A surface molecularly imprinted electrochemical biosensor for detecting spike protein of novel coronavirus and a preparation method and application thereof

Abstract

This invention relates to the fabrication and application of a surface-imprinted electrochemical biosensor using Cu7S4-Au as an embedded signal probe for detecting the spike protein of the novel coronavirus, belonging to the field of electrochemical technology. The invention involves sequentially modifying the electrode surface with copper tetrasulfide-gold nanoparticles, 4-mercaptophenylboronic acid, a template molecule of the SARS-CoV-2 spike protein S protein, and a poly(3-aminophenylboronic acid) PAPBA imprinted polymer formed by polymerizing 3-aminophenylboronic acid (3-APBA). After eluting the SARS-CoV-2 spike protein S protein template molecule with an acidic elution buffer, the surface-imprinted electrochemical biosensor SMI / 4-MPBA / Cu7S4-Au / electrode is obtained. This surface-imprinted electrochemical biosensor can be used for highly sensitive detection of the SARS-CoV-2 spike protein, providing a detection method for the clinical diagnosis of SARS-CoV-2.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical technology, specifically relating to the preparation and application of a surface molecularly imprinted electrochemical biosensor that uses Cu7S4-Au as a built-in signal probe for detecting the spike protein of the novel coronavirus. Technical Background

[0002] Currently, the detection of SARS-CoV-2 is mainly accomplished using reverse transcription-polymerase chain reaction (RT-PCR). This technology can accurately identify viral ribonucleic acid (RNA), but its application is limited by drawbacks such as long analysis time, high instrument costs, and demanding requirements for testing personnel. Antigen detection methods based on SARS-CoV-2 specific antigens (such as nucleocapsid protein (N protein) and spike protein (S protein)) are attracting increasing attention, with the S protein potentially being the most valuable antigenic biomarker.

[0003] Electrochemical biosensors offer advantages such as high sensitivity, low cost, and robustness, providing a reliable solution for COVID-19 clinical diagnosis. Furthermore, molecularly imprinted electrochemical biosensors combine the high specificity of molecularly imprinted polymers with biosensors, showing great potential for application in biomedical analysis. S proteins are typical biomacromolecules, difficult to use as template molecules for traditional bulk imprinting, necessitating the search for a suitable method for molecular imprinting of protein biomacromolecules. Surface molecular imprinting (SMI) technology can precisely address this issue. Copper sulfide nanocrystals (Cu...) x S y Because it possesses 3d electrons, it contains a large number of stable and metastable components. Among them, copper heptasulfide (Cu7S4) has high conductivity, low toxicity, and strong adsorption capacity, making it suitable as a signal probe and highly valuable in the field of electrochemical sensing.

[0004] This invention utilizes gold nanoparticles (AuNPs) loaded onto the surface of Cu7S4 via Au-S bonds, thereby improving electron transfer rate and detection sensitivity. Therefore, Cu7S4 loaded with AuNPs (Cu7S4-Au) is an excellent signal probe in the field of biosensing. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to design and provide a surface-imprinted electrochemical biosensor using Cu7S4-Au as an embedded signal probe for detecting the spike protein of the novel coronavirus, as well as its preparation method and application. The present invention utilizes the affinity of boric acid to form cyclic ester bonds to immobilize the template molecule of the novel coronavirus spike protein; then, 3-aminophenylboronic acid is polymerized using cyclic voltammetry to form an imprinted polymer layer; the spike protein template molecule is removed by the dissociation of the boric acid ester bonds in an acidic solution to obtain the surface-imprinted electrochemical biosensor. This surface-imprinted electrochemical biosensor can be used for highly sensitive detection of the novel coronavirus spike protein, providing a detection method for the clinical diagnosis of the novel coronavirus.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a surface molecularly imprinted electrochemical biosensor that can be used to detect the spike protein of the novel coronavirus. The electrode surface is sequentially modified with copper heptasulfide-gold nanoparticles, 4-mercaptophenylboronic acid, the template molecule of the novel coronavirus spike protein S protein, and a poly-3-aminophenylboronic acid PAPBA imprinted polymer formed by polymerizing 3-aminophenylboronic acid (3-APBA). After eluting the template molecule of the novel coronavirus spike protein S protein with an acidic elution buffer, the surface molecularly imprinted electrochemical biosensor SMI / 4-MPBA / Cu7S4-Au / electrode is obtained.

[0008] Secondly, the present invention provides a method for preparing a surface molecularly imprinted electrochemical biosensor that can be used to detect the spike protein of the novel coronavirus, comprising the following steps:

[0009] (1) Weigh out Cu7S4-Au and disperse it in ultrapure water to form Cu7S4-Au solution. Drop the Cu7S4-Au solution onto a screen-printed carbon electrode and dry it at room temperature to obtain copper tetrasulfide-gold nanoparticles / electrode, i.e. Cu7S4-Au / electrode.

[0010] (2) Weigh out an ethanol solution of 4-mercaptophenylboronic acid, drop it onto the surface of Cu7S4-Au / electrode, and dry it at room temperature to obtain 4-mercaptophenylboronic acid / copper tetrasulfide-gold nanoparticles / electrode, namely 4-MPBA / Cu7S4-Au / electrode.

[0011] (3) Weigh out a 0.1M phosphate buffer solution containing S protein with a pH of 6.0 to 8.0 and drop it onto the surface of the 4-MPBA / Cu7S4-Au / electrode. Incubate the reaction to obtain the S protein / 4-MPBA / Cu7S4-Au / electrode.

[0012] (4) Weigh out a 0.1M phosphate buffer solution containing 3-aminophenylboronic acid and sodium fluoride with a pH of 6.0 to 8.0 and drop it onto the surface of the S protein / 4-MPBA / Cu7S4-Au / electrode. Scan within a scanning potential range of -0.2 to 0.9V to form a poly(3-aminophenylboronic acid) PAPBA imprinted polymer layer, which is the PAPBA / S protein / 4-MPBA / Cu7S4-Au / electrode. Elute the S protein in a 0.2M sulfuric acid solution to obtain the SMI / 4-MPBA / Cu7S4-Au / electrode.

[0013] The preparation method described above, specifically step (1) of the Cu7S4-Au preparation method, includes:

[0014] Polyvinylpyrrolidone was dissolved in a 0.005–0.02 M copper sulfate pentahydrate aqueous solution. After adding a 1.0–2.0 M sodium hydroxide aqueous solution, a blue precipitate was immediately formed. After the first stirring, a 0.05–0.2 M ascorbic acid aqueous solution was added. After the second stirring, a brick-red solution was obtained. A 0.1–0.3 M thiourea aqueous solution was added, and the reaction was carried out. After centrifugation, the precipitate was washed with distilled water and dried under vacuum to obtain Cu7S4 powder. Cu7S4 powder was added to AuNPs solution, and the mixture was stirred for the third time at room temperature. After centrifugation, the precipitate was washed with distilled water and dried under vacuum to obtain Cu7S4-Au.

[0015] In the preparation method described above, the mass-to-volume ratio of polyvinylpyrrolidone, copper sulfate pentahydrate aqueous solution, and sodium hydroxide aqueous solution is 0.1–0.4 g: 50–150 mL: 20–30 mL, and the first stirring time is 0.5–2 min.

[0016] The mass-to-volume ratio of polyvinylpyrrolidone to ascorbic acid aqueous solution is 0.1-0.4 g: 20-30 mL, and the second stirring time is 10-20 min;

[0017] The mass-to-volume ratio of the polyvinylpyrrolidone to the thiourea aqueous solution is 0.1–0.4 g: 2–10 mL.

[0018] The preparation method described herein, wherein the reaction conditions are: a reaction temperature of 80–100°C and a reaction time of 5–8 hours;

[0019] The vacuum drying temperature is 50–80°C;

[0020] The mass-to-volume ratio of Cu7S4 powder to AuNPs solution is 5-15 mg: 20-50 mL;

[0021] The third stirring time is 3 to 6 hours.

[0022] In the preparation method described above, the electrode in step (1) includes a glassy carbon electrode, a metal electrode, or a screen-printed carbon electrode, and the concentration of the Cu7S4-Au solution is 2–4 mg / mL. –1 .

[0023] In the preparation method described above, the concentration of the ethanol solution of 4-mercaptophenylboronic acid in step (2) is 0.05M to 0.2M, preferably 0.1M; the volume ratio of the Cu7S4-Au solution to the ethanol solution of 4-mercaptophenylboronic acid is 5 to 15: 5 to 15.

[0024] In the preparation method described above, the concentration of S protein in the 0.1M phosphate buffer solution containing S protein with a pH of 6.0–8.0 in step (3) is 5–15 μg / mL. –1 The incubation conditions are: incubation temperature 1-5℃, incubation time 0.5-2h, preferably incubation time 1h.

[0025] The preparation method is characterized in that, in step (4), the volume of the 0.1M phosphate buffer solution containing 3-aminophenylboronic acid and sodium fluoride with a pH of 6.0-8.0 is 50-150 μL, wherein the concentration of 3-aminophenylboronic acid is 20-60 mM and the concentration of sodium fluoride is 20-60 mM; the number of scans is 10-30, preferably 20; the volume of the 0.2M sulfuric acid solution is 50-150 μL; and the elution time is 10-30 min, preferably 20 min.

[0026] Thirdly, the present invention provides the application of the surface molecularly imprinted electrochemical biosensor in detecting the spike protein of the novel coronavirus.

[0027] Furthermore, the SMI / 4-MPBA / Cu7S4-Au / electrode was placed in a solution containing 10 μg mL –1 The S protein is incubated in a 0.1 M phosphate buffer solution at pH 7.4 for 10–50 min, preferably 30 min, and a differential pulse voltammogram is recorded within a potential range of –0.2–0.4 V to achieve the detection of the S protein.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The surface molecular imprinting technology of this invention can improve recognition efficiency. The boric acid affinity of this invention involves the covalent interaction of boric acid with molecules containing cis-diols in a weakly alkaline or alkaline environment to form a stable cyclic ester. When the pH becomes acidic, the boric ester dissociates. This reversible property based on the boric ester, combined with surface molecular imprinting technology, greatly improves the selectivity of the sensor, enabling sensitive detection of the S protein. Furthermore, Cu7S4-Au can be transferred through Cu...+ and Cu 2+ The interconversion between them provides a strong differential pulse voltammetric signal, making it a good signal probe in the field of surface molecular imprinted electrochemical biosensors. Attached Figure Description

[0030] Figure 1 This is a transmission electron microscope (TEM) image of Cu7S4-Au prepared in Example 1;

[0031] Figure 2 The X-ray photoelectron spectrum of Cu7S4-Au prepared in Example 1;

[0032] Figure 3 Differential pulse voltammetry plots of the electrodes prepared in Example 2, Cu7S4-Au / electrode, 4-MPBA / Cu7S4-Au / electrode and S protein / 4-MPBA / Cu7S4-Au / electrode;

[0033] Figure 4 The differential pulse voltammetry plots of the PAPBA / S protein / 4-MPBA / Cu7S4-Au / electrode, SMI / 4-MPBA / Cu7S4-Au / electrode, and SMI / 4-MPBA / Cu7S4-Au / electrode prepared in Example 2 after incubation in S protein solution.

[0034] Figure 5 Differential pulse voltammetry plots of S protein solutions containing different concentrations obtained by using the SMI / 4-MPBA / Cu7S4-Au / electrode prepared in Example 2;

[0035] Figure 6 The graph shows the linear relationship between the peak current reduction (ΔI) and the logarithm of the S protein concentration (lgC) when the SMI / 4-MPBA / Cu7S4-Au / electrode prepared in Example 2 is used for S protein detection.

[0036] Figure 7 The differential pulse voltammetry plots of the PAPBA / 4-MPBA / Cu7S4-Au / electrode, the non-molecularly imprinted electrochemical biosensor (NMI / 4-MPBA / Cu7S4-Au / electrode) prepared in Comparative Example 1, and the NMI / 4-MPBA / Cu7S4-Au / electrode after incubation in S protein solution are shown. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are intended to illustrate the present invention and not to further limit the present invention.

[0038] Example 1: Preparation of Cu7S4-Au

[0039] Preparation method of Cu7S4-Au: 0.2 g of polyvinylpyrrolidone was dissolved in 100 mL of 0.01 M copper sulfate pentahydrate aqueous solution at room temperature. After adding 25 mL of 1.5 M sodium hydroxide aqueous solution, a blue precipitate immediately formed, and the mixture was stirred for 1 min. Then, 25 mL of 0.1 M ascorbic acid aqueous solution was added, and the mixture was stirred for 15 min to obtain a brick-red solution. Finally, 5 mL of 0.2 M thiourea aqueous solution was added, and the reaction was carried out at 90 °C for 6 h. The solid obtained by centrifugation was washed with distilled water and dried under vacuum at 60 °C to obtain Cu7S4 powder. 10 mg of Cu7S4 powder was weighed and added to 40 mL of AuNPs solution, stirred at room temperature for 5 h, and the solid obtained by centrifugation was washed with distilled water and dried under vacuum at 60 °C to obtain black solid Cu7S4-Au. Figure 1 The image shown is a transmission electron microscope (TEM) image of Cu7S4-Au. It can be observed that Cu7S4-Au is a hollow nanocage-like structure with AuNPs uniformly distributed on its surface. Figure 2 The X-ray photoelectron spectrum of Cu7S4-Au shows that it contains Cu, Au, S, and C elements. These results demonstrate the successful preparation of Cu7S4-Au.

[0040] Example 2: Fabrication of a surface-imprinted electrochemical biosensor with Cu7S4-Au as the built-in signal probe

[0041] A method for preparing a surface-imprinted electrochemical biosensor using Cu7S4-Au as a built-in signal probe for detecting the spike protein of SARS-CoV-2 includes the following steps:

[0042] (1) Weigh 2.5 mg Cu7S4-Au and dissolve it in 1 mL of ultrapure water. Disperse the solution evenly by ultrasonication. Transfer 10 μL of the 2.5 mg / mL solution to the solution. –1 A Cu7S4-Au solution was drop-coated onto the SPCE surface and dried at room temperature to obtain Cu7S4-Au / SPCE. Figure 3 Unmodified SPCE showed no oxidation peak, while the modified Cu7S4-Au electrode exhibited an oxidation peak at 0.076 V, which is attributed to Cu. + To Cu 2+ The transformation indicates that Cu7S4-Au can provide a good differential pulse voltammetric signal.

[0043] 10 μL of 0.1 M 4-MPBA ethanol solution was drop-coated onto the surface of Cu7S4-Au / SPCE and dried at room temperature to obtain 4-MPBA / Cu7S4-Au / SPCE.

[0044] Transfer another 10 μL containing 10 μg mL –1A 0.1 M phosphate-buffered saline solution of S protein at pH 7.4 was drop-coated onto the surface of 4-MPBA / Cu7S4-Au / SPCE and incubated at 4 °C for 1 h. 4-MPBA binds to the S protein via borate affinity, yielding the S protein / 4-MPBA / Cu7S4-Au / SPCE mixture. Figure 3 After modification with 4-MPBA and S protein, the peak current decreased sequentially. This is attributed to the fact that the non-conductive 4-MPBA and the insulating S protein created an obstructed electron transport layer on the electrode surface, hindering Cu... + To Cu 2+ The transformation.

[0045] (2) Transfer 100 μL of a 0.1 M phosphate buffer solution containing 40 mM 3-APBA and 40 mM sodium fluoride at pH 7.4 and add it dropwise to the electrode surface prepared in step 1. Use cyclic voltammetry to scan at potentials in the range of –0.2 to 0.9 V at 100 mV / s. –1 The scan rate was 20 cycles to form a PAPBA imprint polymer layer, yielding PAPBA / S protein / 4-MPBA / Cu7S4-Au / SPCE. (The text abruptly ends here.) Figure 4 As shown, after PAPBA is deposited on the electrode surface, no obvious redox peak appears. This is because the poorly conductive PAPBA further hinders the Cu deposition process. + To Cu 2+ The transformation.

[0046] 100 μL of 0.2 M sulfuric acid solution was added dropwise to the surface of PAPBA / S protein / 4-MPBA / Cu7S4-Au / SPCE and kept for 20 min to remove the template S protein, thus obtaining SMI / 4-MPBA / Cu7S4-Au / SPCE. Figure 4 The reappearance of the oxidation peak at 0.076 V indicates that the S protein has been eluted from the electrode surface, forming an imprinted cavity, which is beneficial for Cu. + To Cu 2+ The transformation. Transfer 10 μL containing 10 μg mL –1 Incubate S protein in 0.1 M phosphate buffer solution at pH 7.4 with SMI / 4-MPBA / Cu7S4-Au / SPCE at 4°C for 30 min. Figure 4 The peak current decreased significantly because the S protein reoccupied part of the imprinted cavity, thereby inhibiting Cu. + To Cu 2+ The transformation.

[0047] (3) The prepared SMI / 4-MPBA / Cu7S4-Au / SPCE was placed in a container containing S protein (0.005 ng / mL). -1 ~1000ng / mL-1 Incubate in the solution for 30 minutes. Figure 5 As shown, the oxidation peak current gradually decreases with increasing S protein concentration. This is attributed to the S protein gradually occupying the molecularly imprinted cavity on the electrode surface, thus inhibiting Cu oxidation. + To Cu 2+ The transformation.

[0048] (4) When the prepared SMI / 4-MPBA / Cu7S4-Au / SPCE was used to detect different concentrations of S protein, the decrease in peak current showed a linear relationship with the logarithm of the concentration. For example... Figure 6 The linear equation is ΔI(μA)=2.849lgC+11.12(R) 2 =0.9967), with a linear range of 0.005 ng / mL. -1 ~1000ng / mL -1 The detection limit was 1.76 pg / mL. -1 The above results demonstrate the successful preparation and application of this surface molecularly imprinted electrochemical biosensor.

[0049] Example 3:

[0050] A method for preparing a surface-imprinted electrochemical biosensor using Cu7S4-Au as a built-in signal probe for detecting the spike protein of SARS-CoV-2 includes the following steps:

[0051] (1) Weigh 2 mg Cu7S4-Au and dissolve it in 1 mL of ultrapure water. Disperse the solution evenly by ultrasonication. Transfer 5 μL of the solution containing 2.5 mg / mL Cu7S4-Au. –1 Cu7S4-Au solution was drop-coated onto the SPCE surface and dried at room temperature to obtain Cu7S4-Au / SPCE.

[0052] 5 μL of 0.05 M 4-MPBA ethanol solution was drop-coated onto the surface of Cu7S4-Au / SPCE and dried at room temperature to obtain 4-MPBA / Cu7S4-Au / SPCE.

[0053] Transfer another 10 μL containing 5 μg mL –1 A 0.1 M phosphate buffer solution of S protein at pH 6.0 was drop-coated onto the surface of 4-MPBA / Cu7S4-Au / SPCE and incubated at 1 °C for 0.5 h. 4-MPBA binds to S protein through boric acid affinity to obtain S protein / 4-MPBA / Cu7S4-Au / SPCE.

[0054] (3) Transfer 50 μL of a 0.1 M phosphate buffer solution containing 20 mM 3-APBA and 20 mM sodium fluoride at pH 6.0 and add it dropwise to the electrode surface prepared in step 1. Use cyclic voltammetry to scan at potentials in the range of –0.2 to 0.9 V at 100 mV / s.–1 The scan rate was 10 cycles to form a PAPBA imprint polymer layer, which yielded PAPBA / S protein / 4-MPBA / Cu7S4-Au / SPCE.

[0055] 50 μL of 0.2 M sulfuric acid solution was added dropwise to the surface of PAPBA / S protein / 4-MPBA / Cu7S4-Au / SPCE and kept for 10 min to remove the template S protein, thus obtaining SMI / 4-MPBA / Cu7S4-Au / SPCE-1.

[0056] Example 4:

[0057] (1) Weigh 4 mg Cu7S4-Au and dissolve it in 1 mL of ultrapure water. Disperse the solution evenly by ultrasonication. Transfer 15 μL of the solution containing 2.5 mg / mL Cu7S4-Au. –1 Cu7S4-Au solution was drop-coated onto the SPCE surface and dried at room temperature to obtain Cu7S4-Au / SPCE.

[0058] 15 μL of 0.2 M 4-MPBA ethanol solution was dropped onto the surface of Cu7S4-Au / SPCE and dried at room temperature to obtain 4-MPBA / Cu7S4-Au / SPCE.

[0059] Transfer another 10 μL containing 15 μg mL –1 A 0.1 M phosphate buffer solution of S protein at pH 8.0 was drop-coated onto the surface of 4-MPBA / Cu7S4-Au / SPCE and incubated at 5 °C for 2 h. 4-MPBA binds to S protein through boric acid affinity to obtain S protein / 4-MPBA / Cu7S4-Au / SPCE.

[0060] (4) Transfer 150 μL of a 0.1 M phosphate buffer solution containing 60 mM 3-APBA and 60 mM sodium fluoride at pH 8.0 and add it dropwise to the electrode surface prepared in step 1. Use cyclic voltammetry to scan at potentials in the range of –0.2 to 0.9 V at 100 mV / s. –1 The scanning rate was 30 cycles to form a PAPBA imprint polymer layer, which yielded PAPBA / S protein / 4-MPBA / Cu7S4-Au / SPCE.

[0061] 150 μL of 0.2 M sulfuric acid solution was added dropwise to the surface of PAPBA / S protein / 4-MPBA / Cu7S4-Au / SPCE and kept for 30 min to remove the template S protein, thus obtaining SMI / 4-MPBA / Cu7S4-Au / SPCE-2.

[0062] Comparative Example 1:

[0063] To further demonstrate the successful fabrication of surface molecularly imprinted electrochemical biosensors, PAPBA / 4-MPBA / Cu7S4-Au / SPCE and NMI / 4-MPBA / Cu7S4-Au / SPCE were also prepared. The operational steps were the same as steps (1) to (2) in Example 2, except that the S protein template molecule was not added in step (2). Figure 7 The absence of an oxidation peak for Cu7S4 in PAPBA / 4-MPBA / Cu7S4-Au / SPCE is attributed to the PAPBA imprinted polymerization layer hindering the formation of Cu. + To Cu 2+ The transformation occurred because, due to the absence of S protein, the NMI / 4-MPBA / Cu7S4-Au / SPCE obtained by eluting the electrode with 0.2M sulfuric acid solution could not form an imprint cavity, therefore the eluted electrode still did not show a significant oxidation peak. A 10 μL sample containing 10 μg mL of the solution was transferred... –1 Incubation of the S protein in a 0.1 M phosphate buffer solution at pH 7.4 with NMI / 4-MPBA / Cu7S4-Au / SPCE at 4 °C for 30 min showed almost no change in the oxidation peak current signal, indicating the absence of an imprinted cavity. Comparison between the surface-imprinted electrochemical biosensor and the non-molecularly imprinted electrochemical biosensor further demonstrates that the surface-imprinted electrochemical biosensor can be used to detect the S protein.

Claims

1. A surface-imprinted electrochemical biosensor for detecting the spike protein of the SARS-CoV-2 virus, characterized in that, The electrode surface was sequentially modified with copper tetrasulfide-gold nanoparticles, 4-mercaptophenylboronic acid, SARS-CoV-2 spike protein S protein template molecules, and poly(3-aminophenylboronic acid) PAPBA imprinted polymer formed by polymerizing 3-aminophenylboronic acid (3-APBA). After eluting the SARS-CoV-2 spike protein S protein template molecules with acidic elution buffer, the surface molecularly imprinted electrochemical biosensor SMI / 4-MPBA / Cu7S4-Au / electrode was obtained.

2. The method for preparing a surface-imprinted electrochemical biosensor for detecting the spike protein of SARS-CoV-2 as described in claim 1, characterized in that, Includes the following steps: (1) Weigh out Cu7S4-Au and disperse it in ultrapure water to form Cu7S4-Au solution. Drop the Cu7S4-Au solution onto the electrode and dry it at room temperature to obtain copper tetrasulfide-gold nanoparticles / electrode, i.e. Cu7S4-Au / electrode. (2) Weigh out an ethanol solution of 4-mercaptophenylboronic acid, drop it onto the surface of Cu7S4-Au / electrode, and dry it at room temperature to obtain 4-mercaptophenylboronic acid / copper tetrasulfide-gold nanoparticles / electrode, namely 4-MPBA / Cu7S4-Au / electrode; (3) Weigh out 0.1M phosphate buffer solution containing S protein with pH 6.0~8.0 and drop it onto the surface of 4-MPBA / Cu7S4-Au / electrode, and incubate it to obtain S protein / 4-MPBA / Cu7S4-Au / electrode; (4) Weigh out a 0.1M phosphate buffer solution containing 3-aminophenylboronic acid and sodium fluoride with a pH of 6.0~8.0 and drop it onto the surface of the S protein / 4-MPBA / Cu7S4-Au / electrode. Scan within a scanning potential range of -0.2~0.9V to form a poly(3-aminophenylboronic acid) PAPBA imprinted polymer layer, which forms the PAPBA / S protein / 4-MPBA / Cu7S4-Au / electrode. Elute the S protein in 0.2M sulfuric acid solution to remove the S protein, and obtain the SMI / 4-MPBA / Cu7S4-Au / electrode.

3. The preparation method according to claim 2, characterized in that, The preparation method of Cu7S4-Au in step (1) includes: Polyvinylpyrrolidone was dissolved in 0.005-0.02M copper sulfate pentahydrate aqueous solution. After adding 1.0-2.0M sodium hydroxide aqueous solution, a blue precipitate was immediately formed. After the first stirring, 0.05-0.2M ascorbic acid aqueous solution was added. After the second stirring, a brick-red solution was obtained. 0.1-0.3M thiourea aqueous solution was added, and the reaction was carried out. After centrifugation, the precipitate was washed with distilled water and dried under vacuum to obtain Cu7S4 powder. Cu7S4 powder was added to AuNPs solution and mixed for the third time at room temperature. After centrifugation, the precipitate was washed with distilled water and dried under vacuum to obtain Cu7S4-Au.

4. The preparation method according to claim 3, characterized in that, The mass-to-volume ratio of polyvinylpyrrolidone, copper sulfate pentahydrate aqueous solution, and sodium hydroxide aqueous solution is 0.1~0.4g:50~150mL:20~30mL, and the first stirring time is 0.5~2min; The mass-to-volume ratio of polyvinylpyrrolidone to ascorbic acid aqueous solution is 0.1~0.4g:20~30mL, and the second stirring time is 10~20min; The mass-to-volume ratio of the polyvinylpyrrolidone to the thiourea aqueous solution is 0.1~0.4g:2~10mL.

5. The preparation method according to claim 3, characterized in that, The reaction conditions are: a reaction temperature of 80~100℃ and a reaction time of 5~8h; The vacuum drying temperature is 50~80℃; The mass-to-volume ratio of Cu7S4 powder to AuNPs solution is 5~15mg:20~50mL; The third stirring time is 3-6 hours.

6. The preparation method according to claim 2, characterized in that, The electrode mentioned in step (1) includes a glassy carbon electrode, a metal electrode or a screen-printed carbon electrode, and the concentration of the Cu7S4-Au solution is 2~4 mg mL–1.

7. The preparation method according to claim 2, characterized in that, The concentration of the ethanol solution of 4-mercaptophenylboronic acid in step (2) is 0.05M~0.2M; the volume ratio of the Cu7S4-Au solution to the ethanol solution of 4-mercaptophenylboronic acid is 5~15:5~15.

8. The preparation method according to claim 7, characterized in that, The concentration of the ethanol solution of 4-mercaptophenylboronic acid is 0.1M.

9. The preparation method according to claim 2, characterized in that, In step (3), the concentration of S protein in the 0.1M phosphate buffer solution with pH 6.0-8.0 is 5-15 μg / mL. –1 The incubation conditions are: incubation temperature 1~5℃, incubation time 0.5~2h.

10. The preparation method according to claim 9, characterized in that, The incubation time is 1 hour.

11. The preparation method according to claim 2, characterized in that, In step (4), the volume of the 0.1M phosphate buffer solution with a pH of 6.0-8.0 containing 3-aminophenylboronic acid and sodium fluoride is 50-150 μL, wherein the concentration of 3-aminophenylboronic acid is 20-60 mM and the concentration of sodium fluoride is 20-60 mM; the number of scans is 10-30; the volume of the 0.2M sulfuric acid solution is 50-150 μL; and the elution time is 10-30 min.

12. The preparation method according to claim 11, characterized in that, The number of scans is 20, and the elution time is 20 minutes.

13. The application of the surface molecularly imprinted electrochemical biosensor as described in claim 1 in the detection of the spike protein of the novel coronavirus.

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