Preparation and application of electrochemical immunosensor

Abstract

The invention discloses preparation and application of an electrochemical immunosensor, and the electrochemical immunosensor is obtained by taking a screen-printed carbon electrode as a substrate, cleaning the screen-printed carbon electrode through oxygen plasma, assembling an SARS-CoV-2N protein antigen capture antibody, and sealing the screen-printed carbon electrode with a casein PBS (Phosphate Buffer Solution) with the mass concentration of 0.5-1.5%. The sensor is used for detecting the SARS-CoV-2N protein antigen by adopting an electrochemical alternating-current impedance method, the detection linear range is 0.01-50pg / mL, the detection limit is 8.9 pg / mL, the incubation and detection of the antigen and the antibody are controlled within 20min, and the on-site rapid diagnosis of infection can be realized. The electrochemical immunosensor provided by the invention has the advantages of simple preparation process, no label or sandwich treatment, low cost and the like, has high detection sensitivity, and can realize on-site rapid detection. Therefore, the technology has a wide application prospect in various biological detection fields.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the preparation and application of an electrochemical immunosensor. Background Art

[0002] Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) poses a major threat to public health due to its high infectivity, rapid spread, and high mortality rate. Therefore, efficient, rapid, and accurate laboratory diagnosis of SARS-CoV-2 is crucial to ensure that patients receive treatment as soon as possible and to curb the spread of the epidemic. Currently, nucleic acid-based molecular biological detection methods are the main means of virus detection. Among them, real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) has become the gold standard for virus detection during the outbreak due to its high specificity and sensitivity. However, the detection cost of this method is relatively high, mainly because the implementation of this technology requires professional laboratory equipment and high-quality reagents. In addition, the detection process is relatively complicated and includes multiple steps, such as sample extraction, transcription, and amplification, which leads to a long detection time, and a negative result cannot rule out SARS-CoV-2 infection.

[0003] Immunoassay based on antigen-antibody reaction is another major means of SARS-CoV-2 detection. This technology has the advantages of strong specificity, low cost, rapidity, and short turnaround time. For suspected patients and people who need self-testing, the results can be judged visually or with portable devices. This not only makes up for the shortcomings of real-time fluorescence quantitative PCR technology, but also is a useful supplement to molecular biological detection methods. At present, SARS-CoV-2 antigen testing has become an important tool for on-site diagnosis, significantly reducing the cost of testing, alleviating the pressure of large-scale population screening of SARS-CoV-2 infection, and effectively improving the efficiency of SARS-CoV-2 screening.

[0004] Electrochemical immunoassay is an analytical method that combines electrochemical detection and immunoassay. Compared with traditional immunoassay methods, this technology has the advantages of rapid response, high precision and small sample usage. It simplifies the detection process and is easy to automate, so it has broad application prospects in various fields. Researchers Samper and others used a novel electrochemical immunosensor based on a printed carbon electrode modified with anti-SARS-CoV-2N protein antibodies. The sensor captures the SARS-CoV-2N protein and detects it using antibodies labeled with horseradish peroxidase bound to it. The chronoamperometry method was used to quantitatively detect the virus concentration using 3,3′,5,5′-tetramethylbenzidine, and the detection limit of this method in a 20 μL virus sample was less than 50 PFU / mL (reference: IC Samper, CJ McMahon, MS Schenkel, KM Clark, WKhamcharoen, LBR Anderson, JS Terry, EN Gallichotte, GD Ebel, BJ Geiss, DSDandy, CS Henry. Electrochemical Immunoassay for the Detection of SARS-CoV-2 Nucleocapsid Protein in Nasopharyngeal Samples. Anal Chem., 94 (2022), 4712.). Although the detection is relatively complicated, its results meet the requirements of the World Health Organization (WHO) for COVID-19 POCT diagnostic tests. However, the existing electrochemical immunosensor preparation and detection for SARS-CoV-2 antigen detection still have some defects, such as complex preparation process, high cost, poor reproducibility of detection results and long detection time. Zhai et al. used nitrogen-doped porous graphene (N-HRGO) as a nanocarrier and thiocyanate blue (TH) molecules as electrochemical indicators to construct a label-free electrochemical immunosensor for the quantitative detection of SARS-CoV-2 spike protein. Although the detection limit is 0.3 pg / mL, there are still problems such as long detection time and the need to improve the reproducibility of detection (reference: Q Zhai, X Wang, C Hu, Lin Zhu, C Zhang, L Dai. Label-free electrochemical immunosensor for highly sensitive COVID-19 spike protein detection. Sensors and Actuators Reports., 4 (2022), 100124.).Therefore, there is an urgent need to develop an electrochemical immunosensor with simple and inexpensive electrodes, high detection sensitivity, good reproducibility, and suitable for rapid on-site detection, so as to address the defects of existing immunosensors for SARS-CoV-2 antigen detection. Summary of the invention

[0005] Technical issues solved:

[0006] This application aims at the deficiencies of the prior art and solves the technical problems such as the complex preparation process, high cost, poor reproducibility of detection results, long detection time, and the need to improve the detection reproducibility. It provides a preparation and application of an electrochemical immunosensor; it is simple and inexpensive, achieves high sensitivity and good reproducibility for the detection of new coronavirus antigens, and is suitable for rapid on-site detection.

[0007] Technical solution:

[0008] To achieve the above objectives, this application is implemented through the following technical solutions:

[0009] The preparation steps of an electrochemical immunosensor are as follows:

[0010] S1, Cleaning of Screen-printed Carbon Electrode (SPCE):

[0011] S11, rinse with clean water: The SPCE is ultrasonically rinsed with ultrapure water for 5-10 min, and dried with nitrogen gas for later use;

[0012] S12, oxygen plasma cleaning: the SPCE dried in step S11 is placed in an oxygen plasma machine for oxygen plasma cleaning; the working electrode is cleaned for 10 min at a mid-range of 10.5 W, and oxygen is passed once at 1 min, 2.5 min, 4 min, and 5.5 min respectively;

[0013] S2, modification of working electrode:

[0014] S21, coating: dilute the anti-SARS-CoV-2N protein antibody with a concentration of 5.0 mg / mL to 0.1 mg / mL with PBS buffer (10 mM, pH 7.4) to obtain an antibody dilution solution; drop 4 μL of the antibody dilution solution on the working electrode, and place the electrode in a moisturizing sealed container and incubate overnight at 4°C; take it out the next day, blow off the residual liquid on the electrode surface, rinse it twice with PBST, and blow dry it with nitrogen gas;

[0015] The PBST is composed of PBS (10 mM, pH 7.4) buffer with 0.05% (v / v) Tween-20 added;

[0016] S22, blocking: 4-6 μL of PBS buffer containing 1% casein was dripped onto the electrode surface. The electrode was placed in a sealed container at 37°C.

[0017] After incubation for 20 minutes, the surface liquid was removed and blown off, and the sample was rinsed twice with PBST and then blown dry to obtain the electrochemical immunosensor.

[0018] The present application also discloses the application of the electrochemical immunosensor prepared as above in the quantitative detection of the new coronavirus N protein.

[0019] Further, the specific application steps are:

[0020] The first step is to dilute the antigen: SARS-CoV-2N protein is diluted with a mixed solution of potassium ferrocyanide, potassium ferrocyanide, and potassium chloride to concentrations of 50, 10, 5, 1, 0.5, 0.1, 0.05, and 0.01 pg / mL, respectively;

[0021] Step 2: Immune reaction: Take 30 μL of the diluted SARS-CoV-2N protein solution from the first step and drop it onto the surface of the electrochemical immunosensor, and react at room temperature in the dark for 15 minutes.

[0022] The third step is impedance detection: connect the electrochemical immunosensor to the electrochemical workstation, and use electrochemical impedance spectroscopy to detect the changes in the electrode surface before and after binding to the SARS-CoV-2N protein antigen. The established standard curve can be used to quantify the concentration of SARS-CoV-2N protein antigen.

[0023] Furthermore, in the mixed solution of potassium ferrocyanide, potassium ferrocyanide and potassium chloride, the final concentrations of potassium ferrocyanide and potassium ferrocyanide are both 5 mM, and the final concentration of potassium chloride is 0.1 M.

[0024] Furthermore, the electrochemical method detection in the electrochemical workstation is: electrolysis reaction is carried out under the conditions of a frequency range of 0.1 to 10000 Hz, an amplitude of 0.005, and an open circuit voltage of -0.08 V to test the electron transfer resistance on the electrode surface.

[0025] Beneficial effects:

[0026] The present application provides a preparation and application of an electrochemical immunosensor, which has the following beneficial effects compared with the prior art:

[0027] 1. The present invention constructs an electrochemical immunosensor by treating the surface of a screen-printed electrode with oxygen plasma and modifying the electrode surface with antibodies, and determines the concentration of SARS-CoV-2 antigen by directly measuring the change in the electrochemical impedance signal after antigen-antibody specific recognition. Compared with the existing electrochemical immunosensor constructed using screen-printed electrodes, the electrochemical immunosensor of the present invention has a simple production process, is inexpensive, and is suitable for mass production;

[0028] 2. Oxygen plasma cleaning of the electrode surface not only removes surface impurities, improves the hydrophilicity of the electrode, and improves the antibody fixation efficiency, but also helps to improve the electron transfer rate and repeatability of SPCE, thereby improving the sensitivity of electrochemical immunosensors in detecting novel coronavirus antigens, and has certain development potential in biomedical applications;

[0029] 3. The electrochemical immunosensor constructed by the present invention completes antigen-antibody specific recognition and detection within 20 minutes. The SARS-CoV-2 antigen to be detected is proportional to the electrochemical signal. It has the characteristics of high sensitivity (minimum detection limit 8.9 fg / mL), good reproducibility, rapidity (15 minutes) and detection range (0.01 pg / mL to 50 pg / mL), which is conducive to the commercialization of electrochemical immunosensors;

[0030] 4. Zeng et al. modified anti-SARS-CoV-2N antibody (Ab) on the screen-printed electrode with electro-deposited gold nanoparticles to prepare an electrochemical immunosensor. The antigen-antibody reaction between SARS-CoV-2N protein and electrode Ab has a good linear relationship in the range of 0.01 to 1000 ng / mL, and the detection limit is 2.6 pg / mL (R Zeng, M Qiu, Q Wan, Z Huang, X Liu, D Tang, D Knopp. Smartphone-Based Electrochemical Immunoassay for Point-of-Care Detection of SARS-CoV-2 Nucleocapsid Protein. Ana l. Chem. 94 (2022), 12155-15161.), which is more than 200 times more sensitive than the present application.

[0031] 5. The constructed immunosensor showed ultrasensitive response to SARS-COV-2 nucleocapsid protein, with an ideal linear relationship range of 0.001 to 50 ng / mL and a detection limit of 0.38 pg / mL. The prepared sensor has a wide linear range, excellent selectivity, specificity and stability, and provides a valuable reference for the detection of SARS-COV-2. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of the preparation and detection principle of the electrochemical immunosensor for detecting novel coronavirus antigens in this application;

[0033] Figure 21 is the cyclic voltammogram of the untreated SPCE base electrode (curve a) and the O2 Plasma / SPCE electrode (curve b) in Example 1 of the present application;

[0034] Figure 3 It is the electrochemical impedance spectra of the untreated SPCE base electrode (curve a) in Example 1 of the present application, the O2Plasma / SPCE electrode treated with oxygen plasma (curve b), the Ab / O2 Plasma / SPCE electrode modified with anti-novel coronavirus N protein antibodies (curve c), the finally obtained CS / Ab / O2 Plasma / SPCE electrode for detecting the novel coronavirus N protein antigen (curve d), and the NP / CS / Ab / O2 Plasma / SPCE electrode (curve e) after the novel coronavirus N protein antigen is added dropwise to the surface of the electrochemical immunosensor;

[0035] Figure 4 It is a standard curve obtained by detecting the novel coronavirus N protein standard solution of the present application; wherein line a: 50pg / mL, curve b: 10pg / mL, curve c: 5pg / mL, curve d: 1pg / mL, curve e: 0.5pg / mL, curve f: 0.1pg / mL, curve g: 0.05pg / mL, curve h: 0.01pg / mL;

[0036] Figure 5 It is a linear equation between the change in impedance value of this application and the logarithm of the concentration of the novel coronavirus N protein;

[0037] Figure 6 This is a standard curve obtained by comparing the untreated SPCE with the new coronavirus N protein standard solution; curve a: 100pg / mL, curve b: 50pg / mL, curve c: 10pg / mL, curve d: 5pg / mL, curve e: 1pg / mL, curve f: 0.5pg / mL;

[0038] Figure 7 There is a linear relationship between the change in the untreated SPCE impedance value and the logarithm of the new coronavirus N protein concentration. DETAILED DESCRIPTION

[0039] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0040] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased by yourself.

[0041] Standards: SARS-CoV-2 nucleocapsid protein (Nucleocapsid, NP, purity > 96%) was purchased from Anyuan Pharmaceutical Technology (Shanghai) Co., Ltd., and the new coronavirus N protein antibody (Ab) was purchased from Shenzhen Heavy Chain Biotechnology Co., Ltd.

[0042] Potassium ferrocyanide, potassium ferrocyanide and potassium chloride were purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd.

[0043] Casein and phosphate buffered saline (PBS, 10 mM, pH 7.4) were purchased from Shanghai Shenggong Biotechnology Co., Ltd.

[0044] Screen-printed carbon electrodes were purchased from Guangzhou Yuxin Sensing Technology Co., Ltd.

[0045] Embodiment 1:

[0046] Preparation of an electrochemical immunosensor, wherein the electrochemical immunosensor is based on a screen-printed electrode, such as Figure 1 As shown, the preparation method of the working electrode specifically includes the following steps:

[0047] Step 1: Using the screen-printed carbon electrode as the base electrode, the electrode was ultrasonically cleaned in pure water for 5 to 10 minutes and dried with nitrogen;

[0048] The screen-printed electrode cleaned above was placed in an oxygen plasma machine for oxygen plasma cleaning. The cleaning was carried out at a medium level (10.5W) for 10 minutes, and oxygen was passed once at the 1st minute, 2.5th minute, 4th minute and 5.5th minute, respectively, to obtain an O2 Plasma / SPCE electrode;

[0049] Step 2: Anti-SARS-CoV-2N protein antibody Ab was diluted to 0.1 mg / mL with PBS (10 mM, pH 7.4) to obtain Ab dilution solution, 4 μL Ab dilution solution was added dropwise to the surface of O2 Plasma / SPCE electrode, and incubated overnight in a sealed container at 4°C; the electrode was taken out on the next day, the residual liquid on the surface of the electrode was blown off, and the electrode was rinsed twice with PBST and dried with nitrogen to obtain the Ab / O2 plasma / SPCE electrode; the PBST was composed of PBS (10 mM, pH 7.4) buffer solution with 0.05% (v / v) Tween-20;

[0050] Step 3: Take 6 μL of PBS buffer containing 1% casein and drop-coat it on the surface of the Ab / O2 plasma / SPCE electrode, incubate it in a sealed container at 37°C for 20 min, take it out and blow off the surface liquid, rinse the electrode surface twice with PBST, and dry it to obtain an electrochemical immunosensor, i.e., a CS / Ab / O2 plasma / SPCE electrode, and store the electrochemical immunosensor at 4°C for standby use;

[0051] Step 4: The SPCE base electrode was used as the counter electrode and silver as the reference electrode. The O2 plasma / SPCE electrodes treated in the first step were connected to the chemical workstation respectively. Cyclic voltammetry (CV) was used to perform electrochemical tests on the SPCE base electrode and the O2 plasma / SPCE electrode. The test solution contained 5 mM [Fe(CN)6] 3- / 4- (1:1) and 0.1M KCl PBS buffer, scanning range -1.0V ~ 1.0V, scanning speed 50mV / s, test results see Figure 2 .

[0052] like Figure 2 As shown, curve a is the SPCE base electrode, where obvious redox peaks can be seen, and curve b is the O2plasma / SPCE electrode, where the redox peak current increases significantly, the ratio of the two characteristic peak currents is 1, and the potential difference △Ep between the redox peaks is 59mV, indicating that the electrode has a fast electron transfer rate and good electrode performance. This is mainly due to the fact that oxygen plasma treatment improves the conductivity of the electrode, promotes electron transfer in the electrochemical reaction, and thus plays a role in amplifying the current signal.

[0053] Electrochemical impedance spectroscopy (EIS) was further used to characterize the changes in the surface properties of the electrode during the modification process of the electrochemical immunosensor working electrode. The SPCE base electrode, the O2 Plasma / SPCE electrode obtained in the first step, the Ab / O2 Plasma / SPCE electrode obtained in the second step, and the CS / Ab / O2 plasma / SPCE electrode obtained in the third step were exposed to 5 mM [Fe(CN)6] in 30 μL of water. 3- / 4- and 0.1M KCl in PBS buffer; at the same time, 30 μL of 5 mM

[0054] [Fe(CN)6] 3- / 4- The novel coronavirus N protein antigen was diluted with 0.1M KCl and incubated for 15 minutes for direct electrochemical analysis. The three clamps of the chemical workstation were clamped on the working electrode, reference electrode and counter electrode respectively, and the electrochemical impedance spectrum of the working electrode modification process was obtained in the frequency range of 0.1-10000HZ, amplitude of 0.005 and open circuit voltage of -0.08V.

[0055] Figure 3 The impedance spectrum of the electrochemical immunosensor working electrode is gradually modified. The diameter of the semicircle in the high-frequency part of the impedance spectrum is equal to the electron transfer impedance (Ret). Curve a is the SPCE base electrode cleaned with ultrapure water, and the diameter of the semicircle is the largest. Curve b is the O2 Plasma / SPCE electrode cleaned with oxygen plasma, and the semicircle diameter is significantly lower than that of curve a, indicating that oxygen plasma cleaning has an effect on improving electron conduction, which is consistent with Figure 2 The CV spectrum results are consistent. Curve c is the Ab / O2 Plasma / SPCE electrode, which has a significantly increased electron transfer impedance compared to curve b. Curve d is the continued modification of 1% casein (CS) on the electrode, and the semicircle diameter is further increased, indicating that the CS / Ab / SPCE immunosensor has been successfully prepared; Curve e is the SARS-CoV-2N protein antigen bound to the CS / Ab / SPCEs immunosensor, and the semicircle diameter is further increased compared to curve d, indicating that the impedance of the electrode is also further increased.

[0056] The SARS-CoV-2N protein has no electrochemical activity and has no effect on the charge transfer resistance. The above results prove that the new coronavirus antigen and antibody combine successfully, indicating that the electrochemical immunosensor interface has been successfully constructed.

[0057] Embodiment 2:

[0058] Application of an electrochemical immunosensor in the quantitative detection of a novel coronavirus N protein, the method using the electrochemical immunosensor prepared in Example 1, specifically comprising the following steps:

[0059] The first step is to draw a standard curve: using the electrochemical immunosensor prepared in Example 1, the SARS-CoV-2N protein was diluted to a concentration of 50, 10, 5, 1, 0.5, 0.1, 0.05, and 0.01 pg / mL with a mixed solution of 5 mM potassium ferrocyanide and potassium ferrocyanide and 0.1 mM potassium chloride to obtain a gradient concentration of the new coronavirus antigen dilution solution;

[0060] Step 2: 30 μL of gradient concentrations of novel coronavirus antigen dilutions were added dropwise to the electrochemical immunosensor prepared in Example 1 and incubated at room temperature for 15 min;

[0061] Step 3: Connect the electrochemical immunosensor to the chemical workstation and perform electrochemical impedance spectroscopy (EIS) analysis at a frequency range of 0.1 to 10,000 Hz, an amplitude of 0.005, and an open circuit voltage of -0.08 V (see EIS curve for details). Figure 4); It can be seen that as the concentration of the novel coronavirus N protein antigen increases, the semicircle diameter gradually increases. This is because the higher the concentration of the antigen N protein, the non-conductive immune complex formed by the combination of antigen and antibody on the electrode surface affects the efficiency of electron transfer, resulting in an increase in the non-conductive area on the electrode surface, thereby increasing the electrochemical impedance. The equivalent circuit is fitted using ZSimpWin software. Figure 5 Nyquist plot of electrochemical impedance spectroscopy of NPs with different concentrations. ct0 is the impedance value when there is no N protein, R cti is the impedance value after immersion in N protein. The change in impedance value and the logarithm of NP concentration show a good linear relationship between 0.01 and 50 pg / mL, and the linear regression equation is ΔR ct =97.13Ig[Nprotein]+288.8( Figure 5 , N protein represents the concentration of SARS-CoV-2 N protein, r 2 =0.978), detection limit (3 Sb / S) was 9.0 fg / mL, which was lower than the reported detection limit.

[0062] Embodiment 3:

[0063] The electrochemical immunosensor prepared in Example 1 was placed in a mixed solution of potassium ferrocyanide and potassium ferrocyanide with a concentration of 5 mM and potassium chloride with a concentration of 0.1 mM, and the SARS-CoV-2N protein was diluted to a concentration of 1.0 pg / mL and incubated at room temperature for 15 min. Other operations were the same as in Example 1. The electrochemical impedance curve was recorded and the transfer impedance change value was calculated to be 289.6 Ω. According to the linear relationship formula ΔR obtained in Example 2 above, ct =97.13Ig[N protein]+288.8, the concentration can be calculated to be 1.021pg / mL, which is close to the actual concentration of 1pg / mL, with a relative error of 2.1%.

[0064] Comparative Example 1:

[0065] This comparative example provides a three-electrode electrochemical sensor for detecting novel coronavirus antigens. Based on Example 2, the oxygen plasma cleaning of the electrode in Example 1 is replaced by ultrasonic cleaning, and the incubation time is changed to 30 minutes. The other steps are the same as in Example 2. The non-conductive immune complex formed by the combination of antigen and antibody blocks the channel for electron transfer, and the resistance of electron transfer to the electrode surface increases. In the range of 0.5 to 100 pg / mL, the impedance value increases with the increase of the concentration of novel coronavirus N protein ( Figure 6 ), the change in impedance is linearly related to the logarithm of the concentration of the novel coronavirus N protein antigen. The results are as follows Figure 7 , the fitting linear regression equation is ΔRct =361.1Ig[N protein]+1662.24 (N protein represents the concentration of SARS-CoV-2 N protein, r 2 =0.901), the calculated minimum detection limit was 46.6 fg / mL, which is 5 times that of Example 2, indicating that oxygen plasma cleaning is beneficial to improving the sensitivity of electrochemical immunoassay.

Claims

1. Preparation of an electrochemical immunosensor, characterized in that: Here are the steps: S1, Cleaning of Screen-printed Carbon Electrode (SPCE): S11, rinse with clean water: The SPCE is ultrasonically rinsed with ultrapure water for 5-10 min, and dried with nitrogen gas for later use; S12, oxygen plasma cleaning: the SPCE dried in step S11 is placed in an oxygen plasma machine for oxygen plasma cleaning; the working electrode is cleaned for 10 min at a mid-range of 10.5 W, and oxygen is passed once at 1 min, 2.5 min, 4 min, and 5.5 min respectively; S2, modification of working electrode: S21, coating: dilute the anti-SARS-CoV-2N protein antibody with a concentration of 5.0 mg / mL to 0.1 mg / mL with PBS buffer (10 mM, pH 7.4) to obtain an antibody diluent; drop 4 μL of the antibody diluent on the working electrode, place the electrode in a moisturizing sealed container and incubate overnight at 4°C; take it out the next day, blow off the residual liquid on the electrode surface, rinse it twice with PBST, and blow dry it with nitrogen; The PBST is composed of PBS (10 mM, pH 7.4) buffer with 0.05% (v / v) Tween-20 added; S22, sealing: 4-6 μL of PBS buffer containing 1% casein was dripped onto the electrode surface. After the electrode was incubated at 37°C for 20 min in a sealed container, the surface liquid was blown off, and the electrode was rinsed twice with PBST and then blown dry to obtain an electrochemical immunosensor.

2. An application of the electrochemical immunosensor prepared according to claim 1 in the quantitative detection of the novel coronavirus N protein.

3. The use according to claim 2, characterized in that: The specific application steps are: The first step is to dilute the antigen: SARS-CoV-2N protein is diluted with a mixed solution of potassium ferrocyanide, potassium ferrocyanide, and potassium chloride to concentrations of 50, 10, 5, 1, 0.5, 0.1, 0.05, and 0.01 pg / mL, respectively; Step 2: Immune reaction: Take 30 μL of the diluted SARS-CoV-2N protein solution from the first step and drop it onto the surface of the electrochemical immunosensor, and react at room temperature in the dark for 15 minutes. The third step is impedance detection: connect the electrochemical immunosensor to the electrochemical workstation, and use electrochemical impedance spectroscopy to detect the changes in the electrode surface before and after binding to the SARS-CoV-2N protein antigen. The established standard curve can be used to quantify the concentration of SARS-CoV-2N protein antigen.

4. The use according to claim 3, characterized in that: In the mixed solution of potassium ferrocyanide, potassium ferrocyanide and potassium chloride, the final concentrations of potassium ferrocyanide and potassium ferrocyanide are both 5 mM, and the final concentration of potassium chloride is 0.1 M.

5. The use according to claim 3, characterized in that: The electrochemical method detection in the electrochemical workstation is: electrolysis reaction is carried out under the conditions of frequency range 0.1~10000HZ, amplitude 0.005, open circuit voltage -0.08V, and testing the electron transfer resistance on the electrode surface.

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