A functionalized biological modified electrode for rapid detection of pathogens and a preparation method thereof

Abstract

This invention discloses a functionalized biomodified electrode for rapid pathogen detection and its preparation method, relating to the fields of biosensing and electrochemical detection technology. It includes a glassy carbon electrode substrate, the surface of which is sequentially modified with a reduced graphene oxide layer, an electropolymerized polydopamine film, and an electrodeposited gold nanoparticle layer, forming a composite nanomaterial modified electrode. Antibodies are captured and fixed to the electrode surface through covalent interactions. This functionalized biomodified electrode for rapid pathogen detection and its preparation method, using the same electrochemical workstation, first electropolymerizes dopamine to form a film and then electrodeposits gold nanoparticles. This allows for precise control of film thickness, gold particle size, and coverage density by parameters such as polymerization potential, scan cycles, deposition potential, and deposition time. This avoids batch-to-batch fluctuations caused by dissolved oxygen and solution disturbances in traditional chemical methods, reducing the relative standard deviation of peak current detection from different batches of electrodes from 18.7% to 4.2%–4.5%.

Description

Technical Field

[0001] This invention relates to the field of biosensing and electrochemical detection technology, specifically to a functionalized biomodified electrode for rapid pathogen detection and its preparation method. Background Technology

[0002] In the field of biosensing and electrochemical detection, rapid detection technology for pathogens has always been a core pursuit in scientific research and clinical applications. With the advancement of nanotechnology, functionalized biomodified electrodes have shown great potential in pathogen detection due to their unique advantages, such as high sensitivity, strong selectivity and rapid response. These electrodes achieve specific recognition and signal conversion of target pathogens by cleverly combining biorecognition elements (such as antibodies) with electrochemically active nanomaterials, thereby greatly promoting the development of pathogen detection technology.

[0003] However, while pursuing high sensitivity and selectivity, existing technologies still face challenges in the repeatability of electrode preparation and the reliability of detection results. In particular, when using traditional chemical self-polymerization and chemical reduction methods to prepare functionalized biomodified electrodes, environmental factors such as dissolved oxygen concentration and solution disturbance have a significant impact on the thickness of the modified layer on the electrode surface and the loading of nanoparticles, resulting in large fluctuations in the preparation of different batches of electrodes. This batch-to-batch inconsistency severely limits the repeatability of electrode preparation, causing significant differences in the response signals of different batches of electrodes to the same pathogen under the same detection conditions, thus affecting the accuracy and reliability of the detection results. Therefore, improvements are needed. Summary of the Invention

[0004] The purpose of this invention is to provide a functionalized biomodified electrode for rapid pathogen detection and its preparation method, in order to solve the problems of poor repeatability of electrode preparation, low reliability of detection results, and large fluctuations in the thickness of the electrode surface modification layer and the loading of nanoparticles between batches due to environmental factors caused by traditional chemical self-polymerization and chemical reduction methods.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a functionalized biomodified electrode for rapid pathogen detection, comprising a glassy carbon electrode substrate, wherein a reduced graphene oxide layer, a polydopamine layer, and a gold nanoparticle layer are sequentially modified on the surface of the glassy carbon electrode substrate to form a reduced graphene oxide-polydopamine-gold nanoparticle composite nanomaterial modified electrode; a capture antibody is covalently fixed to the surface of the composite nanomaterial modified electrode; the polydopamine layer is an electropolymerized polydopamine film, and the gold nanoparticle layer is an electrodeposited gold nanoparticle layer.

[0006] Furthermore, the electropolymerized polydopamine film is formed by polymerization in an electrolyte solution containing dopamine monomers via potentiostatic method or cyclic voltammetry scanning method, with a polymerization potential range of -0.5V to +0.5V (relative to the reference electrode) and a dopamine monomer concentration of 3 to 5 mg / mL.

[0007] Furthermore, the electrodeposited gold nanoparticle layer is formed by deposition in an electrolyte solution containing chloroauric acid using a constant potential method or a pulsed potential method, with a deposition potential range of -0.2V to -0.5V, a chloroauric acid concentration of 0.5 to 2mM, and a deposition time of 30 to 300 seconds.

[0008] Furthermore, the polydopamine layer is an electrochemical copolymer film of sulfobetaine methacrylate and dopamine, and the concentration of the sulfobetaine methacrylate monomer in the polymerization solution is 5 to 20 mM.

[0009] Furthermore, the capture antibody is directionally immobilized on the surface of the composite nanomaterial-modified electrode via boric acid functional groups, and the boric acid functional groups form cyclic borate ester bonds with the Fc fragment glycosylation sites of the capture antibody.

[0010] Furthermore, the boric acid functional groups are grafted onto the surface of the polydopamine layer via a silane coupling agent containing boric acid functional groups, or are directly covalently linked to the side chains of the polydopamine layer.

[0011] A method for preparing a functionalized biomodified electrode for rapid pathogen detection includes the following steps:

[0012] S1. Reduced graphene oxide is modified on the surface of a glassy carbon electrode substrate to obtain a reduced graphene oxide modified electrode.

[0013] S2. The reduced graphene oxide modified electrode is placed in an electrolyte solution containing dopamine monomer, and a polydopamine film is deposited by electrochemical polymerization to obtain a reduced graphene oxide-polydopamine modified electrode.

[0014] S3. The reduced graphene oxide-polydopamine modified electrode is placed in an electrolyte solution containing chloroauric acid, and gold nanoparticles are generated in situ by electrochemical deposition and loaded onto the surface of the polydopamine film to obtain a reduced graphene oxide-polydopamine-gold nanoparticle composite nanomaterial modified electrode.

[0015] S4. The captured antibody is covalently fixed to the electrode surface obtained in step S3 to obtain the functionalized biomodified electrode.

[0016] Further, in step S2, the electrolyte solution is a phosphate buffer or a Tris-HCl buffer with a pH of 7.0 to 8.5 and a dopamine monomer concentration of 3 to 5 mg / mL; the electrochemical polymerization method is a potentiostatic method (potential +0.1V to +0.5V) or a cyclic voltammetric scan method (potential window -0.5V to +0.5V, scan number 5 to 20 cycles).

[0017] Furthermore, in step S2, the electrolyte solution also contains sulfobetaine methacrylate monomer at a concentration of 5-20 mM. The electrochemical polymerization method causes dopamine to copolymerize with sulfobetaine methacrylate, forming a sulfobetaine methacrylate-polydopamine copolymer film on the electrode surface.

[0018] Furthermore, before step S4, the electrode obtained in step S3 is further modified with boric acid functionalization: the electrode is immersed in a silane coupling agent solution containing boric acid functional groups for reaction, or the boric acid groups are covalently linked to the polydopamine side chain through a chemical reaction; then the capture antibody solution is contacted with the electrode surface and incubated in a buffer solution of pH 7.4-8.5, so that the capture antibody is oriented and immobilized by forming cyclic boric acid ester bonds with the boric acid groups through its Fc fragment glycosylation site.

[0019] Compared with existing technologies, this invention provides a functionalized biomodified electrode for rapid pathogen detection and its preparation method. A polydopamine film is formed by electropolymerization in a dopamine monomer solution using a potentiostatic method or cyclic voltammetry. Subsequently, a gold nanoparticle layer is electrodeposited in a chloroauric acid solution on the same electrochemical workstation using a potentiostatic method or pulsed potential method. This allows for precise nanoscale control of the polydopamine film thickness, gold nanoparticle size distribution, and surface coverage density through electrochemical parameters (polymerization potential, scan number, deposition potential, and deposition time). This avoids batch-to-batch fluctuations in film thickness and particle loading caused by uncontrollable factors such as dissolved oxygen concentration and solution disturbances in traditional chemical self-polymerization and chemical reduction methods. The relative standard deviation of peak current for different batches of electrodes under the same target concentration detection is reduced from 18.7% to 4.2%–4.5%, significantly improving the repeatability of sensor preparation and detection reliability.

[0020] By introducing sulfobetaine methacrylate monomer into an electropolymerization electrolyte solution containing dopamine monomer, and using cyclic voltammetry to induce one-step electrochemical copolymerization of the two on the surface of a reduced graphene oxide-modified electrode, an in-situ sulfobetaine methacrylate-polydopamine copolymer film is generated. The dense hydration layer formed on the surface by the zwitterionic polymer of sulfobetaine methacrylate effectively repels the nonspecific adsorption of proteins, while retaining the function of the polydopamine component in providing covalent fixation sites for antibodies. Thus, without adding an additional blocking step, the nonspecific adsorption response current of the electrode in a buffer solution containing 1 mg / mL bovine serum albumin is reduced from 0.41 μA to 0.07–0.08 μA, achieving the integrated function of antifouling and biomolecule fixation at the same interface.

[0021] By introducing phenylboronic acid functional groups into the electrode surface through composite nanomaterial modification, and utilizing the cyclic borate ester bond formed between the borate group and the cis-diol structure at the N-glycan terminal of the capture antibody Fc fragment, the capture antibody is spatially oriented and immobilized on the electrode surface with the Fc fragment closer to the electrode surface and the Fab antigen-binding fragment facing outwards towards the solution. Compared to conventional EDC / NHS random covalent immobilization methods, this significantly increases the proportion of effective antigen-binding sites on the electrode surface while ensuring the amount of antibody immobilized. This further reduces the detection limit for Staphylococcus aureus from 210 CFU / mL to 8 CFU / mL, and the linear detection range from 10... 3 ~10 5 CFU / mL increased to 10 1 ~10 7 The CFU / mL measurement achieved a significant simultaneous improvement in detection sensitivity and dynamic response range. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0023] Figure 1 A flowchart illustrating the preparation process provided in this embodiment of the invention;

[0024] Figure 2 This is a flowchart illustrating anti-pollution and antibody-directed fixation provided in an embodiment of the present invention. Detailed Implementation

[0025] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0026] As attached Figure 1 and attached Figure 2 As shown:

[0027] Example 1:

[0028] This invention provides a method for preparing a functionalized biomodified electrode for rapid pathogen detection, comprising the following steps:

[0029] Pretreatment of the glassy carbon electrode: Take a glassy carbon electrode with a diameter of 3 mm and mechanically polish it sequentially on a chamois polishing pad using α-Al₂O₃ polishing powders with particle sizes of 1.0 μm, 0.3 μm, and 0.05 μm in a figure-eight pattern. Each polishing stage lasts 3–5 minutes until the electrode surface is mirror-like. Before each change of polishing powder particle size, ultrasonically clean the electrode in deionized water for 30 seconds to thoroughly remove any residual alumina particles from the surface. After completing the three-stage polishing, place the electrode in a 0.5 M H₂SO₄ solution, using an Ag / AgCl (saturated KCl solution) electrode as the reference electrode and a platinum wire electrode as the counter electrode. Perform cyclic voltammetry activation within a potential window of -0.2 V to +1.6 V at a scan rate of 100 mV / s for approximately 20 consecutive scans until the cyclic voltammetry curves stably coincide. After activation, remove the electrode and rinse it thoroughly with deionized water. Then, blow dry the surface moisture with a high-purity nitrogen stream and place it in a clean desiccator for later use.

[0030] Preparation of the reduced graphene oxide modified layer: 10 mg of graphene oxide powder was accurately weighed and added to 10 mL of deionized water. The mixture was ultrasonically treated for 2 hours using an ultrasonic cell disruptor probe under ice bath conditions. The ultrasonic power was set to 300 W, with a 2-second working time followed by a 3-second pause, resulting in a uniformly dispersed, brownish-yellow graphene oxide solution with a concentration of 1 mg / mL. 5 μL of the above graphene oxide dispersion was precisely pipetted onto the surface of the glassy carbon electrode after the first pretreatment step, ensuring that the droplet completely covered the working surface of the electrode without overflowing the edges. The coated electrode was allowed to air dry at room temperature for 4 hours, or dried in a 35°C oven for 1 hour, until a uniform graphene oxide film formed on the surface. The electrode was then used as the working electrode in a three-electrode system. The electrolyte solution was 0.1 M phosphate buffer (pH 7.0). Electrochemical reduction was performed at -1.5 V (relative to the Ag / AgCl reference electrode) using a potentiostatic method for 300 seconds. After reduction, the electrode surface color changed from brownish-yellow to grayish-black, indicating that the graphene oxide had been reduced to reduced graphene oxide. The electrode was removed, the surface was gently rinsed with deionized water, and dried with nitrogen gas to obtain the glassy carbon electrode modified with reduced graphene oxide.

[0031] Preparation of electropolymerized polydopamine film: Preparation of electropolymerization electrolyte solution: Accurately weigh 40 mg of dopamine hydrochloride and dissolve it in 10 mL of 0.1 M phosphate buffer (pH 8.0). Stir thoroughly to dissolve and prepare a dopamine monomer polymerization solution with a concentration of 4 mg / mL. Prepare and store in the dark immediately before use. Immerse the reduced graphene oxide-modified glassy carbon electrode, Ag / AgCl (saturated KCl) reference electrode, and platinum wire electrode obtained in the preparation step of the reduced graphene oxide-modified layer into the above polymerization solution and connect them to an electrochemical workstation. Electropolymerization is performed using cyclic voltammetry. The scanning potential window is set to -0.5 V to +0.5 V, the scanning rate is set to 50 mV / s, and the number of scan cycles is 10. During the scanning process, the dopamine monomer is oxidized to dopaquinone on the electrode surface and further undergoes cyclization, oligomerization, and crosslinking reactions, forming a polydopamine film in situ on the surface of the reduced graphene oxide layer. During the polymerization process, the redox peak current gradually increases with the number of scan cycles, as observed on the cyclic voltammetry curve. After polymerization, the electrode is removed, and its surface is gently rinsed with deionized water to remove unpolymerized free monomers and weakly adsorbed polymers. It is then dried with nitrogen gas to obtain the reduced graphene oxide-electroplated polydopamine modified electrode.

[0032] Preparation of electrodeposited gold nanoparticles: Preparation of electrodeposition solution: Accurately weigh chloroauric acid tetrahydrate and prepare a 1.0 mM HAuCl4 deposition solution using 0.1 M potassium nitrate solution. Store in the dark. The reduced graphene oxide-electroplated polydopamine modified electrode obtained in the preparation step of the electropolymerized polydopamine film was used as the working electrode and immersed in the deposition solution along with the Ag / AgCl reference electrode and the platinum wire counter electrode. A constant potential electrodeposition method was used, with the deposition potential set at -0.3 V and the deposition time at 120 seconds. At this potential, the AuCl4 in the electrolyte... - Ions are reduced to elemental gold on the surface of the polydopamine film, forming gold nanoparticles that are firmly anchored to the active sites of the polydopamine layer. During the deposition process, the electrode surface gradually exhibits a wine-red luster. After deposition, the electrode is removed, thoroughly rinsed with deionized water to remove residual electrolyte, and dried with nitrogen gas to obtain a reduced graphene oxide-polydopamine-gold nanoparticle composite nanomaterial modified electrode.

[0033] Immobilization of the capture antibody: The composite nanomaterial-modified electrode obtained in the preparation step of electrodeposited gold nanoparticles was immersed in a mixed solution containing 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide, and activated at room temperature for 30 minutes to activate the carboxyl functional groups present on the surface of polydopamine and gold nanoparticles. After activation, the electrode was removed and the surface was rapidly rinsed with 0.1 M, pH 7.4 phosphate buffer. Subsequently, 10 μL of a 50 μg / mL anti-Staphylococcus aureus protein A monoclonal antibody solution prepared with 0.1 M, pH 7.4 phosphate buffer was accurately pipetted and dropped onto the working surface of the electrode, ensuring complete coverage. The electrode was placed in a humidified chamber and incubated at 4 °C for 12 hours, allowing the amino groups on the antibody molecules to condense with the activated carboxyl groups on the electrode surface to form amide bonds, thereby covalently immobilizing the capture antibody on the electrode surface. After incubation, the electrode surface was slowly rinsed three times with 0.1M, pH 7.4 phosphate buffer to remove unbound free antibodies. The electrode was then immersed in a 1% bovine serum albumin solution and blocked at room temperature for 1 hour to seal any remaining non-specific protein adsorption sites on the electrode surface. After blocking, the electrode was removed, rinsed with phosphate buffer, and dried with nitrogen to obtain the functionalized biomodified electrode for rapid pathogen detection described in this embodiment.

[0034] Electrochemical detection application of electrodes for target pathogens: Preparation of Staphylococcus aureus standard solutions of different concentrations, with a concentration gradient range of 10. 2 CFU / mL to 10 6 CFU / mL, diluted with 0.1M, pH 7.4 phosphate buffer. The functionalized biomodified electrodes prepared in the antibody immobilization step were immersed in standard solutions of various concentrations and incubated at 37°C for 45 minutes to allow specific binding of the target pathogen to the capture antibody on the electrode surface. After incubation, the electrodes were removed and rinsed three times with phosphate buffer containing 0.05% Tween-20, immersing for 1 minute each time, to remove non-specifically adsorbed impurities.

[0035] The electrodes were immersed in a pre-prepared suspension of reduced graphene oxide-multi-walled carbon nanotubes-palladium@signal-labeled antibody complex. The signal label was prepared according to the method described in the literature: first, multi-walled carbon nanotubes were oxidized with a mixed acid to introduce carboxyl groups; then, they were blended with graphene oxide and hydrothermally reduced to form an rGO-MWCNT composite carrier; next, Pd nanoparticles were loaded onto the carrier surface using a chemical reduction method; finally, an anti-Staphylococcus aureus protein A polyclonal antibody was covalently linked to the complex using an EDC / NHS cross-linking method. The concentration of the label suspension was 20 μg / mL based on antibody mass. Incubation was performed at 37°C for 45 minutes to form a sandwich structure of capture antibody-target pathogen-signal-labeled antibody. After incubation, the electrode surface was thoroughly rinsed with phosphate buffer containing 0.05% Tween-20.

[0036] The electrode, after incubation, was connected to a three-electrode system. The electrolyte solution was a 0.1M phosphate buffer solution (pH 7.4) containing 5mM hydrogen peroxide. Differential pulse voltammetry was used for electrochemical signal detection. The test parameters were set as follows: scan potential range 0V to -0.8V, potential increment 4mV, pulse amplitude 50mV, pulse width 50ms, and pulse period 200ms. The peak current value of hydrogen peroxide electrocatalytic reduction at approximately -0.4V was recorded. A standard working curve was plotted with the logarithm of the target pathogen concentration on the x-axis and the peak current value on the y-axis for the quantitative detection of Staphylococcus aureus in unknown samples. The limit of detection for Staphylococcus aureus using this electrode was 35 CFU / mL (signal-to-noise ratio of 3), and the linear detection range was 1×10⁻⁶. 2 CFU / mL to 1×10 6 The linear regression equation for the CFU / mL standard was I(μA) = 2.31 log C(CFU / mL) + 4.57, with a linear correlation coefficient R² of 0.993. The relative standard deviation of the peak current detected by the five electrodes prepared in different batches at the same concentration of standard was 4.2%.

[0037] Example 2:

[0038] This embodiment is basically the same as the previous embodiment, except that it includes the following steps.

[0039] Pretreatment of the glassy carbon electrode: Take a 3mm diameter glassy carbon electrode and mechanically polish it sequentially on a chamois polishing pad using α-Al₂O₃ polishing powders with particle sizes of 1.0μm, 0.3μm, and 0.05μm in a figure-eight pattern. Each polishing stage lasts 3–5 minutes until the electrode surface is mirror-like. Before each change of polishing powder particle size, ultrasonically clean the electrode in deionized water for 30 seconds. After completing the three-stage polishing, place the electrode in a 0.5M H₂SO₄ solution, using an Ag / AgCl (saturated KCl solution) electrode as the reference electrode and a platinum wire electrode as the counter electrode. Perform cyclic voltammetry activation within a potential window of -0.2V to +1.6V at a scan rate of 100mV / s for approximately 20 consecutive scans until the cyclic voltammetry curves stably coincide. After activation, remove the electrode, rinse thoroughly with deionized water, dry the surface moisture with a high-purity nitrogen stream, and place it in a clean desiccator for later use.

[0040] Preparation of the reduced graphene oxide modified layer: 10 mg of graphene oxide powder was accurately weighed and added to 10 mL of deionized water. The mixture was ultrasonically treated for 2 hours using an ultrasonic cell disruptor probe under ice bath conditions. The ultrasonic power was set to 300 W, with a 2-second working time followed by a 3-second pause, resulting in a uniformly dispersed graphene oxide solution with a concentration of 1 mg / mL. 5 μL of the above graphene oxide dispersion was precisely pipetted onto the surface of the glassy carbon electrode after the first pretreatment step, ensuring complete coverage of the electrode working surface. The coated electrode was allowed to air dry at room temperature for 4 hours until a uniform graphene oxide film formed on the surface. The electrode was then connected to a three-electrode system as the working electrode, using 0.1 M phosphate buffer (pH 7.0) as the electrolyte. Electrochemical reduction was performed at -1.5 V (relative to the Ag / AgCl reference electrode) using a potentiostatic method for 300 seconds. The electrode was removed, gently rinsed with deionized water, and dried with nitrogen gas to obtain the reduced graphene oxide modified glassy carbon electrode.

[0041] Electrochemical copolymerization of sulfobetaine methacrylate and dopamine to prepare an antifouling polydopamine film: Preparation of the electropolymerization copolymer electrolyte solution: Accurately weigh 40 mg of dopamine hydrochloride and dissolve it in 10 mL of 0.1 M phosphate buffer (pH 8.0). Stir thoroughly to obtain a dopamine solution with a concentration of 4 mg / mL. Then, add sulfobetaine methacrylate monomer to this solution to a final concentration of 10 mM. Continue stirring to mix evenly, obtaining a copolymerization precursor solution containing dopamine and sulfobetaine methacrylate. Prepare and store in the dark immediately before use. Immerse the reduced graphene oxide-modified glassy carbon electrode, Ag / AgCl (saturated KCl) reference electrode, and platinum wire electrode obtained in the preparation step of the reduced graphene oxide-modified layer into the above copolymerization precursor solution and connect them to an electrochemical workstation. Electrochemical copolymerization was performed using cyclic voltammetry with a scanning potential window set to -0.5V to +0.5V, a scanning rate of 50mV / s, and 10 scan cycles. During the scan, dopamine monomer and sulfobetaine methacrylate monomer jointly participated in the electrochemical oxidation and subsequent polymerization reaction on the electrode surface, generating a sulfobetaine methacrylate-polydopamine copolymer film in situ. After polymerization, the electrode was removed, and its surface was gently rinsed with deionized water to remove unpolymerized free monomers and weakly adsorbed polymers. The electrode was then dried with nitrogen to obtain the reduced graphene oxide-sulfobetaine methacrylate / polydopamine copolymer modified electrode.

[0042] Preparation of electrodeposited gold nanoparticles: Preparation of electrodeposition solution: Accurately weigh chloroauric acid tetrahydrate and prepare a 1.0 mM HAuCl4 deposition solution using 0.1 M potassium nitrate solution. Store in the dark. The electrode modified with the reduced graphene oxide-sulfobetaine methacrylate / polydopamine copolymer obtained in the electrochemical copolymerization of sulfobetaine methacrylate and dopamine to prepare an antifouling polydopamine film was used as the working electrode, and immersed in the deposition solution along with an Ag / AgCl reference electrode and a platinum wire counter electrode. A constant potential electrodeposition method was used, with the deposition potential set at -0.3 V and the deposition time at 120 seconds. At this potential, the AuCl4 in the electrolyte... - Ions are reduced to elemental gold on the surface of the copolymer film, forming gold nanoparticles that anchor to the active sites of the film. During the deposition process, the electrode surface gradually exhibits a wine-red luster. After deposition, the electrode is removed, thoroughly rinsed with deionized water to remove residual electrolyte, and dried with nitrogen gas to obtain a modified electrode made of reduced graphene oxide-sulfobetaine methacrylate / polydopamine copolymer-gold nanoparticle composite nanomaterial.

[0043] Immobilization of the capture antibody: The composite nanomaterial-modified electrode obtained in the preparation step of electrodeposited gold nanoparticles was immersed in a mixed solution containing 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide, and activated at room temperature for 30 minutes to activate the polydopamine component in the copolymer film and the carboxyl functional groups present on the surface of the gold nanoparticles. After activation, the electrode was removed and the surface was rapidly rinsed with 0.1 M, pH 7.4 phosphate buffer. 10 μL of a 50 μg / mL anti-Staphylococcus aureus protein A monoclonal antibody solution, prepared with 0.1 M, pH 7.4 phosphate buffer, was precisely pipetted onto the working surface of the electrode, ensuring complete coverage. The electrode was placed in a humidified chamber and incubated at 4°C for 12 hours, allowing the amino groups on the antibody molecules to condense with the activated carboxyl groups on the electrode surface to form amide bonds, thereby covalently immobilizing the capture antibody on the electrode surface. After incubation, the electrode surface was slowly rinsed three times with 0.1M, pH 7.4 phosphate buffer to remove unbound free antibodies. The electrode was then immersed in a 1% bovine serum albumin solution and blocked at room temperature for 1 hour to further block any remaining non-specific protein adsorption sites. After blocking, the electrode was removed, rinsed with phosphate buffer, and dried with nitrogen to obtain the functionalized biomodified electrode with an antifouling interface introduced by sulfobetaine methacrylate copolymer as described in this embodiment.

[0044] Electrochemical detection application of electrodes for target pathogens: Preparation of Staphylococcus aureus standard solutions of different concentrations, with a concentration gradient range of 10. 2 CFU / mL to 10 6 CFU / mL, diluted with 0.1M, pH 7.4 phosphate buffer. The functionalized biomodified electrodes prepared in step 5 were immersed in the standard solutions of various concentrations and incubated at 37°C for 45 minutes. After incubation, the electrodes were removed and rinsed three times with phosphate buffer containing 0.05% Tween-20, immersing for 1 minute each time.

[0045] The electrodes were immersed in a suspension of reduced graphene oxide-multi-walled carbon nanotube-palladium@signal-labeled antibody complex, with a concentration of 20 μg / mL based on antibody mass, and incubated at 37°C for 45 minutes. After incubation, the electrode surface was thoroughly rinsed with phosphate buffer containing 0.05% Tween-20.

[0046] The electrode, after incubation, was connected to a three-electrode system. The electrolyte solution was a 0.1M phosphate buffer solution with pH 7.4 containing 5mM hydrogen peroxide. Differential pulse voltammetry was used for electrochemical signal detection, with the following parameters: scan potential range 0V to -0.8V, potential increment 4mV, pulse amplitude 50mV, pulse width 50ms, and pulse period 200ms. The peak current value of hydrogen peroxide electrocatalytic reduction at approximately -0.4V was recorded. A standard working curve was plotted with the logarithm of the target pathogen concentration on the x-axis and the peak current value on the y-axis for the quantitative detection of Staphylococcus aureus in unknown samples. The limit of detection for Staphylococcus aureus using this electrode was 28 CFU / mL (signal-to-noise ratio of 3), and the linear detection range was 1×10⁻⁶. 2 CFU / mL to 1×10 6 The linear regression equation for the CFU / mL concentration was I(μA) = 2.45 log C(CFU / mL) + 4.21, with a linear correlation coefficient R² of 0.996. The nonspecific adsorption response current measured in phosphate buffer containing 1 mg / mL bovine serum albumin was 0.08 μA. The relative standard deviation of the peak currents detected by five electrodes prepared from different batches at the same concentration of standard was 4.5%.

[0047] Example 3:

[0048] This embodiment is basically the same as the previous embodiment, except that it includes the following steps.

[0049] Pretreatment of the glassy carbon electrode: Take a 3mm diameter glassy carbon electrode and mechanically polish it sequentially on a chamois polishing pad using α-Al₂O₃ polishing powders with particle sizes of 1.0μm, 0.3μm, and 0.05μm in a figure-eight pattern. Each polishing stage lasts 3–5 minutes until the electrode surface is mirror-like. Before each change of polishing powder particle size, ultrasonically clean the electrode in deionized water for 30 seconds. After completing the three-stage polishing, place the electrode in a 0.5M H₂SO₄ solution, using an Ag / AgCl (saturated KCl solution) electrode as the reference electrode and a platinum wire electrode as the counter electrode. Perform cyclic voltammetry activation within a potential window of -0.2V to +1.6V at a scan rate of 100mV / s for approximately 20 consecutive scans until the cyclic voltammetry curves stably coincide. After activation, remove the electrode, rinse thoroughly with deionized water, dry the surface moisture with a high-purity nitrogen stream, and place it in a clean desiccator for later use.

[0050] Preparation of the reduced graphene oxide modified layer: 10 mg of graphene oxide powder was accurately weighed and added to 10 mL of deionized water. The mixture was ultrasonically treated for 2 hours using an ultrasonic cell disruptor probe under ice bath conditions. The ultrasonic power was set to 300 W, with a 2-second working time followed by a 3-second pause, resulting in a uniformly dispersed graphene oxide solution with a concentration of 1 mg / mL. 5 μL of the above graphene oxide dispersion was precisely pipetted onto the surface of the glassy carbon electrode after the pretreatment step, ensuring complete coverage of the electrode working surface. The coated electrode was allowed to air dry at room temperature for 4 hours until a uniform graphene oxide film formed on the surface. The electrode was then connected to a three-electrode system as the working electrode, using 0.1 M phosphate buffer (pH 7.0) as the electrolyte. Electrochemical reduction was performed at -1.5 V (relative to the Ag / AgCl reference electrode) using a potentiostatic method for 300 seconds. The electrode was removed, gently rinsed with deionized water, and dried with nitrogen gas to obtain the reduced graphene oxide modified glassy carbon electrode.

[0051] Electrochemical copolymerization of sulfobetaine methacrylate and dopamine to prepare an antifouling polydopamine film: Preparation of the electropolymerization copolymer electrolyte solution: Accurately weigh 40 mg of dopamine hydrochloride and dissolve it in 10 mL of 0.1 M phosphate buffer (pH 8.0). Stir thoroughly to obtain a dopamine solution with a concentration of 4 mg / mL. Then, add sulfobetaine methacrylate monomer to this solution to achieve a final concentration of 10 mM. Continue stirring to mix evenly, obtaining a copolymerization precursor solution containing dopamine and sulfobetaine methacrylate. Prepare and store in the dark immediately before use. Immerse the reduced graphene oxide-modified glassy carbon electrode, Ag / AgCl (saturated KCl) reference electrode, and platinum wire electrode obtained in the preparation step of the reduced graphene oxide-modified layer into the above copolymerization precursor solution and connect them to an electrochemical workstation. Electrochemical copolymerization was performed using cyclic voltammetry with a scanning potential window set to -0.5V to +0.5V, a scanning rate of 50mV / s, and 10 scan cycles. During the scan, dopamine monomer and sulfobetaine methacrylate monomer jointly participated in the electrochemical oxidation and subsequent polymerization reaction on the electrode surface, generating a sulfobetaine methacrylate-polydopamine copolymer film in situ. After polymerization, the electrode was removed, and its surface was gently rinsed with deionized water to remove unpolymerized free monomers and weakly adsorbed polymers. The electrode was then dried with nitrogen to obtain the reduced graphene oxide-sulfobetaine methacrylate / polydopamine copolymer modified electrode.

[0052] Preparation of electrodeposited gold nanoparticles: Preparation of electrodeposition solution: Accurately weigh chloroauric acid tetrahydrate and prepare a 1.0 mM HAuCl4 deposition solution using 0.1 M potassium nitrate solution. Store in the dark. The electrode modified with the reduced graphene oxide-sulfobetaine methacrylate / polydopamine copolymer obtained in the electrochemical copolymerization of sulfobetaine methacrylate and dopamine to prepare an antifouling polydopamine film was used as the working electrode, and immersed in the deposition solution along with an Ag / AgCl reference electrode and a platinum wire counter electrode. A constant potential electrodeposition method was used, with the deposition potential set at -0.3 V and the deposition time at 120 seconds. At this potential, the AuCl4 in the electrolyte... - Ions are reduced to elemental gold on the surface of the copolymer film, forming gold nanoparticles that are anchored to the active sites of the film. After deposition, the electrode is removed, the surface is thoroughly rinsed with deionized water to remove residual electrolyte, and then dried with nitrogen gas to obtain a modified electrode made of reduced graphene oxide-sulfobetaine methacrylate / polydopamine copolymer-gold nanoparticle composite nanomaterial.

[0053] Boric acid functionalization modification of the electrode surface: Preparation of boric acid functionalization modification solution: 3-aminophenylboronic acid silane coupling agent was added to anhydrous ethanol to prepare a 2% (v / v) ethanol solution of 3-aminophenylboronic acid silane coupling agent, and the mixture was stirred magnetically until homogeneous. The composite nanomaterial modified electrode obtained in the preparation step of electrodeposited gold nanoparticles was immersed in the above modification solution and reacted at room temperature for 2 hours. This allowed the silane coupling agent to undergo a condensation reaction with the functional groups such as hydroxyl groups on the surface of polydopamine through silicon-oxygen bonds, covalently grafting phenylboronic acid groups onto the electrode surface. After the reaction was completed, the electrode was removed and rinsed three times each with anhydrous ethanol and deionized water, and dried with nitrogen gas to obtain the boric acid functionalized composite nanomaterial modified electrode.

[0054] Directional immobilization of captured antibodies via boric acid affinity: Preparation of antibody incubation solution: Anti-Staphylococcal protein A monoclonal antibody was prepared to a concentration of 50 μg / mL using 0.1 M, pH 8.5 phosphate buffer. 10 μL of the antibody solution was precisely pipetted and dropped onto the working surface of the boric acid-functionalized electrode obtained in the boric acid functionalization modification step, ensuring complete coverage. The electrode was placed in a humidified chamber and incubated at 4°C for 8 hours. During incubation, the cis-diol structure at the N-terminus of the antibody Fc fragment reversibly covalently binds to the phenylboronic acid groups on the electrode surface, forming stable cyclic borate ester bonds. This allows the antibody to be oriented and immobilized on the electrode surface with the Fc fragment closer to the electrode surface and the Fab antigen-binding fragment facing outwards towards the solution. After incubation, the electrode surface was slowly rinsed three times with 0.1 M, pH 8.5 phosphate buffer to remove unbound free antibody. After rinsing, the electrode was immersed in a 1% bovine serum albumin solution and sealed at room temperature for 30 minutes to seal any non-specific protein adsorption sites that might remain on the electrode surface. After sealing, the electrode was removed, rinsed with 0.1M, pH 7.4 phosphate buffer, and dried with nitrogen to obtain the functionalized biomodified electrode described in this embodiment, which simultaneously possesses anti-fouling interface and antibody-directed immobilization functions.

[0055] Electrochemical detection application of electrodes for target pathogens: Preparation of Staphylococcus aureus standard solutions of different concentrations, with a concentration gradient range of 10. 1 CFU / mL to 10 7 CFU / mL, diluted with 0.1M, pH 7.4 phosphate buffer. The functionalized biomodified electrodes prepared in step 6 were immersed in standard solutions of various concentrations and incubated at 37°C for 45 minutes. After incubation, the electrodes were removed and rinsed three times with phosphate buffer containing 0.05% Tween-20, immersing for 1 minute each time.

[0056] The electrodes were immersed in a suspension of reduced graphene oxide-multi-walled carbon nanotube-palladium@signal-labeled antibody complex, with a concentration of 20 μg / mL based on antibody mass, and incubated at 37°C for 45 minutes. After incubation, the electrode surface was thoroughly rinsed with phosphate buffer containing 0.05% Tween-20.

[0057] The electrode, after incubation, was connected to a three-electrode system. The electrolyte solution was a 0.1M phosphate buffer solution (pH 7.4) containing 5mM hydrogen peroxide. Differential pulse voltammetry was used for electrochemical signal detection. The test parameters were set as follows: scan potential range 0V to -0.8V, potential increment 4mV, pulse amplitude 50mV, pulse width 50ms, and pulse period 200ms. The peak current value of hydrogen peroxide electrocatalytic reduction at approximately -0.4V was recorded. A standard working curve was plotted with the logarithm of the target pathogen concentration on the x-axis and the peak current value on the y-axis for the quantitative detection of Staphylococcus aureus in unknown samples. The limit of detection for Staphylococcus aureus using this electrode was 8 CFU / mL (signal-to-noise ratio of 3), and the linear detection range was 1×10⁻⁶. 1 CFU / mL to 1×10 7 The linear regression equation for the CFU / mL concentration was I(μA) = 3.12 log C(CFU / mL) + 2.87, with a linear correlation coefficient R² of 0.998. The nonspecific adsorption response current measured in phosphate buffer containing 1 mg / mL bovine serum albumin was 0.07 μA. The relative standard deviation of the peak currents detected by five electrodes prepared from different batches at the same concentration of standard was 4.3%.

[0058] Comparative example:

[0059] A method for preparing a functionalized biomodified electrode using a conventional chemical self-polymerization polydopamine deposition, chemical reduction of gold nanoparticles for loading, and conventional random covalent fixation of antibodies, specifically includes the following steps:

[0060] Pretreatment of the glassy carbon electrode: Take a glassy carbon electrode with a diameter of 3 mm and mechanically polish it sequentially on a chamois polishing pad using α-Al₂O₃ polishing powders with particle sizes of 1.0 μm, 0.3 μm, and 0.05 μm in a figure-eight pattern. Each polishing stage lasts 3–5 minutes until the electrode surface is mirror-like. Before each change of polishing powder particle size, ultrasonically clean the electrode in deionized water for 30 seconds. After completing the three-stage polishing, place the electrode in a 0.5 M H₂SO₄ solution, using an Ag / AgCl (saturated KCl solution) electrode as the reference electrode and a platinum wire electrode as the counter electrode. Perform cyclic voltammetry activation within a potential window of -0.2 V to +1.6 V at a scan rate of 100 mV / s for approximately 20 consecutive scans until the cyclic voltammetry curves stably coincide. After activation, remove the electrode, rinse thoroughly with deionized water, dry the surface moisture with a high-purity nitrogen stream, and place it in a clean desiccator for later use.

[0061] Preparation of the reduced graphene oxide modified layer: 10 mg of graphene oxide powder was accurately weighed and added to 10 mL of deionized water. The mixture was ultrasonically treated for 2 hours using an ultrasonic cell disruptor probe under ice bath conditions. The ultrasonic power was set to 300 W, with a 2-second working time followed by a 3-second pause, resulting in a uniformly dispersed graphene oxide solution with a concentration of 1 mg / mL. 5 μL of the above graphene oxide dispersion was precisely pipetted onto the surface of the glassy carbon electrode after the pretreatment step, ensuring complete coverage of the electrode working surface. The coated electrode was allowed to air dry at room temperature for 4 hours until a uniform graphene oxide film formed on the surface. The electrode was then connected to a three-electrode system as the working electrode, using 0.1 M phosphate buffer (pH 7.0) as the electrolyte. Electrochemical reduction was performed at -1.5 V (relative to the Ag / AgCl reference electrode) using a potentiostatic method for 300 seconds. The electrode was removed, gently rinsed with deionized water, and dried with nitrogen gas to obtain the reduced graphene oxide modified glassy carbon electrode.

[0062] Preparation of Traditional Chemically Self-polymerized Polydopamine Film: Preparation of the chemical self-polymerization solution: Accurately weigh 20 mg of dopamine hydrochloride and dissolve it in 10 mL of Tris-HCl buffer (concentration 10 mM, pH 8.5). Stir thoroughly to prepare a 2 mg / mL dopamine self-polymerization solution, to be prepared immediately before use. Completely immerse the reduced graphene oxide-modified glassy carbon electrode obtained in the preparation step of the reduced graphene oxide-modified layer into the above self-polymerization solution. Place the reaction vessel in a light-proof environment and allow it to stand for polymerization at room temperature for 12 hours. During the standing process, dissolved oxygen in the solution acts as an oxidant, causing dopamine monomers to undergo a self-polymerization reaction on the electrode surface, forming a polydopamine film. After the standing polymerization is complete, the solution color changes from colorless to dark brown, and the electrode surface becomes grayish-brown. Remove the electrode and gently rinse the electrode surface with deionized water to remove unpolymerized dopamine monomers and weakly adsorbed polydopamine particles. Dry with nitrogen to obtain the reduced graphene oxide-chemically self-polymerized polydopamine-modified electrode.

[0063] Loading of conventionally chemically reduced gold nanoparticles: Preparation of the chemical reduction deposition solution: Accurately weigh chloroauric acid tetrahydrate and prepare a 1.0 mM HAuCl4 solution with deionized water. Separately prepare a 0.1 M trisodium citrate aqueous solution as a reducing agent. Immerse the reduced graphene oxide-chemically self-polymerized polydopamine modified electrode obtained in the conventional chemical self-polymerization polydopamine film preparation steps into the above HAuCl4 solution. Slowly add trisodium citrate solution dropwise under magnetic stirring until the final concentration of trisodium citrate in the mixture is 2 mM. Continue stirring at room temperature for 30 minutes. During this process, trisodium citrate reduces the AuCl4 content. -Ions are reduced to elemental gold, forming gold nanoparticles that are deposited on the surface of a polydopamine film. After the reaction is complete, the electrode is removed, the surface is thoroughly rinsed with deionized water to remove any residual reaction solution, and then dried with nitrogen gas to obtain a modified electrode made of a composite nanomaterial of reduced graphene oxide-chemically self-polymerized polydopamine-chemically reduced gold nanoparticles.

[0064] Conventional random covalent fixation and blocking of captured antibodies: The composite nanomaterial-modified electrode obtained in the conventional chemical reduction of gold nanoparticles loading step was immersed in a mixed solution containing 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide, and activated at room temperature for 30 minutes to activate the carboxyl functional groups present on the surface of polydopamine and gold nanoparticles. After activation, the electrode was removed and the surface was rapidly rinsed with 0.1 M, pH 7.4 phosphate buffer. 10 μL of a 50 μg / mL anti-Staphylococcus aureus protein A monoclonal antibody solution, prepared with 0.1 M, pH 7.4 phosphate buffer, was precisely pipetted and dropped onto the working surface of the electrode, ensuring complete coverage. The electrode was placed in a humidified chamber and incubated at 4°C for 12 hours, allowing the amino groups on the antibody molecules to condense with the activated carboxyl groups on the electrode surface to form amide bonds, thus achieving random covalent fixation of the antibody. After incubation, the electrode surface was slowly rinsed three times with 0.1M, pH 7.4 phosphate buffer to remove unbound free antibodies. The electrode was then immersed in a 1% bovine serum albumin solution and blocked at room temperature for 1 hour to seal unreacted active sites and non-specific protein adsorption sites on the electrode surface. After blocking, the electrode was removed, rinsed with 0.1M, pH 7.4 phosphate buffer, and dried with nitrogen to obtain the functionalized biomodified electrode prepared using the conventional method described in this comparative example.

[0065] Electrochemical detection application of electrodes for target pathogens: Preparation of Staphylococcus aureus standard solutions of different concentrations, with a concentration gradient range of 10. 3 CFU / mL to 10 5 CFU / mL, diluted with 0.1M, pH 7.4 phosphate buffer. The functionalized biomodified electrodes prepared in step 5 were immersed in the standard solutions of various concentrations and incubated at 37°C for 45 minutes. After incubation, the electrodes were removed and rinsed three times with phosphate buffer containing 0.05% Tween-20, immersing for 1 minute each time.

[0066] The electrodes were immersed in a suspension of reduced graphene oxide-multi-walled carbon nanotube-palladium@signal-labeled antibody complex, with a concentration of 20 μg / mL based on antibody mass, and incubated at 37°C for 45 minutes. After incubation, the electrode surface was thoroughly rinsed with phosphate buffer containing 0.05% Tween-20.

[0067] The electrode, after incubation, was connected to a three-electrode system. The electrolyte solution was a 0.1M phosphate buffer solution (pH 7.4) containing 5mM hydrogen peroxide. Differential pulse voltammetry was used for electrochemical signal detection. The test parameters were set as follows: scan potential range 0V to -0.8V, potential increment 4mV, pulse amplitude 50mV, pulse width 50ms, and pulse period 200ms. The peak current value of hydrogen peroxide electrocatalytic reduction at approximately -0.4V was recorded. A standard working curve was plotted with the logarithm of the target pathogen concentration on the x-axis and the peak current value on the y-axis. The detection limit for Staphylococcus aureus using this electrode was 210 CFU / mL (signal-to-noise ratio of 3), and the linear detection range was 1×10⁻⁶. 3 CFU / mL to 1×10 5 The linear regression equation for the CFU / mL concentration was I(μA) = 1.53 log C(CFU / mL) + 3.28, with a linear correlation coefficient R² of 0.942. The nonspecific adsorption response current measured in phosphate buffer containing 1 mg / mL bovine serum albumin was 0.41 μA. The relative standard deviation of the peak currents detected by five electrodes prepared from different batches at the same concentration of standard was 18.7%.

[0068] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A functionalized bi-modified electrode for rapid detection of pathogens, comprising a glassy carbon electrode substrate, characterized in that, The glassy carbon electrode substrate is sequentially modified with a reduced graphene oxide layer, a polydopamine layer, and a gold nanoparticle layer to form a reduced graphene oxide-polydopamine-gold nanoparticle composite nanomaterial modified electrode; the captured antibody is covalently fixed to the surface of the composite nanomaterial modified electrode; the polydopamine layer is an electropolymerized polydopamine film, and the gold nanoparticle layer is an electrodeposited gold nanoparticle layer.

2. The functionalized biologically modified electrode for rapid detection of pathogens according to claim 1, wherein, The electropolymerized polydopamine film is formed by polymerization in an electrolyte solution containing dopamine monomers via potentiostatic method or cyclic voltammetry scanning method. The polymerization potential range is -0.5V to +0.5V relative to the reference electrode, and the concentration of dopamine monomers is 3 to 5 mg / mL.

3. The functionalized biologically modified electrode for rapid detection of pathogens according to claim 1, wherein, The electrodeposited gold nanoparticle layer is formed by deposition in an electrolyte solution containing chloroauric acid using a constant potential method or a pulsed potential method. The deposition potential range is -0.2V to -0.5V, the chloroauric acid concentration is 0.5 to 2mM, and the deposition time is 30 to 300 seconds.

4. The functionalized biologically modified electrode for rapid detection of pathogens according to claim 1, wherein, The polydopamine layer is an electrochemical copolymer film of sulfobetaine methacrylate and dopamine, wherein the concentration of the sulfobetaine methacrylate monomer in the polymerization solution is 5 to 20 mM.

5. The functionalized biologically modified electrode for rapid detection of pathogens according to claim 1, wherein, The capture antibody is directionally immobilized on the surface of the composite nanomaterial-modified electrode via boric acid functional groups, and the boric acid functional groups form cyclic boric acid ester bonds with the Fc fragment glycosylation sites of the capture antibody.

6. The functionalized biologically modified electrode for rapid detection of pathogens according to claim 5, wherein, The boric acid functional groups are grafted onto the surface of the polydopamine layer via a silane coupling agent containing boric acid functional groups, or are directly covalently linked to the side chains of the polydopamine layer.

7. A method for preparing a functionalized biological modified electrode for rapid detection of pathogens, which is suitable for the functionalized biological modified electrode for rapid detection of pathogens according to any one of claims 1 to 6, characterized in that, Includes the following steps: S1. Reduced graphene oxide is modified on the surface of a glassy carbon electrode substrate to obtain a reduced graphene oxide modified electrode. S2. The reduced graphene oxide modified electrode is placed in an electrolyte solution containing dopamine monomer, and a polydopamine film is deposited by electrochemical polymerization to obtain a reduced graphene oxide-polydopamine modified electrode. S3. The reduced graphene oxide-polydopamine modified electrode is placed in an electrolyte solution containing chloroauric acid, and gold nanoparticles are generated in situ by electrochemical deposition and loaded onto the surface of the polydopamine film to obtain a reduced graphene oxide-polydopamine-gold nanoparticle composite nanomaterial modified electrode. S4. The captured antibody is covalently fixed to the electrode surface obtained in step S3 to obtain the functionalized biomodified electrode.

8. The method according to claim 7, wherein the method is characterized by, In step S2, the electrolyte solution is a phosphate buffer or a Tris-HCl buffer with a pH of 7.0 to 8.5 and a dopamine monomer concentration of 3 to 5 mg / mL; the electrochemical polymerization method is a potentiostatic method with a potential window of +0.1V to +0.5V or a cyclic voltammetric scanning method with a potential window of -0.5V to +0.5V and a scanning number of 5 to 20 cycles.

9. A method for preparing a functionalized biomodified electrode for rapid pathogen detection according to claim 7, characterized in that, In step S2, the electrolyte solution also contains sulfobetaine methacrylate monomer at a concentration of 5-20 mM. The electrochemical polymerization method causes dopamine to copolymerize with sulfobetaine methacrylate, forming a sulfobetaine methacrylate-polydopamine copolymer film on the electrode surface.

10. A method for preparing a functionalized biomodified electrode for rapid pathogen detection according to claim 7, characterized in that, Before step S4, the electrode obtained in step S3 is further modified with boric acid functionalization: the electrode is immersed in a silane coupling agent solution containing boric acid functional groups for reaction, or the boric acid groups are covalently linked to the polydopamine side chain through a chemical reaction; then the capture antibody solution is contacted with the electrode surface and incubated in a buffer solution of pH 7.4-8.5, so that the capture antibody is oriented and immobilized by forming cyclic boric acid ester bonds with the boric acid groups through its Fc fragment glycosylation site.

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