Thiamethoxam colloidal gold test strip, preparation method and application

Abstract

The invention discloses a preparation method and application of a thiamethoxam colloidal gold test strip and the test strip, belongs to the technical field of agricultural detection, and solves the problems that the existing thiamethoxam residue detection sensitivity is insufficient, and low-concentration residues cannot be rapidly and quantitatively judged. According to the method, a nitrocellulose membrane is prepared, a detection line is coated with a thiamethoxam-BSA antigen, and a quality control line is coated with an anti-mouse IgG antibody; when the gold-labeled pad is prepared, carboxylated colloidal gold and an anti-thiamethoxam antibody are coupled to form a labeled probe; when the test strip is assembled, the nitrocellulose membrane, the gold label pad, the sample pad and the absorbent paper are stacked and fixed on the bottom plate to form a chromatography structure. Thiamethoxam in a sample is combined with the labeled probe to form an immune complex, chromatography migrates to a detection line to be competitively combined with a thiamethoxam-BSA antigen, the uncombined probe is captured and develops color through colloidal gold, a quality control line develops color through an anti-mouse IgG antibody, the concentration is judged according to the color development intensity ratio (T / C) of the detection line and the quality control line, and the pesticide exposure risk can be early warned. The sericulture safety production is ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural detection, and in particular relates to a preparation method, application and test strip of a thiamethoxam colloidal gold test strip. Background Art

[0002] Silkworm farming is a major agricultural industry in southern my country, and its production efficiency directly impacts the economic income of silkworm farmers. However, the problem of silkworm poisoning caused by pesticide use is becoming increasingly prominent. Thiamethoxam, a commonly used insecticide, is a major risk factor due to its toxic effects on silkworms. Studies have shown that thiamethoxam residues as low as 0.02 mg / L can inhibit silkworm growth and development. Conventional field application concentrations are far above this threshold, resulting in a significant risk of mulberry leaf contamination. Existing detection technologies have the following limitations in practical applications: First, while traditional instrumental detection methods (such as high-performance liquid chromatography and gas chromatography-mass spectrometry) can achieve high-precision analysis, they are complex, requiring time-consuming steps such as sample extraction and purification. Furthermore, the instrument purchase and maintenance costs are high, making them difficult to promote in grassroots sericulture production areas. Furthermore, these methods typically require detection cycles of several hours to several days, making them difficult to support real-time field monitoring, making it difficult to detect excessive pesticide residues in a timely manner. Secondly, existing rapid test strips mostly rely on visual interpretation, with detection limits generally exceeding 0.02 mg / L, which cannot cover the actual toxicity threshold of thiamethoxam to silkworms. For example, some test strips can only qualitatively determine "positive" or "negative" based on color intensity, and cannot distinguish between low concentrations of 0.005 mg / L to 0.02 mg / L and concentrations exceeding the standard, resulting in an inability to accurately assess risk levels during preventive monitoring. In addition, uneven particle size of colloidal gold-labeled probes or unstable coupling processes may lead to fluctuations in signal intensity, and color development results are easily affected by ambient light, making interpretation highly subjective and reproducible. The root of the above problems lies in the multiple challenges of technical design. First, the content of thiamethoxam in mulberry leaf or silkworm samples is extremely low, and the sample matrix is ​​complex (such as pigments and protein interferences), requiring the detection method to have both high sensitivity and anti-interference capabilities. However, the traditional antibody-antigen binding system is susceptible to nonspecific adsorption at low concentrations, resulting in a decrease in the signal-to-background ratio and increased volatility in the test results. Second, the fluid dynamics characteristics of the test strip chromatography process (such as flow rate and migration time) directly affect the binding efficiency of the immune complex to the test line. If the chromatography parameters are not precisely controlled, it may cause a delayed signal response or some targets to not be captured, thereby affecting the quantitative accuracy. In addition, existing test strips mostly use a single color intensity for interpretation, lack a standardized signal quantification method, and it is difficult to establish a linear relationship between concentration and signal intensity. These technical bottlenecks pose a significant challenge to developing a detection method that combines high sensitivity, rapid response, and reliable quantification. Balancing detection performance with cost and ease of use remains a pressing challenge, especially in resource-limited grassroots production scenarios. Summary of the Invention

[0003] One objective of the present invention is to address the lack of sensitivity and inability of existing thiamethoxam detection methods to rapidly quantify low-concentration residues. Traditional test strips rely on visual interpretation, with detection limits exceeding the toxicity threshold (0.02 mg / L). They also lack the 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. Without a clear concentration optimization method, the coefficient of variation of the quality control line is high, and the test results are unreliable. Solve the signal fluctuation problem caused by uneven colloidal gold particle size and low coupling efficiency. Traditional labeled probes are prone to aggregation or shedding, affecting chromatographic migration rate and detection sensitivity. This solves the problem of inconsistent liquid migration paths caused by unclear chromatography structure assembly parameters. When the membrane pore size, sample pad thickness, and absorbent paper water absorption capacity are not specified, the chromatography flow rate fluctuates significantly, resulting in poor detection repeatability. Solve the problem of uneven distribution density of gold label pad probes and unstable coupling process. If the spray concentration and drying conditions are not optimized, the probe fixed layer is easy to fall off or unevenly distributed. Solve the problem of antigen / antibody activity loss caused by mismatched coating buffer formulas between test and control lines. When buffer types are not differentiated, active substances are easily inactivated during the coating process. This solves the problem of low migration efficiency caused by insufficient chromatographic driving force or fluctuating environmental parameters. When water absorption rate, temperature, and humidity are not controlled, the migration path of immune complexes is inconsistent. This solves the problem that existing application methods cannot quantify low concentrations. When relying on a single color intensity reading, it is impossible to distinguish residual concentrations between 0.005 mg / L and 0.02 mg / L. This solution addresses the issue of poor sample dispersion caused by buffer mismatch. Sample matrix interference can be significant when phosphate concentration and Tween-20 ratio are not specified. Solve the problem of loose fixation of the test strip chromatography structure. Without specifying the base material and adhesive, components are prone to shifting or falling off during assembly. The present invention provides a method for preparing a thiamethoxam colloidal gold test strip, comprising the following steps: preparing a nitrocellulose membrane, coating the thiamethoxam-BSA antigen at the detection line of the nitrocellulose membrane, and coating the anti-mouse IgG antibody at the quality control line; preparing a gold label pad, coupling colloidal gold with an anti-thiamethoxam antibody to form a labeled probe, and fixing the labeled probe on the gold label pad; Assembling the test strip, stacking the nitrocellulose membrane, the gold label 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; Thiamethoxam in the sample combines with the labeled probe on the gold label 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 detection line, it binds to the thiamethoxam-BSA antigen and is captured, and the detection line result is displayed by colloidal gold color development; When the immune complex passes through the quality control line, it combines with the anti-mouse IgG antibody, and the quality control line result is displayed by colloidal gold color development; The color intensity of the test line and the quality control line is used to determine the concentration of thiamethoxam in the sample; The color intensity of the test line and the quality control line is used to determine the concentration of thiamethoxam in the sample by calculating the ratio of the color intensity of the test line to the color intensity of the quality control line; When the ratio is less than 0.5, the concentration of thiamethoxam in the sample is determined to be higher than 0.02 mg / L; When the ratio is between 0.5 and 1.0, the concentration of thiamethoxam in the sample is determined to be within the range of 0.005 to 0.02 mg / L; When the ratio is equal to 1.0, the concentration of thiamethoxam in the sample is determined to be less than 0.005 mg / L; The color intensity was detected by a colloidal gold reader at a wavelength of 520 nm with an integration time of 100-200 ms. Preferably, the coating concentration of the anti-mouse IgG antibody of the present invention is 0.9-1.1 mg / mL, and the coating concentration of the thiamethoxam-BSA antigen is 0.5-0.7 mg / mL; The concentration ratio of the anti-mouse IgG antibody to the thiamethoxam-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 mg / mL, 1.0 mg / mL, and 1.5 mg / mL of anti-mouse IgG antibody. Use a non-contact spotter to spray the solution onto the quality control line of the nitrocellulose membrane at a spray volume of 1.0 μL / cm. Prepare coating solutions with a concentration gradient of 0.2 mg / mL, 0.6 mg / mL, and 1.0 mg / mL of thiamethoxam-BSA antigen and spray them onto the test line area at the same spraying amount; The sprayed nitrocellulose membrane was placed in a drying oven at 37°C for 3 hours to cure; The antibody concentration range of 0.9-1.1 mg / mL with a coefficient of variation of the quality control line signal of less than 5% and the antigen concentration range of 0.5-0.7 mg / mL with a linear correlation coefficient of the ratio of the test line signal to the quality control line signal of greater than 0.98 were screened out by colloidal gold color intensity determination. The coating solutions of the anti-mouse IgG antibody and thiamethoxam-BSA antigen respectively contain 0.05 mol / L carbonate buffer (pH 9.4) and 0.05 mol / L phosphate buffer (pH 7.6), and the spraying speed is 12 mm / s. Preferably, the colloidal gold of the present invention has a particle size of 25-35 nm, the surface carboxyl groups are modified by sodium citrate reduction, and the colloidal gold is coupled with the anti-thiamethoxam antibody at a mass ratio of 1:18-1:22; The coupling process comprises the following steps: Carboxylated colloidal gold and anti-thiamethoxam antibody were mixed in MES buffer (pH 5.5-6.0), and carbodiimide and N-hydroxysuccinimide (7.5 mmol / L) were added to the mixture at a final concentration of 15 mmol / L. The mixture was shaken at 26°C for 2 hours. After the reaction was completed, the cells were washed three times with 0.01 mol / L PBS buffer (pH 7.4) containing 0.1% bovine serum albumin and centrifuged to remove unbound antibodies; The particle size distribution of the colloidal gold was verified by dynamic light scattering, and the coefficient of variation of the particle size was less than 8%; The coupled labeled probe was sprayed onto a gold label pad made of glass fiber at a concentration of 0.8-1.2 mg / mL and a spraying volume of 1.5 μL / mm; The sprayed gold label pad was dried at 38°C for 1.2 hours to form a stable probe fixation layer; The colloidal gold has an absorbance peak of 0.8-1.2 at a wavelength of 520 nm, which matches the detection channel of a colloidal gold reader. Preferably, the selection and assembly of the nitrocellulose membrane, sample pad, and absorbent paper of the present invention meet the following conditions: The pore size of the nitrocellulose membrane is 9-11 μm, and commercially available nitrocellulose membrane materials can be selected. It is assembled in the middle position of the chromatographic structure of the test strip, located downstream of the gold label pad and connected to the absorbent paper; The sample pad can be made of glass fiber with a thickness of 0.3-0.5 mm, assembled at the front end of the test strip, in direct contact with the gold standard pad, and the liquid penetration time is 5-8 seconds; The absorbent paper has a water absorption capacity of 250 mL / m² and can be made of commercially available highly absorbent glass fiber material. It is assembled at the end of the nitrocellulose membrane and fixed to the hard plastic base plate with double-sided tape. The contact width between the nitrocellulose membrane and the sample pad is 3-5 mm, and the contact width with the absorbent paper is 4-6 mm. The consistency of the liquid migration path is verified by chromatography migration experiments, and the coefficient of variation is less than 10%; During the chromatography process, the liquid flow rate of the nitrocellulose membrane is 0.25-0.35 mL / min, the ambient temperature is 22-25° C., and the humidity is 50-60%. Preferably, the preparation of the gold label pad of the present invention comprises the following steps: Colloidal gold and anti-thiamethoxam antibody were mixed in MES buffer at pH 5.0-6.0, and carbodiimide and N-hydroxysuccinimide were added at a final concentration of 10-20 mmol / L and 5-10 mmol / L, respectively. The mixture was shaken and reacted 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 containing 0.1% bovine serum albumin to remove unbound antibodies; The coupled labeled probe was sprayed onto the gold label pad made of glass fiber at a concentration of 0.5-1.5 mg / mL and a spray volume of 1.0-2.0 μL / mm; The sprayed gold label pad is dried at 35-40° C. for 1.0-1.5 hours to form a stable probe fixing layer. Preferably, the coating solution of the thiamethoxam-BSA antigen at the test line of the present invention comprises 0.05-0.1 mol / L phosphate buffer, 0.5%-1.5% sucrose and 0.01%-0.05% Tween-20; the coating solution of the anti-mouse IgG antibody at the quality control line comprises 0.05-0.1 mol / L carbonate buffer, 1%-2% bovine serum albumin and 0.01%-0.03% sodium azide. Preferably, the driving force of the chromatography of the present invention is provided by absorbent paper, the water absorption rate is 0.2-0.4 mL / min, and the chromatography environment temperature is 20-25°C; the migration distance of the immune complex on the nitrocellulose membrane is 40-60 mm, and the migration time is 8-12 minutes. Preferably, the application method of the thiamethoxam colloidal gold test strip of the present invention comprises the following steps: Dissolve the sample to be tested in phosphate buffer at pH 7.2-7.6 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 gold label pad to form an immune complex; When the immune complex moves to the detection line, it competes with the pre-coated thiamethoxam-BSA antigen for binding, and the labeled probe not occupied by thiamethoxam 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 thiamethoxam in the sample was calculated by the color intensity ratio of the test line and the quality control line; When the color intensity of the test line is lower than 50% of the color intensity of the quality control line, the concentration of thiamethoxam in the sample is determined to be higher than 0.02 mg / L. Preferably, the phosphate buffer of the present invention contains 0.05-0.15 mol / L phosphate and 0.5-1.5% Tween-20; the detection limit is 0.02 mg / L, and the quantitative range is 0.02-1.0 mg / L. Preferably, the bottom plate of the present invention is made of hard plastic material with a thickness of 0.5-1.0 mm, and the surface is coated with an adhesive to fix the chromatography structure. Beneficial effects: The color intensity ratio (T / C) of the test line to the control line allows for three-stage determination of thiamethoxam concentration (<0.005 mg / L, 0.005-0.02 mg / L, and >0.02 mg / L). This increases detection sensitivity to 0.005 mg / L, covering the silkworm toxicity threshold and supporting early risk warning. The colloidal gold color development combined with a chromatographic structure reduces detection time to under 15 minutes, making it suitable for rapid field screening. After optimizing the concentration ratio of anti-mouse IgG antibody (0.9-1.1 mg / mL) and thiamethoxam-BSA antigen (0.5-0.7 mg / mL), the coefficient of variation of the quality control line signal dropped below 5%, the linear correlation coefficient of the detection line reached above 0.98, and the consistency of the test strips between batches was significantly improved. After optimizing the coupling ratio of carboxylated colloidal gold (CV<8%) with a particle size of 25-35 nm and antibody (1:18-1:22), the probe migration rate was stabilized, the signal intensity fluctuation was reduced, the background noise was lowered, and the detection sensitivity was increased to 0.005 mg / L. The coordinated control of the nitrocellulose membrane pore size (9-11 μm), sample pad thickness (0.3-0.5 mm), and absorbent paper water absorption capacity (250 mL / m²) stabilizes the chromatography flow rate at 0.25-0.35 mL / min, and the coefficient of variation of the migration path consistency is less than 10%, and the detection repeatability meets actual needs. After optimizing the MES buffer (pH 5.0-6.0) and carbodiimide / NHS coupling process (15 mmol / L / 7.5 mmol / L), the antibody coupling efficiency reached over 90%. The spraying parameters (1.5 μL / mm) and drying conditions (38°C / 1.2 h) ensured uniform probe distribution density and improved chromatographic signal consistency. The differentiated formulations of the test line phosphate buffer (pH 7.6) and the quality control line carbonate buffer (pH 9.4) maintain antigen activity and antibody binding stability, respectively, reducing the activity loss rate during the coating process to less than 5%. The limitations of the chromatographic water absorption rate (0.2-0.4 mL / min) and the ambient temperature (20-25°C) stabilize the migration time of the immune complex (8-12 minutes), improve the linearity of the detection line signal response, and reduce background interference. By dividing the concentration range by the T / C ratio, we can achieve full coverage from extremely low residues (0.005 mg / L) to excessive concentrations (>0.02 mg / L), guiding silkworm farmers in targeted interventions and reducing the misjudgment rate by more than 50%. The formulation of phosphate buffer (0.05-0.15 mol / L) and Tween-20 (0.5-1.5%) was optimized to improve sample dispersion, reduce nonspecific adsorption, and increase the detection signal-to-background ratio. The combination of a hard plastic base (0.5-1.0 mm) and acrylic adhesive ensures a stable chromatography structure, reduces component displacement to less than 2% during assembly, and enhances the long-term storage stability of the test strips.

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

[0005] According to another embodiment of the present invention, a nitrocellulose membrane is prepared, and the detection line is coated with thiamethoxam-BSA antigen, and the control line is coated with anti-mouse IgG antibody. The pore size of the nitrocellulose membrane can be selected from 9 μm, 10 μm, or 11 μm. The concentration of the thiamethoxam-BSA antigen in the coating solution for the test line can be set to 0.5 mg / mL, 0.6 mg / mL, or 0.7 mg / mL, and the concentration of the anti-mouse IgG antibody in the coating solution for the control line can be set to 0.9 mg / mL, 1.0 mg / mL, or 1.1 mg / mL. The spacing between the coating lines can be set to 5 mm, 6 mm, or 7 mm. The coating volume for the test and control lines can be controlled at 1.0 μL / cm, and the spraying speed can be selected from 12 mm / s, 14 mm / s, or 16 mm / s. The nitrocellulose membrane is fixed to the center of a rigid plastic base, with the test and control lines arranged parallel and spaced apart. After coating, the membrane is cured in a 37°C drying oven for 3 hours to form a stable antigen and antibody layer. The coating buffer can be 0.05 mol / L phosphate buffer (pH 7.6) or 0.05 mol / L carbonate buffer (pH 9.4). Sucrose and sodium azide are analytical grade reagents purchased from standard biochemical reagent suppliers. The curing time is controlled by a timer in an incubator, with a drying temperature tolerance of no more than ±1°C. 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 gold label pad is prepared, colloidal gold is coupled with an anti-thiamethoxam antibody to form a labeled probe, and the probe is fixed on the gold label pad. The colloidal gold particle size can be 25 nm, 30 nm, or 35 nm, and the mass ratio of anti-thiamethoxam antibody to colloidal gold can be set at 1:18, 1:20, or 1:22. The coupling reaction is performed in MES buffer at pH 5.5, 5.8, or 6.0 for 2 hours, 2.5 hours, or 3 hours. Cross-linking agents include 15 mmol / L carbodiimide and 7.5 mmol / L N-hydroxysuccinimide, and the reaction temperature is 26°C ± 1°C. After coupling, the cells are washed three times with PBS containing 0.1% bovine serum albumin to remove unbound antibody. The gold label pad, made of glass fiber, is positioned between the sample pad and the nitrocellulose membrane. The conjugated labeled probe is sprayed evenly over the surface of the gold label pad at a rate of 1.5 μL / mm and then dried at 38°C for 1.2 hours. The cleaning step is performed using a centrifuge set at 10,000 rpm for 10 minutes. The colloidal gold particle size distribution is verified by dynamic light scattering, 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, stack the nitrocellulose membrane, gold label pad, sample pad and absorbent paper in sequence and fix them on the surface of the bottom plate to form a chromatographic structure. The base plate can be made of rigid PVC plastic with a thickness of 0.5 mm, 0.8 mm, or 1.0 mm. The sample pad can be made of fiberglass with a thickness of 0.3 mm, 0.4 mm, or 0.5 mm. The absorbent paper can be set to absorbent capacities of 250 mL / m², 280 mL / m², or 300 mL / m². 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 contact width between the sample pad and the gold label pad was 3 mm, 4 mm, or 5 mm, and the contact width between the absorbent paper and the nitrocellulose membrane was 4 mm, 5 mm, or 6 mm. 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 deviations during the test process. The concentration of thiamethoxam in the sample was determined by the color intensity ratio of the test line and the quality control line. The color intensity ratio judgment threshold is 0.5, corresponding to a concentration cutoff of 0.02 mg / L. The integration time of the colloidal gold reader can be set to 100 ms, 150 ms, or 200 ms, and the detection wavelength is 520 nm, with a tolerance of ±5 nm. After the test strip is inserted into the reader, the instrument automatically scans the test line and the quality control line area and calculates the color intensity ratio. If the ratio is 0.3, the concentration is determined to be greater than 0.02 mg / L; if the ratio is 0.7, the concentration is determined to be between 0.005 and 0.02 mg / L. Calibration standards are used at concentrations of 0.005 mg / L, 0.02 mg / L, and 0.1 mg / L. Standard curves are generated through linear regression analysis, with a correlation coefficient required to be greater than 0.98. The reader's optical system is calibrated using a standard colloidal gold solution, with a signal acquisition frequency of 10 times per second. This procedure provides a rapid and objective method for determining concentrations, suitable for on-site screening of agricultural products or environmental samples. Technical Results: By precisely controlling coating parameters, label-probe coupling conditions, and chromatographic structure assembly, rapid detection of thiamethoxam concentrations is achieved. Clear thresholds and standardized operating procedures reduce the risk of human error, making this method suitable for rapid on-site screening.

[0006] According to another embodiment of the present invention, the coating concentration of the anti-mouse IgG antibody is 0.9-1.1 mg / mL, and the coating concentration of the thiamethoxam-BSA antigen is 0.5-0.7 mg / mL; the concentration ratio of the anti-mouse IgG antibody to the thiamethoxam-BSA antigen 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 mg / mL, 1.0 mg / mL, and 1.5 mg / mL of anti-mouse IgG antibody, and spraying it onto the quality control line area of ​​the nitrocellulose membrane using a non-contact spotter at a spray volume of 1.0 μL / cm; preparing a thiamethoxam-BSA antigen concentration gradient of 0.2 mg / mL, 0.6 The coating solutions of 1.0 mg / mL and 1.0 mg / mL were sprayed onto the test line area at the same spraying volume; the sprayed nitrocellulose membrane was placed in a 37°C drying oven to solidify for 3 hours; the antibody concentration range of 0.9-1.1 mg / mL with a coefficient of variation of the quality control line signal less than 5% and the antigen concentration range of 0.5-0.7 mg / mL with a linear correlation coefficient of the ratio of the test line signal to the quality control line signal greater than 0.98 were screened by colloidal gold color intensity measurement; the coating solutions of the anti-mouse IgG antibody and thiamethoxam-BSA antigen contained 0.05 mol / L carbonate buffer (pH 9.4) and 0.05 mol / L phosphate buffer (pH 7.6), respectively, and the spraying speed was 12 mm / s. The coating concentration of anti-mouse IgG antibody can be set at 0.9 mg / mL, 1.0 mg / mL, or 1.1 mg / mL, and the coating concentration of thiamethoxam-BSA antigen can be set at 0.5 mg / mL, 0.6 mg / mL, or 0.7 mg / mL, with a concentration ratio of 1.0:0.6. The antibody coating solution can be 0.05 mol / L carbonate buffer (pH 9.4), and the antigen coating solution can be 0.05 mol / L phosphate buffer (pH 7.6). A non-contact spotter can be used for spraying. The coating should be applied to 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 3 hours. After curing, the color intensity is measured using a colloidal gold reader (detection wavelength 520 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 thiamethoxam-BSA antigen was set at 0.2 mg / mL, 0.6 mg / mL, and 1.0 mg / mL. The spraying parameters were: spray volume 1.0 μL / cm2, spray speed 12 mm / s. The cured nitrocellulose membrane was measured using a colloidal gold reader to determine the coefficient of variation of the color intensity of the control line. An antibody concentration range (0.9-1.1 mg / mL) with a coefficient of variation of less than 5% was selected. The linear correlation coefficient (R²) of the signal ratio between the test line and the control line was also measured. An antigen concentration range (0.5-0.7 mg / mL) with an R² greater than 0.98 was selected. The experiment was repeated three times, and the average value was used as the screening basis. The coating solution for the control line can contain 0.05 mol / L carbonate buffer (pH 9.4), 1.5% bovine serum albumin, and 0.02% sodium azide; the coating solution for the test line can contain 0.05 mol / L phosphate buffer (pH 7.6), 1.0% sucrose, and 0.03% Tween-20. The spray-coated nitrocellulose membrane can be installed in the middle of the test strip's chromatography structure, downstream of the gold pad and connected to the absorbent paper. During chromatography, the ambient temperature can be maintained at 22-25°C, the humidity at 50-60%, and the flow rate at 0.25-0.35 mL / min. Technical Results: By limiting the concentration ratio (1.0:0.6) of anti-mouse IgG antibody (0.9-1.1 mg / mL) to thiamethoxam-BSA antigen (0.5-0.7 mg / mL), the coefficient of variation of the control line's colorimetric 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 0.98. 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. A gradient coating screening method, combining differentiated formulations of carbonate buffer (pH 9.4) and phosphate buffer (pH 7.6), enhanced antibody binding stability and antigen activity, respectively, while minimizing activity loss during the coating process.

[0007] According to another embodiment of the present invention, the colloidal gold particle size can be selected from 25 nm, 30 nm or 35 nm and synthesized by sodium citrate reduction method. During carboxylation modification, the concentration of sodium citrate can be set to 1.0%, 1.5% or 2.0%, the reaction temperature can be controlled to 80°C, 85°C or 90°C, and the reaction time is 15 minutes, 20 minutes or 25 minutes. The concentration of the colloidal gold dispersion after carboxylation can be adjusted to 0.5% w / v, 1.0% w / v or 1.5% w / v. The reaction can be performed using a magnetic stirrer set at 300 rpm, 400 rpm, or 500 rpm. The reaction vessel is a three-necked flask equipped with a condenser reflux device. Sodium citrate can be used as the carboxylation reagent and should be purchased from a chemical reagent supplier. The colloidal gold particle size is verified using dynamic light scattering, with a particle size distribution coefficient of variation of less than 8%. This step ensures uniform colloidal gold particle size and a stable surface carboxyl group density, providing a foundation for subsequent antibody conjugation. The mass ratio of colloidal gold to antibody can be set at 1:18, 1:20, or 1:22. The coupling reaction is performed in MES buffer at pH 5.5, 5.8, or 6.0, with a buffer concentration of 0.05 mol / L, 0.1 mol / L, or 0.15 mol / L. The final concentration of the crosslinker carbodiimide can be set at 15 mmol / L, 17.5 mmol / L, or 20 mmol / L, and the final concentration of N-hydroxysuccinimide can be set at 7.5 mmol / L, 8.5 mmol / L, or 10 mmol / L. The reaction temperature can be controlled at 25°C, 26°C, or 28°C, the shaking frequency at 100 rpm, 150 rpm, or 200 rpm, and the reaction time at 2 hours, 2.5 hours, or 3 hours. The coupling reaction can be performed on a thermostatic shaker, protected from light to prevent fluorescence quenching. After coupling, the centrifuge speed is set to 10,000 rpm, 12,000 rpm, or 15,000 rpm for 10, 15, or 20 minutes. The wash buffer is PBS (pH 7.4) containing 0.1% bovine serum albumin, and the wash cycle is repeated three times. MES buffer and cross-linking agent can be purchased from biochemical reagent companies, and the anti-thiamethoxam antibody is a monoclonal antibody purchased from a biotechnology company. This step achieves efficient coupling of the antibody to colloidal gold, ensuring the activity and stability of the labeled probe. The labeled probe concentration after coupling can be set to 0.8 mg / mL, 1.0 mg / mL, or 1.2 mg / mL, and the spray volume can be controlled to 1.5 μL / mm, 1.8 μL / mm, or 2.0 μL / mm. The gold label pad can be made of glass fiber with a thickness of 0.3 mm, 0.4 mm, or 0.5 mm. The sprayed gold label pad is cured in a drying oven at a temperature of 38°C, 40°C, or 42°C, and a drying time of 1.2 hours, 1.5 hours, or 2 hours. A non-contact spray coating device can be used, with a nozzle aperture of 50 μm, 80 μm, or 100 μm, and the distance between the nozzle and the gold pad surface set to 1.5 mm, 2.0 mm, or 2.5 mm. A stainless steel grid is placed inside the drying oven, and the gold pad is laid flat on the grid surface, avoiding any bending or folding. Spray uniformity is tested using a UV spectrophotometer, with the absorbance coefficient of variation required to be less than 5%. This step ensures even distribution of the probe on the gold pad, improving the consistency of the detection signal. The colloidal gold absorbance peak at 520 nm can be set to 0.8, 1.0, or 1.2, and is detected by UV-Vis spectrophotometry. The colloidal gold reader allows a ±5 nm tolerance for the detection wavelength, and the integration time can be set to 100 ms, 150 ms, or 200 ms. Particle size distribution is verified by dynamic light scattering, with a detection temperature of 25°C ± 1°C and a quartz sample cell. Instrument calibration can be performed using a standard colloidal gold solution, once per batch. Test data is automatically analyzed by software, generating an absorbance curve and particle size distribution report. This step ensures that the optical properties of the colloidal gold match the test equipment, minimizing the impact of batch variability on results. Technical Results: By precisely controlling colloidal gold particle size, coupling conditions, and spraying parameters, efficient preparation of labeled probes is achieved. Rigorous validation procedures ensure the optical properties of colloidal gold are compatible with detection equipment, improving the sensitivity and repeatability of test strips, making them suitable for rapid on-site testing.

[0008] According to another embodiment of the present invention, the mass ratio of colloidal gold to antibody can be set to 1:18, 1:20 or 1:22. The coupling reaction is carried out in MES buffer at pH 5.0, 5.5 or 6.0, and the buffer concentration can be selected from 0.05 mol / L, 0.1 mol / L or 0.15 mol / L. The final concentration of the cross-linking agent 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 reaction temperature can be controlled to 25°C, 26°C or 28°C, the oscillation frequency is 100 rpm, 150 rpm or 200 rpm, and the reaction time is 1.5 hours, 2.0 hours or 2.5 hours. The coupling reaction can be performed on a thermostatic shaker, protected from light to prevent photodegradation of the colloidal gold. The reaction vessel can be a centrifuge tube or glass beaker made of polypropylene or borosilicate glass. MES buffer and crosslinker can be purchased from biochemical reagent companies, and the anti-thiamethoxam antibody is a monoclonal antibody purchased from a biotechnology company. This step ensures efficient coupling of the antibody to the colloidal gold, forming a stable labeled probe. After coupling, wash three times with PBS (pH 7.4) containing 0.1% bovine serum albumin, using a volume twice the reaction mixture for each wash. The centrifuge speed can be set to 8,000 rpm, 10,000 rpm, or 12,000 rpm, and the centrifugation time can be controlled to 5, 10, or 15 minutes. After washing, the labeled probe is dispersed in 0.01 mol / L PBS buffer at concentrations of 0.5 mg / mL, 1.0 mg / mL, or 1.5 mg / mL. Centrifugation can be performed in a benchtop centrifuge using chemically resistant polypropylene tubes. The bovine serum albumin in the wash buffer is analytical grade and purchased from a standard reagent supplier. After centrifugation, the supernatant is analyzed for residual antibody using a UV spectrophotometer to ensure a coupling efficiency greater than 90%. This step removes unbound antibody and improves the purity of the labeled probe. The coupled labeled probe was evenly sprayed onto a glass fiber gold pad at a spray rate of 1.0 μL / mm, 1.5 μL / mm, or 2.0 μL / mm. The gold pad thickness could be 0.3 mm, 0.4 mm, or 0.5 mm, and the width could be 3 mm, 4 mm, or 5 mm. The sprayed gold pad was then cured in a drying oven at 35°C, 38°C, or 40°C, with a controlled drying time of 1.0 hour, 1.2 hours, or 1.5 hours. A non-contact spraying device can be used, with a nozzle aperture of 50 μm, 80 μm, or 100 μm, and the distance between the nozzle and the gold pad surface set to 1.0 mm, 1.5 mm, or 2.0 mm. A stainless steel grid is placed inside the drying oven, and the gold pad is laid flat on the grid surface, avoiding folding or contact with foreign matter. Spraying uniformity is tested using a UV spectrophotometer, with the absorbance coefficient of variation required to be less than 5%. This step ensures even distribution of the probe on the gold pad, improving the consistency of the detection signal. Technical Results: By optimizing coupling reaction conditions, rigorous cleaning procedures, and precise spraying parameters, the labeled probe was efficiently prepared and stably immobilized. The drying and curing process ensured the physical stability of the gold label pad and uniform liquid flow during chromatography. This ultimately improved the test strip's sensitivity and repeatability, making it suitable for rapid on-site testing.

[0009] According to another embodiment of the present invention, the selection and assembly of the nitrocellulose membrane, sample pad and absorbent paper meet the following conditions: the pore size of the nitrocellulose membrane is 9-11 μm, and commercially available nitrocellulose membrane materials can be selected, which are assembled in the middle position of the chromatographic structure of the test strip, located downstream of the gold mark pad and connected to the absorbent paper; the sample pad can be made of glass fiber material with a thickness of 0.3-0.5 mm, assembled at the front end of the test strip, in direct contact with the gold mark pad, and the liquid penetration time is 5-8 seconds; the water absorption capacity of the absorbent paper is 250 mL / m², and commercially available highly absorbent glass fiber material can be selected, assembled at the end of the nitrocellulose membrane, and fixed to the hard plastic base plate by double-sided tape; the contact width between the nitrocellulose membrane and the sample pad is 3-5 mm, and the contact width with the absorbent paper is 4-6 mm. The consistency of the liquid migration path is verified by a chromatographic migration experiment, and the coefficient of variation is less than 10%; during the chromatography process, the liquid flow rate of the nitrocellulose membrane is 0.25-0.35 mL / min, ambient temperature 22-25°C, humidity 50-60%. The pore size of the nitrocellulose membrane can be set to 9 μm, 10 μm, or 11 μm, and commercially available nitrocellulose membrane materials can be used. The sample pad can be made of glass fiber with a thickness of 0.3 mm, 0.4 mm, or 0.5 mm, and the liquid penetration time can be set to 6 seconds. The absorbent paper can have a water absorption capacity of 250 mL / m² and can be made of commercially available highly absorbent glass fiber. The nitrocellulose membrane can be installed in the middle of the test strip's chromatographic structure, downstream of the gold label pad, with a contact width of 5 mm between the absorbent paper and the sample pad. The sample pad can be installed at the front of the test strip, in direct contact with the gold label pad, with a contact width of 4 mm. The contact width between the nitrocellulose membrane and the sample pad can be set to 3 mm, 4 mm, or 5 mm, and the contact width with the absorbent paper can be set to 4 mm, 5 mm, or 6 mm. The thickness of the rigid plastic base can be set to 0.8 mm, and the surface adhesive can be acrylic adhesive with a coating thickness of 0.1 mm. The absorbent paper can be secured to the end of the base using double-sided tape, the width of which can be set to 2 mm. For chromatographic migration experiments, a 0.02 mg / L thiamethoxam standard can be added as the sample. After dripping onto the sample pad, the liquid migration path is recorded, and the path consistency is verified using colloidal gold colorimetric signals. During chromatography, the liquid flow rate through the nitrocellulose membrane can be set to 0.25 mL / min, 0.3 mL / min, or 0.35 mL / min. This flow rate is controlled by adjusting the absorbent capacity of the absorbent paper (250 mL / m²) and the membrane pore size (9-11 μm). The ambient temperature can be maintained at 23°C and the humidity at 55%. After chromatography, the amount of liquid remaining on the nitrocellulose membrane surface can be verified gravimetrically, with a residual threshold of 5 μL. Before weighing, the test strip can be allowed to rest for 2 minutes to avoid external environmental interference. Technical Results: By limiting the pore size of the nitrocellulose membrane (9-11 μm), sample pad thickness (0.3-0.5 mm), and absorbent paper capacity (250 mL / m²), the chromatographic flow rate is stabilized at 0.25-0.35 mL / min, with a coefficient of variation of the liquid migration path less than 10%. Standardized contact widths (3-5 mm and 4-6 mm) minimize chromatographic drift, while a rigid plastic base and double-sided tape secure the assembly securely. Controlled ambient temperature (22-25°C) and humidity (50-60%) further ensure reproducible test results, meeting the practical needs of rapid thiamethoxam residue detection.

[0010] According to another embodiment of the present invention, the test line coating solution is prepared by comprising 0.05-0.1 mol / L phosphate buffer (pH 7.6), 0.5%-1.5% sucrose 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 7.4, 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 0.01%, 0.03%, or 0.05%. A magnetic stirrer is used for mixing, and the stirring time can be controlled to 15 minutes, 20 minutes, or 25 minutes. The pH of the buffer can be calibrated using a pH meter with an accuracy of ±0.1. Phosphate buffer is prepared from a mixture of disodium hydrogen phosphate and potassium dihydrogen phosphate in appropriate proportions. 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 matter. The filtered coating solution is stored at 4°C and is valid for no more than three days. This step ensures uniform and stable composition of the coating solution and prevents nozzle clogging during spraying. The coating solution for the quality control line is prepared by mixing 0.05-0.1 mol / L carbonate buffer (pH 9.4), 1%-2% bovine serum albumin, and 0.01%-0.03% sodium azide. The carbonate buffer concentration 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 sodium azide concentration can be selected to be 0.01%, 0.02%, or 0.03%. Dissolution is aided 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. The coating solution for the test and control lines is sprayed using a non-contact spotter. 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 sprayed nitrocellulose membrane is cured in a drying oven at a temperature of 35°C, 37°C, or 38°C, and the curing time can be set to 2.5 hours, 3.0 hours, or 3.5 hours. A non-contact spotter 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 can be set to 1.0 mm, 1.5 mm, or 2.0 mm. A multi-layer stainless steel grid is placed in the 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. Technical Results: By optimizing the composition and spraying parameters of the coating solution for the test and control lines, the stable immobilization of antigens and antibodies is ensured. Stringent formulation and curing conditions reduce nonspecific binding interference, improving the sensitivity and repeatability of the test strips. Suitable for rapid on-site testing, this method delivers reliable results and simple operation.

[0011] According to another embodiment of the present invention, the driving force of the chromatography is provided by absorbent paper, the water absorption rate is 0.2-0.4 mL / min, and the chromatography environment temperature is 20-25°C; the migration distance of the immune complex on the nitrocellulose membrane is 40-60 mm, and the migration time is 8-12 minutes. The water absorption capacity of the absorbent paper can be set to 200 mL / m², 250 mL / m², or 300 mL / m², and the water absorption rate can be adjusted to 0.2 mL / min, 0.3 mL / min, or 0.4 mL / min. The absorbent paper can be made of commercially available highly absorbent glass fiber material, assembled at the end of the nitrocellulose membrane, and fixed to the hard plastic base with double-sided tape. The chromatographic driving force is achieved by the capillary action of the absorbent paper, and the liquid flow rate is controlled by adjusting the water absorption capacity of the absorbent paper and the pore size of the nitrocellulose membrane (8-12 μm). The ambient temperature can be set to 20°C, 23°C, or 25°C, and the humidity is maintained at 50%-60%. The migration distance of the immune complex on the nitrocellulose membrane can be set to 40 mm, 50 mm, or 60 mm, and the migration time can be set to 8 minutes, 10 minutes, or 12 minutes. The nitrocellulose membrane can be installed in the middle of the test strip's chromatographic structure, downstream of the gold pad and connected to the absorbent paper. During the chromatography process, the migration path of the immune complex can be monitored in real time using a colloidal gold colorimetric signal. The signal acquisition device can be a colloidal gold reader with a detection wavelength of 520 nm and an integration time of 100-200 ms. The uniformity of the migration path is monitored using line scanning with a scan width of 1.0 mm and a spacing of 0.2 mm between detection points. The chromatography environment temperature can be controlled at 20-25°C using a thermostat, and the humidity can be maintained at 50%-60% using a humidity regulator. After chromatography is completed, the amount of residual liquid on the nitrocellulose membrane surface can be verified by weighing using a precision balance, with the residual threshold set at 5 μL. The test strip can be left to rest for 2 minutes before weighing to avoid interference from external humidity. Migration efficiency is assessed by the ratio of the color intensity of the test line to the color intensity of the control line (T / C). When T / C is less than 0.5, the migration path consistency is considered to meet the standard. Technical Results: By limiting the water absorption rate (0.2-0.4 mL / min) and the chromatographic environment temperature (20-25°C), the migration time of the immune complex is stabilized at 8-12 minutes, and the coefficient of variation of the migration path length (40-60 mm) is less than 10%. The synergistic effect of the absorbent paper and nitrocellulose membrane ensures a uniform chromatographic flow rate, reducing signal deviations caused by sample retention or uneven diffusion. Controlling the ambient temperature and humidity further reduces background interference, and the reproducibility of the colloidal gold colorimetric signal acquisition meets practical application requirements.

[0012] According to another embodiment of the present invention, the sample to be tested can be dissolved in a phosphate buffer of pH 7.2, 7.4, or 7.6. The buffer concentration can be selected from 0.05 mol / L, 0.1 mol / L, or 0.15 mol / L, and the amount of Tween-20 added can be 0.5%, 1.0%, or 1.5%. The sample is mixed using a vortex oscillator during dissolution, and the oscillation time can be set to 30 seconds, 60 seconds, or 90 seconds. The amount of sample solution added can be controlled to 50 μL, 100 μL, or 150 μL, and after addition, the sample is allowed to stand for 10 seconds, 20 seconds, or 30 seconds to initiate chromatography. The sample solution can be added dropwise using a pipette with a volume 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 250 mL / m², 280 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. During chromatography, the immune complex competes for binding with the thiamethoxam-BSA antigen at the test line. Labeled probe not occupied by thiamethoxam in the sample binds to the test line and develops color. The antigen coating concentration at the test line can be set to 0.5 mg / mL, 0.6 mg / mL, or 0.7 mg / mL, and the anti-mouse IgG antibody coating concentration at the control line can be set to 0.9 mg / mL, 1.0 mg / mL, or 1.1 mg / mL. The competition binding reaction time can be controlled to 5, 6, or 7 minutes, and the chromatography buffer flow rate can be adjusted to 0.2 mL / min, 0.25 mL / min, or 0.3 mL / min. 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 colorimetric signal is detected using a colloidal gold reader with an excitation wavelength of 520 ± 5 nm. 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 and reduces false positive or negative results. The thiamethoxam concentration in the sample is calculated by the ratio of the color intensity of the test line to the control line. When the color intensity of the test line is less than 50% of the signal intensity of the control line (the ratio threshold is 0.5), the thiamethoxam concentration in the sample is considered to be greater than 0.02 mg / L. The integration time of the colloidal gold reader can be set to 100 ms, 150 ms, or 200 ms, and the detection wavelength allows a deviation of ±5 nm. Data calculation is performed by the built-in software, which uses an algorithm based on a precalibrated standard curve with a concentration range of 0.005 mg / L, 0.02 mg / L, and 0.1 mg / L. A portable colloidal gold reader can be used, with the detection channel matched to 520 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 above 0.02 mg / L; a ratio of 0.7 indicates a concentration within the range of 0.005-0.02 mg / L. A standard curve is generated using linear regression analysis, with a correlation coefficient of greater than 0.98. This procedure provides rapid and objective concentration determination and is suitable for on-site screening of agricultural products or environmental samples. Technical Results: Through standardized sample processing, precise control of chromatography parameters, and colorimetric signal analysis, this system enables rapid and accurate detection of thiamethoxam concentrations. Clear thresholds and standardized operating procedures reduce the risk of human error, making it suitable for rapid on-site screening, with reliable results and easy operation.

[0013] According to another embodiment of the present invention, the phosphate buffer contains 0.05-0.15 mol / L phosphate and 0.5-1.5% Tween-20; the detection limit is 0.02 mg / L, and the quantitative range is 0.02-1.0 mg / L. The concentration of phosphate buffer can be set to 0.05 mol / L, 0.1 mol / L, or 0.15 mol / L, and the concentration of Tween-20 can be set to 0.5%, 1.0%, or 1.5%. To prepare the buffer, weigh potassium dihydrogen phosphate and disodium hydrogen phosphate, mix them in proportion, dissolve them in deionized water, and adjust the pH to 7.4. Tween-20 can be added dropwise and stirred until completely dissolved. The prepared buffer can be stored at 4°C and returned to room temperature before use. To dissolve the sample, weigh 0.5 g of mulberry leaf sample, add 10 mL of buffer, homogenize for 2 minutes, and centrifuge to obtain the supernatant. The detection limit can be verified using a thiamethoxam standard at a concentration of 0.02 mg / L, while the quantification range can be verified using samples at gradient concentrations of 0.02 mg / L, 0.1 mg / L, 0.5 mg / L, and 1.0 mg / L. During testing, the sample solution is added dropwise to the sample pad of the test strip in a volume of 100 μL. After 30 seconds, the chromatography step is initiated. After chromatography, the colloidal gold colorimetric signal can be detected using a reader at a wavelength of 520 nm and an integration time of 150 ms. The detection limit is determined when the color intensity of the test line is less than 50% of the intensity of the control line. The linear correlation coefficient (R²) for the quantification range must be greater than 0.98. The sample pad can be made of 0.4 mm thick glass fiber and attached to the front of the test strip, in direct contact with the gold pad. The sample liquid penetration time can be controlled within 6 seconds. During chromatography, the ambient temperature can be set to 23°C and the humidity can be maintained at 55%. The base of the test strip can be made of 0.8 mm thick hard plastic, coated with acrylic adhesive to secure the chromatography components. After the test is completed, the amount of residual liquid on the nitrocellulose membrane can be verified gravimetrically, with a residual threshold set at 5 μL. Technical Results: The formulation of 0.05-0.15 mol / L phosphate buffer and 0.5-1.5% Tween-20 enhances sample dispersion, reduces nonspecific adsorption, and improves the signal-to-background ratio. The detection limit of 0.02 mg / L and the quantitative range of 0.02-1.0 mg / L cover the toxicity threshold and actual residual concentration range of thiamethoxam in silkworms. A gradient sample validation method, combined with standardized signal acquisition by a colloidal gold reader, ensures reproducible and reliable test results, meeting the needs for rapid monitoring of thiamethoxam residues in mulberry leaves and silkworms.

[0014] According to another embodiment of the present invention, the base plate can be made of a hard plastic material, such as PVC, polycarbonate, or ABS, and can be set to a thickness of 0.5 mm, 0.8 mm, or 1.0 mm. The surface flatness of the material must be less than 0.1 mm, with no burrs on the edges. The base plate can be designed to be 5 mm, 6 mm, or 7 mm wide, with a length that matches the chromatographic structure of the test strip. The base plate can be manufactured using an injection molding process, with a mold accuracy requirement of ±0.05 mm. The rigid plastic sheet is purchased from a standard plastics supplier and can be cut using a CNC cutting machine. The base plate is assembled onto the bottom layer of the test strip and serves as a support structure. Before applying the adhesive, the surface is plasma treated to improve adhesion. This step ensures that the base plate possesses sufficient mechanical strength and dimensional stability to meet the requirements of chromatography assembly. The adhesive can be pressure-sensitive adhesive or double-sided tape, with coating thicknesses of 0.05 mm, 0.1 mm, or 0.15 mm. Application can be done with a roller coater or sprayer. The adhesive coating area should be aligned with the test strip's chromatographic structure, leaving a 0.5 mm, 1.0 mm, or 1.5 mm adhesive-free zone at the edge. Curing conditions can be set to room temperature for 24 hours, baking at 40°C for 30 minutes, or UV curing for 10 seconds. A roller coater can be used for uniform adhesive application, with the coating speed set to 5 mm / s, 10 mm / s, or 15 mm / s. Adhesives are purchased from industrial adhesive suppliers and meet biocompatibility standards. The coated substrate is then laminated with the nitrocellulose membrane, gold label pad, sample pad, and absorbent paper in this order, using pressures set to 0.1 MPa, 0.15 MPa, or 0.2 MPa. This step ensures a tight fit between the layers, preventing interlayer displacement that could lead to fluid flow abnormalities. The baseplate's bending resistance can be verified through a three-point bending test, with a bending radius set to 50 mm, 100 mm, or 150 mm, and a maximum deformation requirement of less than 1%. Dimensional stability testing is conducted at a temperature of 20-30°C and a humidity of 40-80%. After 24 hours, the length change is less than 0.5%. Adhesive peel strength is tested using a tensile testing machine, with thresholds set to 1.0 N / cm, 1.5 N / cm, or 2.0 N / cm. Mechanical testing can be performed using a universal material testing machine at a speed of 5 mm / min. An environmental testing chamber is used to control temperature and humidity, with a temperature fluctuation of ±1°C and a humidity fluctuation of ±5%. Test data is recorded and analyzed using software, with outliers removed and the average calculated. This step ensures the structural integrity of the baseplate during storage and use, preventing deformation or debonding from affecting test results. Technical Results: Through the rational selection of baseplate materials, precise control of adhesive coating parameters, and rigorous performance verification, the stability and durability of the test strip's chromatographic structure are ensured. The baseplate's physical properties and adhesive strength are tailored to testing requirements, reducing detection errors caused by mechanical deformation or interlayer separation, making it suitable for rapid on-site testing.

[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 of Current 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 specification and exemplary embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, further modifications can be easily realized. Therefore, the present invention is not limited to the specific details and illustrations shown and described herein without departing from the general concept defined by the claims and their equivalents.

Claims

1. A method for preparing a thiamethoxam colloidal gold test strip, characterized in that: The following steps are involved: preparing a nitrocellulose membrane, coating the thiamethoxam-BSA antigen at the detection line of the nitrocellulose membrane, and coating the anti-mouse IgG antibody at the quality control line; preparing a gold label pad, coupling colloidal gold with an anti-thiamethoxam antibody to form a labeled probe, and fixing the labeled probe on the gold label pad; Assembling the test strip, stacking the nitrocellulose membrane, the gold label 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; Thiamethoxam in the sample combines with the labeled probe on the gold label 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 detection line, it binds to the thiamethoxam-BSA antigen and is captured, and the detection line result is displayed by colloidal gold color development; When the immune complex passes through the quality control line, it combines with the anti-mouse IgG antibody, and the quality control line result is displayed by colloidal gold color development; The color intensity of the test line and the quality control line is used to determine the concentration of thiamethoxam in the sample; The color intensity of the test line and the quality control line is used to determine the concentration of thiamethoxam in the sample by calculating the ratio of the color intensity of the test line to the color intensity of the quality control line; When the ratio is less than 0.5, the concentration of thiamethoxam in the sample is determined to be higher than 0.02 mg / L; When the ratio is between 0.5 and 1.0, the concentration of thiamethoxam in the sample is determined to be within the range of 0.005 to 0.02 mg / L; When the ratio is equal to 1.0, the concentration of thiamethoxam in the sample is determined to be less than 0.005 mg / L; The color intensity was detected by a colloidal gold reader at a wavelength of 520 nm with an integration time of 100-200 ms.

2. The method for preparing the thiamethoxam colloidal gold test strip according to claim 1, wherein The coating concentration of the anti-mouse IgG antibody is 0.9-1.1 mg / mL, and the coating concentration of the thiamethoxam-BSA antigen is 0.5-0.7 mg / mL; The concentration ratio of the anti-mouse IgG antibody to the thiamethoxam-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 mg / mL, 1.0 mg / mL, and 1.5 mg / mL of anti-mouse IgG antibody. Use a non-contact spotter to spray the solution onto the quality control line of the nitrocellulose membrane at a spray volume of 1.0 μL / cm. Prepare coating solutions with a concentration gradient of 0.2 mg / mL, 0.6 mg / mL, and 1.0 mg / mL of thiamethoxam-BSA antigen and spray them onto the test line area at the same spraying amount; The sprayed nitrocellulose membrane was placed in a drying oven at 37°C for 3 hours to cure; The antibody concentration range of 0.9-1.1 mg / mL with a coefficient of variation of the quality control line signal of less than 5% and the antigen concentration range of 0.5-0.7 mg / mL with a linear correlation coefficient of the ratio of the test line signal to the quality control line signal of greater than 0.98 were screened out by colloidal gold color intensity determination. The coating solutions of the anti-mouse IgG antibody and thiamethoxam-BSA antigen respectively contain 0.05 mol / L carbonate buffer (pH 9.4) and 0.05 mol / L phosphate buffer (pH 7.6), and the spraying speed is 12 mm / s.

3. The method for preparing the thiamethoxam colloidal gold test strip according to claim 1, wherein: The colloidal gold has a particle size of 25-35 nm, and the surface carboxyl groups are modified by sodium citrate reduction method and coupled with anti-thiamethoxam antibody at a mass ratio of 1:18-1:22; The coupling process comprises the following steps: Carboxylated colloidal gold and anti-thiamethoxam antibody were mixed in MES buffer (pH 5.5-6.0), and carbodiimide and N-hydroxysuccinimide (7.5 mmol / L) were added to the mixture at a final concentration of 15 mmol / L. The mixture was shaken at 26°C for 2 hours. After the reaction was completed, the cells were washed three times with 0.01 mol / L PBS buffer (pH 7.4) containing 0.1% bovine serum albumin and centrifuged to remove unbound antibodies; The particle size distribution of the colloidal gold was verified by dynamic light scattering, and the coefficient of variation of the particle size was less than 8%; The coupled labeled probe was sprayed onto a gold label pad made of glass fiber at a concentration of 0.8-1.2 mg / mL and a spraying volume of 1.5 μL / mm; The sprayed gold label pad was dried at 38°C for 1.2 hours to form a stable probe fixation layer; The colloidal gold has an absorbance peak of 0.8-1.2 at a wavelength of 520 nm, which matches the detection channel of a colloidal gold reader.

4. The method for preparing the thiamethoxam colloidal gold test strip according to claim 1, wherein The selection and assembly of the nitrocellulose membrane, sample pad, and absorbent paper meet the following conditions: The pore size of the nitrocellulose membrane is 9-11 μm, and commercially available nitrocellulose membrane materials can be selected. It is assembled in the middle position of the chromatographic structure of the test strip, located downstream of the gold label pad and connected to the absorbent paper; The sample pad can be made of glass fiber with a thickness of 0.3-0.5 mm, assembled at the front end of the test strip, in direct contact with the gold standard pad, and the liquid penetration time is 5-8 seconds; The absorbent paper has a water absorption capacity of 250 mL / m² and can be made of commercially available highly absorbent glass fiber material. It is assembled at the end of the nitrocellulose membrane and fixed to the hard plastic base plate with double-sided tape. The contact width between the nitrocellulose membrane and the sample pad is 3-5 mm, and the contact width with the absorbent paper is 4-6 mm. The consistency of the liquid migration path is verified by chromatography migration experiments, and the coefficient of variation is less than 10%; During the chromatography process, the liquid flow rate of the nitrocellulose membrane is 0.25-0.35 mL / min, the ambient temperature is 22-25° C., and the humidity is 50-60%.

5. The method for preparing the thiamethoxam colloidal gold test strip according to claim 1, wherein The preparation of the gold label pad comprises the following steps: Colloidal gold and anti-thiamethoxam antibody were mixed in MES buffer at pH 5.0-6.0, and carbodiimide and N-hydroxysuccinimide were added at a final concentration of 10-20 mmol / L and 5-10 mmol / L, respectively. The mixture was shaken and reacted 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 containing 0.1% bovine serum albumin to remove unbound antibodies; The coupled labeled probe was sprayed onto the gold label pad made of glass fiber at a concentration of 0.5-1.5 mg / mL and a spray volume of 1.0-2.0 μL / mm; The sprayed gold label pad is dried at 35-40° C. for 1.0-1.5 hours to form a stable probe fixing layer.

6. The method for preparing the thiamethoxam colloidal gold test strip according to claim 1, wherein: The coating solution for the thiamethoxam-BSA antigen at the test line contains 0.05-0.1 mol / L phosphate buffer, 0.5%-1.5% sucrose, and 0.01%-0.05% Tween-20; the coating solution for the anti-mouse IgG antibody at the quality control line contains 0.05-0.1 mol / L carbonate buffer, 1%-2% bovine serum albumin, and 0.01%-0.03% sodium azide.

7. The method for preparing the thiamethoxam colloidal gold test strip according to claim 1, wherein: The driving force of the chromatography is provided by absorbent paper, the water absorption rate is 0.2-0.4 mL / min, and the chromatography environment temperature is 20-25°C; the migration distance of the immune complex on the nitrocellulose membrane is 40-60 mm, and the migration time is 8-12 minutes.

8. A method for using a thiamethoxam colloidal gold test strip, characterized in that: The following steps are involved: The sample to be tested was dissolved in a phosphate buffer solution of pH 7.2-7.6 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 gold label pad to form an immune complex; When the immune complex moves to the detection line, it competes with the pre-coated thiamethoxam-BSA antigen for binding, and the labeled probe not occupied by thiamethoxam 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 thiamethoxam in the sample was calculated by the color intensity ratio of the test line and the quality control line; When the color intensity of the test line is lower than 50% of the color intensity of the quality control line, the concentration of thiamethoxam in the sample is determined to be higher than 0.02 mg / L.

9. The application method of the thiamethoxam colloidal gold test strip according to claim 8, characterized in that: The phosphate buffer contains 0.05-0.15 mol / L phosphate and 0.5-1.5% Tween-20; the detection limit is 0.02 mg / L, and the quantitative range is 0.02-1.0 mg / L.

10. The thiamethoxam colloidal gold test strip according to claim 1 or 8, characterized in that: The bottom plate is made of hard plastic material with a thickness of 0.5-1.0 mm, and the surface is coated with an adhesive to fix the chromatography structure.