Gold nanoflower immunochromatography test strip for detecting acifluorfen and preparation method thereof

Abstract

The invention discloses a gold nanoflower immunochromatography test strip for detecting acifluorfen and a preparation method of the gold nanoflower immunochromatography test strip. A Balb / c mouse murine hybridoma cell strain 4D5 is screened, the preservation number is CGMCC No.46561, the Balb / c mouse murine hybridoma cell strain 4D5 is used for secreting to obtain an anti-acifluorfen monoclonal antibody, the anti-acifluorfen monoclonal antibody is used for preparing a gold nanoflower immune probe, and the gold nanoflower immune probe is used for detecting the content of the acifluorfen. And finally, preparing the gold nanoflower immunochromatography test strip for detecting acifluorfen. The gold nanoflower immunochromatography test strip prepared by the invention is short in determination time, easy to operate and low in cost, has excellent specificity and sensitivity, and can be used for simply, conveniently and quickly detecting acifluorfen.

Description

Technical Field

[0001] This invention belongs to the field of rapid detection of pesticide residues, and relates to a gold nanoflower immunochromatographic test strip for detecting trifluralin and its preparation method. Background Technology

[0002] Trifluorocarbazide is a fluorinated diphenyl ether herbicide. Its aqueous solution can be used to control weeds in fields of crops such as rice, peanuts, soybeans, and corn. It has a significant weeding effect and is widely used in farmland, orchards, non-arable land, and around water bodies.

[0003] The widespread use of trifluralin can lead to significant residues in the environment. These residues can accumulate in animals and humans through the food chain, potentially causing disease or poisoning. This drug is currently classified as a possible human carcinogen. Therefore, strengthening the monitoring and detection of trifluralin residues and establishing rapid, simple, and accurate detection methods are of significant practical importance.

[0004] Literature review revealed that the main methods for detecting trifluralin are gas chromatography, gas chromatography-mass spectrometry, high-performance liquid chromatography (HPLC), and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). However, immunochromatographic detection methods based on anti-trifluralin monoclonal antibodies have not yet been reported. Gold nanoflowers (AuNF) are a type of nanoparticle with irregularly distributed spikes on their surface, resembling flowers. Compared to the most widely used colloidal gold particles, they have a larger specific surface area, allowing them to bind more antibodies and thus further improving the detection sensitivity of immunochromatographic test strips. Summary of the Invention

[0005] In view of the above background, the present invention aims to provide a gold nanoflower immunochromatographic test strip for detecting trifluralin and its preparation method.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A Balb / c mouse hybridoma cell line 4D5 that secretes an anti-trifluthrin monoclonal antibody is characterized in that: the Balb / c mouse hybridoma cell line 4D5 was deposited on July 3, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46561.

[0007] A monoclonal antibody against trifluralin, said monoclonal antibody being secreted by the Balb / c mouse hybridoma cell line 4D5 described above.

[0008] The above-mentioned Balb / c mouse hybridoma cell line 4D5 or anti-trifluralin monoclonal antibody is used in the preparation of products for detecting trifluralin.

[0009] A gold nanoflower immunochromatographic test strip for detecting trifluralin, comprising the following components: a plastic shell, a sample pad, an immunoprobe conjugation pad, an absorbent pad, a nitrocellulose membrane, and a PVC base plate; the immunoprobe conjugation pad is coated with a gold nanoflower immunoprobe labeled with an anti-trifluralin monoclonal antibody; the anti-trifluralin monoclonal antibody is secreted by Balb / c mouse hybridoma cell line 4D5; the Balb / c mouse hybridoma cell line 4D5 was deposited on July 3, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46561.

[0010] The preparation method of the above-mentioned gold nanoflower immunochromatographic test strip for detecting trifluralin includes the following steps: (1) Preparation of colloidal gold: Colloidal gold was prepared by citric acid reduction method; (2) Preparation of gold nanoflowers: Using the colloidal gold prepared in step (1) as gold seeds, gold nanoflowers were prepared by seed growth method. (3) Preparation of gold nanoflower immunoprobe labeled with anti-trifluthrin monoclonal antibody: Potassium carbonate and anti-trifluthrin monoclonal antibody were added to each 1 mL of gold nanoflower prepared in step (2). After the system was stabilized, BSA was added to block the unbound sites. Finally, PEG20000 was added, and after centrifugation and resuspending, gold nanoflower immunoprobe labeled with anti-trifluthrin monoclonal antibody was obtained. (4) Pretreatment of immunoprobe binding pads, sample pads and absorbent pads: Cut untreated immunoprobe binding pads into strips 1 cm wide, place them in a large petri dish, soak them in the pre-prepared blocking solution, and incubate them in a 37°C constant temperature incubator for 2 hours. After removing them from the incubator, filter out the blocking solution and continue to dry them in the incubator at 37°C. Then seal them at room temperature. For subsequent use, cut them to a length of 1 cm and a width of 4 mm. The composition of the blocking solution is: 5 wt% BSA + 1 vol% Tween-20. The sample pads do not need to be treated and should be cut to a length of 1 cm and a width of 4 mm for subsequent use. The absorbent pads do not need to be treated and should be cut to a length of 1.5 cm and a width of 4 mm for subsequent use. (5) Nitrocellulose membrane streaking: Take goat anti-mouse secondary antibody and streak C line on nitrocellulose membrane, take complete antigen AFFF-BSA synthesized by carbodiimide method and streak T line on the same nitrocellulose membrane. The distance between C line and T line on nitrocellulose membrane is 0.5cm. (6) Preparation of immune probe binding pad: The gold nanoflower immune probe labeled with anti-trifluthrin monoclonal antibody prepared in step (3) was dropped onto the pretreated immune probe binding pad and dried at 37°C for later use. (7) Assembly of immunochromatographic test strips: The nitrocellulose membrane after scribing in step (5), the immunoprobe binding pad prepared in step (6), the sample pad pretreated in step (4) and the absorbent pad are assembled on a PVC base plate; the immunoprobe binding pad and the sample pad are overlapped and pasted, with a 2mm gap between the same end, and the absorbent pad and the nitrocellulose membrane overlap by 2mm at both ends. The plastic outer shell is covered, dried and sealed, and stored at 4℃.

[0011] The preparation method of the anti-trifluthrin monoclonal antibody in step (3) is as follows: Balb / c mouse hybridoma cell line 4D5 in the logarithmic growth phase is injected into the peritoneal cavity of paraffin-sensitized Balb / c mice. When the mouse abdomen is distended, the ascites is extracted, separated and purified to obtain the anti-trifluthrin monoclonal antibody.

[0012] The detection principle of the gold nanoflower immunochromatographic test strip for detecting trifluralin described in this invention is as follows: When the test strip is first used for detection, the prepared gold nanoflower immunoprobe labeled with anti-trifluralin monoclonal antibody is first dropped onto the immunoprobe binding pad, and after drying, it is then followed according to the schematic diagram (…). Figure 8 The sample is assembled using the following method, and then dropped onto the sample pad. The liquid flows towards the absorbent paper via chromatography. When it reaches the T-line, any gold nanoflower immunoprobes that have not yet bound to the antigen in the sample bind completely to the antigen at the T-line and remain there, resulting in color development at the T-line. The liquid continues to flow forward. When it reaches the C-line, both those bound to the antigen and those not bound to the sample bind to the C-line until saturation. The bound gold nanoflower immunoprobes remain at the C-line, resulting in color development at the C-line. The complete colorimetric reaction takes 5-10 minutes.

[0013] The beneficial effects of this invention are: (1) This invention is the first to propose a gold nanoflower immunochromatographic test strip technology for detecting trifluralin by combining gold nanoflowers with anti-trifluralin monoclonal antibodies; (2) The lowest visual detection limit for the specific detection of trifluralin using the gold nanoflower immunochromatographic test strip provided by the present invention is 39.0625 ng / mL, the lowest instrument detection limit is 9.76563 ng / mL, and the detection time is only 10 min. Attached Figure Description

[0014] Figure 1 Affinity of anti-trifluthrin monoclonal antibody.

[0015] Figure 2 : Subtype of anti-trifluthrin monoclonal antibody.

[0016] Figure 3 Selection of the optimal concentration for the C line of the gold nanoflower immunochromatographic test strip.

[0017] Figure 4 Selection of the optimal concentration for the T-line of the gold nanoflower immunochromatographic test strip.

[0018] Figure 5 Determination of the optimal probe amount for gold nanoflower immunochromatographic test strips.

[0019] Figure 6 Specificity determination of gold nanoflower immunochromatographic test strips.

[0020] Figure 7 Sensitivity determination of gold nanoflower immunochromatographic test strips.

[0021] Figure 8 Schematic diagram of the structure of gold nanoflower immunochromatographic test strip. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments. The illustrative embodiments and descriptions herein are used to explain the present invention, but are not intended to limit the present invention.

[0023] Example 1: Preparation of mouse hybridoma cells 4D5 The specific preparation process of mouse hybridoma cells 4D5 is as follows: I. Complete Antigen Synthesis The complete antigen was synthesized using the carbodiimide method. The specific steps were as follows: First, 9.326 mg of acifluorofen (hereinafter referred to as AFFF; CAS number 50594-66-6), 30 mg of N-hydroxysuccinimide, and 68.35 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were dissolved in 2 mL of DMSO and reacted at 20 °C and 110 r / min for 12 h. Then, the reaction system was added dropwise to 7 mL of 5.71 mg / mL BSA solution (solvent: 0.01 M, pH 8.5 PBS) and reacted at 25 °C and 110 r / min for 6 h. The reaction system was then transferred to a dialysis bag and dialyzed at 4 °C and 0.01 M PBS (pH 7.4) for 3 days to obtain the complete antigen AFFF-BSA.

[0024] The preparation method of the complete antigen AFFF-OVA is basically the same as that of AFFF-BSA, except that bovine serum albumin (BSA) is replaced with chicken ovalbumin (OVA) in the preparation steps.

[0025] II. Animal Immunization AFFF-BSA was selected as the immunogen, diluted to an appropriate concentration with 0.01M PBS (pH 7.4), and then emulsified with an equal volume of Freund's complete adjuvant. This emulsion was used to initially immunize 6-week-old Balb / c mice that had been acclimatized to their environment for one week. Every two weeks, the same batch of mice was boosted with an emulsion containing the same immunogen and Freund's incomplete adjuvant. Starting with the second immunization, AFFF-OVA was used as the coating antigen for each immunization. The antibody titer and specific recognition ability against trifluralin in the mouse tail vein serum were measured using ELISA. When the serum titer reached 1:8000 and showed good specificity, the spleen of the mouse was harvested for cell fusion after the initial immunization.

[0026] ELISA specific steps: Add 100 μL of the coated antigen diluted to the optimal concentration to an ELISA plate and incubate at 37°C for 2 h or 4°C for 12 h. After washing, block with blocking buffer (200 μL / well) and incubate at 37°C for 2 h or 4°C for 12 h. Add diluted antibody or antiserum to the plate (it can be added directly when determining titer; when determining specificity, take an appropriate amount of hapten and antibody or antiserum dilution and incubate at 37°C for 1 h), and incubate at 37°C for 50 min. After washing, add 100 μL of diluted goat anti-mouse IgG-HRP and incubate at 37°C for 50 min. After washing, add 100 μL / well of chromogenic buffer and incubate at 37°C for 15 min. Finally, add 50 μL of 2M H2SO4 stop solution and measure the optical density (OD) of each well at 450 nm. 450nm ).

[0027] III. Cell Fusion Methods Mouse spleen cells were fused with mouse myeloma cells (SP2 / 0) using the PEG fusion method. After multiple subcloning screenings, a hybridoma cell line that stably secretes high-quality antibodies was obtained.

[0028] Required materials: alcohol swabs (5-6), Dissection board (1 piece) 5mL syringes (2) 50mL centrifuge tubes (4) 15mL centrifuge tubes (2) Three cutters and three tweezers (1 set) Cell culture dishes (3) Filters (2) Filter cartridge nozzles (1 box) 1mL pipette tips (≥10) Waste liquid tank.

[0029] On the day of cell fusion, prepare in advance one bottle of DMEM (>250mL), one tube of DMEM (approximately 40mL), one bottle of 20% FBS-DMEM with added HAT (>200mL, referred to as HAT medium), one mL of PEG1450 (all the above reagents have been preheated to 37°C before use), and 40°C sterile water (>300mL, placed in a beaker and preheated in a water bath). The specific steps for cell fusion are as follows: (1) Take out mice that have been immunized by shock from the mouse room, take blood from their eyeballs, place them in a preheated 37°C water bath, and then euthanize them by dislocation (after fusion, centrifuge them to obtain serum and store it at -20°C), soak them in 75% alcohol, and introduce them into the intercellular space; (2) Turn on the clean bench, light the alcohol lamp, tear open the outer packaging of the three sets of tweezers, absorb as much alcohol as possible from the mouse's fur, and fix the mouse on the dissection board. (3) After being burned by the flame of the alcohol lamp, the first set of scissors cuts open the fur, the second set of scissors opens the peritoneum, and the third set of scissors removes the spleen and removes the surface fat tissue. (4) Take a culture dish and use a 5mL syringe to draw a small amount of DMEM into the lid and the bottom of the dish respectively. Clean the spleen in the lid and then place the spleen in the bottom of the dish. (5) Open the 50mL centrifuge tube, place the filter screen at the centrifuge tube opening, and moisten the filter screen with a little DMEM. (6) Take a syringe, first puncture the spleen with the needle, then press the spleen fully with the handle to allow the spleen cells to enter the culture medium, use a 1mL pipette to draw DMEM through the filter and add it to the centrifuge tube, then repeat the process of taking DMEM to rinse the bottom of the dish several times, adding it through the filter to the centrifuge tube, and replenishing the culture medium to 40mL. (7) Centrifuge the spleen cell suspension at 1100 rpm and room temperature for 7 min (the centrifugation conditions in subsequent steps are the same as this step). At this time, take out SP2 / 0 from the incubator, add a little DMEM to a 50 mL centrifuge tube, blow off SP2 / 0 and add it to the centrifuge tube, and add DMEM to make up to 40 mL for later use. (8) After centrifugation, remove the spleen cells, discard the supernatant, resuspend the precipitate with DMEM and add fluid to 40 mL, and centrifuge the spleen cell suspension together with SP2 / 0 suspension. (9) After centrifugation, compare the amount of precipitate in the two tubes, discard the supernatant, take an appropriate amount of DMEM to resuspend the cells and count them. After calculating the number of cells, take an appropriate amount of SP2 / 0 suspension at a ratio of 1 / 3 to 1 / 10 and add it to the spleen cell suspension. Add DMEM to 40 mL, mix the cell suspension and centrifuge. (10) After centrifugation, place the beaker, PEG1450 and preheated single tube of DMEM into the clean bench, discard the mixed cell supernatant, tap the bottom of the centrifuge tube to disperse the cells, loosen the caps of the two tubes, place the cell precipitate in the centrifuge tube below the liquid surface in the beaker, add PEG1450 dropwise into the centrifuge tube, gently shake the bottom of the tube while adding, and add it within 1 minute, starting slowly and then quickly. (11) After adding PEG1450, let it stand in a water bath for 1 minute, then add DMEM in stages to the centrifuge tube, starting slowly and then quickly (add 1 mL in the first minute, 3 mL in the second minute, 5 mL in the third minute, and 10 mL in the fourth minute). After adding, add DMEM to 40 mL and let it stand at 37°C for 10 minutes. (12) After centrifugation, prepare several cell culture plates, take out the HAT medium for fusion and pour a small amount into the cell culture dish. After centrifugation, discard the supernatant, take an appropriate amount of HAT medium from the dish to resuspend the fused cell pellet, add the resuspended liquid to the whole bottle of HAT medium, let it stand and mix well, plate it, and place it in a carbon dioxide incubator at 37°C and 5% CO2 concentration for culture.

[0030] IV. Antibody Characterization After one week of culture, positive cell wells with high titers and good specificity were screened using ELISA. After multiple rounds of subclonal screening, a Balb / c mouse hybridoma cell line, 4D5, was obtained that stably secretes a high-affinity anti-trifluthrin monoclonal antibody.

[0031] Balb / c mouse hybridoma cell line 4D5 was deposited on July 3, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46561.

[0032] Example 2: Preparation of monoclonal antibody against trifluralin 1. Preparation of ascites (1) Take a 1mL sterile syringe and inject 500μL of paraffin into the peritoneal cavity of Balb / c mice around 12 weeks old to sensitize them. When injecting, insert the syringe vertically into the back of the mouse's peritoneal cavity. Disinfect the injection site with alcohol before and after injection to avoid bleeding. (2) One week after sensitization, take Balb / c mouse hybridoma cell line 4D5 that has just grown to the logarithmic phase and has filled about 80% of the culture dish. Discard the supernatant, use a pipette to take 1 mL of fresh 1640 medium to wash the cells from the bottom of the culture dish, mix them evenly, use a 1 mL sterile syringe to draw the cell suspension from one well of a 6-well plate, and then vertically inject it into the peritoneal cavity of the sensitized mouse. Note that the needle must be inserted into the peritoneal cavity accurately and without obstruction. Do not inject into the peritoneal tissue to avoid tumor formation and waste of the mouse. (3) About a week after injecting cells into the peritoneal cavity of mice, under normal circumstances, the abdominal distension of mice can be clearly observed, and the mice are listless and have messy fur. At this time, it is necessary to start preparing to collect ascites. (4) Prepare sterile injection needles, 1.5mL EP tubes, alcohol swabs, etc. Hold the mouse with one hand and wipe the blood collection site with alcohol swabs. With the other hand, carefully insert the sterile injection needle into the side of the abdominal cavity. Place the sterile injection needle against the EP tube to collect the ascites. You can see the ascites flowing into the tube along the sterile injection needle. The color is reddish, which may be due to excessive ascites production and hemolysis. However, it does not affect the properties of the antibodies in the ascites. When collecting ascites, the mouse should be fixed to prevent the mouse from struggling and causing ascites loss. Each mouse can only collect about 1.5mL of ascites at a time. After the tail blood is collected, put the mouse back into the cage. Ascites can continue to be produced. Ascites can be collected again the next day until the mouse dies. (5) After the mouse ascites fluid was balanced, it was centrifuged at 12000 rpm for 20 min in a 4℃ centrifuge. After taking it out, it can be observed that the liquid in the centrifuge tube is divided into three layers. The top layer is a layer of fat. Carefully take out the middle layer of ascites fluid, put it into a new 1.5mL EP tube, label it, and store it in a -20℃ refrigerator.

[0033] 2. Ascites purification Ascites fluid was diluted 1:10 with equilibration buffer (Na2HPO4 3.5814 g / L, NaCl 4.383 g / L; pH=7.0). The diluted ascites fluid was filtered through a 0.45 μm filter and then passed through a Protein G affinity chromatography column. The procedure was performed according to the instructions for commercial Protein G affinity chromatography. The purified antibody was dialyzed in a dialysis bag with PBS (NaCl 8.0 g / L, KCl 0.2 g / L, Na2HPO4 1.44 g / L, KH2PO4 0.24 g / L; pH=7.4) for 3 days and ultrapure water for 1 day. Finally, it was concentrated with PEG20000 to obtain anti-trifluthrin monoclonal antibody. The anti-trifluthrin monoclonal antibody was stored at -80℃ for later use.

[0034] like Figure 1 and Figure 2As shown, the anti-trifluthrin monoclonal antibody secreted by the Balb / c mouse hybridoma cell line 4D5 has a heavy chain isoform of IgG1 and a light chain isoform of Kappa, with an affinity constant Kaff = 5.88 × 10⁻⁶. 9 L / mol, which is a high-affinity antibody.

[0035] Example 3: Preparation of gold nanoflower immunoprobes 1. Preparation of colloidal gold Dissolve 1 g of chloroauric acid in 100 mL of deionized water to obtain a 1 wt% chloroauric acid aqueous solution; take 1 mL of the 1 wt% chloroauric acid aqueous solution and add it to 100 mL of deionized water, mix well and heat. When the system is about to boil, quickly add 1.5 mL of 1 wt% trisodium citrate aqueous solution and continue heating until the system turns purple-red. Heat for another 3-5 minutes to allow the system to stabilize fully and then let it cool naturally to room temperature. Make up to 100 mL with deionized water to obtain a colloidal gold solution (DLS (average particle size 25.3 nm (PDI = 0.386)), Zeta potential (-18.1 ± 2.1 mV)).

[0036] 2. Preparation of gold nanoflowers Add 750 μL of 1 wt% chloroauric acid aqueous solution to 100 mL of deionized water and stir slowly. Then add 500 μL of the colloidal gold solution prepared in the previous step and 300 μL of 1 wt% trisodium citrate aqueous solution and mix well. Adjust the pH with 1 M sodium hydroxide aqueous solution to control the pH to 7.0-10.0 before and after the reaction. After waiting for 30 seconds, add 1 mL of 0.03 M hydroquinone aqueous solution and increase the stirring speed. Stir until the color of the system changes from clear pale yellow to clear dark blue. After it is completely stable, the gold nanoflower solution (DLS (average particle size 65.9 nm, Zeta potential (-17.7 ± 0.6 mV)) is obtained and stored at 4 °C for later use.

[0037] 3. Preparation of gold nanoflower immunoprobe labeled with anti-trifluthrin monoclonal antibody Take 10 mL of the gold nanoflower solution prepared in the previous step, and add 25 μL of 0.1 M potassium carbonate and 60 μL of 0.5 mg / mL anti-trifluthrin monoclonal antibody sequentially under ice bath conditions. Stir continuously for 45 min, add 2.5 mL of 5 wt% BSA aqueous solution, and stir continuously for 30 min. Finally, add 1.388 mL of 1 wt% PEG20000 aqueous solution and stir continuously for 30 min to obtain the gold nanoflower immunoprobe solution. Centrifuge at low speed for 15 min at room temperature and discard the precipitate to remove a small portion of unstable probes. Centrifuge again at 12000 r / min for 40 min at 4 °C and discard the supernatant. Resuspend each 1 mL of the obtained precipitate in 20 μL of resuspension buffer (0.01 M Tris-HCl + 1 wt% BSA + 10 wt% trehalose, pH = 8.5) to obtain the gold nanoflower immunoprobe solution labeled with anti-trifluthrin monoclonal antibody. Store at 4 °C for later use.

[0038] Example 4: Preparation of gold nanoflower immunochromatographic test strips 1. Pretreatment of immunoprobe binding pads, sample pads, and absorbent pads Untreated immunoprobe binding pads (purchased from Shanghai Jieyi Biotechnology, model: GL0194) were cut into 1cm wide strips, placed in a large petri dish, soaked in pre-prepared blocking solution, and incubated at 37℃ for 2 hours. After removal from the incubator, the blocking solution was filtered off, and the pads were dried at 37℃ (checked every 1 hour until completely dry). After drying, they were sealed at room temperature. For subsequent use, the pads were cut to 1cm in length and 4mm in width. The blocking solution consisted of 5wt% BSA + 1vol% Tween-20. Sample pads (purchased from Shanghai Jieyi Biotechnology, model: GL-b01) did not require treatment and were cut to 1cm in length and 4mm in width for subsequent use. Absorbent pads (purchased from Shanghai Jieyi Biotechnology, model: H5076) did not require treatment and were cut to 1.5cm in length and 4mm in width for subsequent use.

[0039] 2. Selection of the optimal concentration for line C of the gold nanoflower immunochromatographic test strip 1 mg / mL of goat anti-mouse secondary antibody (purchased from Beijing Bio-Sen Biotechnology Co., Ltd., product number: bs-0293G) was diluted with PBS (0.01M) at pH 7.4 to five concentrations: 1 mg / mL, 0.8 mg / mL, 0.6 mg / mL, 0.4 mg / mL, and 0.2 mg / mL. 10 μL of each concentration was taken and streaked onto a nitrocellulose membrane as a C-line. When streaking with a gold sputtering instrument, the streaking speed was 0.8 μL / cm. After the nitrocellulose membrane with the C line drawn was dried in a 37°C incubator for 10 min, it was cut into strips 4 mm wide. Next, 2.5 μL of gold nanoflower immunoprobe solution labeled with anti-trifluthrin monoclonal antibody was added to each of the five probe binding pads. After drying at 37°C, the gold nanoflower immunochromatographic test strips were assembled (assembly method described in the invention description). 100 μL of 0.01M PBS (pH = 7.4) was slowly added to the sample pad, and the mixture was allowed to react at room temperature for 10 min. The color intensity of the C line on the test strips was observed. Finally, based on observation, 200 μg / mL was selected as the optimal concentration for drawing the C line. Figure 3 ).

[0040] 3. Selection of the optimal concentration of the T line on gold nanoflower immunochromatographic test strips The complete antigen AFFF-OVA was diluted with 0.01M PBS (pH=7.4) to five concentrations: 800 μg / mL, 600 μg / mL, 400 μg / mL, 200 μg / mL, and 100 μg / mL. 10 μL of each concentration was taken and streaked onto a nitrocellulose membrane as a T-line. When streaking with a gold sputtering instrument, the streaking speed was 0.8 μL / cm. After the nitrocellulose membrane with the T line drawn was dried in a 37°C incubator for 10 min, it was cut into strips 4 mm wide. Next, 2.5 μL of a gold nanoflower immunoprobe solution labeled with anti-trifluthrin monoclonal antibody was added to each of the five probe binding pads. After drying at 37°C, the gold nanoflower immunochromatographic test strips were assembled (assembly method described in the invention description). 100 μL of 0.01 M PBS (pH = 7.4) was slowly added to the sample pad, and the mixture was allowed to react at room temperature for 10 min. The color intensity of the T line on the test strips was observed. Finally, based on observation, 0.2 mg / mL was selected as the optimal T line concentration. Figure 4 ).

[0041] 4. Determination of the optimal probe amount for gold nanoflower immunochromatographic test strips Based on the optimized C-line streaking concentration (200 μg / mL) and T-line streaking concentration (0.2 mg / mL) mentioned above, streaks were simultaneously applied to nitrocellulose membranes. After streaking, the nitrocellulose membranes were dried in a 37°C incubator for 10 min, then cut into strips 4 mm wide. Next, 1 μL, 1.5 μL, 2 μL, 2.5 μL, and 3 μL of anti-trifluthrin monoclonal antibody-labeled gold nanoflower immunoprobe solution were added to five groups of probe binding pads, respectively. After drying at 37°C, the gold nanoflower immunochromatographic test strips were assembled (assembly method described in the invention description). 100 μL of 0.01 M PBS (pH = 7.4) was slowly added to the sample pads, and the mixture was allowed to react at room temperature for 10 min. The color intensity of the C and T lines on the test strips was observed. Finally, through observation, 2.5 μL was selected as the optimal sample loading volume of the anti-trifluthrin monoclonal antibody-labeled gold nanoflower immunoprobe solution. Figure 5 ).

[0042] Example 5: Performance Verification of Gold Nanoflower Immunochromatographic Test Strips 1. Specificity determination of gold nanoflower immunochromatographic test strips Based on the optimized C-line streaking concentration (200 μg / mL) and T-line streaking concentration (0.2 mg / mL) mentioned above, streaks were simultaneously applied to a nitrocellulose membrane. The streaked nitrocellulose membrane was then dried in a 37°C incubator for 10 min before being cut into strips. Next, 2.5 μL of a gold nanoflower immunochromatographic probe solution labeled with anti-trifluthrin monoclonal antibody was added to the probe binding pad. After drying at 37°C, the gold nanoflower immunochromatographic test strip was assembled (assembly method described in the invention description). Bifenox, Flunixin, Chlomethoxyfen, Nitrofen, Haloxyfop, and AFFF were diluted to a final concentration of 40 μg / mL using 0.01 M PBS (pH = 7.4). 0.01 M PBS (pH = 7.4) was used as a negative control. 100 μL of each solution was added to the sample pad of the test strip, and the strips were incubated at room temperature for 10 min. The disappearance of the T line was observed to determine the specificity of the test strip. The results showed that the test strip only exhibited a competitive reaction with AFFF, resulting in the disappearance of the T line, while no significant cross-reactivity was observed with other similar compounds, indicating good specificity of the test strip. Figure 6 ).

[0043] 2. Sensitivity determination of gold nanoflower immunochromatographic test strips Based on the optimized C-line streaking concentration (200 μg / mL) and T-line streaking concentration (0.2 mg / mL) mentioned above, streaks were simultaneously applied to a nitrocellulose membrane. After streaking, the nitrocellulose membrane was dried in a 37°C incubator for 10 min and then cut into strips. Next, 2.5 μL of gold nanoflower immunoprobe solution labeled with anti-trifluthrin monoclonal antibody was added to the probe binding pad. After drying, the gold nanoflower immunochromatographic test strip was assembled (assembly method is described in the invention description). Trifluralin (AFFF) was diluted with 0.01M PBS (pH=7.4) to final concentrations of 20000 ng / mL, 10000 ng / mL, 5000 ng / mL, 2500 ng / mL, 1250 ng / mL, 625 ng / mL, 312.5 ng / mL, 156.25 ng / mL, 78.125 ng / mL, 39.0625 ng / mL, 19.53125 ng / mL, 9.76563 ng / mL, 4.88281 ng / mL, and 2.44141 ng / mL. Simultaneously, 0.01M PBS was used. PBS (pH=7.4) was used as a negative control. Then, 100 μL of each solution was added to the sample pad of the test strip and allowed to react at room temperature for 10 min. The disappearance of the T line on the test strip was observed. The color intensity of the T line and C line on the test strip was detected by an immunochromatographic quantitative analyzer, and the T / C value was calculated to determine the detection range of the test strip. The results showed that when the concentration of trifluralin in the sample solution was 20,000 ng / mL, the T line disappeared significantly, indicating that the anti-trifluralin monoclonal antibody in the gold nanoparticle immunoprobe was completely bound to the trifluralin in the sample solution. With halving of the concentration, the T line gradually darkened. When the concentration of trifluralin in the sample solution was 39.0625 ng / mL, the T line was still clearly lighter, indicating that the visual detection limit of this immunochromatographic test strip was 39.0625 ng / mL. Quantitative immunochromatographic analysis showed that when the concentration of trifluralin in the sample solution was 9.76563 ng / mL, the T line intensity was significantly lower than the C line, and the corresponding T / C value was also significantly lower than the T / C value of the negative control. Therefore, the instrument detection limit of this immunochromatographic test strip was 9.76563 ng / mL. Figure 7 ).

[0044] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A Balb / c mouse hybridoma cell line 4D5 that secretes an anti-trifluthrin monoclonal antibody, characterized in that: The Balb / c mouse hybridoma cell line 4D5 was deposited on July 3, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46561.

2. A monoclonal antibody against trifluralin, characterized in that: The anti-trifluthrin monoclonal antibody is secreted by the Balb / c mouse hybridoma cell line 4D5 as described in claim 1.

3. The use of the Balb / c mouse hybridoma cell line 4D5 as described in claim 1 or the anti-trifluthrin monoclonal antibody as described in claim 2 in the preparation of products for detecting trifluthrin.

4. A gold nanoflower immunochromatographic test strip for detecting trifluralin, characterized in that: The test strip includes a gold nanoflower immunoprobe, which is formed by electrostatic adsorption of gold nanoflower and anti-trifluthrin monoclonal antibody; the anti-trifluthrin monoclonal antibody is secreted by the Balb / c mouse hybridoma cell line 4D5 as described in claim 1.

5. The gold nanoflower immunochromatographic test strip according to claim 4, characterized in that: The preparation method of the anti-trifluthrin monoclonal antibody includes the following steps: injecting Balb / c mouse hybridoma cell line 4D5 into Balb / c mice that have been pre-sensitized with paraffin, collecting ascites fluid, purifying it, and obtaining the anti-trifluthrin monoclonal antibody.

6. The method for preparing the gold nanoflower immunochromatographic test strip according to any one of claims 4 to 5, characterized in that: Includes the following steps: (1) Preparation of colloidal gold: Colloidal gold was prepared by citric acid reduction method; (2) Preparation of gold nanoflowers: Using the colloidal gold prepared in step (1) as gold seeds, gold nanoflowers were prepared by seed growth method; (3) Preparation of gold nanoflower immunoprobe labeled with anti-trifluthrin monoclonal antibody: Potassium carbonate and anti-trifluthrin monoclonal antibody were added to each 1 mL of gold nanoflower prepared in step (2). After the system stabilized, BSA was added to block the unbound sites. Finally, PEG20000 was added, and after centrifugation and resuspending, gold nanoflower immunoprobe labeled with anti-trifluthrin monoclonal antibody was obtained. (4) Pretreatment of immunoprobe binding pads, sample pads and absorbent pads: Cut the untreated immunoprobe binding pads into strips 1 cm wide, place them in a large petri dish, soak them in the pre-prepared blocking solution, and incubate them in a 37°C constant temperature incubator for 2 hours. After removing them from the incubator, filter out the blocking solution and continue to dry them in the incubator at 37°C. Then seal them at room temperature. For subsequent use, cut them to a length of 1 cm and a width of 4 mm. The sample pads do not need to be treated. For subsequent use, cut them to a length of 1 cm and a width of 4 mm. The absorbent pads do not need to be treated. For subsequent use, cut them to a length of 1.5 cm and a width of 4 mm. (5) Streaking on nitrocellulose membrane: Use goat anti-mouse secondary antibody to streak C lines on nitrocellulose membrane, and use complete antigen AFFF-BSA to streak T lines on the same nitrocellulose membrane; the distance between C lines and T lines on the nitrocellulose membrane is 0.5 cm. (6) Preparation of immune probe binding pad: The gold nanoflower immune probe labeled with anti-trifluthrin monoclonal antibody prepared in step (3) was dropped onto the pretreated immune probe binding pad and dried at 37°C for later use. (7) Assembly of immunochromatographic test strips: The nitrocellulose membrane after scribing in step (5), the immunoprobe binding pad prepared in step (6), the sample pad pretreated in step (4) and the absorbent pad are assembled on the base plate; the immunoprobe binding pad and the sample pad are overlapped and pasted, with a 2mm gap between the same end, and the absorbent pad and the nitrocellulose membrane overlap by 2mm at both ends. The plastic outer shell is covered, dried and sealed, and stored at 4℃.

7. The preparation method according to claim 6, characterized in that: The sealing solution consists of 5% BSA + 1% Tween-20.

8. The preparation method according to claim 6, characterized in that: The concentration indicated by line C is 0.2 mg / mL.

9. The preparation method according to claim 6, characterized in that: The concentration indicated by the T line is 0.2 mg / mL.

10. The preparation method according to claim 6, characterized in that: The amount of gold nanoflower immunoprobe labeled with anti-trifluthrin monoclonal antibody added was 2.5 μL.

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