An electrochemical immunoassay probe, a preparation method and application thereof, and an electrochemical direct immuno electrode

Abstract

The application provides an electrochemical immunoassay probe and a preparation method and application thereof, and an electrochemical direct immuno electrode, and belongs to the technical field of electrochemical detection.The electrochemical immunoassay probe provided by the application can be covalently connected to carbon nanomaterial and used as an immune probe of an electrochemical direct immuno test method.The electrochemical signal generated by the probe has a high signal-to-noise ratio, and can effectively improve the sensitivity of the electrochemical direct immuno test method, which is comparable to the commonly used electrochemical indirect immuno test method.The electrochemical immuno probe is firmly fixed on the surface of the electrode to construct an electrochemical direct immuno electrode, and the reagent-free operation of the electrochemical direct immuno test method can be realized, so that the operation difficulty can be greatly reduced and the required operation steps can be simplified.

Description

Technical Field

[0001] This invention relates to the field of electrochemical detection technology, and in particular to an electrochemical immunoassay probe, its preparation method and application, and an electrochemical direct immunoassay electrode. Background Technology

[0002] Electrochemical immunosensors are a new type of biosensor that combines immunoassay with electrochemical sensing technology. They are used for the analysis and research of trace immunogenic substances and have the advantages of high specificity, high sensitivity, short processing time, and trace detection. They have important application value in disease diagnosis, food hygiene, environmental monitoring and other fields.

[0003] Electrochemical immunoassays can be divided into direct and indirect methods. In the direct method, the sample being tested (e.g., serum, urine) directly binds to a specific antibody. This antibody typically binds specifically to the target antigen, forming an antigen-antibody complex and causing changes in the microstructure of the electrochemical analysis interface. This change results in a signal change in the electrochemical labeling within the analysis environment, allowing detection of the presence and quantity of the antigen-antibody complex. In the indirect method, the target antigen is first immobilized on the electrochemical analysis interface, followed by the addition of the sample. If antibodies specifically binding to the target antigen are present in the sample, they will bind to the antigen at the interface. Next, secondary antibodies, typically labeled with electrochemically active substances, are added and specifically bind to the test antibody. These labeled antibodies bind to the test antibody, forming an antibody-antibody complex. Finally, the presence of the antibody-antibody complex is determined by detecting the intensity of the electrochemical signal.

[0004] Electrochemical direct immunoassay suffers from the problem of large detection systems and inability to miniaturize due to the inability to immobilize probes. Another drawback is its reagent dependence; the immunoelectrode must be in a solution containing dissolved electrochemical immunoassay probes to function properly. This characteristic makes the method cumbersome, requiring a level of expertise and difficulty similar to ELISA, thus making it unsuitable for point-of-care testing (POCT) applications. Summary of the Invention

[0005] In view of this, the present invention aims to provide an electrochemical immunoassay probe, its preparation method and application, and an electrochemical direct immunoassay electrode. The electrochemical immunoassay probe provided by the present invention can be firmly immobilized on the electrode surface, enabling reagent-free operation of the electrochemical direct immunoassay method, significantly reducing operational difficulty and simplifying the required steps.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides an electrochemical immunoassay probe having the structure shown in Formula I:

[0008]

[0009] This invention provides a method for preparing the above-mentioned electrochemical immunoassay probe, comprising the following steps:

[0010] 4-Amino-N-(5-methyl-3-isoxazolyl)benzenesulfonamide was mixed with a mixed acid solution and subjected to an azide reaction to obtain an electrochemical immunoassay probe with the structure shown in Formula I.

[0011] The mixed acid solution is a mixture of nitrous acid and nitric acid.

[0012] Preferably, in the mixed acid solution, the molar ratio of nitrous acid to nitric acid is 1-4:1-4;

[0013] The total concentration of nitrous acid and nitric acid in the mixed acid solution is 0.05–2.2 mol / L.

[0014] Preferably, the azide reaction takes 25 to 300 minutes.

[0015] This invention provides the application of the above-mentioned electrochemical immunoassay probe in the detection of viruses for non-diagnostic purposes.

[0016] The present invention provides an electrochemical direct immunoassay electrode, comprising a screen-printed electrode modified with carbon nanomaterials, and an electrochemical immunoassay probe and a viral antibody modified on the surface of the carbon nanomaterials;

[0017] The electrochemical immunoassay probe is the aforementioned electrochemical immunoassay probe.

[0018] This invention provides a method for preparing an electrochemical direct immunoassay electrode, comprising the following steps:

[0019] A carbon nanomaterial dispersion was loaded onto the working surface of a screen-printed carbon electrode to obtain a screen-printed electrode modified with carbon nanomaterials.

[0020] An electrochemical immunoassay probe, phosphoric acid, nitric acid, and solvent were mixed to obtain a mixed solution.

[0021] The screen-printed electrode modified with the carbon nanomaterial was immersed in the mixed solution to obtain an electrode modified with an electrochemical immunoassay probe.

[0022] The viral antibody solution was loaded onto the electrode surface modified by the electrochemical immunoassay probe to modify and block unbound sites, thus obtaining an electrochemical direct immunoassay electrode.

[0023] Alternatively, an electrochemical immunoassay probe, carbon nanomaterials, phosphoric acid, nitric acid, and solvent can be ultrasonically mixed to obtain a dispersion of carbon nanomaterials modified with an electrochemical immunoassay probe.

[0024] The dispersion of carbon nanomaterials modified with the electrochemical immunoassay probe was loaded onto the working surface of a screen-printed carbon electrode to obtain an electrode modified with the electrochemical immunoassay probe.

[0025] The viral antibody solution is loaded onto the electrode surface modified by the electrochemical immunoassay probe, and the unbound sites are blocked to obtain the electrochemical direct immunoassay electrode.

[0026] Preferably, the volume ratio of phosphoric acid to nitric acid is 1-15:3-20.

[0027] This invention provides a method for electrochemical direct immunoassay of viruses for non-diagnostic purposes, comprising the following steps:

[0028] The sample solution to be tested is loaded onto the surface of the above-mentioned electrochemical direct immunoassay electrode and incubated to obtain the electrode to be tested;

[0029] Differential pulse voltammetry was performed on the electrode under test to obtain the peak current signal intensity value;

[0030] The viral load in the sample solution to be tested is obtained based on the peak current signal intensity value and a predetermined standard curve; the standard curve is a linear relationship curve between the peak current signal intensity value and the logarithm of the viral concentration.

[0031] Preferably, the parameters of the differential pulse voltammetry test include:

[0032] The scanning potential range is -0.5 to 1.0 V;

[0033] The excitation potential increment is 1–16 mV;

[0034] The excitation potential amplitude is 10–100 mV;

[0035] The pulse period is 10–100 ms;

[0036] The pulse width is 5–150 ms;

[0037] The sampling width is 2 to 100 ms.

[0038] This invention provides an electrochemical immunoassay probe (SMBA) with the structure shown in Formula I. The probe molecule provided by this invention can be covalently immobilized on carbon nanomaterials and function as an immunoassay probe in an electrochemical direct immunoassay. In this invention, under a specific acidic environment, the azide group carried by SMBA is in a high-energy activated state, which can spontaneously covalently bind to sp2 or individual sp3 sites on the carbon nanomaterial, ultimately forming a strong CN bond. The SMBA host structure is electrochemically active, generating corresponding redox waveforms at the carbon nanomaterial interface, thus serving as an electrochemical indicator signal. The electrochemical signal generated by the probe provided by this invention has a high signal-to-noise ratio, effectively improving the sensitivity of the electrochemical direct immunoassay, comparable to currently used electrochemical indirect immunoassay methods.

[0039] This invention provides a method for preparing the above-mentioned electrochemical immunoassay probe. This method is simple to operate and easy to achieve industrial-scale mass production.

[0040] This invention provides an electrochemical direct immunoassay electrode, comprising a screen-printed electrode modified with carbon nanomaterials, and an electrochemical immunoassay probe and a viral antibody modified on the surface of the carbon nanomaterials. This invention firmly immobilizes the electrochemical immunoassay probe on the electrode surface to construct an electrochemical direct immunoassay electrode, enabling reagent-free operation of the electrochemical direct immunoassay method. Therefore, it significantly reduces operational difficulty and simplifies the required steps.

[0041] This invention provides a non-diagnostic electrochemical direct immunoassay method for detecting viruses. The method uses the aforementioned electrochemical direct immunoassay electrode to test standard samples and pseudovirus samples of SARS-CoV-2 (SARS-CoV-2 S protein, N protein), influenza A (influenza A virus H protein, N protein), and influenza B (influenza B virus H protein, N protein). Results show that the peak current signal intensity value (I) of the SMBA is linearly correlated with the logarithm (lgC) of the viral antigen concentration in the sample, enabling accurate quantitative analysis at concentrations ranging from 2.7 pg / mL to 50.0 μg / mL, with a limit of detection (LOD) of 0.01 pg / mL.

[0042] Verification showed that the cyclic voltammetric signal of the SMBA electrochemical immunoassay probe provided by this invention did not change under continuous immersion conditions for 14 days, indicating successful modification. A comparison of the electrochemical direct immunoassay electrode of this invention for testing the N protein of SARS-CoV-2 with the classic electrochemical direct immunoassay method demonstrates the advantages of this invention in terms of operational complexity and simplicity of detection equipment. A comparison of the electrochemical direct immunoassay electrode of this invention for testing the N protein of influenza A virus with the classic electrochemical direct immunoassay method demonstrates the advantage of this method in terms of sensitivity. Attached Figure Description

[0043] Figure 1 The FTIR spectrum of 4-azido-N-(5-methyl-3-isoxazolyl)benzenesulfonamide obtained in Example 1;

[0044] Figure 2 The 4-azido-N-(5-methyl-3-isoxazolyl)benzenesulfonamide obtained in Example 1 1 HNMR spectrum;

[0045] Figure 3 The 4-azido-N-(5-methyl-3-isoxazolyl)benzenesulfonamide obtained in Example 1 13 CNMR spectrum;

[0046] Figure 4 The MS spectrum of 4-azido-N-(5-methyl-3-isoxazolyl)benzenesulfonamide obtained in Example 1;

[0047] Figure 5 The yields of 4-azido-N-(5-methyl-3-isoxazolyl)benzenesulfonamide under different ratios of nitrous acid and nitric acid;

[0048] Figure 6 These are the peak current intensity values ​​corresponding to sample solutions of different concentrations;

[0049] Figure 7 This is the standard curve for SMBA-based electrochemical immunoassay. Detailed Implementation

[0050] This invention provides an electrochemical immunoassay probe having the structure shown in Formula I:

[0051]

[0052] This invention provides a method for preparing the above-mentioned electrochemical immunoassay probe, comprising the following steps:

[0053] 4-Amino-N-(5-methyl-3-isoxazolyl)benzenesulfonamide was mixed with a mixed acid solution and subjected to an azide reaction to obtain an electrochemical immunoassay probe with the structure shown in Formula I.

[0054] The mixed acid solution is a mixture of nitrous acid and nitric acid.

[0055] In this invention, the 4-amino-N-(5-methyl-3-isoxazolyl)benzenesulfonamide (SM) is sourced from commercially available products.

[0056] In this invention, the molar ratio of nitrous acid to nitric acid in the mixed acid solution is preferably 1-4:1-4, more preferably 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, and most preferably 1:1, 1:2, or 1:3.

[0057] In this invention, the total concentration of nitrous acid and nitric acid in the mixed acid solution is preferably 0.05 to 2.2 mol / L, more preferably 0.1 to 2 mol / L, and even more preferably 0.5 to 1.5 mol / L.

[0058] As a specific embodiment of the present invention, the mass ratio of the 4-amino-N-(5-methyl-3-isoxazolyl)benzenesulfonamide to the volume ratio of the mixed acid solution is preferably 0.25 g: 100 mL.

[0059] The present invention does not have any special requirements for the mixing method; any mixing method known to those skilled in the art can be used, such as stirring.

[0060] In this invention, the temperature of the azide reaction is preferably 4 to 65°C, more preferably 10 to 30°C; the time is preferably 25 to 300 min, more preferably 150 min.

[0061] This invention provides the application of the aforementioned electrochemical immunoassay probe in the detection of viruses for non-diagnostic purposes. In this invention, the virus is preferably a respiratory virus, specifically one or more of SARS-CoV-2, influenza A virus, and influenza B virus.

[0062] In this invention, the above-mentioned electrochemical immunoassay probe can also be used as an electrochemical nucleic acid probe or an electrochemical aptamer probe.

[0063] The present invention provides an electrochemical direct immunoassay electrode, comprising a screen-printed electrode modified with carbon nanomaterials, and an electrochemical immunoassay probe and a viral antibody modified on the surface of the carbon nanomaterials;

[0064] The electrochemical immunoassay probe is the aforementioned electrochemical immunoassay probe.

[0065] In this invention, the carbon nanomaterial is preferably one or more of carbon nanotubes, graphene, and carbon black; in this invention, the length of the carbon nanotubes is preferably 0.5 to 2 μm, the diameter of the graphene is preferably 2 μm, and the particle size of the carbon black is preferably 60 nm.

[0066] In this invention, the viral antibody is preferably a COVID-19 antibody, an influenza A virus antibody, or an influenza B virus antibody. In this invention, the viral antibody is commercially available. As a specific embodiment of this invention, the viral antibody was purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.

[0067] This invention provides a method for preparing the above-mentioned electrochemical direct immunoelectrode, comprising the following steps:

[0068] A carbon nanomaterial dispersion was loaded onto the working surface of a screen-printed carbon electrode to obtain a screen-printed electrode modified with carbon nanomaterials.

[0069] An electrochemical immunoassay probe, phosphoric acid, nitric acid, and solvent were mixed to obtain a mixed solution.

[0070] The screen-printed electrode modified with the carbon nanomaterial was immersed in the mixed solution to obtain an electrode modified with an electrochemical immunoassay probe.

[0071] The viral antibody solution is loaded onto the electrode surface modified by the electrochemical immunoassay probe, and the unbound sites are blocked to obtain the electrochemical direct immunoassay electrode.

[0072] This invention loads a carbon nanomaterial dispersion onto the working surface of a screen-printed carbon electrode to obtain a screen-printed electrode modified with carbon nanomaterials.

[0073] In this invention, the method for preparing the carbon nanomaterial dispersion preferably includes the following steps:

[0074] Carbon nanomaterials are ultrasonically mixed with a solvent to obtain a carbon nanomaterial dispersion.

[0075] In this invention, the solvent for the carbon nanomaterial dispersion is preferably dimethyl sulfoxide (DMOS), and the concentration is preferably 0.01–0.5 mg / mL, more preferably 0.2 mg / mL. In this invention, the ultrasonic mixing power is preferably 20–100 W, more preferably 40 W, and the time is preferably 60–1000 s, more preferably 500 s.

[0076] In this invention, the loading method is preferably dropwise. After loading is completed, the product is preferably air-dried at room temperature.

[0077] This invention involves mixing an electrochemical immunoassay probe, phosphoric acid, nitric acid, and a solvent to obtain a mixed solution. In this invention, the solvent is preferably one or more of water, N,N-dimethylformamide (DMF), or dimethyl sulfoxide (DMSO).

[0078] In this invention, the concentration of the electrochemical immunoassay probe in the mixed solution is preferably 0.01–10 mmol / L, more preferably 0.1–1 mmol / L. In this invention, the volume concentration of the phosphoric acid is preferably 1–15%, more preferably 5–10%; the volume concentration of the nitric acid is preferably 3–20%, more preferably 10–15%.

[0079] The present invention does not have any special requirements for the mixing method; any mixing method known to those skilled in the art can be used, such as stirring.

[0080] In this invention, a screen-printed electrode modified with the carbon nanomaterial is immersed in the mixed solution to obtain an electrode modified with an electrochemical immunoassay probe. In this invention, the immersion is preferably performed under ice bath conditions, and the immersion time is preferably 30–360 min, more preferably 180 min.

[0081] In this invention, after soaking, the electrode is removed, washed with water, and dried.

[0082] This invention involves loading a viral antibody solution onto the surface of an electrode modified with an electrochemical immunoassay probe, thereby modifying and blocking unbound sites to obtain an electrochemical direct immunoassay electrode. In this invention, the concentration of the viral antibody solution is preferably 0.005–50 μg / mL, more preferably 0.02 μg / mL. In this invention, the loading method is preferably dropwise addition. In this invention, the modification temperature is preferably 4–45°C, more preferably 10–30°C; the modification time is preferably 120–3000 s, more preferably 720 s.

[0083] In this invention, the reagent used to block unbound sites is preferably a BSA solution.

[0084] In this invention, after obtaining the electrochemical direct immunoelectrode, it is preferable to wash and dry it with water.

[0085] Alternatively, the preparation method of the above-mentioned electrochemical direct immunoassay electrode includes the following steps:

[0086] Electrochemical immunoassay probes, carbon nanomaterials, phosphoric acid, nitric acid, and solvents were ultrasonically mixed to obtain a dispersion of carbon nanomaterials modified with electrochemical immunoassay probes.

[0087] The dispersion of carbon nanomaterials modified with the electrochemical immunoassay probe was loaded onto the working surface of a screen-printed carbon electrode to obtain an electrode modified with the electrochemical immunoassay probe.

[0088] The viral antibody solution is loaded onto the electrode surface modified by the electrochemical immunoassay probe, and the unbound sites are blocked to obtain the electrochemical direct immunoassay electrode.

[0089] This invention involves ultrasonically mixing an electrochemical immunoassay probe, carbon nanomaterials, phosphoric acid, nitric acid, and a solvent to obtain a dispersion of carbon nanomaterials modified with the electrochemical immunoassay probe. In this invention, the preferred mass ratio of the electrochemical immunoassay probe to the carbon nanomaterials is 14:1.

[0090] In this invention, the solvent is preferably one or more of water, DMF, and DMSO. In this invention, the concentration of the electrochemical immunoassay probe in the carbon nanomaterial dispersion modified with the electrochemical immunoassay probe is preferably 0.01–10 mmol / L, more preferably 0.1–1 mmol / L. In this invention, the volume concentration of the phosphoric acid is preferably 1–15%, more preferably 5–10%; the volume concentration of the nitric acid is preferably 3–20%, more preferably 10–15%. In this invention, the ultrasonic mixing power is preferably 20–100 W, more preferably 40 W, and the time is preferably 60–1000 s, more preferably 500 s.

[0091] This invention involves loading a dispersion of carbon nanomaterials modified with the electrochemical immunoassay probe onto the working surface of a screen-printed carbon electrode to obtain an electrode modified with the electrochemical immunoassay probe. In this invention, the loading method is preferably dropwise addition. After loading is completed, the electrode is preferably air-dried at room temperature.

[0092] In this invention, a viral antibody solution is loaded onto the surface of an electrode modified with the electrochemical immunoassay probe for modification, thereby blocking unbound sites and obtaining an electrochemical direct immunoassay electrode. The operation method for this step is the same as described above and will not be repeated here.

[0093] This invention provides a method for electrochemical direct immunoassay of viruses for non-diagnostic purposes, comprising the following steps:

[0094] The sample solution to be tested is loaded onto the surface of the above-mentioned electrochemical direct immunoassay electrode and incubated to obtain the electrode to be tested;

[0095] Differential pulse voltammetry was performed on the electrode under test to obtain the peak current signal intensity value;

[0096] The viral load in the sample solution to be tested is obtained based on the peak current signal intensity value and a predetermined standard curve; the standard curve is a linear relationship curve between the peak current signal intensity value and the logarithm of the viral concentration.

[0097] This invention involves loading a sample solution to be tested onto the surface of an electrochemical direct immunoassay electrode and incubating it to obtain the electrode to be tested. In this invention, the sample solution is a solution obtained after mucin degradation treatment. In this invention, the sample type is preferably a nasopharyngeal swab lysis mixture, water, or virus-contaminated items. In this invention, the loading method is preferably dropwise addition.

[0098] In this invention, the incubation temperature is preferably room temperature, and the incubation time is preferably ≥30s.

[0099] This invention performs differential pulse voltammetry (DPV) on the electrode under test to obtain the peak current signal intensity value. In this invention, the parameters for the differential pulse voltammetry preferably include:

[0100] The scanning potential range is -0.5 to 1.0 V;

[0101] The excitation potential increment is 1–16 mV;

[0102] The excitation potential amplitude is 10–100 mV;

[0103] The pulse period is 10–100 ms;

[0104] The pulse width is 5–150 ms;

[0105] The sampling width is 2 to 100 ms.

[0106] This invention obtains the virus content in the sample solution based on the peak current signal intensity value and a predetermined standard curve; the standard curve is a linear relationship curve between the peak current signal intensity value and the logarithm of the virus concentration. In this invention, the method for obtaining the standard curve preferably includes the following steps:

[0107] Provide viral antigen solutions of known concentrations;

[0108] Using viral antigen solutions of known gradient concentrations as test sample solutions, they were loaded onto the surface of the electrochemical direct immunoassay electrode. After incubation, differential pulse voltammetry was performed on the resulting test electrode to obtain peak current signal intensity values ​​corresponding to different concentrations of viral antigen solutions. A standard curve was plotted with the logarithm of the concentration of the viral antigen solution as the abscissa and the peak current signal intensity value as the ordinate.

[0109] In this invention, the viral antigen solutions with known concentrations are preferably 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, and 1 μg / mL, respectively.

[0110] The following detailed description, in conjunction with embodiments, provides an electrochemical immunoassay probe, its preparation method and application, and an electrochemical direct immunoassay electrode provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0111] Example 1: Preparation of Electrochemical Immunoassay Probes

[0112] 0.25 g of 4-amino-N-(5-methyl-3-isoxazolyl)benzenesulfonamide SM was mixed with 100 mL of a mixture of nitrous acid and nitric acid (total concentration of nitrous acid and nitric acid was 0.2 mol / L) and reacted. After 140 min, 4-azido-N-(5-methyl-3-isoxazolyl)benzenesulfonamide (SMBA) was generated.

[0113] The FTIR spectrum of the obtained 4-azido-N-(5-methyl-3-isoxazolyl)benzenesulfonamide is as follows: Figure 1 As shown.

[0114] The obtained 4-azido-N-(5-methyl-3-isoxazolyl)benzenesulfonamide 1 HNMR spectrum as follows Figure 2 As shown.

[0115] The obtained 4-azido-N-(5-methyl-3-isoxazolyl)benzenesulfonamide 13 CNMR spectrum as shown Figure 3 As shown.

[0116] The MS spectrum of the obtained 4-azido-N-(5-methyl-3-isoxazolyl)benzenesulfonamide is as follows: Figure 4 As shown.

[0117] The yields of 4-azido-N-(5-methyl-3-isoxazolyl)benzenesulfonamide under different molar ratios of nitrous acid and nitric acid are shown in Table 1. Figure 5 As shown.

[0118] Table 1. Yield of SMBA at different ratios of nitrite and nitric acid.

[0119] Nitrous acid: nitric acid ratio Yield % 4:1 21.4 3:1 29.2 2:1 37.4 1:1 67.9 1:2 98.1 1:3 43.3 1:4 35.2

[0120] From Table 1 and Figure 5 It can be seen that the ratio of nitrous acid to nitric acid has a significant impact on the yield of SMBA. The highest yield of SMBA and the complete reaction are achieved when the ratio of nitrous acid to nitric acid is 1:2. However, when the ratio of nitrous acid to nitric acid is imbalanced, the yield of SMBA decreases significantly. Therefore, it is necessary to control the ratio of nitrous acid to nitric acid at a level of 1:2 as much as possible.

[0121] Example 2

[0122] A dispersion with a concentration of 0.1 mg / mL was prepared by ultrasonic dispersion of graphene; the above dispersion was drop-coated onto the working surface of a screen-printed carbon electrode and dried at room temperature to obtain an electrode modified with carbon nanomaterials.

[0123] SMBA was mixed with H3PO4 and HNO3 with water to prepare a mixed solution. The concentrations of SMBA, H3PO4 and HNO3 in the mixed solution were 0.8 mM, 2 v / v%, and 4 v / v, respectively.

[0124] The electrode working surface modified with carbon nanomaterials was treated by immersing it in an ice bath for 360 minutes using the above mixed solution, and then washed and dried to complete the modification of carbon nanomaterials with SMBA.

[0125] A mixture of 0.05M PBS (pH 7.4) containing 0.02 μg / mL antibody was added to the surface of the SMBA modified electrode for adsorption. After a certain period of time, the electrode was removed, washed, and then surface-blocked with BSA solution. The electrode was then washed and dried to prepare the SMBA immunoelectrode.

[0126] Example 3

[0127] Standard IFA-N protein antigen (an influenza A antigen, purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) solutions of 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, and 1 μg / mL were prepared as sample solutions. The sample solutions were added to the electrode test surface and incubated at room temperature for at least 30 seconds before differential pulse voltammetry (DPV) was performed. The parameters included a scan potential range of -0.5 to 1.0 V, an excitation potential increment of 8 mV, an excitation potential amplitude of 60 mV, a pulse period of 10 ms, a pulse width of 40 ms, and a sampling width of 15 ms. The results were read, and the peak current intensity value of the SMBA was output as the signal.

[0128] Peak current intensity values ​​corresponding to sample solutions of different concentrations are as follows: Figure 6 As shown, the standard curve for electrochemical immunoassay based on SMBA is as follows: Figure 7 As shown in the figure, the SMBA immunoelectrode provided by this invention can achieve accurate quantitative analysis of viruses ranging from 2.7 pg / mL to 50.0 μg / mL.

[0129] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An electrochemical direct immunoassay electrode, comprising a screen-printed electrode modified with carbon nanomaterials, and an electrochemical immunoassay probe and a viral antibody modified on the surface of the carbon nanomaterials; The electrochemical immunoassay probe has the structure shown in Formula I: Equation I.

2. The electrochemical direct immunoelectrode according to claim 1, characterized in that, The preparation method of the electrochemical immunoassay probe includes the following steps: 4-Amino-N-(5-methyl-3-isoxazolyl)benzenesulfonamide was mixed with a mixed acid solution and subjected to an azide reaction to obtain an electrochemical immunoassay probe with the structure shown in Formula I. The mixed acid solution is a mixture of nitrous acid and nitric acid.

3. The electrochemical direct immunoelectrode according to claim 2, characterized in that, In the mixed acid solution, the molar ratio of nitrous acid to nitric acid is 1~4:1~4; The total concentration of nitrous acid and nitric acid in the mixed acid solution is 0.05~2.2 mol / L.

4. The electrochemical direct immunoelectrode according to claim 2, characterized in that, The azide reaction takes 25 to 300 minutes.

5. The method for preparing the electrochemical direct immunoelectrode according to claim 1, comprising the following steps: A carbon nanomaterial dispersion was loaded onto the working surface of a screen-printed carbon electrode to obtain a screen-printed electrode modified with carbon nanomaterials. An electrochemical immunoassay probe, phosphoric acid, nitric acid, and solvent were mixed to obtain a mixed solution. The screen-printed electrode modified with the carbon nanomaterial was immersed in the mixed solution to obtain an electrode modified with an electrochemical immunoassay probe. The viral antibody solution was loaded onto the electrode surface modified by the electrochemical immunoassay probe to modify and block unbound sites, thus obtaining an electrochemical direct immunoassay electrode. Alternatively, an electrochemical immunoassay probe, carbon nanomaterials, phosphoric acid, nitric acid, and solvent can be ultrasonically mixed to obtain a dispersion of carbon nanomaterials modified with an electrochemical immunoassay probe. The dispersion of carbon nanomaterials modified with the electrochemical immunoassay probe was loaded onto the working surface of a screen-printed carbon electrode to obtain an electrode modified with the electrochemical immunoassay probe. The viral antibody solution is loaded onto the electrode surface modified by the electrochemical immunoassay probe, and the unbound sites are blocked to obtain the electrochemical direct immunoassay electrode.

6. The preparation method according to claim 5, characterized in that, The volume ratio of phosphoric acid to nitric acid is 1~15:3~20.

7. A method for non-diagnostic electrochemical direct immunoassay of viruses, comprising the following steps: The sample solution to be tested is loaded onto the surface of the electrochemical direct immunoassay electrode described in claim 1 and incubated to obtain the electrode to be tested; the sample to be tested is water or a virus-contaminated item. Differential pulse voltammetry was performed on the electrode under test to obtain the peak current signal intensity value; The viral load in the sample solution to be tested is obtained based on the peak current signal intensity value and a predetermined standard curve; the standard curve is a linear relationship curve between the peak current signal intensity value and the logarithm of the viral concentration.

8. The method according to claim 7, characterized in that, The parameters of the differential pulse voltammetry test include: The scanning potential range is -0.5 to 1.0 V; The excitation potential increment is 1~16 mV; The excitation potential amplitude is 10~100 mV; The pulse period is 10~100 ms; The pulse width is 5~150 ms; The sampling width is 2~100 ms.

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