CN109682961B - Test strip for detecting cyphenothrin and application thereof - Google Patents
Test strip for detecting cyphenothrin and application thereof Download PDFInfo
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Abstract
The invention discloses a test strip for detecting cyphenothrin and application thereof. The test strip comprises a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3), a water absorption pad (4) and a bottom plate (7), wherein the reaction membrane is provided with a detection line (5) coated with a cyphenothrin hapten-carrier protein conjugate and a quality control line (6) coated with a goat anti-mouse anti-antibody, and the conjugate release pad (2) is sprayed with a cyphenothrin monoclonal antibody-colloidal gold marker. The invention also provides a method for detecting the cyphenothrin residues in crops such as vegetables, fruits and the like by applying the cyphenothrin test strip. The test strip provided by the invention has the characteristics of simple operation, high sensitivity, high detection speed, low cost and the like, and is suitable for screening and on-site monitoring of a large number of samples.
Description
Technical Field
The invention relates to a test strip for detecting cyphenothrin and application thereof, in particular to a colloidal gold test strip for detecting cyphenothrin, which is particularly suitable for detecting cyphenothrin residues in crops such as vegetables, fruits and the like.
Background
Cyphenothrin, which is sold under the tradenames of Gokilaht, metalaxyl, D-cypermethrin and the like; molecular formula C 24 H 25 NO 3 It is mainly used for preventing and controlling household sanitary pests and pests in storage. It has very low toxicity and is the only pesticide permitted to be used in civil aircraft in the United states, 2 percent of D-cyphenothrin aerosol is approved by WHO to control pests such as mosquitoes, flies and the like in the aircraft cabin, and 0.4 percent of D-cyphenothrin powder can be directly used for controlling head lice of human bodies. The D-cyphenothrin can be processed into aerosol, oil emulsion, aqueous emulsion and powder, and the 10% aqueous emulsion can be used for group facilities such as barracks, restaurants, trains and the like and places with high safety requirements.
Because of different proportions, the sanitary insecticide is a chemical product for preventing and controlling sanitary pests such as mosquitoes, flies, cockroaches and the like, is mainly applied to living environment of human living, and is directly related to the health and life safety of people. The analysis documents of the effective components of the old generation of sanitary insecticides such as tetramethrin, cypermethrin, permethrin and the like are more, and the analysis documents of the effective components of the new generation of sanitary insecticides such as cyphenothrin, imiprothrin and the like are less, and most of the analysis documents are instrumental methods. The establishment of the method is beneficial to improving the supervision means and the supervision level.
Disclosure of Invention
The invention aims to provide the test strip for detecting the cyphenothrin residue, which has the advantages of high sensitivity, simple operation, low cost and short detection time.
The test strip for detecting cyphenothrin residues comprises a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3), an absorbent pad (4) and a bottom plate (7); the reaction membrane is provided with a detection line (5) coated with a cyphenothrin hapten-carrier protein conjugate and a quality control line (6) coated with a goat anti-mouse anti-antibody, and the conjugate release pad (2) is sprayed with a cyphenothrin monoclonal antibody-colloidal gold marker.
The cyhalothrin hapten-carrier protein conjugate is obtained by coupling cyhalothrin hapten and carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein and human serum albumin.
The cyphenothrin monoclonal antibody is prepared by taking a cyphenothrin hapten-carrier protein conjugate as an immunogen and is obtained by secreting a cyphenothrin monoclonal antibody hybridoma cell strain; the goat anti-mouse anti-antibody is obtained by immunizing a goat with a murine antibody.
The sample absorption pad (1), the conjugate release pad (2), the reaction film (3) and the water absorption pad (4) are sequentially adhered to the bottom plate (7), and the conjugate release pads 1/3-1/2 are covered under the sample absorption pad.
The bottom plate can be a PVC bottom plate or other hard non-water-absorbing materials; the sample absorption pad can be suction filter paper or oil filter paper; the conjugate release pad may be a glass wool or polyester material; the water absorption pad is absorbent paper; the reaction membrane can be a nitrocellulose membrane or a cellulose acetate membrane.
Another object of the present invention is to provide a method for preparing the test strip, which comprises the steps of:
1) preparing a conjugate release pad sprayed with a cyphenothrin monoclonal antibody-colloidal gold marker;
2) preparing a reaction membrane with a detection line coated with a cyphenothrin hapten-carrier protein conjugate and a quality control line coated with a 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.
Specifically, the steps include:
1) hapten preparation: reacting cyphenothrin with 1-formyl cyclopropane-2-carboxylic acid to obtain cyphenothrin hapten;
2) coupling the cyhalothrin hapten with carrier protein to obtain a cyhalothrin hapten-carrier protein conjugate;
3) immunizing a mouse by using the cyphenothrin hapten-carrier protein conjugate, and fusing and screening spleen cells and myeloma cells of the mouse to obtain a cyphenothrin monoclonal hybridoma cell strain;
4) extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibodies;
5) preparing colloidal gold by reacting trisodium citrate with chloroauric acid;
6) adding the cyphenothrin monoclonal antibody prepared in the step 3) into the colloidal gold prepared in the step 5) to obtain a cyphenothrin monoclonal antibody-colloidal gold marker;
7) spraying the cyphenothrin 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;
8) coating the reaction membrane with the phenylate cyhalothrin hapten-carrier protein conjugate to form a detection line, and coating the reaction membrane with a goat anti-mouse antibody to form a quality control line;
9) soaking the sample absorption pad in 0.5% bovine serum albumin (volume fraction), pH7.2, 0.1mol/L phosphate buffer solution for 2h, and drying at 37 deg.C for 2 h;
10) and sequentially adhering a sample absorption pad, a conjugate release pad, a reaction film and a water absorption pad on the bottom plate, covering the conjugate release pad with the sample absorption pad, finally cutting into small strips with the width of 3mm, adding a plastic box, carrying out vacuum packaging, and storing for 12 months at the temperature of 4-30 ℃.
The invention also aims to provide a method for detecting cyphenothrin residues in vegetables and crops by using the test strip, which comprises the following steps:
(1) pretreating a sample;
(2) detecting by using a test strip;
(3) and analyzing the detection result.
The cyphenothrin rapid detection test strip adopts a high-specificity antibody antigen reaction and competitive inhibition immunochromatography analysis technology, a cyphenothrin monoclonal antibody-colloidal gold marker is fixed on a conjugate release pad, and the cyphenothrin in a sample is combined with the cyphenothrin monoclonal antibody-colloidal gold marker on the conjugate release pad in the flowing process to form a drug-antibody-colloidal gold marker. The drug in the sample and the cyphenothrin hapten-carrier protein conjugate on the reaction film detection line compete to combine with the cyphenothrin monoclonal antibody-colloidal gold marker, and whether the cyphenothrin residue is contained in the sample liquid to be detected is judged according to the fact that the red strip of the detection line has no or light color.
During detection, a sample is treated and then dropped into a test strip hole, when the concentration of the cyphenothrin in the sample is lower than the detection limit or zero, the monoclonal antibody-colloidal gold marker can be combined with the cyphenothrin hapten-carrier protein conjugate fixed on a reaction membrane in the chromatography process, and a red strip appears in a detection line (T) and a quality control line (C); if the concentration of cyphenothrin in the sample is equal to or above the detection limit, the monoclonal antibody-colloidal gold label will bind to all of the cyphenothrin and no red band will appear at the T-line because the competition reaction will not bind to the cyphenothrin hapten-carrier protein conjugate.
The test strip has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, suitability for various units, simple storage and long quality guarantee period. The method for detecting the cyphenothrin residue by using the test strip is simple, convenient, rapid, visual, accurate, wide in application range, low in cost and easy to popularize and use.
Drawings
FIG. 1 is a diagram of the synthesis of a cyphenothrin hapten.
FIG. 2 is a schematic cross-sectional view of a test strip.
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.
Example 1 preparation of test paper strip for detecting cyphenothrin
The preparation method of the test strip mainly comprises the following steps:
1) preparing a conjugate release pad sprayed with a cyphenothrin monoclonal antibody-colloidal gold marker;
2) preparing a reaction film with a detection line coated with a cyhalothrin hapten-carrier protein conjugate and a quality control line coated with a 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. preparation of cyphenothrin hapten
(1) Taking 0.375g of cyphenothrin (compound a), adding 30ml of tetrahydrofuran for dissolving, stirring for 20min at 0-5 ℃, adding 0.11g of lithium aluminum hydride, returning to the room temperature, and gradually heating and refluxing for reaction for 4 h. The reaction was stopped, 30ml of ice water and 30ml of ethyl acetate × 3 were added, extraction was carried out three times, the organic phases were combined, evaporated to dryness and recrystallized in 80ml of dichloromethane/n-hexane (v/v, 1/10) to give 0.34g of the product compound b in 91.89% yield.
(2) Taking 0.34g of the compound b, adding 50ml of ethanol for dissolution, adding 0.12g of 1-formyl cyclopropane-2-carboxylic acid and 0.1ml of triethylamine, stirring at room temperature for 8h, transferring to 0-5 ℃, stirring for 10min, adding 0.11g of sodium borohydride, and continuing to stir for 1 h. Stopping reaction, evaporating to dryness by rotary evaporation, adding 30ml of water and 30ml of ethyl acetate multiplied by 3, extracting for three times, combining organic phases, evaporating to dryness, putting on a silica gel column, and eluting and separating by dichloromethane/methanol (v/v, 10/1) to obtain 0.38g of hapten product compound d with the yield of 88.37%.
2. Preparation of immunogens
Taking 20mg of compound d, adding 2ml of DMF for dissolving, adding 17mg of NHS and 23mg of EDC, stirring for 1h at room temperature to obtain hapten activation solution A, taking 100mg of bovine serum albumin, adding 0.05mol/L PB buffer solution for dissolving to obtain B solution, dripping A solution into B solution, stirring overnight at 4 ℃, dialyzing and purifying by 0.02mol/L PB buffer solution to obtain immunogen cyphenothrin-BSA, and storing at-20 ℃ for later use.
3. Preparation of coating antigen
Dissolving 12mg of compound d with 2ml of DMF, adding 11mg of NHS and 17mg of EDC, stirring for 1h at room temperature to obtain hapten activation solution A, dissolving ovalbumin 80mg with 0.05mol/L PB buffer solution to obtain B, dripping A into B, stirring overnight at 4 ℃, dialyzing and purifying with 0.02mol/L PB buffer solution to obtain immunogen cyphenothrin-OVA, and storing at-20 ℃ for later use.
4. Preparation of cyphenothrin monoclonal antibody
(1) Animal immunization
Injecting the immunogen obtained in the step 2 into Balb/c mice at an immune dose of 150 mug/mouse to generate antiserum.
(2) Cell fusion and cloning
Taking immune Balb/c mouse spleen cells, fusing the immune Balb/c mouse spleen cells with SP2/0 myeloma cells according to the proportion of 8:1 (number ratio), measuring cell supernatant by adopting an indirect competition ELISA method, and screening positive holes. Cloning the positive hole by using a limiting dilution method until obtaining the hybridoma cell strain which stably secretes the monoclonal antibody.
(3) Cell cryopreservation and recovery
Making hybridoma cell into 1 × 10 with frozen stock solution 6 Cell suspension per ml, stored for long periods in liquid nitrogen. Taking out the frozen tube during recovery, immediately putting the tube into a water bath at 37 ℃ for fast melting, centrifuging to remove frozen liquid, and transferring the tube into a culture bottle for culture.
(4) Preparation and purification of monoclonal antibodies
An incremental culture method: placing the hybridoma cell in cell culture medium, culturing at 37 deg.C, purifying the obtained culture solution by octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibody, and storing at-20 deg.C.
The cell culture medium is prepared by adding calf serum and sodium bicarbonate into RPMI1640 culture medium to make the final concentration of calf serum in the cell culture medium 20% (mass fraction) and the final concentration of sodium bicarbonate in the cell culture medium 0.2% (mass fraction); the pH of the cell culture medium was 7.4.
5. 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.
6. Preparation of cyphenothrin monoclonal antibody-colloidal gold marker
(1) Preparation of colloidal gold
Diluting 1% chloroauric acid to 0.01% (mass fraction) with double distilled deionized water, placing 100ml into a conical flask, heating to boil with a constant temperature electromagnetic stirrer, adding 2.5ml 1% trisodium citrate under continuous stirring at high temperature, stirring at constant speed, heating until the solution is bright red, cooling to room temperature, recovering to original volume with deionized water, and storing at 4 deg.C. The prepared colloidal gold has pure appearance, transparency and no precipitate or floating substances.
(2) Preparation of cyphenothrin monoclonal antibody-colloidal gold marker
Under magnetic stirring, adjusting the pH value of the colloidal gold to 7.0 by using 0.2mol/L potassium carbonate solution, adding 20-50 mu g of cyphenothrin monoclonal antibody into the colloidal gold solution according to the standard of adding each milliliter of the colloidal gold solution, continuously stirring and uniformly mixing for 30min, adding 10% BSA (bovine serum albumin) to enable the final concentration of the BSA in the colloidal gold solution to be 1% (volume fraction), and standing for 10 min. Centrifuging at 12000r/min at 4 deg.C for 40min, discarding supernatant, washing the precipitate with redissolving buffer twice, resuspending the precipitate with redissolving buffer whose volume is 1/10 of the original volume of colloidal gold, and standing at 4 deg.C for use.
Redissolving buffer solution: 0.02mol/L phosphate buffer solution containing casein 0.02-0.1% (mass fraction), tween-800.05-0.2% (mass fraction) and pH7.2.
7. Preparation of conjugate Release pad
The conjugate release pad was soaked in 0.5mol/L phosphate buffer containing bovine serum albumin (concentration of bovine serum albumin in buffer is 0.5%), pH7.2, and the solution was soaked for 1h, and then baked at 37 ℃ for 3 h. And uniformly spraying the prepared cyphenothrin monoclonal antibody-colloidal gold marker on a conjugate release pad by using an Isoflow film spraying instrument, spraying 0.01ml of cyphenothrin monoclonal antibody-colloidal gold marker on each 1cm of conjugate release pad, placing in an environment at 37 ℃ (humidity is less than 20%) for 60min, taking out, and placing in a dry environment (humidity is less than 20%) for storage.
8. Preparation of the reaction film
Coating the cyphenothrin 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 the cyhalothrin hapten-ovalbumin conjugate to 10mg/ml by using a phosphate buffer solution, and coating the conjugate on a detection line (T line) on a nitrocellulose membrane by using an Isoflow point membrane instrument, wherein the coating amount is 0.8 mu l/cm; the goat anti-mouse anti-antibody was diluted to 200. mu.g/ml with 0.01mol/L, pH7.4 phosphate buffer, and coated on a quality control line (C line) 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.
9. Preparation of sample absorbent pad
The sample absorption pad is soaked in 0.5% bovine serum albumin (volume fraction), pH7.2, 0.1mol/L phosphate buffer solution for 2h, and then baked at 37 ℃ for 2h for standby.
10. Assembly of test strips
Sequentially sticking a sample absorption pad, a conjugate release pad, a reaction membrane and a water absorption pad on a PVC base 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 line) and the quality control line (C line) 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 of the end away from the conjugate release pad; the test paper strip is cut into small strips with the width of 3mm 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 Cypermethrin residue in samples
1. Detection with test strip
And (3) sucking the sample solution to be detected by a suction pipe, vertically dripping the sample solution into the sample adding hole, starting timing when the liquid flows, reacting for 5-10 min, and judging a result.
2. Analyzing the results of the detection
The results were read by a colloidal gold analyzer (abbreviated as "analyzer"):
negative (-): indicating that the concentration of the substance to be detected in the sample is lower than the detection limit;
positive (+): indicating that the concentration of the substance to be detected in the sample is equal to or higher than the detection limit;
and (4) invalidation: indicating that retesting is required.
Example 3 sample testing example
1. Limit of detection test
Taking blank cabbage and apple samples, respectively adding cyphenothrin to the samples until the final concentration is 0.5, 1 and 2 mu g/kg, taking test strips for detection, and repeatedly measuring each sample for three times.
When the test paper is used for detecting samples of the Chinese cabbage and the apple, the analyzer shows that the sample is negative when the cyphenothrin is added at a concentration of 0.5 mug/kg; when the cyphenothrin is added at a concentration of 1, 2 mug/kg, the analyzer shows positive.
2. Test for false positive and false negative rates
Taking 20 parts of each of the cabbage and apple positive samples with the known content of the cyphenothrin being 1 mu g/kg and 20 parts of each of the cabbage and apple negative samples with the content of less than 0.5 mu g/kg, detecting by using three batches of test strips, and calculating the negative and positive rates. The results are shown in tables 1 and 2.
TABLE 1 cabbage test sample results
TABLE 2 apple test sample results
The results show that: when 3 test strips produced in batches are used for detecting positive cabbage and apple samples, the results are all positive, the coincidence rate of the positive samples is 100 percent, and the false negative rate is 0; when 20 negative Chinese cabbage and apple samples are detected, the results are all negative, and the negative coincidence rate is 100 percent and the false positive rate is 0. The test strip for detecting the cyphenothrin can quickly detect the cyphenothrin residues in the Chinese cabbage and the apple.
3. Specificity test
The cyphenothrin test paper is used for detecting 500 mu g/kg of cypermethrin, fenvalerate, deltamethrin and other drugs. The result shows that the quality control line and the detection line of the test strip are both colored and negative. The test paper strip has no cross reaction to 500 mu g/kg of cypermethrin, fenvalerate, deltamethrin and other medicaments.
Claims (6)
1. A test strip for detecting cyphenothrin comprises a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3), a water absorption pad (4) and a bottom plate (7), and is characterized in that the reaction membrane is provided with a detection line (5) coated with cyphenothrin hapten-carrier protein conjugate and a quality control line (6) coated with goat anti-mouse anti-antibody, the conjugate release pad (2) is sprayed with a cyphenothrin monoclonal antibody-colloidal gold marker, the cyphenothrin hapten-carrier protein conjugate is obtained by coupling cyphenothrin hapten and carrier protein, the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein and human serum albumin, and the preparation method of the cyphenothrin hapten is as follows:
(1) taking 0.375g of the compound alpha phenothrin, adding 30ml of tetrahydrofuran for dissolving, stirring for 20min at 0-5 ℃, adding 0.11g of lithium aluminum hydride, returning to room temperature, and gradually heating and refluxing for reaction for 4 h; stopping the reaction, adding 30ml of ice water and 30ml of ethyl acetate multiplied by 3, extracting for three times, combining organic phases, evaporating to dryness, and mixing dichloromethane and n-hexane in a volume ratio of 1: 10, 80ml of recrystallization, 0.34g of the product compound b is obtained, the yield is 91.89%;
(2) taking 0.34g of the compound b, adding 50ml of ethanol for dissolving, adding 0.12g of 1-formyl cyclopropane-2-carboxylic acid and 0.1ml of triethylamine, stirring at room temperature for 8h, transferring to 0-5 ℃, stirring for 10min, adding 0.11g of sodium borohydride, and continuing to stir for 1 h; stopping reaction, rotary evaporating and evaporating to dryness, adding 30ml of water and 30ml of ethyl acetate multiplied by 3, extracting for three times, combining organic phases, evaporating to dryness, and putting on a silica gel column, wherein the volume ratio of dichloromethane to methanol is 10: 1, eluting and separating to obtain 0.38g of hapten product compound d with the yield of 88.37%;
the molecular structural formula of the cyhalothrin hapten is as follows:
2. the test strip of claim 1, wherein the sample absorbing pad (1), the conjugate releasing pad (2), the reaction membrane (3) and the absorbent pad (4) are sequentially adhered to the bottom plate (7).
3. The strip of any one of claims 1-2, wherein the conjugate release pad 1/3-1/2 is covered under a sample absorbing pad.
4. The test strip of claim 1, wherein the cyphenothrin monoclonal antibody is prepared by taking a cyphenothrin hapten-carrier protein conjugate as an immunogen, and the goat anti-mouse anti-antibody is obtained by immunizing a goat with a mouse antibody.
5. A method of making the test strip of any one of claims 1-4, comprising the steps of:
1) preparing a conjugate release pad sprayed with a cyphenothrin monoclonal antibody-colloidal gold marker;
2) preparing a reaction membrane with a detection line coated with a cyphenothrin hapten-carrier protein conjugate and a quality control line coated with a 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.
6. A method for detecting cyphenothrin residues in vegetable and fruit crops comprises the following steps:
1) sample pretreatment;
2) performing a test using the test strip of any one of claims 1-4;
3) and analyzing the detection result.
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| CN112067812A (en) * | 2020-08-26 | 2020-12-11 | 北京勤邦生物技术有限公司 | Test strip for detecting polychlorinated biphenyl and application thereof |
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