Paper-based electrochemical luminescence novel coronavirus antigen detection test strip as well as preparation method and application thereof

Abstract

The invention relates to a paper-based electrochemiluminescence novel coronavirus antigen detection test strip as well as a preparation method and application thereof. The test strip comprises a bottom plate, and a paper-based channel layer and an electrode layer are arranged on the bottom plate; the paper-based channel layer comprises a sample pad, a combination pad, a detection pad and a water absorption pad which are sequentially overlapped; the combination pad is coated with an SARS-CoV-2 first antibody marked by ruthenium coupling; the detection pad is coated with an SARS-CoV-2 second antibody; the recognition antigen epitopes of the SARS-CoV-2 first antibody and the SARS-CoV-2 second antibody marked by ruthenium coupling are different; the electrode layer is provided with a plurality of electrodes, and one end of each electrode is connected with the detection pad. Aiming at SARS-CoV-2 detection, a lateral flow immunochromatography technology and an electrochemical luminescence technology are combined, a brand-new detection test strip is designed, and low-cost, rapid and high-sensitivity detection is realized.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and relates to a paper-based electrochemiluminescence novel coronavirus antigen test strip, its preparation method, and its application. Background Technology

[0002] Currently, detection methods for the novel coronavirus SARS-CoV-2 mainly fall into three categories: nucleic acid-based (NAT or molecular), antigen-based, and antibody-based. Nucleic acid-based virological tests have high sensitivity, but their limitations are also significant: cumbersome sampling, long testing and result turnaround times, and high requirements for personnel and equipment. Antibody-based serological tests do not target the virus itself, but rather the antibodies produced by the human immune system after 5-7 days; therefore, they are not suitable for acute COVID-19 infection, and their detection speed has not effectively slowed the spread of SARS-CoV-2. Antigen-based detection methods directly detect viral proteins and have high specificity, but their sensitivity is lower than molecular detection, and they may not capture all active viruses, leading to false positives. Therefore, developing more effective detection methods is of great importance.

[0003] Electrochemiluminescence (ECL) technology combines the advantages of electrochemistry, fluorescence analysis, and chemiluminescence analysis. It boasts advantages such as simple instrumentation, high sensitivity, wide linear range, low cost, and low background. Furthermore, ECL offers benefits like reagent stability and reusability, accurate results, rapid reaction, and good reproducibility. Co-reactants are intermediate substances with strong oxidizing or reducing properties generated during oxidation or reduction reactions in ECL. These intermediates can react with reducing or oxidizing substances in the ECL system to form excited-state molecules, thus promoting the ECL process. Co-reactant-based ECL eliminates the need for an excitation light source, removing stray light interference and improving analytical accuracy. The resulting electrochemical biosensors exhibit advantages such as high sensitivity, rapid response time, low background, low cost, and wide detection range. Immunoassays performed on paper chips, which rely on the immune reaction between antigens and antibodies, have high sensitivity and specificity and can form a double-antibody sandwich complex. By combining immunochromatography with electrochemiluminescence, SARS-CoV-2 virus can be detected. However, these methods are only suitable for single analysis and the results need to be read within a limited time. Their accuracy may be lower than that of laboratory tests, and the samples are not easy to preserve after testing. Reproducibility may vary from batch to batch.

[0004] In conclusion, developing a low-cost, rapid, accurate, and effective method for detecting the SARS-CoV-2 virus remains one of the most pressing issues to be addressed. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, this invention provides a paper-based electrochemiluminescence novel coronavirus antigen test strip, its preparation method, and its application. By combining lateral flow immunochromatography and electrochemiluminescence technologies, a low-cost, rapid, accurate, and effective method for detecting SARS-CoV-2 virus is developed.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a paper-based electrochemiluminescence novel coronavirus antigen detection test strip, the test strip comprising a base plate, wherein a paper-based channel layer and an electrode layer are disposed on the base plate; the paper-based channel layer comprises a sample pad, a conjugate pad, a detection pad and an absorbent pad disposed sequentially overlapping each other; the conjugate pad is coated with a ruthenium-conjugated SARS-CoV-2 primary antibody; the detection pad is coated with a SARS-CoV-2 secondary antibody; the ruthenium-conjugated SARS-CoV-2 primary antibody and the SARS-CoV-2 secondary antibody recognize different antigen epitopes; the electrode layer is provided with a plurality of electrodes, one end of which is connected to the detection pad.

[0008] In this invention, for the detection of SARS-CoV-2, a novel test strip is designed by combining lateral flow immunochromatography and electrochemiluminescence technology. The test strip consists of a ruthenium-labeled SARS-CoV-2 primary antibody coated on the conjugate pad, which binds to the SARS-CoV-2 antigen. Simultaneously, a secondary SARS-CoV-2 antibody, which identifies different epitopes of the antigen, coats the test pad, acting as a capture antibody to trap the conjugate. Electrochemiluminescence detection is then performed based on the electrode layer. Qualitative and quantitative detection of SARS-CoV-2 is achieved based on the luminescence intensity signal, enabling low-cost, rapid, and highly sensitive detection while avoiding false positives.

[0009] Preferably, the matrix material of the sample pad includes any one of polyester fiber membrane RB45, polyester fiber membrane RB65, or glass fiber membrane SB08.

[0010] Preferably, the sample pad is further coated with a sample pad treatment solution.

[0011] Preferably, the sample pad treatment solution contains a PBS solution containing Tris-HCl, BSA, sodium caseinate, trehalose, sucrose, HBR, heterophile antibody blocking agent, and NaCl.

[0012] Preferably, the sample pad treatment solution is a PBS solution containing 0.03–0.06 mol / L Tris-HCl, 0.5%–2% (w / w) BSA, 0.3%–0.6% (w / w) sodium caseinate, 0.3%–0.6% (w / w) trehalose, 1%–3% (w / w) sucrose, 0.3–0.6 mg / mL HBR, 0.3–0.6 mg / mL heterophile antibody blocking agent, and 0.85%–0.95% (w / w) NaCl.

[0013] Preferably, the pH of the sample pad treatment solution is 7.2 to 7.6, such as 7.3, 7.4 or 7.5.

[0014] It is understood that the substrate material of the base plate can be selected according to the actual situation, such as PVC base plate or PS base plate, etc.

[0015] Preferably, the matrix material of the bonding pad includes any one of polyester fiber membrane RB45, polyester fiber membrane RB65, or glass fiber membrane SB08.

[0016] Preferably, the conjunctival pad is further coated with a conjunctival pad treatment solution;

[0017] Preferably, the conjugate pad treatment solution contains a PBS solution of 0.03–0.06 mol / L Tris-HCl, 0.5%–2% BSA, 0.3%–0.6% sodium caseinate, 0.3%–0.6% trehalose, 1%–3% sucrose, 0.3–0.6 mg / mL HBR, 0.3–0.6 mg / mL heterophile antibody blocking agent, and 0.85%–0.95% NaCl.

[0018] Preferably, the pH of the conjunctival pad treatment solution is 7.2 to 7.6, such as 7.3, 7.4 or 7.5.

[0019] Preferably, the matrix material of the detection pad includes any one of a 0.45μm nitrocellulose membrane, a nylon transfer membrane, a Whatman No. 1 filter paper, or a CN95 membrane.

[0020] Preferably, the matrix material of the detection pad is a CN95 membrane.

[0021] Preferably, the length of the detection pad is 1 to 2 cm.

[0022] In this invention, by controlling the use of a specific matrix material for the detection pad, the electrochemiluminescence (ECL) response value can be further improved, thereby enhancing the detection sensitivity.

[0023] Preferably, the mass concentration of the ruthenium-conjugated SARS-CoV-2 primary antibody on the binding pad and the SARS-CoV-2 secondary antibody on the detection pad is 1:(0.01-0.1), including but not limited to 1:0.02, 1:0.025, 1:0.03, 1:0.05, 1:0.06, 1:0.07, 1:0.08 or 1:0.09, etc., preferably 1:(0.03-0.06), and more preferably 1:0.05.

[0024] In this invention, controlling the ratio of specific labeled antibodies and capture antibodies can further improve the electrochemiluminescence (ECL) response value, thereby enhancing detection sensitivity.

[0025] Preferably, the electrode layer comprises screen-printed electrodes.

[0026] It is understood that the substrate material of the absorbent pad can be selected according to the actual situation, such as filter paper.

[0027] It is understood that the length and width of the sample pad, bonding pad, and absorbent pad in this invention can be adjusted according to the actual situation, such as a width of 5 to 8 mm and a length of 10 to 30 mm.

[0028] In a second aspect, the present invention provides a method for preparing the paper-based electrochemiluminescence novel coronavirus antigen test strip described in the first aspect, the method comprising:

[0029] The sample pad matrix material is treated with the sample pad treatment solution to obtain the sample pad;

[0030] Ruthenium tripyridine and SARS-CoV-2 primary antibody were mixed to obtain ruthenium-conjugated SARS-CoV-2 primary antibody. The ruthenium-conjugated SARS-CoV-2 primary antibody was then coated onto the matrix material of the conjugate pad to obtain the conjugate pad.

[0031] The SARS-CoV-2 second antibody is coated onto the matrix material of the test pad to obtain the test pad;

[0032] The sample pad, conjugate pad, detection pad, and absorbent pad are sequentially stacked on the base plate, and one end of the electrode on the electrode layer is connected to the detection pad.

[0033] Preferably, the method for preparing the ruthenium-conjugated SARS-CoV-2 primary antibody specifically includes:

[0034] Antibody modification was performed using Ru-NHS ester. 5 mg of Ru(bpy)2(mcbpy-O-Su-ester)(PF6)2 was equilibrated to room temperature, and 50 μL of DMSO and 450 μL of PBS were added. After shaking until completely dissolved, PBS was added to prepare a 1000 μmol / L stock solution. The solution was wrapped in aluminum foil and placed in a rotary stirrer. The reaction was carried out at 35 rpm for 12 hours at room temperature. 5 mL of 7K MWCO Zeba ester was then added. TM The invention provides, in a third aspect, the application of the paper-based electrochemiluminescence novel coronavirus antigen test strip described in the first aspect in the preparation of novel coronavirus test kits or in the detection of novel coronavirus for non-disease diagnosis purposes. This involves removing free Ru-NHS esters and other small molecule impurities from the reaction products using a rotating desalting column and collecting Ru-NHS ester-labeled SARS-CoV-2Ab1.

[0035] The invention designs a high-sensitivity test strip that can be used for the detection of the novel coronavirus. It can be applied to both disease diagnosis and non-disease diagnosis fields, such as detecting environmental samples and virus prevention and control.

[0036] Fourthly, the present invention provides a method for detecting the novel coronavirus for non-disease diagnosis purposes, the method comprising:

[0037] The sample to be tested is added to the sample pad of the paper-based electrochemiluminescence novel coronavirus antigen test strip described in the first aspect. After the immune reaction, the test pad is subjected to electrochemiluminescence detection.

[0038] Preferably, the detection method further includes the step of mixing the sample to be tested with an electrochemiluminescence co-reactant, or adding the electrochemiluminescence co-reactant to a detection pad.

[0039] This invention has found that using specific electrochemiluminescence co-reactants for detection is more conducive to improving ECL response values ​​and enhancing sensitivity, and that pre-mixing the sample with the electrochemiluminescence co-reactant for detection yields even better results.

[0040] Preferably, the electrochemiluminescence co-reactant includes at least one of TPA (tri-n-propylamine), BDEA (N-butyldiethanolamine), or elecsys buffer (electrochemiluminescence buffer), tri-n-propylamine, and dibutylethanolamine.

[0041] Preferably, the elecsys buffer contains phosphate buffer salt, tripropylamine, preservative, and detergent.

[0042] Preferably, the immune response time is 3 to 9 minutes, including but not limited to 4, 5, 6, 7 or 8 minutes, and preferably 5 to 7 minutes.

[0043] In this invention, it was discovered that controlling specific immune response times can also help improve ECL response values ​​and enhance sensitivity.

[0044] Compared with the prior art, the present invention has at least the following beneficial effects:

[0045] In this invention, a novel test strip is designed. The test pad is coated with a ruthenium-labeled SARS-CoV-2 primary antibody as a labeling antibody, which binds to the SARS-CoV-2 antigen. Simultaneously, a secondary SARS-CoV-2 antibody with a different antigenic epitope is coated on the test pad as a capture antibody to capture the conjugate. Electrochemiluminescence detection is then performed based on the electrode layer. Qualitative and quantitative detection of SARS-CoV-2 is achieved based on the luminescence intensity signal, realizing low-cost, rapid, and highly sensitive detection while avoiding false positives. Furthermore, the test pad matrix material, the ratio of labeling antibody to capture antibody, the immune reaction time, and the electrochemiluminescence co-reactants are all designed synergistically to further improve detection sensitivity. Attached Figure Description

[0046] Figure 1 This is a schematic diagram illustrating antibody labeling and its binding with antigen-antibody.

[0047] Figure 2 This is a schematic diagram illustrating the detection principle.

[0048] Figure 3 A schematic diagram of the structure of a paper-based electrochemiluminescence novel coronavirus antigen detection test strip;

[0049] Figure 4 The graph shows the ECL response values ​​of different luminescent co-reactants participating in the reaction.

[0050] Figure 5 Graph showing the ECL response values ​​of reactions with different proportions of luminescent co-reactants added;

[0051] Figure 6 Graph showing the ECL response values ​​of reactions with different proportions of luminescent co-reactants added;

[0052] Figure 7 Graphs showing the ECL response values ​​for different types of detection pads;

[0053] Figure 8 The graph shows the ECL response values ​​for reactions with different ratios of labeled antibodies and capture antibodies.

[0054] Figure 9 A graph showing the ECL response values ​​at different immunization times;

[0055] Figure 10The graph shows the results of ECL response values ​​for samples of different concentrations.

[0056] Figure 11 The graph shows the linear relationship between sample concentration and ECL response value. Detailed Implementation

[0057] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0058] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0059] This invention combines lateral flow immunochromatography and electrochemiluminescence (ECL) techniques for the detection of novel coronavirus antigens. The main strategies include lateral flow immunochromatography, the double-antibody sandwich method, and the ECL reaction principle. A schematic diagram of antibody labeling and antigen binding is shown below. Figure 1 As shown, ruthenium-labeled SARS-CoV-2 primary antibody is used. After labeling, the antibody binds to the antigen and can be captured by SARS-CoV-2 secondary antibody. After chromatographic separation on a paper substrate, the sample binds to the antibody in the detection zone. When the chromatographic reactants reach the detection zone, they bind to the captured antibody. Then, a photomultiplier tube and signal receiver are used in the detection zone to collect and output the luminescence intensity signal, thereby achieving quantitative analysis of the target substance. This is a method for electrochemiluminescence detection. The schematic diagram of the detection principle is shown below. Figure 2 As shown. Furthermore, in-depth analysis of the impact of multiple steps and factors on the detection results allows for precise control and overall synergy, further improving the detection effectiveness.

[0060] Example 1

[0061] This embodiment prepares a paper-based electrochemiluminescence novel coronavirus antigen detection strip, the structural schematic diagram of which is shown below. Figure 3 As shown.

[0062] 1. Sample pad preparation

[0063] Sample pad preparation: The sample pads were pretreated with sample pad treatment solution (0.05 mol / L Tris-HCl pH 7.4, 1% BSA, 0.5% sodium caseinate, 0.5% trehalose, 2% sucrose, 0.5 mg / mL HBR, 0.5 mg / mL heterophile antibody blocking agent, and 0.9% NaCl, mixed with PBS at pH 7.4). Each 24 mL solution could treat paper of size 250 mm × 300 mm. The sample pad treatment solution was pipetted and sprayed evenly onto the sample pads. The liquid was then evenly dispersed on the paper using a roller. The paper was placed in a vertical drying oven and dried overnight. The dried paper was then cut into strips 0.6 cm wide and 2.5 cm long and stored in a dark, dry environment for later use.

[0064] 2. Preparation of binding pad

[0065] Treat the conjoint pads with a conjoint pad treatment solution (with the same composition as the sample pad treatment solution). Each 24 mL solution can treat paper with a size of 250 mm × 300 mm. Take a sample of the conjoint pad treatment solution, spray it evenly onto the conjoint pads, and then use a roller to evenly distribute the liquid onto the paper. Place the paper in a vertical forced-air drying oven overnight to dry. After drying, cut the paper into strips 0.6 cm wide and 1.0 cm long, and store them in a dark, dry environment for later use.

[0066] SARS-CoV-2 antigen, catalog number: ncov-PS-Ag6, was purchased from Guangdong Feipeng Biotechnology Co., Ltd. (China); the labeling antibody, trade name: 2019-nCoV NP Antibody (7G9), catalog number: DA078; and the capture antibody, trade name: 2019-nCoV NP Antibody (6G9), catalog number: DA027, were all purchased from Suzhou Nearshore Protein Technology Co., Ltd.

[0067] Preparation of Ruthenium-Conjugated SARS-CoV-2 Primary Antibody (as Labeling Antibody): 1. Take 5 mg of Ru(bpy)2(mcbpy-O-Su-ester)(PF6)2 and allow it to reach room temperature. Add 50 μL DMSO and 450 μL PBS to prepare a 10 mmol / L solution. Shake until completely dissolved, then add PBS to prepare a 0.1 μmol / L working solution. 2. Take SARS-CoV-2Ab1 and allow it to dissolve to room temperature. Add PBS and 1% BSA to prepare three antibody dilutions with a final volume of 500 μL and molar concentrations of 10 μmol / L, 2 μmol / L, and 1 μmol / L. Set the molar ratios of Ru-NHS ester dilution to SARS-CoV-2Ab1 dilution to be 10:1, 50:1, and 100:1. Wrap the solution in aluminum foil and react in a rotary shaker for 12 hours. Transfer the reaction mixture to 5 mL of 7K MWCO Zeba solution. TMFree Ru-NHS esters were removed by rotating a desalting column, and the Ru-NHS ester-labeled SARS-CoV-2Ab1 was collected after chromatography.

[0068] The ruthenium-conjugated SARS-CoV-2 primary antibody was coated onto the conjugate pad: the conjugate pad was cut into strips 6 mm wide and 10 mm long, and the labeled antibody was evenly sprayed onto the prepared pad using an HGS510-1 instrument, and then dried again for later use.

[0069] 3. Preparation of the test pad

[0070] Cut the test pad (UniSart CN 95 nitrocellulose membrane purchased from Sartorius (Germany), catalog number: NC-b101) into strips 0.6 cm wide and 1.5 cm long using paper. Dissolve the SARS-CoV-2 secondary antibody (as the capture antibody) (purchased from Suzhou Nearshore Protein Technology Co., Ltd., capture antibody trade name: 2019-nCoV NP Antibody (6G9), catalog number: DA027) at room temperature. Prepare a coating buffer using 10 mM phosphate buffer, 1% sucrose solution, and 100 ml of pure water. Dilute the coating buffer to a 1 mg / mL capture antibody dilution. Spray the capture antibody onto the test pad using an HGS510-1 benchtop gold sprayer at a spray volume of 6 μL / cm. Then, dry the test pad with the capture antibody overnight and store it in a dark, dry environment for later use.

[0071] The mass concentration ratio of capture antibody to labeled antibody on the control coating was 1:0.05.

[0072] 4. Test strip assembly

[0073] Place the prepared paper base material on the experimental table, remove the PVC base plate with adhesive on the bottom, and fix the paper base material onto the PVC base plate in the order of test pad, bonding pad, test pad and absorbent pad.

[0074] The prepared lateral flow immunochromatographic test strip is pasted onto the screen-printed electrode. Note that the detection pad should be in the same position as the black detection area of ​​the electrode chip so that as many signals as possible can be collected during electrochemiluminescence detection, resulting in better detection effect.

[0075] Example 2

[0076] This embodiment describes the preparation of a paper-based electrochemiluminescence novel coronavirus antigen detection strip.

[0077] 1. Sample pad preparation

[0078] Sample pad preparation: The sample pads were pretreated with sample pad treatment solution (0.03 mol / L Tris-HCl pH 7.4, 0.5% BSA, 0.6% sodium caseinate, 0.3% trehalose, 3% sucrose, 0.6 mg / mL HBR, 0.3 mg / mL heterophile antibody blocking agent, and 0.9% NaCl, mixed with PBS at pH 7.4). Each 24 mL solution could treat paper of size 250 mm × 300 mm. The sample pad treatment solution was pipetted and sprayed evenly onto the sample pads. The liquid was then evenly dispersed on the paper using a roller. The paper was placed in a vertical drying oven and dried overnight. The dried paper was then cut into strips 0.6 cm wide and 2.5 cm long and stored in a dark, dry environment for later use.

[0079] 2. Preparation of binding pad

[0080] Treat the conjoint pads with a conjoint pad treatment solution (with the same composition as the sample pad treatment solution). Each 24 mL solution can treat paper with a size of 250 mm × 300 mm. Take a sample of the conjoint pad treatment solution, spray it evenly onto the conjoint pads, and then use a roller to evenly distribute the liquid onto the paper. Place the paper in a vertical forced-air drying oven overnight to dry. After drying, cut the paper into strips 0.6 cm wide and 1.0 cm long, and store them in a dark, dry environment for later use.

[0081] Preparation of ruthenium-conjugated SARS-CoV-2 primary antibody (as labeled antibody): Refer to Example 1.

[0082] Coating the binding pad with a ruthenium-conjugated SARS-CoV-2 primary antibody: Refer to Example 1.

[0083] 3. Preparation of the test pad

[0084] Cut the test pad (UniSart CN 95 nitrocellulose membrane purchased from Sartorius (Germany)) into strips 0.6 cm wide and 1.5 cm long using paper. Dissolve the SARS-CoV-2 secondary antibody (as the capture antibody) at room temperature. Prepare a coating buffer using 10 mM phosphate buffer, 1% sucrose solution, and 100 mL of pure water. Dilute the coating buffer to a 1 mg / mL capture antibody solution. Spray the capture antibody onto the test pad using an HGS510-1 benchtop gold sprayer at a spray volume of 6 μL / cm. Dry the test pad with the captured antibody overnight and store it in a dark, dry environment for later use.

[0085] The mass concentration ratio of capture antibody to labeled antibody on the control coating was 1:0.03.

[0086] 4. Test strip assembly

[0087] Place the prepared paper base material on the experimental table, remove the PVC base plate with adhesive on the bottom, and fix the paper base material onto the PVC base plate in the order of test pad, bonding pad, test pad and absorbent pad.

[0088] The prepared lateral flow immunochromatographic test strip is pasted onto the screen-printed electrode. Note that the detection pad should be in the same position as the black detection area of ​​the electrode chip so that as many signals as possible can be collected during electrochemiluminescence detection, resulting in better detection effect.

[0089] Example 3

[0090] This embodiment describes the preparation of a paper-based electrochemiluminescence novel coronavirus antigen detection strip.

[0091] 1. Sample pad preparation

[0092] Sample pad preparation: The sample pads were pretreated with sample pad treatment solution (0.03 mol / L Tris-HCl pH 7.4, 0.5% BSA, 0.6% sodium caseinate, 0.3% trehalose, 3% sucrose, 0.6 mg / mL HBR, 0.3 mg / mL heterophile antibody blocking agent, and 0.9% NaCl, mixed with PBS at pH 7.4). Each 24 mL solution could treat paper of size 250 mm × 300 mm. The sample pad treatment solution was pipetted and sprayed evenly onto the sample pads. The liquid was then evenly dispersed on the paper using a roller. The paper was placed in a vertical drying oven and dried overnight. The dried paper was then cut into strips 0.6 cm wide and 2.5 cm long and stored in a dark, dry environment for later use.

[0093] 2. Preparation of binding pad

[0094] Treat the conjoint pads with a conjoint pad treatment solution (with the same composition as the sample pad treatment solution). Each 24 mL solution can treat paper with a size of 250 mm × 300 mm. Take a sample of the conjoint pad treatment solution, spray it evenly onto the conjoint pads, and then use a roller to evenly distribute the liquid onto the paper. Place the paper in a vertical forced-air drying oven overnight to dry. After drying, cut the paper into strips 0.6 cm wide and 1.0 cm long, and store them in a dark, dry environment for later use.

[0095] Preparation of ruthenium-conjugated SARS-CoV-2 primary antibody (as labeled antibody): Refer to Example 1.

[0096] Coating the binding pad with a ruthenium-conjugated SARS-CoV-2 primary antibody: Refer to Example 1.

[0097] 3. Preparation of the test pad

[0098] Cut the test pad (UniSart CN 95 nitrocellulose membrane purchased from Sartorius (Germany)) into strips 0.6 cm wide and 1.5 cm long using paper. Dissolve the SARS-CoV-2 secondary antibody (as the capture antibody) at room temperature. Prepare a coating buffer using 10 mM phosphate buffer, 1% sucrose solution, and 100 mL of pure water. Dilute the coating buffer to a 1 mg / mL capture antibody solution. Spray the capture antibody onto the test pad using an HGS510-1 benchtop gold sprayer at a spray volume of 6 μL / cm. Dry the test pad with the captured antibody overnight and store it in a dark, dry environment for later use.

[0099] The mass concentration ratio of capture antibody to labeled antibody on the control coating was 1:0.1.

[0100] 4. Test strip assembly

[0101] Place the prepared paper base material on the experimental table, remove the PVC base plate with adhesive on the bottom, and fix the paper base material onto the PVC base plate in the order of test pad, bonding pad, test pad and absorbent pad.

[0102] The prepared lateral flow immunochromatographic test strip is pasted onto the screen-printed electrode. Note that the detection pad should be in the same position as the black detection area of ​​the electrode chip so that as many signals as possible can be collected during electrochemiluminescence detection, resulting in better detection effect.

[0103] Example 4

[0104] This embodiment describes the preparation of a paper-based electrochemiluminescence novel coronavirus antigen detection strip.

[0105] Compared with Example 1, the only difference is that the CN95 film of the detection pad matrix material is replaced with a nylon transfer C film of the same size (Biodyne C purchased from PALL (USA)).

[0106] Example 5

[0107] This embodiment describes the preparation of a paper-based electrochemiluminescence novel coronavirus antigen detection strip.

[0108] Compared with Example 1, the only difference is that the CN95 membrane matrix material of the test pad is replaced with Whatman No. 1 filter paper of the same size (Whatman No. 1 is purchased from Whatman (USA)).

[0109] Example 6

[0110] This embodiment describes the preparation of a paper-based electrochemiluminescence novel coronavirus antigen detection strip.

[0111] Compared with Example 1, the only difference is that the CN95 membrane of the detection pad matrix material is replaced with a 0.45 μm nitrocellulose membrane of the same size (PT 0.45 μm purchased from Cytiva (USA)).

[0112] Example 7

[0113] This embodiment describes the preparation of a paper-based electrochemiluminescence novel coronavirus antigen detection strip.

[0114] Compared with Example 1, the only difference is that the mass concentration ratio of the capture antibody and the labeled antibody is controlled to be 1:0.025.

[0115] Example 8

[0116] This embodiment describes the preparation of a paper-based electrochemiluminescence novel coronavirus antigen detection strip.

[0117] Compared with Example 1, the only difference is that the mass concentration ratio of the capture antibody and the labeled antibody is controlled to be 1:0.01.

[0118] Example 9

[0119] This embodiment describes the preparation of a paper-based electrochemiluminescence novel coronavirus antigen detection strip.

[0120] Compared with Example 1, the only difference is that the mass concentration ratio of the capture antibody and the labeled antibody is controlled to be 1:0.1.

[0121] Example 10

[0122] This embodiment describes the preparation of a paper-based electrochemiluminescence novel coronavirus antigen detection strip.

[0123] Compared with Example 1, the only difference is that the mass concentration ratio of the capture antibody and the labeled antibody is controlled to be 1:0.05.

[0124] Test Example 1

[0125] This embodiment tests the test strips prepared in each embodiment.

[0126] First, the effect of different co-reactants on the detection response value of ECL was tested. The test procedure included: to investigate the effect of different co-reactants on this experiment, a sample solution of 2000 pg / mL was prepared, and the buffer solution consisted of co-reactants and PBS. The co-reactants in the buffer solution were set up as a PBS buffer blank control group, TPA group, BDEA group, and electrochemiluminescence buffer (Elecsys Buffer) group (brand: Roche Group, 11662988, phosphate buffer 300 mmol / L, tripropylamine 180 mmol / L; detergent ≤0.1%; preservative, pH 6.8). The buffer solution was prepared into corresponding sample dilutions at a mass ratio of 1:1. 150 μL of the sample dilution was added to the sample pad of the test strip prepared in Example 1 for electrochemiluminescence testing.

[0127] Test results as follows Figure 4 As shown, the electrochemiluminescence intensity values ​​of the phosphate buffer blank control group, the tri-n-propylamine group, the N-butyldiethanolamine group, and the electrochemiluminescence buffer group are displayed. The phosphate buffer group has the lowest signal at 542, while the mean values ​​of the other samples with added co-reactants are 2532, 2219, and 3793, respectively (3 parallel tests, n=3). This indicates that compared with the phosphate buffer blank control group, the electrochemiluminescence intensity of the tri-n-propylamine group, the N-butyldiethanolamine group, and the electrochemiluminescence buffer group are significantly increased (p<0.01). Moreover, the electrochemiluminescence buffer group has the best detection effect. Therefore, the electrochemiluminescence buffer is most suitable as a co-reactant for the paper-based electrochemiluminescence side-flow immunochromatographic test strip of this invention to improve the detection fluorescence intensity.

[0128] The effect of the addition ratio of the luminescent co-reactant on the detection ECL response value was tested. The test procedure included:

[0129] To investigate the effect of the proportion of co-reactants (using Elecsys Buffer as an example) on the electrochemiluminescence intensity, 2000 pg / mL samples were prepared with co-reactant volume ratios of 4:1, 2:1, 1:1, 1:2, and 2:3. 150 μL of each of these different ratio mixtures was added to the sample pad of the test strip prepared in Example 1 for electrochemiluminescence testing.

[0130] Test results as follows Figure 5 As shown, by Figure 5It was found that the luminescence values ​​of PBS and co-reactant in the sample diluents at volume ratios of 1:1, 1:2, 2:1, 2:3, and 4:1 were 3519, 2742, 4488, 2439, and 1523, respectively (three parallel tests, n=3). This indicates that the electrochemiluminescence intensity significantly increased when the volume ratio of PBS to co-reactant in the diluent was 2:1 (p<0.01). Therefore, a volume ratio of PBS to co-reactant in the sample diluent of 2:1 is most suitable for the paper-based electrochemiluminescence side-flow immunochromatographic test strip of this invention.

[0131] The effect of the addition method of the luminescent co-reactant (using Elecsys Buffer as an example) on the detection ECL response value was tested. The test process included: setting up a premixed group of co-reactant and SARS-COV-2 and a group in which co-reactant and SARS-COV-2 were added separately. In the premixed group, 150 μL of 2000 pg / mL sample solution was added to the sample pad of the test strip prepared in Example 1, with Elecsys Buffer occupying 1 / 3 of the final volume in the diluent and the remainder made up with PBS. In the separately added group, SARS-COV-2 was diluted with PBS to the same concentration as the premixed group and added to the sample pad of the test strip prepared in Example 1. During detection, 1 / 3 of the total volume of co-reactant was added to the test pad for electrochemiluminescence intensity detection.

[0132] Test results as follows Figure 6 As shown, the results indicate that the mean electrochemiluminescence intensities of the premixed co-reactant with SARS-CoV-2 group and the group in which co-reactant and SARS-CoV-2 were added separately were 2354 and 4428, respectively (3 parallel tests, n=3). This shows that compared with adding co-reactant and SARS-CoV-2 separately, the electrochemiluminescence intensity of the premixed co-reactant with SARS-CoV-2 was significantly increased (p<0.01). Therefore, premixing the co-reactant with SARS-CoV-2 in advance is most suitable for the paper-based electrochemiluminescence lateral flow immunochromatographic test strip of this invention.

[0133] After confirming the optimal luminescent co-reactant and its addition method, the test strips prepared in each embodiment were tested, and the test results are as follows: Figure 7 and Figure 8 As shown, the results indicate that by controlling specific detection pad matrix materials and antibody ratios, the present invention can further improve the detection ECL response value, thereby improving detection sensitivity.

[0134] Taking the test strip of Example 1 as an example, different immune reaction times were tested. The specific experimental procedure included setting up 3-min, 5-min, 7-min, and 9-min groups for the experiment. SARS-CoV-2 was diluted to 20 ng / mL, and 150 μL was added to the sample pad. After reacting in the dark for 3 min, 5 min, 7 min, and 9 min, the samples were tested using an electrochemiluminescence analyzer. Three parallel experiments were performed.

[0135] The results are as follows Figure 9 As shown, the results indicate that the mean electrochemiluminescence values ​​of the 3-min, 5-min, 7-min, and 9-min groups were 3101, 6509, 5248, and 2691 (n=3), respectively. This shows that compared with 3 and 9 min, the electrochemiluminescence intensity was significantly increased when the reaction time was 5-7 min (p<0.05), and 5 min was the optimal time. This indicates that the lateral flow immunochromatographic reaction was completed at 5 min, therefore 5 min is the most suitable detection time.

[0136] Controlling the immune reaction time to 5–7 minutes for electrochemical detection can further improve the detection ECL response value, thereby increasing the detection sensitivity.

[0137] Test Example 2

[0138] This test example uses the test strip prepared in Example 1 to test the detection performance.

[0139] The specific testing procedure included: exploring the limit of detection, detecting Sars-cov-2 concentrations of 0, 7.96, 20, 200, 2000, and 20000 pg / mL, respectively; adding 150 μL of each sample to the sample pad, reacting in the dark for 5 min, and then performing electrochemiluminescence analysis. The measured values ​​were used to calculate the electrochemiluminescence intensity based on a signal-to-noise ratio (S / N) of 3. Figure 10 Substituting these values ​​into the standard curve formula, the limit of detection was found to be 7.96 pg / mL. A linear fit curve was then plotted between the limit of detection and the logarithm of the Sars-cov-2 concentration. Figure 11 ).

[0140] like Figure 10 and Figure 11 As shown, SARS-CoV-2 concentrations of 7.96, 20, 200, 2000, and 20000 pg / mL were detected (C Sars-cov-2 There is a good linear relationship between C and ECL intensity (IECL), with the linear regression equation being IECL = 1327.4lg C. Sars-cov-2 +453.31, correlation coefficient R 2 =0.9927.

[0141] This indicates that it can effectively detect samples with concentrations ranging from 7.96 to 20000 pg / mL.

[0142] In summary, this invention designs a novel test strip. The test pad is coated with a ruthenium-labeled SARS-CoV-2 primary antibody, which acts as the labeling antibody and binds to the SARS-CoV-2 antigen. Simultaneously, a secondary SARS-CoV-2 antibody, which identifies different epitopes of the antigen, is coated on the test pad as a capture antibody conjugate. Electrochemiluminescence detection is then performed based on the electrode layer. Qualitative and quantitative detection of SARS-CoV-2 is achieved based on the luminescence intensity signal, enabling low-cost, rapid, and highly sensitive detection while avoiding false positives. Furthermore, the design of the test pad matrix material, the ratio of labeled antibody to capture antibody, the immune reaction time, and the electrochemiluminescence co-reactants are all optimized to synergistically enhance detection sensitivity.

[0143] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A paper-based electrochemiluminescent novel coronavirus antigen detection test strip, characterized in that: The test strip comprises a bottom plate, on which a paper-based channel layer and an electrode layer are arranged; The paper-based channel layer comprises a sample pad, a conjugation pad, a detection pad and a water-absorbing pad which are overlapped in sequence; the conjugation pad is coated with a SARS-CoV-2 first antibody conjugated with ruthenium; the detection pad is coated with a SARS-CoV-2 second antibody; the SARS-CoV-2 first antibody conjugated with ruthenium and the SARS-CoV-2 second antibody recognize different antigen epitopes; The electrode layer is provided with a plurality of electrodes, one end of each electrode is connected to the detection pad.

2. The paper-based electrochemiluminescent novel coronavirus antigen detection test strip according to claim 1, characterized in that: The matrix material of the sample pad includes any one of polyester fiber membrane RB45, polyester fiber membrane RB65 or glass fiber membrane SB08; Preferably, the sample pad is also coated with a sample pad treatment solution; Preferably, the sample pad treatment solution is a PBS solution containing Tris-HCL, BSA, sodium caseinate, trehalose, sucrose, HBR, heterophilic antibody blocking agent and NaCl; Preferably, the sample pad treatment solution is a PBS solution containing 0.03-0.06 mol / L Tris-HCL, 0.5%-2% BSA, 0.3%-0.6% caseinate sodium, 0.3%-0.6% trehalose, 1%-3% sucrose, 0.3-0.6 mg / mL HBR, 0.3-0.6 mg / mL heterophilic antibody blocking agent and 0.85%-0.95% NaCl.

3. The paper-based electrochemiluminescent novel coronavirus antigen detection test strip according to claim 1 or 2, characterized in that: The matrix material of the binding pad includes any one of glass fiber, polyester fiber membrane RB45, polyester fiber membrane RB65 or polyester fiber membrane SB08; Preferably, the conjugate pad is also coated with a conjugate pad treatment solution; Preferably, the conjugate pad treatment solution contains a PBS solution containing 0.03-0.06 mol / L Tris-HCL, 0.5%-2% BSA, 0.3%-0.6% caseinate sodium, 0.3%-0.6% trehalose, 1%-3% sucrose, 0.3-0.6 mg / mL HBR, 0.3-0.6 mg / mL heterophilic antibody blocking agent and 0.85%-0.95% NaCl.

4. The paper-based electrochemiluminescent novel coronavirus antigen detection test strip according to any one of claims 1 to 3, characterized in that: The matrix material of the detection pad includes any one of 0.45 μm nitrocellulose membrane, nylon transfer C membrane, Whatman No. 1 filter paper or CN95 membrane; Preferably, the matrix material of the detection pad is CN95 film; Preferably, the length of the detection pad is 1 to 2 cm.

5. The paper-based electrochemiluminescent novel coronavirus antigen detection test strip according to any one of claims 1 to 4, characterized in that: The mass concentration ratio of the SARS-CoV-2 first antibody labeled with ruthenium coupling on the binding pad and the SARS-CoV-2 second antibody on the detection pad is 1:(0.01-0.1).

6. The paper-based electrochemiluminescent novel coronavirus antigen detection test strip according to any one of claims 1 to 5, characterized in that: The electrode layer includes screen-printed electrodes.

7. The method for preparing a paper-based electrochemiluminescent novel coronavirus antigen detection test strip according to any one of claims 1 to 6, characterized in that: The preparation method comprises: Using the sample pad treatment solution to treat the matrix material of the sample pad to obtain the sample pad; Mixing terpyridine ruthenium and a SARS-CoV-2 primary antibody to obtain a ruthenium-conjugated SARS-CoV-2 primary antibody, and coating the ruthenium-conjugated SARS-CoV-2 primary antibody onto a matrix material of a conjugate pad to obtain the conjugate pad; Coating a SARS-CoV-2 secondary antibody onto a matrix material of a detection pad to obtain the detection pad; The sample pad, the binding pad, the detection pad and the water absorption pad are overlapped in sequence on the bottom plate, and one end of the electrode on the electrode layer is connected to the detection pad.

8. Use of the paper-based electrochemiluminescent novel coronavirus antigen detection test strip according to any one of claims 1 to 6 in the preparation of a novel coronavirus detection kit or in the detection of novel coronavirus for non-disease diagnosis purposes.

9. A method for detecting a novel coronavirus for purposes other than disease diagnosis, characterized in that: The method comprises: The sample to be tested is added to the sample pad of the paper-based electrochemiluminescent novel coronavirus antigen detection test strip as described in any one of claims 1 to 6, and after the immune reaction, the detection pad is subjected to electrochemiluminescence detection.

10. The method for detecting a novel coronavirus for non-disease diagnosis purposes according to claim 9, characterized in that: The detection method further comprises the steps of mixing the sample to be tested with an electrochemiluminescent co-reactant, or adding the electrochemiluminescent co-reactant to the detection pad; Preferably, the electrochemiluminescent co-reactant comprises at least one of tri-n-propylamine, N-butyldiethanolamine or an electrochemiluminescent buffer; Preferably, the electrochemiluminescent buffer contains phosphate buffer salt, tripropylamine, a preservative and a detergent; Preferably, the immune response lasts for 3 to 9 minutes.

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