Potential resolution type electrochemical luminescence immunosensor and application thereof

Abstract

The invention discloses a potential resolution type electrochemical luminescence immunosensor and application thereof.The potential resolution type electrochemical luminescence immunosensor comprises an anode luminescence probe, a cathode luminescence probe and an electrochemical luminescence electrode, the anode luminescence probe comprises UiO66-NH2-coated luminol connected with an aflatoxin B1 antibody through a chemical bond, and the cathode luminescence probe comprises UiO66-NH2-coated luminol connected with an aflatoxin B1 antibody through a chemical bond; the cathode luminescence probe comprises Py-HOF which is connected with an ochratoxin A antibody through a chemical bond, the electrochemical luminescence electrode comprises an electrode body, and the electrode body is modified with Au (at) MoS2 which is connected with an aflatoxin B1 coating antigen and an ochratoxin A coating antigen through a chemical bond. The potential resolution type electrochemical luminescence immunosensor provided by the invention can realize high-selectivity and high-sensitivity detection of aflatoxin B1 and ochratoxin A in a single sample at the same time, and has important practical significance.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and particularly relates to a potential-resolved electrochemiluminescence immunosensor and its application. Background Art

[0002] In the fields of agricultural production and food processing, mycotoxin contamination poses a major threat to food safety and human health. Typical mycotoxins in food mainly include ochratoxin A (OTA), aflatoxin B1 (AFB1), deoxynivalenol (DON), fumonisin B1 (FB1), T-2 toxin, and zearalenone (ZEN). Among them, ochratoxin A and aflatoxin B1 are listed as key prevention and control targets due to their wide distribution and strong toxicity. Ochratoxin A is mainly produced by the metabolism of fungi of the genus Aspergillus and Penicillium, has thermal stability, and is difficult to be destroyed by conventional cooking or processing methods. It widely contaminates agricultural products such as corn, wheat, coffee beans, and nuts. This toxin has been classified as a Group 2B carcinogen by the International Agency for Research on Cancer (IARC), and long-term intake can cause nephrotoxicity, hepatotoxicity, and immunosuppressive effects. Aflatoxin B1 is produced by strains such as Aspergillus flavus, and its toxicity is hundreds of times stronger than that of potassium cyanide. It has a severe damaging effect on the liver in particular and is clearly listed as a Class 1 carcinogen by IARC. It is commonly found in grains (such as wheat and corn) in temperate and tropical regions with relatively high temperature and humidity.

[0003] Existing toxin detection methods mainly include fluorescence immunoassay (FIA), high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), surface-enhanced Raman spectroscopy (SERS), etc., but they have limitations such as high equipment cost, complex operation, low detection throughput, or the need for professional personnel to operate, and it is difficult to meet the requirements of rapid analysis and multi-component synchronous detection. Electrochemiluminescence technology combines the precise regulation of electrochemical analysis and the high sensitivity advantage of chemiluminescence detection, and has outstanding characteristics such as low instrument cost, fast response speed, low background signal, and easy integration. It has shown application potential in the fields of environmental pollutant monitoring, clinical disease biomarker detection, etc. However, current immunosensors based on the principle of electrochemiluminescence are mostly designed for a single target, while mycotoxins in actual samples often exist in the form of complex contamination. Traditional methods are difficult to achieve synchronous quantitative detection of high-risk toxins such as OTA and AFB1, resulting in an increased risk of missed detection and a reduced detection efficiency. Therefore, developing a multi-component electrochemiluminescence immunosensor that can detect OTA and AFB1 with high specificity, high sensitivity, and synchronously is of great significance for improving the efficiency of food safety detection and ensuring public health. Summary of the Invention

[0004] To solve the above technical problems, the object of the present invention is to provide a potential-resolved electrochemiluminescence immunosensor and its application. The potential-resolved electrochemiluminescence immunosensor uses Au@MoS2 nanomaterials to modify the electrode, UiO66-NH2@luminol and Py-HOF as two different electrochemiluminescence materials, and hydrogen peroxide as the only coreactant, and can simultaneously detect aflatoxin B1 and ochratoxin A.

[0005] The above object of the present invention is achieved by the following technical solutions:

[0006] A potential-resolved electrochemiluminescence immunosensor, comprising an anodic luminescence probe, a cathodic luminescence probe and an electrochemiluminescence electrode;

[0007] The anodic luminescence probe is prepared by the following method:

[0008] S11: Add zirconium tetrachloride, acetic acid and 2-aminoterephthalic acid to N,N-dimethylformamide (DMF), heat and react to obtain UiO66-NH2;

[0009] S12: React the UiO66-NH2 obtained in S11 with luminol to obtain UiO66-NH2@luminol;

[0010] S13: Add aflatoxin B1 antibody to the UiO66-NH2@luminol obtained in S12 for incubation to obtain the anodic luminescence probe;

[0011] The cathodic luminescence probe is prepared by the following method:

[0012] S21: Add 1,3,6,8-tetra(4-carboxyphenyl)pyrene to N,N-dimethylformamide, and then add an organic alcohol solvent, and react to obtain Py-HOF;

[0013] S22: Activate the Py-HOF obtained in S21 with a crosslinking agent, and then add ochratoxin A antibody for incubation to obtain the cathodic luminescence probe;

[0014] The electrochemiluminescence electrode is prepared by the following method:

[0015] S31: Heat and react thiourea and ammonium molybdate tetrahydrate to obtain molybdenum disulfide nanosheets, mix the molybdenum disulfide nanosheets with chloroauric acid aqueous solution, and then add trisodium citrate dihydrate, and react to obtain Au@MoS2;

[0016] S32: Modify the Au@MoS2 obtained in S31 on the electrode body, and incubate the modified electrode body with aflatoxin B1 coated antigen and ochratoxin A coated antigen to obtain the electrochemiluminescence electrode.

[0017] In the potential-resolved electrochemiluminescence immunosensor provided by the present invention, the anodic luminescence probe includes UiO66-NH2@luminol, and the UiO66-NH2@luminol is chemically bonded with an aflatoxin B1 antibody; the cathodic luminescence probe includes Py-HOF, and the Py-HOF is chemically bonded with an ochratoxin A antibody; the electrochemiluminescence electrode includes an electrode body, and the electrode body is modified with Au@MoS2, and the Au@MoS2 is chemically bonded with an aflatoxin B1 coated antigen and an ochratoxin A coated antigen. The present invention successfully prepares two electrochemiluminescence probes and an electrochemiluminescence electrode modified with a nanocomposite material. The potential-resolved electrochemiluminescence immunosensor constructed based on these has a significant advantage of simultaneously detecting two target substances compared with the traditional electrochemiluminescence immunosensor.

[0018] Further, in S11, the temperature of the heating reaction is 110-130 °C, and the time of the heating reaction is 22-26 h.

[0019] Further, in S12, the temperature of the reaction is 70-90 °C, and the time of the reaction is 4-8 h.

[0020] Further, in S13, the mass ratio of UiO66-NH2@luminol to the aflatoxin B1 antibody is (80-120):1, preferably (90-110):1, and more preferably 100:1.

[0021] In the specific embodiment, the preparation method of the anodic luminescence probe includes the following steps:

[0022] S11: Add zirconium tetrachloride (ZrCl4), acetic acid, and 2-aminoterephthalic acid (NH2-BDC) to N,N-dimethylformamide and ultrasonicate for 20-40 min. Put the obtained mixed solution into a polytetrafluoroethylene-lined stainless steel autoclave and heat-react at 110-130 °C for 22-26 h. After the reaction is completed, centrifuge to collect the product, wash it with ethanol, and dry it at 60-70 °C to obtain a yellow powder of UiO66-NH2;

[0023] S12: Dissolve the yellow powder of UiO66-NH2 obtained in S11 and luminol in water, stir and react at 70-90 °C for 4-8 h, cool to room temperature, centrifuge to collect the product, wash it with ethanol, and place it in a vacuum drying oven at 60-70 °C overnight to obtain a powder of UiO66-NH2@luminol;

[0024] S13: Dissolve the UiO66-NH2@luminol powder obtained in S12 in water to obtain a UiO66-NH2@luminol solution. Add an aflatoxin B1 antibody solution to the UiO66-NH2@luminol solution for incubation, so that the aflatoxin B1 antibody is connected to UiO66-NH2@luminol through an amide bond to obtain the anodic luminescence probe.

[0025] Further, in S13, the concentration of UiO66-NH2@luminol in the UiO66-NH2@luminol solution is 0.5 - 1.5 mg / mL.

[0026] Further, in S13, the concentration of the aflatoxin B1 antibody in the aflatoxin B1 antibody solution is 6 - 14 μg / mL, preferably 8 - 14 μg / mL, and more preferably 8 - 12 μg / mL.

[0027] Further, in S13, the volume ratio of the UiO66-NH2@luminol solution to the aflatoxin B1 antibody solution is (0.8 - 1.2):1.

[0028] Further, in S21, the organic alcohol solvent is methanol.

[0029] Specifically, in S21, add 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene to N,N-dimethylformamide at room temperature, and then add methanol and stir. After standing, react to obtain Py-HOF.

[0030] Further, the stirring time is 0.5 - 2 min, and the standing time is 22 - 26 h.

[0031] Further, in S22, the cross-linking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0032] Further, in S22, the mass ratio of Py-HOF to the ochratoxin A antibody is (300 - 400):1, preferably (300 - 350):1, and more preferably 1000:3.

[0033] In a specific embodiment, the preparation method of the cathodic luminescence probe includes the following steps:

[0034] S21: Add 1,3,6,8 - tetra(4 - carboxyphenyl)pyrene (H4TBAPY) to N,N - dimethylformamide, then add methanol, stir for 0.5 - 2 min, age at room temperature for 22 - 26 h, centrifuge to collect the product to obtain yellow crystals of Py - HOF; wash with ethanol and then dry under vacuum at room temperature to obtain yellow powder of Py - HOF;

[0035] S22: Dissolve the yellow powder of Py - HOF obtained in S21 in water, add a cross - linker to the obtained Py - HOF solution for activation, add an ochratoxin A antibody solution to the activated Py - HOF solution for incubation, so that the ochratoxin A antibody is connected to Py - HOF through an amide bond to obtain the cathode luminescence probe.

[0036] Further, in S22, the concentration of Py - HOF in the Py - HOF solution is 1.5 - 2.5 mg / mL.

[0037] Further, in S22, add 1 - ethyl - (3 - dimethylaminopropyl)carbodiimide (EDC) solution and N - hydroxysuccinimide (NHS) solution to the obtained Py - HOF solution for activation.

[0038] Further, the concentration of EDC in the EDC solution is 7.5 - 8.5 mg / mL.

[0039] Further, the concentration of NHS in the NHS solution is 7.5 - 8.5 mg / mL.

[0040] Further, the volume ratio of the Py - HOF solution, EDC solution and NHS solution is 1:1:1.

[0041] Further, in S22, the concentration of ochratoxin A antibody in the ochratoxin A antibody solution is 3 - 7 μg / mL, preferably 5 - 7 μg / mL.

[0042] Further, in S22, the volume ratio of the activated Py - HOF solution to the ochratoxin A antibody solution is (0.8 - 1.2):1.

[0043] Further, in S31, the temperature of the heating reaction is 180 - 220 °C, and the time of the heating reaction is 22 - 26 h.

[0044] Specifically, in S31, heat - react thiourea and ammonium molybdate tetrahydrate to obtain molybdenum disulfide nanosheets, first heat - stir the molybdenum disulfide nanosheets with chloroauric acid aqueous solution to obtain an intermediate product; the intermediate product reacts with trisodium citrate dihydrate to obtain Au@MoS2.

[0045] Furthermore, the temperature of the heating and stirring is 35 - 55°C, and the time of the heating and stirring is 1 - 3 h.

[0046] Furthermore, the temperature of the reaction between the intermediate product and trisodium citrate dihydrate is 90 - 110°C, and the time is 30 - 50 min.

[0047] Furthermore, in S32, the electrode body is a glassy carbon electrode (GCE).

[0048] Furthermore, in S32, the mass ratio of Au@MoS2, aflatoxin B1 - coated antigen, and ochratoxin A - coated antigen is 300:(2 - 3):(1 - 2), preferably 600:5:3.

[0049] Specifically, in S32, Au@MoS2 is directly dropped onto the surface of the polished electrode body. After drying, aflatoxin B1 - coated antigen and ochratoxin A - coated antigen are combined and dropped onto the surface of the modified electrode body for incubation to obtain the electrochemiluminescence electrode.

[0050] Furthermore, in S13, S22, and S32, the incubation temperature is 3 - 5°C, and the incubation time is 10 - 14 h.

[0051] Furthermore, in S13, S22, and S32, after incubation, a bovine serum albumin (BSA) solution is added for blocking.

[0052] Furthermore, the blocking temperature is 3 - 5°C, and the time is 0.5 - 1.5 h.

[0053] Furthermore, the concentration of the bovine serum albumin (BSA) solution added after incubation is 4 - 6 wt.%.

[0054] In the specific embodiment, the preparation method of the electrochemiluminescence electrode includes the following steps:

[0055] S31: Under vigorous stirring, thiourea and ammonium molybdate tetrahydrate ((NH4)6Mo7O 24(NH4)2MoS4·4H2O was dissolved in water, and then the resulting mixture was transferred to a stainless-steel autoclave with a polytetrafluoroethylene liner. The reaction was heated at 180 - 220 °C for 22 - 26 hours, and then naturally cooled to room temperature. The final product was centrifuged, washed with ethanol, and dried at 60 - 70 °C to obtain molybdenum disulfide nanosheets. The molybdenum disulfide nanosheets were added to an aqueous solution of chloroauric acid. After continuously stirring at 35 - 55 °C for 1 - 3 h, the temperature was raised to boiling, and then a solution of trisodium citrate dihydrate (Na3C6H5O7·2H2O) was rapidly injected under boiling and stirring conditions. The reduction reaction was carried out at 90 - 110 °C for 30 - 50 min. The product was centrifuged and washed, and then centrifuged at 5000 - 6000 rpm for 5 - 10 min to collect the Au@MoS2 nanocomposite material, which was placed in a vacuum drying oven at 60 - 70 °C and dried overnight to obtain Au@MoS2 powder;

[0056] S32: The Au@MoS2 powder obtained in S31 was dissolved in water, and the resulting Au@MoS2 suspension was dropped onto the electrode body. Then, a solution of aflatoxin B1 - coated antigen and a solution of ochratoxin A - coated antigen were dropped onto the modified electrode body, so that Au@MoS2 was connected to the aflatoxin B1 - coated antigen and the ochratoxin A - coated antigen through Au - N bonds to obtain the electrochemical luminescence electrode.

[0057] In the electrochemical luminescence electrode provided by the present invention, Au nanoparticles are in - situ grown on the surface of molybdenum disulfide nanosheets to obtain Au@MoS2 nanocomposite materials, which not only improve the electron transfer efficiency but also provide a larger surface area for antigen immobilization.

[0058] Further, in S31, the concentration of chloroauric acid in the aqueous solution of chloroauric acid is 0.5 - 1.5 mg / mL.

[0059] Further, in S31, the concentration of trisodium citrate dihydrate in the solution of trisodium citrate dihydrate is 250 - 350 mg / mL.

[0060] Further, in S32, the concentration of Au@MoS2 in the Au@MoS2 suspension is 0.2 - 1.0 mg / mL, preferably 0.4 - 0.8 mg / mL.

[0061] Further, in S32, the concentration of aflatoxin B1 - coated antigen in the solution of aflatoxin B1 - coated antigen is 4 - 12 μg / mL, preferably 8 - 12 μg / mL.

[0062] Further, in S32, the concentration of ochratoxin A - coated antigen in the solution of ochratoxin A - coated antigen is 1 - 8 μg / mL, preferably 4 - 8 μg / mL.

[0063] Further, in S32, the volume ratio of the Au@MoS2 suspension to the aflatoxin B1-coated antigen solution is (1.8 - 2.2):1.

[0064] Further, in S32, the volume ratio of the Au@MoS2 suspension to the ochratoxin A-coated antigen solution is (1.8 - 2.2):1.

[0065] The present invention protects the application of the above potential-resolved electrochemiluminescence immunosensor in detecting aflatoxin B1 and ochratoxin A.

[0066] Further, the method for detecting aflatoxin B1 and ochratoxin A using a potential-resolved electrochemiluminescence immunosensor includes the following steps:

[0067] S41: Mix and incubate the anode luminescent probe with aflatoxin B1 standard solutions at different concentrations to obtain mixed solution B, mix and incubate the cathode luminescent probe with ochratoxin A standard solutions at different concentrations to obtain mixed solution A, and then mix the mixed solution B and mixed solution A and incubate them with the electrochemiluminescence electrode to obtain the assembled potential-resolved electrochemiluminescence immunosensor;

[0068] S42: Use the assembled potential-resolved electrochemiluminescence immunosensor in S41 as the working electrode, perform cyclic voltammetry scanning using a three-electrode system, record the luminescence intensity-time curve, establish the relationship between the luminescence intensity and the logarithm of the aflatoxin B1 concentration and the logarithm of the ochratoxin A concentration, and obtain the linear regression equation;

[0069] S43: Mix and incubate the anode luminescent probe and the cathode luminescent probe with the test solution respectively, and then incubate the two obtained mixed solutions with the electrochemiluminescence electrode to obtain the assembled potential-resolved electrochemiluminescence immunosensor. Use the assembled potential-resolved electrochemiluminescence immunosensor in this step as the working electrode, perform cyclic voltammetry scanning using a three-electrode system, and based on the measured luminescence intensity-time curve, combine the linear regression equation in S42 to obtain the concentrations of aflatoxin B1 and ochratoxin A in the test solution.

[0070] Further, in S42 and S43, the detection solution of the three-electrode system is a buffer solution containing hydrogen peroxide. Among them, hydrogen peroxide is the only coreactant.

[0071] Further, the buffer solution is a PBS buffer solution, and the concentration of the PBS buffer solution is 0.1 mol / L.

[0072] Further, the volume ratio of hydrogen peroxide to the buffer solution is (1 - 3):250, preferably 1:99.

[0073] The present invention provides a potential-resolved electrochemiluminescence immunosensor based on UiO66-NH2@luminol and Py-HOF as luminophores and hydrogen peroxide as the only coreactant, which realizes the simultaneous sensitive detection of aflatoxin B1 and ochratoxin A in a single sample and has important significance in food safety monitoring.

[0074] Furthermore, the concentration of the aflatoxin B1 standard solution is 0.08 - 1000 ng / mL; the concentration of the ochratoxin A standard solution is 0.006 - 1000 ng / mL.

[0075] In a specific embodiment, the method for detecting aflatoxin B1 and ochratoxin A using an electrochemiluminescence immunosensor includes the following steps:

[0076] S41: Centrifuge the solution of the anode luminescent probe to remove the supernatant, and then mix and incubate the precipitate with different concentrations of aflatoxin B1 standard solutions respectively to obtain mixed solution B; centrifuge the solution of the cathode luminescent probe to remove the supernatant, and then mix and incubate the precipitate with different concentrations of ochratoxin A standard solutions respectively to obtain mixed solution A. Mix the mixed solution B and the mixed solution A and contact them with the electrochemiluminescence electrode, so that the aflatoxin B1 antibody reacts with the aflatoxin B1-coated antigen, and the ochratoxin A antibody reacts with the ochratoxin A-coated antigen. After the reaction is complete, wash the anode luminescent probe, cathode luminescent probe, aflatoxin B1, and ochratoxin A that are not bound to the electrochemiluminescence electrode with a buffer solution to obtain the assembled potential-resolved electrochemiluminescence immunosensor;

[0077] S42: Use the assembled potential-resolved electrochemiluminescence immunosensor in S41 as the working electrode, perform cyclic voltammetry scanning using a three-electrode system, record the luminescence intensity-time curve, establish the relationship between the luminescence intensity and the logarithm of the aflatoxin B1 concentration and the logarithm of the ochratoxin A concentration, and obtain a linear regression equation;

[0078] S43: Mix and incubate the anode luminescent probe and the cathode luminescent probe with the test solution respectively, and then incubate the two obtained mixed solutions with the electrochemiluminescence electrode. After the incubation is complete, wash the electrochemiluminescence electrode with a buffer solution to obtain the assembled potential-resolved electrochemiluminescence immunosensor. Use the assembled potential-resolved electrochemiluminescence immunosensor in this step as the working electrode, perform cyclic voltammetry scanning using a three-electrode system, and based on the measured luminescence intensity-time curve, combine the linear regression equation in S42 to obtain the concentrations of aflatoxin B1 and ochratoxin A in the test solution.

[0079] Further, in S42 and S43, the specific conditions for cyclic voltammetry scanning using a three-electrode system are as follows: in the potential range of -2V to 0.4V, the high voltage of the photomultiplier tube is -650V, the amplification factor is 4, and the scanning rate is 0.5V / s.

[0080] When cyclic voltammetry scanning is performed using a three-electrode system, the ECL signal changes with the change of the concentration of aflatoxin B1 standard solution and the concentration of ochratoxin A standard solution.

[0081] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0082] 1. The electrochemiluminescence immunosensor provided by the present invention includes a cathode luminescence probe, an anode luminescence probe, and an electrochemiluminescence electrode. Among them, UiO66-NH2@luminol is used as the anode luminescence material and has obvious luminescence at the anode but no obvious luminescence at the cathode. Py-HOF is used as the cathode luminescence material and has obvious luminescence at the cathode but no obvious luminescence at the anode. The potential-resolved electrochemiluminescence immunosensor constructed with UiO66-NH2@luminol as the anode luminescence material and Py-HOF as the cathode luminescence material will generate corresponding ECL emissions at different potentials and do not interfere with each other.

[0083] 2. The present invention provides a potential-resolved electrochemical immunosensor based on UiO66-NH2@luminol and Py-HOF as luminophores and hydrogen peroxide as the only coreactant. Using hydrogen peroxide (H2O2) as the coreactant can simultaneously obtain strong cathode and anode luminescence, and can avoid introducing multiple coreactants to interfere with the ECL reaction.

[0084] 3. The present invention uses the specific recognition of aflatoxin B1 antibody and antigen and the specific recognition of ochratoxin A antibody and antigen to quantitatively detect aflatoxin B1 and ochratoxin A. This potential-resolved electrochemiluminescence immunosensor can achieve high-selectivity and high-sensitivity detection of aflatoxin B1 and ochratoxin A in a single sample simultaneously, which has important practical significance. Description of the Drawings

[0085] Figure 1 It is a schematic diagram of the principle for the construction of the potential-resolved electrochemiluminescence immunosensor provided by the present invention and the detection of aflatoxin B1 and ochratoxin A.

[0086] Figure 2TEM and SEM images of the Py-HOF, UiO66-NH2@luminol, and Au@MoS2 nanocomposites in Example 1; where A is the TEM image of Py-HOF, B is the TEM image of UiO66-NH2@luminol, C is the TEM image of Au@MoS2 nanocomposite, D is the SEM image of Py-HOF, E is the SEM image of UiO66-NH2@luminol, and F is the SEM image of Au@MoS2 nanocomposite.

[0087] Figure 3 ECL intensity-potential curve graphs obtained by using an anodic electrochemiluminescence electrode, a cathodic electrochemiluminescence electrode, and an anodic / cathodic electrochemiluminescence electrode as working electrodes for testing; where A is the ECL intensity-potential curve graph obtained by using an anodic electrochemiluminescence electrode as the working electrode for testing, B is the ECL intensity-potential curve graph obtained by using a cathodic electrochemiluminescence electrode as the working electrode for testing, and Figure C is the ECL intensity-potential curve graph obtained by using an anodic / cathodic electrochemiluminescence electrode as the working electrode for testing.

[0088] Figure 4 Test result graphs of ECL tests using a glassy carbon electrode or an anodic / cathodic electrochemiluminescence electrode as the working electrode; where A is the ECL intensity graph, B is the ECL intensity-potential curve graph, the detection condition of curve a is using a glassy carbon electrode as the working electrode and 5 mL of 0.1 mol / L PBS buffer solution added with 50 μL of H2O2 as the detection solution, the detection condition of curve b is using an anodic / cathodic electrochemiluminescence electrode as the working electrode and 5 mL of 0.1 mol / L PBS buffer solution as the detection solution, and the detection condition of curve c is using an anodic / cathodic electrochemiluminescence electrode as the working electrode and 5 mL of 0.1 mol / L PBS buffer solution added with 50 μL of H2O2 as the detection solution.

[0089] Figure 5 Three-dimensional curve graphs of ECL intensity-time and ECL intensity-concentration for aflatoxin B1 standard solutions and ochratoxin A standard solutions with different concentrations.

[0090] Figure 6 Standard curve graph of ECL intensity versus the logarithm of aflatoxin B1 concentration.

[0091] Figure 7 Standard curve graph of ECL intensity versus the logarithm of ochratoxin A concentration.

[0092] Figure 8Optimization diagrams of the concentrations of Au@MoS2, AFB1 Ab and OTA Ab, AFB1 Ae and OTA Ae, and the volume of H2O2; among them, A is the optimization diagram of the concentration of Au@MoS2, B is the optimization diagram of the concentrations of AFB1 Ab and OTA Ab, C is the optimization diagram of the concentrations of AFB1 Ae and OTA Ae, and D is the optimization diagram of the volume of H2O2. Detailed implementation manners

[0093] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this invention belongs. The terms used in the description of this invention herein are for the purpose of describing specific embodiments only and are not intended to limit the invention.

[0094] The following further describes the present invention with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the embodiments given are not intended to limit the present invention.

[0095] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0096] In the following embodiments of the present invention, both the OTA-coated antigen and the OTA antibody were donated by the research group of Professor Deng Anping of Soochow University. Among them, the OTA antibody is a monoclonal antibody secreted by the OTA hybridoma cell line OTA-8B10 (Hybridoma cell line OTA-8B10, deposited at the China Center for Type Culture Collection on April 11, 2023, deposit number CCTCC No: C202384, deposit address: Wuhan University, Bayi Road, Hongshan District, Wuhan, Hubei Province), and the OTA hybridoma cell line was prepared from the OTA antigen. The OTA-Ab has extremely high detection sensitivity (IC 50 in the ELISA method is 0.035 ng / mL) and a relatively high working titer. This OTA-coated antigen was prepared by the carbodiimide method, which is formed by dissolving ochratoxin A (hapten), N-hydroxysuccinimide, and N,N-dicyclohexylcarbodiimide in anhydrous tetrahydrofuran, reacting, drying under nitrogen, dissolving in N,N-dimethylformamide for activation, and then coupling with carrier protein.

[0097] In the following embodiments of the present invention, both the AFB1-coated antigen and the AFB1 antibody were donated by the research group of Professor Deng Anping of Soochow University. Among them, the AFB1 antibody is a monoclonal antibody secreted by the aflatoxin B1 hybridoma cell line AF-6C6A (Hybridoma cell line AF-6C6A, deposited at the China Center for Type Culture Collection on February 16, 2025, with the deposit number CCTCC No: C202561 and the deposit address being Wuhan University, Bayi Road, Hongshan District, Wuhan, Hubei Province). The aflatoxin B1 hybridoma cell line was prepared from the AFB1-coated antigen. The AFB1 antibody has extremely high detection sensitivity (IC 50 by ELISA method is 0.090 ng / mL) and a relatively high working titer. This AFB1-coated antigen was prepared by the carbodiimide method. Aflatoxin B1 (hapten) and aminooxyacetate were dissolved in pyridine and reacted, then dried under nitrogen and dissolved in N,N-dimethylformamide for activation and then coupled with carrier protein.

[0098] Example 1

[0099] A potential-resolved electrochemiluminescence immunosensor, comprising an anode luminescence probe, a cathode luminescence probe and an electrochemiluminescence electrode;

[0100] The preparation method of the anode luminescence probe (UiO66-NH2@luminol-AFB1 Ab probe) comprises the following steps:

[0101] S11: Add 80 mg of ZrCl4, 4.8 mL of acetic acid and 60 mg of NH2-BDC to 32 mL of DMF, ultrasonicate for 30 min, put the obtained mixed solution into a stainless steel autoclave with a polytetrafluoroethylene liner, heat and react at 120 °C for 24 h. After the reaction is completed, cool to room temperature and then centrifuge to collect the product, wash it three times with ethanol, and then dry it at 60 °C for 12 h to obtain yellow UiO66-NH2 powder;

[0102] S12: Dissolve 25 mg of yellow UiO66-NH2 powder and 5 mg of luminol in 20 mL of deionized water, stir and react at 80 °C for 6 h, cool to room temperature and then centrifuge to collect the product, wash it three times with ethanol, and then place it in a vacuum drying oven at 60 °C overnight to obtain UiO66-NH2@luminol powder;

[0103] S13: Dissolve the UiO66-NH2@luminol powder obtained in S12 in water to obtain a UiO66-NH2@luminol solution with a concentration of 1 mg / mL. Add 5 μL of the UiO66-NH2@luminol solution and 5 μL of aflatoxin B1 antibody (AFB1 Ab) solution with a concentration of 10 μg / mL into a centrifuge tube, mix evenly, and place it in an environment at 4 °C for 12 h to connect the aflatoxin B1 antibody to UiO66-NH2@luminol through an amide bond. After the reaction is complete, add 5 μL of 5% BSA dropwise into the centrifuge tube, place it in an environment at 4 °C for blocking for 1 h, and finally centrifuge it and redissolve it in deionized water to obtain the UiO66-NH2@luminol-AFB1 Ab probe.

[0104] The preparation method of the cathodoluminescence probe (Py-HOF-OTA Ab probe) includes the following steps:

[0105] S21: Add 80 mg of H4TBAPY into 12 mL of DMF, then add 48 mL of MeOH, stir for 1 min, age at room temperature for 24 h, centrifuge to collect the product, wash it three times with ethanol, and vacuum dry at room temperature to obtain Py-HOF yellow powder;

[0106] S22: Dissolve the Py-HOF yellow powder obtained in S21 in water. First, add 100 μL of 8 mg / mL EDC and 100 μL of 8 mg / mL NHS to 100 μL of the Py-HOF solution with a concentration of 2 mg / mL obtained to activate the carboxyl group, then shake it in the dark for 1.5 h. After centrifuging the activated Py-HOF solution, redissolve it with 100 μL of ultrapure water. Add 5 μL of the activated Py-HOF solution and 5 μL of ochratoxin A antibody (OTA Ab) solution with a concentration of 6 μg / mL into a centrifuge tube, mix evenly, and place it in an environment at 4 °C for 12 h to connect the ochratoxin A antibody to Py-HOF through an amide bond. After the reaction is complete, add 5 μL of 5% BSA dropwise into the centrifuge tube, place it in an environment at 4 °C for blocking for 1 h, and finally centrifuge it and redissolve it in deionized water to obtain the Py-HOF-OTA Ab probe.

[0107] The preparation method of the electrochemiluminescence electrode includes the following steps:

[0108] S31: Under vigorous stirring, add 2.283 g of thiourea and 1.235 g of (NH4)6Mo7O 24·4H2O was dissolved in 35 mL of deionized water, and then the obtained mixture was transferred to a 100 mL polytetrafluoroethylene-lined stainless-steel autoclave and heated at 200 °C for 24 h. Then it was naturally cooled to room temperature. The final product was centrifuged, washed three times with ethanol, and then dried at 60 °C for 12 h to obtain molybdenum disulfide nanosheets. 50 mg of the molybdenum disulfide nanosheets were added to 30 mL of an aqueous HAuCl4 solution (1.0 mg / mL), and after continuous stirring at 45 °C for 2 h, the temperature was raised to boiling. Then, 220 μL of a Na3C6H5O7·2H2O solution (300 mg / mL) was quickly injected under boiling and stirring conditions, and the reduction reaction was carried out at 100 °C for 40 min. After cooling to room temperature, the product was collected by centrifugation, carefully washed three times with deionized water, and placed in a vacuum drying oven at 60 °C to dry overnight to obtain Au@MoS2 powder;

[0109] S32: Polish the GCE (glassy carbon electrode) with α-Al2O3 on suede to make its surface as smooth as a mirror, and then rinse it with ultrapure water. Dissolve the Au@MoS2 powder obtained in S31 in water, and drop 10 μL of the obtained Au@MoS2 suspension (0.6 mg / mL) onto the surface of the GCE and let it dry naturally. Drop 5 μL of a solution of aflatoxin B1-coated antigen (AFB1 Ae) with a concentration of 10 μg / mL and 5 μL of a solution of ochratoxin A-coated antigen (OTA Ae) with a concentration of 6 μg / mL onto the modified electrode body, and incubate it in the refrigerator for 12 h to connect Au@MoS2 to the aflatoxin B1-coated antigen and the ochratoxin A-coated antigen through Au-N bonds. Subsequently, use BSA to eliminate the non-specific binding effect. After blocking for 1 h, rinse the surface of the electrode with 0.01 mol / L PBS to wash away the unbound aflatoxin B1-coated antigen and ochratoxin A-coated antigen to obtain the electrochemical luminescence electrode.

[0110] Figure 1 It is a schematic diagram of the principle for the construction of the potential-resolved type electrochemical luminescence immunosensor provided by the present invention and the detection of aflatoxin B1 and ochratoxin A.

[0111] Figure 2 It is the transmission electron microscope (TEM) images and scanning electron microscope (SEM) images of the Py-HOF, UiO66-NH2@luminol, and Au@MoS2 nanocomposites in Example 1; among them, A is the TEM image of Py-HOF, B is the TEM image of UiO66-NH2@luminol, C is the TEM image of the Au@MoS2 nanocomposite, D is the SEM image of Py-HOF, E is the SEM image of UiO66-NH2@luminol, and F is the SEM image of the Au@MoS2 nanocomposite.

[0112] Test Example 1

[0113] Perform ECL tests on the anodic luminescence probe and cathodic luminescence probe in Example 1. The test method and test results are as follows:

[0114] (1) Centrifuge the solution of UiO66-NH2@luminol-AFB1 Ab probe, aspirate the supernatant with a pipette, mix the precipitate with deionized water to obtain a mixed solution B, drop the mixed solution B onto the electrochemiluminescence electrode, place it in the refrigerator for incubation. After the incubation process is completed, wash the electrochemiluminescence electrode with PBS buffer to remove the unbound UiO66-NH2@luminol-AFB1 Ab probe on the electrochemiluminescence electrode, and obtain the anodic electrochemiluminescence electrode; centrifuge the solution of Py-HOF-OTA Ab probe, aspirate the supernatant with a pipette, mix the precipitate with deionized water to obtain a mixed solution A, drop the mixed solution A onto the electrochemiluminescence electrode, place it in the refrigerator for incubation. After the incubation process is completed, wash the electrochemiluminescence electrode with PBS buffer to remove the unbound Py-HOF-OTA Ab probe on the electrochemiluminescence electrode, and obtain the cathodic electrochemiluminescence electrode; centrifuge the solutions of UiO66-NH2@luminol-AFB1 Ab probe and Py-HOF-OTA Ab probe respectively, aspirate the supernatant with a pipette, mix the precipitates with deionized water to obtain mixed solutions respectively, mix the two mixed solutions and drop them onto the electrochemiluminescence electrode, place it in the refrigerator for incubation. After the incubation process is completed, wash the electrochemiluminescence electrode with PBS buffer to remove the unbound UiO66-NH2@luminol-AFB1 Ab probe and Py-HOF-OTA Ab probe on the electrochemiluminescence electrode, and obtain the anodic / cathodic electrochemiluminescence electrode.

[0115] (2) Use the anodic electrochemiluminescence electrode obtained in step (1) as the working electrode, the platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode. Perform cyclic voltammetry scanning using a three-electrode system. In the ECL detection solution (5 mL of 0.1 mol / L PBS buffer added with 50 μL of H2O2), the test conditions are in the potential range of -2V to 0.4V, the high voltage of the photomultiplier tube is -650V, the amplification factor is 4, and the scanning rate is 0.5V / s. Record the ECL intensity-potential curve and establish a relationship diagram between the ECL intensity and the potential. The test results are as Figure 3 shown in A. The anodic luminescence probe has an obvious ECL signal at about 0.4V (relative to Ag / AgCl), and there is no obvious signal at the cathode, indicating that the anodic luminescence probe has no influence on the cathode signal.

[0116] (3) Using the cathode electrochemiluminescence electrode obtained in step (1) as the working electrode, a platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode, cyclic voltammetry scanning is carried out using a three-electrode system. In the ECL detection solution (5 mL of 0.1 mol / L PBS buffer solution added with 50 μL of H2O2), the test conditions are within a potential range of -2V to 0.4V, the high voltage of the photomultiplier tube is -650V, the amplification factor is 4, and the scanning rate is 0.5V / s. Record the ECL intensity-potential curve and establish a relationship diagram between ECL intensity and potential. The test results are as Figure 3 shown in B. The cathode luminescence probe has an obvious ECL signal at about -2V (relative to Ag / AgCl), while there is no obvious signal at the anode, thus confirming that the anode signal is not interfered by the cathode luminescence probe.

[0117] (4) Using the anode / cathode electrochemiluminescence electrode obtained in step (1) as the working electrode, a platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode, cyclic voltammetry scanning is carried out using a three-electrode system. In the ECL detection solution (5 mL of 0.1 mol / L PBS buffer solution added with 50 μL of H2O2), the test conditions are within a potential range of -2V to 0.4V, the high voltage of the photomultiplier tube is -650V, the amplification factor is 4, and the scanning rate is 0.5V / s. Record the ECL intensity-potential curve and establish a relationship diagram between ECL intensity and potential. The test results are as Figure 3 shown in C. Both the cathode and the anode have obvious luminescence at the corresponding potentials and do not interfere with each other.

[0118] (5) Using a glassy carbon electrode or the anode / cathode electrochemiluminescence electrode obtained in step (1) as the working electrode, a platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode, cyclic voltammetry scanning is carried out using a three-electrode system. In the ECL detection solution (5 mL of 0.1 mol / L PBS buffer solution added with 50 μL of H2O2 or 5 mL of 0.1 mol / L PBS buffer solution), the test conditions are within a potential range of -2V to 0.4V, the high voltage of the photomultiplier tube is -650V, the amplification factor is 4, and the scanning rate is 0.5V / s. Record the ECL intensity curve and the ECL intensity-potential curve. The test results are as Figure 4As shown, where A is the ECL intensity diagram and B is the ECL intensity-potential curve diagram. The detection conditions for curve a are using a glassy carbon electrode as the working electrode and 5 mL of 0.1 mol / L PBS buffer solution added with 50 μL of H2O2 as the detection solution. The detection conditions for curve b are using an anode / cathode electrochemiluminescence electrode as the working electrode and 5 mL of 0.1 mol / L PBS buffer solution as the detection solution. The detection conditions for curve c are using an anode / cathode electrochemiluminescence electrode as the working electrode and 5 mL of 0.1 mol / L PBS buffer solution added with 50 μL of H2O2 as the detection solution. Figure 4 Curve a of A and B in [reference] shows that a very weak ECL signal is generated at both the cathode and anode of the glassy carbon electrode. Figure 4 Curve b of A and B in [reference] shows that under the condition of using 5 mL of 0.1 mol / L PBS buffer solution as the detection solution, an obvious ECL signal is detected at the cathode, while the ECL activity at the anode can be ignored. Figure 4 Curve c of A and B in [reference] shows that obvious ECL signals are detected at both the cathode and anode, and its ECL intensity exceeds that of curve a and curve b.

[0119] Example 2

[0120] The method for quantitatively detecting aflatoxin B1 and ochratoxin A using the potential-resolved electrochemiluminescence immunosensor of Example 1 includes the following steps:

[0121] S41: Centrifuge the solution of 15 μL UiO66-NH2@luminol-AFB1 Ab probe to remove the supernatant, and then mix and incubate the lower precipitate with 5 μL of aflatoxin B1 standard solutions at different concentrations (0.08 ng / mL, 0.8 ng / mL, 8 ng / mL, 40 ng / mL, 80 ng / mL, 400 ng / mL, 1000 ng / mL) to obtain mixed solution B; centrifuge the solution of 15 μL Py-HOF-OTAAb probe to remove the supernatant, and then mix and incubate the lower precipitate with 5 μL of ochratoxin A standard solutions at different concentrations (0.006 ng / mL, 0.03 ng / mL, 0.6 ng / mL, 6 ng / mL, 60 ng / mL, 300 ng / mL, 1000 ng / mL) to obtain mixed solution A. Mix the mixed solution B and mixed solution A and modify them on the electrochemiluminescence electrode, so that the aflatoxin B1 antibody reacts with the aflatoxin B1 coated antigen, and the ochratoxin A antibody reacts with the ochratoxin A coated antigen. After the reaction is complete, rinse the unbound UiO66-NH2@luminol-AFB1 Ab probe, Py-HOF-OTA Ab probe and the excess AFB1 and OTA on the electrochemiluminescence electrode with 0.01 mol / L PBS buffer to obtain the assembled potential-resolved electrochemiluminescence immunosensor;

[0122] S42: Use the assembled potential-resolved electrochemiluminescence immunosensor described in S41 as the working electrode, a platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode to form a three-electrode system. In the ECL detection solution (5 mL of 0.1 mol / L PBS buffer added with 50 μL H2O2), perform cyclic voltammetry scanning under the instrument parameter conditions of a potential range of -2V to 0.4V, a photomultiplier tube high voltage of -650V, an amplification factor of 4, and a scan rate of 0.5V / s. Record the ECL intensity-time curve, establish the relationship between the ECL intensity and the logarithm of the aflatoxin B1 concentration, and obtain the linear regression equation: I = 8706.8 - 2263.2lgc (ng / mL), the regression coefficient is 0.9957, the detection limit is 26.7 pg / mL, and the linear range is 0.08 - 1000 ng / mL; record the ECL intensity-time curve, establish the relationship between the ECL intensity and the logarithm of the ochratoxin A concentration, and obtain the linear regression equation: I = 9184.5 – 1825.5lg c (ng / mL), the regression coefficient is 0.9949, the detection limit is 2.0 pg / mL, and the linear range is 0.006 - 1000 ng / mL.

[0123] Figure 5It is a three-dimensional curve graph of ECL intensity - time and ECL intensity - concentration for aflatoxin B1 standard solutions and ochratoxin A standard solutions with different concentrations; among them, from a to g, the concentrations of the ochratoxin A standard solutions corresponding to signal 1 are 1000 ng / mL, 300 ng / mL, 60 ng / mL, 6 ng / mL, 0.6 ng / mL, 0.03 ng / mL, 0.006 ng / mL in sequence, and the concentrations of the aflatoxin B1 standard solutions corresponding to signal 2 are 1000 ng / mL, 400 ng / mL, 80 ng / mL, 40 ng / mL, 8 ng / mL, 0.8 ng / mL, 0.08 ng / mL in sequence.

[0124] Figure 6 It is a standard curve graph of ECL intensity and the logarithm value of aflatoxin B1 concentration, Figure 7 It is a standard curve graph of ECL intensity and the logarithm value of ochratoxin A concentration.

[0125] Example 3

[0126] The parameters for quantitatively detecting aflatoxin B1 and ochratoxin A of the potential - resolution type electrochemiluminescence immunosensor in Example 1 were optimized, and the optimization methods and results are as follows:

[0127] (1) Concentration optimization of Au@MoS2

[0128] As the concentration of the Au@MoS2 suspension increases within a specific range, the number of antigens bound to the surface of the electrochemiluminescence electrode also increases accordingly, resulting in a proportional increase in the electrochemiluminescence signal intensity difference (ΔECL). Once the substrate concentration reaches saturation, since the binding sites on the electrode surface have been completely occupied, increasing the concentration further cannot promote more antigen binding. The results are as Figure 8 shown in A of the figure. The concentration of Au@MoS2 reaches the optimal level at 0.6 mg / mL, and at this time, ΔECL reaches the maximum value.

[0129] (2) Concentration optimization of AFB1 Ab and OTA Ab

[0130] When the concentration of AFB1 Ab is fixed below 10 μg / mL and the concentration of OTA Ab is fixed below 6 μg / mL, as the concentrations of OTA Ab and AFB1 Ab increase, ΔECL also increases. This result may be due to the fact that as the antibody concentration increases, the binding force between the antibody and the luminophore enhances. However, once the concentration of AFB1 Ab exceeds 10 μg / mL and the concentration of OTA Ab exceeds 6 μg / mL, the ΔECL intensity will decrease. The reason for the decrease in the ΔECL intensity may be that the efficiency of the immunosensor becomes lower, which is related to the increase in antibody concentration. As Figure 8As shown in B, when the concentration of AFB1 Ab is set to 10 μg / mL and the concentration of OTA Ab is set to 6 μg / mL, the potential-resolved electrochemiluminescence immunosensor exhibits the best performance.

[0131] (3) Optimization of the concentrations of AFB1 Ae and OTA Ae

[0132] When AFB1 Ae is below 10 μg / mL and OTA Ae is below 6 μg / mL, as the concentrations of OTA Ae and AFB1 Ae increase, ΔECL also increases. This can be attributed to the enhanced binding force between the antigen and the substrate when the antigen concentration increases. However, once the concentration of AFB1 Ae exceeds 10 μg / mL and the concentration of OTA Ae exceeds 6 μg / mL, the intensity of ΔECL decreases. This decrease may be due to the fact that antigen excess hinders electron transfer, thereby reducing signal generation and resulting in a decrease in the signal difference. As Figure 8 shown in C, when the concentration of AFB1 Ae is 10 μg / mL and the concentration of OTA Ae is 6 μg / mL, the potential-resolved electrochemiluminescence immunosensor exhibits the best performance.

[0133] (4) Optimization of the volume of H2O2 in 5 mL of PBS buffer solution

[0134] Within the range of 50 μL, the increase in the ΔECL intensity is proportional to the added amount of H2O2. Within this range, H2O2 provides sufficient hydroxyl radicals for the reaction, thereby amplifying the ECL signal. The enhancement of the unlabeled signal exceeds that of the spiked signal, resulting in a significant increase in the ΔECL value. When the added amount of H2O2 exceeds 50 μL, the ΔECL value begins to decrease. This decrease may be due to the fact that the excess H2O2 can no longer effectively provide the required hydroxyl radicals. In addition, with the excess of H2O2, the increase in the unlabeled signal tends to level off, while the enhancement of the spiked signal remains higher compared to the 50 μL level, resulting in a decrease in the ΔECL value. As Figure 8 shown in D, the addition of 50 μL of H2O2 was selected as the optimal condition, under which the ΔECL value is the largest.

[0135] Test Example 2

[0136] To test the applicability of the immunoassay method, corn flour and wheat flour randomly collected from a supermarket were used for the spike recovery experiment. First, 2 g of homogenized samples were weighed into 50 mL centrifuge tubes respectively, and then different volumes of OTA standard solution (1 μg / mL) and AFB1 standard solution (1 μg / mL) were added thereto to obtain an OTA spike concentration of 20 ng / mL and an AFB1 spike concentration of 20 ng / mL in wheat; an OTA spike concentration of 20 ng / mL and an AFB1 spike concentration of 80 ng / mL in corn. Then, 4 mL of methanol aqueous solution (70%) was added to the spiked samples, and the sample solution was mixed evenly by shaking on a vortex oscillator for 20 minutes. The solution was centrifuged at 2500 rpm for 15 min, and the obtained supernatant was transferred to a 10 mL centrifuge tube. Finally, it was appropriately diluted with PBS to the required spike concentration to obtain the test solution. 15 μL of the Py-HOF-OTA Ab probe solution was centrifuged to remove the supernatant, 15 μL of the UiO66-NH2@luminol-AFB1 Ab probe solution was centrifuged to remove the supernatant, and then the lower-layer precipitate was mixed evenly with the test solution respectively to obtain two mixed solutions. The two mixed solutions were mixed and then coated on the electrochemiluminescence electrode in Example 1, and detected using the assembled potential-resolved type electrochemiluminescence immunosensor. The results are shown in Table 1. The recovery rate of the potential-resolved type electrochemiluminescence sensor for OTA in the spiked wheat samples was in the range of 88.38% - 108.78%, and the RSD was in the range of 9.52% - 13.69% (n = 3). The recovery rate of the sensor for AFB1 in the spiked wheat samples was in the range of 86.92% - 102.10%, and the RSD was in the range of 6.77% - 13.10% (n = 3). The recovery rate of the sensor for OTA in the spiked corn samples was in the range of 94.05% - 102.06%, and the RSD was in the range of 12.12% - 13.48% (n = 3). The recovery rate of the sensor for AFB1 in the spiked corn samples was in the range of 95.49% - 107.56%, and the RSD was in the range of 6.08% - 11.65% (n = 3), indicating that this method can be used to accurately detect ochratoxin A and aflatoxin B1 in actual samples.

[0137] Table 1 Determination results of spike recovery of OTA and AFB1 in actual samples by the potential-resolved type ECL immunosensor

[0138]

[0139]

[0140] N.D. = Not detected.

[0141] Therefore, according to the above results, when detecting the contents of ochratoxin A and aflatoxin B1 in an unknown sample, it is only necessary to mix the spiked samples with different spiked contents after treatment with the solution of the Py-HOF-OTA Ab probe and the solution of the UiO66-NH2@luminol-AFB1 Ab probe respectively to obtain two mixed solutions, and then mix these two mixed solutions evenly and coat them on the electrochemiluminescence electrode to obtain the assembled potential-resolved type electrochemiluminescence immunosensor for electrochemistry testing. Substituting the obtained ECL intensity value into the standard curve can calculate the true concentration in the spiked sample.

[0142] Obviously, the above embodiments of the present invention are only examples for clearly explaining the present invention, rather than limitations on the implementation manners of the present invention. Those skilled in the art should understand that other different forms of changes or variations can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A potential-resolved electrochemiluminescence immunosensor, characterized in that: Including anodic luminescent probes, cathodic luminescent probes and electrochemical luminescent electrodes; The anode luminescence probe is prepared by the following method: S11: adding zirconium tetrachloride, acetic acid and 2-aminoterephthalic acid to N,N-dimethylformamide, heating for reaction, and obtaining UiO66-NH2; S12: reacting the UiO66-NH2 obtained in S11 with luminol to obtain UiO66-NH2@luminol; S13: adding aflatoxin B1 antibody to the UiO66-NH2@luminol obtained in S12 for incubation to obtain the anode luminescent probe; The cathode luminescence probe is prepared by the following method: S21: adding 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene to N,N-dimethylformamide, and then adding an organic alcohol solvent to react to obtain Py-HOF; S22: activating the Py-HOF obtained in S21 with a cross-linking agent, adding ochratoxin A antibody for incubation, and obtaining the cathode luminescence probe; The electrochemical luminescent electrode is prepared by the following method: S31: heating thiourea and ammonium molybdate tetrahydrate to react to obtain molybdenum disulfide nanosheets, mixing the molybdenum disulfide nanosheets and an aqueous solution of chloroauric acid, and then adding trisodium citrate dihydrate to react to obtain Au@MoS2; S32: modifying the Au@MoS2 obtained in S31 on the electrode body, combining the modified electrode body with the aflatoxin B1 coated antigen and the ochratoxin A coated antigen and then incubating them to obtain the electrochemiluminescent electrode.

2. The potential-resolved electrochemiluminescence immunosensor according to claim 1, characterized in that: In S13, the mass ratio of UiO66-NH2@luminol and aflatoxin B1 antibody is (80-120):

1.

3. The potential-resolved electrochemiluminescence immunosensor according to claim 1, characterized in that: In S22, the cross-linking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

4. The potential-resolved electrochemiluminescence immunosensor according to claim 1, characterized in that: In S22, the mass ratio of Py-HOF to ochratoxin A antibody is (300-400):

1.

5. The potential-resolved electrochemiluminescence immunosensor according to claim 1, characterized in that: In S32, the mass ratio of Au@MoS2, aflatoxin B1 coated antigen and ochratoxin A coated antigen is 300:(2-3):(1-2).

6. The potential-resolved electrochemiluminescence immunosensor according to claim 1, characterized in that: In S13, S22 and S32, bovine serum albumin solution was added for blocking after incubation.

7. Use of the potential-resolved electrochemiluminescence immunosensor according to any one of claims 1 to 6 in detecting aflatoxin B1 and ochratoxin A.

8. The use according to claim 7, characterized in that: The method for detecting aflatoxin B1 and ochratoxin A using the potential-resolving electrochemiluminescence immunosensor comprises the following steps: S41: mixing and incubating the anodic luminescent probe with aflatoxin B1 standard solutions of different concentrations to obtain a mixed solution B, mixing and incubating the cathodic luminescent probe with ochratoxin A standard solutions of different concentrations to obtain a mixed solution A, mixing the mixed solution B and the mixed solution A and incubating them with an electrochemiluminescent electrode to obtain an assembled potential-resolved electrochemiluminescent immunosensor; S42: Using the assembled potential-resolved electrochemiluminescence immunosensor described in S41 as a working electrode, performing cyclic voltammetry scanning using a three-electrode system, recording the luminescence intensity-time curve, establishing the relationship between the luminescence intensity and the logarithmic value of the aflatoxin B1 concentration and the logarithmic value of the ochratoxin A concentration, and obtaining a linear regression equation; S43: The anodic luminescent probe and the cathodic luminescent probe are mixed and incubated with the test solution respectively, and then the two mixed solutions are incubated with the electrochemiluminescent electrode to obtain an assembled potential-resolved electrochemiluminescent immunosensor. The potential-resolved electrochemiluminescent immunosensor assembled in this step is used as a working electrode, and a cyclic voltammetry scan is performed using a three-electrode system. According to the measured luminescence intensity-time curve, the concentrations of aflatoxin B1 and ochratoxin A in the test solution are obtained in combination with the linear regression equation described in S42.

9. The use according to claim 8, characterized in that: In S42 and S43, the detection solution of the three-electrode system is a buffer solution containing hydrogen peroxide.

10. The use according to claim 9, characterized in that: The volume ratio of the hydrogen peroxide to the buffer solution is (1-3):250.

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