Preparation method of colloidal gold immunochromatographic strip for rapid detection of chlorpyrifos and fipronil in agaricus blazei murill substrate

Abstract

The present application relates to edible mushroom cultivation and biological detection technical field, more specifically, the present application relates to a kind of for tea tree mushroom substrate tox dead klab and fipronil rapid detection colloidal gold immunochromatographic test strip preparation method.The method is labeled with monoclonal antibody by EDC / NHS covalent coupling in carboxyl containing colored latex microspheres, resuspended after spotting and drying in coated micropore bottom;Sample pad is pretreated with buffer salt and tween, detection line and quality control line are set on nitrocellulose membrane and completed assembly cutting strip.Detection is carried out by pre-reaction in coated micropore before entering test strip chromatography color development, realize the rapid screening of tox dead klab and fipronil related target in tea tree mushroom substrate;Multiple detection lines or multiple micropores can be used to realize multiple target parallel detection, operation window is clear, and the result is stable.

Description

Technical Field

[0001] This invention relates to the field of edible fungi cultivation and biological detection technology, and more specifically, to a method for preparing a colloidal gold immunoassay strip for rapid detection of chlorpyrifos and fipronil in the substrate of Agrocybe aegerita. Background Technology

[0002] In the cultivation of tea tree mushrooms, the substrate (cultivation medium, substrate residue, mycelium, and mixtures thereof) is a crucial carrier for disease occurrence and spread. Rapid screening for chlorpyrifos and fipronil-related targets in the substrate is frequently required at the production site to isolate contaminated batches as early as possible, adjust disinfection and watering strategies, and reduce the risk of spread. While existing laboratory methods (such as PCR and ELISA) offer high sensitivity, they typically require specialized equipment, involve complex sample pretreatment, and have long testing cycles, making them unsuitable for rapid on-site decision-making.

[0003] Immunochromatographic test strips, as a rapid on-site detection solution, have advantages such as ease of operation and visual readability. However, in the context of strong matrix interference such as *Agrocybe aegerita* substrate, the following problems still commonly exist: Strong matrix interference and poor reproducibility: Matrix extracts are often turbid, colored, highly viscous, and contain a large amount of colloids and particulate matter, which can easily cause unstable chromatographic flow rate, enhanced non-specific adsorption, and background blemishes, leading to false positives / false negatives and batch-to-batch differences.

[0004] Insufficient stability and signal strength of labeled substances: Conventional colloidal gold or physical adsorption labeling systems are easily affected by pH, salt ions and organic components in complex extraction solutions, resulting in desorption, aggregation or signal attenuation of labeled antibodies, thus limiting sensitivity and consistency.

[0005] Release and reaction timing are uncontrollable: Traditional structures often rely on the immediate release of markers from the binding pad. The amount and timing of marker release after sample entry are greatly affected by temperature, humidity and pad material conditions, resulting in fluctuations in reaction kinetics and making it difficult to stably interpret the results in complex matrices.

[0006] Insufficient on-site engineering: Existing solutions often lack engineering constraints on "quantitative sample loading, pre-reaction time, and effective interpretation window," and differences among operators will further amplify the fluctuations in results.

[0007] Therefore, there is an urgent need for an immunochromatographic test strip preparation scheme that is designed for the tea tree mushroom substrate scenario, has stronger resistance to substrate interference, stable signal and can be engineered for standardized operation, and also takes into account multi-target expansion. Summary of the Invention

[0008] To overcome the aforementioned deficiencies of the prior art, embodiments of the present invention provide a method for preparing a colloidal gold immunoassay strip for rapid detection of chlorpyrifos and fipronil in a tea tree mushroom substrate, thereby solving the problems mentioned in the background art.

[0009] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a colloidal gold immunochromatographic strip for rapid detection of chlorpyrifos and fipronil in a mushroom substrate, comprising the following steps: S1. Establish an immune recognition system for disease targets, wherein the disease targets are target analytes in the tea tree mushroom substrate that are related to disease occurrence, prepare coating antigens or coating substances for detection lines for the disease targets, and prepare monoclonal antibodies that have specific binding ability to the disease targets. S2, providing colored latex microspheres with carboxyl groups on the surface, activating the colored latex microspheres with MES buffer, and covalently coupling the monoclonal antibody with the colored latex microspheres under the action of EDC and NHS to form latex microsphere labeled antibody; S3. After blocking the unbound sites, the latex microsphere-labeled antibody is resuspended in a reconstitution buffer containing Tris-HCl, BSA, sucrose and sodium azide, and the latex microsphere-labeled antibody is quantitatively spotted onto the bottom of the blank coated microwells and dried at 37°C to obtain coated microwells. S4, providing an immunochromatographic test strip consisting of a sample pad, a nitrocellulose membrane, an absorbent pad, and a PVC base plate, wherein the sample pad is made of glass fiber paper and is pretreated with a sample pad treatment solution containing phosphate buffer and Tween 20. S5, a detection line T and a control line C are formed on the nitrocellulose membrane. The T line is coated with the coating antigen or coating substance, and the C line is coated with goat anti-mouse IgG antibody. After drying, the test strip is sequentially pasted onto the PVC base plate along with the sample pad and the absorbent pad, and then cut into strips to obtain the finished test strip. S6, the finished test strip is packaged with the coated microwell, so that the test solution first reacts with the latex microsphere-labeled antibody in the coated microwell before entering the test strip for chromatographic color development, thereby realizing the immunodetection of disease targets in the tea tree mushroom matrix.

[0010] In a preferred embodiment, the sample pad treatment solution is a 0.01M to 0.05M phosphate buffer solution with a pH of 6.5 to 7.5, and contains 0.1% to 0.5% Tween 20.

[0011] In a preferred embodiment, the reconstitution buffer is a 0.01M to 0.05M Tris-HCl buffer with a pH of 8.0 to 9.0, and contains 0.1% to 1% BSA, 5% to 10% sucrose, and 0.02% sodium azide; the blocking step uses a 0.5% to 2% BSA solution as the blocking solution.

[0012] In a preferred embodiment, the amount of latex microsphere-labeled antibody spotted in step S3 is 3µL / well to 5µL / well, and the sample is dried at 37°C and then packaged for storage.

[0013] In a preferred embodiment, step S2 includes: resuspending the stained latex microspheres in 0.1 M MES buffer (pH 6.0), adding EDC and NHS, and reacting at room temperature for 30 min to complete activation; then centrifuging at 11,000 rpm and 4 °C for 15 min to remove the supernatant, resuspending in 0.1 M PBS (pH 7.4), adding monoclonal antibody, and reacting at room temperature for 90 min to complete conjugation; then adding 10% BSA blocking solution, reacting at room temperature for 60 min, and centrifuging to remove the supernatant.

[0014] In a preferred embodiment, the membrane coating solution used in step S5 to form the T-line and the C-line is a 0.01M to 0.05M phosphate buffer solution with a pH of 6.5 to 7.5 and containing 0.5% to 1.0% trehalose.

[0015] In a preferred embodiment, the disease target includes at least two different disease-related target analytes, and a first latex microsphere-labeled antibody and a second latex microsphere-labeled antibody are prepared respectively, and parallel detection is achieved using any of the following methods: A first detection line T1 and a second detection line T2 are set on the same nitrocellulose membrane, and coated with coating antigens or coating substances corresponding to the first disease target and the second disease target, respectively. Alternatively, a first coated microwell and a second coated microwell can be provided, respectively coated with the first latex microsphere-labeled antibody and the second latex microsphere-labeled antibody, and packaged together with the same test strip or two test strips.

[0016] In a preferred embodiment, a colloidal gold immunochromatographic strip assembly for rapid detection of chlorpyrifos and fipronil in a mushroom substrate comprises: An immunochromatographic test strip, wherein the immunochromatographic test strip is composed of a sample pad, a nitrocellulose membrane, an absorbent pad, and a PVC base plate; The nitrocellulose membrane is provided with a detection line (T line) and a control line (C line), and the C line is coated with goat anti-mouse IgG antibody. And coated microwells, wherein the bottom of the coated microwells is coated with monoclonal antibodies labeled with latex microspheres, wherein the monoclonal antibodies labeled with latex microspheres are covalently bound to the colored latex microspheres containing carboxyl groups on the surface via EDC / NHS-mediated amide bonds, and are obtained by spotting and drying after resuspending in a reconstitution buffer containing Tris-HCl, BSA, sucrose and sodium azide. The component is configured such that the test solution first enters the coated microwell and pre-reacts with the monoclonal antibody labeled with the latex microspheres before entering the immunochromatographic test strip for chromatographic color development.

[0017] In a preferred embodiment, a method for rapid detection of chlorpyrifos and fipronil in a *Agrocybe aegerita* matrix using a colloidal gold immunochromatographic strip assembly includes: Prepare the test solution from a sample of *Agrocybe aegerita* matrix; Add 100µL of the test solution to the coated microwell and mix by blowing to fully dissolve the dry material at the bottom of the well. Let it stand for 4 minutes to react. Add all the solution from the coated microwells to the sample well of the test strip and start timing. Read the test results within 5 to 8 minutes. Readings outside the specified time range are invalid.

[0018] The technical effects and advantages of this invention are as follows: 1. By pre-placing labeled antibodies in the coated microwells and performing "microwell pre-reaction", the sample can complete more thorough specific binding and matrix buffering before entering the membrane chromatography, thereby reducing the interference of turbidity and viscosity on chromatography and color development, and improving the stability and repeatability of the results.

[0019] 2. By using EDC / NHS-mediated covalent coupling to link monoclonal antibodies with carboxyl-containing colored latex microspheres, the risk of physical adsorption and desorption is reduced, and the salt resistance and anti-interference ability of the labeling system in complex extraction solutions are enhanced, thereby improving sensitivity and batch-to-batch consistency.

[0020] 3. Pre-treat the sample pad with buffer salts and surfactants to reduce sample adhesion and non-specific adsorption, stabilize the chromatography flow rate, thereby reducing background noise and improving interpretation clarity.

[0021] 4. By combining micropore spotting followed by drying, membrane coating system and assembly process, modular packaging is achieved, which is convenient for on-site carrying and long-term storage, and reduces performance fluctuations caused by transportation and environmental changes.

[0022] 5. By setting up multiple detection lines (T1 / T2) on the same membrane or using a parallel configuration of multiple coated microwells, parallel detection of at least chlorpyrifos and fipronil-related targets can be achieved, meeting the screening needs of the production site, and can be expanded as needed.

[0023] 6. By limiting the sample loading volume, pre-reaction time, and effective interpretation time window, the impact of operator differences on test results is significantly reduced, and the consistency of field applications is improved. Attached Figure Description

[0024] Figure 1 This invention relates to a method for preparing immunoassay strips. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Reference Figure 1 A method for preparing a colloidal gold immunochromatographic strip for rapid detection of chlorpyrifos and fipronil in a substrate of *Agrocybe aegerita* (tea tree mushroom), comprising the following steps: Step S1: Establish an immune recognition system for disease targets. The disease targets are target analytes in the tea tree mushroom substrate that are related to disease occurrence. Prepare coating antigens or coating substances for detection lines targeting the disease targets, and prepare monoclonal antibodies that have specific binding ability to the disease targets. Step S2: Provide colored latex microspheres with carboxyl groups on the surface, activate the colored latex microspheres with MES buffer, and covalently couple monoclonal antibodies with colored latex microspheres under the action of EDC and NHS to form latex microsphere labeled antibodies; Monoclonal antibodies can be prepared by primary and booster immunization of BALB / c mice with complete antigens, and spleen cells are taken from mice with high antibody titers after detecting serum antibody titers. Spleen cells and myeloma cells were fused at a ratio of 5:1 to 10:1 under PEG treatment. The fused cells were seeded in 96-well plates and cultured in HAT / HT medium. Positive clones were screened by indirect competitive ELISA and subclones were obtained by limiting dilution to obtain cell lines that stably secrete monoclonal antibodies. High-purity monoclonal antibodies were further purified by protein A or protein G affinity chromatography for labeling.

[0027] Step S2 includes: resuspending the stained latex microspheres in 0.1 M M ES buffer (pH 6.0), adding EDC and NHS, and reacting at room temperature for 30 min to complete activation; then centrifuging at 11000 rpm and 4℃ for 15 min to remove the supernatant, resuspending in 0.1 M PBS (pH 7.4), adding monoclonal antibody, and rotating and mixing at room temperature for 90 min to complete conjugation; then adding 10% BSA blocking solution, rotating and mixing at room temperature for 60 min, and centrifuging to remove the supernatant.

[0028] As a feasible example, red latex microspheres with carboxyl groups on the surface (e.g., 300 nm particle size, 1% solid content) were selected. 50 μL of red latex microspheres with a solid content of 4% were added to 950 μL of 0.1 M MES (pH 6.0) and mixed well. 10 μL of 10 mg / mL LEDC and 50 μL of 10 mg / mL NHS were added and vortexed. The mixture was then reacted at room temperature for 30 min to complete the activation. The mixture was then centrifuged at 11,000 rpm and 4 °C for 15 min, the supernatant was discarded, and the mixture was resuspended in 1 mL of 0.1 M PBS (pH 7.4). Monoclonal antibody (e.g., 10–50 μg antibody / mg microspheres) was added and the mixture was rotated at room temperature for 90 min to complete the conjugation.

[0029] Step S3: After blocking the unbound sites, the latex microsphere-labeled antibody is resuspended in a reconstitution buffer containing Tris-HCl, BSA, sucrose and sodium azide, and the latex microsphere-labeled antibody is quantitatively spotted onto the bottom of the blank coated microwells and dried at 37°C to obtain coated microwells. The reconstitution buffer is a 0.01M to 0.05M Tris-HCl buffer with a pH of 8.0 to 9.0, containing 0.1% to 1% BSA, 5% to 10% sucrose, and 0.02% sodium azide; the blocking step uses a 0.5% to 2% BSA solution as the blocking solution.

[0030] In step S3, the amount of latex microsphere-labeled antibody applied is 3µL / well to 5µL / well, and the sample is dried at 37°C and then packaged for storage.

[0031] The colorimetric labeling system of the immunochromatographic test strip of the present invention is not limited to the colloidal gold labeling system, but can also use the colored latex microsphere labeling system.

[0032] Preferably, colored latex microspheres with carboxyl groups on their surface are covalently coupled with monoclonal antibodies via EDC / NHS-mediated amide bonds to form latex microsphere-labeled antibodies, thereby improving their stability and anti-interference ability in the complex extract of *Agrocybe aegerita* matrix, and enabling visual interpretation together with the detection line / control line.

[0033] The coating microwells can be independent plastic microwell components or single-well structures of microplates, with the bottom of the wells used to support the dried latex microsphere-labeled antibody membrane. Preferably, the latex microsphere-labeled antibody resuspension is spotted at 3-5 μL / well to the bottom of the blank microwell, dried at 37°C until a uniform dry film layer is formed at the bottom of the well, and the test solution is added and mixed by blowing to fully dissolve the dry film at the bottom of the well, followed by standing for pre-reaction.

[0034] Step S4: Provide an immunochromatographic test strip consisting of a sample pad, a nitrocellulose membrane, an absorbent pad, and a PVC base plate. The sample pad is made of glass fiber paper and is pretreated with a sample pad treatment solution containing phosphate buffer and Tween 20. The sample pad treatment solution is 0.01M to 0.05M phosphate buffer, pH 6.5 to 7.5, and contains 0.1% to 0.5% Tween 20; Step S5: Form the detection line T and the control line C on the nitrocellulose membrane. The T line is coated with antigen or coating material, and the C line is coated with goat anti-mouse IgG antibody. After drying, the test strip is attached to the sample pad and absorbent pad in sequence on the PVC base plate and cut into strips to obtain the finished test strip. In step S5, the membrane coating solution used to form the T-line and C-line is a 0.01M to 0.05M phosphate buffer solution with a pH of 6.5 to 7.5 and containing 0.5% to 1.0% trehalose.

[0035] The detection membrane can be prepared as follows: the coating antigen (e.g., target analyte-BSA conjugate) for the detection line is sprayed onto the nitrocellulose membrane to form the T line, and goat anti-mouse IgG antibody is sprayed onto the membrane to form the C line; After the coating is completed, the nitrocellulose membrane is dried and used as the test membrane. It is then pasted onto the PVC base plate along with the sample pad and the absorbent pad, and cut into strips and mounted.

[0036] Step S6: The test strip is packaged with the coated microwells, so that the test solution first reacts with the latex microsphere-labeled antibody in the coated microwells before entering the test strip for chromatographic color development, thereby realizing the immune detection of disease targets in the tea tree mushroom matrix.

[0037] The disease targets include at least two different disease-related target analytes, and a first latex microsphere labeled antibody and a second latex microsphere labeled antibody are prepared separately, and parallel detection is achieved using any of the following methods: A first detection line T1 and a second detection line T2 are set on the same nitrocellulose membrane, and coated with coating antigens or coating substances corresponding to the first disease target and the second disease target, respectively. Alternatively, a first coated microwell and a second coated microwell can be set up, coated with a first latex microsphere labeled antibody and a second latex microsphere labeled antibody respectively, and packaged with the same test strip or two test strips.

[0038] When performing parallel detection of two target analytes, a first latex microsphere labeled antibody and a second latex microsphere labeled antibody can be prepared and respectively placed in the first coated microwell and the second coated microwell; During detection, the corresponding test solution is added to the corresponding coated microwell for pre-reaction before entering the same test strip or separate test strips for chromatographic color development. The same test strip can be set with a first detection line T1 and a second detection line T2, which are coated with the corresponding coating antigen / coating substance of the two target analytes, respectively, so as to achieve separate line interpretation and parallel detection without interference.

[0039] "Disease targets / analytes" can be detectable indicators in the tea tree mushroom substrate related to the occurrence, spread, or risk warning of diseases, preferably target analytes that can be interpreted by chromatography through immune competition / immune binding.

[0040] As an example, the target analyte can be a small molecule indicator such as chlorpyrifos or fipronil, and it can be used to form a coating antigen (such as a target analyte-BSA conjugate) by conjugating it with a carrier protein for T-line coating. At the same time, monoclonal antibodies with specific binding ability to the target analyte can be prepared for labeling.

[0041] When the target analyte is a small molecule and has insufficient immunogenicity, the target analyte can be structurally modified to introduce active groups to obtain a hapten. The hapten is then conjugated with a carrier protein (such as BSA) in PBS (pH 7.4) to prepare a complete antigen (target analyte - BSA). After the reaction is complete, unreacted small molecules are removed by dialysis, and the conjugation ratio of the conjugate is identified by ultraviolet spectrophotometry to obtain the coated antigen used to detect the T-line coating.

[0042] A colloidal gold immunoassay strip assembly for rapid detection of chlorpyrifos and fipronil in *Agrocybe aegerita* substrate includes: Immunochromatographic test strips consist of a sample pad, a nitrocellulose membrane, an absorbent pad, and a PVC base plate. The nitrocellulose membrane has a detection line (T line) and a control line (C line), with the C line coated with goat anti-mouse IgG antibody. And the coating microwells, the bottom of the coating microwells are coated with monoclonal antibodies labeled with latex microspheres, the monoclonal antibodies labeled with latex microspheres are covalently bound to the colored latex microspheres containing carboxyl groups on the surface through EDC / NHS-mediated amide bonds, and then the samples are resuspended in a reconstitution buffer containing Tris-HCl, BSA, sucrose and sodium azide and dried. The component is configured to allow the test solution to first enter the coated microwells and pre-react with the monoclonal antibody labeled with latex microspheres before entering the immunochromatographic test strip for chromatographic color development.

[0043] A method for rapid detection of chlorpyrifos and fipronil in *Agrocybe aegerita* matrix using a colloidal gold immunochromatographic strip assembly includes: Prepare the test solution from a sample of *Agrocybe aegerita* matrix; Add 100µL of the test solution to the coated microwell and mix by blowing to fully dissolve the dry material at the bottom of the well. Let it stand for 4 minutes to react. Add all the solution from the coated microwells to the sample well of the test strip and start timing. Read the test results within 5 to 8 minutes. Readings outside this time range are invalid.

[0044] The pre-reaction time of the coated microwells is used to ensure that the target analyte in the test solution and the latex microsphere-labeled antibody are in sufficient contact and reach a preliminary binding equilibrium, thereby reducing kinetic fluctuations caused by uneven instantaneous release from the binding pad. The pre-reaction time can be adjusted according to the binding kinetics of the target analyte and the matrix viscosity, and it is preferable to keep it consistent under a fixed operating procedure to ensure that the results are reproducible between different operators.

[0045] The test solution can be prepared as follows: Prepare a sample dilution buffer, which is 0.01–0.1M PBS (pH 7.2–7.4), and add 0.1%–1% BSA, 0.05%–0.2% Tween-20, and 0.02% PBS. ; Dilute the tea tree mushroom sample / matrix with the buffer solution at a ratio of 1:1 to 1:10. For example, take 2g of sample and add it to 3 to 5mL of buffer solution. Manually invert and mix for 2min and let stand for 2min. Take the supernatant or soaking solution as the test solution.

[0046] In competitive immunochromatography mode, the T-line is coated with the target analyte-carrier protein conjugate; when the target analyte is absent or its concentration is below the detection limit in the sample, the latex microsphere-labeled antibody in the microwell binds to the T-line conjugate to form a visible colored band. When the concentration of the target analyte in the sample is high, the target analyte preferentially binds to the latex microsphere-labeled antibody and occupies the binding site, making it difficult for the antibody to bind to the T-line conjugate, and the T-line color becomes lighter or disappears. The C line is coated with goat anti-mouse IgG antibody for effectiveness control; if the C line does not develop color, the antibody is considered invalid.

[0047] To verify the feasibility of this invention in *Agrocybe aegerita* samples, the following example is provided: The sample is cut into pieces to about 1 cm in size, 2.0 g ± 0.1 g of sample is weighed and added to 3 mL of buffer solution, mixed by inversion for 2 min and allowed to stand for 2 min, and the soaking solution is taken as the test solution; Add 100 μL of the test solution to the coated microwell and mix by blowing to dissolve the dry substance at the bottom of the well. Let it stand for 4 minutes for pre-reaction. Add the solution from the microwell to the sample well of the test strip and start timing. Read the result within 5 to 8 minutes. Readings exceeding the time limit are invalid.

[0048] In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a colloidal gold immunochromatographic strip for rapid detection of chlorpyrifos and fipronil in a substrate of *Agrocybe aegerita*, characterized in that; Includes the following steps: Step S1: Establish an immune recognition system for disease targets, wherein the disease targets are target analytes in the tea tree mushroom substrate that are related to disease occurrence, prepare coating antigens or coating substances for detection lines for the disease targets, and prepare monoclonal antibodies that have specific binding ability to the disease targets. Step S2: Provide colored latex microspheres with carboxyl groups on the surface, activate the colored latex microspheres with MES buffer, and covalently couple the monoclonal antibody with the colored latex microspheres under the action of EDC and NHS to form latex microsphere labeled antibody; Step S3: After blocking the unbound sites, the latex microsphere-labeled antibody is resuspended in a reconstitution buffer containing Tris-HCl, BSA, sucrose and sodium azide, and the latex microsphere-labeled antibody is quantitatively spotted onto the bottom of the blank coated microwells and dried at 37°C to obtain coated microwells. Step S4: Provide an immunochromatographic test strip consisting of a sample pad, a nitrocellulose membrane, an absorbent pad, and a PVC base plate, wherein the sample pad is made of glass fiber paper and is pretreated with a sample pad treatment solution containing phosphate buffer and Tween 20. Step S5: Form a detection line T and a control line C on the nitrocellulose membrane. The T line is coated with the coating antigen or coating substance, and the C line is coated with goat anti-mouse IgG antibody. After drying, the test strip is sequentially pasted onto the PVC base plate along with the sample pad and the absorbent pad, and then cut into strips to obtain the finished test strip. Step S6: The finished test strip is packaged with the coated microwells, so that the test solution first reacts with the latex microsphere-labeled antibody in the coated microwells before entering the test strip for chromatographic color development, thereby realizing the immunodetection of disease targets in the tea tree mushroom matrix.

2. The method for preparing a colloidal gold immunochromatographic strip for rapid detection of chlorpyrifos and fipronil in a substrate of *Agrocybe aegerita* according to claim 1, characterized in that: The sample pad treatment solution is 0.01M to 0.05M phosphate buffer, pH 6.5 to 7.5, and contains 0.1% to 0.5% Tween 20.

3. The method for preparing a colloidal gold immunochromatographic strip for rapid detection of chlorpyrifos and fipronil in a substrate of *Agrocybe aegerita* according to claim 1, characterized in that: The reconstitution buffer is a 0.01M to 0.05M Tris-HCl buffer with a pH of 8.0 to 9.0, containing 0.1% to 1% BSA, 5% to 10% sucrose, and 0.02% sodium azide; the blocking step uses a 0.5% to 2% BSA solution as the blocking solution.

4. The method for preparing a colloidal gold immunochromatographic strip for rapid detection of chlorpyrifos and fipronil in a substrate of *Agrocybe aegerita* according to claim 1, characterized in that: The amount of latex microsphere-labeled antibody applied in step S3 is 3µL / well to 5µL / well, and the sample is dried at 37°C and then packaged for storage.

5. The method for preparing a colloidal gold immunochromatographic strip for rapid detection of chlorpyrifos and fipronil in a substrate of *Agrocybe aegerita* according to claim 1, characterized in that: Step S2 includes: resuspending the stained latex microspheres in 0.1 M M ES buffer (pH 6.0), adding EDC and NHS, and reacting at room temperature for 30 min to complete activation; then centrifuging at 11000 rpm and 4℃ for 15 min to remove the supernatant, resuspending in 0.1 M PBS (pH 7.4), adding monoclonal antibody, and rotating and mixing at room temperature for 90 min to complete conjugation; then adding 10% BSA blocking solution, rotating and mixing at room temperature for 60 min, and centrifuging to remove the supernatant.

6. The method for preparing a colloidal gold immunochromatographic strip for rapid detection of chlorpyrifos and fipronil in a substrate of *Agrocybe aegerita* according to claim 1, characterized in that: In step S5, the membrane coating solution used to form the T-line and the C-line is a 0.01M to 0.05M phosphate buffer solution with a pH of 6.5 to 7.5 and containing 0.5% to 1.0% trehalose.

7. The method for preparing a colloidal gold immunochromatographic strip for rapid detection of chlorpyrifos and fipronil in a substrate of *Agrocybe aegerita* according to claim 1, characterized in that: The disease targets include at least two different disease-related target analytes, and a first latex microsphere labeled antibody and a second latex microsphere labeled antibody are prepared separately, and parallel detection is achieved using any of the following methods: A first detection line T1 and a second detection line T2 are set on the same nitrocellulose membrane, and coated with coating antigens or coating substances corresponding to the first disease target and the second disease target, respectively. Alternatively, a first coated microwell and a second coated microwell can be provided, respectively coated with the first latex microsphere-labeled antibody and the second latex microsphere-labeled antibody, and packaged together with the same test strip or two test strips.

8. A colloidal gold immunochromatographic strip assembly for rapid detection of chlorpyrifos and fipronil in *Agrocybe aegerita* substrate, characterized in that, include: An immunochromatographic test strip, wherein the immunochromatographic test strip is composed of a sample pad, a nitrocellulose membrane, an absorbent pad, and a PVC base plate; The nitrocellulose membrane is provided with a detection line (T line) and a control line (C line), and the C line is coated with goat anti-mouse IgG antibody. And coated microwells, wherein the bottom of the coated microwells is coated with monoclonal antibodies labeled with latex microspheres, wherein the monoclonal antibodies labeled with latex microspheres are covalently bound to the colored latex microspheres containing carboxyl groups on the surface via EDC / NHS-mediated amide bonds, and are obtained by spotting and drying after resuspending in a reconstitution buffer containing Tris-HCl, BSA, sucrose and sodium azide. The component is configured such that the test solution first enters the coated microwell and pre-reacts with the monoclonal antibody labeled with the latex microspheres before entering the immunochromatographic test strip for chromatographic color development.

9. A method for rapid detection of chlorpyrifos and fipronil in *Agrocybe aegerita* matrix using the colloidal gold immunochromatographic strip assembly of claim 8, characterized in that... include: Prepare the test solution from a sample of *Agrocybe aegerita* matrix; Add 100µL of the test solution to the coated microwell and mix by blowing to fully dissolve the dry material at the bottom of the well. Let it stand for 4 minutes to react. Add all the solution from the coated microwells to the sample well of the test strip and start timing. Read the test results within 5 to 8 minutes. Readings outside the specified time range are invalid.

Images