A multi-purpose pesticide residue quantitative detection test paper and detection method

Abstract

The present application belongs to the technical field of pesticide detection, and particularly relates to a multi-in-one pesticide residue quantitative detection test paper and a detection method. The test paper is provided with gold nanoparticle labeled antibodies of at least two kinds of pesticides on a colloidal gold combination pad; a chromatography membrane is provided with a quality control line and at least two detection lines, each detection line is coated with an antigen of one kind of pesticide; the pesticides are selected from chlorpyrifos, phorate, dimethoate, acetamiprid, carbendazim, procymidone, fenpropathrin, carbofuran, isoprocarb, chlorothalonil, chlorfenapyr, parathion, tetramethylenedisulfide, triadimefon, isocarbophos or parathion. Through optimization of the pesticide types and preparation methods of the antigens and antibodies, the present application overcomes the problem of mutual interference of detection targets when multiple pesticides are simultaneously detected, has good specificity, high sensitivity, can accurately quantify, can simultaneously detect multiple components, and has simple pretreatment, and thus has good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pesticide detection, and particularly relates to a multi-in-one pesticide residue quantitative detection test paper and a detection method. BACKGROUND

[0002] Pesticide residues refer to micro-pesticide original bodies, toxic metabolites, degradation products and impurities that are not decomposed and remain in organisms, harvests, soil, water bodies and atmosphere for a period after the use of pesticides.

[0003] Pesticides applied to crops, some of which are attached to crops, some of which are scattered in the environment such as soil, atmosphere and water, and some of which are absorbed by plants. Residual pesticides directly reach humans and animals through plant fruits or water, atmosphere, or are ultimately transmitted to humans and animals through the environment and food chain. The pesticide stripper can degrade the pesticide residues on the surface of fruits and vegetables.

[0004] It is urgent to develop high-throughput, rapid, accurate and sensitive pesticide multi-residue analysis technology to analyze various types of different polar residual substances, supervise and control food pollution and ensure food quality.

[0005] The colloidal gold immunochromatography method is a commonly used detection method, and its principle is based on the specific recognition and combination reaction of antigen and antibody. The specific antigen or antibody is fixed on the film in a strip shape, and the colloidal gold labeled reagent (antibody or monoclonal antibody) is adsorbed on the binding pad. When the sample to be detected is added to the sample pad at one end of the test strip, it moves forward through capillary action, dissolves the colloidal gold labeled reagent on the binding pad and reacts with each other. When it moves to the area of the fixed antigen or antibody, the combination of the sample to be detected and the gold labeled reagent is specifically combined with it and is intercepted, and is gathered on the detection belt, and the color development result can be observed by the naked eye. The method has now developed into a diagnostic test strip, which is very convenient to use.

[0006] For the detection of pesticide residues, colloidal gold immunochromatography method can also be used. For example, the Chinese invention patent "CN1203317C Nanometer colloidal gold labeled immunoassay method for carbofuran pesticide" provides a method for detecting carbofuran pesticide by using colloidal gold immunochromatography method. In the method, carbofuran immunogen BFNH is coupled with BSA by using modified active ester method to synthesize artificial immunogen complex, animals are immunized to prepare specific high-titer anti-BFNH antibody, and the nanometer colloidal gold is labeled on the specific anti-carbofuran antibody after the antibody is separated and purified. Then, the labeled combination, carbofuran coupled with ovalbumin and goat anti-mouse IgG are solidified on a carrier respectively. According to the principle of competitive inhibition immunochromatography, semi-quantitative analysis and determination of carbofuran pesticide are carried out. The minimum detection amount of the established detection system for carbofuran pesticide is 0.25 mg / kg, and the rapid detection is completed within 10 minutes.

[0007] However, the existing colloidal gold immunochromatography method is only used for the detection of a single pesticide. If simultaneous detection of multiple pesticides is desired on the same test paper, there is a very high requirement for specificity of the detection, otherwise, mutual interference between pesticide molecules may occur, which leads to great difficulty in the development of multi-in-one pesticide detection test paper and detection method based on colloidal gold immunochromatography method. Therefore, the multi-in-one pesticide detection based on colloidal gold immunochromatography method has not been realized at present. SUMMARY

[0008] In view of the defects of the prior art, the present application provides a multi-in-one pesticide residue quantitative detection test paper and detection method, which aims to overcome the high requirement for specificity in multi-in-one pesticide detection and realize simultaneous detection of multiple pesticides.

[0009] A multi-in-one pesticide residue quantitative detection test paper comprises a sample pad, a colloidal gold combination pad and a chromatography membrane connected in sequence.

[0010] The colloidal gold combination pad is attached with gold nanoparticle labeled antibodies of at least two pesticides.

[0011] The chromatography membrane is provided with a quality control line and at least two detection lines, and each detection line is coated with an antigen of a pesticide.

[0012] The pesticide is selected from chlorpyrifos, phorate, dimethoate, acetamiprid, carbendazim, procymidone, fenpropathrin, carbofuran, isoprocarb, chlorothalonil, cyanofanphos, parathion, piperonyl butoxide, triadimefon, isocarbophos or parathion.

[0013] The antigen of the pesticide is formed by coupling the pesticide molecule with a macromolecular protein, and is synthesized by using one of the following methods:

[0014] Method 1, for pesticides containing carboxyl, using carbodiimide method, activated ester method or mixed anhydride method,

[0015] Method 2, for pesticides containing amino, using diazotization method, carbodiimide method or glutaraldehyde method,

[0016] Method 3, for pesticides containing hydroxyl, using succinic anhydride method, periodic acid oxidation method or sodium chloroacetate method.

[0017] Preferably, the gold nanoparticle-labeled antibody of the pesticide is obtained by combining the antibody of the pesticide with gold nanoparticles, and the antibody of the pesticide is obtained by immunizing animals with the antigen of the pesticide.

[0018] Preferably, the gold nanoparticles are silver-coated gold nanoparticles, and / or the size of the gold nanoparticles is 10-50 nm.

[0019] Preferably, the macromolecular protein is bovine serum albumin or ovalbumin.

[0020] Preferably, the types of pesticides are chlorpyrifos, phorate and dimethoate, the antigen of chlorpyrifos is prepared by carbodiimide method, the antigen of phorate is prepared by carbodiimide method, and the antigen of dimethoate is prepared by carbodiimide method.

[0021] Preferably, the types of pesticides are procymidone, carbofuran, carbendazim and chlorpyrifos, the antigen of procymidone is prepared by succinic anhydride method, the antigen of carbofuran is prepared by diazotization method, the antigen of carbendazim is prepared by activated ester method or mixed anhydride method, and the antigen of chlorpyrifos is prepared by carbodiimide method.

[0022] The application also provides a preparation method of the above-mentioned test paper, comprising the following steps:

[0023] Step 1, preparing the antigen of the pesticide and gold nanoparticles;

[0024] Step 2, performing line coating on the chromatography membrane, each type of pesticide antigen is coated with one detection line, and one quality control line is coated with anti-mouse IgG;

[0025] Step 3, preparing the antibody of the pesticide using the antigen of the pesticide;

[0026] Step 4, combining the antibody of the pesticide with gold nanoparticles to prepare gold nanoparticle-labeled antibody;

[0027] Step 5, spraying the gold nanoparticle-labeled antibody on the colloidal gold combination pad;

[0028] Step 6, the colloidal gold binding pad and the chromatography membrane are taken as components, and other necessary components of the colloidal gold immunochromatography test paper are added to form the multi-in-one pesticide residue quantitative detection test paper.

[0029] Preferably, in step 2, the line coating is performed by a line marker at a speed of 0.5-2 μL / cm, and the concentration of the pesticide antigen in the solution used for coating is 0.1-3 mg / mL.

[0030] Preferably, in step 5, the gold nanoparticle-labeled antibody solution containing the pesticide antibody is sprayed on the colloidal gold binding pad at a dosage of 2-10 μL / cm, and the gold nanoparticle-labeled antibody solution containing the pesticide antibody contains the following components: PBS 2 mM, BSA 0.1-5 wt.%, sucrose 1-20 wt.%, tween-20 0.01-1 wt.%, and the pesticide antibody 0.1-2 mg / mL.

[0031] The present application also provides a method for detecting pesticide residues, comprising the following steps:

[0032] Step 1, a sample is extracted by using an extraction agent, and supernatant is obtained after separation;

[0033] Step 2, the supernatant is diluted by using a diluent to obtain a test sample solution;

[0034] Step 3, the test sample solution is dropped to the sample pad of the above-mentioned test paper for detection.

[0035] Preferably, the extraction agent is a methanol aqueous solution, and / or the diluent contains the following components: PBS 0.2 M, tween-20 0.1-10 wt.%.

[0036] In the present application, the "gold nanoparticle" can be a single-component nanoparticle composed of elemental gold, or a nanoparticle formed by modification or coating on the surface of the elemental gold nanoparticle, such as a silver-coated gold nanoparticle. In the present application, the carbodiimide method, activated ester method, mixed anhydride method, diazonium method, glutaraldehyde method, succinic anhydride method, periodate oxidation method and sodium chloroacetate method all belong to the existing methods for preparing antigens. For various methods, those skilled in the art can refer to the following references for implementation:

[0037] Carbodiimide method: synthesis of phorate artificial antigen and preparation of polyclonal antibody, Wang Chong, Wei Songhong, Li Xinghai, Gao Wei, Hu Sha, Henan Agricultural Science, 2009, No. 3;

[0038] Activated ester method: synthesis of carbaryl artificial antigen and determination of coupling ratio, Qu Qiaoyu, Chen Shanshan, Liu Xiaowei, Li Peiwu, Zhang Qi, China Oil Crop Sciences, October 2013;

[0039] Mixed anhydride method: discussion on influencing factors of conjugation process of estradiol enzyme antigen synthesized by mixed anhydride method, Li Guilin, Sun Guolong, Zhang Chunge, Zhao Qiaohui, Fu Guangyu, Wu Xuewei, Medical Forum Journal, September 2017, Vol. 38, No. 9;

[0040] Diazotization method: research on synthesis of sulfonamide metronidazole immune antigen by diazotization method, Han Zhanjiang, Wang Weihua, Nan Haijuan, Chen Yang, Wu Zheng, Zhang Yanjie, Sun Lei, Zhang Xixiao, Henan University of Science and Technology, Guangdong Trace Element Science, Vol. 15, No. 3, 2008;

[0041] Glutaraldehyde method: synthesis of methylphenylamine and protein conjugate, Chen Yanzhong, Liu Anhua, Liu Xiaoyun, Wang Jihua, Modern Food Science and Technology, 2008, Vol. 24, No. 5;

[0042] succinic anhydride method: preparation of diquat artificial antigen, Hu Xiaofei, Xing Yunrui, Sun Yanning, China Agricultural Bulletin, 2021;

[0043] periodic acid oxidation method: synthesis of astragaloside artificial antigen and preparation of monoclonal antibody, Ren Yajun, Beijing University of Chinese Medicine, 2015;

[0044] sodium chloroacetate method: synthesis and identification of breviscapine artificial antigen, Wan Feng, Kong Hui, Qu Huishu, Zhang Yue, Feng Huibin, Zhao Yan, Wang Qingguo, Chinese Herbal Drugs, Vol. 45, No. 3, February 2014;

[0045] The present application provides a multi-in-one single card pesticide residue quantitative detection pesticide residue test paper and method, through the preparation method of the preferred pesticide type and its antigen and antibody, the problem of mutual interference of detection target when multiple pesticides are detected at the same time is overcome. The present application has the advantages of good specificity, high sensitivity, accurate quantification, simultaneous detection of multiple components, simple pretreatment and good application prospect.

[0046] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and conventional means in the art, other various forms of modification, replacement or change can be made without departing from the above basic technical idea of the present application.

[0047] The above content of the present application is further described in detail through the specific embodiments below. However, this should not be understood as limiting the scope of the above subject matter of the present application to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 The structure and detection method of the detection test paper provided for Example 1 and Example 2 are shown in the schematic diagram.

[0049] Figure 2 Detection results of the test paper provided for Example 1 and Example 2 are shown in the following table. DETAILED DESCRIPTION

[0050] The reagents and materials used in the following examples and experimental examples are commercially available.

[0051] Example 1 Chlorpyrifos, phorate and dimethoate test paper

[0052] The structure and detection method of the test paper of the present example are shown in Figure 1 , and the detection results are shown in Figure 2 , and the preparation method of the test paper is as follows:

[0053] 1. Synthesis of silver-coated gold nanoparticles

[0054] 1.1 Synthesis of gold nanoparticles

[0055] Take 100 mL of 0.01% chloroauric acid aqueous solution and heat to boiling, accurately add 1.8 mL of 1% trisodium citrate aqueous solution under stirring, and the gold yellow chloroauric acid aqueous solution turns red within 2 minutes, and continue to boil for 15 minutes.

[0056] 1.2 Synthesis of silver-coated gold nanoparticles

[0057] Adjust the temperature of the above solution to 40-80°C, add silver nitrate solution to make its content 0.001-0.1%, and add 1.0 mL of 1% trisodium citrate aqueous solution as a reducing agent under stirring, continue to stir for 10 min, and the obtained liquid is the silver-coated gold nanoparticle solution.

[0058] 2. Preparation of antigen

[0059] Pesticide has small molecular weight and no immunogenicity, and must be coupled with a macromolecular substance to form an artificial synthetic antigen (complete antigen) to obtain immunogenicity, so a corresponding protein carrier is needed to be coupled to form a whole antigen with immunogenicity. According to the different active groups (carboxyl, amino, hydroxyl) contained in the pesticide molecule, the corresponding synthesis method is adopted to prepare the required hapten.

[0060] The preparation process of chlorpyrifos artificial antigen is described as follows:

[0061] The carbonic acid diimide method is to first react chlorpyrifos small molecules with carbonic acid diimide to dehydrate the carboxyl group, generate an addition intermediate product, and then react with the amino group on the carrier protein to form an amide bond.

[0062] Operation steps for preparing chlorpyrifos hapten by carbonic acid diimide method:

[0063] 1) Take EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) 100 mg, dissolve it in 2.5 mL of 10 mmol / L PBS solution with pH 8.0 (I solution);

[0064] 2) Take chlorpyrifos 25 mg, dissolve it in 2 mL of 0.2 mol / L NaOH solution (II solution);

[0065] 3) Take bovine serum albumin (BSA) 25 mg, dissolve it in 10 mmol / L PBS (pH 8.0) solution (III solution);

[0066] 4) Mix II solution and III solution, and add I solution (remaining 0.5 mL) drop by drop under magnetic stirring;

[0067] 5) Stir at room temperature for 1 hour in the dark, and add the remaining I solution drop by drop;

[0068] 6) Stir at 4 degrees for 12 hours;

[0069] 7) Stand for 10 hours (at 4 degrees);

[0070] 8) Fully dialyze with distilled water (about 48 hours) to obtain the immunogen.

[0071] Preparation process of phorate artificial antigen:

[0072] 1) Put 11.1 g of phosphorus pentasulfide in a 50 mL reaction bottle, then add 9.66 g of anhydrous ethanol drop by drop, and gradually increase the temperature. When the temperature rises to about 80°C, control the dropping speed, keep the water bath at constant temperature, and stir for 1 h. After the powder is completely dissolved and the solution turns yellow, stop the reaction.

[0073] 2) Extract the reaction mixture with distilled water, and the sulfide exists in the water phase as an impurity to obtain the sulfide.

[0074] 3) Put the sulfide in the reaction bottle, add 10 g of 30% formaldehyde aqueous solution at room temperature, stir for 20 min with a magnetic stirrer, then add 7.9 g of 2-mercaptoethanol, and react at 60°C for 5 h. Concentrate the initially synthesized phorate hapten, pass it through an 83 μm silica gel chromatography column, collect the sample, and obtain the phorate hapten.

[0075] 4) Put the purified phorate hapten and succinic anhydride in the reaction bottle, take DMAP as the catalyst, and react at 70°C for about 6 h to obtain the derivative of the phorate hapten.

[0076] 5) Take 40 mg of the phorate hapten derivative, dissolve it in 1 mL of N,N-dimethylformamide, then add 20 mg of dicyclohexyl carbodiimide, stir at room temperature with a magnetic stirrer, and react overnight.

[0077] 6) The reaction solution is added to a borate buffer solution dissolving 20 mg of BSA, and the reaction is stirred magnetically for 5 h.

[0078] 7) After the reaction is completed, the dialysis bag is filled. The dialysis is performed with a phosphate buffer solution with pH = 7.4 for 3 d, and the dialysis solution is changed 4 times a day. After freeze-drying, the immunogen is obtained, and is stored at 4°C.

[0079] Preparation process of artificial antigen of dimethoate:

[0080] 1) 10 g of dimethoate intermediate O, O-dimethyl-S- (methoxycarbonylmethyl) -dithiophosphonate is added to 50 mL of chloroform.

[0081] 2) 1 g of 1, 4-diaminobutane and 1 g of cyclodextrin are added to 10 mL of water, and the reaction is performed at 40°C for 5 h to produce a dimethoate hapten.

[0082] 3) 40 mg of the dimethoate hapten derivative is dissolved in 1 mL of N, N-dimethylformamide, and then 20 mg of dicyclohexyl carbodiimide is added. The reaction is performed at room temperature with magnetic stirring overnight.

[0083] 4) The reaction solution is added to a borate buffer solution dissolving 20 mg of BSA, and the reaction is stirred magnetically for 5 h.

[0084] 5) After the reaction is completed, the dialysis bag is filled. The dialysis is performed with a phosphate buffer solution with pH = 7.4 for 3 d, and the dialysis solution is changed 4 times a day. After freeze-drying, the immunogen is obtained, and is stored at 4°C.

[0085] 3. Preparation of antibodies

[0086] 3.1 Immunization of mice

[0087] The immunogen is prepared into a solution of 1 μg / μL with PBS buffer solution with pH 8.0, and 6 healthy 7-week-old female mice are numbered and immunized in batches according to the conventional immunization scheme. For the first immunization, the PBS solution of the immunogen is mixed with an equal volume of Freund's complete adjuvant, and the mixed emulsion is extracted for intraperitoneal injection in multiple points, and the injection amount of each mouse is 200 μL. Two weeks later, the second immunization is performed, and the injection method and injection volume are unchanged, but Freund's incomplete adjuvant is used. Thereafter, the immunization is performed according to the second immunization scheme with one week as an interval. One week after the third immunization, a small amount of blood is taken from the tail of the mouse, and the antibody titer of the mouse serum is detected by indirect ELISA. When the titer is at least 1:1000, the same amount of immunogen without adjuvant is injected into the tail vein of the mouse three days before fusion to strengthen the immunization.

[0088] 3.2 Cell fusion

[0089] The day of cell fusion, the titer of the antisera was determined by taking blood from the tail vein of the mice. The serum with high titer and positive antibody was selected for cell fusion. The spleen cells of the immunized mice were fused with mouse myeloma cells under the action of 50% polyethylene glycol (PEG). The fusion scheme was as follows: 50% PEG was added for about 1 min, the first 30 seconds were added drop by drop, the last 30 seconds were added faster, and then the cells were blown for 1 min, and then the cells were left for 1.5 min. Then RPMI-1640 medium was added drop by drop, and the process was completed in about 6 min. Then 10 mL of RPMI-1640 medium was slowly added to terminate the action of PEG, and then the cells were centrifuged for 5 min. The supernatant was discarded, and the cells were suspended in 20 mL of RPMI-1640 medium. The cells were placed in a CO2 incubator for use. The feeder cells were prepared into a suspension of 2×105 cells / mL with RPMI-1640 medium. The feeder cells were mixed with the fused cells at a ratio of 1:1, and then transferred into a culture bottle for culture for 12-24 h. Finally, the cells were collected by centrifugation at 750 g for 5 min, and then suspended in 50 mL of HAT medium. The cells were dispensed into 96-well cell culture plates, 0.2 mL was added to each well, and then the plates were placed in a 37°C, 5% CO2 saturated humidity incubator for culture.

[0090] 3.3 Screening and cloning of positive hybridoma cells

[0091] The medium was changed half the amount every 3-4 days after fusion. After 2-3 days, the myeloma cells were obviously degraded, and after 5-7 days, the myeloma cells were completely dead. The HAT medium was replaced with HT medium, and the medium was changed half the amount every 3-4 days. Hybridoma cell clones appeared about 6-7 days after fusion. The cells were large, round and transparent. The growth of the clones was marked on the cover plate of the culture plate, and the number of wells with hybridoma cell growth was recorded. The hybridomas were screened by indirect ELISA combined with competitive ELISA. First, the antibodies in all the wells with hybridoma growth were screened by indirect ELISA, and then the positive wells were subjected to inhibition experiments by competitive ELISA. The hybridoma cells in the wells with specific antibody positive were cloned and subcloned by limiting dilution method until the positive rate of the supernatant of the monoclonal hybridoma was 100% after cloning and subcloning. The stable monoclonal hybridoma cell line was obtained. The monoclonal hybridoma cell line selected by expansion culture was used to induce ascites in BALB / C mice to prepare a large amount of monoclonal antibody. The antibody was purified by n-octanoic acid-ammonium sulfate precipitation method (CAASP). 80 μL of the monoclonal antibody solution obtained after purification was diluted to 2 mL with PBS buffer. Then the absorbance of the diluted solution at 260 nm and 280 nm was determined by ultraviolet spectrophotometer, and the content of the antibody was calculated.

[0092] 4. Chlorpyrifos, phorate, and dimethoate antibody nanoparticle labeling

[0093] 1) Optimum pH value test

[0094] Take several 1.5 mL EP tubes, respectively, add 1 mL of silver-coated gold nanoparticles solution, adjust the pH to 3, 4, 5, 6, 7, 8, 9, 10 with 25 mM K2CO3; add 0.01 mg of chlorpyrifos, phorate, dimethoate antibody to each tube, mix well, and react at room temperature for 20-40 min; add 100 μL of 10% NaCl solution to each tube, mix well, and react at room temperature for 20-40 min; observe the color change of the solution, and the pH at which the lowest deep red color is maintained is the optimum pH value.

[0095] 2) Optimum labeling amount test

[0096] Take several 1.5 mL EP tubes, respectively, add 1 mL of silver-coated gold nanoparticles solution, adjust the pH to the optimum pH value with 25 mM K2CO3; add 0.002, 0.004, 0.006, 0.008, 0.010 mg of chlorpyrifos, phorate, dimethoate antibody to each tube, mix well, and react at room temperature for 20-40 min; add 100 μL of 10% NaCl solution to each tube, mix well, and react at room temperature for 20-40 min; observe the color change of the solution, and the antibody amount at which the lowest deep red color is maintained is the optimum labeling amount.

[0097] 3) Preparation of immunolabeled nanoparticles

[0098] Take the above silver-coated gold nanoparticle solution, adjust the optimum pH value by adding 25 mM K2CO3 per 100 mL, and add the optimum labeling amount of chlorpyrifos, phorate, dimethoate antibody, respectively, and react for 20-40 min. Add 10 mL of 10% BSA solution and react for 20-40 min. Centrifuge at 12000 r / min for 15 min, remove the supernatant, and dissolve to 1 / 20 of the original solution volume with a reconstitution solution containing 1% BSA and 5% sucrose in 20 mM PBS. Mix the three kinds of nanoparticle labeling reconstitution together, spray on a 2 cm wide glass cellulose membrane at 2-10 μL / cm, and dry at 37°C for 18-24 hours to obtain the silver-coated gold nanoparticle labeled antibody binding pad.

[0099] 5, Chromatography membrane coating process

[0100] Prepare chlorpyrifos, phorate, dimethoate antigens into 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg / mL solutions with 0.01 M pH 7.4 phosphate buffer, and prepare anti-mouse IgG into 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg / mL solutions, and use a membrane marker to perform C, T1, T2, T3 line coating at a speed of 1 μL / cm, and place in a 37°C oven to dry for 12 hours.

[0101] 6. Assembly of the test card

[0102] The assembly of the test card is performed in a room with a temperature of 20-30°C and a humidity of <40%, a plastic base plate is taken, the coated NC membrane is placed in the middle of the plastic base plate and pasted, the silver-coated gold nanoparticle-labeled antibody conjugated pad is placed on one side of the T line of the NC membrane, the silver-coated gold nanoparticle-labeled antibody conjugated pad is pasted on the NC membrane with a thickness of 1 mm, the other side of the silver-coated gold nanoparticle-labeled antibody conjugated pad is pasted on the sample pad, and the sample pad is aligned with the lower edge of the test paper. The sample pad is pasted on the NC membrane with a thickness of 1 mm. Finally, the plastic plate is cut into 5 mm wide test paper strips using a cutting machine, and then the test paper strips are loaded into the plastic card to form the test card.

[0103] 7. Process parameter adjustment of the test paper strip

[0104] The silver-coated gold nanoparticle-labeled antibody conjugated pad with different amounts of gold spraying and the coated reagent are combined and paired to prepare a small sample, and the reagent is tested using a standard sample. According to the T value, C value, T / C value size and test gradient result, the best combination is found.

[0105] 8. Performance test of the test paper strip

[0106] The test paper prepared by the above steps is used for sample detection, and the specific method is as follows:

[0107] 1) Extraction agent: 70% methanol aqueous solution.

[0108] 2) Diluent: 0.02M PBS solution is used to prepare a diluent by adding tween-20 with a final concentration of 1%.

[0109] 3) Preparation, conversion and input of standard curve

[0110] After the process parameters of the test card are determined, mixed standard samples with concentrations of chlorpyrifos, phorate of 0, 20, 50, 100, 200, 500, 800, 1000, 1500 and 2500 μg / kg and with a concentration of dimethoate of 0, 5, 10, 20, 50, 100, 200, 500 and 1000 μg / kg are used to determine the test card. Different concentrations of standard samples show different intensities of T line, and the intensity is determined using a colloidal gold detector. The concentration of chlorpyrifos and carbofuran in the determination sample is obtained by dividing the standard added sample concentration by the recovery rate of 90%; a standard curve is prepared, and the corresponding parameters are input into the detector to complete the instrument curve parameter setting.

[0111] 4) Detection method

[0112] a. The required detection sample is crushed using a crusher and passed through a 20 mesh sieve, or is crushed into a slurry;

[0113] b. Accurately weigh 2 g of the sample to be tested into the extraction bottle using a small balance, add 8 mL of the extraction agent, and react at room temperature for 5 min at a shaking frequency of 2-60 cpm;

[0114] c. Filter using a centrifuge or a filtering device, take the supernatant, and accurately pipette 40 μL of the supernatant into 160 μL of diluent and mix well;

[0115] d. Take out the test card and the above mixture and place them into a dry thermostat for 5 min, with the temperature set at 37°C;

[0116] e. Pipette 120 μL of the above mixture into the sample well of the test card, and continue to react at 37°C for 8 min;

[0117] f. Place the test card after the reaction into the detector, and the concentration of chlorpyrifos, phorate, and dimethoate in the sample can be displayed at one time.

[0118] 5) Sample testing

[0119] Seven samples obtained from the market or testing agencies were tested using the chlorpyrifos, phorate, and dimethoate three-in-one quantitative test card and compared with the gas chromatography method for accuracy analysis. Chlorpyrifos standard sample solutions with concentrations of 50 ppb, 100 ppb, and 200 ppb, phorate standard sample solutions with concentrations of 10 ppb, 20 ppb, and 50 ppb, and dimethoate standard sample solutions with concentrations of 50 ppb, 200 ppb, and 500 ppb were prepared, and then the chlorpyrifos, phorate, and dimethoate three-in-one quantitative test card was used for analysis and detection. Each concentration was detected 6 times to analyze the precision.

[0120] 6) Results

[0121] a. Accuracy test results

[0122]

[0123] The paired sample T test was performed on the data in this group, and the t values of chlorpyrifos, phorate, and dimethoate were 0.056, 0.013, and 0.019, respectively. The two-tailed critical value (0.05, 6) was 2.447, and t < two-tailed critical value (0.05, 6), indicating that there was no significant difference between the test results of the chlorpyrifos, phorate, and dimethoate three-in-one quantitative test card and the test results of the gas chromatography method.

[0124] b. Precision test results

[0125] Chlorpyrifos test results:

[0126]

[0127] The detection results of phorate are as follows:

[0128]

[0129] The detection results of dimethoate are as follows:

[0130]

[0131] From the results and analysis, it can be seen that the variation coefficient of the repeated detection results of chlorpyrifos, phorate and dimethoate three-in-one certain amount detection card is between 10-12%, and the average variation coefficient is less than 15%.

[0132] The detection results show that the chlorpyrifos, phorate and dimethoate three-in-one certain amount detection card has good performance, can quickly and accurately detect the content of chlorpyrifos, phorate and dimethoate in the sample to be detected, is suitable for rapid quantitative detection in laboratory and sampling site, and meets the requirements of customers for rapid quantitative detection of chlorpyrifos, phorate and dimethoate in food.

[0133] Example 2 carbendazim, carbofuran, carbendazim, chlorpyrifos four-in-one certain amount detection test paper

[0134] The preparation method of the detection test paper of the embodiment is as follows:

[0135] 1, silver-coated gold nanoparticles synthesis

[0136] The same as example 1.

[0137] 2, preparation of antigen

[0138] The preparation process of the artificial antigen of carbendazim is described as follows:

[0139] 1) 5 mg of carbendazim and potassium fluoride were dissolved in 200 μL of ethylene glycol, heated in a 90℃ water bath and magnetically stirred for 24 h. A small amount of distilled water was added to the obtained reaction solution, and then the reaction solution was placed in-20℃ for 30 min, and then centrifuged at 10000 r / min for 10 min. The obtained precipitate was dissolved with 200 μL of dimethylformamide, and 2 mg of carbonylimidazole was added and magnetically stirred for 3 h.

[0140] 2) 15 mg of BSA (10 mg of OVA) was dissolved in 400 μL of 0.05 mol / L phosphate buffer solution to prepare a protein solution.

[0141] 3) The solution obtained in 1) was slowly added to the solution in 2), and magnetically stirred at room temperature overnight. Distilled water was used for dialysis for 72 h. The collected conjugate was freeze-dried to obtain the complete antigen. Store at-20℃.

[0142] The preparation process of the artificial antigen of carbofuran is as follows:

[0143] Carbofuran is diazotized by nitrous acid to form aryl diazonium salt. Then the aryl diazonium salt is coupled with ovalbumin (OVA) carrier protein.

[0144] The operation steps of coupling carbofuran with protein by diazotization:

[0145] 1) Prepare a 4 mmol / L carbofuran solution with 0.1 mol / L HCl solution;

[0146] 2) Add 1% NaNO2 (excess) dropwise and continue stirring at 4 degrees;

[0147] Note: The amount of NaNO2 added can be monitored by starch-iodide test paper or by adding 1% starch and 50 mmol / L KI on a white ceramic tile. Free HNO2 can oxidize the oxide to iodine, and iodine can react with starch to become blue-black.

[0148] 3) After the solution turns blue-black, continue the reaction for 15 minutes;

[0149] 4) Dissolve ovalbumin (OVA) with pH 9.0, 200 mmol / L borate or carbonate buffer solution;

[0150] 5) Stir while adding the diazotized hapten (to prevent local acid excess), adjust the pH to 9.5;

[0151] 6) Stir in the refrigerator for 2 hours, constantly adjusting the pH to 9.0;

[0152] 7) Dialyze with PBS for 2 days;

[0153] 8) Store at -20 degrees (concentration of 20 mg / mL).

[0154] Preparation process of artificial antigen of carbendazim:

[0155] 1) Weigh 2g carbendazim into a three-necked flask containing 80mL anhydrous ethanol with a small amount of sodium ethoxide, and add 1.2mL chloroethanol dropwise while stirring, then heat and reflux for 1h.

[0156] 2) When the temperature drops to room temperature, filter, rinse the filter cake with 100mL 50℃ distilled water, and then dry to obtain a light yellow solid, 1-hydroxyethyl-2-benzimidazole methyl carbamate.

[0157] 3) Weigh 0.5894g of 1-hydroxyethyl-2-benzimidazole methyl carbamate and 2.0g succinic anhydride into 5mL anhydrous pyridine. Heat and stir at 60℃ for 10h.

[0158] 4) To the pyridine was added 10 mL of dichloromethane, then the mixture was washed with 10 mL of 5% hydrochloric acid for 3 times, the dichloromethane layer was separated, the residue was dissolved with 10 mL of ether, then washed with 10 mL of water for 3 times, the ether layer was dried with anhydrous magnesium sulfate to obtain the semisuccinic anhydride, i.e. 1-oxycarbonylethyl propionic acid-2-benzimidazole carbamic acid methyl ester solid.

[0159] 5) 1-oxycarbonylethyl propionic acid-2-benzimidazole carbamic acid methyl ester 0.2 mmol was weighed and dissolved in 3 mL of anhydrous DMF (dried overnight with anhydrous MgSO4), and stirring was started.

[0160] 6) 40 μL of tri-n-butylamine and 40 μL (0.24 mmol) of isobutyl chloroformate dissolved in 1.5 mL of anhydrous DMF were added under water bath. The reaction was stirred at 4°C for 1 h, and then was raised to room temperature for 1 h (reaction solution I).

[0161] 7) 120 mg of BSA was dissolved in 12 mL of 0.2 mol / L borate buffer solution with pH 8.7, and the reaction solution I was added dropwise into the protein solution under stirring at room temperature, and was stirred at 15°C overnight.

[0162] 8) The conjugate solution was placed in a dialysis bag, and was dialyzed under stirring at 4°C. The water was changed once every 4 h, and the dialysis was performed for 6 times until the antigenic substance was not detected in the dialysis solution. The product was stored at -20°C.

[0163] The artificial antigen of chlorpyrifos was prepared in the same manner as in Example 1.

[0164] 3, Preparation of antibodies

[0165] The same as in Example 1.

[0166] 4, Labeling of procymidone, carbofuran, carbendazim and chlorpyrifos antibodies with nanoparticles

[0167] 1) Test of optimal pH value

[0168] A plurality of 1.5 mL EP tubes were taken, 1 mL of silver-coated gold nanoparticle solution was added into each tube, and the pH was adjusted to 3, 4, 5, 6, 7, 8, 9 and 10 with 25 mM K2CO3, respectively. 0.01 mg of procymidone, carbofuran, carbendazim and chlorpyrifos antibodies were added into each tube, respectively, and were mixed uniformly, and were placed at room temperature for 20-40 min. 100 μL of 10% NaCl solution was added into each tube, respectively, and was mixed uniformly, and was reacted at room temperature for 20-40 min. The color change of the solution was observed, and the pH value at which the lowest red color was maintained was the optimal pH value.

[0169] 3) Test of optimal labeling amount

[0170] Take several 1.5ml EP tubes, respectively add 1mL silver-coated gold nanoparticles solution, adjust the pH to the optimum pH value with 25mM K2CO3; add 0.002, 0.004, 0.006, 0.008, 0.010mg procymidone, carbofuran, carbendazim, chlorpyrifos antibody to each tube respectively, mix well, and place at room temperature for 20-40min; add 100μL 10% NaCl solution to each tube respectively, mix well, and react at room temperature for 20-40min; observe the color change of the solution, and the minimum deep red antibody amount is the optimum labeling amount.

[0171] 3) Preparation of immunolabeled nanoparticles

[0172] Take the above silver-coated gold nanoparticle solution, adjust the optimum pH value by adding 25mM K2CO3 per 100mL, and add the optimum labeling amount of procymidone, carbofuran, carbendazim, and chlorpyrifos antibody respectively, and react for 20-40min. Add 10mL of 10% BSA solution and react for 20-40min. Centrifuge at 12000r / min for 15min, remove the supernatant, and dissolve to 1 / 20 of the original solution volume with a reconstitution solution containing 1% BSA and 5% sucrose in 20mM PBS. Mix the three nanoparticle labeling reconstitution solutions together, spray 2-10μL / cm on a 2cm wide glass cellulose membrane, and dry at 37℃ for 18-24 hours to obtain the silver-coated gold nanoparticle labeled antibody binding pad.

[0173] 5) Chromatography membrane coating process

[0174] Prepare procymidone, carbofuran, carbendazim, and chlorpyrifos antigens into 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0mg / mL solutions with 0.01M pH7.4 phosphate buffer, and prepare anti-mouse IgG into 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0mg / mL solutions. Use a membrane marker to perform line coating C, T1, T2, T3, T4 at a speed of 1μl / cm, and place in a 37 degree oven to dry for 12 hours.

[0175] 6) Assembly of detection card

[0176] The same as Example 1.

[0177] 7) Test strip process parameter adjustment

[0178] Combine the silver-coated gold nanoparticle labeled antibody binding pad with different amounts of gold spraying and the coated reagent to prepare a small sample, test the reagent using a standard sample, and find the best combination according to the T value, C value, T / C value size, and test gradient results.

[0179] 8) Test strip performance test

[0180] The test paper prepared by the above steps is used for sample detection, and the specific method is as follows:

[0181] 1) Extractant: 70% methanol aqueous solution.

[0182] 2) Diluent: 0.02M PBS solution is used to prepare a diluent by adding tween-20 at a final concentration of 1%.

[0183] 3) Preparation of standard curve, conversion and input

[0184] After determining the test paper process parameters, the detection card is determined by using mixed standard samples with procymidone concentrations of 0, 100, 200, 500, 800, 1000, 2000, 5000, 10000 μg / kg, carbofuran concentrations of 0, 10, 20, 50, 100, 200, 500, 800, 1000 μg / kg, carbendazim concentrations of 0, 20, 50, 100, 200, 500, 1000, 2000, 5000 μg / kg, and chlorpyrifos concentrations of 0, 20, 50, 100, 200, 500, 800, 1000, 1500, 2500 μg / kg. Different concentrations of standard samples show different intensity T lines, and the intensity is determined by using a detector. The concentration value of chlorpyrifos and carbofuran in the determination sample is obtained by dividing the standard sample concentration by the recovery rate of 90%; the standard curve is prepared, and the corresponding parameters are input into the detector to complete the instrument curve parameter setting.

[0185] 4) Detection method

[0186] The same as example 1.

[0187] 5) Sample test

[0188] Seven samples obtained from the market or detection institutions are detected by using a certain amount of detection card combined with procymidone, carbofuran, carbendazim and chlorpyrifos, and the accuracy is compared and analyzed by comparing the results of gas chromatography; standard sample solutions with procymidone concentrations of 200, 500, 1000 μg / kg, carbofuran concentrations of 20, 50, 100 μg / kg, carbendazim concentrations of 50, 100, 200 μg / kg, and chlorpyrifos concentrations of 50, 100, 200 μg / kg are prepared, and a certain amount of detection card combined with procymidone, carbofuran, carbendazim and chlorpyrifos is used for analysis and detection, and each concentration is detected 6 times to analyze the precision.

[0189] 6) Results

[0190] a, accuracy detection results

[0191]

[0192] The paired sample T test method is performed on the data, and the t values of procymidone, carbofuran, carbendazim and chlorpyrifos are 0.022, 0.212, 0.040 and 0.127 respectively, t two-tailed critical (0.05, 6) =2.447, t < t two-tailed critical (0.05, 6), indicating that the detection results of the procymidone, carbofuran, carbendazim and chlorpyrifos four-in-one quantitative detection card have no significant difference with the detection results of the gas chromatography.

[0193] b, precision test results

[0194] Procymidone test results:

[0195]

[0196] Carbofuran test results:

[0197]

[0198] Carbendazim test results:

[0199]

[0200] Chlorpyrifos test results:

[0201]

[0202] From the results and analysis, it can be seen that the coefficient of variation of the repeated detection results of the procymidone, carbofuran, carbendazim and chlorpyrifos four-in-one quantitative detection card is between 10-12.5%, and the average coefficient of variation is less than 15%.

[0203] The test results show that the procymidone, carbofuran, carbendazim and chlorpyrifos four-in-one quantitative detection card has good performance, can quickly and accurately detect the content of procymidone, carbofuran, carbendazim and chlorpyrifos in the sample to be detected at one time, is suitable for rapid quantitative detection in the laboratory and sampling site, and meets the requirements of customers for rapid quantitative detection of procymidone, carbofuran, carbendazim and chlorpyrifos in food.

[0204] The technical scheme of the present application is further described through experiments as follows:

[0205] Experimental Example 1 Comparison of gold nanoparticles and silver-coated gold nanoparticles

[0206] 1. Synthesis of silver-coated gold nanoparticles

[0207] 1.1 Synthesis of gold nanoparticles

[0208] Take 100 ml of 0.01% chloroauric acid aqueous solution and heat to boiling, accurately add 1.8 ml of 1% trisodium citrate aqueous solution under stirring, and the gold yellow chloroauric acid aqueous solution turns red in 2 minutes, and continues to boil for 15 minutes.

[0209] 1.2 Silver-coated gold nanoparticles synthesis

[0210] The above solution is temperature adjusted to 40-80℃, silver nitrate solution is added to make its content 0.001-0.1%, 1.0ml of 1% trisodium citrate aqueous solution is added dropwise under stirring, and the reaction is continued for 10 minutes with stirring. The obtained liquid is silver-coated gold nanoparticle solution.

[0211] 2. Silver-coated gold nanoparticle gray scale and characterization

[0212] Silver-coated gold nanoparticles are nanoparticles with a layer of nanosilver shell formed on the surface of gold nanoparticles through chemical reaction. The nanoparticles have both properties of gold nanoparticles and silver nanoparticles, can effectively and firmly bind to proteins, and have a darker color than gold nanoparticles. After coupling with antibodies, the synthesized silver-coated gold nanoparticles are used to make a binding pad and an immunodetection card. By testing the gray scale value of the card by a scanner, compared with a detection card made of traditional gold nanoparticles, under the same conditions of antibody labeling amount and process, and for the same sample concentration, the gray scale of the color band at the detection line position is 2-5 times that of the silver-coated gold nanoparticles, so the detection sensitivity is improved by 2-5 times.

[0213]

[0214] In the table, the limited value is the maximum content value allowed by the national standard, and the "-" value is too low to be well recognized by the detector.

[0215] 3. Silver-coated gold nanoparticle labeled antibody related data, pH value, labeling amount, stability and colloidal gold comparison

[0216] Chlorpyrifos antibody, carbofuran antibody, and dimethoate antibody are labeled with silver-coated gold nanoparticles and conventional colloidal gold, respectively. The maximum absorption peak of the nanoparticle-antibody conjugate after labeling is between 400-532nm, and the OD value changes little. When the stability is destroyed due to external reasons, colloidal gold aggregates, its color first turns black, and finally becomes colorless. The maximum absorption peak gradually becomes larger than the normal peak value, and at the same time, the OD value gradually becomes smaller to 0. The labeled gold standard is tested as follows:

[0217] 1) The above gold standard is stored in a 4℃ refrigerator, and the changes of gold standard absorption peak and OD value are observed with time increase and measured by a UV spectrophotometer.

[0218] The results are as follows:

[0219]

[0220] From the results, the new silver-coated gold nanoparticles gold label effectively improves the gold label preservation stability. And with the increase of time, the conventional gold label gradually changes to purple color aggregation.

[0221] 2) Add different amounts of 10% NaCl solution to the above gold label, and after 30 min, determine the change of absorption peak and OD value using ultraviolet.

[0222] The results are as follows:

[0223]

[0224] From the results, the new silver-coated gold nanoparticles gold label effectively improves the salt stability of the gold label.

[0225] 3) Add different amounts of 0.1M hydrochloric acid to the above gold label, and after 30 min, determine the change of absorption peak and OD value using ultraviolet.

[0226] The results are as follows:

[0227]

[0228] From the results, the new silver-coated gold nanoparticles gold label effectively improves the acid stability of the gold label.

[0229] 4) Add different amounts of 1M sodium hydroxide to the above gold label, and after 30 min, determine the change of absorption peak and OD value using ultraviolet.

[0230] The results are as follows:

[0231]

[0232] From the results, the new silver-coated gold nanoparticles gold label effectively improves the alkali stability of the gold label.

[0233] From the above results, it can be seen that the new silver-coated gold nanoparticles are more stable in combination with the antibody, and are not affected by the acid-base environment and ion strength, and are resistant to acid, alkali and salt.

[0234] From the above examples and experimental examples, it can be seen that the present application provides a pesticide detection test paper with multi-in-one detection function based on colloidal gold immunochromatography, which has the advantages of good specificity, high sensitivity, accurate quantification, simultaneous detection of multiple components, simple pretreatment, etc. In addition, the present application can effectively improve the stability of the labeled antibody by optimizing the selection of gold nanoparticles. Therefore, the present application has good application prospect.

Claims

1. A multi-in-one pesticide residue quantitative detection test paper, characterized in that: The test paper comprises a sample pad, a colloidal gold combination pad and a chromatography membrane connected in sequence. ​ The colloidal gold combination pad is attached with gold nanoparticle labeled antibodies of the pesticide, and the gold nanoparticles are silver-coated gold nanoparticles. The chromatography membrane is provided with a quality control line and at least two detection lines, and each detection line is coated with an antigen of one pesticide. The pesticide is a combination of chlorpyrifos, phorate and dimethoate, or a combination of procymidone, carbofuran, carbendazim and chlorpyrifos. The antigen of the pesticide is formed by coupling pesticide molecules with macromolecular proteins, and the antigen of chlorpyrifos is prepared by a carbodiimide method, the antigen of phorate is prepared by a carbodiimide method, the antigen of dimethoate is prepared by a carbodiimide method, the antigen of carbofuran is prepared by a diazonium method, and the antigen of carbendazim is prepared by an activated ester method or a mixed anhydride method. The silver-coated gold nanoparticles are prepared by the following method: 100 mL of 0.01% chloroauric acid aqueous solution is heated to boiling, and 1.8 mL of 1% trisodium citrate aqueous solution is accurately added under stirring. The gold yellow chloroauric acid aqueous solution turns red within 2 minutes, and boiling is continued for 15 minutes to prepare a gold nanoparticle solution. The temperature of the gold nanoparticle solution is adjusted to 40-80°C, and silver nitrate solution is added to make the content 0.001-0.1%. Under stirring, 1.0 mL of 1% trisodium citrate aqueous solution is added dropwise as a reducing agent, and stirring is continued for 10 minutes. The obtained liquid is the silver-coated gold nanoparticle solution.

2. The test strip of claim 1, wherein: The gold nanoparticle labeled antibodies of the pesticide are obtained by combining the antibodies of the pesticide with gold nanoparticles, and the antibodies of the pesticide are obtained by immunizing animals with the antigens of the pesticide.

3. The test strip of claim 1, wherein: The size of the gold nanoparticles is 10-50 nm.

4. The test strip of claim 1, wherein: The macromolecular protein is bovine serum albumin or ovalbumin.

5. The method of producing a test strip according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: Step 1, preparation of the antigens of the pesticide and gold nanoparticles; Step 2, scribe coating on the chromatography membrane, one detection line for each antigen of the pesticide, and one quality control line coated with anti-mouse IgG; Step 3, preparation of the antibodies of the pesticide by using the antigens of the pesticide; Step 4, combination of the antibodies of the pesticide with gold nanoparticles to prepare gold nanoparticle labeled antibodies; Step 5, spraying of the gold nanoparticle labeled antibodies on the colloidal gold combination pad; Step 6, combination of the colloidal gold combination pad and the chromatography membrane as components, and other necessary components of the colloidal gold immunochromatography test paper to obtain a multi-in-one pesticide residue quantitative detection test paper.

6. The method of claim 5, wherein: In step 2, scribe coating is performed at a speed of 0.5-2 μL / cm by using a membrane scribe instrument, and the concentration of the antigen of the pesticide in the solution used for coating is 0.1-3 mg / ml. In step 5, the solution of the gold nanoparticle labeled antibodies containing the antibodies of the pesticide is sprayed on the colloidal gold combination pad at a dosage of 2-10 μL / cm, and the solution of the gold nanoparticle labeled antibodies containing the antibodies of the pesticide contains the following components: PBS 2 mM, BSA 0.1-5 wt.%, sucrose 1-20 wt.%, tween-20 0.01-1 wt.%, and the antibodies of the pesticide 0.1-2 mg / mL.

7. The method for detecting the pesticide residue by using the multi-in-one pesticide residue quantitative detection test paper according to any one of claims 1-4 for non-diagnostic purposes, characterized in that, The method comprises the following steps: Step 1, extracting the sample with an extracting agent, and separating to obtain supernatant; Step 2, diluting the supernatant with a diluting agent to obtain a test sample solution; Step 3, dropping the test sample solution to the sample pad of the test paper according to any one of claims 1-4 to perform detection.

8. The method of claim 7, wherein: The extracting agent is a methanol aqueous solution, and / or the diluting agent contains the following components: PBS 0.2 M, tween-20 0.1-10 wt.%.

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