CN109324182B - Fluorescent microsphere immunochromatography test strip for detecting pendimethalin and preparation method and application thereof - Google Patents
Fluorescent microsphere immunochromatography test strip for detecting pendimethalin and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a fluorescent microsphere immunochromatographic test strip for detecting pendimethalin residue, and a preparation method and application thereof. The test strip comprises a base plate, and a sample combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped and adhered on the base plate, wherein a pendimethalin monoclonal antibody marked by fluorescent microspheres is embedded on the sample combination pad, a detection area and a quality control area are fixed on the nitrocellulose membrane, a pendimethalin hapten-carrier protein conjugate is sprayed on the detection area, a goat anti-mouse anti-antibody is sprayed on the quality control area, the pendimethalin monoclonal antibody is prepared by taking the pendimethalin hapten-carrier protein conjugate as an immunogen, and the pendimethalin hapten is obtained by reacting 3- (1-ethyl propylamine) -6-methyl-2, 4-dinitrobenzaldehyde and 3-hydrazino propionic acid. The invention also provides a preparation method of the test strip and a method for detecting pendimethalin residue by applying the test strip. The test strip has the advantages of high sensitivity, quick detection, convenient operation, economy, practicability and the like.
Description
Technical Field
The invention belongs to the field of pesticide residue detection, and particularly relates to a fluorescent microsphere immunochromatographic test strip for detecting pendimethalin residue, a preparation method and application thereof, which are particularly suitable for detecting pendimethalin residue in agricultural products, tobaccos and tobacco products.
Background
Pendimethalin is a selective herbicide for dry field crops, and can be widely applied to weeding in fields of various crops such as corn, soybean, peanut, cotton, direct seeding dry rice, potato, tobacco, vegetables and the like. At present, pendimethalin is the third most common herbicide in the world, has the sales volume second to the biocidal herbicides glyphosate and paraquat, is also the selective herbicide with the largest sales volume in the world, and has wide application in agricultural production in China. Therefore, the problem of pendimethalin residue in crops has also received a high degree of attention. GB 2763-2016 (national food safety Standard food maximum residue limit) specifies that the Maximum Residue Limit (MRL) of pendimethalin in rice, Chinese chives, common head cabbages, common cabbages, spinach, celery and Chinese cabbages is 0.2 mg/kg, and the MRL in brown rice, corns, cottonseeds, garlic and lettuce is 0.1 mg/kg; the MRL of pendimethalin in beans, tea and garlic is 0.1 mg/kg, the MRL in leek is 0.2 mg/kg and the MRL in carrot is 0.5 mg/kg in the United states; the European Union stipulates that pendimethalin has 0.2 mg/kg of MRL in beans and carrots and 0.1 mg/kg of MRL in celery. The international tobacco science research cooperation Center (CORESTA) stipulates that the guiding residual limit of pendimethalin in tobacco is 5 mg/kg, and the maximum residual limit of pendimethalin in tobacco is not established in China.
At present, the detection method of pendimethalin is mainly an instrument detection method, such as gas chromatography, gas chromatography-mass spectrometry, gas chromatography tandem mass spectrometry and the like. However, these analysis methods require expensive large-scale instruments and equipment and professional detection personnel, and are complex in pretreatment process, complex in operation, high in detection cost and slow in analysis speed, so that the requirements of on-site monitoring and rapid screening of pesticide residues in a large number of samples are difficult to meet. 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; and the fluorescence quenching of the dye after the microsphere is formed is greatly reduced, the emission is strong and stable, and the influence of the medium change of the external environment is basically avoided. Therefore, the fluorescent microsphere immunochromatography technology has the advantages of high detection sensitivity, simple and convenient operation and good stability. At present, pendimethalin fluorescent microsphere immunochromatographic test strips do not appear on the market.
Disclosure of Invention
The invention aims to provide a pendimethalin residue detection fluorescent microsphere immunochromatographic test strip which is high in sensitivity, simple to operate, low in cost and short in detection time; 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 detecting pendimethalin.
In order to achieve the purpose, the invention adopts a technical scheme that:
the fluorescent microsphere immunochromatographic test strip for detecting pendimethalin residues comprises a base plate, and a sample combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped and adhered on the base plate, wherein a pendimethalin monoclonal antibody marked by fluorescent microspheres is embedded on the sample combination pad, a detection area and a quality control area are fixed on the nitrocellulose membrane, a pendimethalin hapten-carrier protein conjugate is sprayed on the detection area, a goat anti-mouse antibody is sprayed on the quality control area, the pendimethalin monoclonal antibody is prepared by taking the pendimethalin hapten-carrier protein conjugate as an immunogen, the pendimethalin hapten-carrier protein conjugate is obtained by coupling pendimethalin hapten and carrier protein, the pendimethalin hapten is prepared by 3- (1-ethyl-alanine) -6-methyl-2, 4-dinitrobenzaldehyde reacts with 3-hydrazino propionic acid to obtain the compound, and the molecular structural formula is as follows:
the preparation method of the pendimethalin hapten comprises the following steps:
taking 0.60 g of 3- (1-ethyl propylamine) -6-methyl-2, 4-dinitrobenzaldehyde, adding 50 mL of acetonitrile to dissolve, after clarification, dropwise adding 5 mL of methanol solution for dissolving 0.32 g of 3-hydrazinopropionic acid, and stirring at room temperature for 3 hours; stopping the reaction, removing the organic solvent by rotary evaporation, adding water, adding 0.45 g of sodium hydroxide, dissolving and clarifying, adding 80 mL of ethyl acetate for extraction, separating an organic phase, adding dilute hydrochloric acid into an aqueous phase to adjust the pH to be =4, adding 50 mL of chloroform for extraction, washing with water, concentrating, and recrystallizing with ethanol/petroleum ether in a volume ratio of 1:1 to obtain 0.70 g of a hapten product.
The pendimethalin hapten-carrier protein conjugate is obtained by coupling pendimethalin hapten and carrier protein, wherein the carrier protein can be bovine serum albumin, ovalbumin, hemocyanin, thyroid protein and human serum albumin.
The pendimethalin monoclonal antibody is obtained by secreting a pendimethalin monoclonal antibody hybridoma cell strain; 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 fluorescein isothiocyanate.
The invention adopts another technical scheme that a method for preparing the fluorescent microsphere immunochromatographic test strip for detecting pendimethalin residue is provided, and the method comprises the following steps:
1) preparation of sample conjugate pad: marking pendimethalin monoclonal antibody with commercially available fluorescent microspheres, diluting the pendimethalin monoclonal antibody with a specific buffer system, soaking the sample combined pad in a dilution buffer solution, and performing vacuum freeze drying to prepare the sample combined pad;
2) preparation of nitrocellulose membrane: spraying the pendimethalin 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 binding pad embedded with a fluorescent microsphere labeled pendimethalin 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 to obtain the fluorescent microsphere immunochromatographic test strip.
Specifically, the steps include:
1) hapten preparation: carrying out a series of reactions on 3- (1-ethyl propylamine) -6-methyl-2, 4-dinitrobenzaldehyde and 3-hydrazino propionic acid to obtain a pendimethalin hapten product;
2) coupling pendimethalin hapten with carrier protein to obtain a pendimethalin hapten-carrier protein conjugate;
3) immunizing a mouse by using the pendimethalin hapten-carrier protein conjugate, and fusing and screening spleen cells and myeloma cells of the mouse to obtain a pendimethalin monoclonal hybridoma cell strain;
4) extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibodies;
5) respectively spraying pendimethalin hapten-carrier protein conjugate and goat anti-mouse anti-antibody to a detection area range (T) and a quality control area range (C) of a nitrocellulose membrane;
6) uniformly soaking the sample bonding pad in phosphate buffer solution containing bovine serum albumin (the final concentration of the bovine serum albumin in the buffer system is 0.5% by volume percentage) and having the pH value of 7.2 and the concentration of 0.1 mol/L for 2h, and drying at 37 ℃ for 2 h;
7) marking pendimethalin monoclonal antibody with commercially available fluorescent microspheres, diluting the pendimethalin monoclonal antibody with a specific buffer system, soaking the sample combined pad treated in the step 6) in a dilution buffer solution, and performing vacuum freeze drying for later use;
8) and sequentially overlapping and sticking a sample binding pad embedded with a fluorescent microsphere labeled pendimethalin 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 to obtain the fluorescent microsphere immunochromatographic test strip.
The invention adopts another technical scheme that an application of the fluorescent microsphere immunochromatographic test strip for detecting pendimethalin residue in detecting pendimethalin is provided, which comprises the following steps:
1) pretreating a sample;
2) detecting by using the fluorescent microsphere immunochromatographic test strip for detecting pendimethalin residue;
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 is embedded with the pendimethalin monoclonal antibody marked by the fluorescent microspheres on the sample combination pad, and has the advantages of good hydrophilicity, capability of adsorbing antibody conjugates in large capacity, quick re-wetting, full release of the antibody conjugates, good performance, quick release, good shape and the like, so that errors are reduced, the cost is reduced, and the reaction sensitivity of the whole system is improved;
(2) polystyrene is wrapped on the surface of the fluorescent microsphere, so that the protection of fluorescent substance fluorescein isothiocyanate is realized, the interference of the external environment is reduced, and the stability and the fluorescence life of the fluorescent microsphere are improved;
(3) 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 fluorescent microsphere immunochromatography test strip for detecting pendimethalin in agricultural products, tobaccos and tobacco products exists, and the invention fills the blank. The hapten has proper terminal active groups, the length of a modification site and a spacer arm is properly selected, and the molecular structure of pendimethalin can be simulated to the greatest extent. Meanwhile, 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. In conclusion, the method for detecting pendimethalin residue 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 fluorescent microsphere immunochromatographic test strip, in which: 1. a sample conjugate pad; 2. a nitrocellulose membrane; 3. a water absorbent pad; 4. a detection zone; 5. a quality control region; 6. a base plate;
FIG. 2 is a top view of a fluorescent microsphere immunochromatographic test strip;
FIG. 3 is a synthesis diagram of pendimethalin hapten.
Detailed Description
The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1 composition of fluorescent microsphere immunochromatographic test strip for detecting pendimethalin residue
Referring to fig. 1: the test strip consists of a bottom plate, a sample combination pad, a nitrocellulose membrane and a water absorption pad.
The sample combination pad 1, the nitrocellulose membrane 2 and the water absorption pad 3 are sequentially overlapped and adhered to the bottom plate 6, the tail end of the sample combination pad is connected with the initial end of the nitrocellulose membrane, the tail end of the nitrocellulose membrane is connected with the initial end of the water absorption pad, the initial end of the sample combination pad is aligned with the initial end of the bottom plate, and the tail end of the water absorption pad is aligned with the tail end of the bottom plate.
The detection area 4 and the quality control area 5 are fixed on the nitrocellulose membrane, the pendimethalin hapten-carrier protein conjugate is sprayed on the detection area, and the goat anti-mouse anti-antibody is sprayed on the quality control area.
The bottom plate is a PVC bottom plate; the sample combining pad is glass wool; the absorbent pad is absorbent paper.
Example 2 preparation of fluorescent microsphere immunochromatographic test strip for detecting pendimethalin residue
The preparation method of the test strip mainly comprises the following steps:
1) preparation of sample conjugate pad: marking pendimethalin monoclonal antibody with commercially available fluorescent microspheres, diluting the pendimethalin monoclonal antibody with a specific buffer system, soaking the sample combined pad in a dilution buffer solution, and performing vacuum freeze drying to prepare the sample combined pad;
2) preparation of nitrocellulose membrane: spraying the pendimethalin 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 binding pad embedded with a fluorescent microsphere labeled pendimethalin 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 to obtain the fluorescent microsphere immunochromatographic test strip.
The following steps are detailed:
preparation of the Components
1. Preparation of pendimethalin hapten
Taking 0.60 g of 3- (1-ethyl propylamine) -6-methyl-2, 4-dinitrobenzaldehyde, adding 50 mL of acetonitrile to dissolve, after clarification, dropwise adding 5 mL of methanol solution for dissolving 0.32 g of 3-hydrazinopropionic acid, and stirring at room temperature for 3 hours; stopping the reaction, removing the organic solvent by rotary evaporation, adding water, adding 0.45 g of sodium hydroxide, dissolving and clarifying, adding 80 mL of ethyl acetate for extraction, separating an organic phase, adding dilute hydrochloric acid into an aqueous phase to adjust the pH to be =4, adding 50 mL of chloroform for extraction, washing with water, concentrating, and recrystallizing with ethanol/petroleum ether with the volume ratio of 1:1 to obtain 0.70 g of hapten product with the yield of 90.1%.
Taking the hapten, performing nuclear magnetic resonance hydrogen spectrum identification,1H-NMR（CDCl3300 MHZ) δ 8.505 (10, 1H), 4.363 (11, 1H, quant, J =6.864), 8.326 (13, 1H), 2.247 (14, 3H), 1.525 (16, 2H, qd, J =7.141, J =6.864), 1.525 (17, 2H, qd, J =7.141, J =6.864), 0.822 (21, 3H, t, J =7.141), 0.822 (22, 3H, t, J =7.141), 3.680 (23, 2H, t, J =6.869), 2.679 (24, 2H, t, J = 6.869). Chemical shifts δ =3.680, 2.679 are resonance absorption peaks of hydrogen on the hapten spacer arm, and the presence and position of these two peaks indicate successful hapten synthesis.
2. Preparation of immunogens
The immunogen is obtained by coupling pendimethalin hapten and Bovine Serum Albumin (BSA).
Dissolving 15 mg of hapten in 1 mL of N, N-Dimethylformamide (DMF); adding 8 mg of carbodiimide (EDC), and stirring at room temperature for 24 h to obtain a reaction solution A; weighing 30 mg of BSA, fully dissolving in 4 mL of 0.1 mol/L phosphate buffer solution (PB, pH 7.0), dropwise and slowly adding the reaction solution A into the protein solution, stirring at room temperature for 24 h, dialyzing with 0.01 mol/L Phosphate Buffer Solution (PBS) at 4 ℃ for 3d, changing the dialyzate 3 times every day to remove unreacted micromolecule substances to obtain immunogen, subpackaging, and storing at-20 ℃.
3. Preparation of coating antigen
Coupling pendimethalin hapten with Ovalbumin (OVA) to obtain the coating antigen.
20 mg of hapten is dissolved in 1 mL of DMF; adding 10 mg of Dicyclohexylcarbodiimide (DCC), stirring for 24 h at room temperature, performing suction filtration, and removing insoluble solids to obtain a reaction solution A; weighing OVA 30 mg, fully dissolving in 6 mL of 0.1 mol/L PB (pH value of 7.0), dropwise and slowly adding the reaction solution A into the protein solution, stirring at room temperature for 24 h, dialyzing with 0.01 mol/L PBS at 4 ℃ for 3d, changing the dialyzate for 3 times every day to remove unreacted small molecular substances to obtain a coating antigen, subpackaging, and storing at-20 ℃.
4. Preparation of pendimethalin monoclonal antibody
(1) Obtaining hybridoma cells
First immunization: pendimethalin hapten-BSA conjugate (immunogen) was emulsified well with an equal amount of Freund's complete adjuvant and injected subcutaneously into 6-week-old Balb/c mice, 0.2 mL each;
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 pendimethalin monoclonal antibody, preparing the hybridoma cells in the logarithmic growth phase into cell suspension by using a freezing medium, subpackaging 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 the cryopreserved tube of the pendimethalin monoclonal antibody hybridoma cell strain, immediately putting the cryopreserved tube into a water bath at 37 ℃ for medium-speed thawing, centrifuging to remove a cryopreserved solution, and transferring the cryopreserved 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 octanoic acid-saturated ammonium sulfate method to obtain pendimethalin monoclonal antibody solution (preservation at (-20 deg.C)).
(3) Determination of the potency of monoclonal antibodies
The titer of the antibody is measured to be 1 (300000-700000) by using an indirect competition ELISA method.
Indirect competitive ELISA method: coating an enzyme label plate with a pendimethalin hapten-OVA conjugate, adding a pendimethalin standard solution, a pendimethalin 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 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, dinitroaniline herbicides such as pendimethalin, flumetralin, butralin, trifluralin, and benfluralin are serially diluted, are 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 pendimethalin, less than 1 percent of flumetralin, less than 1 percent of butralin, less than 1 percent of trifluralin and less than 1 percent of benfluralin. The antibody of the invention has no cross reaction to other dinitroaniline herbicides such as flumetralin, butralin, trifluralin, benfluralin and the like, and has specific binding only to pendimethalin.
5. 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.
6. Preparation of fluorescent microsphere labeled pendimethalin 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 10 min 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 10 min at room temperature;
(2) coupling: centrifuging the suspension of the step (1) at 4 ℃ and 10000 r/min for 10 min, 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 pendimethalin monoclonal antibody solution (with the protein concentration of 1 mg/mL), uniformly mixing, and then 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 10 min, 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 10 min, 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.
7. Preparation of sample conjugate pad
(1) Uniformly soaking the sample binding pad in phosphate buffer solution containing bovine serum albumin (the final concentration of BSA in a buffer system is 0.5%, volume percentage content) and having the pH value of 7.2 and 0.1 mol/L for 2h, and drying at 37 ℃ for 2 h;
(2) diluting the stored fluorescence microsphere marked pendimethalin monoclonal antibody with a storage buffer solution, soaking the sample combined pad treated in the step (1) in the dilution buffer solution, and freezing and drying in vacuum for later use.
8. Preparation of cellulose Nitrate (NC) membranes
Diluting pendimethalin hapten-ovalbumin conjugate to 100. mu.g/mL with 0.05 mol/L, pH value of 7.2 PBS buffer, and spraying the diluted pendimethalin hapten-ovalbumin 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 2h at 37 ℃ for later use.
(II) Assembly of test strip
The sample combination pad, the nitrocellulose membrane and the water absorption pad are sequentially overlapped and stuck and fixed on the bottom plate from left to right, the tail end of the sample combination pad is connected with the initial end of the nitrocellulose membrane, the tail end of the nitrocellulose membrane is connected with the initial end of the water absorption pad, the initial end of the sample combination pad is aligned with the initial 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 combination pad, the nitrocellulose membrane and the water absorption pad are cut into small strips with the width of 3.96 mm by a machine and are arranged in a special plastic card to form a. The pendimethalin 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 fluorescent microsphere immunochromatographic test strip for detecting pendimethalin residue
1. Sample pretreatment
Weighing 1.0 +/-0.05 g of homogenized sample to be detected into a 50 mL centrifuge tube, adding 10 mL acetonitrile, whirling for 1 min by using a vortex instrument, and centrifuging for 5 min at room temperature (20-25 ℃) and more than 3000 rpm; taking 2 mL to 10 mL of the supernatant, drying the supernatant in a water bath at 40-50 ℃ by using nitrogen, adding 500 mu L of the sample complex solution, whirling the sample complex solution by using a vortex instrument for 2 min, and uniformly mixing the sample complex solution to be detected.
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 pendimethalin in the tobacco sample according to the intensity of the detected fluorescent signal, and gives out positive and negative judgment according to a preset threshold value.
Negative (-): if the result on the display screen of the fluorescence detector is negative, the result indicates that the sample does not contain pendimethalin or the concentration of the pendimethalin is lower than the detection limit.
Positive (+): if the result on the display screen of the fluorescence detector is positive, the concentration of pendimethalin 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 determination of technical parameters of fluorescent microsphere immunochromatographic test strip for detecting pendimethalin residue
1. Limit of detection test
Adding pendimethalin standard substances into blank tobacco, tea, spinach and celery samples respectively until the final concentrations are 0.05, 0.1 and 0.2 mg/kg, and detecting by using a fluorescent microsphere immunochromatography test strip, wherein the results are as follows: when the concentration of pendimethalin is 0.05 mg/kg, the detection of the fluorescence detector is negative; when the concentration of pendimethalin is 0.1 mg/kg and 0.2 mg/kg, the detection of a fluorescence detector is positive, which shows that the detection limit of pendimethalin in tobacco, tea, spinach and celery is 0.1 mg/kg.
2. Test for false positive and false negative rates
Respectively taking 20 parts of tobacco positive samples with known pendimethalin content larger than the detection limit and 20 parts of negative samples with known pendimethalin content smaller than the detection limit, respectively detecting by using 3 fluorescent microsphere immunochromatographic test strips produced in batches, and calculating the negative and positive rates of the tobacco positive samples and the tobacco negative samples. The results are shown in the following table.
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 fluorescent microsphere immunochromatographic test strip for detecting pendimethalin residue can be used for quickly detecting pendimethalin residue in tobacco leaves.
3. Specificity test
The result of detecting 5 mg/L dinitroaniline herbicides such as flumetralin, butralin, trifluralin, benfluralin and the like by using a pendimethalin test strip shows that the result is negative. The test paper strip has no cross reaction to dinitroaniline herbicides such as pendimethalin, butralin, trifluralin, benfluralin and the like with 5 mg/L and has good specificity.
Claims (7)
1. The utility model provides a detect remaining fluorescence microballon immunochromatography test paper strip of pendimethalin, includes the bottom plate and the sample combination pad, the nitrocellulose membrane and the pad that absorbs water of overlap joint pasting in proper order on the bottom plate, be fixed with detection zone and matter accuse district, its characterized in that on the nitrocellulose membrane: the sample combination pad is embedded with a pendimethalin monoclonal antibody marked by fluorescent microspheres, a detection area is sprayed with a pendimethalin hapten-carrier protein conjugate, a quality control area is sprayed with a goat anti-mouse antibody, the pendimethalin monoclonal antibody is prepared by taking the pendimethalin hapten-carrier protein conjugate as an immunogen, the pendimethalin hapten-carrier protein conjugate is obtained by coupling pendimethalin hapten and carrier protein, the pendimethalin hapten is obtained by reacting 3- (1-ethyl propylamine) -6-methyl-2, 4-dinitrobenzaldehyde with 3-hydrazinopropionic acid, and the molecular structural formula is as follows:
2. the fluorescent microsphere immunochromatographic test strip for detecting pendimethalin residue according to claim 1, which is characterized in that: the preparation reaction process of the pendimethalin hapten is as follows:
3. the fluorescent microsphere immunochromatographic test strip for detecting pendimethalin residues as claimed in claim 1, wherein: 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 fluorescein isothiocyanate.
4. The fluorescent microsphere immunochromatographic test strip for detecting pendimethalin residues as claimed in claim 1, wherein: the carrier protein is any one of bovine serum albumin, ovalbumin, hemocyanin, thyroid protein and human serum albumin.
5. The fluorescent microsphere immunochromatographic test strip for detecting pendimethalin residues as claimed in claim 1, wherein: the goat anti-mouse anti-antibody is obtained by immunizing a goat with a murine antibody.
6. The preparation method of the fluorescent microsphere immunochromatographic test strip for detecting pendimethalin residues as claimed in any one of claims 1 to 5, which is characterized by comprising the following steps:
1) preparation of sample conjugate pad: marking pendimethalin monoclonal antibody with commercially available fluorescent microspheres, diluting the pendimethalin monoclonal antibody with a specific buffer system, soaking the sample combined pad in a dilution buffer solution, and performing vacuum freeze drying to prepare the sample combined pad;
2) preparation of nitrocellulose membrane: spraying the pendimethalin 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 bonding a sample binding pad embedded with a fluorescent microsphere labeled pendimethalin monoclonal antibody, a nitrocellulose membrane fixed with a detection area and a quality control area and a water absorption pad on the bottom plate in a lap joint manner, and shearing the nitrocellulose membrane and the water absorption pad into required widths to obtain the fluorescent microsphere immunochromatographic test strip.
7. The use of the fluorescent microsphere immunochromatographic strip for detecting pendimethalin residue according to any one of claims 1 to 5 in the detection of pendimethalin, characterized by comprising the steps of:
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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| CN110927383A (en) * | 2019-10-08 | 2020-03-27 | 杭州佰昕科技有限公司 | Fluorescent microsphere immunochromatography test strip for detecting olaquindox residue and application thereof |
| CN110927375A (en) * | 2019-10-08 | 2020-03-27 | 杭州佰昕科技有限公司 | Fluorescent microsphere immunochromatography test strip for detecting olaquindox residue and application thereof |
| CN111122856B (en) | 2020-01-10 | 2023-05-12 | 中国农业科学院烟草研究所 | Time-resolved fluorescence immunochromatography test paper card for detecting butralin |
| CN111579790B (en) * | 2020-05-27 | 2023-08-04 | 中国农业科学院烟草研究所 | Test strip for detecting pendimethalin |
| CN112730843A (en) * | 2021-01-26 | 2021-04-30 | 无锡精检生物技术有限公司 | Fluorescence immunoassay quantitative POCT detection method for drug residues in agricultural products |
| CN113637081B (en) * | 2021-08-08 | 2023-08-18 | 江南大学 | Hybridoma cell strain secreting anti-pendimethalin monoclonal antibody and application thereof |
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