Aflatoxin B1 detection kit and detection method based on polyacrylic acid brush probe-enzyme-linked immunosorbent assay

Abstract

The invention discloses an aflatoxin B1 detection kit and a detection method based on polyacrylic acid brush probe-enzyme-linked immunosorbent assay. The aflatoxin B1 detection kit comprises a polyacrylic acid brush probe, a brush probe suspension, an aspergillus flavus B1 monoclonal antibody and a substrate solution, the polyacrylic acid brush type probe is formed by modifying a polyacrylic acid brush type microsphere with horse radish peroxidase and a mouse source universal IgG (Immunoglobulin G) antibody; the brush probe suspension is a PBS buffer solution containing sodium azide. According to the present invention, the polyacrylic acid brush type probe is constructed by simultaneously modifying the polyacrylic acid brush type microspheres with the mouse-derived IgG and the HRP, the polyacrylic acid brush type probe-enzyme-linked immunosorbent assay method can be used to rapidly detect the aflatoxin B1 content, and the detection method has characteristics of high sensitivity, strong specificity and good accuracy, and is beneficial to promotion and application.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, and in particular to a kit and method for detecting aflatoxin B1 based on polyacrylic acid brush probe-enzyme-linked immunosorbent assay (ELISA). Background Technology

[0002] Aflatoxin B1 is a toxic secondary metabolite produced by Aspergillus flavus and Aspergillus parasiticus. The chemical formula of aflatoxin B1 is C1. 17 H 12 O6, with a relative molecular weight of 312.27, exhibits good thermal stability but is hepatotoxic, nephrotoxic, carcinogenic, teratogenic, immunotoxic, and mutagenic to humans, and can cause the conversion of bases GC to TA or AT. Therefore, the International Agency for Research on Cancer (IARC) has classified aflatoxin B1 as a Group I carcinogen, and more than 100 countries worldwide have enacted legislation specifying maximum limits for aflatoxin B1 in agricultural products and food. my country's National Food Safety Standard (GB2761-2017) sets the limit for aflatoxin B1 in corn, peanuts, and bean products at 5 μg / kg-20 μg / kg.

[0003] Existing technologies for the detection of aflatoxin B1 include chromatographic analysis, nucleic acid aptamer or molecular imprinting methods, and immunoassay. Among these, tandem chromatography-mass spectrometry (GC-MS) combines the high separation capability of chromatography with the high sensitivity and selectivity of mass spectrometry, making it the most commonly used standard analytical method for aflatoxin B1. However, this instrumental method requires expensive equipment, has high detection costs, and requires specialized technical personnel to operate, thus not fully meeting the current detection needs of aflatoxin B1 in my country. Compared to antibodies, the core component of immunological detection, nucleic acid aptamers and molecularly imprinted polymers offer advantages such as low cost, stable properties, ease of synthesis, and easy modification by various chemical groups. However, the selectivity of molecularly imprinted polymers in aqueous environments is lower than that of antibodies. There are very few aptamers and molecularly imprinted complexes that meet the high sensitivity and specificity requirements for mycotoxin detection. Currently, there are no commercially available mycotoxin detection products based on nucleic acid aptamers and molecularly imprinted complexes.

[0004] Furthermore, immunological detection is an analytical technique based on the specific recognition reaction of "antigen-antibody". Enzyme-linked immunosorbent assay (ELISA) is a detection method that introduces proteases into an enzyme-labeled plate reaction system, mainly including ELISA, ELISA, and ELISA. ELISA, in particular, is a method that detects mycotoxins by catalyzing the substrate with enzymes, specifically HRP catalyzing o-phenylenediamine (OPD) or tetramethylbenzidine (TMB), causing a color change in the substrate. It is one of the most commonly used methods for detecting mycotoxins. ELISA has advantages such as high throughput, simple instrument requirements, and ease of operation. However, existing ELISA methods share common problems such as low reaction efficiency, limited signal enhancement methods, and insufficient sensitivity to meet the ultrasensitive detection requirements for trace mycotoxins in agricultural products. Traditional immunochromatographic test strips are a solid-phase rapid immunoassay technology using gold nanoparticles (AuNPs) as carriers to label antibodies as tracers. They offer advantages such as simplicity, speed, and suitability for on-site detection. The signal intensity of the detection line is directly presented through the enrichment concentration of AuNPs. Therefore, the detection signal amplification efficiency is low, and the sensitivity is lower than that of enzyme-linked immunosorbent assay (ELISA) and instrumental analysis. The application of fluorescent nanomaterials and enzymes in the construction of immunochromatographic test strips has improved the sensitivity of the detection results. However, the quenching effect of excessive aggregation of fluorescent nanomaterials can lead to a decrease or even disappearance of the fluorescence signal. The relatively low catalytic activity and substrate specificity of nanozymes limit their use in mycotoxin immunochromatography. Electrochemical sensing: Electrodes are modified with antibodies or antigens, and the concentration of mycotoxins in the sample is detected by changes in electron transfer signals caused by the "antigen-antibody" reaction. It has the advantages of good repeatability and high accuracy. Electrochemical sensors based on nanocomposite materials can improve electron transfer efficiency and detection sensitivity. However, due to the complexity of the electrode modification process, electrochemical sensing is not suitable for large-scale end-user applications.

[0005] As can be seen from the above-mentioned detection methods for aflatoxin B1, there is a lack of efficient detection methods with high specificity and sensitivity that can be used by a large number of end users. Therefore, it is necessary to propose a new detection method to solve the shortcomings of existing aflatoxin B1 detection methods. Summary of the Invention

[0006] The purpose of this invention is to provide an aflatoxin B1 detection kit and detection method based on polyacrylic acid brush probe-enzyme-linked immunosorbent assay (ELISA), which solves the problem of the lack of efficient and sensitive aflatoxin B1 detection methods in the prior art.

[0007] To solve the above-mentioned technical problems, the first solution provided by the present invention is an aflatoxin B1 detection kit based on polyacrylic acid brush probe-enzyme-linked immunosorbent assay (ELISA). The aflatoxin B1 detection kit includes a polyacrylic acid brush probe, a brush probe suspension, an aflatoxin B1 monoclonal antibody, and a substrate solution. The polyacrylic acid brush probe is formed by modifying polyacrylic acid brush microspheres with horseradish peroxidase and a mouse universal IgG antibody. The brush probe suspension is a PBS buffer containing sodium azide.

[0008] In some embodiments, the preparation method of the polyacrylic acid brush probe includes the following steps: K1, add ethanethiol and potassium phosphate to anhydrous acetone and stir until homogeneous. Add carbon disulfide and react for 5-20 min. Then add 4-(chloromethyl)phenylmethoxysilane and react under nitrogen for 10-15 h. After concentration and depressurization, dilute with dichloromethane and filter to remove the solvent. The resulting yellow residue is purified by gradient separation of petroleum ether / ethyl acetate on silica gel column chromatography to obtain product (I).

[0009] K2, add product (I) to an ethanol suspension containing SiO2 nanospheres, reflux with nitrogen for 10-15 h, then transfer to N,N-dimethylformamide and centrifuge and resuspend to obtain product (II).

[0010] K3, dodecyl mercaptan and potassium phosphate were added to anhydrous acetone and stirred until homogeneous. Carbon disulfide was added and reacted for 5-20 min. Benzyl bromide was then added and reacted at room temperature for 10-15 h. After removing the solvent by filtration under reduced pressure, the product (III) was obtained by freeze crystallization.

[0011] K4, product (III), acrylic acid and product (II) are mixed evenly, and after several freeze-suction-thaw cycles, DMF is dissolved, centrifuged and washed in sequence to obtain polyacrylic acid brush microspheres.

[0012] K5, polyacrylic acid brush microspheres were mixed with horseradish peroxidase in MES buffer, EDC was added for coupling, the coupling product was then dissolved in MES buffer, EDC / NHS was added to activate the carboxyl groups, the remaining EDC / NHS was removed by centrifugation, and mouse universal IgG antibody was added to couple it to the outer edge of the nanobrush structure of the polyacrylic acid brush microspheres. The reaction was terminated with Tris-HCl solution and unbound sites were blocked with bovine serum albumin to obtain the polyacrylic acid brush probe.

[0013] In some embodiments, in step K1, the ratio of ethanethiol, potassium phosphate, carbon disulfide, and 4-(chloromethyl)phenylmethoxysilane is (8-12) μL: (0.4-0.6) mg: (4-6) μL: (20-30) μL, and the dichloromethane dilution factor is (4-6):1; more preferably, the ratio of ethanethiol, potassium phosphate, carbon disulfide, and 4-(chloromethyl)phenylmethoxysilane is 10 μL: 0.5 mg: 5 μL: 25 μL, and the dichloromethane dilution factor is 5:1.

[0014] In some embodiments, in step K2, the mass ratio of product (I), ethanol suspension containing SiO2 nanospheres, and N,N-dimethylformamide is 1:(4-6):(0.1-0.3), the ethanol suspension containing SiO2 nanospheres is prepared by uniformly dispersing SiO2 nanospheres and ethanol solution at a ratio of 2 mg:(3-8) mL, and the average particle size of SiO2 nanospheres is (20-50) nm; more preferably, the mass ratio of product (I), ethanol suspension containing SiO2 nanospheres, and N,N-dimethylformamide is 1:5:0.2, the ethanol suspension containing SiO2 nanospheres is prepared by uniformly dispersing SiO2 nanospheres and ethanol solution at a ratio of 2 mg:5 mL, and the average particle size of SiO2 nanospheres is 40 nm.

[0015] In some embodiments, in step K3, the ratio of dodecyl mercaptan, potassium phosphate, carbon disulfide, and benzyl bromide is 10 μL: (0.1–0.3) mg: (3–5) μL: (1–4) μL, and the freezing crystallization temperature is -80 °C; more preferably, the ratio of didecyl mercaptan, potassium phosphate, carbon disulfide, and benzyl bromide is 10 μL: 0.2 mg: 4 μL: 2 μL.

[0016] In some embodiments, in step K4, the ratio of product (III), acrylic acid, and product (II) is (0.1–0.3) mg: (1–3) μL: (4–6) mL, and the number of freeze-vacuum-thaw cycles is 3–5; more preferably, the ratio of product (III), acrylic acid, and product (II) is 0.2 mg: 2 μL: 5 mL, and the number of freeze-vacuum-thaw cycles is 3.

[0017] In some embodiments, in step K5, during the coupling reaction of adding EDC, the ratio of polyacrylic acid brush microspheres, horseradish peroxidase, and EDC is 0.2 mg:(8-12) μg:(0.1-0.4) μg; more preferably, the ratio of polyacrylic acid brush microspheres, horseradish peroxidase, and EDC is 0.2 mg:10 μg:0.2 μg. During the EDC / NHS activation reaction, the ratio of polyacrylic acid brush microspheres, EDC, and NHS is 0.2 mg:(0.3-0.5) μg:(0.05-0.15) μg; more preferably, the ratio of polyacrylic acid brush microspheres, EDC, and NHS is 0.2 mg:0.4 μg:0.1 μg. In the process of adding murine universal IgG antibody to the outer edge of the nanobrush structure of polyacrylic acid brush microspheres, the ratio of polyacrylic acid brush microspheres to murine universal IgG antibody is 0.2 mg:(5-10) μg; more preferably, the ratio of polyacrylic acid brush microspheres to murine universal IgG antibody is 0.2 mg:8 μg. The reaction is terminated with a 10 mM Tris-HCl solution, and unbound sites are blocked with 2% bovine serum albumin.

[0018] In some embodiments, the aflatoxin B1 detection kit further includes PBS buffer, washing buffer, stop solution, and aflatoxin B1 standard solution; the PBS buffer is an aqueous solution containing 8‰ NaCl, 0.2‰ KCl, 0.24‰ KH2PO4, and 3.58‰ Na2HPO4•12H2O by mass fraction; the washing buffer is a PBS buffer containing 0.05% Tween 20 by mass fraction; the brush probe suspension is a PBS buffer containing 0.05% sodium azide by mass fraction; and the stop solution is an aqueous solution of 10% H2SO4.

[0019] In some embodiments, the aflatoxin B1 standard solution is an aqueous solution with different aflatoxin B1 concentrations; for example, 8 groups of aqueous solutions with different aflatoxin B1 concentrations are set up: 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.125 ng / mL, 1.5625 ng / mL, and 0 ng / mL.

[0020] In some embodiments, the preparation step of the substrate solution includes: uniformly dispersing citric acid and sodium citrate in water to obtain a mixed solution containing 4.2‰ citric acid and 8.8‰ sodium citrate by mass; adding o-phenylenediamine and H2O2 sequentially to the mixed solution and mixing well, wherein the ratio of the mixed solution, o-phenylenediamine, and H2O2 is 5 mL: 3 mg: 4 μL, to obtain the substrate solution, which is stored at 4 °C for later use.

[0021] To address the aforementioned technical problems, the second solution provided by this invention is a detection method. This detection method uses the aflatoxin B1 detection kit from the first solution described above and includes the following steps: S1, pre-process the sample to be tested to obtain a pre-processed sample.

[0022] S2, add aflatoxin B1 monoclonal antibody and aflatoxin B1 standard solution to the wells of the ELISA plate, and call it the standard group. Add aflatoxin B1 monoclonal antibody and pretreated sample to the wells of the ELISA plate, and call it the test group. Incubate both the standard group and the test group for 8-15 min.

[0023] S3, the incubated standard group and test group were washed with washing solution, and polyacrylic acid brush probe and brush probe suspension were added after washing, and the reaction was carried out for 8-15 min.

[0024] S4. The standard group and the test group were washed again with washing solution after the first reaction. After washing, the substrate solution was added to each group and the reaction was carried out for 12-18 min.

[0025] S5. After the secondary reaction, stop solution was added to the standard group and the test group respectively, and the absorbance value was measured. A standard curve was plotted on the aflatoxin B1 content and absorbance value based on the test data of the standard group. Based on the standard curve and the absorbance value measured by the test group, the aflatoxin B1 content in the test sample was calculated.

[0026] In some embodiments, a 96-well microplate is preferred. In other embodiments, the specifications of the microplate can be adapted and adjusted according to actual needs, and no limitation is made here.

[0027] In some embodiments, the specific pretreatment steps in step S1 are as follows: The finely ground sample to be tested is added to a methanol-water solution and dispersed evenly. Subsequently, it is subjected to ultrasonic treatment and centrifugation. The resulting supernatant is filtered through a membrane and diluted with PBS buffer to obtain the pretreated sample. The ratio of the sample to the methanol-water solution is 1 g: 2 mL, and the methanol-water solution is an aqueous solution containing 70% methanol by volume. The ultrasonic treatment time is 8–15 min, the centrifugation time is 8–15 min, and membrane filtration is performed using a 0.45 μM membrane. More preferably, the ultrasonic treatment time is 10 min, and the centrifugation time is 10 min.

[0028] In some embodiments, in step S2, the standard group includes 3 to 8 groups of aflatoxin B1 standard solutions with different aflatoxin B1 concentrations, such as 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.125 ng / mL, 1.5625 ng / mL, and 0 ng / mL, for a total of 8 groups of aqueous solutions with different aflatoxin B1 concentrations.

[0029] In some embodiments, in step S3, the volume ratio of the polyacrylic acid brush probe and the brush probe suspension added after washing is 1:(1 to 1.2); more preferably, the volume ratio of the polyacrylic acid brush probe and the brush probe suspension added after washing is 1:1.

[0030] In some embodiments, in step S5, a standard curve is plotted based on the test data of the standard group regarding the content of aflatoxin B1 and the absorbance value. This standard curve can be fitted using the formula: y = ax + b, where x represents the logarithm of the aflatoxin B1 concentration, y represents the inhibition rate, and a and b represent fitting constants. Since the content of aflatoxin B1 is negatively correlated with the absorbance value, and the absorbance value is also negatively correlated with the inhibition rate, the content of aflatoxin B1 is positively correlated with the inhibition rate. Therefore, the above standard curve can be characterized by a fitted curve of aflatoxin B1 content versus absorbance value, or by a fitted curve of aflatoxin B1 content versus inhibition rate; no limitation is made here. The above fitting method for the standard curve is only a partial implementation method, not all implementation methods. The specific fitting method can be selected according to actual needs and is not limited here.

[0031] The polyacrylic acid brush probe-ELISA method adopted in the above-mentioned solution of the present invention has the following basic principle: First, a 96-well ELISA plate is coated with a complete antigen, in which the complete antigen competes with the target in the sample for binding to monoclonal antibodies; Second, polyacrylic acid brush microspheres are simultaneously modified with murine IgG and HRP (horseradish peroxidase) to construct a polyacrylic acid brush probe, which then binds to the monoclonal antibody to form a complete antigen-monoclonal antibody-polyacrylic acid brush probe complex; Third, the enzyme catalyzes the substrate to generate a chromogenic substance, and qualitative or quantitative analysis is performed based on the presence and intensity of the chromogenic substance. In particular, in terms of quantitative analysis, the reaction system develops color under HRP catalysis, and the absorbance is read by the ELISA reader after the addition of the stop solution. The absorbance value is negatively correlated with the concentration of aflatoxin B1 in the sample. A standard curve on the aflatoxin B1 content and absorbance value is plotted from the test data of the standard group. Based on the standard curve and the absorbance value measured by the test group, the aflatoxin B1 content in the test sample is calculated.

[0032] In this detection method, the polyacrylic acid brush microspheres, with their flexible brush-like three-dimensional structure and abundant carboxyl groups, can load more bioactive molecules and are less susceptible to steric hindrance, thus significantly shortening the reaction time and increasing the detection speed. Simultaneously, the polyacrylic acid brush microspheres have a large surface area to volume ratio, allowing them to carry more HRP molecules while simultaneously guiding IgG through the nanoprobe, thereby enhancing detection sensitivity. Therefore, this detection method is characterized by high sensitivity, strong specificity, and good accuracy.

[0033] The beneficial effects of this invention are as follows: Unlike the prior art, this invention constructs a polyacrylic acid brush probe by simultaneously modifying polyacrylic acid brush microspheres with mouse-derived IgG and HRP. The polyacrylic acid brush probe-enzyme-linked immunosorbent assay (ELISA) method can rapidly detect the content of aflatoxin B1. Moreover, the detection method has the characteristics of high sensitivity, strong specificity, and good accuracy, which is conducive to its widespread application. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the microstructure of the polyacrylic acid brush probe in this invention; Figure 2 This is a standard curve of the logarithmic concentration of aflatoxin B1 content versus the inhibition rate in Example 3 of the present invention. Detailed Implementation

[0035] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0036] I. Preparation and Detection Methods Example 1 The preparation method of the polyacrylic acid brush probe in Example 1 includes the following steps: (1) Add 10 μL of ethanethiol and 0.5 mg of potassium phosphate to an appropriate amount of anhydrous acetone and stir until homogeneous. Add 5 μL of carbon disulfide and react for 5–20 min. Then add 25 μL of 4-(chloromethyl)phenylmethoxysilane and react under nitrogen for 10–15 h. After concentration and depressurization, dilute with dichloromethane at a ratio of 5:1 and filter to remove the solvent. The resulting yellow residue is purified by gradient separation of petroleum ether / ethyl acetate on silica gel column chromatography to obtain product (I).

[0037] (2) Weigh 2 mg of SiO2 nanospheres and 5 mL of ethanol solution and disperse them evenly to prepare an ethanol suspension containing SiO2 nanospheres. The average particle size of the SiO2 nanospheres is 40 nm. Add product (I) to the ethanol suspension containing SiO2 nanospheres and reflux with nitrogen for 10-15 h. Then transfer it into N,N-dimethylformamide and centrifuge and resuspend it. The mass ratio of product (I), ethanol suspension containing SiO2 nanospheres and N,N-dimethylformamide is 1:5:0.2 to obtain product (II).

[0038] (3) Add 10 μL of dodecyl mercaptan and 0.2 mg of potassium phosphate to an appropriate amount of anhydrous acetone and stir until homogeneous. Add 4 μL of carbon disulfide and react for 5–20 min. Then add 2 μL of benzyl bromide and react at room temperature for 10–15 h. After removing the solvent by filtration under reduced pressure, freeze crystallize at -80 °C to obtain product (III).

[0039] (4) Mix product (III), acrylic acid and product (II) in a ratio of 0.2 mg: 2 μL: 5 mL. After three cycles of freezing-vacuuming-thawing, DMF dissolution, centrifugation and washing are performed in sequence to obtain polyacrylic acid brush microspheres.

[0040] (5) Mix polyacrylic acid brush microspheres and horseradish peroxidase in MES buffer, add EDC for coupling, the ratio of polyacrylic acid brush microspheres, horseradish peroxidase and EDC is 0.2 mg: 10 μg: 0.2 μg; then dissolve the coupling product in MES buffer, add EDC / NHS to activate the carboxyl group, the ratio of polyacrylic acid brush microspheres, EDC and NHS is 0.2 mg: 0.4 μg: 0.1 μg; after centrifugation to remove the remaining EDC / NHS, add mouse universal IgG antibody to couple it to the outer edge of the nanobrush structure of polyacrylic acid brush microspheres, the ratio of polyacrylic acid brush microspheres to mouse universal IgG antibody is 0.2 mg: 8 μg, terminate the reaction with 10 mM Tris-HCl solution and block the unbound sites with 2% bovine serum albumin to obtain polyacrylic acid brush probe.

[0041] Example 2 A kit for detecting aflatoxin B1 was prepared based on the polyacrylic acid brush probe prepared in Example 1.

[0042] Preparation of PBS buffer: Weigh NaCl (8 g), KCl (0.2 g), KH2PO4 (0.24 g), and Na2HPO4·12H2O (3.58 g), add distilled water to 1000 mL to prepare PBS buffer.

[0043] The washing buffer was a PBS buffer containing 0.05% Tween 20 by mass.

[0044] The brush probe suspension is a PBS buffer containing 0.05% sodium azide by mass.

[0045] Aflatoxin B1 standard solutions: 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.125 ng / mL, 1.5625 ng / mL, 0 ng / mL, a total of 8 bottles, designated as the standard group.

[0046] Substrate preparation: Weigh citric acid (4.2 g) and sodium citrate (8.8 g), dissolve in 1000 mL of water, take 20 mL of the solution, add 12 mg of o-phenylenediamine and 16 μL of H2O2, mix well to obtain the substrate solution, and store at 4 °C for later use.

[0047] The stop solution is a 10% aqueous solution of H2SO4.

[0048] The concentration of Aspergillus B1 monoclonal antibody is [missing value].

[0049] The ELISA plate used is a 96-well ELISA plate.

[0050] Example 3 Example 3 uses the aflatoxin B1 detection kit from Example 2 for detection, and the detection method steps are as follows: (1) Pretreatment of the test samples: Take 1g of finely ground corn sample in three groups (each group has a different concentration of aflatoxin B1, which will be referred to as test samples I to III), add 2 mL of 70% methanol aqueous solution and soak for 40 min, sonicate the solution for 10 min, centrifuge at 4000 g for 10 min, filter the supernatant with 0.45 μM membrane and dilute with PBS buffer for later use.

[0051] (2) Add 50 µL of Aspergillus B1 monoclonal antibody and 50 µL of aflatoxin B1 standard solution to each well of the 96-well microplate, and call it the standard group (i.e., the 8 bottles of aflatoxin B1 standard solution in Example 2). Add 50 µL of Aspergillus B1 monoclonal antibody and 50 µL of pretreated sample to each well of the 96-well microplate, and call it the test group (i.e., test samples I to III). Incubate both the standard group and the test group for 10 min.

[0052] (3) Wash the standard group and the test group after incubation with washing solution. After washing, add 100 µL of polyacrylic acid brush probe and 100 µL of brush probe suspension to each well and react for 10 min.

[0053] (4) The standard group and the test group were washed again with washing solution after the first reaction. After washing, 150 µL of substrate solution was added to each well and the second reaction was carried out for 15 min.

[0054] (5) After the secondary reaction, 50 µL of stop solution was added to the standard group and the test group respectively, and the absorbance value was measured. A standard curve was plotted on the aflatoxin B1 content and absorbance value based on the test data of the standard group. Based on the standard curve and the absorbance value measured by the test group, the aflatoxin B1 content in the test sample was calculated.

[0055] Comparative Example 1 Based on the implementation steps of Example 3, the aflatoxin B1 standard solution in Example 3 was replaced with five standard solutions containing fumonisin, zearalenone, ochratoxin A, and T-2 toxin, respectively. The concentration of each toxin after replacement was 500 ng / mL. The content of these five substances, fumonisin, zearalenone, ochratoxin A, and T-2 toxin, was detected using the same polyacrylic acid brush probe-ELISA method as in Example 3.

[0056] II. Test Results and Analysis 1) Structural analysis of polyacrylic acid brush probe For an analysis of the structure of the polyacrylic acid brush probe prepared in Example 1, please refer to [link / reference needed]. Figure 1 , Figure 1 This is a schematic diagram of the microstructure of the polyacrylic acid brush probe in this invention. Figure 1 It is known that polyacrylic acid brush microspheres have a flexible brush-like three-dimensional spatial structure and contain a large number of carboxyl groups, which can load more bioactive molecules and are not easily affected by steric hindrance. After modifying polyacrylic acid brush microspheres with mouse-derived IgG and HRP, the constructed polyacrylic acid brush probe has a large surface area / volume ratio, which enables it to carry more HRP molecules and thus significantly improves the detection sensitivity.

[0057] 2) Feasibility verification A standard curve was plotted based on the test data from the standard group in Example 3 regarding the relationship between aflatoxin B1 content and inhibition rate. The standard curve is shown below. Figure 2 As shown, Figure 2The standard curve fitting equation is y = 35.21x + 43.61. Based on the standard curve and the absorbance values ​​measured in the test groups, the aflatoxin B1 content in samples I–III was calculated (each test group was tested 5 times), and the results are shown in Table 1. As can be seen from Table 1, the actual aflatoxin B1 content in samples I–III is very close to the detection results using the polyacrylic acid brush probe-ELISA method of this invention. The average coefficient of variation for the test results of samples with different aflatoxin B1 concentrations is only 4.40%, thus proving that the kit and detection method described in this invention have high accuracy in detecting aflatoxin B1 content.

[0058] Table 1 Note: a is the mean, n=3 3) Stability verification Based on the aforementioned detection methods for samples I-III, the changes in the detection concentrations of samples I-III over a period of 3 months (stored at 4°C) were recorded weekly, and the results are shown in Table 2. As can be seen from Table 2, during the 3-month period, the actual aflatoxin B1 content in samples I-III remained very close to the detection results using the polyacrylic acid brush probe-ELISA method of this invention. The average coefficient of variation for the detection results of samples with different aflatoxin B1 concentrations was only 4.55%, thus demonstrating that the kit and detection method described in this invention have high detection stability for aflatoxin B1 content.

[0059] Table 2 Note: a is the mean, n=3 4) Specificity verification The results of the tests in Example 3 and Comparative Example 1 were statistically analyzed, and the results are shown in Table 3. As can be seen from the results in Table 3, the polyacrylic acid brush probe-ELISA method of the present invention has a significant detection effect only on aflatoxin B1, and shows no cross-reaction with other potentially coexisting fungal toxins. This demonstrates that the kit and detection method described in this invention have high detection specificity for aflatoxin B1 content.

[0060] Table 3 Note: a No data detected Based on the above test results and analysis, the sensitivity of this detection method is 0.2 ng / kg. The average coefficient of variation for the test results of samples with different aflatoxin B1 concentrations was 4.40%. When all reagents were stored at 4°C for 3 months, the average coefficient of variation for the test results of samples with different aflatoxin B1 concentrations was 4.55%, and no cross-reaction was observed with other potentially coexisting mycotoxins.

[0061] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0062] The above embodiments merely illustrate implementation methods of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. An aflatoxin B1 detection kit based on polyacrylic brush type probe-enzyme linked immunoassay, characterized in that, The aflatoxin B1 detection kit comprises a polyacrylic acid brush probe, a brush probe suspension, an aflatoxin B1 monoclonal antibody and a substrate solution; The polyacrylic acid brush probe is formed by modifying polyacrylic acid brush microspheres with horseradish peroxidase and mouse universal IgG antibodies; The brush probe suspension is a PBS buffer containing sodium azide.

2. The AFB1 detection kit based on polyacrylic brush probe-enzyme linked immunoassay according to claim 1, characterized in that, The preparation method of the polyacrylic acid brush probe comprises the following steps: K1, add ethanethiol and potassium phosphate into anhydrous acetone and stir until uniform, add carbon disulfide and react for 5-20 min, then add 4-(chloromethyl) phenyl methoxysilane and react for 10-15 h under nitrogen, concentrate and reduce pressure, then dilute with dichloromethane and remove the solvent by filtration, the obtained yellow residue is purified by gradient separation on a silica gel column with petroleum ether / ethyl acetate, and the product (I) is obtained; K2, add the product (I) into an ethanol suspension containing SiO2 nanoscale microspheres, reflux for 10-15 h with nitrogen, then move into N,N-dimethylformamide and treat by centrifugation and resuspension to obtain the product (II); K3, add dodecanethiol and potassium phosphate into anhydrous acetone and stir until uniform, add carbon disulfide and react for 5-20 min, then add benzyl bromide and react for 10-15 h at room temperature, remove the solvent by filtration under reduced pressure, and then perform freeze crystallization to obtain the product (III); K4, mix the product (III), acrylic acid and the product (II) uniformly, perform several cycles of freeze-pumping-thaw treatment, then perform DMF dissolution, centrifugation and washing in sequence to obtain polyacrylic acid brush microspheres; K5, mix the polyacrylic acid brush microspheres and horseradish peroxidase in MES buffer, add EDC for coupling, then dissolve the coupling product in MES buffer, add EDC / NHS to activate the carboxyl group, remove the remaining EDC / NHS by centrifugation, add mouse universal IgG antibodies to couple them to the outer edge of the nanobrush structure of the polyacrylic acid brush microspheres, terminate the reaction with Tris-HCl solution, and block the unbound sites with bovine serum albumin to obtain the polyacrylic acid brush probe.

3. The AFB1 detection kit based on polyacrylic brush probe-enzyme linked immunoassay according to claim 2, characterized in that, In the K1 step, the proportions of ethanethiol, potassium phosphate, carbon disulfide and 4-(chloromethyl) phenyl methoxysilane are (8-12) μL:(0.4-0.6) mg:(4-6) μL:(20-30) μL, and the dilution multiple of dichloromethane is (4-6):1; In the K2 step, the volume ratio of the product (I), the ethanol suspension containing SiO2 nanoscale microspheres and N,N-dimethylformamide is 1 μL:(4-6) mL:(0.1-0.3) μL, the ethanol suspension containing SiO2 nanoscale microspheres is prepared by uniformly dispersing SiO2 nanoscale microspheres and ethanol solution at a ratio of 2 mg:(3-8) mL, and the average particle size of the SiO2 nanoscale microspheres is (20-50) nm.

4. The AFB1 detection kit based on polyacrylic brush probe-enzyme linked immunoassay according to claim 2, characterized in that, In the K3 step, the ratio of dodecyl mercaptan, potassium phosphate, carbon disulfide and benzyl bromide is 10 μL: (0.1-0.3) mg: (3-5) μL: (1-4) μL, and the temperature of the freeze crystallization is -80 ℃; In the K4 step, the ratio of the product (III), acrylic acid and the product (II) is (0.1-0.3) mg: (1-3) μL: (4-6) mL, and the number of cycles of the freeze-pumping-thaw cycle is 3-5.

5. The AFB1 detection kit based on polyacrylic brush probe-enzyme linked immunoassay according to claim 2, characterized in that, In the K5 step, in the coupling reaction with EDC, the ratio of the polyacrylic acid brush type microspheres, horseradish peroxidase and EDC is 0.2 mg: (8-12) μg: (0.1-0.4) μg; In the carboxyl group activation reaction with EDC / NHS, the ratio of the polyacrylic acid brush type microspheres, EDC and NHS is 0.2 mg: (0.3-0.5) μg: (0.05-0.15) μg; In the process of coupling the mouse universal IgG antibody to the outer edge of the nanobrush structure of the polyacrylic acid brush type microspheres, the ratio of the polyacrylic acid brush type microspheres and the mouse universal IgG antibody is 0.2 mg: (5-10) μg; The reaction is terminated by using a Tris-HCl solution with a concentration of 10 mM, and the unbound sites are blocked by using a bovine serum albumin with a concentration of 2%.

6. The AFB1 detection kit based on polyacrylic brush probe-enzyme linked immunoassay according to claim 2, characterized in that, The aflatoxin B1 detection kit further comprises a PBS buffer, a washing solution, a termination solution and an aflatoxin B1 standard solution; The PBS buffer is an aqueous solution containing 8‰ NaCl, 0.2‰ KCl, 0.24‰ KH2PO4 and 3.58‰ Na2HPO4·12H2O by mass fraction; The washing solution is a PBS buffer containing 0.05% Tween20 by mass fraction; The brush type probe suspension is a PBS buffer containing 0.05% sodium azide by mass fraction; The termination solution is a 10% H2SO4 aqueous solution; The aflatoxin B1 standard solution is an aqueous solution with different concentrations of aflatoxin B1.

7. The AFB1 detection kit based on polyacrylic brush probe-enzyme linked immunoassay according to claim 1, characterized in that, The preparation step of the substrate solution comprises: Citric acid and sodium citrate are uniformly dispersed in water to obtain a mixed solution containing 4.2‰ citric acid and 8.8‰ sodium citrate by mass fraction; After o-phenylenediamine and H2O2 are sequentially added to the mixed solution and mixed, the mixed solution, o-phenylenediamine and H2O2 are in a ratio of 5 mL: 3 mg: 4 μL to obtain the substrate solution, which is stored at 4 ℃ for standby use.

8. A detection method using the aflatoxin B1 test kit according to any one of claims 1 to 7, characterized by, The method comprises the following steps: S1, pretreating the sample to be tested to obtain a pretreated sample; S2, adding aflatoxin B1 monoclonal antibody and aflatoxin B1 standard solution to the microwells of an enzyme-labeled plate to form a standard group, and adding aflatoxin B1 monoclonal antibody and the pretreated sample to the microwells of the enzyme-labeled plate to form a sample group, and the standard group and the sample group are incubated for 8-15 min; S3, the standard group and the test group after incubation are washed respectively by using washing liquid, and after washing, polyacrylic acid brush type probe and brush type probe suspension are added, and one reaction is carried out for 8-15 min; S4, the standard group and the test group after one reaction are washed again by using washing liquid, and after washing, substrate liquid is added, and two reactions are carried out for 12-18 min; S5, the standard group and the test group after two reactions are added with termination liquid respectively, and absorbance values are measured respectively, a standard curve about aflatoxin Bl content and absorbance value is drawn based on the test data of the standard group, and based on the standard curve and the measured absorbance value of the test group, the content of aflatoxin Bl in the test sample is calculated.

9. The test method as claimed in claim 8, characterized in that In the S1 step, the specific steps of the pretreatment are as follows: The ground test sample is added into methanol aqueous solution for dispersion, and then ultrasonic treatment and centrifugal treatment are carried out in sequence, the obtained supernatant is filtered by membrane, and then diluted by PBS buffer to obtain the pretreated sample; The ratio of the test sample to methanol aqueous solution is 1 g:2 mL, and the methanol aqueous solution is an aqueous solution containing 70% methanol by volume fraction; The ultrasonic treatment time is 8-15 min, the centrifugal treatment time is 8-15 min, and 0.45 μM membrane is used for membrane filtration.

10. The test method as recited in claim 8, wherein, In the S2 step, the standard group contains 3-8 groups of aflatoxin Bl standard liquid with different aflatoxin Bl concentrations; In the S3 step, the volume ratio of polyacrylic acid brush type probe to brush type probe suspension after washing is 1: (1-1.2).

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