Diflubenzuron hapten, antigen, antibody, detection device, and preparation and application thereof

Abstract

The present invention discloses a diflubenzuron hapten, antigen, antibody, detection device, and their preparation and application. It also relates to a diflubenzuron hapten, a diflubenzuron antigen, a diflubenzuron antibody, and a colloidal gold chromatography detection device for diflubenzuron, as well as their preparation and application for detecting diflubenzuron. The present invention utilizes an immunological detection method to specifically detect residual diflubenzuron in a sample. The method has the advantages of good specificity, high sensitivity, ease of operation, and the ability to achieve rapid on-site detection. It can better meet the needs of regulatory authorities and testing agencies for on-site supervision and law enforcement, and provides technical support for relevant law enforcement agencies in rapidly detecting residual diflubenzuron in vegetables and fruits.

Description

Technical Field

[0001] The present invention relates to the technical field of immunological detection of food safety, and more specifically, to a diflubenzuron hapten, antigen, antibody, detection device, and preparation and application thereof. The detection device is particularly suitable for rapid detection of diflubenzuron residues in vegetables and fruits. Background Art

[0002] Diflubenzuron is a benzoylurea chitin synthesis inhibitor that mainly inhibits the activity of insect cuticular chitin synthase and uridine coenzyme, thereby inhibiting insect chitin synthesis and causing their death. It has high activity against mosquito and fly larvae and can also inhibit the hatching of Diptera adult eggs. However, long-term consumption of foods containing diflubenzuron will cause certain damage to the human body. The national standard GB2763-2021 "National Food Safety Standard Maximum Residue Limits of Pesticides in Food" stipulates that the maximum residues of diflubenzuron in fruits and vegetables are 0.2-30 mg / kg. Therefore, it is necessary to control the residues of diflubenzuron in fruits, vegetables, and grains.

[0003] Because vegetable matrices are relatively complex, they need to be pretreated before measurement. The currently used method is mainly solid-phase extraction, but when using it to analyze complex matrix samples or multiple pesticide residues of different properties, it may cause low recovery rates. Detection is mainly carried out by gas chromatography or liquid chromatography, but the process is complicated and the cost is high. It is difficult to achieve the purpose of rapid detection, is not suitable for on-site rapid detection, and cannot meet the needs of on-site supervision and law enforcement by regulatory authorities and testing agencies. Summary of the Invention

[0004] The purpose of the present invention is to provide a hapten, antigen, antibody, detection device and preparation and application thereof.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] According to one aspect of the present invention, a hapten of chlorfenuron is provided, the structure of which is shown in formula (I):

[0007]

[0008] According to another aspect of the present invention, a method for preparing adifenuron hapten comprises the following steps:

[0009] S1. 2-chlorobenzoyl chloride was added dropwise to an aqueous ammonia solution under ice bath, and after 2 h of reaction, the white precipitate was filtered, washed with water and dried to obtain the first intermediate 2-chlorobenzamide;

[0010] S2. p-Nitrophenol and tert-butyl 4-bromobutyrate were dissolved in DMF, potassium carbonate was added after dissolution, and the mixture was stirred at 80°C for 6 hours. After the reaction, the solvent was evaporated to dryness, the mixture was diluted with water, and the mixture was extracted with ethyl acetate. The organic phase was evaporated to dryness and purified by column chromatography to obtain a second intermediate. The structural formula of the second intermediate is shown in Formula (II):

[0011]

[0012] S3. Hydrazine hydrate, Pd / C and ferric chloride are added to the second intermediate dissolved in methanol, and purified to obtain a third intermediate. The structural formula of the third intermediate is shown in formula (III):

[0013]

[0014] S4. The first intermediate was dissolved in 1,2-dichloroethane, and oxalyl chloride was added after dissolution. The mixture was stirred at 70°C for 6 hours, and the solvent was evaporated. 1,2-dichloroethane was added again as a solvent, and the third intermediate was added dropwise. The mixture was kept at 70°C for overnight reaction. After the reaction, the solvent was evaporated and recrystallized to obtain a fourth intermediate. The structural formula of the fourth intermediate is shown in Formula (IV):

[0015]

[0016] S5. The fourth intermediate is dissolved in dichloromethane, trifluoroacetic acid is added after dissolution, and the mixture is stirred at room temperature for 1 hour and then the solvent is evaporated to obtain a hapten having a structure as shown in formula (I).

[0017] In some embodiments, in step S1, the molar ratio of 2-chlorobenzoyl chloride to aqueous ammonia is 1:(1-10);

[0018] In step S2, the molar ratio of p-nitrophenol, tert-butyl 4-bromobutyrate, and potassium carbonate is 1:(1.2-2):(1.5-5). In some embodiments, in step S4, the molar ratio of the first intermediate to oxalyl chloride and the third intermediate is 1:(1-1.2):(1-1.2).

[0019] According to another aspect of the present invention, a diflubenzuron antigen is provided, comprising a diflubenzuron hapten and a carrier protein coupled to the diflubenzuron hapten, wherein the carrier protein is one of bovine serum albumin, ovalbumin, hemocyanin, thyroid protein or human serum albumin.

[0020] According to a fourth aspect of the present invention, there is provided a diflubenzuron antibody, which is prepared by immunizing an animal with a diflubenzuron antigen, and the diflubenzuron antibody is a diflubenzuron monoclonal antibody.

[0021] According to a fifth aspect of the present invention, there is provided a use of a diflubenzuron hapten, a diflubenzuron antigen, and a diflubenzuron antibody in immunological detection of diflubenzuron.

[0022] According to a sixth aspect of the present invention, a colloidal gold chromatography detection device for diflubenzuron is provided. The detection device includes a test strip and a microporous reaction cup. The test strip includes a reaction membrane, a sample absorption pad, a water absorbent pad, and a bottom plate. The reaction membrane is provided with a detection line and a quality control line. The detection line is coated with diflubenzuron antigen, and the quality control line is coated with goat anti-mouse antibody. The microporous reaction cup contains colloidal gold-labeled diflubenzuron antibody.

[0023] According to a seventh aspect of the present invention, a method for detecting diflubenzuron residues in a sample is provided. The method comprises detecting diflubenzuron in the sample using a diflubenzuron colloidal gold chromatography detection device. The method comprises the following steps:

[0024] 1) Sample pretreatment;

[0025] 2) Detection using a diflubenzuron colloidal gold chromatography detection device;

[0026] 3) Analyze the test results.

[0027] In some embodiments, the sample is a vegetable or a fruit.

[0028] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0029] (1) The method for preparing the hapten of chlorfenapyr provided by the present invention uses readily available chemical reagents, has a simple operation process, concise and effective synthesis steps, a high reaction yield, and low detection costs.

[0030] (2) The present invention utilizes the principle of chromatographic immunocolloidal gold to qualitatively detect whether there is residual diflubenzuron in the sample by colorimetry between the test line and the quality control line in the test card. When used, it does not require the use of large instruments such as liquid chromatography or mass spectrometry, thereby achieving the purpose of rapid detection.

[0031] (3) The present invention uses an immunological detection method to perform exclusive detection of residual carbenoxolone in samples, which has the advantages of good specificity, high sensitivity, simple operation, and the ability to achieve rapid on-site detection. It can better meet the needs of on-site supervision and law enforcement by regulatory authorities and testing agencies, and provide technical support for relevant law enforcement departments to quickly detect whether carbenoxolone residues are present in vegetables or fruits. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 1 is the mass spectrum of the diflubenzuron hapten of the present invention.

[0033] Figure 2 The figure is a flow chart of the synthesis of the diflubenzuron hapten of the present invention.

[0034] Figure 3 The figure is a schematic structural diagram of a test strip of the diflubenzuron colloidal gold chromatography detection device of the present invention.

[0035] Figure 4 Schematic diagram of the structure of the microporous reaction cup of the diflubenzuron colloidal gold chromatography detection device of the present invention.

[0036] Figure 5 Schematic diagram of the result determination of the diflubenzuron colloidal gold chromatography detection device of the present invention. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0038] Example 1

[0039] Synthesis and Identification of Diflubenzuron Hapten

[0040] The preparation method of the diflubenzuron hapten comprises the following steps:

[0041] S1. 100ml of 10% ammonia solution was added to a single-necked flask under an ice bath, and 2-chlorobenzoyl chloride 5.0g was slowly added dropwise. After 2h of reaction, the white precipitate was filtered, washed with water, and dried to give the first intermediate 2-chlorobenzamide 4.5g;

[0042] S2. 2.8 g of p-nitrophenol and 4.5 g of tert-butyl 4-bromobutyrate were dissolved in DMF, and 5.5 g of potassium carbonate was added after dissolution. The mixture was stirred at 80 ° C for 6 h. After the reaction, the solvent was evaporated, diluted with water, extracted with ethyl acetate, and the organic phase was evaporated to dryness and purified by column to obtain 5.1 g of the second intermediate;

[0043] S3. Weigh 2.5 g of hydrazine hydrate, 0.52 g of Pd / C, and 0.52 g of ferric chloride, add 50 ml of methanol to dissolve 5.2 g of the second intermediate, stir at 50 ° C for 2 h, and after the reaction is completed, filter, evaporate the solvent, dilute with water, extract with ethyl acetate, and evaporate the organic phase to dryness and then purify to obtain 3.5 g of the third intermediate;

[0044] S4. 1.6 g of the first intermediate was dissolved in 1,2-dichloroethane, and 1.4 g of oxalyl chloride was added after dissolution. The reaction was stirred at 70 ° C for 6 h, and the solvent was evaporated. 1,2-dichloroethane was added again as a solvent, and 2.5 g of the third intermediate was added dropwise. The reaction was kept at 70 ° C overnight. After the reaction, the solvent was evaporated and recrystallized to obtain 3.8 g of the fourth intermediate;

[0045] S5. Dissolve the fourth intermediate in 10 ml of dichloromethane, add 5 ml of trifluoroacetic acid, and stir at room temperature for 1 hour. Then evaporate the solvent to obtain the diflubenzuron hapten.

[0046] The hapten cyclamates were identified by mass spectrometry. The mass spectrum obtained is shown in the attached figure of the specification. Figure 1 From the mass spectrum, we can see that the molecular ion peak of diflubenzuron hapten is m / z 377.1[M+H] + , and it is the highest peak, which is consistent with the molecular weight of the diflubenzuron hapten of 376.09, indicating that the diflubenzuron hapten shown in formula (I) was successfully synthesized.

[0047] Example 2

[0048] Preparation and identification of chlorfenapyr immunization antigen and coating antigen

[0049] 2.1 Preparation of the Diflubenzuron Antigen for Immunization: 25 mg of the diflubenzuron hapten prepared in Example 1 was weighed and dissolved in 2 ml of N,N-dimethylformamide (DMF). 18 mg of N-hydroxysuccinimide (NHS) and 26 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl) were added, and the mixture was reacted at room temperature for 6 h to prepare an activation solution. 60 mg of bovine serum albumin (BSA) was dissolved in 2 ml of 0.05 M boric acid buffer (pH 9.0). 1 ml of DMF and 0.5 ml of the activation solution were added, and the mixture was reacted at room temperature for 4 h. The mixture was then dialyzed against PBS (0.02 mol / L phosphate buffer, pH 7.4), with the solution changed every 4 h for 7 to 8 times. After dialysis, the mixture was centrifuged at 4000 rpm for 5 min. The supernatant was collected to obtain the diflubenzuron hapten-BSA conjugate, i.e., the diflubenzuron antigen for immunization. The supernatant was aliquoted and stored at -20°C.

[0050] 2.2 Preparation of the Diflubenzuron Coating Antigen: 10 mg of the diflubenzuron hapten prepared in Example 2 was weighed and dissolved in 1 ml of anhydrous N,N-dimethylformamide (DMF). 3 μl of diisopropylethylamine and 5 μl of isobutyl chloroformate were then added in sequence, and the mixture was reacted at 0°C for 30 min to prepare Solution A. 70 mg of hen egg white albumin (OVA) was dissolved in 7 ml of 0.1 M boric acid buffer to obtain Solution B. Solution A was added dropwise to Solution B, reacted at room temperature for 4 h, and then dialyzed against PBS (0.02 mol / L, pH 7.4 phosphate buffer), changing the solution every 4 h for 7 to 8 times. After dialysis, the solution was centrifuged at 4000 rpm for 5 min. The supernatant was collected to obtain the diflubenzuron hapten-OVA conjugate, i.e., the diflubenzuron coating antigen. The solution was aliquoted and stored at -20°C.

[0051] Example 3

[0052] Preparation and purification of monoclonal antibodies against diflubenzuron

[0053] 3.1 Animal immunization

[0054] Healthy 6-8 week old BALB / c mice were selected for immunization. The chlorfenapyr immunization antigen prepared in Example 2 was mixed with Freund's complete adjuvant in equal amounts and emulsified. The Balb / c mice were then immunized by multiple subcutaneous injections on the back of the neck (except for sprint immunization). Complete Freund's adjuvant was used for the first immunization at a dose of 150 μg / mouse. After 4 weeks, a booster immunization was performed at a dose of 75 μg / mouse, mixed and emulsified with incomplete Freund's adjuvant. The intervals between subsequent booster immunizations were 3 weeks. For sprint immunization, the dose was halved to 37.5 μg / mouse. The complete antigen was diluted with physiological saline and injected intraperitoneally into the mice. After the third immunization, the mice were tail-cut and blood was collected for testing. The titer and IC of the mouse serum were tested by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). 50 , select high titer, IC 50 Mice with low expression were fused.

[0055] 3.2 Cell fusion and cloning

[0056] Splenocytes of immunized BALB / c mice were obtained and fused with SP2 / 0 myeloma cells at a ratio of 9:1 to obtain a monoclonal hybridoma cell line of carbofuran that stably secreted carbofuran monoclonal antibodies.

[0057] 3.3 Cell cryopreservation and thawing

[0058] The monoclonal hybridoma cells of chlorfenapyr were cryopreserved to prepare 1×10 6 Cell suspensions of 100 cells / mL can be stored in liquid nitrogen for a long term. When thawing, remove the cryovials and immediately thaw in a 37°C water bath. Centrifuge to remove the cryopreservation solution and transfer to culture flasks for culture.

[0059] 3.4 Preparation and purification of monoclonal antibodies

[0060] Incremental culture method: The diflubenzuron monoclonal hybridoma cells are placed in a cell culture medium and cultured at 37°C. The resulting culture fluid is purified using the octanoic acid-saturated ammonium sulfate method to obtain the diflubenzuron monoclonal antibody and stored at -20°C. The cell culture medium comprises RPMI-1640 medium supplemented with calf serum and sodium bicarbonate, with the calf serum content of the cell culture medium being 15-20% by weight and the sodium bicarbonate content of the cell culture medium being 0.1-0.2% by weight. The pH of the cell culture medium is 7.4.

[0061] Example 4

[0062] Preparation of goat anti-mouse antibodies

[0063] Sheep are used as immune animals, and mouse antibodies are used as immunogens to immunize pathogen-free sheep to obtain sheep anti-mouse antibodies.

[0064] Example 5

[0065] Preparation of a colloidal gold chromatography detection device for diflubenzuron

[0066] The preparation method of the diflubenzuron colloidal gold chromatography detection device mainly comprises the following steps:

[0067] 1) Preparing a microporous reaction cuvette containing freeze-dried chlorfenapyr monoclonal antibody-colloidal gold label;

[0068] 2) preparing a reaction membrane having a test line coated with a diflubenzuron hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse antibody;

[0069] 3) Assembling the reaction membrane prepared in 2) with the sample absorption pad, water absorption pad, and base plate to form a test paper;

[0070] 4) Assembling the freeze-dried microporous reaction cup containing the chlorfenapyr monoclonal antibody-colloidal gold marker prepared in 1) and 3) and the test paper.

[0071] The following is a detailed step-by-step description:

[0072] 5.1 Preparation of diflubenzuron monoclonal antibody-colloidal gold label

[0073] (1) Preparation of colloidal gold solution

[0074] Dilute a 1% chloroauric acid solution to 0.01% (mass fraction) with double-distilled deionized water. Place 100 ml of the 0.01% chloroauric acid solution in a conical flask and heat to boiling using a constant-temperature electromagnetic stirrer. Add 2.5 ml of a 1% trisodium citrate solution while continuing to stir at high temperature. Continue stirring and heating at a constant speed until the solution turns a translucent red. Cool to room temperature and return to the original volume with deionized water to obtain a colloidal gold solution, which is stored at 4°C. The prepared colloidal gold solution is pure, translucent, free of precipitates and floating matter, and is wine red when observed in sunlight.

[0075] (2) Preparation of diflubenzuron monoclonal antibody-colloidal gold label

[0076] Under magnetic stirring, the pH of the colloidal gold solution was adjusted to 7.2 with 0.2 mol / L potassium carbonate solution. The diflubenzuron monoclonal antibody prepared in Example 4 was added to the colloidal gold solution at a standard of 20-60 μg of diflubenzuron monoclonal antibody per milliliter of colloidal gold solution. Stirring and mixing were continued for 30 minutes. After standing for 10 minutes, 10% bovine serum albumin (BSA) solution was added to a volume percentage of 1% in the colloidal gold solution. The solution was allowed to stand for 10 minutes. Centrifugation was then performed at 12,000 rpm at 4°C for 40 minutes. The supernatant was discarded, and the precipitate was resuspended in a reconstitution buffer having a volume of 1 / 10 of the initial colloidal gold solution to prepare a diflubenzuron monoclonal antibody-colloidal gold label. The solution was then stored at 4°C until use.

[0077] Reconstitution buffer: 0.02 mol / L phosphate buffer containing 0.1% to 0.3% bovine serum albumin by volume, 0.1% to 0.2% Tween-20 by mass, 3% to 6% trehalose by mass, pH = 7.2.

[0078] 5.2 Preparation of microwell reaction cup

[0079] Add 100 μl of diflubenzuron monoclonal antibody-colloidal gold label to a microporous reaction cup, place the cup in a freeze dryer, pre-freeze for 3 hours at a cold trap temperature of -50°C, and then vacuum dry for 6 hours. The cup can be taken out to obtain a microporous reaction cup with freeze-dried diflubenzuron monoclonal antibody-colloidal gold label. The cup should be sealed and stored. The freeze-dried amount of diflubenzuron monoclonal antibody-colloidal gold label is 0.20-0.50 μg / mL.

[0080] 5.3 Preparation of sample absorption pad

[0081] The sample absorption pad was immersed in 0.05 mol / L phosphate buffer containing bovine serum albumin for 2 hours, and dried at 37°C for 2 hours for later use. The pH of the 0.02 mol / L phosphate buffer was 7.2, and the volume percentage of bovine serum albumin was 0.5%.

[0082] 5.4 Preparation of reaction membrane

[0083] The antimicrobial coating antigen (i.e., antimicrobial hapten-chicken ovalbumin conjugate) is coated on the reaction membrane to form a detection line, and the goat anti-mouse antibody is coated on the reaction membrane to form a quality control line.

[0084] Coating process: Dilute the antimicrobial antigen to a concentration of 1 mg / mL in phosphate buffer and coat it onto the test zone (zone T) of the nitrocellulose membrane using a gold-labeled spray-coater at a coating concentration of 0.5 mg / mL. Dilute the goat anti-mouse antibody to a concentration of 200 μg / mL in phosphate buffer (0.01 mol / L, pH 7.4) and coat it onto the control zone (zone C) of the nitrocellulose membrane using a gold-labeled spray-coater at a coating concentration of 0.5 mg / mL. Dry the coated membrane at 37°C for 6 hours before use in production.

[0085] 5.5 Preparation of Diflubenzuron Colloidal Gold Chromatography Detection Device

[0086] (1) Assembly of test strips

[0087] A sample absorption pad, reaction membrane, and water-absorbing pad are sequentially attached to a base plate. The base plate is a PVC base plate, the sample absorption pad is filter paper, the water-absorbing pad is filter paper, and the reaction membrane is a nitrocellulose membrane. The end of the sample absorption pad is connected to the beginning of the reaction membrane, the end of the reaction membrane is connected to the beginning of the water-absorbing pad, the beginning of the sample absorption pad is aligned with the beginning of the base plate, and the end of the water-absorbing pad is aligned with the end of the base plate. The microporous reaction cuvette has a microporous stopper.

[0088] (2) Assembly of the chlorfenapyr colloidal gold chromatography test paper box

[0089] The test strip obtained in the above step (1) is assembled with the microporous reaction cup into a test strip box, which is stored in an environment of 2-8°C and has a validity period of 12 months.

[0090] Example 6

[0091] A method for detecting diflubenzuron residue in a sample

[0092] 6.1 Preparation of sample extract

[0093] Accurately weigh 9 g of sodium chloride, 6 g of disodium hydrogen phosphate dodecahydrate, and 0.4 g of sodium dihydrogen phosphate dihydrate, dissolve them in water and dilute to 100 ml to obtain the sample extract. The sample extract is a 0.1 mmol / L phosphate buffer solution with a pH of 8.0.

[0094] 6.2 Sample pretreatment

[0095] Accurately weigh 2 g (accurate to 0.01 g) of the prepared vegetable sample into a 15 ml centrifuge tube, add 8 ml of the sample extract, cover the tube, mix with a vortex mixer or manually shake up and down for 30 seconds, let it stand to separate or centrifuge at 4000 r / min for 1 minute, and take the supernatant as the test solution.

[0096] 6.3 Determination steps

[0097] Pipette 200 μl of the test solution into a microwell reaction cup and pipette up and down 5-10 times to mix thoroughly. Incubate at room temperature for 3 minutes. Insert the test strip into the reaction cup and incubate at room temperature for 3 minutes. Remove the test strip, gently scrape off the sample pad at the bottom of the strip, and read the result.

[0098] 6.4 Result determination

[0099] The results were determined by comparing the color depth of the control line (C line) and the test line (T line).

[0100] Positive: When the control area (C) shows a band and the test area (T) does not show color, it is judged as positive, that is, there is diflubenzuron in the sample, which is indicated by "+";

[0101] Negative: When both the control area and the test area show bands, it is judged as negative, that is, there is no diflubenzuron in the sample, which is indicated by "-";

[0102] Invalid: When the quality control area (C) does not show a band, the test paper is invalid, as shown in the attached figure of the instruction manual. Figure 5 shown.

[0103] Example 7

[0104] Sensitivity and false negative rate test of the colloidal gold chromatography detection device for diflubenzuron

[0105] Cabbage, Chinese cabbage and apple without carbenoxolone tested by GB / T 20769-2008 "Determination of 450 pesticides and related chemicals residues in fruits and vegetables by liquid chromatography-tandem mass spectrometry" were selected as blank samples. According to the maximum residue limits (MRLs) of cabbage, Chinese cabbage and apples specified in GB 2763-2021 of 3 mg / kg, 30 mg / kg and 2 mg / kg respectively, the method detection limit of cabbage was set to 3 mg / kg, the method detection limit of Chinese cabbage was set to 30 mg / kg, and the method detection limit of apple was set to 2 mg / kg, i.e., the concentration of concern. The addition levels were 1 times the concentration of concern and 2 times the concentration of concern, and the sensitivity and false negative rate were examined. Samples at two addition concentration levels, 50 samples at each concentration level, were tested according to the detection method in Example 6. The test results are shown in Table 1 below.

[0106] Table 1 Sensitivity and false negative rate test results of diflubenzuron colloidal gold chromatography detection device

[0107]

[0108] The detection method in this embodiment has a sensitivity of ≥95% for the residual diflubenzuron in the sample, and a false negative rate of ≤5%.

[0109] Example 8

[0110] Specificity and false positives of the colloidal gold chromatography detection device for diflubenzuron

[0111] Cabbage, Chinese cabbage, and apple samples were spiked with blank matrix to prepare 50 samples at two concentration levels (0.5 times the detection limit, blank matrix). The samples were tested using the detection method in Example 7. The test results are shown in Table 2 below.

[0112] Table 2 Specificity and false positive test results of the chlorfenuron colloidal gold chromatography detection device

[0113]

[0114]

[0115] As shown in Table 2, the specificity of the detection method in this embodiment is ≥90%, and the false positive rate is ≤10%. These results indicate that the test kit for detecting diflubenzuron of the present invention has good specificity and can accurately detect diflubenzuron in vegetable and fruit samples, thereby enabling rapid detection of diflubenzuron residues in vegetable and fruit samples.

[0116] Example 9

[0117] Shelf Life Determination Using Diflubenzuron Colloidal Gold Chromatography Detection Device

[0118] Three batches of conventionally produced products were used for shelf life experiments. They were kept at room temperature indoors. Twelve devices were taken out at one-month intervals and tested with quality control samples. Negative, 1.5mg / kg, 15mg / kg and 1mg / kg samples were tested three times. The color changes of the products were observed and the shelf life was examined.

[0119] The negative color development began to decline from 13 months, which indicated that the quality of the product had no significant change within 1 year, so the shelf life was determined to be 1 year.

[0120] Although the present invention has been described herein with reference to a number of illustrative embodiments thereof, it will be understood that numerous other modifications and implementations may be devised by those skilled in the art that fall within the scope and spirit of the principles disclosed herein. More specifically, within the scope of the present disclosure, the drawings, and the claims, numerous variations and modifications may be made to the components and / or layout of the subject combination arrangement. In addition to variations and modifications to the components and / or layout, other uses will also be apparent to those skilled in the art.

Claims

1. A hapten containing diflubenzuron, characterized in that Its structure is shown in formula (I):

2. The method for preparing the diflubenzuron hapten according to claim 1, characterized in that: The following steps are involved: S1. 2-chlorobenzoyl chloride was added dropwise to an aqueous ammonia solution under ice bath, and after 2 h of reaction, the white precipitate was filtered, washed with water and dried to obtain the first intermediate 2-chlorobenzamide; S2. p-Nitrophenol and tert-butyl 4-bromobutyrate were dissolved in DMF, potassium carbonate was added after dissolution, and the mixture was stirred at 80°C for 6 hours. After the reaction, the solvent was evaporated to dryness, the mixture was diluted with water, and the mixture was extracted with ethyl acetate. The organic phase was evaporated to dryness and purified by column chromatography to obtain a second intermediate. The structural formula of the second intermediate is shown in Formula (II): S3. Hydrazine hydrate, Pd / C and ferric chloride are added to the second intermediate dissolved in methanol, and purified to obtain a third intermediate. The structural formula of the third intermediate is shown in formula (III): S4. The first intermediate was dissolved in 1,2-dichloroethane, and oxalyl chloride was added after dissolution. The mixture was stirred at 70°C for 6 hours, and the solvent was evaporated. 1,2-dichloroethane was added again as a solvent, and the third intermediate was added dropwise. The mixture was kept at 70°C for overnight reaction. After the reaction, the solvent was evaporated and recrystallized to obtain a fourth intermediate. The structural formula of the fourth intermediate is shown in Formula (IV): S5. The fourth intermediate is dissolved in dichloromethane, trifluoroacetic acid is added after dissolution, and the mixture is stirred at room temperature for 1 hour and then the solvent is evaporated to obtain a hapten having a structure as shown in formula (I).

3. The method for preparing the diflubenzuron hapten according to claim 2, characterized in that: In step S1, the molar ratio of 2-chlorobenzoyl chloride to ammonia water is 1:(1-10); In step S2, the molar ratio of p-nitrophenol, tert-butyl 4-bromobutyrate and potassium carbonate is 1:(1.2-2):(1.5-5).

4. The method for preparing the diflubenzuron hapten according to claim 2, characterized in that: In the step S4, the molar ratio of the first intermediate to oxalyl chloride and the third intermediate is 1:(1-1.2):(1-1.2).

5. A diflubenzuron antigen, characterized in that The diflubenzuron antigen comprises the diflubenzuron hapten according to claim 1 and a carrier protein coupled to the diflubenzuron hapten, wherein the carrier protein is one of bovine serum albumin, ovalbumin, hemocyanin, thyroid protein or human serum albumin.

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