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CN-116297760-B - Surface molecular imprinting electrochemical biosensor for detecting novel coronavirus spike protein, and preparation method and application thereof

CN116297760BCN 116297760 BCN116297760 BCN 116297760BCN-116297760-B

Abstract

The invention relates to preparation and application of a surface molecular imprinting electrochemical biosensor for detecting novel coronavirus spike protein by taking Cu 7 S 4 -Au as a built-in signal probe, and belongs to the technical field of electrochemistry. The invention designs a poly-3-aminophenylboronic acid PAPBA imprinted polymer formed by sequentially modifying heptacopper tetrasulfide-gold nano particles, 4-mercaptophenylboronic acid, novel coronavirus spike protein S protein template molecules and polymerized 3-aminophenylboronic acid 3-APBA on the surface of an electrode, and eluting the novel coronavirus spike protein S protein template molecules by using an acidic eluent to obtain the SMI/4-MPBA/Cu 7 S 4 -Au/electrode of the surface molecular imprinting electrochemical biosensor. The surface molecular imprinting electrochemical biosensor can be used for detecting the spike protein of the novel coronavirus with high sensitivity, and provides a detection method for clinical diagnosis of the novel coronavirus.

Inventors

  • KONG YONG
  • LIU ZIXUAN
  • YIN ZHENGZHI
  • LI JUNYAO
  • CAI WENRONG
  • WU DATONG

Assignees

  • 常州大学

Dates

Publication Date
20260505
Application Date
20230221

Claims (13)

  1. 1. A surface molecular imprinting electrochemical biosensor for detecting novel coronavirus spike protein is characterized in that heptacopper tetrasulfide-gold nano particles, 4-mercaptophenylboronic acid, novel coronavirus spike protein S template molecules and poly 3-aminophenylboronic acid PAPBA imprinted polymer formed by polymerizing 3-aminophenylboronic acid 3-APBA are sequentially modified on the surface of an electrode, and after the novel coronavirus spike protein S template molecules are eluted by acid eluent, the surface molecular imprinting electrochemical biosensor SMI/4-MPBA/Cu 7 S 4 -Au/electrode is obtained.
  2. 2. The method for preparing a surface molecularly imprinted electrochemical biosensor for detecting novel coronavirus spike protein according to claim 1, comprising the following steps: (1) Dispersing Cu 7 S 4 -Au in ultrapure water to form a Cu 7 S 4 -Au solution, dripping the Cu 7 S 4 -Au solution on an electrode, and drying at room temperature to obtain a hepta-gold tetrasulfide nanoparticle/electrode, namely a Cu 7 S 4 -Au/electrode; (2) Weighing an ethanol solution of 4-mercaptophenylboronic acid, dripping the ethanol solution on the surface of the Cu 7 S 4 -Au/electrode, and drying at room temperature to obtain 4-mercaptophenylboronic acid/heptacopper tetrasulfide-Au nano particles/electrodes, namely 4-MPBA/Cu 7 S 4 -Au/electrodes; (3) Weighing 0.1M phosphate buffer solution containing S protein and having pH of 6.0-8.0, dripping the solution on the surface of the 4-MPBA/Cu 7 S 4 -Au/electrode, and performing incubation reaction to obtain the S protein/4-MPBA/Cu 7 S 4 -Au/electrode; (4) And (3) weighing a 0.1M phosphate buffer solution containing 3-aminophenylboric acid and sodium fluoride and having a pH of 6.0-8.0, dripping the solution on the surface of an S protein/4-MPBA/Cu 7 S 4 -Au/electrode, scanning in a scanning potential range of-0.2-0.9V to form a poly 3-aminophenylboric acid PAPBA imprinting polymerization layer, namely forming a PAPBA/S protein/4-MPBA/Cu 7 S 4 -Au/electrode, and placing the electrode in a 0.2M sulfuric acid solution to elute and remove the S protein, thereby obtaining the SMI/4-MPBA/Cu 7 S 4 -Au/electrode.
  3. 3. The method of claim 2, wherein the method of preparing Cu 7 S 4 -Au in step (1) comprises: Weighing polyvinylpyrrolidone, dissolving in a copper sulfate pentahydrate aqueous solution of 0.005-0.02M, adding a sodium hydroxide aqueous solution of 1.0-2.0M to generate blue precipitate immediately, stirring for the first time, adding an ascorbic acid aqueous solution of 0.05-0.2M to obtain a brick red solution, stirring for the second time, adding a thiourea aqueous solution of 0.1-0.3M, reacting, centrifuging, washing the precipitate with distilled water, and vacuum drying to obtain Cu 7 S 4 powder, adding Cu 7 S 4 powder into an AuNPs solution, stirring and mixing for the third time at room temperature, centrifuging, washing the precipitate with distilled water, and vacuum drying to obtain Cu 7 S 4 -Au.
  4. 4. The preparation method of claim 3, wherein the mass to volume ratio of the polyvinylpyrrolidone, the copper sulfate pentahydrate aqueous solution and the sodium hydroxide aqueous solution is 0.1-0.4 g:50-150 mL:20-30 mL, and the time for the first stirring is 0.5-2 min; The mass and volume ratio of the polyvinylpyrrolidone to the ascorbic acid aqueous solution is 0.1-0.4 g:20-30 mL, and the second stirring time is 10-20 min; The mass and volume ratio of the polyvinylpyrrolidone to the thiourea aqueous solution is 0.1-0.4 g:2-10 mL.
  5. 5. The preparation method according to claim 3, wherein the reaction conditions are that the reaction temperature is 80-100 ℃ and the reaction time is 5-8 hours; The temperature of the vacuum drying is 50-80 ℃; the mass-volume ratio of the Cu7S4 powder to the AuNPs solution is 5-15 mg:20-50 mL; and the time of the third stirring is 3-6 hours.
  6. 6. The method of claim 2, wherein the electrode in step (1) comprises a glassy carbon electrode, a metal electrode or a screen-printed carbon electrode, and the concentration of the Cu 7 S 4 -Au solution is 2-4 mg mL "1.
  7. 7. The preparation method of claim 2, wherein the concentration of the ethanol solution of 4-mercaptophenylboronic acid in the step (2) is 0.05-0.2M, and the volume ratio of the Cu 7 S 4 -Au solution to the ethanol solution of 4-mercaptophenylboronic acid is 5-15:5-15.
  8. 8. The method according to claim 7, wherein the concentration of the ethanol solution of 4-mercaptophenylboronic acid is 0.1M.
  9. 9. The preparation method of claim 2, wherein in the step (3), the concentration of S protein in the 0.1M phosphate buffer solution containing the S protein and having a pH of 6.0-8.0 is 5-15 mug mL –1 , and the incubation reaction is carried out at an incubation temperature of 1-5 ℃ for 0.5-2 hours.
  10. 10. The method of claim 9, wherein the incubation time is 1h.
  11. 11. The method according to claim 2, wherein the volume of the 0.1M phosphate buffer solution containing 3-aminophenylboronic acid and sodium fluoride in the step (4) is 50 to 150. Mu.L, the concentration of 3-aminophenylboronic acid is 20 to 60mM, the concentration of sodium fluoride is 20 to 60mM, the number of turns of the scan is 10 to 30, the volume of the 0.2M sulfuric acid solution is 50 to 150. Mu.L, and the elution time is 10 to 30 minutes.
  12. 12. The method of claim 11, wherein the number of turns of the scan is 20 and the elution time is 20 minutes.
  13. 13. Use of the surface molecularly imprinted electrochemical biosensor of claim 1 for detecting novel coronavirus spike proteins.

Description

Surface molecular imprinting electrochemical biosensor for detecting novel coronavirus spike protein, and preparation method and application thereof Technical Field The invention belongs to the technical field of electrochemistry, and particularly relates to preparation and application of a surface molecular imprinting electrochemical biosensor which uses Cu 7S4 -Au as a built-in signal probe for detecting novel coronavirus spike protein. Technical Field At present, the detection of acute respiratory syndrome coronavirus type II (SARS-CoV-2) is mainly accomplished by reverse transcription-polymerase chain reaction (RT-PCR) technology. The technology can accurately identify the ribonucleic acid (RNA) of the virus, but has the defects of long analysis time, high instrument cost and high requirement on detection personnel, and has limited application. Antigen detection methods based on SARS-CoV-2 specific antigens, such as nucleocapsid proteins (N proteins) and spike proteins (S proteins), etc., are of increasing interest, with S proteins being perhaps the most valuable antigen biomarkers. The electrochemical biosensor has the advantages of high sensitivity, low cost, strong durability and the like, and provides a reliable solution for COVID-19 clinical diagnosis. In addition, the molecular imprinting electrochemical biosensor combines the high specificity of the molecular imprinting polymer with the biosensor, and has great application potential in biomedical analysis. S protein is a typical biomacromolecule, and is difficult to be used as a template molecule of a traditional bulk blotting, and a suitable method needs to be found for molecular blotting of protein biomacromolecules, and the surface molecular blotting (SMI) technology can just solve the problem. Copper sulfide nanocrystals (Cu xSy) contain a large number of stable and metastable components due to the 3d electrons. The heptacopper tetrasulfide (Cu 7S4) has high conductivity, low toxicity and strong adsorption capacity, can be used as a signal probe, and has good application value in the field of electrochemical sensing. The invention utilizes gold nano particles (AuNPs) to be loaded on the surface of Cu 7S4 through an Au-S bond, thereby improving the electron transfer rate and the detection sensitivity. Therefore, cu 7S4(Cu7S4 -Au loaded with AuNPs) is a good signaling probe in the field of biosensing. Disclosure of Invention Aiming at the problems in the prior art, the invention aims to provide a surface molecular imprinting electrochemical biosensor which uses Cu 7S4 -Au as a built-in signal probe for detecting novel coronavirus spike protein, and a preparation method and application thereof. The invention utilizes boric acid affinity to form cyclic ester bond to fix new coronavirus spike protein template molecules, then adopts cyclic voltammetry to polymerize 3-aminophenylboronic acid to form a imprinting polymerization layer, and utilizes boric acid ester bond to dissociate in acid solution to remove spike protein template molecules so as to obtain the surface molecular imprinting electrochemical biosensor. The surface molecular imprinting electrochemical biosensor can be used for detecting the spike protein of the novel coronavirus with high sensitivity, and provides a detection method for clinical diagnosis of the novel coronavirus. In order to achieve the above purpose, the present invention adopts the following technical scheme: In a first aspect, the invention provides a surface molecular imprinting electrochemical biosensor for detecting novel coronavirus spike protein, wherein heptacopper tetrasulfide-gold nanoparticles, 4-mercaptophenylboronic acid, novel coronavirus spike protein S template molecules and poly-3-aminophenylboronic acid PAPBA imprinted polymer formed by polymerizing 3-aminophenylboronic acid 3-APBA are sequentially modified on the surface of an electrode, and after the novel coronavirus spike protein S template molecules are eluted by an acidic eluent, the surface molecular imprinting electrochemical biosensor SMI/4-MPBA/Cu 7S4 -Au/electrode is obtained. In a second aspect, the present invention provides a method for preparing a surface molecularly imprinted electrochemical biosensor for detecting novel coronavirus spike proteins, comprising the steps of: (1) Dispersing Cu 7S4 -Au in ultrapure water to form a Cu 7S4 -Au solution, dripping the Cu 7S4 -Au solution on a screen printing carbon electrode, and drying at room temperature to obtain a hepta-gold tetrasulfide nanoparticle/electrode, namely a Cu 7S4 -Au/electrode; (2) Weighing an ethanol solution of 4-mercaptophenylboronic acid, dripping the ethanol solution on the surface of the Cu 7S4 -Au/electrode, and drying at room temperature to obtain 4-mercaptophenylboronic acid/heptacopper tetrasulfide-Au nano particles/electrodes, namely 4-MPBA/Cu 7S4 -Au/electrodes; (3) Weighing 0.1M phosphate buffer solution containing S protein and having pH of 6.0-8.0, dripping the