Method for detecting aflatoxin b1 in edible oil

Abstract

The application provides a detection method of aflatoxin B1 in edible oil, and relates to the technical field of aflatoxin detection. In the application, the nanoenzyme probe reagent PdCoOx-400 is used as a catalytic label for detecting AFB1, and a catalytic reaction of the nanoenzyme probe reagent PdCoOx-400, TMB and H2O2 is utilized to deepen the color change of the detection line of the test strip, so that the sensitivity of detection is improved. Compared with the chroma difference generated by the combination of the PdCoOx-400 probe and the antigen, the signal enhancement strategy can generate a larger signal difference, so that the detection system has higher sensitivity and a wider linear range, and rapid detection with high sensitivity and an ultra-wide range is realized. The detection limit of the method is 20 pg / mL, which is 100 times lower than the maximum detection limit of AFB1 in food regulated by the European Union, so that on-site detection of AFB1 can be realized quickly and reliably.

Description

Technical Field

[0001] The present invention relates to the technical field of aflatoxin detection, and in particular to a method for detecting aflatoxin B1 in edible oil. Background Art

[0002] Food contamination by chemical pollutants (pesticides, heavy metals, antibiotics, etc.) and microbial contaminants (bacteria, fungi, molds, viruses, etc.) has become a major global problem. Fungal growth can lead to the formation of aflatoxins (AFs), which are particularly dangerous due to their toxicity and carcinogenicity. AFs are primarily produced by Aspergillus flavus and Aspergillus parasiticus, two fungi that thrive in warm, humid climates. Aflatoxin B1 (AFB1) is the most abundant and toxic of these substances. Edible vegetable oils (such as peanut oil, corn oil, olive oil, rapeseed oil, soybean oil, and sunflower oil) are examples of foods that become contaminated with AFB1 during production, packaging, transportation, and storage. AFB1 is present in high-volume products such as edible vegetable oils and the human food chain and can cause mental disorders, immunotoxicity, extreme chronic toxicity, carcinogenicity, and mutagenicity. To address these issues, countries and regulatory bodies have established strict guidelines for the management of aflatoxins. For example, the European Union has set maximum residue limits (MRLs) for AFB1 and total AF at 2 and 4 μg / kg, respectively. Therefore, it is very important to develop highly sensitive and simple analytical methods to detect AFs to ensure the safety of products such as vegetable oils.

[0003] Nanozymes are becoming excellent signal reporters for the development of widely used strip sensors. Compared with natural enzymes, nanozymes are generally low-cost, stable, and can be mass-produced. So far, hundreds of nanomaterials with enzyme-like activity have been discovered. Metal-organic framework (MOF) is a porous crystalline material formed by self-assembly of metals or metal nanoclusters and organic ligands. The rich metal ions, organic ligands and diverse coordination modes give this new material excellent physical and chemical properties. MOF derivatives greatly improve stability and provide a new approach to the construction of mesoporous nanozymes. However, the existing single-metal coordination structure is often used, and its catalytic performance is not high, and there has been no use of a bimetallic coordination structure to detect aflatoxin B1 in edible oil. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, the present invention provides a method for detecting aflatoxin B1 in edible oil. Based on the PdCoOx-400 nanozyme strip as a catalytic label for detecting AFB1, a catalytic amplification system formed by PdCoOx-400, TMB (tetramethylbenzidine) and H2O2 (hydrogen peroxide) is utilized to achieve high-sensitivity, ultra-wide-range rapid detection.

[0005] To achieve the above object, the present application is realized by the following technical solutions:

[0006] A detection method of aflatoxin B1 in edible oil, comprising the following steps:

[0007] S1, pretreatment of the sample to be detected: centrifugal extraction of edible oil with acetonitrile as the extraction solvent, and obtaining the sample solution to be detected after filtering the supernatant of the extraction;

[0008] S2, preparation of nanoenzyme probe reagent: mixing nanoenzyme probe reagent PdCoOx-400 with buffer in an enzyme-linked immunosorbent assay microplate to obtain a mixture; the buffer is 5.5mM PBS, and the buffer further contains Tween-20 with a volume fraction of 5%, sucrose with a mass-volume ratio of 0.1g / mL, and BSA with a mass-volume ratio of 0.5g / mL.

[0009] S3, preparation of reactant mixture: adding the sample solution to be detected to the mixture, and incubating the microplate under the condition of 37℃ to obtain a reactant mixture;

[0010] S4, detection: vertically inserting the aflatoxin B1 test strip into the reactant mixture, and adding tetramethylbenzidine solution and hydrogen peroxide solution dropwise on the detection system of the test strip to detect aflatoxin B1.

[0011] Further, in the process of treating the sample to be detected, the ratio of edible oil to acetonitrile is 1-5g:5-10mL;

[0012] The centrifugal condition is 4℃, 8000-12000rpm·min -1 8-12min; preferably 10000rpm·min -1 10min.

[0013] Further, in the process of preparing the nanoenzyme probe reagent, the volume ratio of nanoenzyme probe reagent PdCoOx-400 to buffer is 40μL:110μL.

[0014] Further, in the process of preparing the reactant mixture, the mass ratio of the mixture to the sample to be detected is 150μL:100μL.

[0015] Further, in the detection process, the volume ratio of tetramethylbenzidine solution, hydrogen peroxide solution to reactant mixture is 2μL:2μL:150μL;

[0016] Among them, the concentration of tetramethylbenzidine solution and hydrogen peroxide is 2mM.

[0017] During the test, the qualitative detection of aflatoxin B1 is carried out according to the color change on the test strip as follows:

[0018] If the test line on the test strip showed the same intensity as the negative control, the sample was considered negative, and the sample showing no visible test line was considered positive.

[0019] Furthermore, the nanozyme probe reagent PdCoOx-400 is prepared according to the following steps:

[0020] Preparation of Co-MOF: Benzimidazole and cobalt nitrate hexahydrate dissolved in N,N-dimethylformamide were mixed and stirred once. After stirring, ammonia was added dropwise for a second stirring reaction. The purple particles were collected, washed to neutrality, and then dried to obtain Co-MOF.

[0021] Preparation of PdCo-MOF: Dissolve the Co-MOF sample and palladium nitrate in water at a mass ratio of 1:3 and stir for 10-14 hours. Wash the stirred product until neutral and then freeze-dry to obtain PdCo-MOF.

[0022] Preparation of PdCoOx: PdCo-MOF was placed in a crucible and carbonized in a muffle furnace at 400°C for 1-3 hours to obtain PdCoOx-400;

[0023] Preparation of PdCoOx-400 nanozyme probe reagent: an anti-AFB1 mAb aqueous solution was dropped into a mixed solution formed by equal volumes of PdCoOx-400 solution and PBS buffer solution, and then a BSA aqueous solution was added after stirring. The resulting suspension was stirred to obtain the PdCoOx-400 nanozyme probe reagent.

[0024] Furthermore, during the preparation of Co-MOF:

[0025] The concentration of benzimidazole solution is 0.47g:20mL;

[0026] The concentration of cobalt nitrate hexahydrate solution is 0.44g:7.5mL;

[0027] The primary stirring time is 8-12 min, for example, 8, 9, 10, 11, 12 min, preferably 10 min; the secondary stirring time is 2-4 h, for example, 2, 2.5, 3, 3.5, 4 h, preferably 3 h; the drying temperature is 60-80 ° C, for example, 60, 65, 68, 70, 72, 75, 78, 80 ° C, preferably 70 ° C.

[0028] Furthermore, during the preparation of the PdCoOx-400 nanozyme probe reagent:

[0029] The concentration of PdCoOx-400 solution was 1 mg / mL;

[0030] The concentration of PBS buffer solution was 10 mM and pH was 7.4;

[0031] The concentration of the anti-AFB1 mAb aqueous solution was 0.2 mg / mL;

[0032] The mass fraction of the BSA aqueous solution is 5%; the stirring time is 1-3 hours, for example, 1, 2, or 3 hours, preferably 2 hours.

[0033] Compared with the prior art, the present invention has the following advantages:

[0034] The present invention uses Co-MOF as a precursor and palladium nitrate as a palladium source to prepare bimetallic PdCo oxide with a three-dimensional flower-like nanoparticle structure during air pyrolysis. By introducing bimetallic nodes into the same MOF, defects and significant synergistic effects can be generated, thereby increasing active sites and further improving catalytic performance; and the potential difference between the two metal centers can promote electron transfer, thereby helping to improve catalytic activity. Based on the above reaction, the present invention prepares a nanozyme probe reagent PdCoOx-400, which is used as a catalytic label for detecting aflatoxin B1. The catalytic reaction of PdCoOx-400 with TMB and H2O2 is then used to deepen the color change of the test line of the test strip, thereby improving the sensitivity of the detection. Compared with relying solely on the color difference generated by the binding of the PdCoOx-400 probe to the antigen, this signal enhancement strategy can produce a larger signal difference, making the detection system have higher sensitivity and a wider linear range, achieving high-sensitivity, ultra-wide range rapid detection. The detection limit of this method is 20 pg / mL, which is 100 times lower than the maximum detection limit of aflatoxin B1 in food stipulated by the European Union. It can quickly and reliably realize on-site detection of AFB1. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 The results of edible oil detection in the embodiment of the present invention are shown;

[0037] Among them, A is the detection result of using PdCoOx-400 nanozyme as a probe reagent to detect edible oil supplemented with 20 pg / mL aflatoxin B1; B is the detection result of using PdCoOx-400 nanozyme as a probe reagent and using TMB and H2O2 to amplify the signal to detect edible oil supplemented with 20 pg / mL aflatoxin B1. DETAILED DESCRIPTION

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention. Example 1:

[0039] The synthesis of bimetallic MOF-derived three-dimensional nanoflower structures (PdCoOx-400 nanozymes) includes the following steps:

[0040] (1) Preparation of ZIF-9:

[0041] Dissolve 0.47 g of benzimidazole in 20 mL of DMF and stir for 20 min until solution A is obtained.

[0042] Dissolve 0.44 g of cobalt nitrate hexahydrate in 7.5 mL of DMF and stir to obtain solution B.

[0043] Solution B was slowly added to solution A, and stirred vigorously for 10 min. Then, 0.6 mL of ammonia water was added dropwise, and stirring was continued for 3 h.

[0044] The purple particles were collected, washed with alcohol until neutral, and dried at 70 °C overnight to obtain the Co-MOF sample (ZIF9).

[0045] (2) Preparation of PdCo-MOF:

[0046] Palladium nitrate and ZIF9 were mixed in 50 mL of deionized water at a mass ratio of 1:3 and magnetically stirred for 12 h to allow complete adsorption of the nanoparticles and palladium particles;

[0047] The resulting product was collected, centrifuged at 10,000 rpm for 10 min, and then washed several times with deionized water until neutral. Finally, it was freeze-dried to obtain PdCo-MOF for use.

[0048] (3) Preparation of PdCoOx-400 nanozyme:

[0049] Put 150 mg PdCo-MOF in a crucible and carbonize in a muffle furnace. After keeping at 400℃ for 2 h, PdCoOx-400 nanoszyme is obtained. Example 2:

[0050] PdCoOx-400 nanoszyme catalytic activity identification of TMB, including the following steps:

[0051] Disperse 50 mg of prepared PdCoOx-400 nanoszyme in 100 mL water for 30 min by ultrasonic dispersion method to obtain a catalyst stock solution of 20 μg / mL;

[0052] Disperse 250 μL of the catalyst stock solution in 125 μL of 2 mM TMB solution and 125 μL of 2 mM H2O2 solution, mix well, and finally add 2 mL of HAc-NaAc buffer solution (2 mM, pH 4.0); use the same volume and concentration of TMB solution and H2O2 solution as a control.

[0053] After 10 min of reaction at 37℃, scan the ultraviolet-visible absorption spectrum of the mixed solution and record the absorbance value at 652 nm. After mixing TMB, H2O2 and PdCoOx-400, the oxidation of TMB caused a significant change in absorbance value, with an absorbance value of 0.4. Without PdCoOx-400, the absorbance value is smaller, less than 0.1. Example 3:

[0054] Preparation of PdCoOx-400 nanoszyme probe reagent:

[0055] Add 400 μL of PdCoOx-400 nanoszyme solution (1 mg / mL) to 400 μL of PBS buffer solution (10 mM, pH 7.4) and ultrasonicate in an ice bath for 30 min to obtain a mixture;

[0056] Slowly drop 120 μL of anti- AFB1 mAb aqueous solution (0.2 mg / mL) into the mixture to obtain a mixed solution;

[0057] Gently stir the mixed solution for 2 hours and stand at 4℃ for 2 hours.

[0058] It should be noted that in order to block the unreacted sites on PdCoOx-400, 100 μL of 5% (w / v) BSA aqueous solution is added, and the obtained suspension is gently stirred for 2 hours and used as PdCoOx-400 nanoszyme probe. Example 4:

[0059] Detection of aflatoxin B1 (AFB1), including the following steps:

[0060] The edible oil to be tested was pretreated by taking 2 g of edible oil, treating it with 8 mL of acetonitrile, shaking it for 10 min, and then stirring it at 10000 rpm min. -1 Centrifuge at 4°C for 10 min. Finally, filter the supernatant with a 0.2 μm filter and store at 4°C to obtain the sample extract for further detection and analysis.

[0061] Detection of AFB1: 40 μL of nanozyme probe reagent was mixed with 110 μL of 5.5 mM PBS (containing 5% (v / v) Tween-20, 0.1% (w / v) sucrose, and 0.5% (w / v) BSA) in an enzyme-linked immunosorbent assay (ELISA) microplate. 100 μL of the sample extract to be tested was added during the assay.

[0062] After incubation at 37°C, insert the aflatoxin B1 colloidal gold test strip vertically into the reaction mixture. After 9 minutes, a band should be visible. If the color intensity of the test line in the reaction strip is the same as that of the control strip (e.g. Figure 1 As shown in Figure A), 2 μL of 2 mM TMB and 2 μL of 2 mM H2O2 solution are dripped onto the detection line. One minute later, a new signal can be observed (as shown in Figure A). Figure 1 (As shown in Figure B). Naked eye analysis allows for qualitative detection of aflatoxin B1. Samples with a test line showing the same intensity as the negative control are considered negative, while samples with no visible test line are considered positive.

[0063] The aflatoxin B1 colloidal gold test strips in this embodiment were prepared according to the following steps:

[0064] (1) Preparation of test lines and quality control lines: The following T lines and C lines were sprayed using a film sprayer at a spray volume of 1.0 μL / cm.

[0065] AFB1-BSA was used as the detection line (T line) on the NC membrane (nitrocellulose membrane), and the goat anti-mouse secondary antibody was used as the quality control line (C line). The specific process is as follows:

[0066] AFB1-BSA was diluted to a concentration of 0.5 mg / mL with PBS buffer (pH 7.4, concentration 0.02 mol / L, containing 1% volume fraction of acetonitrile) and sprayed onto the NC membrane to form the T line;

[0067] Goat anti-mouse secondary antibody (1 mg / mL, PBS buffer pH 7.4, 0.02 mol / L) was sprayed 5 mm above the T line, which was the C line.

[0068] The coated NC membrane was dried at 37°C for 2 hours before use. The sample pad was soaked in PBS buffer containing 1% BSA (1% by volume), 2% sucrose (2% by volume), pH 7.4, 0.02 mol / L for 2 hours, and dried at 37°C for 2 hours before use.

[0069] (2) Assemble the colloidal gold test strips: stick the NC membrane, sample pad, and absorbent pad on the PVC base plate so that the sample pad overlaps with the NC membrane by 0.3 cm (in the length direction), and the NC membrane overlaps with the absorbent pad by 0.3 cm (in the length direction). Then use a strip cutter to cut the test strips into 3 mm wide strips, which are the aflatoxin B1 colloidal gold test strips.

[0070] Detection Principle: The PdCoOx-400 nanozyme probe reagent specifically binds to AFB1 in the sample extract and the competing antigen (AFB1-BSA) immobilized on the test line. In the absence of AFB1 (negative), the maximum amount of the PdCoOx-400 nanozyme probe reagent is captured by AFB1-BSA, forming a characteristic colored band on the test line. In the presence of AFB1 (positive), the probe first reacts with free AFB1 in the sample extract, and the remaining PdCoOx-400-mab conjugate binds to AFB1-BSA.

[0071] Regardless of whether the toxin is present in the sample, due to the interaction between anti-AFB1 mAb and goat anti-mouse IgG, the remaining liquid migrates across the aflatoxin B1 colloidal gold test strip by capillary force, and the control line develops color. The quality control line should always be visible as an indicator of good test function. In order to improve the sensitivity of AFB1 detection or broaden the detection range, additional reagents (TMB solution and H2O2 solution) are subsequently added to the test line. Due to the catalytic reaction of PdCoOx-400 with TMB and H2O2, in some embodiments of the present invention, the color of the test line is blue. Compared with the chromaticity difference of the test line, the new signal difference is much larger. Based on this signal enhancement strategy, the constructed detection system has higher sensitivity and a wider linear range.

[0072] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for detecting aflatoxin B1 in edible oil, characterized in that: The following steps are involved: S1, pretreatment of the sample to be tested: centrifugation of the edible oil using acetonitrile as the extraction solvent, filtering the extracted supernatant to obtain the sample solution to be tested; S2, preparation of nanozyme probe reagent: mixing the nanozyme probe reagent PdCoOx-400 with a buffer in an enzyme-linked immunosorbent assay microplate to obtain a mixture; the buffer is 5.5 mM PBS, and the buffer further contains 5% Tween-20 by volume, 0.1 g / mL sucrose by mass / volume ratio, and 0.5 g / mL BSA by mass / volume ratio; the nanozyme probe reagent PdCoOx-400 is prepared according to the following steps: Preparation of Co-MOF: Benzimidazole and cobalt nitrate hexahydrate dissolved in N,N-dimethylformamide were mixed and stirred once. After stirring, ammonia was added dropwise for a second stirring reaction. The purple particles were collected, washed to neutrality, and then dried to obtain Co-MOF. Preparation of PdCo-MOF: Dissolve the Co-MOF and palladium nitrate in water at a mass ratio of 1:3 and stir for 10-14 hours. Wash the stirred product until neutral and then freeze-dry to obtain PdCo-MOF. Preparation of PdCoOx: PdCo-MOF was placed in a crucible and carbonized in a muffle furnace at 400°C for 1-3 hours to obtain PdCoOx-400; Preparation of the PdCoOx-400 nanozyme probe reagent: An anti-AFB1 mAb aqueous solution was added dropwise to a mixture of equal volumes of PdCoOx-400 solution and PBS buffer solution, stirred, and then a BSA aqueous solution was added. The resulting suspension was stirred to obtain the PdCoOx-400 nanozyme probe reagent; S3, preparing a reactant mixture: adding the sample solution to be tested to the mixture, and incubating the microplate at 37° C. to obtain a reactant mixture; S4, detection: insert the aflatoxin B1 test strip vertically into the reactant mixture, add tetramethylbenzidine solution and hydrogen peroxide solution to the detection line of the test strip to detect aflatoxin B1.

2. The method for detecting aflatoxin B1 in edible oil according to claim 1, wherein: During the processing of the sample to be tested, the ratio of edible oil to acetonitrile is 1-5 g: 5-10 mL; Centrifugation conditions: 4°C, 8000-12000 rpm min -1 Centrifuge for 8-12 minutes.

3. The method for detecting aflatoxin B1 in edible oil according to claim 1, wherein: During the preparation of the nanozyme probe reagent, the volume ratio of the nanozyme probe reagent PdCoOx-400 to the buffer solution was 40 μL:110 μL.

4. The method for detecting aflatoxin B1 in edible oil according to claim 1, wherein: During the preparation of the reactant mixture, the volume ratio of the mixture to the sample solution to be tested was 150 μL:100 μL.

5. The method for detecting aflatoxin B1 in edible oil according to claim 1, characterized in that: During the detection process, the volume ratio of tetramethylbenzidine solution, hydrogen peroxide solution and reactant mixture is 2 μL:2 μL:150 μL; The concentrations of tetramethylbenzidine and hydrogen peroxide were both 2 mM.

6. The method for detecting aflatoxin B1 in edible oil according to claim 1, characterized in that: During the preparation of Co-MOF: The concentration of benzimidazole solution is 0.47g:20mL; The concentration of cobalt nitrate hexahydrate solution is 0.44 g; 7.5 mL; The first stirring time is 8-12 minutes, the second stirring time is 2-4 hours, and the drying temperature is 60-80℃.

7. The method for detecting aflatoxin B1 in edible oil according to claim 1, wherein: During the preparation of the PdCoOx-400 nanozyme probe reagent: The concentration of PdCoOx-400 solution was 1 mg / mL; The concentration of PBS buffer solution was 10 mM and pH was 7.4; The concentration of the anti-AFB1 mAb aqueous solution was 0.2 mg / mL; The mass fraction of the BSA aqueous solution is 5%; the stirring time is 1-3h.

Images