CN109061155B - Test strip for detecting metalaxyl and preparation method and application thereof - Google Patents
Test strip for detecting metalaxyl and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a test strip for detecting metalaxyl and a preparation method and application thereof, wherein the test strip comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate; the method is characterized in that: the reaction film is provided with a detection line coated with metalaxyl hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse anti-antibody, and the conjugate release pad is sprayed with a metalaxyl monoclonal antibody-colloidal gold marker. The metalaxyl hapten is obtained by reacting N- (2, 6-dimethylphenyl) alanine methyl ester with (4-nitrophenoxy) acetyl chloride to generate nitrophenylmetalaxyl and then reducing the nitrophenylmetalaxyl by zinc powder. The invention also provides a method for detecting metalaxyl in a sample by applying the test strip. The test strip and the detection method provided by the invention have the advantages of high sensitivity, good specificity, simplicity in operation, high detection speed, low cost and no limitation of detection equipment, and can realize rapid detection and on-site monitoring of metalaxyl in a large batch of samples.
Description
Technical Field
The invention relates to detection of metalaxyl, in particular to a test strip for detecting metalaxyl, a preparation method and application thereof, which are particularly suitable for detecting metalaxyl residue in tobacco leaves.
Background
Metalaxyl (Metalaxyl) is a strong systemic substituted benzamide fungicide, has a chemical name of N- (2-methoxyacetyl) -N- (dimethylphenyl) -rac-aminobenzoic acid, belongs to amide fungicides, has high efficiency and low toxicity, and is mainly used for various downy mildew, late blight, early blight, damping-off, blight, fruit rot and the like caused by oomycetes, phycomycetes and fungi. Mainly inhibits the synthesis of protein in the hypha of the pathogenic bacteria, so that the hypha of the pathogenic bacteria is lack of nutrition and can not grow normally to die. The plant pesticide has strong systemic and osmotic force, can be conducted in the plant body in two directions up and down 30min after the pesticide is applied, has the protection and treatment effects on plant diseases, and has better curative effects on controlling frost virus diseases and epidemic diseases of melons, fruits, vegetables and tobacco.
However, metalaxyl is one of the important pollutants in the environment, and poses potential health threats to human beings and pollutes the ecological environment, so that the residual problem in the production of fruits, vegetables and tobaccos is receiving more and more attention. China sets the maximum residue limit standard of metalaxyl for different crops, wherein the maximum residue limit of cucumber, pepper and tomato is 0.5 mg/kg, the maximum residue limit of brown rice is 0.1 mg/kg, and the maximum residue limit of other grains is 0.05 mg/kg. The international cooperation center for tobacco science research (CORESTA) stipulates that the directive residual limit of metalaxyl in tobacco is 2 mg/kg, and in actual production, 2 mg/kg is taken as the maximum residual quantity judgment standard of tobacco.
At present, the detection methods for metalaxyl residue at home and abroad mainly comprise a gas chromatography-mass spectrometry combined method, a high performance liquid chromatography-mass spectrometry combined method, a gas chromatography and a high performance liquid chromatography. The instrument and the method have the advantages of high detection sensitivity, strong specificity and the like, but the pretreatment of a detection sample is complicated and time-consuming, the sample also needs to be extracted and purified, and meanwhile, the instrument and the detection method need expensive large-scale instruments and equipment and are equipped with professional detection technicians for operation and management, so that the field large-scale detection cannot be carried out, the timeliness is poor, and the popularization is difficult. Therefore, it is an urgent problem to develop a product and a method which are not limited by the detection equipment and can realize rapid detection of a large number of samples.
The invention patent (201510530296.2) discloses a test paper for detecting metalaxyl residue and application thereof, and the biggest difference of the test paper is that the test paper is obviously different from a hapten in structure and a preparation method. It is known that the key to establishing an immunoassay method for small molecular compounds is the ability to prepare antibodies with high affinity and high selectivity to small molecular compounds, and the synthesis of haptens with end groups as functional groups and connecting arms of a certain length is an important process for preparing antibodies against small molecular compounds and establishing a corresponding immunoassay method. Any structural differences between the hapten and the target analyte, such as topological characteristics of molecular size, shape, composition, configuration, conformation, polarity, electron cloud density, etc., can greatly affect the properties of the corresponding antibody. Whether hapten with better performance and effect can be designed and synthesized or not is the focus of the invention, so that the antibody with high sensitivity and good specificity can be obtained.
Disclosure of Invention
The invention aims to overcome the characteristics of high equipment dependence and incapability of realizing rapid detection on a large batch of samples in the conventional metalaxyl detection method, and provides the test strip which is simple to operate, high in sensitivity, high in detection speed, low in cost and not limited by detection equipment, the preparation method and the application thereof so as to realize rapid detection and field monitoring on the large batch of metalaxyl samples.
In order to achieve the purpose of the invention, the invention provides a test strip for detecting metalaxyl, which comprises a sample absorption pad, a conjugate 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 metalaxyl hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody, the conjugate release pad is sprayed with a metalaxyl monoclonal antibody-colloidal gold marker, the metalaxyl monoclonal antibody is prepared by taking the metalaxyl hapten-carrier protein conjugate as an immunogen, the metalaxyl hapten-carrier protein conjugate is obtained by coupling the metalaxyl hapten and carrier protein, the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroxine or human serum albumin, the metalaxyl hapten is obtained by reacting N- (2, 6-dimethylphenyl) alanine methyl ester with (4-nitrophenoxy) acetyl chloride to generate nitrophenyl metalaxyl and then reducing the nitrophenyl metalaxyl with zinc powder, the molecular structural formula is as follows:
the specific preparation method of the metalaxyl hapten comprises the following steps:
1) taking 2.00 g of N- (2, 6-dimethylphenyl) alanine methyl ester, adding pyridine to fully dissolve the N- (2, 6-dimethylphenyl) alanine methyl ester, adding 2.30 g of (4-nitrophenoxy) acetyl chloride, stirring, heating in an oil bath, reacting at 60 ℃ for 2 hours, detecting by TLC (thin layer chromatography), stopping the reaction when the raw materials basically react completely, recovering to room temperature, carrying out rotary evaporation, removing pyridine, adding 60 mL of water, adjusting the pH value to 5 by using 1 mol/L hydrochloric acid, adding 80 mL of ethyl acetate for extraction, drying an organic phase anhydrous sodium sulfate, loading the organic phase on a silica gel column, and eluting and separating by using ethyl acetate/N-hexane with the volume ratio of 1:10 to obtain 3.68 g of an intermediate product, namely;
2) taking 3.60 g of the intermediate product nitrophenyl metalaxyl, and adding ethanol to dissolve the intermediate product nitrophenyl metalaxyl to obtain solution A; taking 1.80 g of zinc powder, adding 10 mL of distilled water, adding 0.5 mL of diluted hydrochloric acid, activating for 20 min at 60 ℃, adding the solution A, continuing stirring for 3h, detecting by TLC, stopping the reaction after all the raw materials are reacted, performing suction filtration to remove the zinc powder, evaporating the filtrate to dryness, adding 50 mL of water, adjusting the pH value to 7 by using sodium carbonate, adding 50 mL of ethyl acetate for extraction, washing an organic phase with water, drying and evaporating anhydrous sodium sulfate, and recrystallizing by using dichloromethane/n-hexane with the volume ratio of 1:2 to obtain 3.22 g of a metalaxyl hapten product.
The goat anti-mouse anti-antibody is obtained by immunizing a goat by taking a murine antibody as an immunogen.
The sample absorption pad, the conjugate release pad, the reaction membrane and the water absorption pad are sequentially adhered to the bottom plate, and the conjugate release pads 1/3-1/2 are covered under the sample absorption pad.
The bottom plate is a PVC bottom plate or other hard non-absorbent materials; the sample absorption pad is absorption filter paper or oil filter paper; the conjugate release pad is made of glass wool or polyester material; the water absorption pad is absorbent paper; the reaction membrane is a nitrocellulose membrane or a cellulose acetate membrane.
A method for preparing the test strip comprises the following steps:
1) preparing a conjugate release pad sprayed with metalaxyl monoclonal antibody-colloidal gold marker;
2) preparing a reaction film with a detection line coated with metalaxyl hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse anti-antibody;
3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.
More specifically, the process for preparing the test strip is as follows:
1) reacting N- (2, 6-dimethylphenyl) alanine methyl ester with (4-nitrophenoxy) acetyl chloride to generate nitrophenylmetalaxyl, and reducing by zinc powder to prepare metalaxyl hapten;
2) coupling metalaxyl hapten and carrier protein to prepare a metalaxyl hapten-carrier protein conjugate;
3) immunizing a mouse by using the metalaxyl hapten-carrier protein conjugate, and fusing and screening spleen cells and myeloma cells of the mouse to obtain a hybridoma cell strain secreting a metalaxyl monoclonal antibody;
4) extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibodies;
5) coating the metalaxyl hapten-carrier protein conjugate and the goat anti-mouse anti-antibody on a detection line (T) and a quality control line (C) of a reaction film respectively;
6) preparing colloidal gold by reacting trisodium citrate with chloroauric acid;
7) adding the prepared metalaxyl monoclonal antibody into the prepared colloidal gold to obtain a metalaxyl monoclonal antibody-colloidal gold marker;
8) spraying metalaxyl monoclonal antibody-colloidal gold marker on a conjugate release pad, drying at 37 ℃ for 1h, taking out, and storing in a dry environment for later use;
9) soaking the sample absorption pad in 0.1 mol/L phosphate buffer solution containing 0.5% bovine serum albumin (mass fraction) and having pH value of 7.2 for 2h, and drying at 37 deg.C for 2 h;
10) a sample absorbing pad, a conjugate releasing pad, a reaction membrane and a water absorbing pad are sequentially adhered on the bottom plate, and the conjugate releasing pad is covered by the sample absorbing pad from the 1/3 area at the starting end. And finally cutting into small strips with the width of 3 mm, adding a plastic box, carrying out vacuum packaging, and storing for 12 months at the temperature of 4-30 ℃. Covering 1/3 of the conjugate release pad with the sample absorption pad can prolong the observation time of the detection result, and make the sample absorption pad sufficiently absorb the detection liquid and sufficiently react with the gold-labeled antibody, thereby reducing errors.
The method for detecting metalaxyl in a sample by applying the test strip comprises the following steps:
1) pretreating a sample;
2) detecting by using a test strip;
3) and analyzing the detection result.
The test strip for detecting metalaxyl adopts a highly specific antibody-antigen reaction and immunochromatographic analysis technology to fix a metalaxyl monoclonal antibody-colloidal gold marker on a conjugate release pad, and metalaxyl in a sample is combined with the metalaxyl monoclonal antibody-colloidal gold marker on the conjugate release pad in the flowing process to form the metalaxyl-antibody-colloidal gold marker. Metalaxyl in the sample competes with a metalaxyl hapten-carrier protein conjugate on a reaction film detection line to combine with a metalaxyl monoclonal antibody-colloidal gold marker, and whether metalaxyl residue exists in the sample liquid to be detected is judged according to the depth of a red strip of the detection line.
During detection, a sample is treated and then dripped into a sample absorption pad, when the concentration of metalaxyl in the sample is lower than the detection limit or zero, the monoclonal antibody-colloidal gold marker is combined with a metalaxyl hapten-carrier protein conjugate fixed on a reaction film in the chromatography process, a red strip is respectively generated at a detection line (T) and a quality control line (C), and the color development of the T line is deeper than that of the C line or is consistent with that of the C line; if the concentration of metalaxyl in the sample is equal to or above the limit of detection, the monoclonal antibody-colloidal gold label will bind to all of the metalaxyl and thus no red band will appear or the color will be lighter than that of line C at line T because the competitive reaction will not bind to the metalaxyl hapten-carrier protein conjugate. As shown in fig. 2.
Negative: when the quality control line (C) shows a red strip, the detection line (T) also shows a red strip, and the color of the line (T) is close to or deeper than that of the line (C), the line (C) is judged to be negative.
Positive: and when the quality control line (C) shows a red strip, the detection line (T) does not show color or the color of the line (T) is lighter than that of the line (C), the test line is judged to be positive.
And (4) invalidation: when the quality control line (C) does not show a red strip, the test strip is judged to be invalid whether the detection line (T) shows a red strip or not.
The molecular structure of the hapten in 201510530296.2 and the modification sites in the selected molecular structure of the hapten are obviously different, and the spatial structures of hapten modifications formed by different modification sites are different, namely, antigenic determinants exposed on the surface of the artificial antigen generated after the artificial antigen is combined with a carrier are different, so that the generated antibody has differences in titer, affinity and specificity.
The hapten has proper terminal active groups, the length of a modification site and a spacer arm is properly selected, and the molecular structure of metalaxyl can be simulated to the greatest extent. Meanwhile, the test strip has the advantages of low cost, simple operation, short detection time, no limitation of detection equipment, suitability for various units, simple storage and long quality guarantee period. The method for detecting metalaxyl by using the test strip of the invention is simple, convenient, rapid, visual, accurate, wide in application range, low in cost and easy to popularize and use.
Drawings
Fig. 1 is a schematic diagram of a cross-sectional structure of a test strip, in which: 1. a sample absorbing pad; 2. a conjugate release pad; 3. a reaction film; 4. a water absorbent pad; 5. detecting lines; 6. a quality control line; 7. a base plate;
FIG. 2 is a diagram showing the test result of the test strip;
FIG. 3 is a synthetic diagram of metalaxyl hapten (the figure is taken as an abstract figure).
Detailed Description
The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. In addition, various changes or modifications of the present invention may be made by those skilled in the art within the scope defined by the appended claims, and these changes or modifications should also fall within the scope of the present invention.
Example 1 preparation of test strip for detecting metalaxyl
The preparation method of the test strip mainly comprises the following steps:
1) preparing a conjugate release pad sprayed with metalaxyl monoclonal antibody-colloidal gold marker;
2) preparing a reaction film with a detection line coated with metalaxyl hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse anti-antibody;
3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a PVC base plate to form the test strip.
The following steps are detailed:
1. synthesis and identification of metalaxyl hapten (synthetic route is shown in figure 3)
Taking 2.00 g of N- (2, 6-dimethylphenyl) alanine methyl ester, adding pyridine to fully dissolve the N- (2, 6-dimethylphenyl) alanine methyl ester, adding 2.30 g of (4-nitrophenoxy) acetyl chloride, stirring, heating in an oil bath, reacting at 60 ℃ for 2 hours, detecting by TLC (thin layer chromatography), stopping the reaction when the raw materials basically react completely, recovering to room temperature, carrying out rotary evaporation, removing pyridine, adding 60 mL of water, adjusting the pH value to 5 by using 1 mol/L hydrochloric acid, adding 80 mL of ethyl acetate to extract, drying an organic phase anhydrous sodium sulfate, loading the organic phase on a silica gel column, eluting and separating by using ethyl acetate/N-hexane with the volume ratio of 1:10 to obtain 3.68 g of an intermediate product, namely nitrophenylmetalaxyl, and;
taking 3.60 g of the intermediate product nitrophenyl metalaxyl, and adding ethanol to dissolve the intermediate product nitrophenyl metalaxyl to obtain solution A; taking 1.80 g of zinc powder, adding 10 mL of distilled water, adding 0.5 mL of diluted hydrochloric acid, activating at 60 ℃ for 20 min, adding the solution A, continuously stirring for 3h, detecting by TLC, stopping the reaction after all the raw materials are reacted, performing suction filtration to remove the zinc powder, evaporating the filtrate to dryness, adding 50 mL of water, adjusting the pH value to 7 by using sodium carbonate, adding 50 mL of ethyl acetate for extraction, washing an organic phase with water, drying and evaporating anhydrous sodium sulfate, recrystallizing by using dichloromethane/n-hexane with the volume ratio of 1:2 to obtain 3.22 g of a metalaxyl hapten product, wherein the yield is 96.99%.
Nuclear magnetic identification1H NMR（CDCl3300 MHz) δ: 4.778 (2H, q, J = 7.047), 3.646 (3H), 1.104 (3H, d, J = 7.047), 4.52 (t, 1H), 7.313 (1H, dd, J = 7.888), 2.260 (t, 6H), 7.41 (1H, dd, J = 7.888), 6.865 (1H, t, J = 7.888), 6.858 (1H, ddd, J =8.804, J =0.545, J = 0.000), 6.272 (t, 2H). In the map, the chemical shift delta =6.272 is the resonance absorption peak of the phenyl ring amino hydrogen on the spacer arm, the chemical shift delta =6.858 and 6.856 are the resonance absorption peaks of the phenyl ring hydrogen on the spacer arm, and the existence of the peaks proves that the spacer arm coupling is successful and the metalaxyl hapten is correct in structure.
2. Synthesis and identification of metalaxyl coupling antigen
Immunogen preparation-metalaxyl hapten is coupled with Bovine Serum Albumin (BSA) to obtain the immunogen.
Adding 0.1 mL of 1 mol/L diluted hydrochloric acid into 8 mg of metalaxyl hapten, adding 0.8 mL of distilled water, stirring at the low temperature of 0-5 ℃ for 20 min, adding 0.1 mL of aqueous solution containing 1.7 mg of sodium nitrite, and continuously stirring for 1h to obtain solution A; and (3) dissolving 50 mg of BSA in 6 mL of 0.1 mol/L sodium carbonate solution, stirring at 0-5 ℃ and balancing the temperature for 20 min to obtain solution B, dripping the solution A into the solution B, and continuing to react for 2 h. Stopping reaction, dialyzing with 0.02 mol/L PBS for 3 d, changing liquid three times per day, and packaging to obtain immunogen, and storing at-20 deg.C.
Preparation of coating antigen-coupling metalaxyl hapten and Ovalbumin (OVA) to obtain the coating antigen.
Taking 6 mg metalaxyl hapten, adding 0.7 mL of 1 mol/L diluted hydrochloric acid, adding 0.8 mL of distilled water, stirring at the low temperature of 0-5 ℃ for 20 min, adding 0.1 mL of aqueous solution containing 1.3 mg of sodium nitrite, and continuously stirring for 1h to obtain solution A; and (3) taking 60 mg of OVA, adding 6 mL of 0.1 mol/L sodium carbonate solution for dissolving, stirring at 0-5 ℃ for balancing the temperature for 20 min to obtain a solution B, dripping the solution A into the solution B, and continuing to react for 2 h. Stopping reaction, dialyzing with 0.02 mol/L PBS for 3 d, changing liquid three times per day, and packaging to obtain coating antigen, and storing at-20 deg.C for use.
And (3) carrying out ultraviolet (200-400 nm) scanning measurement according to the proportion of the hapten, the carrier protein and the coupling product used in the synthetic metalaxyl coupling antigen reaction, and calculating the binding ratio of the hapten, the carrier protein and the coupling product by comparing the absorbance values of the hapten, the carrier protein and the coupling product at 260 nm and 280 nm respectively. The maximum absorption peak of the conjugate metalaxyl hapten-carrier protein is obviously changed compared with the maximum absorption peaks of the metalaxyl hapten and the carrier protein, which indicates that the synthesis of the metalaxyl hapten-carrier protein is successful. The binding ratio of hapten to BSA was calculated to be 13:1 and binding ratio to OVA was calculated to be 10: 1.
3. Preparation of metalaxyl monoclonal antibody
1) Obtaining hybridoma cells
First immunization: the metalaxyl hapten-BSA conjugate (immunogen) was emulsified well with an equal amount of Freund's complete adjuvant and injected subcutaneously into 6-week-old Balb/c mice, 0.2 mL each;
two booster immunizations: from the first immunization, boosting once every two weeks, and replacing Freund's complete adjuvant with Freund's incomplete adjuvant in the same method and dosage as the first immunization;
after one week of last boosting immunization, measuring the titer and inhibition in fundus venous blood sampling, and performing the following last immunization when the titer reaches more than 1: 10000: injecting 0.1 mL of immunogen solution without any adjuvant into the abdominal cavity, killing the mouse after three days, and fusing the spleen with myeloma cells;
and (3) measuring cell supernatant by adopting an indirect competitive enzyme-linked immunoassay method, and screening positive holes. Cloning the positive hole by using a limiting dilution method to obtain and establish a hybridoma cell strain which stably secretes the metalaxyl monoclonal antibody, preparing the hybridoma cells in the logarithmic growth phase into cell suspension by using a freezing medium, subpackaging in a freezing tube, and storing in liquid nitrogen for a long time.
2) Preparation of monoclonal antibodies
Cell recovery: taking out the frozen tube of the monoclonal antibody hybridoma cell strain of metalaxyl, immediately putting the frozen tube into a water bath at 37 ℃ for medium-speed melting, centrifuging to remove frozen stock solution, and transferring the frozen stock solution into a culture bottle for culture;
preparing ascites and purifying antibodies: sterilizing Balb/c mice (8 weeks old) by intraperitoneal injection by in vivo inductionParaffin oil 0.5 mL/mouse, injecting hybridoma cell 5X 10 in abdominal cavity after 7 days5Ascites were collected 7 days later. Purifying by octanoic acid-saturated ammonium sulfate method to obtain metalaxyl monoclonal antibody solution (preservation at-20 deg.C).
3) Determination of the potency of monoclonal antibodies
The titer of the antibody is 1 (100000-300000) by using an indirect competition ELISA method.
Indirect competitive ELISA method: coating an enzyme label plate with a metalaxyl hapten-OVA conjugate, adding a metalaxyl standard substance solution, a metalaxyl monoclonal antibody solution and a horseradish peroxidase-labeled goat anti-mouse anti-antibody solution, reacting for 30min at 25 ℃, pouring out liquid in a hole, washing for 3-5 times with a washing solution, and patting dry with absorbent paper; adding a substrate color developing solution, reacting for 15 min at 25 ℃, and adding a stop solution to stop the reaction; the microplate reader was set to measure the absorbance value per well at a wavelength of 450 nm.
4) Determination of monoclonal antibody specificity
Antibody specificity refers to the comparison of its ability to bind to a specific antigen with the ability to bind to such antigen analogs, often using cross-reactivity as an evaluation criterion. The smaller the cross-reactivity, the higher the specificity of the antibody.
In the experiment, metalaxyl and compounds (propyzamide, alachlor, acetochlor, metolachlor, pretilachlor and butachlor) with similar structures are serially diluted, respectively subjected to indirect competitive ELISA with monoclonal antibodies, a standard curve is prepared, and IC is obtained by analysis50Then, the cross-reactivity was calculated as follows:
the results show that the cross-reactivity of metalaxyl and its structural analogues is: 100 percent of metalaxyl, less than 1 percent of propyzamide, less than 1 percent of alachlor, less than 1 percent of acetochlor, less than 1 percent of metolachlor, less than 1 percent of pretilachlor and less than 1 percent of butachlor. The antibody of the invention has no cross reaction to compounds with similar structures to metalaxyl, such as propyzamide, alachlor, acetochlor, metolachlor, pretilachlor, butachlor and the like, and only has specific binding to the metalaxyl.
4. Preparation of goat anti-mouse anti-antibody
The sheep is taken as an immune animal, and the pathogen-free sheep is immunized by taking the murine antibody as an immunogen to obtain the goat anti-mouse antibody.
5. Preparation of metalaxyl monoclonal antibody-colloidal gold marker
1) Preparation of colloidal gold
Diluting chloroauric acid solution with the mass fraction of 1% to 0.01% by using double-distilled deionized water, placing 100 mL into a conical flask, heating to boiling by using a constant-temperature electromagnetic stirrer, adding 1.5 mL of trisodium citrate solution with the mass fraction of 1% under continuous high-temperature and continuous stirring, continuing stirring at a constant speed and heating until the solution is bright wine red, stopping heating until the solution is cooled to room temperature, recovering the volume of the solution to the original volume by using deionized water, and storing at 4 ℃. The prepared colloidal gold is clear and transparent by naked eye observation, has no turbidity, has no floating object on the liquid surface, and is wine red when observed in sunlight.
2) Preparation of metalaxyl monoclonal antibody-colloidal gold marker
Under magnetic stirring, 0.2 mol/L potassium carbonate solution is used for adjusting the pH value of the colloidal gold to 7.2 (the labeling range of the pH values of different antibodies can be changed from 7 to 8), the metalaxyl monoclonal antibody is added into the colloidal gold solution according to the standard that 20-50 mu g of the antibody is added into each milliliter of the colloidal gold solution, the mixture is stirred and uniformly mixed, the mixture is kept stand for 10 min at room temperature, 10 percent BSA is added to ensure that the final mass fraction of the mixture in the colloidal gold solution is 1 percent, and the mixture is kept stand for 10 min. 12000 r/min, 4 ℃ centrifugation for 40 min, abandoning the supernatant, washing the precipitate twice with a redissolving buffer solution, resuspending the precipitate with the redissolving buffer solution with the volume of 1/10 of the initial volume of the colloidal gold, and standing at 4 ℃ for standby.
Redissolving buffer solution: 0.1-0.3 percent of BSA, 0.05-0.2 percent of Tween-80 and 0.02 mol/L of phosphate buffer solution with the pH value of 7.2.
6. Preparation of conjugate Release pad
The conjugate release pad was soaked in 0.5 mol/L phosphate buffer containing 0.5% BSA (mass fraction) and having a pH of 7.2, soaked for 1h uniformly, and baked at 37 ℃ for 3h for use. Uniformly spraying the prepared metalaxyl monoclonal antibody-colloidal gold marker on a conjugate release pad by using an Isoflow film spraying instrument, spraying 0.01 mL of metalaxyl monoclonal antibody-colloidal gold marker on each 1 cm of conjugate release pad, placing the mixture in an environment at 37 ℃ (humidity is less than 20%) for 60 min, taking out, and placing the mixture in a dry environment (humidity is less than 20%) for storage.
7. Preparation of the reaction film
Coating the metalaxyl hapten-ovalbumin conjugate on a reaction membrane to form a detection line, and coating the goat anti-mouse anti-antibody on the reaction membrane to form a quality control line.
Coating process: diluting metalaxyl hapten-ovalbumin conjugate to 1 mg/mL by using phosphate buffer solution, coating the metalaxyl hapten-ovalbumin conjugate on a detection line (T) on a nitrocellulose membrane by using an Isoflow point membrane instrument, wherein the coating amount is 1.0 mu L/cm; the goat anti-mouse anti-antibody was diluted to 200. mu.g/mL with 0.01 mol/L, pH value of 7.4 phosphate buffer and coated on a quality control line (C) on a nitrocellulose membrane in an amount of 1.0. mu.L/cm using an Isoflow dot-membrane apparatus. And (3) drying the coated reaction membrane for 2 hours at 37 ℃ for later use.
8. Preparation of sample absorbent pad
Soaking the sample absorption pad in 0.1 mol/L phosphate buffer solution containing 0.5% bovine serum albumin (mass fraction) and having pH value of 7.2 for 2h, and drying at 37 deg.C for 2h for later use.
9. Assembly of test strips
Sequentially adhering a sample absorption pad, a conjugate release pad, a reaction membrane and a water absorption pad on a PVC bottom plate; the binder release pad is covered by the sample absorption pad from the 1/3 area at the starting end, the tail end of the binder release pad is connected with the starting end of the reaction film, the tail end of the reaction film is connected with the starting end of the water absorption pad, the starting end of the sample absorption pad is aligned with the starting end of the PVC soleplate, and the tail end of the water absorption pad is aligned with the tail end of the PVC soleplate; the reaction membrane is provided with a detection line and a quality control line, and the detection line (T) and the quality control line (C) are strip-shaped strips which are vertical to the long phase of the test strip; the detection line is located on the side near the end of the conjugate release pad; the quality control line is positioned on the side away from the end of the conjugate release pad; the test paper strip is cut into small strips with the width of 3 mm by a machine, and the small strips are arranged in a special plastic card and can be stored for 12 months at the temperature of 4-30 ℃.
Example 2 detection of metalaxyl in samples
1. Pretreatment of samples
1) The method for pretreating the flue-cured tobacco sample comprises the following steps: weighing 1.0 +/-0.05 g of crushed sample into a 50 mL centrifuge tube, adding 10 mL of 50% methanol aqueous solution, and fully crushing the sample by using a homogenizer; transferring the smashed sample into an injector, and filtering by using a filter membrane; and adding 900 mu L of deionized water into 100 mu L of filtered sample liquid, uniformly mixing and checking.
2) The pretreatment method of the fresh tobacco leaf sample comprises the following steps: weighing 2.0 +/-0.05 g of crushed sample into a 50 mL centrifuge tube, adding 10 mL of 50% methanol aqueous solution, and fully crushing the sample by using a homogenizer; transferring the smashed sample into an injector, and filtering by using a filter membrane; and (3) adding 300 mu L of deionized water into 100 mu L of filtered sample liquid, uniformly mixing, and checking.
2. Detection with test strip
And (3) taking 70 mu L of sample solution to be detected by using a pipettor, vertically dripping the sample solution into the sample adding hole, timing when the liquid flows, reacting for 10 min, and judging the result.
3. Analyzing the results of the detection
Negative (-): the color development of the T line is darker than that of the C line or is consistent with that of the C line, which indicates that the concentration of metalaxyl in the sample is lower than the detection limit, as shown in FIGS. 2a and 2 b.
Positive (+): the color development of the T line is lighter than that of the C line or the T line is not developed, which indicates that the concentration of metalaxyl in the sample is equal to or higher than the detection limit, as shown in FIGS. 2C and 2 d.
And (4) invalidation: the absence of line C indicates an incorrect procedure or the test strip has failed, as shown in fig. 2e and 2 f. In this case, the instructions should be read carefully again and retested with a new test strip.
Example 3 sample testing example
1. Limit of detection test
Taking a blank tobacco leaf sample after baking, and adding metalaxyl into the blank tobacco leaf sample until the final concentration is 1, 2 and 4 mg/kg respectively; taking a blank fresh tobacco leaf sample, adding metalaxyl to the blank fresh tobacco leaf sample until the final concentration is 0.25, 0.5 and 1.0 mg/kg respectively, taking a test strip for detection, and repeatedly measuring each sample for three times.
When the test strip is used for detecting a sample of the flue-cured tobacco leaves, when the adding concentration of metalaxyl is 1 mg/kg, the test strip shows that the color development of a T line is darker than that of a C line or is consistent with that of the C line, and the test strip is negative; when the adding concentration of the metalaxyl is 2 mg/kg and 4 mg/kg, the test strip shows that the color development of the T line is lighter than that of the C line or the color development of the T line is not shown, and the test strip is positive, which shows that the detection limit of the test strip on the metalaxyl in the flue-cured tobacco leaves is 2 mg/kg; when the test strip is used for detecting a fresh tobacco sample, when the adding concentration of metalaxyl is 0.25 mg/kg, the test strip shows that the color development of a T line is darker than that of a C line or is consistent with that of the C line, and the test strip is negative; when the adding concentration of the metalaxyl is 0.5 and 1.0 mg/kg, the test strip shows that the color development of the T line is lighter than that of the C line or the T line is not developed and is positive, which shows that the detection limit of the test strip on the metalaxyl in fresh tobacco leaves is 0.5 mg/kg.
2. Test for false positive and false negative rates
20 parts of baked tobacco positive samples with known metalaxyl content of more than 2 mg/kg and 20 parts of fresh tobacco positive samples with known metalaxyl content of less than 2 mg/kg and 20 parts of baked tobacco negative samples with known metalaxyl content of less than 0.5 mg/kg and 20 parts of fresh tobacco negative samples with known metalaxyl content of less than 0.5 mg/kg are respectively detected by three test strips, and the negative and positive rates of the samples are calculated.
The results show that: when 3 batches of test strips are used for detecting positive samples, the results are all positive, the coincidence rate of the positive samples is 100 percent, and the false negative rate is 0; when the negative samples are detected, the results are all negative, and the coincidence rate of the negative samples is 100 percent, and the false positive rate is 0. The test strip for detecting metalaxyl can be used for quickly detecting the metalaxyl in the tobacco leaves.
3. Specificity test
Metalaxyl structure analogues such as propamocarb, alachlor, acetochlor, metolachlor, pretilachlor and butachlor are diluted to 50 mg/L by phosphate buffer solution with the pH value of 7.2 and 0.2 mol/L, and detection is carried out by using a metalaxyl test strip. The result shows that when the test strip is used for detecting 50 mg/L propachlor, alachlor, acetochlor, metolachlor, pretilachlor and butachlor, the test strip has the negative result that the color development of a T line is darker than that of a C line or is consistent with that of the C line. The test paper strip has no cross reaction to metalaxyl structural analogues such as propyzamide, alachlor, acetochlor, metolachlor, pretilachlor and butachlor.
Claims (5)
1. A test strip for detecting metalaxyl comprises a sample absorption pad, a conjugate release pad, a reaction film, a water absorption pad and a bottom plate; the method is characterized in that: the reaction film is provided with a detection line coated with metalaxyl hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse anti-antibody, and the conjugate release pad is sprayed with a metalaxyl monoclonal antibody-colloidal gold marker; the metalaxyl monoclonal antibody is prepared by taking a metalaxyl hapten-carrier protein conjugate as an immunogen, the metalaxyl hapten-carrier protein conjugate is obtained by coupling metalaxyl hapten and carrier protein, the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein or human serum albumin, the metalaxyl hapten is obtained by reacting N-2, 6-dimethylphenyl-alanine methyl ester with 4-nitrophenoxy-acetyl chloride to generate nitrophenyl metalaxyl and reducing the nitrophenyl metalaxyl by zinc powder, and the molecular structural formula of the metalaxyl monoclonal antibody is as follows:
the reaction formula of the specific preparation reaction process is as follows:
2. the test strip of claim 1, wherein: the sample absorption pad, the conjugate release pad, the reaction membrane and the water absorption pad are sequentially adhered to the bottom plate, and the conjugate release pads 1/3-1/2 are covered under the sample absorption pad.
3. The test strip of claim 1, wherein: the goat anti-mouse anti-antibody is obtained by immunizing a goat by taking a murine antibody as an immunogen.
4. A method of preparing the test strip of any one of claims 1-3, wherein: the method comprises the following steps:
1) preparing a conjugate release pad sprayed with metalaxyl monoclonal antibody-colloidal gold marker;
2) preparing a reaction film with a detection line coated with metalaxyl hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse anti-antibody;
3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.
5. A method for detecting metalaxyl in a tobacco sample by using the test strip of any one of claims 1 to 3, which is characterized in that: the method comprises the following steps:
1) pretreating a sample;
2) detecting with the test strip;
3) and analyzing the detection result.
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