Method for detecting aflatoxin B1 in edible oil

Abstract

The invention provides a method for detecting aflatoxin B1 in edible oil, and relates to the technical field of aflatoxin detection. According to the invention, the nano-enzyme probe reagent PdCoOx-400 is used as a catalytic label for detecting AFB1, and the color change of the detection line of the test strip is deepened by utilizing the catalytic reaction of the nano-enzyme probe reagent PdCoOx-400 with TMB and H2O2, so that the detection sensitivity is improved. Compared with the chromaticity difference generated by only depending on the combination of the PdCoOx-400 probe and the antigen, the signal enhancement strategy can generate larger signal difference, so that the detection system has higher sensitivity and wider linear range, and high-sensitivity and ultra-wide-range rapid detection is realized. The detection limit of the method is 20 pg / mL and is 100 times lower than the highest detection limit of AFB1 in food specified by European Union, and field detection of AFB1 can be rapidly and reliably achieved.

Description

Technical Field

[0001] The 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] At present, food contamination by chemical pollutants (pesticides, heavy metals, antibiotics, etc.) and microbial pollutants (bacteria, fungi, molds, viruses, etc.) has become a major global problem. Among them, fungal growth can lead to the emergence of aflatoxins (AFs), which are more dangerous due to their toxicity and carcinogenicity. AFs are mainly produced by Aspergillus flavus and Aspergillus parasiticus, two fungi that grow in warm and humid climates. Among them, AFB1 (aflatoxin B1) is the most abundant and toxic one. Edible vegetable oils (such as peanut oil, corn oil, olive oil, rapeseed oil, soybean oil, and sunflower oil) are foods contaminated by AFB1 during production, packaging, transportation, and storage. AFB1 is present in high-consumption products such as edible vegetable oils and the human food chain, and can cause problems such as mental disorders, immunotoxicity, extreme chronic toxicity, carcinogenic toxicity, and mutagenicity. Considering these issues, countries and relevant regulatory organizations have formulated strict guidelines for the management of aflatoxins. For example, the EU has set the maximum residue limits (MRLs) for AFB1 and total AF concentrations 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 mass-producible. So far, hundreds of nanomaterials with enzyme-like activities have been discovered. Metal-organic framework (MOF) is a porous crystalline material formed by self-assembly of metals or metal nanoclusters with organic ligands. Rich metal ions, organic ligands and diverse coordination modes give this new material excellent physical and chemical properties. Derivatives of MOF 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 is no detection of aflatoxin B1 in edible oil using a bimetallic coordination structure. Summary of the invention

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

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions: A method for detecting aflatoxin B1 in edible oil comprises the following steps: S1, pretreatment of the sample to be tested: centrifugation of the edible oil was performed using acetonitrile as the extraction solvent, and the extracted supernatant was filtered to obtain a sample solution to be tested; S2, preparation of nanozyme probe reagent: mix the nanozyme probe reagent PdCoOx-400 with a buffer solution in an enzyme-linked immunosorbent assay microplate to obtain a mixture; the buffer solution is 5.5 mM PBS, and the buffer solution also contains 5% Tween-20 by volume, 0.1 g / mL sucrose by mass-to-volume ratio, and 0.5 g / mL BSA by mass-to-volume ratio.

[0006] S3, preparing a reactant mixture: adding a 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.

[0007] Furthermore, during the processing of the sample to be tested, the ratio of edible oil to acetonitrile is 1-5 g: 5-10 mL; The centrifugal conditions were 4°C, 8000-12000 rpm·min -1 Centrifuge for 8-12 minutes; preferably 10000 rpm·min -1 Centrifuge for 10 minutes.

[0008] Furthermore, 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.

[0009] Furthermore, during the preparation of the reactant mixture, the mass ratio of the mixture to the sample to be tested was 150 μL:100 μL.

[0010] Furthermore, in 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 the tetramethylbenzidine solution and hydrogen peroxide are both 2 mM.

[0011] During the detection process, the qualitative detection of aflatoxin B1 is carried out according to the color change on the test strip: Samples showing a test line on the test strip with the same intensity as the negative control were considered negative, and samples showing no visible test line were considered positive.

[0012] Furthermore, the nanozyme probe reagent PdCoOx-400 is prepared according to the following steps: Preparation of Co-MOF: benzimidazole and cobalt nitrate hexahydrate solution dissolved in N,N-dimethylformamide were mixed and stirred once, and then ammonia water was dripped into the mixture for a second stirring reaction, and purple particles were collected, washed to neutrality, and then dried to obtain Co-MOF; Preparation of PdCo-MOF: dissolving the Co-MOF sample and palladium nitrate in water at a mass ratio of 1:3 and stirring for 10-14 hours, washing the stirred product to neutrality and then freeze-drying to obtain PdCo-MOF; Preparation of PdCoOx: PdCo-MOF was placed in a crucible, carbonized in a muffle furnace, and kept at 400°C for 1-3h to obtain PdCoOx-400; Preparation of PdCoOx-400 nanozyme probe reagent: drop the anti-AFB1 mAb aqueous solution into a mixed solution formed by equal volumes of PdCoOx-400 solution and PBS buffer solution, add BSA aqueous solution after stirring, and stir the obtained suspension to obtain the PdCoOx-400 nanozyme probe reagent.

[0013] Furthermore, during the preparation of Co-MOF: The concentration of benzimidazole solution is 0.47 g:20 mL; The concentration of cobalt nitrate hexahydrate solution is 0.44g:7.5mL; 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.

[0014] Furthermore, 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 1-3 h, for example, 1, 2, 3 h, preferably 2 h.

[0015] Compared with the prior art, the present invention has the following advantages: The present invention uses Co-MOF as a precursor and palladium nitrate as a palladium source to prepare bimetallic PdCo oxides in an air pyrolysis process, which have a three-dimensional flower-like nanoparticle structure. By introducing bimetallic nodes in 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, and then utilizes the catalytic reaction of PdCoOx-400 with TMB and H2O2 to deepen the color change of the detection line of the test strip, thereby improving the sensitivity of the detection. Compared with the chromaticity difference generated by the binding of the PdCoOx-400 probe to the antigen, this signal enhancement strategy can produce a larger signal difference, so that the detection system has a higher sensitivity and a wider linear range, and realizes high-sensitivity, ultra-wide range rapid detection. The detection limit of this method is 20pg / 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

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces 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 creative work.

[0017] Figure 1 The results of edible oil detection in the embodiment of the present invention are shown; Among them, A is the detection result of edible oil added with 20pg / mL aflatoxin B1 using PdCoOx-400 nanozyme as a probe reagent; B is the detection result of edible oil added with 20pg / mL aflatoxin B1 using PdCoOx-400 nanozyme as a probe reagent and amplifying the signal with TMB and H2O2. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Embodiment 1:

[0019] Synthesis of bimetallic MOF-derived three-dimensional nanoflower structures (PdCoOx-400 nanozymes) including the following steps: (1) Preparation of ZIF-9: Dissolve 0.47 g of benzimidazole in 20 mL of DMF and stir for 20 min until dissolved into solution A; Dissolve 0.44 g of cobalt nitrate hexahydrate in 7.5 mL of DMF and stir to obtain solution B; Solution B was slowly added to solution A, and stirred vigorously. After stirring for 10 min, 0.6 mL of ammonia water was added dropwise, and stirring was continued for 3 h. The purple particles were collected, washed with alcohol until neutral, and dried at 70 °C overnight to obtain the Co-MOF sample (ZIF9).

[0020] (2) Preparation of PdCo-MOF: 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; The obtained product was collected, centrifuged at 10000 rpm for 10 min, and then washed with deionized water several times until neutral. Finally, PdCo-MOF was obtained by freeze drying for use.

[0021] (3) Preparation of PdCoOx-400 nanozyme: 150 mg of PdCo-MOF was placed in a crucible and carbonized in a muffle furnace. After being kept at 400 °C for 2 h, PdCoOx-400 nanozyme was obtained. Embodiment 2:

[0022] The catalytic activity of PdCoOx-400 nanozyme on TMB was identified, including the following steps: 50 mg of the prepared PdCoOx-400 nanozyme was dispersed in 100 mL of water by ultrasonic dispersion for 30 min to obtain a catalyst stock solution of 20 μg / mL; 250 μL of catalyst stock solution was dispersed in 125 μL of 2 mM TMB solution and 125 μL of 2 mM H2O2 solution, mixed evenly, and finally added into 2 mL of HAc-NaAc buffer (2 mM, pH 4.0); TMB solution and H2O2 solution of the same volume and concentration were used as controls.

[0023] After reacting at 37℃ for 10min, the UV-visible absorption spectrum of the mixed solution was scanned and the absorbance value at 652nm was recorded. After the mixed culture of TMB, H2O2 and PdCoOx-400, the oxidation of TMB caused a significant change in the absorbance value, which was 0.4. Without PdCoOx-400, the absorbance value was smaller, less than 0.1. Embodiment 3:

[0024] Preparation of PdCoOx-400 nanozyme probe reagent: 400 μL of PdCoOx-400 nanozyme solution (1 mg / mL) was added to 400 μL of PBS buffer solution (10 mM, pH 7.4), and ultrasonicated for 30 min in an ice bath to obtain a mixture; 120 μL of anti-AFB1 mAb aqueous solution (0.2 mg / mL) was slowly dripped into the mixture to obtain a mixed solution; The mixed solution was gently stirred for 2 hours and allowed to stand at 4°C for 2 hours.

[0025] It should be noted that in order to block the unreacted sites on PdCoOx-400, 100 μL of 5% (w / v) BSA aqueous solution was added, and the resulting suspension was slightly stirred for 2 hours and then retained to obtain the PdCoOx-400 nanozyme probe. Embodiment 4:

[0026] The detection of aflatoxin B1 (AFB1) includes the following steps: Pre-treatment of the edible oil to be tested: 2 g of edible oil was treated with 8 mL of acetonitrile, shaken for 10 min, and then stirred 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. Detection of AFB1: 40 μL of nanozyme probe reagent was mixed with 110 μL of flow buffer 5.5 mM PBS (containing 5% (v / v, volume fraction) Tween-20, 0.1% (w / v, mass volume ratio) sucrose and 0.5% (w / v, mass volume ratio) BSA) in an enzyme-linked immunosorbent assay (ELISA) microplate, and 100 μL of the sample extract to be tested was added during the detection; After incubation at 37°C, insert the aflatoxin B1 colloidal gold test strip vertically into the reaction mixture. After 9 minutes, the strip is 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 2μL 2mM TMB and 2μL 2mM H2O2 solution were dripped onto the detection line. One minute later, a new signal was observed (as shown in Figure 1The naked eye analysis can achieve qualitative detection of aflatoxin B1. Samples with a test line of the same intensity as the negative control are considered negative, and samples with no visible test line are considered positive.

[0027] The aflatoxin B1 colloidal gold test strip in this embodiment is prepared according to the following steps: (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.

[0028] AFB1-BSA was used as the detection line (T line) on the NC membrane (nitrocellulose membrane), and goat anti-mouse secondary antibody was used as the quality control line (C line). The specific process is as follows: 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% acetonitrile by volume) and sprayed on the NC membrane to form the T line; Sheep 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.

[0029] The coated NC membrane was placed at 37°C to dry for 2 hours and then used. The sample pad was placed in a PBS buffer containing 1% BSA (1% by mass volume ratio), 2% sucrose (2% by mass volume ratio), pH 7.4, 0.02 mol / L for 2 hours and then dried at 37°C for 2 hours and then used.

[0030] (2) Assembling 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), and then use a strip cutter to cut the test strips into 3 mm wide, which are the aflatoxin B1 colloidal gold test strips.

[0031] Detection principle: Since the PdCoOx-400 nanozyme probe reagent can specifically bind to AFB1 in the sample extract and the competing antigen (AFB1-BSA) fixed 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 the free AFB1 in the sample extract, and the remaining PdCoOx-400-mab conjugate binds to AFB1-BSA.

[0032] Regardless of whether the toxin is present in the sample, due to the interaction between the anti-AFB1 mAb and the 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.

[0033] 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 the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. 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 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 was performed using acetonitrile as the extraction solvent, and the supernatant was filtered 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 also 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; S3, preparing a reactant mixture: adding a 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, characterized in that: During the processing of the sample to be tested, the ratio of edible oil to acetonitrile is 1-5 g: 5-10 mL; The centrifugal conditions were 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, characterized in that: 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, characterized in that: During the preparation of the reactant mixture, the mass ratio of the mixture to the sample 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: The nanozyme probe reagent PdCoOx-400 is prepared according to the following steps: Preparation of Co-MOF: benzimidazole and cobalt nitrate hexahydrate solution dissolved in N,N-dimethylformamide were mixed and stirred once, and then ammonia water was dripped into the mixture for a second stirring reaction, and purple particles were collected, washed to neutrality, and then dried to obtain Co-MOF; Preparation of PdCo-MOF: dissolving the Co-MOF sample and palladium nitrate in water at a mass ratio of 1:3 and stirring for 10-14 hours, washing the stirred product to neutrality and then freeze-drying to obtain PdCo-MOF; Preparation of PdCoOx: PdCo-MOF was placed in a crucible, carbonized in a muffle furnace, and kept at 400°C for 1-3h to obtain PdCoOx-400; Preparation of PdCoOx-400 nanozyme probe reagent: drop the anti-AFB1 mAb aqueous solution into a mixed solution formed by equal volumes of PdCoOx-400 solution and PBS buffer solution, add BSA aqueous solution after stirring, and stir the obtained suspension to obtain the PdCoOx-400 nanozyme probe reagent.

7. The method for detecting aflatoxin B1 in edible oil according to claim 6, characterized in that: During the preparation of Co-MOF: The concentration of benzimidazole solution is 0.47 g:20 mL; 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°C.

8. The method for detecting aflatoxin B1 in edible oil according to claim 6, characterized in that: 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 BSA aqueous solution is 5%; the stirring time is 1-3h.

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