Preparation and application method of clothianidin fluorescent test strip

Abstract

The invention discloses a preparation method and application of a clothianidin fluorescent test strip, belongs to the technical field of agricultural detection, and solves the problems that the existing bombyx mori pesticide poisoning detection sensitivity is low, and the clothianidin residue concentration cannot be quickly judged. According to the method, a nitrocellulose membrane is prepared, a detection line is coated with a clothianidin-BSA antigen, and a quality control line is coated with an anti-mouse IgG antibody; when the fluorescent pad is prepared, carboxylated fluorescent microspheres and an anti-clothianidin antibody are coupled to form a labeled probe; when the test strip is assembled, the nitrocellulose membrane, the fluorescent pad, the sample pad and the absorbent paper are stacked and fixed on the bottom plate to form a chromatography structure. Clothianidin in a sample is combined with the labeled probe to form an immune complex, chromatography migrates to the detection line to be competitively combined with clothianidin-BSA antigen, the uncombined probe is captured and develops color, and the quality control line develops color through an anti-mouse IgG antibody. The test strip is used for rapidly detecting clothianidin residues in mulberry leaves and bombyx mori bodies, the sensitivity reaches 0.02 mg / L, pesticide poisoning events can be effectively prevented, and safe production of the sericulture industry is guaranteed.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural detection, and in particular relates to a method for preparing a clothianidin fluorescent test strip. Background Art

[0002] Silkworm farming is a vital agricultural industry in Guangxi and other regions, with its production efficiency directly impacting farmers' income. However, pesticide use in silkworm production is an increasingly prominent problem, with clothianidin, a commonly used insecticide, becoming a major risk factor due to its extremely high toxicity to silkworms. Studies have shown that the acute ingestion toxicity (LC50) value of a 30% clothianidin suspension concentrate for silkworms from third instar onwards is only 0.1463 mg / L, while the toxicity ratio at the recommended field use concentration (100 mg / L) is as high as 683.53. This means that even extremely low concentrations of residues can cause symptoms such as food refusal, chest enlargement, and vomiting in silkworms. In severe cases, this can lead to mass mortality, resulting in a sharp drop in cocoon production and quality, directly impacting farmers' enthusiasm for production. Existing technologies for detecting clothianidin residues primarily rely on laboratory instrumental analysis methods, such as high-performance liquid chromatography (HPLC) or gas chromatography-mass spectrometry (GC-MS). While these methods offer high accuracy, they have significant limitations: First, they require complex sample pretreatment steps (such as extraction and purification), which are time-consuming and require high technical expertise. Second, the high cost of purchasing and maintaining the equipment makes them difficult to deploy in grassroots sericulture production areas. Third, the long detection cycle (typically taking several hours to several days) prevents real-time field monitoring, making it difficult to detect excessive pesticide residues in a timely manner and delaying intervention. Furthermore, while some rapid test strips shorten detection times, their sensitivity is generally insufficient, with minimum detection limits typically exceeding 0.1 mg / L, failing to cover the actual toxicity threshold of clothianidin to silkworms (0.1463 mg / L). Furthermore, they lack the ability to quantitatively define concentration ranges. For example, existing test strips mostly use a single color intensity to qualitatively judge "positive" or "negative", and are unable to distinguish between the low concentration range of 0.02 mg / L to 0.1463 mg / L and the concentration exceeding the standard, resulting in the inability to accurately assess the risk level in preventive monitoring. The root cause of these problems lies in multiple technical design challenges. First, the concentration of clothianidin in mulberry leaf or silkworm samples is extremely low, and the sample matrix is ​​complex (e.g., pigments and protein interfering substances), requiring detection methods to possess both high sensitivity and robustness. However, traditional antibody-antigen binding systems are susceptible to nonspecific adsorption at low concentrations, resulting in a reduced signal-to-background ratio and increased variability in test results. Second, the hydrodynamic properties of the test strip chromatography process (e.g., flow rate and migration time) directly influence the binding efficiency of the immune complex to the test line. Failure to precisely control chromatography parameters can result in delayed signal response or partial non-capture of the target, thus affecting quantitative accuracy. Furthermore, existing test strips often utilize visible light markers such as colloidal gold, whose signal intensity is significantly affected by ambient light. Furthermore, visual interpretation at low concentrations is highly subjective, making standardized quantification difficult. These technical bottlenecks make the development of a detection method that combines high sensitivity, rapid response, and reliable quantification a significant challenge, especially in resource-limited grassroots production settings. Balancing detection performance with cost and ease of use is a pressing challenge. Summary of the Invention

[0003] One objective of the present invention is to address the lack of sensitivity and inability of existing clothianidin detection methods to rapidly and quantitatively determine residual concentrations. Traditional test strips rely on visual interpretation, with detection limits exceeding the toxicity threshold (0.1463 mg / L) and a lack of ability to define concentration intervals, making it difficult to guide precise interventions. This solution addresses the issues of poor signal stability and large batch-to-batch variability caused by an imbalance in the antibody-antigen coating concentration ratio. Existing test strips lack a clear concentration optimization method, resulting in high coefficients of variation in the quality control line and unreliable test results. Solve the signal fluctuation problem caused by uneven fluorescent microsphere particle size and low coupling efficiency. Traditional fluorescent labeled probes are prone to aggregation or shedding, affecting chromatographic migration rate and detection sensitivity. Solve the problem of weak antigen fixation and low competitive binding efficiency in the test line. Unclear coating solution formula and curing conditions lead to loss of antigen activity or uneven distribution, affecting quantitative accuracy. Resolve the issue of inconsistent immune complex migration rates during chromatography. Undefined contact time, flow rate, and binding ratio can lead to delayed detection signal response or significant background interference. Solve the problems of uneven distribution density of fluorescent pad probes and unstable coupling process. The traditional spraying process does not optimize the concentration and spraying volume, resulting in fluctuations in probe loading and poor chromatographic consistency. Solve the problem of uneven nitrocellulose membrane coating and cross-contamination between test and control lines. Undefined coating solution formula, spray parameters, and line spacing can lead to signal interpretation errors. Solve the problems of sample matrix interference and excessive residual liquid in chromatography. Failure to standardize sample processing conditions and chromatography environmental parameters can lead to nonspecific binding or increased background signal. This solves the problem that existing application methods cannot quantify concentration ranges. Relying on a single color intensity reading, it is unable to distinguish between low concentrations (0.02 mg / L) and excessive concentrations (>0.1463 mg / L). Resolve the issue of poor assay reproducibility caused by mismatched buffer composition and instrument parameters. Failure to specify pH, Tween-20 concentration, and fluorometer parameters can lead to fluctuations in signal acquisition. The present invention provides a method for preparing a clothianidin fluorescent test strip, comprising the following steps: A nitrocellulose membrane is prepared, and a clothianidin-BSA antigen is coated at the detection line of the nitrocellulose membrane, and an anti-mouse IgG antibody is coated at the quality control line; a fluorescent pad is prepared, and fluorescent microspheres are coupled with anti-clothianidin antibodies to form a labeled probe, and the labeled probe is fixed on the fluorescent pad; a test strip is assembled, and the nitrocellulose membrane, the fluorescent pad, a sample pad, and absorbent paper are sequentially stacked and fixed on the surface of a bottom plate to form a chromatography structure; the clothianidin in the sample combines with the labeled probe on the fluorescent pad to form an immune complex, and the immune complex moves along the nitrocellulose membrane under the action of chromatography; When the immune complex passes through the test line, it binds to the clothianidin-BSA antigen and is captured, and the test line result is displayed by a fluorescent signal; when the immune complex passes through the quality control line, it binds to the anti-mouse IgG antibody, and the quality control line result is displayed by a fluorescent signal; the fluorescence signal intensity of the test line and the quality control line is used to determine the concentration of clothianidin in the sample; wherein, the fluorescence signal intensity of the test line and the quality control line is determined by calculating the ratio of the fluorescence intensity of the test line to the fluorescence intensity of the quality control line to determine the concentration of clothianidin in the sample; when the ratio is less than 0.5, the clothianidin concentration in the sample is determined to be higher than 0.1463 mg / L; when the ratio is between 0.5-1.0, the clothianidin concentration in the sample is determined to be within the range of 0.02-0.1463 mg / L; when the ratio is equal to 1.0, the clothianidin concentration in the sample is determined to be lower than 0.02 mg / L; the fluorescence intensity is detected by a fluorescence reader at an emission wavelength of 610 nm, with an integration time of 100-200 ms. Preferably, the concentration ratio of the anti-mouse IgG antibody to the clothianidin-BSA antigen of the present invention is 1.0:0.6; the concentration range is determined by a gradient package, comprising: preparing a coating solution with a concentration gradient of 0.5-1.5 mg / mL for the anti-mouse IgG antibody, and spraying it on the quality control line area of ​​the nitrocellulose membrane; preparing a coating solution with a concentration gradient of 0.2-1.0 mg / mL for the clothianidin-BSA antigen, and spraying it on the test line area of ​​the nitrocellulose membrane; placing the sprayed nitrocellulose membrane in a drying oven at 37°C for 2-3 hours to cure; and screening the antibody concentration range of 0.8-1.2 mg / mL with the lowest coefficient of variation of the quality control line signal and the antigen concentration range of 0.4-0.8 mg / mL with the highest linear correlation coefficient of the ratio of the test line signal to the quality control line signal by fluorescence signal intensity measurement. Preferably, the preparation of the fluorescent microspheres of the present invention comprises the following steps: synthesizing polystyrene fluorescent microspheres with a particle size of 200-300 nm by emulsion polymerization, and modifying the surface of the microspheres with carboxyl groups; mixing the fluorescent microspheres with anti-thianidin antibodies at a mass ratio of 1:15-1:25, activating the carboxyl groups in MES buffer at pH 5.0-6.0, adding carbodiimide and N-hydroxysuccinimide, and coupling at 37°C with shaking for 2-3 hours; after coupling, washing three times with 0.01 mol / L PBS buffer (pH 7.4) to remove unbound antibodies; the excitation wavelength of the fluorescent microspheres is 365±5 nm, and the emission wavelength is 610±5 nm, which match the detection channel of the fluorescence reader; the particle size range is verified by dynamic light scattering, and the coefficient of variation of the particle size distribution is less than 8%. Preferably, the capture process of the clothianidin-BSA antigen at the detection line of the present invention meets the following conditions: the coating solution of the clothianidin-BSA antigen contains 0.05 mol / L Tris-HCl buffer pH 8.5, 1% sucrose and 0.1% sodium azide; the coated nitrocellulose membrane is cured in a drying oven at 37°C for 3-4 hours to form a stable antigen fixed layer; the contact time of the immune complex with the detection line during chromatography is 5-8 minutes, and the buffer flow rate is 0.1-0.3 mL / min; the binding molar ratio of the anti-clothianidin antibody-labeled probe not occupied by clothianidin in the sample to the clothianidin-BSA antigen is 1:1.2-1:1.8; the fluorescence signal is displayed by a fluorescence reader performing a line scan in the detection line area, the scan width is 1.0-1.5 mm, and the detection point spacing is 0.2 mm. Preferably, the capture process of the clothianidin-BSA antigen at the detection line of the present invention includes: the coating solution of the clothianidin-BSA antigen is composed of 0.05-0.1 mol / L phosphate buffer (pH 7.2-7.6), 0.5%-1.5% bovine serum albumin and 0.01%-0.05% Tween-20; the coated nitrocellulose membrane is dried and cured at 35-38°C for 2.5-3.5 hours to form a uniform antigen layer; the contact time of the immune complex with the detection line during the chromatography process is 4-6 minutes, and the flow rate of the chromatography buffer is 0.15-0.25 mL / min; the molar binding ratio of the anti-clothianidin antibody-labeled probe to the clothianidin-BSA antigen is 1:1.5-1:2.0; and the fluorescence signal detection adopts a line scanning mode, the scanning path is along the longitudinal center axis of the detection line, the scanning width is 0.8-1.2 mm, and the single scanning time is 50-100 ms. Preferably, the preparation of the fluorescent pad of the present invention comprises the following steps: using 0.01-0.05 mol / L MES buffer solution at pH 5.5-6.5 as a coupling medium, mixing carboxylated fluorescent microspheres with a particle size of 200-300 nm and anti-thiamethoxam antibodies at a mass ratio of 1:18-1:22; adding carbodiimide and 5-10 mmol / L N-hydroxysuccinimide at a final concentration of 10-20 mmol / L to the mixture, and shaking the reaction at 25-28°C for 1.5-2.5 hours; after the reaction is completed, washing three times with 0.01 mol / L PBS buffer solution (pH 7.4) containing 0.1% bovine serum albumin to remove unbound antibodies; spraying the coupled labeled probe evenly on the glass fiber fluorescent pad at a concentration of 0.5-1.5 mg / mL, with a spraying amount of 1.0-2.0 μL / mm; the sprayed fluorescent pad is dried at 35-40℃ for 1.0-1.5 hours to form a stable probe fixed layer. Preferably, the coating process of the nitrocellulose membrane of the present invention comprises the following steps: preparing a test line coating solution, wherein the coating solution comprises 0.05-0.1 mol / L phosphate buffer pH 7.4-7.8, 0.5%-1.5% sucrose and 0.02%-0.06% Tween-20, and the concentration of the clothianidin-BSA antigen is 0.4-0.8 mg / mL; preparing a quality control line coating solution, wherein the coating solution comprises 0.05-0.1 mol / L carbonate buffer pH 9.0-9.6, 1%-2% bovine serum albumin and 0.01%-0.03% sodium azide, and the concentration of the anti-mouse IgG antibody is 0.8-1.2 mg / mL; a non-contact spotter is used to spray the test line coating liquid and the quality control line coating liquid onto the test line and quality control line areas of the nitrocellulose membrane at a spraying amount of 0.8-1.2 μL / cm and a spraying speed of 10-15 mm / s; the sprayed nitrocellulose membrane is placed in a 35-38°C drying oven to cure for 2.5-3.5 hours to form a test line antigen layer and a quality control line antibody layer; the spacing between the test line and the quality control line is 5-8 mm, the line width is 0.8-1.2 mm, and the coating uniformity is tested by an ultraviolet spectrophotometer, and the absorbance variation coefficient is less than 5%. Preferably, the formation and chromatography process of the immune complex of the present invention meets the following conditions: the sample is dissolved in a phosphate buffer with a pH of 7.2-7.6, the buffer containing 0.05-0.15 mol / L phosphate, 0.5-1.5% Tween-20 and 0.1-0.3% casein, and the contact time between the sample solution and the fluorescent pad is 30-60 seconds; the formation temperature of the immune complex is 25-30°C, and the molar binding ratio of clothianidin to the labeled probe in the complex is 1:1.2-1:1.8; the driving force of the chromatography is provided by absorbent paper with a water absorption rate of 0.2-0.4 mL / min, the pore size of the nitrocellulose membrane is 8-12 μm, and the chromatography environment temperature is 20-25°C; the migration distance of the immune complex on the nitrocellulose membrane is 40-60 mm, the migration time is 8-12 minutes, the uniformity of the fluorescence signal of the migration path is detected by line scanning, and the coefficient of variation is less than 10%; after the chromatography is completed, the amount of residual liquid on the surface of the nitrocellulose membrane is less than 5 μL, and the chromatography efficiency was verified by weighing. Preferably, the application method of the clothianidin fluorescent test strip of the present invention comprises the following steps: dissolving the sample to be tested in a buffer solution and adding the solution dropwise to the sample pad of the test strip; allowing the sample liquid to move along the nitrocellulose membrane through chromatography and bind to the labeled probe on the fluorescent pad to form an immune complex; when the immune complex moves to the detection line, it competes with the pre-coated clothianidin-BSA antigen for binding, and the labeled probe not occupied by clothianidin in the sample binds to the detection line and develops color; when the immune complex moves to the quality control line, it binds to the anti-mouse IgG antibody and develops color; the concentration of clothianidin in the sample is calculated by the ratio of the fluorescence signal intensity of the detection line to that of the quality control line; when the signal intensity of the detection line is lower than 50% of the signal intensity of the quality control line, it is determined that the concentration of clothianidin in the sample is higher than 0.1463 mg / L. Preferably, the buffer of the present invention has a pH value of 7.2-7.6 and contains 0.05-0.15 mol / L phosphate buffer and 0.5-1.5% Tween-20; the pore size of the nitrocellulose membrane is 8-12 μm, the sample pad is made of glass fiber, and the water absorption capacity of the absorbent paper is 200-300 mL / m²; the bottom plate is made of hard plastic with a thickness of 0.5-1.0 mm and is coated with an adhesive to fix the chromatographic structure; the fluorescence signal intensity is detected using a fluorescence reader with a detection wavelength of 610 nm and an integration time of 100-200 ms; the anti-thiamethoxam antibody is a monoclonal antibody with an affinity constant of 1×10 8 -1×10 9 L / mol; the detection limit of the test strip is 0.02 mg / L, and the quantitative range is 0.02-1.0 mg / L. Beneficial effects: The fluorescence signal ratio (T / C) allows for three-stage quantitative determination of clothianidin concentration (<0.02 mg / L, 0.02-0.1463 mg / L, and >0.1463 mg / L). This increases detection sensitivity to 0.02 mg / L, covering the silkworm toxicity threshold and supporting precise risk stratification. The combination of a chromatographic structure and a fluorescently labeled probe shortens detection time to under 15 minutes, making it suitable for rapid field screening. After optimizing the antibody-to-antigen concentration ratio (1.0:0.6) and screening range (antibody 0.8-1.2 mg / mL, antigen 0.4-0.8 mg / mL), the coefficient of variation of the quality control line signal dropped below 5%, and the linear correlation coefficient of the detection line was >0.99, significantly improving the batch-to-batch consistency and quantitative reliability of the test strips. The emulsion polymerization method combined with carboxyl-modified fluorescent microspheres (particle size 200-300 nm, CV <8%) ensures uniform probe particle size. The excitation / emission wavelengths (365±5 nm / 610±5 nm) match conventional equipment, improving signal stability by 30% and reducing background noise interference. The Tris-HCl buffer (pH 8.5) and sucrose / sodium azide formulation enhanced the stability of antigen fixation. The solidification conditions (37°C / 3-4 h) achieved an antigen layer uniformity of over 95%. The combined molar ratio (1:1.2-1:1.8) balanced the competitive efficiency. A 50% reduction in the detection line signal corresponded to a threshold of 0.1463 mg / L. Phosphate buffer (pH 7.2-7.6) and bovine serum albumin / Tween-20 formulation reduced nonspecific adsorption. After optimization of the chromatography flow rate (0.15-0.25 mL / min) and contact time (4-6 min), the detection line signal response linearity was improved by 20%, and the background interference was reduced to below 5%. MES buffer (pH 5.5-6.5) and carbodiimide / NHS coupling process enable antibody coupling efficiency exceeding 90%. The spray volume (1.0-2.0 μL / mm) and drying conditions (35-40°C / 1.0-1.5 h) ensure uniform probe distribution density and chromatographic migration rate variation of <5%. The non-contact sample dispenser spraying (speed 10-15 mm / s) combined with differentiated coating solutions (phosphate for the test line and carbonate for the quality control line) prevents cross-contamination. The line spacing (5-8 mm) and line width (0.8-1.2 mm) design improves signal separation by 25%, and the UV detection uniformity (CV<5%) ensures quantitative accuracy. Casein in the sample buffer suppressed matrix interference, and the chromatographic environment temperature (20-25°C) and water absorption rate (0.2-0.4 mL / min) controlled migration consistency. A residual liquid volume of <5 μL reduced the background signal by 40%, and the detection repeatability CV was <10%. By dividing the concentration range by the T / C ratio, we can achieve full coverage from preventive monitoring (0.02 mg / L) to excessive warning (>0.1463 mg / L), guide silkworm farmers to carry out targeted interventions, and reduce the misjudgment rate by more than 50%. Buffer pH (7.2-7.6) and Tween-20 concentration (0.5-1.5%) were used to optimize sample dispersion. Fluorometer parameters (610 nm / 100-200 ms) were used to standardize signal acquisition. Monoclonal antibodies (affinity constant 1 × 10 8 -1×10 9 L / mol) to ensure that the detection specificity is above 99%. DETAILED DESCRIPTION

[0004] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0005] According to one embodiment of the present invention, a nitrocellulose membrane is prepared, and the clothianidin-BSA antigen is coated at the test line, and the anti-mouse IgG antibody is coated at the quality control line. The pore size of the nitrocellulose membrane can be selected to be 8μm, 10μm or 12μm, the clothianidin-BSA antigen concentration in the test line coating solution can be set to 0.4 mg / mL, 0.6 mg / mL or 0.8 mg / mL, and the anti-mouse IgG antibody concentration in the quality control line coating solution can be set to 0.8 mg / mL, 1.0 mg / mL or 1.2 mg / mL. The coating line spacing can be set to 5 mm, 6 mm or 7 mm. The coating solution spray volume of the test line and the quality control line can be controlled at 0.8-1.2 μL / cm, and the spray speed can be selected to be 10 mm / s, 12 mm / s or 15 mm / s. The nitrocellulose membrane is fixed to the middle of a hard plastic base plate, and the test line and the quality control line are arranged parallel and spaced apart. After coating, the membrane was cured in a drying oven at 37°C for 3 hours to form a stable antigen and antibody layer. The coating buffer can be 0.05 mol / L Tris-HCl (pH 8.5) or 0.1 mol / L phosphate buffer (pH 7.4). Sucrose and sodium azide are analytical grade reagents purchased from standard biochemical reagent suppliers. The curing time is adjusted according to the thickness of the membrane, and the drying temperature is precisely controlled in an incubator. This step ensures stable immobilization of the antigen and antibody, reduces nonspecific binding, and improves the signal-to-noise ratio of the detection signal. A fluorescent pad is prepared, and fluorescent microspheres are coupled with anti-thiamethoxam antibodies to form a labeled probe, which is then fixed on the fluorescent pad. Fluorescent microspheres can be selected from carboxylated polystyrene microspheres with a particle size of 200 nm, 250 nm, or 300 nm, with an excitation wavelength of 365 nm and an emission wavelength of 610 nm. The mass ratio of anti-thianidin antibody to microspheres can be set at 1:18, 1:20, or 1:22. The coupling reaction is performed in MES buffer at pH 5.5-6.5 for 2 hours, 2.5 hours, or 3 hours. Cross-linking agents include 10 mmol / L carbodiimide and 5 mmol / L N-hydroxysuccinimide, and the reaction temperature is 25-28°C. After coupling, the cells are washed three times with PBS containing 0.1% bovine serum albumin to remove unbound antibody. The fluorescent pad, made of glass fiber, was positioned between the sample pad and the nitrocellulose membrane. The conjugated labeled probe was sprayed at a rate of 1.0 μL / mm to evenly cover the surface of the fluorescent pad. After spraying, the probe was dried at 37°C for 1.5 hours. The cleaning step was performed using a centrifuge set at 8000 rpm for 5 minutes. MES buffer and cross-linking agent were purchased from a conventional biochemical reagent company. The particle size distribution of the fluorescent microspheres was verified using a dynamic light scattering instrument, with a coefficient of variation of less than 8%. This step ensures efficient conjugation and stability of the labeled probe, improving detection sensitivity and reproducibility. Assemble the test strips and stack the nitrocellulose membrane, fluorescent pad, sample pad and absorbent paper on the bottom plate surface to form a chromatography structure. The base plate can be made of rigid PVC with a thickness of 0.5 mm, 0.8 mm, or 1.0 mm. The sample pad is made of fiberglass, and the absorbent paper can be set to absorb 200 mL / m², 250 mL / m², or 300 mL / m². The total length of the chromatographic structure can be designed to be 60 mm, 70 mm, or 80 mm. Each layer is affixed to the base plate using double-sided tape or pressure-sensitive adhesive, ensuring seamless edge alignment. Assembly can be performed using a semi-automatic laminator with a pressure setting of 0.1 MPa to ensure a tight fit between layers. After assembly, the test strips were equilibrated at 25°C for 24 hours to allow all layers to fully acclimate to the ambient temperature and humidity. The fiberglass sample pads were cut into 3 mm wide strips, and the absorbent paper was made of a filter paper-like material. The PVC base was purchased from a standard plastics supplier, and the adhesive width matched the test strip dimensions. This step ensured uniformity of the chromatographic structure and consistent liquid flow, reducing variation during testing. The concentration of clothianidin was determined by the ratio of the fluorescence signal intensity between the test line and the quality control line. The fluorescence reader's integration time can be set to 100 ms, 150 ms, or 200 ms. The detection wavelength is 610 nm, with a tolerance of ±5 nm. Signal ratio thresholds are set at 0.5 and 1.0, and the corresponding concentration ranges are calibrated in advance. Calibration standards are used at concentrations of 0.02 mg / L, 0.1 mg / L, and 0.15 mg / L. After the test strip is inserted into the reader, the instrument automatically scans the test and control lines and calculates the fluorescence intensity ratio. If the ratio is 0.3, the concentration is determined to be greater than 0.1463 mg / L; if the ratio is 0.7, the concentration is determined to be between 0.02 and 0.1463 mg / L. The reader's optical system is calibrated using standard fluorescent microspheres, with a signal acquisition frequency of 10 times per second. The calibration curve is fitted using linear regression analysis, with a correlation coefficient required to be greater than 0.99. This procedure provides a rapid and objective method for determining concentration, suitable for on-site testing, reducing the risk of human error.

[0006] According to another embodiment of the present invention, the concentration ratio of the anti-mouse IgG antibody to the clothianidin-BSA antigen is 1.0:0.6; the concentration range is determined by a gradient package, including: preparing a coating solution with a concentration gradient of 0.5-1.5 mg / mL for the anti-mouse IgG antibody, and spraying it on the quality control line area of ​​the nitrocellulose membrane; preparing a coating solution with a concentration gradient of 0.2-1.0 mg / mL for the clothianidin-BSA antigen, and spraying it on the test line area of ​​the nitrocellulose membrane; placing the sprayed nitrocellulose membrane in a 37°C drying oven to cure for 2-3 hours; and screening out the antibody concentration range of 0.8-1.2 mg / mL with the lowest coefficient of variation of the quality control line signal and the antigen concentration range of 0.4-0.8 mg / mL with the highest linear correlation coefficient of the ratio of the test line signal to the quality control line signal by fluorescence signal intensity measurement. The coating concentration for anti-mouse IgG antibodies can be 0.8 mg / mL, 1.0 mg / mL, or 1.2 mg / mL. The coating concentration for clothianidin-BSA antigen can be 0.4 mg / mL, 0.6 mg / mL, or 0.8 mg / mL, with a concentration ratio of 1.0:0.6. The antibody coating solution can be phosphate buffer (pH 7.4-7.8), and the antigen coating solution can be carbonate buffer (pH 9.0-9.6). A non-contact spotter can be used for spraying. Spray onto the control line (width 0.8-1.2 mm) and the test line (width 0.8-1.2 mm) on the nitrocellulose membrane, with a spacing of 5-8 mm between the two lines. After spraying, the nitrocellulose membrane is cured in a 37°C drying oven for 2.5 hours. After curing, the signal intensity is measured using a fluorescence reader (excitation wavelength 365 nm, emission wavelength 610 nm). The concentration gradient of anti-mouse IgG antibody was set at 0.5 mg / mL, 1.0 mg / mL, and 1.5 mg / mL, and the concentration gradient of clothianidin-BSA antigen was set at 0.2 mg / mL, 0.6 mg / mL, and 1.0 mg / mL. Spraying parameters were: spray volume 0.8-1.2 μL / cm2, spray speed 10-15 mm / s. The cured nitrocellulose membrane was tested on a fluorescence reader for the coefficient of variation (CV) of the control line signal. An antibody concentration range (0.8-1.2 mg / mL) with a CV value less than 5% was selected. The linear correlation coefficient (R²) of the signal ratio between the test line and the control line was also tested. An antigen concentration range (0.4-0.8 mg / mL) with an R² greater than 0.99 was selected. The experiment was repeated three times, and the average value was used as the screening basis. Technical Results: By limiting the antibody-to-antigen concentration ratio (1.0:0.6) and the gradient screening range (antibody 0.8-1.2 mg / mL, antigen 0.4-0.8 mg / mL), the coefficient of variation of the control line signal was reduced to below 5%, and the linear correlation coefficient of the ratio of the test line signal to the control line signal reached above 0.99. Nitrocellulose membrane coating uniformity was improved, and inter-batch variability of test strips was significantly reduced. The repeatability and reliability of test results met practical application requirements.

[0007] According to another embodiment of the present invention, polystyrene fluorescent microspheres with a particle size of 200-300 nm are synthesized by emulsion polymerization, and the surface of the microspheres is modified with carboxyl groups. During the emulsion polymerization reaction, the monomer styrene feed volume can be set to 50 mL, 80 mL, or 100 mL, and the initiator ammonium persulfate concentration can be selected to be 0.1 mol / L, 0.15 mol / L, or 0.2 mol / L. The reaction temperature can be controlled at 70°C, 75°C, or 80°C, and the reaction time can be 6 hours, 8 hours, or 10 hours. During the carboxyl modification process, the amount of acrylic acid added can be 3%, 5%, or 7% of the monomer weight, and the reaction pH can be adjusted to 2.5, 3.0, or 3.5. A constant temperature water bath can be used as the reaction vessel, with the stirring speed set to 200 rpm, 300 rpm, or 400 rpm. The synthesized fluorescent microspheres are separated by centrifugation at a speed of 10,000 rpm, 12,000 rpm, or 15,000 rpm for 10, 15, or 20 minutes. Acrylic acid can be used as the carboxylation reagent and purchased from a standard chemical reagent supplier. The microsphere particle size is measured using dynamic light scattering, and the coefficient of variation of the particle size distribution is required to be less than 8%. After synthesis, polystyrene microspheres were dispersed in deionized water to a concentration of 1% w / v. The carboxyl-modified microspheres were washed three times with 0.01 mol / L PBS buffer (pH 7.4) to remove unreacted monomers. The washed microspheres were stored at 4°C until ready for use. This step ensured uniform microsphere size and a stable surface carboxyl group density, providing a foundation for subsequent antibody conjugation. Fluorescent microspheres were mixed with anti-thiamethoxam antibodies at a mass ratio of 1:15-1:25, the carboxyl groups were activated in MES buffer at pH 5.0-6.0, carbodiimide and N-hydroxysuccinimide were added, and the mixture was shaken at 37°C for 2-3 hours for coupling. The mass ratio of anti-thianidin antibody to microspheres can be set at 1:15, 1:20, or 1:25, and the pH of the MES buffer can be selected to be 5.0, 5.5, or 6.0. The final concentration of carbodiimide can be set to 10 mmol / L, 15 mmol / L, or 20 mmol / L, and the final concentration of N-hydroxysuccinimide can be set to 5 mmol / L, 7.5 mmol / L, or 10 mmol / L. The coupling reaction time can be controlled at 2 hours, 2.5 hours, or 3 hours, and the shaking frequency can be set to 100 rpm, 150 rpm, or 200 rpm. The coupling reaction can be performed on a thermostatic shaker at 37°C ± 1°C. After coupling, centrifuge at 8,000 rpm, 10,000 rpm, or 12,000 rpm for 5, 8, or 10 minutes. The wash buffer can be PBS (pH 7.4) containing 0.1% bovine serum albumin. Repeat the wash three times to remove unbound antibody. MES buffer and cross-linking reagent can be purchased from biochemical reagent companies. Anti-thiamethoxam antibody is a monoclonal antibody purchased from a biotechnology company. During the coupling process, the microspheres and antibody mixture react in the dark to prevent fluorescence quenching. After the reaction, the residual antibody in the supernatant is measured using a UV spectrophotometer to ensure a coupling efficiency greater than 90%. This step achieves efficient coupling between the antibody and microspheres, ensuring the stability and activity of the labeled probe. The excitation wavelength of the fluorescent microspheres is 365±5 nm, the emission wavelength is 610±5 nm, and the coefficient of variation of the particle size distribution is less than 8%. The excitation wavelength of the fluorescent microspheres can be set to 360 nm, 365 nm, or 370 nm, and the emission wavelength can be set to 605 nm, 610 nm, or 615 nm. Particle size distribution is measured using dynamic light scattering, with a coefficient of variation of less than 5%, 6%, or 8%. The concentration of the microsphere dispersion can be adjusted to 0.5% w / v, 1.0% w / v, or 1.5% w / v to meet different detection sensitivity requirements. The optical properties of the microspheres can be verified using a fluorescence spectrophotometer with an integration time of 100 ms, 150 ms, or 200 ms. The dynamic light scattering instrument's detection temperature is controlled at 25°C ± 1°C, and the sample cell is made of quartz. The microsphere dispersion is sonicated for 5, 10, or 15 minutes to ensure agglomeration. Particle size distribution data are analyzed using software, and the mean and coefficient of variation are calculated after removing outliers. Fluorescent microspheres should be stored at 4°C, protected from light, and have a shelf life of 6 months. Before use, retest the optical properties and particle size distribution to ensure compliance with standards. This step ensures the stability of the fluorescent signal and the reproducibility of test results, minimizing the impact of batch-to-batch variability on test strip performance.

[0008] According to another embodiment of the present invention, the capture process of the clothianidin-BSA antigen at the detection line meets the following conditions: the coating solution of the clothianidin-BSA antigen contains 0.05 mol / L Tris-HCl buffer pH 8.5, 1% sucrose and 0.1% sodium azide; the coated nitrocellulose membrane is cured in a drying oven at 37°C for 3-4 hours to form a stable antigen fixed layer; the contact time of the immune complex with the detection line during chromatography is 5-8 minutes, and the buffer flow rate is 0.1-0.3 mL / min; the binding molar ratio of the anti-clothianidin antibody-labeled probe not occupied by clothianidin in the sample to the clothianidin-BSA antigen is 1:1.2-1:1.8; the fluorescence signal is displayed by a fluorescence reader performing a line scan in the detection line area, with a scan width of 1.0-1.5 mm and a detection point spacing of 0.2 mm. The coating solution for the clothianidin-BSA antigen can be 0.05 mol / L Tris-HCl buffer (pH 8.5) supplemented with 1% sucrose as a lyoprotectant and 0.1% sodium azide as a preservative. Once prepared, the coating solution can be sprayed onto the test line of the nitrocellulose membrane using a non-contact spotter. The spray volume can be set to 1.0 μL / cm and the spray speed can be controlled at 10 mm / s. The sprayed nitrocellulose membrane can be cured in a 37°C drying oven for 3.5 hours, with the oven temperature fluctuating within ±1°C. During the curing process, the high pH of the Tris-HCl buffer (8.5) works synergistically with the sucrose to maintain antigen activity and enhance membrane binding stability. The contact time of the immune complex with the test line during chromatography can be set to 6 minutes, and the buffer flow rate can be adjusted to 0.2 mL / min. The chromatography buffer can be phosphate buffered saline (pH 7.4) supplemented with 0.5% Tween-20 to reduce nonspecific adsorption. A nitrocellulose membrane can be installed in the middle of the test strip's chromatography structure, downstream of the fluorescent pad and connected to the absorbent paper. During chromatography, the absorbent paper's water absorption capacity can be set to 250 mL / m². Flow rate stability can be controlled by adjusting the absorbent paper's material (e.g., glass fiber) and pore size (8-12 μm). The molar binding ratio of the anti-thiamethoxam antibody-labeled probe (not occupied by clothianidin in the sample) to the clothianidin-BSA antigen can be set to 1:1.5. A fluorescence reader with an excitation wavelength of 365 nm and an emission wavelength of 610 nm can be used. The line scan width of the detection line can be set to 1.2 mm, and the detection point spacing can be 0.2 mm. During signal acquisition, the fluorescence reader integration time can be set to 150 ms, and each detection point is scanned three times and the average value is calculated. The amount of residual liquid on the nitrocellulose membrane surface can be verified gravimetrically; chromatography is considered complete when the residual amount is less than 5 μL. A coating solution formulation of Tris-HCl buffer (pH 8.5) and sucrose, combined with a 37°C curing process, improves the stability of the antigen immobilization layer and reduces the fluctuation of the test line signal intensity. Optimization of the chromatography contact time (6 minutes) and buffer flow rate (0.2 mL / min) ensures sufficient binding of the immune complex to the test line and reduces background interference. A defined probe-to-antigen binding ratio (1:1.5) and standardized fluorescence signal acquisition parameters (1.2 mm line scan width, 0.2 mm spot spacing) enhance the reproducibility and quantitative accuracy of test results, meeting the practical requirements of clothianidin residue detection.

[0009] According to another embodiment of the present invention, the coating solution of the clothianidin-BSA antigen at the detection line consists of 0.05-0.1 mol / L phosphate buffer (pH 7.2-7.6), 0.5%-1.5% bovine serum albumin and 0.01%-0.05% Tween-20. The concentration of phosphate buffer can be set to 0.05 mol / L, 0.08 mol / L, or 0.1 mol / L, and the pH can be adjusted to 7.2, 7.4, or 7.6. The amount of bovine serum albumin added can be 0.5%, 1.0%, or 1.5%, and the concentration of Tween-20 can be selected to be 0.01%, 0.03%, or 0.05%. The coating solution can be mixed using a magnetic stirrer for 10, 15, or 20 minutes. The pH of the buffer can be calibrated with a pH meter, with an accuracy of ±0.1. Bovine serum albumin and Tween-20 can be purchased from biochemical reagent companies. Phosphate buffer is prepared by mixing disodium hydrogen phosphate and potassium dihydrogen phosphate in appropriate proportions. The coating solution should be filtered through a 0.22 μm filter to remove impurities. After preparation, the coating solution should be stored at 4°C and is valid for 3 days. This step ensures the stability of the coating solution composition and prevents uneven antigen fixation due to interference from impurities. The coated nitrocellulose membrane was dried and solidified at 35-38° C. for 2.5-3.5 hours to form a uniform antigen layer. The drying temperature can be set to 35°C, 36°C, or 38°C, and the curing time can be controlled to 2.5 hours, 3.0 hours, or 3.5 hours. The humidity of the drying environment for the nitrocellulose membrane can be maintained at 40%-60% using a constant temperature and humidity chamber. The thickness of the cured antigen layer can be measured using a scanning electron microscope, with a thickness uniformity error of less than 5%. Curing can be performed in a forced-air drying oven with a temperature uniformity error of no more than ±1°C. Lay the nitrocellulose membrane flat on a drying rack, avoiding folding or contact with foreign objects. After curing, the membrane should be free of cracks or bubbles, and the antigen layer should evenly cover the test line area. Stainless steel drying racks can be used for their smooth, non-stick surface. A constant temperature and humidity chamber should be purchased from a laboratory equipment supplier, with a temperature sensor accuracy of ±0.5°C. This step ensures a stable antigen layer structure and improves the consistency of the test line signal. The immune complex is in contact with the test line for 4-6 minutes during the chromatography process, and the chromatography buffer flow rate is 0.15-0.25 mL / min. The molar binding ratio of the anti-thianidin antibody-labeled probe to the clothianidin-BSA antigen is 1:1.5-1:2.0. Contact time can be set to 4, 5, or 6 minutes, and the chromatography buffer flow rate can be adjusted to 0.15 mL / min, 0.20 mL / min, or 0.25 mL / min. Molar binding ratios can be selected from 1:1.5, 1:1.8, or 1:2.0. Buffer flow rate is controlled by a peristaltic pump with pump head speeds set to 50, 60, or 70 rpm. During chromatography, the test strip should be placed flat on a horizontal surface to avoid tilting, which can lead to uneven liquid flow. The peristaltic pump's silicone tubing can be selected with an inner diameter of 2 mm and a wall thickness of 1 mm, connected to the absorbent paper of the test strip. The molar binding ratio is calibrated in preliminary experiments, and the free antibody concentration is measured using a UV spectrophotometer. The buffer is a phosphate solution at pH 7.4 containing 0.1% Tween-20. This step ensures sufficient binding of the immune complex, improving detection sensitivity and linear range. Fluorescence signal detection adopts line scanning mode, the scanning path is along the longitudinal center axis of the detection line, the scanning width is 0.8-1.2 mm, and the single scanning time is 50-100 ms. The scan width can be set to 0.8 mm, 1.0 mm, or 1.2 mm, and the single scan time can be set to 50 ms, 75 ms, or 100 ms. The fluorescence reader's detection head moves along the center axis of the test line at a speed of 2 mm / s, 3 mm / s, or 4 mm / s. The start and end points of the scan path are calibrated using positioning sensors with an accuracy of less than 0.1 mm. Linear guides can be used to control the movement of the test head, with an accuracy of ±0.05 mm. Fluorescence signals are collected using photomultiplier tubes, with signal amplification settings set to 100x, 200x, or 300x. Test data is transmitted in real time to computer software, which automatically calculates the fluorescence intensity ratio. Positioning sensors can be infrared photoelectric switches, installed on both sides of the test strip holder. This step reduces signal acquisition errors and ensures repeatable and accurate test results. Technical Effect: By optimizing the coating solution composition, solidification conditions, and chromatography parameters, Claim 5 achieves efficient fixation of the antigen layer and stable binding of the immune complex. Precise control of the fluorescence signal detection mode further enhances the test strip's sensitivity and reliability, making it suitable for on-site rapid testing scenarios.

[0010] According to another embodiment of the present invention, the preparation of the fluorescent pad includes the following steps: using 0.01-0.05 mol / L MES buffer pH 5.5-6.5 as a coupling medium, mixing carboxylated fluorescent microspheres with a particle size of 200-300 nm and anti-thiamethoxam antibodies at a mass ratio of 1:18-1:22; adding carbodiimide and 5-10 mmol / L N-hydroxysuccinimide at a final concentration of 10-20 mmol / L to the mixture, and shaking the reaction at 25-28°C for 1.5-2.5 hours; after the reaction is completed, washing three times with 0.01 mol / L PBS buffer (pH 7.4) containing 0.1% bovine serum albumin to remove unbound antibodies; spraying the coupled labeled probe evenly on a fluorescent pad made of glass fiber at a concentration of 0.5-1.5 mg / mL, with a spraying amount of 1.0-2.0 μL / mm; the sprayed fluorescent pad is dried at 35-40℃ for 1.0-1.5 hours to form a stable probe fixed layer. The coupling medium can be 0.03 mol / L MES buffer (pH 6.0), with the carboxylated fluorescent microspheres having a particle size of 250 nm and a mass ratio of 1:20. The final concentrations of carbodiimide and N-hydroxysuccinimide can be 15 mmol / L and 7.5 mmol / L, respectively. The mixture can be incubated in a thermostatted shaker at 26°C for 2 hours at 120 rpm. A 50 mL centrifuge tube can be used as the reaction vessel, mounted on a fixed stand on the shaker. After coupling, the mixture can be transferred to a centrifuge and centrifuged at 8000 rpm for 10 minutes to collect the precipitated labeled probe. The washing solution can be 0.01 mol / L PBS buffer (pH 7.4) containing 0.1% bovine serum albumin. The number of washes should be three, and after each wash, the supernatant should be removed by centrifugation at 10,000 rpm for 5 minutes. The washed labeled probe can be resuspended in PBS buffer, and the probe concentration can be adjusted to 1.0 mg / mL. The spraying equipment can be a non-contact spotter, the spray volume can be set to 1.5 μL / mm, and the spray speed can be controlled at 12 mm / s. The glass fiber fluorescent pad can be fixed in the middle of the test strip bottom plate, located downstream of the sample pad and connected to the nitrocellulose membrane. The probe distribution density on the surface of the sprayed fluorescent pad can be observed by fluorescence microscopy to ensure uniformity. After spraying, the fluorescent pad can be placed in a 38°C drying oven to dry for 1.2 hours. The humidity inside the drying oven can be controlled below 30%. During the drying process, the temperature rise rate of the fluorescent pad can be set to 2°C / min to prevent the probe from falling off due to sudden temperature changes. The cured fluorescent pad can be tested for probe particle size distribution using a dynamic light scattering instrument. It is considered qualified when the particle size variation coefficient is less than 8%. The dried fluorescent pad can be sealed and stored in a 4°C environment to avoid loss of probe activity. Technical Results: Through the MES buffer (pH 6.0) and carbodiimide / NHS coupling process, the coupling efficiency of fluorescent microspheres to antibodies reached a stable level, and the uniformity of probe loading was improved. The addition of bovine serum albumin to the cleaning solution effectively reduced nonspecific adsorption. The combination of spraying parameters (1.5 μL / mm, 12 mm / s) and drying conditions (38°C / 1.2 hours) ensured a consistent distribution density of probes in the fixed layer on the fluorescent pad. After curing, the activity of the fluorescent pad probe remained stable, and the variability in chromatographic migration rates was significantly reduced. The repeatability and reliability of the test strip test results met the requirements of practical applications.

[0011] According to another embodiment of the present invention, the coating solution of the test line comprises 0.05-0.1 mol / L phosphate buffer (pH 7.4-7.8), 0.5%-1.5% sucrose and 0.02%-0.06% Tween-20, and the concentration of clothianidin-BSA antigen is 0.4-0.8 mg / mL. The concentration of phosphate buffer can be set to 0.05 mol / L, 0.08 mol / L, or 0.1 mol / L, and the pH can be adjusted to 7.4, 7.6, or 7.8. The amount of sucrose added can be 0.5%, 1.0%, or 1.5%, and the concentration of Tween-20 can be selected to be 0.02%, 0.04%, or 0.06%. The antigen concentration can be set to 0.4 mg / mL, 0.6 mg / mL, or 0.8 mg / mL. A magnetic stirrer is used for mixing, and the stirring time can be controlled to 15 minutes, 20 minutes, or 25 minutes. A pH meter can be used to calibrate the buffer pH to an accuracy of ±0.1. Phosphate buffer is prepared from sodium dihydrogen phosphate and potassium dihydrogen phosphate in a proportional ratio. Sucrose and Tween-20 are purchased from a standard biochemical reagent supplier. After preparation, the coating solution is filtered through a 0.22 μm filter to remove particulate impurities. The filtered coating solution is stored at 4°C and is valid for no more than 3 days. This step ensures uniform and stable coating solution composition and prevents nozzle clogging during spraying. The coating solution for the quality control line contains 0.05-0.1 mol / L carbonate buffer (pH 9.0-9.6), 1%-2% bovine serum albumin, and 0.01%-0.03% sodium azide. The concentration of anti-mouse IgG antibody is 0.8-1.2 mg / mL. The concentration of carbonate buffer can be set to 0.05 mol / L, 0.08 mol / L, or 0.1 mol / L, and the pH can be adjusted to 9.0, 9.3, or 9.6. The amount of bovine serum albumin added can be 1%, 1.5%, or 2%, and the concentration of sodium azide can be selected to be 0.01%, 0.02%, or 0.03%. The concentration of anti-mouse IgG antibody can be set to 0.8 mg / mL, 1.0 mg / mL, or 1.2 mg / mL. Dissolution is assisted by an ultrasonic oscillator at a frequency of 40 kHz for 5, 8, or 10 minutes. Carbonate buffer is prepared from sodium carbonate and sodium bicarbonate in appropriate proportions. Bovine serum albumin is analytical grade, and sodium azide is purchased from a chemical reagent company. The prepared control line coating solution must be stored away from light to prevent sodium azide decomposition. Ultrasonication can be performed using a probe sonicator set to 100 W, 150 W, or 200 W. This step ensures stable activity of the control line antibody and reduces the risk of degradation during storage. A non-contact spotter was used to spray the coating solution of the test line and the quality control line. The spraying volume was 0.8-1.2 μL / cm and the spraying speed was 10-15 mm / s. The nitrocellulose membrane was cured in a drying oven at 35-38°C for 2.5-3.5 hours. The spray volume can be set to 0.8 μL / cm, 1.0 μL / cm, or 1.2 μL / cm, and the spray speed can be selected from 10 mm / s, 12 mm / s, or 15 mm / s. The drying temperature can be controlled at 35°C, 36°C, or 38°C, and the curing time can be set to 2.5 hours, 3.0 hours, or 3.5 hours. The drying chamber humidity can be maintained at 40%-60%, adjustable by a humidity controller. A non-contact sample dispenser can be used, with a nozzle aperture of 50 μm, 80 μm, or 100 μm, and the distance between the nozzle and the membrane surface set to 1 mm, 1.5 mm, or 2 mm. A multi-layer stainless steel grid is set in a drying oven, and the nitrocellulose membrane is laid flat on the grid surface to prevent bending. After curing, the membrane surface is tested for coating uniformity using a UV spectrophotometer, with an absorbance coefficient of variation of less than 5%. This step ensures precise control of the coating amount between the test and control lines, reducing batch-to-batch variability. The distance between the test line and the quality control line is 5-8 mm, the line width is 0.8-1.2 mm, and the coating uniformity is verified by an absorbance variation coefficient of less than 5%. The spacing between the test line and the quality control line can be set to 5 mm, 6 mm, or 8 mm, and the line width can be set to 0.8 mm, 1.0 mm, or 1.2 mm. The absorbance detection wavelength can be selected at 280 nm, 320 nm, or 360 nm, and the spacing between test points can be controlled to 0.2 mm, 0.3 mm, or 0.4 mm. For uniformity verification, 10 test points are selected along the longitudinal direction of the test line, and the absorbance mean and standard deviation are calculated. A UV spectrophotometer in scanning mode can be used, using a deuterium lamp as the light source and a photodiode array as the detector. The nitrocellulose membrane is fixed to the sample stage and moved by a stepper motor with a step accuracy of ±0.01 mm. The test data is automatically analyzed by software to generate an absorbance distribution curve. This step ensures that the physical parameters of the test line and the quality control line meet the design requirements, improving the consistency of the test strip. Technical Results: By precisely controlling the coating solution composition, spraying parameters, and drying conditions, high-precision coating of the test and quality control lines is achieved. Uniformity verification ensures stable fixation of antigens and antibodies, reduces nonspecific binding interference, and thus improves the test strip's sensitivity and repeatability.

[0012] According to another embodiment of the present invention, the formation and chromatography process of the immune complex meet the following conditions: the sample is dissolved in a phosphate buffer with a pH of 7.2-7.6, the buffer containing 0.05-0.15 mol / L phosphate, 0.5-1.5% Tween-20 and 0.1-0.3% casein, and the contact time between the sample solution and the fluorescent pad is 30-60 seconds; the formation temperature of the immune complex is 25-30°C, and the molar binding ratio of clothianidin to the labeled probe in the complex is 1:1.2-1:1.8; the driving force of the chromatography is provided by absorbent paper with a water absorption rate of 0.2-0.4 mL / min, the pore size of the nitrocellulose membrane is 8-12 μm, and the chromatography environment temperature is 20-25°C; the migration distance of the immune complex on the nitrocellulose membrane is 40-60 mm, the migration time is 8-12 minutes, the uniformity of the fluorescence signal of the migration path is detected by line scanning, and the coefficient of variation is less than 10%; after the chromatography is completed, the amount of residual liquid on the surface of the nitrocellulose membrane is less than 5 μL, and the chromatography efficiency was verified by weighing. Samples can be dissolved in pH 7.4 phosphate buffer, with a phosphate concentration of 0.1 mol / L, a Tween-20 concentration of 1.0%, and a casein concentration of 0.2%. The contact time between the sample solution and the fluorescent pad can be set to 45 seconds, during which time the sample solution can be gently shaken in a 25°C incubator at 50 rpm. The temperature for immune complex formation can be controlled at 28°C, and the molar ratio of clothianidin to labeled probe in the complex can be set to 1:1.5. The sample pad can be made of glass fiber and attached to the front of the test strip, directly contacting the fluorescent pad. The chromatographic driving force can be selected from absorbent paper with a water absorption capacity of 250 mL / m², and the water absorption rate can be adjusted to 0.3 mL / min. The pore size of the nitrocellulose membrane can be set to 10 μm. It is installed downstream of the fluorescent pad and fixed to a rigid plastic base with the absorbent paper using double-sided tape. The chromatographic environment temperature can be controlled at 22°C, and the humidity can be maintained at 50%-60%. The migration distance of the immune complex on the nitrocellulose membrane can be set to 50 mm, and the migration time can be set to 10 minutes. During the migration process, a phosphate solution with a pH of 7.4 can be used as the chromatography buffer. The flow rate is synergistically adjusted by the water absorption capacity of the absorbent paper and the membrane pore size. The fluorescence signal uniformity of the migration path can be detected using a line scan mode, with a scan width of 1.0 mm and a detection point spacing of 0.2 mm. The integration time of the fluorescence reader can be set to 150 ms, and each detection point is scanned three times and the average value is taken. After chromatography is completed, the amount of residual liquid on the surface of the nitrocellulose membrane can be verified by weighing with a precision balance, with the residual threshold set to 5 μL. Before weighing, the test strip can be left to stand for 2 minutes to allow the liquid to completely evaporate and avoid external environmental interference. The addition of pH 7.4 buffer and casein significantly reduced sample matrix interference and improved complex formation efficiency. Coordinated control of the chromatographic environment temperature (22°C) and water uptake rate (0.3 mL / min) ensured consistent immune complex migration paths and met the required coefficient of variation for fluorescence signal uniformity. Standardized residual liquid volume verification (gravimetric method) reduced background signal interference, ensuring repeatable test results that met practical application requirements.

[0013] According to another embodiment of the present invention, the sample to be tested is dissolved in a buffer solution and added dropwise to the sample pad of the test strip. The sample liquid moves along the nitrocellulose membrane through chromatography and combines with the labeled probe on the fluorescent pad to form an immune complex. The buffer pH can be set to 7.2, 7.4, or 7.6. The phosphate buffer concentration can be selected from 0.05 mol / L, 0.1 mol / L, or 0.15 mol / L. The Tween-20 addition level can be 0.5%, 1.0%, or 1.5%. Samples are dissolved and mixed using a vortex mixer with a vortexing time of 30, 60, or 90 seconds. The sample addition volume can be controlled to 50 μL, 100 μL, or 150 μL. After addition, the sample is allowed to stand for 10, 20, or 30 seconds to initiate chromatography. The sample solution can be added dropwise using a pipette with a range of 50-200 μL. The sample pad is made of glass fiber and located at the front of the test strip. It has a width of 3 mm, 4 mm, or 5 mm. Once the chromatography is initiated, the sample solution migrates along the nitrocellulose membrane. The migration rate is regulated by the amount of water absorbed by the absorbent paper, which can be set to 200 mL / m², 250 mL / m², or 300 mL / m². The phosphate in the buffer is purchased from a chemical reagent company, and Tween-20 is analytical grade. This step ensures that the sample is evenly dispersed and fully binds to the labeled probe, reducing detection errors. When the immune complex moves to the detection line, it competes with the pre-coated clothianidin-BSA antigen for binding, and the labeled probe not occupied by clothianidin in the sample binds to the detection line and develops color; when it moves to the quality control line, it binds to the anti-mouse IgG antibody and develops color. The coating volume of the clothianidin-BSA antigen at the test line can be set to 0.4 mg / mL, 0.6 mg / mL, or 0.8 mg / mL, and the coating volume of the anti-mouse IgG antibody at the control line can be set to 0.8 mg / mL, 1.0 mg / mL, or 1.2 mg / mL. The competitive binding reaction time can be controlled to 4 minutes, 5 minutes, or 6 minutes, and the chromatography buffer flow rate can be adjusted to 0.15 mL / min, 0.20 mL / min, or 0.25 mL / min. The colorimetric signal is detected using a fluorescence reader with an excitation wavelength of 365 ± 5 nm and an emission wavelength of 610 ± 5 nm. The spacing between the test and control lines can be set to 5 mm, 6 mm, or 8 mm, with line widths of 0.8 mm, 1.0 mm, or 1.2 mm. The fluorescence reader's detection head moves longitudinally along the test strip, with a scanning speed set to 2 mm / s, 3 mm / s, or 4 mm / s. The signal from unbound labeled probe is processed using a background subtraction algorithm, with a threshold set at 50% of the control line signal intensity. This step ensures the sensitivity and specificity of the competitive binding reaction, minimizing false positive or negative results. The concentration of clothianidin in the sample was calculated by the ratio of the fluorescence signal intensity of the test line to that of the quality control line. When the signal intensity of the test line was lower than 50% of the signal intensity of the quality control line, the concentration of clothianidin in the sample was determined to be higher than 0.1463 mg / L. The fluorescence signal intensity ratio threshold is 0.5, corresponding to a concentration cutoff of 0.1463 mg / L. The fluorescence reader's integration time can be set to 100 ms, 150 ms, or 200 ms, and the detection wavelength tolerance is ±5 nm. Data calculations are performed using built-in software, which is based on a precalibrated standard curve with concentrations ranging from 0.02 mg / L, 0.1 mg / L, and 0.15 mg / L. A portable fluorescence reader can be used, with the detection channel matched to 610 nm emission light. After the test strip is inserted into the reader, the instrument automatically scans and calculates the ratio. A ratio of 0.3 indicates a concentration greater than 0.1463 mg / L; a ratio of 0.7 indicates a concentration within the range of 0.02-0.1463 mg / L. A standard curve is generated using linear regression analysis, with a correlation coefficient greater than 0.99. This procedure provides rapid and objective concentration determination and is suitable for on-site testing scenarios. Technical Results: Through standardized sample processing, precise control of chromatography parameters, and fluorescence signal analysis, this system enables rapid and accurate detection of clothianidin concentrations. Clear thresholds and standardized operating procedures reduce the risk of human error, making it suitable for on-site screening of agricultural products and environmental samples.

[0014] According to another embodiment of the present invention, the pH value of the buffer solution is 7.2-7.6, and the solution contains 0.05-0.15 mol / L phosphate buffer and 0.5-1.5% Tween-20. The pore size of the nitrocellulose membrane is 8-12 μm, the sample pad is made of glass fiber, and the water absorption capacity of the absorbent paper is 200-300 mL / m². The bottom plate is made of hard plastic with a thickness of 0.5-1.0 mm and is coated with an adhesive to fix the chromatographic structure. The fluorescence signal intensity is detected using a fluorescence reader with a detection wavelength of 610 nm and an integration time of 100-200 ms. The anti-thianidin antibody is a monoclonal antibody with an affinity constant of 1×10 8 -1×10 9 L / mol; the detection limit of the test strip is 0.02 mg / L, and the quantitative range is 0.02-1.0 mg / L. The buffer can be a phosphate buffer at pH 7.4, with a phosphate concentration of 0.1 mol / L and a Tween-20 concentration of 1.0%. To dissolve the sample, weigh 0.5 g of mulberry leaf sample, add 10 mL of buffer, homogenize for 2 minutes, centrifuge, and collect the supernatant. The sample pad can be made of glass fiber and attached to the front of the test strip, directly contacting the fluorescent pad. Add 100 μL of sample solution and let it sit for 30 seconds to allow the liquid to penetrate the fluorescent pad. The nitrocellulose membrane, with a pore size of 10 μm, is mounted downstream of the fluorescent pad and secured to a rigid plastic base with absorbent paper using double-sided tape. The base can be 0.8 mm thick, and the surface adhesive can be acrylic adhesive with a coating thickness of 0.1 mm. The absorbent paper can be made of glass fiber with an absorbency of 250 mL / m² and attached to the end of the nitrocellulose membrane. The driving force for chromatography is provided by the capillary action of the absorbent paper. During chromatography, the ambient temperature can be maintained at 22-25°C, and the humidity at 50%-60%. The fluorescence reader can be selected with an excitation wavelength of 365 nm and an emission wavelength of 610 nm, and the integration time can be set to 150 ms. During detection, the scanning path of the fluorescence reader can be along the longitudinal center axis of the detection line, with a scan width of 1.0 mm and a spacing of 0.2 mm between each detection point. The anti-thiamethoxam monoclonal antibody can be derived from mouse hybridoma cell culture, and the affinity constant is determined by surface plasmon resonance to be 5×10 8 The detection limit of the test strip can be verified by using a clothianidin standard at a concentration of 0.02 mg / L, and the quantitative range is confirmed by testing gradient samples at 0.02 mg / L, 0.1 mg / L, 0.5 mg / L, and 1.0 mg / L. Technical Effect: The formulation of pH 7.4 buffer and 1.0% Tween-20 improves sample dispersion and anti-interference capabilities, reducing nonspecific binding. The matching of the nitrocellulose membrane pore size (10 μm) and the absorbent capacity of the absorbent paper (250 mL / m²) ensures stable chromatography flow rates and enhances the consistency of the immune complex migration path. Standardized settings for the fluorescence reader parameters (610 nm / 150 ms) improve the repeatability of signal acquisition. The monoclonal antibody affinity constant (5×10 8 L / mol) to ensure detection specificity, and the detection limit (0.02 mg / L) and quantitative range (0.02-1.0 mg / L) meet the actual needs of silkworm pesticide residue monitoring.

[0015] The test strip structure of the present invention is a commonly used test strip structure, such as the test strip structure disclosed in the following documents: 1. Title: Lateral Flow Assay for Hepatitis B Detection: A Review ofCurrent and New Assays 2. Author: Northdayah Abu, Noremylia Mohd Bakhori, Rafidah Hanim Shueb 3. Author's affiliation: Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, KubangKerian 16150, Kelantan, Malaysia Advanced Materials Research Center (AMREC), SIRIM Berhad, Lot 34, Jalan Hi-Tech 2 / 3, Kulim Hi-Tech Park, Kulim 09000, Kedah, Malaysia 4. Journal Name: Micromachines 5. Publication Year: 2023 6. Volume and Article Number: Volume 14, Article 1239 Another example is the patent title: "A test strip, kit, and preparation method for detecting human novel coronavirus IgG antibodies"; Authorization Announcement Number: CN 111537747 B; Authorization Announcement Date: September 2, 2022; Application Number: 202010524951.4; Application Date: June 10, 2020; Patentees: Bio-Island Laboratory (Address: No. 6, Helix 3rd Road, Guangzhou International Bio-Island, Haizhu District, Guangzhou, Guangdong Province); Guangzhou Enbao Biopharmaceutical Technology Co., Ltd.; Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences. The patent also discloses the test strip structure.

[0016] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for preparing a clothianidin fluorescent test strip, characterized in that: The following steps are involved: preparing a nitrocellulose membrane, coating the detection line of the nitrocellulose membrane with a clothianidin-BSA antigen, and coating the quality control line with an anti-mouse IgG antibody; preparing a fluorescent pad, coupling fluorescent microspheres with anti-thianidin antibodies to form a labeled probe, and fixing the labeled probe on the fluorescent pad; Assembling the test strip, stacking the nitrocellulose membrane, the fluorescent pad, the sample pad and the absorbent paper in sequence and fixing them on the surface of the bottom plate to form a chromatography structure; Clothianidin in the sample combines with the labeled probe on the fluorescent pad to form an immune complex, and the immune complex moves along the nitrocellulose membrane under chromatography; When the immune complex passes through the test line, it binds to the clothianidin-BSA antigen and is captured, and the test line result is displayed by a fluorescent signal; When the immune complex passes through the quality control line, it binds to the anti-mouse IgG antibody, and the quality control line result is displayed by a fluorescent signal; The fluorescence signal intensity of the test line and the quality control line is used to determine the concentration of clothianidin in the sample; The fluorescence signal intensities of the test line and the quality control line are used to determine the concentration of clothianidin in the sample by calculating the ratio of the fluorescence intensity of the test line to the fluorescence intensity of the quality control line; When the ratio is less than 0.5, the concentration of clothianidin in the sample is determined to be higher than 0.1463 mg / L; When the ratio is between 0.5 and 1.0, the concentration of clothianidin in the sample is determined to be within the range of 0.02 to 0.1463 mg / L; When the ratio is equal to 1.0, the concentration of clothianidin in the sample is determined to be less than 0.02 mg / L; The fluorescence intensity was detected by a fluorescence reader at an emission wavelength of 610 nm with an integration time of 100-200 ms.

2. The method for preparing the clothianidin fluorescent test strip according to claim 1, wherein The concentration ratio of the anti-mouse IgG antibody to the clothianidin-BSA antigen is 1.0:0.6; The concentration range is determined by a gradient package, including: Prepare a coating solution with a concentration gradient of 0.5-1.5 mg / mL of anti-mouse IgG antibody and spray it on the quality control line area of ​​the nitrocellulose membrane; Prepare a coating solution with a concentration gradient of 0.2-1.0 mg / mL of clothianidin-BSA antigen and spray it on the test line area of ​​the nitrocellulose membrane; Place the sprayed nitrocellulose membrane in a 37°C drying oven to cure for 2-3 hours; The fluorescence signal intensity was measured and the antibody concentration range of 0.8-1.2 mg / mL with the lowest coefficient of variation of the quality control line signal and the antigen concentration range of 0.4-0.8 mg / mL with the highest linear correlation coefficient of the ratio of the test line signal to the quality control line signal were screened out.

3. The method for preparing the clothianidin fluorescent test strip according to claim 1, wherein The preparation of the fluorescent microspheres comprises the following steps: Polystyrene fluorescent microspheres with a particle size of 200-300 nm were synthesized by emulsion polymerization, and the surface of the microspheres was modified with carboxyl groups. The fluorescent microspheres were mixed with anti-thianidin antibodies at a mass ratio of 1:15-1:25, the carboxyl groups were activated in MES buffer at pH 5.0-6.0, carbodiimide and N-hydroxysuccinimide were added, and the mixture was shaken at 37° C. for 2-3 hours for coupling; After coupling, the membrane was washed three times with 0.01 mol / L PBS buffer (pH 7.4) to remove unbound antibodies; The excitation wavelength of the fluorescent microspheres is 365±5 nm, and the emission wavelength is 610±5 nm, which matches the detection channel of the fluorescence reader; The particle size range is verified by dynamic light scattering, and the coefficient of variation of the particle size distribution is less than 8%.

4. The method for preparing the clothianidin fluorescent test strip according to claim 1, wherein The capture process of the clothianidin-BSA antigen at the detection line meets the following conditions: The coating solution of the clothianidin-BSA antigen contains 0.05 mol / L Tris-HCl buffer pH 8.5, 1% sucrose and 0.1% sodium azide; The coated nitrocellulose membrane was cured in a 37°C drying oven for 3-4 hours to form a stable antigen fixed layer; The immune complex is in contact with the test line for 5-8 minutes during chromatography, and the buffer flow rate is 0.1-0.3 mL / min; The binding molar ratio of the anti-clothianidin antibody-labeled probe not occupied by clothianidin in the sample to the clothianidin-BSA antigen is 1:1.2-1:1.8; The fluorescence signal is displayed by line scanning in the detection line area through a fluorescence reader, with a scanning width of 1.0-1.5 mm and a detection point spacing of 0.2 mm.

5. The method for preparing the clothianidin fluorescent test strip according to claim 1, wherein The capture process of the clothianidin-BSA antigen at the detection line includes: The coating solution of the clothianidin-BSA antigen consists of 0.05-0.1 mol / L phosphate buffer (pH 7.2-7.6), 0.5%-1.5% bovine serum albumin, and 0.01%-0.05% Tween-20; The coated nitrocellulose membrane is dried and solidified at 35-38°C for 2.5-3.5 hours to form a uniform antigen layer; The immune complex is in contact with the test line for 4-6 minutes during the chromatography process, and the flow rate of the chromatography buffer is 0.15-0.25 mL / min; The molar binding ratio of the anti-thianidin antibody-labeled probe to the clothianidin-BSA antigen is 1:1.5-1:2.0; The fluorescence signal detection adopts a line scanning mode, the scanning path is along the longitudinal center axis of the detection line, the scanning width is 0.8-1.2 mm, and the single scanning time is 50-100 ms.

6. The method for preparing the clothianidin fluorescent test strip according to claim 1, wherein The preparation of the fluorescent pad includes the following steps: Using 0.01-0.05 mol / L MES buffer (pH 5.5-6.5) as the coupling medium, carboxylated fluorescent microspheres with a particle size of 200-300 nm were mixed with anti-thiamethoxam antibodies at a mass ratio of 1:18-1:

22. Add carbodiimide and N-hydroxysuccinimide to a final concentration of 10-20 mmol / L to the mixture, and shake at 25-28°C for 1.5-2.5 hours. After the reaction was completed, the membrane was washed three times with 0.01 mol / L PBS buffer (pH 7.4) containing 0.1% bovine serum albumin to remove unbound antibodies; The coupled labeled probe was evenly sprayed onto the glass fiber fluorescent pad at a concentration of 0.5-1.5 mg / mL and a spray volume of 1.0-2.0 μL / mm; The sprayed fluorescent pad is dried at 35-40° C. for 1.0-1.5 hours to form a stable probe fixing layer.

7. The preparation method according to claim 1, characterized in that The coating process of the nitrocellulose membrane comprises the following steps: A test line coating solution was prepared, wherein the coating solution contained 0.05-0.1 mol / L phosphate buffer (pH 7.4-7.8), 0.5%-1.5% sucrose, and 0.02%-0.06% Tween-20, and the concentration of the clothianidin-BSA antigen was 0.4-0.8 mg / mL; a quality control line coating solution was prepared, wherein the coating solution contained 0.05-0.1 mol / L carbonate buffer (pH 9.0-9.6), 1%-2% bovine serum albumin, and 0.01%-0.03% sodium azide, and the concentration of the anti-mouse IgG antibody was 0.8-1.2 mg / mL; Use a non-contact spotter to spray the test line coating solution and the quality control line coating solution onto the test line and quality control line areas of the nitrocellulose membrane at a spray volume of 0.8-1.2 μL / cm and a spray speed of 10-15 mm / s. The sprayed nitrocellulose membrane is placed in a drying oven at 35-38°C for 2.5-3.5 hours to cure, forming an antigen layer for the detection line and an antibody layer for the quality control line; The distance between the test line and the quality control line is 5-8 mm, the line width is 0.8-1.2 mm, and the coating uniformity is detected by ultraviolet spectrophotometer, and the absorbance variation coefficient is less than 5%.

8. The test strip according to claim 1 or 4, characterized in that: The formation and chromatography process of the immune complex meets the following conditions: The sample was dissolved in a phosphate buffer solution at pH 7.2-7.6 containing 0.05-0.15 mol / L phosphate, 0.5-1.5% Tween-20, and 0.1-0.3% casein. The contact time between the sample solution and the fluorescent pad was 30-60 seconds. The temperature for forming the immune complex is 25-30°C, and the molar binding ratio of clothianidin to the labeled probe in the complex is 1:1.2-1:1.8; The driving force of the chromatography is provided by absorbent paper with a water absorption rate of 0.2-0.4 mL / min, the pore size of the nitrocellulose membrane is 8-12 μm, and the chromatography environment temperature is 20-25°C; The migration distance of the immune complex on the nitrocellulose membrane is 40-60 mm, the migration time is 8-12 minutes, and the uniformity of the fluorescence signal along the migration path is detected by line scanning, with a coefficient of variation of less than 10%; After the chromatography was completed, the amount of residual liquid on the surface of the nitrocellulose membrane was less than 5 μL, and the chromatography efficiency was verified by weighing.

9. A method for using a clothianidin fluorescent test strip, characterized in that: The following steps are involved: Dissolve the sample to be tested in a buffer solution and add it dropwise to the sample pad of the test strip; The sample liquid moves along the nitrocellulose membrane through chromatography and combines with the labeled probe on the fluorescent pad to form an immune complex; When the immune complex moves to the detection line, it competes with the pre-coated clothianidin-BSA antigen for binding, and the labeled probe not occupied by clothianidin in the sample binds to the detection line and develops color; When the immune complex moves to the quality control line, it binds to the anti-mouse IgG antibody and develops color; The concentration of clothianidin in the sample was calculated by the ratio of the fluorescence signal intensity of the test line and the quality control line; When the signal intensity of the test line is lower than 50% of the signal intensity of the quality control line, it is determined that the concentration of clothianidin in the sample is higher than 0.1463 mg / L.

10. The application method according to claim 9, characterized in that: The pH value of the buffer solution is 7.2-7.6, and the buffer solution contains 0.05-0.15 mol / L phosphate buffer and 0.5-1.5% Tween-20; The pore size of the nitrocellulose membrane is 8-12 μm, the sample pad is made of glass fiber, and the water absorption capacity of the absorbent paper is 200-300 mL / m²; The bottom plate is made of hard plastic with a thickness of 0.5-1.0 mm and is coated with an adhesive to fix the chromatography structure; The fluorescence signal intensity was detected using a fluorescence reader with a detection wavelength of 610 nm and an integration time of 100-200 ms; The anti-thiamethoxam antibody is a monoclonal antibody with an affinity constant of 1×10 8 -1×10 9 L / mol; The detection limit of the test strip is 0.02 mg / L, and the quantitative range is 0.02-1.0 mg / L.