CN109061151B - Time-resolved fluorescence immunochromatographic test strip for detecting quinclorac and preparation method and application thereof - Google Patents
Time-resolved fluorescence immunochromatographic test strip for detecting quinclorac and preparation method and application thereof Download PDFInfo
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Abstract
A time-resolved fluorescence immunochromatographic test strip for detecting quinclorac, 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 quinclorac 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 quinclorac hapten-carrier protein conjugate is sprayed on the detection area, a goat anti-mouse antibody is sprayed on the quality control area, and the quinclorac hapten is obtained by reacting quinclorac with (1, 3-dioxacyclohexyl ethyl) magnesium bromide to generate malonaldehyde quinclorac and then reacting with trifluoroacetic acid. 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 on-site monitoring of quinclorac in a large batch of samples.
Description
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 quinclorac in tobacco-planting soil, tobacco and tobacco products, and a preparation method and application thereof.
Background
The quinclorac belongs to hormone-type quinoline carboxylic acid herbicides, is mainly used for preventing and killing monocotyledonous weeds in rice fields, and particularly has extremely high activity on barnyard grass. The composition can be absorbed by germinated seed, root and leaf, has the characteristics of hormone herbicide, and has similar action symptom to auxin. Quinclorac generates obvious phytotoxicity to tobacco production, and particularly in rice and tobacco rotation areas, the accumulation of quinclorac seriously affects the yield and quality of tobacco. At present, the detection method for the residual dichloroquinacrine at home and abroad mainly comprises a high performance liquid chromatography-mass spectrometry combined method, a high performance liquid chromatography and the like. The instrument and the method have the advantages of high detection sensitivity, accurate qualitative and quantitative determination and the like, but the pretreatment of a detected sample is complicated and time-consuming, the sample needs further purification treatment, and meanwhile, the instrument and the detection method need expensive large-scale instruments and equipment and are equipped with professional detection technicians for operation and management, so that large-scale field detection cannot be carried out, the timeliness is poor, and the popularization at the basic level is difficult. 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
The invention aims to provide a time-resolved fluorescence immunochromatographic test strip for detecting quinclorac, which has the advantages of high sensitivity, simple and convenient operation, quick detection and low cost, and aims to overcome the defects of the prior art; the invention also aims to provide a preparation method of the test strip; the invention further aims to provide the application of the test strip in the detection of quinclorac.
In order to achieve the purpose, the invention adopts a technical scheme that:
the time-resolved fluorescence immunochromatographic test strip for detecting quinclorac 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 quinclorac 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 quinclorac 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 quinclorac monoclonal antibody is prepared by taking a quinclorac hapten-carrier protein conjugate as an immunogen; the quinclorac hapten-carrier protein conjugate is obtained by coupling quinclorac hapten and carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroid protein or human serum albumin, the quinclorac hapten is obtained by reacting quinclorac and (1, 3-dioxacyclohexylethyl) magnesium bromide to generate malonaldehyde quinclorac, and then reacting with trifluoroacetic acid, and the molecular structural formula is as follows:
the preparation method of the quinclorac hapten specifically comprises the following steps:
1) taking 1.00 g of quinclorac, adding 50 mL of dry anhydrous tetrahydrofuran to dissolve and clarify, dropwise adding 10 mL of anhydrous tetrahydrofuran solution containing 1.10 g of (1, 3-dioxa cyclohexyl ethyl) magnesium bromide, and stirring at 0 ℃ for 2 h; quickly mixing with 100 mL of 0-5 ℃ ammonium chloride aqueous solution, immediately adding 100 mL of ether for extraction, oscillating, standing for layering, drying organic phase anhydrous sodium sulfate, performing rotary evaporation, concentrating, evaporating to dryness, applying to a silica gel column, and performing elution and separation by using dichloromethane/methanol with the volume ratio of 10:1 to obtain 1.41 g of an intermediate, namely the propionylquinclorac;
2) taking 1.40 g of the acetal quinclorac, adding 10 mL of trifluoroacetic acid for dissolving, adding 10 mL of water, and stirring at 50 ℃ for reacting for 2 h; TLC detection, the raw materials are basically completely reacted; stopping the reaction, cooling to room temperature, removing trifluoroacetic acid by rotary evaporation, adding 50 mL of water, adjusting the pH value to 5 by using 0.1 mol/L sodium hydroxide, extracting by using 50 mL of ethyl acetate, concentrating, and recrystallizing by using ethanol/n-hexane with the volume ratio of 1:1 to obtain 1.06 g of quinclorac hapten.
The goat anti-mouse anti-antibody is obtained by immunizing a goat with a murine antibody.
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 quinclorac is provided, which comprises the following steps:
1) preparation of conjugate release pad: marking quinclorac monoclonal antibody by using commercially available fluorescent microspheres, diluting the 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 monoclonal antibody;
2) preparation of nitrocellulose membrane: spraying the quinclorac 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 quinclorac monoclonal antibody marked by fluorescent microspheres, a nitrocellulose membrane fixed with a detection area and a quality control area and a water absorption pad on the bottom plate, and shearing the nitrocellulose membrane and the water absorption pad into a required width, namely the time-resolved fluorescence immunochromatographic test strip.
Specifically, the steps include:
1) reacting quinclorac with (1, 3-dioxa cyclohexyl ethyl) magnesium bromide to generate acetal quinclorac, and reacting with trifluoroacetic acid to obtain a quinclorac hapten product;
2) coupling quinclorac hapten with carrier protein to prepare a quinclorac hapten-carrier protein conjugate;
3) immunizing a mouse by using the quinclorac hapten-carrier protein conjugate, and fusing and screening spleen cells and myeloma cells of the mouse to obtain a hybridoma cell strain secreting the quinclorac monoclonal antibody;
4) extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibodies;
5) respectively spraying the quinclorac 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 a nitrocellulose membrane;
6) soaking the sample absorption pad in 0.5% bovine serum albumin (volume fraction), 0.1 mol/L phosphate buffer solution with pH of 7.2 for 2h, and drying at 37 deg.C for 2 h;
7) marking quinclorac monoclonal antibody by using commercially available fluorescent microspheres, diluting the monoclonal antibody by using a specific buffer system, soaking the conjugate release pad in a dilution buffer solution, and freeze-drying in vacuum for later use;
8) and a sample absorption pad, a conjugate release pad embedded with a fluorescent microsphere labeled quinclorac monoclonal antibody, a nitrocellulose membrane and a water absorption pad which are fixed with a detection area and a quality control area are sequentially overlapped and adhered on the bottom plate, and the nitrocellulose membrane and the water absorption pad are cut into required widths, 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 quinclorac in the detection of quinclorac is provided, which comprises the following steps:
1) pretreating a sample;
2) detecting by using the time-resolved fluorescence immunochromatographic test strip for detecting quinclorac;
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 quinclorac 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, rapid rewetting, full release of the antibody conjugate, good performance, rapid release, good shape and the like, thereby reducing errors, reducing the 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 quinclorac in tobacco planting soil, tobacco and tobacco products exists, and the invention fills the gap. 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 quinclorac by using the test strip is simple, convenient, rapid, visual and accurate, does not need large-scale instruments, has low cost and is 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 is a scheme showing the synthesis of quinclorac 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 quinclorac
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 quinclorac hapten-carrier protein conjugate (a quinclorac 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 quinclorac
The preparation method of the time-resolved fluorescence immunochromatographic test strip for detecting quinclorac mainly comprises the following steps:
1) preparation of conjugate release pad: marking quinclorac monoclonal antibody by using commercially available fluorescent microspheres, diluting the 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 monoclonal antibody;
2) preparation of nitrocellulose membrane: spraying the quinclorac 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 quinclorac monoclonal antibody marked by fluorescent microspheres, a nitrocellulose membrane fixed with a detection area and a quality control area and a water absorption pad on the bottom plate, and shearing the nitrocellulose membrane and the water absorption pad into a required width, namely the time-resolved fluorescence immunochromatographic test strip.
The following steps are detailed:
preparation of the Components
1. Synthesis and identification of quinclorac hapten-carrier protein conjugate
Quinclorac 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 quinclorac hapten
Taking 1.00 g of quinclorac, adding 50 mL of dry anhydrous tetrahydrofuran to dissolve and clarify, dropwise adding 10 mL of anhydrous tetrahydrofuran solution containing 1.10 g of (1, 3-dioxa cyclohexyl ethyl) magnesium bromide, and stirring at 0 ℃ for 2 h; quickly mixing with 100 mL of ammonium chloride aqueous solution at 0-5 ℃, immediately adding 100 mL of diethyl ether for extraction, oscillating, standing for layering, drying organic phase anhydrous sodium sulfate, performing rotary evaporation, concentrating, evaporating to dryness, applying to a silica gel column, and performing elution and separation by using dichloromethane/methanol with the volume ratio of 10:1 to obtain 1.41 g of intermediate propionylquinclorac, wherein the yield is 95.92%;
taking 1.40 g of the acetal quinclorac, adding 10 mL of trifluoroacetic acid for dissolving, adding 10 mL of water, and stirring at 50 ℃ for reacting for 2 h; TLC detection, the raw materials are basically completely reacted; stopping the reaction, cooling to room temperature, removing trifluoroacetic acid by rotary evaporation, adding 50 mL of water, adjusting the pH value to 5 by using 0.1 mol/L sodium hydroxide, extracting by using 50 mL of ethyl acetate, concentrating, and recrystallizing by using ethanol/n-hexane with the volume ratio of 1:1 to obtain 1.06 g of quinclorac hapten with the yield of 90.60%.
Nuclear magnetic identification of 1H NMR (CDCl)3300 MHZ) δ 11.01 (1H, t), 8.223 (1H, dd, J =1.577, J =0.443), 3.205 (2H, t, J =6.096), 2.841 (2H, dt, J =6.370, J =6.096), 8.315 (1H, dd, J =8.532, J =1.577), 9.673 (1H, t, J =6.370), 7.738 (1H, dd, J =8.532, J =0.443), the chemical shift δ =9.673 is the aldehyde-based hydrogen resonance absorption peak on the spacer arm, the δ =2.84, 3.205 is the methylene hydrogen resonance absorption peak on the spacer arm, the presence of these peaks proves the success of spacer arm coupling.
(2) Preparation of immunogens
The quinclorac hapten is coupled with Bovine Serum Albumin (BSA) to obtain the immunogen.
Taking 11 mg of quinclorac hapten, adding 0.3 mL of ethanol for dissolving, and clarifying to obtain solution A; and dissolving 50 mg of bovine serum albumin in 0.05 mol/L sodium carbonate solution to obtain solution B, dropwise adding the solution A into the solution B, and stirring at room temperature for reaction for 16 h. Dialyzing and purifying 0.02M PBS for three days, and changing the solution for 3 times per day to obtain the quinclorac-BSA conjugate antigen which is the immunogen, and subpackaging at-20 ℃ for later use.
(3) Preparation of coating antigen
And coupling quinclorac hapten with Ovalbumin (OVA) to obtain the coating antigen.
Taking 5 mg of quinclorac hapten, adding 0.2 mL of methanol for dissolving, and clarifying to obtain solution A; dissolving egg white albumin 50 mg in 0.05 mol/L sodium carbonate solution to obtain solution B, dropwise adding the solution A into the solution B, and stirring at room temperature for 16 h. Dialyzing and purifying 0.02M PBS for three days, changing the solution for 3 times per day to obtain the quinclorac-OVA conjugate antigen which is the coating antigen, and subpackaging at-20 ℃ for later use.
2. Preparation of quinclorac monoclonal antibody
(1) Animal immunization
Injecting quinclorac hapten-BSA conjugate (immunogen) into Balb/c mice, wherein the immunization dose is 150 mu g/mouse, so that antiserum is generated;
(2) cell fusion and cloning
Taking immune Balb/c mouse spleen cells, fusing the immune Balb/c mouse spleen cells with SP2/0 myeloma cells according to the proportion of 8:1 (quantitative ratio), measuring cell supernatant by adopting an indirect competitive ELISA method, and screening positive holes. Cloning the positive hole by using a limiting dilution method until obtaining the hybridoma cell strain which stably secretes the monoclonal antibody.
(3) Cell cryopreservation and recovery
Making hybridoma cell into 1 × 10 with frozen stock solution6Cell suspension per mL, preserved for long period in liquid nitrogen. Taking out the frozen tube during recovery, immediately putting the tube into a water bath at 37 ℃ for fast melting, centrifuging to remove frozen liquid, and transferring the tube into a culture bottle for culture.
(4) Preparation and purification of monoclonal antibodies
An incremental culture method: placing the hybridoma cell in cell culture medium, culturing at 37 deg.C, purifying the obtained culture solution by octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibody, and storing at-20 deg.C.
The cell culture medium is prepared by adding calf serum and sodium bicarbonate into RPMI1640 culture medium to make the final concentration of calf serum in the cell culture medium 20% (mass fraction) and the final concentration of sodium bicarbonate in the cell culture medium 0.2% (mass fraction); the pH of the cell culture medium was 7.4.
(5) Determination of the potency of monoclonal antibodies
The titer of the antibody is 1 (100000-800000) by using an indirect competition ELISA method.
Indirect competitive ELISA method: coating an enzyme label plate with a quinclorac hapten-OVA conjugate, adding a quinclorac standard solution, a quinclorac monoclonal antibody solution and a horse radish peroxidase-labeled goat anti-mouse anti-antibody solution, reacting at 25 ℃ for 30 min, pouring out liquid in a hole, washing for 3-5 times by using a washing solution, and patting dry by using 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.
(6) 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, quinclorac, quinmerac, pendimethalin, butralin and flumetralin are serially diluted, respectively subjected to indirect competitive ELISA with monoclonal antibodies, a standard curve is prepared, IC50 is obtained through analysis, and then the cross reaction rate is calculated according to the following formula:
the results show that the cross-reactivity rate of each analog is: 100 percent of quinclorac, less than 1 percent of cloquintocet-mexyl, less than 1 percent of quinmerac, less than 1 percent of pendimethalin, less than 1 percent of butralin and less than 1 percent of flumetralin. The antibody of the invention has no cross reaction to other herbicides such as cloquintocet-mexyl, quinmerac, pendimethalin, butralin, flumetralin and the like, and only has specific binding to quinclorac.
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 quinclorac monoclonal antibody marked by fluorescent microsphere
(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 quinclorac 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 quinclorac monoclonal antibody marked by the fluorescent microspheres 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 quinclorac 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 solution on 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 2h 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 quinclorac fluorescent microsphere immunochromatographic test paper card is stored in a shady, cool and dark dry mode at the temperature of 2-8 ℃, and the effective period is 12 months.
Example 3 application of time-resolved fluorescence immunochromatographic test strip for detecting quinclorac
1. Sample pretreatment
Weighing 1.0 +/-0.05 g of sieved soil sample into a 15 mL polystyrene centrifuge tube, adding 5 mL deionized water, carrying out vortex for 3 min, and centrifuging at 3000 rpm at room temperature (20-25 ℃) for 5 min; and (3) taking 500 mu L of supernatant into a 2 mL polystyrene centrifuge tube, adding 500 mu L of deionized water for dilution, and uniformly mixing to be tested.
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
(1) Semi-quantitative detection
After the test is finished, the instrument automatically calculates the concentration value of quinclorac 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, indicating that the sample does not contain quinclorac or the concentration of the quinclorac 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 the quinclorac 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.
(2) Quantitative detection
After the test is finished, the instrument obtains the ratio of the time-resolved fluorescence intensity of the detection area on the fluorescent test strip to the time-resolved fluorescence intensity of the quality control area, obtains the content of the quinclorac in the extracting solution of the sample to be tested based on the relation curve between the preset ratio of the time-resolved fluorescence intensity of the detection area on the fluorescent test strip to the time-resolved fluorescence intensity of the quality control area and the concentration of the quinclorac, and finally obtains the content of the quinclorac in the sample to be tested through conversion.
Example 4 sample testing example
1. Limit of detection test
Adding quinclorac standard substances into the blank soil samples respectively until the final concentration is 25, 50 and 100 mu g/kg, and detecting by using a time-resolved fluorescence immunochromatographic test strip, wherein the result is as follows: when the concentration of the quinclorac is 25 mug/kg, the detection of the fluorescence detector is negative; when the concentration of the quinclorac is 50 and 100 mug/kg, the detection of the fluorescence detector is positive, which shows that the detection limit of the test strip on the quinclorac in the tobacco is 50 mug/kg.
2. Test for false positive and false negative rates
Taking 20 parts of positive tobacco samples with known quinclorac content of more than 50 mug/kg and 20 parts of negative tobacco samples without known quinclorac, respectively detecting by using 3 time-resolved fluorescence immunochromatographic test strips produced in batches, and calculating the positive and negative rates. The results are shown in Table 1.
TABLE 1 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 quinclorac can be used for quickly detecting quinclorac in tobacco.
3. Specificity test
The herbicide with the added concentration of 1 mg/kg, such as cloquintocet-mexyl, quinmerac, pendimethalin, butralin, flumetralin and the like, is detected by a quinclorac test strip. The result shows that the quality control line and the detection line of the test strip are both colored and negative. The test paper strip has no cross reaction to 1 mg/kg of herbicides such as cloquintocet-mexyl, quinmerac, pendimethalin, butralin, flumetralin and the like, and has good specificity.
Claims (6)
1. A time-resolved fluorescence immunochromatographic test strip for detecting quinclorac comprises a base plate, and a sample absorption pad, a conjugate release pad, a cellulose nitrate membrane and a water absorption pad which are sequentially overlapped and adhered on the base plate, and is characterized in that the conjugate release pad is embedded with a quinclorac monoclonal antibody marked by fluorescent microspheres, the cellulose nitrate membrane is fixed with a detection area and a quality control area, the detection area is sprayed with a quinclorac hapten-carrier protein conjugate, and the quality control area is sprayed with a goat anti-mouse anti-antibody; the quinclorac monoclonal antibody is prepared by taking a quinclorac hapten-carrier protein conjugate as an immunogen; the quinclorac hapten-carrier protein conjugate is obtained by coupling quinclorac hapten and carrier protein, wherein the quinclorac hapten is obtained by reacting quinclorac with (1, 3-dioxacyclohexylethyl) magnesium bromide to generate malonaldehyde quinclorac, and then reacting with trifluoroacetic acid, and the molecular structural formula of the quinclorac hapten-carrier protein conjugate is as follows:
the preparation reaction process of the quinclorac hapten is as follows:
2. the time-resolved fluorescence immunochromatographic test strip for detecting quinclorac 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.
3. The time-resolved fluorescence immunochromatographic test strip for detecting quinclorac according to claim 2, which is characterized in that: the fluorescent substance is a lanthanide series.
4. The time-resolved fluorescence immunochromatographic test strip for detecting quinclorac 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.
5. The preparation method of the time-resolved fluorescence immunochromatographic test strip for detecting quinclorac in any one of claims 1 to 4 is characterized by comprising the following steps:
1) preparation of conjugate release pad: marking quinclorac monoclonal antibody with fluorescent microsphere, diluting the monoclonal antibody with storage buffer solution, soaking the conjugate release pad in the dilution buffer solution, and preparing the monoclonal antibody after vacuum freeze drying;
2) preparation of nitrocellulose membrane: spraying the quinclorac 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 quinclorac monoclonal antibody marked by fluorescent microspheres, a nitrocellulose membrane fixed with a detection area and a quality control area and a water absorption pad on the bottom plate, and shearing the nitrocellulose membrane and the water absorption pad into a required width, namely the time-resolved fluorescence immunochromatographic test strip.
6. The use of the time-resolved fluorescence immunochromatographic strip for detecting quinclorac in any one of claims 1 to 4, 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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