Colorimetric-fluorescence immunochromatography kit based on double-targeting probe mediation and detection method and application of colorimetric-fluorescence immunochromatography kit

Abstract

The invention discloses a colorimetric / fluorescence immunochromatography kit based on double-targeting probe mediation, a detection method and application. The colorimetric / fluorescence immunochromatography kit is used for simultaneously detecting various target objects. The test strip comprises a sample pad, a nitrocellulose membrane, an absorption pad and a polyvinyl chloride backing bottom plate, wherein the nitrocellulose membrane is sprayed with two monoclonal antibodies for identifying different target objects; the preparation method of the detection probe comprises the following steps: taking a nanoparticle solution, simultaneously adding monoclonal antibody solutions for identifying different target objects, co-incubating, and then adding bovine serum albumin to obtain the nanoparticle-labeled monoclonal antibody detection probe for identifying different target objects. The immunochromatography test strip is high in specificity, convenient to use, simple to operate, low in cost and capable of realizing simultaneous detection of various target objects.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical devices, and specifically relates to a colorimetric-fluorescence immunochromatography kit mediated by dual-targeting probes, and a detection method and application thereof. Background Art

[0002] In recent years, chronic diseases such as cardiovascular disease and cancer have become common and frequently occurring diseases in humans. Early diagnosis and early treatment are crucial to the cure of such diseases. Biomarkers are a class of biological molecules that can evaluate normal biological processes, pathogenic processes, or therapeutic processes, including nucleic acids, proteins, and cell surface receptors. They have been proven to be of great value in disease screening and prognosis prediction. However, single markers usually have poor specificity, and disease diagnosis that relies on a single marker is prone to a high false positive rate. Therefore, simultaneous evaluation of multiple marker levels can effectively improve diagnostic accuracy and help patients receive early treatment.

[0003] At present, researchers have developed a series of disease diagnosis methods for the simultaneous detection of multiple markers, including multiplex real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), microarray and high-throughput sequencing. Although these methods are highly sensitive and specific, and can improve diagnostic efficiency, they require expensive equipment, lengthy operating procedures and skilled technicians for detection and analysis. These disadvantages make them unsuitable for immediate diagnosis of diseases. Compared with the above methods, lateral flow immunochromatography (LFIA) is currently the most promising multiplex detection device due to its low price, simple operation and short time consumption. Existing multiplex LFIA can be divided into the following two categories: (1) using the same nanomaterial to label detection antibodies for different markers, and setting up multiple detection lines, and making qualitative judgments based on whether the detection lines are colored; (2) using different nanomaterials to label different detection antibodies as probes, only setting up 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 complicated and time-consuming.

[0004] Multi-targeted nanoprobes, which are nanoparticles with multiple targeted molecules modified simultaneously, including small molecules, peptides and antibodies, have been widely used in the field of disease treatment. However, the use of such probes in disease diagnosis has rarely been reported. If multi-targeted probes are combined with LFIA, the complex problem of probe preparation in LFIA can be effectively improved, while ensuring detection specificity and reducing detection costs, providing new ideas for the application of LFIA in multi-marker detection. Summary of the invention

[0005] In order to solve the problems existing in the above-mentioned LFIA technology, the present invention discloses a colorimetric / fluorescent immunochromatography kit, a detection method and an application based on dual-targeted probe mediation. The immunochromatographic test strip has strong specificity, is easy to use, simple to operate and low in cost, and can realize the simultaneous detection of multiple targets.

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

[0007] The present invention provides an in vitro diagnostic kit, comprising a test paper and a detection probe mediated by a dual-targeting probe:

[0008] A test strip based on dual-targeted probe mediation, the test strip includes a sample pad, a nitrocellulose membrane, an absorption pad, and a polyvinyl chloride backing substrate. The nitrocellulose membrane is provided with two detection lines and a quality control line from left to right. The two detection lines are respectively sprayed with a detection antibody spray liquid for a first target and a detection antibody spray liquid for a second target, and the quality control line is sprayed with a secondary antibody spray liquid; the distance between the two detection lines is 3-5 mm, the distance between the quality control line and the second detection line is 3-5 mm, and the distance between the quality control line and the end of the nitrocellulose membrane is 5-10 mm.

[0009] Among them: the sample pad, nitrocellulose membrane, absorption pad, and polyvinyl chloride backing substrate together constitute the immunochromatographic test strip, and the sample pad, nitrocellulose membrane, and absorption pad are sequentially pasted on the polyvinyl chloride backing substrate from left to right, and adjacent pads overlap at the connection.

[0010] Detection probe, the detection probe is a detection probe of a monoclonal antibody that recognizes the first target and the second target and is labeled with a nanoparticle

[0011] Furthermore, the preparation of the detection antibody spray liquid of the first target comprises the following steps: taking the monoclonal antibody of the first target and adding it to a solvent to prepare the detection antibody spray liquid of the first target, and the concentration of the detection antibody spray liquid of the first target is 0.8-1.2 mg / mL; the preparation of the detection antibody spray liquid of the second target comprises the following steps: taking the monoclonal antibody of the second target and adding it to a solvent to prepare the detection antibody spray liquid of the second target, and the concentration of the detection antibody spray liquid of the second target is 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 to prepare the secondary antibody spray liquid, and the concentration of the secondary antibody spray liquid is 0.8-1.2 mg / mL.

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

[0013] Furthermore, the preparation method of the detection probe includes the following steps: taking a nanoparticle solution, adding a phosphate buffered saline mixture of a monoclonal antibody that recognizes a first target and a monoclonal antibody that recognizes a second target, incubating for 1-6 hours, and then adding a bovine serum albumin solution to obtain a nanoparticle-labeled detection probe of two monoclonal antibodies that recognize different targets.

[0014] Furthermore, the first target or the second target includes a disease marker or a pathogenic microorganism; preferably, the first target or the 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 buffered saline mixture 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 targets bind to the detection probes to form immune complexes, which are then captured by the detection antibodies on the detection line, thereby aggregating the nanoparticles on the detection line for color development. The excess detection probes bind to the secondary antibodies on the quality control line, aggregating the nanoparticles on the quality control line for color development. After a period of time, the signal on the detection line is observed with the naked eye to perform qualitative and semi-quantitative analysis of the target; the RGB value of the detection line is measured, and the target content is quantitatively analyzed based on the RGB value-target concentration standard curve.

[0018] The present invention also provides a method for preparing a colorimetric-fluorescent immunochromatographic test strip mediated by a dual-targeting probe, comprising the following steps:

[0019] S1-1, cut the absorbent paper into absorbent pads with a width of 20-30 mm;

[0020] S1-2, prepare the detection antibody of the first target and the detection antibody of the second target into 0.8-1.2 mg / mL spray solution respectively, spray them on the nitrocellulose membrane at intervals to obtain two detection lines with a distance of 3-5 mm;

[0021] S1-3, prepare the secondary antibody into a spray solution with a concentration of 0.8-1.2 mg / mL, and spray it on the nitrocellulose membrane obtained in step S1-2, with a distance of 3-5 mm from the second detection line and a distance of 5-10 mm from the end of the nitrocellulose membrane to obtain a treated nitrocellulose membrane;

[0022] S1-4, cutting the polyester film into a sample pad with a width of 20-30 mm, and soaking it in a phosphate buffered saline solution (pH 7.4) containing 3-5% by mass of sucrose, 0.05-2% by mass of Triton-100, and 0.05-0.1 mol / L of tris(hydroxymethyl)aminomethane hydrochloride for 2-4 hours, and then drying it at 35-42° C. for 1-3 hours to obtain a treated sample pad;

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

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

[0025] S2-1, preparing nanoparticle-labeled detection probes for monoclonal antibodies that recognize two different targets;

[0026] S2-2, taking the detection probe prepared in step S2-1, adding it to the sample to be tested, mixing evenly, and letting it stand to obtain an immune complex formed by the target and the corresponding detection probe;

[0027] S2-3. Add the immune complex obtained in step S2-2 to the sample pad of the test strip. The sample liquid moves to the absorption pad under the action of capillary force. The target can perform specific antigen-antibody recognition with the detection antibody coated on the detection line on the nitrocellulose membrane, and the nanoparticles are aggregated on the detection line. After the quality control line is colored, the RGB value of the T line is analyzed by Color Picker software and compared with the standard curve to achieve quantitative judgment of the concentration of the target in the sample to be tested.

[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 of two monoclonal antibodies that recognize different targets and are labeled with nanoparticles in step S2-1 is the same as the preparation method of the detection probes above.

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

[0030] When the nanoparticles are time-resolved fluorescent microspheres (TRFNs), the preparation of the detection probe of the monoclonal antibodies for recognizing two different targets labeled by the nanoparticles includes the following steps: taking a nanoparticle solution, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / N-hydroxysuccinimide to react at 25-37° C. for 0.5-2 hours, adding a phosphate buffered saline mixture of the monoclonal antibody for recognizing the first target and the monoclonal antibody for recognizing the second target, incubating for 4-6 hours, and then adding a bovine serum albumin solution to obtain the detection probe of the two monoclonal antibodies for recognizing different targets labeled by the nanoparticles.

[0031] Wherein, 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 the detection probes of the monoclonal antibodies that recognize two different targets and are labeled with the nanoparticles includes the following steps: taking a nanoparticle solution, adding a phosphate buffered saline mixture of the monoclonal antibody that recognizes the first target and the monoclonal antibody that recognizes the second target, incubating for 1-2 hours, and then adding a bovine serum albumin solution to obtain the detection probes of the two monoclonal antibodies that recognize different targets and are labeled with the nanoparticles.

[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 standing time is 5-20 min.

[0034] This dual-targeted probe-mediated colorimetric / fluorescent immunochromatographic test strip is used to achieve simultaneous LFIA detection of multiple targets. The nanoparticles used are PDAN and TRFNs, respectively. Their surfaces can be simultaneously labeled with monoclonal antibodies targeting different targets. As the concentration of the target changes, the detection line displays different signals, which can be qualitatively and semi-quantitatively judged by naked eye observation, greatly improving the simplicity of the overall detection and reducing the detection cost. It is a designer-friendly test strip.

[0035] The beneficial effects of the present invention are:

[0036] (1) The dual-targeted probe-mediated colorimetric / fluorescent immunochromatography kit of the present invention is simple to assemble, easy to use, can quickly and efficiently complete qualitative and quantitative detection, and has high sensitivity and high specificity; the multi-targeting of the probe can detect multiple targets at the same time, realizing the instant detection of a wide spectrum of targets, greatly saving the preparation cost, and having universal applicability in the field of multi-target detection;

[0037] (2) The dual-targeted probe-mediated colorimetric / fluorescent immunochromatographic detection method of the present invention takes alpha-fetoprotein and carcinoembryonic antigen as examples of the target, and takes PDAN and TRFNs as examples of the nanoparticles, respectively. The lowest detectable concentration of the target is 2 ng / ml, and all detections can be completed within 25 minutes. The test results can be observed with the naked eye, and visual qualitative and semi-quantitative analysis can be achieved. The present invention has strong specificity for the target, high visual sensitivity, is easy to use, has a simple and fast detection process, and safe and reliable detection results. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0042] Figure 5 is the fluorescence spectrum of TRFNs;

[0043] Figure 6 The visual test results of alpha-fetoprotein and carcinoembryonic antigen were 2 ng / mL for the colorimetric immunochromatographic test strips based on PDAN;

[0044] Figure 7 The results of the fluorescent immunochromatographic test strips based on TRFNs for the detection of alpha-fetoprotein and carcinoembryonic antigen were visualized at 2 ng / mL;

[0045] Figure 8 The color-shifting fluorescent immunochromatographic test strips based on TRFNs and Arg / ATT-AuNCs were used to detect alpha-fetoprotein and carcinoembryonic antigen, and the visualized detection result was 2 ng / mL.

[0046] Fig. 9 It is a comparison chart of the test results of Example 1 and Comparative Example 1, and Example 2 and Comparative Example 2.

[0047] Explanation of the accompanying drawings: 1 is a sample pad, 2 is a nitrocellulose membrane, 3 is a first detection line, 4 is a second detection line, 5 is a quality control line, 6 is an absorption pad, and 7 is a polyvinyl chloride plate. DETAILED DESCRIPTION

[0048] The present invention will be further explained below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0049] Example 1

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

[0051] LFIA test strips (such as Figure 1 As shown) includes three parts: sample pad, nitrocellulose membrane and absorption pad. Cut the absorbent paper into 22mm wide absorption pads, and the absorption pads do not need further treatment. Cut the polyester film into 25mm wide sample pads, and soak them in phosphate buffered saline (pH 7.4) of 4% sucrose, 0.08% Triton-100, and 0.08mol / L tris(hydroxymethyl)aminomethane hydrochloride for 2.5h, and then dry them at 37°C for 2h; the sample pad, nitrocellulose membrane and absorption pad are sequentially pasted on the flat plate and overlapped with each other by 2.5mm to assemble into lateral flow chromatography test paper. The surface of the nitrocellulose membrane is provided with two detection lines and a quality control line, and the detection antibody spraying liquid of the target to be detected (the detection antibody spraying of the first target, the detection antibody spraying liquid of the second target) is sprayed onto the nitrocellulose membrane at a rate of 1μL / cm to form a detection line, and the secondary antibody spraying liquid is sprayed onto the nitrocellulose membrane at a rate of 1μL / cm to form a quality control line.

[0052] Preparation of the first target detection antibody spray solution: 0.8-1.2 mg of anti-alpha-fetoprotein monoclonal antibody is added to 1 mL of phosphate buffered saline to prepare an anti-alpha-fetoprotein monoclonal antibody spray solution;

[0053] Preparation of the second target detection antibody spray solution: 0.8-1.2 mg of anti-CEA monoclonal antibody is added into 1 mL of phosphate buffered saline to prepare anti-CEA monoclonal antibody spray solution;

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

[0055] The distance between the two test lines is 3 mm, the distance between the quality control line and the second test line is 4 mm, and the distance between the quality 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 water and 9 mL of pure water were mixed at 800 rpm for 30 min, and then 25 mg of dopamine hydrochloride was added and stirred for 30 h to obtain a PDAN solution.

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

[0059] Take 1.5 mg of anti-alpha-fetoprotein monoclonal antibody and add it to 1 mL of phosphate buffered saline to prepare a 1.5 mg / mL anti-alpha-fetoprotein monoclonal antibody solution. Take 1 mg of anti-carcinoembryonic antigen monoclonal antibody and add it to 1 mL of phosphate buffered saline to prepare a 1 mg / mL anti-carcinoembryonic antigen monoclonal antibody solution for use.

[0060] The PDAN-labeled dual-targeted detection probe was prepared according to the following method: 0.5 mL of 1 mg / mL PDAN solution was taken, and 20 μL of phosphate buffered saline solution containing 10 μg of anti-alpha-fetoprotein monoclonal antibody and 10 μg of anti-carcinoembryonic antigen monoclonal antibody was added, the mixed solution was stirred at room temperature for 2 h, and then 10% bovine serum albumin solution was added to block the unreacted sites, the volume ratio of bovine serum albumin solution to nanoparticle solution was 1:10, centrifuged, and the obtained precipitate was added with 0.5 mL of phosphate buffered saline 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 quality control line is colored for a period of time, the RGB value of the detection line is determined by Color Picker software and compared 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] LFIA test strips include three parts: sample pad, nitrocellulose membrane and absorption pad. The absorbent paper is cut into a 22mm wide absorption pad, and the absorption pad does not need further treatment. The polyester film is cut into a 25mm wide sample pad, and soaked in a phosphate buffered saline solution (pH 7.4) of 4% sucrose, 0.08% Triton-100, and 0.08mol / L tris(hydroxymethyl)aminomethane hydrochloride for 2.5h, and then dried at 37°C for 2h; the sample pad, nitrocellulose membrane and absorption pad are sequentially pasted on the flat plate and overlapped with each other by 2.5mm to assemble into a lateral flow chromatography test paper. The surface of the nitrocellulose membrane is provided with two detection lines and a quality control line, and the detection antibody spraying liquid of the target to be detected (the detection antibody spraying of the first target, the detection antibody spraying liquid of the second target) is sprayed onto the nitrocellulose membrane at a rate of 1μL / cm to form a detection line, and the secondary antibody spraying liquid is sprayed onto the nitrocellulose membrane at a rate of 1μL / cm to form a quality control line.

[0065] Preparation of the first target detection antibody spray solution: 0.8-1.2 mg of anti-alpha-fetoprotein monoclonal antibody is added to 1 mL of phosphate buffered saline to prepare an anti-alpha-fetoprotein monoclonal antibody spray solution;

[0066] Preparation of the second target detection antibody spray solution: 0.8-1.2 mg of anti-CEA monoclonal antibody is added into 1 mL of phosphate buffered saline to prepare anti-CEA monoclonal antibody spray solution;

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

[0068] The distance between the two test lines is 3 mm, the distance between the quality control line and the second test line is 4 mm, and the distance between the quality 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 at 1200 rpm, and then 1 mL of europium chelate was added. Under N2 protection, the mixture was heated to 75°C and reacted for 4 hours, and then 200 μL of acrylic acid was added and the reaction was continued for 4 hours to obtain a TRFNs solution.

[0071] (3) TRFNs labeled detection probe:

[0072] Take 1.5 mg of anti-alpha-fetoprotein monoclonal antibody and add it to 1 mL of phosphate buffered saline to prepare a 1.5 mg / mL anti-alpha-fetoprotein monoclonal antibody solution. Take 1 mg of anti-carcinoembryonic antigen monoclonal antibody and add it to 1 mL of phosphate buffered saline to prepare a 1 mg / mL anti-carcinoembryonic antigen monoclonal antibody solution for use.

[0073] The dual-target detection probe labeled with TRFNs was prepared according to the following method: 1 mL of 1 mg / mL TRFNs solution was added to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / N-hydroxysuccinimide for reaction for 2 h, and then 40 μL of phosphate buffered saline solution containing 20 μg of anti-alpha-fetoprotein monoclonal antibody and 20 μg of anti-carcinoembryonic antigen monoclonal antibody was added, the mixed solution was stirred at room temperature for 4 h, and then 10% bovine serum albumin solution was added to block the unreacted sites, the volume ratio of bovine serum albumin solution to nanoparticle solution was 1:10, centrifuged, and the obtained precipitate was added with 1 mL of phosphate buffered saline 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 quality control line is colored for a period of time, the RGB value of the detection line is determined by Color Picker software and compared 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] 40 mg of sodium hydroxide, 60 mg of 6-aza-2-thiopyrimidine and 50 mg of tetrachloroauric acid were added to 5 mL of water, and the mixture was dissolved by ultrasound. After stirring at room temperature for 1 h, the product was collected and purified with a dialysis bag with a molecular weight of 3 kDa to obtain a yellow product solution. 7 mg of L-arginine was dissolved in 1 mL of water, and 9 mL of the obtained yellow product solution was added. The pH was adjusted to 10 with sodium hydroxide, and after stirring at 37 ° C for 24 h, it was purified with a dialysis bag with a molecular weight of 3 kDa to obtain Arg / ATT-AuNCs solution for use.

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

[0079] Take 200 μL Arg / ATT-AuNCs solution, add 800 μL tris(hydroxymethylaminomethane) hydrochloric acid buffer, mix, add 10 μL 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. Use an ultrafiltration tube with a molecular weight of 100 kDa to remove the unreacted bovine serum albumin 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 absorption pad. The absorbent paper is cut into a 20 mm wide absorption pad, which does not require further treatment. The polyester film is cut into a 20 mm wide sample pad and soaked in a phosphate buffered saline solution (pH 7.4) of 3% sucrose, 0.05% Triton-100, and 0.05 mol / L tris(hydroxymethyl)aminomethane hydrochloride) for 2 hours, and then dried at 37°C for 1 hour; the sample pad, nitrocellulose membrane, and absorption pad are sequentially pasted on a flat plate and overlapped by 2 mm to assemble into a lateral flow chromatography test strip, such as Figure 1As shown. Two detection lines and a quality control line are set on the surface of the nitrocellulose membrane. The detection antibody of the target object to be detected is mixed with the Arg / ATT-AuNCs coated with bovine serum albumin and then sprayed onto the nitrocellulose membrane at a rate of 1 μL / cm to form the detection line, and the secondary antibody spray liquid is sprayed onto the nitrocellulose membrane at a rate of 1 μL / cm to form the quality control line.

[0082] The preparation of the first target detection antibody spray solution includes the following steps: taking 0.8-1.2 mg of anti-alpha-fetoprotein monoclonal antibody and adding 1 mL of bovine serum albumin-coated Arg / ATT-AuNCs 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 of the second target includes the following steps: taking 0.8-1.2 mg of monoclonal antibody against carcinoembryonic antigen and adding 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 of the secondary antibody are as follows: 0.8-1.2 mg of the secondary antibody is added to 1 mL of phosphate buffered saline to prepare a secondary antibody spray 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 quality control line and the second test line is 4 mm, and the distance between the quality 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 at 1200 rpm, and then 1 mL of europium chelate was added. Under N2 protection, the mixture was heated to 75°C and reacted for 4 hours, and then 200 μL of acrylic acid was added and the reaction was continued for 4 hours to obtain a TRFNs solution.

[0088] (5) TRFNs labeled detection probe:

[0089] Take 1.5 mg of anti-alpha-fetoprotein monoclonal antibody and add it to 1 mL of phosphate buffered saline to prepare a 1.5 mg / mL anti-alpha-fetoprotein monoclonal antibody solution. Take 1 mg of anti-carcinoembryonic antigen monoclonal antibody and add it to 1 mL of phosphate buffered saline to prepare a 1 mg / mL anti-carcinoembryonic antigen monoclonal antibody solution for use.

[0090] The dual-target detection probe labeled with TRFNs was prepared according to the following method: 1 mL of 1 mg / mL TRFNs solution was added to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / N-hydroxysuccinimide for reaction for 2 h, and then 40 μL of phosphate buffered saline solution containing 20 μg of anti-alpha-fetoprotein monoclonal antibody and 20 μg of anti-carcinoembryonic antigen monoclonal antibody was added, the mixed solution was stirred at room temperature for 4 h, and then 10% bovine serum albumin solution was added to block the unreacted sites, the volume ratio of bovine serum albumin solution to nanoparticle solution was 1:10, centrifuged, and the obtained precipitate was added with 1 mL of phosphate buffered saline 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 quality control line is colored for a period of time, the color development result is observed with the naked eye for semi-quantitative analysis; the RGB value of the detection line is determined using Color Picker software, the R / G value is calculated, and compared with the standard curve to perform quantitative analysis of the target concentration in the sample to be tested.

[0092] Comparative Example 1

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

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

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

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

[0097] Take 1.5 mg of anti-alpha-fetoprotein monoclonal antibody and add it to 1 mL of phosphate buffered saline to prepare a 1.5 mg / mL anti-alpha-fetoprotein monoclonal antibody solution. Take 1 mg of anti-carcinoembryonic antigen monoclonal antibody and add it to 1 mL of phosphate buffered saline to prepare a 1 mg / mL anti-carcinoembryonic antigen monoclonal antibody solution for use.

[0098] The PDAN-labeled detection probe for recognizing alpha-fetoprotein was prepared according to the following method: 0.5 mL of 1 mg / mL PDAN solution was taken, 10 μL of phosphate buffered saline solution containing 10 μg of anti-alpha-fetoprotein monoclonal antibody was added, the mixed solution was stirred at room temperature for 2 h, and then 10% bovine serum albumin solution was added to block the unreacted sites, the volume ratio of bovine serum albumin solution to nanoparticle solution was 1:10, centrifuged, and the obtained precipitate was added with 0.5 mL of phosphate buffered saline 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 according to the following method: 0.5 mL of 1 mg / mL PDAN solution was taken, 10 μL of phosphate buffered saline solution containing 10 μg of monoclonal antibody against carcinoembryonic antigen was added, the mixed solution was stirred at room temperature for 2 hours, 10% bovine serum albumin solution was added to block unreacted sites, centrifuged, and the obtained precipitate was added with 0.5 mL of phosphate buffered saline 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) (a total of 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 quality control line has been colored for a period of time, the RGB value of the detection line is determined using the Color Picker software and compared with the standard curve to perform quantitative analysis of the target concentration in the sample to be tested.

[0101] (5) The test results of the above test strips were compared with those of Example 1. Under the same detection sensitivity, the amounts 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 for simultaneous detection of multiple targets (TRFNs)

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

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

[0109] (3) TRFNs labeled detection probe:

[0110] Take 1.5 mg of anti-alpha-fetoprotein monoclonal antibody and add it to 1 mL of phosphate buffered saline to prepare a 1.5 mg / mL anti-alpha-fetoprotein monoclonal antibody solution. Take 1 mg of anti-carcinoembryonic antigen monoclonal antibody and add it to 1 mL of phosphate buffered saline to prepare a 1 mg / mL anti-carcinoembryonic antigen monoclonal antibody solution for use.

[0111] The detection probe for recognizing alpha-fetoprotein labeled by TRFNs was prepared according to the following method: 1 mL of 1 mg / mL TRFNs solution was added to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / N-hydroxysuccinimide for reaction for 2 h, and then 20 μL of phosphate buffered saline solution containing 20 μg of anti-alpha-fetoprotein monoclonal antibody was added, the mixed solution was stirred at room temperature for 4 h, and then 10% bovine serum albumin solution was added to block the unreacted sites, and the mixture was centrifuged, and the obtained precipitate was added to 1 mL of phosphate buffered saline solution containing 10% sucrose and 1% serum albumin, and stored at 4°C for later use;

[0112] The detection probe for recognizing carcinoembryonic antigen labeled by TRFNs was prepared according to the following method: 1 mL of 1 mg / mL TRFNs solution was added to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / N-hydroxysuccinimide for reaction for 2 h, and then 20 μL of phosphate buffered saline solution containing 20 μg of monoclonal antibody against carcinoembryonic antigen was added, the mixed solution was stirred at room temperature for 4 h, and then 10% bovine serum albumin solution was added to block the unreacted sites, the volume ratio of bovine serum albumin solution to nanoparticle solution was 1:10, centrifuged, and the obtained precipitate was added to 1 mL of phosphate buffered saline 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) (a total of 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 quality control line has been colored for a period of time, the RGB value of the detection line is determined using the Color Picker software and compared with the standard curve to perform quantitative analysis of the target concentration in the sample to be tested.

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

[0115] Table 2

[0116]

[0117] Example 4

[0118] Take 4 portions of 50 μL, 500 ng / mL of the sample to be tested, add the detection probes prepared in Example 1, Example 2, Comparative Example 1, and Comparative Example 2, respectively, mix well, let stand for 10 minutes, 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 quality control line develops color for a period of time, observe the test results with the naked eye. The results are as follows: Fig. 9 shown.

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

[0120] like Figure 3 , which is a transmission electron micrograph of TRFNs prepared in Example 2 of the present invention, indicating that the synthesized TRFNs are uniformly dispersed spherical particles with no obvious aggregation phenomenon and a particle size of about 200 nm.

[0121] like Figure 4 , which is the UV-visible spectrum of the PDAN prepared in Example 1 of the present invention, indicating that the synthesized PDAN exhibits broad-spectrum absorption in the range of 300-800 nm.

[0122] like Figure 5 As shown, this is the fluorescence spectrum of TRFNs prepared in Example 2 of the present invention. The maximum excitation wavelength and emission wavelength of TRFNs are respectively, and it exhibits red fluorescence.

[0123] like Figure 6 As shown, the test results of alpha-fetoprotein and carcinoembryonic antigen detected by colorimetric immunochromatographic test strips based on dual-targeted probes in Example 1. Take 50 μL of the sample to be tested, add 10 μL of the prepared detection probe, mix well, let stand for 10 minutes, and then drop the mixed solution onto the sample pad of the test strip. The sample liquid migrates to the nitrocellulose membrane under the action of capillary force. After the quality control line is colored for a period of time, the color development results are observed with the naked eye for analysis. For samples with an alpha-fetoprotein concentration greater than or equal to 20 ng / mL and a carcinoembryonic antigen concentration greater than or equal to 2 ng / mL, the test strip detection line shows a black-brown band after detection, and the detection limits of alpha-fetoprotein and carcinoembryonic antigen are 20 ng / mL and 2 ng / mL, respectively; for samples with an alpha-fetoprotein concentration less than 20 ng / mL or a carcinoembryonic antigen concentration less than 2 ng / mL, the test strip detection line does not show a band after detection.

[0124] like Figure 7Shown is the test result of detecting alpha-fetoprotein and carcinoembryonic antigen by fluorescent immunochromatographic test strip based on dual-targeted probes in Example 2. Take 50 μL of the sample to be tested, add 10 μL of the prepared detection probe, mix well, let stand for 10 minutes, and then drop the mixed solution onto the sample pad of the test strip. The sample liquid migrates to the nitrocellulose membrane under the action of capillary force. After the quality control line is colored for a period of time, the color development result is 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 shows red fluorescence after detection, and the detection limit is 2 ng / mL; for samples with a concentration less than 2 ng / mL, the test strip detection line does not show fluorescence after detection.

[0125] like Figure 8 As shown, the test results of Example 3 for detecting alpha-fetoprotein and carcinoembryonic antigen using a hue-changing fluorescent immunochromatographic test strip based on dual-targeted probes. Take 50 μL of the sample to be tested, add 10 μL of the prepared detection probe, mix well, let stand for 10 minutes, and then drop the mixed solution onto the sample pad of the test strip. The sample liquid migrates to the nitrocellulose membrane under the action of capillary force. After the quality control line is colored for a period of time, the color development result is observed with the naked eye for analysis. For samples with a concentration greater than or equal to 2 ng / mL, the fluorescent color of the test strip detection line after detection is significantly different from the negative, and the detection limit is 2 ng / mL; for samples with a concentration less than 2 ng / mL, the test strip detection line still shows green fluorescence after detection.

[0126] like Fig. 9 As shown, the test strip results of comparative examples 1 and 2 are compared with the test strip results of examples 1 and 2. At the same detection concentration, the test strip prepared based on the dual-targeting probe of the present invention has a slightly higher signal intensity, and consumes less nanomaterials and probe volume, which proves that the invention can significantly save preparation costs.

[0127] It should be noted that the above content only illustrates the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications all fall within the protection scope of the claims of the present invention.

Claims

1. An in vitro diagnostic kit, characterized in that: Include: A test strip mediated by a dual-targeted probe, the test strip comprising a sample pad, a nitrocellulose membrane, an absorption pad, and a polyvinyl chloride backing substrate, the nitrocellulose membrane being provided with two detection lines and a quality control line from left to right, the two detection lines being sprayed with a detection antibody spray liquid of a first target and a detection antibody spray liquid of a second target, respectively, and the quality control line being sprayed with a secondary antibody spray liquid; The detection probe is a detection probe of a monoclonal antibody that is labeled with nanoparticles and recognizes the first target and the second target.

2. An in vitro diagnostic kit according to claim 1, characterized in that: The preparation of the detection antibody spray liquid of the first target object includes the following steps: taking the monoclonal antibody of the first target object and adding it to a solvent to prepare the detection antibody spray liquid of the first target object; the preparation of the detection antibody spray liquid of the second target object includes the following steps: taking the monoclonal antibody of the second target object and adding it to a solvent to prepare the detection antibody spray liquid of the second target object; the preparation step of the secondary antibody is: taking the secondary antibody and adding it to a phosphate buffer to prepare the secondary antibody spray liquid.

3. An in vitro diagnostic kit according to claim 2, characterized in that: The solvent is phosphate buffered saline solution or bovine serum albumin-coated Arg / ATT-AuNCs.

4. An in vitro diagnostic kit according to claim 1, characterized in that: The preparation method of the detection probe comprises the following steps: taking a nanoparticle solution, adding a phosphate buffered saline mixture of a monoclonal antibody that recognizes a first target and a monoclonal antibody that recognizes a second target, incubating for 1-6 hours, and then adding a bovine serum albumin solution to obtain a nanoparticle-labeled detection probe that can simultaneously recognize two different targets.

5. An in vitro diagnostic kit according to claim 4, characterized in that: The nanoparticles are one or more of colorimetric nanomaterials and fluorescent nanomaterials.

6. An in vitro diagnostic kit according to claim 4, characterized in that: The first target or the second target includes disease markers and pathogenic microorganisms.

7. An in vitro diagnostic kit according to claim 4, 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 buffered saline mixture is 25:1-50:1; and the volume ratio of the bovine serum albumin solution to the nanoparticle solution is 1:

10.

8. An in vitro diagnostic kit according to claim 1, characterized in that: The method for preparing the test strip based on dual-targeting probe mediation comprises the following steps: S1-1, cut the absorbent paper into absorbent pads with a width of 20-30 mm; S1-2, prepare the detection antibody of the first target and the detection antibody of the second target into the detection antibody spray liquid of the first target and the detection antibody spray liquid of the second target respectively, spray them on 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 spray solution, and spray it on the nitrocellulose membrane obtained in step S1-2, with a distance of 3-5 mm from the second detection line and a distance of 5-10 mm from the end of the nitrocellulose membrane, to obtain a treated nitrocellulose membrane; S1-4, cutting the polyester film into a sample pad with a width of 20-30 mm, and soaking it in a phosphate buffered saline solution containing 3-5% sucrose, 0.05-2% Triton-100, and 0.05-0.1 mol / L tris(hydroxymethyl)aminomethane hydrochloride for 2-4 hours, and then drying it at 35-42° C. for 1-3 hours to obtain a treated sample pad; S1-5. Paste the treated nitrocellulose membrane onto a polyvinyl chloride backing substrate, and then paste the treated sample pad and the absorption pad obtained in step S1-1 in sequence from one side of the polyvinyl chloride backing substrate to the other side, with the sample pad and the absorption pad overlapping the nitrocellulose membrane by 2-4 mm respectively.

9. A detection method of the kit according to any one of claims 1 to 5, characterized in that: The following steps are involved: S2-1, preparing nanoparticle-labeled detection probes for monoclonal antibodies that recognize two different targets; S2-2, taking the detection probe prepared in step S2-1, adding it to the sample to be tested, mixing evenly, and letting it stand to obtain an immune complex formed by the target and the corresponding detection probe; S2-3, adding the immune complex obtained in step S2-2 to the sample pad of the test strip, and after the quality control line is colored, using Color Picker software to analyze the RGB value of the T line and compare it with the standard curve to achieve quantitative judgment of the concentration of the target substance in the sample to be tested.

10. The detection method of a 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 standing time is 5-20 minutes.

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