Fluorescent microsphere immunochromatography test strip for detecting aflatoxin B1, preparation method and detection method

Abstract

The invention discloses a fluorescent microsphere immunochromatography test strip for detecting aflatoxin B1, a preparation method and a detection method. The test strip comprises a bottom plate, and filter paper, a sample pad, a glass fiber membrane, a nitrocellulose membrane and absorbent paper are sequentially arranged on the upper side face of the bottom plate in a lap joint mode; a core-shell structure nanoparticle-fluorescent microsphere labeled aflatoxin B1 antibody A is attached to the glass fiber membrane, the fluorescent microsphere serves as a core, and the aflatoxin B1 antibody A is attached to the outer side face of the fluorescent microsphere in a coupled mode to serve as a shell; a detection line coated with an aflatoxin B1 antibody B and a quality control line coated with a secondary antibody or an aflatoxin B1 synthetic antigen are arranged on the nitrocellulose membrane. According to the detection method disclosed by the invention, the concentration of the aflatoxin B1 in the sample test solution is calculated by analysis software of a fluorescence analyzer, so that the result data of the content of the aflatoxin B1 in the sample to be detected, which is obtained by conversion, is high in reliability, good in accuracy and good in stability.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent immunochromatographic detection, and in particular to a fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1, a preparation method and a detection method. Background Art

[0002] Aflatoxin (AF) is a secondary metabolite of Aspergillus flavus and Aspergillus sparasiticus. There are many types of aflatoxin, and more than 12 types have been isolated and identified. Common ones include aflatoxin B1, B2, G1, G2, M1, M2, B2a, G2a, BM2a and GM2a. Among them, aflatoxin B1 (AFB1) is the most widely distributed, the most toxic and the most harmful. AF has very good thermal stability and can only be decomposed at 280°C, so it is difficult to destroy its molecular structure and toxicity at conventional processing temperatures. AFB1 is a derivative of dihydrofuranoxanaphthophenone, and its molecular structure contains a bifuran ring and anoxanaphthophenone (coumarin). It ranks first in carcinogenicity among known chemical substances. Its acute toxicity is 10 times that of potassium cyanide, and its chronic toxicity can induce carcinogenesis. Its carcinogenicity is 75 times that of dimethylnitrosamine. AFB1 is highly toxic to humans and some animals, and its toxic effect is mainly damage to the liver. Human primary liver cancer is also likely to be related to it. AFB1 has a high detection rate in natural foods and medicines, especially in dry storage materials. The General Administration of Quality Supervision, Inspection and Quarantine stipulates that AFB1 is one of the mandatory inspection items for most foods. The Chinese Pharmacopoeia also stipulates that AFB1 is one of the mandatory inspection items for many kinds of Chinese herbal medicines such as animal and spicy ones.

[0003] At present, there are many methods for detecting AFB1, and the commonly used ones are thin layer chromatography (TLC), high performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), liquid chromatography-mass spectrometry (HPLC-MS), etc. These methods usually take a long time to detect, and require specific detection equipment, which is inconvenient to operate, has high detection costs, and has a limited scope of application. Immunochromatography is often used for rapid qualitative or semi-quantitative detection due to its low cost and simple operation, such as the colloidal gold test paper detection method for luteinizing hormone, clenbuterol hydrochloride, and aflatoxin. Products based on immunochromatography technology usually only require naked eye observation or simple instruments to quickly obtain test results, and can achieve rapid on-site detection, which can often be used as a preliminary screening method for disease or safety factors and impurity detection. Therefore, the development of products based on immunochromatography technology will be beneficial to on-site inspection of Chinese herbal medicines or process quality control.

[0004] Immunochromatography obtains the test results through enzyme reaction or directly using the intuitive experimental phenomena displayed by visually observable markers. The markers include colloidal gold, latex, colloidal selenium, gelatin, etc., and colloidal gold is the most commonly used. However, colloidal gold immunochromatography is greatly interfered by the background and is prone to false positives; colloidal gold and target proteins are combined by electrostatic adsorption, which has poor stability and is prone to false negatives; colloidal gold immunochromatography has low detection sensitivity and can only be used for qualitative or semi-quantitative detection in actual detection.

[0005] In immunochromatographic detection technology, in addition to using colloidal gold as a marker, related patents have reported immunochromatographic detection using fluorescent nanoparticles as markers. For example, the invention patent application with publication number CN1645146A discloses an immunochromatographic method using fluorescent rare earth nanoparticles as markers and the preparation of a test strip thereof. This method can improve the sensitivity of immunochromatographic detection, but this method does not solve the problem of weak antigenicity of small chemical molecules; the invention patent applications with publication numbers CN116656688A and CN116590384A respectively disclose aptamers that specifically recognize AFB1 and optimization methods thereof. This method avoids the antigenicity problem of AFB1 and is based on DNA chain reaction for labeling. However, there are many factors affecting the binding of AFB1 to single-stranded DNA, and the detection results lack stability, which limits its scope of application.

[0006] The present invention aims to propose a fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1, a preparation method and a detection method, in order to provide a new method for the quantitative detection of aflatoxin B1 in samples such as Chinese herbal medicines. Summary of the invention

[0007] The purpose of the present invention is to overcome the defects existing in the prior art and provide a fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1, a preparation method and a detection method. The fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1 of the present invention has a simple structural design, high feasibility of preparation and implementation, low cost, convenient and fast operation, easy promotion and popularization, and good practicality; the aflatoxin B1 antibody A labeled with the fluorescent microspheres is a core-shell structure nanoparticle, and the fluorescent microspheres, as the inner core of the core-shell structure nanoparticles, are not easily affected by external environmental factors (such as solution interference), and help to increase the stability of the fluorescence intensity and fluorescence lifetime emitted by the fluorescent substance on the fluorescent microspheres, thereby expanding the sample range that the fluorescent microsphere immunochromatographic test strip of the present invention can detect and is applicable to, and compared with the colloidal gold immunochromatographic technology, the accuracy and reliability of the detection results are significantly improved, and the detection sensitivity is also greatly improved. The method of the present invention uses a fluorescent microsphere immunochromatographic test strip to quantitatively detect aflatoxin B1 in a sample. The method is simple to operate, and a series of aflatoxin B1 standard solution concentrations corresponding to the aflatoxin B1 fluorescence intensity-concentration standard curves are stored in advance in the analysis software of the fluorescence analyzer, so that the concentration of aflatoxin B1 in the sample test solution is calculated by the analysis software of the fluorescence analyzer, which significantly reduces the manual calculation labor, helps to eliminate human error factors, and ensures that the converted aflatoxin B1 content result data in the sample to be tested has high reliability, good accuracy and good stability.

[0008] One of the purposes of the present invention is to design a fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1, comprising a bottom plate, on the side of which filter paper, a sample pad, a glass fiber membrane, a nitrocellulose membrane and absorbent paper are sequentially overlapped;

[0009] The glass fiber membrane is attached with aflatoxin B1 antibody A labeled with fluorescent microspheres, and the aflatoxin B1 antibody A labeled with fluorescent microspheres is a core-shell structure nanoparticle, wherein the fluorescent microspheres serve as the core of the core-shell structure nanoparticle, and the aflatoxin B1 antibody A is coupled and attached to the outer side of the fluorescent microspheres as the shell of the core-shell structure nanoparticle;

[0010] The nitrocellulose membrane is provided with a detection line coated with aflatoxin B1 antibody B and a quality control line coated with a secondary antibody or aflatoxin B1 synthetic antigen.

[0011] The preferred technical solution is that the fluorescent microspheres are prepared by reacting NaHCO3, K2S2O8, fluorescent substances, and styrene in a solvent, and the particle size range is 50 to 300 nm;

[0012] The aflatoxin B1 antibody A is attached to the outer side of the fluorescent microsphere through a coupling reaction in a buffer system containing boric acid-sodium tetraborate-morpholineethanesulfonic acid, and is blocked by a macromolecular protein.

[0013] A further preferred technical solution is that the fluorescent substance is one or more of rhodamine B methyl ester, rhodamine isothiocyanate, fluorescein isothiocyanate, fluorescein derivatives, 6-fusinylfluorescein phthalamide, coumarin organic fluorescent dyes or their dopants and quantum dots, and the macromolecular protein is bovine serum albumin or ovalbumin.

[0014] A further preferred technical solution is that the bottom plate is a PVC plate, and the sample pad is a polyester film or a glass fiber film.

[0015] The second object of the present invention is to design a method for preparing the fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1, comprising the following steps:

[0016] S1: Weigh a certain amount of NaHCO3 and K2S2O8 and dissolve them in deionized water to form an inorganic salt aqueous solution; weigh a certain amount of fluorescent substance and dissolve it in a mixed solution of anhydrous ethanol and styrene to form a solution containing fluorescent substance; add the solution containing fluorescent substance to the prepared inorganic salt aqueous solution, shake it well, and introduce nitrogen into the mixed solution system for a certain time, then place it in a constant temperature water bath at a certain temperature and shake it for a certain time, take out and wash the precipitate obtained by centrifugation with anhydrous ethanol and deionized water for several times to obtain fluorescent microspheres;

[0017] S2: Weigh an appropriate amount of the fluorescent microspheres prepared in step S1, and vortex mix with a certain amount of boric acid-sodium tetraborate buffer, add a certain amount of morpholineethanesulfonic acid buffer and mix evenly, shake at room temperature for a period of time, centrifuge and discard the supernatant, add a certain amount of boric acid-sodium tetraborate buffer to resuspend the precipitate, add a certain amount of aflatoxin B1 antibody A, mix evenly, perform coupling reaction at room temperature for a period of time, then add a certain amount of macromolecular protein to block the reaction for a period of time, centrifuge and discard the supernatant, add a certain amount of latex to the precipitate, mix evenly, and obtain aflatoxin B1 antibody A latex labeled with fluorescent microspheres;

[0018] S3: spraying the aflatoxin B1 antibody A latex labeled with fluorescent microspheres prepared in step S2 onto the glass fiber membrane and drying;

[0019] S4: coating aflatoxin B1 antibody B on a nitrocellulose membrane to form a detection line, and coating a secondary antibody or aflatoxin B1 synthetic antigen on a nitrocellulose membrane to form a quality control line;

[0020] S5: Filter paper, sample pad, glass fiber membrane, nitrocellulose membrane and absorbent paper are sequentially attached to the side of the bottom plate in overlapping order to obtain a fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1.

[0021] The preferred technical scheme is that the specific operation of step S1 is as follows: weigh 20 mg NaHCO3 and 45 mg K2S2O8 and dissolve them in 35 mL deionized water to form an inorganic salt aqueous solution; weigh a certain mass of fluorescent substance and dissolve it in a mixed solution of 10 mL anhydrous ethanol and 5 mL styrene to form a solution containing the fluorescent substance; add the solution containing the fluorescent substance to the prepared inorganic salt aqueous solution, shake it well, and introduce nitrogen into the mixed solution system for 5 to 10 minutes, then place it in a constant temperature water bath at 60 to 80°C and shake it for 24 hours, then take it out, centrifuge it at a speed of 10,000 rpm, discard the supernatant, and wash the precipitate with anhydrous ethanol and deionized water for 3 times respectively to obtain fluorescent microspheres;

[0022] The specific operation of step S2 is as follows: weigh 1 mg of the fluorescent microspheres prepared in step S1, add 400 uL of boric acid-sodium tetraborate buffer and vortex mix, add 600 uL of morpholineethanesulfonic acid buffer and mix evenly, shake at room temperature for 0.5 to 1 hour, centrifuge at 12000 rpm to discard the supernatant, add 400 uL of boric acid-sodium tetraborate buffer to resuspend the precipitate, add a certain amount of aflatoxin B1 antibody A and mix evenly, wherein the content of aflatoxin B1 antibody A is 100 ug / mL, couple at room temperature for 3 hours, then add 400 uL of macromolecular protein solution to block the reaction for a period of time, centrifuge and discard the supernatant, add 10 mL of latex to the precipitate, mix evenly, and obtain aflatoxin B1 antibody A latex labeled with fluorescent microspheres, wherein the mass fraction of the solute in the macromolecular protein solution is 1%;

[0023] The specific operation of step S3 is: spraying the fluorescent microsphere-labeled aflatoxin B1 antibody A latex prepared in step S2 on the glass fiber membrane using a BIODOT Dispensing System, wherein the spraying amount of the fluorescent microsphere-labeled aflatoxin B1 antibody A is 4uL / cm, and drying in a vacuum environment at 25°C for 2h;

[0024] In the step S4, the coating concentrations of aflatoxin B1 antibody B, secondary antibody or aflatoxin B1 synthetic antigen are all 0.6-0.8 mg / mL, and the spraying amounts are all 0.6-0.8 uL / cm.

[0025] A further preferred technical solution is that in step S2, the pH of the boric acid-sodium tetraborate buffer solution is 8.0, and the pH of the morpholineethanesulfonic acid buffer solution is 6.0.

[0026] The third object of the present invention is to design a method for quantitatively detecting aflatoxin B1 in a sample using a fluorescent microsphere immunochromatographic test strip, wherein the fluorescent microsphere immunochromatographic test strip used is the fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1, and the method comprises the following steps:

[0027] H1: Draw a standard curve of aflatoxin B1: prepare a series of concentrations of aflatoxin B1 standard solutions, use a number of fluorescent microsphere immunochromatographic test strips for detecting aflatoxin B1 from the same batch to detect the fluorescence intensity of each concentration of aflatoxin B1 standard solution one by one, and draw a standard curve of aflatoxin B1 fluorescence intensity-concentration with the corresponding fluorescence intensity as the ordinate and the concentration of the aflatoxin B1 standard solution as the abscissa, and save it in the analysis software system of the fluorescence analyzer;

[0028] H2: Extract the sample suspected of containing aflatoxin B1 from the sample to be tested, and place the sample in a volumetric flask to quantitatively prepare the sample test solution;

[0029] H3: Take an appropriate amount of the sample test solution prepared in step H2 and add it dropwise to the sample pad of the fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1, incubate at room temperature for 10 to 15 minutes, then place the fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1 in a fluorescence analyzer to test the fluorescence intensity of the test line and the quality control line, and automatically calculate the fluorescence intensity correction coefficient z at the quality control line position through the analysis software system of the fluorescence analyzer, then substitute the fluorescence intensity at the test line position and the fluorescence intensity correction coefficient z into the standard curve formula stored in the analysis software system of the fluorescence analyzer, calculate the concentration of aflatoxin B1 in the sample test solution, and then convert it to obtain the content of aflatoxin B1 in the sample to be tested.

[0030] The advantages and beneficial effects of the present invention are:

[0031] 1. A fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1 of the present invention has a simple structural design, high feasibility of preparation and implementation, low cost, convenient and fast operation, easy promotion and popularization, and good practicality; the aflatoxin B1 antibody A labeled with fluorescent microspheres is a core-shell structure nanoparticle, and the fluorescent microspheres, as the inner core of the core-shell structure nanoparticles, are not easily affected by external environmental factors (such as solution interference), and help to increase the stability of the fluorescence intensity and fluorescence lifetime emitted by the fluorescent substance on the fluorescent microspheres, thereby expanding the range of samples that can be detected by the fluorescent microsphere immunochromatographic test strip of the present invention. Compared with the colloidal gold immunochromatographic technology, the accuracy and reliability of the detection results are significantly improved, and the detection sensitivity is also greatly improved.

[0032] 2. The method for preparing a fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1 of the present invention has simple operation steps, high feasibility of preparation and implementation, and strong practicality.

[0033] 3. The method of the present invention for quantitatively detecting aflatoxin B1 in a sample using a fluorescent microsphere immunochromatographic test strip is simple to operate. A series of aflatoxin B1 standard solution concentrations corresponding to aflatoxin B1 fluorescence intensity-concentration standard curves are stored in advance in the analysis software of the fluorescence analyzer, so that the concentration of aflatoxin B1 in the sample test solution is calculated by the analysis software of the fluorescence analyzer, which significantly reduces the workload of manual calculations and helps to eliminate human error factors, ensuring that the converted aflatoxin B1 content result data in the sample to be tested has high reliability, good accuracy and good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a schematic structural diagram of a fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1 according to the present invention.

[0035] In the figure: 1, filter paper; 2, sample pad; 3, glass fiber membrane; 4, nitrocellulose membrane; 5, test line; 6, quality control line; 7, absorbent paper; 8, bottom plate. DETAILED DESCRIPTION

[0036] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0037] like Figure 1 A fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1 of the present invention comprises a bottom plate 8, on the upper side of which a filter paper 1, a sample pad 2, a glass fiber membrane 3, a nitrocellulose membrane 4 and a water-absorbing paper 7 are sequentially overlapped;

[0038] The glass fiber membrane 3 is attached with aflatoxin B1 antibody A labeled with fluorescent microspheres, and the aflatoxin B1 antibody A labeled with fluorescent microspheres is a core-shell structure nanoparticle, wherein the fluorescent microspheres serve as the core of the core-shell structure nanoparticle, and the aflatoxin B1 antibody A is coupled and attached to the outer side of the fluorescent microspheres as the shell of the core-shell structure nanoparticle;

[0039] The nitrocellulose membrane 4 is provided with a detection line 5 coated with aflatoxin B1 antibody B and a quality control line 6 coated with a secondary antibody or aflatoxin B1 synthetic antigen.

[0040] Preferably, the fluorescent microspheres are prepared by reacting NaHCO3, K2S2O8, fluorescent substances and styrene in a solvent, and have a particle size range of 50 to 300 nm; the aflatoxin B1 antibody A is attached to the outer side of the fluorescent microspheres through a coupling reaction in a buffer system containing boric acid-sodium tetraborate-morpholineethanesulfonic acid, and is blocked by macromolecular proteins.

[0041] Further preferably, the fluorescent substance is one or more of rhodamine B methyl ester, rhodamine isothiocyanate, fluorescein isothiocyanate, fluorescein derivatives, 6-fusinylfluorescein phthalamide, coumarin organic fluorescent dyes or their dopants and quantum dots, and the macromolecular protein is bovine serum albumin or ovalbumin.

[0042] More preferably, the bottom plate is a PVC plate, and the sample pad is a polyester film or a glass fiber film.

[0043] The method for preparing a fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1 of the present invention comprises the following steps:

[0044] S1: Weigh a certain amount of NaHCO3 and K2S2O8 and dissolve them in deionized water to form an inorganic salt aqueous solution; weigh a certain amount of fluorescent substance and dissolve it in a mixed solution of anhydrous ethanol and styrene to form a solution containing fluorescent substance; add the solution containing fluorescent substance to the prepared inorganic salt aqueous solution, shake it well, and introduce nitrogen into the mixed solution system for a certain time, then place it in a constant temperature water bath at a certain temperature and shake it for a certain time, take out and wash the precipitate obtained by centrifugation with anhydrous ethanol and deionized water for several times to obtain fluorescent microspheres;

[0045] S2: Weigh an appropriate amount of the fluorescent microspheres prepared in step S1, and vortex mix with a certain amount of boric acid-sodium tetraborate buffer, add a certain amount of morpholineethanesulfonic acid buffer and mix evenly, shake at room temperature for a period of time, centrifuge and discard the supernatant, add a certain amount of boric acid-sodium tetraborate buffer to resuspend the precipitate, add a certain amount of aflatoxin B1 antibody A, mix evenly, perform coupling reaction at room temperature for a period of time, then add a certain amount of macromolecular protein to block the reaction for a period of time, centrifuge and discard the supernatant, add a certain amount of latex to the precipitate, mix evenly, and obtain aflatoxin B1 antibody A latex labeled with fluorescent microspheres;

[0046] S3: spraying the aflatoxin B1 antibody A latex labeled with fluorescent microspheres prepared in step S2 onto the glass fiber membrane and drying;

[0047] S4: coating aflatoxin B1 antibody B on a nitrocellulose membrane to form a detection line, and coating a secondary antibody or aflatoxin B1 synthetic antigen on a nitrocellulose membrane to form a quality control line;

[0048] S5: Filter paper, sample pad, glass fiber membrane, nitrocellulose membrane and absorbent paper are sequentially attached to the side of the bottom plate in overlapping order to obtain a fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1.

[0049] Preferably, the specific operation of step S1 is as follows: weigh 20 mg NaHCO3 and 45 mg K2S2O8 and dissolve them in 35 mL deionized water to form an inorganic salt aqueous solution; weigh a certain mass of fluorescent substance and dissolve it in a mixed solution of 10 mL anhydrous ethanol and 5 mL styrene to form a solution containing the fluorescent substance; add the solution containing the fluorescent substance to the prepared inorganic salt aqueous solution, shake it well, and introduce nitrogen into the mixed solution system for 5 to 10 minutes, then place it in a constant temperature water bath at 60 to 80°C and shake it for 24 hours, then take it out, centrifuge it at a speed of 10000 rpm, discard the supernatant, and wash the precipitate with anhydrous ethanol and deionized water for 3 times respectively to obtain fluorescent microspheres;

[0050] The specific operation of step S2 is as follows: weigh 1 mg of the fluorescent microspheres prepared in step S1, add 400 uL of boric acid-sodium tetraborate buffer and vortex mix, add 600 uL of morpholineethanesulfonic acid buffer and mix evenly, shake at room temperature for 0.5 to 1 hour, centrifuge at 12000 rpm to discard the supernatant, add 400 uL of boric acid-sodium tetraborate buffer to resuspend the precipitate, add a certain amount of aflatoxin B1 antibody A and mix evenly, wherein the content of aflatoxin B1 antibody A is 100 ug / mL, couple at room temperature for 3 hours, then add 400 uL of macromolecular protein solution to block the reaction for a period of time, centrifuge and discard the supernatant, add 10 mL of latex to the precipitate, mix evenly, and obtain aflatoxin B1 antibody A latex labeled with fluorescent microspheres, wherein the mass fraction of the solute in the macromolecular protein solution is 1%;

[0051] The specific operation of step S3 is: spraying the fluorescent microsphere-labeled aflatoxin B1 antibody A latex prepared in step S2 on the glass fiber membrane using a BIODOT Dispensing System, wherein the spraying amount of the fluorescent microsphere-labeled aflatoxin B1 antibody A is 4uL / cm, and drying in a vacuum environment at 25°C for 2h;

[0052] In the step S4, the coating concentrations of aflatoxin B1 antibody B, secondary antibody or aflatoxin B1 synthetic antigen are all 0.6-0.8 mg / mL, and the spraying amounts are all 0.6-0.8 uL / cm.

[0053] Further preferably, in step S2, the pH of the boric acid-sodium tetraborate buffer solution is 8.0, and the pH of the morpholineethanesulfonic acid buffer solution is 6.0.

[0054] A method for quantitatively detecting aflatoxin B1 in a sample using a fluorescent microsphere immunochromatographic test strip, wherein the fluorescent microsphere immunochromatographic test strip used is the fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1, and comprises the following steps:

[0055] H1: Draw a standard curve of aflatoxin B1: prepare a series of concentrations of aflatoxin B1 standard solutions, use a number of fluorescent microsphere immunochromatographic test strips for detecting aflatoxin B1 from the same batch to detect the fluorescence intensity of each concentration of aflatoxin B1 standard solution one by one, and draw a standard curve of aflatoxin B1 fluorescence intensity-concentration with the corresponding fluorescence intensity as the ordinate and the concentration of the aflatoxin B1 standard solution as the abscissa, and save it in the analysis software system of the fluorescence analyzer;

[0056] H2: Extract the sample suspected of containing aflatoxin B1 from the sample to be tested, and quantitatively prepare the sample test solution in a volumetric flask;

[0057] H3: Take an appropriate amount of the sample test solution prepared in step H2 and add it dropwise to the sample pad of the fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1, incubate at room temperature for 10 to 15 minutes, then place the fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1 in a fluorescence analyzer to test the fluorescence intensity of the test line and the quality control line, and automatically calculate the fluorescence intensity correction coefficient z at the quality control line position through the analysis software system of the fluorescence analyzer, then substitute the fluorescence intensity at the test line position and the fluorescence intensity correction coefficient z into the standard curve formula stored in the analysis software system of the fluorescence analyzer, calculate the concentration of aflatoxin B1 in the sample test solution, and then convert it to obtain the content of aflatoxin B1 in the sample to be tested.

[0058] The principle of the fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1 of the present invention for detecting aflatoxin B1 content is as follows: when the sample test solution is added to the sample pad 2, the test substance (aflatoxin B1) first combines with the aflatoxin B1 antibody A labeled with the fluorescent microspheres on the glass fiber membrane to form a primary immune complex (fluorescent microsphere-antibody A-AFB1); under the capillary action of the sample test solution, the primary immune complex and the free aflatoxin B1 antibody A labeled with the fluorescent microspheres enter the glass fiber membrane together with the liquid matrix. When passing through the detection line 5, the aflatoxin B1 antibody B on the detection line 5 combines with the B site on the analyte (aflatoxin B1) in the primary immune complex to form a secondary immune complex (fluorescent microsphere-antibody A-AFB1-antibody B), and is fixed on the detection line 5, while the free aflatoxin B1 antibody A labeled with fluorescent microspheres does not combine with antibody B, and continues to flow under capillary action. When passing through the quality control line 6, the free aflatoxin B1 antibody A labeled with fluorescent microspheres combines with the secondary antibody and is fixed on the quality control line 6. The fluorescence intensity at the detection line 5 and the quality control line 6 on the test paper strip is detected by a fluorescence analyzer, wherein the fluorescence intensity on the detection line 5 is proportional to the concentration of the analyte (aflatoxin B1).

[0059] Example 1

[0060] (I) Preparation of fluorescent microsphere immunochromatographic test strips for detecting aflatoxin B1

[0061] S1: Preparation of Rhodamine B methyl ester-polystyrene fluorescent microspheres

[0062] Weigh 20 mg NaHCO3 and 45 mg K2S2O8 and dissolve them in 35 mL deionized water to form an inorganic salt aqueous solution; weigh 5 mg of rhodamine B methyl ester and dissolve it in a mixed solution of 10 mL anhydrous ethanol and 5 mL styrene to form a solution containing rhodamine B methyl ester; add the solution containing rhodamine B methyl ester to the prepared inorganic salt aqueous solution, shake well, and pass nitrogen gas into the mixed solution system for 5 minutes, then place it in a constant temperature water bath at 70°C and shake it for 24 hours, then take it out, centrifuge it at 10000 rpm for 10 minutes, discard the supernatant, and wash the precipitate with anhydrous ethanol and deionized water for 3 times respectively to obtain rhodamine B methyl ester-polystyrene fluorescent microspheres.

[0063] S2: Preparation of aflatoxin B1 antibody A latex labeled with rhodamine B methyl ester-polystyrene fluorescent microspheres

[0064] Weigh 1 mg of rhodamine B methyl ester-polystyrene fluorescent microspheres, add 400uL of boric acid-sodium tetraborate buffer (pH8.0) and vortex mix, add 600uL of morpholineethanesulfonic acid buffer (pH 6.0) and mix evenly, shake at room temperature for 0.5-1h, centrifuge at 12000rpm for 1h and discard the supernatant, add 400uL of boric acid-sodium tetraborate buffer (pH 8.0) to resuspend the precipitate, add a certain amount of aflatoxin B1 antibody A and mix evenly, wherein the content of aflatoxin B1 antibody A is 100ug / mL, couple at room temperature for 3h, then add 400uL of bovine serum albumin solution to block the reaction for a period of time, centrifuge and discard the supernatant, add 10mL of latex to the precipitate, mix evenly, and obtain rhodamine B methyl ester-polystyrene fluorescent microsphere labeled aflatoxin B1 antibody A latex, wherein the solute mass fraction in the bovine serum albumin solution is 1%.

[0065] S3: Spray the prepared fluorescent microsphere-labeled aflatoxin B1 antibody A latex onto the glass fiber membrane using a BIODOT Dispensing System, wherein the spraying amount of rhodamine B methyl ester-polystyrene fluorescent microsphere-labeled aflatoxin B1 antibody A is 4 uL / cm, and dry it in a vacuum environment at 25°C for 2 hours.

[0066] S4: Coating the test line and quality control line on the nitrocellulose membrane:

[0067] The concentration of aflatoxin B1 antibody B was adjusted to 0.8 mg / mL with 0.01 M phosphate buffer (containing 5% sucrose and 0.05% Tween-20, pH 7.4), and the resulting solution was sprayed on a nitrocellulose membrane to form a detection line with a spraying amount of 0.8 uL / cm; the concentration of the anti-mouse antibody was adjusted to 0.6 mg / mL with 0.01 M phosphate buffer (containing 5% sucrose and 0.05% Tween-20, pH 7.4), and the resulting solution was sprayed on a nitrocellulose membrane to form a quality control line with a spraying amount of 0.8 uL / cm; the mixture was dried at 37°C and stored in a dry environment at room temperature for later use.

[0068] S5: Filter paper 1, sample pad 2, glass fiber membrane 3, nitrocellulose membrane 4 and absorbent paper 7 are sequentially overlapped on the side of the PVC bottom plate 8, and then cut into several fluorescent microsphere immunochromatographic test strips for detecting aflatoxin B1 according to a width of 4 mm for later use.

[0069] (II) Establishment of aflatoxin B1 fluorescence intensity-concentration standard curve

[0070] Weigh a certain amount of aflatoxin B1 reference substance, and prepare a 300ug / L aflatoxin B1 reference substance stock solution with a 70% methanol aqueous solution containing sodium chloride (wherein, 75mL of the 70% methanol aqueous solution contains 3g of sodium chloride), then take several portions of the 300ug / L aflatoxin B1 reference substance stock solution and dilute them in sequence with the 70% methanol aqueous solution containing sodium chloride to prepare aflatoxin B1 reference substance solutions with mass concentrations of 0, 1.5, 3.0, 4.5, 6.0, 7.5, 9.0, 10.5, and 12.0ug / L; then, take several fluorescent microsphere immunochromatographic test strips for detecting aflatoxin B1 prepared in step (a), and take 50μL of a series of prepared aflatoxin B1 reference substance solutions and drop them one by one into the corresponding The sample was added to the sample pad 2 of the fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1, incubated at room temperature for 10 minutes, and then placed in a fluorescence analysis, and fluorescence detection and analysis were performed under the conditions of an excitation wavelength of 524 nm and an emission detection wavelength of 580 nm. Three fluorescent microsphere immunochromatographic test samples were set in parallel for each concentration of aflatoxin B1 reference solution; a standard curve of aflatoxin B1 fluorescence intensity-concentration was drawn with the mass concentration of a series of prepared aflatoxin B1 reference solutions as the horizontal axis and the corresponding fluorescence intensity as the vertical axis, and the standard curve was saved in the software analysis system of the fluorescence analyzer, r=0.9927, indicating that the method for quantitatively detecting aflatoxin B1 in a sample by the fluorescent microsphere immunochromatographic test strip of the present invention, the concentration of aflatoxin B1 is proportional to the measured fluorescence intensity in the range of 0-12.0 ug / L.

[0071] (III) Determination of aflatoxin B1 in Chinese herbal medicine Zanthoxylum bungeanum

[0072] Take 10 batches of commercially available Chinese medicine prickly ash, remove the seeds, crush, sieve, accurately weigh 15.0 g of prickly ash powder, and then place them in 10 homogenizing bottles respectively; add 3 g of sodium chloride and 75 mL of a 70% methanol aqueous solution to each homogenizing bottle, stir at 12000 rpm for 2 min, and centrifuge at 4000 rpm for 5 min, accurately measure 15.0 mL of supernatant from each homogenizing bottle and place it in a 50 mL volumetric bottle, dilute with water to constant volume, and shake well to form a sample test solution; centrifuge the sample test solution at 4000 rpm for 10 min, take 50 μL of the supernatant and add it to the fluorescent microsphere immunochromatographic test strip sample pad 2 for detecting aflatoxin B1 prepared in step (a), and simmer at room temperature. After incubation for 10 minutes, the mixture was placed in a fluorescence analyzer and fluorescence detection and analysis were performed under the conditions of an excitation wavelength of 524 nm and an emission detection wavelength of 580 nm. Three fluorescent microsphere immunochromatographic test samples were set in parallel for each sample test solution corresponding to the Chinese medicine Zanthoxylum bungeanum. The fluorescence intensity correction coefficient z at the quality control line position on each test strip was automatically calculated by the analysis software system of the fluorescence analyzer, and then the fluorescence intensity at the detection line position and the fluorescence intensity correction coefficient z were substituted into the standard curve formula saved in the analysis software system of the fluorescence analyzer to calculate the concentration of aflatoxin B1 in the sample test solution, and then the content of aflatoxin B1 in the Chinese medicine Zanthoxylum bungeanum sample to be tested was obtained by conversion. The test results are shown in Table 1.

[0073] Example 2

[0074] (I) Preparation of fluorescent microsphere immunochromatographic test strips for detecting aflatoxin B1

[0075] S1: Preparation of FITC-polystyrene fluorescent microspheres

[0076] Weigh 20 mg NaHCO3 and 45 mg K2S2O8 and dissolve them in 35 mL deionized water to form an inorganic salt aqueous solution; weigh 5 mg fluorescein isothiocyanate and dissolve it in a mixed solution of 10 mL anhydrous ethanol and 5 mL styrene to form a solution containing fluorescein isothiocyanate; add the solution containing fluorescein isothiocyanate to the prepared inorganic salt aqueous solution, shake well, and pass nitrogen gas into the mixed solution system for 10 minutes, then place it in a constant temperature water bath at 50°C and shake for 24 hours, then take it out, centrifuge it at 10000 rpm for 15 minutes, discard the supernatant, and wash the precipitate with anhydrous ethanol and deionized water for 3 times respectively to obtain fluorescein isothiocyanate-polystyrene fluorescent microspheres.

[0077] S2: Preparation of aflatoxin B1 antibody latex labeled with fluorescein isothiocyanate-polystyrene fluorescent microspheres A Weigh 1 mg of fluorescein isothiocyanate-polystyrene fluorescent microspheres, add 400 uL of boric acid-sodium tetraborate buffer (pH 8.0) and vortex to mix, then add 600 uL of morpholineethanesulfonic acid buffer (pH 6.0) mix evenly, shake at room temperature for 0.5 to 1 h, and centrifuge at 12000 rpm for 1 h to discard the supernatant, add 400 uL boric acid-sodium tetraborate buffer (pH 8.0) to resuspend the precipitate, add a certain amount of aflatoxin B1 antibody A to mix evenly, wherein the content of aflatoxin B1 antibody A is 100 ug / mL, and react at room temperature for 3 h, then add 400 uL bovine serum albumin solution to block the reaction for a period of time, centrifuge and discard the supernatant, add 10 mL of latex to the precipitate, mix evenly, and obtain fluorescein isothiocyanate-polystyrene fluorescent microspheres labeled aflatoxin B1 antibody A latex, wherein the solute mass fraction in the ovalbumin solution is 1%.

[0078] S3: Spray the prepared fluorescent microsphere-labeled aflatoxin B1 antibody A latex onto the glass fiber membrane using a BIODOT Dispensing System, wherein the spraying amount of the fluorescein isothiocyanate-polystyrene fluorescent microsphere-labeled aflatoxin B1 antibody A is 4 uL / cm, and dry it in a vacuum environment at 25°C for 2 hours.

[0079] S4: Coating the test line and quality control line on the nitrocellulose membrane:

[0080] The concentration of aflatoxin B1 antibody B was adjusted to 0.6 mg / mL with 0.01 M phosphate buffer (containing 5% sucrose and 0.05% Tween-20, pH 7.4), and the resulting solution was sprayed on a nitrocellulose membrane to form a detection line with a spraying amount of 0.8 uL / cm; the concentration of anti-mouse antibody was adjusted to 0.8 mg / mL with 0.01 M phosphate buffer (containing 5% sucrose and 0.05% Tween-20, pH 7.4), and the resulting solution was sprayed on a nitrocellulose membrane to form a quality control line with a spraying amount of 0.6 uL / cm; dried at 37°C and stored in a dry environment at room temperature for later use.

[0081] S5: Filter paper 1, sample pad 2, glass fiber membrane 3, nitrocellulose membrane 4 and absorbent paper 7 are sequentially overlapped on the side of the PVC bottom plate 8, and then cut into several fluorescent microsphere immunochromatographic test strips for detecting aflatoxin B1 according to a width of 4 mm for later use.

[0082] (II) Establishment of aflatoxin B1 fluorescence intensity-concentration standard curve

[0083] Weigh a certain amount of aflatoxin B1 reference substance, and prepare a 300ug / L aflatoxin B1 reference substance stock solution with a 70% methanol aqueous solution containing sodium chloride (wherein, 75mL of the 70% methanol aqueous solution contains 3g of sodium chloride), then take several portions of the 300ug / L aflatoxin B1 reference substance stock solution and dilute them in sequence with the 70% methanol aqueous solution containing sodium chloride to prepare aflatoxin B1 reference substance solutions with mass concentrations of 0, 1.5, 3.0, 4.5, 6.0, 7.5, 9.0, 10.5, and 12.0ug / L; then, take several fluorescent microsphere immunochromatographic test strips for detecting aflatoxin B1 prepared in step (a), and take 50μL of a series of prepared aflatoxin B1 reference substance solutions and drop them one by one into the corresponding The sample was added to the sample pad 2 of the fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1, incubated at room temperature for 10 minutes, and then placed in a fluorescence analysis, and fluorescence detection and analysis were performed under the conditions of an excitation wavelength of 490 nm and an emission detection wavelength of 525 nm. Three fluorescent microsphere immunochromatographic test samples were set in parallel for each concentration of aflatoxin B1 reference solution; a standard curve of aflatoxin B1 fluorescence intensity-concentration was drawn with the mass concentration of a series of prepared aflatoxin B1 reference solutions as the horizontal axis and the corresponding fluorescence intensity as the vertical axis, and the curve was saved in the software analysis system of the fluorescence analyzer, and r=0.9956, indicating that the method for quantitatively detecting aflatoxin B1 in a sample by the fluorescent microsphere immunochromatographic test strip of the present invention, the concentration of aflatoxin B1 is proportional to the measured fluorescence intensity in the range of 0-12.0 ug / L.

[0084] (III) Determination of aflatoxin B1 in Chinese herbal medicine Zanthoxylum bungeanum

[0085] Take 10 batches of commercially available Chinese medicine prickly ash, remove the seeds, crush, sieve, accurately weigh 15.0 g of prickly ash powder, and then place them in 10 homogenizing bottles respectively; add 3 g of sodium chloride and 75 mL of a 70% methanol aqueous solution to each homogenizing bottle, stir at 12000 rpm for 2 min, and centrifuge at 4000 rpm for 5 min, accurately measure 15.0 mL of supernatant from each homogenizing bottle and place it in a 50 mL volumetric bottle, dilute with water to constant volume, and shake well to form a sample test solution; centrifuge the sample test solution at 4000 rpm for 10 min, take 50 μL of the supernatant and add it to the fluorescent microsphere immunochromatographic test strip sample pad 2 for detecting aflatoxin B1 prepared in step (a), and simmer at room temperature. After incubation for 10 minutes, the mixture was placed in a fluorescence analyzer and fluorescence detection and analysis were performed under the conditions of an excitation wavelength of 490 nm and an emission detection wavelength of 525 nm. Three fluorescent microsphere immunochromatographic test samples were set in parallel for each sample test solution corresponding to the Chinese medicine Zanthoxylum bungeanum. The fluorescence intensity correction coefficient z at the quality control line position on each test strip was automatically calculated by the analysis software system of the fluorescence analyzer. Then, the fluorescence intensity at the detection line position and the fluorescence intensity correction coefficient z were substituted into the standard curve formula saved in the analysis software system of the fluorescence analyzer to calculate the concentration of aflatoxin B1 in the sample test solution, and then the content of aflatoxin B1 in the Chinese medicine Zanthoxylum bungeanum sample to be tested was obtained by conversion. The test results are shown in Table 1.

[0086] Comparative Example 1

[0087] Accurately weigh 10 batches of commercially available Chinese medicine Zanthoxylum bungeanum that are the same as step (iii) in Example 1, remove the seeds, crush, sieve, accurately weigh 15.0 g of Zanthoxylum bungeanum powder, and then place them in 10 homogenizing bottles respectively; add 3 g of sodium chloride and 75 mL of 70% methanol aqueous solution to each homogenizing bottle, stir at 12000 rpm for 2 min, centrifuge at 4000 rpm for 5 min, accurately measure 15.0 mL of supernatant from each homogenizing bottle and place it in a 50 mL measuring bottle, dilute with water to volume, and shake to form a sample Sample test solution; centrifuge the sample test solution at 4000rpm for 10min, accurately measure 20.0mL of supernatant, pass it through the immunoaffinity column, elute with 20ml of deionized water at a flow rate of 3mL / min, discard the eluent, and then allow air to enter the immunoaffinity column to squeeze out the water; then elute the immunoaffinity column with an appropriate amount of methanol, collect the eluent, put it in a 2mL volumetric flask, add methanol to make up to volume, shake well, and filter with a microporous filter membrane (pore size of 0.22μm). The filtrate is the comparison sample test solution 1.

[0088] Accurately pipette 5uL, 10uL, 15uL, 20uL, and 25uL of the 300ug / L aflatoxin B1 reference substance stock solution prepared in step (ii) of a series of Example 1, and inject them into a liquid chromatograph for liquid chromatography analysis and detection, respectively, to determine the peak area, and draw a standard curve with the peak area as the ordinate and the injection volume as the abscissa; in addition, accurately pipette 50uL of the above-mentioned comparison sample test solution 1, inject it into the liquid chromatograph, determine the peak area, and calculate the corresponding aflatoxin B1 content according to the standard curve equation. The test results are compared with those in Example 1. The results are shown in Table 1.

[0089] Table 1 Mass concentration of aflatoxin B1 in the sample test solutions corresponding to 10 batches of Chinese medicine Zanthoxylum bungeanum in Examples 1 to 2 and Comparative Example 1 (ug / L)

[0090]

[0091] Note: All data results in Table 1 are the average values ​​of three parallel samples.

[0092] From the data results in Table 1, it can be seen that the Spearman correlation coefficient (Spearman) ρ=0.948** between the mass concentration result data of aflatoxin B1 in the sample test solutions corresponding to 10 batches of Chinese medicine Zanthoxylum bungeanum measured in Example 1 and the mass concentration result data of aflatoxin B1 in the sample test solutions corresponding to 10 batches of Chinese medicine Zanthoxylum bungeanum measured in Comparative Example 1 indicates that the detection results of the two methods are significantly correlated; and the result data measured in Example 2 are very close to the result data measured in Example 1, indicating that the fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1 prepared by the method of the present invention can be used to quantitatively detect the concentration content of aflatoxin B1 in Chinese herbal medicine pieces, and a method for quantitatively detecting aflatoxin B1 in a sample using a fluorescent microsphere immunochromatographic test strip proposed by the present invention can be used for the quantitative detection of aflatoxin B1 in Chinese medicine Zanthoxylum bungeanum, thereby achieving the purpose of the invention.

[0093] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1, characterized in that: It comprises a bottom plate, on the side of which filter paper, a sample pad, a glass fiber membrane, a nitrocellulose membrane and absorbent paper are sequentially overlapped and arranged; The glass fiber membrane is attached with aflatoxin B1 antibody A labeled with fluorescent microspheres, and the aflatoxin B1 antibody A labeled with fluorescent microspheres is a core-shell structure nanoparticle, wherein the fluorescent microspheres serve as the core of the core-shell structure nanoparticle, and the aflatoxin B1 antibody A is coupled and attached to the outer side of the fluorescent microspheres as the shell of the core-shell structure nanoparticle; The nitrocellulose membrane is provided with a detection line coated with aflatoxin B1 antibody B and a quality control line coated with a secondary antibody or aflatoxin B1 synthetic antigen.

2. The fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1 according to claim 1, characterized in that: The fluorescent microspheres are prepared by reacting NaHCO3, K2S2O8, fluorescent substances and styrene in a solvent, and the particle size range is 50 to 300 nm; The aflatoxin B1 antibody A is attached to the outer side of the fluorescent microsphere through a coupling reaction in a buffer system containing boric acid-sodium tetraborate-morpholineethanesulfonic acid, and is blocked by a macromolecular protein.

3. The fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1 according to claim 2, characterized in that: The fluorescent substance is one or more of rhodamine B methyl ester, rhodamine isothiocyanate, fluorescein isothiocyanate, fluorescein derivatives, 6-fusinylfluorescein phthalamide, coumarin organic fluorescent dyes or their dopants and quantum dots, and the macromolecular protein is bovine serum albumin or ovalbumin.

4. The fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1 according to claim 3, characterized in that: The bottom plate is a PVC plate, and the sample pad is a polyester film or a glass fiber film.

5. The method for preparing the fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1 according to claim 4, characterized in that: The steps are as follows: S1: Weigh a certain amount of NaHCO3 and K2S2O8 and dissolve them in deionized water to form an inorganic salt aqueous solution; weigh a certain amount of fluorescent substance and dissolve it in a mixed solution of anhydrous ethanol and styrene to form a solution containing fluorescent substance; add the solution containing fluorescent substance to the prepared inorganic salt aqueous solution, shake it well, and introduce nitrogen into the mixed solution system for a certain time, then place it in a constant temperature water bath at a certain temperature and shake it for a certain time, take out and wash the precipitate obtained by centrifugation with anhydrous ethanol and deionized water for several times to obtain fluorescent microspheres; S2: Weigh an appropriate amount of the fluorescent microspheres prepared in step S1, and vortex mix with a certain amount of boric acid-sodium tetraborate buffer, add a certain amount of morpholineethanesulfonic acid buffer and mix evenly, shake at room temperature for a period of time, centrifuge and discard the supernatant, add a certain amount of boric acid-sodium tetraborate buffer to resuspend the precipitate, add a certain amount of aflatoxin B1 antibody A, mix evenly, perform coupling reaction at room temperature for a period of time, then add a certain amount of macromolecular protein to block the reaction for a period of time, centrifuge and discard the supernatant, add a certain amount of latex to the precipitate, mix evenly, and obtain aflatoxin B1 antibody A latex labeled with fluorescent microspheres; S3: spraying the aflatoxin B1 antibody A latex labeled with fluorescent microspheres prepared in step S2 onto the glass fiber membrane and drying; S4: coating aflatoxin B1 antibody B on a nitrocellulose membrane to form a detection line, and coating a secondary antibody or aflatoxin B1 synthetic antigen on a nitrocellulose membrane to form a quality control line; S5: Filter paper, sample pad, glass fiber membrane, nitrocellulose membrane and absorbent paper are sequentially attached to the side of the bottom plate in overlapping order to obtain a fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1.

6. The method for preparing the fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1 according to claim 5, characterized in that: The specific operation of step S1 is as follows: weigh 20 mg NaHCO3 and 45 mg K2S2O8 and dissolve them in 35 mL deionized water to form an inorganic salt aqueous solution; weigh a certain mass of fluorescent substance and dissolve it in a mixed solution of 10 mL anhydrous ethanol and 5 mL styrene to form a solution containing the fluorescent substance; add the solution containing the fluorescent substance to the prepared inorganic salt aqueous solution, shake it well, and introduce nitrogen into the mixed solution system for 5 to 10 minutes, then place it in a constant temperature water bath at 60 to 80° C. and shake it for 24 hours, then take it out, centrifuge it at a speed of 10,000 rpm, discard the supernatant, and wash the precipitate with anhydrous ethanol and deionized water for 3 times in sequence to obtain fluorescent microspheres; The specific operation of step S2 is as follows: weigh 1 mg of the fluorescent microspheres prepared in step S1, add 400 uL of boric acid-sodium tetraborate buffer and vortex mix, add 600 uL of morpholineethanesulfonic acid buffer and mix evenly, shake at room temperature for 0.5 to 1 hour, centrifuge at 12000 rpm to discard the supernatant, add 400 uL of boric acid-sodium tetraborate buffer to resuspend the precipitate, add a certain amount of aflatoxin B1 antibody A and mix evenly, wherein the content of aflatoxin B1 antibody A is 100 ug / mL, couple at room temperature for 3 hours, then add 400 uL of macromolecular protein solution to block the reaction for a period of time, centrifuge and discard the supernatant, add 10 mL of latex to the precipitate, mix evenly, and obtain aflatoxin B1 antibody A latex labeled with fluorescent microspheres, wherein the mass fraction of the solute in the macromolecular protein solution is 1%; The specific operation of step S3 is: spraying the fluorescent microsphere-labeled aflatoxin B1 antibody A latex prepared in step S2 on the glass fiber membrane using a BIODOT Dispensing System, wherein the spraying amount of the fluorescent microsphere-labeled aflatoxin B1 antibody A is 4uL / cm, and drying in a vacuum environment at 25°C for 2h; In the step S4, the coating concentrations of aflatoxin B1 antibody B, secondary antibody or aflatoxin B1 synthetic antigen are all 0.6-0.8 mg / mL, and the spraying amounts are all 0.6-0.8 uL / cm.

7. The method for preparing the fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1 according to claim 6, characterized in that: In step S2, the pH of the boric acid-sodium tetraborate buffer solution is 8.0, and the pH of the morpholineethanesulfonic acid buffer solution is 6.

0.

8. A method for quantitatively detecting aflatoxin B1 in a sample using a fluorescent microsphere immunochromatographic test strip, characterized in that: The fluorescent microsphere immunochromatographic test strip used is the fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1 as described in any one of claims 1 to 4, comprising the following steps: H1: Draw a standard curve of aflatoxin B1: prepare a series of concentrations of aflatoxin B1 standard solutions, use a number of fluorescent microsphere immunochromatographic test strips for detecting aflatoxin B1 from the same batch to detect the fluorescence intensity of each concentration of aflatoxin B1 standard solution one by one, and draw a standard curve of aflatoxin B1 fluorescence intensity-concentration with the corresponding fluorescence intensity as the ordinate and the concentration of the aflatoxin B1 standard solution as the abscissa, and save it in the analysis software system of the fluorescence analyzer; H2: Extract the sample suspected of containing aflatoxin B1 from the sample to be tested, and quantitatively prepare the sample test solution in a volumetric flask; H3: Take an appropriate amount of the sample test solution prepared in step H2 and add it dropwise to the sample pad of the fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1, incubate at room temperature for 10 to 15 minutes, then place the fluorescent microsphere immunochromatographic test strip for detecting aflatoxin B1 in a fluorescence analyzer to test the fluorescence intensity of the test line and the quality control line, and automatically calculate the fluorescence intensity correction coefficient z at the quality control line position through the analysis software system of the fluorescence analyzer, then substitute the fluorescence intensity at the test line position and the fluorescence intensity correction coefficient z into the standard curve formula stored in the analysis software system of the fluorescence analyzer, calculate the concentration of aflatoxin B1 in the sample test solution, and then convert it to obtain the content of aflatoxin B1 in the sample to be tested.

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