CN118348239A - Test strip for detecting bifenazate and preparation method and application thereof - Google Patents

Abstract

The invention discloses a test strip for detecting bifenazate and a preparation method and application thereof, and belongs to the technical field of bifenazate detection. The test strip comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate, wherein the reaction membrane is provided with a detection line coated with a biphenylhydrazine ester hapten carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody, and the conjugate release pad is sprayed with a biphenylhydrazine ester monoclonal antibody colloidal gold marker. The invention also provides an application of the test strip in detecting bifenazate residues in fruit and vegetable samples. The test strip provided by the invention has the advantages of simplicity in operation, high sensitivity, high detection speed, low cost, suitability for large-batch sample screening and the like, and can meet the requirements of food supervision departments for on-site monitoring and detection.

Description

Test strip for detecting bifenazate and preparation method and application thereof

Technical Field

The invention relates to the technical field of bifenazate detection, in particular to a test strip for detecting bifenazate, a preparation method and application thereof.

Background

Bifenazate (BIFENAZATE) is a novel selective foliar acaricide which has no systemic property, is mainly used for preventing and treating spider mites in the active period, but has ovicidal effect on other mites, especially spider mites. It has good control effect on agricultural mites such as citrus red mites, rust ticks, yellow mites, short-hair mites, hawthorn spider mites, tetranychus cinnabarinus, tetranychus urticae and the like. Bifenthrin acts mainly on the complex III site of the cell's mitochondria, inhibiting the cell's mitochondrial energy transfer. The mites start to paralyze and overstimulation after 3 to 10 hours after contacting the medicament, feeding and spawning are stopped within 1 to 2 days, and the mites die gradually within 3 to 4 days. Is effective for all development stages of mites, and has ovicidal activity and knockdown activity (48-72 h) for adult mites. However, as the bifenazate is directly sprayed on crops, the bifenazate is used in a large amount and easily pollutes water sources, and potential pollution is caused to the environment and even human bodies. According to the requirements of GB 2763-2021 national food safety standard maximum residue limit of pesticides: the maximum residual limit of bifenazate in the fruit and vegetable sample is 0.2-7 mg/kg.

At present, the method for detecting the bifenazate at home and abroad mainly adopts analysis methods such as gas chromatography, gas chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry and the like, has the defects of complicated sample pretreatment, long detection time, expensive instrument and the like, cannot be widely applied, and does not meet the requirement of on-site detection on accurate detection and screening of a large number of samples in a short time at low cost. The immunological detection analysis technology has the advantages of high sensitivity, high specificity, rapidness, simple and convenient operation and the like, is widely applied to the field of drug residue detection, and has a lot of advantages compared with the detection methods such as instruments and the like. Therefore, the immunoassay provides a new analytical detection method for the bifenazate residue research.

Disclosure of Invention

The invention aims to provide a test strip capable of detecting bifenazate residues in fruit and vegetable samples, and provides a detection method which is efficient, accurate, simple and convenient and suitable for on-site monitoring and screening of a large number of samples.

The invention aims at realizing the following technical scheme:

In a first aspect, the invention provides a test strip for detecting bifenazate, which comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate, wherein the reaction membrane is provided with a detection line and a quality control line, the detection line is coated with a bifenazate hapten-carrier protein conjugate, and the conjugate release pad is sprayed with a bifenazate monoclonal antibody-colloidal gold marker.

Preferably, the biphenylhydrazine ester hapten-carrier protein conjugate is obtained by coupling a biphenylhydrazine ester hapten with a carrier protein.

Preferably, the synthesis method of the bifenazate hapten comprises the following steps: the method comprises the steps of taking bifenazate as a starting material, heating the bifenazate and 1, 4-cyclohexane diisocyanate with higher activity in pyridine and triethylamine environments, and introducing cyclohexane isocyanate at an imine position through an acylation reaction; the molecular structural formula of the bifenazate hapten is shown as formula I:

preferably, the biphenylhydrazine ester monoclonal antibody is prepared by taking biphenylhydrazine ester hapten-carrier protein conjugate as immunogen.

Preferably, the quality control line is coated with goat anti-mouse anti-antibody.

Preferably, the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroxine or human serum albumin.

Preferably, the sample absorbing pad, the conjugate releasing pad, the reaction membrane and the absorbent pad are sequentially adhered to the bottom plate.

Preferably, 1/3 to 1/2 of the conjugate release pad is covered under the sample absorbing pad.

Preferably, the base plate may be a PVC base plate or other rigid, non-absorbent material.

Preferably, the sample absorbing pad may be a suction filter paper or a oil filter paper.

Preferably, the conjugate release pad may be a glass wool or polyester material.

Preferably, the absorbent pad is absorbent paper.

Preferably, the reaction membrane may be a nitrocellulose membrane or a cellulose acetate membrane.

In a second aspect, the present invention provides a method for preparing the test strip, which includes the following steps:

(1) Preparing a conjugate release pad sprayed with a bifenazate monoclonal antibody-colloidal gold marker;

(2) Preparing a reaction membrane with a detection line coated with a biphenylhydrazine ester hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody:

(3) And (3) assembling the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the bottom plate which are prepared in the steps (1) and (2) into the test strip.

Preferably, the colloidal gold is prepared by reacting trisodium citrate with chloroauric acid.

Preferably, the biphenylhydrazine ester monoclonal antibody is obtained by immunizing a mouse by the biphenylhydrazine ester hapten-carrier protein conjugate, fusing and screening spleen cells of the mouse and myeloma cells of the mouse to obtain a hybridoma cell strain secreting the biphenylhydrazine ester monoclonal antibody, and culturing and purifying the hybridoma cell strain to obtain the biphenylhydrazine ester monoclonal antibody.

Preferably, the prepared bifenazate monoclonal antibody is added into the prepared colloidal gold to obtain the bifenazate monoclonal antibody-colloidal gold marker.

Preferably, the goat anti-mouse antibody is derived from a healthy goat immunized with mouse IgG.

Preferably, the sample absorbing pad is obtained by soaking the sample absorbing pad in phosphate buffer solution containing 0.5% bovine serum albumin and having pH of 7.2 and 0.1moL/L for 2 hours and drying the sample absorbing pad at 37 ℃ for 2 hours.

Preferably, a sample absorbing pad, a conjugate releasing pad, a reaction membrane and a water absorbing pad are sequentially stuck on the bottom plate, and 1/3 area of the conjugate releasing pad from the initial end is covered by the sample absorbing pad; cutting into strips with the width of 2.88mm, adding a plastic box, vacuum packaging, and preserving for 12 months at the temperature of 4-30 ℃.

In a third aspect, the invention provides an application of the test strip or the test strip prepared by the preparation method in detecting bifenazate residues in fruit and vegetable samples.

The invention provides a test strip for detecting bifenazate, a preparation method and application thereof. The quick biphenylhydrazine ester detection test strip adopts the high-specificity antibody antigen reaction and immunochromatography analysis technology, fixes the biphenylhydrazine ester monoclonal antibody-colloidal gold label on the conjugate release pad, and combines the biphenylhydrazine ester in the sample with the biphenylhydrazine ester monoclonal antibody-colloidal gold label on the conjugate release pad in the flowing process to form the drug-antibody-colloidal gold label. The drugs in the sample compete with the biphenylhydrazine ester hapten-carrier protein conjugate on the reaction membrane detection line for combining with the biphenylhydrazine ester monoclonal antibody-colloidal gold marker, and whether the biphenylhydrazine ester residue is contained in the sample liquid to be detected is judged according to the red stripe depth of the detection line.

During detection, after the sample is treated, dripping into a test strip clamping hole, when the concentration of the bifenazate in the sample is lower than the detection limit or is zero, combining the monoclonal antibody-colloidal gold marker with the bifenazate hapten-carrier protein conjugate fixed on the reaction membrane in the chromatography process, wherein a red strip appears on the detection line (T) and the quality control line (C), and the color development of the T line is deeper than or consistent with that of the C line; if the concentration of bifenazate in the sample is equal to or higher than the detection limit, the monoclonal antibody-colloidal gold-labeled substance will bind to the bifenazate completely, so that no red band or less coloration than the C-line will occur at the T-line because of the competition reaction without binding to the bifenazate hapten-carrier protein conjugate. As shown in fig. 3.

Negative: and when the quality control line (C) shows red stripes, the detection line (T) also shows red stripes, and the color of the (T) line is close to or deeper than that of the (C), the judgment is negative.

Positive: and judging positive when the quality control line (C) shows red stripes and the detection line (T) does not develop color or the color of the detection line (T) is lighter than that of the detection line (C).

Invalidation: when the quality control line (C) does not display red stripes, whether the detection line (T) displays red stripes or not, the test strip is judged to be invalid.

Compared with other prior art, the invention has the following beneficial effects:

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 shelf life. The method for detecting the bifenazate residue by using the test strip is simple, convenient, quick, visual and accurate, wide in application range, low in cost and easy to popularize and use.

Drawings

FIG. 1 is a schematic structural diagram of a bifenazate hapten.

FIG. 2 is a schematic diagram of a cross-sectional structure of a test strip according to the present invention.

Reference numerals illustrate: 1. a sample absorbing pad; 2. a conjugate release pad; 3. a reaction membrane; 4. a water absorbing pad; 5. a detection line; 6. a quality control line; 7. a bottom plate.

FIG. 3 is a graph showing the test result of the test strip according to the present invention.

Detailed Description

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

A test strip for detecting bifenazate is prepared by the following steps:

1) Preparing a conjugate release pad sprayed with a bifenazate monoclonal antibody-colloidal gold marker;

2) Preparing a reaction membrane with a detection line coated with a biphenylhydrazine ester hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse antibody;

3) And (3) assembling the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the bottom plate which are prepared in the steps 1) and 2) into the test strip.

The method comprises the following specific steps:

1. Synthesis of biphenylhydrazine ester hapten (structural view of biphenylhydrazine ester hapten is shown in figure 1)

Taking 6.007g of bifenazate, adding 40mL of pyridine for dissolution, adding 5.96g of 1, 4-cyclohexane diisocyanate, adding 2.5mL of triethylamine, heating to 80 ℃ for reaction for 4 hours, stopping the reaction, performing rotary evaporation, removing pyridine and triethylamine to obtain oily matter, wherein the volume ratio of the added 1:15 in 80mL of diethyl ether/n-hexane to obtain cyclohexane isocyanate-biphenyl hydrazine ester hapten products.

2. Preparation of immunogens

Taking 15mg of the bifenazate hapten prepared in the step 1, adding 1mL of DMSO for dissolving and clarifying to obtain hapten solution, and marking the hapten solution as A solution; taking 50mg of bovine serum albumin BSA, adding 6mL of carbonate buffer solution of 0.1moL/LpH to dissolve to obtain solution B, dropwise adding the solution A into the solution B, reacting for 4 hours at room temperature, stopping the reaction, dialyzing and purifying for 3 days with 0.02moL/LPBS, changing the solution 3 times a day, centrifuging and subpackaging to obtain the bifenazate-BSA conjugate, namely the immunogen, subpackaging and preserving at the temperature of minus 20 ℃.

3. Preparation of coating Material

Taking 10mg of the bifenazate hapten prepared in the step 1, adding 1mL of DMSO for dissolving and clarifying to obtain hapten solution, and marking the hapten solution as A solution; taking 50mg of Ovalbumin (OVA), adding 6mL of carbonate buffer solution with the concentration of 0.1moL/L and the pH of 9.5 for dissolution to obtain solution B, dropwise adding the solution A into the solution B, reacting at room temperature for 4 hours, stopping the reaction, dialyzing and purifying for 3 days with the concentration of 0.02moL/LPBS, changing the solution for 3 times every day, centrifuging and split charging to obtain the bifenazate-OVA conjugate, namely the coating source.

4. Preparation of Bifenazate monoclonal antibodies

(1) Immunization of animals

The immunogen obtained in the step 2 is injected into Balb/c mice, and the immune dose is 150 mug/mouse, so that antisera are generated.

(2) Cell fusion and cloning

Spleen cells of immunized Balb/c mice were taken at an ratio of 8:1 (quantitative ratio) and SP2/0 myeloma cells are fused, cell supernatant is measured by adopting indirect competition ELISA, and positive holes are screened. Cloning the positive hole by limiting dilution method until obtaining hybridoma cell strain for stably secreting monoclonal antibody.

(3) Cell cryopreservation and resuscitation

The hybridoma cells were prepared as a cell suspension of 1X 10 ⁶ cells/mL in a frozen solution and stored in liquid nitrogen for a long period of time. 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) Preparation and purification of monoclonal antibodies

Incremental culture method: 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 as follows: adding calf serum and sodium bicarbonate to the RPMI 1640 medium to make the final concentration of the calf serum in the cell culture medium be 20% (mass fraction) and make the final concentration of the sodium bicarbonate in the cell culture medium be 0.2% (mass fraction); the pH was 7.4.

5. Preparation of goat anti-mouse antibody

And (3) taking a healthy goat (without pathogen) as an immunized animal, and taking a murine IgG antibody as an immunogen to immunize the goat so as to obtain the goat anti-mouse antibody.

6. Preparation of biphenylhydrazine ester monoclonal antibody-colloidal gold marker

(1) Preparation of colloidal gold

Diluting 1% chloroauric acid into 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 high temperature and continuous stirring, stopping stirring and heating until the solution is transparent red, cooling to room temperature, recovering to original volume with deionized water, and preserving at 4deg.C. The prepared colloidal gold has pure appearance, is transparent, has no sediment or floating matters, and has a wine red color when observed in sunlight.

(2) Preparation of biphenylhydrazine ester monoclonal antibody-colloidal gold marker

Under the magnetic stirring, adjusting the pH value of the colloidal gold to 7.2 by using 0.2moL/L potassium carbonate solution, adding the bifenazate monoclonal antibody into the colloidal gold solution according to the standard of adding 20-50 mu g of antibody into each milliliter of the colloidal gold solution, and continuously stirring and uniformly mixing for 30min; after 10min of standing, 10% BSA was added to give a final concentration of 1% in the colloidal gold solution, and the mixture was left for 10min. Centrifuging at 12000r/min and 4deg.C for 40min, discarding supernatant, washing the precipitate twice with redissolving buffer, re-suspending the precipitate with redissolving buffer with volume 1/10 of that of initial colloidal gold, and standing at 4deg.C for use.

Reconstitution buffer: 0.02moL/L phosphate buffer containing 0.2% BSA (mass fraction), 0.1% Tween-80 (mass fraction), pH 7.2.

7. Preparation of conjugate release pads

The conjugate release pad was soaked in phosphate buffer containing 0.5% BSA, pH 7.2, 0.5moL/L, and soaked uniformly for 1h, and baked at 37℃for 3 h. And uniformly spraying the prepared biphenylhydrazine ester monoclonal antibody-colloidal gold marker on a conjugate release pad by using a film spraying instrument, spraying 0.01mL of biphenylhydrazine ester monoclonal antibody-colloidal gold marker on each 1cm of conjugate release pad, placing the conjugate release pad in a 37 ℃ environment (humidity < 20%) for 24 hours, taking out the conjugate release pad, and placing the conjugate release pad in a dry environment (humidity < 20%) for storage for later use.

8. Preparation of sample absorbent pad

The sample absorbing pad is placed in phosphate buffer solution containing 0.5% bovine serum albumin with pH of 7.2 and 0.1moL/L for soaking for 2 hours, and dried at 37 ℃ for 8 hours for standby.

9. Preparation of reaction film

The biphenylhydrazine ester hapten-ovalbumin conjugate is coated on a reaction membrane to form a detection line, and the goat anti-mouse anti-antibody is coated on the reaction membrane to form a quality control line.

The coating process comprises the following steps: diluting the biphenylhydrazine hapten-ovalbumin conjugate to 0.7mg/mL by using a phosphate buffer solution, and coating the biphenylhydrazine hapten-ovalbumin conjugate on a detection line (T line) on a nitrocellulose membrane by using a spot film tester, wherein the coating amount is 1.0 mu L/cm; the goat anti-mouse antibody was diluted to 0.5mg/mL with 0.01moL/L, pH 7.4.4 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 a spot film apparatus. And (5) drying the coated reaction film for 8 hours at 37 ℃ for standby.

10. Assembly of test strips

According to the cross-section structure of the test strip shown in FIG. 2, a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3) and a water absorption pad (4) are sequentially stuck on a PVC bottom plate (7); the 1/3 area of the initial end of the conjugate release pad is covered by the sample absorption pad, the tail end of the conjugate release pad is connected with the initial end of the reaction membrane, the tail end of the reaction membrane is connected with the initial end of the water absorption pad, the initial end of the sample absorption pad is aligned with the initial end of the PVC bottom plate, and the tail end of the water absorption pad is aligned with the tail end of the PVC bottom plate; the reaction film is provided with a detection line (5) and a quality control line (6), and the detection line (T line) and the quality control line (C line) are strip-shaped strips which are perpendicular to the length of the test strip; the detection line is positioned at one side close to the tail end of the conjugate release pad; the quality control line is positioned at one side far away from the tail end of the conjugate release pad; cutting the test paper strip into strips with the width of 2.88mm by a machine, putting the strips into a specially made plastic card, and storing the strips in the environment of 4-30 ℃ for 12 months.

Example 2

The detection of bifenazate in fruit and vegetable samples comprises the following specific processes:

1. Sample pretreatment

Wiping off soil from a fresh sample, and shearing the fresh sample into fragments smaller than 1cm square; weighing (2.00+/-0.05) g of a sample into a 50mL centrifuge tube, adding 8mL of phosphate buffer solution, covering a cover, manually oscillating for 30s, standing for 2min, and obtaining a supernatant as sample liquid 1; and adding 400 mu L of phosphate buffer solution into 100 mu L of sample solution 1, and uniformly mixing to obtain the sample solution to be tested.

2. Detection by test strips

Sucking 70 mu L of sample liquid to be detected by a micropipette and vertically dripping the 70 mu L of sample liquid into a sample adding hole; the flow of the liquid was started to time, the reaction was continued for 5 minutes, and the result was judged.

3. Analyzing the detection result

Negative (-). The T line color development is darker than or consistent with the C line color development, indicating that the concentration of bifenazate in the sample is below the detection limit, as shown in FIGS. 3a and b.

Positive (+): the T-line developed less than the C-line developed or the T-line developed no color, indicating that the concentration of bifenazate in the sample was equal to or higher than the detection limit, as shown in fig. 3C and d.

Invalidation: the absence of line C indicates an incorrect procedure or that the test strip has failed due to deterioration, as shown by e and f in fig. 3.

Example 3

Sample detection examples, the specific procedure is as follows:

1. Limit of detection test

Blank tomato, capsicum, bean, orange, strawberry, apple, grape and pineapple samples were taken, bifenazate was added to the samples to a final concentration of 0.1mg/kg, 0.2mg/kg and 0.4mg/kg, and the test strips prepared in example 1 were taken and tested according to the procedure of example 2, and each sample was repeatedly assayed three times.

When a test strip is used for detecting samples of tomatoes, peppers, beans, oranges, strawberries, apples, grapes and pineapples, when the sample is free of bifenazate and the addition concentration of the bifenazate is 0.1mg/kg, the test strip shows that the color development of a T line is deeper than or consistent with that of a C line, and the test strip is negative; when the adding concentration of the bifenazate is 0.2mg/kg and 0.4mg/kg, the test strip shows that the color development of the T line is lighter than that of the C line or the color development of the T line is not positive, which indicates that the detection limit of the test strip on the bifenazate in fruits and vegetables is 0.2mg/kg.

2. False positive rate and false negative rate test

Taking 20 parts of blank tomato, capsicum, bean, orange, strawberry, apple, grape and pineapple samples, respectively, adding bifenazate into the blank tomato, capsicum, bean, orange, strawberry, apple, grape and pineapple samples with the final concentration of 0.2mg/kg, respectively detecting the blank tomato, capsicum, bean, orange, strawberry, apple, grape and pineapple samples by using test strips produced in 3 batches, and calculating the negative-positive rate of the blank tomato, capsicum, bean, orange, strawberry, apple, grape and pineapple samples.

The results show that: when positive samples are detected by using test strips produced in 3 batches, the results are positive, the positive coincidence rate is 100%, and the false negative rate is 0; when a negative sample was detected, the result was all negative, and it was found that the negative coincidence rate was 100% and the false positive rate was 0. The test strip for detecting bifenazate can be used for rapidly detecting bifenazate residues in fruit and vegetable samples.

3. Specificity test

When the test strip is used for detecting 1mg/kg of bipyrazate structural and functional analogues such as pyriproxyfen, bromopropylate, spirotetramat, chlorfenapyr, pyriminostrobin and the like, the test strip shows that the color development of the T line is deeper than that of the C line or consistent with that of the C line, and the test strip is negative, so that the test strip has no cross reaction to the medicines.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (7)

1. The test strip for detecting the bifenazate comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate, wherein the reaction membrane is provided with a detection line and a quality control line;

the biphenylhydrazine ester hapten-carrier protein conjugate is obtained by coupling a biphenylhydrazine ester hapten and a carrier protein;

The synthesis method of the bifenazate hapten comprises the following steps: using bifenazate as a starting material, heating the bifenazate and 1, 4-cyclohexane diisocyanate in pyridine and triethylamine environment, and introducing cyclohexane isocyanate at an imine position through an acylation reaction; the molecular structural formula of the bifenazate hapten is shown as formula I:

The biphenylhydrazine ester monoclonal antibody is prepared by taking a biphenylhydrazine ester hapten-carrier protein conjugate as an immunogen.

2. The test strip of claim 1, wherein the quality control line is coated with goat anti-mouse antibody.

3. The test strip of claim 1, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroxine or human serum albumin.

4. The test strip of claim 1, wherein the sample absorbing pad, the conjugate releasing pad, the reaction membrane, and the absorbent pad are sequentially attached to the base plate.

5. The test strip of claim 1, wherein the conjugate release pad is 1/3 to 1/2 covered under the sample absorbent pad.

6. A method for preparing the test strip according to any one of claims 1 to 5, comprising the steps of:

(1) Preparing a conjugate release pad sprayed with a bifenazate monoclonal antibody-colloidal gold marker;

(2) Preparing a reaction membrane with a detection line coated with a biphenylhydrazine ester hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody:

(3) And (3) assembling the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the bottom plate which are prepared in the steps (1) and (2) into the test strip.

7. Use of the test strip according to any one of claims 1 to 5 or the test strip prepared by the preparation method according to claim 6 in detecting bifenazate residues in fruit and vegetable samples.