CN109061149B - Time-resolved fluorescence immunochromatographic test strip for detecting butralin and preparation method and application thereof - Google Patents

Abstract

The invention discloses a time-resolved fluorescence immunochromatographic test strip for detecting butralin and a preparation method and application thereof. The test strip comprises a base plate, and a sample absorption pad, a conjugate release pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped and adhered on the base plate, wherein a butralin monoclonal antibody marked by fluorescent microspheres is embedded on the conjugate release pad, a detection area and a quality control area are fixed on the nitrocellulose membrane, a butralin hapten-carrier protein conjugate is sprayed on the detection area, a goat anti-mouse anti-antibody is sprayed on the quality control area, and the butralin hapten is obtained by reacting 4-chloro-3, 5-dinitrophenylacetic acid and butylamine. The invention also provides a preparation method of the test strip and a method for detecting butralin in a sample by applying the test strip. The test strip and the detection method provided by the invention have the advantages of simple operation, high sensitivity, high detection speed and low cost, and can realize rapid detection and field monitoring of butralin in a large batch of samples.

Description

Time-resolved fluorescence immunochromatographic test strip for detecting butralin and preparation method and application thereof

Technical Field

The invention belongs to the field of pesticide residue detection, and particularly relates to a time-resolved fluorescence immunochromatographic test strip for detecting butralin in tobacco and tobacco products, and a preparation method and application thereof.

Background

Butralin (Butralin) also known as dimethomon, dibutylelin, nitroaniline, bidanine, and nystatin, chemically named as N-sec-butyl-4-tert-butyl-2, 6-dinitroaniline, with molecular formula C₁₄H₂₁N₃O₄. Butralin is a dinitroaniline herbicide, is an orange crystal in pure product, is easy to dissolve in an organic solvent and insoluble in water, has volatility, is easy to decompose after being illuminated, and has low toxicity to animals such as human beings, livestock and the like. The herbicide is a selective pre-emergent herbicide, and has the effect similar to that of trifluralin. After entering the plant body, the pesticide mainly inhibits the cell division of meristematic tissue, thereby inhibiting the growth of the sprouts and the roots of weeds, and leading to the death of the weeds. Butralin can be applied to field crops to inhibit the growth of weeds, can also be used for removing miscellaneous algae in water bodies in aquaculture, is widely applied to tobacco production and is mainly used for inhibiting the growth of tobacco leaf buds. The international cooperative center for tobacco science research (CORESTA) stipulates that the directive residual limit of butralin in tobacco is 5 mg/kg. At present, the commonly used detection methods include gas chromatography, high performance liquid chromatography, gas chromatography-tandem mass spectrometry, liquid chromatography-tandem mass spectrometry and the like. The methods all need advanced detection instruments, are expensive in detection cost, complex in steps and time-consuming, have high requirements on the professional performance of operators, and are not suitable for high-throughput rapid screening and detection of the primary enterprises and public institutions. Therefore, it is an urgent problem to develop a product and a method which are not limited by the detection equipment and can realize rapid detection of a large number of samples.

The fluorescent microsphere immunochromatography technology is developed on the fluorescent dye labeling technology, is a combination of an immunoaffinity technology, an immunolabeling technology and an immunochromatography technology as an immunological detection method, and has the advantages of rapidness, simple and convenient operation and the like. Compared with the traditional marker, the luminous intensity of the fluorescent microsphere can be enhanced along with the enhancement of the intensity of exciting light, so that the fluorescent microsphere marker is expected to improve the detection limit of the immunochromatography technology; under the action of the microsphere shell structure, the fluorescent microsphere has a relatively stable morphological structure, uniform granularity, good monodispersity, good stability, high luminous efficiency, good repeatability and better biocompatibility; after the microsphere is formed, the fluorescence quenching of the dye is greatly reduced, the emission is strong and stable, and the influence of the change of an external environment medium is basically avoided. Therefore, compared with the detection method, the fluorescent microsphere immunochromatography technology has the advantages of high detection sensitivity, simple and convenient operation and good stability.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the time-resolved fluorescence immunochromatographic test strip for detecting butralin, which has the advantages of high sensitivity, simple and convenient operation, quick detection and low cost; the invention also aims to provide a preparation method of the test strip; the invention further aims to provide application of the test strip in butralin detection.

In order to achieve the purpose, the invention adopts a technical scheme that:

the time-resolved fluorescence immunochromatographic test strip for detecting butralin comprises a base plate, and a sample absorption pad, a conjugate release pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped and adhered to the base plate, wherein a butralin monoclonal antibody marked by fluorescent microspheres is embedded on the conjugate release pad, a detection area and a quality control area are fixed on the nitrocellulose membrane, a butralin hapten-carrier protein conjugate is sprayed on the detection area, and a goat anti-mouse anti-antibody is sprayed on the quality control area.

The butralin monoclonal antibody is prepared by taking a butralin hapten-carrier protein conjugate as an immunogen; the butralin hapten-carrier protein conjugate is obtained by coupling butralin hapten and carrier protein, the carrier protein is bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroid protein or human serum albumin, the butralin hapten is obtained by reacting 4-chloro-3, 5-dinitrophenylacetic acid and butylamine, and the molecular structural formula is as follows:

。

the preparation method of the butralin hapten specifically comprises the following steps:

taking 1.00 g of 4-chloro-3, 5-dinitrophenylacetic acid, adding 50 mL of ethanol for dissolving, adding 0.45 g of sodium carbonate, stirring, adding 0.31 g of butylamine, heating in an oil bath at the temperature of 80 ℃, stirring for 3 hours, and detecting that the raw materials are basically completely reacted; stopping the reaction, cooling to room temperature, carrying out rotary evaporation, removing ethanol, adding 80 mL of water, adjusting the pH value to 4 by using 1 mol/L hydrochloric acid to obtain a large amount of turbidity, adding 80 mL of ethyl acetate for extraction, washing with water, applying to a silica gel column, and carrying out elution separation by using dichloromethane/methanol with the volume ratio of 5:1 to obtain 1.05 g of butralin hapten product.

The fluorescent microspheres are microspheres with the diameter of 100-300 nm and are prepared by wrapping fluorescent materials with polystyrene, the surfaces of the microspheres are connected with-COOH groups, and the fluorescent materials are lanthanide series.

The invention adopts another technical scheme that a method for preparing the time-resolved fluorescence immunochromatographic test strip for detecting butralin is provided, and the method comprises the following steps:

1) preparation of conjugate release pad: marking a butralin monoclonal antibody by using a commercially available fluorescent microsphere, diluting the butralin monoclonal antibody by using a specific buffer system, soaking a conjugate release pad in a dilution buffer solution, and performing vacuum freeze drying to prepare the conjugate release pad;

2) preparation of nitrocellulose membrane: spraying the butralin hapten-carrier protein conjugate to a detection area range on a nitrocellulose membrane to prepare a detection area; spraying goat anti-mouse anti-antibody to the range of the quality control area on the nitrocellulose membrane to prepare the quality control area;

3) assembling and shearing: and sequentially overlapping and sticking a sample absorption pad, a conjugate release pad embedded with a fluorescent microsphere labeled butralin monoclonal antibody, a nitrocellulose membrane fixed with a detection area and a quality control area and a water absorption pad on the bottom plate, and shearing into required width, namely the time-resolved fluorescence immunochromatographic test strip.

Specifically, the steps include:

1) reacting 4-chloro-3, 5-dinitrophenylacetic acid with butylamine to prepare butralin hapten;

2) coupling butralin hapten with carrier protein to prepare a butralin hapten-carrier protein conjugate;

3) immunizing a mouse by using a butralin hapten-carrier protein conjugate, and fusing and screening splenocytes of the mouse and myeloma cells of the mouse to obtain a hybridoma cell strain secreting a butralin monoclonal antibody;

4) extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibodies;

5) respectively spraying the butralin hapten-carrier protein conjugate and the goat anti-mouse anti-antibody to a detection area range (T) and a quality control area range (C) of the nitrocellulose membrane;

6) soaking the sample absorption pad in 0.5% bovine serum albumin (volume fraction), pH 7.2, 0.1 mol/L phosphate buffer solution for 2 h, and drying at 37 deg.C for 2 h;

7) marking a butralin monoclonal antibody by using a commercially available fluorescent microsphere, diluting the butralin monoclonal antibody by using a specific buffer system, soaking a conjugate release pad in a dilution buffer solution, and carrying out vacuum freeze drying for later use;

8) and sequentially overlapping and adhering a sample absorption pad, a conjugate release pad embedded with a fluorescent microsphere labeled butralin monoclonal antibody, a nitrocellulose membrane and a water absorption pad fixed with a detection area and a quality control area on the bottom plate, and shearing into required width, namely the time-resolved fluorescence immunochromatographic test strip.

The invention adopts another technical scheme that an application of the time-resolved fluorescence immunochromatographic test strip for detecting butralin in butralin detection is provided, and the application comprises the following steps:

1) pretreating a sample;

2) detecting by using the time-resolved fluorescence immunochromatographic test strip for detecting butralin;

3) and analyzing the detection result by using a fluorescence detector.

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

(1) strong specificity and high sensitivity: the test strip embeds the butralin monoclonal antibody marked by the fluorescent microspheres on the conjugate release pad, and has the advantages of good hydrophilicity, capability of adsorbing the antibody conjugate in a large capacity, quick rewetting, full release of the antibody conjugate, good performance, quick release, good shape and the like, thereby reducing errors, reducing cost and increasing the reaction sensitivity of the whole system.

(2) The time-resolved fluorescence has larger stock displacement, so that the interference of specific stray light caused by exciting light on detection is reduced, and the fluorescence detection stability is improved; the service life is long, and the interference of fluorescent substances in the environment to an object to be detected is eliminated; the excitation wavelength is wide, the emission spectrum range is narrow, the background fluorescence intensity is reduced, and the resolution ratio is improved.

(3) Polystyrene is wrapped on the surface of the fluorescent microsphere, so that the lanthanide series of the fluorescent substance is protected, the interference of the external environment is reduced, and the stability and the fluorescent life of the fluorescent microsphere are improved.

(4) The surface of the fluorescent microsphere is modified with active groups-COOH, and the antibody is marked by adopting a chemical coupling method to form stable combination of the antibody and the microsphere.

At present, no time-resolved fluorescence immunochromatographic test strip for detecting butralin in tobacco and tobacco products exists, and the invention fills the blank. The test strip has the advantages of low cost, simple operation, short detection time, suitability for various units, simple storage and long quality guarantee period, and the method for detecting butralin by using the test strip is simple, convenient, rapid, visual, accurate, wide in application range, low in cost and easy to popularize and use.

Drawings

FIG. 1 is a schematic diagram of a cross-sectional structure of a time-resolved fluorescence immunochromatographic test strip;

FIG. 2 shows a scheme for synthesis of butralin hapten (the figure is taken as an abstract figure).

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1 constitution of time-resolved fluorescence immunochromatographic test strip for detecting butralin

Test paper strip

Referring to fig. 1: the test strip consists of a bottom plate, a sample absorption pad, a conjugate release pad, a nitrocellulose membrane and a water absorption pad;

the sample absorption pad 1, the conjugate release pad 2, the nitrocellulose membrane 3 and the water absorption pad 4 are sequentially overlapped and adhered to the bottom plate 7, the conjugate release pad is covered by the sample absorption pad from an area 1/3 at the starting end, the tail end of the conjugate release pad is connected with the starting end of the nitrocellulose membrane, the tail end of the nitrocellulose membrane is connected with the starting end of the water absorption pad, the starting end of the sample absorption pad is aligned with the starting end of the PVC bottom plate, and the tail end of the water absorption pad is aligned with the tail end of the PVC bottom plate;

a detection area 5 and a quality control area 6 are fixed on the nitrocellulose membrane, a butralin hapten-carrier protein conjugate (butralin hapten-ovalbumin conjugate) is sprayed on the detection area, and a goat anti-mouse anti-antibody is sprayed on the quality control area;

the bottom plate is a PVC bottom plate; the conjugate release pad is glass wool; the absorbent pad is absorbent paper.

Example 2 preparation of time-resolved fluorescence immunochromatographic test strip for detecting butralin

The preparation method of the time-resolved fluorescence immunochromatographic test strip for detecting butralin mainly comprises the following steps:

1) preparation of conjugate release pad: marking a butralin monoclonal antibody by using a commercially available fluorescent microsphere, diluting the butralin monoclonal antibody by using a specific buffer system, soaking a conjugate release pad in a dilution buffer solution, and performing vacuum freeze drying to prepare the conjugate release pad;

2) preparation of nitrocellulose membrane: spraying the butralin hapten-carrier protein conjugate to a detection area range on a nitrocellulose membrane to prepare a detection area; spraying goat anti-mouse anti-antibody to the range of the quality control area on the nitrocellulose membrane to prepare the quality control area;

3) assembling and shearing: and sequentially overlapping and sticking a sample absorption pad, a conjugate release pad embedded with a fluorescent microsphere labeled butralin monoclonal antibody, a nitrocellulose membrane fixed with a detection area and a quality control area and a water absorption pad on the bottom plate, and shearing into required width, namely the time-resolved fluorescence immunochromatographic test strip.

The following steps are detailed:

preparation of the Components

1. Synthesis and identification of butralin hapten-carrier protein conjugate

Butralin is a small molecular substance, has immunoreactivity and no immunogenicity, cannot induce an organism to generate immune response, and has immunogenicity only after being coupled with a macromolecular carrier protein.

(1) Preparation of butralin hapten

Taking 1.00 g of 4-chloro-3, 5-dinitrophenylacetic acid, adding 50 mL of ethanol for dissolving, adding 0.45 g of sodium carbonate, stirring, adding 0.31 g of butylamine, heating in an oil bath at the temperature of 80 ℃, stirring for 3 hours, and detecting that the raw materials are basically completely reacted; stopping the reaction, cooling to room temperature, carrying out rotary evaporation, removing ethanol, adding 80 mL of water, adjusting the pH value to 4 by using 1 mol/L hydrochloric acid to obtain a large amount of turbidity, adding 80 mL of ethyl acetate for extraction, washing with water, applying to a silica gel column, and carrying out elution separation by using dichloromethane/methanol with the volume ratio of 5:1 to obtain 1.05 g of butralin hapten product, wherein the yield is 92.11%.

Nuclear magnetic identification¹H NMR（CDCl₃, 300MHz）δ：11.01（1H, d, J=0.000），8.281（1H, d, J=0.000），4.011（1H, tq, J=6.914, J=6.757），8.281（1H, d, J=0.000），3.822（t, 2H），2.791（1H, ddd, J=7.114, J=6.914），1.591（2H, d, J=6.757），1.251（3H, t, J=7.114），0.901（3H, t, J=7.114）。

In the spectrum, the chemical shifts delta =1.591, 1.251, 0.901 and 2.791 are resonance absorption peaks of methyl and methylene hydrogen on the raw material butylamine, and the chemical shifts delta =4.011 are resonance absorption peaks of imine hydrogen formed after the reaction of the raw material butylamine, and the existence of the peaks proves that the spacer arm coupling is successful and the butralin hapten structure is correct.

(2) Preparation of immunogens

Coupling butralin hapten with Bovine Serum Albumin (BSA) to obtain the immunogen.

Dissolving butralin hapten 11 mg in dimethyl sulfoxide (DMSO) 0.2 mL, adding triethylamine 20 mu L, stirring, mixing uniformly, adding isobutyl chloroformate 6 mg, and stirring for 2 h to obtain hapten activating solution A; dissolving 50 mg BSA in 0.1 mol/L PB buffer solution with the pH value of 7.2 to obtain solution B, slowly dripping the solution A into the solution B, continuously stirring for 5 h, stopping reaction, dialyzing with 0.02 mol/L PBS for 3 d, changing the solution 3 times every day to obtain butralin-BSA conjugate which is the immunogen, and subpackaging at-20 ℃ for later use.

(3) Preparation of coating antigen

Coupling butralin hapten with Ovalbumin (OVA) to obtain the coating antigen.

Taking 6 mg of butralin hapten, adding 0.2 mL of N, N-Dimethylformamide (DMF) for dissolving, adding 4.5 mg of carbodiimide (EDC), stirring, clarifying, adding 2.24 mg of N-succinimide (NHS), stirring at room temperature and activating for 2 h to obtain solution A; dissolving OVA 50 mg in 6 mL of 0.05 mol/L PB buffer solution with pH value of 7.2 to obtain solution B, slowly dripping solution A into solution B, and stirring at room temperature for reaction for 5 h. Stopping reaction, dialyzing with 0.02 mol/L PBS buffer solution for 3 d, changing the solution 3 times per day to obtain butralin-OVA conjugate which is the coating antigen, subpackaging, and storing at-20 ℃ for later use.

(4) Identification

And (3) carrying out ultraviolet (200 nm-400 nm) scanning measurement according to the proportion of the hapten, the carrier protein and the coupling product used in the reaction for synthesizing the butralin coupling antigen, and calculating the binding ratio of the hapten, the carrier protein and the coupling product by comparing the absorbance values of the hapten, the carrier protein and the coupling product at 260 nm and 280 nm respectively. Compared with the maximum absorption peaks of butralin hapten and carrier protein, the maximum absorption peak of the conjugate butralin hapten-carrier protein is obviously changed, which indicates that the synthesis of the butralin hapten-carrier protein is successful. The binding ratio of hapten to BSA and OVA was calculated to be 12:1 and 9: 1.

2. Preparation of butralin monoclonal antibody

(1) Obtaining hybridoma cells

First immunization: fully emulsifying a butralin hapten-BSA conjugate (immunogen) with an equal amount of Freund complete adjuvant, and injecting 0.2 mL of 6-week-old Balb/c mice into each animal subcutaneously;

two booster immunizations: from the first immunization, boosting once every two weeks, and replacing Freund's complete adjuvant with Freund's incomplete adjuvant in the same method and dosage as the first immunization;

after one week of last boosting immunization, measuring the titer and inhibition in fundus venous blood sampling, and performing the following last immunization when the titer reaches more than 1: 10000: injecting 0.1 mL of immunogen solution without any adjuvant into the abdominal cavity, killing the mouse after three days, and fusing the spleen with myeloma cells;

and (3) measuring cell supernatant by adopting an indirect competitive enzyme-linked immunoassay method, and screening positive holes. Cloning the positive hole by using a limiting dilution method to obtain and establish a hybridoma cell strain which stably secretes the butralin monoclonal antibody, preparing the hybridoma cells in the logarithmic growth phase into cell suspension by using a freezing medium, sub-packaging the cell suspension in a freezing tube, and storing the cell suspension in liquid nitrogen for a long time.

(2) Preparation of monoclonal antibodies

Cell recovery: taking out a cryopreservation tube of the butralin monoclonal antibody hybridoma cell strain, immediately putting the cryopreservation tube into a water bath at 37 ℃ for fast thawing, centrifuging to remove a cryopreservation solution, and transferring the frozen cryopreservation solution into a culture bottle for culture;

preparing ascites and purifying antibodies: injecting sterilized paraffin oil 0.5 mL/mouse in Balb/c mouse (8 weeks old) into abdominal cavity by in vivo induction method, injecting hybridoma cells 5 × 10 into abdominal cavity 7 days later⁵Ascites were collected 7 days later. Purifying by an octanoic acid-saturated ammonium sulfate method to obtain a butralin monoclonal antibody solution (stored at the temperature of minus 20 ℃).

(3) Determination of the potency of monoclonal antibodies

The titer of the antibody is 1 (150000-400000) by using an indirect competition ELISA method.

Indirect competitive ELISA method: coating an enzyme label plate with a butralin hapten-OVA conjugate, adding a butralin standard substance solution, a butralin monoclonal antibody solution and a horse radish peroxidase labeled goat anti-mouse anti-antibody solution, reacting for 30 min at 25 ℃, pouring out liquid in a hole, washing for 3-5 times with a washing solution, and patting dry with absorbent paper; adding a substrate color developing solution, reacting for 15 min at 25 ℃, and adding a stop solution to stop the reaction; the microplate reader was set to measure the absorbance value per well at a wavelength of 450 nm.

(4) Determination of monoclonal antibody specificity

Antibody specificity refers to the comparison of its ability to bind to a specific antigen with the ability to bind to such antigen analogs, often using cross-reactivity as an evaluation criterion. The smaller the cross-reactivity, the higher the specificity of the antibody.

In the experiment, butralin and other dinitroaniline herbicides (flumetralin, isoprotulin, pendimethalin, benfluralin and trifluralin) which are common in tobacco are serially diluted, respectively subjected to indirect competitive ELISA with monoclonal antibodies, a standard curve is prepared, and IC is obtained through analysis₅₀Then, the cross-reactivity was calculated as follows:

the results show that the cross-reaction rate of butralin and other dinitroaniline herbicides common in tobacco is as follows: 100% of butralin, less than 1% of flumetralin, less than 1% of isoprotulin, less than 1% of pendimethalin, less than 1% of benfluralin and less than 1% of trifluralin. The antibody has no cross reaction to other common dinitroaniline herbicides such as flumetralin, isoprotulin, pendimethalin, benfluralin, trifluralin and the like, and only has specific binding to butralin.

3. Preparation of goat anti-mouse anti-antibody

The sheep is taken as an immune animal, and the pathogen-free sheep is immunized by taking the murine antibody as an immunogen to obtain the goat anti-mouse antibody.

4. Preparation of fluorescence microsphere labeled butralin monoclonal antibody

(1) And (3) activation: suspending 100 mu L of microsphere suspension which is embedded with fluorescent dye and modified with carboxyl functional groups on the surface and is sold in market in 900 mu L of activation buffer solution, centrifuging for 10min at 4 ℃ at 10000 r/min, then discarding supernatant, resuspending microspheres in 1 mL of activation buffer solution, washing the microspheres for 2 times by the method, adding a proper amount of activating agent, uniformly mixing, and then oscillating and activating for 10min at room temperature;

(2) coupling: centrifuging the suspension of the step (1) at 4 ℃ and 10000 r/min for 10min, then discarding the supernatant, suspending the suspension in a coupling buffer solution, washing the microspheres for 2 times by the method, adding 10-20 mu L of butralin monoclonal antibody solution (with the protein concentration of 1 mg/mL), uniformly mixing, and oscillating and coupling at room temperature for 120 min;

(3) and (3) sealing: centrifuging the suspension of (2) at 4 ℃ and 10000 r/min for 10min, then discarding the supernatant, suspending in a closed buffer solution, washing the microspheres for 1 time by the method, uniformly mixing, and oscillating at room temperature and sealing for 30 min;

(4) and (3) storage: centrifuging the suspension of (3) at 4 ℃ 10000 r/min for 10min, then discarding the supernatant, suspending in a storage buffer solution, washing the microspheres for 1 time by the method, mixing uniformly, and storing at 4 ℃ in a dark place.

The activating buffer solution is a 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution with the pH value of 5.5-6.5 and the mol/L of 0.05.

The activating agent is water-soluble carbodiimide, wherein the molar mass ratio of EDC to NHS to COOH = (1.5-3) to (8-20) to 1, and the activating agent is diluted to a required concentration by using an activating buffer solution before use.

The coupling buffer is borate buffer with the pH value of 7.5-8.50.05 mol/L (solvent with free amine is avoided).

The blocking buffer solution is a PB buffer solution which contains 0.1-0.4 mol/L primary amine (hydroxylamine hydrochloride, ethanolamine or aminoethanol) and 1% -10% BSA and has a pH value of 7.4.

The storage buffer solution contains 0.01 percent of NaN₃0.1% BSA at pH 7.4.

5. Preparation of conjugate Release pad

Diluting the stored fluorescence microsphere marked butralin monoclonal antibody with a storage buffer solution, soaking the conjugate release pad in the dilution buffer solution, and freeze-drying in vacuum for later use.

6. Preparation of cellulose Nitrate (NC) membranes

Diluting the butralin hapten-ovalbumin conjugate to 100 mu g/mL by using 0.05 mol/L, pH PBS buffer solution with the value of 7.2, and spraying the conjugate to a detection area (T) on an NC membrane by using an Isoflow point membrane instrument, wherein the spraying amount is 1.0 mu L/cm; the goat anti-mouse anti-antibody was diluted to 200. mu.g/mL with 0.01 mol/L, pH value of 7.4 PBS buffer, and sprayed on the quality control area (C) on the NC membrane in an Isoflow point membrane machine in an amount of 1.0. mu.L/cm. And (3) drying the prepared NC membrane for 2 h at 37 ℃ for later use.

7. Preparation of sample absorbent pad

The sample absorption pad is soaked in 0.5 percent bovine serum albumin (volume fraction), pH value of 7.2 and 0.1 mol/L phosphate buffer solution for 2 hours and dried for 2 hours at 37 ℃.

(II) Assembly of test strip

A sample absorption pad, a conjugate release pad, a nitrocellulose membrane and a water absorption pad are sequentially overlapped and stuck and fixed on a bottom plate from left to right, the conjugate release pad is covered by the sample absorption pad from the area 1/3 at the starting end, the tail end of the conjugate release pad is connected with the starting end of the nitrocellulose membrane, the tail end of the nitrocellulose membrane is connected with the starting end of the water absorption pad, the starting end of the sample absorption pad is aligned with the starting end of the bottom plate, the tail end of the water absorption pad is aligned with the tail end of the bottom plate, and then the sample absorption pad is cut into small strips with the width of 3.96 mm by a machine and is arranged in a special plastic card to form a test paper card. The butralin fluorescent microsphere immunochromatographic test paper card is dried and stored at the temperature of 2-8 ℃ in shade and dark, and the effective period is 12 months.

Example 3 application of time-resolved fluorescence immunochromatographic test strip for detecting butralin

1. Tobacco sample pretreatment

Weighing 2.0 +/-0.05 g of crushed tobacco sample into a polystyrene centrifuge tube; adding 10 mL of 50% methanol aqueous solution, and sufficiently crushing the mixture by using a homogenizer to obtain a sample solution; and transferring 75 mu L of sample liquid, mixing with 425 mu L of deionized water uniformly, and detecting.

2. Detection with test strips

Sucking 100 mu L of sample solution to be detected, vertically dropping the sample solution into a sample adding hole of a test paper card, starting timing when the liquid flows, and reacting for 10 min; inserting the test paper card into a carrier of a KFT-100A type fluorescence detector, selecting an item to be detected by touching a display screen, pressing a 'start detection' button, automatically carrying out scanning test on the test paper card by the fluorescence detector, and reading or printing a detection result on a display screen of the fluorescence detector.

3. Analysis of detection results

After the test is finished, the instrument automatically calculates the concentration value of butralin in the extracting solution according to the ratio of the time-resolved fluorescence intensity of the detection area to the time-resolved fluorescence intensity of the quality control area, and gives out positive and negative judgment according to a preset threshold value.

Negative (-): and if the result on the display screen of the fluorescence detector is negative, the result indicates that the sample does not contain butralin or the concentration of the butralin is lower than the detection limit.

Positive (+): and if the result on the display screen of the fluorescence detector is positive, indicating that the concentration of butralin in the sample is equal to or higher than the detection limit.

And (4) invalidation: if the quality control area does not detect the intensity of the fluorescence signal, the incorrect operation process or the failure of the test paper card is indicated.

Example 4 sample testing example

1. Limit of detection test

Respectively adding butralin standard substances into blank tobacco samples until the final concentration is 2.5, 5 and 10 mg/kg, and detecting by using a time-resolved fluorescence immunochromatography test strip, wherein the result is as follows: when the concentration of butralin is 2.5 mg/kg, the detection of a fluorescence detector is negative; when the concentration of butralin is 5 and 10 mg/kg, the detection of the fluorescence detector is positive, which shows that the detection limit of the test strip on butralin in tobacco is 5 mg/kg.

2. Test for false positive and false negative rates

And (3) taking 20 parts of positive tobacco samples with known butralin content of more than 5 mg/kg and 20 parts of negative tobacco samples without butralin, respectively detecting by using 3 time-resolved fluorescence immunochromatographic test strips produced in batches, and calculating the negative and positive rates of the positive and negative samples. The results are shown in Table 2.

TABLE 2 test results for positive and negative samples

The results show that: when 3 batches of test strips are used for detecting positive samples, the results are all positive, the positive coincidence rate is 100 percent, and the false negative rate is 0; when negative samples are detected, the results are all negative, and the negative coincidence rate is 100 percent and the false positive rate is 0. The time-resolved fluorescence immunochromatographic test strip for detecting butralin can be used for quickly detecting butralin in tobacco.

3. Specificity test

Diluting other dinitroaniline herbicides common in tobacco such as flumetralin, isoproturon, pendimethalin, benfluralin and trifluralin to 500 mg/L by using a phosphate buffer solution with the pH value of 7.2 and 0.2 mol/L, and detecting by using butralin test paper strips. The result shows that when the test strip is used for detecting 500 mg/L of flumetralin, isoprotulin, pendimethalin, benfluralin and trifluralin, the T line color development of the test strip is darker than or consistent with the C line color development, and the test strip is negative. The test strip shows that the test strip has no cross reaction to other dinitroaniline herbicides commonly seen in tobaccos such as flumetralin, isoprotulin, pendimethalin, benfluralin and trifluralin.

Claims (7)

1. A time-resolved fluorescence immunochromatographic test strip for detecting butralin comprises a base plate, and a sample absorption pad, a conjugate release pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped and adhered on the base plate, and is characterized in that a butralin monoclonal antibody marked by fluorescent microspheres is embedded on the conjugate release pad, a detection area and a quality control area are fixed on the nitrocellulose membrane, a butralin hapten-carrier protein conjugate is sprayed on the detection area, and a goat anti-mouse anti-antibody is sprayed on the quality control area; the butralin monoclonal antibody is prepared by taking a butralin hapten-carrier protein conjugate as an immunogen; the butralin hapten-carrier protein conjugate is obtained by coupling butralin hapten and carrier protein, the butralin hapten is obtained by reacting 4-chloro-3, 5-dinitrophenylacetic acid and butylamine, and the molecular structural formula is as follows:

。

2. the time-resolved fluorescence immunochromatographic strip for detecting butralin according to claim 1, which is characterized in that: the preparation reaction process of the butralin hapten is as follows:

。

3. the time-resolved fluorescence immunochromatographic test strip for detecting butralin according to claim 1, which is characterized in that: the fluorescent microspheres are microspheres with the diameter of 100-300 nm and fluorescent materials wrapped by polystyrene, and the surfaces of the microspheres are connected with-COOH groups.

4. The time-resolved fluorescence immunochromatographic strip for detecting butralin according to claim 3, which is characterized in that: the fluorescent substance is a lanthanide series.

5. The time-resolved fluorescence immunochromatographic test strip for detecting butralin according to claim 1, which is characterized in that: the carrier protein is bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroid protein or human serum albumin.

6. The preparation method of the time-resolved fluorescence immunochromatographic test strip for detecting butralin in any one of claims 1 to 5, which is characterized by comprising the following steps:

1) preparation of conjugate release pad: labeling butralin monoclonal antibody with fluorescent microsphere, diluting with storage buffer solution, soaking conjugate release pad in dilution buffer solution, and vacuum freeze drying to prepare;

2) preparation of nitrocellulose membrane: spraying the butralin hapten-carrier protein conjugate to a detection area range on a nitrocellulose membrane to prepare a detection area; spraying goat anti-mouse anti-antibody to the range of the quality control area on the nitrocellulose membrane to prepare the quality control area;

3) assembling and shearing: and sequentially overlapping and sticking a sample absorption pad, a conjugate release pad embedded with a fluorescent microsphere labeled butralin monoclonal antibody, a nitrocellulose membrane fixed with a detection area and a quality control area and a water absorption pad on the bottom plate, and shearing into required width, namely the time-resolved fluorescence immunochromatographic test strip.

7. The application of the time-resolved fluorescence immunochromatographic test strip for detecting butralin in any one of claims 1 to 5 is characterized by comprising the following steps:

1) pretreating a sample;

2) detecting with the test strip;

3) and analyzing the detection result by using a fluorescence detector.

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