A parathion pesticide colloidal gold label based on nanoantibodies and its application

Abstract

The invention provides a colloidal gold marker for parathion pesticide based on nano-antibodies and its application. The present invention develops for the first time the combination of parathion nano-antibodies and colloidal gold to prepare parathion nano-antibody-colloidal gold markers for the detection of parathion pesticides. The detection stability and sensitivity of the parathion nano-antibody-colloidal gold marker are good; the nano-antibody colloidal gold test strips for parathion pesticides prepared therefrom have the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, easy storage and long shelf life. The test strips provided by the present invention have a sensitivity of 0.0013-0.085 mg / kg for the detection of parathion pesticide residues, and a detection limit of 0.00069 mg / kg. They can be used for the rapid detection of parathion pesticides in agricultural products such as vegetables and fruits. The method is simple, fast, intuitive, accurate, widely applicable, low cost and easy to promote and use.

Description

Technical Field

[0001] The present invention belongs to the technical field of pesticide residue immunoassay detection, and more specifically, relates to a parathion pesticide colloidal gold marker based on nano-antibodies and its application. Background Art

[0002] Parathion is a broad-spectrum, highly toxic organophosphate insecticide and acaricide with strong contact and stomach poisoning effects, and a certain fumigation effect. Due to its pest prevention and control effects on crops such as fruits, vegetables and grains, parathion pesticides are widely used in agricultural production. Its ecological safety risks and potential safety hazards to humans and other non-target organisms are currently also attracting much attention. Parathion can inhibit the activity of acetylcholinesterase, leading to a large accumulation of the neurotransmitter acetylcholine in the body, which in turn causes a series of neurological reactions, resulting in nausea, headache, weakness, chest tightness and other poisoning symptoms, and severe cases can lead to death. If parathion remains and accumulates in the environment and food, it will inevitably cause direct or potential serious harm to human health. Parathion is a highly toxic pesticide variety and is currently banned from use on vegetables and fruits. Therefore, the development of rapid detection methods and technologies for parathion pesticide residues is of great practical significance.

[0003] At present, the main detection methods for the residues of parathion pesticides are instrumental methods such as high performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC / MS). Although the instrumental detection method has accurate and sensitive detection results, its complex operation process, expensive instruments and high detection costs make instrumental detection unsuitable for on-site instant detection. Colloidal gold immunochromatography technology is an instant detection technology that combines immunoassay and chromatography technology and has been used since the 1980s. The principle is that the sample containing the analyte flows through the entire test strip through capillary action and reacts with the coated substance (antigen or antibody) on the reaction membrane of the test strip with high specificity and high affinity. The immune complex is trapped on the test strip for color development, so that the test result can be obtained. At present, the antibodies used in colloidal gold immunochromatography technology are almost all monoclonal antibodies or polyclonal antibodies. For example, the prior art CN 106970217 B discloses an immunochromatographic method for quantitative detection of organophosphorus pesticides, which uses monoclonal antibodies as fixed detection lines and acetylcholinesterase as modified indicators to prepare immunochromatographic methods for quantitative detection of organophosphorus pesticides. This method is for multi-residue detection of organophosphorus pesticides; although the detection method using antigen antibodies can only detect a single target, the maximum residue limit of parathion pesticides in my country is relatively strict, and the maximum residue limit is 0.01 mg / kg. The immunochromatographic method for parathion detection using monoclonal antibodies or polyclonal antibodies cannot meet the national standard requirements. Compared with traditional antibodies, nanobodies (Nanobody, Nbs) have the advantages of small relative molecular mass, simple humanization, high affinity, high stability, microbial expression, low immunogenicity, strong penetration, and good solubility, and have attracted much attention in basic medical research and disease diagnosis and treatment. So far, there are no reports on the use of nanoantibodies for the detection and analysis of parathion pesticides. Therefore, it is necessary to develop more new products and methods that are simpler, more convenient, and low-cost and can be used for the detection of parathion pesticide residues, so that the detection methods of parathion can meet the national standards. 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 existing problems and provide a parathion pesticide colloidal gold marker based on nano antibodies and its application.

[0005] The first object of the present invention is to provide a parathion pesticide colloidal gold label based on nano antibodies.

[0006] The second object of the present invention is to provide the application of the parathion nanobody colloidal gold label.

[0007] The third object of the present invention is to provide a nano antibody colloidal gold test strip for detecting parathion pesticide.

[0008] The fourth object of the present invention is to provide application of the test strip.

[0009] A fifth object of the present invention is to provide a method for detecting parathion pesticide in crops.

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

[0011] A parathion pesticide colloidal gold marker based on a nano-antibody, the preparation method of which is as follows: adding 1-10 μg / mL of parathion nano-antibody to a colloidal gold solution with a pH value of 8-10, mixing and standing, then adding 20-40 μL of 10% BSA, standing and centrifuging, discarding the supernatant, and resuspending the precipitate with a colloidal gold re-dissolving buffer to obtain the parathion nano-antibody colloidal gold marker; the colloidal gold solution is prepared with 1% chloroauric acid and 1% trisodium citrate in a volume ratio of 1:6; the colloidal gold re-dissolving buffer has a formula of: 0.01-0.5 M Tris-HCl, 0.1-2% BSA, 0.1-2% Tween-20, 0.1-2% polyvinyl pyrrolidone and 1-15% sucrose.

[0012] The present invention combines parathion nanoantibodies with colloidal gold for the first time, and the prepared parathion nanoantibody colloidal gold marker can be used to detect parathion pesticides; by optimizing and screening various conditions in the preparation process of the parathion nanoantibody colloidal gold marker, a phosphorus nanoantibody colloidal gold marker with good stability and high sensitivity is finally prepared.

[0013] Preferably, the pH value of the colloidal gold solution is 8.5-9.5, the amount of parathion nanobody added is 3-7 μg / mL, and the amount of 10% BSA added is 30-40 μL.

[0014] Preferably, the particle size of the colloidal gold solution is 10 to 20 nm.

[0015] Preferably, the centrifugation conditions are 9000 rpm, 4°C, and 15 min.

[0016] Preferably, the resuspended precipitate is resuspended in a colloidal gold resolubilization buffer with a volume of 1 / 5 of the colloidal gold.

[0017] Preferably, the colloidal gold resolubilization buffer has a formula of: 0.05M Tris-HCl, 0.2% to 1% BSA, 2% to 10% sucrose, 0.1% to 1% Tween-20 and 0.3% to 1% polyvinyl pyrrolidone.

[0018] The invention provides the application of parathion nano antibody colloidal gold marker in detecting parathion pesticide by immunochromatography technology.

[0019] The invention provides application of a parathion nano-antibody colloidal gold marker in the preparation and detection of a parathion pesticide product.

[0020] The invention provides a nanometer antibody colloidal gold test paper strip for detecting parathion pesticide, which contains the parathion nanometer antibody colloidal gold marker on a binding release pad.

[0021] Preferably, it also includes a sample pad, a reaction membrane, a water absorbent pad and a bottom plate.

[0022] Preferably, the reaction membrane has a detection line coated with a parathion pesticide hapten-carrier protein conjugate, and a quality control line coated with a rabbit anti-camel antibody.

[0023] The nano antibody colloidal gold test strip for detecting parathion pesticide provided by the present invention adopts a parathion nano antibody colloidal gold marker to bind to a test object, and detects the parathion pesticide by performing a competitive inhibition reaction with the parathion pesticide hapten-carrier protein and rabbit anti-camel anti-anti. The sample solution to be tested is added to the test strip from the sample pad, and the sample solution flows through the entire test strip through the capillary force. The substance to be tested in the sample first binds to the parathion nano antibody colloidal gold marker on the binding release pad to form a drug-antibody-colloidal gold marker. After that, the mixed solution flows through the reaction membrane, and the drug competes with the parathion pesticide hapten-carrier protein on the detection line to bind to the parathion nano antibody colloidal gold marker, and whether there is parathion pesticide residue in the sample solution to be tested is judged according to whether the detection line band is colored or the color depth.

[0024] The above test strips are prepared by the following method:

[0025] S1. Prepare a binding release pad sprayed with parathion nanoantibody colloidal gold marker;

[0026] S2. preparing a reaction membrane having a test line coated with a parathion pesticide hapten-carrier protein conjugate and a quality control line coated with a rabbit anti-camel anti-antibody;

[0027] S3. Assemble the combined release pad, reaction membrane, sample pad, water absorbent pad and bottom plate prepared in step S1 and step S2 into a test strip.

[0028] Preferably, the colloidal gold resolubilization buffer has a formula of: 0.05M Tris-HCl, 0.2% to 1% BSA, 2% to 10% sucrose, 0.1% to 1% Tween-20 and 0.3% to 1% polyvinyl pyrrolidone.

[0029] Preferably, the preparation of the parathion pesticide hapten solution is prepared according to the method described in the literature (Xu et al.Analytica Chimica Acta, 2009, 647(1):90-96); the parathion pesticide hapten-carrier protein is obtained by chemically coupling the parathion pesticide hapten and ovalbumin using chemical reagents DMF and NHS.

[0030] Preferably, the rabbit anti-camelid antibody solution is an antibody solution obtained by injecting parathion nanobody as an immunogen into a rabbit and finally separating it from the rabbit serum.

[0031] Preferably, the parathion pesticide hapten-ovalbumin conjugate is coated on the reaction membrane to form a detection line, and the rabbit anti-camel antibody is coated on the reaction membrane to form a quality control line.

[0032] When the test strip prepared by the present invention is used for detection, the sample solution is added to the test strip from the sample pad and passes through the entire test strip through the capillary force. When the concentration of the parathion pesticide in the sample treatment solution is lower than the detection limit or zero, the parathion nano-antibody colloidal gold marker will combine with the parathion pesticide hapten-carrier protein and the rabbit anti-camel anti-antibody during the chromatography process to develop color on the detection line and the quality control line respectively; when the concentration of the parathion pesticide in the sample treatment solution is equal to or higher than the detection limit, the pesticide in the sample will completely combine with the parathion nano-antibody colloidal gold marker, so that the parathion hapten-carrier protein on the detection line will not combine with the parathion nano-antibody colloidal gold marker due to the competitive reaction and will not develop color.

[0033] The invention provides application of the test strip in detecting parathion pesticide residues in fruit and vegetable crops.

[0034] The invention provides a method for detecting parathion pesticide in crops, adopting the above-mentioned test strip to detect samples, and judging the residual situation of the parathion pesticide according to the color development result of the test strip.

[0035] The present invention has the following beneficial effects:

[0036] The present invention combines parathion nano-antibody with colloidal gold to prepare a parathion nano-antibody colloidal gold marker for the first time. The parathion nano-antibody colloidal gold marker has good stability for parathion pesticide detection and high detection sensitivity. The prepared colloidal gold test strip for parathion pesticide detection based on the nano-antibody 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 test strip provided by the present invention has a sensitivity of 0.0013-0.085 mg / kg for parathion pesticide residue detection, a detection limit of 0.00069 mg / kg to meet the national standard detection requirements, a reaction time of 5-8 minutes, and is suitable for rapid detection of parathion pesticides in agricultural products such as vegetables and fruits. The method is simple, rapid, intuitive, accurate, widely applicable, low cost, and easy to promote and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Five colloidal gold solutions of different proportions were prepared;

[0038] Figure 2The results of the combination of colloidal gold solution and parathion nanobody at five different ratios are shown;

[0039] Figure 3 The colloidal gold-parathion nanoantibody probes were prepared for five different colloidal gold solutions to detect the parathion drug solution;

[0040] Figure 4 pH optimization for the preparation of colloidal gold label of parathion nanobody;

[0041] Figure 5 Optimization of the amount of nanoantibody added for the preparation of colloidal gold labeled parathion nanoantibody;

[0042] Figure 6 Optimization of BSA content for the preparation of colloidal gold labeled parathion nanobody;

[0043] Figure 7 Optimization of colloidal gold reconstitution buffer for the preparation of colloidal gold labeled parathion nanobody;

[0044] Figure 8 The structure diagram of the test strip of the present invention is shown in FIG. 1 (wherein 1 is a sample pad, 2 is a conjugate release pad, 3 is a reaction membrane, 4 is a water-absorbing pad, 5 is a detection line of a conjugate of parathion pesticide hapten and a carrier protein, 6 is a quality control line coated with rabbit anti-camel antibodies, and 7 is a bottom plate);

[0045] Fig. 9 This is a schematic diagram of the test results of the nano antibody colloidal gold test strip for parathion pesticide of the present invention;

[0046] Fig.10 The test results of different parathion standard pesticide concentration test strips;

[0047] Fig.11 It is the standard curve for different concentration gradient detection;

[0048] Fig.12 Stability test of nano-antibody colloidal gold test strips for parathion pesticide;

[0049] Fig.13 Detection of actual samples of nano-antibody colloidal gold test strips for parathion pesticide. DETAILED DESCRIPTION

[0050] 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.

[0051] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0052] Example 1 Preparation and Optimization of Parathion Nanobody Colloidal Gold Label

[0053] 1. Preparation of colloidal gold

[0054] Ultrapure water was placed in a conical flask, 1% chloroauric acid was added, and it was heated to boiling with a constant temperature electromagnetic stirrer. 1% trisodium citrate was added under continuous high temperature and continuous stirring. The color of the solution first changed from yellow to colorless, then to black and then slowly to wine red, and the reaction was continued for 10 minutes when the color of the solution no longer changed. After cooling to room temperature, the volume was restored to the original volume with ultrapure water and stored at 4°C. The prepared colloidal gold had a pure, transparent appearance, without precipitation and floating matter.

[0055] In the process of preparing colloidal gold solution, the particle size of the colloidal gold solution finally obtained will change with the different content of trisodium citrate added. In the current prior art, the volume ratio of 1% chloroauric acid and 1% trisodium citrate used in the preparation of colloidal gold solution used for monoclonal antibodies is usually 1:1, 1:1.25 and 1:1.5. In order to prepare colloidal gold solution suitable for nano antibodies, 1% chloroauric acid and 1% trisodium citrate in different volume ratios are used to prepare colloidal gold solution, and the volume ratio of 1% chloroauric acid and 1% trisodium citrate is 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 combined with parathion nano antibodies, and the combination of colloidal gold solution and nano antibodies is judged according to the results.

[0056] The five colloidal gold solutions with different volume ratios were prepared as follows: Figure 1 As shown in Figure 2, the binding results of colloidal gold solution with parathion nanoantibody at five different volume ratios are shown in Figure 2. Figure 2 As shown, the colloidal gold solution will aggregate under unsuitable conditions, and the color will change from wine red to black with precipitation; after adding parathion nanoantibody solution to the colloidal gold solution commonly used for monoclonal antibodies prepared at a volume ratio of 1:1, 1:1.25, and 1:1.5, the color of the solution changed from red to black, indicating that the colloidal gold solution commonly used for monoclonal antibodies prepared at a volume ratio of 1:1, 1:1.25, and 1:1.5 is not suitable for the binding of parathion nanoantibodies. After the colloidal gold solution with a volume ratio of 1:2 was combined with the parathion nanoantibody, the solution also changed from red to purple, indicating that the colloidal gold solution prepared at a volume ratio of 1:2 was also unstable after combining with the parathion nanoantibody; and after adding the parathion nanoantibody to the colloidal gold solution prepared at a volume ratio of 1:6, the color of the solution did not change significantly, indicating that the mixed solution can remain stable.

[0057] The colloidal gold-parathion nanoantibody probes were prepared using the five colloidal gold solutions with different volume ratios to detect the parathion drug solution. The results are as follows: Figure 3 As shown, the binding stability and detection effect of colloidal gold solution and parathion nanoantibody in a volume ratio of 1:6 are the best.

[0058] 2. Preparation of colloidal gold label of parathion nanoantibody

[0059] The parathion nanoantibody solution used in the present invention is prepared by referring to the description in the literature and combining conventional methods (Zhang Yuqi et al., Analytical Chemistry, 2019, 47(9):1419-1426). The pH value of the colloidal gold solution is adjusted with 0.2 mol / L potassium carbonate solution, 1-10 μg of parathion nanoantibody is added to each milliliter of colloidal gold solution, shaken and mixed, and then allowed to stand for 30 minutes, 10% BSA is added to make the final concentration in the colloidal gold solution 0.5% (volume fraction), and allowed to stand for 30 minutes. Centrifuge, discard the supernatant, and resuspend the precipitate with 1 / 5 of the volume of colloidal gold in colloidal gold resolubilization buffer, and place at 4°C for use.

[0060] 3. Optimization of colloidal gold label for parathion nanoantibody

[0061] During the entire process of labeling the parathion nanoantibody colloidal gold marker, the pH, nanoantibody addition amount, BSA addition amount, centrifugation conditions and the final colloidal gold complex solution system were optimized, and the optimization result was determined based on the sensitivity and stability of the final nanoantibody colloidal gold marker.

[0062] (1) pH optimization: The pH of the colloidal gold solution was adjusted using a 0.2 M potassium carbonate solution. By adding 20, 25, 30, 35, and 40 μL potassium carbonate solution, the pH values ​​were 8.12, 8.45, 8.78, 9.12, and 9.67, respectively, to obtain colloidal gold solutions of different pH values. Then, different colloidal gold complexes were obtained according to the reaction process in step 2 above, and the appropriate pH was determined based on the sensitivity and stability of the nanoantibody colloidal gold label obtained.

[0063] The five different pH colloidal gold-parathion nanoantibody probes were used to detect parathion drug solutions. The results are as follows: Figure 4 As shown, the colloidal gold-nanoantibody complex obtained when the potassium carbonate solution addition amount is 30 μL has the best effect, and the colloidal gold and parathion nanoantibody binding stability and detection effect are the best when the pH is 8.5-9.5.

[0064] (2) Optimization of the amount of nanoantibody added: According to the optimized pH of 9.12, 3, 5, 7, and 10 μL of 1 mg / mL parathion nanoantibody solution were added respectively to obtain different colloidal gold complexes. The appropriate amount of parathion nanoantibody added was determined based on the sensitivity and stability of the final nanoantibody colloidal gold label.

[0065] The colloidal gold-parathion nanoantibody probes with four different contents of parathion nanoantibody solutions were used to detect parathion drug solutions. The results are as follows: Figure 5 As shown, the binding stability and detection effect of colloidal gold and parathion nanoantibody are the best when the content of parathion nanoantibody solution is 3-7 μg / mL.

[0066] (3) Optimization of BSA content: Using the above optimized conditions, 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 nanobody colloidal gold label.

[0067] The four colloidal gold-parathion nanoantibody probes with different BSA contents were used to detect parathion drug solution. The results are as follows: Figure 6 As shown, the binding stability and detection effect of colloidal gold and parathion nanoantibody are the best when the BSA content is 30-40 μL.

[0068] (4) Optimization of colloidal gold reconstitution buffer: Optimization of colloidal gold reconstitution buffer, including colloidal gold reconstitution solution system (using different buffer solutions): PBS, Tris-HCl, BB, PB; different Tris-HCl concentrations of 0.5, 0.2, 0.1, 0.05, 0.02M; different BSA contents of 0.1, 0.2, 0.5, 1, 2%; sucrose content of 1, 2, 5, 10, 15%; Tween-20 content of 0.1, 0.2, 0.5, 1, 2%; and PVP content of 0.1, 0.2, 0.5, 1, 2%. Similarly, the sensitivity and stability of the nanoantibody-colloidal gold marker were used to determine the appropriate reconstitution solution formula.

[0069] After optimizing the formula of colloidal gold reconstitution buffer, colloidal gold-parathion nanoantibody probes prepared with colloidal gold reconstitution buffer were used to detect parathion drug solution. The results are as follows: Figure 7 As shown, when the colloidal gold resolubilization buffer is formulated as: 0.05M Tris-HCl, BSA 0.2%-1%, sucrose 2-10%, Tween-20 0.1%-1% and polyvinyl pyrrolidone 0.3%-1%, the colloidal gold prepared has the best binding stability and detection effect with parathion nanoantibody.

[0070] (5) Optimization of centrifugal conditions: Based on the above optimized conditions, the centrifugal conditions were optimized, including centrifugal temperature, centrifugal speed and centrifugal time. The following settings were made: temperature including 0, 4, 16, 25, 37°C; speed including 8000, 9000, 10000, 12000 rpm; centrifugal time including 10, 15, 20 min. The appropriate centrifugal conditions were also determined based on the sensitivity and stability of the nanobody-colloidal gold marker obtained in the end.

[0071] The colloidal gold-parathion nanoantibody probe was used to detect parathion drug solution under different centrifugal conditions. When the centrifugal conditions were 9000rpm, 4℃, and 15min, the parathion nanoantibody binding stability and detection effect were the best.

[0072] In summary, after optimizing the preparation of parathion nanoantibody colloidal gold label, the final optimization results are: pH 8.5-9.5, parathion nanoantibody addition amount 3-7μg / mL, 10% BSA content 30-40μL, centrifugation conditions 9000rpm, 4℃, 15min. The colloidal gold reconstitution buffer formula is 0.05M Tris-HCl, BSA 0.2%-1%, sucrose 2-10%, Tween-20 0.1%-1% and PVP 0.3%-1%; the parathion nanoantibody colloidal gold label prepared at this time has good stability and high sensitivity.

[0073] Example 2 Preparation of Nano Antibody Colloidal Gold Test Strips for Detecting Parathion Pesticide

[0074] 1. Preparation of bonding release pad

[0075] First, the binding release pad was soaked in the colloidal gold complex solution for 30 minutes and then dried in a 45°C oven. Then, the parathion nanoantibody colloidal gold label solution prepared in Example 1 was sprayed on the treated binding release pad at a rate of 5 μL / cm per hole using an AUTOKUN film spray gold labeling machine. Then, the sprayed binding release pad was placed in a 37°C oven, sealed and dried for 12 hours, and a desiccant was added.

[0076] 2. Preparation of reaction membrane

[0077] The parathion pesticide hapten solution used in the present invention is prepared by referring to the method described in the literature (Xuet al.Analytica Chimica Acta, 2009, 647(1):90-96); the parathion pesticide hapten-carrier protein is obtained by chemically coupling the parathion pesticide hapten and ovalbumin using chemical reagents DMF and NHS.

[0078] The rabbit anti-camel antibody solution used in the present invention is an antibody solution obtained by injecting parathion nano-antibody as an immunogen into a rabbit body and finally separating it from the rabbit serum.

[0079] The parathion pesticide hapten-ovalbumin conjugate was coated on the reaction membrane to form a detection line, and the rabbit anti-camelus antibody was coated on the reaction membrane to form a quality control line.

[0080] Coating process: dilute the parathion pesticide hapten-ovalbumin conjugate to 0.2 mg / mL with phosphate buffer, and coat it on the test line (T line) on the nitrocellulose membrane with an AUTOKUN film-spraying gold labeling machine, with a coating amount of 0.8 μL / cm; dilute the rabbit anti-camel anti-antibody to 1 mg / mL with phosphate buffer, and coat it on the quality control line (C line) on the nitrocellulose membrane with an AUTOKUN film-spraying gold labeling machine, with a coating amount of 0.8 μL / cm. Dry the coated reaction membrane at 37°C for 4 hours and set it aside.

[0081] 3. Preparation of sample pad

[0082] The sample pad was immersed in a 0.2 mol / L phosphate buffer solution containing 1% bovine serum albumin (volume fraction), 1% sucrose (volume fraction), 0.3% polyvinyl pyrrolidone (volume fraction), 0.5% Triton (volume fraction), pH 7.4 for 1 hour, and then dried at 37°C for 4 hours for use.

[0083] 4. Assembly of test strips

[0084] Paste the reaction membrane, binding release pad, sample pad and water absorbent pad on the PVC bottom plate in order; the end of the sample pad is connected to the beginning of the binding release pad, the end of the binding release pad is connected to the beginning of the reaction membrane, the end of the reaction membrane is connected to the beginning of the water absorbent pad, and the binding release pad and the water absorbent pad have a 2mm overlap with the reaction membrane, and the sample pad has a 2mm overlap with the binding release pad. The beginning of the sample pad is aligned with the beginning of the PVC bottom plate, and the end of the water absorbent pad is aligned with the end of the PVC bottom plate; there are detection lines and quality control lines on the reaction membrane, and the detection line (T line) and the quality control line (C line) are both strips perpendicular to the length of the test strip; the detection line is located on the side close to the end of the sample pad; the quality control line is located on the side close to the beginning of the water absorbent pad; the assembled test strip is as follows Figure 8 The test strips are cut into small strips of 4 mm width by a machine and packed in a special sealed bag, which can be stored for 12 months at 4-30°C.

[0085] Example 3 Detection of parathion pesticide residues by nano-antibody colloidal gold test strips

[0086] 1. Detection limit test

[0087] Parathion standard pesticide was added to PBS buffer to a final concentration of 0.2 mg / kg, 0.1 mg / kg, 0.05 mg / kg, 0.025 mg / kg, 0.0125 mg / kg, 0.00625 mg / kg, 0.003125 mg / kg, 0.00156 mg / kg and 0.00078 mg / kg, respectively, and then the test strips prepared in Example 2 were taken for detection, and each sample was tested three times.

[0088] Drop the sample solution vertically onto the sample pad. Start timing when the liquid flows. Let the reaction last for 5 to 8 minutes. Determine the result. The schematic diagram of the test strip test result is shown in the figure. Fig. 9 As shown. The results can be accurately read by the colloidal gold analyzer. Based on the data, a standard curve can be obtained by Origin software.

[0089] The test strip test results are shown in Figure 10. As the concentration of the parathion standard pesticide decreases, the color of the test line of the parathion standard pesticide test results on the test strip gradually deepens. The standard curve obtained by testing different concentration gradients is as follows: Fig.11 As shown, the results corresponding to the curve indicate that the detection range of this method for detecting parathion pesticide residues is 0.0013-0.085 mg / kg, and the detection limit is 0.00069 mg / kg, which meets the national standard detection requirements.

[0090] 2. Specificity test

[0091] The nano antibody colloidal gold test strips of parathion pesticide prepared by the present invention are used to detect 5 mg / kg of quinalphos, methyl parathion, isocarbophos, fenitrothion, profenofos and carbofuran. The pesticides used are all diluted with PBS buffer solution to a concentration of 5 mg / kg.

[0092] Add the sample solution vertically onto the sample pad, start timing when the liquid flows, react for 5 to 8 minutes, and determine the result.

[0093] Read the test results through the colloidal gold analyzer:

[0094] Negative (-): indicates that the concentration of the substance to be tested in the sample is lower than the detection limit;

[0095] Positive (+): indicates that the concentration of the substance to be tested in the sample is equal to or higher than the detection limit;

[0096] Invalid: Indicates that retesting is required.

[0097] The results showed that both the quality control line and the test line of the test strip were colored and negative, indicating that the test strip had no cross reaction to 5mg / kg quinalphos, triazophos, methyl parathion, isocarbothion, fenitrothion, profenofos and carbofuran.

[0098] 3. Stability test

[0099] The nano-antibody colloidal gold test strips for parathion pesticide prepared in Example 2 were placed in a sealed bag with a built-in desiccant and stored in an oven at 45°C. They were taken out after 3 days, 7 days, 14 days, 21 days, 28 days, and 30 days, and the parathion standard was diluted with PBS to a solution of 0.01 mg / kg, and then the stability of the test strips was tested.

[0100] The results are as follows Fig.12 As shown in the figure, the test results did not change significantly after being stored at 45℃ for 3, 7, 14, 21 and 28 days. After being stored at 45℃ for 30 days, the re-dissolution effect of the colloidal gold-labeled antibody on the gold label pad decreased, the color of the T line and C line became lighter, and the sensitivity decreased slightly. Storage at 45℃ for 30 days is equivalent to storage at room temperature for 12 months.

[0101] Example 4 Detection and Analysis of Parathion Pesticide Residues in Fruit and Vegetable Crops

[0102] The commercially purchased cabbage, cucumber and orange samples were washed and dried, and 10g (±0.1g) of homogenized samples were weighed into a 50mL plastic centrifuge tube. Each sample was divided into two groups, negative samples and positive samples. Negative samples were not treated in any way, and positive samples were added with parathion standard until the drug concentration reached 0.01mg / kg. Then 10mL of acetonitrile was added to both negative and positive samples, and vortexed to mix; the extraction package was added, and the vortex was continued for 2min, centrifuged at 5000rpm / min for 3min, and allowed to stand at room temperature. The supernatant solution was added to the purification tube, vortexed for 2min, centrifuged at 5000rpm / min for 3min, and the supernatant was taken, nitrogen blown at 37 degrees, and then re-dissolved with EDTA solution.

[0103] Take the test strip prepared in Example 2 for testing, vertically drip the treated sample solution onto the sample pad, start timing when the liquid flows, react for 5 to 8 minutes, and determine the result.

[0104] Read the test results through the colloidal gold analyzer:

[0105] Negative (-): indicates that the concentration of the substance to be tested in the sample is lower than the detection limit;

[0106] Positive (+): indicates that the concentration of the substance to be tested in the sample is equal to or higher than the detection limit;

[0107] Invalid: Indicates that retesting is required.

[0108] The result diagram of the test strip prepared by the present invention is as follows: Fig.13As shown, when testing cabbage, cucumber and orange samples, we can see that both T line and C line in negative samples are colored, while the color of C line in positive samples is obviously stronger than that of T line. This shows that the detection results of the parathion detection method of the present invention for actual samples can also meet the requirements.

[0109] 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 parathion pesticide colloidal gold label based on nano antibodies, characterized in that: The preparation method is as follows: adding 3 to 7 μg / mL parathion nano-antibody to a colloidal gold solution with a pH value of 8.5 to 9.5, mixing and standing, then adding 30 to 40 μL of 10% BSA, standing and centrifuging, discarding the supernatant, and resuspending the precipitate with a colloidal gold resolubilization buffer to obtain a parathion nano-antibody colloidal gold marker; The colloidal gold solution is prepared by using 1% chloroauric acid and 1% trisodium citrate in a volume ratio of 1:6; The colloidal gold re-dissolving buffer solution comprises: 0.5M Tris-HCl, 0.2% to 2% BSA, 0.1% to 1% Tween-20, 0.3% to 1% polyvinyl pyrrolidone and 2% to 10% sucrose.

2. Use of the parathion nanoantibody colloidal gold marker according to claim 1 in immunochromatography technology or in detecting parathion pesticides.

3. Use of the parathion nano-antibody colloidal gold marker according to claim 1 in the preparation and detection of parathion pesticide products.

4. A nano antibody colloidal gold test strip for detecting parathion pesticide, characterized in that: The parathion nanobody colloidal gold label according to claim 1 is contained on the binding and releasing pad.

5. The test strip according to claim 4, characterized in that: Also includes sample pads, reaction membranes, absorbent pads and base plates.

6. The test strip according to claim 5, characterized in that: The reaction membrane is provided with a detection line coated with a parathion pesticide hapten-carrier protein conjugate and a quality control line coated with a rabbit anti-camel antibody.

7. Use of the test strip according to any one of claims 4 to 6 in detecting parathion pesticide residues in fruit and vegetable crops.

8. A method for detecting parathion pesticide in crops, characterized in that: The sample is tested using the test strip according to any one of claims 4 to 6, and the residual situation of the parathion pesticide is determined according to the color development result of the test strip.

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