Electrochemiluminescence immunosensor and application thereof in detection of aflatoxin B1

Abstract

The invention discloses an electrochemical luminescence immunosensor and application thereof in detection of aflatoxin B1, the electrochemical luminescence immunosensor comprises an electrochemical luminescence probe and an electrochemical luminescence electrode, the electrochemical luminescence probe comprises a CoSOH nanosheet, and the CoSOH nanosheet is connected with an aflatoxin B1 antibody through a chemical bond; the electrochemical luminescence electrode comprises an electrode body, the electrode body is modified with In-MOF, and the In-MOF is connected with an aflatoxin B1 coating antigen through a chemical bond. The electrochemical luminescence immunosensor has the advantages of simple instrument, convenience in operation, high detection efficiency, simplicity and convenience in sample treatment, high sensitivity, wide linear range, high universality, low cost and the like, and has extremely high practical value.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemiluminescence biosensing detection, and particularly relates to an electrochemiluminescence immunosensor and its application in the detection of aflatoxin B1. Background Art

[0002] Aflatoxins, especially aflatoxin B1 (AFB1), as a class of highly toxic natural compounds among mycotoxins, pose a serious threat to food safety. AFB1 is mainly produced by filamentous fungi such as Aspergillus flavus and Aspergillus parasiticus during the planting, storage, and processing of crops, and can contaminate a variety of foods, including but not limited to grains, wines, peanuts, and soy products, and enter the human body through the food chain. AFB1 has extremely strong carcinogenicity and toxicity, and its exposure can cause various toxic effects in experimental animals and livestock, including hepatotoxicity, nephrotoxicity, neurotoxicity, intestinal toxicity, splenic toxicity, and cardiotoxicity. Therefore, there is an urgent need for an accurate, efficient, and easy-to-operate AFB1 detection method to minimize the adverse effects caused by aflatoxin B1.

[0003] Currently, the detection techniques for AFB1 mainly focus on high-performance liquid chromatography, liquid chromatography-mass spectrometry, and enzyme-linked immunosorbent assay. These methods perform excellently in terms of analytical performance, but face many challenges in actual commercial applications, such as complex and cumbersome sample pretreatment processes, expensive required instrument equipment, and occasional false positive results. These problems limit the wide popularization and in-depth application of these detection techniques.

[0004] In view of this, it is particularly important to develop a highly sensitive detection system for AFB1 in agricultural products. Electrochemiluminescence (ECL) technology has attracted wide attention in the analysis field due to its advantages such as simplicity, easy controllability, and high sensitivity. In particular, electrochemiluminescence immunoassay (ECLIA) combines the advantages of electrochemiluminescence and immunoassay, showing unique analytical advantages and being regarded as a highly potential immunoassay method. In the ECL system, the energy resonance transfer (ECL-RET) phenomenon, as a novel ECL biosensing strategy, is achieved through the close-range energy transfer between the donor and the acceptor. When there is a large overlap between the ECL emission spectrum of the donor and the ultraviolet absorption spectrum of the acceptor, and the distance between the two meets specific conditions, the ECL emission of the donor will be quenched. The ECL-RET method has attracted the close attention of many researchers because it does not require an excitation light source, effectively avoiding problems such as light scattering, autofluorescence, and high background signals, and at the same time, with its characteristics of high sensitivity and low background interference.

[0005] However, high potential may have adverse effects in ECL bioanalysis, such as causing irreversible damage to biological samples and leading to passivation of the electrode surface, thereby reducing the ECL intensity. Therefore, achieving low-potential ECL detection is crucial for improving the performance of ECL bioanalysis. There is an urgent need to develop a low-potential ECL-RET detection method for aflatoxin B1 to improve its detection sensitivity and reduce the operation difficulty. Summary of the Invention

[0006] To solve the above technical problems, the object of the present invention is to provide an electrochemiluminescence immunosensor and its application in the detection of aflatoxin B1. A competitive ECL immunosensor based on energy resonance transfer is developed, using In-MOF as the energy donor and CoSOH nanosheets as the energy acceptor for ultrasensitive quantitative detection of aflatoxin B1.

[0007] The above object of the present invention is achieved by the following technical solutions:

[0008] In the first aspect of the present invention, an electrochemiluminescence immunosensor is provided, including an electrochemiluminescence probe and an electrochemiluminescence electrode;

[0009] The electrochemiluminescence probe includes CoSOH nanosheets, and the CoSOH nanosheets are chemically bonded with aflatoxin B1 antibody (AFB1-antiboby, hereinafter referred to as AFB1-Ab);

[0010] The electrochemiluminescence electrode includes an electrode body, and the electrode body is modified with In-MOF, and the In-MOF is chemically bonded with aflatoxin B1 coated antigen (AFB1-antigen, hereinafter referred to as AFB1-Ag).

[0011] Further, the preparation method of the electrochemiluminescence probe includes the following steps:

[0012] (1) Dissolve cobalt nitrate hexahydrate and urea in an alcohol solution, carry out a solvothermal reaction at 100-120 °C, and disperse the reaction product in Na 2 S solution, and centrifuge to obtain CoSOH nanosheets;

[0013] (2) Incubate the CoSOH nanosheets obtained in step (1) with aflatoxin B1 antibody to obtain the electrochemiluminescence probe (Ab-CoSOH).

[0014] Further, in step (1), the alcohol solution is an ethylene glycol solution.

[0015] Further, the ethylene glycol solution is obtained by mixing ethylene glycol and water.

[0016] Further, in step (1), the time of the solvothermal reaction is 4 - 5 h.

[0017] Further, in step (1), the 2 Na 2 concentration of Na

[0018] 2S in the Na 2 2S solution is 0.1 - 0.5 M.

[0019] Further, in step (1), the reaction product is dispersed in the Na 2 2S solution at room temperature.

[0020] Further, in step (2), the mass ratio of the CoSOH nanosheets to the aflatoxin B1 antibody is (20 - 400):1, preferably (80 - 100):1.

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

[0022] (1) Dissolve cobalt nitrate hexahydrate and urea in water and ethylene glycol, then seal in a high-pressure reaction kettle for solvothermal reaction. After the reaction is complete, soak the reaction product in the Na 2 2S solution for stirring, and centrifuge to obtain CoSOH nanosheets;

[0023] (2) Incubate the CoSOH nanosheets with the aflatoxin B1 antibody so that the aflatoxin B1 antibody is connected to the CoSOH nanosheets through Co-N bonds to obtain the electrochemiluminescence probe.

[0024] In a specific embodiment, in step (2), the CoSOH nanosheets are dispersed in water to obtain a CoSOH suspension. React the CoSOH suspension with the AFB1-Ab solution so that the aflatoxin B1 antibody is connected to the CoSOH nanosheets through Co-N bonds. After the reaction is complete, add BSA for blocking to obtain the electrochemiluminescence probe (Ab-CoSOH).

[0025] Further, the concentration of the CoSOH suspension is 0.1 - 1.4 mg / mL, preferably 0.4 - 0.8 mg / mL.

[0026] Further, the concentration of the AFB1-Ab solution is 3 - 12 μg / mL, preferably 5 - 10 μg / mL.

[0027] Further, the volume ratio of the CoSOH suspension to the AFB1-Ab solution is (0.8 - 1.2):1.

[0028] Further, the preparation method of the electrochemiluminescence electrode comprises the following steps:

[0029] S1. Dissolve indium nitrate and 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene (H 4 TBAPy) in an acidic organic solvent solution, and react at 80 - 90 °C to obtain In-MOF;

[0030] S2. Activate the In-MOF obtained in S1 with a cross-linking agent and then centrifuge it. Disperse the obtained precipitate in a Nafion solution to obtain an In-MOF suspension. Modify the electrode body with the In-MOF suspension, and then incubate the modified electrode body with an aflatoxin B1-coated antigen to obtain the electrochemiluminescence electrode.

[0031] Further, in S1, the mass ratio of indium nitrate to 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene is (1 - 1.5):1.

[0032] Further, in S1, the acidic organic solvent solution is obtained by mixing dioxane, N,N-dimethylformamide, water, and concentrated hydrochloric acid.

[0033] Further, the volume ratio of dioxane, N,N-dimethylformamide, water, and concentrated hydrochloric acid is (150 - 250):(50 - 150):(50 - 150):1.

[0034] Further, in S1, the reaction time is 10 - 12 h.

[0035] Further, in S2, the dosage ratio of the obtained precipitate to the Nafion solution is (0.5 - 3) mg:(30 - 50) μL.

[0036] Further, in S2, the concentration of Nafion in the Nafion solution is 0.5 - 0.7 wt.%.

[0037] Further, in S2, the cross-linking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0038] Further, in S2, the electrode body is a glassy carbon electrode.

[0039] Further, in S2, the incubation temperature is 4 - 5 °C.

[0040] Further, in S2, the mass ratio of In-MOF to the aflatoxin B1-coated antigen is (70 - 250):1, preferably (100 - 150):1.

[0041] Further, in S2, after the modified electrode body is incubated with aflatoxin B1 coated antigen, bovine serum albumin (BSA) is added for blocking to eliminate non-specific adsorption of the electrode body.

[0042] In a specific embodiment, the preparation method of the electrochemiluminescence electrode comprises the following steps:

[0043] S1. Indium nitrate nonahydrate and 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene are dissolved in an acidic organic solvent solution, and the reaction is carried out at 80 - 90 °C. After cooling to room temperature, In-MOF is obtained.

[0044] S2. The In-MOF obtained in S1 is activated with a crosslinking agent and then centrifuged. The obtained precipitate is dispersed in a Nafion solution to obtain an In-MOF suspension. The In-MOF suspension is modified on the electrode body, and then the modified electrode body is incubated with aflatoxin B1 coated antigen, so that the aflatoxin B1 coated antigen is connected to the carboxyl group on the In-MOF through an amide bond, and the electrochemiluminescence electrode is obtained.

[0045] In a specific embodiment, in S2, an AFB1-Ag solution is added dropwise to the modified electrode body for incubation.

[0046] Further, the volume ratio of the In-MOF suspension to the AFB1-Ag solution is (0.8 - 1.2):1.

[0047] Further, the concentration of the AFB1-Ag solution is 4 - 14 μg / mL, preferably 6 - 10 μg / mL.

[0048] In the electrochemiluminescence electrode of the chemiluminescence immunosensor constructed by the present invention, indium ions coordinate with 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene to form In-MOF. The 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene luminescent groups are assembled into a rigid MOF framework through coordination bonds, which not only expands the distance between pyrene nuclei, eliminates the aggregation-caused quenching (ACQ) effect, but also hinders the intramolecular movement of the ligand, reduces non-radiative relaxation, and thus realizes significant coordination-enhanced ECL. The In-MOF is combined with aflatoxin B1 coated antigen through a chemical bond, and the aflatoxin B1 antibody is connected to the CoSOH nanosheet probe through an amide bond. The electrochemiluminescence immunosensor constructed by the present invention uses In-MOF as the donor material and CoSOH nanosheet as the acceptor material, and has a higher sensitivity (more than 100 times) than the traditional ELISA method.

[0049] The second aspect of the present invention provides an application of the electrochemiluminescence immunosensor described in the first aspect in detecting aflatoxin B1.

[0050] Furthermore, the method for detecting aflatoxin B1 using an electrochemiluminescence immunosensor includes the following steps:

[0051] Step 1: Mix and incubate an electrochemiluminescence probe with standard solutions of aflatoxin B1 at different concentrations, and then contact the resulting mixed solution with an electrochemiluminescence electrode to allow the aflatoxin B1 antibody to react with the aflatoxin B1 coated antigen, obtaining an assembled electrochemiluminescence immunosensor;

[0052] Step 2: Use the assembled electrochemiluminescence immunosensor described in Step 1 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 obtain a linear regression equation;

[0053] Step 3: Mix and incubate an electrochemiluminescence probe with the test solution, and then contact the resulting mixed solution with an electrochemiluminescence electrode to allow the aflatoxin B1 antibody to react with the aflatoxin B1 coated antigen, obtaining an assembled electrochemiluminescence immunosensor. Use the assembled 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 with the linear regression equation described in Step 2 to obtain the concentration of aflatoxin B1 in the test solution.

[0054] Furthermore, in Step 2, the potential range for cyclic voltammetry scanning using the three-electrode system is 0 - 0.65V.

[0055] Furthermore, in Step 2, the specific conditions for cyclic voltammetry scanning using the three-electrode system are: within the electrochemical window range of 0 - 0.65V, the high voltage of the photomultiplier tube is -550 - 700V, the amplification factor is 4, and the scanning rate is 0.15V / s.

[0056] When performing cyclic voltammetry scanning using the three-electrode system, the ECL signal changes with the change in the concentration of the aflatoxin B1 standard solution.

[0057] Furthermore, in Step 2 and Step 3, the detection buffer solution of the three-electrode system is a PBS buffer solution containing 1,4-diazabicyclo[2.2.2]octane (DABCO). Among them, DABCO is a co-reactant.

[0058] Furthermore, the concentration of 1,4-diazabicyclo[2.2.2]octane in the PBS buffer solution containing DABCO is 0.04 - 0.15mol / L, preferably 0.08 - 0.15mol / L, and more preferably 0.10 - 0.12mol / L.

[0059] In a specific embodiment, the method for detecting aflatoxin B1 using an electrochemiluminescence immunosensor includes the following steps:

[0060] Step 1: Centrifuge the solution of the electrochemiluminescence probe to remove the supernatant, then mix the precipitate with standard solutions of aflatoxin B1 at different concentrations and incubate. Then, contact the resulting mixed solution with the electrochemiluminescence electrode to allow the aflatoxin B1 antibody to react with the aflatoxin B1 coated antigen. After the reaction is complete, wash the electrochemiluminescence probe and aflatoxin B1 that are not bound to the electrochemiluminescence electrode with a buffer solution to obtain the assembled electrochemiluminescence immunosensor.

[0061] Step 2: Use the assembled electrochemiluminescence immunosensor described in Step 1 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 obtain the linear regression equation.

[0062] Step 3: Centrifuge the solution of the electrochemiluminescence probe to remove the supernatant, then mix the precipitate with the test solution and incubate. Then, contact the resulting mixed solution with the electrochemiluminescence electrode to allow the aflatoxin B1 antibody to react with the aflatoxin B1 coated antigen to obtain the assembled electrochemiluminescence immunosensor. Use the assembled 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 with the linear regression equation described in Step 2 to obtain the concentration of aflatoxin B1 in the test solution.

[0063] Further, in Step 1, the concentration of the electrochemiluminescence probe in the solution of the electrochemiluminescence probe is 0.7 - 1 mg / mL.

[0064] Further, in Step 1, the volume ratio of the solution of the electrochemiluminescence probe to the standard solution of aflatoxin B1 is (0.8 - 1.2):1.

[0065] When detecting aflatoxin B1 using the electrochemiluminescence immunosensor provided by the present invention, the quantitative basis is that aflatoxin B1 competes with AFB1-Ag for limited AFB1-Ab.

[0066] When there is no AFB1 analyte, AFB1-Ab and AFB1-Ag are connected by specific binding. Due to the quenching effect of the receptor CoSOH nanosheets, the ECL signal of the donor In-MOF is relatively low. When there is an AFB1 analyte, aflatoxin B1 competes with AFB1-Ag for the limited AFB1-Ab, and less Ab-CoSOH is connected to the electrode. At this time, the ECL signal intensity becomes higher. As the AFB1 analyte gradually increases, the Ab-CoSOH connected to the electrode gradually decreases, and the ECL signal gradually increases. Due to the significant quenching effect and simple assembly process, this electrochemiluminescence immunosensor has excellent sensitivity, precision, and reproducibility, which has important practical significance for the detection of aflatoxin B1.

[0067] Advantages of the present invention:

[0068] 1. The electrochemiluminescence immunosensor provided by the present invention includes an electrochemiluminescence probe and an electrochemiluminescence electrode. Among them, In-MOF is used as an energy donor, and CoSOH nanosheets are used as an energy receptor and an electrochemiluminescence probe. The constructed electrochemiluminescence immunosensor has strong and stable ECL emission at a low excitation potential (below 1.0 V), which can avoid electrode passivation and interference from redox substances in biological substances, and helps to protect the biological activities of antigens, antibodies, and target analytes.

[0069] 2. When the electrochemiluminescence immunosensor of the present invention is used to detect aflatoxin B1, the detection buffer solution of the three-electrode system is a PBS buffer solution containing the coreactant DABCO. This coreactant is rarely used in existing ECL systems, which provides the possibility for constructing a new type of electron transfer path ECL immunosensor.

[0070] 3. The present invention respectively uses aflatoxin B1 antibody and coated antigen to modify the energy receptor and energy donor, and develops an immunoassay method for sensitive detection of aflatoxin B1 based on electrochemiluminescence energy resonance transfer according to the above principle. The energy resonance transfer mechanism between In-MOF and CoSOH, as well as the specific immune reaction between aflatoxin B1 antibody and coated antigen, are used to quantitatively or qualitatively detect aflatoxin B1, improving the selectivity for the target analyte. This electrochemiluminescence immunosensor has the advantages of simple instrument, convenient operation, efficient detection, simple sample treatment, high sensitivity, wide linear range, strong popularity, and low cost, and has extremely high practical value. Description of the Drawings

[0071] Figure 1 It is a schematic diagram of the construction of the electrochemiluminescence immunosensor provided by the present invention and the principle of detecting aflatoxin B1.

[0072] Figure 2TEM images of In-MOF and CoSOH nanosheets; among them, A is the TEM image of In-MOF, and B is the TEM image of CoSOH nanosheets.

[0073] Figure 3 UV-Vis absorption spectrum of CoSOH nanosheets and ECL emission spectrum of In-MOF; among them, curve a is the UV-Vis absorption spectrum curve of CoSOH nanosheets, and curve b is the ECL emission spectrum curve of In-MOF.

[0074] Figure 4 Schematic diagram of ECL-RET-based detection principle and comparison diagram of quenching effects of CoSOH nanosheets and other materials; among them, A is the schematic diagram of ECL-RET-based detection principle, B is the quenching effect diagram of In-MOF with AuPt@ZIF-67 as the quencher, C is the quenching effect diagram of In-MOF with CoOOH as the quencher, and D is the quenching effect diagram of In-MOF with Au@NiFeMOF as the quencher.

[0075] Figure 5 ECL intensity-potential diagram of the electrochemiluminescence immunosensor in Example 2.

[0076] Figure 6 Concentration optimization diagrams of CoSOH, AFB1-Ab, AFB1-Ag, and DABCO; among them, A is the concentration optimization diagram of CoSOH, B is the concentration optimization diagram of AFB1-Ab, C is the concentration optimization diagram of AFB1-Ag, and D is the concentration optimization diagram of DABCO.

[0077] Figure 7 ECL intensity-concentration curve diagram of aflatoxin B1 standard solutions with different concentrations and standard curve diagram of luminescence intensity and logarithm of aflatoxin B1 concentration; among them, A is the ECL intensity-concentration curve diagram, and B is the standard curve diagram of luminescence intensity and logarithm of aflatoxin B1 concentration. Detailed implementation methods

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this invention belongs. The terms used in the description of this invention herein are only for the purpose of describing specific embodiments and are not intended to limit this invention.

[0079] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the embodiments cited are not intended to limit the present invention.

[0080] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified.

[0081] In the following examples 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) (preserved in the China Center for Type Culture Collection, preservation number CCTCC NO.C202561, preservation address is Wuhan University, Bayi Road, Hongshan District, Wuhan City, Hubei Province, preservation date is February 16, 2025) (joint preservation number CCTCC NO.C202561, preserved in the China Center for Type Culture Collection, preservation address is Wuhan University, Bayi Road, Hongshan District, Wuhan City, 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 in the ELISA method is 0.09 ng / mL) and a relatively high working titer. This AFB1-coated antigen was prepared by reacting aflatoxin B1 (hapten) with aminooxyacetate dissolved in pyridine, blowing dry with nitrogen, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride for activation, and then cross-linking with the free -NH 2 in bovine serum albumin.

[0082] Example 1

[0083] An electrochemiluminescence immunosensor includes an electrochemiluminescence probe and an electrochemiluminescence electrode. The electrochemiluminescence probe is prepared by the following method:

[0084] (1) Dissolve 0.73 g of Co(NO 3 ) 2 ·6H 2 O and 0.6 urea in 2.5 mL of deionized water and 17.5 mL of ethylene glycol, and stir to form a homogeneous solution. Then, transfer the obtained pink solution to a 45 mL autoclave and react at 120 °C for 4 h. Immerse the obtained product in a 1 mol / L Na 2 S solution, stir vigorously for 12 h. Finally, collect the black product, wash it several times with distilled water after centrifugation, and freeze-dry it at -40 °C for 48 h to obtain CoSOH nanosheets.

[0085] (2) The CoSOH nanosheets were dispersed in water to obtain a CoSOH suspension with a concentration of 0.7 mg / mL. 10 μL of the CoSOH suspension (0.7 mg / mL) was reacted with 10 μL of AFB1-Ab (8 μg / mL) overnight. The aflatoxin B1 antibody was connected to the CoSOH nanosheets through Co-N bonds. After the reaction was complete, 10 μL of 5% BSA was added and blocked for 1 h to obtain an electrochemiluminescence probe (Ab-CoSOH).

[0086] The electrochemiluminescence electrode was prepared by the following method:

[0087] S1. 12 mg of In(NO 3 ) 3 ·9H 2 O and 10 mg of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene were dissolved in 4 mL of DMF / dioxane / H 2 O (volume ratio 2:1:1) and 10 μL of concentrated HCl, and sealed in a glass bottle. It was heated at 85 °C for 12 h and then cooled to room temperature. The resulting yellow block crystals were washed with DMF and dried in vacuo to obtain In-MOF solid powder.

[0088] S2. The In-MOF solid powder was dissolved in water to obtain an In-MOF suspension with a concentration of 1 mg / mL. 200 μL of NHS and EDC at 8 mg / mL were added to 200 μL of the In-MOF suspension (1 mg / mL), activated at 4 °C for 1 h, and after centrifugation, 200 μL of Nafion solution (0.5 wt%) was added to the precipitate and dispersed evenly to obtain an In-MOF suspension.

[0089] On the suede, the glassy carbon electrode (GCE) was polished with α-Al 2 O 3 powder to make it mirror-like, rinsed successively with ethanol and ultrapure water, dried with nitrogen, then 10 μL of the In-MOF suspension was added and dried in air. Then 10 μL of AFB1-Ag (8 μg / mL) was added to the dried glassy carbon electrode and incubated overnight at 4 °C to immobilize AFB1-Ag through amide bonds. Subsequently, 10 μL of 5% BSA was added to block the remaining active sites and eliminate non-specific binding. After 1 h, it was rinsed with PBS and the electrochemiluminescence electrode was stored at 4 °C.

[0090] Example 2

[0091] The method for detecting aflatoxin B1 using the electrochemiluminescence immunosensor of Example 1 includes the following steps:

[0092] Step 1. First, centrifuge 30 μL of the Ab-CoSOH probe solution to remove the supernatant, and then take the lower precipitate and add it to 10 μL of aflatoxin B1 standard solutions with different concentrations (0.002 ng / mL, 0.02 ng / mL, 0.2 ng / mL, 2 ng / mL, 10 ng / mL, 100 ng / mL, 1000 ng / mL). Then, coat the above mixture on the electrochemiluminescence electrode. During this process, AFB1-Ag and AFB1 compete for the limited binding sites on AFB1-Ab. After 1 h, rinse with PBS to remove the unbound Ab-CoSOH and excess AFB1 that did not bind to AFB1-Ag, and obtain the assembled electrochemiluminescence immunosensor.

[0093] Step 2. Use the above-assembled electrochemical immunosensor 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 an ECL detection buffer solution (PBS buffer solution with a pH of 7.4 containing 0.1 M co-reactant 1,4-diazabicyclo[2.2.2]octane), within the electrochemical window range of 0 - 0.65 V, with a photomultiplier tube high voltage of -550 V and a scan rate of 0.15 V / s, perform cyclic voltammetry scanning. Record the luminescence intensity-time curve, establish the linear relationship between the ECL luminescence intensity and the logarithm of the aflatoxin B1 concentration, and obtain the corresponding linear regression equation: I = 10118.5 + 1752.3lg c(ng / mL), with a regression coefficient of 0.9961. The linear detection range of this electrochemiluminescence immunosensor is 0.002 - 1000 ng / mL, and the detection limit is 0.42 pg / mL.

[0094] Figure 1 It is a schematic diagram of the construction of the electrochemiluminescence immunosensor provided by the present invention and the principle of aflatoxin B1 detection.

[0095] Figure 2 It is the TEM image of In-MOF and CoSOH nanosheets; among them, A is the TEM image of In-MOF, and B is the TEM image of CoSOH nanosheets.

[0096] In order to explore whether resonance energy transfer occurs between In-MOF and CoSOH nanosheets, the ECL emission wavelength of In-MOF and the ultraviolet absorption wavelength of CoSOH nanosheets were detected, and the detection results are as Figure 3 shown, Figure 3 It is the ultraviolet-visible absorption spectrum of CoSOH nanosheets and the ECL emission spectrum of In-MOF; among them, curve a is the ultraviolet-visible absorption spectrum curve of CoSOH nanosheets, and curve b is the ECL emission spectrum curve of In-MOF. From Figure 3It can be seen that the ECL emission wavelength range is 450 - 550 nm (curve b), and the CoSOH nanosheets have absorption at wavelengths from 300 to 700 nm (curve a). There is a large overlap between the two spectra, which provides the possibility for ECL-RET.

[0097] Figure 4 Figure 4 is the schematic diagram of the detection principle based on ECL-RET and the comparison diagram of the quenching effects of CoSOH nanosheets and other materials. Among them, A is the schematic diagram of the detection principle based on ECL-RET, B is the quenching effect diagram of In-MOF when AuPt@ZIF-67 is used as the quencher, C is the quenching effect diagram of In-MOF when CoOOH is used as the quencher, and D is the quenching effect diagram of In-MOF when Au@NiFeMOF is used as the quencher. As can be seen from Figure 4 Figure 4A, when the AFB1 analyte is present (curve II), the immunosensor can obtain a high ECL signal; while when the AFB1 analyte is absent (curve I), the probe is connected to the electrode through specific antigen-antibody binding. Due to the quenching effect of the receptor CoSOH, the ECL signal of In-MOF decreases significantly. The above results indicate the occurrence of the ECL-RET phenomenon. As can be seen from Figure 4 Figure 4B, C, and D, when the AFB1 analyte is absent, the quenching effect of CoSOH nanosheets is significantly better than that of other materials.

[0098] Figure 5 Figure 5 is the ECL intensity-potential diagram of the electrochemiluminescence immunosensor in Example 2. As can be seen from Figure 5 Figure 5, the electrochemiluminescence immunosensor has the maximum ECL emission at 0.6 V. The low ECL excitation potential can avoid electrode passivation and the interference of redox substances in biological substances, which helps to protect the biological activities of antigens, antibodies, and target analytes.

[0099] Figure 6 Figure 6 is the concentration optimization diagram of CoSOH, AFB1-Ab, AFB1-Ag, and DABCO. Among them, A is the concentration optimization diagram of CoSOH, B is the concentration optimization diagram of AFB1-Ab, C is the concentration optimization diagram of AFB1-Ag, and D is the concentration optimization diagram of DABCO. As can be seen from Figure 6 Figure 6A, when the concentration of CoSOH starts to increase from 0.2 mg / mL, the signal difference (ΔECL) of the electrochemiluminescence immunosensor also increases. When the concentration increases to 0.7 mg / mL, ΔECL reaches the highest value. Subsequently, when the concentration is greater than 0.5 mg / mL, due to the excessive quenching effect of CoSOH, ΔECL decreases. At the same time, the antibody and antigen concentrations are also key factors affecting the performance of the electrochemiluminescence immunosensor. As can be seen from Figure 6As can be seen in Figure B, the ΔECL signal is the highest when the AFB1-Ab concentration is 8 μg / mL, and then starts to decline. This can be attributed to the limitations on the electrode surface and the poor conductivity of biological macromolecules, resulting in a decrease in the electron transfer rate on the electrode surface and a decrease in the signal. Similarly, the optimal concentration of AFB1-Ag is also 8 μg / mL. In addition, the ECL detection solution also has an important impact on the performance of the electrochemiluminescence immunosensor. As Figure 5 shown in Figure D, ΔECL increases with the increase in the DABCO concentration and reaches the maximum value at 0.1 mol / L, and then remains basically unchanged, indicating that the optimal concentration of DABCO is 0.1 mol / L. Therefore, the optimal optimization conditions for the electrochemiluminescence immunosensor are a CoSOH concentration of 0.7 mg / mL, AFB1-Ab and AFB1-Ag concentrations of 0.8 μg / mL each, and the dosage of the coreactant DABCO of 0.1 mol / L.

[0100] Figure 7 are the ECL intensity-concentration curve graph and the standard curve graph of the luminescence intensity versus the logarithm of the AFB1 concentration for AFB1 standard solutions at different concentrations; among them, A is the ECL intensity-concentration curve graph, and B is the standard curve graph of the luminescence intensity versus the logarithm of the AFB1 concentration.

[0101] Test Example 1

[0102] To test the applicability of the immunoassay method, wheat flour and corn flour were randomly purchased from local supermarkets in Suzhou, China for the spike recovery experiment. The actual samples have been ground to achieve complete homogenization. First, 2 g of the homogenized samples were weighed into 50 mL centrifuge tubes, and different volumes of AFB1 standard solution (1000 ng / mL) were added to the samples; then 10 mL of acetonitrile solution (acetonitrile:water = 8:2) was added, shaken well and oscillated at room temperature for 1 h, and then centrifuged at 3500 r min -1 for 15 min, the supernatant was taken, and the supernatant was diluted to the required concentration with PBS to obtain the test solution. 30 μL of the Ab-CoSOH probe solution was centrifuged to remove the supernatant, then 10 μL of the test solution was added and mixed evenly, and then coated on the electrochemiluminescence electrode in Example 1. The assembled electrochemiluminescence immunosensor was used for detection. The results are shown in Table 1. The recoveries of AFB1 in the spiked samples by this sensor were 96.1%-104.7% and 98.2%-111.2% respectively, and the RSDs were 1.85%-9.17% and 4.81%-7.66% (n = 3), indicating that this method can be used to accurately detect AFB1 in actual samples.

[0103] Table 1 Determination results of spike recovery of AFB1 in actual samples by the ECL immunosensor

[0104]

[0105] Therefore, according to the above results, when detecting the content of aflatoxin B1 in an unknown sample, it is only necessary to mix the sample with the Ab-CoSOH probe solution, and then coat the uniformly mixed solution on the electrochemiluminescence electrode to obtain the assembled electrochemiluminescence immunosensor for testing and then compare it with the corresponding standard curve.

[0106] The present invention develops a competitive electrochemiluminescence immunosensor based on electrochemiluminescence energy resonance transfer. Using In-MOF as the energy donor, it has excellent ECL emission at a low excitation potential, which helps to protect the biological activity of the detection target and at the same time can reduce the interference of background signals. The CoSOH nanosheets have a wide ultraviolet absorption and have good quenching ability for In-MOF. The electrochemiluminescence immunosensor provided by the present invention has good sensitivity and accuracy, providing a simple and convenient method for the detection of aflatoxin B1 in agricultural products.

[0107] Obviously, the above embodiments of the present invention are only examples for clearly explaining the present invention, and are not limitations on the implementation manners of the present invention. Those skilled in the art should understand that other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to enumerate all the implementation manners here. Any modifications, equivalent replacements, and improvements 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. An electrochemiluminescent immunosensor, comprising an electrochemiluminescent probe and an electrochemiluminescent electrode, characterized in that: The electrochemiluminescent probe comprises a CoSOH nanosheet, and the CoSOH nanosheet is connected to an aflatoxin B1 antibody via a chemical bond; The electrochemiluminescent electrode comprises an electrode body, the electrode body is modified with In-MOF, and the In-MOF is connected with aflatoxin B1 coated antigen via chemical bonds.

2. The electrochemiluminescent immunosensor according to claim 1, characterized in that: The preparation method of the electrochemiluminescent probe comprises the following steps: (1) dissolving cobalt nitrate hexahydrate and urea in an alcohol solution, performing a solvothermal reaction at 100-120° C., dispersing the reaction product in a Na2S solution, and centrifuging to obtain CoSOH nanosheets; (2) Incubating the CoSOH nanosheets obtained in step (1) with aflatoxin B1 antibodies to obtain the electrochemiluminescent probe.

3. The electrochemiluminescent immunosensor according to claim 2, characterized in that: In step (2), the mass ratio of the CoSOH nanosheets to the aflatoxin B1 antibody is (20-400):

1.

4. The electrochemiluminescent immunosensor according to claim 1, characterized in that: The preparation method of the electrochemical luminescent electrode comprises the following steps: S1. dissolving indium nitrate and 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene in an acidic organic solvent solution, and reacting at 80-90° C. to obtain In-MOF; S2. The In-MOF obtained in S1 is activated with a cross-linking agent and then centrifuged, and the obtained precipitate is dispersed in a Nafion solution to obtain an In-MOF suspension, the In-MOF suspension is modified on the electrode body, and then the modified electrode body is incubated with aflatoxin B1-coated antigen to obtain the electrochemiluminescent electrode.

5. The electrochemiluminescent immunosensor according to claim 4, characterized in that: In S1, the acidic organic solvent solution is obtained by mixing dioxane, N,N-dimethylformamide, water and concentrated hydrochloric acid.

6. The electrochemiluminescent immunosensor according to claim 4, characterized in that: In S2, the mass ratio of the In-MOF to the aflatoxin B1 coated antigen is (70-250):

1.

7. Use of the electrochemiluminescence immunosensor according to any one of claims 1 to 6 in detecting aflatoxin B1.

8. The use according to claim 7, characterized in that: The method for detecting aflatoxin B1 using an electrochemiluminescence immunosensor comprises the following steps: Step 1: Mix and incubate the electrochemiluminescent probe and aflatoxin B1 standard solutions of different concentrations, and then contact the obtained mixed solution with an electrochemiluminescent electrode to react the aflatoxin B1 antibody with the aflatoxin B1 coated antigen to obtain an assembled electrochemiluminescent immunosensor; Step 2: Using the electrochemiluminescent immunosensor assembled in step 1 as the 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 aflatoxin B1 concentration, and obtaining a linear regression equation; Step three, the electrochemiluminescent probe and the solution to be tested are mixed and incubated, and then the obtained mixed solution is contacted with the electrochemiluminescent electrode to react the aflatoxin B1 antibody with the aflatoxin B1 coated antigen to obtain the assembled electrochemiluminescent immunosensor. The electrochemiluminescent immunosensor assembled in this step is used as the working electrode, and a cyclic voltammetry scan is performed using a three-electrode system. The concentration of aflatoxin B1 in the solution to be tested is obtained based on the measured luminescence intensity-time curve combined with the linear regression equation described in step two.

9. The use according to claim 8, characterized in that: In step 2 and step 3, the detection buffer of the three-electrode system is a PBS buffer solution containing 1,4-diazabicyclo[2.2.2]octane.

10. The use according to claim 9, characterized in that: The concentration of 1,4-diazabicyclo[2.2.2]octane in the PBS buffer solution containing 1,4-diazabicyclo[2.2.2]octane is 0.08-0.15 mol / L.

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