A molecular imprinting micro-electrochemical biosensor for detecting novel coronavirus spike protein and its preparation method and application

Abstract

The present invention discloses a molecular imprinting micro electrochemical biosensor that can be used to detect the spike protein of the new coronavirus, and its preparation method and application, which belong to the field of electrochemical technology. The molecular imprinting micro electrochemical biosensor of the present invention includes the following steps: sequentially modifying gold nanoparticles on the surface of an AN electrode, reducing graphene oxide, doping polymethylene blue with poly-1-vinyl-3-butylimidazolium bromide ionic liquid, and imprinting a polymer with a polyionic liquid having a spike protein imprinting cavity, to obtain MIP / PMB / P[Bvim]Br / rGO / AuNPs / AN. The present invention utilizes the fact that when the spike protein is bound to the molecular imprinting cavity or eluted from the cavity, the number of imprinting cavities decreases or increases accordingly, resulting in a corresponding change in the current signal of polymethylene blue. According to the linear correlation between the logarithm of the reduction rate of the polymethylene blue current signal and the concentration of the new coronavirus spike protein, specific detection of the new coronavirus spike protein is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of electrochemical technology and relates to a molecular imprinting micro-electrochemical biosensor that can be used to detect novel coronavirus spike protein, as well as a preparation method and application thereof. Technical Background

[0002] In order to rapidly detect cases and their contacts, rapid and easy-to-use diagnostic tools that can test a large number of samples in a short period of time are urgently needed. Existing SARS-CoV-2 detection methods are mainly reverse transcription-polymerase chain reaction (RT-PCR) and detection of specific antibodies in serum. RT-PCR is time-consuming, expensive, and requires well-trained operators, so it cannot be used for rapid screening. For the detection of specific antibodies in serum, it is not of much clinical significance because specific antibodies will be produced in vaccinated individuals and test positive. Since the spike protein (SP) of SARS-CoV-2 can bind to the human angiotensin-converting enzyme receptor to enter target cells, SP has become an important marker for antigen detection.

[0003] Molecular imprinting electrochemical biosensors combine molecular imprinting technology with electrochemical sensing technology, and use molecular imprinting polymers (MIP) to specifically identify the analyte, which can enhance the selectivity of electrochemical biosensors. Molecular imprinting electrochemical biosensors have the advantages of high sensitivity, good selectivity, and fast analysis speed. They have been widely used in the detection of various markers in serum. Since proteins have complex conformations, it is necessary to find a suitable method for molecular imprinting of proteins. Surface imprinting can effectively reduce embedding and easily achieve elution of imprinted molecules, and is an effective method for protein imprinting. Summary of the invention

[0004] In view of the problems existing in the above-mentioned prior art, the purpose of the present invention is to design and provide a molecular imprinting micro-electrochemical biosensor that can be used to detect the spike protein of the new coronavirus, as well as its preparation method and application. The present invention adopts PMB as a built-in electrochemical redox probe, and utilizes the electrostatic interaction and imprinting effect between SP and P[Bvim]Br. When SP binds to the molecular imprinting cavity or is eluted from the cavity, the number of imprinting cavities decreases or increases accordingly, resulting in a corresponding change in the current signal of PMB. According to the linear correlation between the reduction rate of the PMB current signal and the logarithm of the SP concentration, specific detection of SP is achieved.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] On the one hand, the present invention provides a molecular imprinting micro-electrochemical biosensor that can be used to detect the novel coronavirus spike protein, comprising: sequentially modifying the surface of an AN electrode with gold nanoparticles (i.e., AuNPs), reducing graphene oxide (i.e., rGO), doping polymethylene blue (i.e., PMB / P[Bvim]Br) with poly-1-vinyl-3-butylimidazolium bromide ionic liquid, and imprinting a polymer with a polyionic liquid having a spike protein (i.e., SP) imprinting cavity to obtain MIP / PMB / P[Bvim]Br / rGO / AuNPs / AN.

[0007] The method for preparing a molecular imprinting micro-electrochemical biosensor that can be used to detect the spike protein of the new coronavirus, wherein the AN electrode includes an acupuncture needle as the AN electrode.

[0008] In a second aspect, the present invention provides a method for preparing a molecular imprinting micro-electrochemical biosensor for detecting a novel coronavirus spike protein, comprising the following steps:

[0009] (1) An electrode was taken, the surface oxide layer was removed by grinding, and the electrode was ultrasonically cleaned with ethanol and ultrapure water, respectively. The electrode was placed in a chloroauric acid solution for constant potential deposition to obtain AuNPs / AN, and then placed in a graphene oxide solution (i.e., GO) for constant potential electrochemical reduction to obtain rGO / AuNPs / AN. The electrode was placed in a phosphate buffer solution (i.e., [Bvim]Br) mixed with methylene blue (i.e., MB) and 1-vinyl-3-butylimidazolium bromide ionic liquid, and ultrasonically dispersed uniformly. Cyclic voltammetry was used to scan the electrode to obtain PMB / P[Bvim]Br / rGO / AuNPs / AN, where P[Bvim]Br represents poly-1-vinyl-3-butylimidazolium bromide and PMB represents polymethylene blue;

[0010] (2) PMB / P[Bvim]Br / rGO / AuNPs / AN was incubated in a solution containing spike protein, and cyclic voltammetry was used to scan in 1-vinyl-3-butylimidazolium bromide ionic liquid buffer solution. The formed P[Bvim]Br was used as an imprinting polymer to embed the spike protein to obtain P[Bvim]Br / SP / PMB / P[Bvim]Br / rGO / AuNPs / AN. The spike protein was eluted with a phosphate buffer solution containing methanol to obtain MIP / PMB / P[Bvim]Br / rGO / AuNPs / AN, where MIP stands for molecular imprinting polymer.

[0011] The method for preparing a molecular imprinting micro-electrochemical biosensor for detecting the spike protein of the new coronavirus, wherein the conditions for obtaining AuNPs / AN by constant potential deposition in step (1) are: potential range -0.4V to -0.8V, preferably -0.8V; deposition time 75 to 125s, preferably 100s; the conditions for obtaining rGO / AuNPs / AN by constant potential electrochemical reduction are: potential range -1.2V to -1.6V, preferably -1.5V; deposition time 480 to 720s, preferably 600s; the conditions for the cyclic voltammetry scan are: potential -0.8 to 1.2V, scan rate 25 to 100mV s -1 The preferred scan rate is 50 mV s -1 ; The number of deposition turns is 5 to 25, and the preferred number of deposition turns is 15.

[0012] The method for preparing a molecular imprinting micro-electrochemical biosensor for detecting the spike protein of the new coronavirus, wherein the concentration of the chloroauric acid solution in step (1) is 1 to 5 mM, preferably 2.5 mM; the concentration of the graphene oxide solution is 1 to 5 mg mL -1 , the preferred concentration of graphene oxide solution is 1 mg mL -1 ; The pH of the phosphate buffer solution is 8.2; the molar ratio of the methylene blue to the 1-vinyl-3-butylimidazolium bromide ionic liquid is 1 to 5:1, and the preferred molar ratio is 2:1.

[0013] The method for preparing a molecular imprinting micro-electrochemical biosensor for detecting the spike protein of the new coronavirus, wherein the conditions for scanning by cyclic voltammetry in the 1-vinyl-3-butylimidazolium bromide ionic liquid buffer solution in step (2) are: potential -0.5 to 1.2 V, scanning rate 25 to 100 mV s -1 , preferably at a scan rate of 50 mV s -1 The number of deposition turns is 3 to 8, and the preferred number of deposition turns is 5.

[0014] The method for preparing a molecular imprinting microelectrochemical biosensor for detecting the spike protein of the new coronavirus, wherein the concentration of the solution containing the spike protein in step (2) is 20 to 100 μg mL -1 , the preferred concentration is 50 μg mL -1 The phosphate buffer solution containing methanol is a phosphate buffer solution with a pH of 5.0 and containing 5% methanol. The elution time is 20 to 60 minutes, and the preferred elution time is 40 minutes.

[0015] In the third aspect, the present invention provides the application of the molecular imprinting micro-electrochemical biosensor in detecting the novel coronavirus spike protein.

[0016] In the fourth aspect, the present invention provides a detection method for the molecular imprinting micro-electrochemical biosensor, wherein the molecular imprinting micro-electrochemical biosensor is placed in a solution containing or not containing spike protein and incubated for 10 to 60 minutes, and the square wave voltammogram of the sensor is recorded in the potential range of -0.5 to 0 V to achieve detection of SP.

[0017] The detection method is characterized in that the incubation time is 30 minutes.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention adopts the surface molecular imprinting method to improve the recognition efficiency; the present invention adopts 1-vinyl-3-butylimidazolium bromide ionic liquid as the electrochemical polymerization ionic liquid, so that it has high stability, biocompatibility and conductivity, and the thickness of the preparation process is controllable; the present invention adopts AuNPs and rGO nanomaterials to modify the electrode, which significantly improves the sensitivity of the electrochemical biosensor.

[0020] The present invention uses polymethylene blue as a built-in electrochemical signal probe. Due to the electrostatic interaction and imprinting effect between the spike protein and the ionic liquid, when the spike protein binds to the molecular imprinting cavity or is eluted from the cavity, the number of imprinting cavities decreases or increases accordingly, resulting in a corresponding change in the current signal of polymethylene blue. According to the linear correlation between the logarithm of the reduction rate of the polymethylene blue current signal and the concentration of the new coronavirus spike protein, specific detection of the new coronavirus spike protein is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Cyclic voltammograms of AN, AuNPs / AN, and rGO / AuNPs / AN in Example 1 in a 0.1 M potassium chloride solution containing 5 mM potassium ferrocyanide / potassium ferrocyanide;

[0022] Figure 2 Square wave voltammograms of PMB / P[Bvim]Br / rGO / AuNPs / AN, P[Bvim]Br / SP / PMB / P[Bvim]Br / rGO / AuNPs / AN, MIP / PMB / P[Bvim]Br / rGO / AuNPs / AN and MIP / PMB / P[Bvim]Br / rGO / AuNPs / AN after recombining with SP prepared in Example 1;

[0023] Figure 3 It is a scanning electron microscopy image of P[Bvim]Br / SP / PMB / P[Bvim]Br / rGO / AuNPs / AN in Example 1;

[0024] Figure 4 is a scanning electron microscopy image of MIP / PMB / P[Bvim]Br / rGO / AuNPs / AN in Example 1;

[0025] Figure 5 The square wave voltammograms of the molecular imprinting micro electrochemical biosensor prepared in Example 1 for detecting SP solutions with different concentrations, and the inset is a partial enlarged view;

[0026] Figure 6 This is a linear relationship diagram between the reduction rate of PMB peak current (ΔI / I0) and the logarithmic value of SP concentration (lgC) when the molecular imprinting micro electrochemical biosensor prepared in Example 1 is used to detect SP;

[0027] Figure 7 The specific results of the molecular imprinting micro-electrochemical biosensor prepared in Example 1;

[0028] Figure 8 Square wave voltammograms of the non-molecular imprinting micro-electrochemical biosensor prepared in Comparative Example 1 before elution, after elution and after re-binding to SP. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following embodiments.

[0030] Embodiment 1:

[0031] A method for preparing a molecular imprinting micro-electrochemical biosensor for detecting novel coronavirus SP comprises the following steps:

[0032] (1) After AN was polished to remove the surface oxide layer, it was ultrasonically cleaned with ethanol and ultrapure water, respectively. AuNPs / AN was deposited in 2.5 mM chloroauric acid solution by -0.8 V constant potential method for 100 s. -1 rGO / AuNPs / AN was obtained by electrochemical reduction in GO solution at -1.5 V constant potential for 600 s.

[0033] like Figure 1 As shown in the figure, there is no redox peak of potassium ferrocyanide / potassium ferrocyanide on AN, while a pair of obvious redox peaks appear on AuNPs / AN, which is attributed to the good conductivity and large specific surface area of ​​AuNPs. Compared with AuNPs / AN, the redox peak current of potassium ferrocyanide / potassium ferrocyanide on rGO / AuNPs / AN is reduced, which is attributed to the poor conductivity of rGO compared with AuNPs.

[0034] (2) 0.0186 g MB and 0.0053 g [Bvim] Br were weighed and dissolved in 25 mL 0.1 M pH = 8.2 phosphate buffer solution, and ultrasonically dispersed uniformly. Cyclic voltammetry was performed in the potential range of -0.8 to 1.2 V at 50 mV s -1 The rate was scanned for 15 cycles to obtain PMB / P[Bvim]Br / rGO / AuNPs / AN.

[0035] like Figure 2 As shown, an oxidation peak of PMB appears at -0.27 V in PMB / P[Bvim]Br / rGO / AuNPs / AN, indicating that PMB is an ideal electrochemical probe. The present invention uses the change of PMB current signal to indicate the concentration of SP.

[0036] (3) PMB / P[Bvim]Br / rGO / AuNPs / AN were prepared in a solution containing 50 μg mL -1 SP / PMB / P[Bvim]Br / rGO / AuNPs / AN was obtained and cyclic voltammetry was performed in a 20 mM [Bvim]Br buffer solution at a potential range of -0.5 to 1.2 V at 80 mV s -1 The rate was scanned for 5 cycles, and the formed P[Bvim]Br was used as the imprinted polymer to embed SP, obtaining P[Bvim]Br / SP / PMB / P[Bvim]Br / rGO / AuNPs / AN, which was eluted in a pH = 5.0 phosphate buffer solution containing 5% methanol for 40 min to obtain MIP / PMB / P[Bvim]Br / rGO / AuNPs / AN.

[0037] like Figure 2 As shown in the figure, P[Bvim]Br / SP / PMB / P[Bvim]Br / rGO / AuNPs / AN did not show obvious redox peaks, which was attributed to the presence of SP and P[Bvim]Br imprinted polymers hindering the electron transfer process of PMB. After the template molecule SP was eluted, the oxidation peak of PMB reappeared at -0.27V in the formed MIP / PMB / P[Bvim]Br / rGO / AuNPs / AN, indicating that after SP was eluted from the electrode surface, a molecular imprinting cavity that can be used for electron transfer was formed. After MIP / PMB / P[Bvim]Br / rGO / AuNPs / AN recombined with SP, the signal of PMB decreased, which was attributed to the reoccupancy of the molecular imprinting cavity. As shown in the figure, Figure 3 and 4 As shown, the surface of P[Bvim]Br / SP / PMB / P[Bvim]Br / rGO / AuNPs / AN is relatively smooth, but when the template molecule SP is eluted from the electrode surface, the electrode surface becomes obviously rough.

[0038] Embodiment 2:

[0039] A method for preparing a molecular imprinting micro-electrochemical biosensor for detecting novel coronavirus SP comprises the following steps:

[0040] (1) Stainless steel acupuncture needles (AN) were used as substrate electrodes. The needles were polished and ultrasonically cleaned with ethanol and ultrapure water, respectively. AuNPs / AN were deposited in 1 mM chloroauric acid solution by -0.8 V constant potential method for 75 s. -1 rGO / AuNPs / AN was obtained by electrochemical reduction in GO solution at -1.6 V constant potential for 480 s.

[0041] (2) 0.0186 g MB and 0.0053 g [Bvim] Br were weighed and dissolved in 25 mL 0.1 M pH = 8.2 phosphate buffer solution, and ultrasonically dispersed uniformly. Cyclic voltammetry was performed in the potential range of -0.8 to 1.2 V at 20 mV s -1 The rate was scanned for 5 cycles to obtain PMB / P[Bvim]Br / rGO / AuNPs / AN.

[0042] (3) PMB / P[Bvim]Br / rGO / AuNPs / AN were prepared in a solution containing 20 μg mL -1 SP / PMB / P[Bvim]Br / rGO / AuNPs / AN was obtained and cyclic voltammetry was performed in a 20 mM [Bvim]Br buffer solution at a potential range of -0.5 to 1.2 V at 25 mV s -1 The rate was scanned for 3 cycles, and the formed P[Bvim]Br was used as the imprinted polymer to embed SP, obtaining P[Bvim]Br / SP / PMB / P[Bvim]Br / rGO / AuNPs / AN, which was eluted in a pH = 5.0 phosphate buffer solution containing 5% methanol for 20 min to obtain MIP / PMB / P[Bvim]Br / rGO / AuNPs / AN.

[0043] Embodiment 3:

[0044] A method for preparing a molecular imprinting micro-electrochemical biosensor for detecting novel coronavirus SP comprises the following steps:

[0045] (1) Stainless steel acupuncture needles (AN) were used as substrate electrodes. The needles were polished and ultrasonically cleaned with ethanol and ultrapure water, respectively. AuNPs / AN were deposited in 5 mM chloroauric acid solution by -0.8 V constant potential method for 125 s. -1rGO / AuNPs / AN was obtained by electrochemical reduction in GO solution at -1.2 V constant potential for 720 s. Figure 1 As shown, there is no redox peak of potassium ferrocyanide / potassium ferrocyanide on AN, while a pair of obvious redox peaks appear on AuNPs / AN, which is attributed to the good conductivity and large specific surface area of ​​AuNPs. Compared with AuNPs / AN, the redox peak current of potassium ferrocyanide / potassium ferrocyanide on rGO / AuNPs / AN is reduced, which is attributed to the poor conductivity of rGO compared with AuNPs.

[0046] (2) 0.0186 g MB and 0.0053 g [Bvim] Br were weighed and dissolved in 25 mL 0.1 M pH = 8.2 phosphate buffer solution, and ultrasonically dispersed uniformly. Cyclic voltammetry was performed in the potential range of -0.8 to 1.2 V at 100 mV s -1 The rate was scanned for 25 cycles to obtain PMB / P[Bvim]Br / rGO / AuNPs / AN.

[0047] (3) PMB / P[Bvim]Br / rGO / AuNPs / AN were added to a solution containing 100 μg mL -1 SP / PMB / P[Bvim]Br / rGO / AuNPs / AN were obtained and cyclic voltammetry was performed in a 20 mM [Bvim]Br buffer solution at a potential range of -0.5 to 1.2 V at 100 mV s -1 The rate was scanned for 8 cycles, and the formed P[Bvim]Br was used as the imprinted polymer to embed SP, obtaining P[Bvim]Br / SP / PMB / P[Bvim]Br / rGO / AuNPs / AN, which was eluted in a pH = 5.0 phosphate buffer solution containing 5% methanol for 60 min to obtain MIP / PMB / P[Bvim]Br / rGO / AuNPs / AN.

[0048] Embodiment 4:

[0049] The molecular imprinting micro electrochemical biosensor prepared in Example 1 was used to prepare a 100 μL molecular imprinting micro electrochemical biosensor in a solution containing SP (0 ng mL -1 ~1000ng mL -1 ) solution and incubate for 40 min. Figure 5 As shown in the figure, with the increase of SP concentration, the PMB oxidation peak current gradually decreases, which is attributed to the re-occupancy of SP on the electrode surface molecular imprinting cavity, inhibiting the electron transfer process of PMB.

[0050] When the prepared molecular imprinting micro-electrochemical biosensor detects SP, the peak current reduction rate is linearly related to the logarithm of the concentration. Figure 6As shown, the linear equation is ΔI / I0(μA)=0.0516lgC+0.0648(R 2 =0.9940), the linear range is 0.1ng mL -1 ~1000ng mL -1 , the detection limit was 0.038ng mL -1 The above results demonstrate that the molecular imprinting microelectrochemical biosensor can be used for the accurate determination of SP.

[0051] By detecting 1 μg mL -1 SP and the same concentration of bovine serum albumin, immunoglobulin G, immunoglobulin M, glutathione, L-tryptophan, L-phenylalanine and the mixture of the above substances and SP were used to study the specificity of molecular imprinting microelectrochemical biosensor. Figure 7 As shown, the change in ΔI / I0 value is much lower than the value of the sensor detecting SP alone, indicating that the molecular imprinting microelectrochemical biosensor has good specificity.

[0052] Comparative Example 1:

[0053] To further demonstrate the successful preparation of molecular imprinting micro-electrochemical biosensors, we also prepared non-molecular imprinting micro-electrochemical biosensors NIP / PMB / P[Bvim]Br / rGO / AuNPs / AN, and the operation steps were the same as steps (1) to (3) of Example 1, except that the template molecule SP was not added in step (3). Figure 8 As shown, the electrode did not show oxidation peak of PMB before and after elution and after rebinding, which was attributed to the lack of formation of imprinted cavity conducive to electron transfer. By comparing the molecular imprinting microelectrochemical biosensor with the non-molecular imprinting microelectrochemical biosensor, it was further proved that the molecular imprinting microelectrochemical biosensor can be used to accurately detect SP.

Claims

1. A molecular imprinting micro-electrochemical biosensor for detecting the spike protein of the new coronavirus, characterized in that: The method comprises sequentially modifying the surface of an AN electrode with gold nanoparticles, reducing graphene oxide, doping polymethylene blue with poly-1-vinyl-3-butylimidazolium bromide ionic liquid, and polyionic liquid imprinting polymer with a spike protein imprinting cavity to obtain MIP / PMB / P[Bvim]Br / rGO / AuNPs / AN.

2. A method for preparing a molecularly imprinted microelectrochemical biosensor for detecting the novel coronavirus spike protein according to claim 1, characterized in that: The AN electrode includes an acupuncture needle as the AN electrode.

3. The method for preparing a molecular imprinting micro-electrochemical biosensor for detecting the novel coronavirus spike protein according to claim 1, characterized in that: The following steps are involved: (1) An electrode was taken, the surface oxide layer was removed by grinding, and the electrode was ultrasonically cleaned with ethanol and ultrapure water, respectively. The electrode was placed in a chloroauric acid solution for constant potential deposition to obtain AuNPs / AN, and then placed in a graphene oxide solution for constant potential electrochemical reduction to obtain rGO / AuNPs / AN. The electrode was placed in a phosphate buffer solution mixed with methylene blue and 1-vinyl-3-butylimidazolium bromide ionic liquid, and ultrasonically dispersed uniformly. Cyclic voltammetry was used to scan the electrode to obtain PMB / P[Bvim]Br / rGO / AuNPs / AN. (2) PMB / P[Bvim]Br / rGO / AuNPs / AN was incubated in a solution containing spike protein, and cyclic voltammetry was used to scan in 1-vinyl-3-butylimidazolium bromide ionic liquid buffer solution. The formed P[Bvim]Br was used as an imprinting polymer to embed the spike protein to obtain P[Bvim]Br / SP / PMB / P[Bvim]Br / rGO / AuNPs / AN. The spike protein was eluted with a phosphate buffer solution containing methanol to obtain MIP / PMB / P[Bvim]Br / rGO / AuNPs / AN.

4. The method for preparing a molecular imprinting micro-electrochemical biosensor for detecting the novel coronavirus spike protein according to claim 3, characterized in that: The conditions for constant potential deposition to obtain AuNPs / AN in step (1) are: potential range -0.4V to -0.8V, deposition time 75 to 125s; the conditions for constant potential electrochemical reduction to obtain rGO / AuNPs / AN are: potential range -1.2V to -1.6V, deposition time 480 to 720s; the conditions for cyclic voltammetry scanning are: potential -0.8 to 1.2V, scan rate 25 to 100mV s -1 , the number of deposition circles is 5 to 25.

5. A method for preparing a molecular imprinting micro-electrochemical biosensor for detecting the novel coronavirus spike protein as claimed in claim 3, characterized in that: The conditions for obtaining AuNPs / AN by constant potential deposition in step (1) are: potential of -0.8 V, deposition time of 100 s; the conditions for obtaining rGO / AuNPs / AN by constant potential electrochemical reduction are: potential of -1.5 V, deposition time of 600 s; the conditions for cyclic voltammetry scanning are: potential of -0.8 to 1.2 V, scan rate of 50 mVs -1 , the number of deposition circles is 15.

6. A method for preparing a molecular imprinting micro-electrochemical biosensor for detecting the novel coronavirus spike protein according to claim 3, characterized in that: The concentration of the chloroauric acid solution in step (1) is 1 to 5 mM; the concentration of the graphene oxide solution is 1 to 5 mg mL -1 ; The pH of the phosphate buffer solution is 8.2; the molar ratio of the methylene blue to the 1-vinyl-3-butylimidazolium bromide ionic liquid is 1 to 5:

1.

7. A method for preparing a molecularly imprinted micro-electrochemical biosensor for detecting the spike protein of the new coronavirus as claimed in claim 3, characterized in that: The concentration of the chloroauric acid solution in step (1) is 2.5 mM; the concentration of the graphene oxide solution is 1 mg mL -1 ; The molar ratio of methylene blue to 1-vinyl-3-butylimidazolium bromide ionic liquid is 2:

1.

8. The method for preparing a molecular imprinting micro-electrochemical biosensor for detecting the novel coronavirus spike protein according to claim 3, characterized in that: The conditions for scanning by cyclic voltammetry in the 1-vinyl-3-butylimidazolium bromide ionic liquid buffer solution in step (2) are: potential -0.5 to 1.2 V, scanning rate 25 to 100 mV s -1 , the number of deposition circles is 3 to 8.

9. The method for preparing a molecular imprinting micro-electrochemical biosensor for detecting the novel coronavirus spike protein according to claim 3, characterized in that: The conditions for scanning by cyclic voltammetry in the 1-vinyl-3-butylimidazolium bromide ionic liquid buffer solution in step (2) are: potential -0.5 to 1.2 V, scanning rate 50 mV s -1 , the number of deposition circles is 5.

10. The method for preparing a molecular imprinting micro-electrochemical biosensor for detecting the novel coronavirus spike protein according to claim 3, characterized in that: The concentration of the solution containing the spike protein in step (2) is 20 to 100 μg mL -1 ; The phosphate buffer solution containing methanol is a phosphate buffer solution with a pH of 5.0 and containing 5% methanol, and the elution time is 20 to 60 minutes.

11. A method for preparing a molecular imprinting micro-electrochemical biosensor for detecting the novel coronavirus spike protein according to claim 3, characterized in that: The concentration of the solution containing the spike protein in step (2) is 50 μg mL -1 ; The elution time is 40min.

12. Use of the molecular imprinting microelectrochemical biosensor as described in claim 1 or 2 in detecting the novel coronavirus spike protein.

13. The detection method of molecular imprinting micro electrochemical biosensor according to claim 1 or 2, characterized in that: The molecular imprinting micro-electrochemical biosensor was placed in a solution containing or not containing the spike protein for an incubation time of 10 to 60 min, and the square wave voltammogram of the sensor was recorded in the potential range of -0.5 to 0 V to achieve the detection of SP.

14. The detection method according to claim 13, characterized in that: The incubation time is 30 min.

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