Aflatoxin colorimetric immunoassay method based on AuPdCu probe
Through the colorimetric immunoassay method based on AuPdCu probe and combined with immunochromatography, the problem of detection of aflatoxin B1 in food is solved, and the on-site instant detection of aflatoxin B1 in food is realized, with the advantages of high sensitivity and simplicity of operation.
Patent Information
- Application Number
- CN202411717585.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-27
- Publication Date
- 2025-02-25
AI Technical Summary
The prior art is difficult to realize instant on-site detection of aflatoxin B1 in food, and lacks efficient, simple and low-cost detection methods.
Using the colorimetric immunoassay method based on the AuPdCu probe, AuPdCu nanocomposites were prepared, and the aflatoxin B1 monoclonal antibody was labeled, and AuPdCu nanochromatogenic probes were prepared, and the detection was carried out in combination with immunochromatography.
It realizes the on-site instant detection of aflatoxin B1 in food, with the advantages of high sensitivity, simplicity of operation and low cost, and can quickly and accurately detect the presence of AFB1 on site.
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Figure CN119510744A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of food analysis, and more specifically, to an aflatoxin colorimetric immunoassay method based on AuPdCu probe. Background Art
[0002] Aflatoxin (AF) refers to a type of bifuranoid toxin produced by certain strains of Aspergillus flavus and Aspergillus parasiticus. According to the different fluorescence colors, RF values and structures, they are named aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1, aflatoxin M2, aflatoxin P1, aflatoxin R1, aflatoxin GM and aflatoxin alcohol. AF can be detected in many types of food, such as seeds of grains and oil crops, milk and dairy products, and meat. On October 27, 2017, the World Health Organization's International Agency for Research on Cancer published a list of carcinogens, in which AF was identified as a Class 1 carcinogen, among which B1 is the most carcinogenic and can induce liver cancer in animals. For some animals, it may cause acute poisoning and death at a concentration of 10 μg / kg. Therefore, AFB1 is used as a contamination indicator in domestic food testing to detect the degree of AF contamination in food.
[0003] At present, the detection methods for aflatoxins in food can be divided into chemical analysis, instrumental analysis, immunoassay and other detection methods. Immunochromatographic test strip detection technology combines the advantages of immunoassay technology and chromatography technology. The integration of the two technologies makes immunochromatographic technology have the advantages of high efficiency, simplicity and low cost. At present, this method has been widely used in the field of food safety testing. And the combination with nanomaterials and biological immunity has enabled more immune test strips with multiple detection functions to be continuously developed, and the development prospects of immunochromatographic test strips are broad.
[0004] Therefore, there is an urgent need for a colorimetric immunoassay for aflatoxin based on AuPdCu probe. Summary of the invention
[0005] The purpose of the present invention is to provide a colorimetric immunoassay method for aflatoxin based on AuPdCu probe to solve the above-mentioned problems in the prior art and to achieve on-site instant detection of AFB1 in food.
[0006] The present invention provides a colorimetric immunoassay method for aflatoxin based on AuPdCu probe, which comprises:
[0007] Preparation of AuPdCu nanocomposites;
[0008] AuPdCu nanocomposite was used to label aflatoxin B1 monoclonal antibody to prepare AuPdCu nanochromogenic probe.
[0009] The concentration of aflatoxin B1 was determined by immunochromatography using AuPdCu nanochromogenic probe.
[0010] The above-mentioned aflatoxin colorimetric immunoassay method based on AuPdCu probe, wherein, preferably, the preparation of AuPdCu nanocomposite material specifically comprises:
[0011] Accurately weigh 0.1 mg CuCl2 and add it to 10 mL ultrapure water to prepare a 1% CuCl2 solution for later use;
[0012] Accurately weigh 0.1 mg of Na2PdCl4 powder and add it to 5 mL of ultrapure water to prepare a 1% Na2PdCl4 solution for later use;
[0013] Accurately weigh 0.1 mg of trisodium citrate solid and add it to 10 mL of ultrapure water to prepare a trisodium citrate solution with a mass concentration of 1% for later use;
[0014] Add 200 μL of 1% HAuCl4 solution, 100 μL of 1% CuCl2 solution, and 200 μL of 1% Na2PdCl4 solution into a 50 mL beaker, and add 20 mL of ultrapure water;
[0015] The mixed solution in the beaker was heated to boiling while stirring, and a sodium citrate solution with a volume of 1.6 mL and a mass concentration of 1% was added. The stirring was continued until the solution gradually turned black. The beaker was stirred in a boiling state for 20 min until the color turned into a stable black. Finally, the obtained AuPdCu nanocomposite solution was fixed to 10 mL and placed in a 4°C refrigerator away from light for later use.
[0016] The AuPdCu probe-based aflatoxin colorimetric immunoassay method as described above, wherein preferably, the AuPdCu nanocomposite material is used to label aflatoxin B1 monoclonal antibody to prepare the AuPdCu nanocolorimetric probe, specifically comprising:
[0017] Weigh 8.0 g NaCl, 2.9 g Na2HPO4·12H2O, 0.2 g KCl, and 0.2 g KH2PO4, add 800 mL ultrapure water, then add concentrated hydrochloric acid to adjust the pH to 7.4, and finally dilute to 1 L to prepare a phosphate buffer solution for later use;
[0018] The AuPdCu nanocomposite solution was diluted with a phosphate buffer solution having a concentration of 0.01 mol / L, wherein the volume ratio of the AuPdCu nanocomposite solution to the phosphate buffer solution was 1:2;
[0019] Pipette 20 μL of the diluted AuPdCu solution and add it to a 1.5 mL centrifuge tube. Then add 2.0 μL of aflatoxin B1 monoclonal antibody with a mass concentration of 0.1 μg / mL and let it stand for 30 min.
[0020] The obtained solution was mixed with a 10% bovine serum albumin solution to make the final concentration of bovine serum albumin in the solution 1%, and the mixture was allowed to stand for 30 min to prepare the probe AuPdCu-mAb.
[0021] The obtained probe solution was placed in a 4°C refrigerator and protected from light for future use.
[0022] The AuPdCu probe-based aflatoxin colorimetric immunoassay method as described above, wherein preferably, the AuPdCu nano-chromogenic probe is used to determine the concentration of aflatoxin B1 by immunochromatography, specifically comprising:
[0023] Treating the analyte with acetonitrile-water solution and phosphate buffer solution to obtain a sample extract;
[0024] Pipette 5 μL~20 μL of probe AuPdCu-mAb and add it to a 1.5 mL centrifuge tube;
[0025] Add 100 μL of sample extract to the centrifuge tube;
[0026] After mixing the two solutions in the centrifuge tube, insert the pre-made immunochromatographic test paper, and read the color intensity value using a colloidal gold detector after 5 minutes to 30 minutes to obtain the concentration test result of aflatoxin B1.
[0027] The AuPdCu probe-based aflatoxin colorimetric immunoassay method as described above, wherein preferably, the acetonitrile-water solution and phosphate buffer solution are used to treat the analyte to obtain the sample extract, specifically comprising:
[0028] Accurately weigh 5 g of sample into a centrifuge tube;
[0029] Add 10 mL of acetonitrile-water solution to the centrifuge tube, where the volume ratio of acetonitrile to water is 7:3;
[0030] Vortex the centrifuge tube using a mixer for 1 min;
[0031] The centrifuge tube was ultrasonicated in a water bath for 30 min;
[0032] filter;
[0033] The filtrate was diluted 10 times with 0.01 mol / L phosphate buffer solution to obtain the sample extract, which was stored in a 4°C refrigerator for later use.
[0034] As described above, the AuPdCu probe-based aflatoxin colorimetric immunoassay method, wherein preferably, the method for preparing the immunochromatographic test paper comprises:
[0035] Soak the sample pad and conjugate pad fully in phosphate buffer;
[0036] Place the sample pad and conjugate pad soaked with buffer in a forced air drying oven at 37°C for 4 hours;
[0037] Cut the nitrocellulose membrane into 20 cm in length and width;
[0038] Use a film-scribing and gold-spraying machine to solidify the T and C lines in the test strip on the nitrocellulose membrane;
[0039] Paste the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad on the PVC base plate in order;
[0040] The test strips were cut using a strip cutter, wherein the cutting width was set to 3 mm;
[0041] The prepared test strips were stored in a plastic bag and kept in a 4°C refrigerator for future use.
[0042] The above-mentioned AuPdCu probe-based aflatoxin colorimetric immunoassay method, wherein preferably, the solidification of the T line and the C line in the test strip on the nitrocellulose membrane by using a film-scratch gold-spraying machine specifically includes:
[0043] 20 μL of 1 μg / mL aflatoxin antigen and 1 μg / mL goat anti-mouse secondary antibody diluted with phosphate buffer solution were respectively aspirated and dispensed onto the nitrocellulose membrane at 1 μL / cm as T line and C line.
[0044] As described above, the AuPdCu probe-based aflatoxin colorimetric immunoassay method, wherein preferably, the AuPdCu probe-based aflatoxin colorimetric immunoassay method further comprises: determining the detection sensitivity of aflatoxin B1 by immunochromatography.
[0045] The AuPdCu probe-based aflatoxin colorimetric immunoassay method as described above, wherein preferably, the determination of the detection sensitivity of aflatoxin B1 by immunochromatography specifically includes:
[0046] The color intensity of the T line without the addition of the standard was measured, and the ratio of the color intensity of the spiked T line to the color intensity of the unspiked T line was calculated to obtain the detection sensitivity of aflatoxin B1 determined by the immunochromatographic method.
[0047] The invention provides an aflatoxin colorimetric immunoassay method based on AuPdCu probe, prepares a tri-metal nanocomposite material AuPdCu as a probe marker, uses the tri-metal nanocomposite material to label an AFB1 monoclonal antibody to obtain a probe AuPdCu-mAb, establishes a colorimetric immunoassay rapid detection method for AFB1, and can realize on-site instant detection of AFB1 in food. BRIEF DESCRIPTION OF THE DRAWINGS
[0048] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described below with reference to the accompanying drawings, in which:
[0049] Figure 1 A flowchart of an embodiment of the aflatoxin colorimetric immunoassay method based on AuPdCu probe provided by the present invention;
[0050] Figure 2 This is a transmission electron microscope photo of AuPdCu nanomaterials;
[0051] Figure 3 Schematic diagram of the volume optimization process of AuPdCu-mAb probe;
[0052] Figure 4 Schematic diagram of the detection time optimization process of AuPdCu-mAb probe;
[0053] Figure 5 This is the sensitivity experimental curve of the AuPdCu labeled probe;
[0054] Figure 6 This is the sensitivity experimental curve of the Au NPs labeled probe. DETAILED DESCRIPTION
[0055] Various exemplary embodiments of the present disclosure will now be described in detail with reference to the accompanying drawings. The description of the exemplary embodiments is merely illustrative and is in no way intended to limit the present disclosure and its application or use. The present disclosure can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are provided to make the present disclosure thorough and complete and to fully express the scope of the present disclosure to those skilled in the art. It should be noted that unless otherwise specifically stated, the relative arrangement of the components and steps, the composition of the materials, the numerical expressions and the numerical values set forth in these embodiments should be interpreted as being merely exemplary and not as limiting.
[0056] The words "first", "second" and similar words used in this disclosure do not indicate any order, quantity or importance, but are only used to distinguish different parts. The words "include" or "comprises" and similar words mean that the elements before the word include the elements listed after the word, and do not exclude the possibility of including other elements. "Up", "down" and the like are only used to indicate relative positional relationships. When the absolute position of the described object changes, the relative positional relationship may also change accordingly.
[0057] In the present disclosure, when a specific component is described as being located between a first component and a second component, there may or may not be an intermediate component between the specific component and the first component or the second component. When a specific component is described as being connected to other components, the specific component may be directly connected to the other components without an intermediate component, or may not be directly connected to the other components but have an intermediate component.
[0058] All terms (including technical terms or scientific terms) used in the present disclosure have the same meanings as those understood by ordinary technicians in the field to which the present disclosure belongs, unless otherwise specifically defined. It should also be understood that terms defined in general dictionaries, such as general dictionaries, should be interpreted as having meanings consistent with their meanings in the context of the relevant technology, and should not be interpreted in an idealized or extremely formal sense, unless explicitly defined herein.
[0059] Technologies, methods, and equipment known to ordinary technicians in the relevant art may not be discussed in detail, but where appropriate, the technologies, methods, and equipment should be considered part of the specification.
[0060] The following are some of the reagents and instruments used in the examples.
[0061] Main reagents: chloroauric acid (HAuCl4) (Sinopharm Chemical Reagent Co., Ltd.); cupric chloride (CuCl2) (Guangdong Guanghua Technology Co., Ltd.); sodium chloropalladate (Na2PdCl4) (BiDe Pharmaceuticals); aflatoxin antibody, aflatoxin antigen, aflatoxin standard (Haining Zhanchang Experimental Equipment); goat anti-mouse secondary antibody (IgG), bovine serum albumin (BSA) (Luoyang Bioton Experimental Materials Center).
[0062] Main instruments and equipment: BNCH 78-1 constant temperature magnetic stirrer (Wenzhou Biaonuo Instrument Co., Ltd.); TECNAI G2SPIRIT BIO03040112 transmission electron microscope (FEI Company, USA); HGS201 programmable strip cutter, HGS510 film spraying gold labeling machine, JIC-S1011 colloidal gold detector (Hangzhou Fenghang Technology Co., Ltd.).
[0063] like Figure 1As shown, the aflatoxin colorimetric immunoassay method based on AuPdCu probe provided in this embodiment includes the following steps in actual implementation:
[0064] Step S1, preparing AuPdCu nanocomposite material.
[0065] In one embodiment of the aflatoxin colorimetric immunoassay method based on AuPdCu probe of the present invention, the step S1 may specifically include:
[0066] Step S11, accurately weigh 0.1 mg CuCl2, add it to 10 mL ultrapure water, and prepare a CuCl2 solution with a mass concentration of 1% for later use.
[0067] Step S12: Accurately weigh 0.1 mg of Na2PdCl4 powder and add it to 5 mL of ultrapure water to prepare a Na2PdCl4 solution with a mass concentration of 1% for later use.
[0068] Step S13, accurately weigh 0.1 mg of trisodium citrate solid, add it to 10 mL of ultrapure water, and prepare a trisodium citrate solution with a mass concentration of 1% for later use.
[0069] Step S14, add 200 μL of HAuCl4 solution with a mass concentration of 1%, 100 μL of CuCl2 solution with a mass concentration of 1%, and 200 μL of Na2PdCl4 solution with a mass concentration of 1% into a 50 mL beaker, and add 20 mL of ultrapure water.
[0070] Step S15, heating the mixed solution in the beaker to boiling under stirring, adding 1.6 mL of sodium citrate solution with a mass concentration of 1%, and continuing to stir until the solution gradually turns black, stirring the beaker in a boiling state for 20 min until the color turns into a stable black; finally, the obtained AuPdCu nanocomposite solution is fixed to 10 mL and placed in a 4°C refrigerator away from light for later use.
[0071] Figure 2 The transmission electron micrograph of AuPdCu nanomaterials is shown in Figure 2 It can be seen that the AuPdCu tri-metallic composite material structure is nanoparticles with a diameter of 15 nm ~20 nm.
[0072] Step S2: using the AuPdCu nanocomposite material to label aflatoxin B1 monoclonal antibody to prepare an AuPdCu nanocolorimetric probe.
[0073] In one embodiment of the aflatoxin colorimetric immunoassay method based on AuPdCu probe of the present invention, the step S2 may specifically include:
[0074] Step S21, weigh 8.0 g NaCl, 2.9 g Na2HPO4·12H2O, 0.2 g KCl and 0.2 g KH2PO4, add 800 mL ultrapure water, then add concentrated hydrochloric acid to adjust the pH to 7.4, and finally dilute to 1 L to prepare a phosphate buffer solution (PBS) for later use.
[0075] Step S22, diluting the AuPdCu nanocomposite solution with a phosphate buffer solution with a concentration of 0.01 mol / L, wherein the volume ratio of the AuPdCu nanocomposite solution to the phosphate buffer solution is 1:2.
[0076] Step S23, aspirate 20 μL of the diluted AuPdCu solution, add it to a 1.5 mL centrifuge tube, add 2.0 μL of aflatoxin B1 monoclonal antibody with a mass concentration of 0.1 μg / mL, and let it stand for 30 min.
[0077] Step S24, mixing the obtained solution with a bovine serum albumin (BSA) solution with a mass concentration of 10% to make the final concentration of the bovine serum albumin in the solution be 1%, and letting it stand for 30 minutes to prepare the probe AuPdCu-mAb.
[0078] Step S25, placing the obtained probe solution in a 4°C refrigerator and protecting from light for future use.
[0079] Step S3: using AuPdCu nano-chromogenic probe, the concentration of aflatoxin B1 (AFB1) is determined by immunochromatography.
[0080] In one embodiment of the aflatoxin colorimetric immunoassay method based on AuPdCu probe of the present invention, the step S3 may specifically include:
[0081] Step S31: using acetonitrile-water solution and phosphate buffer solution to treat the analyte to obtain a sample extract.
[0082] In one embodiment of the aflatoxin colorimetric immunoassay method based on AuPdCu probe of the present invention, the step S31 may specifically include:
[0083] Step S311, accurately weigh 5 g of sample into a centrifuge tube.
[0084] In some embodiments of the present invention, 5 g of rice (sample 1), corn (sample 2) and tobacco leaves (sample 3) are weighed respectively.
[0085] Step S312, adding 10 mL of acetonitrile-water solution into the centrifuge tube, wherein the volume ratio of acetonitrile to water is 7:3.
[0086] Step S313: vortex the centrifuge tube using a mixer for 1 min.
[0087] Step S314, subjecting the centrifuge tube to water bath ultrasound for 30 min.
[0088] Step S315: Filter.
[0089] Step S316, dilute the filtrate 10 times with a phosphate buffer solution with a concentration of 0.01 mol / L to obtain a sample extract, and store it in a refrigerator at 4°C for future use.
[0090] Step S32: Pipette 5 μL to 20 μL of the probe AuPdCu-mAb (obtained in step S2) and add it to a 1.5 mL centrifuge tube.
[0091] Figure 3 The optimization process of the dosage volume of AuPdCu-mAb probe is shown. Figure 3 It was shown that a probe volume of 20 μL corresponds to the optimal detection condition.
[0092] Step S33: Add 100 μL of sample extract into the centrifuge tube.
[0093] Step S34, after mixing the two solutions in the centrifuge tube, insert the pre-made immunochromatographic test paper, and read the color intensity value using a colloidal gold detector after 5 minutes to 30 minutes to obtain the concentration test result of aflatoxin B1.
[0094] Figure 4 The detection time optimization process of AuPdCu-mAb probe is shown. Figure 4 It shows that the detection time of 25 min corresponds to the optimal detection condition. In some embodiments of the present invention, 20 μL of the probe AuPdCu-mAb is respectively drawn and added to a 1.5 mL centrifuge tube, and then 100 μL of the sample extract corresponding to sample 1, sample 2 and sample 3 is respectively added, mixed, and inserted into the immunochromatographic test paper, and the color intensity value is read using a colloidal gold detector after 25 min. The test results are shown in Table 1.
[0095] Table 1 Detection results of AFB1 in samples 1-3
[0096] Sample No. 1 2 3 <![CDATA[AFB1 content (ng / mL)]]> Not detected Not detected Not detected
[0097] In some embodiments of the present invention, the method for preparing the immunochromatographic test paper comprises:
[0098] Step S341, fully soak the sample pad and the conjugate pad in phosphate buffer.
[0099] Among them, phosphate buffer (PB) was prepared by adding 0.58 g Na2HPO4·12H2O and 0.06 g NaH2PO4·2H2O into 100 mL ultrapure water.
[0100] Step S342: Place the sample pad and conjugate pad soaked with the buffer solution in a forced air drying oven at 37° C. for 4 hours.
[0101] Step S343, cutting a nitrocellulose membrane (NC membrane) with a length and width of 20 cm.
[0102] Step S344: solidify the T line and C line in the test strip on the nitrocellulose membrane using a film-scribing and gold-spraying machine.
[0103] Specifically, 20 μL of 1 μg / mL aflatoxin antigen and 1 μg / mL goat anti-mouse secondary antibody (IgG) diluted with phosphate buffer solution were respectively aspirated and dispensed onto the nitrocellulose membrane at 1 μL / cm as T line and C line.
[0104] Step S345, stick the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad on the PVC bottom plate in order.
[0105] Step S346: cutting the test strips using a strip cutter, wherein the cutting width is set to 3 mm.
[0106] Step S347: Use a plastic bag to store the prepared test strips and store them in a 4° C. refrigerator for future use.
[0107] The present invention adopts a competitive method to establish a colorimetric immunoassay rapid detection method for AFB1, and prepares an AFB1 immunochromatographic test strip by using aflatoxin antigen and goat anti-mouse secondary antibody (IgG) as the detection line and quality control line of the immunochromatographic test strip.
[0108] Furthermore, in some embodiments of the present invention, the aflatoxin colorimetric immunoassay method based on AuPdCu probe further comprises:
[0109] Step S4, determining the detection sensitivity of aflatoxin B1 by immunochromatography.
[0110] Specifically, the color intensity of the T line without the addition of the standard was measured, and the ratio of the color intensity of the spiked T line to the color intensity of the unspiked T line (T / T0) was calculated to obtain the detection sensitivity of aflatoxin B1 determined by the immunochromatographic method.
[0111] Figure 5 and Figure 6 The sensitivity experimental curves of the AuPdCu and Au NPs labeled probes of the present invention are shown respectively. Figure 5 This indicates that the detection limit of the detection method of the present invention is 0.006 ng / mL. Figure 6 The results showed that the detection limit of the traditional colloidal gold (Au NPs) method was 0.024 ng / mL. Therefore, the detection sensitivity of the present invention was 4 times higher than that of the traditional colloidal gold labeled probe, and the operation was simple and the reproducibility was good.
[0112] The embodiment of the present invention provides an aflatoxin colorimetric immunoassay method based on AuPdCu probe, prepares a tri-metal nanocomposite material AuPdCu as a probe marker, and uses it to label AFB1 monoclonal antibody to obtain the probe AuPdCu-mAb, establishes a colorimetric immunoassay rapid detection method for AFB1, and can realize on-site instant detection of AFB1 in food.
[0113] So far, various embodiments of the present disclosure have been described in detail. In order to avoid obscuring the concept of the present disclosure, some details known in the art are not described. Based on the above description, those skilled in the art can fully understand how to implement the technical solution disclosed here.
[0114] Although some specific embodiments of the present disclosure have been described in detail by way of examples, it should be understood by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the present disclosure. It should be understood by those skilled in the art that the above embodiments may be modified or some technical features may be replaced by equivalents without departing from the scope and spirit of the present disclosure. The scope of the present disclosure is defined by the appended claims.
Claims
1. A colorimetric immunoassay method for aflatoxin based on AuPdCu probe, characterized in that: include: Preparation of AuPdCu nanocomposites; AuPdCu nanocomposite was used to label aflatoxin B1 monoclonal antibody to prepare AuPdCu nanochromogenic probe; The concentration of aflatoxin B1 was determined by immunochromatography using AuPdCu nanochromogenic probe.
2. The aflatoxin colorimetric immunoassay method based on AuPdCu probe according to claim 1, characterized in that: The preparation of the AuPdCu nanocomposite material specifically comprises: Accurately weigh 0.1 mg CuCl2 and add it to 10 mL ultrapure water to prepare a 1% CuCl2 solution for later use; Accurately weigh 0.1 mg of Na2PdCl4 powder and add it to 5 mL of ultrapure water to prepare a 1% Na2PdCl4 solution for later use; Accurately weigh 0.1 mg of trisodium citrate solid and add it to 10 mL of ultrapure water to prepare a trisodium citrate solution with a mass concentration of 1% for later use; Add 200 μL of 1% HAuCl4 solution, 100 μL of 1% CuCl2 solution, and 200 μL of 1% Na2PdCl4 solution into a 50 mL beaker, and add 20 mL of ultrapure water; The mixed solution in the beaker was heated to boiling while stirring, and a sodium citrate solution with a volume of 1.6 mL and a mass concentration of 1% was added. The stirring was continued until the solution gradually turned black. The beaker was stirred in a boiling state for 20 min until the color turned into a stable black. Finally, the obtained AuPdCu nanocomposite solution was fixed to 10 mL and placed in a 4°C refrigerator away from light for later use.
3. The aflatoxin colorimetric immunoassay method based on AuPdCu probe according to claim 1, characterized in that: The AuPdCu nanocomposite material is used to label aflatoxin B1 monoclonal antibody to prepare the AuPdCu nanocolorimetric probe, which specifically includes: Weigh 8.0 g NaCl, 2.9 g Na2HPO4·12H2O, 0.2 g KCl, and 0.2 g KH2PO4, add 800 mL ultrapure water, then add concentrated hydrochloric acid to adjust the pH to 7.4, and finally dilute to 1 L to prepare a phosphate buffer solution for later use; The AuPdCu nanocomposite solution was diluted with a phosphate buffer solution having a concentration of 0.01 mol / L, wherein the volume ratio of the AuPdCu nanocomposite solution to the phosphate buffer solution was 1:2; Pipette 20 μL of the diluted AuPdCu solution and add it to a 1.5 mL centrifuge tube. Then add 2.0 μL of aflatoxin B1 monoclonal antibody with a mass concentration of 0.1 μg / mL and let it stand for 30 min. The obtained solution was mixed with a 10% bovine serum albumin solution to make the final concentration of bovine serum albumin in the solution 1%, and the mixture was allowed to stand for 30 min to prepare the probe AuPdCu-mAb. The obtained probe solution was placed in a 4°C refrigerator and protected from light for future use.
4. The aflatoxin colorimetric immunoassay method based on AuPdCu probe according to claim 1, characterized in that: The method of using AuPdCu nano-chromogenic probe and immunochromatography to determine the concentration of aflatoxin B1 specifically includes: Treating the analyte with acetonitrile-water solution and phosphate buffer solution to obtain a sample extract; Pipette 5 μL~20 μL of probe AuPdCu-mAb and add it to a 1.5 mL centrifuge tube; Add 100 μL of sample extract to the centrifuge tube; After mixing the two solutions in the centrifuge tube, insert the pre-made immunochromatographic test paper, and read the color intensity value using a colloidal gold detector after 5 minutes to 30 minutes to obtain the concentration test result of aflatoxin B1.
5. The aflatoxin colorimetric immunoassay method based on AuPdCu probe according to claim 4, characterized in that: The method of treating the analyte with an acetonitrile-water solution and a phosphate buffer solution to obtain a sample extract specifically comprises: Accurately weigh 5 g of sample into a centrifuge tube; Add 10 mL of acetonitrile-water solution to the centrifuge tube, where the volume ratio of acetonitrile to water is 7:3; Vortex the centrifuge tube using a mixer for 1 min; The centrifuge tube was ultrasonicated in a water bath for 30 min; filter; The filtrate was diluted 10 times with 0.01 mol / L phosphate buffer solution to obtain the sample extract, which was stored in a 4°C refrigerator for later use.
6. The aflatoxin colorimetric immunoassay method based on AuPdCu probe according to claim 4, characterized in that: The method for preparing the immunochromatographic test paper comprises: Soak the sample pad and conjugate pad fully in phosphate buffer; Place the sample pad and conjugate pad soaked with buffer in a forced air drying oven at 37°C for 4 hours; Cut the nitrocellulose membrane into 20 cm in length and width; Use a film-scribing and gold-spraying machine to solidify the T and C lines in the test strip on the nitrocellulose membrane; Paste the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad on the PVC base plate in order; The test strips were cut using a strip cutter, wherein the cutting width was set to 3 mm; The prepared test strips were stored in a plastic bag and kept in a 4°C refrigerator for future use.
7. The aflatoxin colorimetric immunoassay method based on AuPdCu probe according to claim 6, characterized in that: The method of using a film-scribing and gold-spraying machine to solidify the T line and the C line in the test strip on the nitrocellulose membrane specifically includes: 20 μL of 1 μg / mL aflatoxin antigen and 1 μg / mL goat anti-mouse secondary antibody diluted with phosphate buffer solution were respectively aspirated and dispensed onto the nitrocellulose membrane at 1 μL / cm as T line and C line.
8. The aflatoxin colorimetric immunoassay method based on AuPdCu probe according to claim 1, characterized in that: The AuPdCu probe-based aflatoxin colorimetric immunoassay method further includes: determining the detection sensitivity of aflatoxin B1 by using immunochromatography.
9. The aflatoxin colorimetric immunoassay method based on AuPdCu probe according to claim 8, characterized in that: The determination of the detection sensitivity of aflatoxin B1 by immunochromatography specifically includes: The color intensity of the T line without the addition of the standard was measured, and the ratio of the color intensity of the spiked T line to the color intensity of the unspiked T line was calculated to obtain the detection sensitivity of aflatoxin B1 determined by the immunochromatographic method.