A double-targeting probe-mediated colorimetric-fluorescent immunochromatographic kit, a detection method and application thereof

Abstract

The application discloses a kind of based on double-targeting probe mediated colorimetric / fluorescent immunochromatography kit, detection method and application, for the simultaneous detection of a variety of targets;Test strip includes sample pad, nitrocellulose membrane, absorbent pad, polyvinyl chloride backing substrate, nitrocellulose membrane is sprayed with two kinds of monoclonal antibody for recognizing different targets;The preparation of the detection probe includes the following steps: take nanoparticle solution, add monoclonal antibody solution for recognizing different targets simultaneously, after co-incubation, add bovine serum albumin, and the detection probe of nanoparticle labeled monoclonal antibody for recognizing different targets is obtained;The immunochromatography test strip is strong in specificity, convenient to use, simple to operate and low in cost, and can realize the simultaneous detection of a variety of targets.

Description

Technical Field

[0001] This invention belongs to the field of medical device technology, specifically relating to a colorimetric-fluorescence immunochromatographic reagent kit mediated by dual-target probes, its detection method, and its application. Background Technology

[0002] In recent years, chronic diseases such as cardiovascular disease and cancer have become common and prevalent diseases in humans. Early diagnosis and treatment are crucial for the cure of these diseases. Biomarkers are a class of biomolecules that can assess normal biological processes, pathogenic processes, or therapeutic processes. They include nucleic acids, proteins, and cell surface receptors, and have been proven to have important value in disease screening and prognosis prediction. However, single biomarkers often have poor specificity, and disease diagnosis relying on a single biomarker is prone to a high false positive rate. Therefore, simultaneously assessing the levels of multiple biomarkers can effectively improve diagnostic accuracy and help patients receive early treatment.

[0003] Researchers have developed a series of disease diagnostic methods for the simultaneous detection of multiple biomarkers, including multiplex real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), microarrays, and high-throughput sequencing. Although these methods are highly sensitive and specific, improving diagnostic efficiency, they require expensive equipment, lengthy procedures, and skilled technicians for detection and analysis. These drawbacks make them unsuitable for point-of-care diagnosis. Compared with the above methods, lateral flow immunochromatography (LFIA) is the most promising multiplex detection device due to its low cost, ease of operation, and short processing time. Existing multiplex LFIA can be divided into the following two categories: (1) using the same nanomaterial to label detection antibodies against different biomarkers and setting multiple detection lines, making qualitative judgments based on whether the detection lines show color; (2) using different nanomaterials to label different detection antibodies as probes, setting only a single detection line, and making qualitative judgments based on the different color signals of the detection lines. Although multiplex LFIA is more convenient to use, researchers need to prepare different probes for different analytes, which makes the design and preparation of probes complex and time-consuming.

[0004] Multi-targeted nanoprobes, which simultaneously modify the surface of nanoparticles with multiple target molecules, including small molecules, peptides, and antibodies, have been widely used in disease treatment. However, their application in disease diagnosis is rarely reported. Combining multi-targeted probes with LFIA can effectively improve the complexity of probe preparation in LFIA, reduce detection costs while ensuring detection specificity, and provide a new approach for the application of LFIA in the detection of multiple biomarkers. Summary of the Invention

[0005] To address the problems existing in the aforementioned LFIA technology, this invention discloses a colorimetric / fluorescent immunochromatographic reagent kit, detection method, and application based on dual-target probe-mediated colorimetric / fluorescent immunochromatographic assay. This immunochromatographic test strip is highly specific, easy to use, simple to operate, and low in cost, and can achieve simultaneous detection of multiple target analytes.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] This invention provides an in vitro diagnostic kit comprising a test strip and a detection probe based on dual-target probe mediation:

[0008] The test strip is based on a dual-target probe-mediated design. The test strip includes a sample pad, a nitrocellulose membrane, an absorbent pad, and a polyvinyl chloride backing plate. The nitrocellulose membrane has two detection lines and one control line arranged from left to right. The two detection lines are respectively coated with detection antibody solutions for the first and second target analytes, while the control line is coated with secondary antibody solution. The distance between the two detection lines is 3-5 mm, the distance between the control line and the second detection line is 3-5 mm, and the distance between the control line and the end of the nitrocellulose membrane is 5-10 mm.

[0009] The immunochromatographic test strip consists of a sample pad, a nitrocellulose membrane, an absorbent pad, and a polyvinyl chloride backing plate. The sample pad, nitrocellulose membrane, and absorbent pad are pasted onto the polyvinyl chloride backing plate from left to right, with adjacent pads overlapping at the joints.

[0010] The detection probe is a detection probe of a monoclonal antibody labeled with nanoparticles that recognizes a first target and a second target.

[0011] Further, the preparation of the detection antibody spray solution for the first target analyte includes the following steps: taking a monoclonal antibody of the first target analyte and adding it to a solvent to prepare a detection antibody spray solution for the first target analyte, the concentration of the detection antibody spray solution for the first target analyte being 0.8-1.2 mg / mL; the preparation of the detection antibody spray solution for the second target analyte includes the following steps: taking a monoclonal antibody of the second target analyte and adding it to a solvent to prepare a detection antibody spray solution for the second target analyte, the concentration of the detection antibody spray solution for the second target analyte being 0.8-1.2 mg / mL; the preparation step of the secondary antibody is: taking the secondary antibody and adding it to a phosphate buffer solution to prepare a secondary antibody spray solution, the concentration of the secondary antibody spray solution being 0.8-1.2 mg / mL.

[0012] Furthermore, the solvent is a phosphate buffer solution or Arg / ATT-AuNCs coated with bovine serum albumin.

[0013] Furthermore, the preparation method of the detection probe includes the following steps: take a nanoparticle solution, add a phosphate buffer mixture of a monoclonal antibody that recognizes a first target and a monoclonal antibody that recognizes a second target, incubate for 1-6 hours, and then add bovine serum albumin solution to obtain two detection probes labeled with nanoparticles that recognize different targets.

[0014] Furthermore, the first or second target includes disease markers and pathogenic microorganisms; preferably, the first or second target is alpha-fetoprotein or carcinoembryonic antigen.

[0015] Furthermore, the nanoparticles are one or more of colorimetric nanomaterials and fluorescent nanomaterials. Preferably, the nanoparticles are one or more of polydopamine (PDAN) and time-resolved fluorescent microspheres (TRFNs).

[0016] Furthermore, the concentration of the nanoparticle solution is 1-10 mg / mL, the concentration of the monoclonal antibody is 1-3 mg / mL, the volume ratio of the nanoparticle solution to the phosphate buffer solution is 25:1-50:1, and the volume ratio of the bovine serum albumin solution to the nanoparticle solution is 1:10.

[0017] During the detection process, the two target analytes bind to the detection probes, forming immune complexes. These complexes are then captured by the detection antibodies on the detection lines, causing the nanoparticles to aggregate and develop color on the detection lines. Excess detection probes bind to the secondary antibody on the control line, causing the nanoparticles to aggregate and develop color on the control line. After a period of time, the signals on the detection lines are observed with the naked eye to perform qualitative and semi-quantitative analysis of the target analytes. The RGB values ​​of the detection lines are measured, and the content of the target analytes is quantitatively analyzed based on the RGB value-target analyte concentration standard curve.

[0018] This invention also provides a method for preparing a colorimetric-fluorescent immunochromatographic test strip based on dual-target probes, comprising the following steps:

[0019] S1-1. Cut absorbent paper into absorbent pads 20-30mm wide;

[0020] S1-2. Prepare detection antibodies for the first target and the second target into 0.8-1.2 mg / mL spray solutions, and spray them onto the nitrocellulose membrane at intervals to obtain two detection lines with a distance of 3-5 mm.

[0021] S1-3. Prepare a spraying solution with a concentration of 0.8-1.2 mg / mL for the secondary antibody. Spray the solution onto the nitrocellulose membrane obtained in step S1-2. The distance between the secondary antibody and the second detection line is 3-5 mm, and the distance between the secondary antibody and the end of the nitrocellulose membrane is 5-10 mm, thus obtaining the treated nitrocellulose membrane.

[0022] S1-4. Cut the polyester film into sample pads 20-30 mm wide, and soak them in a phosphate buffer solution (pH 7.4) containing 3-5% sucrose, 0.05-2% Triton-100, and 0.05-0.1 mol / L tris(hydroxymethyl)aminomethane hydrochloride for 2-4 hours. Then dry them at 35-42℃ for 1-3 hours to obtain the treated sample pads.

[0023] S1-5. Adhere the treated nitrocellulose membrane to the polyvinyl chloride backing substrate, and then sequentially adhere the treated sample pad and the absorption pad obtained in step S1-1 from one side of the polyvinyl chloride backing substrate to the other side, with the sample pad and absorption pad overlapping the nitrocellulose membrane by 2-4 mm respectively.

[0024] This invention also provides a detection method based on a dual-target probe-mediated colorimetric-fluorescence immunochromatographic test kit, comprising the following steps:

[0025] S2-1. Preparation of a detection probe for nanoparticle-labeled monoclonal antibodies that recognize two different target analytes;

[0026] S2-2. Take the detection probe prepared in step S2-1, add it to the sample to be tested, mix it evenly, and let it stand to obtain the immune complex formed by the target and the corresponding detection probe.

[0027] S2-3. The immune complex obtained in step S2-2 is dropped onto the sample pad of the test strip. The sample solution moves towards the absorbent pad under the action of capillary force. The target can be specifically recognized by the detection antibody coated on the detection line on the nitrocellulose membrane, and the nanoparticles are aggregated on the detection line. After the control line develops color, the RGB value of the T line is analyzed using Color Picker software and compared with the standard curve to achieve quantitative judgment of the concentration of the target in the test sample.

[0028] Furthermore, the concentration of the detection probe obtained in step S2-1 is 1-5 mg / mL, and the preparation of the detection probes for the two monoclonal antibodies labeled with nanoparticles that recognize different targets in step S2-1 is the same as the preparation method of the detection probe above.

[0029] As a specific embodiment of the present invention:

[0030] When the nanoparticles are time-resolved fluorescent microspheres (TRFNs), the preparation of detection probes for nanoparticle-labeled monoclonal antibodies recognizing two different targets includes the following steps: Take the nanoparticle solution, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / N-hydroxysuccinimide, react at 25-37℃ for 0.5-2h, add a phosphate buffer mixture of monoclonal antibodies recognizing the first target and monoclonal antibodies recognizing the second target, incubate for 4-6h, and then add bovine serum albumin solution to obtain detection probes for nanoparticle-labeled monoclonal antibodies recognizing two different targets.

[0031] The volume ratio of the nanoparticle solution to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / N-hydroxysuccinimide is 20:1.

[0032] When the nanoparticles are polydopamine (PDAN), the preparation of detection probes for nanoparticle-labeled monoclonal antibodies that recognize two different targets includes the following steps: take the nanoparticle solution, add a phosphate buffer mixture of monoclonal antibodies that recognize the first target and monoclonal antibodies that recognize the second target, incubate for 1-2 hours, and then add bovine serum albumin solution to obtain detection probes for nanoparticle-labeled monoclonal antibodies that recognize two different targets.

[0033] Furthermore, in step S2-2, the volume ratio of the detection probe to the sample to be tested is 1:3-1:5, and the settling time is 5-20 min.

[0034] This dual-target probe-mediated colorimetric / fluorescent immunochromatographic test strip enables simultaneous detection of multiple targets using LFIA. The nanoparticles used are PDAN and TRFNs, respectively, whose surfaces can be simultaneously labeled with monoclonal antibodies against different targets. As the concentration of the target changes, the detection line displays different signals, which can be qualitatively and semi-quantitatively determined by naked-eye observation. This greatly improves the overall ease of detection and reduces the detection cost, making it a designer-friendly test strip.

[0035] The beneficial effects of this invention are as follows:

[0036] (1) The colorimetric / fluorescent immunochromatographic reagent kit based on dual-target probes of the present invention is simple to assemble and easy to use. It can quickly and efficiently complete qualitative and quantitative detection, and has high sensitivity and high specificity. The multi-targeting of the probes can detect multiple targets at the same time, realizing the instantaneous detection of broad-spectrum targets, which greatly saves preparation costs and has universal applicability in the field of multi-target detection.

[0037] (2) The colorimetric / fluorescent immunochromatographic detection method based on dual-target probes of this invention, using alpha-fetoprotein and carcinoembryonic antigen as examples of target analytes and PDAN and TRFNs as examples of nanoparticles, can detect target analytes at a minimum concentration of 2 ng / ml. All detections can be completed within 25 minutes, and the test results can be observed with the naked eye, achieving visualized qualitative and semi-quantitative analysis. This invention exhibits high specificity for target analytes, high visualization sensitivity, ease of use, a simple and rapid detection process, and safe and reliable detection results. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the side-flow chromatography test strip of the present invention;

[0039] Figure 2 This is a scanning electron microscope image of the PDAN of the present invention;

[0040] Figure 3 This is a transmission electron microscope image of the TRFNs of the present invention;

[0041] Figure 4 This is the UV-Vis absorption spectrum of PDAN;

[0042] Figure 5 The fluorescence spectrum of TRFNs;

[0043] Figure 6 The results of PDAN-based colorimetric immunochromatographic test strips for detecting alpha-fetoprotein and carcinoembryonic antigen were visualized, with a result of 2 ng / mL.

[0044] Figure 7 The results of detecting alpha-fetoprotein and carcinoembryonic antigen using a TRFN-based fluorescent immunochromatographic test strip were visualized, with a result of 2 ng / mL.

[0045] Figure 8 The results of detecting alpha-fetoprotein and carcinoembryonic antigen using a color-changing fluorescent immunochromatographic test strip based on TRFNs and Arg / ATT-AuNCs were visualized, with a result of 2 ng / mL.

[0046] Figure 9 This is a comparison chart of the detection results of Example 1 and Comparative Example 1, and Example 2 and Comparative Example 2.

[0047] Explanation of the attached diagram labels: 1 is the sample pad, 2 is the nitrocellulose membrane, 3 is the first detection line, 4 is the second detection line, 5 is the quality control line, 6 is the absorption pad, and 7 is the polyvinyl chloride plate. Detailed Implementation

[0048] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0049] Example 1

[0050] (1) Preparation of immunochromatographic test strips:

[0051] LFIA test strips (such as...) Figure 1 The test strip (shown) consists of three parts: a sample pad, a nitrocellulose membrane, and an absorbent pad. Absorbent paper is cut into 22mm wide absorbent pads, which do not require further treatment. A polyester membrane is cut into 25mm wide sample pads and soaked in a phosphate buffer solution (pH 7.4) containing 4% sucrose, 0.08% Triton-100, and 0.08 mol / L tris(hydroxymethyl)aminomethane hydrochloride for 2.5 hours, then dried at 37°C for 2 hours. The sample pad, nitrocellulose membrane, and absorbent pad are then sequentially pasted onto a plate, overlapping each other by 2.5mm, to assemble a side-flow chromatography test strip. The surface of the nitrocellulose membrane has two detection lines and one control line. The detection antibody solutions for the target analytes (antibody solutions for the first and second target analytes) are sprayed onto the nitrocellulose membrane at a rate of 1 μL / cm to form the detection lines, and the secondary antibody solution is sprayed onto the nitrocellulose membrane at a rate of 1 μL / cm to form the control line.

[0052] Preparation of the first target antibody spray solution: Take 0.8-1.2 mg of anti-alpha-fetoprotein monoclonal antibody and add it to 1 mL of phosphate buffer solution to prepare the anti-alpha-fetoprotein monoclonal antibody spray solution;

[0053] Preparation of the detection antibody spray solution for the second target: Take 0.8-1.2 mg of monoclonal antibody against carcinoembryonic antigen and add it to 1 mL of phosphate buffer solution to prepare the monoclonal antibody spray solution against carcinoembryonic antigen;

[0054] Preparation of secondary antibody: Take 0.8-1.2 mg of secondary antibody and add it to 1 mL of phosphate buffer solution to prepare secondary antibody spray solution.

[0055] The distance between the two test lines is 3 mm, the distance between the control line and the second test line is 4 mm, and the distance between the control line and the end of the nitrocellulose membrane is 5 mm.

[0056] (2) Preparation of PDAN:

[0057] 4 mL of anhydrous ethanol, 70 μL of ammonia and 9 mL of pure water were mixed at 800 rpm for 30 min. Then 25 mg of dopamine hydrochloride was added and the mixture was stirred for another 30 h to obtain the PDAN solution.

[0058] (3) PDAN-labeled detection probes:

[0059] Prepare a 1.5 mg / mL anti-alpha-fetoprotein monoclonal antibody solution by dissolving 1.5 mg of anti-alpha-fetoprotein monoclonal antibody in 1 mL of phosphate buffered saline. Prepare a 1 mg / mL anti-carcinoembryonic antigen monoclonal antibody solution by dissolving 1 mg of anti-carcinoembryonic antigen monoclonal antibody in 1 mL of phosphate buffered saline.

[0060] The PDAN-labeled dual-target detection probe was prepared as follows: 0.5 mL of 1 mg / mL PDAN solution was taken, and 20 μL of phosphate buffer solution containing 10 μg of monoclonal antibody against alpha-fetoprotein and 10 μg of monoclonal antibody against carcinoembryonic antigen was added. The mixture was stirred at room temperature for 2 h, and then 10% bovine serum albumin solution was added to block unreacted sites. The volume ratio of bovine serum albumin solution to nanoparticle solution was 1:10. After centrifugation, the precipitate was placed in 0.5 mL of phosphate buffer solution containing 10% sucrose and 1% serum albumin and stored at 4 °C for later use.

[0061] (4) Detection method of immunochromatographic test strip: Take 50 μL of the sample to be tested, add 10 μL of the detection probe prepared in step (3), mix well, let stand for 10 min, and then drop the mixed solution onto the sample pad of the test strip. The sample solution migrates to the nitrocellulose membrane under the action of capillary force. After the control line develops color for a period of time, use Color Picker software to measure the RGB value of the detection line and compare it with the standard curve to perform quantitative analysis of the concentration of the target substance in the sample to be tested.

[0062] Example 2

[0063] (1) Preparation of immunochromatographic test strips:

[0064] The LFIA test strip consists of three parts: a sample pad, a nitrocellulose membrane, and an absorbent pad. Absorbent paper is cut into 22mm wide absorbent pads, which do not require further treatment. A polyester membrane is cut into 25mm wide sample pads and soaked in a phosphate buffer solution (pH 7.4) containing 4% sucrose, 0.08% Triton-100, and 0.08 mol / L tris(hydroxymethyl)aminomethane hydrochloride for 2.5 hours, then dried at 37°C for 2 hours. The sample pad, nitrocellulose membrane, and absorbent pad are then sequentially adhered to a plate, overlapping each other by 2.5mm, to assemble the side-flow chromatography test strip. The nitrocellulose membrane has two detection lines and one control line on its surface. The detection antibody solutions for the target analytes (antibody solutions for the first and second target analytes) are sprayed onto the nitrocellulose membrane at a rate of 1 μL / cm to form the detection lines, and the secondary antibody solution is sprayed onto the nitrocellulose membrane at a rate of 1 μL / cm to form the control line.

[0065] Preparation of the first target antibody spray solution: Take 0.8-1.2 mg of anti-alpha-fetoprotein monoclonal antibody and add it to 1 mL of phosphate buffer solution to prepare the anti-alpha-fetoprotein monoclonal antibody spray solution;

[0066] Preparation of the detection antibody spray solution for the second target: Take 0.8-1.2 mg of monoclonal antibody against carcinoembryonic antigen and add it to 1 mL of phosphate buffer solution to prepare the monoclonal antibody spray solution against carcinoembryonic antigen;

[0067] Preparation of secondary antibody: Take 0.8-1.2 mg of secondary antibody and add it to 1 mL of phosphate buffer solution to prepare secondary antibody spray solution.

[0068] The distance between the two test lines is 3 mm, the distance between the control line and the second test line is 4 mm, and the distance between the control line and the end of the nitrocellulose membrane is 5 mm.

[0069] (2) Preparation of TRFNs:

[0070] 1 mL of styrene monomer, 11 mL of 3% potassium persulfate aqueous solution, and 16 mL of anhydrous ethanol were mixed thoroughly with stirring at 1200 rpm, followed by the addition of 1 mL of europium chelate. The mixture was heated to 75 °C under N2 protection and reacted for 4 h. Then, 200 μL of acrylic acid was added, and the reaction was continued for another 4 h to obtain the TRFNs solution.

[0071] (3) Detection probes labeled with TRFNs:

[0072] Prepare a 1.5 mg / mL anti-alpha-fetoprotein monoclonal antibody solution by dissolving 1.5 mg of anti-alpha-fetoprotein monoclonal antibody in 1 mL of phosphate buffered saline. Prepare a 1 mg / mL anti-carcinoembryonic antigen monoclonal antibody solution by dissolving 1 mg of anti-carcinoembryonic antigen monoclonal antibody in 1 mL of phosphate buffered saline.

[0073] The TRFNs-labeled dual-target detection probe was prepared as follows: 1 mL of 1 mg / mL TRFNs solution was added, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / N-hydroxysuccinimide was reacted for 2 h. Then, 40 μL of phosphate buffer solution containing 20 μg of monoclonal antibody against alpha-fetoprotein and 20 μg of monoclonal antibody against carcinoembryonic antigen was added. The mixture was stirred at room temperature for 4 h. Then, 10% bovine serum albumin solution was added to block unreacted sites. The volume ratio of bovine serum albumin solution to nanoparticle solution was 1:10. After centrifugation, the precipitate was placed in 1 mL of phosphate buffer solution containing 10% sucrose and 1% serum albumin and stored at 4 °C for later use.

[0074] (4) Detection method of immunochromatographic test strip: Take 50 μL of the sample to be tested, add 10 μL of the detection probe prepared in step (3), mix well, let stand for 10 min, and then drop the mixed solution onto the sample pad of the test strip. The sample solution migrates to the nitrocellulose membrane under the action of capillary force. After the control line develops color for a period of time, use Color Picker software to measure the RGB value of the detection line and compare it with the standard curve to perform quantitative analysis of the concentration of the target substance in the sample to be tested.

[0075] Example 3

[0076] (1) Preparation of Arg / ATT-AuNCs:

[0077] Add 40 mg sodium hydroxide, 60 mg 6-aza-2-thiopyrimidine, and 50 mg tetrachloroauric acid to 5 mL of water. After sonicating to dissolve the mixture, stir at room temperature for 1 h and collect the product. Purify the product using a dialysis bag with a molecular weight of 3 kDa to obtain a yellow product solution. Dissolve 7 mg L-arginine in 1 mL of water, then add 9 mL of the obtained yellow product solution. Adjust the pH to 10 with sodium hydroxide. Stir at 37 °C for 24 h, then purify using a dialysis bag with a molecular weight of 3 kDa to obtain the Arg / ATT-AuNCs solution for later use.

[0078] (2) Preparation of Arg / ATT-AuNCs coated with bovine serum albumin:

[0079] Take 200 μL of Arg / ATT-AuNCs solution, add 800 μL of tris(hydroxymethyl)aminomethane hydrochloride buffer, mix, then add 10 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / N-hydroxysuccinimide and react for 30 min. Then add 0.1 mL of 10% bovine serum albumin solution and continue the reaction for 2 h. Remove unreacted bovine serum albumin using an ultrafiltration tube with a molecular weight of 100 kDa to obtain bovine serum albumin-coated Arg / ATT-AuNCs.

[0080] (3) Preparation of immunochromatographic test strips:

[0081] The LFIA test strip consists of three parts: a sample pad, a nitrocellulose membrane, and an absorbent pad. The absorbent paper is cut into 20mm wide absorbent pads, which do not require further treatment. The polyester membrane is cut into 20mm wide sample pads and soaked in a phosphate buffer solution (pH 7.4) containing 3% sucrose, 0.05% Triton-100, and 0.05 mol / L tris(hydroxymethyl)aminomethane hydrochloride for 2 hours, then dried at 37°C for 1 hour. The sample pad, nitrocellulose membrane, and absorbent pad are then sequentially pasted onto a plate, overlapping each other by 2mm, to assemble the side-flow chromatography test strip. Figure 1As shown. The surface of the nitrocellulose membrane is provided with two detection lines and one control line. The detection antibody of the target analyte is mixed with Arg / ATT-AuNCs coated with bovine serum albumin and sprayed onto the nitrocellulose membrane at a rate of 1 μL / cm to form the detection line. The secondary antibody spray solution is sprayed onto the nitrocellulose membrane at a rate of 1 μL / cm to form the control line.

[0082] The preparation of the detection antibody spray solution for the first target analyte includes the following steps: 0.8-1.2 mg of anti-alpha-fetoprotein monoclonal antibody is added to 1 mL of Arg / ATT-AuNCs coated with bovine serum albumin to prepare an anti-alpha-fetoprotein monoclonal antibody spray solution with a concentration of 0.8-1.2 mg / mL.

[0083] The preparation of the detection antibody spray solution for the second target includes the following steps: Take 0.8-1.2 mg of monoclonal antibody against carcinoembryonic antigen and add it to 1 mL of Arg / ATT-AuNCs coated with bovine serum albumin to prepare a monoclonal antibody spray solution against carcinoembryonic antigen with a concentration of 0.8-1.2 mg / mL.

[0084] The preparation steps for the secondary antibody are as follows: Take 0.8-1.2 mg of the secondary antibody and add it to 1 mL of phosphate buffer solution to prepare a secondary antibody spraying solution with a concentration of 0.8-1.2 mg / mL.

[0085] The distance between the two test lines is 3 mm, the distance between the control line and the second test line is 4 mm, and the distance between the control line and the end of the nitrocellulose membrane is 5 mm.

[0086] (4) Preparation of TRFNs:

[0087] 1 mL of styrene monomer, 11 mL of 3% potassium persulfate aqueous solution, and 16 mL of anhydrous ethanol were mixed thoroughly with stirring at 1200 rpm, followed by the addition of 1 mL of europium chelate. The mixture was heated to 75 °C under N2 protection and reacted for 4 h. Then, 200 μL of acrylic acid was added, and the reaction was continued for another 4 h to obtain the TRFNs solution.

[0088] (5) Detection probes labeled with TRFNs:

[0089] Prepare a 1.5 mg / mL anti-alpha-fetoprotein monoclonal antibody solution by dissolving 1.5 mg of anti-alpha-fetoprotein monoclonal antibody in 1 mL of phosphate buffered saline. Prepare a 1 mg / mL anti-carcinoembryonic antigen monoclonal antibody solution by dissolving 1 mg of anti-carcinoembryonic antigen monoclonal antibody in 1 mL of phosphate buffered saline.

[0090] The TRFNs-labeled dual-target detection probe was prepared as follows: 1 mL of 1 mg / mL TRFNs solution was added, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / N-hydroxysuccinimide was reacted for 2 h. Then, 40 μL of phosphate buffer solution containing 20 μg of monoclonal antibody against alpha-fetoprotein and 20 μg of monoclonal antibody against carcinoembryonic antigen was added. The mixture was stirred at room temperature for 4 h. Then, 10% bovine serum albumin solution was added to block unreacted sites. The volume ratio of bovine serum albumin solution to nanoparticle solution was 1:10. After centrifugation, the precipitate was placed in 1 mL of phosphate buffer solution containing 10% sucrose and 1% serum albumin and stored at 4 °C for later use.

[0091] (6) Detection method of immunochromatographic test strip: Take 50 μL of the sample to be tested, add 10 μL of the detection probe prepared in step (3), mix well, let stand for 10 min, and then drop the mixed solution onto the sample pad of the test strip. The sample solution migrates to the nitrocellulose membrane under the action of capillary force. After the control line develops color for a period of time, observe the color development result with naked eye for semi-quantitative analysis; use Color Picker software to determine the RGB value of the detection line, calculate the R / G value, compare with the standard curve, and perform quantitative analysis of the concentration of the target substance in the sample to be tested.

[0092] Comparative Example 1

[0093] Two single-target probes are used for simultaneous detection of multiple targets (PDAN).

[0094] (1) Preparation of immunochromatographic test strips: Same as in Example 1;

[0095] (2) Preparation of PDAN: Same as in Example 1;

[0096] (3) PDAN-labeled detection probes:

[0097] Prepare a 1.5 mg / mL anti-alpha-fetoprotein monoclonal antibody solution by dissolving 1.5 mg of anti-alpha-fetoprotein monoclonal antibody in 1 mL of phosphate buffered saline. Prepare a 1 mg / mL anti-carcinoembryonic antigen monoclonal antibody solution by dissolving 1 mg of anti-carcinoembryonic antigen monoclonal antibody in 1 mL of phosphate buffered saline.

[0098] The PDAN-labeled detection probe for recognizing alpha-fetoprotein was prepared as follows: 0.5 mL of 1 mg / mL PDAN solution was added, along with 10 μL of phosphate buffer solution containing 10 μg of anti-alpha-fetoprotein monoclonal antibody. The mixture was stirred at room temperature for 2 h, and then 10% bovine serum albumin solution was added to block unreacted sites. The volume ratio of bovine serum albumin solution to nanoparticle solution was 1:10. After centrifugation, the precipitate was placed in 0.5 mL of phosphate buffer solution containing 10% sucrose and 1% serum albumin and stored at 4 °C for later use.

[0099] The PDAN-labeled detection probe for recognizing carcinoembryonic antigen was prepared as follows: 0.5 mL of 1 mg / mL PDAN solution was added, along with 10 μL of phosphate buffer solution containing 10 μg of monoclonal antibody against carcinoembryonic antigen. The mixture was stirred at room temperature for 2 h, and then 10% bovine serum albumin solution was added to block unreacted sites. After centrifugation, the precipitate was placed in 0.5 mL of phosphate buffer solution containing 10% sucrose and 1% serum albumin and stored at 4 °C for later use.

[0100] (4) Detection method of immunochromatographic test strip: Take 50 μL of the sample to be tested, add 10 μL of each of the two detection probes prepared in step (3) (total 20 μL), mix well, let stand for 10 min, and then drop the mixed solution onto the sample pad of the test strip. The sample solution migrates to the nitrocellulose membrane under the action of capillary force. After the control line develops color for a period of time, use Color Picker software to measure the RGB value of the detection line and compare it with the standard curve to perform quantitative analysis of the concentration of the target substance in the sample to be tested.

[0101] (5) The detection results of the above test strips were compared with those of Example 1. Under the same detection sensitivity, the amount of PDAN and probe used in the two methods were compared. The results are shown in Table 1.

[0102] Table 1

[0103]

[0104]

[0105] Comparative Example 2

[0106] Two single-target probes are used for the simultaneous detection of multiple targets (TRFNs).

[0107] (1) Preparation of immunochromatographic test strips: Same as in Example 2;

[0108] (2) Preparation of TRFNs: Same as in Example 2;

[0109] (3) Detection probes labeled with TRFNs:

[0110] Prepare a 1.5 mg / mL anti-alpha-fetoprotein monoclonal antibody solution by dissolving 1.5 mg of anti-alpha-fetoprotein monoclonal antibody in 1 mL of phosphate buffered saline. Prepare a 1 mg / mL anti-carcinoembryonic antigen monoclonal antibody solution by dissolving 1 mg of anti-carcinoembryonic antigen monoclonal antibody in 1 mL of phosphate buffered saline.

[0111] The TRFN-labeled detection probe for recognizing alpha-fetoprotein was prepared as follows: 1 mL of 1 mg / mL TRFN solution was added, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / N-hydroxysuccinimide was added and reacted for 2 h. Then, 20 μL of phosphate buffer solution containing 20 μg of anti-alpha-fetoprotein monoclonal antibody was added. The mixture was stirred at room temperature for 4 h. Then, 10% bovine serum albumin solution was added to block unreacted sites. After centrifugation, the precipitate was placed in 1 mL of phosphate buffer solution containing 10% sucrose and 1% serum albumin and stored at 4 °C for later use.

[0112] The TRFN-labeled detection probe for recognizing carcinoembryonic antigen was prepared as follows: 1 mL of 1 mg / mL TRFN solution was added, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / N-hydroxysuccinimide was added and reacted for 2 h. Then, 20 μL of phosphate buffer solution containing 20 μg of monoclonal antibody against carcinoembryonic antigen was added. The mixture was stirred at room temperature for 4 h. Then, 10% bovine serum albumin solution was added to block unreacted sites. The volume ratio of bovine serum albumin solution to nanoparticle solution was 1:10. After centrifugation, the precipitate was placed in 1 mL of phosphate buffer solution containing 10% sucrose and 1% serum albumin and stored at 4 °C for later use.

[0113] (4) Detection method of immunochromatographic test strip: Take 50 μL of the sample to be tested, add 10 μL of each of the two detection probes prepared in step (3) (total 20 μL), mix well, let stand for 10 min, and then drop the mixed solution onto the sample pad of the test strip. The sample solution migrates to the nitrocellulose membrane under the action of capillary force. After the control line develops color for a period of time, use Color Picker software to measure the RGB value of the detection line and compare it with the standard curve to perform quantitative analysis of the concentration of the target substance in the sample to be tested.

[0114] (5) The detection results of the above test strips were compared with those of Example 1. Under the same detection sensitivity, the amount of TRFNs and probes used in the two methods were compared. The results are shown in Table 2.

[0115] Table 2

[0116]

[0117] Example 4

[0118] Take four 50 μL, 500 ng / mL test samples, add the detection probes prepared in Example 1, Example 2, Comparative Example 1, and Comparative Example 2 respectively, mix well, let stand for 10 min, and then drop the mixed solution onto the sample pad of the test strip. The sample solution migrates to the nitrocellulose membrane under the action of capillary force. After the control line develops color for a period of time, observe the detection results with the naked eye. The results are as follows. Figure 9 As shown.

[0119] like Figure 2 The image shown is a scanning electron microscope image of the PDAN prepared in Example 1 of the present invention, which shows that the synthesized PDAN is a spherical particle with slight aggregation and a particle size of about 180 nm.

[0120] like Figure 3 The image shown is a transmission electron microscope (TEM) image of the TRFNs prepared in Example 2 of this invention. It shows that the synthesized TRFNs are uniformly dispersed spherical particles with no obvious aggregation and a particle size of about 200 nm.

[0121] like Figure 4 The image shows the UV-Vis spectrum of the PDAN prepared in Example 1 of this invention, indicating that the synthesized PDAN exhibits broad-spectrum absorption in the 300-800 nm range.

[0122] like Figure 5 The image shows the fluorescence spectrum of the TRFNs prepared in Example 2 of this invention. The maximum excitation wavelength and emission wavelength of the TRFNs are respectively, and they exhibit red fluorescence.

[0123] like Figure 6 The image shows the detection results of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) using a colorimetric immunochromatographic test strip based on dual-target probes in Example 1. 50 μL of the sample to be tested was taken, and 10 μL of the prepared detection probe was added. The mixture was mixed and allowed to stand for 10 min. The solution was then dropped onto the sample pad of the test strip. The sample solution migrated to the nitrocellulose membrane under capillary force. After the control line developed color for a period of time, the colorimetric results were observed with the naked eye for analysis. For samples with AFP concentrations greater than or equal to 20 ng / mL and CEA concentrations greater than or equal to 2 ng / mL, a dark brown band appeared on the test strip after detection. The detection limits for AFP and CEA were 20 ng / mL and 2 ng / mL, respectively. For samples with AFP concentrations less than 20 ng / mL or CEA concentrations less than 2 ng / mL, no band appeared on the test strip after detection.

[0124] like Figure 7The image shows the detection results of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) using a fluorescent immunochromatographic test strip based on dual-target probes in Example 2. 50 μL of the sample to be tested was taken, and 10 μL of the prepared detection probe was added. The mixture was mixed and allowed to stand for 10 min. The solution was then dropped onto the sample pad of the test strip. The sample solution migrated towards the nitrocellulose membrane under capillary force. After the control line developed color for a period of time, the colorimetric results were observed with the naked eye for analysis. For samples with a concentration greater than or equal to 2 ng / mL, the test strip detection line showed red fluorescence after detection, with a detection limit of 2 ng / mL. For samples with a concentration less than 2 ng / mL, the test strip detection line did not show fluorescence after detection.

[0125] like Figure 8 The image shows the detection results of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) using a color-changing fluorescent immunochromatographic test strip based on dual-target probes in Example 3. 50 μL of the sample to be tested was taken, and 10 μL of the prepared detection probe was added. The mixture was mixed and allowed to stand for 10 min. The solution was then dropped onto the sample pad of the test strip. The sample solution migrated to the nitrocellulose membrane under capillary force. After the control line developed color for a period of time, the color development result was observed with the naked eye for analysis. For samples with a concentration greater than or equal to 2 ng / mL, the fluorescence color of the test strip's detection line was significantly different from that of the negative result, with a detection limit of 2 ng / mL. For samples with a concentration less than 2 ng / mL, the test strip's detection line remained green after detection.

[0126] like Figure 9 As shown, the test strip results of Comparative Examples 1 and 2 are compared with those of Examples 1 and 2. At the same detection concentration, the test strip prepared based on the dual-target probe of this invention exhibits slightly higher signal intensity and consumes less nanomaterial and probe volume, demonstrating that this invention can significantly reduce preparation costs.

[0127] It should be noted that the above content merely illustrates the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. For those skilled in the art, various improvements and modifications can be made without departing from the principle of the present invention, and all such improvements and modifications fall within the scope of protection of the claims of the present invention.

Claims

1. An in vitro diagnostic reagent kit, characterized in that, Include: Based on a dual-target probe-mediated test strip, the test strip includes a sample pad, a nitrocellulose membrane, an absorbent pad, and a polyvinyl chloride backing plate. The nitrocellulose membrane has two detection lines and one control line arranged from left to right. The two detection lines are respectively sprayed with the detection antibody solution for the first target and the detection antibody solution for the second target, and the control line is sprayed with secondary antibody solution. The detection probe is a detection probe of a monoclonal antibody labeled with nanoparticles that recognizes a first target and a second target. The preparation of the detection antibody spray solution for the first target analyte includes the following steps: taking a monoclonal antibody of the first target analyte and adding it to a solvent to prepare a detection antibody spray solution for the first target analyte; the preparation of the detection antibody spray solution for the second target analyte includes the following steps: taking a monoclonal antibody of the second target analyte and adding it to a solvent to prepare a detection antibody spray solution for the second target analyte; the preparation step of the secondary antibody is: taking the secondary antibody and adding it to a phosphate buffer solution to prepare a secondary antibody spray solution; The solvent is Arg / ATT-AuNCs coated with bovine serum albumin; The preparation method of the detection probe includes the following steps: take a nanoparticle solution, add a phosphate buffer mixture of monoclonal antibody recognizing the first target and monoclonal antibody recognizing the second target, incubate for 1-6 hours, and then add bovine serum albumin solution to obtain a nanoparticle-labeled detection probe that can simultaneously recognize two different targets.

2. The in vitro diagnostic reagent kit according to claim 1, characterized in that, The nanoparticles are one or more of colorimetric nanomaterials and fluorescent nanomaterials.

3. The in vitro diagnostic reagent kit according to claim 1, characterized in that, The first or second target includes disease markers and pathogenic microorganisms.

4. The in vitro diagnostic reagent kit according to claim 1, characterized in that, The concentration of the nanoparticle solution is 1-10 mg / mL, the concentration of the monoclonal antibody is 1-3 mg / mL, the volume ratio of the nanoparticle solution to the phosphate buffer solution is 25:1-50:1, and the volume ratio of the bovine serum albumin solution to the nanoparticle solution is 1:

10.

5. The in vitro diagnostic reagent kit according to claim 1, characterized in that, The method for preparing the test strip based on dual-target probes includes the following steps: S1-1. Cut absorbent paper into absorbent pads 20-30 mm wide; S1-2. The detection antibodies for the first target and the second target are respectively prepared into detection antibody spraying solutions for the first target and the second target. They are then sprayed onto the nitrocellulose membrane at intervals to obtain two detection lines with a distance of 3-5 mm. S1-3. Prepare the secondary antibody into a spraying solution and spray it onto the nitrocellulose membrane obtained in step S1-2. The distance between the secondary antibody and the second detection line is 3-5 mm, and the distance between the secondary antibody and the end of the nitrocellulose membrane is 5-10 mm, thus obtaining the treated nitrocellulose membrane. S1-4. Cut the polyester film into sample pads 20-30 mm wide, and soak them in a phosphate buffer solution containing 3-5% sucrose, 0.05-2% Triton-100, and 0.05-0.1 mol / L tris(hydroxymethyl)aminomethane hydrochloride for 2-4 h, and then bake them at 35-42℃ for 1-3 h to obtain the treated sample pads. S1-5. Adhere the treated nitrocellulose membrane to the polyvinyl chloride backing substrate, and then sequentially adhere the treated sample pad and the absorption pad obtained in step S1-1 from one side of the polyvinyl chloride backing substrate to the other side. The sample pad and the absorption pad overlap the nitrocellulose membrane by 2-4 mm.

6. A detection method for a reagent kit as described in any one of claims 1-5, characterized in that, Includes the following steps: S2-1. Preparation of a detection probe for nanoparticle-labeled monoclonal antibodies that recognize two different target analytes; S2-2. Take the detection probe prepared in step S2-1, add it to the sample to be tested, mix it evenly, and let it stand to obtain the immune complex formed by the target and the corresponding detection probe. S2-3. Add the immune complex obtained in step S2-2 to the sample pad of the test strip. After the control line develops color, analyze the RGB value of the T line using Color Picker software and compare it with the standard curve to achieve quantitative judgment of the concentration of the target substance in the test sample.

7. The detection method of the reagent kit according to claim 6, characterized in that, In step S2-2, the volume ratio of the detection probe to the sample to be tested is 1:3-1:5, and the settling time is 5-20 min.

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