A test strip and method for detecting carbofuran

Abstract

The present invention discloses a test strip and method for detecting carbofuran. The test strip comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorbent pad and a bottom plate, the reaction membrane has a detection line coated with a carbofuran hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody, and the conjugate release pad is sprayed with a carbofuran monoclonal antibody-colloidal gold marker. The present invention also provides a method for detecting carbofuran in plant-derived food using the above-mentioned test strip. The test strip provided by the present invention has the advantages of simple operation, high sensitivity, fast detection speed, low cost, and suitability for large-scale sample screening, and can meet the needs of my country's food regulatory authorities in carrying out on-site monitoring and detection.

Description

Technical Field

[0001] The invention relates to a test strip and method for detecting carbofuran, and in particular to a colloidal gold test strip for detecting carbofuran, which is particularly suitable for detecting carbofuran in plant-derived foods such as vegetables and fruits. Background Art

[0002] Carbofuran, also known as furadan and cypermethrin, belongs to the carbamate pesticides, is a systemic broad-spectrum insecticide and acaricide, with contact and stomach poisoning effects. Its mechanism of action is similar to that of organophosphorus, in that it inhibits the activity of acetylcholinesterase, relies on the affinity between the inhibitor and the enzyme activity range to form an inhibitor complex, thereby destroying the normal conduction of nerve impulses, causing abnormal excitement, convulsions, paralysis and death, etc. It can be used to control most pests of crops such as rice, peanuts, sugarcane, cotton, etc., mainly for systemic insecticide effects.

[0003] Unlike other carbamate insecticides, carbofuran is irreversibly bound to acetylcholinesterase, so it is highly toxic. Once exposed to the environment, it will remain for a long time and is highly toxic to birds, bees and aquatic animals such as fish. The U.S. Environmental Protection Agency (EPA) has classified it as a highly toxic pesticide. In 2006, the EPA believed that the use of carbofuran posed a high ecological and occupational risk, and that the widespread use of carbofuran on potatoes, sweet corn and gourds greatly increased the dietary risk. In 2008, it conducted a risk assessment again. From December 31, 2009, the residue value of carbofuran was completely cancelled, meaning that carbofuran was a banned pesticide. my country's national standard GB 2763 "National Food Safety Standard Maximum Residue Limits of Pesticides in Food" also stipulates that the maximum residue limit of carbofuran in vegetables and fruits is 0.02 mg / kg.

[0004] At present, the detection of carbofuran mainly adopts analytical methods such as high performance liquid chromatography, liquid chromatography-mass spectrometry, and gas chromatography-mass spectrometry. These methods must be operated under laboratory conditions, and the sample pretreatment is cumbersome and time-consuming. Expensive instruments and equipment are also required. The detection cost is high, the time is long, and the operation is complicated. There are great limitations in the actual application process, and it is difficult to meet the needs of rapid detection of a large number of samples and on-site samples. Therefore, the development of a simple and fast colloidal gold test strip suitable for the detection of carbofuran in food can meet the needs of on-site screening and monitoring of a large number of samples, and can better meet the detection work of my country's food regulatory authorities. Summary of the invention

[0005] The purpose of the present invention is to provide a colloidal gold test strip capable of detecting carbofuran in plant-derived food, and to provide a detection method which is efficient, accurate, simple and suitable for on-site monitoring and large-scale sample screening.

[0006] The test strip for detecting carbofuran provided by the present invention comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate; the reaction membrane is provided with a detection line coated with a carbofuran hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse anti-antibody; the conjugate release pad is sprayed with a carbofuran monoclonal antibody-colloidal gold marker.

[0007] The carbofuran monoclonal antibody is prepared by using the carbofuran hapten-carrier protein conjugate as an immunogen.

[0008] The carbofuran hapten-carrier protein conjugate is obtained by coupling the carbofuran hapten with a carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein or human serum albumin. The carbofuran hapten is obtained by using 2-n-butylaminopropionic acid as a starting material, and then reacting with 3-hydroxycarbofuran after sulfidation and chlorination. The molecular structure is as follows:

[0009]

[0010] The sample absorption pad, the conjugate release pad, the reaction membrane and the water absorption pad are sequentially pasted on the bottom plate, and 1 / 3 to 1 / 2 of the conjugate release pad is covered under the sample absorption pad.

[0011] The bottom plate is a PVC bottom plate or other hard non-absorbent material; the sample absorption pad is made of polyester fiber or glass fiber; the conjugate release pad is a polyester cellulose membrane or a glass cellulose membrane; the water-absorbing pad is a water-absorbing filter paper; and the reaction membrane is a nitrocellulose membrane or a cellulose acetate membrane.

[0012] Another object of the present invention is to provide a method for preparing the above-mentioned test strip, which comprises the steps of:

[0013] 1) preparing a release pad sprayed with a conjugate of carbofuran monoclonal antibody-colloidal gold marker;

[0014] 2) preparing a reaction membrane having a detection line coated with a carbofuran hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;

[0015] 3) Assemble the conjugate release pad, reaction membrane, sample absorption pad, water absorption pad and bottom plate prepared in 1) and 2) into a test strip.

[0016] Specifically, the steps include:

[0017] 1) Using 2-n-butylaminopropionic acid as a starting material, after sulfidation and chlorination, it reacts with 3-hydroxycarbofuran to prepare the carbofuran hapten;

[0018] 2) coupling the carbofuran hapten with a carrier protein to prepare a carbofuran hapten-carrier protein conjugate;

[0019] 3) Immunizing mice with carbofuran hapten-carrier protein conjugate, fusing mouse spleen cells and mouse myeloma cells, and screening to obtain hybridoma cell lines that secrete carbofuran monoclonal antibodies;

[0020] 4) extracting mouse IgG and immunizing healthy goats to obtain goat anti-mouse antibodies;

[0021] 5) coating the carbofuran hapten-carrier protein conjugate and goat anti-mouse anti-antibody on the detection line (T) and quality control line (C) of the reaction membrane respectively;

[0022] 6) preparing colloidal gold by reacting trisodium citrate with chloroauric acid;

[0023] 7) adding the prepared carbofuran monoclonal antibody to the prepared colloidal gold to obtain a carbofuran monoclonal antibody-colloidal gold marker;

[0024] 8) Spray the carbofuran monoclonal antibody-colloidal gold marker onto the conjugate release pad, dry it at 37° C. for 2 h, then take it out and store it in a dry environment for later use;

[0025] 9) Soak the sample absorption pad in 0.02 mol / L phosphate buffer containing 1% bovine serum albumin and pH 7.2 for 2 h, and dry at 37°C for 2 h for later use;

[0026] 10) Paste the sample absorption pad, conjugate release pad, reaction membrane, and water absorption pad on the bottom plate in order, and cover 1 / 3 of the conjugate release pad from the starting end with the sample absorption pad. Finally, cut into 3.95mm wide strips, put them in a special plastic card shell, seal them with aluminum foil bags, and store them at 4-30℃ for 12 months.

[0027] Another object of the present invention is to provide a method for detecting carbofuran in plant-derived food using the above test strip, which comprises the steps of:

[0028] (1) Sample pretreatment;

[0029] (2) Testing with test strips;

[0030] (3) Analyze the test results.

[0031] The carbofuran rapid detection test strip of the present invention adopts highly specific antibody-antigen reaction and immunochromatographic analysis technology, fixes carbofuran monoclonal antibody-colloidal gold marker on the conjugate release pad, and the carbofuran in the sample is combined with the carbofuran monoclonal antibody-colloidal gold marker on the conjugate release pad during the flow process to form carbofuran-antibody-colloidal gold marker. The carbofuran in the sample competes with the carbofuran hapten-carrier protein conjugate on the reaction membrane detection line to bind to the carbofuran monoclonal antibody-colloidal gold marker, and whether the sample liquid to be tested contains carbofuran is determined according to the depth of the red band on the detection line.

[0032] During the test, the sample is treated and then dripped into the hole of the test strip. When the concentration of carbofuran in the sample is lower than the detection limit or zero, the monoclonal antibody-colloidal gold marker will bind to the carbofuran hapten-carrier protein conjugate fixed on the reaction membrane during the chromatography process, and a red strip will appear on the test line (T) and the quality control line (C), and the color of the T line is darker than that of the C line or consistent with the color of the C line; if the concentration of carbofuran in the sample is equal to or higher than the detection limit, the monoclonal antibody-colloidal gold marker will completely bind to the carbofuran, so that no red strip will appear at the T line due to the competitive reaction, or the color will be lighter than that of the C line. Figure 3 shown.

[0033] Negative: When the control line (C) shows a red strip, the test line (T) also shows a red strip, and the color of the (T) line is close to or darker than the (C) line, it is judged as negative.

[0034] Positive: When the control line (C) shows a red strip, and the test line (T) does not show color or the color of the (T) line is lighter than that of the (C) line, it is judged as positive.

[0035] Invalid: When the control line (C) does not show a red strip, the test strip is considered invalid regardless of whether the test line (T) shows a red strip or not.

[0036] The test strip of the present invention has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, suitable for use by various units, simple storage and long shelf life. The method of using the test strip of the present invention to detect carbofuran is simple, rapid, intuitive, accurate, widely applicable, low cost and easy to promote and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the synthesis diagram of the hapten of carbofuran.

[0038] Figure 2 This is a schematic diagram of the cross-sectional structure of a test strip, in which: 1. Sample absorption pad; 2. Conjugate release pad; 3. Reaction membrane; 4. Water absorbent pad; 5. Test line; 6. Quality control line; 7. Bottom plate.

[0039] Figure 3 This is a diagram for determining the test strip test results. DETAILED DESCRIPTION

[0040] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0041] Example 1 Preparation of a test strip for detecting carbofuran

[0042] The preparation method of the test strip mainly comprises the following steps:

[0043] 1) preparing a release pad sprayed with a conjugate of carbofuran monoclonal antibody-colloidal gold marker;

[0044] 2) preparing a reaction membrane having a detection line coated with a carbofuran hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;

[0045] 3) Assemble the conjugate release pad, reaction membrane, sample absorption pad, water absorption pad and bottom plate prepared in 1) and 2) into a test strip.

[0046] The following is a detailed step-by-step description:

[0047] 1. Synthesis of carbofuran hapten (synthesis route see attached Figure 1 )

[0048] Take 1.31g of 2-n-butylaminopropionic acid, add 100mL of 1,2-dichloroethane to dissolve, add 5mL of triethylamine, stir, cool to 5°C, add 0.87g of sulfur monochloride, react at 0-5°C for 2h, add 50mL of water, stir well, let stand, separate the water phase, add anhydrous sodium sulfate to the lower mother liquor to dry, filter out the solid sodium sulfate, and cool to 5°C; add 1.43g of sulfuryl chloride and react for 4h; add 3.6g of 3-hydroxycarbofuran, add 5mL of triethylamine, react at 0-5°C for 4h, add 100mL of 1,2-dichloroethane, add 100mL of water, stir well, let stand, separate the water phase, rotary evaporate the organic phase, evaporate to dryness to obtain a red oily substance, put it on a silica gel column, and elute and separate it with a dichloromethane-methanol mixed solution with a volume ratio of 10:1 to obtain 3.7g of carbofuran hapten with a yield of 92.5%.

[0049] 2. Preparation of immunogen

[0050] Take 15 mg of carbofuran hapten, add 1 mL of N,N-dimethylformamide (DMF) to dissolve and clarify, add 9.8 mg of N-hydroxysuccinimide (NHS) and 11 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), fully dissolve and mix, react at room temperature for 2 hours, and obtain hapten activation liquid A; take 50 mg of bovine serum albumin (BSA), add 6 mL of 0.1 mol / L pH 9.5 CB buffer to dissolve, and obtain liquid B; add liquid A drop by drop into liquid B, react at room temperature for 4 hours, stop the reaction, dialyze and purify with 0.02 mol / L PBS for 3 days, change the liquid 3 times a day, centrifuge and pack, and obtain carbofuran hapten-BSA conjugate, which is the immunogen.

[0051] 3. Preparation of coating

[0052] Take 9 mg of carbofuran hapten, add 1 mL of DMF to dissolve and clarify, add 6.1 mg of NHS and 8.1 mg of EDC, fully dissolve and mix, react at room temperature for 2 hours, and obtain hapten activation liquid A; take 50 mg of ovalbumin (OVA), add 6 mL of 0.1 mol / L pH 9.5 CB buffer to dissolve, and obtain liquid B; add liquid A drop by drop into liquid B, react at room temperature for 4 hours, stop the reaction, dialyze and purify with 0.02 mol / L PBS for 3 days, change the liquid 3 times a day, centrifuge and pack, and obtain carbofuran hapten-OVA conjugate, which is the coating source.

[0053] 4. Preparation of monoclonal antibodies against carbofuran

[0054] (1) Animal immunization

[0055] The immunogen obtained in step 2 was injected into Balb / c mice at an immunization dose of 150 μg / mouse to produce antiserum.

[0056] (2) Cell fusion and cloning

[0057] Spleen cells of immune Balb / c mice were taken and fused with SP2 / 0 myeloma cells at a ratio of 8:1 (quantity ratio), and the cell supernatant was measured by indirect competitive ELISA to screen positive wells. The positive wells were cloned by limiting dilution method until a hybridoma cell line that stably secreted monoclonal antibodies was obtained.

[0058] (3) Cell cryopreservation and recovery

[0059] Hybridoma cells were cryopreserved in 1×10 6 Cell suspensions of 100 cells / mL were stored in liquid nitrogen for a long time. When thawing, the cryopreserved tubes were taken out and immediately placed in a 37°C water bath for rapid thawing. After centrifugation to remove the cryopreservation solution, the cells were transferred to culture bottles for culture.

[0060] (4) Preparation and purification of monoclonal antibodies

[0061] Incremental culture method: hybridoma cells are placed in cell culture medium and cultured at 37°C. The obtained culture fluid is purified using the caprylic acid-saturated ammonium sulfate method to obtain monoclonal antibodies, which are then stored at -20°C.

[0062] The cell culture medium is prepared by adding calf serum and sodium bicarbonate to RPMI1640 culture medium, so that the final concentration of calf serum in the cell culture medium is 20% (mass fraction), and the final concentration of sodium bicarbonate in the cell culture medium is 0.2% (mass fraction); the pH value of the cell culture medium is 7.4.

[0063] 5. Preparation of goat anti-mouse antibodies

[0064] Sheep are used as immune animals, and mouse antibodies are used as immunogens to immunize pathogen-free sheep to obtain sheep anti-mouse antibodies.

[0065] 6. Preparation of carbofuran monoclonal antibody-colloidal gold label

[0066] (1) Preparation of colloidal gold

[0067] Dilute 1% chloroauric acid to 0.01% (mass fraction) with double distilled deionized water, take 100 mL and place it in a conical flask, heat it to boiling with a constant temperature electromagnetic stirrer, add 1.5 mL 1% trisodium citrate under continuous high temperature and continuous stirring, continue to stir and heat at a uniform speed until the solution turns translucent red, stop, cool to room temperature, restore to the original volume with deionized water, and store at 4°C. The prepared colloidal gold has a pure, translucent appearance, no precipitation and floating matter, and the color is wine red when observed under sunlight.

[0068] (2) Preparation of carbofuran monoclonal antibody-colloidal gold label

[0069] Under magnetic stirring, adjust the pH value of colloidal gold to 7.2 with 0.2 mol / L potassium carbonate solution, add the above-mentioned carbofuran monoclonal antibody to the colloidal gold solution according to the standard of adding 5-50 μg antibody per ml of colloidal gold solution, continue stirring and mixing for 30 minutes; after standing for 10 minutes, add 10% BSA to make the final concentration in the colloidal gold solution 1%, and stand for 10 minutes. Centrifuge at 12000r / min and 4℃ for 40 minutes, discard the supernatant, wash the precipitate twice with resolubilization buffer, resuspend the precipitate with resolubilization buffer with a volume of 1 / 10 of the initial colloidal gold volume, and place at 4℃ for use.

[0070] Reconstitution buffer: 0.02 mol / L phosphate buffer containing 0.1% to 0.5% BSA by mass, 2% to 4% sucrose by mass, and pH 7.2.

[0071] 7. Preparation of conjugate release pad

[0072] The conjugate release pad was soaked in 0.02 mol / L phosphate buffer containing 0.5% BSA, 5% sucrose, pH 7.4, and evenly soaked for 2 hours, and dried at 37°C for use. The prepared carbofuran monoclonal antibody-colloidal gold marker was evenly sprayed on the conjugate release pad using a Bio dot film scribing instrument, and 0.01 mL of carbofuran monoclonal antibody-colloidal gold marker was sprayed per 1 cm of the conjugate release pad, and then placed in a 37°C environment (humidity <20%) for 2 hours, taken out, and placed in a dry environment (humidity <20%) for storage.

[0073] 8. Preparation of sample absorption pad

[0074] The sample absorption pad was soaked in 0.02 mol / L phosphate buffer solution containing 1% BSA and pH 7.2 for 2 h, and dried at 37°C for 2 h for later use.

[0075] 9. Preparation of reaction membrane

[0076] The carbofuran hapten-OVA conjugate was coated on the reaction membrane to form a detection line, and the goat anti-mouse antibody was coated on the reaction membrane to form a quality control line.

[0077] Coating process: Dilute the carbofuran hapten-OVA conjugate to 1 mg / mL with 0.01 mol / L, pH 7.2 phosphate buffer, and coat it on the test line (T line) on the nitrocellulose membrane with a Bio dot film stripper, with a coating amount of 1.0 μL / cm; dilute the goat anti-mouse anti-antibody to 200 μg / mL with 0.01 mol / L, pH 7.2 phosphate buffer, and coat it on the quality control line (C line) on the nitrocellulose membrane with a Bio dot film stripper, with a coating amount of 1.0 μL / cm. Dry the coated reaction membrane at 37°C for 16 hours for use.

[0078] 10. Assembly of test strips

[0079] According to the attached Figure 2The cross-sectional structure of the test strip is shown, wherein a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3), and a water absorption pad (4) are sequentially pasted on a PVC bottom plate (7); 1 / 3 of the conjugate release pad from the starting end is covered by the sample absorption pad, the end of the conjugate release pad is connected to the starting end of the reaction membrane, the end of the reaction membrane is connected to the starting end of the water absorption pad, the starting end of the sample absorption pad is aligned with the starting end of the PVC bottom plate, and the end of the water absorption pad is aligned with the end of the PVC bottom plate; a detection line (5) and a quality control line (6) are provided on the reaction membrane, and both the detection line (T line) and the quality control line (C line) are strips perpendicular to the length of the test strip; the detection line is located on the side close to the end of the conjugate release pad; the quality control line is located on the side away from the end of the conjugate release pad; the test strip is cut into small strips of 3.95 mm width by a machine, placed in a special plastic card shell, sealed with an aluminum foil bag, and stored in an environment of 4 to 30° C., with a validity period of 12 months.

[0080] Example 2 Detection of Carbofuran in Plant-derived Foods

[0081] 1. Sample pretreatment

[0082] Take a fresh sample and wipe off the dirt, cut it into pieces less than 1 cm square; weigh (2.00±0.05) g of sample into a 15 mL polystyrene centrifuge tube, add 6 mL of 0.02 mol / L, pH 7.4 PBS buffer, cover the tube, shake it up and down manually for 1 min, let it stand for 1 min, and the upper liquid is the sample solution to be tested.

[0083] 2. Test with test strips

[0084] Use a micropipette to draw 100 μL of the sample solution to be tested and drop it vertically into the sample well; start timing when the liquid flows, react for 10 minutes, and determine the result.

[0085] 3. Analyze the test results

[0086] Negative (-): The color of T line is darker than that of C line or the same as that of C line, indicating that the concentration of carbofuran in the sample is lower than the detection limit. Figure 3 a, 3b.

[0087] Positive (+): The color of the T line is lighter than that of the C line or the T line does not show color, indicating that the concentration of carbofuran in the sample is equal to or higher than the detection limit. Figure 3 c. 3d.

[0088] Invalid: No C line appears, indicating incorrect operation process or the test strip has deteriorated and become invalid, such as Figure 3 e, 3f.

[0089] Example 3 Sample detection example

[0090] 1. Detection limit test

[0091] Take blank Brussels sprouts, cowpea, Chinese cabbage, apple, pear and orange samples, add carbofuran to the final concentrations of 0.01 mg / kg, 0.02 mg / kg and 0.04 mg / kg respectively, take test strips for testing, and repeat the measurement three times for each sample.

[0092] When the test strips were used to test Brussels sprouts, cowpeas, Chinese cabbage, apples, pears, and orange samples, when there was no chlorpyrifos and its added concentration was 0.01 mg / kg, the T line shown on the test strip was darker than or consistent with the C line, indicating a negative result; when the chlorpyrifos added concentration was 0.02 mg / kg or 0.04 mg / kg, the T line shown on the test strip was lighter than or did not show any color, indicating a positive result, indicating that the detection limit of this test strip for chlorpyrifos in plant-based foods is 0.02 mg / kg.

[0093] 2. False positive rate and false negative rate test

[0094] 20 blank Brussels sprouts, cowpea, Chinese cabbage, apple, pear, and orange samples and 20 positive Brussels sprouts, cowpea, Chinese cabbage, apple, pear, and orange samples added with carbofuran to a final concentration of 0.02 mg / kg were taken and tested using test strips produced in three batches, and their positive and negative rates were calculated.

[0095] The results show that when the test strips produced in three batches are used to detect positive samples, the results are all positive, indicating that the positive coincidence rate is 100% and the false negative rate is 0; when the negative samples are detected, the results are all negative, indicating that the negative coincidence rate is 100% and the false positive rate is 0. This indicates that the test strip for detecting carbofuran of the present invention can be used to quickly detect carbofuran in plant-derived foods.

[0096] 3. Specificity test

[0097] When this test strip is used to detect 10mg / kg of other carbamate pesticides such as aldicarb, methomyl, carbaryl, isoprocarb, fenbutyl, and mefenamic acid, the color of the T line on the test strip is darker than that of the C line or is consistent with the color of the C line, which is negative, indicating that this test strip has no cross reaction to these drugs.

[0098] 4. Comparison test with instrument method

[0099] Take 20 samples of Brussels sprouts, cowpea, Chinese cabbage, apple, pear and orange, numbered 1-20, and use this test strip to compare and test with the instrument detection method. The instrument method refers to: "NY / T 761-2008 Determination of organophosphorus, organochlorine, pyrethroid and carbamate pesticide residues in vegetables and fruits" (detection limit is 0.01mg / kg). The content of carbofuran in Brussels sprouts, cowpea, Chinese cabbage, apple, pear and orange samples of this test strip is lower than 0.02mg / kg, which is regarded as not detected and represented by "-". For samples higher than 0.02mg / kg, the instrument method uses the actual result, and the test strip detection result is represented by "+". The results are shown in the table below.

[0100] Table 1 Comparison of test results of Brussels sprouts sample test strips and instrumental methods (mg / kg)

[0101]

[0102] Table 2 Comparison of test results of cowpea sample test strips and instrumental methods (mg / kg)

[0103]

[0104] Table 3 Comparison of the test results of Chinese cabbage sample strips and instrumental methods (mg / kg)

[0105]

[0106]

[0107] Table 4 Comparison of test results of apple sample test strips and instrumental methods (mg / kg)

[0108]

[0109] Table 5 Comparison of test results of pear sample test strips and instrumental methods (mg / kg)

[0110]

[0111] Table 6 Comparison of test results of orange sample test strips and instrumental methods (mg / kg)

[0112]

[0113] It can be seen from the above table that the test strip test results are consistent with the instrument test results.

[0114] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A test strip for detecting carbofuran, comprising a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorbent pad and a bottom plate, wherein the reaction membrane has a detection line coated with a carbofuran hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse anti-antibody, and the conjugate release pad is sprayed with a carbofuran monoclonal antibody-colloidal gold marker; the carbofuran monoclonal antibody is prepared using the carbofuran hapten-carrier protein conjugate as an immunogen; the carbofuran hapten-carrier protein conjugate is obtained by coupling the carbofuran hapten with a carrier protein, and the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein or human serum albumin, characterized in that The synthesis method of the carbofuran hapten is: Take 1.31g of 2-n-butylaminopropionic acid, add 100mL of 1,2-dichloroethane to dissolve, add 5mL of triethylamine, stir, cool to 5℃, add 0.87g of sulfur monochloride, react at 0~5℃ for 2h, add 50mL of water, stir well, let stand, separate the water phase, add anhydrous sodium sulfate to the lower mother liquor to dry, filter to remove solid sodium sulfate, cool to 5℃; add 1.43g of sulfuryl chloride, react for 4h; add 3.6g of 3-hydroxycarbofuran, add 5mL of triethylamine, react at 0~5℃ for 4h, add 100mL of 1,2-dichloroethane, add 100mL of water, stir well, let stand, separate the water phase, rotary evaporate the organic phase, evaporate to dryness, obtain a red oily substance, put it on a silica gel column, and elute and separate it with a dichloromethane-methanol mixed solution with a volume ratio of 10:1 to obtain the carbofuran hapten, whose molecular structure is:

2. The test strip as claimed in claim 1, characterized in that The sample absorption pad, the conjugate release pad, the reaction membrane and the water absorption pad are sequentially pasted on the bottom plate.

3. A test strip as described in any one of claims 1-2, characterized in that 1 / 3 to 1 / 2 of the conjugate release pad is covered under the sample absorption pad.

4. A method for preparing the test strip according to any one of claims 1 to 3, comprising the steps of: 1) preparing a release pad sprayed with a conjugate of carbofuran monoclonal antibody-colloidal gold marker; 2) preparing a reaction membrane having a detection line coated with a carbofuran hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody; 3) Assemble the conjugate release pad, reaction membrane, sample absorption pad, water absorption pad and bottom plate prepared in 1) and 2) into a test strip.

5. A method for detecting carbofuran in plant-derived food, comprising the steps of: 1) Sample pretreatment; 2) Testing with the test strip according to any one of claims 1 to 3; 3) Analyze the test results.

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