CN106918705B - Test paper for detecting fenpropathrin and application thereof - Google Patents
Test paper for detecting fenpropathrin and application thereof Download PDFInfo
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Abstract
The invention discloses test paper for detecting fenpropathrin and application thereof. The test paper 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 sample absorption pad (1), the conjugate release pad (2), the reaction membrane (3) and the water absorption pad (4) are sequentially adhered to the bottom plate (7), the reaction membrane (3) is provided with a detection line (5) coated with fenpropathrin hapten-carrier protein conjugate and a quality control line (6) coated with goat anti-mouse anti-antibody, and the conjugate release pad (2) is coated with a fenpropathrin monoclonal antibody-colloidal gold marker. The test paper provided by the invention can be used for detecting fenpropathrin residues in tea, is simple to operate, high in sensitivity, short in detection time and low in cost, and is suitable for screening and on-site monitoring of a large number of samples.
Description
Technical Field
The invention belongs to the technical field of rapid detection, and particularly relates to test paper for detecting fenpropathrin, which is particularly suitable for detecting fenpropathrin in tea samples.
Background
Fenpropathrin (fenprothiorin), alpha-cyano-phenoxybenzyl-2, 3-tetramethyl cyclopropane carboxylic acid ester, also known as clomazone, is a pseudopyrethrin insecticidal acaricide. Fenpropathrin belongs to nerve agents, and can act on the nervous system of insects to cause the insects to be over excited and paralyzed to death. In recent years, pyrethroid pesticides have been widely used because of their high efficacy in killing insects, and fenpropathrin is sometimes detected in foods, and has moderate toxicity to higher animals.
At present, the detection of fenpropathrin residues mainly comprises liquid chromatography, gas chromatography-mass spectrometry and the like, and the instrument method has the advantages of high detection sensitivity, strong specificity and the like, but the pretreatment of a detection sample is tedious and time-consuming, the instrument detection needs expensive large-scale instruments and equipment, and professional detection technicians are equipped, so that the popularization and the application of the instrument method are limited. The colloidal gold immunochromatography has the advantages of good specificity, high sensitivity, simple and convenient operation, low detection cost, suitability for batch sample detection and the like, and is an ideal rapid screening means.
Disclosure of Invention
The invention aims to provide test paper for detecting fenpropathrin, which has the advantages of high detection sensitivity, simple and convenient operation, low cost and short detection time, and application thereof.
The test paper for detecting fenpropathrin provided by the invention 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 sample absorption pad 1, the conjugate release pad 2, the reaction membrane 3 and the water absorption pad 4 are sequentially adhered to the bottom plate 7, the reaction membrane 3 is provided with a detection line 5 coated with a fenpropathrin 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 coated with a fenpropathrin monoclonal antibody-colloidal gold marker.
The conjugate release pad 2 is stacked under the sample absorption pad 1, and 1/3 of the conjugate release pad 2 is covered under the sample absorption pad 1.
The fenpropathrin hapten-carrier protein conjugate is obtained by coupling a fenpropathrin hapten with carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin or thyroxine.
The fenpropathrin hapten is synthesized by the reaction of fenpropathrin with m-hydroxybenzaldehyde, potassium cyanide, fenpropathrin chloride and p-hydroxy phenylpropionic acid, the structural formula of the fenpropathrin hapten is shown as the formula (I),
the fenpropathrin monoclonal antibody is prepared by taking fenpropathrin hapten-carrier protein conjugate as an immunogen; the goat anti-mouse antibody is obtained by immunizing a goat with a mouse-derived antibody.
The invention also provides a preparation method of the test paper, which mainly comprises the following steps:
a) Preparing a bond release pad 2 coated with fenpropathrin monoclonal antibody-colloidal gold marker;
b) Preparing a reaction membrane 3 with a detection line 5 coated with fenpropathrin hapten-carrier protein conjugate and a quality control line 6 coated with goat anti-mouse anti-antibody;
c) The conjugate release pad 2, the reaction membrane 3, the sample absorption pad 1, the water absorption pad 4 and the bottom plate 7 prepared in a) and b) are assembled into a test paper.
The test paper for detecting fenpropathrin adopts a highly specific antibody antigen reaction and immunochromatographic analysis technology, a fenpropathrin monoclonal antibody-colloidal gold marker is fixed on a conjugate release pad, and fenpropathrin in a sample is firstly mixed with the fenpropathrin monoclonal antibody-colloidal gold marker on the conjugate release pad in the flowing process to form a medicine-antibody-colloidal gold marker. The medicine in the sample and the fenpropathrin hapten-carrier protein conjugate on the reaction membrane detection line compete for combining with a fenpropathrin monoclonal antibody-colloidal gold marker, and the residual fenpropathrin content in the sample liquid to be detected is judged according to the existence or the darkness of the red strip of the detection line, wherein the detection limit of the tea green sample is 1mg/kg, and the detection limit of the dry tea sample is 2mg/kg.
During detection, a sample is dripped into a test paper card hole after treatment, when the concentration of fenpropathrin in the sample is lower than the detection limit or is zero, a fenpropathrin monoclonal antibody-colloidal gold marker is combined with a fenpropathrin hapten-carrier protein conjugate fixed on a reaction membrane in the chromatography process, and a red strip appears in a detection line (T line) and a quality control line (C line) respectively; if the concentration of fenpropathrin in the sample is equal to or higher than the detection limit, the fenpropathrin monoclonal antibody-colloidal gold-labeled will bind to the fenpropathrin entirely, so that no red band will appear at the T-line due to competition reaction with the fenpropathrin hapten-carrier protein conjugate.
The test paper has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, simple storage, long quality guarantee period, wide application range, easy popularization and application and the like.
Drawings
FIG. 1 is a scheme showing the synthesis of fenpropathrin hapten according to the invention;
FIG. 2 fenpropathrin hapten identification chart (hydrogen spectrogram);
FIG. 3 fenpropathrin hapten identification chart (carbon spectrogram);
FIG. 4 is a schematic cross-sectional view of a test strip according to the present invention;
FIG. 5 is a graph showing the test result of the test paper according to the present invention.
Detailed Description
The invention is further illustrated below in conjunction with specific examples. It is to be understood that these examples are for illustration of the invention only and are not intended to limit the scope of the invention. The reagents of the invention are conventional reagents unless otherwise specified.
Example 1 preparation of test paper for detecting fenpropathrin
The preparation method of the test paper mainly comprises the following steps:
a) Preparing a bond release pad 2 coated with fenpropathrin monoclonal antibody-colloidal gold marker;
b) Preparing a reaction membrane 3 with a detection line 5 coated with fenpropathrin hapten-carrier protein conjugate and a quality control line 6 coated with goat anti-mouse anti-antibody;
c) The conjugate release pad 2, the reaction membrane 3, the sample absorption pad 1, the water absorption pad 4 and the bottom plate 7 prepared in a) and b) are assembled into a test paper.
The following is a stepwise detailed description:
1. preparation of fenpropathrin hapten, and synthetic route is shown in figure 1
1) Synthesis of m-phenoxybenzonitrile alcohol
Dissolving 1.22g of m-hydroxybenzaldehyde in 50mL of tetrahydrofuran, slowly adding 0.65g of potassium cyanide and 0.76mL of deionized water under ice bath stirring, continuously stirring for 30min, slowly dropwise adding 2.1mL of 2mol/L of concentrated hydrochloric acid, continuously stirring for 1h, detecting the reaction process by TLC, adding 50mL of deionized water into the reaction solution after the reaction is finished, extracting with dichloromethane, drying an organic phase by anhydrous sodium sulfate, and removing a solvent by reduced pressure distillation to obtain a yellow oily liquid product;
2) M-hydroxybenzoyl cyanide connected with fenpropathrin chloride
Dissolving the product obtained in the last step in 50mL of acetone, stirring and cooling in an ice bath, dissolving 3.54g of fenpropathrin chloride in 50mL of dichloromethane, adding the mixture into an acetone solution of the product, adding 2.14mL of pyrimidine, continuing the ice bath reaction of the mixture for 3h, washing the reaction solution with 3mol/L hydrochloric acid and deionized water in sequence after the reaction is completed, drying with anhydrous sodium sulfate, and distilling under reduced pressure to obtain yellow oily liquid;
3) Synthesis of fenpropathrin hapten
Dissolving the product in 100mL of 98% concentrated sulfuric acid, adding 1.66g of parahydroxybenzoic acid, heating and refluxing at 100 ℃ for 6h, detecting by TLC, pouring the reaction liquid into ice after the reaction is completed, adjusting the pH to 5.0 by using solid sodium bicarbonate, extracting by using ethyl acetate, drying by using anhydrous sodium sulfate, purifying the product by using silica gel column chromatography, collecting target components, confirming by nuclear magnetic resonance hydrogen spectrum that a peak near the left side of 12ppm in FIG. 2 is hydrogen on the tail end carboxyl of a hapten connecting arm, a cluster of peaks near 7ppm is hydrogen on a fenpropathrin benzene ring and hydrogen on cyano alpha carbon, and in a carbon spectrum shown in FIG. 3, a peak between 110 and 140ppm is carbon on two benzene rings and cyano on fenpropathrin, a peak at the left side of 170-180ppm is carbon on the tail end carboxyl of the connecting arm, and a peak at the right side is carbon on the ester structure in fenpropathrin molecule, thus indicating that hapten synthesis is successful.
2. Preparation of immunogen-coupling of fenpropathrin hapten and Bovine Serum Albumin (BSA) to obtain immunogen
40mg of fenpropathrin hapten is weighed and dissolved in 3mL of DMF solution, 80mgEDC and 80mgNHS (both dissolved in 3mL of deionized water) are added for activation for 30min, activated fenpropathrin is added into 180-400 mg of BSA (dissolved in 7mL of deionized water) for coupling to prepare immunogen, 0.02mol/L of phosphate buffer solution is used for dialysis for 3 days, dialyzate is replaced in the morning and evening every day to remove unreacted micromolecule substances, so that the immunogen is obtained, and the immunogen is packaged and stored at the temperature of minus 20 ℃ for standby.
3. Preparation of coating antigen-coupling of fenpropathrin hapten and Ovalbumin (OVA) to obtain coating antigen
Weighing 20mg of fenpropathrin hapten, dissolving the fenpropathrin hapten by using 1.5 mM (L-D-F), and cooling the solution to 10 ℃ to obtain a reaction solution I; adding 15 mu L of isobutyl chloroformate into the reaction solution I, and stirring at 10 ℃ for reaction for 30min; 45mg of ovalbumin was taken and treated with 3mL of 50mmol/L Na 2 CO 3 Dissolving, reacting for 4 hours at 10 ℃, and transferring to a refrigerator at 4 ℃ for overnight; dialyzing with 0.01mol/L phosphate buffer solution at 4deg.C for 3 days to remove unreacted small molecular substances to obtain coating antigen, packaging, and storing at-20deg.C.
4. Preparation of fenpropathrin monoclonal antibody
1) Immunization of animals
The immunogen obtained in the step 2 is injected into Balb/c mice, and the immune dose is 120 mug/mouse, so that antisera are generated.
2) Cell fusion and cloning
After the measurement result of the mouse serum is higher, spleen cells are taken and fused with SP2/0 myeloma cells according to the proportion of 8:1 (quantitative ratio), cell supernatant is measured by adopting indirect competition ELISA, and positive holes are screened. Cloning the positive hole by using a limiting dilution method until obtaining a hybridoma cell strain secreting the fenpropathrin monoclonal antibody.
3) Cell cryopreservation and resuscitation: freezing the monoclonal hybridoma cell strain into 1×10 6 Cell suspensions of individual/mL were stored in liquid nitrogen for long periods. And (3) taking out the frozen storage tube during recovery, immediately putting into a 37 ℃ water bath for medium-speed thawing, centrifuging to remove frozen storage liquid, and transferring into a culture flask for culture.
4) Production and purification of monoclonal antibodies: the hybridoma cells are placed in a cell culture medium, cultured at 37 ℃, and the obtained culture solution is purified by an octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibodies, and the monoclonal antibodies are preserved at-20 ℃.
The cell culture medium is prepared by adding calf serum and sodium bicarbonate into RPMI1640 culture medium, wherein the final concentration of the calf serum in the cell culture medium is 20% (mass percent) and the final concentration of the sodium bicarbonate in the cell culture medium is 0.2% (mass percent); the pH of the cell culture medium was 7.4.
5. Preparation of goat anti-mouse antibody
Sheep is used as immune animals, and a murine antibody is used as immunogen to immunize pathogen-free sheep, so that the goat anti-mouse antibody is obtained.
6. Preparation of fenpropathrin monoclonal antibody-colloidal gold marker
1) Preparation of colloidal gold
Diluting 1% chloroauric acid into 0.01% (mass percent) by using double distilled deionized water, placing 100mL into a conical flask, heating to boiling by using a constant-temperature electromagnetic stirrer, adding 2.5mL of 1% trisodium citrate under continuous high temperature and continuous stirring, stopping stirring and heating at a constant speed until the solution is transparent red, cooling to room temperature, recovering the original volume by using deionized water, and preserving at 4 ℃. The prepared colloidal gold has pure appearance, is transparent, and has no sediment or floaters.
2) Preparation of fenpropathrin monoclonal antibody-colloidal gold marker
Under the magnetic stirring, regulating the pH value of the colloidal gold to 7.2 by using 0.2mol/LTris-HCl solution, adding 40-80 mu g of fenpropathrin monoclonal antibody into the colloidal gold solution according to the standard of adding 40-80 mu g of antibody into each milliliter of colloidal gold solution, continuously stirring and uniformly mixing for 30min, adding 10% BSA and uniformly mixing to ensure that the final concentration of the fenpropathrin monoclonal antibody in the colloidal gold solution is 1% (volume percent), standing for 10min, centrifuging for 40min at 12000r/min and 4 ℃, discarding supernatant, washing the precipitate twice by using a re-dissolving buffer, and re-suspending the precipitate by using the re-dissolving buffer with the volume of 1/10 of the initial colloidal gold volume, and standing for standby at 4 ℃.
Reconstitution buffer: 0.01mol/LTris-HCl buffer solution containing 0.1-0.3% (volume percentage), 0.05-0.2% (mass percentage) Tween-80 and pH 7.2.
7. Preparation of conjugate release pads
The conjugate release pad was soaked in phosphate buffer containing bovine serum albumin (0.5% concentration of bovine serum albumin in buffer) and having a pH of 7.2,0.5mol/L, and soaked uniformly for 1h, and baked at 37℃for 3 h. Uniformly coating the prepared fenpropathrin monoclonal antibody-colloidal gold marker on a conjugate release pad by using a ZX1000 type film spraying instrument produced by Baidao corporation, coating 0.01mL of the fenpropathrin monoclonal antibody-colloidal gold marker on each 1cm of conjugate release pad, placing in a 37 ℃ environment (humidity is less than 20%) for 60min, taking out, sealing, placing in a dry environment (humidity is less than 20%) and preserving for later use. The conjugate release liner is made of nitrocellulose membrane.
8. Preparation of reaction film
The fenpropathrin hapten-ovalbumin conjugate is coated on a reaction membrane to form a detection line 5, and the goat anti-mouse anti-antibody is coated on the reaction membrane to form a quality control line 6. The reaction membrane is a nitrocellulose membrane.
The coating process comprises the following steps: dilute fenpropathrin hapten-ovalbumin conjugate to 10mg/mL by using 0.1mol/L phosphate buffer solution with pH7.4, coat the same on a detection line (T line) on a nitrocellulose membrane by using ZX1000 type spot film instrument manufactured by Baidao corporation in an amount of 1.0 mug/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the With 0.01mol/L phosphate pH7.4Diluting goat anti-mouse antibody to 200 mug/mL with the flushing liquid, coating the goat anti-mouse antibody on a quality control line (C line) on a nitrocellulose membrane by a spot film tester, wherein the coating amount is 1.0 mug/cm 2 . And (5) drying the coated reaction film for 2 hours at 37 ℃ for standby.
9. Preparation of sample absorbent pad
The sample absorption pad is placed in phosphate buffer solution containing 0.5% of bovine serum albumin (volume percentage) and pH 7.2.1 mol/L for 2 hours, and is baked at 37 ℃ for 2 hours for standby. The sample absorbing pad can be made of glass fiber, non-woven fabric and blood filtering film, preferably non-woven fabric. 10. The test paper is assembled, see FIG. 4
Sequentially adhering a sample absorption pad 1, a conjugate release pad 2, a reaction membrane 3 and a water absorption pad 4 on a bottom plate 7; the 1/3 area of the initial end of the conjugate release pad 2 is covered by the sample absorption pad 1, the tail end of the conjugate release pad 2 is connected with the initial end of the reaction membrane 3, the tail end of the reaction membrane 3 is connected with the initial end of the water absorption pad 4, the initial end of the sample absorption pad 1 is aligned with the initial end of the bottom plate 7, and the tail end of the water absorption pad 4 is aligned with the tail end of the bottom plate 7; the reaction film 3 is provided with a detection line 5 and a quality control line 6, and the detection line 5 and the quality control line 6 are strip-shaped strips which are perpendicular to the length of the test paper; the detection line 5 is located on the side near the end of the conjugate release pad 2; the quality control line 6 is positioned at one side far away from the tail end of the conjugate release pad 2; cutting the test paper into small strips with the width of 3mm by a machine, and placing the small strips in a special plastic card to form the test paper card with a sample adding hole and an observation window, wherein the test paper card can be stored for 12 months at the temperature of 4-30 ℃.
EXAMPLE 2 detection of fenpropathrin residue in tea samples
1. Pretreatment of tea samples
1) The sample should be restored to the room temperature of 20-25 ℃ before detection;
2) Weighing 2.0+/-0.05 g of sample into a polystyrene centrifuge tube;
3) Adding 10mL of sample extracting solution, and uniformly mixing by a vortex instrument; the method comprises the steps of carrying out a first treatment on the surface of the
4) The dilution method of the tea leaves and the dry tea is as follows:
tea green: 100. Mu.L of sample solution+400. Mu.L of sample dilution;
dry tea: 100. Mu.L of sample solution+900. Mu.L of sample dilution;
the sample dilution was 0.2moL/L phosphate buffer.
2. Detection was performed using the test paper of example 1
And sucking the tea sample solution to be detected by using a suction pipe, vertically dripping 2-3 drops of the tea sample solution into a sample adding hole, starting timing when the liquid flows, reacting for 5min, judging the result, and judging that the tea sample solution is invalid in other time.
3. Analysis of the detection results, see FIG. 5
Negative (-). Both the T-line and the C-line are colored, indicating that the concentration of fenpropathrin drug in the tea sample is below the limit of detection, as shown in fig. 5a.
Positive (+): the T line shows no color development and the C line shows color development, indicating that the fenpropathrin drug concentration in the sample is equal to or higher than the detection limit, as shown in FIG. 5b.
Invalidation: the absence of line C indicates an incorrect procedure or that the test paper has failed due to deterioration, as shown in fig. 5C. In this case, a new test strip card is applied for retesting.
4. Limit of detection test
Taking blank dry tea samples, respectively adding fenpropathrin to the blank dry tea samples to reach final concentrations of 1, 2 and 4mg/kg, taking test paper for detection, and repeating the detection for three times for each sample.
When the test paper is used for detecting the tea samples, when the addition concentration of fenpropathrin is 1mg/kg, two macroscopic red lines are displayed on the test paper and are negative; when the addition concentration of fenpropathrin is 2mg/kg and 4mg/kg, the test paper quality control line is displayed, but the detection line is not displayed and is positive; the test line of fenpropathrin in the test paper dry tea sample is 2mg/kg.
Blank theanine samples were taken, fenpropathrin was added thereto to a final concentration of 0.5, 1 and 2mg/kg, and test paper was taken for detection, and each sample was repeatedly assayed three times.
When the test paper is used for detecting a tea green sample, when the addition concentration of fenpropathrin is 0.5mg/kg, two macroscopic red lines are displayed on the test paper and are negative; when the addition concentration of fenpropathrin is 1 and 2mg/kg, the test paper quality control line is displayed, but the detection line is not displayed and is positive; the test paper shows that the detection line of fenpropathrin in the test paper tea green sample is 1mg/kg.
5. False positive rate and false negative rate test
Taking 20 parts of tea green positive samples with the known fenpropathrin content of more than 1mg/kg, 20 parts of dry tea positive samples with the known fenpropathrin content of more than 2mg/kg and 20 parts of dry tea negative samples with the known fenpropathrin content of less than 1mg/kg, detecting by using three batches of test paper, and calculating the false positive rate and the false negative rate of the test paper. The results are shown in tables 1 to 4.
TABLE 1 detection of positive sample results
| Batch of | Positive tea green sample (20 parts) |
| 1 | 20 parts of positive |
| 2 | 20 parts of positive |
| 3 | 20 parts of positive |
TABLE 2 detection of positive sample results
| Batch of | Positive dry tea sample (20 parts) |
| 1 | 20 parts of positive |
| 2 | 20 parts of positive |
| 3 | 20 parts of positive |
TABLE 3 detection of negative sample results
| Batch of | Negative tea green sample (20 parts) |
| 1 | |
| 2 | |
| 3 | |
TABLE 4 detection of negative sample results
| Batch of | Negative dry tea sample (20 parts) |
| 1 | |
| 2 | |
| 3 | |
When 3 batches of produced test paper are used for detecting positive samples, the results are all positive, the coincidence rate of the positive samples is 100%, when 20 negative tea green samples are detected, all negative samples are negative, and when 20 negative dry tea samples are detected, only one batch of positive samples are detected. The test paper disclosed by the invention can be used for rapidly detecting fenpropathrin in tea green and dry tea.
6. Specificity test
Specificity is commonly expressed as the rate of cross-reactivity, which refers to the ability of an antibody to bind to structurally distinct epitopes. The test paper is used for detecting samples of the commonly detected carbendazim, triadimenol and metalaxyl drug with the concentration of 500 mug/L. The result shows that when the test paper provided by the invention is used for detecting 500 mug/L of carbendazim, triadimenol and metalaxyl drugs, the quality control line and the detection line of the test paper are both developed, and the test paper is negative, so that the test paper has no cross reaction on the drugs.
Claims (5)
1. The test paper for detecting fenpropathrin 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 sample absorption pad (1), the conjugate release pad (2), the reaction membrane (3) and the water absorption pad (4) are sequentially adhered to the bottom plate (7), the test paper is characterized in that the reaction membrane is provided with a detection line (5) coated with a fenpropathrin hapten-carrier protein conjugate and a quality control line (6) coated with a goat anti-mouse anti-antibody, the conjugate release pad (2) is coated with a fenpropathrin monoclonal-colloidal gold marker, the fenpropathrin hapten-carrier protein conjugate is obtained by coupling fenpropathrin hapten with carrier protein, the carrier protein is bovine serum albumin, egg albumin, hemocyanin or thyroxine, the fenpropathrin hapten is obtained by synthesizing fenpropathrin with m-hydroxybenzaldehyde, potassium cyanide, fenpropathrin chloride and parahydroxyphenoxypropionic acid through reaction, the fenpropiophenone hapten is shown as formula I,
the fenpropathrin hapten is specifically prepared by the following steps:
a) Synthesis of m-phenoxybenzonitrile alcohol
Dissolving 1.22g of m-hydroxybenzaldehyde in 50mL of tetrahydrofuran, slowly adding 0.65g of potassium cyanide and 0.76mL of deionized water under ice bath stirring, continuously stirring for 30min, slowly dropwise adding 2.1mL of 2mol/L of concentrated hydrochloric acid, continuously stirring for 1h, detecting the reaction process by TLC, adding 50mL of deionized water into the reaction solution after the reaction is finished, extracting with dichloromethane, drying an organic phase by anhydrous sodium sulfate, and removing a solvent by reduced pressure distillation to obtain a yellow oily liquid product;
b) M-hydroxybenzoyl cyanide connected with fenpropathrin chloride
Dissolving the product obtained in the step a) in 50mL of acetone, stirring and cooling in an ice bath, dissolving 3.54g of fenpropathrin chloride in 50mL of dichloromethane, adding the solution into the acetone solution of the product, adding 2.14mL of pyrimidine, continuing the ice bath reaction of the mixture for 3h, washing the reaction solution with 3mol/L hydrochloric acid and deionized water in sequence after the reaction is completed, drying with anhydrous sodium sulfate, and distilling under reduced pressure to obtain yellow oily liquid;
c) Synthesis of fenpropathrin hapten
Dissolving the product of the step b) in 100mL of 98% concentrated sulfuric acid, adding 1.66g of parahydroxybenzoic acid, heating and refluxing at 100 ℃ for 6 hours, pouring the reaction liquid into ice after TLC detection reaction is completed, adjusting pH to 5.0 with solid sodium bicarbonate, extracting with ethyl acetate, drying with anhydrous sodium sulfate, purifying the product with silica gel column chromatography, and collecting to obtain fenpropathrin hapten.
2. Test paper according to claim 1, characterized in that the conjugate release pad (2) is stacked under the sample absorption pad (1) and that 1/3 of the conjugate release pad (2) is covered under the sample absorption pad (1).
3. The test paper according to claim 1 or 2, wherein the fenpropathrin monoclonal antibody is prepared by using fenpropathrin hapten-carrier protein conjugate as an immunogen, and the goat anti-mouse antibody is obtained by immunizing a goat with a mouse-derived antibody.
4. A method of preparing the test strip of any one of claims 1 to 3, comprising the steps of:
a) Preparing a bond release pad (2) coated with fenpropathrin monoclonal antibody-colloidal gold marker;
b) Preparing a reaction membrane (3) with a detection line (5) coated with fenpropathrin hapten-carrier protein conjugate and a quality control line (6) coated with goat anti-mouse anti-antibody;
c) And (3) assembling the conjugate release pad (2), the reaction membrane (3), the sample absorption pad (1), the water absorption pad (4) and the bottom plate (7) which are prepared from the steps a) and b) into the test paper.
5. A method for detecting fenpropathrin residues in a tea sample using the test paper of any one of claims 1 to 3, comprising the steps of:
a) Pretreatment of tea samples;
b) Detecting with the test paper according to any one of claims 1 to 3;
c) And analyzing the detection result.
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| CN109682961B (en) * | 2018-09-17 | 2022-09-20 | 北京勤邦生物技术有限公司 | Test strip for detecting cyphenothrin and application thereof |
| CN110540526B (en) * | 2019-08-09 | 2022-08-30 | 广西大学 | Carane acidyl 1,2, 3-triazole compound and preparation method and application thereof |
| CN111735951B (en) * | 2020-06-03 | 2022-11-18 | 北京勤邦生物技术有限公司 | Test strip for detecting fenpropathrin and application thereof |
| CN111812316B (en) * | 2020-06-03 | 2022-11-18 | 北京勤邦生物技术有限公司 | Application of fenpropathrin artificial antigen in enzyme linked immunosorbent assay kit |
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| CN105807056A (en) * | 2016-03-21 | 2016-07-27 | 贵州勤邦食品安全科学技术有限公司 | Test paper card for detecting efficient cyhalothrin residue |
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| FR2593503B1 (en) * | 1986-01-29 | 1988-04-29 | Roussel Uclaf | IODINE-MARKED PYRETHRINOIDS, THEIR PREPARATION PROCESS AND THEIR APPLICATION TO RADIOIMMUNOLOGICAL ASSAYS |
| US5108900A (en) * | 1989-03-06 | 1992-04-28 | Regents Of The University Of California | Monoclonal antibodies to synthetic pyrethroids and method for detecting the same |
| CN1137091C (en) * | 2000-12-06 | 2004-02-04 | 南开大学 | Prepn. of fenpropathrin |
| EP1854797A1 (en) * | 2006-05-02 | 2007-11-14 | Isdin, S.A. | Process for preparing cyameluric chloride |
| CN101017165B (en) * | 2007-03-09 | 2011-04-27 | 中国水产科学研究院淡水渔业研究中心 | Method for preparing fenpropathrin artificial antigen |
| SI3428148T1 (en) * | 2007-05-15 | 2021-04-30 | Boehringer Ingelheim Animal Health USA Inc. | Aryloazol-2-yl cyanoethylamino compounds, method of making and method of using thereof |
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