increase the risk of poisoning. Therefore, accurate detection of selenium content in environmental media (soil, water, atmospheric particulate matter), food raw materials, and biomarkers (whole blood, hair, urine) has irreplaceable scientific value for delineating selenium nutrition zoning, developing dietary guidelines, implementing food safety supervision, and preventing and controlling local diseases.

[0003] Currently, there are many methods for detecting selenium, such as inductively coupled plasma mass spectrometry (ICP-MS), atomic absorption spectrometry (AAS), atomic fluorescence spectrometry (AFS), gas chromatography (GC), and high-performance liquid chromatography (HPLC), etc. However, these methods have many defects and shortcomings: (1) High equipment requirements: methods such as ICP-MS require expensive and complex large-scale instrument equipment, the cost of purchasing the instrument is high, the maintenance difficulty is large, and the requirements for the laboratory environment are harsh, which limits its application in basic laboratories and on-site detection. (2) Complex operation: the sample pretreatment process of such methods is tedious, usually requiring multiple steps such as digestion, extraction, and enrichment, the operation process is time-consuming, and errors are easily introduced. For example, in atomic absorption spectrometry, the digestion process of the sample needs to strictly control the temperature, time, and reagent amount, otherwise it will affect the accuracy of the detection result. (3) Long detection period: from sample collection to the final detection result, the whole process needs a long time, which cannot meet the requirements of actual application scenarios that require high detection speed, such as emergency detection and real-time monitoring. (4) Limited sensitivity: for trace selenium detection in complex mechanisms, the sensitivity of some traditional methods is difficult to meet the requirements, and it is difficult to accurately detect extremely low concentrations of selenium, resulting in inaccurate detection results or false negatives. (5) Poor sample adaptability: in complex mechanisms, the sample composition is complex, and there are many interfering substances, the anti-interference ability of traditional detection methods is weak, and it is easy to be disturbed by other elements or compounds, affecting the accurate determination of selenium. Although electrochemical detection technology has the advantages of nanomolar level sensitivity, second level response speed, portable equipment, and low cost, it still faces severe challenges in the analysis of trace selenium in complex matrices: proteins, polysaccharides, and lipids in biological samples are easily adsorbed on the electrode surface to form an insulating layer, hindering electron transfer and selenium ion diffusion, resulting in signal attenuation and reduced reproducibility; coexisting elements such as arsenic (As), antimony (Sb), and tellurium (Te) produce overlapping redox signals due to similar electrochemical behavior, seriously interfering with the specific recognition of selenium; existing methods have insufficient detection capability for ultra-low selenium samples (detection limit is mostly in the μg / L level) such as high-altitude plants and deep groundwater, resulting in underestimation of the true selenium level. In addition, in the detection process, trace elements released from the material of the sampling container, tool residual reagents can easily introduce cross contamination; light, temperature fluctuations and pH changes during storage can accelerate the conversion of selenium valence states (such as Se(IV) and Se(VI)), causing the detection value to deviate from the actual content. The development of new electrochemical methods with high sensitivity, strong anti-interference ability, and convenient operation to realize rapid and accurate detection of trace selenium in complex matrices has important practical significance for improving selenium nutrition evaluation efficiency, strengthening food safety supervision, and promoting the progress of local disease prevention and control technology. SUMMARY

[0004] One of the purposes of the present application is to provide a multi-layer modified ultra-micro array electrode to solve the problem that electrochemical detection cannot specifically detect selenium; The second object of the present application is to provide a method for detecting selenium ions by using a multilayer modified ultramicroarray electrode, so as to solve the problems of insufficient sensitivity and poor selectivity in the detection of ultratrace selenium in a high interference environment.

[0005] The object of the present application can be achieved by the following technical solutions. In a first aspect, a multilayer modified ultramicroarray electrode is prepared by the following steps: S1. After polishing the surface of a glassy carbon electrode, first immerse it in a sulfuric acid solution for cyclic voltammetry activation until stable; then immerse it in a potassium ferricyanide solution and scan it by cyclic voltammetry until the peak current tends to be stable; S2. The glassy carbon electrode treated in S1 is used as a working electrode, and is immersed in a chloroauric acid solution together with a counter electrode and a reference electrode to obtain a gold film modified electrode by electrodeposition; S3. The gold film modified electrode is immersed in an L-cysteine solution together with a counter electrode and a reference electrode, and is modified by cyclic voltammetry to obtain a multilayer modified ultramicroarray electrode.

[0006] Further, in S1, the polishing treatment includes: polishing a glassy carbon electrode with a diameter of 3mm to a mirror surface with 0.05μm aluminum oxide powder, and washing the surface dirt with purified water after each polishing.

[0007] Further, in S1, the concentration of the sulfuric acid solution is 0.1M; In the sulfuric acid solution, the scanning range of cyclic voltammetry activation is-1.0~1V, and the cyclic voltammogram is repeatedly scanned until it is stable.

[0008] Further, in S1, the concentration of the potassium ferricyanide solution is 5mM; In the potassium ferricyanide solution, the scanning rate of cyclic voltammetry is 50mV / s, and the scanning range is-1.0~0.6V. The peak potential difference in the cyclic voltammetry graph obtained under laboratory conditions is ≤80mV or less, and the electrode can be used, otherwise the electrode needs to be treated again until it meets the above requirements.

[0009] Further, in S2, the concentration of the chloroauric acid solution is 1mM, the solute is HAuCl4, and the solvent is a 0.1M hydrochloric acid solution.

[0010] Further, in S2, the constant potential for electrodeposition is-0.8V, the scanning time is 200s, and the standing time is 30s.

[0011] Further, in S3, the concentration of the L-cysteine solution is 10mM, and the pH is 5.5±0.1; In the L-cysteine solution, the electrodes are kept in the same horizontal plane, and the modified electrodes are tested by cyclic voltammetry CV test, with a scanning range of initial potential-0.5V, high potential 1.5V, low potential-0.5V (initial / high / low potential-0.5V / 1.5V / -0.5V), a scanning rate of 0.1V / s, 40 scanning circles, and a standing time of 2s.

[0012] Further, in S2, the counter electrode is a platinum electrode, and the reference electrode is a silver / silver chloride electrode.

[0013] In a second aspect, a method for detecting selenium ions by using a multilayer modified ultramicroarray electrode comprises the following steps: Step 1: The multilayer modified ultramicroarray electrode, the counter electrode and the reference electrode are immersed in a cleaning solution, and the cleaning is performed by cyclic voltammetry scanning. Step 2: The three-electrode system after cleaning is used to test a selenium ion test solution.

[0014] Further, in Step 1, the cleaning solution is an acetic acid-sodium acetate solution with pH≤4.5, which is prepared by mixing 0.01-0.05M acetic acid solution and 0.05-0.1M sodium acetate solution. In the cleaning solution, the electrodes are kept in the same horizontal plane, and the scanning range of cyclic voltammetry is initial potential-1V, high potential 1V, low potential-1V, the scanning rate is 0.08V / s, the scanning circle number is 10, and the standing time is 2s.

[0015] Further, in Step 2, the selenium ion test solution uses a mixture of 0.01M hydrochloric acid and 0.5mM ethylenediaminetetraacetic acid as a solvent, and a selenium ion test sample is added.

[0016] Compared with the prior art, the present application has the following beneficial effects: 1. The present application provides a multilayer modified ultramicroarray electrode, which uses L-cysteine (L-Cys) as a modifier. The sulfhydryl group (-SH) in the molecular structure of L-Cys can form a firm Au-S covalent bond with the surface of the gold film, thereby constructing an ordered and stable modified layer. Meanwhile, the amino group (-NH2) and the carboxyl group (-COOH) in the molecule can specifically recognize and coordinate with selenium ions (especially Se(IV)) through hydrogen bonds and electrostatic attraction, thereby significantly enhancing the selective adsorption capacity for selenium. This dual action (anchoring multilayer modification + specific recognition) can improve the enrichment efficiency of selenium on the electrode surface by more than 10 times. Combined with the high resolution of differential pulse voltammetry, the detection limit is as low as 0.1μg / L, which is 1-2 orders of magnitude lower than that of the traditional unmodified gold electrode or other modification systems. Therefore, the present application can successfully realize the precise capture and detection of ultratrace selenium in complex matrices. The popularization and application of the present application can provide a standardized and highly reliable selenium element analysis solution for the fields of environmental pollutant monitoring, food quality and safety control, clinical diagnosis and detection, and the like, and has important scientific value and application prospect.

[0017] 2, the method for detecting selenium ions by using multi-layer modified ultra-micro array electrode, platinum electrode and silver / silver chloride electrode as a three-electrode system is a non-mercury film modified high-density integrated selenium ultra-trace detection method with regular array structure. Through the precise design of the surface chemical properties and structure of the electrode material and the breakthrough improvement of the detection mechanism, high sensitivity and high resolution detection of trace selenium in various complex biological matrices is realized. The method discards the inherent defects of traditional mercury film electrode and pre-plating process. Traditional single electrode can only measure one point at a time, while microarray can measure hundreds or even thousands of points at the same time, so as to obtain spatial distribution information. Without complex pretreatment, the key technical bottlenecks of insufficient sensitivity and poor selectivity in the detection of ultra-trace selenium in high interference environment are successfully solved. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a linear detection graph of selenium ion concentration of the application; Figure 2 It is a stability test graph of the application; Figure 3 It is an anti-interference test graph of the application. DETAILED DESCRIPTION

[0019] The specific embodiments of the application will be described in detail below, but it should be understood that the protection scope of the application is not limited by the specific embodiments.

[0020] The terms used in the embodiments of the application are only for the purpose of describing specific embodiments, and are not intended to limit the application. The singular forms "a", "an" and "the" used in the embodiments of the application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0021] It should be understood that in various embodiments of the application, the size of the serial number of each process does not mean the order of execution, and part or all of the steps can be executed in parallel or in sequence, and the execution order of each process should be determined according to its function and inherent logic, and should not constitute any limitation on the implementation process of the embodiments of the application.

[0022] The weight of the related components mentioned in the specification of the embodiments of the application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of each component, therefore, as long as the content of the related components in the specification of the embodiments of the application is enlarged or reduced in proportion, it is within the scope disclosed in the specification of the embodiments of the application. Specifically, the mass mentioned in the specification of the embodiments of the application can be μg, mg, g, kg and other mass units commonly known in the chemical field.

[0023] Unless otherwise defined, all terms used in the description herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. The description herein uses professional and technical terms merely for the purpose of describing specific embodiments and is not intended to limit the scope of protection of the present application.

[0024] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or prepared by existing methods.

[0025] The first aspect of the present application provides a multi-layer modified ultramicro array electrode, specifically prepared by the following steps: S1. After polishing the surface of the glassy carbon electrode, first immerse it in a sulfuric acid solution for cyclic voltammetry activation until stable; then immerse it in a potassium ferricyanide solution and scan by cyclic voltammetry until the peak current tends to be stable; S2. Take the glassy carbon electrode treated in S1 as the working electrode, immerse it in a chloroauric acid solution together with a counter electrode and a reference electrode, and obtain a gold film modified electrode by electrodeposition; S3. Immerse the gold film modified electrode in an L-cysteine solution together with a counter electrode and a reference electrode, and obtain a multi-layer modified ultramicro array electrode by cyclic voltammetry modification.

[0026] The polishing treatment obtains a smooth and clean glassy carbon electrode surface, reduces the interference of surface impurities on subsequent electrochemical reactions, and ensures the consistency of electrode performance; cyclic voltammetry scanning in sulfuric acid further cleans the electrode while generating active groups such as hydroxyl groups, enhancing the conductivity of the electrode and improving the electrochemical response sensitivity of the electrode; cyclic voltammetry scanning in potassium ferricyanide solution determines the electrode surface state by peak potential difference. Take the pretreated glassy carbon electrode as the working electrode, a platinum electrode as the counter electrode, and a silver / silver chloride electrode as the reference electrode to form a three-electrode system, and perform electrodeposition in a chloroauric acid solution to form a continuous and uniform gold film on the surface of the glassy carbon electrode. 3+ The electrons on the electrode surface are reduced to Au atoms to form a continuous and uniform gold film; the gold film has good conductivity and biocompatibility, which can significantly improve the electron transfer rate of the electrode, and the Au-S bonds on the surface of the gold film can specifically bind to the sulfhydryl groups (-SH) of the subsequent L-cysteine, providing an anchoring site for the next modification.

[0027] Cyclic voltammetry modification with L-cysteine is the core of constructing a stable modification layer on the surface of the gold film modified electrode, forming a multi-layer structure of "glassy carbon electrode-gold film-L-cysteine", and the active groups (amino, carboxyl) of L-cysteine can specifically interact with selenium ions to provide recognition sites for detecting selenium ions. The multi-layer structure improves the adsorption capacity and selectivity of the electrode for selenium ions, and reduces the interference of other ions. The counter electrode and the reference electrode ensure accurate control of the potential during the modification process, avoiding uneven or falling off of the modification layer due to potential fluctuations.

[0028] In a specific embodiment, in S1, the polishing treatment includes: polishing the 3mm-diameter glassy carbon electrode with 0.05μm aluminum oxide powder to a mirror surface, and washing the surface dirt with purified water after each polishing. The fine-particle abrasive removes the oxide layer, scratches and contaminants on the electrode surface, to obtain a smooth "mirror surface" surface, reduce the thickness unevenness of the subsequent modification layer, and reduce the electronic transmission resistance.

[0029] In a specific embodiment, in S1, the concentration of the sulfuric acid solution is 0.1mol / L (M); in the sulfuric acid solution, the scan range for cyclic voltammetry activation is -1.0~1V, and the cyclic voltammogram is repeatedly scanned until it is stable. In the 0.1mol / L sulfuric acid, the cyclic scanning range is -1.0~1V, the surface is further cleaned through electrochemical reaction, active groups such as hydroxyl groups are generated, and the electrode conductivity is enhanced.

[0030] In a specific embodiment, in S1, the concentration of the potassium ferricyanide solution is 5mM; in the potassium ferricyanide solution, the scan rate for cyclic voltammetry is 50mV / s, and the scan range is -1.0~0.6V. The peak potential difference in the cyclic voltammetry graph obtained under laboratory conditions is ≤80mV, and it is as close to 64mV as possible. The electrode can be used, otherwise the electrode needs to be treated again until it meets the above requirements. In the potassium ferricyanide solution, the scan range is -1.0~0.6V at a scan rate of 50mV / s, and the performance qualified electrode is screened out through the peak potential difference to ensure the consistency of the subsequent modification.

[0031] In a specific embodiment, in S2, the concentration of the chloroauric acid solution is 1mM, the solute is HAuCl4, and the solvent is 0.1M hydrochloric acid solution. The hydrochloric acid as the solvent can effectively prevent the hydrolysis of chloroauric acid, and ensure the stability of the chloroauric acid solution.

[0032] In a specific embodiment, in S2, the constant potential for electrodeposition is -0.8V, the scan time is 200s, and the standing time is 30s. The constant potential of -0.8V is applied, Au 3+ The electrons on the electrode surface are reduced to Au atoms, and a continuous and uniform gold film is formed through 200s deposition and 30s standing.

[0033] In a specific embodiment, in S3, the concentration of the L-cysteine solution is 10 mmol / L (mM), and the pH is 5.5±0.1; the L-cysteine solution is used to maintain the electrode at the same level, and the modified electrode is tested by cyclic voltammetry (CV) test, with a scanning range of initial potential-0.5 V, high potential 1.5 V, and low potential-0.5 V (initial / high / low potential-0.5 V / 1.5 V / -0.5 V), a scanning rate of 0.1 V / s, 40 scanning cycles, and a standing time of 2 s. In the 10 mM L-cysteine solution (pH=5.5±0.1), the scanning range is-0.5 V / 1.5 V / -0.5 V, the rate is 0.1 V / s, the scanning is 40 cycles, and the thiol group (-SH) of L-cysteine is used to form a firm Au-S covalent bond with the Au on the surface of the gold film modified electrode, while the amino group (-NH2) is exposed on the surface. The cyclic voltammetry promotes the ordered arrangement of molecules to form a multi-layer modified structure; and the 2 s standing time allows the molecules to be fully adsorbed.

[0034] In a specific embodiment, in S3, the counter electrode is a platinum electrode, and the reference electrode is a silver / silver chloride electrode. The platinum electrode provides an electron transfer channel and avoids self-reaction, and the silver / silver chloride electrode provides a stable potential reference to ensure the accuracy of the modified potential.

[0035] The second aspect of the embodiment of the present application provides a method for detecting selenium ions by using the multi-layer modified ultra-micro array electrode provided in the first aspect, which comprises the following steps: Step 1: The multi-layer modified ultra-micro array electrode, the counter electrode, and the reference electrode are immersed in a cleaning solution, and are scanned by cyclic voltammetry. Step 2: The three-electrode system after cleaning is used to test the selenium ion test solution.

[0036] Under the premise of not damaging the modified layer, the cleaning solution dissolves and removes the residual air pollutants and solution impurities on the surface of the electrode, so that only selenium ions can specifically bind to the modified layer during detection; the cyclic voltammetry scanning restores the initial activity of the electrode, reduces the background signal interference, and improves the accuracy of the detection results. The L-cysteine modification layer on the surface of the modified electrode forms a coordination bond with selenium ions through the amino group and the carboxyl group. The combined selenium ions change the electron transfer rate on the surface of the electrode, resulting in changes in the peak current / peak potential in the cyclic voltammogram, and the change amount is positively correlated with the concentration of selenium ions.

[0037] In a specific embodiment, in step 1, the cleaning solution is a pH≤4.5 acetic acid-sodium acetate solution prepared by mixing 0.01-0.05 M acetic acid solution and 0.05-0.1 M sodium acetate solution; the electrode is maintained at the same level in the cleaning solution, and the scanning range of cyclic voltammetry is initial potential-1 V, high potential 1 V, and low potential-1 V, the scanning rate is 0.08 V / s, the scanning cycles are 10, and the standing time is 2 s.

[0038] The acetic acid-sodium acetate buffer (pH≤4.5) can adjust the surface charge state of the electrode, so as to create a suitable environment for subsequent selenium ion combination; through cyclic potential scanning, the residual impurities or uncombined molecules on the surface of the electrode are removed, that is, the interfering substances on the surface of the electrode are removed, so as to avoid the influence of the interfering substances on the recognition and detection of selenium ions, and to ensure the stability of the baseline while keeping the modified layer stable.

[0039] In a specific embodiment, in step 2, the selenium ion test solution adopts a mixed solution composed of 0.01M hydrochloric acid and 0.5mM ethylenediaminetetraacetic acid in a volume ratio of 1:1 as a solvent, and selenium ion test samples are added. The hydrochloric acid provides an acidic environment, and the EDTA can be coordinated with other metal ions in the test solution to eliminate the interference of the other metal ions on the detection of selenium ions, thereby improving the selectivity of the detection of selenium ions. The cleaned three-electrode system is immersed in the test solution, and the selenium ions are combined on the surface of the electrode by coordination with L-cysteine, and the combination process will cause the change of the electrochemical signal (current) on the surface of the electrode. The content of selenium ions can be quantitatively or qualitatively analyzed by detecting the signal change.

[0040] The present application realizes the accurate detection of selenium ions by using the specific adsorption and electrochemical signal response of the multilayer modified electrode to selenium ions.

[0041] The following specific embodiments are further described, wherein the preparation method of the chloroauric acid solution is as follows: the HAuCl4 is dissolved in 0.1M hydrochloric acid solution to prepare 1mM chloroauric acid solution as a gold film modification solution; the preparation method of the L-cysteine solution is as follows: the L-cysteine (L-Cys) is dissolved in ultrapure water to prepare 10 mM L-cysteine solution as an L-Cys modification solution, and the pH is adjusted to 5.5±0.1 to avoid the influence of the protonation of the amino group on the combination efficiency; the preparation method of the cleaning solution is as follows: 0.025M acetic acid solution and 0.075M sodium acetate solution are prepared, and the acetic acid-sodium acetate solution with pH=4 is prepared.

[0042] Example 1

[0043] A multilayer modified ultramicro array electrode is prepared by the following steps: S1. A glassy carbon electrode with a diameter of 3mm is polished to a mirror surface with 0.05μm aluminum oxide powder, and after each polishing, the surface dirt is washed away with purified water. After the purified water is washed, it is immersed in a 0.1M sulfuric acid solution for cyclic voltammetry activation, and the scanning range is-1.0~1V. The electrode is repeatedly scanned until the cyclic voltammogram is stable. After stabilization, the glassy carbon electrode is immersed in a 5mM potassium ferricyanide solution to test the electrode performance by cyclic voltammetry curve, and the scanning rate is 50mV / s and the scanning range is-1.0~0.6V. The peak potential difference in the cyclic voltammetry curve obtained under laboratory conditions is below 80mV, the peak current tends to be stable, and the electrode can be used. Otherwise, the electrode needs to be reprocessed until it meets the above requirements.

[0044] S2. The glassy carbon electrode treated in S1 is used as the working electrode, and a platinum electrode is used as the counter electrode and a silver / silver chloride electrode is used as the reference electrode, and they are immersed in a 1 mM chloroauric acid solution, and the electrodes are kept at the same level, and the electrodeposition modification is performed by using the current-time method, the constant potential is -0.8 V, the scanning time is 200 s, and the standing time is 30 s, and a gold film with uniform size and nanoparticle structure is formed on the surface of the working electrode, and a gold film modified electrode is obtained; S3. The gold film modified electrode is immersed in a 10 mM L-cysteine solution together with the counter electrode and the reference electrode, and the electrodes are kept at the same level, and the modified electrode is tested by using the cyclic voltammetry CV method, the scanning range is -0.5 V / 1.5 V / -0.5 V, the scanning rate is 0.1 V / s, the scanning number is 40, and the standing time is 2 s; after the cyclic voltammetry modification, a multilayer modified ultramicroarray electrode is obtained.

[0045] Example 2

[0046] A method for detecting selenium ions by using a multilayer modified ultramicroarray electrode, comprising the following steps: Step 1: The multilayer modified ultramicroarray electrode of Example 1 is used as the working electrode, and the counter electrode and the reference electrode are immersed in a cleaning solution, and the electrodes are kept at the same level, and the cleaning is performed by using the cyclic voltammetry CV method, the scanning range is -1 V / 1 V / -1 V, the scanning rate is 0.08 V / s, the scanning number is 10, and the standing time is 2 s; Step 2: The selenium element standard sample of the Center for Disease Control and Prevention is diluted into selenium ion test solutions with concentration gradients by using a mixed solution of 0.01 M hydrochloric acid and 0.5 mM ethylenediaminetetraacetic acid as a solvent, and the three-electrode system after cleaning is used to test the selenium ion test solutions, and the working electrode needs to be cleaned after each test is completed.

[0047] The gradient concentration levels of the selenium ion test solution are shown in Table 1.

[0048] Table 1

[0049] Example 3

[0050] Linear test: The test method in Example 2 is used, 9 concentration gradient biological samples (selenium ion test solutions) are selected, 10 repeated detections are performed for each concentration gradient, the obtained detection current signal results and the average value (unit: μA) are taken, the detection results are shown in Table 2, and a linear relationship is constructed, and the selenium ion concentration linear graph is shown in Figure 1 The horizontal coordinate is the gradient concentration, and the vertical coordinate is the current value (10 -6 A); and the correlation coefficient R2 =0.997, indicating that the method has a good linear relationship.

[0051] Table 2

[0052] Example 3

[0053] Repeatability test: Using the detection method in Example 2, five concentration gradient biological samples (selenium ion test solution) were selected, and each gradient was tested for 20 times of repeatability, and the current (unit: μA) detection results are shown in Table 3. The coefficient of variation (CV value) of the calculated results is less than 10%, indicating that the repeatability of selenium ion detection is good.

[0054] Table 3

[0055] Example 4

[0056] Limit of detection and limit of quantification test: Using the detection method in Example 2, when the sample concentration is lower than 0.1 μg / L, the current value result shows that it is not detected, and when it is greater than 0.1 μg / L μM, the current value increases with the increase of concentration. The current (unit: μA) detection results are shown in Table 4.

[0057] Table 4

[0058] Example 5

[0059] Stability test: Using the detection method in Example 2, the stability data of the current signal (unit: μA) value of the multilayer composite ultra-micro array electrode in detecting 0.5 μM selenium element biological samples within 30 days was tested, as shown in Table 5. Figure 2 From the results, it can be seen that the multilayer composite ultra-micro array electrode in the present application has good stability within 30 days.

[0060] Example 6

[0061] Anti-interference test: The 6 μg / L selenium element standard, 3 μg / L iron element standard, 10 μg / L lead element standard, 10 μg / L zinc element standard, 5 μg / L copper element standard, 10 μg / L tellurium element standard and the mixed element standard composed of the selenium element standard, the iron element standard, the lead element standard, the zinc element standard, the copper element standard and the tellurium element standard are diluted into the same concentration of the to-be-tested element sample by a mixed solution of 0.01M hydrochloric acid and 0.5mM ethylenediaminetetraacetic acid in a volume ratio of 1:1; the detection method in Example 2 is used for testing, and the test results are shown in Table 1. Figure 3 From the results, it can be known that the detection method in the application has good specificity for selenium element ions.

[0062] Comparative Example 1

[0063] A modified ultramicro array electrode is prepared by the following steps: S1. A gold electrode with a diameter of 3mm is polished to a mirror surface with 0.05 μm aluminum oxide powder, and after each polishing, the surface dirt is washed away with purified water. After the purified water is washed, it is immersed in a 0.1M sulfuric acid solution for cyclic voltammetry activation, with a scanning range of -1.0~1V, and repeated scanning until the cyclic voltammogram is stable. After stabilization, the gold electrode is immersed in a 5mM potassium ferricyanide solution to test the electrode performance by cyclic voltammetry curve, with a scanning rate of 50mV / s and a scanning range of -1.0~0.6V. The peak potential difference in the cyclic voltammetry curve obtained under laboratory conditions is below 80mV, the peak current tends to be stable, and the electrode can be used, otherwise the electrode needs to be reprocessed until it meets the above requirements. S2. The gold electrode treated in S1 is used as a working electrode, and is immersed in a 10mM L-cysteine solution together with a platinum electrode as a counter electrode and a silver / silver chloride electrode as a reference electrode, keeping the electrodes at the same level. The modified electrode is tested by cyclic voltammetry CV, with a scanning range of -0.5 / 1.5 / -0.5V, a scanning rate of 0.1V / s, and 40 scanning cycles, and a standing time of 2s. After cyclic voltammetry modification, the modified ultramicro array electrode is obtained.

[0064] Comparative Example 2

[0065] A gold film modified electrode is prepared by the following steps: S1. A 3mm diameter glassy carbon electrode is polished with 0.05μm aluminum oxide powder to mirror surface, after each polishing, the surface dirt is washed away with purified water, after the purified water rinse, it is immersed in a 0.1M sulfuric acid solution for cyclic voltammetry activation, the scan range is -1.0~1V, repeatedly scan until the cyclic voltammogram is stable; after stabilization, the glassy carbon electrode is immersed in a 5mM potassium ferricyanide solution to test the electrode performance with cyclic voltammogram, the scan rate is 50mV / s, the scan range is -1.0~0.6V, the peak potential difference in the cyclic voltammetry curve obtained under laboratory conditions is below 80mV, the peak current tends to be stable, the electrode can be used, otherwise the electrode needs to be reprocessed until it meets the above requirements; S2. The glassy carbon electrode treated in S1 is used as the working electrode, and a platinum electrode as the counter electrode and a silver / silver chloride electrode as the reference electrode are immersed in a 1mM chloroauric acid solution, the electrodes are kept in the same horizontal plane, and the electrodeposition modification is carried out by current-time method, the constant potential is -0.8V, the scan time is 200s, and the standing time is 30s, a gold film with uniform size and nano-particle structure is formed on the surface of the working electrode to obtain a gold film modified electrode.

[0066] Comparative Example 3

[0067] The working electrode in the three-electrode system of the detection method of Example 2 is replaced with the modified ultra-micro array electrode in Comparative Example 1 to detect a selenium ion test solution with a concentration of 0.15μg / L, and the detection results of the current value (unit: μA) are shown in Table 5.

[0068] Comparative Example 4

[0069] The working electrode in the three-electrode system of the detection method of Example 2 is replaced with the gold film modified electrode in Comparative Example 2 to test a selenium ion test solution with a concentration of 0.15μg / L, and the detection results of the current value (unit: μA) are shown in Table 5.

[0070] Table 5

[0071] As can be seen from Table 5, in Comparative Example 3, the gold electrode surface is directly modified, and the current value is weaker than that of the gold film electrodeposition modification detection, the reason is that the gold nano-particles of uniform size produced by gold film electrodeposition can increase the contact area with L-cysteine, thereby the sensitivity is higher than that of the gold electrode; in Comparative Example 4, due to the lack of gold film, L-cysteine cannot form Au-S bond to realize self-assembly, and the current value shows no detection.

[0072] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting; thus the use of any

[0073] The foregoing is considered as illustrative only of the principles of the application. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the application to the exact construction and practice described. Accordingly, all such variations are intended to be included within the scope of the present application as defined in the claims below and their equivalents.

Claims

1. A multilayer modified ultramicroarray electrode characterized in that, Preparation by the following steps: S1. After polishing the surface of the glassy carbon electrode, first immerse it in sulfuric acid solution for cyclic voltammetry activation until stable; then immerse it in potassium ferricyanide solution, and scan by cyclic voltammetry until the peak current tends to be stable; S2. Take the glassy carbon electrode after S1 processing as the working electrode, immerse it in chloroauric acid solution together with the counter electrode and the reference electrode, and obtain the gold film modified electrode by electrodeposition; S3. Immerse the gold film modified electrode together with the counter electrode and the reference electrode in L-cysteine solution, and obtain the multilayer modified ultramicroarray electrode by cyclic voltammetry modification.

2. The multilayer modified ultramicroarray electrode according to claim 1, wherein, In S1, the polishing treatment includes: polishing the glassy carbon electrode with a diameter of 3 mm to a mirror surface with 0.05 μm aluminum oxide powder, and washing the surface dirt with purified water after each polishing.

3. The multilayer modified ultramicroarray electrode according to claim 1, wherein In S1, the concentration of the sulfuric acid solution is 0.1 M; The scanning range of cyclic voltammetry activation in the sulfuric acid solution is -1.0~1 V, and the cyclic voltammetry graph is repeatedly scanned until it is stable.

4. The multilayer modified ultramicroarray electrode according to claim 1, wherein In S1, the concentration of the potassium ferricyanide solution is 5 mM; The scanning rate of cyclic voltammetry in the potassium ferricyanide solution is 50 mV / s, the scanning range is -1.0~0.6 V, and the peak potential difference in the cyclic voltammetry graph is ≤80 mV.

5. The multilayer modified ultramicroarray electrode according to claim 1, wherein In S2, the concentration of the chloroauric acid solution is 1 mM, the solute is HAuCl4, and the solvent is 0.1 M hydrochloric acid solution.

6. The multilayer modified ultramicroarray electrode according to claim 1, wherein In S2, the constant potential for electrodeposition is -0.8 V, the scanning time is 200 s, and the standing time is 30 s.

7. The multilayer modified ultramicroarray electrode according to claim 1, wherein In S3, the concentration of the L-cysteine solution is 10 mM, and the pH is 5.5±0.1; In the L-cysteine solution, the electrodes are kept at the same level, the scanning range of cyclic voltammetry modification is initial potential -0.5 V, high potential 1.5 V, and low potential -0.5 V, the scanning rate is 0.1 V / s, the scanning number is 40, and the standing time is 2 s.

8. The method for detecting selenium ions by using the multilayer modified ultramicroarray electrode according to any one of claims 1-7, characterized in that, Comprising the following steps: Step 1: immerse the multilayer modified ultramicroarray electrode, the counter electrode, and the reference electrode in the cleaning solution, and scan by cyclic voltammetry for cleaning; Step 2: test the selenium ion test solution by the three-electrode system after cleaning.

9. The method for detecting selenium ions by using the multilayer modified ultramicroarray electrode according to claim 8, characterized in that, In Step 1, the cleaning solution is acetic acid-sodium acetate solution with pH≤4.5, which is prepared by mixing 0.01~0.05 M acetic acid solution and 0.05~0.1 M sodium acetate solution; In the cleaning solution, the electrodes are kept at the same level, the scanning range of cyclic voltammetry is initial potential -1 V, high potential 1 V, and low potential -1 V, the scanning rate is 0.08 V / s, the scanning number is 10, and the standing time is 2 s.

10. The method for detecting selenium ions by using the multilayer modified ultramicroarray electrode according to claim 8, characterized in that, In Step 2, the selenium ion test solution uses a mixture of 0.01 M hydrochloric acid and 0.5 mM ethylenediaminetetraacetic acid as the solvent, and adds the selenium ion test sample.

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