A chemiluminescent immunosensor for detecting zearalenone and its application

Abstract

The present invention discloses a chemiluminescent immunosensor for detecting zearalenone and its application. The chemiluminescent immunosensor comprises a chemiluminescent probe and a chemiluminescent immunosensor substrate. The preparation method of the chemiluminescent probe is as follows: CuCo 2 O 4 The nanomaterial solution is incubated with the ZEN antibody to obtain the chemiluminescent immunoprobe; the preparation method of the chemiluminescent immunosensor substrate is: activating carboxyl resin beads to obtain activated resin beads, incubating the activated resin beads with the ZEN coated antigen, and obtaining the chemiluminescent immunosensor substrate for detecting zearalenone. The chemiluminescent immunosensor for detecting zearalenone provided by the present invention has excellent sensitivity, stability and reproducibility, and uses flow injection chemiluminescent immunoassay to detect the content of ZEN in foods such as corn and wheat, so as to achieve rapid and ultra-sensitive detection of ZEN, and has the advantages of simple operation, cost-effectiveness, high sensitivity, etc., and has good practical value and practical significance.

Description

Technical Field

[0001] The invention relates to the technical field of chemiluminescent immunosensors, and in particular to a chemiluminescent immunosensor for detecting zearalenone and an application thereof. Background Art

[0002] Zearalenone (ZEN) is a mycotoxin produced by Fusarium fungi. Contamination of food and feed with ZEN has become an important global problem. ZEN has toxic effects, mainly manifested in reproductive toxicity, hepatotoxicity, immunotoxicity, genotoxicity, and carcinogenicity. In addition, the chemical structure of ZEN is very stable and is not easily destroyed even at high temperatures. Therefore, ZEN has become the main target for monitoring the quality and safety of cereal foods and feeds. To date, the detection of ZEN is usually carried out by high-performance liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). Although the chromatography method is simple to operate and has a fast detection speed, it requires expensive instruments and equipment, a large amount of organic solvents, and has poor selectivity and sensitivity. In addition, although the traditional ELISA is a sensitive detection method, it requires professional personnel to operate in the laboratory in most cases, and the subsequent steps are numerous and very time-consuming. Based on these backgrounds, it is urgent to develop methods with higher selectivity, higher sensitivity, and higher automation to deal with trace detection of ZEN in food. Summary of the invention

[0003] In order to solve the above technical problems, one object of the present invention is to provide a chemiluminescent immunosensor for detecting zearalenone, based on CuCo 2 O 4 The nanomaterials form immune probes with antibodies through Co-N bonds, and the carboxyl-modified resin beads form immune sensor substrates with coated antigens through amide bonds. According to the principle of competitive immunity, the analyte and the coated antigen will compete for the specific binding sites on the antibody, causing the immune sensor to show quantitative differences, thereby generating different chemiluminescence (CL) signals, ultimately realizing the detection of ZEN in actual samples.

[0004] Another object of the present invention is to provide the use of the above-mentioned chemiluminescent immunosensor for detecting zearalenone in detecting ZEN, using a CuCo based on DES solvent. 2 O 4 Flow injection chemiluminescent immunoassay of nanomaterials to detect ZEN.

[0005] The technical solution for detecting ZEN in the present invention is to combine chemiluminescent reaction with immunoassay technology based on chemiluminescent immunoassay (CLIA) method, which has become a new method for highly specific detection of detection objects based on chemiluminescent signals, and has been widely used in clinical diagnosis, food safety and environmental analysis. On this basis, the new detection method (FI-CLIA) combining flow injection and chemiluminescent immunoassay has more advantages such as simple operation, easy automation, strong specificity, high sensitivity, wide linear range, etc.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] A chemiluminescent immunosensor for detecting zearalenone, comprising a chemiluminescent probe and a chemiluminescent immunosensor substrate, wherein the chemiluminescent probe is prepared by the following method: 2 O 4 The nanomaterial solution is incubated with the ZEN antibody to obtain the chemiluminescent immunoprobe;

[0008] The chemiluminescent immunosensor substrate is prepared by the following method: activating carboxyl resin beads to obtain activated resin beads, and incubating the activated resin beads with ZEN coated antigens to obtain the chemiluminescent immunosensor substrate.

[0009] The present invention provides a CuCo-based 2 O 4 Nanomaterials and carboxyl resin beads based chemiluminescent immunosensor, CuCo 2 O 4 As nanoenzymes, nanomaterials have excellent catalytic properties and good chemical stability; their chemiluminescent immunosensor substrates use carboxyl resin beads with good biocompatibility and a large specific surface area as solid phase carriers to load more coated antigens, with high loading efficiency and stable properties.

[0010] Harsh physiological conditions, difficult preparation requirements and unstable enzyme activity are the inherent fragility of natural enzymes. In order to find alternatives, chemists have been committed to developing nanomaterials that can completely mimic natural enzymes, commonly known as "nanozymes". With the rapid development of nanoscience, more and more new nanomaterials have been found to have excellent peroxidase properties. These peroxidase-like enzymes not only exhibit catalytic properties similar to natural enzymes, but some also have higher stability and higher specific surface area. In recent years, nanozymes with peroxidase activity have made significant progress, such as: precious metals, metal oxides, bimetallic oxides, metal sulfides, metal organic frameworks and carbon-based nanomaterials all show good peroxidase activity. The present invention adopts CuCo 2 O 4Nanomaterials as nanozymes, bimetallic oxide nanozymes can improve the current low catalytic performance of single metal ions and have wide applications in tumor treatment, cancer imaging, biosensors, etc.

[0011] Furthermore, the CuCo 2 O 4 The nanomaterial is prepared by the following method: dissolving copper salt and cobalt salt in a deep eutectic solvent (DES) to obtain a mixed solution, adding water to the mixed solution, and drying to obtain the CuCo 2 O 4 Nanomaterials.

[0012] Deep eutectic solvents (DES) are a new type of green solvents. Eutectic mixtures can be simply prepared by mixing hydrogen bond acceptors (HBA) with hydrogen bond donors (HBD) at 70-80°C. Compared with other solvents, they show better biocompatibility, biodegradability and lower toxicity. So far, DES have been widely used in the development of biosensors. They can be combined with different materials to improve the sensing performance of biosensor devices.

[0013] Furthermore, the copper salt is Cu(NO 3 ) 2 ·3H 2 O, the cobalt salt is Co(NO 3 ) 2 6H 2 O.

[0014] Furthermore, the copper salt and the cobalt salt are dissolved in DES at a temperature of 70 to 80° C. for a time of 1 to 3 hours.

[0015] Furthermore, the water is ultrapure water.

[0016] In a specific embodiment, water is added to the mixed solution to obtain a blue substance, and the blue substance is centrifuged, washed, and dried to obtain the CuCo 2 O 4 Nanomaterials.

[0017] Specifically, ultrapure water and ethanol were used for washing.

[0018] Specifically, the drying conditions are vacuum drying, the temperature is 60-70° C., and the time is 10-12 hours.

[0019] Furthermore, the DES is prepared from alkali and alcohol compounds.

[0020] Furthermore, the preparation method of the DES is: adding a base to an alcohol compound to obtain a mixed solution; stirring the mixed solution by a constant temperature magnetic stirrer to obtain the deep eutectic solvent (DES).

[0021] Furthermore, the stirring temperature is 60-80° C., and the stirring time is 20-30 min.

[0022] Furthermore, the base is potassium hydroxide (KOH), and the alcohol compound is 1,2-propylene glycol.

[0023] Furthermore, the CuCo 2 O 4 The concentration of the nanomaterial solution is 1.4-1.6 mg / mL.

[0024] Furthermore, the CuCo 2 O 4 The mass ratio of the nanomaterial to the ZEN antibody is (200-300):1, preferably 250:1.

[0025] Furthermore, the mass ratio of the carboxyl resin beads to the ZEN coated antigen is (150-250):1, preferably 213:1.

[0026] Furthermore, the zearalenone antibody is a monoclonal antibody secreted by the zearalenone hybridoma cell line ZEN-2D2.

[0027] Furthermore, the zearalenone coated antigen is prepared from the zearalenone hapten, and the structural formula of the zearalenone hapten is

[0028]

[0029] Furthermore, the zearalenone hapten is coupled with ovalbumin (OVA) to obtain the zearalenone coated antigen.

[0030] Furthermore, the activation method is to add an activation reagent, and the activation reagent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0031] Furthermore, the concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 8-9 mg / mL.

[0032] Furthermore, the concentration of the N-hydroxysuccinimide is 8-9 mg / mL.

[0033] Furthermore, the activation time is 2 to 3 hours.

[0034] The chemiluminescent immunosensor for detecting zearalenone provided by the present invention not only introduces low-cost, biocompatible and large specific surface area carboxyl resin beads as solid phase carriers to load more coated antigens, but also for the first time uses CuCo synthesized based on a new type of green solvent - deep eutectic solvent (with excellent biocompatibility, biodegradability and low toxicity). 2 O 4 Nanomaterials are used as antibody loads to label antibodies. The chemiluminescent immunosensor constructed has a simple preparation method and has good accuracy, selectivity and sensitivity. The chemiluminescent immunosensor was used for the first time to detect ZEN in food.

[0035] The present invention also protects the use of the above-mentioned chemiluminescent immunosensor for detecting zearalenone in detecting zearalenone.

[0036] The present invention uses peroxidase-like nanomaterials to replace natural enzyme molecules to react with Luminol-H 2 O 2 The chemiluminescence system was used for catalytic amplification, and a new type of flow injection chemiluminescence immunosensor based on non-natural enzyme catalysis was proposed for the detection of real samples.

[0037] The present invention adopts flow injection chemiluminescence immunoassay to detect the content of ZEN in food. Compared with the existing ZEN detection methods, this analysis method has strong controllability in time, space and luminescence intensity, and also has the advantages of simple operation, cost-effectiveness and high sensitivity. It provides a new method for the detection of ZEN, realizes rapid and ultra-sensitive detection of ZEN, and has good practical value and practical significance.

[0038] Further, the detection of ZEN by flow injection chemiluminescence immunoassay includes the following steps:

[0039] S1. Mixing the chemiluminescent probe with ZEN standard solutions of different concentrations and incubating the mixture to obtain a complex solution;

[0040] S2. adding a chemiluminescent immunosensor substrate to the complex solution to obtain an immunosensor composition after reaction;

[0041] S3. placing the immunosensor composition in a detector, adding 3-amino-phthalic acid hydrazide, hydrogen peroxide and a buffer solution, recording the luminescence intensity-time curve, establishing a relationship between the luminescence intensity of the immunosensor composition and the concentration of the ZEN standard solution, and obtaining a linear regression equation;

[0042] S4. Repeat steps S1 to S3 for the solution to be tested, and obtain the concentration of ZEN in the solution to be tested through the luminescence intensity of the immunosensor composition according to the linear regression equation.

[0043] When the chemiluminescent immunosensor for detecting zearalenone provided by the present invention detects ZEN, the ZEN solution competes with the ZEN coated antigen for limited ZEN antibodies. When ZEN is not added, the antigen and antibody are directly connected and there is no competitive reaction, so the ZEN coated antigen can bind well to the antibody, thereby showing a strong CL signal. When the ZEN standard solution concentration is added, the coated antigen and the ZEN standard solution compete for the limited binding sites on the antibody, resulting in a reduction in the number of immune probes connected to the immunosensor substrate, and the CL signal is reduced. Due to the strong specific recognition ability of the antigen and antibody, the CL immunosensor has excellent sensitivity, stability and reproducibility, which is of great significance for the detection of ZEN and similar small molecules.

[0044] Beneficial effects of the present invention:

[0045] 1. The chemiluminescent immunosensor for detecting zearalenone provided by the present invention not only introduces low-cost, biocompatible and large specific surface area carboxyl resin beads as solid phase carriers to load more coated antigens, but also for the first time uses CuCo synthesized based on a new type of green solvent - deep eutectic solvent (with excellent biocompatibility, biodegradability and low toxicity). 2 O 4 Nanomaterials are used as antibody loads to label antibodies. The chemiluminescent immunosensor constructed has a simple preparation method and has good accuracy, selectivity and sensitivity. The chemiluminescent immunosensor was used for the first time to detect ZEN in food.

[0046] 2. The present invention adopts flow injection chemiluminescence immunoassay to detect the content of ZEN in food. Compared with the existing ZEN detection methods, this analysis method has strong controllability in time, space and luminescence intensity. It also has the advantages of simple operation, cost-effectiveness and high sensitivity. It provides a new method for the detection of ZEN, realizes rapid and ultra-sensitive detection of ZEN, and has good practical value and practical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The present invention provides a schematic diagram of the construction of a chemiluminescent immunosensor for detecting zearalenone and the mechanism of detecting ZEN by flow injection chemiluminescent immunoassay.

[0048] Figure 2 CuCo 2 O 4Scanning electron microscope (SEM) and transmission electron microscope (TEM) images; A and B are CuCo 2 O 4 SEM images of C and D are CuCo 2 O 4 TEM image of.

[0049] Figure 3 : are SEM images of carboxyl resin beads and carboxyl resin beads loaded with ZEN-coated antigens; wherein A is the SEM image of carboxyl resin beads, and B is the SEM image of carboxyl resin beads loaded with ZEN-coated antigens.

[0050] Figure 4 The CL luminescence intensity-time curve and linear regression curve of ZEN standard solutions with different concentrations; A is the CL luminescence intensity-time curve of ZEN standard solutions with different concentrations. From left to right, the concentrations of ZEN standard solutions corresponding to each of the three adjacent peaks are (a) 0.0001 ng·mL -1 , (b) 0.005 ng·mL -1 , (c) 0.01 ng·mL -1 , (d) 0.1 ng·mL -1 , (e) 0.5 ng·mL -1 , (f) 10 ng·mL -1 and (g) 150ng·mL -1 , B is the linear regression curve of CL luminescence intensity of ZEN standard solution with different concentrations and the logarithm of ZEN concentration.

[0051] Figure 5 CuCo 2 O 4 , ZEN coated antigen and ZEN antibody concentration optimization diagram; where A is CuCo 2 O 4 A is the concentration optimization diagram of ZEN, B is the concentration optimization diagram of ZEN coated antigen, and C is the concentration optimization diagram of ZEN antibody.

[0052] Figure 6 CuCo 2 O 4 TMB or H 2 O 2 Michaelis-Menten model curve as substrate; A and C are CuCo 2 O 4 Michaelis-Menten curve with TMB as substrate and corresponding Lineweaver-Burk plot, B and D are CuCo 2 O 4 H 2 O2 Michaelis-Menten plot as substrate and the corresponding Lineweaver-Burk plot. DETAILED DESCRIPTION

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0054] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0055] In the following examples of the present invention, ZEN coated antigen and ZEN monoclonal antibody were donated by the research group of Professor Deng Anping of Soochow University. Among them, ZEN monoclonal antibody is a monoclonal antibody secreted by ZEN hybridoma cell line ZEN-2D2 (deposited in China Center for Type Culture Collection, with the deposit number CCTCC No.C202383, and the deposit address is Wuhan University, Bayi Road, Hongshan District, Wuhan City, Hubei Province). Antibody preparation first determines the appropriate modification site in the ZEN molecular structure based on the hapten molecule design theory and the characteristics of the ZEN molecular structure, synthesizes two ZEN modifiers with different linker arm lengths and carboxyl terminals, and connects them to the carrier protein, and then uses hybridoma antibody preparation technology to prepare highly sensitive and highly specific anti-ZEN monoclonal antibodies through animal immunization, fusion, screening and other steps. The IC of the ELISA method for determining ZEN established based on this antibody is 50 The value is 0.43ng·mL -1 The cross-reaction rates with aflatoxin and patulin are less than 0.1%.

[0056] The zearalenone coating antigen is obtained by coupling the zearalenone hapten with ovalbumin (OVA), and the reaction formula is as follows:

[0057]

[0058] In the following examples of the present invention, carboxyl resin beads were provided by Nanjing McPhee High Efficiency Separation Carrier Co., Ltd.

[0059] A chemiluminescent immunosensor for detecting zearalenone, comprising a chemiluminescent probe and a chemiluminescent immunosensor substrate, wherein the chemiluminescent probe is prepared by the following method: 2 O 4 The nanomaterial solution is incubated with the ZEN antibody (ZEN-Ab) to obtain the chemiluminescent immunoprobe;

[0060] The CuCo 2 O 4 The nanomaterial is prepared by the following method: dissolving copper salt and cobalt salt in a deep eutectic solvent (DES) to obtain a mixed solution, adding water to the mixed solution, and drying to obtain the CuCo 2 O 4 Nanomaterials.

[0061] The chemiluminescent immunosensor substrate is prepared by the following method: activating carboxyl resin beads to obtain activated resin beads, and incubating the activated resin beads with ZEN coated antigens (ZEN-Ae) to obtain the chemiluminescent immunosensor substrate.

[0062] The purpose of the present invention is to construct a CuCo based on DES solvent to address the shortcomings of the existing ZEN detection method. 2 O 4 The CL immunosensing method of nanozymes is used to detect the content of ZEN in foods such as corn and wheat, providing a new method for the detection of ZEN.

[0063] In one embodiment of the present invention, the copper salt is Cu(NO 3 ) 2 ·3H 2 O, the cobalt salt is Co(NO 3 ) 2 6H 2 O.

[0064] In one embodiment of the present invention, the DES is prepared from KOH and 1,2-propylene glycol.

[0065] In one embodiment of the present invention, the copper salt and the cobalt salt are dissolved in DES at a temperature of 70 to 80° C. for a time of 1 to 3 hours.

[0066] In one embodiment of the present invention, the CuCo 2 O 4 The concentration of the nanomaterial solution is 1.4-1.6 mg / mL.

[0067] In one embodiment of the present invention, the CuCo 2 O 4 The mass ratio of the nanomaterial to the ZEN antibody is (200-300):1, preferably 250:1.

[0068] In one embodiment of the present invention, the mass ratio of the carboxyl resin beads to the ZEN coated antigen is (150-250):1, preferably 213:1.

[0069] On this basis, flow injection chemiluminescence immunoassay was used to detect ZEN, including the following steps:

[0070] S1. Mixing the chemiluminescent probe with ZEN standard solutions of different concentrations and incubating the mixture to obtain a complex solution;

[0071] S2. adding a chemiluminescent immunosensor substrate to the complex solution to obtain an immunosensor composition after reaction;

[0072] S3. placing the immunosensor composition in a detector, adding 3-amino-phthalic acid hydrazide, hydrogen peroxide and a buffer solution, recording the luminescence intensity-time curve, establishing a relationship between the luminescence intensity of the immunosensor composition and the concentration of the ZEN standard solution, and obtaining a linear regression equation;

[0073] S4. Repeat steps S1 to S3 for the solution to be tested, and obtain the concentration of ZEN in the solution to be tested through the luminescence intensity of the immunosensor composition according to the linear regression equation.

[0074] Figure 1 The schematic diagram of the construction of the chemiluminescent immunosensor provided by the present invention and the mechanism of detecting ZEN by flow injection chemiluminescent immunoassay is shown in FIG. Figure 1 It can be seen that the ZEN solution competes with the ZEN coated antigen for the limited ZEN antibody. When ZEN is not added, the antigen and antibody are directly connected and there is no competition reaction. Therefore, the ZEN coated antigen can bind well to the antibody, thereby showing a strong CL signal. When the ZEN standard solution concentration is added, the coated antigen and the ZEN standard solution compete for the limited binding sites on the antibody, resulting in a decrease in the number of immune probes connected to the immunosensor substrate, and the CL signal decreases.

[0075] Figure 2 CuCo 2 O 4 Scanning electron microscope (SEM) and transmission electron microscope (TEM) images; A and B are CuCo 2 O 4 SEM images of C and D are CuCo 2 O 4 TEM image of.

[0076] Figure 3 : are SEM images of carboxyl resin beads and carboxyl resin beads loaded with ZEN-coated antigens; wherein A is the SEM image of carboxyl resin beads, and B is the SEM image of carboxyl resin beads loaded with ZEN-coated antigens.

[0077] Example 1

[0078] A chemiluminescent immunosensor for detecting zearalenone, comprising a chemiluminescent probe (CuCo2 O 4 -Ab) and a chemiluminescent immunosensor substrate, wherein the chemiluminescent probe is prepared by the following method:

[0079] Configure a certain concentration of CuCo 2 O 4 Nanomaterial solution, slowly add a certain concentration of ZEN antibody stock solution into CuCo 2 O 4 Nanomaterial solution (CuCo 2 O 4 The mass ratio of nanomaterial to ZEN antibody was 250:1), and after gently mixing, a mixed solution was obtained, and the mixed solution was placed in a 4°C refrigerator for incubation overnight. The next day, 5% bovine serum albumin (BSA) was added to the mixed solution, and after 2 hours, the unbound ZEN antibody and BSA were removed by centrifugation at 4500rpm for 5 minutes, and finally 0.01M PBS was added thereto, mixed and stored in a 4°C refrigerator for later use.

[0080] The CuCo 2 O 4 The nanomaterial was prepared by the following method: 0.34 g KOH was added to 16.16 mL 1,2-propylene glycol to obtain a mixed solution, and the mixed solution was stirred at 60° C. for 25 min using a constant temperature magnetic stirrer to obtain DES. 3 ) 2 ·3H 2 O and Co(NO 3 ) 2 6H 2 O was dispersed in 5 mL of DES in a molar ratio of 1:2 to obtain a mixed solution, and then the mixed solution was stirred at 70°C for 2 h in an oil bath. After the reaction was completed, 5 mL of deionized water was added to the mixed solution to obtain a blue substance. Finally, the blue substance was washed several times with deionized water and ethanol by centrifugation, and dried at 60°C for 10 h to obtain CuCo 2 O 4 Nanomaterials.

[0081] The chemiluminescent immunosensor substrate is prepared by the following method:

[0082] First, the carboxyl resin beads were dissolved in an ethanol-water mixture (V 无水乙醇 :V 水 =1:4) and placed in a 4°C refrigerator for later use. 0.17 g of carboxyl resin beads were placed in a 5 mL centrifuge tube and firstly 8 mg mL -1 EDC and 8mg mL -1NHS activated the carboxyl resin beads on a shaker in the dark. After 2 hours, the activated resin beads were washed several times with 0.01M PBS. After that, the ZEN coated antigen stock solution was diluted to a certain concentration with 0.01M PBS solution, and added to the centrifuge tube containing the washed carboxyl resin beads (the mass ratio of carboxyl resin beads to ZEN coated antigen was 213:1), and finally incubated in a 4°C refrigerator overnight. The next day, the resin beads modified with ZEN coated antigen were washed once with 0.01M PBS, and then 1mL of 2% casein (Casein) was added to each tube. After shaking for 2 hours, they were washed 3 times with PBST and PBS respectively, and then 0.01M PBS buffer was added to the centrifuge tube and stored in a 4°C refrigerator for later use.

[0083] Example 2

[0084] The chemiluminescent immunosensor for detecting zearalenone in Example 1 is used to detect ZEN by flow injection chemiluminescent immunoassay, and the detection includes the following steps:

[0085] S1. Add 110 μL of CuCo 2 O 4 -Ab solution was centrifuged to remove the supernatant, and then the lower precipitate was added to 200 μL of different concentrations ((a) 0.0001 ng mL -1 , (b) 0.005 ng·mL -1 , (c) 0.01 ng·mL -1 , (d) 0.1 ng·mL -1 , (e) 0.5 ng·mL -1 , (f) 10 ng·mL -1 and (g) 150ng·mL -1 ) in a ZEN standard solution, oscillate at a medium speed for 1 h to obtain a complex solution;

[0086] S2. Add chemiluminescent immunosensor substrate to the above complex solution. In this process, ZEN-Ae and ZEN can bind to ZEN-Ab. After 1 hour of reaction, use 0.01M PBS to wash away the CuCo that is not bound to ZEN-Ae. 2 O 4 -Ab and excess ZEN to obtain an immunosensor composition;

[0087] S3. Place the above immunosensor composition in a detector and add 0.1M Luminol (also known as luminol, chemical name 3-amino-phthalic acid hydrazide, CAS No. 521-31-3) and 8mM H 2 O 2The three chemiluminescent substrate solutions (0.01 M PBS buffer solution as carrier solution) were injected simultaneously, and the immunosensor was positioned by controlling the speed of the peristaltic pump. The pump speed of peristaltic pump 1 (P1) was selected to be 2 mL min -1 The pump speed of peristaltic pump 2 (P2) is 0.5 mL min -1 The chemiluminescent signal intensity detected by the photomultiplier tube (PMT) (-800V, ×2) was linearly negatively correlated with the concentration of the ZEN standard solution. The signal was converted by a computer and displayed as an amplified electrical signal. The luminescence intensity-time curve was recorded, and a linear relationship between the CL luminescence intensity of the immunosensor composition and the logarithmic value of the concentration of the ZEN standard solution was established to obtain the linear regression equation I CL =8117.3-1141.8logC(R 2 =0.9921).

[0088] The detection range of the chemiluminescent immunosensor for detecting zearalenone provided by the present invention is 0.0001 to 100 ng·mL -1 When the signal-to-noise ratio was 3, the detection limit (LOD) was 0.036 pg·mL -1 .

[0089] Figure 4 The CL luminescence intensity-time curve and linear regression curve of ZEN standard solutions with different concentrations; A is the CL luminescence intensity-time curve of ZEN standard solutions with different concentrations. From left to right, the concentrations of ZEN standard solutions corresponding to each of the three adjacent peaks are (a) 0.0001 ng·mL -1 , (b) 0.005 ng·mL -1 , (c) 0.01 ng·mL -1 , (d) 0.1 ng·mL -1 , (e) 0.5 ng·mL -1 , (f) 10 ng·mL -1 and (g) 150ng·mL -1 , B is the linear regression curve of CL luminescence intensity of ZEN standard solution with different concentrations and the logarithm of ZEN concentration.

[0090] Figure 5 CuCo 2 O 4 , ZEN coated antigen and ZEN antibody concentration optimization diagram; where A is CuCo 2 O 4 A is the concentration optimization diagram of ZEN coated antigen, and C is the concentration optimization diagram of ZEN antibody. Figure 5 As can be seen in A, the ΔCL intensity increases with the CuCo 2 O4 The ΔCL intensity gradually increases with the increase of concentration. When the concentration exceeds 1.5 mg / mL, the ΔCL intensity decreases sharply. This is because as CuCo 2 O 4 As the concentration increases, the effective concentration of the immunoprobe increases, the binding efficiency of the immunoprobe to the sensor substrate increases, and CuCo 2 O 4 Luminol-H 2 O 2 The catalytic amplification of the system is enhanced, but when the concentration of the nanomaterials reaches a certain value, the nanomaterials will aggregate. As the degree of aggregation increases, the coupling efficiency of the ZEN-coated antigen and the ZEN antibody will eventually decrease. Figure 5 As can be seen in Figure B, as the concentration of ZEN-coated antigen increases, the ΔCL value gradually increases. When the concentration of ZEN-coated antigen reaches 8 μg mL -1 When ΔCL reaches the maximum value, it indicates that 8 μg·mL -1 is the optimal parameter for the concentration of ZEN coating antigen. Figure 5 As shown in Figure C, the concentration of ZEN antibody is also very critical. As the concentration of ZEN antibody increases, the ΔCL value increases first and then decreases. 2 O 4 The material has a low antibody loading rate, a small number of immune probes are formed, and a low chemiluminescent signal. In addition, the higher the ZEN antibody concentration, the better, because a higher ZEN antibody concentration will increase the antibody loading rate, but it will also increase the background signal, ultimately affecting the performance of the sensor.

[0091] Example 3

[0092] The typical chromogenic substrates 3,3,5,5-tetramethyl-benzidine (TMB) and H 2 O 2 Steady-state kinetics were performed to evaluate the CuCo 2 O 4 of peroxidase activity.

[0093] By changing the TMB and H 2 O 2 The concentration of , determined the apparent steady-state kinetic parameters that follow the typical Michaelis-Menton model curve (Michaelis-Menten curve and corresponding Lineweaver-Burk plot), such as Figure 6 As shown. CuCo 2 O 4 and the Michaelis constant (K m ) and the maximum reaction rate (Vmax ) is achieved by the double reciprocal plotting method (Lineweaver-Burk plot), and the results are shown in Table 1:

[0094] Table 1 CuCo 2 O 4 Comparison of steady-state kinetic parameters (K m ,V max )

[0095]

[0096] Generally speaking, K m The smaller the value, the higher the affinity between the catalyst and the substrate. According to Table 1, the CuCo 2 O 4 K m The value is lower than that of HRP, which proves that CuCo 2 O 4 The affinity for the substrate TMB is higher than that for HRP. 2 O 2 CuCo 2 O 4 K m The value is higher than that of HRP, which indicates that CuCo 2 O 4 With H 2 O 2 The reaction will consume more H 2 O 2 , proves that CuCo 2 O 4 Nanomaterials can catalyze H 2 O 2 , which is CuCo 2 O 4 Materials in Luminol-H 2 O 2 It provides strong support for the catalytic amplification effect in the system.

[0097] Test Example 1

[0098] In order to evaluate the detection ability of the chemiluminescent immunosensor for detecting zearalenone of the present invention for actual samples, a spike recovery experiment was carried out on actual samples. 0.1 ng·mL -1 , 0.2ng·mL -1 and 0.5 ng·mL -1The samples containing corn and wheat were added. Wheat flour and corn flour were selected as real samples for detection, and the actual application ability of the sensor was evaluated by the recovery rate. The final experimental results are shown in Table 2. The recovery rates of wheat flour and corn flour are between 90.49% and 112.5%, and the errors are within the acceptable range. The above results show that the analytical method and chemiluminescent immunosensor used in the present invention are feasible and have good practical application value for the detection of ZEN in actual samples.

[0099] Table 2 Recovery experiment of spiked ZEN in real samples

[0100]

[0101] ND = not detected.

[0102] ZEN is a typical estrogen metabolite with a non-steroidal molecular structure. It mainly acts on the reproductive system and can cause hyperestrogenism in livestock and poultry. It often contaminates agricultural and grain products. Eating contaminated products may cause various toxic effects on humans and animals. Therefore, accurate and quantitative analysis of ZEN in agricultural products is very necessary for food safety and animal husbandry.

[0103] The present invention not only introduces low-cost, biocompatible and large specific surface area carboxyl resin beads as solid phase carriers to load more coated antigens in flow injection chemiluminescence immunoassay, but also for the first time uses CuCo synthesized based on a new type of green solvent - deep eutectic solvent (with excellent biocompatibility, biodegradability and low toxicity). 2 O 4 Nanomaterials are used as antibody loads to label antibodies. The chemiluminescent immunosensor constructed on this basis has a simple preparation method and good accuracy, selectivity and sensitivity. The chemiluminescent immunosensor was used for the first time to detect ZEN in feed. Under the optimal experimental conditions, the detection range of ZEN was 0.0001 to 100 ng·mL -1 The detection limit (LOD) was 0.036 pg·mL -1 (S / N=3). As a new type of green solvent, the combination of DES and nanomaterials will provide some new ideas for biosensing, which is conducive to its further in-depth research. At the same time, the sensor also provides a reference idea for the quantitative detection of other small molecules.

[0104] The present invention proposes a new detection method for the detection of ZEN in food and feed. Compared with the existing technology, it has strong controllability in time, space and luminescence intensity, can realize rapid and ultra-sensitive detection of ZEN, has the advantages of simple operation, cost-effectiveness, high sensitivity, etc., and has good practical value and practical significance.

[0105] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A chemiluminescent immunosensor for detecting zearalenone, comprising a chemiluminescent probe and a chemiluminescent immunosensor substrate, characterized in that: The chemiluminescent probe is prepared by the following method: incubating a CuCo2O4 nanomaterial solution with a zearalenone antibody to obtain the chemiluminescent probe; The chemiluminescent immunosensor substrate is prepared by the following method: activating carboxyl resin beads to obtain activated resin beads, and incubating the activated resin beads with zearalenone-coated antigens to obtain the chemiluminescent immunosensor substrate; The CuCo2O4 nanomaterial is prepared by the following method: dissolving a copper salt and a cobalt salt in a deep eutectic solvent to obtain a mixed solution, adding water to the mixed solution, and drying to obtain the CuCo2O4 nanomaterial; the copper salt is copper nitrate, and the cobalt salt is cobalt nitrate; the deep eutectic solvent is prepared from an alkali and an alcohol compound; the alkali is potassium hydroxide, and the alcohol compound is 1,2-propylene glycol.

2. The chemiluminescent immunosensor for detecting zearalenone according to claim 1, characterized in that: The concentration of the CuCo2O4 nanomaterial solution is 1.4-1.6 mg / mL.

3. The chemiluminescent immunosensor for detecting zearalenone according to claim 1, characterized in that: The mass ratio of the CuCo2O4 nanomaterial to the zearalenone antibody is (200-300):

1.

4. The chemiluminescent immunosensor for detecting zearalenone according to claim 1, characterized in that: The mass ratio of the carboxyl resin beads to the zearalenone-coated antigen is (150-250):

1.

5. The chemiluminescent immunosensor for detecting zearalenone according to claim 1, characterized in that: The zearalenone antibody is a monoclonal antibody secreted by the zearalenone hybridoma cell line ZEN-2D2.

6. The chemiluminescent immunosensor for detecting zearalenone according to claim 1, characterized in that: The zearalenone coated antigen is prepared from the zearalenone hapten, and the structural formula of the zearalenone hapten is 。 7. Use of the chemiluminescent immunosensor for detecting zearalenone according to any one of claims 1 to 6 in detecting zearalenone.

8. The use according to claim 7, characterized in that: The detection of zearalenone includes the following steps: S1. mixing the chemiluminescent probe with different concentrations of zearalenone standard solutions and incubating them to obtain a complex solution; S2. adding a chemiluminescent immunosensor substrate to the complex solution to obtain an immunosensor composition after the reaction; S3. placing the immunosensor composition in a detector, adding 3-amino-phthalhydrazide, hydrogen peroxide and a buffer solution, recording the luminescence intensity-time curve, establishing a relationship between the luminescence intensity of the immunosensor composition and the concentration of the zearalenone standard solution, and obtaining a linear regression equation; S4. Repeat steps S1 to S3 for the test solution, and obtain the concentration of zearalenone in the test solution through the luminescence intensity of the immunosensor composition according to the linear regression equation.

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