Preparation method and application of fenitrothion nano-antibody-colloidal gold marker

Abstract

The present invention discloses a preparation method and application of a cypermethrin nano-antibody-colloidal gold marker. The present invention provides a preparation method of a cypermethrin nano-antibody-colloidal gold marker, and the prepared cypermethrin nano-antibody-colloidal gold marker has high sensitivity and strong stability. The nano-antibody-colloidal gold test strip for detecting cypermethrin pesticide provided by the present invention has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, rapid detection suitable for the market, easy storage, and long shelf life. The nano-antibody-colloidal gold test strip prepared by the present invention has a sensitivity of 0.013-0.309 mg / kg in the residual detection of cypermethrin pesticide, and the reaction time is 5-8 min. The method for detecting the residue of cypermethrin pesticide is simple, rapid, intuitive, accurate, widely applicable, low cost, easy to promote and use, and is suitable for the screening and on-site monitoring of a large number of samples on the market.

Description

Technical Field

[0001] The present invention belongs to the technical field of pesticide residue immunoassay detection, and more specifically, relates to a preparation method and application of a fenitrothion nano-antibody-colloidal gold marker. Background Art

[0002] Fenitrothion, also known as fenitrothion, is an organophosphorus broad-spectrum insecticide that has a wide range of targets, generally targeting pest control of rice, wheat, oil crops, fruits and vegetables, and trees. Fenitrothion is a moderately toxic pesticide. Irrational application of fenitrothion will lead to accumulation of fenitrothion residues on crops. Excessive intake has certain toxic effects on humans and animals: it leads to single-bond accumulation in the nervous system, causing symptoms such as nausea, vomiting, abdominal pain, diarrhea, and coma. Acute poisoning can also endanger life. In recent years, the excess of fenitrothion in the screening of food samples in my country has been the most serious, and the detection rate has also exceeded that of similar organophosphorus pesticides. Therefore, it is of great significance to develop a more economical, efficient, and sensitive fenitrothion rapid detection technology to achieve monitoring of fenitrothion.

[0003] At present, the standard detection methods for cypermethrin are mainly instrumental analysis and immunoassay. The instrumental analysis method has accurate and sensitive detection results and can realize the simultaneous detection of multiple analytes, but it requires complicated and cumbersome sample pretreatment to reduce the interference of sample matrix on the analysis results, and requires the use of expensive reagents and equipment as well as professional and skilled technicians. These factors make the instrumental analysis method unsuitable for large-scale instant detection in the market. Immunochromatography is to fix specific antibodies to a certain zone of nitrocellulose membrane first. When one end of the dry nitrocellulose is immersed in the sample (liquid), the sample will move forward along the membrane due to capillary action. When it moves to the area where the antibody is fixed, the corresponding antigen in the sample will specifically bind to the antibody. If immunocolloidal gold or immunoenzyme staining is used, the area can show a certain color, thereby realizing specific immunodiagnosis. The immunoassay method has the advantages of strong specificity, high sensitivity, low detection cost, short detection time and low requirements on personnel. For example, in the prior art, CN106290830A discloses an immunochromatographic test paper for rapid detection of cypermethrin based on upconversion fluorescent nanoparticles and a preparation method thereof, and uses upconversion fluorescent nanoparticles to label antibody samples for detection of cypermethrin pesticides; there are also monoclonal antibodies or polyclonal antibodies combined with colloidal gold for immunochromatographic detection, but this method is for the detection of residues of various organophosphorus pesticides. However, the antibodies currently used in colloidal gold immunochromatographic technology are almost all monoclonal antibodies and polyclonal antibodies, but due to the high production cost of monoclonal antibodies and polyclonal antibodies and the large batch difference between the two antibodies, the colloidal gold test strips obtained have different effects, while nano antibodies themselves are stable, low in production cost and can be obtained quickly. There is no report on the use of nano antibodies as markers and combined with immunochromatographic technology to analyze small molecule pesticide residues, and there is no report on the use of nano antibodies combined with colloidal gold in the detection of cypermethrin pesticide residues. Therefore, researching and developing more, more efficient and sensitive products or methods that can effectively detect the residues of cypermethrin pesticides has important theoretical research and practical application significance for the development of rapid pesticide multi-residue detection technology. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the above-mentioned problems and provide a preparation method and application of a fenitrothion nano-antibody-colloidal gold marker.

[0005] The first object of the present invention is to provide a method for preparing a fenitrothion nanobody-colloidal gold marker.

[0006] The second object of the present invention is to provide a fenitrothion nanobody-colloidal gold label.

[0007] The third object of the present invention is to provide the application of the fenitrothion nanobody-colloidal gold label.

[0008] The fourth object of the present invention is to provide a nano antibody-colloidal gold test strip for detecting the pesticide fenitrothion.

[0009] A fifth object of the present invention is to provide application of the test strip.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] The invention screens out a colloidal gold solution that can stably combine with a fenitrothion nano antibody through research, and combines the fenitrothion nano antibody with colloidal gold through optimization screening to prepare a fenitrothion nano antibody-colloidal gold marker with high sensitivity and strong stability.

[0012] The invention provides a preparation method of a fenitrothion nano antibody-colloidal gold marker. The method comprises the following steps: adding the fenitrothion nano antibody to a colloidal gold solution, mixing and standing the mixture, blocking the mixture, standing the mixture for centrifugation, discarding the supernatant, and resuspending the precipitate with a colloidal gold resolution to obtain the fenitrothion nano antibody-colloidal gold marker. The colloidal gold solution is prepared by mixing 1% chloroauric acid and 1% trisodium citrate in a volume ratio of 1:2. The amount of the fenitrothion nano antibody is 5-10 μg / mL. The blocking reaction is carried out by using 25-35 μL of 10% bovine serum albumin for 25-40 minutes. The formula of the colloidal gold resolution is as follows: 0.02-0.5 MBB, 0.1%-2% BSA, 1%-15% sucrose, 0.1%-2% Triton-100 and 0.1%-2% PVP.

[0013] Preferably, the cypermethrin nanobody is prepared with reference to the prior art (Wang Yu, Zhang Yifeng, Shen Yudong, et al. Preparation of anti-cypermethrin biotinylated nanoantibodies and their application in immunoassay methods [J]. Modern Food Science and Technology, 2021, Vol. 37 (8): 286-294, 285.).

[0014] Preferably, the colloidal gold solution has a pH value of 6.5 to 7.5.

[0015] Preferably, the formula of the colloidal gold complex solution is: 0.2M BB, BSA: 0.5% to 2%, sucrose: 2% to 10%, Triton-100: 0.5% to 2%, PVP: 0.2% to 1%.

[0016] Preferably, the precipitate is resuspended with a colloidal gold rehydration solution at a volume of 1 / 10 of the colloidal gold.

[0017] Preferably, the centrifugation conditions are 4°C, 8000-10000 rpm, and a centrifugation time of 10-20 min.

[0018] The invention provides the fenitrothion nano antibody-colloidal gold marker prepared as described above.

[0019] The invention provides application of the fenitrothion nano antibody-colloidal gold marker in detecting the fenitrothion pesticide.

[0020] The invention provides application of the fenitrothion nano-antibody-colloidal gold marker in preparing a nano-antibody-colloidal gold test strip for detecting fenitrothion.

[0021] The invention provides a nano antibody-colloidal gold test strip for detecting the fenitrothion pesticide, which contains the fenitrothion nano antibody-colloidal gold marker.

[0022] The nano antibody colloidal gold test strip for detecting the fenitrothion pesticide provided by the present invention comprises a sample absorption pad, a binding release pad, a reaction membrane, a water absorption pad and a bottom plate, wherein the reaction membrane is provided with a detection line coated with a fenitrothion pesticide hapten-carrier protein conjugate and a quality control line coated with a rabbit anti-camel anti-antibody, and the binding release pad is sprayed with a fenitrothion nano antibody-colloidal gold marker.

[0023] The above test strips are prepared by the following steps:

[0024] S1. preparing a fenitrothion nanobody-colloidal gold marker by the above method;

[0025] S2. Soak the binding release pad in the colloidal gold complex solution and then dry it, and spray the fenitrothion nanobody-colloidal gold marker prepared in step S1 on the treated binding release pad, dry it, and seal it for dry storage;

[0026] S3. coating the hapten-lactoferrin conjugate of the pesticide fenitrothion on the reaction membrane to form a detection line, and coating the rabbit anti-camel antibody on the reaction membrane to form a quality control line;

[0027] S4. Soak the sample absorption pad in the buffer solution and dry it for later use;

[0028] S5. Assemble the sample absorption pad, the binding release pad, the reaction membrane, and the water absorption pad on the bottom plate in sequence to obtain the nano-antibody colloidal gold test strip for detecting the pesticide fenitrothion.

[0029] Preferably, the amount of the fenitrothion nanobody added in step S1 is 3-10 μg / mL.

[0030] More preferably, the amount of the fenitrothion nanobody added is 5 to 10 μg / mL.

[0031] Preferably, in step S1, 20 to 40 μL of 10% bovine serum albumin is used for blocking reaction for 25 to 40 minutes.

[0032] More preferably, 25 to 35 μL of bovine serum albumin is used for blocking reaction for 30 minutes.

[0033] Preferably, the concentration of the fenitrothion pesticide hapten-lactoferrin conjugate used in step S2 is 0.1-0.5 mg / mL, and the coating amount is 0.5-1.0 μL / cm; the concentration of the rabbit anti-camel antibody is 0.5-2 mg / mL, and the coating amount is 0.5-1.0 μL / cm.

[0034] More preferably, the concentration of the fenitrothion pesticide hapten-lactoferrin conjugate used is 0.25 mg / mL, and the coating amount is 0.8 μL / cm; the concentration of the rabbit anti-camel antibody is 1 mg / mL, and the coating amount is 0.8 μL / cm.

[0035] Preferably, the buffer in step S4 is a phosphate buffer containing 0.2% sodium chloride, 0.5% sucrose, 0.3% polyvinyl pyrrolidone and 0.5% Tween-20, with a pH of 7.4.

[0036] Preferably, the fenitrothion pesticide hapten-carrier protein is obtained by coupling the hapten of the fenitrothion pesticide with lactoferrin.

[0037] More preferably, the cypermethrin pesticide hapten solution is prepared according to the description method in the technical literature (Chen et al. Analyst, 2021, 146(3): 864-873.), and the cypermethrin antibody solution is prepared according to the description in the technical literature (Wang Yu et al., Modern Food Science, 2021, 37(8): 286-294) combined with conventional methods.

[0038] Preferably, the rabbit anti-camelid antibody is obtained by immunizing a rabbit with a nanobody.

[0039] Preferably, the bottom plate is made of a non-absorbent PVC plate, one side of the plastic plate is coated with a self-adhesive sticker, and the bottom plate serves to fix and support other components of the test paper.

[0040] Preferably, the sample absorption pad can be filter paper or oil filter paper.

[0041] Preferably, the absorbent pad is glass fiber paper, filter paper or absorbent paper.

[0042] Preferably, the reaction membrane may be a nitrocellulose membrane or a cellulose acetate membrane.

[0043] The invention provides a nano antibody colloidal gold test strip for detecting a cypermethrin pesticide. The test strip utilizes a highly specific reaction between a cypermethrin nano antibody and the cypermethrin pesticide and an immunochromatographic technique of competitive inhibition. A sample solution is added to the test strip from a sample absorption pad, and the sample solution flows through the entire test strip through a capillary force. A substance to be tested in the sample first combines with a cypermethrin nano antibody-colloidal gold marker on a binding release pad to form a drug-antibody-colloidal gold marker. Then, the mixed solution flows through a reaction membrane, and the drug competes with a cypermethrin pesticide hapten-carrier protein on a detection line to bind to the cypermethrin nano antibody-colloidal gold marker. The cypermethrin pesticide residue in the sample solution to be tested is judged according to whether the detection line is colored or the color depth.

[0044] The present invention provides application of the nano antibody-colloidal gold test strip for detecting the fenitrothion pesticide in detecting the fenitrothion pesticide residue.

[0045] The present invention has the following beneficial effects:

[0046] The present invention provides a method for preparing a fenitrothion nano-antibody-colloidal gold marker. The fenitrothion nano-antibody-colloidal gold marker prepared by the present invention has high sensitivity and strong stability. The nano-antibody-colloidal gold test strip for detecting fenitrothion pesticide provided by the present invention has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, rapid detection suitable for the market, easy storage, and long shelf life. The nano-antibody-colloidal gold test strip for detecting fenitrothion pesticide prepared by the present invention has a sensitivity of 0.013-0.309 mg / kg in the residual detection of fenitrothion pesticide, and the reaction time is 5-8 minutes. The method for detecting the residue of fenitrothion pesticide is simple, rapid, intuitive, accurate, widely applicable, low cost, easy to promote and use, and is suitable for the screening and on-site monitoring of a large number of samples on the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Five colloidal gold solutions with different volume ratios were prepared;

[0048] Figure 2 The results of the combination of colloidal gold solution and fenitrothion nanobody with five different volume ratios are shown;

[0049] Figure 3 Colloidal gold-fenthion nanoantibody probes were prepared for five different colloidal gold solutions to test the results of fenthion drug solutions;

[0050] Figure 4 pH optimization for the preparation of fenitrothion nanobody-colloidal gold label;

[0051] Figure 5To optimize the amount of nanoantibody added for the preparation of fenitrothion nanoantibody-colloidal gold label;

[0052] Figure 6 Optimization of BSA content for the preparation of fenitrothion nanobody-colloidal gold label;

[0053] Figure 7 Optimization of colloidal gold reconstitution buffer for the preparation of fenitrothion nanobody-colloidal gold label;

[0054] Figure 8 It is a schematic diagram of the structure of the test strip;

[0055] Fig. 9 It is a schematic diagram of the test strip test results;

[0056] Fig.10 The test strips provide the test results for different organophosphorus pesticides;

[0057] Fig.11 is the standard curve of fenitrothion pesticide at different concentrations;

[0058] Fig.12 The test results of different concentrations of standard pesticides of fenitrothion are shown;

[0059] Fig.13 This is the test strip stability test result;

[0060] Fig.14 Test the test strips on different samples. DETAILED DESCRIPTION

[0061] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0062] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0063] Example 1 Preparation and Optimization of Fenitrothion Nanobody-Colloidal Gold Label

[0064] 1. Solution preparation

[0065] Colloidal gold complex solution: 0.02-0.5M BB, BSA: 0.1%-2%, sucrose: 1%-15%, Triton-100: 0.1%-2% and PVP: 0.1%-2%.

[0066] The cypermethrin nanoantibody used in the present invention is prepared with reference to the prior art (Wang Yu, Zhang Yifeng, Shen Yudong, et al. Preparation of anti-cypermethrin biotinylated nanoantibodies and their application in immunoassay methods [J]. Modern Food Science and Technology, 2021, Vol. 37 (8): 286-294, 285.).

[0067] 2. Preparation and optimization of colloidal gold

[0068] In the prior art, monoclonal antibodies are often tested by preparing colloidal gold solutions with 1% chloroauric acid and 1% trisodium citrate in volume ratios of 1:1, 1:1.25, and 1:1.5. Since the present invention uses a fenitrothion nano-antibody, in order to prepare a colloidal gold solution suitable for the fenitrothion nano-antibody, 1% chloroauric acid and 1% trisodium citrate in different volume ratios are used to prepare the colloidal gold solution. In the preparation process of the colloidal gold solution, the particle size of the colloidal gold solution finally obtained will change with the different content of trisodium citrate added. At the same time, the volume ratios of 1% chloroauric acid and 1% trisodium citrate are set to 1:1, 1:1.25, 1:1.5, 1:2, and 1:6 to prepare five colloidal gold solutions with different particle sizes, which are respectively labeled and combined with the fenitrothion nano-antibody, and the combination of the colloidal gold solution and the fenitrothion nano-antibody is judged according to the results.

[0069] Specifically, 1% chloroauric acid and 1% trisodium citrate were added to ultrapure water to prepare the colloidal gold solution. By changing the ratio of chloroauric acid and trisodium citrate, colloidal gold solutions with different particle sizes can be obtained. Five different volume ratios of 1:1, 1:1.25, 1:1.5, 1:2, and 1:6 were used to prepare colloidal gold solutions with different particle sizes. First, 1mL of 1% chloroauric acid was added to 100mL of ultrapure water and heated with a magnetic stirrer until boiling. Then, 1, 1.25, 1.5, 2, and 6mL of 1% trisodium citrate were added respectively. Stirring at a uniform speed continued. The color of the solution changed from light yellow to colorless, then to black and then gradually to wine red. After reacting for 10 minutes, the heating was stopped, and the solution was restored to 100mL with ultrapure water after cooling to room temperature, and stored at 4°C. The five prepared colloidal gold solutions with different volume ratios are as follows: Figure 1 As shown, the prepared colloidal gold is pure, transparent, and free of precipitation and floating matter.

[0070] The results of the binding of colloidal gold solution with the fenitrothion nanobody at five different volume ratios are shown in Figure 2. Figure 2As shown in the figure, after adding the cypermethrin nanobody to the conventional colloidal gold volume ratio (1:1, 1:1.25 and 1:1.5) solution, the color of the colloidal gold solution changed from red to purple and black, indicating that the mixed solution of colloidal gold and nanobody was not stable enough. The colloidal gold solution with a volume ratio of 1:2 and 1:6 was added with the nanobody to be stable. The colloidal gold-cypermethrin nanobody probe was prepared with five different volume ratios of colloidal gold solutions to detect the cypermethrin drug solution. The results are shown in the figure. Figure 3 As shown, the following examples all select a colloidal gold solution of 1% chloroauric acid and 1% trisodium citrate in a volume ratio of 1:2 for preparation and use in subsequent steps.

[0071] 3. Preparation and optimization of fenitrothion nanobody-colloidal gold label

[0072] The colloidal gold solution prepared as above was used, and the pH value of the colloidal gold solution was adjusted with potassium carbonate solution. Then, the fenitrothion nano-antibody was added to the colloidal gold solution with adjusted pH and allowed to react for 30 minutes. Then, 10% BSA was added for blocking reaction for 30 minutes. After blocking, the supernatant was discarded by centrifugation, and the precipitate was resuspended with a colloidal gold solution with a volume of 1 / 10 of the colloidal gold, and stored at 4°C.

[0073] The present invention optimizes the preparation conditions of the fenitrothion nanobody-colloidal gold marker, and the optimization result is determined according to the sensitivity and stability of the finally obtained fenitrothion nanobody-colloidal gold marker. The specific optimization conditions are as follows:

[0074] (1) pH optimization: The pH of the colloidal gold solution was adjusted using a 0.2 M potassium carbonate solution, and colloidal gold solutions of different pH values ​​were obtained by adding 5, 10, 15, 20, and 25 μL of potassium carbonate solution. Different colloidal gold complexes were then obtained according to the above reaction process, and the appropriate pH was determined based on the sensitivity and stability of the final obtained cypermethrin nanobody-colloidal gold marker.

[0075] The pH optimization results are as follows Figure 4 As shown, when 5 to 15 μL potassium carbonate is used and the pH is 6.5 to 7.5, the effect of the finally obtained fenitrothion nano-antibody-colloidal gold marker can meet the detection requirements.

[0076] (2) Optimization of the amount of nanoantibody added: According to the optimized pH, 3, 5, 7, and 10 μL of 1 mg / mL cypermethrin nanoantibody solution were added to obtain different colloidal gold complexes. The appropriate amount of cypermethrin nanoantibody added was determined based on the sensitivity and stability of the final cypermethrin nanoantibody-colloidal gold label.

[0077] The optimization results of the amount of nanoantibodies added are as follows Figure 5As shown, when 5 to 10 μL is used, the effect of the final obtained fenitrothion nano-antibody-colloidal gold marker can meet the detection requirements.

[0078] (3) Optimization of BSA content: According to the above optimized conditions, 20, 25, 30, 35, and 40 μL of 10% BSA were used for blocking in the blocking process. The appropriate amount of BSA added was also determined based on the sensitivity and stability of the final cypermethrin nanobody-colloidal gold marker.

[0079] The results of BSA content optimization are as follows Figure 6 As shown, when 25 to 35 μL 10% BSA is used, the effect of the finally obtained fenitrothion nano-antibody-colloidal gold label can meet the detection requirements.

[0080] (4) Optimization of centrifugation conditions: According to the above optimized conditions, the centrifugation conditions including centrifugation temperature, centrifugation speed and centrifugation time are optimized. The temperature includes 0, 4, 16, 25, and 37°C; the speed includes 8000, 9000, 10000, and 12000 rpm; and the centrifugation time includes 10, 15, and 20 min. Similarly, the appropriate centrifugation conditions are determined based on the sensitivity and stability of the final obtained cypermethrin nanobody-colloidal gold marker.

[0081] When the centrifugation conditions are 4° C., 8000-10000 rpm, and the centrifugation time is 10-20 min, the detection effect of the finally obtained fenitrothion nano-antibody-colloidal gold marker meets the requirements.

[0082] (5) Optimization of colloidal gold complex solution formula: The colloidal gold complex solution was optimized according to the above optimal conditions, including the selection of the system, the concentration of BB, the content of BSA, the content of sucrose, the content of Triton-100 and the content of PVP. The system selection includes BB, PB, Tris and EDTA solution, and the concentration of the system includes 0.02, 0.05, 0.1, 0.2, and 0.5M; the amount of sucrose added includes 1%, 2%, 5%, 10%, and 15%; the content of the remaining additives includes 0.1%, 0.2%, 0.5%, 1%, and 2%. The sensitivity and stability of the cypermethrin nanoantibody-colloidal gold marker were also used to determine the appropriate complex solution formula.

[0083] The optimization results of colloidal gold complex solution formula are as follows Figure 7 As shown, when using 0.2M BB solution, sucrose addition amount is 2-10%, BSA addition amount is 0.5-2%, PVP addition amount is 0.2-1% and Triton-100 addition amount is 0.5-2%, the effect of the finally obtained fenitrothion nano-antibody-colloidal gold marker can meet the detection requirements.

[0084] During the optimization process, the colloidal gold solution will aggregate under unsuitable conditions, and the color will change from wine red to black with precipitation. Finally, according to the above optimization results, the optimal preparation conditions of the fenitrothion nano-antibody-colloidal gold marker prepared by the present invention are determined as follows: pH 6.5-7.5, the amount of fenitrothion nano-antibody added is 5-10 μg / mL, 10% BSA 25-35 μL, the centrifugation conditions are 4 ° C, 8000-10000 rpm, and the centrifugation time is 10-20 min. When the colloidal gold complex solution formula adopts 0.2M BB, BSA: 0.5%-2%, sucrose: 2%-10%, Triton-100: 0.5%-2%, PVP: 0.2%-1%, the effect of the finally obtained fenitrothion nano-antibody-colloidal gold marker can meet the detection requirements.

[0085] Example 2 Preparation of Nanobody-Colloidal Gold Test Strips for Detecting Fenitrothion Pesticide

[0086] 1. Preparation of bonding release pad

[0087] The binding release pad was soaked in the colloidal gold complex solution for 30 min and dried in a 45°C oven. The prepared cypermethrin nanoantibody-colloidal gold marker solution was sprayed on the binding release pad treated with the colloidal gold complex solution at an amount of 5 μL / cm per well using an AUTOKUN film spray gold labeling machine (Hangzhou Fenghang Technology Co., Ltd., HGS510). The sprayed binding release pad was placed in an oven at 37°C for 12 h to dry. After drying, the sprayed binding release pad was sealed and stored with a desiccant.

[0088] 2. Preparation of reaction membrane

[0089] The preparation method of the hapten of the cypermethrin pesticide used in the present invention is prepared with reference to the prior art (Chen et al. Analyst, 2021, 146(3): 864-873.).

[0090] The hapten of the pesticide fenitrothion-lactoferrin was obtained by chemical coupling with chemical reagents DMF and NHS, and the rabbit anti-camel antibody was obtained by immunizing rabbits with fenitrothion nanoantibodies.

[0091] (1) Coating of the test line: The prepared fenitrothion pesticide hapten-lactoferrin conjugate was coated on a reaction membrane to form a test line. The fenitrothion pesticide hapten-lactoferrin conjugate was diluted to 0.25 mg / mL with phosphate buffer and coated on a test line (T line) on a nitrocellulose membrane using an AUTOKUN film spray gold label machine. The coating amount was 0.8 μL / cm; the coated reaction membrane was placed at 37° C. and dried for 4 h.

[0092] (2) Coating of the quality control line: Coat the rabbit anti-camelid antibody on the reaction membrane to form a quality control line. Dilute the rabbit anti-camelid antibody to 1 mg / mL with phosphate buffer and coat it on the quality control line (C line) on the nitrocellulose membrane using an AUTOKUN film spray gold labeler. The coating amount is 0.8 μL / cm. Dry the coated reaction membrane at 37°C for 4 hours and set it aside for use.

[0093] 3. Preparation of sample absorption pad

[0094] The sample absorption pad was placed in a pH 7.4 0.2 mol / L phosphate buffer containing 0.2% sodium chloride, 0.5% sucrose, 0.3% polyvinyl pyrrolidone and 0.5% Tween-20 by volume, soaked for 1 hour, and dried at 37°C for 4 hours for use.

[0095] 4. Assembly of test strips

[0096] The reaction membrane, binding release pad, sample absorption pad and water absorption pad prepared above are sequentially pasted on the PVC bottom plate; the starting end of the sample absorption pad is aligned with the starting end of the PVC bottom plate, the end of the sample absorption pad is connected to the starting end of the binding release pad, the end of the binding release pad is connected to the starting end of the reaction membrane, and the sample absorption pad overlaps with the binding release pad by 2 mm; the end of the reaction membrane is connected to the starting end of the water absorption pad, and the end of the water absorption pad is aligned with the end of the PVC bottom plate; and the binding release pad and the water absorption pad both overlap with the reaction membrane by 2 mm. The assembled test strip is as shown in FIG. Figure 8 As shown. The reaction membrane has a detection line (T line) and a quality control line (C line), both of which are strips perpendicular to the length of the test strip. The detection line is located on the side close to the end of the sample absorption pad, and the quality control line is located on the side close to the head end of the water absorption pad. After the test strip is assembled according to the above steps, each part is assembled and pressed tightly, and then cut into small strips of 4mm width by a machine, and packed in a special sealed bag, it can be stored for 12 months at 4-30℃.

[0097] Example 3 Limit test of nanobody-colloidal gold test strip for detecting fenitrothion pesticide

[0098] 1. Specificity test

[0099] In order to detect the specificity of the nano antibody-colloidal gold test strip for detecting the cypermethrin pesticide prepared by the present invention, different pesticides were used for determination. 6.5 mg / kg of quinalphos, triazophos, isocarbophos, profenofos, methiocarb and carbofuran were used for detection, and the drugs were diluted to 6.5 mg / kg using PBS solution.

[0100] Add the sample solution vertically from the sample pad of the test strip, start timing when the liquid flows, react for 5 to 8 minutes, and read the results through the colloidal gold analyzer. When the concentration of the test substance in the sample is lower than the detection limit, the result is negative, and when the concentration of the test substance in the sample is equal to or higher than the detection limit, the result is positive. If the result is invalid, it means that retesting is required.

[0101] The flow reaction principle of the test strip is as follows Fig. 9 As shown, when the test result is negative, the color of the test line (T line) and the quality control line (C line) will change, becoming redder and darker; when the test result is positive, the test line (T line) will remain unchanged or become lighter, while the color of the quality control line (C line) will change, becoming redder and darker; when the color of both the test line (T line) and the quality control line (C line) does not change, or only the test line (T line) becomes redder and darker, it is an invalid result, which means that retesting is required.

[0102] The test strips prepared by the present invention are used to test different pesticides. Fig.10 As shown, when the test strip detects the pesticide cypermethrin, only the color of the quality control line deepens, indicating a positive result; when detecting other organophosphorus pesticides, both the quality control line and the test line are colored, indicating a negative result. This indicates that the test strip has no cross-reaction to the detection of 6.5mg / kg quinalphos, triazophos, isocarbophos, profenofos, methiocarb and carbofuran, and can only specifically detect the pesticide cypermethrin.

[0103] 2. Detection limit test

[0104] In order to detect the detection limit and sensitivity of the nano antibody-colloidal gold test strip for detecting the cypermethrin pesticide prepared by the present invention, the cypermethrin standard pesticide was added to the PBS buffer solution to a final concentration of 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.0156, 0.0078, 0.0039 mg / kg, and then the test strip prepared in Example 1 was taken for detection, and each sample was tested three times. The sample solution was vertically dripped onto the sample absorption pad, and the timing was started when the liquid flowed, and the reaction was 5 to 8 minutes, and the result was determined.

[0105] The test results can be accurately read by the colloidal gold analyzer. Based on the data, a standard curve can be obtained through the Origin software. Fig.11 As shown in the figure. The test results of different concentrations of standard pesticides of cypermethrin are shown in the figure. Fig.12 As shown, the results corresponding to the standard curve show that the detection range of this method for detecting the pesticide residue of fenitrothion is 0.013-0.309 mg / kg, and the detection limit is 0.00598 mg / kg, which meets the national standard detection requirements.

[0106] 3. Stability test

[0107] In order to detect the stability of the nano antibody-colloidal gold test strip for detecting the fenitrothion pesticide prepared by the present invention, the test strip is placed for a long time to detect its accuracy and stability.

[0108] The test strips prepared by the method of the present invention were placed in a sealed bag with a built-in desiccant, stored in a 45°C oven, and taken out after 3 days, 7 days, 14 days, 21 days, and 30 days to test the stability of the test strips.

[0109] The results are as follows Fig.13 As shown, there was no significant change in the test results when stored at 45°C for 3 days, 7 days, 14 days, 21 days and 30 days. Storage at 45°C for 30 days is equivalent to storage at room temperature for 12 months.

[0110] Example 4 Actual sample detection

[0111] Buy different cabbage, cucumber and orange samples from the market and supermarket, wash and dry the samples, then weigh 10g (±0.1g) of homogenized samples into 50mL plastic centrifuge tubes, divide the samples into negative samples and positive samples, and do not do additional treatment for negative samples. For positive samples, add cypermethrin standard to the drug concentration of 0.02mg / kg. Then add 10mL of acetonitrile to both negative and positive samples, vortex to mix; then add the extraction package, continue vortexing for 2min, centrifuge at 5000rpm / min for 3min, and stand at room temperature. Take the supernatant solution and add it to the purification tube, vortex for 2min, centrifuge at 5000rpm / min for 3min, take the supernatant, blow with nitrogen at 37 degrees, and then use EDTA solution to re-dissolve.

[0112] Take the test strip prepared in Example 2 for testing, vertically drip the treated sample solution onto the sample absorption pad, start timing when the liquid flows, react for 5 to 8 minutes, and determine the result.

[0113] Read the test results through the colloidal gold analyzer:

[0114] Negative (-): indicates that the concentration of the substance to be tested in the sample is lower than the detection limit;

[0115] Positive (+): indicates that the concentration of the substance to be tested in the sample is equal to or higher than the detection limit;

[0116] Invalid: Indicates that retesting is required.

[0117] The test strips prepared by the present invention were used to test different samples, and the test results were as follows: Fig.14As shown, when testing cabbage, cucumber and orange samples, we can see that both the T line and the C line in the negative samples are colored, while the color of the C line in the positive samples is significantly stronger than that of the T line, indicating that the test results can also meet the requirements.

[0118] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A fenitrothion nanobody-colloidal gold label, characterized in that: The preparation method comprises the following steps: adding a fenitrothion nano antibody to a colloidal gold solution, mixing and standing the mixture, blocking the mixture, standing the mixture for centrifugation, discarding the supernatant, and resuspending the precipitate with a colloidal gold resolution to obtain a fenitrothion nano antibody-colloidal gold marker; the colloidal gold solution is prepared by mixing 1% chloroauric acid and 1% trisodium citrate in a volume ratio of 1:2, and the pH value of the colloidal gold solution is 6.5-7.5; the amount of the fenitrothion nano antibody added is 5-10 μg / mL; the blocking reaction is carried out for 25-40 minutes using 25-35 μL of 10% bovine serum albumin; the formula of the colloidal gold resolution is: 0.02-0.5 MBB, 0.1%-2% BSA, 1%-15% sucrose, 0.1%-2% Triton-100, and 0.1%-2% PVP.

2. The fenitrothion nanobody-colloidal gold label according to claim 1, characterized in that: The formula of the colloidal gold complex solution is: 0.2M BB, BSA: 0.5%-2%, sucrose: 2%-10%, Triton-100: 0.5%-2%, PVP: 0.2%-1%.

3. Use of the fenitrothion nanobody-colloidal gold label according to claim 1 in detecting fenitrothion pesticide.

4. Use of the fenitrothion nanobody-colloidal gold label according to claim 1 in preparing a nanobody-colloidal gold test strip for detecting fenitrothion.

5. A nano antibody-colloidal gold test strip for detecting the pesticide fenitrothion, characterized in that: Contains the fenitrothion nanobody-colloidal gold label as claimed in claim 1.

6. The test strip according to claim 5, characterized in that: The test strips are prepared by the following steps: S1. preparing a fenitrothion nanobody-colloidal gold marker by the method of claim 1; S2. Soak the binding release pad in the colloidal gold complex solution and then dry it, and spray the fenitrothion nanobody-colloidal gold marker prepared in step S1 on the treated binding release pad, dry it, and seal it for dry storage; S3. coating the hapten-lactoferrin conjugate of the pesticide fenitrothion on the reaction membrane to form a detection line, and coating the rabbit anti-camel antibody on the reaction membrane to form a quality control line; S4. Soak the sample absorption pad in the buffer solution and dry it for later use; S5. Assemble the sample absorption pad, the binding release pad, the reaction membrane, and the water absorption pad on the bottom plate in sequence to obtain the nano-antibody colloidal gold test strip for detecting the pesticide fenitrothion.

7. The test strip according to claim 6, characterized in that: The concentration of the fenitrothion pesticide hapten-lactoferrin conjugate used in step S2 is 0.1-0.5 mg / mL, and the coating amount is 0.5-1.0 μL / cm; the concentration of the rabbit anti-camel antibody is 0.5-2 mg / mL, and the coating amount is 0.5-1.0 μL / cm.

8. Use of the nano antibody-colloidal gold test strip for detecting fenitrothion pesticide according to claim 5 in detecting fenitrothion pesticide residues.

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