A method for detecting aflatoxin in edible oils

Abstract

This invention provides a method for detecting aflatoxin in edible oils, belonging to the field of aflatoxin detection. The method includes the following steps: functionalization treatment, in-situ compounding, preparation of a detection electrode, establishment of a standard curve, and sample detection. This invention's method for detecting aflatoxin in edible oils effectively overcomes the shortcomings of existing aflatoxin detection methods, such as complex pretreatment, low detection efficiency, and expensive detection equipment. Furthermore, it improves the loading, distribution uniformity, and stability of the luminescent material on the electrode surface, enhances electron transport efficiency, and optimizes antibody immobilization methods, thereby improving the luminescent signal intensity, response speed, sensitivity, accuracy, and reproducibility of the electrode in detecting aflatoxin in edible oils.

Description

Technical Field

[0001] This invention relates to the field of aflatoxin detection, and in particular to a method for detecting aflatoxin in edible oils. Background Technology

[0002] Aflatoxins are a class of fungal toxins, primarily secondary metabolites produced by *Aspergillus flavus*, *Aspergillus parasiticus*, and *Aspergillus simonii*. Currently, 18 toxins belonging to this class have been isolated and identified, including AFB1, AFB2, AFB2a, AFG1, AFG2, AFG2a, AFM1, and AFM2. Among them, aflatoxin B1 has the highest toxicity and carcinogenicity, approximately 10 times that of potassium cyanide and 68 times that of arsenic. Its carcinogenicity is 75 times that of dimethylnitrosamine, 900 times that of butter yellow, and 4000 times that of 3,4-benzopyrene. It also has teratogenic and immunosuppressive effects and is mainly distributed in plants and animals. Ingestion of aflatoxins by humans or animals can cause chronic poisoning with hemorrhagic necrosis of internal organs and can lead to cancer. The World Health Organization classified it as a Group 1 carcinogen in 1993. Meanwhile, since aflatoxin is fat-soluble, mold growth in oil crops such as peanuts and soybeans is the main route for aflatoxin contamination of food. Furthermore, aflatoxin B1 is highly stable and heat-resistant, making it difficult to destroy by general cooking methods. Low-dose intake of aflatoxin B1 can lead to poisoning in humans. Therefore, the detection and analysis of aflatoxin in edible oils is of great significance.

[0003] Currently, the main detection methods for aflatoxin include thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis, and fluorescence spectrometry. Among these, HPLC and ELISA are more widely used, but they still have the following technical drawbacks: the samples require cumbersome pretreatment and purification, resulting in complex pretreatment, low detection efficiency, expensive equipment, and unsuitability for large-scale detection.

[0004] Electrochemiluminescence immunosensors are widely used for the detection of various harmful substances due to their simple instrumentation, ease of operation, absence of background signal, rapid detection, and high sensitivity, and they also provide a new direction for the detection of aflatoxin. In existing technologies, organic light-emitting materials with aggregation-induced emission properties (such as 9,10-diphenylanthracene luminescent materials) are used in the construction of electrochemiluminescence immunosensors and the detection of aflatoxin due to their improved solubility in aqueous phase, high luminescence efficiency, and good biocompatibility. While they can avoid the drawbacks of conventional aflatoxin detection methods, such as complex pretreatment, low detection efficiency, and expensive equipment, they still have the following shortcomings in practical applications: 1) The loading density, distribution uniformity and stability of 9,10-diphenylanthracene luminescent material on the electrode surface are limited. Not only is it easy to fall off during use, but it also directly limits the intensity of its luminescent signal. As a result, its quantitative accuracy is poor when the concentration of aflatoxin in the edible oil to be tested is at the low end of the linear range.

[0005] 2) The 9,10-diphenylanthracene luminescent material has a simple interface structure on the electrode surface, resulting in high electron transfer impedance and low electron transport efficiency. Its response speed in actual detection needs to be further improved.

[0006] 3) During the process of using glutaraldehyde to crosslink and fix antibodies, it is difficult to accurately control the spatial orientation of the antibodies. Some antibody antigen-binding sites are easily masked or modified by crosslinking agents and thus inactivated, thereby reducing the effective antibody loading per unit area on the electrode surface and affecting the sensitivity and reproducibility of the sensor.

[0007] Based on this, a method for detecting aflatoxin in edible oils is provided. This method can effectively overcome the shortcomings of existing aflatoxin detection methods, such as complex pretreatment, low detection efficiency, and expensive detection equipment. It further improves the loading, distribution uniformity, and stability of the luminescent material on the electrode surface, enhances electron transport efficiency, and optimizes antibody immobilization methods. This, in turn, improves the luminescent signal intensity, response speed, sensitivity, accuracy, and reproducibility of the electrode in detecting aflatoxin in edible oils. This method has significant technical significance and research value. Summary of the Invention

[0008] To address the technical problems existing in the prior art, this invention provides a method for detecting aflatoxin in edible oils. This method effectively overcomes the shortcomings of existing aflatoxin detection methods, such as complex pretreatment, low detection efficiency, and expensive detection equipment. Furthermore, it improves the loading, distribution uniformity, and stability of the luminescent material on the electrode surface, enhances electron transport efficiency, and optimizes antibody immobilization methods. Consequently, it improves the luminescent signal intensity, response speed, sensitivity, accuracy, and reproducibility of the electrode in detecting aflatoxin in edible oils.

[0009] To solve the above technical problems, the technical solution adopted by the present invention is as follows: A method for detecting aflatoxin in edible oil includes the following steps: functionalization treatment, in-situ compounding, preparation of detection electrode, establishment of standard curve, and sample detection; The functionalization process involves adding 9,10-diphenylanthracene luminescent particles to an aqueous solution of N,N-dimethylformamide and dispersing them evenly. Then, at room temperature, an aqueous solution of polyethyleneimine is added dropwise and dispersed evenly to obtain a functionalized dispersion. The in-situ composite method involves dispersing zinc nitrate hexahydrate and nickel nitrate hexahydrate evenly in anhydrous methanol to obtain a metal source solution; dispersing 2-methylimidazole evenly in anhydrous methanol to obtain a ligand solution; mixing the functionalized dispersion with anhydrous methanol to obtain a functionalized diluent; and sequentially adding the metal source solution and ligand solution to the functionalized diluent at room temperature, stirring at room temperature, centrifuging to separate the solids, washing the solids, and dispersing them in deionized water to obtain a composite dispersion. The method for preparing the detection electrode is as follows: a composite dispersion is drop-coated onto the surface of a glassy carbon electrode and dried; then a glutaraldehyde solution is drop-coated onto the electrode surface, incubated at room temperature, and then washed; then aflatoxin B1 monoclonal antibody is drop-coated onto the electrode surface, incubated, and then washed; then bovine serum albumin is drop-coated onto the electrode surface, allowed to stand, and then washed to obtain the detection electrode. A three-electrode system was established using the aforementioned detection electrode to detect aflatoxin B1 standard working solutions of varying concentrations. A standard curve was established based on the relationship between the detected light signal intensity and the concentration of the aflatoxin B1 standard working solution. The sample solution to be tested was dropped onto the surface of the detection electrode, and the light signal intensity was detected. The aflatoxin B1 content of the sample solution to be tested was obtained based on the standard curve.

[0010] Preferably, in the functionalization treatment, the mass-to-volume ratio of 9,10-bisphenylanthracene luminescent particles to N,N-dimethylformamide aqueous solution is 10-12 mg:1 mL; The volume ratio of polyethyleneimine aqueous solution to N,N-dimethylformamide aqueous solution is 1:22-25; The concentration of polyethyleneimine is 8-12 μg / mL.

[0011] Preferably, in the in-situ composite, the total metal ion concentration in the metal source solution is 0.1-0.15 mol; the molar ratio of zinc nitrate hexahydrate to nickel nitrate hexahydrate in the metal source solution is 8.7-9:1-1.3. The concentration of 2-methylimidazole in the ligand solution is 0.8-0.9 mol.

[0012] Preferably, in the in-situ composite, the volume ratio of the functionalized dispersion to anhydrous methanol in the functionalized diluent is 1:9-9.2; The volume ratio of the metal source solution, ligand solution, and functionalized diluent is 1:1:1-1.1.

[0013] Preferably, in the in-situ compounding process, the stirring speed at room temperature is 300-400 rpm, and the stirring time at room temperature is 90-120 min; The concentration of solids in the composite dispersion is 10-12 mg / mL.

[0014] Preferably, in the preparation of the detection electrode, the room temperature incubation time after the glutaraldehyde solution is drop-coated is 1-2 hours; The incubation temperature for aflatoxin B1 monoclonal antibody after drop-coating is 4-6℃, and the incubation time is 10-12h. The standing time after bovine serum albumin is applied to the electrode surface is 50-70 minutes.

[0015] Preferably, in the preparation of the detection electrode, the concentration of glutaraldehyde solution is 1-1.2 wt%; the concentration of aflatoxin B1 monoclonal antibody is 40-50 μg / mL; and the concentration of bovine serum albumin is 0.9-1 wt%.

[0016] Furthermore, the method for establishing the standard curve is as follows: A gradient concentration of aflatoxin B1 standard working solution is dropped onto the surfaces of different detection electrodes. Each detection electrode is used as the working electrode, a platinum wire electrode as the counter electrode, and a calomel electrode as the reference electrode to establish a three-electrode system. Tripropylamine solution is used as the electrolyte. The detection voltage is controlled at 900-1000V, and the scanning voltage at 100-110mV / s. Cyclic voltages are applied to each working electrode using cyclic voltammetry to obtain the light signal intensity. A standard curve is established based on the relationship between the light signal intensity and the concentration of the aflatoxin B1 standard working solution.

[0017] Furthermore, the sample detection method involves adding a drop of the sample solution to the surface of the detection electrode, establishing a three-electrode system using the detection electrode as the working electrode, a platinum wire electrode as the counter electrode, and a calomel electrode as the reference electrode, using tripropylamine solution as the electrolyte, controlling the detection voltage at 900-1000V and the scanning voltage at 100-110mV / s, applying a cyclic voltage to the working electrode using cyclic voltammetry to obtain the light signal intensity, and obtaining the aflatoxin B1 content of the sample solution according to the standard curve.

[0018] Furthermore, it also includes the following steps: sample processing; The sample processing method is as follows: the edible oil to be tested, sodium chloride, and methanol solution are mixed and centrifuged. The lower layer of centrifuged liquid is mixed with an equal volume of n-hexane and centrifuged again. The lower layer of centrifuged liquid is then mixed evenly with phosphate buffer to obtain a sample dilution. The sample dilution is treated with an aflatoxin immunoaffinity column and then eluted with methanol to obtain an eluent. The eluent is then evaporated by nitrogen purging and reconstituted with phosphate buffer to obtain the sample solution to be tested. The weight ratio of the edible oil, sodium chloride, and methanol solution to be tested is 5-5.5:0.8-1:18-20.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) In the method for detecting aflatoxin in edible oil of the present invention, in the preparation of the detection electrode, based on the existing 9,10-diphenylanthracene luminescent particles with aggregation-induced emission characteristics, polyethyleneimine is used to functionalize them, and then Ni-doped ZIF-8 metal-organic framework is further composited by in-situ growth. The high specific surface area and three-dimensional pore structure of Ni-ZIF-8 are used to effectively increase the luminescent loading per unit electrode area, and the luminescence efficiency is enhanced by the metal-organic framework. At the same time, the electrochemical activity of ZIF-8 is regulated by Ni metal doping to form a conductive pathway, shorten the electron transport distance between the luminescent particle and the electrode, thereby shortening the detection response time and improving the stability of continuous scanning. Furthermore, the binding performance with antibodies is improved by the active groups and active sites of the metal-organic framework, and the antigen capture efficiency is improved by combining its porous structure and high specific surface area characteristics, thereby increasing the antigen loading. And the coordination effect of the metal-organic framework is achieved by cross-linking with glutaraldehyde, thereby realizing the directional fixation of antibodies, reducing the situation where the antigen binding sites are blocked or modified by cross-linking agents and thus inactivated, and improving the sensitivity and reproducibility of the detection electrode. The aforementioned technical means work together synergistically to effectively overcome the shortcomings of existing aflatoxin detection methods, such as complex pretreatment, low detection efficiency, and expensive detection equipment. At the same time, they can further improve the loading, distribution uniformity, and stability of the luminescent material on the electrode surface, improve electron transmission efficiency, optimize antibody immobilization methods, and thus improve the luminescent signal intensity, response speed, sensitivity, accuracy, and reproducibility of the electrode in the detection of aflatoxin in edible oils.

[0020] (2) The method for detecting aflatoxin in edible oil of the present invention has high sensitivity, strong anti-interference ability and good stability for detecting aflatoxin in edible oil, with an intra-batch RSD (precision) of 4.1-4.3%; the relative error rate between the detection results and the HPLC / MS determination results is 2.0-2.5%; at the same time, the spiked recovery rate is 94.3-97.0%, and the accuracy is consistent at different concentration levels; the detection limit for aflatoxin B1 in edible oil is 0.8 fg / mL.

[0021] (3) In the method for detecting aflatoxin in edible oil of the present invention, the intra-batch relative standard deviation (RSD) of the prepared detection electrode is 4.5%, the preparation method of the detection electrode is stable and controllable, and the prepared detection electrode has good response consistency; and after the detection electrode is continuously repeated for 25 cycles of cyclic voltammetry scanning, the light signal retention rate can still reach 96.1%; at the same time, the signal retention rate of the prepared detection electrode can still reach 91.3% after being stored in an environment of 4°C for 10 days; and the prepared detection electrode can effectively avoid the interference of common interfering substances (aflatoxin B2, aflatoxin G1, ochratoxin A, zearalenone, deoxynivalenol) on the detection results.

[0022] (4) In the method for detecting aflatoxin in edible oils of the present invention, the detection electrode prepared can increase the initial ECL signal intensity by 1.87 times compared with the traditional detection electrode that only uses 9,10-diphenylanthracene luminescent particles. ct Charge transfer resistance can be reduced by 43%, and antibody loading can be increased by 1.54 times. Detailed Implementation

[0023] To provide a clearer understanding of the technical features, objectives, and effects of this invention, specific embodiments are now described. It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, "first," "second," etc., are used to distinguish similar objects and are not used to describe a particular order or sequence. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0025] This invention provides a method for detecting aflatoxin in edible oil, comprising the following steps: preparing luminescent particles, functionalization treatment, in-situ composite, preparing detection electrode, establishing standard curve, sample processing, and sample detection.

[0026] The method for preparing the luminescent particles is as follows: 9,10-diphenylanthracene is added to tetrahydrofuran and ultrasonically dispersed to obtain a dispersion; the dispersion is injected into 2-2.5 times its volume of deionized water, ultrasonically dispersed for 20-30 min, and then freeze-dried to obtain luminescent particles based on 9,10-diphenylanthracene.

[0027] In the preparation of the luminescent particles, the mass-to-volume ratio of 9,10-bisphenylanthracene to tetrahydrofuran is 1 mg: 1-1.5 mL.

[0028] The functionalization process involves adding luminescent particles to an aqueous solution of N,N-dimethylformamide (DMF volume percentage of 35-40%), ultrasonically dispersing for 40-60 minutes, and then, at room temperature, adding an aqueous solution of polyethyleneimine (polyethyleneimine concentration of 8-12 μg / mL) with stirring. After the addition is complete, ultrasonically disperse for 10-20 minutes to obtain a functionalized dispersion, which is then stored at 4-6°C in the dark for later use.

[0029] In the functionalization process, the mass-to-volume ratio of the luminescent particles to the aqueous solution of N,N-dimethylformamide is 10-12 mg:1 mL. The volume ratio of the aqueous solution of polyethyleneimine to the aqueous solution of N,N-dimethylformamide is 1:22-25.

[0030] The in-situ composite method involves adding zinc nitrate hexahydrate and nickel nitrate hexahydrate in a molar ratio of 8.7-9:1-1.3 to anhydrous methanol, stirring until homogeneous, to obtain a metal source solution with a total metal ion concentration of 0.1-0.15 mol, which is then set aside. 2-Methylimidazole is added to anhydrous methanol, stirred until homogeneous, to obtain a ligand solution with a 2-methylimidazole concentration of 0.8-0.9 mol, which is then set aside. The functionalized dispersion is added to 9-9.2 times its volume of anhydrous methanol, ultrasonically dispersed until homogeneous, to obtain the functionalized dilute... Prepare the diluent and set aside for later use. Under stirring conditions of 300-400 rpm at room temperature, add the metal source solution dropwise to the functionalized diluent. After the addition is complete, continue stirring for 20-30 min. Then, continue adding the ligand solution dropwise. After the addition is complete, stir at room temperature for 90-120 min. Centrifuge at 10000-12000 rpm to collect the solids. Wash the solids sequentially with anhydrous methanol and deionized water, and then disperse them evenly in deionized water to obtain a composite dispersion with a solid concentration of 10-12 mg / mL.

[0031] In the in-situ composite, the volume ratio of the metal source solution, ligand solution, and functionalized diluent is 1:1:1-1.1.

[0032] The method for preparing the detection electrode is as follows: 5-6 μL of composite dispersion is drop-coated onto the surface of a clean glassy carbon electrode. After evaporation and drying in air, 18-22 μL of glutaraldehyde solution (1-1.2 wt%) is drop-coated onto the electrode surface. After incubation at room temperature for 1-2 hours, the electrode is washed with deionized water. 8-10 μL of aflatoxin B1 monoclonal antibody (40-50 μg / mL) is then drop-coated onto the electrode surface. The electrode is placed at 4-6℃ and incubated for 10-12 hours, followed by washing with phosphate buffer at pH 7.2-7.4. 18-20 μL of bovine serum albumin (BSA) (0.9-1 wt%) is then drop-coated onto the electrode surface. After standing at room temperature for 50-70 minutes, the electrode is washed with phosphate buffer at pH 7.2-7.4. The detection electrode is then prepared and stored at 4-6℃ for later use.

[0033] The method for establishing the standard curve is as follows: In a ventilated environment, a standard stock solution of aflatoxin B1 with a concentration of 1 mg / mL is prepared. Using phosphate buffer solution with a pH of 7.4 as a diluent, the standard stock solution of aflatoxin B1 is gradually diluted to a gradient concentration of 0-100 ng / mL to form aflatoxin B1 standard working solution. The specific concentrations are: 0.01 pg / mL, 0.1 pg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, and 100 ng / mL.

[0034] Different concentrations of aflatoxin B1 standard working solutions were added dropwise to the surfaces of the previously prepared detection electrodes. After incubation at room temperature for 40-50 min, the surfaces of each detection electrode were rinsed with phosphate buffer solution (pH 7.2-7.4) to remove unbound aflatoxin B1. A three-electrode system was then established, using each detection electrode as the working electrode, a platinum wire electrode as the counter electrode, and a calomel electrode as the reference electrode, and connected to an electrochemiluminescence detection device. This three-electrode system was inserted into an electrolytic cell containing 18-23 mL of PBS buffer solution containing tripropylamine. The detection voltage was controlled at 900-1000 V, and the scanning voltage at 100-110 mV / s. Cyclic voltammetry was used to scan within the working potential range of 0-1.6 V to obtain the light signal intensity. A standard curve was plotted based on the relationship between the light signal intensity and the corresponding concentration of the aflatoxin B1 standard working solution: the light signal intensity of the blank standard was denoted as D0, and the light signal intensity of the standard solutions containing different concentrations of aflatoxin B1 was denoted as D... i The difference in the decrease of the response optical signal intensity is ΔD = D0 - D i Plot a standard curve with ΔD as the ordinate and lgC (where C is the concentration of aflatoxin B1 standard working solution) as the abscissa, and fit the linear regression equation of the standard curve.

[0035] In establishing the standard curve, the PBS buffer solution of tripropylamine contained 70-75 mmol / L tripropylamine, 0.09-0.11 mol / L potassium chloride, and the balance was phosphate buffer solution with pH=7.4.

[0036] The sample processing method is as follows: The edible oil to be tested, sodium chloride, and methanol solution (volume concentration 75-80%) are mixed evenly at a weight ratio of 5-5.5:0.8-1:18-20. The mixture is then centrifuged at 4000-5000 rpm for 10-15 minutes. The lower layer of the centrifuged liquid is collected and mixed evenly with an equal volume of n-hexane. This mixture is then centrifuged at 4000-5000 rpm for 10-15 minutes. The lower layer of the centrifuged liquid is then collected and mixed evenly with 4.5-5 times its volume of phosphate buffer (pH 7.4). Obtain the sample diluent; after the sample diluent is treated with an aflatoxin immunoaffinity column, the immunoaffinity column is washed with phosphate buffer (pH 7.4) to remove impurities, and then eluted with 0.06-0.08 times the volume of the sample diluent in methanol to obtain the eluent; the eluent is placed in a nitrogen purging apparatus and purged with nitrogen at 45-50℃ until nearly dry, and then reconstituted with 0.65-0.7 times the volume of the eluent in phosphate buffer (pH 7.4) to ensure that the detection value falls within the linear range of the standard curve, thus obtaining the test sample solution.

[0037] The sample detection method is as follows: The sample solution to be tested is dropped onto the surface of another detection electrode prepared earlier. After incubation at room temperature for 40-50 minutes, the surfaces of each detection electrode are rinsed with phosphate buffer solution at pH 7.2-7.4 to remove unbound aflatoxin B1. Then, a three-electrode system is established using each detection electrode as the working electrode, a platinum wire electrode as the counter electrode, and a calomel electrode as the reference electrode, and connected to an electrochemiluminescence detection device. This three-electrode system is inserted into an electrolytic cell containing 18-23 mL of PBS buffer solution containing tripropylamine. Under the same detection parameters as those used to establish the standard curve, the light signal intensity D is detected. s ; Calculate the difference in intensity reduction of the response optical signal, ΔD = D0 - D s Substituting ΔD into the linear regression equation of the standard curve, the concentration C of aflatoxin B1 in the sample was calculated. s .

[0038] The PBS buffer solution of tripropylamine used in the sample detection was the same as that used to establish the standard curve.

[0039] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described below in conjunction with some specific embodiments.

[0040] Example 1 This embodiment provides a method for preparing a detection electrode for detecting aflatoxin in edible oils, the specific steps of which are as follows: 1. Preparation of luminescent particles 9,10-Diphenylanthracene was added to tetrahydrofuran and ultrasonically dispersed to obtain a dispersion. The dispersion was then injected into twice the volume of deionized water, ultrasonically dispersed for 25 min, and freeze-dried to obtain luminescent particles based on 9,10-diphenylanthracene.

[0041] The mass-to-volume ratio of 9,10-bisphenylanthracene to tetrahydrofuran is 1 mg:1.2 mL.

[0042] 2. Functionalization The luminescent particles were added to an aqueous solution of N,N-dimethylformamide (DMF volume percentage of 40%), and ultrasonically dispersed for 50 min. Then, at room temperature, an aqueous solution of polyethyleneimine (polyethyleneimine concentration of 11 μg / mL) was added dropwise with stirring. After the addition was complete, the solution was ultrasonically dispersed for 15 min to obtain a functionalized dispersion, which was stored at 4°C in the dark for later use.

[0043] The mass-to-volume ratio of the luminescent particles to the N,N-dimethylformamide aqueous solution was 10 mg: 1 mL.

[0044] The volume ratio of the aqueous solution of polyethyleneimine to the aqueous solution of N,N-dimethylformamide is 1:22.

[0045] 3. In-situ composite Zinc nitrate hexahydrate and nickel nitrate hexahydrate in a molar ratio of 8.9:1.1 were added to anhydrous methanol and stirred until homogeneous to obtain a metal source solution with a total metal ion concentration of 0.12 mol, which was set aside for later use. 2-Methylimidazole was added to anhydrous methanol and stirred until homogeneous to obtain a ligand solution with a 2-methylimidazole concentration of 0.85 mol, which was set aside for later use. The functionalized dispersion was added to 9 times its volume of anhydrous methanol and ultrasonically dispersed until homogeneous to obtain a functionalized diluent, which was set aside for later use. Under stirring at 350 rpm at room temperature, the metal source solution was added dropwise to the functionalized diluent. After the addition was complete, stirring was continued for 25 min. Then, the ligand solution was added dropwise. After the addition was complete, stirring was continued at room temperature for 110 min. The solids were collected by centrifugation at 12000 rpm. The solids were washed sequentially with anhydrous methanol and deionized water, and then dispersed homogeneously in deionized water to obtain a composite dispersion with a solid concentration of 12 mg / mL.

[0046] The volume ratio of the metal source solution, ligand solution, and functionalized diluent is 1:1:1.

[0047] 4. Preparation of detection electrodes 5 μL of the composite dispersion was drop-coated onto the clean glassy carbon electrode surface and dried in air. Then, 20 μL of glutaraldehyde solution (1.2 wt%) was drop-coated onto the electrode surface and incubated at room temperature for 1.5 h, followed by washing with deionized water. Next, 9 μL of aflatoxin B1 monoclonal antibody (50 μg / mL) was drop-coated onto the electrode surface and incubated at 4 °C for 12 h, followed by washing with phosphate buffer at pH 7.4. Finally, 20 μL of bovine serum albumin (BSA) (1 wt%) was drop-coated onto the electrode surface and allowed to stand at room temperature for 60 min, followed by washing with phosphate buffer at pH 7.4. The detection electrode was then prepared and stored at 4 °C for later use.

[0048] Example 2 This embodiment provides a method for preparing a detection electrode for detecting aflatoxin in edible oils, the specific steps of which are as follows: 1. Preparation of luminescent particles 9,10-Diphenylanthracene was added to tetrahydrofuran and ultrasonically dispersed to obtain a dispersion. The dispersion was then injected into 2.5 times its volume of deionized water, ultrasonically dispersed for 30 min, and freeze-dried to obtain luminescent particles based on 9,10-diphenylanthracene.

[0049] The mass-to-volume ratio of 9,10-bisphenylanthracene to tetrahydrofuran is 1 mg:1.5 mL.

[0050] 2. Functionalization The luminescent particles were added to an aqueous solution of N,N-dimethylformamide (DMF volume percentage of 35%) and ultrasonically dispersed for 60 min. Then, at room temperature, an aqueous solution of polyethyleneimine (polyethyleneimine concentration of 8 μg / mL) was added dropwise with stirring. After the addition was complete, the solution was ultrasonically dispersed for 20 min to obtain a functionalized dispersion, which was stored at 4°C in the dark for later use.

[0051] The mass-to-volume ratio of the luminescent particles to the N,N-dimethylformamide aqueous solution was 10 mg: 1 mL.

[0052] The volume ratio of the aqueous solution of polyethyleneimine to the aqueous solution of N,N-dimethylformamide is 1:25.

[0053] 3. In-situ composite Zinc nitrate hexahydrate and nickel nitrate hexahydrate in a molar ratio of 9:1 were added to anhydrous methanol and stirred until homogeneous to obtain a metal source solution with a total metal ion concentration of 0.15 mol, which was set aside for later use. 2-Methylimidazole was added to anhydrous methanol and stirred until homogeneous to obtain a ligand solution with a 2-methylimidazole concentration of 0.9 mol, which was set aside for later use. The functionalized dispersion was added to 9.2 times its volume of anhydrous methanol and ultrasonically dispersed until homogeneous to obtain a functionalized diluent, which was set aside for later use. Under stirring at 400 rpm at room temperature, the metal source solution was added dropwise to the functionalized diluent. After the addition was complete, stirring was continued for 30 min. Then, the ligand solution was added dropwise. After the addition was complete, stirring was continued at room temperature for 90 min. The solids were collected by centrifugation at 12000 rpm. The solids were washed sequentially with anhydrous methanol and deionized water, and then dispersed homogeneously in deionized water to obtain a composite dispersion with a solid concentration of 12 mg / mL.

[0054] The volume ratio of the metal source solution, ligand solution, and functionalized diluent is 1:1:1.1.

[0055] 4. Preparation of detection electrodes 5 μL of the composite dispersion was drop-coated onto the clean glassy carbon electrode surface and dried by evaporation in air. Then, 18 μL of glutaraldehyde solution (1.2 wt%) was drop-coated onto the electrode surface and incubated at room temperature for 2 h, followed by washing with deionized water. Next, 8 μL of aflatoxin B1 monoclonal antibody (50 μg / mL) was drop-coated onto the electrode surface and incubated at 4 °C for 10 h, followed by washing with phosphate buffer at pH 7.4. Finally, 18 μL of bovine serum albumin (BSA) (0.9 wt%) was drop-coated onto the electrode surface and allowed to stand at room temperature for 50 min, followed by washing with phosphate buffer at pH 7.4. The detection electrode was then prepared and stored at 4 °C for later use.

[0056] Example 3 This embodiment provides a method for detecting aflatoxin in edible oil, using the detection electrode prepared in Example 1. The specific steps are as follows: 1. Establish a standard curve In a well-ventilated environment, a standard stock solution of aflatoxin B1 with a concentration of 1 mg / mL was prepared. Using phosphate buffer solution with a pH of 7.4 as a diluent, the aflatoxin B1 standard stock solution was serially diluted to a working standard solution of aflatoxin B1 with concentrations ranging from 0 to 100 ng / mL. The specific concentrations were: 0.01 pg / mL, 0.1 pg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, and 100 ng / mL.

[0057] Different concentrations of aflatoxin B1 standard working solutions were added dropwise to the surfaces of the previously prepared detection electrodes. After incubation at room temperature for 50 min, the surfaces of each detection electrode were rinsed with phosphate buffer solution at pH 7.4 to remove unbound aflatoxin B1. A three-electrode system was then established, using each detection electrode as the working electrode, a platinum wire electrode as the counter electrode, and a calomel electrode as the reference electrode, and connected to an electrochemiluminescence detection device. This three-electrode system was inserted into an electrolytic cell containing 22 mL of tripropylamine in PBS buffer solution. The detection voltage was controlled at 1000 V, and the scanning voltage at 100 mV / s. Cyclic voltammetry was used to scan within the working potential range of 0-1.6 V to obtain the light signal intensity. A standard curve was plotted based on the relationship between the light signal intensity and the corresponding concentration of the aflatoxin B1 standard working solution: the light signal intensity of the blank standard was denoted as D0, and the light signal intensity of the standard solutions containing different concentrations of aflatoxin B1 was denoted as D... i The difference in the decrease of the response optical signal intensity is ΔD = D0 - D i Plot a standard curve with ΔD as the ordinate and lgC (where C is the concentration of aflatoxin B1 standard working solution) as the abscissa. The linear regression equation for the standard curve is then fitted: ΔD = 32041.96 - 2615.08lgC, with a linear correlation coefficient R0. 2 =0.9935.

[0058] The tripropylamine PBS buffer solution contains 75 mmol / L tripropylamine, 0.1 mol / L potassium chloride, and the remainder is phosphate buffer at pH 7.4.

[0059] 2. Sample processing Peanut oil and corn oil were selected as the edible oils to be tested. They were mixed with sodium chloride and methanol solution (80% volume concentration) at a weight ratio of 5.5:1:20. The mixture was centrifuged at 5000 rpm for 15 min. The lower layer of the centrifuged liquid was mixed with an equal volume of n-hexane and centrifuged at 5000 rpm for 15 min. The lower layer of the centrifuged liquid was mixed with 5 times the volume of phosphate buffer (pH 7.4) to obtain the sample dilution. The sample dilution was treated with an aflatoxin immunoaffinity column. The immunoaffinity column was washed with phosphate buffer (pH 7.4) to remove impurities. Then, it was eluted with 0.06 times the volume of the sample dilution in methanol to obtain the eluent. The eluent was placed in a nitrogen purging apparatus and purged with nitrogen at 45°C until nearly dry. It was then reconstituted with 0.65 times the volume of the eluent in phosphate buffer (pH 7.4) to obtain sample solution No. 1 (peanut oil) and sample solution No. 2 (corn oil).

[0060] 3. Sample testing Sample solutions 1 and 2 were added dropwise to the surface of the newly prepared detection electrodes. After incubation at room temperature for 45 min, the surfaces of each detection electrode were rinsed with phosphate buffer (pH 7.4) to remove unbound aflatoxin B1. A three-electrode system was then established, using each detection electrode as the working electrode, a platinum wire electrode as the counter electrode, and a calomel electrode as the reference electrode, and connected to the electrochemiluminescence detection device. This three-electrode system was inserted into an electrolytic cell containing 22 mL of tripropylamine in PBS buffer solution. Under the same conditions as the established standard curve (detection voltage 1000 V, scan voltage 100 mV / s), cyclic voltammetry was used to scan within the working potential range of 0-1.6 V to detect the light signal intensity D. s ; Calculate the difference in intensity reduction of the response optical signal, ΔD = D0 - D s Substituting ΔD into the linear regression equation of the standard curve, the concentration C of aflatoxin B1 in the sample was calculated. s Each sample solution was measured three times consecutively in parallel.

[0061] The concentration of aflatoxin B1 in the tested edible oil (peanut oil) was 0.51 μg / kg, with an intra-batch RSD (precision) of 4.3%. The concentration of aflatoxin B1 in the tested edible oil (peanut oil) was determined to be 0.5 μg / kg using HPLC-MS / MS. Based on the HPLC-MS / MS result, the relative error rate was calculated to be 2.0%.

[0062] The concentration of aflatoxin B1 in the tested edible oil (corn oil) was 1.23 μg / kg, with an intra-batch RSD (precision) of 4.1%. The concentration of aflatoxin B1 in the tested edible oil (peanut oil) was determined to be 1.20 μg / kg by HPLC-MS / MS. Based on the HPLC-MS / MS results, the relative error rate was calculated to be 2.5%.

[0063] 4. Detection of spiked recovery rate Take three equal volumes of sample solution No. 1 (peanut oil). Add 1 μg / L of aflatoxin B1 standard working solution to the first sample solution, controlling the final spiking concentration to 0.5 μg / L. Using the aforementioned three-electrode system and under the aforementioned detection parameters, detect the spiked sample extract. Based on the detection results of the spiked sample extract, the recovery rate is calculated to be 94.3%. Add 10 μg / L of aflatoxin B1 standard working solution to the second sample solution, controlling the final spiking concentration to 5.0 μg / L. L. Using the aforementioned three-electrode system and under the aforementioned detection parameters, the spiked sample extract was tested. Based on the test results of the spiked sample extract, the recovery rate was calculated to be 96.8%. Aflatoxin B1 standard working solution with a concentration of 20 μg / L was added to the third sample solution (sample No. 1), and the final spiked concentration was controlled to be 10.0 μg / L. Using the aforementioned three-electrode system and under the aforementioned detection parameters, the spiked sample extract was tested. Based on the test results of the spiked sample extract, the recovery rate was calculated to be 97.0%.

[0064] 5. Detection limit Using the aforementioned three-electrode system and under the aforementioned detection parameters, a phosphate buffer solution (blank sample) with a pH of 7.4 was tested, and the standard deviation S0 of the blank signal was obtained. The AFB1 standard working solution with a concentration of 1 ng / mL was gradually diluted, and the limit of detection was calculated using a signal-to-noise ratio S / N = 3. The method detection limit for aflatoxin B1 in edible oils using the method of this invention was found to be 0.8 fg / mL, which meets the requirements for ultra-trace detection.

[0065] Furthermore, to evaluate the response consistency of the detection electrodes, the detection electrode preparation method of Example 1 was repeated 5 times to prepare 5 detection electrodes. Then, a 10 pg / mL aflatoxin B1 standard working solution was used as the test solution. Using the aforementioned three-electrode system and under the aforementioned detection parameter conditions, each newly prepared detection electrode was used to detect the test solution, and the light signal intensity D of the test solution was recorded. s Each detection electrode was tested in parallel three times, and the average value was taken as the response value of that electrode. The intra-batch relative standard deviation (RSD) of the aforementioned five detection electrodes was calculated to be 4.5%, indicating that the preparation method of the detection electrodes is stable and controllable, and the prepared detection electrodes have good response consistency.

[0066] Furthermore, to evaluate the continuous detection stability of the detection electrode, after preparing one detection electrode using the detection electrode preparation method of Example 1, the aforementioned three-electrode system was used to continuously and repeatedly perform cyclic voltammetry scans (without intervals) on aflatoxin B1 standard working solution with a concentration of 10 pg / mL under the aforementioned detection parameter conditions; the light signal intensity D obtained from the first detection was used as the basis for the measurement. s1 Using this as a benchmark, calculate the light signal retention rate of the detection electrode in the 25th detection: (Light signal intensity D obtained in the 25th detection) s25 / Light signal intensity D obtained in the first detection s1 ()×100%. Through testing, the detection electrode of this invention still achieved a light signal retention rate of 96.1% in the 25th detection, indicating good continuous detection stability of the detection electrode.

[0067] Furthermore, to evaluate the long-term stability of the detection electrodes, four detection electrodes were prepared using the method described in Example 1. These electrodes were placed in a humid environment at 4°C and stored covered with gauze soaked in phosphate buffer. On days 1, 3, 7, and 10 of storage, one electrode was removed. Using the aforementioned three-electrode system and under the aforementioned detection parameters, a 10 pg / mL aflatoxin B1 standard working solution was detected. The intensity D of the detected light signal was recorded. s The intensity D of the light signal detected on day 1. s1 Based on this, calculate the signal retention rate of the detection electrode during the 10th day of detection: (Signal intensity D obtained during the 10th day of detection) s10 / Signal strength D obtained on day 1 s1 ()×100%. Tests showed that the signal retention rate of the detection electrode of this invention remained at 91.3% after 10 days of storage at 4°C, indicating good long-term stability.

[0068] Furthermore, to evaluate the anti-interference performance of the detection electrode, a 10 pg / mL aflatoxin B1 standard working solution was used as the test substrate. Interfering substances (aflatoxin B2, aflatoxin G1, ochratoxin A, zearalenone, and deoxynivalenol) were added to the test substrate, and the concentrations of each interfering substance were controlled to be 10, 20, 50, 100, and 200 times that of aflatoxin in the aflatoxin B1 standard working solution. Then, the aforementioned three-electrode system was used to perform detection under the aforementioned detection parameter conditions, and the intensity D of the detected light signal was recorded. n .

[0069] Using the light signal intensity D0 of aflatoxin B1 standard working solution with a concentration of 10 pg / mL as a benchmark, calculate the signal change rate after adding the interfering substance: [(light signal intensity D0 after adding the interfering substance)] n [(Reference light signal intensity D0) / Reference light signal intensity D0] × 100%; The highest allowable concentration ratio of the interfering substance that causes a change in light signal within ±5% is recorded as the highest allowable concentration ratio of that substance. Experiments showed that the highest allowable concentration ratios of aflatoxin B2, aflatoxin G1, ochratoxin A, zearalenone, and deoxynivalenol against aflatoxin B1 all exceeded 100 times.

[0070] Comparative Example 1 The detection electrode preparation method of Example 1 is adopted, except that the in-situ composite step is omitted, and the solid concentration of the functionalized dispersion obtained in the functionalization treatment step is adjusted to 12 mg / mL, which is used as the composite dispersion in the detection electrode preparation step.

[0071] To compare the initial ECL signal intensity of Example 1 and Comparative Example 1, 5 μL of the composite dispersion used in the preparation of the detection electrode in Example 1 and Comparative Example 1 were respectively drop-coated onto the surface of different glassy carbon electrodes and dried in air to form detection electrodes. Each electrode was then inserted into an electrolytic cell containing the same tripropylamine solution as described above. The detection voltage was controlled at 1000 V and the scanning voltage at 100 mV / s. The initial ECL signal intensity was recorded using cyclic voltammetry (0-1.6 V, 100 mV / s). Based on the initial ECL signal intensity of the electrode in Comparative Example 1, the improvement factor of the initial ECL signal intensity of Example 1 compared to Comparative Example 1 was calculated to be 1.87 times.

[0072] To compare the electron transport efficiency of Example 1 and Comparative Example 1, the detection electrode (without fixed antibody) of Example 1 and the detection electrode (without fixed antibody) of Comparative Example 1 were respectively subjected to 0.1 mol / L KCl + 5 mmol / L [Fe(CN)6] 3- / 4- As the probe solution, electrochemical impedance spectroscopy was performed at open-circuit potential, controlling the frequency range from 0.1 to 100 kHz and the amplitude at 5 mV; the equivalent circuit model R+(R) was used. ct ||CPE) is used to fit the impedance data, where R is the solution resistance, R ct As a charge transfer resistor, CPE is a constant-phase element. Taking the R of electrode 1 in Comparative Example 1 as an example... ct Based on the charge transfer resistance, the R value of the electrode in Example 1 was calculated. ct The charge transfer resistance was reduced by 43% compared to Comparative Example 1.

[0073] To compare the antibody immobilization efficiency of Example 1 and Comparative Example 1, five detection electrodes (without fixed antibody) from each of Example 1 and Comparative Example 1 were used. Using a fluorescent labeling method, aflatoxin B1 monoclonal antibody was first labeled with fluorescein isothiocyanate (FITC) (F / P molar ratio 1:2), and then drop-coated onto the electrode surface at the same concentration (50 μg / mL). The electrodes were incubated at 4-6°C for 10-12 hours, followed by washing with phosphate buffer (pH 7.4). Then, each electrode was immersed in 0.1 mol / L NaOH solution to elute the FITC-antibody, and the fluorescence intensity of the eluent was measured (excitation wavelength 495 nm, emission wavelength 520 nm). The antibody loading was calculated based on the standard curve. Using the antibody loading of the electrode in Comparative Example 1 as a baseline, the antibody loading of the electrode in Example 1 was calculated to be 1.54 times higher than that in Comparative Example 1.

[0074] Unless otherwise stated, all percentages used in this invention are mass percentages.

[0075] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting aflatoxin in edible oil, characterized in that, The process includes the following steps: functionalization, in-situ recombination, preparation of detection electrodes, establishment of standard curves, and sample detection. The functionalization process involves adding 9,10-diphenylanthracene luminescent particles to an aqueous solution of N,N-dimethylformamide and dispersing them evenly. Then, at room temperature, an aqueous solution of polyethyleneimine is added dropwise and dispersed evenly to obtain a functionalized dispersion. The in-situ composite method involves dispersing zinc nitrate hexahydrate and nickel nitrate hexahydrate evenly in anhydrous methanol to obtain a metal source solution; dispersing 2-methylimidazole evenly in anhydrous methanol to obtain a ligand solution; mixing the functionalized dispersion with anhydrous methanol to obtain a functionalized diluent; and sequentially adding the metal source solution and ligand solution to the functionalized diluent at room temperature, stirring at room temperature, centrifuging to separate the solids, washing the solids, and dispersing them in deionized water to obtain a composite dispersion. The method for preparing the detection electrode is as follows: a composite dispersion is drop-coated onto the surface of a glassy carbon electrode and dried; then a glutaraldehyde solution is drop-coated onto the electrode surface, incubated at room temperature, and then washed; then aflatoxin B1 monoclonal antibody is drop-coated onto the electrode surface, incubated, and then washed; then bovine serum albumin is drop-coated onto the electrode surface, allowed to stand, and then washed to obtain the detection electrode. A three-electrode system was established using the aforementioned detection electrode to detect aflatoxin B1 standard working solutions of varying concentrations. A standard curve was established based on the relationship between the detected light signal intensity and the concentration of the aflatoxin B1 standard working solution. The sample solution to be tested was dropped onto the surface of the detection electrode, and the light signal intensity was detected. The aflatoxin B1 content of the sample solution to be tested was obtained based on the standard curve.

2. The method for detecting aflatoxin in edible oil according to claim 1, characterized in that, In the functionalization process, the mass-to-volume ratio of 9,10-bisphenylanthracene luminescent particles to N,N-dimethylformamide aqueous solution is 10-12 mg:1 mL. The volume ratio of polyethyleneimine aqueous solution to N,N-dimethylformamide aqueous solution is 1:22-25; The concentration of polyethyleneimine is 8-12 μg / mL.

3. The method for detecting aflatoxin in edible oil according to claim 1, characterized in that, In the in-situ recombination, the total metal ion concentration in the metal source solution is 0.1-0.15 mol; the molar ratio of zinc nitrate hexahydrate and nickel nitrate hexahydrate in the metal source solution is 8.7-9:1-1.

3. The concentration of 2-methylimidazole in the ligand solution is 0.8-0.9 mol.

4. The method for detecting aflatoxin in edible oil according to claim 1, characterized in that, In the in-situ composite process, the volume ratio of the functionalized dispersion to anhydrous methanol in the functionalized diluent is 1:9-9.

2. The volume ratio of the metal source solution, ligand solution, and functionalized diluent is 1:1:1-1.

1.

5. The method for detecting aflatoxin in edible oil according to claim 1, characterized in that, In the in-situ composite process, the stirring speed at room temperature is 300-400 rpm, and the stirring time at room temperature is 90-120 min. The concentration of solids in the composite dispersion is 10-12 mg / mL.

6. The method for detecting aflatoxin in edible oil according to claim 1, characterized in that, In the preparation of the detection electrode, the room temperature incubation time after the glutaraldehyde solution is dropped onto the electrode is 1-2 hours. The incubation temperature for aflatoxin B1 monoclonal antibody after drop-coating is 4-6℃, and the incubation time is 10-12h. The standing time after bovine serum albumin is applied to the electrode surface is 50-70 minutes.

7. The method for detecting aflatoxin in edible oil according to claim 1, characterized in that, In the preparation of the detection electrode, the concentration of glutaraldehyde solution is 1-1.2 wt%; the concentration of aflatoxin B1 monoclonal antibody is 40-50 μg / mL; and the concentration of bovine serum albumin is 0.9-1 wt%.

8. The method for detecting aflatoxin in edible oil according to claim 1, characterized in that, The method for establishing the standard curve is as follows: A gradient concentration of aflatoxin B1 standard working solution is dropped onto the surfaces of different detection electrodes. Each detection electrode is used as the working electrode, a platinum wire electrode as the counter electrode, and a calomel electrode as the reference electrode to establish a three-electrode system. Tripropylamine solution is used as the electrolyte. The detection voltage is controlled at 900-1000V, and the scanning voltage at 100-110mV / s. Cyclic voltages are applied to each working electrode using cyclic voltammetry to obtain the light signal intensity. A standard curve is then established based on the relationship between the light signal intensity and the concentration of the aflatoxin B1 standard working solution.

9. The method for detecting aflatoxin in edible oil according to claim 1, characterized in that, The sample detection method involves adding a drop of the sample solution to the surface of the detection electrode. A three-electrode system is established, using the detection electrode as the working electrode, a platinum wire electrode as the counter electrode, and a calomel electrode as the reference electrode. Tripropylamine solution is used as the electrolyte. The detection voltage is controlled at 900-1000V, and the scanning voltage is controlled at 100-110mV / s. Cyclic voltage is applied to the working electrode using cyclic voltammetry to obtain the light signal intensity. The aflatoxin B1 content of the sample solution is obtained according to the standard curve.

10. The method for detecting aflatoxin in edible oil according to claim 1, characterized in that, It also includes the following steps: Sample processing; The sample processing method is as follows: the edible oil to be tested, sodium chloride, and methanol solution are mixed and centrifuged. The lower layer of centrifuged liquid is mixed with an equal volume of n-hexane and centrifuged again. The lower layer of centrifuged liquid is then mixed evenly with phosphate buffer to obtain a sample dilution. The sample dilution is treated with an aflatoxin immunoaffinity column and then eluted with methanol to obtain an eluent. The eluent is then evaporated by nitrogen purging and reconstituted with phosphate buffer to obtain the sample solution to be tested. The weight ratio of the edible oil, sodium chloride, and methanol solution to be tested is 5-5.5:0.8-1:18-20.