A quality control line coating solution for colloidal gold method test strips, a quality control line, a test strip and applications thereof

Abstract

The present invention provides a quality control line coating solution, a quality control line, a test strip, and applications thereof for colloidal gold test strips. The quality control line provided by the present invention uses inexpensive hydrogels to replace commonly used goat anti-mouse, rabbit anti-mouse, and other antibodies in quality control lines. The optimal hydrogel concentration is determined by improving the quality control line of the test strip. The quality control line technology is applicable to the sandwich method and the competitive method in colloidal gold test strip detection, and can effectively intercept colloidal gold and develop color. The general quality control line technology provided by the present invention uses hydrogels for the quality control line of test strip detection, which not only greatly reduces the cost, but also has good universality and can be widely used in colloidal gold test strip detection.

Description

Technical Field

[0001] The invention belongs to the field of lateral flow chromatography technology detection, and particularly relates to a quality control line coating solution, a quality control line, a test strip and applications thereof for colloidal gold method test strips. Background Art

[0002] Colloidal gold lateral flow detection technology uses a nitrocellulose membrane (NC membrane) as a carrier. When the sample to be tested is added to the sample pad at one end of the test strip, it flows laterally due to capillary action. The analyte binds to the colloidal gold on the binding pad and then moves to the NC membrane. It is captured, aggregated, and colored by the antibody or antigen fixed to the detection line (T line) on the NC membrane. The remaining colloidal gold not bound to the T line crosses the T line and is captured and intercepted by the quality control line (C line). The presence and depth of the T and C lines enable qualitative and semi-quantitative detection of the analyte.

[0003] Colloidal gold lateral flow assays are primarily categorized into sandwich and competitive methods. The former involves the binding of gold-labeled anti-I to the analyte antigen, which then binds to anti-II on the T-line, forming a "sandwich" structure. The latter involves the analyte binding to gold-labeled anti-I, which then passes through the T-line without binding to anti-I on the T-line, resulting in competitive inhibition. However, both methods require the binding of colloidal gold to anti-I on the C-line to determine the effectiveness of the assay.

[0004] Currently, traditional colloidal gold lateral flow test strips often use goat anti-mouse or rabbit anti-mouse as the capture protein on the C line. Different anti-I antibodies require the selection of appropriate antibodies, which has disadvantages such as high cost, limited availability, and demanding storage conditions. These issues significantly increase the cost of using the test strips, necessitating the development of a low-cost, widely applicable, and universal technology for quality control lines for colloidal gold test strips. Summary of the Invention

[0005] The present invention provides a solution to the above-mentioned technical problems. This technology uses hydrogel as a quality control line to capture colloidal gold of different sizes. It is applicable to the sandwich and competitive methods used in colloidal gold test strips, effectively intercepting colloidal gold and developing color. The involved testing method is simple and easy to operate, and can be widely used in colloidal gold test strips.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] A quality control line coating solution for a colloidal gold method test strip comprises a hydrogel.

[0008] Preferably, the hydrogel is a natural hydrogel or a synthetic hydrogel.

[0009] Preferably, the hydrogel includes but is not limited to one or a mixture of two or more of polyacrylic acid hydrogel, polyacrylamide hydrogel, gelatin hydrogel, carrageenan hydrogel, polyvinyl alcohol hydrogel or polyvinyl alcohol-sodium alginate hydrogel.

[0010] Preferably, the hydrogel is agarose hydrogel, and the mass fraction of the hydrogel is less than 40%.

[0011] Preferably, the mass fraction of the agarose hydrogel is less than 10%, preferably 1-5%, more preferably 1%, 2%, 3%, 4%, 5%, and most preferably 3%.

[0012] Preferably, the colloidal gold is unlabeled gold nanoparticles, and the size thereof is 10 to 100 nm, preferably 20 to 80 nm, and more preferably 20, 40, 60, or 80 nm.

[0013] A quality control line for a colloidal gold test strip, the quality control line being prepared by the following method:

[0014] preparing a hydrogel solution;

[0015] The prepared hydrogel solution is pipetted and dripped onto the chromatographic membrane as the quality control line, and then vacuum dried to obtain the quality control line.

[0016] Preferably, the hydrogel solution is an agarose hydrogel solution, and the specific steps of preparing the agarose hydrogel solution are: adding agarose powder to water, heating to boiling and maintaining for 10 to 12 minutes, thereby obtaining the agarose hydrogel solution.

[0017] Preferably, the agarose hydrogel loading capacity on the quality control line is 8 to 12 μL / cm.

[0018] Preferably, when the hydrogel solution is an agarose hydrogel solution, the drying conditions are: vacuum drying at 40-60° C. for 3-5 minutes.

[0019] A colloidal gold test strip comprises a backing plate and a sample pad, a binding pad, a chromatography membrane and a water absorbent pad sequentially arranged on the backing plate; the chromatography membrane is provided with a detection line and a quality control line, and the quality control line adopts the above-mentioned quality control line.

[0020] The quality control line is used in the sandwich method and the competitive method in the detection of colloidal gold test paper.

[0021] Preferably, the sandwich method includes detection of a biotin-avidin detection system or a novel coronavirus detection system, but is not limited to antigen-antibody detection, and can also be used to detect nucleic acids, polysaccharides or lectin systems.

[0022] The competition method is used for host-guest system detection, for example, including a cucurbituril-adamantanamine competition detection system. The host macrocyclic molecule is a cucurbituril, pillararenes, or cyclodextrin; the guest molecule includes all molecules that can recognize the host.

[0023] Preferably, the specific steps of the application are as follows:

[0024] Preparation of quality control lines: The quality control lines are substances capable of intercepting colloidal gold, including antibodies, nucleic acids, etc. In this patent, the quality control lines are natural or artificial hydrogels of varying mass fractions, including but not limited to one or more of agarose hydrogel, polyacrylic acid hydrogel, polyacrylamide hydrogel, gelatin hydrogel, carrageenan hydrogel, polyvinyl alcohol hydrogel, or polyvinyl alcohol-sodium alginate hydrogel. Different masses (volumes) and types of raw materials are dissolved in a certain volume of water to obtain hydrogels of varying concentrations (0-50% by weight) and types.

[0025] Preparation of test line coating solution: The coating solution is biotin, protein, nucleic acid or small molecule that can specifically bind to the analyte. The scope of this patent includes but is not limited to biotin that can bind to avidin, antibodies that can specifically bind to antigens, or guest molecules that can bind to macrocyclic host molecules. A certain concentration of the above antibodies or molecules is dissolved in a specific solution and then printed on a chromatographic membrane at a certain concentration (0-30 mg / L).

[0026] Coating of the test line: Print the coating solution prepared above at a distance of 3-5 mm below the quality control line, with a loading capacity of 0-20 μL / cm;

[0027] Colloidal gold labeling: Colloidal gold labels nucleic acid aptamers, avidin, antibodies, macrocyclic molecules, etc. The scope of this patent includes but is not limited to avidin and antibodies. A certain concentration of avidin or antibody is mixed with a certain concentration of colloidal gold and incubated for a certain period of time (for gold-labeled macrocyclic molecules, a co-reduction method is used); then, protein is backfilled to cover as much of the exposed gold surface as possible, and finally, the colloidal gold-labeled solution is obtained by enrichment and dispersion.

[0028] Preparation of gold-labeled pad: Take a certain volume of the colloidal gold-labeled solution and spray it on the gold-labeled pad, and then dry it.

[0029] Preparation of the test strip: Based on the above, a sample pad is attached to one end of a backing plate. One end of the sample pad is tightly pressed against a gold label pad, one end of the gold label pad is tightly pressed against a chromatography membrane, and the other end of the chromatography membrane is tightly pressed against a sample suction pad to obtain a test strip. The backing plate includes, but is not limited to, a PVC backing plate. The sample pad includes, but is not limited to, untreated or specially treated glass cellulose pads or cellulose acetate. The gold label pad includes, but is not limited to, untreated or specially treated glass cellulose pads or polyester. The sample suction pad includes, but is not limited to, untreated or specially treated cellulose filter membrane. The chromatography membrane includes, but is not limited to, untreated or specially treated nitrocellulose membrane. The backing plate, sample pad, gold label pad, chromatography membrane, and sample suction pad have the same width of 3-5 mm and are 50-60 mm, 15-20 mm, 4-8 mm, 2-30 mm, and 15-25 mm in length, respectively.

[0030] Preferably, in the biotin-avidin detection system, the quality control line coating solution is agarose hydrogel, and the concentration is preferably 3%.

[0031] Preferably, in the biotin-avidin detection system, the detection line coating solution is biotin modified with bovine serum albumin; preferably, the biotin concentration is 3-5 mg / mL, and the carrying capacity is 1-1.5 μL / cm.

[0032] Preferably, in the biotin-avidin detection system, the conjugate pad is streptavidin labeled with colloidal gold; preferably, the size of the colloidal gold is 40 to 50 nm, and the concentration of streptavidin is 10 to 20 μg / mL.

[0033] Preferably, in the cucurbituril-adamantanamine competitive detection system, the binding pad is cucurbituril labeled with colloidal gold.

[0034] Preferably, in the cucurbituril-amantadine competitive detection system, the quality control line coating solution is agarose hydrogel, and the concentration is preferably 3%.

[0035] Preferably, in the cucurbituril-adamantanamine competitive detection system, the detection line is adamantane acetic acid-BSA conjugate, and preferably, its concentration is 18-22 mg / mL.

[0036] Preferably, in the cucurbituril-adamantanamine competitive detection system, the developing agent is Tris-HCl or PB solution, and preferably, the volume thereof is 60 to 80 μL, respectively.

[0037] Preferably, in the novel coronavirus detection system, the quality control line coating solution is agarose hydrogel, and the concentration is preferably 3%.

[0038] Preferably, in the novel coronavirus detection system, the carrying capacity of the agarose hydrogel on the quality control line is 8 to 12 μL / cm.

[0039] Preferably, in the novel coronavirus detection system, the drying temperature of the agarose hydrogel on the quality control line is vacuum drying at 40 to 60°C, and the drying time is 3 to 5 minutes.

[0040] Preferably, in the novel coronavirus detection system, the colloidal gold solution is a colloidal gold-labeled novel coronavirus antigen recombinant protein anti-I (15C3), and preferably, its antibody concentration is 10 to 20 μg / mL.

[0041] Preferably, in the new coronavirus detection system, the coating solution of the detection line is the antibody solution corresponding to the antigen. Preferably, the carrying capacity of the coating solution on the detection line is 1 to 2 μL / cm. Preferably, the coating solution concentration of the detection line is 2 mg / mL of the antibody solution corresponding to the antigen.

[0042] Preferably, in the novel coronavirus detection system, the developing agent is a PBS solution containing Tween-20. Preferably, the concentration of the developing agent PBS is 10 mM and the concentration of Tween-20 is 0.1% (volume fraction, v / v).

[0043] Compared with the prior art, the present invention has the following beneficial effects: the quality control line technology provided by the present invention uses inexpensive hydrogels to replace the goat anti-mouse and rabbit anti-mouse antibodies commonly used in quality control lines. By improving the quality control line of the test strip, the optimal hydrogel concentration is determined. The general quality control line technology described is applicable to the sandwich method and competitive method used in colloidal gold test strip detection, and can effectively intercept colloidal gold and develop color. The quality control line technology provided by the present invention, which uses hydrogels as the quality control line for test strip detection, not only greatly reduces costs, but also has good universality and can be widely used in colloidal gold test strip detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 TEM image and particle size statistics of 20 nm colloidal gold involved in Example 1 of the present invention;

[0045] Figure 2 TEM image and particle size statistics of 40nm colloidal gold involved in Example 1 of the present invention;

[0046] Figure 3 TEM image and particle size statistics of 60nm colloidal gold involved in Example 1 of the present invention;

[0047] Figure 4 TEM image and particle size statistics of 80nm colloidal gold involved in Example 1 of the present invention;

[0048] Figure 5is the UV-visible absorption spectrum of the colloidal gold involved in Example 1 of the present invention;

[0049] Figure 6 The detection results and grayscale values ​​of 20 nm colloidal gold intercepted by agarose hydrogels of different concentrations in Example 2 of the present invention are shown;

[0050] Figure 7 The detection results and grayscale values ​​of 40 nm colloidal gold intercepted by agarose hydrogels of different concentrations in Example 3 of the present invention are shown;

[0051] Figure 8 The detection results and grayscale values ​​of 60 nm colloidal gold intercepted by agarose hydrogels of different concentrations in Example 4 of the present invention are shown;

[0052] Figure 9 The test results and grayscale values ​​of 80 nm colloidal gold intercepted by agarose hydrogels of different concentrations in Example 5 of the present invention are shown;

[0053] Figure 10 The test results and grayscale values ​​of polyacrylic acid hydrogels of different concentrations used as quality control lines to intercept 40 nm colloidal gold in Example 6 of the present invention;

[0054] Figure 11 The test results and grayscale values ​​of polyacrylamide hydrogels of different concentrations intercepting 40 nm colloidal gold as the quality control line in Example 7 of the present invention are shown;

[0055] Figure 12 The test results and grayscale values ​​of gelatin hydrogels of different concentrations as the quality control line intercepting 40 nm colloidal gold in Example 8 of the present invention are shown;

[0056] Figure 13 The test results and grayscale values ​​of carrageenan hydrogels with different concentrations as the quality control line intercepting 40 nm colloidal gold in Example 9 of the present invention are shown;

[0057] Figure 14 The test results and grayscale values ​​of polyvinyl alcohol hydrogels of different concentrations used as quality control lines to intercept 40 nm colloidal gold in Example 10 of the present invention;

[0058] Figure 15 The test results and grayscale values ​​of polyvinyl alcohol-sodium alginate hydrogels of different concentrations intercepting 40 nm colloidal gold as the quality control line in Example 11 of the present invention are shown;

[0059] Figure 16 Schematic diagram of the test strip structure in Example 12 of the present invention, and the test results of 3% agarose hydrogel in the biotin-avidin system;

[0060] Figure 17 TEM image and particle size statistics of GNP@CB[7] involved in Example 13 of the present invention;

[0061] Figure 18 Schematic diagram of the test strip structure in Example 13 of the present invention, and the results of 3% agarose hydrogel intercepting GNP@CB[7] in the host-guest competition detection system;

[0062] Figure 19 Schematic diagram of the test strip structure in Example 13 of the present invention, and the results of detecting amantadine using 3% agarose hydrogel in a host-guest competition detection system;

[0063] Figure 20 Schematic diagram of the test strip structure in Example 14 of the present invention and the results of detecting the new coronavirus antigen recombinant protein using 4% agarose hydrogel in the antigen-antibody immune system. DETAILED DESCRIPTION

[0064] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0065] Example 1

[0066] Preparation of colloidal gold of different sizes

[0067] 1) Preparation of 20nm colloidal gold solution: Add 1mL of 25mM chloroauric acid solution to 150mL of 2.2mM sodium citrate solution, heat to boiling, then naturally cool to 90℃, add 1mL of 25mM chloroauric acid solution, and react for 30min to obtain the solution. Figure 1 As shown, TEM results show that gold nanoparticles (hereinafter referred to as GNPs) were obtained, and particle size statistics showed that their size was about 20 nm.

[0068] 2) Preparation of 40nm colloidal gold solution: After removing 55mL of the 20nm colloidal gold solution, the remaining solution was used as a seed. 55mL of 2.2mM sodium citrate solution was added to the seed solution, heated to boiling, and then naturally cooled to 90°C. Then, 1mL of 25mM chloroauric acid solution was added and reacted for 30min to obtain the solution. Figure 2 As shown, TEM results show that GNPs were obtained, and particle size statistics show that their size is about 40 nm.

[0069] 3) Preparation of 60nm colloidal gold solution: After removing 55mL of the 40nm colloidal gold solution, the remaining solution was used as a seed. 55mL of 2.2mM sodium citrate solution was added to the seed solution, heated to boiling, and then naturally cooled to 90°C. Then, 1mL of 25mM chloroauric acid solution was added and reacted for 30min to obtain the solution. Figure 3As shown, TEM results show that GNPs were obtained, and particle size statistics show that their size is around 60 nm.

[0070] 4) Preparation of 80nm colloidal gold solution: After removing 55mL of the 60nm colloidal gold solution, the remaining solution was used as a seed. 55mL of 2.2mM sodium citrate solution was added to the seed solution, heated to boiling, and then naturally cooled to 90°C. Then, 1mL of 25mM chloroauric acid solution was added and reacted for 30min to obtain the solution. Figure 4 As shown, TEM results show that GNPs were obtained, and particle size statistics show that their size is about 80 nm.

[0071] 5) UV-visible absorption spectra of colloidal gold of different sizes Figure 5 As shown in Figure 3, as the size of GNPs increases, the maximum absorption peak of the UV-visible absorption spectrum red-shifts, further proving the successful preparation of the above GNPs.

[0072] Example 2

[0073] Agarose hydrogels of different concentrations were used as quality control lines to intercept 20 nm colloidal gold solution

[0074] 1) Preparation of agarose hydrogels: 0.1, 0.2, 0.3, 0.4, and 0.5 g of agarose powder (for biological use) were added to 9.9, 9.8, 9.7, 9.6, and 9.5 mL of ultrapure water, respectively. The mixture was heated to boiling for 12 min to obtain 1%, 2%, 3%, 4%, and 5% agarose hydrogels (wt %, the same below), respectively.

[0075] 2) Prepare a control line: Use a micropipette to transfer 5 μL of the agarose hydrogel solution to the chromatographic membrane while it is still hot, and dropwise apply the solution onto the membrane to form a control line. The control line should be 5 mm long and 2 mm wide. The membrane should then be vacuum dried at 60°C for 3 min.

[0076] 3) Preparation of test strips for intercepting colloidal gold of different sizes: A sample pad (17 × 5 mm) was attached to one end of a PVC backing plate (60 × 5 mm) of the test strip. One end of the sample pad was tightly pressed against an NC membrane (25 × 5 mm), and the other end of the NC membrane was tightly pressed against a sample suction pad (22 × 5 mm).

[0077] 4) Detection method: 10 μL of the 20 nm colloidal gold solution in Example 1 was dripped onto the lower end of the NC membrane. 60 μL of ultrapure water was then dripped onto the sample pad as a developing solvent. Lateral flow chromatography was performed for 5 min. A photo was taken and the grayscale value on the quality control line was read.

[0078] 5) The interception of 20nm colloidal gold solution by agarose hydrogels of different concentrations is shown in Figure 5. Figure 6As shown, the results showed that 1%-5% agarose hydrogel can effectively intercept 20nm colloidal gold, among which 3% agarose hydrogel has the best interception effect.

[0079] Example 3

[0080] Agarose hydrogels of different concentrations were used as quality control lines to intercept 40 nm colloidal gold solution.

[0081] This embodiment is substantially the same as embodiment 2, except that:

[0082] 4) Detection method: 10 μL of the 40 nm colloidal gold solution in Example 1 was dripped onto the lower end of the NC membrane. 60 μL of ultrapure water was then dripped onto the sample pad as a developing agent. Lateral flow chromatography was performed for 5 min. A photo was taken and the grayscale value on the quality control line was read.

[0083] 5) The interception of 40nm colloidal gold solution by agarose hydrogels of different concentrations is shown in Figure 5. Figure 7 As shown, the results showed that 1%-5% agarose hydrogel can effectively intercept 40nm colloidal gold, among which 3% agarose hydrogel has the best interception effect.

[0084] Example 4

[0085] Agarose hydrogels of different concentrations were used as quality control lines to intercept 60 nm colloidal gold solution

[0086] This embodiment is substantially the same as embodiment 2, except that:

[0087] 4) Detection method: 10 μL of the 60 nm colloidal gold solution in Example 1 was dripped onto the lower end of the NC membrane. 60 μL of ultrapure water was then dripped onto the sample pad as a developing solvent. Lateral flow chromatography was performed for 5 min. A photo was taken and the grayscale value on the quality control line was read.

[0088] 5) The interception of 60nm colloidal gold solution by agarose hydrogels of different concentrations is shown in Figure 5. Figure 8 As shown, the results showed that 1%-5% agarose hydrogel can effectively intercept 60nm colloidal gold, among which 3% agarose hydrogel has the best interception effect.

[0089] Example 5

[0090] Agarose hydrogels of different concentrations were used as quality control lines to intercept 80 nm colloidal gold solution

[0091] This embodiment is substantially the same as embodiment 2, except that:

[0092] 4) Detection method: 10 μL of the 80 nm colloidal gold solution from Example 1 was dripped onto the lower end of the NC membrane. 60 μL of ultrapure water was then dripped onto the sample pad as a developing solvent. Lateral flow chromatography was performed for 5 min. A photograph was taken and the grayscale value on the quality control line was read.

[0093] 5) The interception of 80nm colloidal gold solution by agarose hydrogels of different concentrations is shown in Figure 5. Figure 9 As shown, the results showed that 1%-5% agarose hydrogel can effectively intercept 80nm colloidal gold, among which 3% agarose hydrogel has the best interception effect.

[0094] Example 6

[0095] Polyacrylic acid hydrogels of different concentrations were used as quality control lines to intercept 40 nm colloidal gold solution

[0096] 1) Preparation of polyacrylic acid hydrogel: 3, 4, 5, and 6 g of acrylic acid, 0.1, 0.133, 0.167, and 0.2 g of N,N-methylenebisacrylamide, 0.042, 0.056, 0.07, and 0.084 g of ammonium persulfate, and 0.042, 0.056, 0.07, and 0.084 g of sodium bisulfite were added to 10 mL of ultrapure water and stirred to obtain 23, 29, 33, and 38% polyacrylic acid hydrogels (wt %, the same below), respectively;

[0097] 2) Preparation of a control line: Use a micropipette to pipette 5 μL of the polyacrylic acid hydrogel solution onto the chromatographic membrane as a control line; the control line is 5 mm long and 2 mm wide. The membrane is then allowed to stand at 25°C for 12 hours, and then vacuum-dried at 25°C for 10 minutes.

[0098] 3) Preparation of a test strip for intercepting 40 nm colloidal gold solution: A sample pad (17 × 5 mm) was affixed to one end of a PVC backing plate (60 × 5 mm) of the test strip. One end of the sample pad was tightly pressed against an NC membrane (25 × 5 mm), and the other end of the NC membrane was tightly pressed against a sample suction pad (22 × 5 mm).

[0099] 4) Detection method: 10 μL of the 40 nm colloidal gold solution in Example 1 was dripped onto the lower end of the NC membrane. 60 μL of ultrapure water was then dripped onto the sample pad as a developing agent. Lateral flow chromatography was performed for 5 min. A photo was taken and the grayscale value on the quality control line was read.

[0100] 5) The interception of 40nm colloidal gold solution by polyacrylic acid hydrogels with different concentrations is shown in the figure. Figure 10 As shown in the figure, the results showed that 23% to 38% polyacrylic acid hydrogels could intercept 40nm colloidal gold, among which 33% polyacrylic acid hydrogel had the best interception effect.

[0101] Example 7

[0102] Polyacrylamide hydrogels of different concentrations were used as quality control lines to intercept 40 nm colloidal gold solution

[0103] 1) Preparation of polyacrylamide hydrogel: 1.0, 1.5, 2.0, 2.5, 3.0 g of acrylamide, 0.0025, 0.0038, 0.005, 0.0062, 0.0075 g of N,N-methylenebisacrylamide, 0.1, 0.15, 0.2, 0.25, 0.3 g of alginic acid, 0.025, 0.0038, 0.005, 0.0062 , 0.0075 g ammonium persulfate, 0.017, 0.0255, 0.034, 0.0425, 0.051 g calcium sulfate, 0.005, 0.0075, 0.01, 0.0125, 0.015 g tetramethylethylenediamine were added to 5 mL ultrapure water, stirred evenly, and 17, 23, 29, 33, 38% polyacrylamide hydrogels (wt%, the same below) were obtained, respectively;

[0104] 2) Preparation of a control line: Use a micropipette to pipette 5 μL of the polyacrylamide hydrogel solution onto the chromatographic membrane as a control line; the control line is 5 mm long and 2 mm wide. The membrane is then allowed to stand at 25°C for 12 hours, and then vacuum-dried at 25°C for 10 minutes.

[0105] 3) Preparation of a test strip for intercepting 40 nm colloidal gold solution: A sample pad (17 × 5 mm) was affixed to one end of a PVC backing plate (60 × 5 mm) of the test strip. One end of the sample pad was tightly pressed against an NC membrane (25 × 5 mm), and the other end of the NC membrane was tightly pressed against a sample suction pad (22 × 5 mm).

[0106] 4) Detection method: 10 μL of the 40 nm colloidal gold solution in Example 1 was dripped onto the lower end of the NC membrane. 60 μL of ultrapure water was then dripped onto the sample pad as a developing solvent. Lateral flow chromatography was performed for 10 min. A photo was taken and the grayscale value on the quality control line was read.

[0107] 5) The interception of 40nm colloidal gold solution by polyacrylamide hydrogels with different concentrations is as follows Figure 11 As shown, the results showed that 17%-38% polyacrylamide hydrogels could intercept 40nm colloidal gold, among which 33% polyacrylamide hydrogel had the best interception effect.

[0108] Example 8

[0109] Different concentrations of gelatin hydrogels were used as quality control lines to intercept 40nm colloidal gold solution

[0110] 1) Preparation of gelatin hydrogels: 0.35, 0.53, 0.89, and 1.04 g of gelatin powder were added to 3.15, 2.96, 3.57, and 3.13 mL of ultrapure water, respectively. The mixture was heated to 70°C and maintained for 12 min to obtain 10%, 15%, 20%, and 25% gelatin hydrogels (wt %, the same below), respectively.

[0111] 2) Preparation of a control line: Use a micropipette to pipette 5 μL of the gelatin hydrogel solution and dropwise add it onto the chromatographic membrane while it is still hot to form a control line. The control line should be 5 mm long and 2 mm wide. The membrane should then be allowed to stand at 25°C for 2 hours and then vacuum dried at 25°C for 10 minutes.

[0112] 3) Preparation of a 40 nm colloidal gold interception test strip: A sample pad (17 × 5 mm) was affixed to one end of a PVC backing plate (60 × 5 mm) of the test strip. One end of the sample pad was tightly pressed against an NC membrane (25 × 5 mm), and the other end of the NC membrane was tightly pressed against a sample suction pad (22 × 5 mm).

[0113] 4) Detection Method: 10 μL of the 40 nm colloidal gold solution from Example 1 was dripped onto the lower end of the NC membrane. 60 μL of ultrapure water was then dripped onto the sample pad as a developing agent. Lateral flow chromatography was performed on a 10% gelatin hydrogel for 5 min, and on gelatin hydrogels with concentrations of 15%, 20%, and 25% for 30 min. A photograph was taken and the grayscale value on the quality control line was read.

[0114] 5) The interception of 40nm colloidal gold solution by gelatin hydrogels of different concentrations is as follows Figure 12 As shown, the results showed that 10%-25% gelatin hydrogels could intercept 40nm colloidal gold, among which 10% gelatin hydrogel had the best interception effect.

[0115] Example 9

[0116] Carrageenan hydrogels with different concentrations were used as quality control lines to intercept 40 nm colloidal gold solution

[0117] 1) Preparation of carrageenan hydrogels: 0.02, 0.04, 0.05, and 0.07 g of carrageenan powder were added to 3.98, 3.96, 3.48, and 3.43 mL of ultrapure water, respectively. The mixture was heated to 40°C and stirred for 20 min, then heated to 90°C and maintained for 12 min to obtain 0.5%, 1%, 1.5%, and 2% carrageenan hydrogels (wt %, the same below), respectively.

[0118] 2) Preparation of a control line: Use a micropipette to pipette 5 μL of the carrageenan hydrogel solution onto the chromatographic membrane while hot, to serve as a control line. The control line is 5 mm long and 2 mm wide. The membrane is then allowed to stand at 25°C for 12 hours, followed by vacuum drying at 25°C for 10 minutes.

[0119] 3) Preparation of a 40 nm colloidal gold interception test strip: A sample pad (17 × 5 mm) was affixed to one end of a PVC backing plate (60 × 5 mm) of the test strip. One end of the sample pad was tightly pressed against an NC membrane (25 × 5 mm), and the other end of the NC membrane was tightly pressed against a sample suction pad (22 × 5 mm).

[0120] 4) Detection method: 10 μL of the 40 nm colloidal gold solution in Example 1 was dripped onto the lower end of the NC membrane. 60 μL of ultrapure water was then dripped onto the sample pad as a developing solvent. Concentrated lateral flow chromatography was performed for 20 min. A photo was taken and the grayscale value on the quality control line was read.

[0121] 5) The interception of 40nm colloidal gold solution by carrageenan hydrogels with different concentrations is shown in Figure 5. Figure 13 As shown, the results showed that 0.5%-2% carrageenan hydrogels could intercept 40nm colloidal gold, among which 1% gelatin hydrogel had the best interception effect.

[0122] Example 10

[0123] Polyvinyl alcohol hydrogels of different concentrations were used as quality control lines to intercept 40nm colloidal gold solution

[0124] 1) Preparation of polyvinyl alcohol hydrogels: 0.03, 0.083, 0.151, 0.25, and 0.30 g of polyvinyl alcohol 1799 powder were added to 3.17, 4.06, 3.62, 3.92, and 3.45 mL of ultrapure water, respectively. The mixture was heated to 100°C and maintained for 12 min to obtain 1%, 2%, 4%, 6%, and 8% polyvinyl alcohol hydrogels (wt %, the same below), respectively.

[0125] 2) Preparation of a control line: Use a micropipette to pipette 5 μL of the polyvinyl alcohol hydrogel solution and drip it onto the chromatographic membrane while it is still hot, to serve as a control line. The control line should be 5 mm long and 2 mm wide. The membrane should then be placed at -20°C for 12 hours, and then vacuum-dried at 25°C for 10 minutes.

[0126] 3) Preparation of a 40 nm colloidal gold interception test strip: A sample pad (17 × 5 mm) was affixed to one end of a PVC backing plate (60 × 5 mm) of the test strip. One end of the sample pad was tightly pressed against an NC membrane (25 × 5 mm), and the other end of the NC membrane was tightly pressed against a sample suction pad (22 × 5 mm).

[0127] 4) Detection method: 10 μL of the 40 nm colloidal gold solution in Example 1 was dripped onto the lower end of the NC membrane. 60 μL of ultrapure water was then dripped onto the sample pad as a developing solvent. Concentrated lateral flow chromatography was performed for 5 min. A photo was taken and the grayscale value on the quality control line was read.

[0128] 5) The interception of 40nm colloidal gold solution by polyvinyl alcohol hydrogels with different concentrations Figure 14 As shown, the results show that 1%-8% polyvinyl alcohol hydrogel can intercept 40nm colloidal gold, among which 8% polyvinyl alcohol hydrogel has the best interception effect.

[0129] Example 11

[0130] Different concentrations of polyvinyl alcohol-sodium alginate hydrogels were used as quality control lines to intercept 40nm colloidal gold solution

[0131] 1) Preparation of polyvinyl alcohol-sodium alginate hydrogel: 0.0203, 0.062, 0.11, 0.153, 0.234 g of polyvinyl alcohol 1799 powder were added to 2, 2, 2.09, 2.033, 2.106 mL of ultrapure water, respectively, and heated to 100 ° C and maintained for 5 min to obtain polyethylene glycol solutions with mass fractions of 1%, 3%, 5%, 7%, and 10% (wt%, the same below); 0.3239 g of sodium alginate powder was added to 6.154 mL of ultrapure water and completely dissolved at 60 ° C to prepare a 5% sodium alginate solution, and then 5 0.2, 0.6, 1, 1.4, and 2 mL of 0.5% sodium alginate solution were diluted to 2 mL with ultrapure water to obtain 0.5%, 1.5%, 2.5%, 3.5%, and 5% sodium alginate solutions; 2 mL of the above 0.5-5% sodium alginate solution was added to 2 mL of 1-10% polyethylene glycol solution, and stirred at 100°C for 2 min to obtain 0.5-0.25, 1.5-0.75, 2.5-1.25, 3.5-1.75, and 5-2.5 wt% polyvinyl alcohol-sodium alginate hydrogels (the mass fraction ratio of polyethylene glycol to sodium alginate was 2:1);

[0132] 2) Preparation of a control line: Use a micropipette to pipette 5 μL of the polyvinyl alcohol-sodium alginate hydrogel solution onto the chromatographic membrane while still hot, to serve as a control line. The control line is 5 mm long and 2 mm wide. The membrane is then placed at -20°C for 12 hours and then vacuum-dried at 25°C for 10 minutes.

[0133] 3) Preparation of a 40 nm colloidal gold interception test strip: A sample pad (17 × 5 mm) was affixed to one end of a PVC backing plate (60 × 5 mm) of the test strip. One end of the sample pad was tightly pressed against an NC membrane (25 × 5 mm), and the other end of the NC membrane was tightly pressed against a sample suction pad (22 × 5 mm).

[0134] 4) Detection method: 10 μL of the 40 nm colloidal gold solution in Example 1 was dripped onto the lower end of the NC membrane. 60 μL of ultrapure water was then dripped onto the sample pad as a developing solvent. Concentrated lateral flow chromatography was performed for 5 min. A photo was taken and the grayscale value on the quality control line was read.

[0135] 5) The interception of 40nm colloidal gold solution by polyvinyl alcohol-sodium alginate hydrogels with different concentrations is shown in the figure. Figure 15 As shown, the results show that 0.5-0.25% to 5-2.5% vinyl alcohol-sodium alginate hydrogels can intercept 40nm colloidal gold, among which 2.5%-1.25% vinyl alcohol-sodium alginate hydrogel has the best interception effect.

[0136] Example 12

[0137] A test strip for colloidal gold biotin-avidin system

[0138] 1) Preparation of a control line: Use a micropipette to pipette 5 μL of the 3% agarose hydrogel solution onto the chromatographic membrane while still hot, to form a control line; the control line should be 3 mm long and 2 mm wide. The solution should then be vacuum-dried at 60°C for 3 min.

[0139] 2) Prepare the test line coating solution:

[0140] a. In an ice bath, add 30 mg of NHS-activated biotin to 3 mL of DMSO solution, add 241.6 mg of bovine serum albumin to 40 mL of 10 mM PB solution (pH 8), slowly add the biotin solution dropwise to the BSA solution, and stir at room temperature for 2 h to react to obtain a BSA-biotin solution.

[0141] b. The BSA-biotin solution was dialyzed in a 3 kDa dialysis bag for 48 h, during which ultrapure water was replaced every 12 h. Finally, the solution in the dialysis bag was taken out and freeze-dried for 72 h to obtain a white flocculent substance, namely BSA-biotin;

[0142] c. Take the 4 mg BSA-biotin and add it to 1 mL of 10 mM PB solution to obtain a 4 mg / mL BSA-biotin coating solution.

[0143] 3) Coating the test line: Take the chromatographic membrane prepared in step 1) of this embodiment, adjust the distance between the test line and the quality control line to 5 mm, set the membrane dot parameters, start the membrane streaking apparatus, and coat the BSA-biotin coating solution on the test line. The carrying capacity of the test line is 1 μL / cm.

[0144] 4) Preparation of colloidal gold-labeled streptavidin:

[0145] a. Preparation of reconstitution solution: 8.7 mg boric acid, 5.7 mg borax, 50 mg BSA, 50 mg polyvinylpyrrolidone (K30), 1 g sucrose, 50 mg NaCl, and 100 mg surfactant Tetronic 1307 (S9) were added to 100 mL ultrapure water.

[0146] b. Add 10 μg / mL of the labeled streptavidin to 1 mL of the unlabeled 40 nm (Abs=2) colloidal gold solution of Example 1, incubate for 30 min, then add 20 μL of 10% BSA solution for blocking. After incubation for 10 min, centrifuge at 8000 rpm for 10 min at 4°C, remove the supernatant, and add 200 μL of the reconstitution solution to the precipitate to obtain the product.

[0147] 5) Preparation of gold label pad: 7.5 μL of colloidal gold-labeled streptavidin prepared in step 4) was evenly spread on the glass fiber surface and dried at 37°C.

[0148] 6) Preparation of a colloidal gold-biotin-avidin test strip: A sample pad (18 × 3 mm) is attached to one end of a PVC backing plate (60 × 3 mm) of the test strip. One end of the sample pad is tightly pressed against the gold label pad (6 × 3 mm) prepared in step 5). One end of the gold label pad is tightly pressed against the NC membrane (25 × 3 mm) prepared in step 3). The other end of the NC membrane is tightly pressed against a sample suction pad (19 × 3 mm).

[0149] 7) As a control, a test strip for a colloidal gold-biotin-avidin system was prepared by attaching a sample pad to one end of the PVC backing of the test strip, tightly pressing one end of the sample pad against the gold label pad prepared in step 5), tightly pressing one end of the gold label pad against the NC membrane prepared in step 1), and tightly pressing the other end of the NC membrane against the sample pad.

[0150] 8) Detection method: Take the test paper prepared in steps 6) and 7) above, add 50 μL of Tris-HCl buffer solution as a developing agent onto the sample pad, perform lateral flow chromatography for 20 minutes, and read the result colorimetrically.

[0151] 9) Detection of test strips for colloidal gold biotin-avidin system Figure 16 As shown, the results showed that in the biotin-avidin detection system, the test strip with 3% agarose hydrogel as the quality control line can intercept streptavidin-coupled gold nanoparticles.

[0152] Example 13

[0153] A test strip for detecting amantadine based on host-guest competition method

[0154] 1) Preparation of a control line: Use a micropipette to pipette 5 μL of the 3% agarose hydrogel solution onto the chromatographic membrane while still hot to serve as a control line; the control line is 3 mm long and 2 mm wide. The membrane is then vacuum dried at 60°C for 3 min.

[0155] 2) Prepare the test line coating solution:

[0156] a. Dissolve a 1:5:5 molar ratio of adamantane acetic acid:EDC·HCl:sulfo-NHS in 5 mL of 10 mM PB solution (pH 6.0) and stir in the dark for 60 min to obtain an activated adamantane acetic acid solution mixture.

[0157] b. Dissolve 439.9 mg of BSA in 20 mL of 10 mM PB solution to obtain a BSA solution.

[0158] c. The activated adamantane acetic acid solution mixture in a was mixed with the BSA solution in b, and the mixture was reacted at room temperature for 60 min, and then dialyzed using a 3 kDa dialysis bag for 48 h. The solution in the dialysis bag was then freeze-dried for 72 h to obtain;

[0159] d. Dissolve 20 mg of BAS-adamantane acetic acid in 1 mL of ultrapure water to obtain a BAS-adamantane acetic acid coating solution.

[0160] 3) Coating the test line: Take the chromatographic membrane from step 1) of this example, adjust the distance between the test line and the quality control line to 5 mm, set the membrane dot parameters, start the membrane streaking instrument, and coat the BAS-adamantane acetic acid coating solution from 2) on the test line. The test line has a loading capacity of 1.5 μL / cm.

[0161] 4) Preparation of colloidal gold labeled cucurbit[7]uril:

[0162] a. Mix 20 mL of 5 mM CB[7] solution with 20 mL of 5 mM KAuCl4·2H2O solution. After standing for 5 min, add 40 mL of 0.2 M NaOH solution to make the mixed solution pH = 13. Then, ultrasonicate for 5 min and stand at 37 °C for 48 h to obtain a solution containing GNP@CB[7] precipitate. Remove the supernatant, add ultrapure water, ultrasonicate for 2 min, disperse and wash, continue precipitation for 12 h, repeat washing 4 times, add ultrapure water for the last time and dilute to Abs = 0.7 to obtain GNP@CB[7] solution.

[0163] b. Add BSA to 1.5 mL of the above GNP@CB[7] solution to block the solution and make the BSA concentration 1%. Incubate at room temperature for 40 min, centrifuge at 3000 rpm for 8 min at 4 °C, take the supernatant and disperse it in 200 μL Tris buffer solution (containing 0.5% BSA, 0.5% Tween-20, and 2% sucrose), and sonicate for 5 min to obtain GNP@CB[7]-BSA solution. Figure 17 As shown, TEM results show that GNP@CB[7] was prepared and the particle size statistics showed that its size was 8 nm.

[0164] 5) Preparation of gold label pad: Take 8 μL of colloidal gold-labeled cucurbit[7]uril prepared in step 4) and spread it evenly on the glass fiber surface, and dry it at 37°C.

[0165] 6) Preparation of a test strip for detecting amantadine based on a host-guest competition method: a sample pad (18 × 3 mm) is attached to one end of a PVC backing plate (60 × 3 mm) of the test strip, one end of the sample pad is tightly pressed against the gold label pad (6 × 3 mm) prepared in step 5), one end of the gold label pad is tightly pressed against the NC membrane (25 × 3 mm) prepared in step 3), and the other end of the NC membrane is tightly pressed against a sample suction pad (19 × 3 mm).

[0166] 7) As a control, a test strip for detecting amantadine based on the host-guest competition method was prepared: a sample pad was attached to one end of the PVC backing plate of the test strip, one end of the sample pad was tightly pressed against the gold label pad prepared in step 5), one end of the gold label pad was tightly pressed against the NC membrane prepared in step 1), and the other end of the NC membrane was tightly pressed against the sample suction pad, thereby obtaining a test strip; Figure 18 As shown in the figure, in the host-guest competition detection system, the test strip with 3% agarose hydrogel as the quality control line can intercept CB[7]-coupled gold nanoparticles.

[0167] 8) Detection Method: Take the test strips from steps 6) and 7) above, add 60 μL of Tris-HCl buffer solution as the developing solvent to the sample pad, perform lateral flow chromatography for 10 minutes, and read the result colorimetrically. Take two test strips from steps 6) and 7) above, add 60 μL of Tris-HCl buffer solution without and with 10 μM amantadine as the developing solvent, respectively, to the sample pad, perform lateral flow chromatography for 10 minutes, and read the result colorimetrically.

[0168] 9) The detection results of a test strip for detecting amantadine based on the host-guest competition method are as follows Figure 19 As shown in the results, in the host-guest competition method for detecting the adamantane system, the test strip with 3% agarose hydrogel as the quality control line can intercept GNP@CB[7], and the color of the test line of the test strip with adamantane added is lighter than that without adamantane added, indicating a positive test result.

[0169] Example 14

[0170] A test strip for detecting new coronavirus antigen recombinant protein

[0171] 1) Preparation of a control line: Use a micropipette to transfer 10 μL of the 3% agarose hydrogel solution to the chromatographic membrane while still hot, to form a control line. The control line is 3 mm long and 2 mm wide. The membrane is then vacuum-dried at 60°C for 3 min.

[0172] 2) Preparation of the test line coating solution and the detection limit: Take the chromatographic membrane in step 1) of this embodiment, adjust the distance between the test line and the quality control line to 5 mm, set the membrane drawing parameters, the load capacity of the test line is 1.5 μL / cm, start the membrane drawing instrument, and coat 2 mg / mL of the PB solution of the new crown antigen recombinant protein anti-II (11F2) (concentration is 10 mM) on the test line.

[0173] 3) Colloidal gold-labeled recombinant protein anti-I protein (GNP-15C3)

[0174] a. Preparation of reconstitution solution: 8.7 mg boric acid, 5.7 mg borax, 50 mg BSA, 50 mg polyvinylpyrrolidone (K30), 1 g sucrose, 50 mg NaCl, and 100 mg surfactant Tetronic 1307 (S9) were added to 100 mL ultrapure water to obtain the solution;

[0175] b. Preparation of colloidal gold-labeled SARS-CoV-2 antigen recombinant protein anti-I solution: Add SARS-CoV-2 antigen recombinant protein anti-I protein to 1 mL of 40 nm (adjust Abs = 2) colloidal gold solution in Example 1 to a concentration of 10 μg / mL. After oscillation, add 20 μL of 10% BSA solution, oscillate evenly, centrifuge at 8000 r / min at 4°C for 10 min, remove the supernatant, and add 200 uL of SARS-CoV-2 recombinant solution.

[0176] 4) Preparation of gold label pad: Take 7.5 μL of the colloidal gold-labeled SARS-CoV-2 antigen recombinant protein anti-I solution from step 3) and evenly spread it on the glass cellulose surface. Dry it at 37°C for 2 hours.

[0177] 5) Preparation of a test strip for detecting recombinant protein of the new coronavirus antigen: stick a sample pad (18×3mm) on one end of the PVC backing plate (60×3mm) of the test strip, tightly press one end of the sample pad onto the gold label pad (6×3mm) in step 4), tightly press one end of the gold label pad onto the NC membrane (25×3mm) in step 2), and tightly press the other end of the NC membrane onto the sample suction pad (19×3mm).

[0178] 6) Detection method: Take the test paper from step 5) above and add different concentrations of SARS-CoV-2 antigen recombinant protein to the sample pad. Develop with 10 mM PBS and 0.1% Tween-20 as the developing agent. Perform lateral flow chromatography for 20 minutes and read the sample colorimetrically.

[0179] 7) The detection results of a test strip for detecting the recombinant protein of the new coronavirus antigen are as follows Figure 20As shown, the results show that in the system for detecting the recombinant protein of the new coronavirus antigen, the test strip with 3% agarose hydrogel as the quality control line can intercept the colloidal gold coupled with the new coronavirus N protein 15C3, and can detect the recombinant protein of the new coronavirus antigen at concentrations of 0.5, 1, and 10 ng / L.

[0180] The above results show that the general technology designed and constructed by the present invention for the quality control line of colloidal gold test paper can achieve the effective capture of colloidal gold of different sizes, and can also be applied to a variety of detection systems including but not limited to biotin-avidin detection, host-guest competition detection, and antigen-antibody immunoassay; compared with agarose hydrogel, various other hydrogels have similar hydrophilicity and porous structure, can effectively intercept colloidal gold, and can also be used for quality control lines on test strips for various detection systems; the general technology for quality control lines of test strips involved in the present invention can further broaden the types of analytes applicable to it, can effectively solve the limitations and shortcomings of existing test paper quality control lines, and is of great significance to the development of low-cost and timely detection technology.

[0181] The above embodiments are merely examples for illustrative purposes only and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications may be made based on the above descriptions. It is not necessary and impossible to enumerate all implementation methods here. Therefore, any obvious variations or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A quality control line coating solution for colloidal gold test strips, characterized in that: The quality control line coating solution includes a hydrogel, which is one or a mixture of two or more of agarose hydrogel, polyacrylic acid hydrogel, polyacrylamide hydrogel, gelatin hydrogel, carrageenan hydrogel, polyvinyl alcohol hydrogel or polyvinyl alcohol-sodium alginate hydrogel, and the mass fraction of the hydrogel is less than 40%.

2. A quality control line for a colloidal gold test strip, characterized in that: The quality control line is prepared by the following method: preparing a hydrogel solution, wherein the hydrogel is one or a mixture of two or more of agarose hydrogel, polyacrylic acid hydrogel, polyacrylamide hydrogel, gelatin hydrogel, carrageenan hydrogel, polyvinyl alcohol hydrogel, or polyvinyl alcohol-sodium alginate hydrogel, and the mass fraction of the hydrogel is less than 40%; The prepared hydrogel solution is pipetted and dripped onto the chromatographic membrane as the quality control line, and then vacuum dried to obtain the quality control line.

3. The quality control line according to claim 2, wherein: The hydrogel solution is an agarose hydrogel solution. The specific steps of preparing the agarose hydrogel solution are: adding agarose powder to water, heating and maintaining for 10 to 12 minutes, thereby obtaining the agarose hydrogel solution.

4. A colloidal gold test strip comprising a backing plate and a sample pad, a conjugate pad, a chromatography membrane, and a water-absorbing pad sequentially arranged on the backing plate; the chromatography membrane is provided with a detection line and a quality control line, and the quality control line is the quality control line according to claim 2 or 3.

5. Application of the quality control line as claimed in claim 2 or 3 in a sandwich method and a competitive method in colloidal gold test paper detection.

6. The use according to claim 5, characterized in that The sandwich method is used to detect the biotin-avidin system, the new coronavirus detection system, nucleic acid, polysaccharide or lectin system; the competition method is used to detect the host-guest system, and the host-guest system includes a host macrocyclic molecule and a guest molecule.

7. The use according to claim 5, characterized in that The specific steps of the application are: preparing a quality control line, preparing a coating solution for the detection line, coating the detection line, labeling with colloidal gold, preparing a gold label pad, and preparing a test strip.

8. The use according to claim 6, characterized in that The main macrocyclic molecule is cucurbituril, pillararene or cyclodextrin.

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