Preparation method and application of ultra-sensitive electrochemical immunosensor for detecting MX1 protein based on nano composite material

Abstract

The invention relates to the technical field of electrochemical immunosensors, in particular to a preparation method and application of an ultra-sensitive electrochemical immunosensor for detecting MX1 protein based on a nano composite material, and the preparation method comprises the following steps: S1, pretreating a glassy carbon electrode; s2, modifying with thiolated graphene oxide; s3, gold nanoparticle modification; s4, carrying out antibody immobilization; and S5, carrying out bovine serum albumin sealing treatment. The kit has ultrahigh sensitivity and high specificity, the detection method is simple, convenient and rapid, multiple signal amplification mechanisms and wide clinical application prospects are achieved, and early diagnosis and clinical monitoring of virus infection are facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of electrochemical immunosensors, and in particular to a preparation method and application of an ultra-sensitive electrochemical immunosensor for MX1 ​​protein detection based on a nanocomposite material. Background Art

[0002] In the biomedical field, interferon-induced MX1 protein is an important viral infection marker, and its detection is of great significance for early diagnosis of viral infection, monitoring of disease progression, and evaluation of treatment efficacy. In particular, in pediatric respiratory diseases (such as influenza virus, respiratory syncytial virus, and adenovirus infection), MxA protein detection can provide early infection information before symptoms appear, which helps to initiate antiviral treatment in a timely manner. At the same time, the etiology of pediatric respiratory infections is complex, including viral, bacterial, and other pathogen infections. As a specific viral infection marker, MxA protein can help distinguish viral from bacterial respiratory infections and avoid unnecessary abuse of antibiotics. Through the detection of MxA protein levels, different types of infections can be effectively distinguished clinically, targeted treatment can be guided, and diagnostic accuracy can be improved. Portable electrochemical sensors or microfluidic paper-based analytical devices based on MxA protein can achieve rapid on-site detection, especially in environments with limited medical resources, providing convenient diagnostic tools. This point-of-care diagnostic device can obtain test results in a short time (such as within a few minutes), providing timely information support for the diagnosis and treatment of children with acute respiratory infections.

[0003] However, the existing MX1 protein detection methods have the following major problems in terms of sensitivity, specificity and ease of operation: (1) The existing MX1 protein detection methods (such as ELISA, Western Blot and qRT-PCR) have obvious limitations in terms of sensitivity, specificity and ease of operation. ELISA detection requires complex sample processing and multi-step experiments, making it difficult to achieve sensitive detection of low-concentration MX1 proteins; Western Blot operations are cumbersome and time-consuming, making it difficult to quickly apply in clinical practice; although qRT-PCR can indirectly detect MX1 mRNA levels, it cannot reflect the actual expression of the protein and has high requirements for sample processing. In addition, traditional electrochemical immunosensors are also insufficient in sensitivity and specificity due to problems such as small electrode surface area, insufficient antibody fixation, and nonspecific adsorption. (2) Based on the above limitations, the present invention proposes an ultrasensitive electrochemical immunosensor based on a nanocomposite material for achieving high-sensitivity and high-specificity detection of MX1 proteins. By using a thiolated graphene oxide modified electrode with a high active surface area, combined with a streptavidin-biotin system and a sandwich structure with multiple signal amplification mechanisms, the sensor can significantly increase the fixed amount of antibodies and the signal amplification effect, achieving a minimum detection limit of fg / mL. At the same time, the optimized electrode surface modification strategy and simplified operation process significantly improve the detection accuracy and ease of operation of the sensor in complex biological matrices, meeting the actual needs of early diagnosis of viral infection and disease monitoring, and has good clinical application prospects. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for preparing and applying an ultrasensitive electrochemical immunosensor for detecting MX1 protein based on nanocomposite materials. By introducing a variety of nanomaterials and multiple signal amplification mechanisms, the problems of insufficient sensitivity, complex operation, limited specificity and poor signal amplification effect in the prior art are solved, and ultrasensitive, high specificity and rapid detection of MX1 protein are achieved, thereby providing an effective detection method for early diagnosis of viral infection and disease monitoring.

[0005] To achieve the above object, the present invention provides the following technical solutions: A method for preparing an ultrasensitive electrochemical immunosensor for detecting MX1 protein based on a nanocomposite material comprises the following steps: S1. Pretreatment of glassy carbon electrode: Use 0.05-0.07 µm alumina slurry on a polishing cloth to polish the surface of the glassy carbon electrode until the surface is smooth and mirror-like, then repeatedly clean the electrode surface with deionized water, and then clean it with deionized water and anhydrous ethanol in an ultrasonic cleaner for 5-10 minutes to remove surface residues, and blow dry the electrode surface under a nitrogen flow to obtain a pretreated glassy carbon electrode; S2. Thiolated graphene oxide modification: applying a 1-3 mg / mL thiolated graphene oxide dispersion droplet on the surface of a clean pretreated glassy carbon electrode, and drying at room temperature to form a uniform thiolated graphene oxide film to obtain a thiolated graphene oxide modified electrode; S3. Gold nanoparticle modification: 0.1-1 mg / mL streptavidin-coated gold nanoparticle solution was dropped onto the surface of the thiolated graphene oxide modified electrode, incubated at room temperature for 1-2 h to allow the gold nanoparticles to be evenly distributed on the electrode surface, and the electrode was rinsed with deionized water to remove unbound gold nanoparticles to obtain a gold nanoparticle modified electrode; S4. Antibody immobilization: A biotinylated anti-MX1 monoclonal antibody solution with a concentration of 1-3 µg / mL was drop-coated on the surface of the gold nanoparticle-modified electrode, incubated at room temperature for 1-2 h, and the electrode surface was rinsed with PBS buffer to remove unbound antibodies, thereby obtaining an electrode modified with antibodies; S5. Bovine serum albumin blocking treatment: immerse the electrode modified with the antibody in a bovine serum albumin solution with a mass fraction of 1-5% and incubate it at room temperature for 0.5-1h to block the active sites on the electrode surface not occupied by the antibody. Gently wash the electrode surface with PBS buffer to remove unbound bovine serum albumin and antibody solution residues to obtain an ultrasensitive electrochemical immunosensor for MX1 ​​protein detection based on nanocomposite materials.

[0006] Preferably, the pH of the PBS buffer used in the detection method is 7.4-7.8.

[0007] Preferably, in step S3, the preparation process of the streptavidin-coated gold nanoparticles is as follows: S301. A 0.01-0.03% chloroauric acid solution is heated to boiling, and a sodium citrate solution is added and stirred and heated for 10-15 minutes. The solution changes color to wine red and is cooled to room temperature to obtain gold nanoparticles; S302. Centrifuge and wash the gold nanoparticles with deionized water, then mix with 1-3 mg / mL polyethylene glycol-thiol solution, and stir at room temperature for 1.5-2h for surface modification to obtain polyethylene glycol-thiol solution-modified gold nanoparticles; S303. Activate 1-2 mg / mL of streptavidin solution with 1-2 mg / mL of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-2 mg / mL of N-hydroxysuccinimide in 0.1-0.3 M MES buffer, and react for 0.5-1 h to obtain activated streptavidin; S304. Mixing the activated streptavidin with the gold nanoparticles modified with polyethylene glycol-thiol solution, stirring at room temperature for 1.5-2h, and coupling to form streptavidin-coated gold nanoparticles; S305. Add 1-3% BSA solution to block the unreacted active sites and incubate for 0.5-1h. Finally, centrifuge to remove unbound streptavidin and BSA, resuspend the streptavidin-coated gold nanoparticles in PBS buffer to a final concentration of 0.1-1 mg / mL, and store at 2-4°C to obtain streptavidin-coated gold nanoparticles.

[0008] Preferably, in step S301, the concentration of the sodium citrate solution is 0.01-0.03 M.

[0009] Preferably, in step S302, the mass ratio of the gold nanoparticles to the polyethylene glycol-thiol solution is 1:1.

[0010] An ultrasensitive electrochemical immunosensor for MX1 ​​protein detection based on nanocomposite materials and its application in the detection of MX1 protein.

[0011] Preferably, the application method comprises the following steps: S101. MX1 protein capture: The nanocomposite-based ultrasensitive electrochemical immunosensor for MX1 ​​protein detection was immersed in a solution containing the MX1 protein to be detected, incubated for 0.5-1 h, and washed with PBS buffer to obtain an electrode that removed unbound MX1 protein; S102. Formation of sandwich structure: 1-3 mg / mL silver nanoparticle-anti-MxA monoclonal antibody B-HRP solution is added dropwise to the electrode surface from which unbound MX1 protein is removed, and incubated for 0.5-1 h to form a sandwich structure. The electrode surface is gently washed with PBS buffer to remove unbound complexes, ensuring that the signal probe and the target protein form a stable sandwich structure, thereby obtaining an electrode with a sandwich structure. S103. Electrochemical signal amplification and detection: The sandwich structure electrode was immersed in an electrolyte solution of 1-3 mM hydroquinone and 1-3 mM hydrogen peroxide. The electrochemical signal was generated by horseradish peroxidase catalytic reaction. The current response of the electrode was measured using differential pulse voltammetry. The current peak was proportional to the concentration of MX1 protein. Quantitative analysis was performed based on the standard curve.

[0012] Preferably, in step S102, the preparation steps of the silver nanoparticle-anti-MxA monoclonal antibody B-HRP solution are as follows: S201. placing a 0.5 mg / mL silver nanoparticle suspension in a centrifuge, discarding the supernatant, then resuspending the particles with deionized water, and repeating the centrifugation and washing steps 3 times to remove impurities and stabilizers remaining on the surface, and finally resuspending the washed silver nanoparticles in 0.1 M MES buffer with a pH of 6.0 to obtain a silver nanoparticle suspension; S202. N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are added to the silver nanoparticle suspension to a final concentration of 2 mM and 5 mM, respectively. The mixed solution is gently stirred at room temperature for 20-30 minutes to activate the carboxyl functional groups on the surface of the silver nanoparticles. After the activation is completed, the mixture is centrifuged again to remove the unreacted N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and the silver nanoparticles are resuspended in MES buffer. The above washing steps are repeated twice, and the final silver nanoparticles are suspended in MES buffer for later use; S203. diluting the anti-MxA monoclonal antibody B to 1-3 mg / mL with MES buffer, and then adding the diluted antibody to the activated silver nanoparticle suspension, the mass ratio of the antibody to the silver nanoparticles is 1:10, the mixed solution is gently stirred at room temperature for 1-2 hours and then centrifuged to remove unbound antibodies, and the silver nanoparticle-antibody complex is resuspended with PBS buffer, and the centrifugation and washing steps are repeated 3 times, and finally the complex is resuspended in PBS buffer; S204. Dilute horseradish peroxidase with MES buffer to 1 mg / mL, and add N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to the horseradish peroxidase solution at final concentrations of 2 mM and 5 mM, respectively. Gently stir at room temperature for 20-30 minutes to activate the carboxyl groups on the horseradish peroxidase molecules. Then add the activated horseradish peroxidase solution to the silver nanoparticle-antibody complex suspension, and gently stir for 3-4 hours to ensure that the horseradish peroxidase and the active sites on the silver nanoparticle-antibody complex undergo covalent coupling reaction. After the coupling reaction is completed, centrifuge to remove unbound horseradish peroxidase, resuspend the silver nanoparticle-antibody-horseradish peroxidase complex with PBS buffer, repeat the centrifugation and washing steps 3 times, and resuspend the final complex in PBS buffer; S205. Add the silver nanoparticle-antibody-horseradish peroxidase complex suspension to a bovine serum albumin solution with a mass fraction of 1-5%, gently stir at room temperature for 0.5-1h, and after sealing, centrifuge again to remove excess bovine serum albumin, resuspend the complex with PBS buffer, and finally store it in a refrigerator at 2-4°C to avoid freezing to prevent particle aggregation or loss of antibody activity. After the above steps are completed, take out a portion and mix it with deionized water to obtain a silver nanoparticle-anti-MxA monoclonal antibody B-HRP solution.

[0013] Preferably, the speed of the centrifuge during the preparation of the silver nanoparticle-anti-MxA monoclonal antibody B-HRP solution is 10000-12000 rpm, and the time is 15-20 min.

[0014] Preferably, in step S204, the mass ratio of the activated horseradish peroxidase solution to the silver nanoparticle-antibody complex suspension is 1:10.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. Ultra-high sensitivity: The electrode is modified with thiolated graphene oxide (T-GO) to increase the active surface area and conductivity of the electrode, significantly increase the fixed amount of antibodies and protein capture efficiency. Combined with the streptavidin-biotin system and sandwich structure signal probe, multiple signal amplification is achieved, and the minimum detection limit can reach the fg / mL level, which significantly improves the detection sensitivity and can detect low concentrations of MX1 protein in the early stages of viral infection.

[0016] 2. High specificity: Through the use of streptavidin-coated gold nanoparticles and biotinylated specific anti-MX1 monoclonal antibodies, nonspecific adsorption is reduced to ensure the specificity of detection. At the same time, the modification strategy and protective layer design of the electrode surface are optimized to further reduce background signal interference and improve detection accuracy in complex biological matrices.

[0017] 3. Simple method and rapid detection: The detection method is relatively simple. The entire detection process can be completed by modifying the electrode once, eliminating the cumbersome sample processing steps and complex experimental equipment required in traditional methods. The detection time is greatly shortened, which is suitable for rapid on-site detection and immediate diagnosis.

[0018] 4. Multiple signal amplification mechanism: The multiple signal amplification mechanism of the sandwich structure is adopted, and the synergistic effect of silver nanoparticles and HRP complexes can significantly amplify the electrochemical signal and improve the detection sensitivity and accuracy. This method effectively overcomes the limitations of the single signal amplification mechanism of traditional electrochemical sensors.

[0019] 5. Broad clinical application prospects: The present invention can be used for rapid screening of early viral infections, disease monitoring and evaluation of treatment effects, especially in the fields of public health and clinical diagnosis, and has broad application prospects. At the same time, the technology platform can be expanded to the detection of other proteins or biomarkers, and has good versatility and scalability. In summary, the present invention is superior to the existing technology in terms of sensitivity, specificity, ease of operation and signal amplification mechanism, and provides an innovative solution for the rapid and ultra-sensitive detection of MX1 protein, which is conducive to the early diagnosis and clinical monitoring of viral infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a step diagram of the preparation method of the ultrasensitive electrochemical immunosensor for MX1 ​​protein detection based on nanocomposite materials of the present invention; Figure 2 Schematic diagram of the structure of gold nanoparticles coated with streptavidin of the present invention; Figure 3 Schematic diagram of the structure of the biotinylated anti-MX1 monoclonal antibody A (mAb) of the present invention; Figure 4 This is a schematic diagram of the structure of silver nanoparticle-anti-MxA monoclonal antibody B-HRP of the present invention; Figure 5 The structural hierarchical relationship of the glassy carbon electrode surface modified with thiolated graphene oxide, gold nanoparticles and biotinylated antibodies in sequence; Figure 6 This is a linear curve diagram of the response current and the MX1 protein concentration when the ultrasensitive electrochemical immunosensor for MX1 ​​protein detection based on the nanocomposite material prepared in Example 1 of the present invention is used, wherein the ordinate is the response current value and the abscissa is the MX1 protein concentration. DETAILED DESCRIPTION

[0021] The present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] See also Figure 1-6 , the present invention provides a technical solution: Example 1 Preparation of modified electrode: S1. Pretreatment of glassy carbon electrode: Use 0.05µm alumina slurry on a polishing cloth to polish the surface of the glassy carbon electrode until the surface is smooth and mirror-like, then repeatedly clean the electrode surface with deionized water, and clean it in an ultrasonic cleaner with deionized water and anhydrous ethanol for 5 minutes in turn to remove surface residues, and blow dry the electrode surface under a nitrogen flow to obtain a pretreated glassy carbon electrode.

[0023] S2. Thiolated graphene oxide modification: Apply 1 mg / mL of thiolated graphene oxide dispersion droplets on the surface of the clean pretreated glassy carbon electrode and dry at room temperature until a uniform thiolated graphene oxide film is formed to obtain a thiolated graphene oxide modified electrode.

[0024] S3. Gold nanoparticle modification: 0.1 mg / mL streptavidin-coated gold nanoparticle solution was dropped onto the surface of the thiolated graphene oxide modified electrode and incubated at room temperature for 1 h to allow the gold nanoparticles to be evenly distributed on the electrode surface. The electrode was rinsed with deionized water to remove unbound gold nanoparticles to obtain a gold nanoparticle modified electrode.

[0025] S4. Antibody immobilization: 1 µg / mL biotinylated anti-MX1 monoclonal antibody solution was dropped onto the gold nanoparticle modified electrode surface and incubated at room temperature for 1 h. The electrode surface was rinsed with PBS buffer at pH 7.4 to remove unbound antibodies and obtain an electrode modified with antibodies.

[0026] S5. Bovine serum albumin blocking treatment: The electrode modified with the antibody was immersed in a 1% bovine serum albumin solution and incubated at room temperature for 0.5 h to block the active sites on the electrode surface that were not occupied by the antibody. The electrode surface was gently washed with a PBS buffer with a pH of 7.4 to remove the unbound bovine serum albumin and antibody solution residues, and an ultrasensitive electrochemical immunosensor for MX1 ​​protein detection based on nanocomposite materials was obtained.

[0027] Electrochemical detection: S6. MX1 protein capture: The nanocomposite-based ultrasensitive electrochemical immunosensor for MX1 ​​protein detection was immersed in a solution containing the MX1 protein to be detected, incubated for 0.5 h, and washed with PBS buffer at pH 7.4 to obtain an electrode that removed unbound MX1 protein.

[0028] S7. Sandwich structure formation: 1 mg / mL silver nanoparticle-anti-MxA monoclonal antibody B-HRP solution was added dropwise to the electrode surface from which unbound MX1 protein was removed, and incubated for 0.5 h to form a sandwich structure. The electrode surface was gently washed with PBS buffer at pH 7.4 to remove unbound complexes and ensure that the signal probe and the target protein formed a stable sandwich structure, thereby obtaining an electrode with a sandwich structure.

[0029] S8. Electrochemical signal amplification and detection: The electrodes forming the sandwich structure were immersed in an electrolyte solution of 1 mM hydroquinone and 1 mM hydrogen peroxide, and the electrochemical signal was generated by HRP catalytic reaction. The current response of the electrode was measured using differential pulse voltammetry. The current peak was proportional to the concentration of MX1 protein, and quantitative analysis was performed based on the standard curve.

[0030] The preparation process of the streptavidin-coated gold nanoparticles is as follows: S301. Heat 0.01% chloroauric acid solution to boiling, add 0.01 M sodium citrate solution, continue stirring and heating for 10 min, the solution color turns to wine red and then cools to room temperature to obtain gold nanoparticles; S302. Centrifuge and wash the gold nanoparticles with deionized water, then mix with 1 mg / mL polyethylene glycol-thiol solution in a volume ratio of 1:1, and stir at room temperature for 1.5 h for surface modification to obtain polyethylene glycol-thiol solution-modified gold nanoparticles; S303. Activate 1 mg / mL streptavidin solution with 1 mg / mL EDC and 1-mg / mL NHS in 0.1 M MES buffer for 0.5 h to obtain activated streptavidin; S304. Mixing the activated streptavidin with the gold nanoparticles modified with polyethylene glycol-thiol solution, stirring at room temperature for 1.5 h, and coupling to form streptavidin-coated gold nanoparticles; S305. Add 1% BSA solution to block the unreacted active sites and incubate for 0.5 h. Finally, centrifuge to remove unbound streptavidin and BSA, resuspend the streptavidin-coated gold nanoparticles in PBS buffer to a final concentration of 0.1 mg / mL and store at 2°C.

[0031] The preparation steps of the silver nanoparticle-anti-MxA monoclonal antibody B-HRP solution are as follows: S701. Place the 0.5 mg / mL silver nanoparticle suspension in a centrifuge and centrifuge at 10,000 rpm for 15 min, and discard the supernatant. Then resuspend the particles with deionized water, and repeat the centrifugation and washing steps 3 times to remove impurities and stabilizers remaining on the surface. The washed silver nanoparticles are finally resuspended in 0.1 M, pH 6.0 MES buffer to obtain a silver nanoparticle suspension.

[0032] S702. Add N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the silver nanoparticle suspension at final concentrations of 2 mM and 5 mM, respectively. The mixed solution was gently stirred at room temperature for 20 minutes to activate the carboxyl functional groups on the surface of the silver nanoparticles. After activation, centrifuge again at 10,000 rpm for 15 minutes to remove unreacted EDC and NHS, and resuspend the silver nanoparticles with MES buffer. Repeat the above washing steps twice, and suspend the final silver nanoparticles in MES buffer for use.

[0033] S703. Anti-MxA monoclonal antibody B was diluted to 1 mg / mL with MES buffer, and then the diluted antibody was added to the activated silver nanoparticle suspension, with a mass ratio of antibody to silver nanoparticles of 1:10. The mixed solution was gently stirred at room temperature for 1 hour, and then centrifuged at 10,000 rpm for 15 minutes to remove unbound antibodies, and the silver nanoparticle-antibody complex was resuspended with PBS buffer at pH 7.4, and the centrifugation and washing steps were repeated 3 times, and finally the complex was resuspended in PBS buffer.

[0034] S704. Horseradish peroxidase (HRP) was diluted to 1 mg / mL with MES buffer, and EDC and NHS were added to the HRP solution with final concentrations of 2 mM and 5 mM, respectively, and gently stirred for 20 minutes at room temperature to activate the carboxyl groups on the HRP molecules. The activated HRP solution was then added to the silver nanoparticle-antibody complex suspension at a mass ratio of 1:10, and gently stirred for 3 hours to ensure that HRP covalently coupled with the active sites on the silver nanoparticle-antibody complex. After the coupling reaction was completed, the unbound HRP was removed by centrifugation at 10,000 rpm for 15 minutes, and the silver nanoparticle-antibody-HRP complex was resuspended with PBS buffer at pH 7.4. The centrifugation and washing steps were repeated 3 times, and the final complex was resuspended in PBS buffer.

[0035] S705. Add the silver nanoparticle-antibody-HRP complex suspension to a 1% bovine serum albumin solution and stir gently at room temperature for 0.5h. After blocking, centrifuge again at 10,000rpm for 15min to remove excess bovine serum albumin, resuspend the complex with PBS buffer, and finally store it in a refrigerator at 2°C to avoid freezing to prevent particle aggregation or loss of antibody activity. After the above steps are completed, take out a portion and mix it with deionized water to obtain a silver nanoparticle-anti-MxA monoclonal antibody B-HRP solution.

[0036] Example 2 Preparation of modified electrode: S1. Pretreatment of glassy carbon electrode: Use 0.07 µm alumina slurry on a polishing cloth to polish the surface of the glassy carbon electrode until the surface is smooth and mirror-like, then repeatedly clean the electrode surface with deionized water, and clean it with deionized water and anhydrous ethanol in an ultrasonic cleaner for 10 minutes in turn to remove surface residues, and blow dry the electrode surface under a nitrogen flow to obtain a pretreated glassy carbon electrode.

[0037] S2. Thiolated graphene oxide modification: 3 mg / mL of thiolated graphene oxide dispersion was dropwise applied on the surface of the clean pretreated glassy carbon electrode and dried at room temperature until a uniform thiolated graphene oxide film was formed to obtain a thiolated graphene oxide modified electrode.

[0038] S3. Gold nanoparticle modification: 1 mg / mL streptavidin-coated gold nanoparticle solution was dropped onto the surface of the thiolated graphene oxide modified electrode and incubated at room temperature for 2 h to allow the gold nanoparticles to be evenly distributed on the electrode surface. The electrode was rinsed with deionized water to remove unbound gold nanoparticles to obtain a gold nanoparticle modified electrode.

[0039] S4. Antibody immobilization: 3 µg / mL biotinylated anti-MX1 monoclonal antibody solution was dropped onto the gold nanoparticle modified electrode surface and incubated at room temperature for 2 h. The electrode surface was rinsed with PBS buffer at pH 7.8 to remove unbound antibodies and obtain an electrode modified with antibodies.

[0040] S5. Bovine serum albumin blocking treatment: The electrode modified with the antibody was immersed in a 5% bovine serum albumin solution and incubated at room temperature for 1 hour to block the active sites on the electrode surface that were not occupied by the antibody. The electrode surface was gently washed with a PBS buffer with a pH of 7.8 to remove the unbound bovine serum albumin and antibody solution residues, and an ultrasensitive electrochemical immunosensor for MX1 ​​protein detection based on nanocomposite materials was obtained.

[0041] Electrochemical detection: S6. MX1 protein capture: The nanocomposite-based ultrasensitive electrochemical immunosensor for MX1 ​​protein detection was immersed in a solution containing the MX1 protein to be detected, incubated for 1 h, and washed with PBS buffer at pH 7.8 to obtain an electrode that removes unbound MX1 protein.

[0042] S7. Sandwich structure formation: 3 mg / mL silver nanoparticle-anti-MxA monoclonal antibody B-HRP solution was added dropwise to the electrode surface from which unbound MX1 protein was removed, and incubated for 1 hour to form a sandwich structure. The electrode surface was gently washed with PBS buffer at pH 7.8 to remove unbound complexes and ensure that the signal probe and the target protein formed a stable sandwich structure, thereby obtaining an electrode with a sandwich structure.

[0043] S8. Electrochemical signal amplification and detection: The sandwich structure electrode was immersed in an electrolyte solution of 3 mM hydroquinone and 3 mM hydrogen peroxide, and the electrochemical signal was generated by HRP catalytic reaction. The current response of the electrode was measured using differential pulse voltammetry. The current peak was proportional to the concentration of MX1 protein, and quantitative analysis was performed according to the standard curve.

[0044] The preparation process of the streptavidin-coated gold nanoparticles is as follows: S301. Heat 0.03% chloroauric acid solution to boiling, add 0.03 M sodium citrate solution, continue stirring and heating for 15 min, the solution color turns to wine red and then cools to room temperature to obtain gold nanoparticles; S302. Centrifuge and wash the gold nanoparticles with deionized water, then mix with 3 mg / mL polyethylene glycol-thiol solution at a volume ratio of 1:1, and stir at room temperature for 2 h to perform surface modification, thereby obtaining polyethylene glycol-thiol solution-modified gold nanoparticles; S303. 2 mg / mL of EDC and 2 mg / mL of NHS were added to activate 2 mg / mL of streptavidin solution in 0.3 M MES buffer and reacted for 1 h to obtain activated streptavidin; S304. Mixing the activated streptavidin with the gold nanoparticles modified with polyethylene glycol-thiol solution, stirring at room temperature for 2 h, and coupling to form streptavidin-coated gold nanoparticles; S305. Add 3% BSA solution to block the unreacted active sites and incubate for 1 hour. Finally, centrifuge to remove unbound streptavidin and BSA, resuspend the streptavidin-coated gold nanoparticles in PBS buffer to a final concentration of 1 mg / mL and store at 4°C.

[0045] The preparation steps of the silver nanoparticle-anti-MxA monoclonal antibody B-HRP solution are as follows: S701. Place the 0.5 mg / mL silver nanoparticle suspension in a centrifuge and centrifuge at 12,000 rpm for 20 minutes, and discard the supernatant. Then resuspend the particles with deionized water, and repeat the centrifugation and washing steps 3 times to remove impurities and stabilizers remaining on the surface. The washed silver nanoparticles are finally resuspended in 0.1 M, pH 6.0 MES buffer to obtain a silver nanoparticle suspension.

[0046] S702. Add N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the silver nanoparticle suspension at final concentrations of 2 mM and 5 mM, respectively. The mixed solution was gently stirred at room temperature for 30 minutes to activate the carboxyl functional groups on the surface of the silver nanoparticles. After activation, centrifuge again at 12,000 rpm for 20 minutes to remove unreacted EDC and NHS, and resuspend the silver nanoparticles with MES buffer. Repeat the above washing steps twice, and suspend the final silver nanoparticles in MES buffer for use.

[0047] S703. Anti-MxA monoclonal antibody B was diluted to 3 mg / mL with MES buffer, and then the diluted antibody was added to the activated silver nanoparticle suspension, with a mass ratio of antibody to silver nanoparticles of 1:10. The mixed solution was gently stirred at room temperature for 2 hours, and then centrifuged at 12000 rpm for 20 minutes to remove unbound antibodies, and the silver nanoparticle-antibody complex was resuspended with PBS buffer at pH 7.8, and the centrifugation and washing steps were repeated 3 times, and finally the complex was resuspended in PBS buffer.

[0048] S704. Horseradish peroxidase (HRP) was diluted to 1 mg / mL with MES buffer, and EDC and NHS were added to the HRP solution with final concentrations of 2 mM and 5 mM, respectively, and gently stirred for 30 minutes at room temperature to activate the carboxyl groups on the HRP molecules. The activated HRP solution was then added to the silver nanoparticle-antibody complex suspension at a mass ratio of 1:10, and gently stirred for 4 hours to ensure that HRP covalently coupled with the active sites on the silver nanoparticle-antibody complex. After the coupling reaction was completed, the unbound HRP was removed by centrifugation at 12,000 rpm for 20 minutes, and the silver nanoparticle-antibody-HRP complex was resuspended with PBS buffer at pH 7.8. The centrifugation and washing steps were repeated 3 times, and the final complex was resuspended in PBS buffer.

[0049] S705. Add the silver nanoparticle-antibody-HRP complex suspension to a 5% bovine serum albumin solution and stir gently at room temperature for 1 hour. After blocking, centrifuge again at 12,000 rpm for 20 minutes to remove excess bovine serum albumin, resuspend the complex with PBS buffer, and finally store it in a refrigerator at 4°C to avoid freezing to prevent particle aggregation or loss of antibody activity. After the above steps are completed, take out a portion and mix it with deionized water to obtain a silver nanoparticle-anti-MxA monoclonal antibody B-HRP solution.

[0050] Example 3 Preparation of modified electrode: S1. Pretreatment of glassy carbon electrode: Use 0.06µm alumina slurry on a polishing cloth to polish the surface of the glassy carbon electrode until the surface is smooth and mirror-like, then repeatedly clean the electrode surface with deionized water, and clean it in an ultrasonic cleaner with deionized water and anhydrous ethanol for 6 minutes to remove surface residues, and blow dry the electrode surface under a nitrogen flow to obtain a pretreated glassy carbon electrode.

[0051] S2. Thiolated graphene oxide modification: Apply 2 mg / mL of thiolated graphene oxide dispersion droplets on the clean pretreated glassy carbon electrode surface and dry at room temperature until a uniform thiolated graphene oxide film is formed to obtain a thiolated graphene oxide modified electrode.

[0052] S3. Gold nanoparticle modification: 0.5 mg / mL streptavidin-coated gold nanoparticle solution was dropped onto the surface of the thiolated graphene oxide modified electrode and incubated at room temperature for 1.5 h to allow the gold nanoparticles to be evenly distributed on the electrode surface. The electrode was rinsed with deionized water to remove unbound gold nanoparticles to obtain a gold nanoparticle modified electrode.

[0053] S4. Antibody immobilization: 2 µg / mL biotinylated anti-MX1 monoclonal antibody solution was dropped onto the gold nanoparticle modified electrode surface and incubated at room temperature for 1.5 h. The electrode surface was rinsed with PBS buffer at pH 7.5 to remove unbound antibodies and obtain an electrode modified with antibodies.

[0054] S5. BSA blocking treatment: The electrode modified with the antibody was immersed in a 2% BSA solution and incubated at room temperature for 0.6 h to block the active sites on the electrode surface that were not occupied by the antibody. The electrode surface was gently washed with a PBS buffer at pH 7.5 to remove the unbound BSA and antibody solution residues, and an ultrasensitive electrochemical immunosensor for MX1 ​​protein detection based on nanocomposite materials was obtained.

[0055] Electrochemical detection: S6. MX1 protein capture: The nanocomposite-based ultrasensitive electrochemical immunosensor for MX1 ​​protein detection was immersed in a solution containing the MX1 protein to be detected, incubated for 0.6 h, and washed with PBS buffer at pH 7.5 to obtain an electrode that removed unbound MX1 protein.

[0056] S7. Sandwich structure formation: 2 mg / mL silver nanoparticle-anti-MxA monoclonal antibody B-HRP solution was added dropwise to the electrode surface from which unbound MX1 protein was removed, and incubated for 0.6 h to form a sandwich structure. The electrode surface was gently washed with PBS buffer at pH 7.5 to remove unbound complexes and ensure that the signal probe and the target protein formed a stable sandwich structure, thereby obtaining an electrode with a sandwich structure.

[0057] S8. Electrochemical signal amplification and detection: The sandwich structure electrode was immersed in an electrolyte solution of 2 mM hydroquinone and 2 mM hydrogen peroxide, and the electrochemical signal was generated by HRP catalytic reaction. The current response of the electrode was measured using differential pulse voltammetry. The current peak was proportional to the concentration of MX1 protein, and quantitative analysis was performed according to the standard curve.

[0058] The preparation process of the streptavidin-coated gold nanoparticles is as follows: S301. 0.02% chloroauric acid solution was heated to boiling, and 0.02 M sodium citrate solution was added. After continuous stirring and heating for 11 min, the solution turned into wine red and then cooled at room temperature to obtain gold nanoparticles. S302. Centrifuge and wash the gold nanoparticles with deionized water, then mix with 2 mg / mL polyethylene glycol-thiol solution at a volume ratio of 1:1, and stir at room temperature for 1.6 h for surface modification to obtain polyethylene glycol-thiol solution-modified gold nanoparticles; S303. Activate 1.5 mg / mL streptavidin solution with 1.5 mg / mL EDC and 1.5 mg / mL NHS in 0.2 M MES buffer, react for 0.6 h, and obtain activated streptavidin; S304. Mixing the activated streptavidin with the gold nanoparticles modified with polyethylene glycol-thiol solution, stirring at room temperature for 1.6 h, and coupling to form streptavidin-coated gold nanoparticles; S305. Add 2% BSA solution to block the unreacted active sites and incubate for 0.6 h. Finally, centrifuge to remove unbound streptavidin and BSA, resuspend the streptavidin-coated gold nanoparticles in PBS buffer to a final concentration of 0.5 mg / mL and store at 3°C.

[0059] The preparation steps of the silver nanoparticle-anti-MxA monoclonal antibody B-HRP solution are as follows: S701. Place the 0.5 mg / mL silver nanoparticle suspension in a centrifuge and centrifuge at 11,000 rpm for 16 minutes, and discard the supernatant. Then resuspend the particles with deionized water, and repeat the centrifugation and washing steps 3 times to remove impurities and stabilizers remaining on the surface. The washed silver nanoparticles are finally resuspended in 0.1 M, pH 6.0 MES buffer to obtain a silver nanoparticle suspension.

[0060] S702. Add N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the silver nanoparticle suspension at final concentrations of 2 mM and 5 mM, respectively. The mixed solution was gently stirred at room temperature for 25 minutes to activate the carboxyl functional groups on the surface of the silver nanoparticles. After activation, centrifuge again at 11,000 rpm for 16 minutes to remove unreacted EDC and NHS, and resuspend the silver nanoparticles with MES buffer. Repeat the above washing steps twice, and suspend the final silver nanoparticles in MES buffer for use.

[0061] S703. Anti-MxA monoclonal antibody B was diluted to 2 mg / mL with MES buffer, and then the diluted antibody was added to the activated silver nanoparticle suspension, with a mass ratio of antibody to silver nanoparticles of 1:10. The mixed solution was gently stirred at room temperature for 1.5 hours, and then centrifuged at 11000 rpm for 16 minutes to remove unbound antibodies, and the silver nanoparticle-antibody complex was resuspended with PBS buffer at pH 7.5, and the centrifugation and washing steps were repeated 3 times, and finally the complex was resuspended in PBS buffer.

[0062] S704. Horseradish peroxidase (HRP) was diluted to 1 mg / mL with MES buffer, and EDC and NHS were added to the HRP solution with final concentrations of 2 mM and 5 mM, respectively, and gently stirred for 25 minutes at room temperature to activate the carboxyl groups on the HRP molecules. The activated HRP solution was then added to the silver nanoparticle-antibody complex suspension at a mass ratio of 1:10, and gently stirred for 3.5 hours to ensure that HRP covalently coupled with the active sites on the silver nanoparticle-antibody complex. After the coupling reaction was completed, centrifuged at 11,000 rpm for 16 minutes to remove unbound HRP, and the silver nanoparticle-antibody-HRP complex was resuspended with PBS buffer at pH 7.5. The centrifugation and washing steps were repeated 3 times, and the final complex was resuspended in PBS buffer.

[0063] S705. Add the silver nanoparticle-antibody-HRP complex suspension to a 2% bovine serum albumin solution and stir gently at room temperature for 0.6h. After blocking, centrifuge again at 11,000 rpm for 16min to remove excess bovine serum albumin, resuspend the complex with PBS buffer, and finally store it in a refrigerator at 3°C ​​to avoid freezing to prevent particle aggregation or loss of antibody activity. After the above steps are completed, take out a portion and mix it with deionized water to obtain a silver nanoparticle-anti-MxA monoclonal antibody B-HRP solution.

[0064] Example 4 Preparation of modified electrode: S1. Pretreatment of glassy carbon electrode: Use 0.06 µm alumina slurry on a polishing cloth to polish the surface of the glassy carbon electrode until the surface is smooth and mirror-like, then repeatedly clean the electrode surface with deionized water, and clean it in an ultrasonic cleaner with deionized water and anhydrous ethanol for 8 minutes to remove surface residues, and blow dry the electrode surface under a nitrogen flow to obtain a pretreated glassy carbon electrode.

[0065] S2. Thiolated graphene oxide modification: Apply 2 mg / mL of thiolated graphene oxide dispersion droplets on the clean pretreated glassy carbon electrode surface and dry at room temperature until a uniform thiolated graphene oxide film is formed to obtain a thiolated graphene oxide modified electrode.

[0066] S3. Gold nanoparticle modification: 0.8 mg / mL streptavidin-coated gold nanoparticle solution was dropped onto the surface of the thiolated graphene oxide modified electrode and incubated at room temperature for 1.5 h to allow the gold nanoparticles to be evenly distributed on the electrode surface. The electrode was rinsed with deionized water to remove unbound gold nanoparticles to obtain a gold nanoparticle modified electrode.

[0067] S4. Antibody immobilization: 2 µg / mL biotinylated anti-MX1 monoclonal antibody solution was dropped onto the gold nanoparticle modified electrode surface and incubated at room temperature for 1.5 h. The electrode surface was rinsed with PBS buffer at pH 7.7 to remove unbound antibodies and obtain an electrode modified with antibodies.

[0068] S5. Bovine serum albumin blocking treatment: The electrode modified with the antibody was immersed in a 4% bovine serum albumin solution and incubated at room temperature for 0.8 h to block the active sites on the electrode surface that were not occupied by the antibody. The electrode surface was gently washed with a PBS buffer at pH 7.5 to remove the unbound bovine serum albumin and antibody solution residues, and an ultrasensitive electrochemical immunosensor for MX1 ​​protein detection based on nanocomposite materials was obtained.

[0069] Electrochemical detection: S6. MX1 protein capture: The nanocomposite-based ultrasensitive electrochemical immunosensor for MX1 ​​protein detection was immersed in a solution containing the MX1 protein to be detected, incubated for 0.8 h, and washed with PBS buffer at pH 7.7 to obtain an electrode that removes unbound MX1 protein.

[0070] S7. Sandwich structure formation: 2 mg / mL silver nanoparticle-anti-MxA monoclonal antibody B-HRP solution was added dropwise to the electrode surface from which unbound MX1 protein was removed, and incubated for 0.8 h to form a sandwich structure. The electrode surface was gently washed with PBS buffer at pH 7.7 to remove unbound complexes and ensure that the signal probe and the target protein formed a stable sandwich structure, thereby obtaining an electrode with a sandwich structure.

[0071] S8. Electrochemical signal amplification and detection: The sandwich structure electrode was immersed in an electrolyte solution of 2 mM hydroquinone and 2 mM hydrogen peroxide, and the electrochemical signal was generated by HRP catalytic reaction. The current response of the electrode was measured using differential pulse voltammetry. The current peak was proportional to the concentration of MX1 protein, and quantitative analysis was performed according to the standard curve.

[0072] The preparation process of the streptavidin-coated gold nanoparticles is as follows: S301. 0.02% chloroauric acid solution was heated to boiling, and 0.02 M sodium citrate solution was added. After continuous stirring and heating for 14 min, the solution turned into wine red and then cooled at room temperature to obtain gold nanoparticles. S302. Centrifuge and wash the gold nanoparticles with deionized water, then mix with 2 mg / mL polyethylene glycol-thiol solution at a volume ratio of 1:1, and stir at room temperature for 1.8 h to perform surface modification, thereby obtaining polyethylene glycol-thiol solution-modified gold nanoparticles; S303. 1.5 mg / mL of EDC and 1.5 mg / mL of NHS were added to activate 1.5 mg / mL of streptavidin solution in 0.2 M MES buffer and reacted for 0.8 h to obtain activated streptavidin; S304. Mixing the activated streptavidin with the gold nanoparticles modified with polyethylene glycol-thiol solution, stirring at room temperature for 1.8 h, and coupling to form streptavidin-coated gold nanoparticles; S305. Add 2% BSA solution to block the unreacted active sites and incubate for 0.8 h. Finally, centrifuge to remove unbound streptavidin and BSA, resuspend the streptavidin-coated gold nanoparticles in PBS buffer to a final concentration of 0.8 mg / mL and store at 3°C.

[0073] The preparation steps of the silver nanoparticle-anti-MxA monoclonal antibody B-HRP solution are as follows: S701. Place the 0.5 mg / mL silver nanoparticle suspension in a centrifuge and centrifuge at 11,000 rpm for 18 minutes, and discard the supernatant. Then resuspend the particles with deionized water, and repeat the centrifugation and washing steps 3 times to remove impurities and stabilizers remaining on the surface. The washed silver nanoparticles are finally resuspended in 0.1 M, pH 6.0 MES buffer to obtain a silver nanoparticle suspension.

[0074] S702. Add N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the silver nanoparticle suspension at final concentrations of 2 mM and 5 mM, respectively. The mixed solution was gently stirred at room temperature for 28 minutes to activate the carboxyl functional groups on the surface of the silver nanoparticles. After activation, centrifuge again at 11,000 rpm for 18 minutes to remove unreacted EDC and NHS, and resuspend the silver nanoparticles with MES buffer. Repeat the above washing steps twice, and suspend the final silver nanoparticles in MES buffer for use.

[0075] S703. Anti-MxA monoclonal antibody B was diluted to 2 mg / mL with MES buffer, and then the diluted antibody was added to the activated silver nanoparticle suspension, with a mass ratio of antibody to silver nanoparticles of 1:10. The mixed solution was gently stirred at room temperature for 1.5 hours, and then centrifuged at 11000 rpm for 18 minutes to remove unbound antibodies, and the silver nanoparticle-antibody complex was resuspended with PBS buffer at pH 7.7, and the centrifugation and washing steps were repeated 3 times, and finally the complex was resuspended in PBS buffer.

[0076] S704. Horseradish peroxidase (HRP) was diluted to 1 mg / mL with MES buffer, and EDC and NHS were added to the HRP solution with final concentrations of 2 mM and 5 mM, respectively, and gently stirred for 28 minutes at room temperature to activate the carboxyl groups on the HRP molecules. The activated HRP solution was then added to the silver nanoparticle-antibody complex suspension at a mass ratio of 1:10, and gently stirred for 3.5 hours to ensure that HRP covalently coupled with the active sites on the silver nanoparticle-antibody complex. After the coupling reaction was completed, the unbound HRP was removed by centrifugation at 11,000 rpm for 18 minutes, and the silver nanoparticle-antibody-HRP complex was resuspended with PBS buffer at pH 7.7. The centrifugation and washing steps were repeated 3 times, and the final complex was resuspended in PBS buffer.

[0077] S705. Add the silver nanoparticle-antibody-HRP complex suspension to a 4% bovine serum albumin solution and stir gently at room temperature for 0.8h. After blocking, centrifuge again at 11,000 rpm for 18min to remove excess bovine serum albumin, resuspend the complex with PBS buffer, and finally store it in a refrigerator at 3°C ​​to avoid freezing to prevent particle aggregation or loss of antibody activity. After the above steps are completed, take out a portion and mix it with deionized water to obtain a silver nanoparticle-anti-MxA monoclonal antibody B-HRP solution.

[0078] Performance Test: The present invention obtains the quantitative analysis of the standard curve Figure 6 : Linear curve of response current and MX1 protein concentration. It can be seen from the linear curve that Example 1 can achieve high sensitivity and high specificity in measuring MX1 protein. This strongly proves that the present invention is superior to the prior art in terms of sensitivity, specificity, ease of operation and signal amplification mechanism, and provides an innovative solution for rapid and ultra-sensitive detection of MX1 protein, which is helpful for early diagnosis and clinical monitoring of viral infection.

[0079] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing an ultrasensitive electrochemical immunosensor for MX1 ​​protein detection based on nanocomposite materials, characterized in that: The following steps are involved: S1. Pretreatment of glassy carbon electrode: Use 0.05-0.07 µm alumina slurry on a polishing cloth to polish the surface of the glassy carbon electrode until the surface is smooth and mirror-like, then repeatedly clean the electrode surface with deionized water, and then clean it with deionized water and anhydrous ethanol in an ultrasonic cleaner for 5-10 minutes to remove surface residues, and blow dry the electrode surface under a nitrogen flow to obtain a pretreated glassy carbon electrode; S2. Thiolated graphene oxide modification: applying a 1-3 mg / mL thiolated graphene oxide dispersion droplet on the surface of a clean pretreated glassy carbon electrode, and drying at room temperature to form a uniform thiolated graphene oxide film to obtain a thiolated graphene oxide modified electrode; S3. Gold nanoparticle modification: 0.1-1 mg / mL streptavidin-coated gold nanoparticle solution was dropped onto the surface of the thiolated graphene oxide modified electrode, incubated at room temperature for 1-2 h to allow the gold nanoparticles to be evenly distributed on the electrode surface, and the electrode was rinsed with deionized water to remove unbound gold nanoparticles to obtain a gold nanoparticle modified electrode; S4. Antibody immobilization: A biotinylated anti-MX1 monoclonal antibody solution with a concentration of 1-3 µg / mL was drop-coated on the surface of the gold nanoparticle-modified electrode, incubated at room temperature for 1-2 h, and the electrode surface was rinsed with PBS buffer to remove unbound antibodies, thereby obtaining an electrode modified with antibodies; S5. Bovine serum albumin blocking treatment: immerse the electrode modified with the antibody in a bovine serum albumin solution with a mass fraction of 1-5% and incubate it at room temperature for 0.5-1h to block the active sites on the electrode surface not occupied by the antibody. Gently wash the electrode surface with PBS buffer to remove unbound bovine serum albumin and antibody solution residues to obtain an ultrasensitive electrochemical immunosensor for MX1 ​​protein detection based on nanocomposite materials.

2. The method for preparing an ultrasensitive electrochemical immunosensor for MX1 ​​protein detection based on nanocomposite materials according to claim 1, characterized in that: The pH of the PBS buffer used in the detection method was 7.4-7.

8.

3. The method for preparing an ultrasensitive electrochemical immunosensor for MX1 ​​protein detection based on nanocomposite materials according to claim 1, characterized in that: In step S3, the preparation process of the streptavidin-coated gold nanoparticles is as follows: S301. A 0.01-0.03% chloroauric acid solution is heated to boiling, and a sodium citrate solution is added and stirred and heated for 10-15 minutes. The solution changes color to wine red and is cooled to room temperature to obtain gold nanoparticles; S302. Centrifuge and wash the gold nanoparticles with deionized water, then mix with 1-3 mg / mL polyethylene glycol-thiol solution, and stir at room temperature for 1.5-2h for surface modification to obtain polyethylene glycol-thiol solution-modified gold nanoparticles; S303. Activate 1-2 mg / mL of streptavidin solution with 1-2 mg / mL of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-2 mg / mL of N-hydroxysuccinimide in 0.1-0.3 M MES buffer, and react for 0.5-1 h to obtain activated streptavidin; S304. Mixing the activated streptavidin with the gold nanoparticles modified with polyethylene glycol-thiol solution, stirring at room temperature for 1.5-2h, and coupling to form streptavidin-coated gold nanoparticles; S305. Add 1-3% BSA solution to block the unreacted active sites and incubate for 0.5-1h. Finally, centrifuge to remove unbound streptavidin and BSA, resuspend the streptavidin-coated gold nanoparticles in PBS buffer to a final concentration of 0.1-1 mg / mL, and store at 2-4°C to obtain streptavidin-coated gold nanoparticles.

4. The method for preparing an ultrasensitive electrochemical immunosensor for MX1 ​​protein detection based on nanocomposite materials according to claim 3, characterized in that: In step S301, the concentration of the sodium citrate solution is 0.01-0.03M.

5. The method for preparing an ultrasensitive electrochemical immunosensor for MX1 ​​protein detection based on nanocomposite materials according to claim 3, characterized in that: In step S302, the mass ratio of the gold nanoparticles to the polyethylene glycol-thiol solution is 1:

1.

6. Use of the ultrasensitive electrochemical immunosensor for detecting MX1 protein based on the nanocomposite material according to any one of claims 1 to 5 in detecting MX1 protein.

7. The use according to claim 6, characterized in that: The method of application comprises the following steps: S101. MX1 protein capture: The nanocomposite-based ultrasensitive electrochemical immunosensor for MX1 ​​protein detection was immersed in a solution containing the MX1 protein to be detected, incubated for 0.5-1 h, and washed with PBS buffer to obtain an electrode that removed unbound MX1 protein; S102. Formation of sandwich structure: 1-3 mg / mL silver nanoparticle-anti-MxA monoclonal antibody B-HRP solution is added dropwise to the electrode surface from which unbound MX1 protein is removed, and incubated for 0.5-1 h to form a sandwich structure. The electrode surface is gently washed with PBS buffer to remove unbound complexes, ensuring that the signal probe and the target protein form a stable sandwich structure, thereby obtaining an electrode with a sandwich structure. S103. Electrochemical signal amplification and detection: The sandwich structure electrode was immersed in an electrolyte solution of 1-3 mM hydroquinone and 1-3 mM hydrogen peroxide. The electrochemical signal was generated by horseradish peroxidase catalytic reaction. The current response of the electrode was measured using differential pulse voltammetry. The current peak was proportional to the concentration of MX1 protein. Quantitative analysis was performed based on the standard curve.

8. The use according to claim 7, characterized in that: In step S102, the preparation steps of the silver nanoparticle-anti-MxA monoclonal antibody B-HRP solution are as follows: S201. placing a 0.5 mg / mL silver nanoparticle suspension in a centrifuge, discarding the supernatant, then resuspending the particles with deionized water, and repeating the centrifugation and washing steps 3 times to remove impurities and stabilizers remaining on the surface, and finally resuspending the washed silver nanoparticles in 0.1 M MES buffer with a pH of 6.0 to obtain a silver nanoparticle suspension; S202. N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are added to the silver nanoparticle suspension to a final concentration of 2 mM and 5 mM, respectively. The mixed solution is gently stirred at room temperature for 20-30 minutes to activate the carboxyl functional groups on the surface of the silver nanoparticles. After the activation is completed, the mixture is centrifuged again to remove the unreacted N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and the silver nanoparticles are resuspended in MES buffer. The above washing steps are repeated twice, and the final silver nanoparticles are suspended in MES buffer for later use; S203. diluting the anti-MxA monoclonal antibody B to 1-3 mg / mL with MES buffer, and then adding the diluted antibody to the activated silver nanoparticle suspension, the mass ratio of the antibody to the silver nanoparticles is 1:10, the mixed solution is gently stirred at room temperature for 1-2 hours and then centrifuged to remove unbound antibodies, and the silver nanoparticle-antibody complex is resuspended with PBS buffer, and the centrifugation and washing steps are repeated 3 times, and finally the complex is resuspended in PBS buffer; S204. Dilute horseradish peroxidase with MES buffer to 1 mg / mL, and add N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to the horseradish peroxidase solution at final concentrations of 2 mM and 5 mM, respectively. Gently stir at room temperature for 20-30 min to activate the carboxyl groups on the horseradish peroxidase molecules. Then add the activated horseradish peroxidase solution to the silver nanoparticle-antibody complex suspension, and gently stir for 3-4 h to ensure that the horseradish peroxidase and the active sites on the silver nanoparticle-antibody complex undergo covalent coupling reaction. After the coupling reaction is completed, centrifuge to remove unbound horseradish peroxidase, resuspend the silver nanoparticle-antibody-horseradish peroxidase complex with PBS buffer, repeat the centrifugation and washing steps 3 times, and resuspend the final complex in PBS buffer; S205. Add the silver nanoparticle-antibody-horseradish peroxidase complex suspension to a bovine serum albumin solution with a mass fraction of 1-5%, gently stir at room temperature for 0.5-1h, and after sealing, centrifuge again to remove excess bovine serum albumin, resuspend the complex with PBS buffer, and finally store it in a refrigerator at 2-4°C to avoid freezing to prevent particle aggregation or loss of antibody activity. After the above steps are completed, take out a portion and mix it with deionized water to obtain a silver nanoparticle-anti-MxA monoclonal antibody B-HRP solution.

9. The use according to claim 8, characterized in that: During the preparation of the silver nanoparticle-anti-MxA monoclonal antibody B-HRP solution, the centrifuge speed during the centrifugation treatment is 10000-12000 rpm, and the time is 15-20 minutes.

10. The use according to claim 8, characterized in that: In step S204, the mass ratio of the activated horseradish peroxidase solution to the silver nanoparticle-antibody complex suspension is 1:10.

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