Traditional Chinese medicinal material aflatoxin B1 residue screening method based on multicolor evolution fluorescence immunochromatography

Abstract

The invention belongs to the technical field of immunoassay rapid detection, and relates to a method for screening residues of traditional Chinese medicinal material aflatoxin B1 based on multicolor evolution fluorescence immunochromatography, which comprises the following steps: coupling red quantum dot fluorescent microspheres with an AFB1 antibody to prepare a detection probe; coupling or mixing the green quantum dot fluorescent microspheres with an AFB1 antigen to prepare an internal standard probe; respectively coating an internal standard probe and goat anti-mouse IgG on an NC membrane to complete preparation of a T line and a C line; preparing a test solution containing a detection probe, dripping the test solution on a test paper sample pad, and observing fluorescence colors of a T line and a C line by ultraviolet; and judging whether the residual quantity of the traditional Chinese medicine AFB1 exceeds the limit specification or not according to the T-line fluorescence color. By introducing the internal standard probe, a ratio fluorescence immunoassay system is constructed, so that the sensitivity and the accuracy of an analysis result can be improved, and the interference of a sample matrix is reduced; the used detection test paper is small in matrix influence and wide in application range, and can be used for rapid limited screening of AFB1 residues in traditional Chinese medicinal materials.

Description

Technical Field

[0001] The invention belongs to the technical field of rapid immunoassay detection, and specifically relates to a method for screening aflatoxin B1 residues in traditional Chinese medicines based on multicolor evolution fluorescence immunochromatography. Background Art

[0002] Aflatoxins are a group of fungal secondary metabolites with similar chemical structures. Studies have shown that aflatoxins have serious hepatotoxicity and can even cause teratogenicity, carcinogenicity, and mutagenicity. Among them, aflatoxin B1 (AFB1) is the most toxic and widely contaminated, and is classified as a Class I carcinogen. Aflatoxin contamination may occur in every link of Chinese medicine, including planting, harvesting, processing, and storage, affecting the quality of Chinese medicine and the safety of clinical use.

[0003] In view of the extreme toxicity of AFB1, the 2020 edition of the Chinese Pharmacopoeia stipulates that the maximum residue limit (MRL) of AFB1 in Chinese medicinal materials is 5μg / kg. Carrying out rapid detection and on-site limit screening technology for aflatoxin contamination in Chinese medicines, and real-time and efficient monitoring of aflatoxin contamination levels in Chinese medicines are of great significance to ensuring the quality and safety of Chinese medicines, breaking through international trade barriers, maintaining the reputation of my country's traditional Chinese medicine cultural industry, and enhancing its competitiveness in the international market.

[0004] In recent years, colloidal gold immunochromatographic test strips based on the principle of antigen-antibody specific recognition have provided an effective way to achieve on-site detection of AFB1. The concentration level can be judged by observing the depth of the color of the test line. Generally, the test can be completed within 10 minutes without the need for special instruments. It is particularly suitable for on-site screening based on limited quantities, and provides a powerful means for source control, on-site control, and full control from field to clinic of AFB1 contamination in traditional Chinese medicine.

[0005] However, since the existing colloidal gold test paper detects targets based on changes in color intensity (depth), the human eye has limited recognition capabilities and low visual resolution, resulting in low detection sensitivity and a narrow detection range. In addition, complex components in traditional Chinese medicine, such as pigments, interfere with the color development of colloidal gold. The immunochromatographic test paper based on gold nanoparticles is seriously affected by the matrix effect and has large background interference, which makes the colloidal gold test paper prone to false negative results in the detection of traditional Chinese medicine AFB1, seriously affecting the accuracy of the detection and greatly restricting its scope of application. Summary of the invention

[0006] In order to solve the problems of low sensitivity, matrix interference, low visualization resolution, etc. of aflatoxin B1 colloidal gold test paper in the prior art, the present invention proposes a method for screening aflatoxin B1 residues in traditional Chinese medicines based on multicolor evolution fluorescence immunochromatography. The present invention has developed a visualized immunochromatographic fluorescence test paper with a continuous color evolution effect, which is suitable for rapid limited screening of aflatoxin B1 residues in traditional Chinese medicines. Since the sensitivity of the human eye to changes in color hue (such as red, yellow, and blue) is much greater than the sensitivity to changes in color intensity (depth), therefore, compared with existing colloidal gold immunochromatographic test papers, the multicolor evolution test paper of the present invention is easier to obtain high sensitivity and a wide detection range, thereby overcoming the problems of low sensitivity and narrow detection range of existing colloidal gold immunochromatographic test papers.

[0007] The technical solution of the present invention is:

[0008] The present invention provides a method for screening aflatoxin B1 residues in traditional Chinese medicines based on multicolor evolution fluorescence immunochromatography, comprising the following steps:

[0009] (1) coupling red quantum dot fluorescent microspheres (QDM-625) with AFB1 antibodies to prepare detection probes; coupling or mixing green quantum dot fluorescent microspheres (QDM-530) with AFB1 antigens to prepare internal standard probes;

[0010] (2) preparing a multicolor evolving fluorescent test paper, including a sample pad, a NC membrane, and an absorption pad; coating the internal standard probe on the surface of the NC membrane at a density of 0.5 to 1 μL / cm, drying at 37°C for 12 h, and completing the T line, wherein the concentration of the AFB1 antigen in the internal standard probe is 0.25 to 1 mg / mL, and the concentration of the green quantum dot fluorescent microspheres is 0.01 to 1 mg / mL;

[0011] Goat anti-mouse IgG with a concentration of 0.25-1 mg / mL was coated on the surface of the NC membrane at a density of 0.5-1 μL / cm and dried at 37°C for 12 h to complete the preparation of C line;

[0012] (3) Weigh a certain mass of Chinese herbal medicine powder, add methanol, sonicate, centrifuge, take the supernatant, mix it with the detection probe, and obtain the test solution. Drop the test solution onto the sample pad of the test paper and observe the fluorescence color of the T line and C line under ultraviolet light. Determine whether the AFB1 residue in the Chinese herbal medicine exceeds the limit based on the fluorescence color of the T line.

[0013] The principle diagram of the present invention is as follows Figure 1As shown, the principle is: select red and green quantum dot fluorescent microspheres, couple them with antibodies and artificial antigens respectively, and construct ratio fluorescence detection probes and internal standard probes. Assemble the immunochromatographic test paper, spray the detection probe on the conjugate pad (or mix it with the sample solution), fix the internal standard probe on the detection line (T line) of the cellulose membrane (NC membrane), and fix the control line (C line) with goat anti-mouse IgG secondary antibody. Adjust the probe concentration to obtain a fluorescent immunochromatographic test paper based on the two primary colors of "red and green".

[0014] After the sample solution is dripped onto the sample addition area (sample pad), under the action of chromatography, the red fluorescent detection probe binds to the green fluorescent internal standard probe on the T line, resulting in the superposition of red and green fluorescence, which leads to the change of the observed fluorescence color. The excess detection probe will bind to the secondary antibody on the C line, making the C line present a bright red fluorescence. If the sample solution contains the analyte, part of the detection probe will bind to the analyte, and the detection probe bound to the internal standard probe will be reduced, resulting in the weakening of the red fluorescence component of the T line. Based on the principle of competitive reaction, as the concentration of the target increases, under the irradiation of ultraviolet light, the color of the detection area gradually changes from red to green, and by comparing the color with the standard colorimetric card, the visual rapid detection of the analyte can be achieved.

[0015] Furthermore, the red quantum dot fluorescent microspheres are monodisperse polymer nanospheres with a particle size of 100 to 200 nm, which are embedded with red fluorescent quantum dots and have carboxyl groups on the surface, and an emission wavelength of 620 to 630 nm;

[0016] The green quantum dot fluorescent microspheres are monodisperse polymer nanospheres with a particle size of 100 to 200 nm and embedded with green fluorescent quantum dots, and have carboxyl groups on the surface and an emission wavelength of 525 to 535 nm.

[0017] Furthermore, in the step (1), the red quantum dot fluorescent microspheres after ultrasound are mixed with MES buffer, and then EDC / NHS mixed solution is added, reacted at 37°C, centrifuged, and the supernatant is discarded; then resuspended with MES buffer, AFB1 antibody is added, reacted at 37°C for 30 to 60 minutes, then microsphere blocking solution is added, reacted at 37°C for 30 to 60 minutes, centrifuged, and the supernatant is discarded; after washing with phosphate solution, it is dispersed in the resuspension solution to obtain a detection probe, and stored at 4°C for use;

[0018] The same method steps are used to couple green quantum dot fluorescent microspheres with AFB1 antigen to prepare internal standard probes; or green quantum dot fluorescent microspheres with a concentration of 0.01-1 mg / mL are mixed with AFB1 antigens with a concentration of 0.25-1 mg / mL to construct internal standard probes.

[0019] Furthermore, in step (2), the sample pad is pretreated by soaking it in a phosphate buffer containing 1% BSA, 5% sucrose and 0.6% Tween-20, wherein the concentration of the phosphate buffer is 0.01 M and the pH is 7.4, and the sample pad is dried at 60°C after soaking for 5 minutes.

[0020] Furthermore, in step (2), the sample pad, NC membrane and absorption pad are overlapped by 2 mm and laminated onto a PVC backing plate in sequence. The assembled card is cut into 4 mm wide strips by an automatic cutting machine and sealed for storage.

[0021] Furthermore, in the step (3), a certain mass of Chinese medicinal material powder is accurately weighed, methanol is added, vortexed and then ultrasonicated, the supernatant is centrifuged and mixed with a 1% Tween-PB solution containing a detection probe to obtain a test solution; the test solution is dropped onto the sample pad of the test paper, and after 10 minutes, the fluorescence color at the T line and C line is observed under ultraviolet light.

[0022] Furthermore, in step (3), whether the AFB1 residue in the traditional Chinese medicine exceeds the limit is judged according to the color of the T-line fluorescence: red or orange is judged to be within the limit, green or blue-green is judged to be exceeded, and yellow is judged to be near the limit.

[0023] Beneficial effects of the present invention:

[0024] (1) The present invention uses quantum dot microspheres (QDM) instead of colloidal gold / gold nanoparticles to label AFB1 antibodies and prepare detection probes; the quantum dot microspheres used use a polymer to encapsulate a large number of quantum dots on the outer layer, which has a higher fluorescence intensity and is more tolerant to solution pH and ionic strength. Since the intensity of fluorescence can be observed / detected in a direction at right angles to the excitation light, it is not affected by the excitation light and has a much higher sensitivity than the UV-visible absorption spectrum.

[0025] The sensitivity of the fluorescent probe prepared by the present invention is 10 to 1000 times higher than that of the traditional detection method marked with colored substances, which can reach ng / L. The sensitivity is comparable to that of large-scale precision instruments such as LC-MS / MS and GC-MS / MS, but the cost, ease of operation, portability, etc. are better than those of large-scale instruments, and rapid on-site detection can be achieved.

[0026] In addition, due to the increased sensitivity, the corresponding allowable sample dilution multiple increases, thereby simplifying the sample processing method to be tested, reducing the influence of matrix components on antigen-antibody binding and fluorescence, and greatly increasing the types of matrices applicable to the fluorescent test paper.

[0027] (2) In the screening method of the present invention, a ratiometric fluorescence immunoassay system is constructed by introducing an internal standard probe. When the target is added, only the fluorescence signal intensity of the detection probe is enhanced or decreased, while the fluorescence signal of the internal standard probe does not change, thereby effectively reducing the fluorescence intensity deviation caused by factors such as instruments and environment, thereby improving the sensitivity and accuracy of the analysis results and reducing the interference of the sample matrix.

[0028] (3) The test strip provided by the present invention is easy to use, simple to operate, fast in detection speed, low in cost and highly sensitive. The sample is simply extracted, mixed with a sample diluent containing a detection probe at room temperature, and then dripped onto the sample pad of the test strip. After 10 minutes, the result can be interpreted by naked eye observation under ultraviolet light. When the test strip is used, the matrix has little influence, and the application range is wide. It can be used for rapid limited screening of AFB1 residues in traditional Chinese medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the principle of multi-color evolution fluorescent immunochromatographic test strips for detecting target objects;

[0030] Figure 2 This is the sensitivity evaluation of the multicolor evolution test strip; in the figure, the concentrations of 0-10AFB1 standards are 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.3, 0.4 and 0.5 ng / mL respectively;

[0031] Figure 3 This is the application of multicolor evolution test strips in the limited screening of AFB1 in traditional Chinese medicine; in the figure, the addition amounts of 1 to 4 AFB1 are 0, 2.5, 5, and 7.5 μg / kg respectively;

[0032] Figure 4 The figure shows the application results of the multi-color evolution test paper in actual samples and spiked samples. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] In order to further understand the present invention, the present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0035] The present invention provides a method for screening aflatoxin B1 residues in traditional Chinese medicines based on multicolor evolution fluorescence immunochromatography, comprising the following steps:

[0036] (1) Preparation of detection probe and internal standard probe

[0037] (1.1) Activation: 100-200 μg of ultrasonic red quantum dot fluorescent microspheres were mixed with 0.05 M MES buffer at pH 5.5, and then EDC / NHS mixed solution was added to react at 37°C, centrifuged, and the supernatant was discarded.

[0038] (1.2) Coupling antibody or antigen: Resuspend in MES buffer, add 2-10 μg AFB1 antibody or antigen, and react at 37°C for 30-60 min.

[0039] (1.3) Blocking: Add 100-200 μL of microsphere blocking solution (phosphate buffer containing 1-5% BSA) and react at 37°C for 30-60 min.

[0040] (1.4) Resuspension: Centrifuge the blocked solution, discard the supernatant, wash twice with phosphate solution, and finally disperse in 0.5-2 mL of resuspension solution (phosphate buffer containing 1% BSA and 5% sucrose); store at 4°C in the dark for future use.

[0041] The same steps as above were used to couple green quantum dot fluorescent microspheres with AFB1 antigen to prepare the internal standard probe; green quantum dot fluorescent microspheres with a concentration of 0.01 to 1 mg / mL and AFB1 antigen with a concentration of 0.25 to 1 mg / mL were mixed to construct the internal standard probe.

[0042] (2) Preparation of multicolor evolution fluorescent immunochromatographic test strips

[0043] Sample pad pretreatment: soak in phosphate buffer (0.01 M, pH 7.4) containing 1% BSA, 5% sucrose and 0.6% Tween-20 for 5 min and then dry at 60°C.

[0044] Preparation of T line and C line: The internal standard probe (AFB1 antigen 0.25-1 mg / mL, green quantum dot fluorescent microspheres 0.01-1 mg / mL) and goat anti-mouse IgG (0.25-1 mg / mL) were coated on the surface of the NC membrane at a density of 0.5-1 μL / cm, respectively, and then dried at 37 °C for 12 h.

[0045] (3) Construction of multi-color evolution fluorescent test paper detection system

[0046] Accurately weigh 2-10g of Chinese herbal medicine powder in a centrifuge tube, add 10-50mL of methanol, vortex mix and ultrasonicate for 3-10min, centrifuge to obtain 20-100μL of supernatant, and mix with 80-400μL of sample diluent (1% Tween-PB solution) containing 2-20μg / mL detection probe to obtain the test solution. Drop the test solution onto the sample pad of the test strip, and observe the fluorescence color at the T line and C line under ultraviolet light after 10min.

[0047] The fluorescence color of the T line is used to determine whether the AFB1 residue in the traditional Chinese medicine exceeds the limit. Red and orange indicate that the residue does not exceed the limit, green indicates that the residue exceeds the limit, and yellow indicates that the residue is close to the limit.

[0048] Example 1

[0049] This embodiment provides a method for screening aflatoxin B1 residues in traditional Chinese medicines based on multicolor evolution fluorescence immunochromatography, and the specific steps are as follows:

[0050] (1) Disperse 100 μg of ultrasonic QDM-625 into 200 μL of 0.05M MES buffer at pH 5.5, add 20 μL of EDC / NHS mixture (2 mg / mL), react at 37°C for 25 min, centrifuge at 14000 rmin for 8 min, and discard the supernatant. Resuspend with MES buffer, add 6 μg of AFB1 antibody, react at 37°C for 60 min, then add 100 μL of microsphere blocking solution (MES buffer containing 1% BSA), react at 37°C for 60 min, centrifuge at 14000 rmin for 8 min, and discard the supernatant. Wash twice with PBS solution (0.01M pH 7.4), and finally disperse and fix the volume in 1 mL of resuspension solution (PBS containing 1% BSA and 5% sucrose). Prepare the detection probe QDM-mAb and store at 4°C for future use.

[0051] The same steps as above were used to couple QDM-530 with AFB1 antigen to prepare the internal standard probe; alternatively, QDM-530 at a concentration of 0.1 mg / mL and AFB1 antigen at a concentration of 0.75 mg / mL were mixed to construct the internal standard probe.

[0052] (2) Treat the sample pad with a PBS solution containing 1% BSA, 5% sucrose and 0.6% Tween-20, and dry at 60°C. Coat the internal standard probe (AFB1 antigen 0.75 mg / mL, QDM-5300.1 mg / mL) and goat anti-mouse IgG (0.5 mg / mL) on the surface of the NC membrane at a density of 1 μL / cm, respectively, and then dry at 37°C for 12 hours to complete the preparation of T lines and C lines. Then, overlap the sample pad, NC membrane, and absorption pad by 2 mm and laminate them onto the PVC backing board in sequence. The assembled card is cut into 4 mm wide strips with an automatic cutting machine, sealed with a ziplock bag and stored in a desiccator at room temperature.

[0053] (3) Accurately weigh 2.0 g of Chinese medicinal material powder into a centrifuge tube, add 10 mL of methanol, vortex mix, and sonicate for 5 min. Centrifuge to obtain 20 μL of the supernatant, and mix it with 80 μL of sample diluent (1% Tween-PB solution) containing 10 μg / mL detection probe to obtain the test solution.

[0054] Drop the test solution onto the sample pad of the test strip. After 10 minutes, observe the fluorescent color at the T line and C line under ultraviolet light.

[0055] Test Example 1

[0056] The test strips were assembled according to the optimized conditions, and the AFB1 standard solution containing 8 μg / mL detection probe was prepared with sample diluent (1% Tween-PB solution). The AFB1 concentrations were 0 ng / mL, 0.01 ng / mL, 0.02 ng / mL, 0.04 ng / mL, 0.06 ng / mL, 0.08 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL ( Figure 2 Then take 100 μL and drop it on the sample pad. After 10 minutes, observe the fluorescence color at the T line and C line under ultraviolet light. The results are as follows Figure 2 As shown in the figure, the T-line fluorescence changes from red to orange to yellow to green as the AFB1 concentration increases. When the concentration is less than 0.08 ng / mL, the T-line is red or orange-red; when the concentration is between 0.08 and 0.2 ng / mL, the T-line is yellow or yellow-green; when it is greater than 0.2 ng / mL, the T-line is green or blue-green. Therefore, the semi-quantification of AFB1 concentration can be achieved by the T-line fluorescence color. This shows that the screening method has high detection sensitivity.

[0057] Example 2

[0058] Using lotus seeds as an example, limited screening of AFB1 residues in traditional Chinese medicines

[0059] The 2020 edition of the "Chinese Pharmacopoeia" stipulates that the maximum residue limit (MRL) of AFB1 in traditional Chinese medicines is 5μg / kg.

[0060] According to the sensitivity and fluorescence color changes of the above multicolor evolution test paper, combined with sample pretreatment, the detection of the sample solution with a limit concentration falls within the yellow fluorescence interval. When the T line is red or orange-red, it can be judged that the residual amount of AFB1 in the sample is below the MRL; when the T line is yellow or yellow-green, the residual amount of AFB1 in the sample is near the MRL; when the T line is green or blue-green, the residual amount of AFB1 in the sample exceeds the MRL.

[0061] The present invention uses lotus seeds as a representative to investigate the application of multicolor evolution test paper in the AFB1 limit screening of traditional Chinese medicine. Lotus seed sample powder verified to be free of aflatoxin residues was weighed, and AFB1 standard was added at 0, 0.5, 1, and 1.5 times the MRL.

[0062] Then, 2.0 g of lotus seed powder with four addition levels was weighed into a centrifuge tube, 10 mL of methanol was added, vortexed and ultrasonicated for 5 min, and 20 μL of the supernatant was centrifuged and mixed with 80 μL of sample diluent (1% Tween-PB solution) containing 10 μg / mL detection probe to obtain the test solution.

[0063] Drop the test solution onto the sample pad of the test strip, and after 10 minutes, observe the fluorescence color at the T line and C line under ultraviolet light. Figure 3 As shown in the figure, the T lines of the samples with four addition levels are red, orange, yellow and green respectively. It can be judged that the AFB1 residues in the samples are below the limit, near the limit and above the limit respectively.

[0064] The multicolor evolution test strip of the present invention can be used to quickly judge and screen out Chinese medicines whose AFB1 residue levels exceed the limit standard. Samples judged by the test strip to be near the limit or exceeding the limit can be further confirmed by LC-MS / MS.

[0065] Test Example 2

[0066] In order to further verify the application of the multicolor evolution fluorescent test paper in the limited screening of AFB1 in traditional Chinese medicine, actual lotus seed samples were collected and tested with the fluorescent test paper, and blank lotus seed samples were added with different contents of AFB1 and then tested with the fluorescent test paper.

[0067] The results are as follows Figure 4 As shown in the figure, as the spiked concentration increases, the fluorescence of the T line of the test strip gradually changes from red to orange, yellow and green. When the AFB1 content in the sample is near the MRL (5μg / kg), the T line shows bright yellow fluorescence; when it exceeds the MRL, the T line shows green fluorescence; when it is below the MRL, the T line shows red or orange fluorescence.

[0068] It can be seen that the multicolor evolving fluorescent test paper can be conveniently used for limited screening of AFB1 in traditional Chinese medicines, greatly improving the screening efficiency and saving costs.

[0069] The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, modification, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for screening aflatoxin B1 residues in traditional Chinese medicine based on multicolor evolution fluorescence immunochromatography, characterized in that: The following steps are involved: (1) coupling red quantum dot fluorescent microspheres with AFB1 antibodies to prepare detection probes; coupling or mixing green quantum dot fluorescent microspheres with AFB1 antigens to prepare internal standard probes; (2) preparing a multicolor evolving fluorescent test paper, including a sample pad, a NC membrane, and an absorption pad; coating the internal standard probe on the surface of the NC membrane at a density of 0.5 to 1 μL / cm, drying at 37°C for 12 h, and completing the T line, wherein the concentration of the AFB1 antigen in the internal standard probe is 0.25 to 1 mg / mL, and the concentration of the green quantum dot fluorescent microspheres is 0.01 to 1 mg / mL; Goat anti-mouse IgG with a concentration of 0.25-1 mg / mL was coated on the surface of the NC membrane at a density of 0.5-1 μL / cm and dried at 37°C for 12 h to complete the preparation of C line; (3) Weigh a certain mass of Chinese herbal medicine powder, add methanol, sonicate, centrifuge, take the supernatant, mix it with the detection probe, and obtain the test solution. Drop the test solution onto the sample pad of the test paper and observe the fluorescence color of the T line and C line under ultraviolet light. Determine whether the AFB1 residue in the Chinese herbal medicine exceeds the limit based on the fluorescence color of the T line.

2. The screening method according to claim 1, characterized in that The red quantum dot fluorescent microspheres are monodisperse polymer nanospheres with a particle size of 100 to 200 nm that are embedded with red fluorescent quantum dots, have carboxyl groups on the surface, and emit a wavelength of 620 to 630 nm; The green quantum dot fluorescent microspheres are monodisperse polymer nanospheres with a particle size of 100 to 200 nm and embedded with green fluorescent quantum dots, and have carboxyl groups on the surface and an emission wavelength of 525 to 535 nm.

3. The screening method according to claim 1, characterized in that In the step (1), the red quantum dot fluorescent microspheres after ultrasound are mixed with MES buffer, and then EDC / NHS mixed solution is added, reacted at 37°C, centrifuged, and the supernatant is discarded; then resuspended with MES buffer, AFB1 antibody is added, reacted at 37°C for 30-60 minutes, then microsphere blocking solution is added, reacted at 37°C for 30-60 minutes, centrifuged, and the supernatant is discarded; after washing with phosphate solution, the microspheres are dispersed in the resuspension solution to obtain the detection probe, which is stored at 4°C for future use; The same method steps are used to couple green quantum dot fluorescent microspheres with AFB1 antigen to prepare an internal standard probe; or green quantum dot fluorescent microspheres with a concentration of 0.01-1 mg / mL are mixed with AFB1 antigen with a concentration of 0.25-1 mg / mL to construct an internal standard probe.

4. The screening method according to claim 1, characterized in that: In the step (2), the sample pad is pretreated by soaking it in a phosphate buffer containing 1% BSA, 5% sucrose and 0.6% Tween-20, wherein the concentration of the phosphate buffer is 0.01 M and the pH is 7.

4. After soaking for 5 minutes, the sample pad is dried at 60°C.

5. The screening method according to claim 1, characterized in that: In the step (2), the sample pad, NC membrane and absorption pad are overlapped by 2 mm and laminated onto a PVC backing plate in sequence. The assembled card is cut into 4 mm wide strips by an automatic cutting machine and sealed for storage.

6. The screening method according to claim 1, characterized in that: In the step (3), a certain mass of Chinese medicinal material powder is accurately weighed, methanol is added, vortexed and ultrasonicated, and the supernatant is centrifuged and mixed with a 1% Tween-PB solution containing a detection probe to obtain a test solution; Drop the test solution onto the sample pad of the test paper. After 10 minutes, observe the fluorescent color at the T line and C line under ultraviolet light.

7. The screening method according to claim 1, characterized in that: In step (3), whether the AFB1 residue in the traditional Chinese medicine exceeds the limit is determined based on the color of the T-line fluorescence: red or orange indicates that it does not exceed the limit, green or cyan indicates that it exceeds the limit, and yellow indicates that it is near the limit.

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