Electrochemical immunosensor, and electrochemical immunoassay kit and preparation method therefor and method for using same

Abstract

Disclosed in the present invention are an electrochemical immunosensor, an electrochemical immunoassay kit and a preparation method therefor. The electrochemical immunosensor comprises: a working electrode, a counter electrode, and a reference electrode, wherein the working electrode is a gold-vertical graphene electrode that uses a titanium wire or a tantalum wire or a tungsten wire as a substrate, and bovine serum albumin and an antibody are incubated on the gold-vertical graphene electrode; the counter electrode is a platinum wire; the reference electrode is a saturated calomel electrode. The present invention can further optimize the electrochemical properties of graphene, and improve the conductivity, stability and corrosion resistance of graphene, so that graphene can still maintain good performance during high-frequency signal acquisition and long-term use.

Description

Electrochemical immunosensor, electrochemical immunoassay kit and its preparation and application methods

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 202510234136.7, filed on February 28, 2025. The entire contents of the foregoing application are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of immunology, specifically to an electrochemical immunosensor, an electrochemical immunoassay kit, and methods for their preparation and application. Background Technology

[0004] In recent years, graphene has been widely studied as a candidate material for microelectrodes due to its excellent conductivity, mechanical strength, and biocompatibility. Graphene possesses a single-atom-layer thickness, a large surface area, and excellent conductivity, enabling rapid electrical signal transmission, and exhibits low electrode resistance and high electrochemical activity. Furthermore, graphene's two-dimensional structure endows it with excellent flexibility and strength, allowing it to adapt to electrode arrays of different shapes and curvatures, making it particularly suitable for the design of flexible microelectrode arrays.

[0005] Electrochemical immunoassay employs electrochemical immunosensors. Electrochemical immunosensors are a novel type of biosensor based on the specific reaction between antigens and antibodies. Due to their high sensitivity, low cost, and rapid response, they have attracted considerable attention in the field of clinical diagnostics.

[0006] However, traditional electrochemical immunosensors have limitations. Their complex composition makes them difficult to mass-produce industrially, resulting in low stability. Summary of the Invention

[0007] According to one aspect of the present invention, an electrochemical immunosensor is provided, comprising: a working electrode, a counter electrode, and a reference electrode, wherein the working electrode is a gold-vertical graphene electrode with titanium, tantalum, or tungsten as the substrate, and bovine serum albumin and antibodies are incubated on the gold-vertical graphene electrode; the counter electrode is a platinum sheet or platinum wire; and the reference electrode is a saturated calomel electrode.

[0008] According to another aspect of the present invention, an electrochemical immunoassay kit is provided, comprising: the aforementioned electrochemical immunosensor; an electrolytic cell, first used to bind toxins in the blood to be tested with antibodies incubated on the working electrode, and then used to contain a probe solution and perform differential pulse voltammetry testing of the redox channel using an electrochemical tester, wherein the probe solution comprises potassium chloride, potassium ferricyanide, and potassium ferrocyanide; and a shell, formed by injection molding or three-dimensional printing of acrylonitrile-butadiene-styrene copolymer, for ensuring the stability of the electrochemical immunosensor and the sealing of the electrolytic cell.

[0009] According to another aspect of the present invention, a method for preparing an electrochemical immunoassay kit is provided, comprising: preparing an electrochemical immunosensor including a working electrode, a counter electrode, and a reference electrode, wherein the working electrode is a gold-vertical graphene electrode with titanium, tantalum, or tungsten as the substrate material, the counter electrode is a platinum sheet or platinum wire, and the reference electrode is a saturated calomel electrode; incubating an antibody and bovine serum albumin on the gold-vertical graphene electrode; and using an acrylonitrile-butadiene-styrene copolymer or polycarbonate material, injection molding or three-dimensional printing is used to obtain a test kit shell to ensure the stability of the electrochemical immunosensor; the test kit shell includes an electrolytic cell, which is first used to bind toxins in the blood to be tested with the antibody incubated on the working electrode, and then used to contain a probe solution and perform differential pulse voltammetry testing of the redox channel using an electrochemical tester, wherein the probe solution includes potassium chloride, potassium ferricyanide, and potassium ferrocyanide.

[0010] According to another aspect of the present invention, a method for applying an electrochemical immunoassay kit is provided, comprising: 1) preparing N standard solutions, each standard solution being a mixture of antigen and phosphate buffer, wherein the antigen concentration in the N standard solutions is different, and calculating ΔI by using each standard solution as a sample solution; 2) establishing a coordinate system, with the lg value of antigen concentration and ΔI as the horizontal and vertical axes, substituting the lg value of antigen concentration and ΔI of the N standard solutions into the coordinate system, and fitting a linear regression equation; 3) using the test solution as a sample solution, calculating the ΔI of the test solution, substituting the ΔI of the test solution into the linear regression equation to obtain the lg value of antigen concentration in the test solution, and then calculating the antigen concentration in the test solution.

[0011] This invention can further optimize the electrochemical performance of graphene, improve its conductivity, stability and corrosion resistance, and enable it to maintain good performance during high-frequency signal acquisition and long-term use. Attached Figure Description

[0012] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0013] Figure 1 is a schematic diagram of the gold-vertical graphene electrode before bending (middle left) and after bending (right) according to Embodiment 1 of the present invention;

[0014] Figure 2 is a schematic diagram of the surface SEM of the gold-vertical graphene electrode according to Embodiment 1 of the present invention;

[0015] Figure 3 is a schematic diagram of a cross-sectional SEM image of the gold-vertical graphene electrode according to Embodiment 1 of the present invention;

[0016] Figure 4(a) shows the Raman spectrum of the gold-vertical graphene electrode prepared according to Example 1 of the present invention;

[0017] Figure 4(b) is the EDS spectrum of the gold-vertical graphene electrode prepared according to Example 1 of the present invention;

[0018] Figure 5 shows the X-ray photoelectron spectroscopy (XPS) of the gold-vertical graphene electrode prepared according to Example 1 of the present invention;

[0019] Figure 6 is a schematic diagram of the electrochemical immunosensor according to Embodiment 2 of the present invention;

[0020] Figure 7 is the STEP curve of the electrode sensor after 180s of electrochemical deposition according to Embodiment 2 of the present invention;

[0021] Figure 8 is a surface SEM image of the gold-vertical graphene electrode prepared according to Example 3 of the present invention;

[0022] Figure 9 is a graph showing the cyclic voltammetry (CV) scan rate variation according to Embodiment 4 of the present invention;

[0023] Figure 10 is a schematic diagram of the linear regression equations for the oxidation channel and the reduction channel according to Embodiment 4 of the present invention;

[0024] Figure 11(a) shows the DPV curve of the oxidation-reduction channel tested using the test kit according to Embodiment 4 of the present invention;

[0025] Figure 11(b) is the DPV curve of the redox channel tested in solution according to Example 4 of the present invention;

[0026] Figure 12 is a Nyquist curve according to Embodiment 5 of the present invention;

[0027] Figure 13 is the DPV curve of the electrochemical immunosensor according to Example 6 of the present invention;

[0028] Figure 14(a) is the DPV curve of the BSA-Ab@gold-vertical graphene electrode according to Example 7 of the present invention;

[0029] Figure 14(b) shows the DPV curve of the electrochemical immunosensor according to Example 7 of the present invention;

[0030] Figure 15 is a curve showing the difference between the redox channels under different antibody incubation times according to Example 7 of the present invention;

[0031] Figure 16 shows the DPV curves of the electrochemical immunosensor (TT-) according to Examples 4-7 of the present invention;

[0032] Figure 17 is a schematic diagram comparing the DPV curves and ΔI of the alternative electrochemical immunoassay kit according to Embodiment 8 of the present invention; and

[0033] Figure 18 is a schematic diagram of the application of the electrochemical immunoassay kit according to Embodiment 9 of the present invention. Detailed Implementation

[0034] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0035] Example 1

[0036] According to one embodiment of the present invention, an electrochemical immunosensor is provided, comprising: a working electrode, a counter electrode, and a reference electrode, wherein the working electrode is a gold-vertical graphene electrode with titanium, tantalum, or tungsten as the substrate, and bovine serum albumin and antibodies are incubated on the gold-vertical graphene electrode; the counter electrode is a platinum sheet or platinum wire; and the reference electrode is a saturated calomel electrode.

[0037] Traditional electrochemical immunosensors have limitations; their complex composition makes them difficult to mass-produce industrially, resulting in low stability. In this invention, the electrochemical performance of graphene is further optimized, improving its conductivity, stability, and corrosion resistance, enabling it to maintain good performance during high-frequency signal acquisition and long-term use.

[0038] According to an embodiment of the present invention, a gold-vertical graphene electrode with titanium as the substrate comprises: a titanium strip formed by pressing titanium wire, the titanium strip having a thickness of 70 μm and a length of 3 cm; a carbonization layer deposited on the surface of the titanium strip; a vertically grown graphene layer, the graphene layer being a nanosheet with a width of 0.7 mm and a thickness of 0.1 mm (as shown in Figure 1), and a working area length of 1 cm, the dimensions of which conform to the structure of a microelectrode; and gold nanoparticles embedded in the nanosheet, the gold nanoparticles being 0.41% of the vertical graphene. Furthermore, as shown in Figure 1, the vertical graphene electrode maintains the flexibility of the metal component; the vertical graphene electrode can be bent nearly 180° without significant morphological changes. Due to its unique structure and properties, the flexible vertical graphene electrode has significant advantages in in vivo detection, such as high biocompatibility, high spatial resolution, low invasiveness, and long-term stability. Vertical graphene electrodes are small in size and can accurately detect local electrophysiological signals, making them suitable for high-resolution neural activity recording. They are also small and flexible, resulting in minimal trauma and rapid recovery during implantation. They are widely used in neuroscience, clinical diagnosis, and cardiovascular monitoring, making them an ideal tool for in vivo detection.

[0039] In this embodiment of the invention, the gold-vertical graphene electrode retains the large surface area of ​​two-dimensional graphene material and incorporates doped gold as a catalyst, resulting in higher electrocatalytic activity and higher electron mobility for the graphene. Gold nanoparticles synergistically optimize the electrode's catalytic activity and stability, while the introduction of gold enhances electron transfer efficiency. This three-dimensional porous structure enables the electrode to maintain high conductivity while possessing excellent biocompatibility and antibody loading capacity.

[0040] Figure 2 is a schematic diagram of the surface SEM of the gold-vertical graphene electrode according to Embodiment 1 of the present invention, and Figure 3 is a schematic diagram of the cross-sectional SEM of the gold-vertical graphene electrode according to Embodiment 1 of the present invention. The vertical graphene electrode was observed under a scanning electron microscope. As shown in Figure 2, the vertical graphene nanosheets are two-dimensional nanosheets, and their cross-sectional SEM is shown in Figure 3.

[0041] The Raman spectrum of the gold-vertical graphene electrode prepared in Example 1 is shown in Figure 4. Figure 4 shows the characteristic G peak and 2D peak of graphene. The intensity ratio (I²D / IG) of the 2D peak to the G peak is 1.36, and the full width at half maximum (FWHM) of the 2D peak is approximately 47.35 cm⁻¹, indicating that the gold-vertical graphene electrode prepared in Example 1 is composed of graphene nanosheets. A relatively sharp D peak (1357 cm⁻¹) is present, which is related to the exposure of many atomic defects due to the vertical growth of graphene. The EDS spectrum of the gold-vertical graphene electrode is shown in Figure 4(b). Figure 4(b) shows that Au particles account for 0.4 At%, while C, O, and Ti atoms account for 77.81 At%, 0.47 At%, and 21.32 At%, respectively.

[0042] The C1s, Au 4f, Ti 2p, and O1s of the gold-vertical graphene electrode prepared in Example 1 were characterized by X-ray photoelectron spectroscopy (XPS), as shown in Figure 5. In the Au 4f spectrum, the two peaks at 84.4 eV and 88.1 eV belong to Au 4f7 / 2 and Au 4f5 / 2, respectively, indicating that the gold-vertical graphene electrode prepared in Example 1 contains Au nanoparticles.

[0043] Example 2

[0044] This invention also provides an electrochemical immunoassay kit. The kit includes: any of the aforementioned electrochemical immunosensors; an electrolytic cell, first used to bind toxins in the blood to be tested with antibodies incubated on the working electrode, and then used to contain a probe solution and perform differential pulse voltammetry testing of the redox channel using an electrochemical testing instrument, wherein the probe solution includes potassium chloride, potassium ferricyanide, and potassium ferrocyanide; and a shell, injection molded or 3D printed using an acrylonitrile-butadiene-styrene copolymer, to ensure the stability of the electrochemical immunosensor and the sealing of the electrolytic cell.

[0045] This test kit utilizes 3D printing technology, using thermoplastic polymer ABS (acrylonitrile-butadiene-styrene) as the consumable material. The kit measures 2.5cm wide, 5cm long, and 1cm high. A recessed area, 1.5cm wide, 2.1cm long, and 0.8cm deep, is used for adding solution or blood. The electrolytic cell has a volume of 2–5mL, with 2mL of probe solution per vial. 3D printing facilitates innovative design and experimental verification, driving technological advancement. 3D-printed chemical test kits offer significant advantages in customization, cost control, complex structure manufacturing, environmental friendliness, and experimental efficiency, making them an ideal choice for the field of electrochemical testing.

[0046] In this electrochemical immunoassay kit, the electrochemical immunosensor includes: a gold-vertical graphene electrode and an insulating fixation carrier, a titanium wire, and a platinum wire. The three wires are fixed in place by the test kit, as shown in Figure 6.

[0047] The C1s, Au 4f, Ti 2p, and O1s X-ray photoelectron spectroscopy (XPS) spectra of the gold-vertical graphene electrode prepared in Example 2 were characterized, as shown in Figure 7. In the Au 4f spectrum, the two peaks at 84.4 eV and 88.1 eV belong to Au 4f, respectively. 7 / 2 and Au 4f 5 / 2 This indicates that the gold-vertical graphene electrode prepared in Example 1 contains Au nanoparticles.

[0048] Example 3

[0049] This invention provides a method for preparing an electrochemical immunoassay kit, comprising: preparing an electrochemical immunosensor including a working electrode, a counter electrode, and a reference electrode, wherein the working electrode is a gold-vertical graphene electrode with titanium, tantalum, or tungsten as the substrate, the counter electrode is a platinum sheet or platinum wire, and the reference electrode is a saturated calomel electrode; incubating an antibody and bovine serum albumin on the gold-vertical graphene electrode; using an acrylonitrile-butadiene-styrene copolymer or polycarbonate material, injection molding or 3D printing is used to obtain a test kit shell to ensure the stability of the electrochemical immunosensor; the test kit shell includes an electrolytic cell, which is first used to bind toxins in the blood to be tested with the antibody incubated on the working electrode, and then used to contain a probe solution and perform differential pulse voltammetry testing of the redox channel using an electrochemical tester, wherein the probe solution includes potassium chloride, potassium ferricyanide, and potassium ferrocyanide.

[0050] According to an embodiment of the present invention, the preparation of the gold-vertical graphene electrode includes: Step 1, pressing a titanium wire with a diameter of 300 μm into a titanium strip with a diameter of 40 μm, polishing off the oxide layer on the surface with 180-grit sandpaper, immersing it in ultrapure water and ultrasonically cleaning it for 10-20 min, and drying it by irradiating it with a 285W heat lamp for 1-2 min; Step 2, placing it in the reaction chamber of an electronically assisted hot-wire chemical vapor deposition system, suspending 5 tantalum wires on a hot-wire copper frame, closing the reaction chamber, tightening the vent valve, and starting vacuuming; then introducing 18 mL / min... -1 Methane gas and 300 mL·min -1 When the pressure in the reaction chamber reaches 2000 Pa, the AC filament power supply is turned on, and a 120A current is applied to both ends of the five tantalum wires through the copper frame, at which point the voltage is 10V. The pressure in the reaction chamber is then stabilized between 5000 and 5132 Pa. After stabilization, the carbonization time is started for 30 minutes. After carbonization, the gas circuit switch is changed to set the methane gas flow rate to 20 mL / min. -1 The hydrogen gas flow rate is 40 mL / min. -1 Adjust the DC bias power supply to raise the current to 6A, at which point the voltage is 30V; Step 3: Immerse in tetrachloroauric acid aqueous solution and use multipotential step electrochemical deposition method to remove Au from tetrachloroauric acid. 3+ Electrochemical reduction yielded gold nanoparticles, which were then loaded onto the surface of a gold-vertical graphene electrode. The electrodeposition process involved 30 cycles of multi-potential step electrochemical deposition, with each cycle consisting of a 1-second hold at 2V followed by a 5-second hold at -2V, resulting in the gold-vertical graphene electrode. The tetrachloroauric acid concentration in the aqueous solution was 0.1 mM. Electrochemical deposition of Au was performed for 180 seconds. The surface SEM image of the gold-vertical graphene microelectrode prepared in Example 3 is shown in Figure 8.

[0051] In this embodiment of the invention, a high degree of controllability of the electrode structure was achieved by precisely controlling the graphene growth parameters. The island-like distribution of gold nanoparticles significantly enhances catalytic activity. The final electrode sensitivity reaches 0.09 fg / mL, which is two orders of magnitude higher than that of traditional processes.

[0052] According to an embodiment of the present invention, incubating the antibody on the gold-vertical graphene electrode comprises: placing the gold-vertical graphene electrode into a centrifuge tube containing 150 μL-200 μL of tetanus antibody solution, wherein the concentration of the tetanus antibody solution is 1000-5000 ng·mL. -1 The centrifuge tubes were placed in an electric thermostatic incubator at 37°C and incubated for 60 minutes. After incubation, the tubes were washed with phosphate buffer to remove excess tetanus antibody solution from the surface and then allowed to stand at 37°C for 10 minutes.

[0053] The antibody immobilization process in this invention, through finely optimized experimental parameters and a unique buffer system design, achieves highly efficient targeted immobilization of antibody molecules and a significant improvement in electrode performance. 1000-5000 ng / mL is used. -1 The antibody concentration range ensures sufficient antibody coverage while avoiding steric hindrance between molecules, thus optimizing antibody binding efficiency. The 37°C incubation temperature precisely simulates the physiological environment, maintaining the integrity of the antibody structure while promoting full exposure of its active sites. The 20–60 minute incubation time window ensures sufficient binding of antibody molecules to functional groups (such as carboxyl / amino groups) on the electrode surface, while also considering operational efficiency. Particularly noteworthy is the dual technical advantage of the potassium ferrocyanide / potassium ferrocyanide phosphate buffer system used in this embodiment: firstly, the redox couple in the buffer ([Fe(CN)6])... 3- / 4- In subsequent electrochemical detection, it can be directly used as a probe molecule, avoiding interfacial disturbances caused by buffer changes. Furthermore, the suitable ionic strength (0.09–0.11 M) provided by potassium chloride effectively shields the electrostatic repulsion between antibody molecules, promoting their uniform distribution on the electrode surface. The entire process, through a "static incubation in centrifuge tubes + gentle rinsing" operation, achieves controllable adjustment of antibody immobilization density (up to 1.2 × 10⁻⁶) while maintaining the integrity of the vertical graphene three-dimensional structure. 12 molecules / cm 2 Furthermore, the antibody activity retention rate exceeded 90%. This standardized fixation method controlled the performance difference of electrodes prepared in different batches to within 5%, significantly improving the reproducibility and reliability of the detection, and laying a solid foundation for subsequent high-sensitivity detection (0.09 fg / mL).

[0054] This invention provides an electrochemical immunoassay kit for real-time rapid detection of tetanus. The electrode uses graphene as the base material, enabling efficient signal monitoring and possessing high application value. Tetanus is an acute infectious disease caused by Clostridium tetani entering the human body through skin or mucous membrane wounds, growing and multiplying in an anaerobic environment, producing toxins, and causing paroxysmal muscle spasms. The harmful effects of tetanus mainly manifest in impacts on the nervous, respiratory, circulatory, digestive, and musculoskeletal systems. Specifically, tetanus is a disease caused by Clostridium tetani. After entering the human body, Clostridium tetani multiplies rapidly in an anaerobic environment, producing toxins that cause symptoms such as a sardonic smile, opisthotonus, and convulsions. In severe cases, it can lead to increased intracranial pressure and cerebral edema. Tetanus irritates the respiratory tract, causing difficulty breathing, and if left untreated, can lead to suffocation. The toxins produced by Clostridium tetani affect the circulatory system, potentially causing symptoms such as decreased blood pressure and rapid pulse, and in severe cases, shock. The toxins also affect the digestive system, causing abdominal pain and bloating, and in severe cases, gastrointestinal ulcers. During a tetanus attack, the patient experiences persistent, rigid muscle contractions, rendering them unable to move independently, and in severe cases, causing muscle necrosis.

[0055] Currently, tetanus detection still relies primarily on clinical diagnosis, which has a significant lag. By the time patients develop obvious clinical symptoms, the disease has often progressed to an irreversible stage, missing the optimal intervention window. If adults remain untreated, allowing the disease to progress, the mortality rate increases to approximately 60%. In children and the elderly, whose immune systems are weakened, tetanus symptoms are more severe, and the likelihood of complications such as pneumonia and cardiovascular disease increases, making treatment more difficult. In these cases, the mortality rate may also rise accordingly, reaching 60%-70%.

[0056] According to an embodiment of the present invention, incubating bovine serum albumin on the gold-vertical graphene electrode includes: weighing 0.03 g of bovine serum albumin powder into a vial, adding 2 mL of phosphate buffer to prepare a bovine serum albumin solution with a concentration of 1.5 wt%; using a pipette to aspirate 150 μL-200 μL of the bovine serum albumin solution and transferring it to a centrifuge tube; immersing the gold-vertical graphene electrode in the bovine serum albumin solution in the centrifuge tube; placing the centrifuge tube in a thermostatic incubator at 37°C for 15 min; washing with phosphate buffer to remove excess bovine serum albumin solution from the surface; and allowing it to stand at 37°C for 10 min.

[0057] The bovine serum albumin (BSA) described in this invention contains proteins that can bind to non-specific sites on the surface of gold nanoparticles, occupying these sites and thus reducing the binding of antigens to non-specific sites on the surface of gold nanoparticles during subsequent incubation, reducing non-specific adsorption, and increasing the accuracy of test results. Specifically, this sealing process significantly improves the detection specificity and stability of the test kit through precisely controlled reaction conditions and optimized solution formulation. A BSA concentration of 1.5 wt.% can form a dense monolayer on the electrode surface, effectively covering more than 99% of non-specific binding sites, reducing background noise to less than 1 / 5 of traditional sealing methods. An incubation temperature of 37°C combined with an incubation time of 10 minutes allows BSA molecules to fully bind to the electrode surface through hydrophobic interactions and hydrogen bonds, forming a stable sealing layer while avoiding masking of active sites due to excessive aggregation. A solution volume of 500 μL ensures complete wetting of all surface microstructures of the electrode, including the nano-gap between vertical graphene sheets and the microgrooves of tantalum strips. This blocking process enhances the electrode's resistance to interference by more than three times in complex biological samples (such as whole blood), reducing non-specific adsorption to less than 0.5% while maintaining target antigen-antibody binding efficiency above 95%. Notably, this process is perfectly compatible with the pre-treatment antibody immobilization step; BSA molecules only block regions where no antibody has bound, without displacing or destroying the immobilized antibody molecules. Electrodes blocked with BSA exhibit less than 5% performance degradation after 30 days of storage at 4°C, significantly extending the test kit's shelf life. This standardized blocking protocol keeps the coefficient of variation between different batches of electrodes below 3%, providing highly reliable results for clinical testing.

[0058] Example 4

[0059] This invention also provides an application method for an electrochemical immunoassay kit, comprising: 1) preparing N standard solutions, each standard solution being a mixture of antigen and phosphate buffer, wherein the antigen concentration in the N standard solutions is different, and calculating ΔI by using each standard solution as a sample solution; 2) establishing a coordinate system, with the lg value of antigen concentration and ΔI as the horizontal and vertical axes, substituting the lg value of antigen concentration and ΔI of the N standard solutions into the coordinate system, and fitting a linear regression equation; 3) using the test solution as a sample solution, calculating the ΔI of the test solution, substituting the ΔI of the test solution into the linear regression equation, obtaining the lg value of antigen concentration in the test solution, and then calculating the antigen concentration in the test solution.

[0060] According to an embodiment of the present invention, the method for calculating ΔI for each sample solution includes: ① incubating the antigen on the electrochemical immunosensor according to claim 1 using the sample solution to obtain the sensor; performing differential pulse voltammetry on the sensor to obtain the current value corresponding to the peak value of the DPV curve I2; ② calculating ΔI for the sample solution, ΔI = I1 - I2, where I1 is the current value corresponding to the peak value of the DPV curve obtained by performing differential pulse voltammetry on the electrochemical immunosensor.

[0061] According to an embodiment of the present invention, 19 standard solutions are prepared, each standard solution containing 1.6 mg / mL. -1 A mixture of tetanus toxin solution antigen and phosphate buffer, with an antigen concentration of 0.1 fg / mL. -1 0.3 fg·mL -1 1 fg·mL -1 3 fg·mL -1 10 fg·mL -1 30 fg·mL -1 100 fg·mL -1 300 fg·mL -1 1 pg·mL -1 3 pg·mL -1 10 pg·mL -1 30 pg·mL -1 100 pg·mL -1 300 pg·mL -1 1 ng·mL -1 3ng·mL -1 10 ng·mL -1 30 ng / mL -1 and 100 ng·mL -1 Establish a coordinate system, with the lg value of antigen concentration and ΔI as the x and y axes respectively. Substitute the lg values ​​of antigen concentration and ΔI of N standard solutions into the coordinate system, and fit the oxidative linear regression equation ΔI to obtain the equation. pa =4.35log c1+8.37, thus obtaining the reconstructed linear regression equation ΔI pc = -4.19log c1 - 11.04; where c1 is the antigen concentration; using the electrochemical immunosensor as the working electrode, oxidation differential pulse voltammetry was performed, and the oxidation peak current I1 of the differential pulse voltammetry test curve of the oxidation channel was obtained as 341.75 μA; using the electrochemical immunosensor as the working electrode, reduction differential pulse voltammetry was performed, and the reduction peak current I1 of the differential pulse voltammetry test curve of the reduction channel was obtained as -333.08 μA; 1.6 mg·mL -1The tetanus toxin solution was diluted with phosphate buffer to a concentration of 100 ng / mL. -1 The test solution is used as the sample solution. The current value corresponding to the peak value of the oxidation differential pulse voltammetry curve and the current value I2 corresponding to the peak value of the reduction differential pulse voltammetry curve are calculated. The ΔI = I1 - I2 of the sample solution is calculated. The calculated ΔI is then substituted into the oxidation linear regression equation ΔI. pa = 4.35log c1 + 8.37, where log c1 is the logarithmic value of TT concentration. Substitute the calculated ΔI into the reconstructed linear regression equation ΔI pc = -4.19log c1 - 11.04, where log c1 is the lg value of the TT concentration. Based on the lg value of the antigen concentration in the test solution, the antigen concentration in the test solution is then calculated.

[0062] In this invention, by simultaneously utilizing two independent electrochemical processes—oxidation and reduction—signal stability, result reliability, and electrode regenerability are significantly improved, offering multiple advantages. First, the dual-channel redox design enables simultaneous detection of the electrochemical signals of target molecules during both oxidation and reduction processes, providing more comprehensive information. Second, this technology effectively distinguishes target molecules from other interfering substances by comparing oxidation and reduction signals, reducing interference from non-specific adsorption or background signals, thereby improving detection selectivity. Furthermore, dual-channel redox detection exhibits strong anti-interference capabilities, reducing the impact of environmental factors (such as temperature and pH) and instrument fluctuations on detection results through internal calibration, thus improving data reliability and repeatability. Additionally, dual-channel detection technology is typically combined with miniaturized and portable devices, suitable for rapid on-site detection, meeting the needs of environmental monitoring, food safety, and clinical diagnostics for efficient and convenient testing. Finally, dual-channel redox detection significantly improves electrode regenerability; continuous detection of both oxidation and reduction channels effectively removes contaminants from the electrode surface, further enabling implantable long-term measurement.

[0063] The implementation process of Embodiment 4 above will be explained in detail below through specific examples.

[0064] This invention demonstrates oxidative DPV and reduced DPV tests. The initial potential was set to -0.6V, the final potential to 1V, and the scan rate to 100mV / s. The CV curves for different working electrodes are shown in Figure 9. Compared to the vertical graphene electrode (VG), the gold-vertical graphene electrode after antibody incubation (Ab-), and the gold-vertical graphene electrode after bovine serum albumin incubation (BSA-), the gold-vertical graphene electrode (Au-VG) exhibited the largest peak current. For the electrochemical immunosensor, the peak current corresponding to "TT-" in Figure 9 was the smallest, indicating that as the immune response progresses, the amount of protein increases, and since proteins are non-conductive, the peak current gradually decreases.

[0065] (1) When the differential pulse voltammetry test is used for oxidation DPV testing, the oxidation channel DPV curves obtained from the differential pulse voltammetry test of 16 standard solutions are shown as downward-opening curves in Figure 9. The linear regression equation (oxidation channel linear regression equation) is shown in Figure 10. By substituting ΔI of the test solution into the linear regression equation, the lg value of the antigen concentration in the test solution is obtained, and then the antigen concentration in the test solution is calculated to be 0.104 pg / mL. -1 The deviation from the actual value of the antigen concentration in the test solution is within 5%.

[0066] (2) When the differential pulse voltammetry test is a reduction DPV test, the reduction channel DPV curves obtained from the differential pulse voltammetry test of 16 standard solutions are shown as upward-opening curves in Figure 9. The linear regression equation (reduction channel linear regression equation) is shown in Figure 10. By substituting ΔI of the test solution into the linear regression equation, the lg value of the antigen concentration in the test solution is obtained, and then the antigen concentration in the test solution is calculated to be 0.103 pg / mL. -1 The deviation from the actual value of the antigen concentration in the test solution is within 5%.

[0067] The test kit prepared in this embodiment was used for electrochemical testing. Gold-vertical graphene was used as the working electrode, platinum wire as the counter electrode, and titanium wire as the reference electrode. 2-4 ml of probe solution was added to the groove to perform DPV testing of the redox channel, as shown in Figure 11(a), proving the feasibility of the test kit. The DPV testing of the redox channel using the probe solution in the three-electrode system is shown in Figure 11(b). After data processing, it was found that in the three-electrode system, I... 氧化1 =479.24μA, I 还原1 = -472.99μA; I in the test kit 氧化2 =347.28μA, I 还原2 = -323.49μA; therefore, the correction factor for the oxidation channel is 72.46%, and the correction factor for the reduction channel is 68.39%.

[0068] Example 5

[0069] A series of tests were performed on the immune assembly electrode, including cyclic voltammetry (CV) and electrochemical impedance spectroscopy.

[0070] Place the three-electrode system into the probe solution, start the electrochemical workstation, select AC Impedance mode, and set the parameters as follows: open-circuit voltage of 0.17–0.23 V, and AC signal frequency applied to the working electrode of 10 Hz. -2 ~10 6 The Nyquist curve was obtained by measuring Hz. The Nyquist curve is shown in Figure 12. It can be seen from Figure 12 that the semicircle diameter of "Ab-" is significantly smaller than that of "BSA-" and "TT-", indicating that the resistance to tetanus toxin is the lowest during incubation.

[0071] Example 6

[0072] Take 15 ml of potassium ferricyanide solution and place it in a beaker. Connect the beaker to an electrochemical workstation (CHI660E) using a three-electrode system. As shown in Figure 6, the green clip connects to the working electrode (gold-vertical graphene electrode), the red clip connects to the counter electrode (platinum wire electrode), and the white electrode connects to the reference electrode (calomel electrode).

[0073] First, electrode activation was performed. 15 ml of 0.2 M NaOH aqueous solution was placed in a beaker, and the three-electrode system was immersed in the NaOH aqueous solution for CV testing. The scan rate was set to 1 V / s, and 50 segments were scanned continuously. Activation was then complete.

[0074] Figure 13 shows the DPV curve of the electrochemical immunosensor according to Example 6 of the present invention. In the figure, Au-VG represents the gold-vertical graphene electrode, BSA- represents the gold-vertical graphene electrode after incubation with antibody and bovine serum albumin, and TT- represents the electrochemical immunosensor.

[0075] The DPV (oxidation DPV) test was performed on the electrochemical immunosensor as the working electrode to obtain the oxidation channel DPV curve. As shown in the upper half of Figure 13, the peak current value of the oxidation channel DPV curve of the gold-vertical graphene electrode (Au-VG) is 479.24 μA, the peak current value of the oxidation channel DPV curve of the gold-vertical graphene electrode after incubation with antibody and bovine serum albumin (BSA-) is 341.75 μA, and the peak current value of the oxidation channel DPV curve of the electrochemical immunosensor (TT-) is 316.64 μA.

[0076] The electrochemical immunosensor was used as the working electrode for DPV testing (reduction DPV testing), and the reduction channel DPV curves were obtained. As shown in the lower half of Figure 13, the peak current value of the oxidation channel DPV curve (Au-VG) of the gold-vertical graphene electrode was -472.99 μA, the peak current value of the oxidation channel DPV curve (BSA-) of the gold-vertical graphene electrode after incubation with antibody and bovine serum albumin was -333.08 μA, and the peak current value of the oxidation channel DPV curve (TT-) of the electrochemical immunosensor was -310.40 μA.

[0077] For the oxidation channel, ΔI = 341.75 - 316.64 = 25.11 μA. For the reduction channel, ΔI = -333.08 - (-310.40) = -22.68 μA. This demonstrates the stability and reliability of the electrochemical dual-channel measurement.

[0078] Example 7

[0079] This embodiment further illustrates the preparation method of the electrochemical immunoassay kit. The difference from the previous embodiment lies in the incubation time of the gold-vertical graphene electrode, which is 10, 30, 60, 90, and 120 min, respectively.

[0080] In this embodiment, oxidation DPV and reduction DPV tests were performed on the BSA-Ab@gold-vertical graphene electrode to obtain oxidation channel DPV curves and reduction channel DPV curves, as shown in Figure 14(a). Then, oxidation DPV and reduction DPV tests were performed on the electrochemical immunosensor to obtain oxidation channel DPV curves and reduction channel DPV curves, as shown in Figure 14(b).

[0081] The peak current value of the oxidation channel DPV curve of the BSA-Ab@gold-vertical graphene electrode was subtracted from the peak current value of the corresponding electrochemical immunosensor's oxidation channel DPV curve to obtain ΔI, as shown in "Oxidation" in Figure 15. Similarly, the peak current value of the reduction channel DPV curve of the BSA-Ab@gold-vertical graphene electrode was subtracted from the peak current value of the corresponding immunosensor's reduction channel DPV curve to obtain ΔI, as shown in "Reduction" in Figure 15. As shown in Figure 15, the absolute value of ΔI initially increases and then slowly decreases with increasing antibody incubation time. Therefore, the optimal antibody delivery time is 60 min.

[0082] Furthermore, for Examples 4 to 7 of this invention, the DPV curves (reduction channel DPV curves and oxidation channel DPV curves) of the electrochemical immunosensor (TT-) corresponding to the BSA-Ab@gold-vertical graphene electrode and 19 standard solutions are shown in Figure 16. The oxidation linear regression equation and the reduction linear regression equation are shown in Figure 10. As can be seen from Figure 10, the electrochemical immunosensor prepared by the gold-vertical graphene microelectrode of this invention can detect concentrations of 0.1 fg / mL. -1 ~100ng mL -1 The tetanus toxoid was tested.

[0083] Example 8

[0084] This embodiment further illustrates the preparation method of the electrochemical immunoassay kit. The difference from the previous embodiment is that the gold-vertical graphene electrode (Au-VG) is replaced with the gold-copper co-doped vertical graphene electrode (AuCu-) prepared in Example 1 of CN119915879A.

[0085] As shown in Figure 17(a), “BSA-Ab@AuCu-VG” represents the gold-copper co-doped vertical graphene electrode after incubation of antibody and bovine serum albumin, and “1 pg mL” represents the value of the electrode. -1 "TT-AuCu-VG" represents the gold-copper electrochemical immunosensor in this embodiment, "BSA-Ab@Au-VG" represents the gold-vertical graphene electrode after incubation with antibody and bovine serum albumin, and "1 pg mL" represents the total weight of the electrodes. -1 "TT-Au-VG" represents the electrochemical immunosensor in the aforementioned embodiments.

[0086] Subtracting the peak current value of the oxidation channel DPV curve of the BSA-Ab@gold-vertical graphene electrode from the peak current value of the oxidation channel DPV curve of the electrochemical immunosensor yields a ΔI of 22.42 μA, as shown in the upper part of "Au-VG" in Figure 17(b). Subtracting the peak current value of the reduction channel DPV curve of the BSA-Ab@gold-vertical graphene electrode from the peak current value of the reduction channel DPV curve of the immunosensor yields a ΔI of -23.45 μA, as shown in the lower part of "Au-VG" in Figure 17(b). Subtracting the peak current value of the oxidation channel DPV curve of the BSA-Ab@gold-copper co-doped vertical graphene electrode from the peak current value of the oxidation channel DPV curve of the gold-copper electrochemical immunosensor yields a ΔI of 8.87 μA, as shown in the upper part of "AuCu-VG" in Figure 17(b). Subtracting the peak current value of the reduction channel DPV curve of the gold-copper electrochemical immunosensor from the peak current value of the reduction channel DPV curve of the BSA-Ab@gold-copper co-doped vertical graphene electrode yields a ΔI of -4.29 μA, as shown in the lower part of "AuCu-VG" in Figure 17(b). This indicates that the current response difference for detecting tetanus toxoid with the BSA-Ab@gold-vertical graphene electrode of this invention is significantly greater than that with the BSA-Ab@gold-copper co-doped vertical graphene electrode. In other words, the detection capability of the BSA-Ab@gold-copper co-doped vertical graphene electrode for tetanus toxoid is inferior to that of the BSA-Ab@gold-vertical graphene electrode of this invention.

[0087] Example 9

[0088] To evaluate the practical application potential and anti-interference performance of the electrochemical immunosensor, this invention also simulates the complex matrix environment of human serum and systematically investigates the specificity of the BSA-Ab@gold-vertical graphene electrode for the detection of tetanus toxoid (TT) solution.

[0089] The BSA-Ab@gold-vertical graphene electrode was incubated with a test solution, which was a mixture of solvent and solute. The solvent was PBS (pH 7.4), and the solute was one of the following: CEA (carcinoembryonic antigen), CA125 (carbohydrate antigen 125), CA153 (carbohydrate antigen 15-3), AFP (alpha-fetoprotein), TT, and CA199 (carbohydrate antigen 199). The concentration of the solute in the test solution was 5 ng / mL. -1 CEA, 35 U mL -1 CA125, 25U mL -1 CA153, 25 μg mL -1 AFP, 37U mL -1 CA199 and 5fg mL -1For TT, the BSA-Ab@gold-vertical graphene electrode was immersed in centrifuge tubes containing 150μL of test solution. The centrifuge tubes were placed in an electric thermostatic incubator at 37℃ for 25min. After incubation, the tubes were rinsed with phosphate buffer (PBS) to remove excess solution from the surface and then allowed to stand at 37℃ for 10min.

[0090] The oxidation DPV and reduction DPV tests were performed on the BSA-Ab@gold-vertical graphene electrode and the electrode after solute incubation, respectively. The peak current value of the oxidation channel DPV curve of the BSA-Ab@gold-vertical graphene electrode was subtracted from the peak current value of the oxidation channel DPV curve of each electrode after solute incubation, resulting in ΔI, as shown by the bar above the horizontal axis in Figure 18. Similarly, the peak current value of the reduction channel DPV curve of the BSA-Ab@gold-vertical graphene electrode was subtracted from the peak current value of the reduction channel DPV curve of each electrode after solute incubation, resulting in ΔI, as shown by the bar below the horizontal axis in Figure 18. As shown in Figure 18, it can be seen that the reaction current was not significantly changed when the substance was incubated alone, confirming that the BSA-Ab@gold-vertical graphene electrode has satisfactory specificity for TT detection.

[0091] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An electrochemical immunosensor, characterized in that, include: Working electrode, counter electrode, and reference electrode, among which, The working electrode is a gold-vertical graphene electrode with titanium, tantalum or tungsten as the substrate material, and bovine serum albumin and antibodies are incubated on the gold-vertical graphene electrode. The counter electrode is a platinum sheet or platinum wire; The reference electrode is a saturated calomel electrode.

2. The electrochemical immunosensor according to claim 1, characterized in that, Gold-vertical graphene electrodes based on titanium substrates include: A titanium strip made of titanium wire, the titanium strip having a thickness of 70 μm and a length of 3 cm; A carbide layer deposited on the surface of the titanium strip; Vertically grown graphene layers, wherein the graphene layers are nanosheets with a width of 0.7 mm and a thickness of 0.1 mm; and The gold nanoparticles embedded in the nanosheet are 0.41% of the vertical graphene.

3. An electrochemical immunoassay kit, characterized in that, include: The electrochemical immunosensor according to claim 1 or 2; An electrolytic cell is first used to bind toxins in the blood sample to antibodies incubated at the working electrode, and then used to contain a probe solution for differential pulse voltammetry testing of an oxidation-reduction channel using an electrochemical testing instrument. The probe solution includes potassium chloride, potassium ferrocyanide, and potassium ferrocyanide. The outer shell is made of acrylonitrile-butadiene-styrene copolymer by injection molding or 3D printing to ensure the stability of the electrochemical immunosensor and the sealing of the electrolytic cell.

4. A method for preparing an electrochemical immunoassay kit according to claim 3, characterized in that, include: An electrochemical immunosensor comprising a working electrode, a counter electrode, and a reference electrode is fabricated, wherein the working electrode is a gold-vertical graphene electrode with titanium, tantalum, or tungsten as the substrate, the counter electrode is a platinum sheet or platinum wire, and the reference electrode is a saturated calomel electrode. Antibodies and bovine serum albumin were incubated on the gold-vertical graphene electrode. The test box shell is obtained by injection molding or 3D printing using acrylonitrile-butadiene-styrene copolymer or polycarbonate material to ensure the stability of the electrochemical immunosensor. The test kit housing includes an electrolytic cell, which is first used to bind toxins in the blood to be tested with antibodies incubated on the working electrode, and then used to contain a probe solution and perform differential pulse voltammetry testing of the oxidation-reduction channel using an electrochemical tester, wherein the probe solution includes potassium chloride, potassium ferricyanide and potassium ferrocyanide.

5. The method according to claim 4, characterized in that, The preparation of the gold-vertical graphene electrode includes: Step 1: Press a titanium wire with a diameter of 300μm into a titanium strip with a diameter of 40μm, polish off the oxide layer on the surface with 180-grit sandpaper, soak it in ultrapure water and clean it by ultrasonication for 10-20 minutes, and dry it by irradiation with a 285W baking lamp for 1-2 minutes. Step 2: Place the device into the reaction chamber of the electronically assisted hot-wire chemical vapor deposition system, suspend the five tantalum wires on the hot-wire copper frame, close the reaction chamber, tighten the vent valve, and begin evacuation; then introduce 18 mL / min... -1 Methane gas and 300 mL·min -1 When the pressure in the reaction chamber reaches 3999 Pa, the AC filament power supply is turned on, and a 120A current is applied to both ends of the five tantalum wires through the copper frame, at which point the voltage is 10V. The pressure in the reaction chamber is then stabilized between 5000 and 5132 Pa. After stabilization, the carbonization time is started for 30 minutes. After carbonization, the gas circuit switch is changed to set the methane gas flow rate to 20 mL / min. -1 The hydrogen gas flow rate is 40 mL / min. -1 Adjust the DC bias power supply to raise the current to 6A, at which point the voltage is 30V. Step 3: Immerse in tetrachloroauric acid aqueous solution and use multipotential step electrochemical deposition method to remove Au from tetrachloroauric acid. 3+ Gold nanoparticles were electrochemically reduced and loaded onto the surface of a gold-vertical graphene electrode. The electrodeposition time ranged from 60 s to 600 s. The electrodeposition method was performed using a multi-potential step electrochemical deposition method with 30 cycles. Each cycle included a 1 s electrodeposition at a potential of 2 V followed by a 5 s electrodeposition at a potential of -2 V. The resulting gold-vertical graphene electrode was obtained. The concentration of tetrachloroauric acid in the tetrachloroauric acid aqueous solution was 0.1 mM.

6. The method according to claim 5, characterized in that, Incubating antibodies on the gold-vertical graphene electrode includes: The gold-vertical graphene electrode was placed in a centrifuge tube containing 150 μL-200 μL of tetanus antibody solution, wherein the concentration of the tetanus antibody solution was 1000-5000 ng·mL. -1 ; The centrifuge tubes were placed in an electric thermostatic incubator at 37°C and incubated for 60 minutes. After incubation, the tubes were washed with phosphate buffer to remove excess tetanus antibody solution from the surface and then allowed to stand at 37°C for 10 minutes.

7. The method according to claim 6, characterized in that, Incubation of bovine serum albumin on the gold-vertical graphene electrode includes: Weigh 0.03g of bovine serum albumin powder into a vial, add 2mL of phosphate buffer, and prepare a bovine serum albumin solution with a concentration of 1.5wt%. Use a pipette to draw 150μL-200μL of bovine serum albumin solution and transfer it to a centrifuge tube. Immerse the gold-vertical graphene electrode in the bovine serum albumin solution in the centrifuge tube. The centrifuge tubes were placed in an electric thermostatic incubator at 37°C and incubated for 15 minutes. They were then washed with phosphate buffer to remove excess bovine serum albumin solution from the surface and allowed to stand at 37°C for 10 minutes.

8. A method for applying an electrochemical immunoassay kit, characterized in that, include: 1) Prepare N standard solutions, each of which is a mixture of antigen and phosphate buffer. The antigen concentration in the N standard solutions is different. Calculate ΔI by using each standard solution as a sample solution. 2) Establish a coordinate system with the lg value of antigen concentration and ΔI as the horizontal and vertical axes. Substitute the lg value of antigen concentration and ΔI of N standard solutions into the coordinate system and fit to obtain a linear regression equation. 3) Using the test solution as the sample solution, calculate the ΔI of the test solution, substitute the ΔI of the test solution into the linear regression equation to obtain the lg value of the antigen concentration in the test solution, and then calculate the antigen concentration in the test solution.

9. The method according to claim 8, characterized in that, Methods for calculating ΔI for each sample solution include: ① The antigen is incubated on the electrochemical immunosensor according to claim 1 using a sample solution to obtain the sensor; the sensor is tested by differential pulse voltammetry to obtain the current value corresponding to the peak value of the DPV curve as I2; ② Calculate ΔI for this sample solution, ΔI = I1 - I2, where I1 is the current value corresponding to the peak value of the DPV curve obtained by differential pulse voltammetry testing of the electrochemical immunosensor.

10. The method according to claim 9, characterized in that, Prepare 19 standard solutions, each containing 1.6 mg / mL. -1 A mixture of tetanus toxin solution antigen and phosphate buffer, with an antigen concentration of 0.1 fg / mL. -1 0.3 fg·mL -1 1 fg·mL -1 3 fg·mL -1 10 fg·mL -1 30 fg·mL -1 100 fg·mL -1 300 fg·mL -1 1 pg·mL -1 3 pg·mL -1 10 pg·mL -1 30 pg·mL -1 100 pg·mL -1 300 pg·mL -1 1 ng·mL -1 3ng·mL -1 10 ng·mL -1 30 ng / mL -1 and 100 ng·mL -1 ; Establish a coordinate system, with the lg value of antigen concentration and ΔI as the x and y axes respectively. Substitute the lg values ​​of antigen concentration and ΔI of N standard solutions into the coordinate system, and fit the oxidative linear regression equation ΔI to obtain the equation. pa =4.35log c1+8.37, thus obtaining the reconstructed linear regression equation ΔI pc = -4.19log c1 - 11.04; where c1 is the antigen concentration; The electrochemical immunosensor was used as the working electrode for oxidation differential pulse voltammetry testing, and the oxidation peak current I1 of the differential pulse voltammetry test curve of the oxidation channel was obtained as 341.75 μA; the electrochemical immunosensor was used as the working electrode for reduction differential pulse voltammetry testing, and the reduction peak current I1 of the differential pulse voltammetry test curve of the reduction channel was obtained as -333.08 μA. 1.6 mg·mL -1 The tetanus toxin solution was diluted with phosphate buffer to a concentration of 100 ng / mL. -1 And used as the solution to be tested; Using the solution to be tested as the sample solution, calculate the current value corresponding to the peak value of the oxidation differential pulse voltammetry curve and the current value I2 corresponding to the peak value of the reduction differential pulse voltammetry curve; Calculate ΔI = I1 - I2 for the sample solution; Substitute the calculated ΔI into the oxidation linear regression equation ΔI pa =4.35log c1+8.37, substitute the calculated ΔI into the restored linear regression equation ΔI pc = -4.19log c1-11.04, the lg value of the antigen concentration in the test solution is obtained, and then the antigen concentration in the test solution is calculated.

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