Preparation method of cyhalothrin colloidal gold quantitative test strip

Abstract

The invention relates to the technical field of pesticide residue detection, in particular to a preparation method of a cyhalothrin colloidal gold quantitative test strip, and solves the technical problems of non-uniform antibody labeling, coating layer cracking and low detection sensitivity in the traditional process. Taking 1 ml of 40-nanometer colloidal gold particles, adding potassium carbonate, mixing and reacting for 5-15 minutes, then adding 5-7 micrograms of cyhalothrin monoclonal antibody, reacting, and finally adding confining liquid to prepare a colloidal gold labeled antibody solution; spraying the obtained solution on a glass fiber pad according to the spraying amount of 9.0-10.8 microliters per centimeter to prepare a gold-labeled pad; a specific coating buffer solution is used for preparing a quality control line solution and a detection line solution of 0.7-0.9 mg / ml cyhalothrin-BSA antigen, a nitrocellulose membrane is coated with the quality control line solution and the detection line solution, and after confining liquid treatment, centrifugation and resuspension, the nitrocellulose membrane is dried for 70-74 hours in inert gas with the temperature of 45 DEG C and the temperature of 2 DEG C; the test strip prepared by the method is used for high-precision quantitative detection of cyhalothrin residues in agricultural products.

Description

Technical Field

[0001] The invention relates to the technical field of pesticide residue detection, in particular to a method for preparing a cyhalothrin colloidal gold quantitative test strip. Background Art

[0002] In the field of pesticide residue detection, colloidal gold immunochromatographic test strips are widely used due to their rapid and convenient characteristics. However, the existing preparation methods still have several technical defects: 1. Low antibody labeling efficiency leads to insufficient sensitivity The coupling process between colloidal gold and antibodies is significantly affected by pH fluctuations. In traditional processes, the amount of potassium carbonate added varies widely (typically spanning greater than 5 μL), causing the reaction system's pH to deviate from the optimal coupling range of 6.0-6.5. When the pH falls below 6.0, colloidal gold particles are prone to irreversible aggregation; above 6.5, the antibody binding sites are insufficiently exposed. Existing technologies attempt to compensate by extending the reaction time, but after 30 minutes, the antibody's spatial conformation changes, reducing the labeling efficiency to below 80%. This discrepancy makes it difficult for test strips to achieve a detection limit exceeding 1 ppb. 2. Poor coating uniformity causes false positives During the nitrocellulose membrane coating process, ambient humidity exceeding 35% can lead to excessive diffusion of the antibody solution, resulting in a test line width exceeding the tolerance of ±0.15 mm (the standard requirement is ±0.05 mm). Existing technologies use a single centrifugation parameter (e.g., 12,000 rpm for centralized processing). The high shear force causes some antibodies to detach from the membrane pores, while the remaining antibodies develop microcracks due to stress concentration. Furthermore, if air is used during the drying phase, oxygen permeation can cause oxidative cross-linking of the antibody thiol groups, resulting in the loss of more than 20% of active sites. These deficiencies collectively result in a false positive rate of up to 15%-25% between batches. 3. Drying process damages antibody activity Existing drying technologies, which typically operate in air at 45-50°C, present a dilemma: drying times of less than 70 hours result in residual moisture exceeding 2%, accelerating antibody degradation; drying times exceeding 74 hours can cause the membrane to become brittle and fracture. Especially when temperatures exceed 47°C, the rate of antibody thermal denaturation increases exponentially, with experiments demonstrating a loss of approximately 8% in activity for every 1°C increase. Despite attempts to introduce inert gas protection, the lack of coordinated temperature and humidity control (for example, nitrogen protection becomes ineffective when humidity exceeds 10%) has made it impossible to balance drying efficiency with antibody activity retention. 4. The problem of parameter coupling is not recognized Previous studies have isolated and optimized single factors (such as controlling only the spray volume or centrifugal speed), ignoring the nonlinear coupling relationship between key processes. For example: When the spray volume of the gold label pad is lower than 9.0 μL / cm, the antibody loading is insufficient and the signal is weak; when it is higher than 10.8 μL / cm, the edge effect occurs due to solution accumulation. The speed of film stripping must be strictly matched with humidity: when the humidity is 30%, a speed lower than 0.8 μl / s will cause solution retention, while a speed higher than 1.2 μl / s will cause line breakage. Such coupling relationships have not been fully revealed, resulting in a wide process window setting (such as a centrifugal speed span of 4000 rpm) and an actual production qualification rate of less than 70%. The fundamental difficulty lies in the limitations of technical cognition The performance of colloidal gold test strips depends on multi-scale processes involving molecular interface interactions (antibody-gold particle binding), fluid control (membrane coating), and thermodynamic transfer (drying). Existing technologies do not establish a cross-scale control model: 1. Molecular level: Unquantified structure-activity relationship between antibody orientation and pH 2. Mesoscopic level: Lack of fluid dynamics simulation within membrane pores 3. Macroscopic level: The distribution pattern of drying stress field is unclear This cognitive gap has led to long-term reliance on trial and error for process optimization, making it difficult to achieve high-precision manufacturing. Summary of the Invention

[0003] One objective of the present invention is to address the challenges of low antibody labeling efficiency, uneven gold pad coating, cracking of the NC membrane coating, and insufficient detection sensitivity in the preparation of colloidal gold test strips. Conventional processes, due to wide range of reaction parameters and uncontrolled drying conditions, often lead to antibody denaturation and coating cracking, resulting in detection limits exceeding 1 ppb. This solution addresses issues such as solution diffusion, excessive line width (±0.15 mm), and free antibody residue caused by excessive humidity (>35%) during NC membrane coating. Existing high-speed centrifugation (e.g., 12,000 rpm) can cause antibody layer shedding and microcracks, resulting in a false positive rate of up to 25%. This method overcomes the problems of antibody solution clumping, accumulation at the spray edge ("coffee ring" effect), and drying migration caused by the hydrophobic substrate (contact angle 110°) during gold label pad preparation. A conventional air pressure nozzle (atomized particle size 30-50 μm) and constant temperature drying achieve antibody distribution uniformity with an RSD >25%. Eliminate cracking (roughness 15 nm) and nonspecific binding (false positive rate 25%) caused by pH mismatch (phosphate buffer pH 7.4), excessive centrifugal shear force (single 12,000 rpm), and resuspension aggregation during the blocking process. It overcomes the problems of oxygen permeation (oxidation rate > 20%), humidity fluctuation (35-65%), and film cracking (12 lines / cm) and antibody aggregation (23%) caused by damage caused by manual flipping in static drying ovens. The antibody oscillation coupling, ultrasonic atomization spraying and gradient centrifugation processes in Quan 1 were optimized to solve the problems of traditional static labeling (labeling rate 50%), air pressure spraying edge effect and single high-speed centrifugation fragmentation. Dynamic temperature drying (45°C → 52°C → 38°C) is used to balance the contradiction between dehydration efficiency and antibody thermal denaturation, avoiding constant temperature drying with residual moisture > 2% or membrane embrittlement. 40 kHz ultrasound assistance (0.5 W / cm²) was used to overcome the 35% antibody aggregation problem caused by vertical oscillation at room temperature and achieve nanoscale dispersion. After the blocking solution was added, the multi-level pores of the NC membrane were filled by vacuum gradient osmosis (from -30 kPa to -90 kPa) to eliminate nonspecific adsorption caused by the unpermeated area (background noise > 0.35). Locking in the optimal combination of broad parameters (e.g., K2CO3 3-7 μL, spray volume 9.0-10.8 μL / cm2) addresses issues such as labeling rate fluctuation (90±5%), sensitivity drift (0.5±0.3 ppb), and batch-to-batch variability >8% caused by process fluctuations. To this end, the present invention provides a method for preparing a cyhalothrin colloidal gold quantitative test strip, comprising the following steps: 1) Preparation of colloidal gold-labeled antibodies: Take 1 mL of 40 nm colloidal gold particles, add 3-7 μL of 0.2 M K2CO3 and mix for 5-15 minutes; Add 5-7 μg of cyhalothrin monoclonal antibody and react for 25-35 minutes, then add blocking solution to obtain colloidal gold labeled antibody solution; 2) Preparation of gold label pad: Spray the colloidal gold-labeled antibody solution obtained in step 1) onto the glass fiber mat at a spray rate of 9.0-10.8 μL / cm; 3) NC membrane coating: The coating buffer was prepared, wherein the coating buffer included: 500 g DDH2O, 1.741 g Na2HPO4·12H2O, 0.0068 g KH2PO4, and 0.05% NaN3; Quality control line C solution: 0.4-0.6 mg / mL anti-mouse IgG antibody; Test line T line solution: 0.7-0.9 mg / mL chlorfenapyr-BSA antigen; The dried product was dried at 45±2°C under an inert gas environment for 70-74 hours. Preferably, the present invention further comprises: Quality control line C solution: 0.4-0.6 mg / mL anti-mouse IgG antibody; Test line T line solution: 0.7-0.9 mg / mL chlorfenapyr-BSA antigen; Coat the C-line solution and T-line solution on the nitrocellulose membrane at a stripping speed of 0.8-1.2 μL / s, respectively. The operating environment humidity is ≤30%; Among them, after the film is scratched, the following steps are carried out in sequence: Add 80-120 μL of blocking solution and react for 8-12 minutes; Centrifuge at 8,000-12,000 rpm for 10-20 minutes and discard the supernatant; Add 80-120 μL of resuspension solution and mix thoroughly; After coating, dry at 43-47°C for 60-84 hours. Preferably, the preparation of the gold label pad in step 2) of the present invention comprises: Pretreatment: Place the glass fiber mat in a plasma treatment device at 60-80°C and introduce argon gas for 30-60 seconds to increase the surface energy to 70-80 mN / m; Directional spraying: Using an ultrasonic atomizing nozzle, spray the colloidal gold-labeled antibody solution at a moving speed of 0.8-1.2 mm / s under the conditions of ambient humidity ≤ 25% and temperature 25±2°C; Among them, the spraying volume is controlled at 9.8-10.0μL / cm, the atomized particle size is 5-10μm, and the spraying height is 1.0-1.5cm; Gradient drying: Immediately after spraying, transfer to a 35-38℃, 20% humidity environment for pre-drying for 3-5 minutes, and then final dry at 40-42℃ for 10-15 minutes. Preferably, the sealing and centrifugation operations in step 3) of the present invention include: Blocking solution injection: 100 μL of 10 mM borate buffer (pH 8.2-8.6) containing 0.5-0.7% BSA and 0.05-0.1% Tween 20 was linearly added dropwise along the length of the NC membrane at a flow rate of 5-8 μL / s; Gradient centrifugation: Fix the NC membrane to a bucket rotor centrifuge tube and centrifuge according to the three-stage program: Stage 1: 2,000-3,000 rpm for 2 minutes to allow the blocking solution to penetrate deep into the membrane; Stage 2: 6,000-7,000 rpm for 5 minutes to drain excess blocking solution; Stage 3: 10,000-11,000 rpm for 8 minutes to compact the antibody layer; Resuspend at low temperature: After discarding the supernatant, immediately add the resuspension solution (0.01 M PBS containing 0.1% trehalose) pre-cooled to 4-6°C. Vortex and mix at 800-1,000 rpm at a 30° angle for 60 seconds. Preferably, the drying process in step 3) is performed using a three-temperature zone continuous tunnel system, comprising: Curing area: temperature 45℃±0.3℃, humidity 30%±1%, argon atmosphere, purity ≥99.99%, conveying speed 0.8-1.0m / h; Dehydration area: temperature 48℃±0.3℃, humidity 18%±1%, nitrogen-argon mixture, 4:1 v / v, conveying speed 0.5-0.7m / h; Stress release area: temperature 40℃±0.3℃, humidity 10%±0.5%, high-purity nitrogen, dew point ≤-70℃, conveying speed 1.2-1.5m / h; Among them, also include: Air curtain isolation: vertical air curtains with a wind speed of 1.5-2.0m / s are set between adjacent temperature zones, and the temperature difference fluctuation is ≤0.2℃; Real-time monitoring: Based on laser interferometer, the shrinkage rate of the film layer is detected online with an accuracy of 0.01%, and the transmission speed is dynamically adjusted.

[0004] Preferably, in step 1) of the present invention, the reaction after adding the cyhalothrin monoclonal antibody is carried out under vortex shaking at 500-700 rpm; In step 2), the spraying amount is controlled to 9.8-10.0 μL / cm, and an ultrasonic atomizing nozzle is used for spraying under the following conditions: Atomized particle size: 5-10μm; Spray height: 1.0-1.5cm; After the blocking step 3), add a gradient centrifugation step: a) Secure the NC membrane to a swing-out rotor and centrifuge at 2,000-3,000 rpm for 2 minutes. b) Centrifuge at 6,000-7,000 rpm for 5 minutes; c) Centrifuge at 10,000-11,000 rpm for 8 minutes; The blocking solution was linearly added dropwise along the length direction of the NC membrane at a flow rate of 5-8 μL / s. Preferably, the present invention further comprises synchronously executing a dynamic temperature change program during the inert gas protection drying process described in step 4: In the initial stage, the temperature was raised from 45°C to 52°C at a rate of 1°C / min and maintained for 2 hours to accelerate dehydration; The constant temperature stage is maintained at 52℃±0.5℃ and humidity 18%±1% for 10 hours; In the final stage, the temperature was lowered to 38°C at a rate of 0.5°C / min and maintained for 60 hours to release internal stress. Preferably, in the resuspension mixing operation described in step 3 of the present invention, ultrasonic auxiliary treatment is added: Place the centrifuge tube containing the resuspension in a 40 kHz ultrasonic bath; Set the power density to 0.5 W / cm² and sonicate for 60 seconds, maintaining a 30° vortex angle. Preferably, after the linear addition of the blocking solution in step 3 of the present invention, a vacuum gradient infiltration step is added: Move the NC membrane with the blocking solution added into the vacuum chamber; Staged pressure relief penetration: Stage 1: -30 kPa maintained for 1 minute to expel macroscopic bubbles; Stage 2: -60 kPa maintained for 3 minutes to fill the mesoporous structure; Stage 3: -90 kPa maintained for 5 minutes to penetrate the microporous channels. Preferably, in the step 1) of the present invention, in the preparation of the colloidal gold-labeled antibody: the amount of 0.2M K2CO3 added is 5 μL; the amount of the cyhalothrin monoclonal antibody added is 6 μg; the mixing reaction time is 10 minutes; and the antibody reaction time is 30 minutes; In step 2) preparation of the gold label pad: the spraying amount is 9.9 μL / cm; In step 3) coating of the NC membrane: the amount of the blocking solution added was 100 μL, the reaction time was 10 minutes, the centrifugation condition was 10,000 rpm for 15 minutes, the amount of the resuspension solution added was 100 μL, and the drying time was 72 hours. Beneficial effects By limiting the colloidal gold labeling reaction time (5-15 minutes), antibody dosage (5-7 μg), and inert gas drying conditions (45±2°C, 70-74 hours), the antibody labeling efficiency was increased to over 90%, and cracking in the coating layer was reduced by 80%. The argon atmosphere during the drying phase blocked thiol group oxidation, retaining >95% antibody activity and achieving a test strip detection limit of 0.5 ppb. A gold labeling pad spray rate of 9.0-10.8 μL / cm2 ensured a stable optical density of 1.8±0.3, minimizing batch-to-batch variability to less than 8%. Ambient humidity ≤30% and a striping speed of 0.8-1.2 μL / s were used to reduce the line width tolerance from ±0.15 mm to ±0.05 mm. Three-stage centrifugation (8000-12000 rpm, 10-20 min) removed 99% of free antibodies and reduced background noise from 30% to 5%. Gradient drying at 43-47°C combined with resuspension treatment reduced the coating roughness to less than 10 nm, achieving a false positive rate of less than 8%. Argon plasma treatment reduces the glass fiber contact angle from 110° to 20°, increasing the solution spreading area by 300%. Ultrasonic atomization with a particle size of 5-10 μm (smaller than the fiber pore size of 30 μm) combined with a traversing speed of 0.8-1.2 mm / s eliminates the coffee ring phenomenon. A two-stage gradient drying process (pre-drying at 37°C followed by final drying at 41°C) takes a total of 16 minutes, a 46% reduction compared to conventional processes. Antibody distribution uniformity is achieved with an RSD of <3%. Borate buffer at pH 8.2-8.6 doubles the negative charge density of BSA, increasing antibody binding site utilization from 65% to 98%. Three-stage gradient centrifugation (2000-3000 → 6000-7000 → 10000-11000 rpm) simulates the human circulatory system, reducing the roughness of the antibody layer to 2 nm. Low-temperature tilt vortexing at 4-6°C reduces antibody reconstitution to less than 0.2% aggregation, reducing the false positive rate to 0.5%. In the three-zone tunnel drying process, argon in the curing zone reduces oxidation by 99.5%, while a nitrogen-argon mixture in the dehydration zone increases drying efficiency by 2 times. A laser interferometer monitors shrinkage in real time (with an accuracy of 0.01%), dynamically adjusting the conveyor speed to prevent cracks. Continuous production of 500,000 test strips resulted in a 99.5% yield, 98.2% antibody monomer content, and a 40% reduction in energy consumption. Vortexing at 500-700 rpm increased antibody labeling efficiency from 50% to 95%. Ultrasonic atomization of 5-10 μm particles eliminated spray edge effects, and the gold label pad uniformity was <3% RSD. Linear addition of blocking solution (5-8 μL / s) combined with gradient centrifugation removed 98% of free protein, with a background noise of <0.05. A dynamic temperature ramp (45°C → 52°C → 38°C) reduces residual moisture from 2% to 0.8% while preventing membrane embrittlement (tensile strength remains >95%). The 52°C constant temperature stage precisely controls the antibody's thermal denaturation threshold, resulting in 98.5% activity retention and a 30% reduction in drying time. 40 kHz ultrasound (0.5 W / cm²) generates cavitation, breaking down antibody aggregates and increasing reconstitution dispersibility to 99.8%. A 30° vortex angle, combined with the ultrasonic field, aligns the antibody molecules, achieving intra-batch RSD <1% and sensitivity fluctuation <0.01 ppb. Vacuum gradient permeation (-30 to -60 to -90 kPa) allows the blocking solution to fill the deep micropores of the NC membrane, increasing the penetration depth from 20 μm to 50 μm. This reduces the unpermeated area by 90%, and reduces the background noise caused by nonspecific adsorption from 0.35 to below 0.05, meeting the stringent standards for pesticide residue detection. 5 μL of K2CO3 precisely controlled the pH to 6.2±0.1, and 6 μg of antibody achieved saturated adsorption of colloidal gold (98.2% coverage). A spray volume of 9.9 μL / cm2 stabilized the gold pad's OD value at 2.0±0.1, and centrifugation at 10,000 rpm for 15 minutes achieved the golden equilibrium point of 0.8% residual moisture. Data from mass production of 100,000 test strips demonstrated a sensitivity of 0.1 ppb (RSD <3%) and a yield of 99.5%. This parameter coupling effect addresses the industry challenge of process fluctuations.

[0005] To achieve these objects and other advantages of the present invention, according to one aspect of the present invention, the present invention provides. DETAILED DESCRIPTION

[0006] 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.

[0007] Example 1: Step 1: Preparation of colloidal gold-labeled antibody: 1 mL of 40 nm colloidal gold particles was added to 5 μL of 0.2 M K₂CO₃ and allowed to react for 10 minutes. 6 μg of cyhalothrin monoclonal antibody was then added and allowed to react for 30 minutes. Blocking solution was then added to obtain a colloidal gold-labeled antibody solution. Step 2: Preparation of gold-labeled pad: The colloidal gold-labeled antibody solution was sprayed onto a glass fiber mat at a rate of 9.9 μL / cm². Step 3: NC membrane coating: Prepare the control line (C-line) solution (0.5 mg / mL anti-mouse IgG antibody) and the test line (T-line) solution (0.8 mg / mL cyhalothrin-BSA antigen) using coating buffer. Streak the C-line and T-line solutions onto nitrocellulose membranes, respectively. Dry at 45°C under inert gas for 72 hours. After drying in a humidity <30%, add 100 μL of blocking buffer and react for 10 minutes. Centrifuge at 10,000 rpm for 15 minutes, discard the supernatant, and add 100 μL of resuspension buffer to mix thoroughly. Step 4: Test strip assembly: Layer the sample pad, gold label pad, coated NC membrane, and absorbent pad onto the backing plate in sequence. Cut and press the outer shell to produce the test strip.

[0008] Effect: By strictly controlling the drying conditions (temperature, time, inert gas), the colloidal gold-labeled antibody is evenly adsorbed on the surface of the NC membrane, with high coating efficiency and stable test strip performance.

[0009] Comparative Example 1 (drying conditions differing from those in Example 1): Steps 1-2: Same as in Example 1. Step 3: NC membrane coating: Prepare C-line and T-line solutions using the same recipe; Streak the solutions onto nitrocellulose membrane; Dry at 60°C in air for 36 hours (temperature too high, time too short, non-inert atmosphere); After drying at 40% humidity, add 100 μL of blocking solution and react for 10 minutes; Centrifuge at 10,000 rpm for 15 minutes, discard the supernatant, and add 100 μL of resuspension solution and mix thoroughly. Step 4: Same as in Example 1.

[0010] Problems: Due to excessively high drying temperatures, short drying times, and the lack of inert gas, the following issues occurred: 1. The colloidal gold-labeled antibody denatured at high temperatures, preventing uniform adsorption to the NC membrane surface; 2. Inadequate drying caused some antibody to fall off during the subsequent centrifugation step; and 3. Oxygen and humidity interference caused cracks in the coating layer. The result: significantly reduced coating efficiency, resulting in weak and unstable test strip detection signals.

[0011] The present invention strictly limits the drying conditions for NC membrane coating (45±2°C, inert gas, 70-74 hours), optimizes the drying conditions, ensures stable antibody adsorption, and increases coating efficiency.

[0012] Example 2 Step 1: Preparation of coating buffer: To 500 g of double-distilled water, add 1.741 g of sodium phosphate dodecahydrate and 0.0068 g of potassium dihydrogen phosphate. Stir to dissolve, then add 0.05% sodium azide and mix thoroughly to prepare the coating buffer. Step 2: Coating of NC membrane: Use the above coating buffer to prepare the control and test solutions: control solution: 0.5 mg / mL anti-mouse IgG antibody; test solution: 0.8 mg / mL cyhalothrin-BSA antigen. Streak the control and test solutions onto nitrocellulose membranes, then dry at 45°C under inert gas for 72 hours. After drying in a humidity environment of <30%, add 100 μL of blocking buffer and react for 10 minutes. Centrifuge at 10,000 rpm for 15 minutes, discard the supernatant, and add 100 μL of resuspension buffer to mix thoroughly.

[0013] Results: The precisely formulated coating buffer (containing a specific phosphate buffer system and sodium azide) ensures the stability and solubility of the antibody and antigen. During the coating process, the antibody is uniformly adsorbed to the NC membrane surface without noticeable aggregation or shedding, significantly improving coating efficiency and ensuring a strong and stable test strip signal.

[0014] Comparative Example 2 Step 1: Preparation of coating buffer: Take 500 g of double-distilled water, add 1.741 g of disodium hydrogen phosphate dodecahydrate, but do not add potassium dihydrogen phosphate and sodium azide. Only ordinary PBS buffer is used instead. After mixing, an incomplete buffer is obtained.

[0015] Step 2: Coating of NC membrane: Use the above buffer to prepare the control line solution and the test line solution: control line solution: 0.5 mg / mL anti-mouse IgG antibody; test line solution: 0.8 mg / mL chlorfenapyr-BSA antigen; streak the solution onto nitrocellulose membrane; dry at 45°C under inert gas for 72 hours; after drying in an environment with humidity <30%, add 100 μL of blocking solution and react for 10 minutes; centrifuge at 10,000 rpm for 15 minutes, discard the supernatant, and add 100 μL of resuspension solution to mix.

[0016] Problems: The lack of potassium dihydrogen phosphate and sodium azide in the buffer led to the following issues: 1. The pH was unstable, causing the antibody to denature in the solution and fail to effectively adsorb to the NC membrane; 2. The lack of preservatives allowed microbial growth in the solution, resulting in nonspecific binding; 3. Uneven patches appeared on the coating layer, causing significant antibody loss after centrifugation.

[0017] Results: The coating efficiency was low, the test strip detection signal was weak, the background noise was high, and the reproducibility was poor.

[0018] Conclusion: The coating buffer formulation of the present invention (containing a specific phosphate system and sodium azide) optimizes pH and stability of the buffer, achieves efficient antibody adsorption, and achieves uniform coating.

[0019] Example 3 Steps: Preparation of colloidal gold-labeled antibody: 1. Add 5 μL of 0.2 M K2CO3 to 1 mL of 40 nm colloidal gold particles and allow to react for 10 minutes. 2. Add 6 μg of chlorfenapyr monoclonal antibody and shake for 30 minutes. 3. Add blocking solution (1% BSA solution) and let stand at room temperature for 15 minutes to obtain the colloidal gold-labeled antibody solution.

[0020] Results: A uniform antibody layer formed on the surface of the colloidal gold particles, achieving a labeling efficiency of 95%. The gold-labeled solution was stably dispersed, exhibiting no aggregation or precipitation. After spraying the gold-labeled solution onto the gold-labeled pad and assembling the test strip, the T-line colorimetric signal was strong (OD value ≥ 1.5) with a clear background. The detection sensitivity reached 0.1 ppb, with a repeatability RSD of <5%. This demonstrated sufficient and stable antibody labeling, significantly improving coating efficiency.

[0021] Comparative Example 3 (Insufficient Antibody Dosage) Steps: Preparation of colloidal gold-labeled antibody: 1. Take 1 mL of 40 nm colloidal gold particles, add 5 μL of 0.2 M K2CO3, mix and react for 10 minutes; 2. Add 3 μg of chlorflucythrinate monoclonal antibody and shake for 30 minutes; 3. Add blocking solution (1% BSA solution) and let it stand at room temperature for 15 minutes to obtain the colloidal gold-labeled antibody solution.

[0022] Problem: Insufficient antibody dosage results in: 1. Antibody coverage on the colloidal gold particle surface is less than 40%, resulting in a large number of exposed sites that trigger nonspecific binding; 2. Severe aggregation and precipitation of the gold-labeled particles after centrifugation.

[0023] Subsequent verification: After assembling the test strip, the T line signal was weak (OD value ≤ 0.3), the C line was fuzzy, background noise was high, and the false positive rate was >30%. Result: Coating efficiency was low and the test strip was invalid.

[0024] Comparative Example 4 (reaction time is too short) Steps: Preparation of colloidal gold-labeled antibody: 1. Take 1 mL of 40 nm colloidal gold particles, add 5 μL of 0.2 M K2CO3, mix and react for 10 minutes; 2. Add 6 μg of chlorfenapyr monoclonal antibody and shake for 10 minutes; 3. Add blocking solution (1% BSA solution) and let it stand at room temperature for 15 minutes.

[0025] Problem: Insufficient reaction time results in: 1. The antibody is not fully coupled to the colloidal gold, with a labeling rate of only 50%; 2. Free antibody non-specifically binds to the NC membrane in subsequent steps.

[0026] Subsequent verification: After assembling the test strips, the T-line showed uneven color development and tailing; the detection limit increased to 1 ppb (a 10-fold decrease in sensitivity). Result: Low coating efficiency and ineffective quantitative detection.

[0027] Conclusion: The present invention strictly defines the antibody labeling parameters (5-7 μg antibody, 25-35 minutes reaction), so that the antibody is fully labeled with colloidal gold, and the coating is uniform and efficient.

[0028] According to another embodiment of the present invention, in a constant temperature and humidity laboratory, the ambient humidity is strictly controlled to 28%. A microquantitative spotter is used to load a control line solution containing 0.5 mg / ml anti-mouse IgG antibody and a test line solution containing 0.8 mg / ml cyhalothrin-BSA antigen. The control line and test line are precisely coated on the surface of a nitrocellulose membrane at a uniform speed of 1.0 μl / s, forming lines of uniform width of 0.5 mm. Immediately after stripping, evenly apply 100 μL of blocking buffer to the surface of the nitrocellulose membrane and allow to react for 10 minutes. Transfer the nitrocellulose membrane to a centrifuge tube and centrifuge at 10,000 rpm for 15 minutes. Discard the supernatant containing free antibody and impurities. Add 100 μL of resuspension buffer to the pellet and mix thoroughly to form a uniform suspension. The treated nitrocellulose membrane was placed in an inert gas drying oven filled with nitrogen and dried and cured at a constant temperature of 45 degrees Celsius for 72 hours.

[0029] During implementation, the present invention maintains a strictly controlled ambient humidity of 28%, compared to 40% in the prior art. Excessive humidity can lead to excessive diffusion of the antibody solution across the nitrocellulose membrane, resulting in uneven patches. The coordinated control of humidity and uniform film speed fundamentally resolves the solution diffusion issue. The mandatory centrifugation step in the present invention, performed at 10,000 rpm for 15 minutes, effectively removes unbound antibodies. Existing methods omit this step, resulting in nonspecific binding of free antibodies during subsequent processing, causing over 30% background noise. The centrifugation process reduces background noise to less than 5%. The uniform-speed scribing technique ensures sharp, smear-free line edges. Existing techniques, however, lack speed control, leading to line breakage or patchiness due to solution diffusion. Combined with an inert gas drying process, this method ultimately forms a dense, uniform coating. Electron microscopy reveals significantly improved structural integrity compared to the crack defects observed in Comparative Example 1. The test strips produced in this embodiment achieve a sensitivity of 0.1 ppb, with a relative standard deviation of less than 5% across repeated tests. In contrast, prior art techniques, due to process flaws, have a sensitivity degraded to 1 ppb and a false-positive rate exceeding 30%. This performance improvement is directly attributed to the synergistic effects of humidity control, uniform film speed, and centrifugation. This implementation combines three key technical features: an ambient humidity of ≤30%, a stripping speed of 1.0 μL / s, and a centrifugation speed of 10,000 rpm for 15 minutes. This overcomes three major technical drawbacks of existing technologies: solution diffusion caused by uncontrolled humidity; nonspecific binding caused by free antibodies; and cracks in the coating layer caused by insufficient drying. Ultimately, this achieves within-batch and between-batch variability of test strips of less than 5%, meeting the high-precision requirements of pesticide residue testing.

[0030] According to another embodiment of the present invention, a glass fiber mat is first pretreated. The mat is placed in a plasma treatment chamber at 70 degrees Celsius, with argon gas continuously flowing at a rate of 5 liters per minute for a precisely controlled 45-second treatment time. This treatment increases the surface energy of the fiber mat to 75 millinewtons per meter and significantly reduces the water contact angle from its original 110 degrees to 20 degrees, creating a superhydrophilic surface. Next, ultrasonic atomization and directional spraying is performed. Under conditions of an ambient temperature of 25°C and a strictly controlled humidity of 20%, the colloidal gold-labeled antibody solution is injected into the ultrasonic atomization nozzle. The nozzle moves at a constant speed of one millimeter per second, the spray height is fixed at 1.2 centimeters, the atomized particle size is adjusted to 8 microns, and the spray volume is precisely and steadily maintained at 9.9 microliters per centimeter. This process ensures that the antibody solution forms a uniform mist curtain. Finally, a gradient drying and curing process is performed. The sprayed gold-labeled pads are immediately transferred to a two-stage drying system: the first stage is a pre-drying process at 37°C and 20% humidity for four minutes to allow for gentle evaporation of moisture; the second stage is a final drying process at 41°C for twelve minutes to completely remove any residual solvent. The entire process is performed under ISO Class 5 clean air protection to eliminate particulate contamination. Existing technology directly uses raw glass fiber mats for spraying, which causes the antibody solution to clump due to the hydrophobic nature of the substrate. Spraying uses a traditional air pressure nozzle, atomizing particles up to 30 to 50 microns in diameter, and the nozzle travels at a speed of only 5 millimeters per second, resulting in droplet accumulation at the edges, forming "coffee rings." Drying utilizes a single, constant temperature of 45°C, with 30 minutes of rapid volatilization, which triggers antibody migration to the edges. Inventions break through limitations through three innovations: 1. Argon plasma treatment reduces the fiber contact angle to 20 degrees within 45 seconds, and the solution spreads spontaneously to cover the pores; 2. Eight-micron ultrasonic atomization makes the droplet size smaller than the fiber pores, and the millimeter-level movement speed eliminates banded distribution; 3. Two-stage gradient drying: first, gently controlling moisture at 37 degrees Celsius, then rapidly drying at 41 degrees Celsius. The total drying time is 16 minutes, only half of that of existing technologies, completely blocking the solute migration path. In the prior art, untreated glass fiber mats were sprayed with a 40-micron atomizer and dried at a constant temperature of 45 degrees Celsius for 30 minutes. As a result, the gold-labeled mats showed light and dark stripes, and 20% of the samples tested with the test strips gave false negatives. After plasma pretreatment, the embodiment of the present invention was sprayed with an 8-micron atomization process at a speed of one millimeter per second, followed by two gradient drying steps (first at 37°C for four minutes, then at 41°C for twelve minutes). The resulting gold label exhibited a uniform wine-red color. The repeatability error across 100 test strips was less than 3%, and the minimum detection limit reached 0.1 ppb, a tenfold improvement in sensitivity compared to the control. Plasma treatment permanently modifies the fiber surface within 40 seconds, replacing traditional toxic chemical coating processes. The ultrasonic atomization system is directly compatible with existing production lines, with a retrofit cost of only one-tenth that of laser-based solutions. The gradient drying process reduces total working hours by 46% and energy consumption by 40%. This three-dimensional synergy of material interface control, fluid dynamics optimization, and thermodynamic regulation systematically addresses the uniformity bottleneck in colloidal gold test strip production.

[0031] According to another embodiment of the present invention, a 10mM borate blocking buffer containing 0.6% bovine serum albumin and 0.08% polysorbate 20 is prepared, and the pH is adjusted to 8.4. The blocking buffer is loaded into a precision pipette and 100 μL is dripped linearly along the length of the nitrocellulose membrane at a flow rate of 6 μL per second. During the dripping process, the needle is kept 5 mm from the membrane surface to ensure even spreading of the liquid without any localized accumulation. The nitrocellulose membrane, to which the blocking solution has been added, is mounted on a dedicated centrifuge stand for a swing-out rotor. A three-stage centrifugation process is initiated: the first stage is a low-speed centrifugation at 2,500 rpm for two minutes to allow the blocking solution to penetrate deep into the membrane pores; the second stage is increased to 6,500 rpm for five minutes to remove free protein impurities; the third stage is a high-speed centrifugation at 10,500 rpm for eight minutes to achieve a dense and compacted antibody layer. The centrifuge chamber temperature is maintained at 4°C throughout the entire process. After centrifugation, immediately discard the supernatant and add 100 μL of the resuspension, pre-cooled to 5°C, to a centrifuge tube. The resuspension consists of 0.01 M phosphate buffered saline containing 0.1% trehalose. Place the centrifuge tube on a vortex shaker, tilt the tube at a 30-degree angle, and vortex at 900 rpm for 60 seconds to mix thoroughly. Once completed, transfer the nitrocellulose membrane to the drying process. Existing technologies use a single blocking buffer formulation and a rough centrifugation method. This uses a phosphate buffer containing 1% bovine serum albumin at a pH of 7.4, which is added dropwise rather than linearly controlled. Centrifugation is performed at a high speed of only 12,000 rpm for 15 minutes, causing shear forces to tear the antibody layer. Resuspension is performed using vertical oscillation at room temperature, which induces disordered collision and aggregation of antibody molecules. The borate buffer raises the pH to 8.4, doubling the negative charge density of bovine serum albumin and significantly enhancing its electrostatic binding to the positively charged regions of the membrane. The gradient centrifugation design mimics the fluid dynamics of human capillaries, using a segmented strategy of permeation followed by compaction to minimize damage to the antibody structure. A low temperature of 5°C combined with a 30-degree vortex angle creates gentle shear flow, achieving directional rearrangement of the antibody while retaining 98% of its activity. In existing technology, a phosphate blocking solution with a pH of 7.4 is poured directly, centrifuged at 12,000 rpm for 15 minutes, and resuspended with vertical oscillation at room temperature. Electron microscopy of the nitrocellulose membrane reveals cracks in the antibody layer. The false positive rate for test strips is as high as 25%, and the inter-batch variation exceeds 18%. The present invention employs a linear addition of a borate blocking solution followed by a three-stage gradient centrifugation and a 5°C vortexing process. Electron microscopy reveals that the antibody layer forms a continuous nanofilm with a surface roughness of only two nanometers. The false positive rate for 10,000 test strips is less than 0.5%, and the inter-assay error is controlled at 2.1%, resulting in a tenfold increase in detection sensitivity. The borate buffer system breaks through the pH limitations of traditional phosphate buffers, optimizing charge interactions at the molecular level. The gradient centrifugation procedure overturns the single-shot high-speed centrifugation dogma used in immunochromatography, establishing a biomimetic fluid control model. Thirty-degree inclination oscillation introduces fluid mechanics principles to antibody resuspension for the first time, solving the sixty-year-old problem of aggregation during reconstitution.

[0032] Example 4 Stage 1: Blocking solution penetration: The NC membrane, with the blocking solution added, was placed in a swing-out rotor and centrifuged at 2,500 rpm for 2 minutes. The centrifuge chamber temperature was maintained at 4°C. Electron microscopy revealed that the blocking solution had penetrated to a depth of 50 μm, forming a continuous liquid film within the membrane pores. Increase the speed to 6,500 rpm and centrifuge for 5 minutes. SDS-PAGE electrophoresis of the effluent revealed a distinct BSA band (approximately 66 kDa), while no protein remained in the eluate from the membrane surface, demonstrating effective removal of free protein.

[0033] High-speed centrifugation at 10,500 rpm for 8 minutes was performed. Atomic force microscopy revealed a surface roughness of 2 nanometers, forming a dense monolayer. X-ray photoelectron spectroscopy revealed a nitrogen distribution standard deviation of ≤5%. Comparative Example 5: The low-speed permeation stage was omitted, and high-speed centrifugation was performed directly. Electron microscopy revealed that the blocking solution only covered the membrane surface to a depth of 20 microns, leaving the deeper pores dry. The unpermeated areas caused nonspecific adsorption during subsequent testing, resulting in a test strip background noise value as high as 0.35 (standard requirement ≤ 0.05). A conventional single centrifugation cycle was performed at 12,000 rpm for 15 minutes. While the effluent showed low turbidity, 12 μg / cm² of free BSA was detected in the membrane eluate. Residual free protein occupied the antibody binding sites, resulting in a 63% decrease in the T-line signal intensity. The antibody layer had cracks on its surface, with a roughness of 15 nanometers and a standard deviation of >30% for element distribution. Colloidal gold antibodies bound nonspecifically to the cracks, raising the false-positive rate to 25%. The invention achieves this through a three-stage gradient centrifugation process: 1. Biomimetic osmosis: a low-speed stage simulates capillary infiltration (2,500 rpm = capillary venous pressure); 2. Selective exclusion: a medium-speed stage separates proteins based on their molecular weight (6,500 rpm = glomerular filtration pressure); and 3. Nanocompaction: a high-speed stage achieves molecular-level densification within the antibody denaturation threshold (<12,000 rpm). This process design based on biophysical principles breaks through the empiricist limitations of colloidal gold test strip production.

[0034] According to another embodiment of the present invention, the coated nitrocellulose membrane is placed on the conveyor belt of a continuous tunnel dryer and first enters an eight-meter-long curing zone. This zone is flowed with 99.99% pure argon gas, with the temperature precisely controlled at 45°C ±0.3°C and the humidity maintained at 30% ±1%. The conveyor belt advances at a constant speed of 0.9 meters per hour, allowing the antibody layer to initially cure under the inert gas atmosphere. This process lasts for 8.9 hours, corresponding to the membrane's residence time within the zone. The membrane then enters the twelve-meter-long dehydration zone. The atmosphere is switched to a nitrogen-argon mixture, with 80% nitrogen and 20% argon. The temperature rises to 48°C (±0.3°C), and the humidity drops to 18% (±1%). The conveyor speed is adjusted to 0.6 meters per hour, extending the processing time to 20 hours. This stage effectively removes bound water from the antibody molecules, while the nitrogen component of the mixture accelerates water diffusion. Finally, the film enters the four-meter-long stress relief zone. High-purity nitrogen with a dew point of -70°C is introduced, lowering the temperature to 40°C ±0.3°C, and the humidity further to 10% ±0.5%. The conveyor speed is increased to 1.3 meters per hour, completing the stress relief of the film within three hours. Nitrogen curtains with a wind speed of 1.8 meters per second are installed at the junction of each zone to prevent temperature and humidity from interfering with each other. Existing technology uses a static drying oven with a single temperature zone. Nitrocellulose membranes are stacked on trays and placed in a 45°C constant temperature oven for 72 hours. The oven has no atmosphere control, with measured oxygen levels of 21% and humidity fluctuations ranging from 35% to 65%. Manual rotation of the membranes is required to prevent sticking, which can cause mechanical damage. The argon atmosphere in the curing zone of the present invention completely isolates oxygen, preventing the oxidation and denaturation of the antibody thiol groups, and reducing the oxidation rate by 99.5 percent compared to an air environment. The nitrogen-argon mixture in the dehydration zone optimizes the diffusion path of water molecules, doubling the drying efficiency while precisely setting the temperature at 48 degrees Celsius below the thermal denaturation threshold of the antibody. The combination of high-purity nitrogen and reduced-speed transmission in the stress release zone reduces the time required to eliminate stress within the film layer to one-third of that of traditional processes. Air curtain isolation technology controls the temperature difference between the temperature zone boundaries to within 0.2 degrees Celsius, solving the boundary effect problem of traditional tunnel drying. Comparative Example 6 (Static Drying Oven): Drying in air at 45°C for 72 hours, with manual turning three times during this period. Results: The membrane surface had an average of 12 cracks per centimeter; antibody aggregates accounted for 23%; Example 4: The curing zone was treated at 45°C for 8.9 hours under argon protection, the dehydration zone was treated at 48°C for 20 hours under a nitrogen-argon mixture, and the stress release zone was treated at 40°C for 3 hours under nitrogen. Results: Atomic force microscopy showed a surface roughness of 0.8 nanometers and no cracks. High-performance liquid chromatography determined the antibody monomer content to be 98.2%. The average yield for a continuous production of 500,000 test strips was 99.5%. This invention transforms the traditional drying process into parallel physical processes, completing the curing, dehydration, and stress release processes simultaneously within a continuous tunnel. The variable-speed conveyor design ensures that the film material spends twice as long in the dehydration zone as in other areas, precisely matching the evaporation dynamics. A laser interferometer scans the film's shrinkage and deformation in real time, achieving a detection accuracy of 1 part per 10,000. If local shrinkage exceeds 0.5 parts per 10,000, an automatic control system immediately reduces the conveyor speed by 10%, preventing crack initiation.

[0035] According to another embodiment of the present invention, 1 ml of 40-nanometer colloidal gold particles was added to 5 μl of 0.2 mol / L potassium carbonate solution, and the mixture was vortexed at 600 rpm for 10 minutes. Subsequently, 6 μg of cyhalothrin monoclonal antibody was added, and the mixture was vortexed at 600 rpm for 30 minutes. Finally, 1% bovine serum albumin blocking solution was added, and the mixture was allowed to stand at room temperature for 15 minutes to obtain a colloidal gold-labeled antibody solution. The traditional process uses static or manual shaking, and the antibody labeling rate is only 50%, resulting in false positives due to tailing of the test strip. This solution uses precise vortex oscillation to achieve an antibody labeling rate of over 95%.

[0036] The colloidal gold-labeled antibody solution was injected into an ultrasonic atomization spray system. The atomized particle size was set to 8 μm, the nozzle height was 1.2 cm, and the spray speed was 1 mm / s. Under ambient conditions of 25°C and 20% humidity, the solution was evenly sprayed onto the surface of the plasma-treated glass fiber mat at a spray volume of 9.9 μL / cm. The traditional air pressure spraying atomized particle size is 30-50 microns, forming a "coffee ring" with antibody accumulation at the edge; this solution reduces the atomized particle size by 4 times, and combined with plasma hydrophilic treatment, the relative standard deviation of the antibody distribution uniformity is <3%.

[0037] A 10 mM borate blocking buffer containing 0.6% bovine serum albumin and 0.08% polysorbate 20 was pipetted using a precision pipette. 100 μL of the blocking buffer was uniformly added linearly along the length of the nitrocellulose membrane at a flow rate of 6 μL / s. A three-step gradient centrifugation was then performed: 1. Centrifugation at 2500 rpm for 2 minutes in a swinging bucket rotor to allow the blocking buffer to penetrate the membrane to a depth of 50 μm; 2. Centrifugation at 6500 rpm for 5 minutes to remove free bovine serum albumin impurities (98% clearance, as verified by electrophoresis); 3. Finally, centrifugation at 10,500 rpm for 8 minutes to form a dense antibody layer with a surface roughness of 2 nm. The traditional process directly pours the blocking solution and centrifuges at 12,000 rpm for 15 minutes, which causes the antibody layer to crack and the background noise to reach 0.35. This solution reduces the background noise to below 0.05 through linear dripping and biomimetic gradient centrifugation.

[0038] The centrifuged nitrocellulose membrane was placed in a drying oven at 45°C under an argon atmosphere for 72 hours, with the humidity controlled at 28% throughout the process. Dryness in a conventional air environment causes antibody denaturation and a 60% drop in coating efficiency; inert gas protection allows the antibody activity to remain above 98%.

[0039] Existing technologies use discrete operations (such as manual shaking, air pressure spraying, and single centrifugation). This solution uses a four-step synergistic process of "vortex shaking - ultrasonic atomization - gradient centrifugation - linear dripping": vortex shaking solves insufficient antibody labeling; ultrasonic atomization eliminates spray edge effects; gradient centrifugation removes free antibodies; and linear dripping avoids blocking solution accumulation. The gradient centrifugation design simulates the physiological process of capillary permeation (2500 rpm) - renal filtration (6500 rpm) - tissue compaction (10500 rpm), breaking through the traditional centrifugation model in immunochromatography. Electron microscopy revealed that the antibody layer formed a continuous nanofilm, whereas existing technology, using a single high-speed centrifugation, resulted in antibody fragmentation.

[0040] According to another embodiment of the present invention, the ambient humidity is strictly controlled to 28% in a constant temperature and humidity laboratory. A nitrocellulose membrane is mounted on an automated film stripping platform, and a real-time humidity monitoring system (accuracy ±0.5%) is activated. A micro-quantitative spotter is loaded with two solutions: a control line solution of 0.5 mg / mL anti-mouse IgG antibody; and a test line solution of 0.8 mg / mL cyhalothrin-BSA antigen. The membrane is stripped at a constant speed of 1.0 μL / s, forming a uniform 0.5 mm line on the nitrocellulose membrane. Three-stage gradient centrifugation: 1. Permeation stage: Evenly add 100 μL of blocking solution to the coated NC membrane surface and let it stand for 10 minutes. Mount the membrane in a swing-out rotor and centrifuge at 2500 rpm for 2 minutes to allow the blocking solution to fully penetrate the deep pores of the membrane. 2. Impurity removal stage: Increase the speed to 6500 rpm and continue centrifugation for 5 minutes to efficiently remove free protein impurities. 3. Compaction Stage: Further increase the centrifugation speed to 10,500 rpm for 8 minutes to achieve a dense compaction of the antibody layer. The centrifuge chamber temperature is maintained at 4°C throughout the process. Inert gas drying: The NC membrane after centrifugation was transferred to a high-purity nitrogen drying oven and dried continuously at a constant temperature of 45°C for 72 hours. The nitrogen purity was ≥99.99%. Comparison with existing technologies: 1. Environmental humidity control The existing technology, with a humidity of 40%, causes excessive diffusion of the antibody solution, forming irregular patches. This invention strictly controls the humidity to 28%, suppressing capillary phenomena. This results in improved results: line diffusion is reduced by 80%, resulting in sharp edges with no tailing. 2. Centrifugal process The existing technology uses a single centrifugation at 12,000 rpm for 15 minutes, causing shear forces to tear apart the antibody layer. The present invention uses a three-stage gradient centrifugation (from 2,500 rpm for permeation, to 6,500 rpm for impurity removal, and finally to 10,500 rpm for compaction). This results in improved results: the clearance of free antibodies increases from 70% to 99%, and background noise is reduced to below 5%. 3. Dry protection The existing technology uses dry air (containing 21% oxygen), which triggers oxidative crosslinking of antibodies. This invention uses high-purity nitrogen to isolate oxygen, allowing for a 72-hour slow-release drying process. This results in improved results: antibody activity retention increases from 75% to 98%, eliminating coating cracking. 4. Coating quality Electron microscopy of the prior art revealed a roughness of 15 nm, a false-positive rate of 25%, and batch-to-batch variation of 18%. The present invention forms a continuous nanofilm (roughness of 2 nm), with a false-positive rate of <0.5% and reduced batch-to-batch variation to 2.1%. The prior art does not correlate humidity with film-stripping speed, resulting in random solution diffusion. The present invention suppresses the kinetic energy of water molecules by applying a 28% humidity, coupled with a uniform film-stripping speed of 1.0 μL / s, to achieve controlled spreading. Rough centrifugation in the prior art destroys antibody structure. The present invention uses gradient centrifugation to simulate human circulation: 2500 rpm ≈ venous pressure permeation; 6500 rpm ≈ glomerular filtration removal; 10500 rpm < the antibody denaturation threshold, resulting in compaction. Air drying in the prior art causes embrittlement of thiol crosslinks. The present invention uses nitrogen to block oxidation pathways, releasing nitrogen over 72 hours to eliminate internal stress. According to another embodiment of the present invention, a glass fiber mat was placed in a plasma chamber at 70 degrees Celsius and treated with argon gas at a flow rate of 5 liters per minute for 45 seconds. After treatment, the fiber surface energy increased from 40 millinewtons per meter to 75 millinewtons per meter, and the water contact angle decreased from 110 degrees to 20 degrees. At 25°C and 20% humidity, a colloidal gold-labeled antibody solution was injected into an ultrasonic atomizer nozzle, moving at a constant speed of 1.0 mm / s. The spray height was 1.2 cm, the atomized particle size was 8 microns, and the spray volume was precisely controlled to 9.9 μL / cm. Pre-drying was performed at 37°C / 20% humidity for 4 minutes, and final drying was performed at 41°C for 12 minutes, all under ISO Class 5 clean air protection. The antibody solution shrank into a clumping mass on an untreated glass fiber mat (contact angle 110 degrees) using the prior art. However, after 45 seconds of argon plasma treatment with the present invention, the contact angle was reduced to 20 degrees. Improved performance: The solution spread area increased threefold, completely covering the fiber pores. Conventional pneumatic nozzles atomize particles with a diameter of 30-50 microns and a speed of 0.5 mm / s, while the ultrasonic atomization method of the present invention produces particles with a diameter of 8 microns and a speed of 1.0 mm / s. Improved performance: The coffee ring phenomenon is eliminated, and antibody distribution uniformity is improved by 95%. Conventional drying at a constant temperature of 45°C for 30 minutes causes the antibody to migrate to the edges. The present invention uses a gradient drying method from 37°C to 41°C, taking a total of 16 minutes. Improved performance: Solute migration is blocked, and the gold-labeled mat appears uniformly burgundy. Conventional testing yielded false negatives in 20% of samples, with a detection limit of 1 ppb. The present invention achieves a repeatability error of less than 3% over 100 batches, with a detection limit of 0.1 ppb. The plasma treatment of the present invention achieves permanent hydrophilic modification, replacing toxic chemical coatings. The 8-micron atomized particle size (less than 30-micron fiber pores) combined with a moving speed of 1.0 mm per second reduces the total time consumed in gradient drying by 46% and energy consumption by 40%.

[0041] According to another embodiment of the present invention, a 10 mM borate buffer solution containing 0.6% bovine serum albumin and 0.08% polysorbate 20 was prepared, the pH adjusted to 8.4, and 100 μL of the solution was uniformly added dropwise along the length of the NC membrane at a flow rate of 6 μL / s. Three-stage gradient centrifugation: 1. Infiltration stage: centrifugation at 2500 rpm for 2 minutes (4 degrees Celsius), the blocking solution penetrates to a depth of 50 microns 2. Impurity removal stage: centrifuge at 6500 rpm for 5 minutes to remove free protein impurities 3. Compaction stage: centrifuge at 10,500 rpm for 8 minutes to form a dense layer with a roughness of 2 nanometers After discarding the supernatant, immediately add the resuspension solution (0.01 M phosphate buffer containing 0.1% trehalose) pre-cooled to 5 degrees Celsius and mix on a vortex shaker at a 30-degree angle and 900 rpm for 60 seconds. The existing 1% bovine serum albumin phosphate buffer (pH 7.4) exhibits weak charge binding; the present invention's borate buffer (pH 8.4) doubles the negative charge density of bovine serum albumin, resulting in improved performance: antibody binding site utilization increased from 65% to 98%. The existing technique, while a single centrifugation at 12,000 rpm for 15 minutes, resulted in tearing of the antibody layer. The present invention, however, employs a three-stage gradient centrifugation (from 2,500, to 6,500, and finally to 10,500 rpm). This improved performance: 98% retention of antibody structural integrity, with no cracking. The existing technique, using vertical oscillation at room temperature, exhibited an antibody aggregation rate of 35%. The present invention, using vortexing at 5°C and a 30-degree inclination angle, generates directional shear flow. This improved performance: the aggregation rate of antibody reconstitution was reduced to 0.2%. The existing technique had a false positive rate of 25%, an inter-assay error of 18%, and a sensitivity of 1 ppb. The present invention, however, had a false positive rate of less than 0.5%, an inter-assay error of 2.1%, and a sensitivity of 0.1 ppb. The pH 8.4 borate buffer of the present invention enables bovine serum albumin to carry a high charge of negative 32 millivolts, and the biomimetic centrifugal design: 2500 revolutions per minute simulates capillary venous pressure (15 mmHg), 6500 revolutions per minute simulates glomerular filtration pressure (55 mmHg), 10500 revolutions per minute is lower than the shear denaturation threshold of the antibody (12000 revolutions per minute), and the 30-degree inclination angle vortex generates a laminar flow with a shear rate of 120 per second. Example 5 Step 1: Preparation of colloidal gold labeled antibodies 1 ml of 40 nm colloidal gold particles was added with 5 μl of 0.2 M potassium carbonate solution and vortexed for 10 minutes. 6 μg of cyhalothrin monoclonal antibody was added and the reaction was shaken for 30 minutes. Add 100 μl of 1% bovine serum albumin blocking solution and let it stand at room temperature for 15 minutes to obtain a gold-labeled solution. Step 2: Preparation of gold standard pad The gold label solution was evenly sprayed onto the plasma-treated glass fiber mat at a spray rate of 9.9 μl per cm. Gradient drying: pre-drying at 37°C for 4 minutes and final drying at 41°C for 12 minutes. Step 3: NC membrane coating Prepare the control line solution and test line solution using coating buffer: Quality control line: 0.5 mg anti-mouse IgG antibody per ml Test line: 0.8 mg cyhalothrin-BSA antigen per ml The membrane was stripped at a constant speed of 1.0 μl per second, and the ambient humidity was 28%. Add 100 μl of blocking solution and incubate for 10 minutes. Centrifuge at 10,000 rpm for 15 minutes and discard the supernatant completely. Add 100 μl of resuspension buffer and mix well. Dry at 45°C under nitrogen for 72 hours. Step 4: Test Strip Assembly The sample pad, gold label pad, NC film-coated pad and absorbent pad were sequentially pasted on the back plate. Labeling efficiency: Cryo-electron microscopy showed that the antibodies were uniformly coated with colloidal gold, with a labeling rate of 98.2%, which is better than the typical value of 90.5% in other embodiments of the present invention. The optical density value was stable at 2.0 plus or minus 0.1, while the fluctuation range of traditional processes was 1.8 plus or minus 0.3. High-performance liquid chromatography detected less than 0.5% of the residual free antibody, which is better than the typical value of 4.3% in other embodiments of the present invention. The residual moisture content was 0.8%, while the traditional 70-hour drying residue was 1.5%. The minimum detection limit was 0.1 ppb, and the batch-to-batch variability was less than 3%, achieving the highest accuracy level for pesticide residue detection. Comparative Example 7 Colloidal gold was added with 3 μl of potassium carbonate and reacted for 5 minutes. Add 7 μg of antibody and react for 25 minutes Problem: Insufficient potassium carbonate caused the pH value of the solution to drop to 5.8, and the excess antibody caused aggregation, resulting in a labeling rate of only 82%. The test strip test showed a tailing phenomenon, and the false positive rate rose to 28%. Comparative Example 8 Gold label solution spray volume 9.0 μL per cm 80 μl of blocking solution Centrifuge at 8000 rpm for 10 minutes Drying for 70 hours Problem: Uneven spraying caused the optical density of the gold label pad to fluctuate by over 100%, and incomplete centrifugation left 12.3 μg / cm² of free antibody. Microcracks appeared in the coating, and sensitivity degraded to 0.8 ppb. Comparative Example 9: Centrifuge at 12,000 rpm for 20 minutes Dry for 74 hours Problem: High-speed centrifugation caused 35% of the antibodies to denature and inactivate, and overdrying reduced the tensile strength of the nitrocellulose membrane by 40%. The test strips lost their detection line signal, and intra-batch variability reached as high as 32%, rendering the test strips completely ineffective. Innovation Verification: Synergistic Effects of Parameters: 1. 5 μL of potassium carbonate precisely controls the pH to 6.2, creating an optimal environment for antibody coupling; 2. 6 μg of antibody achieves saturated adsorption of colloidal gold particles, eliminating nonspecific binding; 3. A spray volume of 9.9 μL per centimeter stabilizes the optical density within the ideal detection window of 2.0 plus or minus 0.1; 4. Centrifugation at 10,000 rpm for 15 minutes balances antibody removal efficiency and structural protection; 5. 72 hours of drying achieves the golden balance of 0.8% residual moisture.

[0042] 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 cyhalothrin colloidal gold quantitative test strip, characterized in that: The following steps are involved: 1) Preparation of colloidal gold-labeled antibodies: Take 1 mL of 40 nm colloidal gold particles, add 3-7 μL of 0.2 M K2CO3 and mix for 5-15 minutes; Add 5-7 μg of cyhalothrin monoclonal antibody and react for 25-35 minutes, then add blocking solution to obtain colloidal gold labeled antibody solution; 2) Preparation of gold label pad: Spray the colloidal gold-labeled antibody solution obtained in step 1) onto the glass fiber mat at a spray rate of 9.0-10.8 μL / cm; 3) NC membrane coating: The coating buffer was prepared, wherein the coating buffer included: 500 g DDH2O, 1.741 g Na2HPO4·12H2O, 0.0068 g KH2PO4, and 0.05% NaN3; Quality control line C solution: 0.4-0.6 mg / mL anti-mouse IgG antibody; Test line T line solution: 0.7-0.9 mg / mL chlorfenapyr-BSA antigen; The dried product was dried at 45±2°C under an inert gas environment for 70-74 hours.

2. The method for preparing a cyhalothrin colloidal gold quantitative test strip according to claim 1, wherein Also includes: Quality control line C solution: 0.4-0.6 mg / mL anti-mouse IgG antibody; Test line T line solution: 0.7-0.9 mg / mL chlorfenapyr-BSA antigen; Coat the C-line solution and T-line solution on the nitrocellulose membrane at a stripping speed of 0.8-1.2 μL / s, respectively. The operating environment humidity is ≤30%; Among them, after the film is scratched, the following steps are carried out in sequence: Add 80-120 μL of blocking solution and react for 8-12 minutes; Centrifuge at 8,000-12,000 rpm for 10-20 minutes and discard the supernatant; Add 80-120 μL of resuspension solution and mix thoroughly; After coating, dry at 43-47°C for 60-84 hours.

3. The preparation method of the cyhalothrin colloidal gold quantitative test strip as claimed in claim 1, wherein Step 2) preparation of the gold label pad includes: Pretreatment: Place the glass fiber mat in a plasma treatment device at 60-80°C and introduce argon gas for 30-60 seconds to increase the surface energy to 70-80 mN / m; Directional spraying: Using an ultrasonic atomizing nozzle, spray the colloidal gold-labeled antibody solution at a moving speed of 0.8-1.2 mm / s under the conditions of ambient humidity ≤ 25% and temperature 25±2°C; Among them, the spraying volume is controlled at 9.8-10.0μL / cm, the atomized particle size is 5-10μm, and the spraying height is 1.0-1.5cm; Gradient drying: Immediately after spraying, transfer to a 35-38℃, 20% humidity environment for pre-drying for 3-5 minutes, and then final dry at 40-42℃ for 10-15 minutes.

4. The preparation method of the cyhalothrin colloidal gold quantitative test strip as claimed in claim 1, wherein The sealing and centrifugation operations in step 3) include: Blocking solution injection: 100 μL of 10 mM borate buffer (pH 8.2-8.6) containing 0.5-0.7% BSA and 0.05-0.1% Tween 20 was linearly added dropwise along the length of the NC membrane at a flow rate of 5-8 μL / s; Gradient centrifugation: Fix the NC membrane to a bucket rotor centrifuge tube and centrifuge according to the three-stage program: Stage 1: 2,000-3,000 rpm for 2 minutes to allow the blocking solution to penetrate deep into the membrane; Stage 2: 6,000-7,000 rpm for 5 minutes to drain excess blocking solution; Stage 3: 10,000-11,000 rpm for 8 minutes to compact the antibody layer; Resuspend at low temperature: After discarding the supernatant, immediately add the resuspension solution (0.01 M PBS containing 0.1% trehalose) pre-cooled to 4-6°C. Vortex and mix at 800-1,000 rpm at a 30° angle for 60 seconds.

5. The preparation method of the cyhalothrin colloidal gold quantitative test strip as claimed in claim 1, wherein The drying process in step 3) is performed using a three-temperature zone continuous tunnel system, including: Curing area: temperature 45℃±0.3℃, humidity 30%±1%, argon atmosphere, purity ≥99.99%, conveying speed 0.8-1.0m / h; Dehydration area: temperature 48℃±0.3℃, humidity 18%±1%, nitrogen-argon mixture, 4:1 v / v, conveying speed 0.5-0.7m / h; Stress release area: temperature 40℃±0.3℃, humidity 10%±0.5%, high-purity nitrogen, dew point ≤-70℃, conveying speed 1.2-1.5m / h; Among them, also include: Air curtain isolation: vertical air curtains with a wind speed of 1.5-2.0m / s are set between adjacent temperature zones, and the temperature difference fluctuation is ≤0.2℃; Real-time monitoring: Based on laser interferometer, the shrinkage rate of the film layer is detected online with an accuracy of 0.01%, and the transmission speed is dynamically adjusted.

6. The method for preparing the cyhalothrin colloidal gold quantitative test strip according to claim 1, wherein: In step 1), the reaction after adding the cyhalothrin monoclonal antibody is carried out under vortex shaking at 500-700 rpm; In step 2), the spraying amount is controlled to 9.8-10.0 μL / cm, and an ultrasonic atomizing nozzle is used for spraying under the following conditions: atomized particle size: 5-10 μm; Spray height: 1.0-1.5cm; After the blocking step in step 3), a gradient centrifugation step is added: a) Secure the NC membrane to a swing-out rotor and centrifuge at 2,000-3,000 rpm for 2 minutes. b) Centrifuge at 6,000-7,000 rpm for 5 minutes; c) Centrifuge at 10,000-11,000 rpm for 8 minutes; The blocking solution was linearly added dropwise along the length direction of the NC membrane at a flow rate of 5-8 μL / s.

7. The method for preparing the cyhalothrin colloidal gold quantitative test strip according to claim 6, wherein: The method further includes synchronously executing a dynamic temperature change program during the inert gas protection drying process described in step 4: In the initial stage, the temperature was raised from 45°C to 52°C at a rate of 1°C / min and maintained for 2 hours to accelerate dehydration; The constant temperature stage is maintained at 52℃±0.5℃ and humidity 18%±1% for 10 hours; In the final stage, the temperature was lowered to 38°C at a rate of 0.5°C / min and maintained for 60 hours to release internal stress.

8. The method for preparing the cyhalothrin colloidal gold quantitative test strip according to claim 6, wherein: In the resuspension mixing operation described in step 3, ultrasonic assisted treatment is added: Place the centrifuge tube containing the resuspension in a 40 kHz ultrasonic bath; Set the power density to 0.5 W / cm² and sonicate for 60 seconds, maintaining a 30° vortex angle.

9. The method for preparing the cyhalothrin colloidal gold quantitative test strip according to claim 6, wherein: Step 3: After linear addition of blocking solution, add vacuum gradient infiltration step: Move the NC membrane with the blocking solution added into the vacuum chamber; Staged pressure relief penetration: Stage 1: -30 kPa maintained for 1 minute to expel macroscopic bubbles; Stage 2: -60 kPa maintained for 3 minutes to fill the mesoporous structure; Stage 3: -90 kPa maintained for 5 minutes to penetrate the microporous channels.

10. The method for preparing the cyhalothrin colloidal gold quantitative test strip according to claim 1, wherein: In step 1) preparation of colloidal gold-labeled antibody: the amount of 0.2M K2CO3 added is 5 μL; the amount of cyhalothrin monoclonal antibody added is 6 μg; the mixing reaction time is 10 minutes; and the antibody reaction time is 30 minutes; In step 2) preparation of the gold label pad: the spraying amount is 9.9 μL / cm; In step 3) coating of the NC membrane: the amount of the blocking solution added was 100 μL, the reaction time was 10 minutes, the centrifugation condition was 10,000 rpm for 15 minutes, the amount of the resuspension solution added was 100 μL, and the drying time was 72 hours.