An immunochromatographic reaction composition for simultaneously detecting quinclorac and atrazine, and a preparation method and application thereof

Abstract

The application belongs to the technical field of immune detection, and discloses an immune chromatography reaction composition for simultaneously detecting quinclorac and atrazine as well as a preparation method and application thereof. The preparation method is as follows: an anti-quinclorac monoclonal antibody and colloidal gold nanoparticles are coupled respectively, an anti-atrazine monoclonal antibody and time-resolved fluorescent microspheres are coupled respectively, and the two kinds of labeled combinations are fixed on a combination pad after being treated by a freeze-drying protection solution; a detection line T1 coated with a quinclorac-carrier protein coupling product, a detection line T2 coated with an atrazine-carrier protein coupling product and a quality control line C are arranged on a chromatography membrane; after a test strip is assembled and a developing solution is prepared, the test strip is sealed and packaged. The colloidal gold and the time-resolved fluorescent label are integrated in the same test strip, the double-mode simultaneous detection of visual qualitative judgment of quinclorac and fluorescent quantitative detection of atrazine is realized, the two kinds of signals do not interfere with each other, and the test strip has the convenience of on-site rapid screening and the precision of laboratory confirmation.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and more particularly to an immunochromatographic reaction composition for the simultaneous detection of dichloroquinolinic acid and atrazine, its preparation method, and its application. Background Technology

[0002] Quinclorac and atrazine are commonly used herbicides in tobacco fields. Both have long residual periods in the soil, easily causing phytotoxicity to subsequent crops and posing a risk of groundwater pollution. Effective monitoring of these two herbicide residues is crucial for guiding rational herbicide application and protecting the agricultural ecological environment.

[0003] Immunochromatographic test strips for dichloroquinoline acid or atrazine have been reported, such as the time-resolved fluorescence immunochromatographic test strip for detecting dichloroquinoline acid disclosed in Chinese patent application CN201811104565.9. However, most existing test strips target only a single target, and both colloidal gold and fluorescent labels have their limitations: colloidal gold test strips are easy to operate and the results are visually interpretable, but they have low sensitivity and are difficult to quantify accurately; fluorescent test strips have high sensitivity and can achieve quantification, but they rely on instrument readings and cannot meet the needs of rapid on-site qualitative screening. Although a few multi-target detection test strips can detect two targets simultaneously, they still use the same labeling material, providing only homogeneous signals for the two targets, making it difficult to meet the differentiated needs of different application scenarios for result types. Furthermore, integrating two different types of markers into the same test strip presents technical challenges such as optical interference between the markers, inconsistent release behavior, and cross-reactivity. Currently, there is no immunochromatographic composition capable of simultaneously detecting dichloroquinolinic acid and atrazine using both visual qualitative and quantitative methods.

[0004] Therefore, there is an urgent need to develop an immunochromatographic reaction composition that can achieve both visual qualitative interpretation of dichloroquinoline acid and quantitative fluorescence detection of atrazine on the same test strip, without interference between the two signals. Summary of the Invention

[0005] The purpose of this invention is to provide an immunochromatographic reaction composition for the simultaneous detection of dichloroquinolinic acid and atrazine, its preparation method and application. The method involves co-carrying colloidal gold-labeled anti-dichloroquinolinic acid monoclonal antibody and time-resolved fluorescent microsphere-labeled anti-atrazine monoclonal antibody on the same binding pad, and sequentially setting corresponding detection lines on the chromatographic membrane to achieve simultaneous detection of heterogeneous signals of dual targets on the same test strip.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing an immunochromatographic reaction composition for the simultaneous detection of dichloroquinolinic acid and atrazine, comprising the following steps: (1) The anti-dichloroquinoline monoclonal antibody was conjugated with colloidal gold nanoparticles to obtain the first labeled conjugate; (2) The anti-atrazine monoclonal antibody was coupled with time-resolved fluorescent microspheres to obtain a second labeled conjugate; the emission wavelength of the time-resolved fluorescent microspheres was 610~620 nm, which was more than 100 nm different from the absorption peak wavelength of the colloidal gold nanoparticles. (3) The first and second labeled conjugates were mixed with the freeze-drying protective solution and fixed onto the conjugate pad, and then dried to obtain the conjugate pad; (4) Detection line T1, detection line T2 and control line C are set sequentially along the chromatography direction on the chromatography membrane. Detection line T1 is coated with dichloroquinoline acid-carrier protein conjugate, detection line T2 is coated with atrazine-carrier protein conjugate, and control line C is coated with goat anti-mouse IgG antibody. The distance between T1 and T2 is 3~8 mm. (5) Place the sample pad, conjugation pad, chromatography membrane and absorption pad on the backing plate in sequence, cut them into strips to obtain test strips; (6) Prepare the developing solution, wherein the developing solution is a phosphate buffer containing bovine serum albumin and Tween-20; (7) Seal the test strip and the developing solution together to obtain the immunochromatographic reaction composition for the simultaneous detection of dichloroquinolinic acid and atrazine.

[0007] Furthermore, the anti-dichloroquinoline monoclonal antibody mentioned in step (1) is a monoclonal antibody obtained by immunizing mice with an artificial antigen prepared by conjugating dichloroquinoline hapten with a carrier protein. The carrier protein is bovine serum albumin, ovalbumin, or keyhole hemocyanin. The dichloroquinoline hapten is prepared by introducing a straight-chain alkyl active arm containing 3 to 6 carbon atoms at the 3-position carboxyl group of dichloroquinoline acid through an amide bond. The anti-atrazine monoclonal antibody mentioned in step (2) is a monoclonal antibody obtained by immunizing mice with an artificial antigen prepared by conjugating atrazine hapten with a carrier protein. The carrier protein is bovine serum albumin, ovalbumin or keyhole hemocyanin. The atrazine hapten is prepared by introducing a straight-chain carboxyl alkyl active arm containing 3 to 6 carbon atoms at the 6-position chlorine atom of atrazine through nucleophilic substitution.

[0008] Furthermore, the colloidal gold nanoparticles mentioned in step (1) have a particle size of 15~45 nm, and the coupling method is electrostatic adsorption, which is carried out by adjusting the pH to within the range of ±0.5 of the antibody isoelectric point; The time-resolved fluorescent microspheres mentioned in step (2) are polystyrene fluorescent microspheres coated with europium chelates, with a particle size of 100~300 nm, and the coupling method is covalent coupling by carbodiimide method or active ester method.

[0009] Furthermore, the lyophilization protection solution described in step (3) contains 1-3% w / v sucrose, 0.5-2% w / v casein and 0.02-0.1% w / v Tween-20 in 0.02 mol / L Tris-HCl buffer solution, with a pH of 8.0-8.5; The mass ratio of the first labeled conjugate to the second labeled conjugate is 1:0.8~1.2; the fixation method is 2~5 μL / cm 2 After spraying the coating, vacuum dry at 35~40℃ for 1~2 hours.

[0010] Furthermore, in step (4), the coating concentration of detection line T1 is 0.5~2.0 mg / mL, the coating concentration of detection line T2 is 0.3~1.5 mg / mL, and the coating concentration of control line C is 0.5~2.0 mg / mL; after spraying, it is dried in a forced-air drying oven at 35~40℃ for 0.5~1 hour.

[0011] Furthermore, in step (5), the sample pad is a glass fiber membrane, the binding pad is a polyester fiber membrane, the chromatography membrane is a nitrocellulose membrane, and the absorbent pad is cotton pulp paper or absorbent filter paper.

[0012] Furthermore, the developing solution in step (6) contains 1-2% w / v bovine serum albumin, 0.1-0.5% w / v Tween-20 in 0.02-0.05 mol / L phosphate buffer, with a pH of 7.2-7.6.

[0013] The present invention provides an immunochromatographic reaction composition for the simultaneous detection of dichloroquinolinic acid and atrazine, prepared by the above-described method.

[0014] This invention provides an immunochromatographic reaction composition for the simultaneous detection of quinclorac and atrazine, and its application in the preparation of a kit for the simultaneous detection of quinclorac and atrazine residues in tobacco field soil or water.

[0015] This invention provides a dual-mode immunochromatographic method for the simultaneous qualitative detection of dichloroquinoline acid and the quantitative detection of atrazine using the above-mentioned immunochromatographic reaction composition for the simultaneous detection of dichloroquinoline acid and atrazine, comprising the following steps: S1. Mix the sample solution to be tested with the developing solution at a volume ratio of 1:1~4 and then drop the mixture onto the sample pad. S2. The chromatography reaction is completed under capillary action, and the reaction time is 10-20 minutes. S3. Observe the red color development of colloidal gold at the detection line T1 under natural light to determine the presence or absence of dichloroquinoline acid. S4. Excitation with a 365 nm ultraviolet light source is used, and the fluorescence intensity at the detection line T2 is read at 615 nm using a fluorescence reader to achieve quantitative detection of atrazine. S5. Confirm the color development and fluorescence signal at control line C using both visual inspection and a fluorescence reader to determine the validity of the test.

[0016] Compared with the prior art, the present invention has the following beneficial effects: First, this study is the first to apply colloidal gold labeling and time-resolved fluorescence labeling to the same immunochromatographic test strip, enabling both visual qualitative analysis of quincloracine and quantitative fluorescence detection of atrazine in the same reaction system. Quincloracine has relatively high residue limits in tobacco soil; the colloidal gold visual qualitative analysis mode is sufficient for screening, simple to operate, requires no instruments, and is suitable for field use. Atrazine has low residue limits but poses a risk of cumulative toxicity; the time-resolved fluorescence quantitative mode provides accurate concentration data, offering a basis for scientific decision-making.

[0017] Secondly, by optimizing the mass ratio of the two heterogeneous labels, the detection line spacing, the formulation of the lyophilization protective solution, and the components of the developing solution, the aggregation and cross-interference between the heterogeneous labels were effectively suppressed. In particular, the protective layer formed by the lyophilization protective solution on the surface of the labeled particles ensures that the colloidal gold and fluorescent microspheres maintain independent dispersion and release characteristics in the coexisting system, thus ensuring the specificity and accuracy of dual-target detection.

[0018] Third, the control line C can be recognized by both colloidal gold-labeled antibodies and fluorescent microsphere-labeled antibodies, enabling separate verification of the effectiveness of the two detection pathways. In traditional single-labeled test strips, the control line can only verify the activity of the label; however, in this invention, line C simultaneously provides both a visible red signal and a fluorescent signal, which can separately verify whether the colloidal gold channel and the fluorescent channel are functioning properly, avoiding the risk of missed detection that may occur with single-channel quality control. Detailed Implementation

[0019] This invention provides a method for preparing an immunochromatographic reaction composition for the simultaneous detection of dichloroquinolinic acid and atrazine, comprising the following steps: (1) The anti-dichloroquinoline monoclonal antibody was conjugated with colloidal gold nanoparticles to obtain the first labeled conjugate; (2) The anti-atrazine monoclonal antibody was coupled with time-resolved fluorescent microspheres to obtain a second labeled conjugate; the emission wavelength of the time-resolved fluorescent microspheres was 610~620 nm, which was more than 100 nm different from the absorption peak wavelength of the colloidal gold nanoparticles, to ensure that the two signals did not interfere with each other in the spectrum. (3) The first and second labeled conjugates were mixed with the freeze-drying protective solution and fixed onto the conjugate pad, and then dried to obtain the conjugate pad; (4) Detection line T1, detection line T2 and control line C are sequentially set on the chromatography membrane along the chromatography direction. Detection line T1 is coated with dichloroquinoline acid-carrier protein conjugate, detection line T2 is coated with atrazine-carrier protein conjugate, and control line C is coated with goat anti-mouse IgG antibody. The distance between T1 and T2 is 3~8 mm. This distance can ensure that the two markers react fully on their respective detection lines without generating steric hindrance and signal crosstalk. (5) Place the sample pad, conjugation pad, chromatography membrane and absorption pad on the backing plate in sequence, cut them into strips to obtain test strips; (6) Prepare the developing solution, wherein the developing solution is a phosphate buffer containing bovine serum albumin and Tween-20; (7) Seal the test strip and the developing solution together to obtain the immunochromatographic reaction composition for the simultaneous detection of dichloroquinolinic acid and atrazine.

[0020] In this invention, the anti-dichloroquinoline acid monoclonal antibody in step (1) is a monoclonal antibody obtained by immunizing mice with an artificial antigen prepared by conjugating dichloroquinoline acid hapten with a carrier protein. The carrier protein is bovine serum albumin, ovalbumin, or keyhole hemocyanin. The dichloroquinoline acid hapten is prepared by introducing a straight-chain alkyl active arm containing 3 to 6 carbon atoms at the 3-carboxyl group of dichloroquinoline acid through an amide bond. By site-specific modification of the dichloroquinoline acid molecule, a flexible linker arm of appropriate length is introduced while retaining the characteristic structure of the quinoline ring, so that the hapten can be fully exposed to the immune system after conjugation with the carrier protein, thereby inducing the production of monoclonal antibodies with high affinity and high specificity.

[0021] In this invention, the anti-atrazine monoclonal antibody in step (2) is a monoclonal antibody obtained by immunizing mice with an artificial antigen prepared by conjugating atrazine hapten with a carrier protein. The carrier protein is bovine serum albumin, ovalbumin, or keyhole hemocyanin. The atrazine hapten is prepared by introducing a straight-chain carboxyl alkyl active arm containing 3 to 6 carbon atoms at the chlorine atom of atrazine through nucleophilic substitution. By introducing a carboxyl-containing active arm at the 6-position of the triazine ring of atrazine, the characteristic antigenic determinant of atrazine is retained, and a reaction site for conjugation with the carrier protein is provided.

[0022] In this invention, the colloidal gold nanoparticles described in step (1) have a particle size of 15-45 nm, preferably 25 nm; the coupling method is electrostatic adsorption, which is carried out by adjusting the pH to within ±0.5 of the antibody's isoelectric point, so that the antibody carries an appropriate amount of positive charge under this pH condition, and undergoes efficient electrostatic adsorption with the negatively charged colloidal gold particles. After coupling, blocking protein is added to block unbound sites, centrifuged for purification, resuspended in storage buffer, and stored at 4°C in the dark.

[0023] In this invention, the time-resolved fluorescent microspheres in step (2) are polystyrene fluorescent microspheres coated with europium chelates, with a particle size of 100~300 nm, preferably 200 nm; the coupling method is covalent coupling by carbodiimide method or active ester method, and the carboxyl groups on the surface of the microspheres are activated by EDC / NHS to form stable amide bonds with the amino groups on the antibody.

[0024] In this invention, the lyophilization protection solution in step (3) contains 1-3% w / v sucrose, 0.5-2% w / v casein and 0.02-0.1% w / v Tween-20 in 0.02 mol / L Tris-HCl buffer with a pH of 8.0-8.5. It can form a protective layer on the surface of the labeled particles, effectively avoiding non-specific aggregation of colloidal gold and fluorescent microspheres due to surface charge differences, while ensuring that the two labeled substances have consistent release kinetics during chromatography. The mass ratio of the first labeled conjugate to the second labeled conjugate is 1:0.8~1.2, preferably 1:1; the fixation method is 2~5μL / cm 2 After spraying the coating, vacuum dry at 35~40℃ for 1~2 hours.

[0025] In this invention, in step (4), the coating concentration of detection line T1 is 0.5~2.0 mg / mL, preferably 1 mg / mL; the coating concentration of detection line T2 is 0.3~1.5 mg / mL, preferably 1 mg / mL; the coating concentration of control line C is 0.5~2.0 mg / mL, preferably 1 mg / mL; and after spraying, it is dried in a forced-air dryer at 35~40℃ for 0.5~1 hour.

[0026] In this invention, the sample pad in step (5) is a glass fiber membrane used to receive and filter the sample solution; the conjugation pad is a polyester fiber membrane used to carry the labeled conjugate and control its release; the chromatography membrane is a nitrocellulose membrane used to provide a solid-phase carrier for the immunorecognition reaction; and the absorbent pad is cotton pulp paper or absorbent filter paper used to provide capillary driving force.

[0027] In this invention, the developing solution in step (6) contains 1-2% w / v bovine serum albumin and 0.1-0.5% w / v Tween-20 in a 0.02-0.05 mol / L phosphate buffer solution with a pH of 7.2-7.6. Bovine serum albumin effectively blocks non-specific adsorption sites on the chromatography membrane, reducing background signal. Tween-20, as a non-ionic surfactant, improves the fluidity of the sample solution, promoting the uniform release and migration of the labeled conjugate.

[0028] The present invention provides an immunochromatographic reaction composition for the simultaneous detection of dichloroquinolinic acid and atrazine, prepared by the above-described method.

[0029] This invention provides an immunochromatographic reaction composition for the simultaneous detection of quinclorac and atrazine, and its application in the preparation of a kit for the simultaneous detection of quinclorac and atrazine residues in tobacco field soil or water.

[0030] This invention provides a dual-mode immunochromatographic method for the simultaneous qualitative detection of dichloroquinoline acid and the quantitative detection of atrazine using the above-mentioned immunochromatographic reaction composition for the simultaneous detection of dichloroquinoline acid and atrazine, comprising the following steps: S1. Mix the sample solution to be tested with the developing solution at a volume ratio of 1:1~4 and then drop the mixture onto the sample pad. S2. The chromatography reaction is completed under capillary action, and the reaction time is 10-20 minutes, preferably 15 minutes; S3. Observe the red color development of colloidal gold at the detection line T1 under natural light to determine the presence or absence of dichloroquinoline acid. S4. Excitation with a 365 nm ultraviolet light source is used, and the fluorescence intensity at the detection line T2 is read at 615 nm using a fluorescence reader to achieve quantitative detection of atrazine. S5. Confirm the color development and fluorescence signal at control line C using both visual inspection and a fluorescence reader to determine the validity of the test.

[0031] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1 Step (1): Preparation of anti-dichloroquinolinic acid monoclonal antibody-colloidal gold nanoparticle conjugate Synthesis of dichloroquinoline acid hapten: 3.14 g (10 mmol) of dichloroquinoline acid was weighed and dissolved in 50 mL of anhydrous dichloromethane. 1.26 g (11 mmol) of N-hydroxysuccinimide and 2.27 g (11 mmol) of dicyclohexylcarbodiimide were added, and the mixture was stirred at room temperature for 4 hours. The precipitate was removed by filtration to obtain an active ester solution of dichloroquinoline acid. Separately, 1.31 g (10 mmol) of 6-aminohexanoic acid was weighed and dissolved in 10 mL of dimethylformamide. This solution was slowly added dropwise to the above active ester solution, and the mixture was stirred overnight at room temperature after the addition was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: chloroform / methanol = 10 / 1) to give 2.86 g of a white solid product, with a yield of 75%. Mass spectrometry analysis revealed that the molecular ion peak was [M+H]+=383.2, which is consistent with the theoretical molecular weight of the target hapten (dichloroquinolinic acid-3-amidohexanoic acid) of 382.2.

[0033] Preparation of artificial antigen: Weigh 14.3 mg (0.0375 mmol) of the hapten and dissolve it in 0.5 mL of dimethylformamide. Add 6.2 mg (0.0375 mmol) of N,N'-dicyclohexylcarbodiimide and 4.3 mg (0.0375 mmol) of N-hydroxysuccinimide. Stir the reaction at room temperature for 2 hours, centrifuge, and collect the supernatant. Separately, dissolve 50 mg of bovine serum albumin in 5 mL of 0.01 M phosphate buffer (pH 7.4). Slowly add the hapten active ester solution dropwise while cooling in an ice bath, and stir the reaction overnight. Dialyze the reaction solution against phosphate buffer at 4 °C for 72 hours, changing the dialysate 6 times during this period. After lyophilization, obtain the dichloroquinoline acid-bovine serum albumin complete antigen and store it at -20 °C for later use.

[0034] Preparation of monoclonal antibodies: Six- to eight-week-old female BALB / c mice were immunized with the complete antigen. The initial immunization used complete Freund's adjuvant emulsified antigen, administered via subcutaneous injection at multiple sites, at a dose of 50 μg / mouse. Two weeks later, booster immunizations were administered using incomplete Freund's adjuvant at the same dose, for a total of three booster immunizations. Three days before cell fusion, a pulse immunization was performed via intraperitoneal injection of 100 μg / mouse of adjuvant-free antigen. Spleen cells from immunized mice were fused with SP2 / 0 myeloma cells at a 5:1 ratio using polyethylene glycol 1500. Hybridoma cells were screened on HAT medium. Positive clones were screened using indirect ELISA and competitive ELISA. Subcloning was performed using limiting dilution to obtain a hybridoma cell line, 2D3, that stably secretes anti-dichloroquinoline monoclonal antibody. The isoelectric point of this monoclonal antibody was determined to be 8.2. Hybridoma cells were injected into the peritoneal cavity of BALB / c mice presensitized with liquid paraffin, and the ascites fluid was collected and purified by Protein G affinity column to obtain monoclonal antibodies with a purity greater than 95%, which were identified as IgG1.

[0035] Colloidal gold conjugation: Take 500 mL of 0.01% chloroauric acid aqueous solution, heat to boiling, quickly add 4.0 mL of 1% trisodium citrate aqueous solution, maintain boiling and stir for 15 minutes, cool, and then dilute to 500 mL with pure water to obtain a colloidal gold solution with a particle size of approximately 25 nm. Take 10 mL of the colloidal gold solution and adjust the pH to 7.7-8.7 with 0.1 M potassium carbonate (i.e., within the range of the antibody isoelectric point 8.2 ± 0.5). While stirring, add 0.12 mg of anti-dichloroquinoline monoclonal antibody (pre-diluted to 1 mL with 1 mM phosphate buffer) dropwise, and continue stirring for 30 minutes. Add 10% bovine serum albumin solution to a final concentration of 1% to block unbound sites, and stir for 15 minutes. Centrifuge at 10,000 rpm and 4°C for 30 minutes, discard the supernatant, and resuspend the precipitate in 0.02 M Tris-HCl buffer (pH 8.5) containing 1% bovine serum albumin, 0.05% Tween-20, and 5% sucrose to 1 mL. Store at 4°C protected from light to obtain the first labeled conjugate.

[0036] Step (2): Preparation of anti-atrazine monoclonal antibody-time-resolved fluorescent microsphere conjugate Synthesis of atrazine hapten: 2.16 g (10 mmol) of atrazine was dissolved in 30 mL of anhydrous ethanol, and 1.78 g (15 mmol) of 3-mercaptopropionic acid and 2.07 g (15 mmol) of potassium carbonate were added. The mixture was heated under nitrogen protection and refluxed for 12 hours. After cooling to room temperature, the solvent was removed by vacuum distillation. The residue was dissolved in water, and the pH was adjusted to 2-3 with 1 M hydrochloric acid. The mixture was extracted three times with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give 2.23 g of a pale yellow oil, with a yield of 78%. The peaks were identified by 1H NMR spectroscopy as follows: δ 1.21-1.31 (triplet, 3H, -CH3), δ 2.65-2.72 (multiplet, 2H, -CH2-COOH), δ 3.19-3.27 (triplet, 2H, -S-CH2-), δ 5.85 (single, 1H, -NH-), and δ 10.32 (single, 1H, -COOH), indicating that the propionic acid active arm was successfully introduced at the 6-position of atrazine.

[0037] Preparation of artificial antigen: Weigh 12.5 mg (0.044 mmol) of hapten and dissolve it in 0.5 mL of dimethylformamide. Add 7.3 mg (0.044 mmol) of N,N'-dicyclohexylcarbodiimide and 5.1 mg (0.044 mmol) of N-hydroxysuccinimide. Stir at room temperature for 2 hours, centrifuge, and collect the supernatant. Separately, dissolve 45 mg of ovalbumin in 4.5 mL of 0.01 M phosphate buffer (pH 7.4). Slowly add the above active ester solution under ice bath conditions, stir and react overnight, dialyze, and lyophilize to obtain the atrazine-ovalbumin complete antigen.

[0038] Preparation of monoclonal antibodies: BALB / c mice were immunized with the same immunization and fusion protocol using the atrazine-ovalbumin complete antigen. The initial immunization used complete Freund's adjuvant emulsified antigen, administered via multiple subcutaneous injections at a dose of 50 μg / mouse. Two weeks later, booster immunizations were administered using incomplete Freund's adjuvant at the same dose, for a total of three booster immunizations. Three days before cell fusion, a pulse immunization was performed via intraperitoneal injection of 100 μg / mouse of adjuvant-free antigen. Spleen cells from immunized mice were fused with SP2 / 0 myeloma cells. After screening and subcloning, a stable hybridoma cell line, AT-5B7, secreting anti-atrazine monoclonal antibodies was obtained. The antibody, IgG1, was obtained from ascites fluid purified using a Protein G affinity column.

[0039] Fluorescent microsphere conjugation: 100 μL of commercial europium chelate time-resolved fluorescent microspheres (200 nm diameter, excitation wavelength 365 nm, emission wavelength 615 nm) were washed twice with 0.05 M borate buffer (pH 8.0). 50 μL of freshly prepared EDC / NHS solution (10 mg / mL each of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide) was added, and the microspheres were slowly shaken at room temperature for 30 minutes. The supernatant was discarded by centrifugation, and the microspheres were washed with borate buffer. The activated microspheres were resuspended in 200 μL of borate buffer, and 0.1 mg of anti-atrazine monoclonal antibody was added. The reaction was carried out overnight at 4°C with slow shaking. Blocking buffer containing 1% bovine serum albumin and 0.05% Tween-20 was added, and the microspheres were incubated at room temperature for 1 hour. After centrifugation and washing, the precipitate was resuspended in 200 μL with 0.02 M Tris-HCl buffer (pH 8.5) containing 1% bovine serum albumin, 5% sucrose, and 0.05% Tween-20. The precipitate was stored at 4°C. The coupling efficiency was 82.3%, yielding the second labeled conjugate.

[0040] Step (3): Preparation of the binding pad The first and second labeled conjugates were mixed with a lyophilization protectant (0.02 M Tris-HCl buffer containing 2% sucrose, 1% casein, and 0.05% Tween-20, pH 8.5) at a volume ratio of 1:1. Then, the two were mixed thoroughly at a mass ratio of 1:1 (colloidal gold conjugate to fluorescent microsphere conjugate), and uniformly sprayed onto a polyester fiber membrane (8 mm wide) at a spraying rate of 3 μL / cm using a sprayer. The membrane was then vacuum dried at 37°C for 2 hours to obtain the conjugation pad.

[0041] Step (4): Preparation of chromatography membrane The dichloroquinolinic acid-ovalbumin conjugate (prepared in the same way as the artificial antigen in step (1), except that bovine serum albumin was replaced with ovalbumin) was diluted to 1.2 mg / mL with 0.01 M phosphate buffer (pH 7.4) as the coating solution for the T1 line; the atrazine-bovine serum albumin conjugate (prepared in the same way as the artificial antigen in step (2), except that ovalbumin was replaced with bovine serum albumin) was diluted to 0.8 mg / mL as the coating solution for the T2 line; and the goat anti-mouse IgG antibody was diluted to 1.0 mg / mL as the coating solution for the C line. The T1 line, T2 line and C line were sequentially sprayed onto the nitrocellulose membrane using a gold spraying apparatus, with a spraying amount of 1 μL / cm for each line. The distance between the T1 and T2 lines was 5 mm, and the distance between the T2 and C lines was 6 mm. The membrane was dried at 37°C for 1 hour to obtain the chromatographic membrane.

[0042] Step (5): Assemble the test strips The sample pad (glass fiber membrane, 20 mm), the binding pad prepared in step (3), the chromatography membrane (25 mm) prepared in step (4), and the absorbent pad (cotton pulp paper, 17 mm) are sequentially overlapped on the backing plate, with adjacent parts overlapping by 2 mm. The strips are then cut into 4 mm wide test strips using a strip cutter to obtain the test strips.

[0043] Step (6): Prepare the developing solution Prepare a 0.03 M phosphate buffer solution containing 1.5% w / v bovine serum albumin and 0.2% w / v Tween-20, pH 7.4. Sterilize the solution by filtration through a 0.22 μm filter membrane and dispense 1 mL per tube to obtain the developing solution.

[0044] Step (7): Seal and package the test strip obtained in step (5) and the developing solution obtained in step (6) together to obtain the immunochromatographic reaction composition.

[0045] Example 2 The difference between this embodiment and embodiment 1 lies in the process parameters in step (3), while the other steps are the same as in embodiment 1.

[0046] Step (3) Preparation of the conjugate pad: The first and second labeled conjugates were mixed with a lyophilization protection solution (0.02 M Tris-HCl buffer containing 1% sucrose, 0.5% casein, and 0.02% Tween-20, pH 8.0) at a volume ratio of 1:1. Then, the two were mixed evenly at a mass ratio of 1:0.8 between the colloidal gold conjugate and the fluorescent microsphere conjugate. The mixture was then sprayed evenly onto a polyester fiber membrane at a spraying rate of 2 μL / cm using a spraying device. The membrane was then vacuum dried at 35°C for 2 hours to obtain the conjugate pad.

[0047] Example 3 The difference between this embodiment and embodiment 1 lies in the process parameters in step (3), while the other steps are the same as in embodiment 1.

[0048] Step (3) Preparation of the conjugate pad: The first and second labeled conjugates were mixed with a lyophilization protection solution (0.02 M Tris-HCl buffer containing 3% sucrose, 2% casein, and 0.1% Tween-20, pH 8.5) at a volume ratio of 1:1. Then, the two were mixed evenly at a mass ratio of 1:1.2 between the colloidal gold conjugate and the fluorescent microsphere conjugate. The mixture was then evenly sprayed onto a polyester fiber membrane using a spraying device at a spraying rate of 5 μL / cm and vacuum dried at 40°C for 1 hour to obtain the conjugate pad.

[0049] Example 4 The difference between this embodiment and Embodiment 1 is the coating concentration in step (4), while the other steps are the same as in Embodiment 1.

[0050] Step (4) Preparation of the chromatographic membrane: Dilute the dichloroquinoline acid-ovalbumin conjugate to 0.5 mg / mL with 0.01 M phosphate buffer as the coating solution for the T1 line; dilute the atrazine-bovine serum albumin conjugate to 0.3 mg / mL as the coating solution for the T2 line; dilute the goat anti-mouse IgG antibody to 0.5 mg / mL as the coating solution for the C line. Spray the T1 line, T2 line and C line sequentially onto the nitrocellulose membrane using a gold spraying apparatus, with a spraying volume of 1 μL / cm for each line. The distance between the T1 and T2 lines is 3 mm, and the distance between the T2 and C lines is 6 mm. Dry the membrane at 35°C for 1 hour to obtain the chromatographic membrane.

[0051] Example 5 The difference between this embodiment and Embodiment 1 is the coating concentration in step (4), while the other steps are the same as in Embodiment 1.

[0052] Step (4) Preparation of the chromatographic membrane: Dichloroquinoline-ovalbumin conjugate was diluted to 2.0 mg / mL with 0.01 M phosphate buffer as the coating solution for the T1 line; atrazine-bovine serum albumin conjugate was diluted to 1.5 mg / mL as the coating solution for the T2 line; and goat anti-mouse IgG antibody was diluted to 2.0 mg / mL as the coating solution for the C line. The T1 line, T2 line, and C line were sequentially coated on the nitrocellulose membrane using a gold spraying apparatus, with a coating volume of 1 μL / cm for each line. The distance between the T1 and T2 lines was 8 mm, and the distance between the T2 and C lines was 6 mm. The membrane was dried at 40°C for 0.5 hours to obtain the chromatographic membrane.

[0053] Example 6 The difference between this embodiment and Embodiment 1 is the formulation of the developing solution in step (6), while the other steps are the same as in Embodiment 1.

[0054] Step (6) Preparation of developing solution: Prepare 0.02 M phosphate buffer containing 1.0% w / v bovine serum albumin and 0.1% w / v Tween-20, pH 7.2, filter sterilize with a 0.22 μm filter membrane, and dispense 1 mL / tube to obtain developing solution.

[0055] Example 7 The difference between this embodiment and Embodiment 1 is the formulation of the developing solution in step (6), while the other steps are the same as in Embodiment 1.

[0056] Step (6) Preparation of developing solution: Prepare 0.05 M phosphate buffer containing 2.0% w / v bovine serum albumin and 0.5% w / v Tween-20, pH 7.6, filter sterilize with a 0.22 μm filter membrane, and dispense 1 mL / tube to obtain developing solution.

[0057] Performance testing The following performance tests were performed using the immunochromatographic reaction composition prepared in Example 1 as an example.

[0058] Test 1: Prepare dichloroquinoline acid standard solutions (concentration gradient: 0, 25, 50, 75, 100, 200, 500 ng / mL) and atrazine standard solutions (concentration gradient: 0, 0.1, 0.25, 0.5, 1, 2, 5 ng / mL). Mix 100 μL of each standard solution with 300 μL of developing buffer. Add 200 μL of the mixture to the sample pad of the test strip. Perform chromatography at room temperature for 15 minutes and then interpret the results. Each concentration group was repeated 6 times. The visual qualitative results of dichloroquinoline acid are shown in Table 1, and the fluorescence detection results of atrazine are shown in Table 2.

[0059] Table 1. Results of qualitative detection of dichloroquinoline acid by visual inspection (n=6)

[0060] Table 2. Results of fluorescence detection of atrazine (n=6)

[0061] Based on the data in Tables 1 and 2, the visual detection limit for dichloroquinoline is 100 ng / mL. The regression equation for the fluorescence detection standard curve of atrazine is Y = -28.6 lgX + 72.4 (R²). 2 =0.995), where Y is the fluorescence inhibition rate (%) and X is the atrazine concentration (ng / mL). The limit of detection was calculated as 0.28 ng / mL based on the concentration corresponding to an inhibition rate of 20%, and the limit of quantitation (inhibition rate of 50%) was 1.2 ng / mL.

[0062] Test 2: Standard solutions of dichloroquinoline acid structural analogs (chloroquinoline acid, quinoxalic acid), atrazine analogs (simazine, promethazine), and other commonly used tobacco herbicides (nicosulfuron, pendimethalin) were prepared, each with a concentration of 1000 ng / mL. Positive controls containing only dichloroquinoline acid (200 ng / mL) and only atrazine (5 ng / mL) were also prepared. The tests were performed according to the method in Test Example 1, and the results are shown in Table 3.

[0063] Table 3 Results of cross-reactivity test

[0064] As shown in Table 3, a positive signal is generated only when the corresponding target compound is detected, and the cross-reactivity rate of structural analogs and other herbicides is less than 5%, indicating that the composition has good specificity.

[0065] Test 3: Three groups of samples were prepared: Group A contained only 200 ng / mL of quinclorac acid; Group B contained only 5 ng / mL of atrazine; and Group C contained both 200 ng / mL of quinclorac acid and 5 ng / mL of atrazine. Six replicates were prepared for each group, and the results were obtained according to the method in Test Example 1. The results are shown in Table 4.

[0066] Table 4. Experimental results of dual-channel cross-interference

[0067] As shown in Table 4, the visual line disappearance of Group A and Group C is consistent at T1 line, and there is no significant difference in fluorescence inhibition rate between Group B and Group C at T2 line (relative deviation <5%). Furthermore, the signal of Line C is normal in both detection modes, indicating that the two markers and detection channels do not interfere with each other.

[0068] Test 4: Soil samples from tobacco fields, confirmed by high-performance liquid chromatography (HPLC) to be free of quinclorac and atrazine, were air-dried and passed through a 60-mesh sieve. 10 g of soil sample was weighed, and mixed standard solutions were added to different concentration levels. After mixing, the samples were allowed to stand at room temperature for 2 hours. 20 mL of extraction buffer (methanol: 0.01 M phosphate buffer = 7:3, v / v) was added, and the samples were vortexed for 5 minutes, centrifuged at 4000 rpm for 10 minutes, and the supernatant was diluted 5 times with phosphate buffer before analysis. Spiking levels and test results are shown in Table 5.

[0069] Table 5 Results of the spiked soil recovery experiment in tobacco fields (n=6)

[0070] Table 5 shows that the macroscopic qualitative results for quinclorac were all positive, consistent with the theoretical results. The recovery rate for quantitative detection of atrazine was between 85.3% and 96.7%, with intra-batch coefficient of variation of 5.2% to 8.9% and inter-batch coefficient of variation of 7.1% to 11.3%, meeting the methodological requirements for pesticide residue detection.

[0071] Test 5: The test strips and developing solutions prepared in the same batch of Example 1 were sealed and stored at 4℃ and 37℃ respectively. Samples were taken at 0, 1, 3, 6 and 12 months (testing at 37℃ for 12 weeks) to detect 200 ng / mL dichloroquinoline acid and 2 ng / mL atrazine standard. Each group had 3 replicates. The results are shown in Table 6.

[0072] Table 6. Results of test strip stability test

[0073] As shown in Table 6, after 12 months of storage at 4℃, there was no significant change in the degree of disappearance of the T1 line by the naked eye, and the fluorescence inhibition rate of the T2 line decreased by less than 10%; after 12 weeks of accelerated storage at 37℃, the performance indicators decreased by less than 15%, indicating that the product has good stability at room temperature.

[0074] The above embodiments and performance test data fully demonstrate that the immunochromatographic reaction composition provided by the present invention can simultaneously achieve visual qualitative screening of dichloroquinoline acid and fluorescence quantitative detection of atrazine in tobacco field soil and water. It has advantages such as high sensitivity, strong specificity, simple operation and intuitive results, and is suitable for combined use in the field and laboratory, and has high practical application value.

[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing an immunochromatographic reaction composition for the simultaneous detection of dichloroquinolinic acid and atrazine, characterized in that, Includes the following steps: (1) The anti-dichloroquinoline monoclonal antibody was conjugated with colloidal gold nanoparticles to obtain the first labeled conjugate; (2) The anti-atrazine monoclonal antibody was coupled with time-resolved fluorescent microspheres to obtain a second labeled conjugate; the emission wavelength of the time-resolved fluorescent microspheres was 610~620 nm, which was more than 100 nm different from the absorption peak wavelength of the colloidal gold nanoparticles. (3) The first and second labeled conjugates were mixed with the freeze-drying protective solution and fixed onto the conjugate pad, and then dried to obtain the conjugate pad; (4) Detection line T1, detection line T2 and control line C are set sequentially along the chromatography direction on the chromatography membrane. Detection line T1 is coated with dichloroquinoline acid-carrier protein conjugate, detection line T2 is coated with atrazine-carrier protein conjugate, and control line C is coated with goat anti-mouse IgG antibody. The distance between T1 and T2 is 3~8 mm. (5) Place the sample pad, conjugation pad, chromatography membrane and absorption pad on the backing plate in sequence, cut them into strips to obtain test strips; (6) Prepare the developing solution, wherein the developing solution is a phosphate buffer containing bovine serum albumin and Tween-20; (7) Seal the test strip and the developing solution together to obtain the immunochromatographic reaction composition for the simultaneous detection of dichloroquinolinic acid and atrazine.

2. The preparation method according to claim 1, characterized in that, The anti-dichloroquinoline monoclonal antibody mentioned in step (1) is a monoclonal antibody obtained by immunizing mice with an artificial antigen prepared by conjugating dichloroquinoline hapten with a carrier protein. The carrier protein is bovine serum albumin, ovalbumin, or keyhole hemocyanin. The dichloroquinoline hapten is prepared by introducing a straight-chain alkyl active arm containing 3 to 6 carbon atoms at the 3-position carboxyl group of dichloroquinoline acid through an amide bond. The anti-atrazine monoclonal antibody mentioned in step (2) is a monoclonal antibody obtained by immunizing mice with an artificial antigen prepared by conjugating atrazine hapten with a carrier protein. The carrier protein is bovine serum albumin, ovalbumin or keyhole hemocyanin. The atrazine hapten is prepared by introducing a straight-chain carboxyl alkyl active arm containing 3 to 6 carbon atoms at the 6-position chlorine atom of atrazine through nucleophilic substitution.

3. The preparation method according to claim 2, characterized in that, The colloidal gold nanoparticles mentioned in step (1) have a particle size of 15~45 nm and are coupled by electrostatic adsorption, which is carried out by adjusting the pH to within ±0.5 of the antibody isoelectric point; The time-resolved fluorescent microspheres mentioned in step (2) are polystyrene fluorescent microspheres coated with europium chelates, with a particle size of 100~300 nm, and the coupling method is covalent coupling by carbodiimide method or active ester method.

4. The preparation method according to any one of claims 1 to 3, characterized in that, The lyophilization protection solution described in step (3) contains 1-3% w / v sucrose, 0.5-2% w / v casein and 0.02-0.1% w / v Tween-20 in 0.02 mol / L Tris-HCl buffer solution, with a pH of 8.0-8.5; The mass ratio of the first labeled conjugate to the second labeled conjugate is 1:0.8~1.2; the fixation method is 2~5 μL / cm 2 After spraying the coating, vacuum dry at 35~40℃ for 1~2 hours.

5. The preparation method according to claim 4, characterized in that, In step (4), the coating concentration of detection line T1 is 0.5~2.0 mg / mL, the coating concentration of detection line T2 is 0.3~1.5 mg / mL, and the coating concentration of control line C is 0.5~2.0 mg / mL; after spraying, it is dried in a forced-air dryer at 35~40℃ for 0.5~1 hour.

6. The preparation method according to claim 5, characterized in that, The sample pad in step (5) is a glass fiber membrane, the binding pad is a polyester fiber membrane, the chromatography membrane is a nitrocellulose membrane, and the absorbent pad is cotton pulp paper or absorbent filter paper.

7. The preparation method according to claim 1, 3, 5 or 6, characterized in that, The developing solution in step (6) contains 1-2% w / v bovine serum albumin, 0.1-0.5% w / v Tween-20 in 0.02-0.05 mol / L phosphate buffer, with a pH of 7.2-7.

6.

8. An immunochromatographic reaction composition for the simultaneous detection of dichloroquinolinic acid and atrazine, prepared by the method according to any one of claims 1 to 7.

9. The use of the immunochromatographic reaction composition of claim 8 for the simultaneous detection of quinclorac and atrazine in the preparation of a kit for the simultaneous detection of quinclorac and atrazine residues in tobacco field soil or water.

10. A dual-mode immunochromatographic method for simultaneously qualitatively detecting dichloroquinoline acid and quantitatively detecting atrazine using the immunochromatographic reaction composition of claim 8 for the simultaneous detection of dichloroquinoline acid and atrazine, characterized in that, Includes the following steps: S1. Mix the sample solution to be tested with the developing solution at a volume ratio of 1:1~4 and then drop the mixture onto the sample pad. S2. The chromatography reaction is completed under capillary action, and the reaction time is 10-20 minutes. S3. Observe the red color development of colloidal gold at the detection line T1 under natural light to determine the presence or absence of dichloroquinoline acid. S4. Excitation with a 365 nm ultraviolet light source is used, and the fluorescence intensity at the detection line T2 is read at 615 nm using a fluorescence reader to achieve quantitative detection of atrazine. S5. Confirm the color development and fluorescence signal at control line C using both visual inspection and a fluorescence reader to determine the validity of the test.

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