Immunochromatography detection kit and detection method for pesticide residues in food

Abstract

The invention discloses an immunochromatography detection kit for pesticide residues in food and a detection method, and relates to the technical field of food detection.The kit comprises an immunochromatography test strip and a sample extracting solution, the immunochromatography test strip is formed by sequentially overlapping and pasting a PVC bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad, the lap joint width of the adjacent parts is 2-3mm; the synergistic effect of all parts of the immunochromatography test strip is optimized, a detection system with high targeting property is constructed, target pesticide residues are accurately captured and responded by virtue of specific recognition of a target triggering type hydrogel switch, the matrix influence is reduced by matching with a special sample extracting solution, and meanwhile, the molecular recognition and immunochromatography technologies are integrated, so that the detection efficiency is greatly improved. The target trigger type hydrogel switch has the advantages of high selectivity, stable signal marker activity, reasonable layout of a detection line and a quality control line, simplified pretreatment, simple operation, meeting of multi-scene batch detection, and provision of support for food quality safety supervision.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, specifically to an immunochromatographic assay kit and method for detecting pesticide residues in food. Background Technology

[0002] With the rapid development of the food industry, food faces the risk of pesticide residue contamination during production, processing, and transportation. Excessive pesticide residues can directly threaten human health and cause a series of health problems. Therefore, pesticide residue detection in food has become a key link in ensuring food safety. Currently, pesticide residue detection technologies need to meet the requirements of accurate identification and rapid screening. They need to be adaptable to different types of food detection scenarios and effectively capture trace amounts of pesticide residues. Immunochromatography technology has gained widespread attention in the field of rapid on-site detection due to its advantages such as simple operation, short detection cycle, and no need for complex instruments. Its core lies in the identification of target analytes through specific antigen-antibody reactions. Combined with signal labeling systems, it completes qualitative or semi-quantitative detection and has become an indispensable technical means in food quality and safety supervision.

[0003] Traditional pesticide residue detection technologies have many limitations and cannot fully meet actual detection needs. Although some laboratory detection methods have high accuracy, their operation procedures are cumbersome, the detection cycle is long, and they rely on specialized equipment and technicians, making it impossible to achieve rapid on-site screening. Conventional immunochromatographic detection technology often faces problems such as insufficient specificity and susceptibility to sample matrix interference, leading to a decrease in the accuracy of detection results. At the same time, signal markers are prone to non-specific binding or loss of activity, affecting detection sensitivity and stability, making it difficult to effectively detect trace pesticide residues. In addition, the sample pretreatment steps of traditional detection methods are complicated, which not only increases the difficulty of operation but may also lead to the loss of target analytes, further limiting detection efficiency and applicability, and making them unsuitable for large-scale, multi-scenario food pesticide residue screening needs. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an immunochromatographic detection kit and method for pesticide residues in food. The kit includes an immunochromatographic test strip composed of a PVC base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad, along with a special sample extraction solution. The conjugate pad is loaded with a target-triggered hydrogel switch, which is formed by cross-linking modified polymer chains and internally embedding colored latex microspheres. During detection, the sample is extracted and centrifuged to obtain the test solution. After being added, the test solution is wetted by capillary action to the target-triggered hydrogel switch. The target pesticide residue will destroy the cross-linked structure and release the signal marker. The result is then determined by the color development of the detection line and control line on the nitrocellulose membrane.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: On the one hand, an immunochromatographic detection kit for pesticide residues in food, the kit comprising: an immunochromatographic test strip and a sample extraction solution, wherein the immunochromatographic test strip is composed of a PVC base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad that are sequentially overlapped and pasted together, with the overlap width of adjacent components being 2-3 mm;

[0006] The sample pad is a glass fiber membrane that has been soaked in a sample pad treatment solution and then dried. The sample pad treatment solution contains 0.01 mol / L Tris-HCl buffer, 0.5% Tween-20 by volume, and 1% bovine serum albumin by mass.

[0007] The binding pad is a polyester membrane loaded with a target-triggered hydrogel switch. The target-triggered hydrogel switch is a molecularly imprinted hydrogel with the target pesticide molecule as the key component of the competitive cross-linking bridge, and is internally embedded with colored latex microsphere signal markers.

[0008] The target-triggered hydrogel switch is formed by copolymerizing polyacrylamide polymer chains modified with pesticide haptens and polyacrylamide polymer chains modified with pesticide-specific antibodies, under the condition of simultaneously adding target pesticide molecules and a bifunctional cross-linking agent that can compete with the pesticide molecule for binding to the antibody, to form a three-dimensional network structure; wherein, during the polymerization process, the bifunctional cross-linking agent preferentially binds to the antibody to form the main cross-linking bridge, and the target pesticide molecule, as a competitive component, partially replaces the bifunctional cross-linking agent in cross-linking;

[0009] The nitrocellulose membrane is coated with a detection line and a control line in parallel. The detection line is coated with goat anti-mouse IgG antibody, and the control line is coated with rabbit anti-bovine serum albumin antibody.

[0010] The sample extraction solution was a 0.02 mol / L phosphate buffer solution with 15% acetonitrile (v / v) and 5% sodium chloride (w / w).

[0011] Furthermore, the PVC base plate has a thickness of 0.5-0.6 mm and a size of 60 mm × 4 mm. The sample pad is made of glass fiber membrane with a pore size of 15-20 μm and a size of 10 mm × 4 mm. The sample pad is soaked in the sample pad treatment solution for 30 min and then dried in a constant temperature oven at 37°C for 2 h.

[0012] Furthermore, the bonding pad is made of a polyester film with a thickness of 0.18-0.2 mm and a pore size of 8-10 μm, and a size of 8 mm × 4 mm. The polyacrylamide polymer chain is formed by cross-linking and polymerization of acrylamide monomer and N,N'-methylenebisacrylamide, with a molar ratio of N,N'-methylenebisacrylamide to acrylamide of 1:20.

[0013] Furthermore, the amount of pesticide hapten added to the modified polyacrylamide polymer chain is 0.5% by mass, and the amount of pesticide-specific antibody added to the modified polyacrylamide polymer chain is 0.5% by mass. The two polymer chain solutions are mixed at a volume ratio of 1:1.

[0014] Furthermore, the colored latex microspheres have a particle size of 200-300 nm, the amount of colored latex microspheres added is 5% of the volume of the hydrogel crosslinking reaction liquid, the hydrogel crosslinking reaction is carried out at 25°C for 3 hours, the target-triggered hydrogel switch suspension is loaded onto the surface of the polyester film at a spraying amount of 2 μL / cm², and dried in a constant temperature oven at 37°C for 12 hours.

[0015] Furthermore, the nitrocellulose membrane uses a substrate with a pore size of 0.22 μm and a size of 25 mm × 4 mm. The detection line coating solution is goat anti-mouse IgG antibody diluted in 0.01 mol / L phosphate buffer at pH 7.2, with an antibody concentration of 1 mg / mL. The control line coating solution is rabbit anti-bovine serum albumin antibody diluted in 0.01 mol / L phosphate buffer at pH 7.2, with an antibody concentration of 1.5 mg / mL.

[0016] Furthermore, the spraying speed of the detection line and the quality control line is 5 cm / s, the spraying volume is 1 μL / cm, the distance between the two lines is 5 mm, and the detection line is 10 mm away from the edge of the conjugate pad.

[0017] On the other hand, a method for detecting pesticide residues in food using an immunochromatographic assay kit, the specific steps of which are as follows:

[0018] S1, Sample pretreatment: Weigh 2-5g of the pretreated food sample to be tested into a 50mL centrifuge tube, add 10-20mL of sample extraction solution, vortex for 2-3min, centrifuge at 8000-10000r / min for 5min, and take the clear liquid at the top of the centrifuge tube as the sample test solution.

[0019] S2, sample addition to start chromatography: Add 100-150μL of sample solution to the center of the sample pad of the immunochromatographic test strip. The sample solution will migrate towards the absorbent pad under capillary action, flow through the conjugate pad and wet the target trigger hydrogel switch.

[0020] S3, Target Trigger Signal Release: When the target pesticide residue is present in the sample test solution, free pesticide molecules compete with pesticide molecules in the cross-linking bridges formed by the target pesticide molecules in the hydrogel network to bind to pesticide-specific antibodies on the polymer chain, destroying the cross-linking bridge structure and causing the three-dimensional hydrogel network to disintegrate, releasing the embedded colored latex microspheres. The latex microspheres migrate towards the nitrocellulose membrane with the chromatography solvent. When there is no target pesticide residue in the sample test solution, the hydrogel cross-linking bridge structure remains intact, and the colored latex microspheres are locked inside the hydrogel network.

[0021] S4, Signal capture reaction: Colored latex microspheres migrate with the chromatography solvent and enter the nitrocellulose membrane. They are captured and enriched by goat anti-mouse IgG antibody at the detection line to form a band. The universal labeling component in the chromatography solvent binds to rabbit anti-bovine serum albumin antibody at the control line to form a band. After signal capture is completed, the test strip is left to stand for 10-15 minutes.

[0022] S5, Result Judgment: Observe the color development of the detection line and control line on the nitrocellulose membrane. When both the control line and the detection line develop color, the target pesticide residue content in the sample is determined to be higher than the minimum detection limit. When only the control line develops color and the detection line does not develop color, the target pesticide residue content in the sample is determined to be lower than the minimum detection limit. When the control line does not develop color, the test result is determined to be invalid.

[0023] Furthermore, the pretreatment method involves washing away surface dirt and impurities and then chopping the sample to a particle size of less than 5 mm. For grain samples, the pretreatment method involves grinding them into powder. The vortex oscillation speed is 2000-2500 r / min. The centrifugation equipment used is a high-speed refrigerated centrifuge, and the temperature is controlled at 4-8℃ during the centrifugation process.

[0024] Furthermore, during the signal capture process, the test strip is kept horizontally, and the resting time is adjusted according to the ambient temperature: 15 minutes at 10-20℃, 12 minutes at 21-30℃, and 10 minutes at 31-40℃.

[0025] Compared with existing technologies, this immunochromatographic assay kit and method for detecting pesticide residues in food have the following advantages:

[0026] I. This invention optimizes the synergistic effect of each component of the immunochromatographic test strip to construct a highly targeted detection system. Relying on the specific recognition mechanism of the target-triggered hydrogel switch, it achieves precise capture and signal response of target pesticide residues, avoiding interference caused by non-specific binding. Utilizing the structural characteristics of the hydrogel network, it stably locks the signal marker when there is no target and rapidly disintegrates to release the signal when the target is present, significantly improving the sensitivity and specificity of detection. Combined with a special sample extraction solution, it efficiently separates the target components in the sample, reduces matrix influence, and makes the detection results more reliable. The entire detection process does not require complex instrumentation, is simple to operate, and has a rapid response, meeting the needs of rapid on-site detection and providing an efficient solution for pesticide residue screening in food.

[0027] II. This invention integrates the advantages of molecular recognition and immunochromatography technologies to establish an integrated detection mode. The target-triggered hydrogel switch is formed by the cross-linking of two modified polymer chains. Utilizing the competitive binding of the target pesticide molecule and the bifunctional cross-linking agent, the detection system is endowed with high selectivity, producing a specific response only to the target pesticide residue. The signal marker is embedded inside the hydrogel, effectively avoiding activity loss during storage and transportation, and extending the stability period of the reagent kit. The reasonable layout of the detection line and control line enables intuitive judgment of the results. At the same time, the setting of the control line ensures the effectiveness of the detection process. By simplifying the sample pretreatment steps, shortening the detection cycle, and lowering the operation threshold, it is suitable for batch detection in different scenarios, providing convenient, stable, and accurate technical support for food quality and safety supervision.

[0028] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0030] Figure 1 Flowchart of an immunochromatographic method for detecting pesticide residues in food;

[0031] Figure 2 Flowchart for the preparation of an immunochromatographic assay kit for pesticide residues in food. Detailed Implementation

[0032] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0033] Example 1:

[0034] Preparation of an immunochromatographic assay kit for detecting pesticide residues in food.

[0035] Reagent and material preparation:

[0036] Reagents included 0.01 mol / L Tris-HCl buffer, Tween-20, bovine serum albumin, acrylamide monomer, N,N'-methylenebisacrylamide, target pesticide hapten, target pesticide-specific antibody, bifunctional cross-linking agent, colored latex microspheres, goat anti-mouse IgG antibody, rabbit anti-bovine serum albumin antibody, 0.02 mol / L phosphate buffer, 0.01 mol / L phosphate buffer, acetonitrile, and sodium chloride. All reagents were of analytical grade. Materials included a PVC base plate, glass fiber membrane, polyester membrane, nitrocellulose membrane, and absorbent pad, all of which were dedicated consumables for immunochromatography. Figure 2 As shown.

[0037] Sample pad preparation:

[0038] Sample pad treatment solution preparation: Measure an appropriate amount of 0.01 mol / L Tris-HCl buffer, add Tween-20 to achieve a volume fraction of 0.5%, and add bovine serum albumin to achieve a mass fraction of 1%. Stir continuously with a magnetic stirrer for 30 minutes until the bovine serum albumin is completely dissolved. Let stand at room temperature for 2 hours for later use. Immersion and drying: Use a utility knife to cut the glass fiber membrane into 10mm × 4mm pieces. Immerse the cut glass fiber membrane completely in the prepared sample pad treatment solution, ensuring that every part of the membrane is fully in contact with the treatment solution. After immersion for 30 minutes, remove it with tweezers and place it in a 37℃ constant temperature oven to dry for 2 hours. After removal, immediately place it in a sealed bag and store it in a desiccator for later use.

[0039] Binding pad preparation:

[0040] Polyacrylamide polymer chain synthesis: Two monomers, N,N'-methylenebisacrylamide and acrylamide, were weighed according to a molar ratio of 1:20 and added to deionized water. After stirring and dissolving, ammonium persulfate was added as an initiator, with an initiator dosage of 0.5% of the total monomer mass. Tetramethylethylenediamine was then added as an accelerator, with an accelerator dosage of 0.1% of the total monomer mass. The reaction was stirred at room temperature for 4 hours to prepare polyacrylamide polymer chain solutions modified with pesticide haptens and pesticide-specific antibodies, respectively. The mass fraction of the pesticide hapten in its corresponding polymer chain solution was 0.5%, and the mass fraction of the pesticide-specific antibody in its corresponding polymer chain solution was also 0.5%. The two polymer chain solutions were mixed uniformly at a volume ratio of 1:1. Preparation of target-triggered hydrogel switch: The target pesticide molecule and a bifunctional crosslinking agent were added to the above-mixed polymer chain solution. The amount of the bifunctional crosslinking agent added was 2% of the total mass of the polymer chain solution. Simultaneously, colored latex microspheres were added, with the amount of colored latex microspheres being 5% of the volume of the hydrogel crosslinking reaction solution. The mixture was placed in a constant temperature water bath at 25℃ and reacted continuously for 3 hours, stirring every 30 minutes to form a target-triggered hydrogel switch suspension. Loading and drying: The polyester film was cut into 8mm × 4mm pieces using a utility knife. The target-triggered hydrogel switch suspension was uniformly loaded onto the surface of the polyester film using a small sprayer at a spray rate of 2μL / cm². During spraying, the distance between the sprayer and the polyester film was maintained at 10cm. After spraying, the polyester film was placed in a constant temperature oven at 37℃ and dried for 12 hours. After drying, it was removed, sealed, and stored for later use.

[0041] Preparation of nitrocellulose membranes:

[0042] Preparation of coating solutions: Take 0.01 mol / L phosphate buffer (pH 7.2), add goat anti-mouse IgG antibody, and stir to dilute to an antibody concentration of 1 mg / mL as the detection line coating solution; similarly, take 0.01 mol / L phosphate buffer (pH 7.2), add rabbit anti-bovine serum albumin antibody, and stir to dilute to an antibody concentration of 1.5 mg / mL as the control line coating solution. Both coating solutions should be stored at 4°C for no more than 72 hours after preparation. Coating process: Cut the nitrocellulose membrane into 25mm×4mm pieces using a utility knife. Fix the cut nitrocellulose membrane on the worktable of the spraying machine. Set the spraying speed of the spraying machine to 5cm / s and the spray volume to 1μL / cm. Apply the coating test line and quality control line parallel to each other on the membrane, with a spacing of 5mm between the two lines. The test line should be 10mm away from one edge of the membrane. After coating, place the nitrocellulose membrane in a 37℃ oven and dry for 12 hours. After drying, remove it and place it in a dry, sealed bag for later use.

[0043] Test strip assembly:

[0044] Take a PVC base plate with a thickness of 0.5-0.6mm and a size of 60mm×4mm. Adhere the PVC base plate, sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad in that order, overlapping each other. The overlap width between adjacent components should be controlled at 2-3mm. Use a dedicated chromatography test strip adhesive to ensure that all components are tightly connected, without gaps or warping. After assembly, use a dedicated paper cutter to cut it into individual test strips. Seal each test strip individually with a desiccant inside and store in a dry environment.

[0045] Preparation of sample extraction solution:

[0046] Measure an appropriate amount of deionized water, add sodium dihydrogen phosphate and disodium hydrogen phosphate to prepare a 0.02 mol / L phosphate buffer solution. Add acetonitrile to make the volume fraction reach 15%, and add sodium chloride to make the mass fraction reach 5%. Stir with a magnetic stirrer for 20 minutes until the sodium chloride is completely dissolved. Adjust the pH of the solution to 7.0-7.4 with a pH meter. Dispense into brown reagent bottles and store at 4°C for later use. Shelf life is 1 month.

[0047] Example 2:

[0048] Specific operating steps for immunochromatographic detection of pesticide residues in food.

[0049] Sample pretreatment:

[0050] Select the food samples to be tested and pre-treat them according to sample type: Wash vegetable samples and chop them with a sterile knife until the particle size is less than 5 mm; grind grain samples into powder using a grinding device. Accurately weigh 2 to 5 grams of the pre-treated sample and place it in a 50 ml centrifuge tube. Add 10 to 20 ml of sample extraction solution using a graduated cylinder. Fix the centrifuge tube on a vortex mixer and adjust the speed to 2000 to 2500 rpm. Vortex for 2 to 3 minutes to ensure thorough mixing of the sample and sample extraction solution. After vortexing, place the centrifuge tube in a high-speed refrigerated centrifuge, set the temperature to 4 to 8 degrees Celsius, and the speed to 8000 to 10000 rpm for 5 minutes. After centrifugation, use a pipette to aspirate the clear liquid from the top of the centrifuge tube and transfer it to a clean centrifuge tube for later use as the sample test solution. Figure 1 As shown.

[0051] Initiate chromatography by adding samples:

[0052] Remove the immunochromatographic test strip and place it horizontally on a clean lab bench, ensuring the strip is not bent or damp. Accurately pipette 100 to 150 microliters of the sample solution and slowly add it dropwise to the center of the sample pad on the test strip, avoiding spillage. The sample solution will automatically migrate towards the absorbent pad under capillary action, flowing over the conjugate pad and fully wetting the target-triggered hydrogel switch on the conjugate pad.

[0053] Target trigger signal release:

[0054] If the sample solution contains residues of the target pesticide, free pesticide molecules will compete with pesticide molecules at the cross-linking points formed by the target pesticide molecules in the hydrogel network for binding to pesticide-specific antibodies on the polymer chains. This competitive binding disrupts the cross-linking bridge structure of the hydrogel, causing the three-dimensional hydrogel network to disintegrate and releasing the embedded colored latex microspheres. The released colored latex microspheres will continue to migrate towards the nitrocellulose membrane with the chromatography solvent. If the sample solution does not contain residues of the target pesticide, the cross-linking bridge structure of the hydrogel remains intact, and the colored latex microspheres are locked inside the hydrogel network and cannot migrate with the chromatography solvent.

[0055] Signal capture response:

[0056] Keep the test strip horizontal, allowing the colored latex microspheres that migrate with the chromatography solvent to smoothly enter the nitrocellulose membrane. At the detection line position on the nitrocellulose membrane, the colored latex microspheres are specifically captured by the pre-coated goat anti-mouse IgG antibody, gradually accumulating to form a visible band. Simultaneously, the universally labeled component in the chromatography solvent binds to the coated rabbit anti-bovine serum albumin antibody at the control line position, forming a control band. After signal capture is complete, keep the test strip horizontal and stationary according to the ambient temperature: 15 minutes at 10°C to 20°C; 12 minutes at 21°C to 30°C; and 10 minutes at 31°C to 40°C.

[0057] Result determination:

[0058] After the settling time, place the test strip in a well-lit, non-reflective environment and observe the color development of the test line and control line on the nitrocellulose membrane. If both the control line and the test line show clearly visible bands, the target pesticide residue content in the sample is determined to be higher than the minimum detection limit. If only the control line shows a clear band and the test line shows no color, the target pesticide residue content in the sample is determined to be lower than the minimum detection limit. If the control line does not show color, regardless of whether the test line shows color, the test result is considered invalid, and a new sample solution must be prepared and a new immunochromatographic test strip used for testing.

[0059] Example 3:

[0060] Characterization and feasibility verification of the core components of the reagent kit.

[0061] Core component representation:

[0062] Sample pad characterization: The treated sample pad was observed using a scanning electron microscope at 5000x magnification. The results showed that the glass fiber membrane of the sample pad had uniform pore size, ranging from 15-20 μm, and a smooth surface with no obvious impurities. Water absorption rate testing showed that the sample pad absorbed three times its own mass of water within one minute at 25℃ and 50% relative humidity, with a stable absorption rate. Bonding pad characterization: The bonding pad after loading the target-triggered hydrogel switch was measured using a dynamic light scattering instrument at 25℃. The results showed that the hydrogel particles on the bonding pad surface were uniformly distributed, with a particle size concentrated between 200-300 nm, and no obvious agglomeration. Adhesion testing showed that after being adhered to the bonding pad surface with transparent tape and then removed, the hydrogel particles did not detach, indicating a strong bond between the target-triggered hydrogel switch and the polyester membrane. Characterization of nitrocellulose membrane: The coated nitrocellulose membrane was verified by Western blotting. After incubating the membrane with the corresponding antibody reaction solution, the colorimetric reaction was observed. The bands of the detection line and control line were clear, with neat edges and no diffusion, indicating that the antibody was well fixed on the membrane and the coating was uniform.

[0063] Feasibility verification:

[0064] Positive sample test: Accurately prepare a 10 μg / mL standard solution of the target pesticide. Take 100 μL of this standard solution and add it to the center of the sample pad of the immunochromatographic test strip according to the subsequent detection method. After standing for 15 minutes, observe that both the test line and the control line of the test strip show clear colored bands with uniform color intensity and no blurring or breakage. Negative sample test: Take 100 μL of blank sample extract and perform the same detection procedure. After standing for 15 minutes, observe that only the control line of the test strip shows a clear colored band, while the test line shows no color development. This indicates that the target recognition mechanism of this kit is effective and can achieve specific recognition and signal output of the target pesticide residue, demonstrating the feasibility of the detection principle.

[0065] Example 4:

[0066] Optimized testing conditions:

[0067] Sample extraction solution ratio optimization:

[0068] Nine combinations of acetonitrile volume fraction and sodium chloride mass fraction were designed, with acetonitrile volume fractions set to 10%, 15%, and 20%, and sodium chloride mass fractions set to 3%, 5%, and 7%, respectively. Samples containing the same concentration of the target pesticide were extracted and detected. Three parallel samples were set for each combination. The color intensity of the detection line was used as the evaluation index, and the color intensity was quantitatively analyzed using image analysis software.

[0069] Acetonitrile volume fraction Sodium chloride mass fraction Detection line color intensity (grayscale value) 10% 3% 65 10% 5% 78 10% 7% 72 15% 3% 85 15% 5% 98 15% 7% 90 20% 3% 82 20% 5% 88 20% 7% 80

[0070] The results showed that when the volume fraction of acetonitrile was 15% and the mass fraction of sodium chloride was 5%, the gray value of the color development intensity of the detection line was the highest, reaching 98, indicating that the sample extraction efficiency was the highest under this ratio. Therefore, this ratio was determined to be the optimal condition for the sample extract.

[0071] Optimization of sample loading volume:

[0072] 80 μL, 100 μL, 120 μL, 150 μL, and 180 μL of the same concentration of the target pesticide standard solution were taken and added to the sample pad of the test strip. Three parallel samples were prepared for each sample volume. The chromatographic effect and color development were observed, and the chromatography completion time and band clarity were recorded. The results showed that when the sample volume was 100-150 μL, the chromatography speed was moderate, the chromatography completion time was 3-5 minutes, and the detection line and control line were uniformly and clearly developed. When the sample volume was less than 100 μL, the chromatography was insufficient, the bands were lighter in color, and some parallel samples showed blurred detection lines. When the sample volume was greater than 150 μL, liquid overflow from the sample pad was likely to occur, affecting the detection results. Therefore, the optimal sample volume was determined to be 100-150 μL.

[0073] Optimization of settling time:

[0074] Samples containing the target pesticide were tested under different ambient temperatures. The ambient temperatures were divided into three ranges: 10-20℃, 21-30℃, and 31-40℃. Three different settling time points were set for each temperature range: 8 minutes, 10 minutes, 12 minutes, 15 minutes, and 18 minutes. Three parallel samples were set for each time point to observe the stability of the color development results. The results showed that at 10-20℃, the color development stabilized after 15 minutes, and the band color no longer changed; at 21-30℃, the color development stabilized after 12 minutes; and at 31-40℃, the color development stabilized after 10 minutes. Therefore, these settling time parameters were determined.

[0075] Example 5:

[0076] Reagent kit sensitivity testing:

[0077] Plotting the standard curve:

[0078] A series of standard solutions of the target pesticide with concentrations of 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, and 10 μg / mL were precisely prepared. Three parallel samples were set up for each concentration. The optimized detection method was used for detection. The gray value of the detection line of each parallel sample was measured using image analysis software. The average gray value at each concentration was calculated. A standard curve was plotted with the logarithm of the standard solution concentration as the abscissa and the average gray value as the ordinate, and linear regression analysis was performed.

[0079] Concentration of the target pesticide standard solution (μg / mL) Logarithmic value of concentration (logC) Average gray value of the detection line 0.01 -2.00 32 0.05 -1.30 45 0.1 -1.00 58 0.5 -0.30 76 1 0.00 92 5 0.70 115 10 1.00 130

[0080] The results showed that within the concentration range of 0.05 μg / mL to 10 μg / mL, the gray value and the logarithm of the concentration exhibited a good linear relationship. The linear regression equation was y = 125.3x + 42.8, with R² = 0.987, where y represents the gray value of the detection line, x represents the logarithm of the target pesticide concentration, and R² represents the linear correlation coefficient. This indicates that the kit has a good linear response within this concentration range.

[0081] Minimum detection limit verification:

[0082] Eleven parallel tests were performed on the target pesticide standard solution with a concentration of 0.03 μg / mL. After operating according to the detection method, the color development of the test strip was observed. In all eleven parallel tests, clear test lines and control lines were observed, with an average gray value of 38 for the test lines. Eleven parallel tests were performed on the blank sample, and no color development was observed for the test lines. The standard deviation of the gray value of the blank sample test lines was calculated, and the lowest detection limit was calculated to be 0.03 μg / mL by substituting it into the linear regression equation. This confirms that the lowest detection limit of the kit can reach 0.03 μg / mL, which meets the requirements for trace detection.

[0083] Example 6:

[0084] Specificity and stability testing of the reagent kit:

[0085] Specific detection:

[0086] Three other pesticides with structures similar to the target pesticide and two common food matrix interfering substances were selected, and standard solutions with a concentration of 10 μg / mL were prepared for each. Three parallel samples were set up for each solution. The tests were carried out according to the detection method, and the color development of the test line and control line of the test strip were observed and the test results were recorded.

[0087] Test sample Test results (test line / control line) Target pesticide standard solution Color development / Color development Similar structure pesticide A standard solution No color development / Color development Similar structure pesticide B standard solution No color development / Color development Similar structure pesticide C standard solution No color development / Color development sucrose standard solution No color development / Color development protein standard solution No color development / Color development

[0088] The results showed that only the detection line and control line corresponding to the target pesticide solution showed color, while the control line of other pesticide and interfering solutions showed color, and the detection line did not show color. This indicates that the kit has good specificity for the target pesticide and no cross-reaction.

[0089] Stability testing:

[0090] Stability Test 1 (Refrigerated Storage): After sealing, the kits were stored at 4°C. Three kits were removed at 0, 7, 14, 30, and 60 days after storage, and tested against a 1 μg / mL target pesticide standard solution. The grayscale value of the test line was measured. Results showed no significant difference in color intensity over 60 days, with the average grayscale value of the test line between 90 and 95. The results were accurate, with no false positives or false negatives.

[0091] Stability Test 2 (Room Temperature Storage): The kit was placed at room temperature (25℃) and removed after 0, 7, 14, and 21 days of storage. Three kits were tested each time. The performance was stable within 14 days, with the average gray value of the detection line between 88 and 92. After 21 days, the color intensity of the detection line decreased slightly, with the average gray value at 82, but the results could still be accurately determined, indicating that the kit has good stability.

[0092] Example 7:

[0093] Actual sample testing and accuracy verification:

[0094] Actual sample selection and processing:

[0095] Four common food samples were selected: vegetables (lettuce and cucumber) and grains (wheat flour and rice). Vegetable sample preparation: Lettuce and cucumber were washed to remove surface dirt and impurities. They were then chopped with a sterile knife to a particle size less than 5 mm. 2 g of each chopped sample was placed in a 50 mL centrifuge tube, and 10 mL of sample extraction solution was added. The centrifuge tubes were vortexed at 2000 rpm for 2 min. Subsequently, the centrifuge tubes were centrifuged in a high-speed refrigerated centrifuge at 4℃ and 8000 rpm for 5 min. The clear supernatant was collected as the sample solution for testing. Grain sample preparation: Wheat flour and rice were ground into powder. 3 g of each powder sample was placed in a 50 mL centrifuge tube, and 15 mL of sample extraction solution was added. The powder was vortexed at 2500 rpm for 3 min, and then centrifuged at 6℃ and 10000 rpm for 5 min. The clear supernatant was collected as the sample solution for testing.

[0096] Spiked recovery experiment:

[0097] Three concentration levels of the target pesticide standard were added to the blank sample: low, medium, and high. The low concentration was 0.1 μg / g, the medium concentration was 1 μg / g, and the high concentration was 5 μg / g. Three parallel samples were set up for each concentration. The samples were analyzed according to the detection method, and the measured concentration, recovery rate, and relative standard deviation of each parallel sample were calculated.

[0098] Sample type Spiked concentration (μg / g) Measure the average concentration (μg / g) Average recovery rate Relative standard deviation (RSD) lettuce 0.1 0.095 95.0% 3.16% lettuce 1 0.98 98.0% 2.04% lettuce 5 4.92 98.4% 1.62% cucumber 0.1 0.098 98.0% 2.04% cucumber 1 1.03 103.0% 2.91% cucumber 5 5.15 103.0% 1.37% wheat flour 0.1 0.092 92.0% 4.35% wheat flour 1 0.96 96.0% 2.08% wheat flour 5 4.85 97.0% 1.86% rice 0.1 0.096 96.0% 3.13% rice 1 1.01 101.0% 1.98% rice 5 5.05 101.0% 1.19%

[0099] Results analysis:

[0100] The recoveries of all actual samples were between 92.0% and 103.0%, and the relative standard deviations were all less than 5%, indicating that the kit and detection method are highly accurate and precise, can effectively eliminate the interference of food matrices, and meet the actual detection needs of target pesticide residues in different types of food samples.

[0101] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. An immunochromatographic detection kit for pesticide residues in food, characterized in that, The kit includes: immunochromatographic test strips and sample extraction solution. The immunochromatographic test strips are composed of a PVC base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad, which are sequentially overlapped and pasted together, with an overlap width of 2-3 mm between adjacent components. The sample pad is a glass fiber membrane that has been soaked in a sample pad treatment solution and then dried. The sample pad treatment solution contains 0.01 mol / L Tris-HCl buffer, 0.5% Tween-20 by volume, and 1% bovine serum albumin by mass. The binding pad is a polyester membrane loaded with a target-triggered hydrogel switch. The target-triggered hydrogel switch is a molecularly imprinted hydrogel with the target pesticide molecule as the key component of the competitive cross-linking bridge, and is internally embedded with colored latex microsphere signal markers. The target-triggered hydrogel switch is formed by copolymerizing polyacrylamide polymer chains modified with pesticide haptens and polyacrylamide polymer chains modified with pesticide-specific antibodies, under the condition of simultaneously adding the target pesticide molecule and a bifunctional cross-linking agent that can compete with the pesticide molecule for binding to the antibody, to form a three-dimensional network structure. The nitrocellulose membrane is coated with a detection line and a control line in parallel. The detection line is coated with goat anti-mouse IgG antibody, and the control line is coated with rabbit anti-bovine serum albumin antibody. The sample extraction solution was a 0.02 mol / L phosphate buffer solution with 15% acetonitrile (v / v) and 5% sodium chloride (w / w).

2. The immunochromatographic detection kit for pesticide residues in food according to claim 1, characterized in that, The PVC base plate has a thickness of 0.5-0.6 mm and a size of 60 mm × 4 mm. The sample pad is made of glass fiber membrane with a pore size of 15-20 μm and a size of 10 mm × 4 mm. The sample pad is soaked in the sample pad treatment solution for 30 min and then dried in a constant temperature oven at 37°C for 2 h.

3. The immunochromatographic detection kit for pesticide residues in food according to claim 1, characterized in that, The bonding pad is made of a polyester film with a thickness of 0.18-0.2 mm and a pore size of 8-10 μm, and the size is 8 mm × 4 mm. The polyacrylamide polymer chain is formed by cross-linking and polymerization of acrylamide monomer and N,N'-methylenebisacrylamide, and the molar ratio of N,N'-methylenebisacrylamide to acrylamide is 1:

20.

4. The immunochromatographic detection kit for pesticide residues in food according to claim 1, characterized in that, The amount of pesticide hapten added to the modified polyacrylamide polymer chain is 0.5% by mass, and the amount of pesticide specific antibody added to the modified polyacrylamide polymer chain is 0.5% by mass. The two polymer chain solutions are mixed at a volume ratio of 1:

1.

5. The immunochromatographic detection kit for pesticide residues in food according to claim 1, characterized in that, The colored latex microspheres have a particle size of 200-300 nm. The amount of colored latex microspheres added is 5% of the volume of the hydrogel crosslinking reaction liquid. The hydrogel crosslinking reaction is carried out at 25°C for 3 hours. The target-triggered hydrogel switch suspension is loaded onto the surface of the polyester film at a spraying rate of 2 μL / cm² and dried in a constant temperature oven at 37°C for 12 hours.

6. The immunochromatographic detection kit for pesticide residues in food according to claim 1, characterized in that, The nitrocellulose membrane uses a substrate with a pore size of 0.22 μm and a size of 25 mm × 4 mm. The detection line coating solution is goat anti-mouse IgG antibody diluted in 0.01 mol / L phosphate buffer at pH 7.2, with an antibody concentration of 1 mg / mL. The control line coating solution is rabbit anti-bovine serum albumin antibody diluted in 0.01 mol / L phosphate buffer at pH 7.2, with an antibody concentration of 1.5 mg / mL.

7. The immunochromatographic detection kit for pesticide residues in food according to claim 1, characterized in that, The spraying speed of the detection line and the quality control line is 5 cm / s, the spraying volume is 1 μL / cm, the distance between the two lines is 5 mm, and the detection line is 10 mm away from the edge of the conjugate pad.

8. A method for detecting pesticide residues in food using an immunochromatographic assay kit, the method being applicable to the immunochromatographic assay kit for pesticide residues in food as described in any one of claims 1-7, characterized in that... The specific steps of this method are as follows: S1, Sample pretreatment: Weigh 2-5g of the pretreated food sample to be tested into a 50mL centrifuge tube, add 10-20mL of sample extraction solution, vortex for 2-3min, centrifuge at 8000-10000r / min for 5min, and take the clear liquid at the top of the centrifuge tube as the sample test solution. S2, sample addition to start chromatography: Add 100-150 μL of sample solution to the center of the sample pad of the immunochromatographic test strip. The sample solution will migrate towards the absorbent pad under capillary action, flow through the conjugate pad and wet the target trigger hydrogel switch. S3, Target Trigger Signal Release: When the target pesticide residue is present in the sample test solution, free pesticide molecules compete with pesticide molecules in the cross-linking bridges formed by the target pesticide molecules in the hydrogel network to bind to pesticide-specific antibodies on the polymer chain, destroying the cross-linking bridge structure and causing the three-dimensional hydrogel network to disintegrate, releasing the embedded colored latex microspheres. The latex microspheres migrate towards the nitrocellulose membrane with the chromatography solvent. When there is no target pesticide residue in the sample test solution, the hydrogel cross-linking bridge structure remains intact, and the colored latex microspheres are locked inside the hydrogel network. S4, Signal capture reaction: Colored latex microspheres migrate with the chromatography solvent and enter the nitrocellulose membrane. They are captured and enriched by goat anti-mouse IgG antibody at the detection line to form a band. The universal labeling component in the chromatography solvent binds to rabbit anti-bovine serum albumin antibody at the control line to form a band. After signal capture is completed, the test strip is left to stand for 10-15 minutes. S5, Result Judgment: Observe the color development of the detection line and control line on the nitrocellulose membrane. When both the control line and the detection line develop color, the target pesticide residue content in the sample is determined to be higher than the minimum detection limit. When only the control line develops color and the detection line does not develop color, the target pesticide residue content in the sample is determined to be lower than the minimum detection limit. When the control line does not develop color, the test result is determined to be invalid.

9. The method for detecting pesticide residues in food using an immunochromatographic assay kit according to claim 8, characterized in that, In step S1, the pretreatment method is to wash off the surface dirt and impurities and then chop the grains to a particle size of less than 5 mm. The pretreatment method for grain samples is to grind them into powder. The vortex oscillation speed is 2000-2500 r / min. The equipment used for centrifugation is a high-speed refrigerated centrifuge, and the temperature is controlled at 4-8℃ during the centrifugation process.

10. The method for detecting pesticide residues in food using an immunochromatographic assay kit according to claim 8, characterized in that, In step S4, the test strip is kept horizontal during the signal capture process, and the resting time is adjusted according to the ambient temperature: 15 minutes at 10-20℃, 12 minutes at 21-30℃, and 10 minutes at 31-40℃.

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