CN114349780A - Flusilazole hapten, antigen, antibody, detection device and detection method thereof - Google Patents
Flusilazole hapten, antigen, antibody, detection device and detection method thereof Download PDFInfo
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- CN114349780A CN114349780A CN202210255908.1A CN202210255908A CN114349780A CN 114349780 A CN114349780 A CN 114349780A CN 202210255908 A CN202210255908 A CN 202210255908A CN 114349780 A CN114349780 A CN 114349780A
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Abstract
The invention discloses a flusilazole hapten, an antigen, an antibody, a detection device and a detection method thereof, and relates to a flusilazole hapten for immunization, a flusilazole hapten for coating, a flusilazole antigen, a flusilazole antibody, a flusilazole colloidal gold chromatography detection device and a method for detecting flusilazole residue. The flusilazole colloidal gold chromatography detection device disclosed by the invention can be used for quickly detecting flusilazole residues in fruits, vegetables and grain grains, and a solid-phase extraction column is not required to be purified during application, so that the aim of quickly detecting the flusilazole residues is fulfilled.
Description
Technical Field
The invention relates to the field of pesticide safety detection, in particular to flusilazole hapten, antigen, antibody, detection device and detection method thereof.
Background
Flusilazole (Flusilazole) with the chemical name of bis (4-fluorophenyl) methyl (1H-1, 2, 4-triazole-1-methyl) silane is a novel silicon-containing triazole bactericide, is mainly used as a sterol demethylation inhibitor, and can destroy cell membranes and kill germs. Flusilazole has excellent preventing and treating effect on diseases caused by fungi of Ascomycetes, Basidiomycetes and fungi imperfecti, such as gray mold, scab, stemphylium leaf spot, etc.
The maximum residue limit standard of flusilazole pesticides in fruits formulated in China is 0.2mg/kg, but the current flusilazole detection method has no standard. The detection method of flusilazole mainly comprises liquid chromatography and liquid chromatography-tandem mass spectrometry, but the detection process is complex, a solid-phase extraction column needs to be purified, the cost is high, the purpose of rapid detection cannot be achieved, rapid detection on site cannot be realized, and the requirements of supervision departments and detection institutions on site supervision and law enforcement cannot be met.
Disclosure of Invention
The invention aims to provide a flusilazole hapten, an antigen, an antibody, a detection device and a detection method thereof.
According to one aspect of the present invention, there is provided an immunizing flusilazole hapten, which has the following structure:
according to another aspect of the present invention, there is provided a flusilazole hapten for coating, which has the following structure:
according to yet another aspect of the present invention, there is provided a flusilazole antigen comprising a flusilazole hapten for immunization and a carrier protein coupled to the flusilazole hapten for immunization.
In some embodiments, the carrier protein is one of bovine serum albumin, bovine lactoferrin, chicken ovalbumin, or hemocyanin.
According to the third aspect of the invention, the flusilazole antibody is provided, and is obtained by immunizing animals with flusilazole hapten or flusilazole antigen, taking blood, separating and collecting hyperimmune serum, and extracting and purifying.
In some embodiments, the flusilazole antibody is a flusilazole polyclonal antibody.
According to a fourth aspect of the present invention, there is provided the use of a flusilazole hapten for immunization or a flusilazole hapten, a flusilazole antigen, a flusilazole antibody for coating in the immunological detection of flusilazole residues.
According to a fifth aspect of the invention, a flusilazole colloidal gold chromatography detection device is provided, which comprises a test strip and a reaction cup, and is characterized in that the test strip comprises a reaction membrane, the reaction membrane is provided with a detection line and a quality control line, the detection line is coated with a coating antigen, the coating antigen comprises a flusilazole hapten for coating and a carrier protein coupled with the flusilazole hapten for coating, and the reaction cup contains a flusilazole antibody marked by colloidal gold.
According to a sixth aspect of the present invention, a method for detecting flusilazole residues in a sample is provided, wherein flusilazole in the sample is detected by using a flusilazole colloidal gold chromatography detection device.
In some embodiments, the sample is one of a fruit, a vegetable, or a grain.
The invention has the beneficial effects that: the flusilazole colloidal gold chromatography detection device disclosed by the invention can be used for quickly detecting flusilazole residues in fruits, vegetables and grain grains, and a solid-phase extraction column is not required to be purified during application, so that the purpose of quick detection is achieved.
Drawings
FIG. 1 is a mass spectrum of an immunohapten of flusilazole in accordance with the present invention.
FIG. 2 is a mass spectrum of flusilazole-coated hapten of the present invention.
FIG. 3 is a flow chart of the synthesis of the fluorosilicone immune hapten of the present invention.
FIG. 4 is a flow chart of the synthesis of flusilazole-coated haptens of the present invention.
FIG. 5 is a schematic structural diagram of the flusilazole colloidal gold chromatography detection device of the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example 1
In this example, for fluorophenylmagnesium bromide, 1.0M solution in THF specification of 4-fluorophenylmagnesium bromide supplied by national drug group chemical reagent Co., Ltd, cuprous cyanide of analytical purity supplied by national drug group chemical reagent Co., Ltd, chloromethylmethyldichlorosilane of 95% (chloromethyl) methyldichlorosilane supplied by national drug group chemical reagent Co., Ltd, hydrochloric acid of 36% -38% hydrochloric acid supplied by national drug group chemical reagent Co., Ltd, ethyl acetate of analytical purity ethyl acetate supplied by national drug group chemical reagent Co., Ltd, anhydrous sodium sulfate of 99% anhydrous sodium sulfate supplied by national drug group chemical reagent Co., Ltd, and 1, 2, 4-triazole-3-carboxylic acid methyl ester of 97% 1, 2, 4-triazole-3-carboxylic acid methyl ester supplied by national drug group chemical reagent Co., Ltd are selected, n, N-dimethylformamide is analytically pure N, N-dimethylformamide supplied by national drug group chemical reagent company Limited, sodium hydride is 60% sodium hydride supplied by national drug group chemical reagent company Limited, sublimed sulfur is chemically pure sublimed sulfur supplied by national drug group chemical reagent company Limited, potassium carbonate is analytically pure potassium carbonate supplied by national drug group chemical reagent company Limited, tert-butyl 4-bromobutyrate is 97% tert-butyl 4-bromobutyrate supplied by national drug group chemical reagent company Limited, dichloromethane is analytically pure dichloromethane supplied by national drug group chemical reagent company Limited, trifluoroacetic acid is analytically pure trifluoroacetic acid supplied by national drug group chemical reagent company Limited,
the reagents of this example 1 were used in the following examples 2 to 11.
Example 2 Synthesis of Fluorosilazole hapten for immunization
The preparation method of the flusilazole hapten for immunization comprises the following steps:
s1, taking a 100ml single-neck bottle, measuring 17ml of p-fluorophenyl magnesium bromide, adding 9mg of cuprous cyanide, stirring for 5min in an ice-water bath, dropwise adding 3.9ml of chloromethyl methyl dichlorosilane, then transferring to a 30 ℃ oil bath for continuing to react for 1h, then carrying out an ice-water bath for 10min, dropwise adding 5ml of 1M hydrochloric acid, stirring for 0.5h, keeping the ice-water bath, adding a proper amount of distilled water and ethyl acetate, fully stirring, separating liquid, drying with anhydrous sodium sulfate, concentrating, mixing the sample, and passing the mixture through a column filled with 200-mesh silica gel powder to obtain a first intermediate;
s2, taking another 100ml single-mouth bottle, filling 2.4g of all the first intermediates, weighing 1.19g of 1, 2, 4-triazole-3-methyl carboxylate, adding 20ml of DMF, stirring uniformly at room temperature, adding 0.68g of 60% sodium hydride, transferring to an oil bath, stirring, reacting at 105 ℃ for 3h, cooling the reaction solution, concentrating, adding water for diluting, adjusting the pH to 7-8 with 6M hydrochloric acid, extracting twice with ethyl acetate, concentrating, mixing with a sample, purifying with a column filled with 200-mesh silica gel powder to obtain the flusilazole hapten for immunization, and identifying the molecular weight of EI-MS (negative) M/z by mass spectrometry: 358.1[ M-H ] -.
Example 3 Synthesis of Fluorosilazole hapten for coating
The preparation method of the flusilazole hapten for coating comprises the following steps:
A1. weighing 1g of flusilazole original drug and 0.82g of sublimed sulfur in a 100ml single-mouth bottle, adding 20ml of DMF, carrying out oil bath at 156 ℃ for overnight reaction, concentrating the reaction solution, adding water for dilution, extracting with ethyl acetate, drying with anhydrous sodium sulfate, and concentrating to obtain a second intermediate;
A2. taking another 100ml single-mouth bottle, filling all the second intermediates, weighing 1.5eq potassium carbonate and 1.2eq 4-tert-butyl bromobutyrate, adding 15ml DMF, carrying out oil bath reaction at 80 ℃ for 3h, cooling the reaction solution, concentrating, adding water for dilution, extracting with ethyl acetate, directly mixing the sample, and purifying by using a column filled with 200-mesh silica gel powder to obtain a third intermediate, wherein in the step, 1eq is calculated by flusilazole;
A3. and filling another 100ml single-mouth bottle with all the third intermediates, adding 5ml dichloromethane and 2.5ml trifluoroacetic acid, stirring and reacting for 2 hours at room temperature, concentrating the reaction solution, adding water for dilution, extracting with ethyl acetate, purifying by using a column filled with 200-mesh silica gel powder to obtain a coating flusilazole hapten, and identifying EI-MS (negative) m/z by mass spectrometry: 432.1[ M-H ] -.
Example 4 Synthesis of Fluorosilazole antigen for immunization
The preparation method of the flusilazole antigen for immunization comprises the following steps:
dissolving 0.1mmol of flusilazole hapten for immunity prepared in example 1 in 2ml of DMF, stirring, adding 0.2mmol of DCC and 0.1mmol of NHS, magnetically stirring at 4 ℃ for reaction overnight, centrifuging, and taking supernatant as solution A; weighing 140mg of bovine lactoferrin, dissolving in 10ml of PBS (pH8.0) with the concentration of 0.1mol/L, adding 1ml of DMF, stirring and dissolving to prepare solution B; gradually dropping the solution A into the solution B under magnetic stirring, reacting for 12h at 4 ℃, centrifuging, taking supernatant, dialyzing for 3 days at 4 ℃ with normal saline, replacing the dialysate for 3 times per day to obtain flusilazole antigen for immunization, subpackaging the flusilazole antigen for immunization in 0.5ml centrifuge tubes at the concentration of 10mg/ml, and freezing in a refrigerator at-20 ℃ for later use.
Example 5 Synthesis of Flusilazole antigen for detection line (T line) of nitrocellulose Membrane
The preparation method of the flusilazole antigen for the detection line comprises the following steps:
dissolving 0.1mmol of the coated flusilazole hapten prepared in example 3 in 2ml of DMF, stirring, adding 0.2mmol of DCC and 0.1mmol of NHS, magnetically stirring at 4 ℃ for reacting overnight, centrifuging, and taking supernatant as solution C; weighing 120mg OVA, dissolving in 10ml PBS (pH8.0) with the concentration of 0.1mol/L, adding 1ml DMF, stirring and dissolving to obtain solution D; gradually dropping the solution C into the solution D under magnetic stirring, reacting for 12h at 4 ℃, centrifuging, taking supernatant, dialyzing for 3 days at 4 ℃ with normal saline, replacing the dialysate for 3 times per day to obtain flusilazole antigen for the detection line, subpackaging the flusilazole antigen for the detection line in 0.5ml centrifuge tubes at the concentration of 10mg/ml, and freezing in a refrigerator at-20 ℃ for later use. The detection line uses the flusilazole antigen as a coating antigen, namely comprises the flusilazole hapten for coating and carrier protein coupled with the flusilazole hapten.
Example 6 preparation and purification of Fluorosilazole polyclonal antibody
3 new zealand white rabbits with the weight of more than 2.5kg are respectively immunized by using the flusilazole antigen for immunization prepared in the embodiment 4, the immunizing dose is 50-100 mug/time, and the back is injected subcutaneously in multiple points; the first immunization, namely emulsifying the flusilazole antigen for immunization and equivalent Freund complete adjuvant; enhancing immunity, emulsifying fluorosilicone antigen for immunization and equivalent Freund incomplete adjuvant, continuously immunizing for 4-5 times, separating for 4-8 weeks each time, measuring the titer by an ELISA method to be more than 105 after 10-15 days after the last immunization, collecting blood, separating and collecting hyperimmune serum, extracting IgG antibody by a saturated ammonium sulfate salting-out method, purifying by a Protein A column chromatography to obtain fluorosilicone polyclonal antibody, and freezing and storing at-20 ℃ for later use.
Example 7 preparation of Flusilazole colloidal gold chromatography detection device
7.1 preparation of colloidal gold
Taking 1ml of 1% chloroauric acid solution, adding 99ml of ultrapure water to prepare a chloroauric acid solution with the concentration of 0.01%, heating to boil, adding 1.6ml of 1% trisodium citrate into the boiled 0.01% chloroauric acid solution at one time, continuously heating until the solution is changed from light yellow to blue black, finally changing to bright red, continuously heating for 5min after the color is stable, cooling at room temperature, and supplementing water loss to the original volume to obtain a colloidal gold solution;
7.2 preparation of colloidal gold-labeled polyclonal antibody
Adjusting the pH value of the colloidal gold solution to 8.0, uniformly stirring by using a constant-speed stirrer, simultaneously dropwise adding the flusilazole polyclonal antibody prepared in the embodiment 6, adding PEG with the amount equivalent to that of the antibody after 1h, fully reacting for 30min, adding BSA with the amount equivalent to that of the antibody, continuously stirring for 30min after the addition, centrifuging for 30min at the rotating speed of 9000rpm/min to obtain uniform gold-labeled antibody precipitate, adding PNPB (para-nepheloid phosphate) for re-suspension to obtain the colloidal gold-labeled polyclonal antibody for later use;
7.3 preparation of colloidal gold chromatography detection device
Sequentially overlapping and adhering the sample pad, the nitrocellulose membrane sprayed with flusilazole-OVA (detection line) and goat anti-rabbit IgG (quality control line) and the absorbent paper on the bottom plate along the same direction; adding colloidal gold labeled polyclonal antibody into the reaction cup, and freeze-drying.
Example 8 method for detecting flusilazole residue in sample
8.1 pretreatment of the sample: adding 5g of crushed cucumber to be detected into an extraction bottle containing 5ml of phosphate buffer solution (containing 10% ethanol in buffer solution) with pH7.4, screwing a bottle cover, and shaking for 30 seconds, and mixing uniformly to obtain a liquid to be detected;
8.2, detection: using the flusilazole colloidal gold chromatography detection device prepared in example 7 to perform detection, unscrewing the upper cover of the extraction bottle, sucking 200 μ l of supernatant into a reaction cup, sucking up and down for 5 times, mixing uniformly, starting the first-step reaction at 20-40 ℃, and timing for 3 min; inserting the test strip into a reaction cup, starting the second-step reaction at 20-40 ℃, and timing for 3 min; taking out the test strip, slightly scraping off a sample pad at the lower end of the test strip, and judging the result;
8.3 interpretation of results: the results of the test strips were interpreted according to table 1 below;
table 1 test strip result interpretation table
Comparison with the result of the verification method
The results of the detection method of this example were compared with the results of the measurement by liquid chromatography-mass spectrometry/mass spectrometry, and the results were: the detection method is consistent with the results of the liquid chromatography-mass spectrometry/mass spectrometry, which shows that the method for detecting flusilazole residues in the sample has higher accuracy.
Example 9 specificity test of Flusilazole colloidal gold chromatography detection device
Adding flusilazole in an amount of 25 mug/kg, simeconazole in an amount of 5mg/kg, imazalil in an amount of 5mg/kg, propiconazole in an amount of 5mg/kg, difenoconazole in an amount of 5mg/kg and triazole in an amount of 5mg/kg to a negative apple sample to obtain a plurality of apple samples to be detected containing different pesticide components, and detecting the apple samples to be detected by adopting the flusilazole colloidal gold chromatography detection device prepared in example 7 and the detection method in example 8.
The detection result shows that only the apple sample to be detected added with flusilazole is positive, but the apple sample to be detected added with simeconazole, imazalil, propiconazole, difenoconazole and triazole is not detectable, which indicates that the detection device has good specificity on flusilazole.
Example 10 sensitivity of Flusilazole colloidal gold chromatography detection device
And (3) respectively adding flusilazole into the negative wheat samples according to the addition amounts of 10 mug/kg, 20 mug/kg, 25 mug/kg and 30 mug/kg to obtain a plurality of wheat samples to be detected with different flusilazole contents, and detecting the wheat samples to be detected by adopting the flusilazole colloidal gold chromatography detection device prepared in the embodiment 7 and the detection method in the embodiment 8.
The detection result shows that the wheat sample to be detected with the flusilazole addition amount of 25 mug/kg and 30 mug/kg is detected as positive, and the wheat sample to be detected with the addition amount of 10 mug/kg and 20 mug/kg is detected as negative. Through the addition experiment, the flusilazole colloidal gold chromatography detection device disclosed by the invention has the detection limit of 25 mu g/kg on flusilazole. And when the content of flusilazole in the sample to be detected is lower than 25 mu g/kg, the detection result is negative. The flusilazole colloidal chromatography gold detection device has high sensitivity to flusilazole.
Example 11 shelf life test of flusilazole colloidal gold chromatography detection device
Selecting three batches of conventionally produced products to respectively perform quality guarantee period experiments, placing the products in an indoor room temperature environment for keeping, taking 12 devices every 1 month, detecting by using a quality control sample, respectively performing negative blank control and standard adding experiments of 25 mu g/kg, 50 mu g/kg, 100 mu g/kg and 200 mu g/kg, repeating the steps for three times, observing data change, and inspecting the time of the quality guarantee period. The negative color development is reduced from 13 months, and the product quality has no obvious change within 1 year, so that the shelf life of the flusilazole colloidal gold chromatographic detection device is determined to be 1 year.
The above description is only for the embodiments of the present invention, and it is obvious to those skilled in the art that various changes and modifications can be made without departing from the inventive concept of the present invention, and these changes and modifications are all within the scope of the present invention.
Claims (10)
1. An immunization fluorosilicone hapten which is characterized by having the following structure:
2. a flusilazole hapten for coating is characterized by having the following structure:
3. a flusilazole antigen comprising the flusilazole hapten for immunization of claim 1 and a carrier protein coupled to the flusilazole hapten for immunization.
4. The flusilazole antigen of claim 3, wherein the carrier protein is one of bovine serum albumin, bovine lactoferrin, chicken ovalbumin, or hemocyanin.
5. The flusilazole antibody is characterized in that the flusilazole antibody is obtained by immunizing animals with the flusilazole hapten for immunization according to claim 1 or the flusilazole antigen for immunization according to claim 3, taking blood, separating and collecting hyperimmune serum, and extracting and purifying the hyperimmune serum.
6. The flusilazole antibody of claim 5, wherein the flusilazole antibody is a flusilazole polyclonal antibody.
7. Use of the ImmunoFlusilazole hapten of claim 1 or the coated flusilazole hapten of claim 2, the flusilazole antigen of claim 3 or 4, the flusilazole antibody of claim 5 or 6 for the immunological detection of flusilazole residues.
8. A flusilazole colloidal gold chromatography detection device comprises a test strip and a reaction cup, and is characterized in that the test strip comprises a reaction membrane, the reaction membrane is provided with a detection line and a quality control line, the detection line is coated with a coating antigen, the coating antigen comprises the coated flusilazole hapten of claim 2 and carrier protein coupled with the coated flusilazole hapten, and the reaction cup contains the colloidal gold-labeled flusilazole antibody of claim 5 or 6.
9. A method for detecting flusilazole residues in a sample, which is characterized in that the flusilazole colloidal gold chromatography detection device of claim 8 is used for detecting flusilazole in the sample.
10. The method of claim 9, wherein the sample is one of a fruit, a vegetable, or a grain.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114656421A (en) * | 2022-05-23 | 2022-06-24 | 深圳市易瑞生物技术股份有限公司 | Indoxacarb hapten, indoxacarb antigen, indoxacarb antibody, detection device, preparation method and application thereof |
| CN115215811A (en) * | 2022-09-15 | 2022-10-21 | 深圳市易瑞生物技术股份有限公司 | Prothioconazole hapten, antigen, antibody, detection device, preparation and application thereof |
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| US4729986A (en) * | 1986-04-24 | 1988-03-08 | E. I. Du Pont De Nemours And Company | Fungicidal triazoles and imidazoles |
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| US4729986A (en) * | 1986-04-24 | 1988-03-08 | E. I. Du Pont De Nemours And Company | Fungicidal triazoles and imidazoles |
| DE19520095A1 (en) * | 1995-06-01 | 1996-12-05 | Bayer Ag | New 1-((tri- substd.)silyl)methyl-1,2,4-triazole-5-thiol derivs. |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114656421A (en) * | 2022-05-23 | 2022-06-24 | 深圳市易瑞生物技术股份有限公司 | Indoxacarb hapten, indoxacarb antigen, indoxacarb antibody, detection device, preparation method and application thereof |
| CN115215811A (en) * | 2022-09-15 | 2022-10-21 | 深圳市易瑞生物技术股份有限公司 | Prothioconazole hapten, antigen, antibody, detection device, preparation and application thereof |
| CN115215811B (en) * | 2022-09-15 | 2022-12-13 | 深圳市易瑞生物技术股份有限公司 | Prothioconazole hapten, antigen, antibody, detection device, preparation and application thereof |
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