A lateral flow chromatographic test strip and its use

Abstract

The application provides a lateral flow chromatography test paper and application thereof. The lateral flow chromatography test paper comprises a quality control line and an absorption pad. The lateral flow chromatography test paper further comprises an intercept line arranged between the quality control line and the absorption pad, and the material of the intercept line is one or a mixture of two or more of gelatin, carrageenan, polyacrylamide hydrogel, polyacrylic acid hydrogel, polyvinyl alcohol hydrogel or polyvinyl alcohol-sodium alginate hydrogel solution. The lateral flow chromatography test paper can improve the sensitivity of commercial test paper strips, and can be applied to new crown antigen detection, human chorionic gonadotropin detection (HCG) and cardiac troponin detection. The lateral flow chromatography test paper provided by the application can significantly improve the sensitivity by adding a cheap hydrogel intercept line after the quality control line, has good universality, and can be widely applied to various colloidal gold method test paper detection and immunofluorescence test paper detection.

Description

Technical Field

[0001] This invention belongs to the field of lateral flow chromatography detection technology, and specifically relates to a lateral flow chromatography test strip and its application. Background Technology

[0002] Lateral flow chromatography (LCC) is widely used in disease diagnosis, food safety, and environmental monitoring. Compared to traditional detection methods such as high-performance liquid chromatography (HPLC) and polymerase chain reaction (PCR), which are costly, time-consuming, and require professional operators, LCC test strips are low-cost, easy to operate, and portable. Commercial LCC test strips typically include a sample pad, a gold-labeled pad, a detection (T) line, a control (C) line, an absorbent pad, and a backing plate. Changing the conjugated antibody on the gold-labeled pad and the capture antibody on the T line allows for the detection of different analytes. Therefore, LCC test strips have been used to detect various targets such as nucleic acids, proteins, viruses, bacteria, and heavy metals. However, low sensitivity limits its widespread application.

[0003] Currently, researchers are improving the sensitivity of lateral flow chromatography detection through methods such as substrate material modification, probe signal enhancement, and sample concentration. However, these techniques require expensive chemical reagents and complex processing, increasing detection costs. Furthermore, the product development stage requires extensive screening and testing, further increasing costs. Slowing down the flow rate of lateral flow chromatography to enhance sensitivity is an effective method, but existing methods suffer from drawbacks such as long material synthesis times, complex operations, and difficulties in mass production. Therefore, simple and low-cost modification of existing, commercially viable test strips to improve their sensitivity is a preferred approach to effectively address these issues. Summary of the Invention

[0004] This invention provides a side-flow chromatography test strip and its application to solve the above-mentioned technical problems. This test strip addresses the issues of poor sensitivity in commercially available test strips, as well as the problems of long development time, complex operation, and high cost in methods for improving test strip sensitivity.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A lateral flow chromatography test strip, the lateral flow chromatography test strip includes a control line and an absorbent pad, the lateral flow chromatography test strip further includes an intercept line disposed between the control line and the absorbent pad, the material of the intercept line being one or a mixture of two or more of gelatin, carrageenan, polyacrylamide hydrogel, polyacrylic acid hydrogel, polyvinyl alcohol hydrogel or polyvinyl alcohol-sodium alginate hydrogel solution.

[0007] Preferably, the mass fraction of the hydrogel solution is less than or equal to 40%. The hydrogel is a natural hydrogel or a synthetic hydrogel.

[0008] Preferably, the hydrogel solution is prepared as follows:

[0009] When the hydrogel solution is a gelatin solution, the method is as follows: add gelatin powder to pure water (preferably ultrapure water), heat to 60-80°C, and maintain for 10-15 minutes to obtain the solution;

[0010] When the hydrogel solution is a carrageenan solution, the method is as follows: add carrageenan powder to a KCl solution with a KCl concentration of 0.15-0.25% (w / v), heat the first time at 35-40°C and stir for 15-25 min, heat the second time at 85-95°C and hold for 10-15 min, and the hydrogel is obtained.

[0011] When the hydrogel solution is a polyacrylamide hydrogel solution, the method is as follows: acrylamide, N,N-methylenebisacrylamide, sodium alginate, ammonium persulfate, calcium sulfate, and tetramethylethylenediamine are added to ultrapure water respectively and stirred evenly to obtain the solution.

[0012] Preferably, the gelatin has a mass fraction of less than or equal to 20%, more preferably 4-20%, more preferably 4%, 8%, 12%, 16%, or 20%, and most preferably 16%. The carrageenan has a mass fraction of less than or equal to 3%, preferably 1-3%, more preferably 1%, 1.5%, 2%, 2.5%, or 3%, and most preferably 1.5% or 2%. The polyacrylamide hydrogel has a mass fraction of less than or equal to 40%, preferably 20-40%, more preferably 20%, 30%, 35%, or 40%, and most preferably 35%.

[0013] Preferably, the intercept line is prepared by the following method: coating the hydrogel solution onto the control line at a distance of 1-2 mm from the absorbent pad, allowing it to stand, and then vacuum drying to obtain the intercept line.

[0014] Preferably, the area of ​​the coating is 2-6 mm in length, 2-5 mm in width, and has a load capacity of 10-20 μL / cm.

[0015] Preferably, the settling time is 20 to 40 minutes.

[0016] Preferably, the vacuum drying time is 5 to 20 minutes and the temperature is 20 to 60°C.

[0017] The application of the lateral flow chromatography test strip in the detection of COVID-19 antigen by colloidal gold method, the detection of HCG by colloidal gold method, or the detection of cardiac troponin by fluorescence method.

[0018] Preferably, in the detection of the COVID-19 antigen protein, the COVID-19 antigen protein dissolution and unfolding agent is used as the sample to be tested.

[0019] Preferably, in the HCG detection, the HCG protein dissolution developing solvent is used as the sample to be tested.

[0020] Preferably, in the cardiac troponin detection, a serum solution containing cardiac troponin is dissolved in a developing solvent as the sample to be tested.

[0021] Preferably, the developing solvent is a PBS solution containing Tween-20. The PBS solution of Tween-20 is obtained by dissolving Tween-20 in a 10 mM PBS solution with a pH of 7.4. The volume concentration of Tween-20 is 0.1%.

[0022] Preferably, in the application of the COVID-19 antigen protein detection, the optimal concentrations of gelatin, carrageenan, or polyacrylamide hydrogel on the intercept line are 16%, 1.5-2.5%, and 35% (wt%), respectively.

[0023] Preferably, in the application of the colloidal gold method for detecting HCG, the optimal concentrations of gelatin, carrageenan, or polyacrylamide hydrogel on the intercept line are 12%, 1.5%, and 35% (wt%), respectively.

[0024] Preferably, in the application of the fluorescence method for detecting cardiac troponin, the optimal concentrations of gelatin, carrageenan, or polyacrylamide hydrogel on the intercept line are 16%, 1.5%, and 35% (wt%), respectively.

[0025] Compared with existing technologies, the beneficial effects of this invention are as follows: The lateral flow chromatography test strip provided by this invention features an intercept line between the control line and the absorption pad of a commercial test strip, enhancing its sensitivity. The intercept line of the lateral flow chromatography test strip of this invention is coated with inexpensive hydrogel, and the optimal concentration of hydrogel is determined through improvements to the intercept line. The lateral flow chromatography test strip is suitable not only for the detection of COVID-19 antigen and HCG in colloidal gold method test strips, but also for the detection of cardiac troponin in fluorescence method test strips. The improved technology for enhancing the sensitivity of commercial test strips provided by this invention does not require extensive screening, substrate modification, or other complex methods and processes. It only requires modification of existing commercial test strips, making it highly operable, widely applicable, and low-cost, and can be widely used in colloidal gold and fluorescence detection methods. Attached Figure Description

[0026] Figure 1 This refers to the results of the Tianjin Bio-Sys test strip with a 16% gelatin intercept line used in Embodiment 1 of the present invention for the colloidal gold method detection of COVID-19 antigen;

[0027] Figure 2 The results of the Tianjin Bio-Sys test strip with a 1.5% carrageenan intercept line for the colloidal gold method detection of COVID-19 antigen, as described in Embodiment 2 of the present invention;

[0028] Figure 3 The results of the Tianjin Bio-Sys test strip with a 35% polyacrylamide hydrogel interception line for the colloidal gold method detection of COVID-19 antigen, as described in Example 3 of this invention;

[0029] Figure 4 This refers to the results of the Beijing Lepu test strip with a 16% gelatin intercept line used in Example 4 of this invention for the colloidal gold method detection of COVID-19 antigen;

[0030] Figure 5 This is the result of the Beijing Lepu test strip with a 2% carrageenan intercept line used in Example 5 of the present invention for the colloidal gold method detection of COVID-19 antigen;

[0031] Figure 6 This refers to the results of the Beijing Lepu test strip with a 35% polyacrylamide hydrogel interception line used in Embodiment 6 of the present invention for the colloidal gold method detection of COVID-19 antigen.

[0032] Figure 7 The results of the Shantou David early pregnancy test strip with a 12% gelatin intercept line in Embodiment 7 of the present invention for the colloidal gold method detection of HCG antigen;

[0033] Figure 8 The results of the Shantou David early pregnancy test strips with different settings of 1.5% carrageenan intercept line in Example 8 of the present invention for the colloidal gold method detection of HCG antigen;

[0034] Figure 9 The results of the Shantou David early pregnancy test strip with a 35% polyacrylamide hydrogel interception line in Example 9 of this invention for the colloidal gold method detection of HCG antigen.

[0035] Figure 10 The results of the immunofluorescence assay for cardiac troponin using Wondfo Biotech paper strips with a 4% gelatin interception line in Example 10 of this invention;

[0036] Figure 11 The results of the immunofluorescence assay for cardiac troponin using Wondfo Biotech paper strips with a 0.5% carrageenan intercept line in Example 11 of this invention are shown.

[0037] Figure 12 The results of the immunofluorescence assay for cardiac troponin using Wondfo Biotech paper strips with a 25% polyacrylamide hydrogel interception line in Example 11 of this invention;

[0038] Figure 13 This is a schematic diagram of the side-flow chromatography test paper of the present invention. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0040] like Figure 13 As shown, this is a lateral flow chromatography test strip provided by the present invention. The lateral flow chromatography test strip includes a sample pad, a gold-labeled pad, a detection (T) line, a control (C) line, an intercept line, an absorbent pad, and a backing plate. The gold-labeled pad is modified with conjugated antibodies, and the T line is modified with capture antibodies to detect different analytes. The intercept line is positioned between the control line and the absorbent pad. The intercept line is made of one or more of the following materials: gelatin, carrageenan, polyacrylamide hydrogel, polyacrylic acid hydrogel, polyvinyl alcohol hydrogel, or a polyvinyl alcohol-sodium alginate hydrogel solution. It is understood that an intercept line can also be added directly to a commercial test strip. Figure 13 This is a schematic diagram of the structure of a gel-modified commercial test strip (taking the COVID-19 antigen test strip as an example). The following examples are all lateral flow chromatography test strips obtained by adding an intercept line to a commercial test strip, but are not limited to this, and the lateral flow chromatography test strip of the present invention can also be prepared independently.

[0041] Example 1

[0042] This embodiment provides a lateral flow chromatography test strip, which is a modification of the Tianjin Bio-Rad test strip used for colloidal gold detection of SARS-CoV-2 antigen. The intercept line of the lateral flow chromatography test strip in this embodiment is made of gelatin.

[0043] The specific preparation method of the side-flow chromatography test paper is as follows:

[0044] 1) Preparation of gelatin hydrogels: 0.3, 0.5, 0.7, and 0.9 g of gelatin powder were added to 3.667, 3.698, 3.652, and 3.553 mL of ultrapure water, respectively. The mixture was heated to 70 °C and held for 12 min to obtain gelatin hydrogels of 8%, 12%, 16%, and 20% (wt%), respectively.

[0045] 2) Preparation of the intercept line: Take 5 μL of the above gelatin solution and drop it onto the chromatographic membrane of the Tianjin Bio-Sys COVID-19 test strip as the intercept line; the intercept line is 1-2 mm away from the absorbent pad after the quality control line, with a length of 3 mm and a width of 3 mm; then let it stand at 25℃ for 2 h, and finally place it in a vacuum dryer at 25℃ for 5-10 min to obtain the side-flow chromatographic test strip.

[0046] The side-flow chromatography test strip is used in the colloidal gold method for detecting SARS-CoV-2 antigen as follows:

[0047] 1) Preparation of test solutions: Dilute the COVID-19 antigen solution with Tween-20 in PBS to 10 ng / mL. Then, take 1, 5, 10, 50, and 100 μL of this solution and dilute it to 1 mL with Tween-20 in PBS, respectively, to obtain test solutions with COVID-19 antigen protein concentrations of 0.01, 0.05, 0.1, 0.5, 1, and 10 ng / mL. The Tween-20 in PBS is prepared by dissolving Tween-20 in a 10 mM PBS solution with a pH of 7.4. The volume concentration of Tween-20 is 0.1%.

[0048] 2) Detection method: Take 50 μL of the test solution containing 0.01, 0.05, 0.1, 0.5, 1, and 10 ng / mL of SARS-CoV-2 antigen, respectively, and drop them onto the sample plate of the Tianjin Bio-Cys COVID-19 test strip without gelatin modification as a blank; then, drop the above test solutions of different concentrations onto the sample pad of the lateral flow chromatography test strip modified based on the Tianjin Bio-Cys COVID-19 test strip of this invention. After lateral flow chromatography, take a picture and read the gray value on the test line.

[0049] 3) Results and Statistics: The gray values ​​at the T-line on the sideflow chromatography test strips after modification (or without modification) with different concentrations of gelatin intercept lines were statistically analyzed, and the detection limits after modification with different gelatin intercept lines were calculated, as shown in Table 1 (Table 1 shows the statistical analysis of the detection limits corresponding to different concentrations of hydrogel intercept lines on the colloidal gold method commercial test strips involved in Examples 1-9 of this invention). Figure 1 As shown, the results indicate that the detection limit of the Tianjin Bio-Cys test strip in the blank group was 0.1043 ng / mL; while the detection limit of the side-flow chromatography test strip of the present invention is improved. When the gelatin solution concentration is 16%, there is an optimal detection limit of 0.0429 ng / mL, and the sensitivity is significantly enhanced.

[0050] Table 1. Statistics on the detection limits of the lateral flow chromatography test strips obtained by setting different concentrations of hydrogel intercept lines on the colloidal gold method commercial test strips according to the present invention.

[0051]

[0052] Example 2

[0053] This embodiment provides a lateral flow chromatography test strip, which is a modification of the Tianjin Bio-Cys test strip used for colloidal gold detection of COVID-19 antigens. The intercept line material of the lateral flow chromatography test strip in this embodiment is carrageenan.

[0054] The specific preparation method of the side-flow chromatography test paper is as follows:

[0055] 1) Preparation of carrageenan hydrogels: 0.0558, 0.0764, 0.0812, and 0.988 g of carrageenan powder were added to 3.664, 3.743, 3.166, and 3.194 mL of 0.2% KCl (w / v) solution, respectively. The solution was heated to 40℃ and stirred for 20 min, then heated to 90℃ and held for 12 min to obtain 1.5%, 2%, 2.5%, and 3% carrageenan hydrogels (wt%), respectively.

[0056] 2) Preparation of the intercept line: Take 5 μL of the above carrageenan solution and drop it onto the chromatographic membrane of the Tianjin Bio-Sys COVID-19 test strip as the intercept line; the intercept line is 1-2 mm away from the absorbent pad after the control line, with a length of 3 mm and a width of 3 mm; then let it stand at 25℃ for 2 h, and finally place it in a vacuum dryer at 25℃ for 5-10 min to obtain the side-flow chromatographic test strip.

[0057] The side-flow chromatography test strip is used in the colloidal gold method for detecting SARS-CoV-2 antigen as follows:

[0058] 1) Preparation of the test solution: Same as the preparation steps for the test solution in Example 1;

[0059] 2) Detection method: Take 50 μL of the test solution containing 0.01, 0.05, 0.1, 0.5, 1 and 10 ng / mL of SARS-CoV-2 antigen, respectively, and drop them onto the sample pad of the lateral flow chromatography test strip modified based on the Tianjin Bio-Cys SARS-CoV-2 test strip of this invention. After lateral flow chromatography, take a picture and read the gray value on the test line.

[0060] 3) Results and Statistics: The gray values ​​at the T-line on the side-flow chromatography test strips modified with different concentrations of carrageenan intercept lines were statistically analyzed, and the detection limits for different carrageenan intercept line modifications were calculated, as shown in Table 1 and... Figure 2 As shown, the results indicate that the detection limit of the side-flow chromatography test strip of the present invention is improved. When the carrageenan solution concentration is 1.5%, there is an optimal detection limit of 0.0477 ng / mL, which is significantly enhanced in sensitivity compared with the detection limit of the blank control (0.1043 ng / mL, see Example 1).

[0061] Example 3

[0062] This embodiment provides a lateral flow chromatography test strip, which is a modification of the Zimbio Ocythias test strip used for colloidal gold detection of SARS-CoV-2 antigen. The intercept line material of the lateral flow chromatography test strip in this embodiment is polyacrylamide hydrogel.

[0063] The specific preparation method of the side-flow chromatography test paper is as follows:

[0064] 1) Preparation of polyacrylamide hydrogels: 0.5, 0.857, 1.077, and 1.33 g of acrylamide, 0.001, 0.0017, 0.0021, and 0.0026 g of N,N-methylenebisacrylamide, 0.04, 0.0685, 0.0861, and 0.106 g of alginic acid, 0.001, 0.0017, 0.0021, and 0.0026 g of ammonium persulfate, 0.068, 0.0116, 0.0146, and 0.018 g of calcium sulfate, and 0.002, 0.0034, 0.0043, and 0.0053 g of tetramethylethylenediamine were added to 2 mL of ultrapure water and stirred evenly to obtain 20%, 30%, 35%, and 40% polyacrylamide hydrogels (wt%), respectively.

[0065] 2) Preparation of the intercept line: Take 5 μL of the above polyacrylamide hydrogel solution and drop it onto the chromatography membrane of the Tianjin Bio-Sys COVID-19 test strip as the intercept line; the intercept line is 1-2 mm away from the absorbent pad after the control line, with a length of 3 mm and a width of 3 mm; then let it stand at 25℃ for 2 h, and finally place it in a vacuum dryer at 25℃ for 5-10 min to obtain the side-flow chromatography test strip.

[0066] The side-flow chromatography test strip is used in the colloidal gold method for detecting SARS-CoV-2 antigen as follows:

[0067] 1) Preparation of the test solution: Same as the preparation steps for the test solution in Example 1;

[0068] 2) Detection method: Take 50 μL of the test solution containing 0.01, 0.05, 0.1, 0.5, 1 and 10 ng / mL of SARS-CoV-2 antigen, respectively, and drop them onto the sample pad of the lateral flow chromatography test strip modified based on the Tianjin Bio-Cys SARS-CoV-2 test strip of this invention. After lateral flow chromatography, take a picture and read the gray value on the test line.

[0069] 3) Results and Statistics: The gray values ​​at the T-line on the sideflow chromatography test strips modified with different concentrations of polyacrylamide hydrogel intercept lines were statistically analyzed, and the detection limits after modification with different polyacrylamide hydrogel intercept lines were calculated, as shown in Table 1 and... Figure 3 As shown, the results indicate that the detection limit of the side-flow chromatography test strip of the present invention is improved. When the concentration of polyacrylamide hydrogel is 35%, there is an optimal detection limit of 0.0439 ng / mL, which is significantly enhanced in sensitivity compared with the detection limit of the blank control (0.1043 ng / mL, see Example 1).

[0070] Example 4

[0071] This embodiment provides a lateral flow chromatography test strip, which is a modification of the Beijing Lepu test strip used for colloidal gold detection of COVID-19 antigen. The intercept line of the lateral flow chromatography test strip in this embodiment is made of gelatin.

[0072] The specific preparation method of the side-flow chromatography test paper is as follows:

[0073] 1) Preparation of gelatin hydrogel: Same as the preparation steps of gelatin hydrogel in Example 1;

[0074] 2) Preparation of the intercept line: Take 5 μL of the above gelatin hydrogel solution and drop it onto the chromatographic membrane of the Beijing Lepu COVID-19 test strip as the intercept line; the intercept line is 1-2 mm away from the absorbent pad after the quality control line, with a length of 3 mm and a width of 3 mm; then let it stand at 25℃ for 2 h, and finally place it in a vacuum dryer at 25℃ for 5-10 min to obtain the side-flow chromatographic test strip.

[0075] The side-flow chromatography test strip is used in the colloidal gold method for detecting SARS-CoV-2 antigen as follows:

[0076] 1) Preparation of the test solution: Same as the preparation steps for the test solution in Example 1;

[0077] 2) Detection method: Take 50 μL of the test solution containing 0.01, 0.05, 0.1, 0.5, 1, and 10 ng / mL of SARS-CoV-2 antigen, respectively, and drop them onto the sample plate of the Beijing Lepu SARS-CoV-2 test strip without gelatin modification as a blank control; then, drop the above test solutions of different concentrations onto the sample pad of the lateral flow chromatography test strip modified based on the Beijing Lepu SARS-CoV-2 test strip of this invention. After lateral flow chromatography, take a picture and read the gray value on the test line.

[0078] 5) Results and Statistics: The gray values ​​at the T-line on the sideflow chromatography test strips after modification (or without modification) with different concentrations of gelatin intercept lines were statistically analyzed, and the detection limits after modification with different gelatin intercept lines were calculated, as shown in Table 1 and... Figure 4 As shown, the results indicate that the detection limit of the Beijing Lepu test strip in the blank group was 0.0936 ng / mL; while the detection limit of the side-flow chromatography test strip of the present invention is improved. When the gelatin solution concentration is 16%, there is an optimal detection limit of 0.0276 ng / mL, and the sensitivity is significantly enhanced.

[0079] Example 5

[0080] This embodiment provides a lateral flow chromatography test strip, which is a modification of the Beijing Lepu test strip used for colloidal gold detection of COVID-19 antigen. The intercept line of the lateral flow chromatography test strip in this embodiment is made of carrageenan.

[0081] The specific preparation method of the side-flow chromatography test paper is as follows:

[0082] 1) Preparation of carrageenan hydrogel: Same as the preparation steps of carrageenan hydrogel in Example 2;

[0083] 2) Preparation of the intercept line: Take 5 μL of the above carrageenan solution and drop it onto the chromatography membrane of the Beijing Lepu test strip as the intercept line; the intercept line is 1-2 mm away from the absorbent pad after the quality control line, with a length of 3 mm and a width of 3 mm; then let it stand at 25℃ for 2 h, and finally place it in a vacuum dryer at 25℃ for 5-10 min to obtain the side-flow chromatography test strip.

[0084] The side-flow chromatography test strip is used in the colloidal gold method for detecting SARS-CoV-2 antigen as follows:

[0085] 1) Preparation of the test solution: Same as the preparation steps for the test solution in Example 1;

[0086] 2) Detection method: Take 50 μL of the test solution containing 0.01, 0.05, 0.1, 0.5, 1 and 10 ng / mL of SARS-CoV-2 antigen, respectively, and drop them onto the sample pad of the lateral flow chromatography test strip modified based on the Beijing Lepu SARS-CoV-2 test strip of this invention. After lateral flow chromatography, take a picture and read the gray value on the test line.

[0087] 5) Results and Statistics: The gray values ​​at the T-line on the side-flow chromatography test strips modified with different concentrations of carrageenan intercept lines were statistically analyzed, and the detection limits for different carrageenan intercept line modifications were calculated, as shown in Table 1 and... Figure 5 As shown, the results indicate that the detection limit of the side-flow chromatography test strip of the present invention is improved. When the carrageenan solution concentration is 2%, there is an optimal detection limit of 0.0462 ng / mL, which is significantly enhanced in sensitivity compared with the detection limit of the blank control (0.0936 ng / mL, see Example 2).

[0088] Example 6

[0089] This embodiment provides a lateral flow chromatography test strip, which is a modification of the Beijing Lepu test strip used for colloidal gold detection of COVID-19 antigen. The intercept line material of the lateral flow chromatography test strip in this embodiment is polyacrylamide hydrogel.

[0090] The specific preparation method of the side-flow chromatography test paper is as follows:

[0091] 1) Preparation of polyacrylamide hydrogel: Same as the preparation steps of polyacrylamide hydrogel in Example 3;

[0092] 2) Preparation of the intercept line: 5 μL of the above polyacrylamide hydrogel solution was added dropwise onto the chromatographic membrane of the Beijing Lepu COVID-19 test strip to serve as the intercept line. The intercept line was positioned 1-2 mm after the control line, 3 mm in length and 3 mm in width, 1-2 mm away from the absorption pad. The strip was then allowed to stand at 25°C for 2 hours, and finally vacuum-dried at 25°C for 5-10 minutes to obtain the side-flow chromatographic test strip.

[0093] The side-flow chromatography test strip is used in the colloidal gold method for detecting SARS-CoV-2 antigen as follows:

[0094] 1) Preparation of the test solution: Same as the preparation steps for the test solution in Example 1;

[0095] 2) Detection method: Take 50 μL of the test solution containing 0.01, 0.05, 0.1, 0.5, 1 and 10 ng / mL of SARS-CoV-2 antigen, respectively, and drop them onto the sample pad of the lateral flow chromatography test strip modified based on the Beijing Lepu SARS-CoV-2 test strip of this invention. After lateral flow chromatography, take a picture and read the gray value on the test line.

[0096] 3) Results and Statistics: The gray values ​​at the T-line on the sideflow chromatography test strips modified with different concentrations of polyacrylamide hydrogel intercept lines were statistically analyzed, and the detection limits after modification with different gelatin intercept lines were calculated, as shown in Table 1 and... Figure 6 As shown, the results indicate that the detection limit of the side-flow chromatography test strip of the present invention is improved. When the concentration of polyacrylamide hydrogel is 35%, there is an optimal detection limit of 0.0410 ng / mL, which is significantly enhanced in sensitivity compared with the detection limit of the blank control (0.0936 ng / mL, see Example 2).

[0097] Example 7

[0098] This embodiment provides a lateral flow chromatography test strip, which is a modification of the Shantou David early pregnancy test strip used for colloidal gold detection of HCG. The intercept line of the lateral flow chromatography test strip in this embodiment is made of gelatin.

[0099] The specific preparation method of the side-flow chromatography test paper is as follows:

[0100] 1) Preparation of gelatin hydrogel: Same as the preparation steps of gelatin hydrogel in Example 1;

[0101] 2) Preparation of the intercept line: Take 5 μL of the above gelatin solution and drop it onto the chromatographic membrane of the Shantou David early pregnancy HCG test strip as the intercept line; the intercept line is 1-2 mm away from the absorbent pad after the control line, with a length of 3 mm and a width of 3 mm; then let it stand at 25℃ for 2 h, and finally place it in a vacuum dryer at 25℃ for 5-10 min to obtain the side-flow chromatographic test strip.

[0102] The side-flow chromatography test strip is used in the colloidal gold method for HCG detection as follows:

[0103] 1) Preparation of test solutions: Dilute the HCG antigen solution with Tween-20 in PBS to 10 ng / mL. Then, take 1, 5, 10, 50, and 100 μL of this solution and dilute it to 1 mL with Tween-20 in PBS, respectively, to obtain test solutions with HCG antigen concentrations of 0.01, 0.05, 0.1, 0.5, 5, and 10 ng / mL. The Tween-20 in PBS is prepared by dissolving Tween-20 in a 10 mM PBS solution with a pH of 7.4. The volume concentration of Tween-20 is 0.1%.

[0104] 2) Detection method: Take 50 μL of the test solution containing HCG protein concentrations of 0.01, 0.05, 0.1, 0.5, 5, and 10 ng / mL, respectively, and drop them onto the sample pad of the Shantou David Early Pregnancy HCG Test Strip without gelatin modification as a blank; then use the above test solutions of different concentrations to drop onto the sample pad of the lateral flow chromatography test strip modified based on the Shantou David Early Pregnancy HCG Test Strip of this invention. After lateral flow chromatography, take a picture and read the gray value on the detection line.

[0105] 3) Results and Statistics: The gray values ​​at the T-line on the sideflow chromatography test strips after modification (or without modification) with different concentrations of gelatin intercept lines were statistically analyzed, and the detection limits for different gelatin intercept line modifications were calculated, as shown in Table 1 and... Figure 7 As shown, the results indicate that the detection limit of the Shantou David early pregnancy HCG test strip in the blank group was 0.0970 ng / mL; while the detection limit of the side-flow chromatography test strip of the present invention was improved. When the gelatin solution concentration was 12%, the optimal detection limit was reached at 0.0230 ng / mL, and the sensitivity was significantly enhanced.

[0106] Example 8

[0107] This embodiment provides a lateral flow chromatography test strip, which is a modification of the Shantou David early pregnancy test strip used for colloidal gold detection of HCG. The intercept line of the lateral flow chromatography test strip in this embodiment is made of carrageenan.

[0108] The specific preparation method of the side-flow chromatography test paper is as follows:

[0109] 1) Preparation of carrageenan: Same as the preparation steps of carrageenan in Example 2;

[0110] 2) Preparation of the intercept line: Take 5 μL of the above carrageenan solution and drop it onto the chromatographic membrane of the Shantou David early pregnancy HCG test strip as the intercept line; the intercept line is 1-2 mm away from the absorbent pad after the control line, with a length of 3 mm and a width of 3 mm; then let it stand at 25℃ for 2 h, and finally place it in a vacuum dryer at 25℃ for 5-10 min to obtain the side-flow chromatographic test strip.

[0111] The side-flow chromatography test strip is used in the colloidal gold method for HCG detection as follows:

[0112] 1) Preparation of the test solution: Same as the preparation steps for the test solution in Example 7;

[0113] 2) Detection method: Take 50 μL of the test solution containing HCG protein concentrations of 0.01, 0.05, 0.1, 0.5, 5, and 10 ng / mL respectively, and drop them onto the sample pad of the lateral flow chromatography test strip modified based on the Shantou David early pregnancy HCG test strip of this invention. After lateral flow chromatography, take a picture and read the gray value on the detection line.

[0114] 3) Results and Statistics: The gray values ​​at the T-line on the side-flow chromatography test strips modified with different concentrations of carrageenan intercept lines were statistically analyzed, and the detection limits for different concentrations of carrageenan intercept line modification were calculated, as shown in Table 1 and... Figure 8 As shown, the results indicate that the detection limit of the side-flow chromatography test strip of the present invention is improved. When the carrageenan solution concentration is 1.5%, there is an optimal detection limit of 0.0280 ng / mL, which is significantly enhanced compared with the detection limit of the blank control (0.0970 ng / mL, see Example 7).

[0115] Example 9

[0116] This embodiment provides a lateral flow chromatography test strip, which is a modification of the Shantou David early pregnancy test strip used for colloidal gold detection of HCG. The intercept line material of the lateral flow chromatography test strip in this embodiment is polyacrylamide hydrogel.

[0117] The specific preparation method of the side-flow chromatography test paper is as follows:

[0118] 1) Preparation of polyacrylamide hydrogel: Same as the polyacrylamide hydrogel steps in Example 3;

[0119] 2) Preparation of the intercept line: Take 5 μL of the above polyacrylamide hydrogel solution and drop it onto the chromatographic membrane of the Shantou David early pregnancy HCG test strip as the intercept line; the intercept line is 1-2 mm away from the absorbent pad after the control line, with a length of 3 mm and a width of 3 mm; then let it stand at 25℃ for 2 h, and finally place it in a vacuum dryer at 25℃ for 5-10 min to obtain the side-flow chromatographic test strip.

[0120] The side-flow chromatography test strip is used in the colloidal gold method for HCG detection as follows:

[0121] 1) Preparation of the test solution: Same as the preparation steps for the test solution in Example 7;

[0122] 2) Detection method: Take 50 μL of the test solution containing HCG protein concentrations of 0.01, 0.05, 0.1, 0.5, 5, and 10 ng / mL respectively, and drop them onto the sample pad of the lateral flow chromatography test strip modified based on the Shantou David early pregnancy HCG test strip of this invention. After lateral flow chromatography, take a picture and read the gray value on the detection line.

[0123] 3) Results and Statistics: The gray values ​​at the T-line on the sideflow chromatography test strips modified with different concentrations of polyacrylamide hydrogel intercept lines were statistically analyzed, and the detection limits after modification with different concentrations of polyacrylamide hydrogel intercept lines were calculated, as shown in Table 1 and... Figure 9 As shown, the results indicate that the detection limit of the side-flow chromatography test strip of the present invention is improved. When the concentration of the polyacrylamide hydrogel solution is 35%, there is an optimal detection limit of 0.0410 ng / mL, which is significantly enhanced in sensitivity compared with the detection limit of the blank control (0.0970 ng / mL, see Example 7).

[0124] Example 10

[0125] This embodiment provides a lateral flow chromatography test strip, which is a modification of Wondfo Biotech test strips used for the fluorescence detection of cardiac troponin. The intercept line material of the lateral flow chromatography test strip in this embodiment is gelatin.

[0126] The specific preparation method of the side-flow chromatography test paper is as follows:

[0127] 1) Preparation of gelatin: The preparation steps are roughly the same as those in Example 1, except that the concentrations of the prepared gelatin solutions are 2%, 4%, 6%, and 8%, respectively.

[0128] 2) Preparation of the intercept line: Take 5 μL of the above gelatin solution and drop it onto the chromatographic membrane of Wondfo Biotech cardiac troponin test strip as the intercept line; the intercept line is 1-2 mm away from the absorbent pad after the control line, with a length of 3 mm and a width of 3 mm; then let it stand at 25℃ for 2 h, and finally place it in a vacuum dryer at 25℃ for 5-10 min to obtain the side-flow chromatographic test strip.

[0129] The side-flow chromatography test strip is used in the fluorescence detection of cardiac troponin as follows:

[0130] 1) Preparation of test solutions: Serum solutions containing cardiac troponin were diluted in 0.85% physiological saline to obtain cardiac troponin serum concentrations of 0.01, 0.05, 0.1, 0.5, 1, and 5 ng / mL. 75 μL of each of these solutions was then added to the buffer solution in the kit, allowed to stand for 1 min, and mixed thoroughly to obtain the test solutions. The buffer solution contains fluorescently labeled cTnI monoclonal antibody and fluorescently labeled anti-rabbit IgG. The cTnI antigen in the sample and the fluorescently labeled cTnI monoclonal antibody in the buffer solution bind to form a reaction complex.

[0131] 2) Detection method: Take 75 μL of the above test solution containing different concentrations of cTnI serum and drop it onto the sample pad of the Wondfo Biotech cardiac troponin test strip without gelatin modification as a blank; then take 75 μL of the above test solution with different concentrations and drop it onto the sample pad of the lateral flow chromatography test strip modified based on the Wondfo Biotech cardiac troponin test strip of this invention. After lateral flow chromatography, read the value using an immunofluorescence detector.

[0132] 3) Results and Statistics: The proportions of fluorescence values ​​on the T and C lines of the lateral flow chromatography test strips after modification (or without modification) with different concentrations of gelatin intercept lines were statistically analyzed, and the detection limits after modification with different gelatin intercept lines were calculated, as shown in Table 2 (Table 2 shows the statistical analysis of the detection limits of cardiac troponin on commercial immunofluorescence test strips with different concentrations of hydrogel intercept lines in Examples 10-11 of this invention). Figure 10 As shown, the results indicate that the detection limit of the Wondfo Biotech cardiac troponin test strip in the blank group was 0.0230 ng / mL; while the detection limit of the side-flow chromatography test strip of the present invention was improved, with an optimal detection limit of 0.0048 ng / mL when the gelatin solution concentration was 4%, and the sensitivity was significantly enhanced.

[0133] Table 2. Statistical analysis of the detection limits for cardiac troponin on commercial immunofluorescence test strips with different concentrations of hydrogel interception lines.

[0134]

[0135] Example 11

[0136] This embodiment provides a lateral flow chromatography test strip, which is a modification of Wondfo Biotech test strips used for the fluorescence detection of cardiac troponin. The intercept line material of the lateral flow chromatography test strip in this embodiment is carrageenan.

[0137] The specific preparation method of the side-flow chromatography test paper is as follows:

[0138] 1) Preparation of carrageenan: The steps for preparing carrageenan are roughly the same as in Example 2, except that the concentrations of the prepared carrageenan solutions are 0.2%, 0.5%, 1%, and 2%, respectively.

[0139] 2) Preparation of the intercept line: Take 5 μL of the above carrageenan solution and drop it onto the chromatographic membrane of the Wondfo Biotech cardiac troponin test strip as the intercept line; the intercept line is 1-2 mm away from the absorbent pad after the control line, with a length of 3 mm and a width of 3 mm; then let it stand at 25℃ for 2 h, and finally place it in a vacuum dryer at 25℃ for 5-10 min to obtain the side-flow chromatographic test strip.

[0140] The side-flow chromatography test strip is used in the fluorescence detection of cardiac troponin as follows:

[0141] 1) Preparation of the test solution: Same as the preparation steps of the test solution in Example 10;

[0142] 2) Detection method: Take 75 μL of the above test solution containing different concentrations of cTnI serum, and take 75 μL of the above test solution with different concentrations, and drop them onto the sample pad of the lateral flow chromatography test strip modified based on the Wondfo Biotech cardiac troponin test strip of this invention. After lateral flow chromatography, read the value using an immunofluorescence detector.

[0143] 3) Results and Statistics: The proportions of fluorescence values ​​on the T and C lines of the side-flow chromatography test strips modified with different concentrations of carrageenan intercept lines were statistically analyzed, and the detection limits after different carrageenan intercept line modifications were calculated, as shown in Table 2 and... Figure 11 As shown, the detection limit of the side-flow chromatography test strip of the present invention is improved. When the concentration of carrageenan solution is 0.5%, there is an optimal detection limit of 0.0089 ng / mL, which is significantly enhanced in sensitivity compared with the detection limit of the blank control (0.0230 ng / mL, see Example 10).

[0144] Example 12

[0145] This embodiment provides a lateral flow chromatography test strip, which is a modification of Wondfo Biotech test strips used for the fluorescence detection of cardiac troponin. The intercept line material of the lateral flow chromatography test strip in this embodiment is polyacrylamide hydrogel.

[0146] The specific preparation method of the side-flow chromatography test paper is as follows:

[0147] 1) Preparation of polyacrylamide hydrogel: Same as the preparation steps of polyacrylamide hydrogel in Example 3;

[0148] 2) Preparation of the intercept line: Take 5 μL of the above polyacrylamide hydrogel solution and drop it onto the chromatography membrane of Wondfo Biotech cardiac troponin test strip as the intercept line; the intercept line is 1-2 mm away from the absorbent pad after the control line, with a length of 3 mm and a width of 3 mm; then let it stand at 25℃ for 2 h, and finally place it in a vacuum dryer at 25℃ for 5-10 min to obtain the side-flow chromatography test strip.

[0149] The side-flow chromatography test strip is used in the fluorescence detection of cardiac troponin as follows:

[0150] 1) Preparation of the test solution: Same as the preparation steps of the test solution in Example 10;

[0151] 2) Detection method: Take 75 μL of the above test solution containing different concentrations of cTnI serum, and take 75 μL of the above test solution with different concentrations, and drop them onto the sample pad of the lateral flow chromatography test strip modified based on the Wondfo Biotech cardiac troponin test strip of this invention. After lateral flow chromatography, read the value using an immunofluorescence detector.

[0152] 3) Results and Statistics: The proportions of fluorescence values ​​on the T and C lines of the sideflow chromatography test strips modified with different concentrations of polyacrylamide hydrogel intercept lines were statistically analyzed, and the detection limits after modification with different polyacrylamide hydrogel intercept lines were calculated, as shown in Table 2 and... Figure 12 As shown, the detection limit of the side-flow chromatography test strip of the present invention is improved. When the concentration of polyacrylamide hydrogel solution is 25%, there is an optimal detection limit of 0.0092 ng / mL, which is significantly enhanced in sensitivity compared with the detection limit of the blank control (0.0230 ng / mL, see Example 10).

[0153] The above results demonstrate that the improved method for enhancing the sensitivity of commercial test strips designed and constructed in this invention can improve the sensitivity of test strips from different manufacturers and can be applied to various detection systems, including but not limited to colloidal gold method for detecting SARS-CoV-2 antigen and HCG antigen, and fluorescence immunoassay for detecting cardiac troponin. Other hydrogels, compared with gelatin, carrageenan, and polyacrylamide hydrogels, have similar hydrophilicity and porous structures, effectively reducing chromatography time and can also be used to improve the sensitivity of various detection systems. The improved method of adding a hydrogel interception line to the test strip to enhance its sensitivity, as described in this invention, can further broaden the types of test strips applicable and the types of analytes, effectively addressing the limitations and deficiencies of existing test strip sensitivity, and is of great significance for developing low-cost, high-sensitivity, and timely detection technologies.

[0154] The above embodiments are merely illustrative examples and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations; therefore, any obvious variations or modifications derived therefrom remain within the scope of protection of this invention.

Claims

1. A lateral flow chromatography test strip for detecting SARS-CoV-2 antigen using colloidal gold method, detecting HCG using colloidal gold method, or detecting cardiac troponin using fluorescence method, wherein the lateral flow chromatography test strip comprises a control line and an absorbent pad, characterized in that, The lateral flow chromatography test strip also includes an intercept line disposed between the control line and the absorption pad. The intercept line is made of gelatin, carrageenan, or polyacrylamide hydrogel solution. In the colloidal gold method for detecting COVID-19 antigen, the mass concentrations of the gelatin, carrageenan, or polyacrylamide hydrogel solution on the intercept line are 12-20%, 1.5-2%, and 30-40%, respectively. In the colloidal gold method for detecting HCG, the mass concentrations of the gelatin, carrageenan, or polyacrylamide hydrogel solution on the intercept line are 12-20%, 1-2.5%, and 35-40%, respectively. In the fluorescence method for detecting cardiac troponin, the mass concentrations of the gelatin, carrageenan, or polyacrylamide hydrogel solution on the intercept line are 4-8%, 0.5-1.5%, and 20-30%, respectively.

2. The side-flow chromatography test paper as described in claim 1, characterized in that, The preparation method of the hydrogel solution is as follows: When the hydrogel solution is a gelatin solution, the method is as follows: add gelatin powder to pure water, heat to 60-80 ℃, and maintain for 10-15 min to obtain the solution; When the hydrogel solution is a carrageenan solution, the method is as follows: add carrageenan powder to a KCl solution with a KCl concentration of 0.15~0.25% (w / v), first heating at 35~40 ℃ and stirring for 15~25 min, second heating at 85~95 ℃ and holding for 10~15 min, and the hydrogel is obtained; When the hydrogel solution is a polyacrylamide hydrogel solution, the method is as follows: acrylamide, N,N-methylenebisacrylamide, sodium alginate, ammonium persulfate, calcium sulfate, and tetramethylethylenediamine are added to ultrapure water respectively and stirred evenly to obtain the solution.

3. The side-flow chromatography test paper as described in claim 1, characterized in that, The intercept line is prepared by the following method: the hydrogel solution is coated on the control line at a distance of 1-2 mm from the absorbent pad, left to stand, and then vacuum dried to obtain the intercept line.

4. The side-flow chromatography test paper as described in claim 3, characterized in that, The coating area has a length of 2 to 6 mm, a width of 2 to 5 mm, and a load capacity of 10 to 20 µL / cm.

5. The side-flow chromatography test paper as described in claim 3, characterized in that, The settling time is 20 to 40 minutes.

6. The side-flow chromatography test paper as described in claim 3, characterized in that, The vacuum drying time is 5 to 20 minutes, and the temperature is 25 to 60 ℃.

7. The application of the lateral flow chromatography test strip as described in claim 1 in the preparation of test strips for the colloidal gold method to detect SARS-CoV-2 antigen, the colloidal gold method to detect HCG, or the fluorescence method to detect cardiac troponin, wherein in the application for the colloidal gold method to detect SARS-CoV-2 antigen, the mass concentrations of the gelatin, carrageenan, or polyacrylamide hydrogel solution on the intercept line are 12-20%, 1.5-2%, and 30-40%, respectively; in the application for the colloidal gold method to detect HCG, the mass concentrations of the gelatin, carrageenan, or polyacrylamide hydrogel solution on the intercept line are 12-20%, 1-2.5%, and 35-40%, respectively; and in the application for the fluorescence method to detect cardiac troponin, the mass concentrations of the gelatin, carrageenan, or polyacrylamide hydrogel solution on the intercept line are 4-8%, 0.5-1.5%, and 20-30%, respectively.

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