Sample pad treatment solution and its preparation method, sample pads, colloidal gold immunochromatographic test strips and kits

Abstract

This invention discloses a sample pad treatment solution and its preparation method, a sample pad, a colloidal gold immunochromatographic test strip, and a reagent kit, belonging to the field of colloidal gold immunochromatographic technology. The sample pad treatment solution comprises the following components: surfactant, blocking agent, protective agent, Evans blue, and pH buffer. The sample pad treatment solution provided by this invention is suitable for pretreating the sample pad of a colloidal gold immunochromatographic test strip. During the testing of target analytes using the colloidal gold immunochromatographic test strip, Evans blue can form a weak complex with unbound colloidal gold nanoparticles through electrostatic and hydrophobic interactions. This complex has strong hydrophilicity and excellent chromatographic flowability in nitrocellulose membranes, allowing it to rapidly carry colloidal gold labels to the absorbent pad, reducing or avoiding the retention of colloidal gold nanoparticles on the membrane surface, thereby improving the membrane whitening effect of the colloidal gold immunochromatographic test strip.

Description

Technical Field

[0001] This invention relates to the field of colloidal gold immunochromatography technology, specifically to a sample pad treatment solution and its preparation method, a sample pad, a colloidal gold immunochromatographic test strip, and a reagent kit. Background Technology

[0002] Colloidal gold immunochromatography is a rapid detection technique based on antigen-antibody specific binding reactions and colloidal gold labeling. It boasts advantages such as ease of operation, rapid detection, low cost, and no need for specialized instruments, making it widely applicable in clinical diagnostics, food safety testing, and environmental monitoring. The colloidal gold immunochromatographic test strip is the core carrier of this technology. Its basic structure typically includes a sample pad, a conjugation pad, a nitrocellulose membrane (NC membrane), an absorbent pad, and a PVC backing. The NC membrane has a test line (T line) and a control line (C line) fixed on it. During the detection process, after the sample droplet is added to the sample pad, it precipitates towards the absorbent pad under capillary action. The target analyte in the sample binds to the colloidal gold-labeled probe on the conjugation pad, forming a complex. This complex continues to precipitate to the NC membrane, where it binds to the specific ligand of the test line and is retained, forming a visible red band. Unbound colloidal gold-labeled probes continue to precipitate to the absorbent pad and are adsorbed. Ideally, within the set interpretation time (usually 5-15 minutes), the unbound colloidal gold-labeled probe should be completely absorbed into the absorbent pad, and there should be no obvious colloidal gold residue on the NC film surface except for the detection line and the quality control line, that is, "clean whitening" is achieved. At this time, the detection results are clear and easy to interpret.

[0003] However, in actual product development and application, a common problem arises when the interpretation time arrives: significant colloidal gold residue remains on the NC membrane surface (manifested as an overall reddish tinge or red afterimage), indicating incomplete bleaching. This issue leads to decreased color contrast between the detection and control lines, and in severe cases, makes it impossible to accurately distinguish between positive and negative results, significantly impacting the accuracy and reliability of the test results. This is a common technical challenge in the development of colloidal gold immunochromatography products. To address the incomplete bleaching problem, existing technologies typically employ methods such as optimizing the pore size and material of the NC membrane, adjusting the formulation of the sample pad and binding pad treatment solutions (e.g., adjusting the type of buffer, pH value, ionic strength, surfactant concentration, etc.), optimizing the particle size and labeling conditions of colloidal gold particles, and adjusting the material and size of the absorbent pad. However, the improvement effects of these methods are often limited, and their adaptability to different detection items is poor, making it difficult to achieve universally applicable bleaching optimization. Some methods may also affect the binding activity of antigen and antibody, leading to a decrease in detection sensitivity.

[0004] Therefore, how to improve the whitening effect of the colloidal gold immunochromatographic test strip without affecting the detection performance is a technical problem that urgently needs to be solved. Summary of the Invention

[0005] In view of the deficiencies of the existing technology, the purpose of this invention is to provide a colloidal gold immunochromatographic test strip, its preparation method and application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a sample pad treatment solution comprising a surfactant, a blocking agent, a protective agent, Evans blue, and a pH buffer solution; wherein the surfactant has a mass percentage content of 0.5-2%, the blocking agent has a mass percentage content of 1-3%, and the Evans blue has a mass percentage content of 0.02-0.1%.

[0007] The sample pad treatment solution provided by this invention is suitable for pretreating the sample pads of colloidal gold immunochromatographic test strips so that the sample pads are adsorbed with surfactants, blocking agents and Evans blue.

[0008] The working principle of the colloidal gold immunochromatographic test strip is as follows: The sample is dropped onto the sample pad of the test strip. Under capillary action, the sample moves forward along the strip, dissolving Evans blue in the sample pad and the colloidal gold label loaded on the binding pad, and continues to move to the test line area. If the target analyte (i.e., target antibody or target antigen) is present in the sample, the antigen or antibody coated on the test line will bind to the target analyte and simultaneously capture the colloidal gold label, forming an immune complex, causing the test line to appear as a red band. If the target analyte is absent in the sample, the colloidal gold label cannot bind to the test line, and therefore no color develops. The sample and colloidal gold label continue to move to the control line area. The antibody or antigen coated on the control line will bind to the colloidal gold label. Regardless of whether the target analyte is detected, the control line should show a red band to verify the effectiveness of the test strip reaction process.

[0009] Evans blue (chemical formula C) 34 H 24 N6Na4O 14S4 is a water-soluble acidic dye with a large molecular weight and a specific molecular structure. The inventors hypothesize that Evans blue can form a weak complex with unbound colloidal gold nanoparticles through electrostatic and hydrophobic interactions. This complex is highly hydrophilic and exhibits excellent chromatographic flow in nitrocellulose membranes, allowing it to rapidly carry colloidal gold labels to the absorbent pad, reducing or preventing the retention of colloidal gold nanoparticles on the membrane surface, thereby improving the membrane whitening effect of the colloidal gold immunochromatographic test strip. In addition, the blue of Evans blue can form a complementary color with the red of colloidal gold. Even if there are trace amounts of colloidal gold residue on the membrane surface, its red color will be neutralized and masked by the blue of Evans blue, visually eliminating the redness of the membrane surface. Evans blue itself does not specifically bind to antigens or antibodies, nor does it affect the binding reaction, therefore it will not adversely affect the detection performance of the test strip.

[0010] In the sample pad treatment solution, the surfactant may be any one or a combination of any two of the following mass percentages: 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, and 2.0%; and the blocking agent may be 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, and 1.5%. The Evans blue content can be any one or any two of the following: 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%.

[0011] Preferably, the Evans blue content in the sample pad treatment solution is 0.04-0.06% by mass. When the Evans blue content in the sample pad treatment solution is within this preferred range, using the sample pad treated with this solution for colloidal gold immunochromatographic test strips results in better whitening effects on the colloidal gold immunochromatographic test strips.

[0012] Preferably, the surfactant includes at least one of Tween 20 and Triton X-100.

[0013] Preferably, the blocking agent includes at least one of bovine serum albumin and polyvinylpyrrolidone.

[0014] Preferably, the sample pad treatment solution further includes a protective agent, wherein the mass percentage of the protective agent in the sample pad treatment solution is 0.5-1.5%, and the protective agent includes sucrose.

[0015] Preferably, the pH buffer includes at least one of Tris-HCl buffer, PBS buffer, and borate buffer, and the pH value of the pH buffer is 7.2 to 8.0.

[0016] Preferably, the sample pad treatment solution further includes a protective agent, which comprises sucrose, and the protective agent has a mass percentage of 0.5-1.5% in the sample pad treatment solution. The introduction of the protective agent can improve the stability and absorbency of the sample pad.

[0017] In the sample pad treatment solution, the mass percentage of the protective agent can be any one or any combination of 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, and 1.5%.

[0018] Preferably, the sample pad treatment solution further includes sodium chloride, wherein the mass percentage of sodium chloride in the sample pad treatment solution is 0.5-1%.

[0019] In the sample pad treatment solution, the mass percentage of sodium chloride can be any one or a combination of any two of the following: 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, and 1.0%.

[0020] In a second aspect, the present invention provides a method for preparing the sample pad treatment solution as described in the first aspect, comprising the following steps: Surfactant, blocking agent, and protective agent were added to pH buffer solution in sequence, stirred to dissolve and mix evenly; then Evans blue was added and stirred until completely dissolved. After filtration, the sample pad treatment solution was obtained.

[0021] Thirdly, the present invention provides a sample pad, which is treated with the sample pad treatment solution as described in the first aspect.

[0022] Preferably, the method of treating the sample pad with the sample pad treatment solution as described in the first aspect includes at least one of immersion and spraying. The immersion method includes the following steps: immersing the substrate of the sample pad in the sample pad treatment solution, taking it out, drying it, and obtaining the sample pad. The spraying method includes the following steps: spraying the sample pad treatment solution onto the substrate of the sample pad, drying it, and obtaining the sample pad.

[0023] Preferably, the substrate of the sample pad is glass fiber or polyester fiber.

[0024] Fourthly, the present invention provides a colloidal gold immunochromatographic test strip, comprising the sample pad as described in the third aspect.

[0025] Preferably, the colloidal gold immunochromatographic test strip further includes a conjugate pad, a nitrocellulose membrane, and an absorbent pad, wherein the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad are arranged sequentially. The conjugate pad is loaded with a colloidal gold label, which is an antibody or antigen labeled with colloidal gold nanoparticles. The nitrocellulose membrane is provided with a control line and a detection line. The control line is positioned close to the absorbent pad relative to the detection line. The detection line is coated with an antibody or antigen corresponding to the target analyte, and the control line is coated with an antibody or antigen corresponding to the colloidal gold label.

[0026] In a further preferred embodiment, the colloidal gold immunochromatographic test strip also includes a base plate, on which the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad are all disposed.

[0027] More preferably, the base plate is made of polyvinyl chloride.

[0028] More preferably, the substrate of the bonding pad is glass fiber or polyester fiber.

[0029] More preferably, the substrate of the absorbent pad is absorbent filter paper.

[0030] The preparation method of the colloidal gold immunochromatographic test strip includes the following steps: The sample pad, conjugation pad, nitrocellulose membrane, and absorbent pad are sequentially overlapped and pasted onto the base plate in the direction of chromatography to obtain the colloidal gold immunochromatographic test strip.

[0031] Fifthly, the present invention provides a reagent kit comprising at least one of the sample pad as described in the third aspect and the colloidal gold immunochromatographic test strip as described in the fourth aspect.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows: The sample pad treatment solution provided by this invention is suitable for pretreating the sample pad of colloidal gold immunochromatographic test strips, so that the sample pad adsorbs surfactants, blocking agents, protective agents, and Evans blue. During the testing of target analytes using colloidal gold immunochromatographic test strips, Evans blue can form a weak complex with unbound colloidal gold nanoparticles through electrostatic and hydrophobic interactions. This complex has strong hydrophilicity and excellent chromatographic flowability in nitrocellulose membranes, which can carry colloidal gold labels to migrate rapidly to the absorbent pad, reducing or avoiding the retention of colloidal gold nanoparticles on the membrane surface, thereby improving the membrane whitening effect of the colloidal gold immunochromatographic test strip. Moreover, Evans blue itself does not specifically bind to antigens or antibodies and will not adversely affect the detection performance of the test strip. Detailed Implementation

[0033] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments and comparative examples. The purpose of this description is to provide a detailed understanding of the invention, not to limit its scope. All other embodiments obtained by those skilled in the art without inventive effort are within the protection scope of this invention. Unless otherwise specified, the experimental reagents and instruments involved in the implementation of this invention are commonly used reagents and instruments.

[0034] The source information and performance parameters of the components described in each embodiment and comparative example are shown in Table 1 below.

[0035] Table 1 Unless otherwise specified, all components and raw materials used in the embodiments and comparative examples of this invention are commercially available, and the same type of components and raw materials are used in each parallel experiment.

[0036] Examples 1-9 and Comparative Examples 1-2 Examples and comparative examples of the sample pad treatment solution of the present invention are provided. The sample pad treatment solution is composed of the components shown in Table 2, and the mass percentage of each component in the sample pad treatment solution is shown in Table 2.

[0037] The sample pad treatment solution is prepared as follows: all components except Evans blue are added sequentially to pH buffer solution, stirred to dissolve and mix evenly; then Evans blue is added, stirred until completely dissolved, and filtered to obtain the sample pad treatment solution.

[0038] Table 2 (Unit: mass percentage, %) Application Example 1 This set of application examples provides a colloidal gold immunochromatographic test strip, the preparation method of which is as follows: The sample pad substrate (glass fiber) was immersed in the sample pad treatment solution for 15 minutes, then removed and dried in an oven at 40°C for 18 hours to obtain the sample pad; the sample pad treatment solutions used in Application Examples 1A to 1K are shown in Table 3.

[0039] Using a conventional colloidal gold labeling method, a colloidal gold solution was taken, and the pH was adjusted to 8.0 with 0.2M K2CO3 solution. Anti-HCG monoclonal antibody (labeled antibody) was added at a ratio of 10 mL:0.1 mg:0.1 g of colloidal gold solution volume, antibody mass, and bovine serum albumin (BSA) mass. The reaction was allowed to proceed for 30 min, followed by blocking with BSA for 30 min. After centrifugation and discarding the supernatant, the colloidal gold label was obtained. The colloidal gold label was then resuspended in 0.05M PB buffer solution (pH 7.4) (PB buffer solution also contains 5 mg / ml BSA and 1 mg / ml arginine) to 1 / 10 of the original colloidal gold solution volume, yielding the colloidal gold label solution. The colloidal gold label solution was sprayed onto a conjugate pad substrate (polyester fiber) using a spraying device at a spray rate of 2.0 μl / cm. The substrate was then dried in an oven at 37℃ for 12 h to obtain the conjugate pad. Anti-HCG monoclonal antibody (capture antibody) was diluted to 1 mg / mL with PB buffer (0.01 M, pH 7.4) and coated onto a nitrocellulose membrane at a rate of 0.8 μL / cm using a coating applicator to form a detection line (T line). Goat anti-mouse IgG antibody was diluted to 1 mg / mL with PB buffer (0.01 M, pH 7.4) and coated onto a nitrocellulose membrane at a rate of 0.8 μL / cm using a coating applicator to form a control line (C line). The nitrocellulose membrane was then dried in an oven at 37°C for 24 h to obtain a nitrocellulose membrane with detection and control lines. The sample pad, conjugate pad, nitrocellulose membrane, and absorbent filter paper are sequentially glued onto a PVC base plate. After assembly, the strips are cut into 3mm wide test strips using a cutting machine to obtain colloidal gold immunochromatographic test strips.

[0040] This application example group also underwent whitening effect testing, sensitivity testing, and specificity testing. The test results are shown in Table 3, and the test methods are as follows: (1) Whitening effect test: ① Test conditions: room temperature 25℃, relative humidity 40%-60%; ②The test method is as follows: Take 10 test strips respectively, add 100μL of negative urine sample to each test strip, let it stand for 5 minutes, and observe the whitening of the NC membrane surface; ③ The evaluation criteria are as follows: no obvious red residue on the membrane surface indicates that the whitening is qualified, and obvious red residue indicates that it is unqualified; the number of test strips with clean membrane surface and no colloidal gold residue, the number of test strips with slight residual image on the membrane surface, and the number of test strips with obvious colloidal gold residue on the membrane surface are counted respectively. (2) Sensitivity test: ① Test samples: Positive urine samples with HCG concentrations of 2.5 mIU / mL, 5 mIU / mL, 10 mIU / mL, and 20 mIU / mL were prepared using human chorionic gonadotropin (HCG) and urine matrix, respectively. The urine matrix was used as the negative urine sample. ② Test method: Five test strips were tested in parallel for each concentration. 100 μL of sample was added to the sample pad of the test strip, and the color development of the test line was observed after 5 minutes. ③ Evaluation criteria: The lowest concentration at which the detection line is clearly visible and colored is the lowest detection limit.

[0041] (3) Specificity test: ① Test samples: Urine samples containing luteinizing hormone (LH 100mIU / mL), thyroid-stimulating hormone (TSH 100mIU / mL), and follicle-stimulating hormone (FSH 100mIU / mL) were prepared using luteinizing hormone, thyroid-stimulating hormone, and follicle-stimulating hormone (FSH) and urine matrix, respectively. The urine matrix was used as the negative urine sample. ② Test method: Five test strips were tested in parallel for each sample. 100 μL of sample was added to the sample pad of the test strip, and the color development of the test line and control line was observed after 10 min. ③ Evaluation criteria: If the test line does not develop color (only the quality control line develops color), the specificity is qualified.

[0042] The test results are shown in Table 3.

[0043] Table 3 As shown in Table 3, the introduction of an appropriate amount of Evans blue into the sample pad treatment solution used in Application Examples 1A to 1I significantly improved the whitening effect of the test strips. Compared with Application Examples 1A to 1I, the sample pad treatment solution used in Application Example 1J did not contain Evans blue, resulting in a significantly worse whitening effect on the nitrocellulose membrane of the test strips. In Application Example 1K, the sample pad treatment solution contained too much Evans blue, leading to a worse whitening effect on the nitrocellulose membrane of the test strips.

[0044] The lowest detection limit for HCG obtained from all application examples was 5 mIU / mL, indicating that the introduction of Evans blue does not affect the detection sensitivity of the test strip for HCG.

[0045] When samples with different matrices were added, the test lines of the test strips obtained in each application example did not develop color, while the control lines developed clearly. This indicates that the introduction of Evans blue does not affect the detection specificity of the test strips.

[0046] Application Example 2 This set of application examples provides a colloidal gold immunochromatographic test strip, the preparation method of which is as follows: The sample pad substrate (glass fiber) was immersed in the sample pad treatment solution for 15 minutes, then removed and dried in an oven at 40°C for 18 hours to obtain the sample pad; the sample pad treatment solutions used in Application Examples 2A to 2K are shown in Table 4.

[0047] Using a conventional colloidal gold labeling method, a colloidal gold solution was taken, and the pH was adjusted to 8.0 with 0.2M K2CO3 solution. A monoclonal antibody against the SARS-CoV-2 N protein (labeled antibody) was added at a ratio of 10 mL:0.1 mg:0.1 g (colloidal gold solution volume, antibody mass, bovine serum albumin (BSA) mass). The reaction was allowed to proceed for 30 min, followed by blocking with BSA for 30 min. After centrifugation and discarding the supernatant, the colloidal gold label was obtained. The colloidal gold label was then resuspended in 0.05M PB buffer (pH 7.4) (PB buffer also contains 5 mg / ml BSA and 1 mg / ml arginine) to 1 / 10 of the original colloidal gold solution volume, yielding the colloidal gold label solution. The colloidal gold label solution was then sprayed onto a conjugate pad substrate (polyester fiber) using a sprayer at a spray rate of 2.0 μl / cm. The substrate was then dried in an oven at 37℃ for 12 h to obtain the conjugate pad. Monoclonal antibody against the SARS-CoV-2 N protein (capture antibody) was diluted to 1 mg / mL with PB buffer (0.01 M, pH 7.4) and coated onto a nitrocellulose membrane at a rate of 0.8 μL / cm using a coating applicator to form a detection line (T line). Goat anti-mouse IgG antibody was diluted to 1 mg / mL with PB buffer (0.01 M, pH 7.4) and coated onto a nitrocellulose membrane at a rate of 0.8 μL / cm using a coating applicator to form a control line (C line). The nitrocellulose membrane was then dried in an oven at 37°C for 24 h to obtain a nitrocellulose membrane with detection and control lines. The sample pad, conjugate pad, nitrocellulose membrane, and absorbent filter paper are sequentially glued onto a PVC base plate. After assembly, the strips are cut into 3mm wide test strips using a cutting machine to obtain colloidal gold immunochromatographic test strips.

[0048] This application case also underwent whitening effect testing, sensitivity testing, and specificity testing. The test results are shown in Table 4, and the test methods are as follows: (1) Whitening effect test: ① Test conditions: room temperature 25℃, relative humidity 40%~60%; ②The test method is as follows: Take 10 test strips respectively, add 100μL of negative urine sample to each test strip, let it stand for 5 minutes, and observe the whitening of the NC membrane surface; ③ The evaluation criteria are as follows: no obvious red residue on the membrane surface indicates that the whitening is qualified, and obvious red residue indicates that it is unqualified; the number of test strips with clean membrane surface and no colloidal gold residue, the number of test strips with slight residual image on the membrane surface, and the number of test strips with obvious colloidal gold residue on the membrane surface are counted respectively. (2) Sensitivity test: ① Test samples: Positive pharyngeal swab samples with a SARS-CoV-2 N protein concentration of 25 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL were prepared using SARS-CoV-2 N protein and pharyngeal swab sampling solution, respectively. The pharyngeal swab sampling solution was used as the negative pharyngeal swab sample. ② Test method: Five test strips were tested in parallel for each concentration. 100 μL of sample was added to the sample pad of the test strip, and the color development of the test line was observed after 5 minutes. ③ Evaluation criteria: The lowest concentration at which the T-line is clearly visible is the lowest detection limit.

[0049] (3) Specificity test: ① Test samples: Pharyngeal swab samples containing influenza A / B antigen (100 ng / mL) and respiratory syncytial virus antigen (100 ng / mL) were prepared using influenza A / B antigen, respiratory syncytial virus antigen, and pharyngeal swab sampling solution, respectively. Pharyngeal swab sampling solution was used as the negative pharyngeal swab sample. ② Test method: Five test strips were tested in parallel for each sample. 100 μL of sample was added, and the color development of the test line and control line was observed after 10 min. ③ Evaluation criteria: No color development on the T line (only the C line is colored) indicates that the specificity is qualified.

[0050] Table 4 As shown in Table 4, the introduction of an appropriate amount of Evans blue into the sample pad treatment solution used in Application Examples 2A to 2I significantly improved the whitening effect of the test strips. Compared with Application Examples 2A to 2I, the sample pad treatment solution used in Application Example 2J did not contain Evans blue, resulting in a significantly worse whitening effect on the nitrocellulose membrane of the test strips. In Application Example 2K, the sample pad treatment solution contained too much Evans blue, further deteriorating the whitening effect on the nitrocellulose membrane of the test strips.

[0051] The lowest detection limit for the SARS-CoV-2 N protein obtained from all application examples was 50 ng / mL, indicating that the introduction of Evans blue did not affect the detection sensitivity of the test strip for the SARS-CoV-2 N protein.

[0052] When samples with different matrices were added, the test lines of the test strips obtained in each application example did not develop color, while the control lines developed clearly. This indicates that the introduction of Evans blue does not affect the detection specificity of the test strips.

[0053] Application Example 3 This set of application examples provides a colloidal gold immunochromatographic test strip, the preparation method of which is as follows: The sample pad substrate (glass fiber) was immersed in the sample pad treatment solution for 15 minutes, then removed and dried in an oven at 40°C for 18 hours to obtain the sample pad; the sample pad treatment solutions used in Application Examples 3A to 3K are shown in Table 5. Using a conventional colloidal gold labeling method, a colloidal gold solution was taken, and the pH was adjusted to 8.0 with 0.2M K2CO3 solution. Anti-aflatoxin B1 monoclonal antibody was added at a ratio of 10 mL:0.1 mg:0.1 g of colloidal gold solution volume, antibody mass, and bovine serum albumin (BSA) mass. The reaction was allowed to proceed for 30 min, followed by blocking with BSA for 30 min. After centrifugation and discarding the supernatant, the colloidal gold label was obtained. The colloidal gold label was then resuspended in 0.05M PB buffer (pH 7.4) (PB buffer also contains 5 mg / ml BSA and 1 mg / ml arginine) to 1 / 10 of the original colloidal gold solution volume, yielding the colloidal gold label solution. The colloidal gold label solution was sprayed onto a conjugate pad substrate (polyester fiber) using a sprayer at a spray rate of 2.0 μl / cm. The substrate was then dried in an oven at 37℃ for 12 h to obtain the conjugate pad. Aflatoxin B1 artificial antigen was diluted to 1 mg / mL with PB buffer (0.01 M, pH 7.4) and coated onto a nitrocellulose membrane at a rate of 0.8 μL / cm using a coating applicator to form a detection line (T line). Goat anti-mouse IgG antibody was diluted to 1 mg / mL with PB buffer (0.01 M, pH 7.4) and coated onto a nitrocellulose membrane at a rate of 0.8 μL / cm using a coating applicator to form a control line (C line). The nitrocellulose membrane was then dried in an oven at 37°C for 24 h to obtain a nitrocellulose membrane with both detection and control lines. The sample pad, conjugate pad, nitrocellulose membrane, and absorbent filter paper are sequentially glued onto a PVC base plate. After assembly, the strips are cut into 3mm wide test strips using a cutting machine to obtain colloidal gold immunochromatographic test strips.

[0054] This application example also underwent whitening effect testing, sensitivity testing, and matrix effect testing. The test results are shown in Table 5, and the test methods are as follows: (1) Whitening effect test: ① Test conditions: room temperature 25℃, relative humidity 40%-60%; ②The test method is as follows: Take 10 test strips of each type, add 100μL of corn extract to each test strip, let it stand for 5 minutes, and observe the whitening of the NC membrane surface; ③ The evaluation criteria are as follows: no obvious red residue on the membrane surface indicates that the whitening is qualified, and obvious red residue indicates that it is unqualified; the number of test strips with clean membrane surface and no colloidal gold residue, the number of test strips with slight residual image on the membrane surface, and the number of test strips with obvious colloidal gold residue on the membrane surface are counted respectively. (2) Sensitivity test: ① Test samples: Positive corn extract samples with aflatoxin B1 concentrations of 1 μg / kg, 2.5 μg / kg, 5 μg / kg, and 10 μg / kg were prepared using aflatoxin B1 and corn extract, respectively. Corn extract was used as the negative sample. ② Test method: Five test strips were tested in parallel for each concentration. 100 μL of sample was added to the sample pad of the test strip, and the color development of the test line was observed after 5 minutes. ③ Evaluation criteria: The lowest concentration at which the detection line is clearly visible and colored is the lowest detection limit.

[0055] (3) Matrix effect test: ① Test samples: Rice extract containing aflatoxin B1 (2.5 μg / kg), wheat extract containing aflatoxin B1 (2.5 μg / kg), and peanut extract containing aflatoxin B1 (2.5 μg / kg) were prepared using aflatoxin B1, corn extract, wheat extract, and peanut extract, respectively. ② Test method: Five test strips were tested in parallel for each sample. 100 μL of sample was added to the sample pad of the test strip. After 5 minutes, the color development of the test line and the control line and the appearance of the film were observed. ③ Evaluation criteria: The detection line is clearly visible and the film surface is cleanly faded, with no colloidal gold residue.

[0056] Table 5 As shown in Table 5, the introduction of an appropriate amount of Evans blue into the sample pad treatment solution used in Application Examples 3A to 3I significantly improved the whitening effect of the test strips. Compared with Application Examples 3A to 3I, the sample pad treatment solution used in Application Example 3J did not contain Evans blue, resulting in a significantly worse whitening effect on the nitrocellulose membrane of the test strips. In Application Example 3K, the sample pad treatment solution contained too much Evans blue, leading to a worse whitening effect on the nitrocellulose membrane of the test strips.

[0057] The lowest detection limit for aflatoxin B1 obtained from all application examples was 2.5 μg / kg, indicating that the introduction of Evans blue did not affect the detection sensitivity of the test strip for aflatoxin B1.

[0058] When samples with different matrices were added, the test lines of the test strips obtained in each application example showed normal color development, and the membrane surface faded cleanly. This indicates that the test strips obtained in each example are suitable for the detection of samples with different matrices.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A sample pad treatment solution, characterized in that, The sample pad treatment solution comprises the following components: surfactant, blocking agent, protective agent, Evans blue, and pH buffer; wherein the surfactant has a mass percentage of 0.5-2%, the blocking agent has a mass percentage of 1-3%, and the Evans blue has a mass percentage of 0.02-0.1%.

2. The sample pad treatment solution as described in claim 1, characterized in that, The surfactant includes at least one of Tween 20 and Triton X-100; And / or, the blocking agent includes at least one of bovine serum albumin and polyvinylpyrrolidone; And / or, the pH buffer includes at least one of Tris-HCl buffer, PBS buffer, and borate buffer; And / or, the pH value of the pH buffer solution is 7.2 to 8.

0.

3. The sample pad treatment solution as described in claim 1, characterized in that, It also includes the following components: a protective agent comprising sucrose, wherein the protective agent has a mass percentage of 0.5 to 1.5% in the sample pad treatment solution.

4. The sample pad treatment solution as described in claim 1, characterized in that, It also includes the following component: sodium chloride, wherein the mass percentage of sodium chloride in the sample pad treatment solution is 0.5-1%.

5. A method for preparing the sample pad treatment solution as described in any one of claims 1 to 4, characterized in that, Includes the following steps: Mix all components except Evans blue thoroughly, then add Evans blue and stir until Evans blue dissolves. Filter to obtain the sample pad treatment solution.

6. A sample pad, characterized in that, The sample pad is treated with the sample pad treatment solution as described in any one of claims 1 to 2.

7. The sample pad as described in claim 6, characterized in that, The substrate of the sample pad is glass fiber or polyester fiber.

8. A colloidal gold immunochromatographic test strip, characterized in that, Includes the sample pad as described in any one of claims 6 to 7.

9. The colloidal gold immunochromatographic test strip as described in claim 8, characterized in that, It also includes a conjugate pad, a nitrocellulose membrane, and an absorbent pad, wherein the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad are arranged sequentially. The conjugate pad is loaded with a colloidal gold label, which is an antibody or antigen labeled with colloidal gold nanoparticles. The nitrocellulose membrane is provided with a control line and a detection line. The control line is positioned close to the absorbent pad relative to the detection line. The detection line is coated with an antibody or antigen corresponding to the target analyte. The control line is coated with an antibody or antigen corresponding to the colloidal gold label.

10. A reagent kit, characterized in that, It includes at least one of the sample pads as described in any one of claims 6 to 7 and the colloidal gold immunochromatographic test strips as described in any one of claims 8 to 9.

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