Colorimetric-fluorescent bimodal nanoprobe for detecting cMyBP-C and micro-fluidic chip

Abstract

The invention discloses a colorimetric-fluorescent bimodal nanoprobe for detecting cMyBP-C and a micro-fluidic chip, and belongs to the technical field of biomedical detection and in-vitro diagnosis. The colorimetric-fluorescent bimodal nanoprobe which takes UCNPs as a core and MOF as a shell and is loaded with horse radish peroxidase and a labeled antibody is prepared by providing a layer-by-layer self-assembly technology; the micro-fluidic chip is prepared according to the probe, the micro-fluidic chip can realize cMyBP-C colorimetric and fluorescence signal bimodal detection, and the technical problems of limited sensitivity, susceptibility to background interference, limited linear range, photobleaching phenomenon, signal instability and the like in single signal detection are solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection and in vitro diagnosis, and in particular relates to a colorimetric-fluorescence dual-modal nanoprobe and a microfluidic chip for detecting cMyBP-C. Background Art

[0002] Cardiovascular disease is a type of disease that seriously affects human health and is the number one killer of Chinese people's health. It is estimated that there are about 330 million cardiovascular patients in my country. Cardiovascular disease is a high incidence and younger trend, and is an important disease that affects national health. At the same time, cardiovascular disease can induce a variety of diseases, especially major diseases such as myocardial injury. The causes of myocardial injury are complex and may be caused by insufficient blood or oxygen supply to myocardial cells in various situations, other diseases, drug toxicity, strenuous exercise, harsh living environment, and unhealthy living habits, resulting in myocardial cell necrosis, apoptosis, or pyroptosis. Since mature myocardial cells cannot regenerate, the disease worsens and heart failure occurs.

[0003] When myocardial damage occurs, the left ventricle accumulates heart-specific proteins and releases them into the blood throughout the body. These specific proteins combine with inflammatory indicators and other blood indicators to form a specific biomarker group. The clinical significance shown is different depending on the time and concentration of their release into the blood. Monitoring the release time and concentration of these specific biomarkers can reveal the extent of myocardial damage.

[0004] Cardiac myosin binding protein-C (cMyBP-C) is a thick filament structural protein unique to cardiomyocytes. It exists in cardiomyocytes in a phosphorylated form. In the ultra-early stage of acute myocardial infarction, intact cMyBP-C and its hydrolysis products are continuously released into the blood in large quantities, and their blood concentration increases sharply. Due to its myocardial specificity and large molecular weight, the detection of serum cMyBP-C concentration can help diagnose myocardial injury.

[0005] At present, the main methods for detecting cardiac markers are chemiluminescence, first-generation colloidal gold or second-generation immunofluorescence. Among them, chemiluminescence has the advantages of high sensitivity and wide detection range, but its instruments and equipment are expensive, large in size, have high laboratory requirements, and require strong professional operation, and are not suitable for scenarios such as instant detection. The first-generation colloidal gold based on the chromatography platform can realize qualitative visual detection of the target through colorimetric signals. The second-generation immunofluorescence method can realize quantitative detection of the target through fluorescent signals, but it is limited by the inherent physical properties of the chromatography membrane, such as different pore sizes and inability to ensure liquid flow consistency, resulting in poor detection accuracy (CV ≥ 15%), low sensitivity, and the test results fluctuate greatly with different incubation times.

[0006] In addition, single colorimetric or fluorescent signal detection faces multiple challenges, such as ① sensitivity limitation: single colorimetric detection cannot accurately detect low-concentration or weak signal changes, which limits its application in trace analysis; ② susceptibility to background interference: the background color, turbidity or autofluorescent substances of the sample may interfere with the detection signal, resulting in inaccurate results; ③ linear range limitation: colorimetric and fluorescent detection have specific linear ranges respectively. Beyond the specific range, the relationship between signal intensity and concentration may no longer be linear, affecting the accuracy of quantitative analysis; ④ photobleaching phenomenon: in fluorescence detection, fluorescent substances may undergo photobleaching under continuous irradiation, affecting the stability and repeatability of the signal; ⑤ signal stability and variability: the fluorescence signal may be affected by changes in environmental conditions, such as pH, temperature, ionic strength, etc., resulting in signal instability and variability.

[0007] Lanthanide-doped upconversion nanoparticles (UCNPs) show great potential in the field of fluorescence detection for in vitro diagnostics due to their unique photophysical properties. UCNPs can convert low-energy near-infrared light into high-energy ultraviolet / visible light, which not only enhances the penetration depth of light in biological tissues, but also effectively reduces the interference of autofluorescence of biological tissues. The chemical stability and low toxicity of UCNPs make them suitable for biomedical applications, and the high photostability ensures signal reliability. UCNPs have broad application prospects in the fields of biosensing, disease diagnosis and environmental monitoring.

[0008] At present, there is no report on the use of UCNPs-based colorimetric-fluorescent dual-modal probes for detecting cMyBP-C. Based on this, the present invention provides a UCNPs-based colorimetric-fluorescent dual-modal nanoprobe for detecting cMyBP-C, which realizes dual-modal detection of cMyBP-C colorimetric and fluorescent signals, and solves technical problems such as sensitivity limitation and susceptibility to background interference in single signal detection. Summary of the invention

[0009] One object of the present invention is to provide a colorimetric-fluorescent dual-modal nanoprobe for detecting cMyBP-C and a preparation method thereof. The present invention forms a core-shell complex with UCNPs as the core and MOF shell by "layer-by-layer self-assembly", and then loads horseradish peroxidase and antibodies to achieve dual-modal detection of cMyBP-C colorimetric and fluorescent signals, solving technical problems such as sensitivity limitation, susceptibility to background interference, linear range limitation, photobleaching, and signal instability in single signal detection. The second object of the present invention is to provide the use of the colorimetric-fluorescent dual-modal nanoprobe in the preparation of products for detecting cMyBP-C. The third object of the present invention is to provide a microfluidic chip based on the colorimetric-fluorescent dual-modal nanoprobe. The fourth object of the present invention is to provide a cMyBP-C dual-modal detection method.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] In the first aspect, the present invention provides a colorimetric-fluorescent dual-modal nanoprobe for detecting cMyBP-C, characterized in that the dual-modal nanoprobe has upconversion nanoparticles (UCNPs) as the core and metal-organic frameworks (MOFs) as the shell, the shell is loaded with horseradish peroxidase (HRP), and the shell surface is coupled with a labeled antibody (Ab) to form a colorimetric-fluorescent dual-modal nanoprobe, which is denoted as UCNP@MOF@Ab / HRP.

[0012] Furthermore, the labeled antibody is an anti-cMyBP-C antibody.

[0013] In a specific embodiment of the present invention, the anti-cMyBP-C antibody is selected from anti-cMyBP-C monoclonal antibody, polyclonal antibody or antigen-specific binding fragment, and the antibody can be purchased through commercial channels.

[0014] The up-conversion nanoparticles are composed of rare earth ions doped into nanocrystals NaYF4, Y2O3 or NaGdF4, and the rare earth ions include sensitizing ions and activating ions.

[0015] The sensitizing ion is selected from Nd 3+ , Yb 3+ The molar content of the sensitizing ion is 0.01-60%.

[0016] In a preferred embodiment of the present invention, the sensitizing ion is Yb 3+ .

[0017] The activating ion is selected from Pr 3+ 、Nd 3+ 、Sm 3+ , Tb 3+ 、Ho 3+ , Er 3+ 、Tm 3+ The molar content of the activating ions is 0.01-20%.

[0018] In a preferred embodiment of the present invention, the activating ion is selected from Er 3+ 、Tm 3+ 、Ho 3+ One of them.

[0019] In the most preferred embodiment of the present invention, the upconversion nanoparticles are NaYF4:Yb / Tm, the amount of Yb is 20-25% of the molar amount of NaYF4, and the amount of Tm is 0.3-0.6% of the molar amount of NaYF4.

[0020] More preferably, the upconversion nanoparticles are upconversion nanoparticles stabilized by PEG-phosphate ligands, and specifically, the PEG-phosphate is PEG(1000)-phosphate.

[0021] The metal-organic framework described in the present invention is a porous material formed by the coordination of metal nodes and multi-dentate organic ligands.

[0022] The metal node is selected from Zn 2+ , Cu 2+ , Fe 3+ 、Al 3+ 、Zr 4+ One or more of the above, the molar content of the metal node is 0.01-60%.

[0023] The multidentate organic ligand is selected from one or more of terephthalic acid, dimethyl terephthalate and 2-imidazole carboxylic acid, and the molar content of the organic ligand is 0.01-40%.

[0024] In a specific embodiment of the present invention, the metal-organic framework is Zr-MOF, specifically UIO-66-NH2.

[0025] The mass of horseradish peroxidase loaded in the metal-organic framework shell is 2-4% of the mass of the nanocomposite.

[0026] In a second aspect, the present invention provides a method for preparing a colorimetric-fluorescent dual-modality nanoprobe for detecting cMyBP-C, characterized in that the preparation method comprises the following steps:

[0027] (1) Preparation of UCNPs

[0028] The rare earth raw material is dissolved in deionized water, a certain amount of high-temperature solvent is added, and the water is evaporated; the reaction is carried out at 150-170°C for 1-1.5 hours under an argon atmosphere to obtain a transparent yellow solution, which is then cooled to room temperature; the solution is added dropwise to a methanol solution containing NH4F and NaOH, and the methanol solvent is evaporated; the temperature is raised to 290-320°C under a nitrogen atmosphere to react for 1-2 hours, and the solution is cooled to room temperature; the solution is ethanol precipitated, centrifuged, and washed to obtain upconversion nanoparticles, which are stored in cyclohexane for later use;

[0029] (2) Preparation of PEG-phosphate ligand-stabilized UCNPs

[0030] The prepared upconversion nanoparticles are dispersed in anhydrous ethanol containing PEG-phosphate ligands, slowly heated to 60-70° C., reacted for 8-10 hours, cooled to room temperature, and centrifuged to obtain upconversion nanoparticles stabilized by PEG-phosphate ligands;

[0031] (3) Preparation of UCNP@MOF

[0032] The PEG-phosphate ligand-stabilized UCNPs prepared above were dispersed in DMF, and ZrOCl2·8H2O solution was added dropwise, and the mixture was reacted at 60-65°C for 30-40 min. Then, BDC-NH2 solution was added dropwise, and the mixture was heated to 100-120°C and reacted for 1-1.5 h. A thin layer of UIO-66-NH2 shell was grown on the surface of UCNPs. The above UIO-66-NH2 growth method was repeated to obtain an ideal shell thickness, and UCNP@MOF was prepared, which was dispersed in DMF for later use.

[0033] (4) Preparation of UCNP@MOF@Ab / HRP

[0034] The UCNP@MOF dispersion prepared above was mixed with an equal volume of Tris HCl buffer, HRP was added to a final concentration of 1.0-1.5 mg / mL, incubated at room temperature for 8-24 hours, centrifuged, washed, and the HRP-loaded nanoparticles were redispersed, activated by adding NHS and EDC, and reacted with labeled antibodies for 3-4 hours, and blocking solution was added, centrifuged, and labeled antibody protection solution was added to resuspend for use to prepare UCNP@MOF@Ab / HRP.

[0035] Preferably, the high temperature solvent in step (1) is one or a combination of two or more of oleic acid, octadecene, oleylamine, tri-n-octylphosphine oxide, and trioctylphosphine.

[0036] In a specific embodiment of the present invention, the high temperature solvent is a combination of oleic acid and octadecene.

[0037] Preferably, the rare earth raw material described in step (1) is selected from: one or a combination of two or more of YCl3, YbCl3, TmCl3, and ErCl3.

[0038] In a specific embodiment of the present invention, the rare earth raw material is selected from a combination of YCl3, YbCl3, and TmCl3, wherein the molar ratio of YCl3, YbCl3, and TmCl3 is (1.6-1.8):(0.4-0.5):(0.007-0.008).

[0039] Preferably, in step (2), the PEG-phosphate is PEG (1000) -phosphate, and the mass ratio of the up-conversion nanoparticles to the PEG (1000) -phosphate is (30-40 mg): 1 g.

[0040] The molar concentration of the ZrOCl2·8H2O solution and the BDC-NH2 solution in step (3) is 5-6 mM, and freshly prepared solutions are preferred.

[0041] In a third aspect, the present invention provides a use of a colorimetric-fluorescent dual-modal nanoprobe in preparing a product for detecting cMyBP-C.

[0042] The products include but are not limited to reagents, test kits, chips, test strips, membrane strips or detection platforms.

[0043] In a specific embodiment of the present invention, the product for detecting cMyBP-C is a microfluidic chip.

[0044] In a fourth aspect, the present invention provides a colorimetric-fluorescent dual-modal microfluidic chip for detecting cMyBP-C, characterized in that the microfluidic chip comprises the colorimetric-fluorescent dual-modal nanoprobe described in the first aspect of the present invention.

[0045] Furthermore, the main structure of the microfluidic chip includes a base plate and an upper cover. The base plate structure is finely processed by one-time injection molding. A welding line of 0.05-1.00 mm is set on the base plate. The surface of the upper cover is flat and smooth. After welding, the base plate and the upper cover can form a microchannel with capillary driving force.

[0046] According to the sample flow direction, the bottom plate structure includes a sample addition area, a sample filtration area, a reaction area, a time-controlled valve, a detection area and a waste liquid pool connected in sequence, wherein a blood filter membrane is installed in the sample filtration area, and the sample is filtered through the blood filter membrane, and the reaction area fixes the colorimetric-fluorescence dual-modal nanoprobe described in the first aspect of the present invention by spotting, and the spotting volume is 1μL-2μL / piece.

[0047] The detection area includes a detection line and a quality control line. The detection line area is fixed with capture antibodies by spotting, and the spotting volume is 0.2 μL-1 μL / sheet; the quality control line area is fixed with goat anti-mouse IgG by spotting.

[0048] Furthermore, the capture antibody is an anti-cMyBP-C antibody that can bind to the UCNP@MOF@Ab / HRP-antigen complex to form a double antibody sandwich structure.

[0049] The upper cover structure has an elliptical sample loading port and an air hole. The elliptical sample loading port is located at the front half of the bottom plate sample loading area, and the air hole is located at the front end of the bottom plate detection area.

[0050] Preferably, the reaction zone, the time-controlled valve and the detection zone are composed of flow channels and micron-scale micro-column structures.

[0051] In a specific embodiment of the present invention, the reaction zone is sequentially provided with three array structures according to the sample flow direction, firstly a high capillary action zone with a relatively dense array structure (the array structure is arranged obliquely), followed by a short section of low capillary action zone without an array structure, and finally a medium capillary action zone with a relatively sparse array structure (the array structure is arranged horizontally).

[0052] In a specific embodiment of the present invention, the timing valve is provided with dense transverse grooves for blocking the fluid, reducing the liquid flow rate, and prolonging the reaction time of the sample to be tested and the reagent; there is a triangular high capillary action area at the end of the timing valve, which guides the fluid to always enter the microfluidic detection area at the same position.

[0053] In a specific embodiment of the present invention, the area of ​​the microfluidic detection zone is 100-105mm 2 The region includes 11,000-12,000 microstructures, and the diameter of the microstructure is 50±5 μm, which can significantly increase the specific surface area of ​​the flow channel, increase the antigen capture efficiency, and improve the dynamic detection range, sensitivity, and repeatability of the microfluidic chip.

[0054] The advantage of the reaction zone of the microfluidic chip provided by the present invention is that the sample will first enter and fill the high capillary action zone of the reaction zone, the faster-running fluid will be slightly blocked by the low capillary action zone, waiting for the liquid behind to catch up, and then the liquid will enter the medium capillary action zone in a relatively uniform distribution state and contact and mix with the pre-buried reagent in the area (the dual-modal nanoprobe described in the first aspect of the present invention), and be integrated through the laterally arranged microarray structure, and finally enter the time-controlled valve with a relatively flat gas-liquid interface to guide the fluid to always enter the microfluidic detection area at the same position, thereby ensuring the consistency of the sample flow rate and direction and the stability of the reaction system.

[0055] The microfluidic chip is coated with a nano-scale hydrophilic coating of controllable thickness on the surface of the chip bottom plate by ultrasonic atomization spraying technology. The nano-scale hydrophilic coating is used to change the hydrophilic angle of the chip bottom plate to regulate the magnitude of the capillary driving force and avoid nonspecific adsorption of biological samples on the chip surface.

[0056] The nano-scale hydrophilic coating is prepared by dissolving 4, 3 or 2 of 0.1%-5% PVP (polyvinyl pyrrolidone), 0.01%-1% sodium alginate, 0.05%-0.3% polyethylene glycol diamine, 0.01%-0.5% methyl carbamate and 0.1%-6% PVA (polyvinyl alcohol) in ethanol, wherein the "%" refers to the mass percentage of each component relative to ethanol, and the mass concentration of ethanol is 50%-100%.

[0057] The detection line and the accusation line area of ​​the microfluidic detection zone use a crosslinking agent to crosslink the embedded antibody and the nano-scale hydrophilic coating of the bottom plate. The crosslinking agent is a mixed solvent of a silane coupling agent and a high molecular organic matter. The embedded antibody is a capture antibody and goat anti-mouse IgG.

[0058] The cross-linking agent is prepared by dissolving 3, 2 or 1 of 0.05%-5% aminosilane, 0.05%-0.3% polyethylene glycol diamine and 0.1%-6% PVA (polyvinyl alcohol) in ethanol, wherein the "%" refers to the mass percentage of each component relative to ethanol, and the mass concentration of ethanol is 75%-100%. The cross-linking agent provided by the present invention is rich in amino groups and can stably cross-link the carboxyl end of the capture antibody, so that the detection line and the quality control line will not dissolve and fall off with the flow of the blood sample.

[0059] Preferably, the cross-linking agent is prepared by dissolving 0.05%-5% aminosilane, 0.05%-0.3% polyethylene glycol diamine and 0.1%-6% PVA (polyvinyl alcohol) in ethanol.

[0060] The working principle of the microfluidic chip provided by the present invention for detecting cMyBP-C is: using immunomicrofluidic technology to achieve colorimetric-fluorescence dual signal quantitative detection of cMyBP-C in the test sample. The detection process is as follows: adding a sample to be tested to a sample adding area of ​​a microfluidic chip, filtering the sample through a blood filter membrane in a sample filtering area; when the sample contains cMyBP-C, cMyBP-C reaches the reaction area through capillary force, and combines with the anti-cMyBP-C antibody on the colorimetric-fluorescent dual-modal nanoprobe provided by the present invention fixed in the area to form an antigen-antibody complex; under the capillary action, the antigen-antibody complex continues to move forward, and when encountering a detection line (T line) fixed with a capture antibody, the antigen in the antigen-antibody complex combines with the capture antibody to form a double sandwich structure, and the antigen-antibody complex is captured by the area, and a fluorescent color band is formed under near-infrared light irradiation, and a color signal will appear at the same time after a color developer is added; part of the colorimetric-fluorescent dual-modal nanoprobe that has not been bound will continue to move, and when it moves to the quality control line (C line) coated with goat anti-mouse IgG, it forms a primary antibody-secondary antibody conjugate with the goat anti-mouse IgG antibody, develops fluorescence in the quality control area, and a color signal will appear at the same time after a color developer is added. When the sample does not contain cMyBP-C, the colorimetric-fluorescent dual-modal nanoprobe fixed in the reaction zone will not bind to the capture antibody on the detection line (T line), the detection line will have no fluorescence and color signal, and will move directly to the quality control line (C line), forming a primary antibody-secondary antibody conjugate with the goat anti-mouse IgG antibody, showing fluorescence in the quality control area, and a color signal will appear at the same time after the color developer is added.

[0061] In a fifth aspect, the present invention provides a cMyBP-C dual-modality detection method based on a microfluidic chip, wherein the detection method is not for the purpose of disease diagnosis and treatment, and is characterized in that the method comprises the following steps:

[0062] S1: using the colorimetric-fluorescence dual-modality microfluidic chip described in the fourth aspect of the present invention to establish standard curves of colorimetric intensity and fluorescence intensity versus cMyBP-C concentration;

[0063] S2: Dilute the biological sample with a diluent and load it onto the sample loading area of ​​the microfluidic chip described in the fourth aspect of the present invention. Load the AEC staining reagent 5-8 minutes later. Measure the colorimetric intensity and fluorescence intensity of the test line and the quality control line 5-7 minutes later, and compare them with the standard curve. Calculate the dilution multiple to obtain the cMyBP-C concentration in the biological sample.

[0064] The diluent is selected from NaCl (final concentration 136.89 mM), KCl (final concentration 2.67 mM), Na2HPO4 (final concentration 8.1 mM), KH2PO4 (final concentration 1.76 mM), BSA (final concentration 0.5%) or Proclin-300 (final concentration 0.1%).

[0065] In a specific embodiment of the present invention, the standard curve is prepared by the following method: a cMyBP-C standard of known concentration is mixed with a diluent and diluted to different concentration gradients, 280 μL of the samples to be tested with different concentration gradients are respectively loaded onto the sample loading area of ​​the microfluidic chip, 3-amino-9-ethylcarbazole (AEC) staining reagent is loaded again after 5-8 minutes, and the colorimetric intensity and fluorescence intensity of the test line and the quality control line are measured after 5-7 minutes, and a standard curve 1 is established according to the colorimetric intensity and the cMyBP-C concentration, and a standard curve 2 is established according to the fluorescence intensity and the cMyBP-C concentration.

[0066] Compared with the prior art, the technical solution provided by the present invention has the following technical advantages:

[0067] 1) The present invention uses layer-by-layer self-assembly technology to coat the MOF (UIO-66-NH2) shell on the surface of UCNP to achieve efficient loading of HRP and efficient labeling of antibodies. UCNP can output fluorescence signals through near-infrared light excitation to avoid interference from the sample matrix; the HRP loaded on MOF catalyzes the corresponding substrate to output color signals, so that the chip has both colorimetric and fluorescence dual-modal detection methods. The combination of colorimetric and fluorescent signals can effectively distinguish target signals from background interference signals, and mutual verification of the two helps to reduce false positive and false negative results and improve the accuracy of detection; dual-modality can expand the linear range of detection. In the low concentration range, the fluorescent signal provides high-sensitivity detection; in the high concentration range, the colorimetric signal provides stable quantitative analysis, thereby achieving a wider quantitative linear range.

[0068] 2) The present invention adopts self-driven microfluidic technology to solve some technical problems inherent in existing chromatography platforms, such as poor detection accuracy (CV≥15%), low sensitivity, and large fluctuations in detection results with different incubation times.

[0069] 3) The present invention adopts technologies such as nano-scale hydrophilic coating and silane coupling agent to ensure the stability of the chip bottom plate surface treatment and capture antibody, thereby reducing the impact of environmental conditions changes on the fluorescent signal and ensuring the stability and repeatability of the signal. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Schematic diagram of the preparation method of dual-modal nanoprobe.

[0071] Figure 2 Characterization of the core-shell structure of UCNP@UIO-66-NH2; (A) OA-encapsulated UCNPs, (B) PEG-phosphate-modified UCNPs, (C) TEM and particle size distribution of UCNP@UIO-66-NH2 (the UIO-66-NH2 shell is indicated by a red arrow) and (D) elemental mapping analysis of UCNP@UIO-66-NH2.

[0072] Figure 3 cMyBP-C colorimetric and fluorescence dual-modal microfluidic detection chip; (A) overall schematic diagram of the microfluidic chip, (B) schematic diagram of the microstructure of the microfluidic chip base.

[0073] Figure 4 Photos and fluorescence imaging of the colorimetric-fluorescence dual-modal microfluidic chip detecting cMyBP-C.

[0074] Figure 5 Standard curve of cMyBP-C concentration and colorimetric signal intensity.

[0075] Figure 6 Standard curve of cMyBP-C concentration and fluorescence signal intensity. DETAILED DESCRIPTION

[0076] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0077] Example 1 Preparation of colorimetric-fluorescent dual-modal nanoprobe UCNP@MOF@Ab / HRP

[0078] In this embodiment, the preparation method of the dual-mode nanoprobe is as follows: Figure 1 As shown, specifically:

[0079] Step 1. Preparation of UCNPs stabilized by PEG-phosphate ligands

[0080] YCl3·6H2O (482 mg), YbCl3·6H2O (155 mg) and TmCl3·6H2O (2.8 mg) were dissolved in 2 mL of deionized water and added to a mixture of oleic acid (15 mL) and 1-octadecene (30 mL). The above solution was stirred at room temperature for 1 hour under argon to eliminate oxygen in the solution. The solution was then slowly heated to 120°C to remove moisture from the solution and reacted at 156°C for about 1 hour until a uniform transparent yellow solution was obtained. After the solution was cooled to room temperature under argon, 10 mL of a methanol solution containing NH4F (296 mg) and NaOH (200 mg) was added dropwise, and the mixture was then heated to 70°C and maintained for 20 minutes to evaporate the methanol. Subsequently, the solution was heated to 290°C, reacted for 2 hours, and then cooled to room temperature. 20 mL of anhydrous ethanol was added to the above reaction solution, and after sufficient shaking, the reaction solution was centrifuged at 9000 rpm for 10 minutes to collect the centrifuged product, i.e., UCNPs. After repeated washing with cyclohexane and ethanol, the final product was redispersed in 20 mL of cyclohexane and freeze-dried for further use.

[0081] The 30 mg UCNPs powder synthesized above was dispersed in 2 mL of anhydrous ethanol containing 1 g PEG (1000) - phosphate ligand. The above solution was slowly heated to 70 ° C and reacted for 8 hours. The reaction solution was then cooled to room temperature and centrifuged at 7000 rpm for 15 minutes to obtain PEG-phosphate ligand-stabilized UCNPs. After the reaction product was repeatedly washed twice with cyclohexane (2 ml), it was repeatedly washed four times with anhydrous ethanol (2 mL) to remove the free PEG (1000) - phosphate ligand in the reaction system. Finally, the final product was redispersed in 2 mL N, N-dimethylformamide (DMF) for subsequent UIO-66-NH2 shell coating.

[0082] Step 2: Synthesis of UCNP@UIO-66-NH2 core-shell complex

[0083] In this experiment, the UIO-66-NH2 shell was coated on the surface of UCNPs by the "layer-by-layer self-assembly" method. The specific operation steps are as follows: 2 mL of the PEG (1000)-phosphate ligand-stabilized UCNPs dispersion was added to 8 mL of DMF, and then 5 mL of freshly prepared ZrOCl2·8H2O solution (5 mM) was added dropwise. The reaction was stirred at 60 ° C for 40 minutes to allow the UCNPs surface to efficiently adsorb Zr 4+ions. Subsequently, 5 mL of freshly prepared BDC-NH2 solution (5 mM) was added dropwise to the solution, which was heated to 120 °C and reacted for 1 hour. A thin layer of UIO-66-NH2 shell was grown on the surface of UCNPs, and the intermediate product was collected by centrifugation at 9000 rpm for 10 minutes. To further increase the thickness of the MOF shell, the above-mentioned UIO-66-NH2 shell growth method can be repeated using the "layer-by-layer self-assembly" method to form MOF shells of different thicknesses on the surface of UCNPs. Finally, the final product was washed alternately with ethanol and deionized water for 6 times and redispersed in 10 mL of DMF for short-term stable storage.

[0084] The present invention coats the surface of UCNPs once, twice, three times, and four times according to the above method, and a MOF shell of different thicknesses is formed on the surface of UCNPs. The inventors found that the core-shell complex coated once and twice, respectively, has poor stability of nanoparticles and poor dispersibility in aqueous solution due to the thin thickness of the MOF shell, and a certain degree of agglomeration phenomenon occurs; the thickness of the MOF shell coated four times is about 14.5nm, and the dispersibility is good, but the thickness of the MOF coating layer on the surface of UCNPs is uneven under the TEM microscope, and the thickness uniformity is not as good as that coated three times. Finally, the present invention preferably uses the above method to coat the surface of UCNPs three times.

[0085] The UCNP@UIO-66-NH2 core-shell composite prepared by repeated coating three times was characterized. Figure 2 As shown, oleic acid-coated UCNPs (NaYF4: Yb / Tm) exhibited an obvious hexagonal phase structure ( Figure 2 A). After the above method was repeated three times using "layer-by-layer self-assembly" coating, the thickness of the coated MOF shell was about 10.7nm ( Figure 2 C), which is an ideal thickness; element mapping, elements Y, F, Tm and Zr are evenly distributed on the nanoparticles ( Figure 2 D), indicating the formation of a core-shell composite with NaYF4:Yb / Tm as the core and UIO-66-NH2 as the shell, named UCNP@MOF.

[0086] Step 3: Loading of HRP and modification of antibodies with UCNP@UIO-66-NH2

[0087] UCNP@UIO-66-NH2 was dispersed in Tris-HCl buffer, HRP was added to a final concentration of 1.0 mg / mL, and the mixture was placed on a shaker for incubation for 8 hours to obtain a core-shell complex loaded with HRP, named UCNP@MOF / HRP. Subsequently, UCNP@MOF / HRP was dispersed in 100 μL MES buffer solution, 1.5 μL NHS solution was added to the shaker for 1 min, and then 1.5 μL LEDC solution was added, placed on a 37°C constant temperature shaker, reacted for 30 min, and redispersed in 200 μL HEPES buffer by centrifugation. 100 μg of labeled antibody (anti-cMyBP-C antibody) was added to the above solution, placed on a 37°C constant temperature shaker, reacted for 120 min, and then 30 μL of blocking solution was added, ultrasonically mixed for 1 min, and the reaction continued for 60-120 min. After the reaction was completed, centrifuged at 9000 rpm for 15 minutes to collect the constructed probe. Finally, the collected product was washed three times with HEPES buffer to remove free antibodies, centrifuged to remove the supernatant, and resuspended in 500 μL labeled antibody protection solution to obtain UCNP@MOF@Ab / HRP.

[0088] Example 2 Preparation of colorimetric-fluorescence dual-mode microfluidic chip

[0089] The colorimetric-fluorescence dual-mode microfluidic chip consists of an upper cover and a chip bottom plate. The upper cover structure has an elliptical sample loading port and an air hole, wherein the elliptical sample loading port is located in the front half of the bottom plate sample loading area, and the air hole is located at the front end of the bottom plate detection area. The bottom plate structure is finely processed by one-time injection molding, a 0.05-1.00mm welding line is arranged on the bottom plate, and a nano-scale hydrophilic coating is sprayed on the surface of the bottom plate by ultrasonic atomization spraying technology. The nano-scale hydrophilic coating material is prepared by the following method: PVP with an amount of 2% by mass of anhydrous ethanol and PVA with an amount of 2% by mass of anhydrous ethanol are added to anhydrous ethanol in sequence, and stirred for 10-15 minutes; polyethylene glycol diamine with an amount of 0.3% by mass of anhydrous ethanol and methyl carbamate with an amount of 0.3% by mass of anhydrous ethanol are added continuously, and stirred for 10-15 minutes; finally, sodium alginate with an amount of 1% by mass of anhydrous ethanol is added, and stirred for 10-15 minutes to prepare a nano-scale hydrophilic coating material.

[0090] The bottom plate is provided with a sample adding area, a sample filtering area, a reaction area, a time-controlled valve, a detection area and a waste liquid pool connected in sequence. The sample adding area is used to add the sample to be tested; the sample filtering area is provided with a blood filter membrane to filter the sample through the blood filter membrane.

[0091] According to the sample flow direction, the reaction zone is sequentially provided with three array structures: first, a high capillary action zone with a relatively dense array structure (array structure arranged obliquely), followed by a short section of low capillary action zone without array structure, and then a medium capillary action zone with a relatively sparse array structure (array structure arranged horizontally); the UCNP@MOF@Ab / HRP prepared in Example 1 is fixed in the reaction zone by spotting, with a spotting amount of 2.1 μL / piece, and dried at room temperature.

[0092] Dense transverse grooves are set in the timing valve area to block the fluid, reduce the liquid flow rate, and prolong the reaction time of the sample and reagent to be tested. A triangular high capillary action area is set at the end of the timing valve to guide the fluid to always enter the microfluidic detection area at the same position.

[0093] The area of ​​the microfluidic detection zone is 105 mm 2 , the region includes 11000-12000 microstructures, the diameter of the microstructure is 50±5μm, which can significantly increase the specific surface area of ​​the flow channel and increase the antigen capture efficiency. A detection line and a quality control line are set in the detection area, and a crosslinker is first sprayed in the detection line and quality control line areas, and dried at room temperature; cMyBP-C capture antibody is fixed in the detection line area by spotting, and the spotting amount is 0.42μL / piece, and goat anti-mouse IgG is fixed in the quality control line area, and the spotting amount is 0.42μL / piece, and dried at room temperature. In the experimental stage, the embodiment of the present invention is configured to obtain two crosslinking agents, namely crosslinker 1 and crosslinker 2. The crosslinker 1 is prepared by the following method: aminosilane with anhydrous ethanol mass 4% and PVA with anhydrous ethanol mass 2% are added to anhydrous ethanol in sequence, stirred for 10-15 minutes, and polyethylene glycol diamine with anhydrous ethanol mass 0.3% is added, and stirred for 10-15 minutes to prepare a crosslinker. The preparation method of the cross-linker 2 is the same as that of the cross-linker 1, the only difference being that its composition does not contain polyethylene glycol diamine. Cross-linker 1 and cross-linker 2 are sprayed on two base plates respectively, and the influence of the cross-linker on the stability of the detection line is verified respectively. The final verification results show that cross-linker 1 has a better cross-linking effect between the antibody and the hydrophilic layer of the base plate, and the detection line and the quality control line have good stability. When the chip sprayed with cross-linker 2 has a large amount of sample, the detection line and the quality control line will be offset with the flow of the sample, which affects the interpretation of the final signal intensity. The present invention preferably uses cross-linker 1 as a cross-linker for preparing microfluidic chips. The technicians analyzed that polyethylene glycol diamine, as a linear molecule containing amino groups, increases the compatibility of the cross-linker with the hydrophilic coating, and can also increase the capture effect of the cross-linker on the antibody.

[0094] The waste liquid pool is used to absorb the sample liquid at the end of the chromatography. Finally, the upper cover is installed, the welding program of the ultrasonic welding instrument is run, and the welding detection card can complete the preparation of the microfluidic chip.

[0095] The microfluidic chip prepared in this embodiment is as follows Figure 3 As shown in the figure, it can be seen that the chip surface is processed with a variety of functional units to ensure the consistency of sample flow rate and direction and the stability of the reaction system. Among them, a is the sample addition area, b is the reaction area, c is the time-controlled valve, and d is the microfluidic detection area. The sample loading area (a) is designed with transverse and longitudinal guide groove structures, which can achieve the drainage of samples; the reaction area (b) is designed with three array structures. As mentioned above, the sample will first enter and fill the high capillary action area of ​​the reaction area, and the faster-running fluid will be slightly blocked by the low capillary action area, waiting for the liquid behind to catch up. Then the liquid will enter the medium capillary action area in a relatively uniform distribution state and contact and mix with the reagents embedded in the area, and will be further integrated through the transversely arranged microarray structure, and finally enter the timing valve (c) with a relatively flat gas-liquid interface; the bottom of the timing valve is designed with dense transverse grooves to block the fluid, reduce the liquid flow rate, and prolong the reaction time of the sample and reagent to be tested. At the same time, a triangular high capillary action area is designed at the end of the timing valve area, which will guide the fluid to always enter the microfluidic detection area (d) at the same position; at 105mm 2 The microfluidic detection area is processed with more than 11,000 microstructures with a diameter of about 50μm, which can significantly increase the specific surface area of ​​the flow channel and increase the capture efficiency of antigens, thereby improving the dynamic detection range, sensitivity and repeatability of the microfluidic chip.

[0096] Example 3 Application of colorimetric-fluorescence dual-mode microfluidic chip

[0097] Take 20 μL of 5 ng / mL cMyBP-C corporate reference material, mix it with 480 μL of diluent, take 280 μL and load it into the sample loading area of ​​the microfluidic chip. After 8 minutes, load 100 μL of AEC staining reagent again. After 5 minutes, observe the color change of the test line and quality control line in the detection area of ​​the microfluidic chip, and use a fluorescence microscope to observe the upconversion fluorescence of the test line and quality control line.

[0098] The results are as follows Figure 4 As shown, it can be seen that the T line has a detection signal output through both color and fluorescence signals, which proves that the colorimetric-fluorescence dual-modal microfluidic chip prepared by the present invention can be used to detect cMyBP-C, which is feasible.

[0099] Example 4 Detection Performance Verification of Colorimetric-Fluorescence Dual-Mode Microfluidic Chip

[0100] Take 20 μL of cMyBP-C enterprise reference products with concentrations of 50 ng / mL, 10 ng / mL, 5 ng / mL, 1 ng / mL, 0.5 ng / mL, 0.1 ng / mL, 0.05 ng / mL, 0.01 ng / mL, 0.005 ng / mL, and 0 ng / mL, respectively, and mix with 480 μL of diluent to prepare a series of sample solutions containing cMyBP-C with a concentration gradient; take 280 μL and load it to the sample loading area of ​​the microfluidic chip, and load 100 μL of AEC staining reagent again after 8 minutes. After 5 minutes, use the colorimetric and fluorescence detection instruments equipped with the microfluidic chip to measure the colorimetric intensity and fluorescence intensity of the test line and the quality control line, respectively. Use cMyBP-C concentration as the horizontal axis and colorimetric intensity and fluorescence intensity as the vertical axis to establish standard curves, and analyze the linear equation, correlation coefficient, linear range, detection limit and other performance indicators of the standard curves corresponding to the colorimetric and fluorescence signals. The results are shown in Table 1. Figure 5-6 shown.

[0101] Table 1 Corresponding linear equations, correlation coefficients, linear ranges and detection limits for colorimetric and fluorescence signals

[0102]

[0103] From Table 1 and Figure 5-6 The displayed standard curve, linear equation, correlation coefficient, and linear range data show that there is a significant linear relationship between the colorimetric and fluorescent signals and the cMyBP-C concentration, with a correlation coefficient of >0.99. The colorimetric and fluorescent signal intensities of the samples to be tested can be used to quantitatively detect the cMyBP-C concentration in the samples based on the provided standard curve. In addition, the detection limit data show that the colorimetric-fluorescence dual-modal signal can extend the linear range of detection. In the low concentration range, the fluorescent signal provides high-sensitivity detection; in the high concentration range, the colorimetric signal provides stable quantitative analysis, thereby achieving a wider quantitative linear range.

[0104] Comparative Example 1 Preparation of fluorescent nanoprobe UCNP@MOF@Ab

[0105] The preparation method is the same as that in Example 1, except that in step 3, HRP is not loaded on the surface of UCNP@UIO-66-NH2, and the antibody is directly coupled. The specific method is: UCNP@UIO-66-NH2 is dispersed in 100 μL MES buffer solution, 1.5 μL NHS solution is added to the oscillator to mix for 1 min, and then 1.5 μL EDC solution is added, placed on a 37°C constant temperature shaker, reacted for 30 min, and re-dispersed in 200 μL HEPES buffer by centrifugation. Take 100 μg of labeled antibody (anti-cMyBP-C antibody) and add it to the above solution, place it on a 37°C constant temperature shaker, react for 120 min, then add 30 μL of blocking solution, ultrasonically mix for 1 min, and continue to react for 60-120 min. After the reaction is completed, centrifuge at 9000 rpm for 15 minutes to collect the constructed probe. Finally, the collected product was washed three times with HEPES buffer to remove free antibodies, the supernatant was removed by centrifugation, and 500 μL of labeled antibody protection solution was added for resuspending to obtain UCNP@MOF@Ab.

[0106] Comparative Example 2 Preparation of a Microfluidic Chip without Color Signal

[0107] The preparation method is the same as that in Example 2, except that the reaction area of ​​the chip bottom plate is fixed by spotting the UCNP@MOF@Ab prepared in Comparative Example 1, and the spotting amount is 2.1 μL / chip. The settings of other structural areas are exactly the same as those in Example 2.

[0108] Take 20 μL of the cMyBP-C enterprise reference product with a concentration of 0.01 ng / mL, mix it with 480 μL of diluent, prepare a low-concentration cMyBP-C sample solution, configure 100 copies in total, and set 100 blank controls at the same time. The 200 samples are randomly arranged, and the microfluidic chips prepared in Example 2 and Comparative Example 2 are respectively used for qualitative detection according to the method provided in Example 3. Among them, the microfluidic chip prepared in Comparative Example 2 is used for detection, and the AEC staining reagent is not required, and the detection result is only a fluorescent signal. The final sample positive rate is: the positive rate of the microfluidic chip prepared in Example 2 is 50%, and the positive rate of the microfluidic chip prepared in Comparative Example 2 is 53%. The test results show that the microfluidic chip prepared in Example 2 has a higher detection accuracy. This is because for low-concentration samples, only the fluorescent signal is used as the basis for interpretation, and the background signal in the system may interfere with the target signal, resulting in a false positive result. The addition of a colorimetric signal can correct the false positive data. Therefore, the combination of colorimetric and fluorescent signals can effectively distinguish target signals from background interference signals, and the mutual verification between the two helps reduce false positive and false negative results and improve the accuracy of detection.

[0109] Comparative Example 3 Preparation of microfluidic chips with different reaction zone array structures

[0110] The preparation method is the same as that in Example 2, the only difference being the array structure of the reaction zone of the chip base plate, wherein the array structure is: first a high capillary action zone having a relatively dense array structure (the array structure is arranged obliquely), followed by a medium capillary action zone having a relatively sparse array structure (the array structure is arranged horizontally), and there is no low capillary action zone. The arrangement of other structural areas is exactly the same as that in Example 2.

[0111] Comparative Example 4 Preparation of a microfluidic chip without a time-controlled valve

[0112] The preparation method is the same as that in Example 2, the only difference being that the structural setting of the chip base plate does not include a time-controlled valve. The chip base plate includes a sample addition area, a sample filtration area, a reaction area, a detection area and a waste liquid pool connected in sequence, and the specific structure of the areas is the same as that in Example 2.

[0113] The microfluidic chips prepared in Comparative Examples 3 and 4 were used to test samples containing cMyBP-C (>100 sample volume), and the test results showed that the microfluidic chips prepared in Comparative Examples 3 and 4 had no effect on the qualitative test results of the samples, and the qualitative test results were accurate. However, when the microfluidic chips prepared in Comparative Examples 3 and 4 were used to prepare the standard curve, the technicians found that the correlation coefficient of the obtained standard curve was significantly lower than that of the microfluidic chip prepared in Example 2, and the correlation coefficient of the colorimetric signal standard curve decreased from 0.9934 to 0.9615 and 0.9462, respectively, and the correlation coefficient of the fluorescence signal standard curve decreased from 0.9996 to 0.9482 and 0.9133, respectively. The technicians analyzed that the three array structures set in the reaction zone and the array structure set in the time-controlled valve of the present invention have good control of the chromatography speed, ensuring that the antigen in the sample to be tested is fully combined with UCNP@MOF@Ab / HRP, increasing the stability of the formed antigen-antibody complex, and ensuring that the antigen-antibody complex is combined with the capture antibody on the detection line at a uniform speed to form a stable colorimetric-fluorescent signal. If the array structure of the reaction zone is changed at will, or the time-controlled valve structure is cancelled, the antigen in the sample to be tested cannot be fully combined with UCNP@MOF@Ab / HRP, the formed antigen-antibody complex structure is unstable, and it is combined with the capture antibody at an uneven speed, affecting the stability of the colorimetric-fluorescent signal.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A colorimetric-fluorescent dual-modal nanoprobe for detecting cMyBP-C, characterized in that: The dual-modal nanoprobe has upconversion nanoparticles (UCNPs) as the core and metal-organic frameworks (MOFs) as the shell. The shell is loaded with horseradish peroxidase (HRP), and the shell surface is coupled with a labeled antibody (Ab) to form a colorimetric-fluorescent dual-modal nanoprobe, denoted as UCNP@MOF@Ab / HRP, wherein the labeled antibody is an anti-cMyBP-C antibody.

2. The dual-modal nanoprobe according to claim 1, characterized in that: The anti-cMyBP-C antibody is selected from anti-cMyBP-C monoclonal antibody, polyclonal antibody or antigen-specific binding fragment.

3. The dual-modal nanoprobe according to claim 1, characterized in that: The up-conversion nanoparticles are composed of rare earth ions doped into nanocrystals NaYF4, Y2O3 or NaGdF4, wherein the rare earth ions include sensitizing ions and activating ions; the sensitizing ions are selected from Nd 3+ , Yb 3+ One or a combination of two of the following; the activating ion is selected from Pr 3+ 、Nd 3+ 、Sm 3+ , Tb 3 + 、Ho 3+ , Er 3+ 、Tm 3+ One or a combination of two or more of the above; the metal-organic framework is a porous material formed by the coordination of metal nodes and multi-dentate organic ligands.

4. A method for preparing a colorimetric-fluorescent dual-modality nanoprobe for detecting cMyBP-C according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Preparation of UCNPs The rare earth raw material is dissolved in deionized water, a certain amount of high-temperature solvent is added, and the water is evaporated; the reaction is carried out at 150-170°C for 1-1.5 hours under an argon atmosphere to obtain a transparent yellow solution, which is then cooled to room temperature; the solution is added dropwise to a methanol solution containing NH4F and NaOH, and the methanol solvent is evaporated; the temperature is raised to 290-320°C under a nitrogen atmosphere to react for 1-2 hours, and the solution is cooled to room temperature; the solution is ethanol precipitated, centrifuged, and washed to obtain upconversion nanoparticles, which are stored in cyclohexane for later use; (2) Preparation of PEG-phosphate ligand-stabilized UCNPs The prepared upconversion nanoparticles are dispersed in anhydrous ethanol containing PEG-phosphate ligands, slowly heated to 60-70° C., reacted for 8-10 hours, cooled to room temperature, and centrifuged to obtain upconversion nanoparticles stabilized by PEG-phosphate ligands; (3) Preparation of UCNP@MOF The PEG-phosphate ligand-stabilized UCNPs prepared above were dispersed in DMF, and ZrOCl2·8H2O solution was added dropwise, and the mixture was reacted at 60-65°C for 30-40 min. Then, BDC-NH2 solution was added dropwise, and the mixture was heated to 100-120°C and reacted for 1-1.5 h. A thin layer of UIO-66-NH2 shell was grown on the surface of UCNPs. The above UIO-66-NH2 growth method was repeated to obtain an ideal shell thickness, and UCNP@MOF was prepared, which was dispersed in DMF for later use. (4) Preparation of UCNP@MOF@Ab / HRP The UCNP@MOF dispersion prepared above was mixed with an equal volume of Tris HCl buffer, HRP was added to a final concentration of 1.0-1.5 mg / mL, incubated at room temperature for 8-24 hours, centrifuged, washed, and the HRP-loaded nanoparticles were redispersed, activated by adding NHS and EDC, and the labeled antibody was reacted for 3-4 hours, a blocking solution was added, centrifuged, and the antibody protection solution was added to resuspend for use to prepare UCNP@MOF@Ab / HRP.

5. The preparation method according to claim 4, characterized in that: The high-temperature solvent in step (1) is one or a combination of two or more of oleic acid, octadecene, oleylamine, tri-n-octylphosphine oxide, and trioctylphosphine; the rare earth raw material is selected from one or a combination of two or more of YCl3, YbCl3, TmCl3, and ErCl3.

6. Use of the colorimetric-fluorescent dual-modal nanoprobe according to claim 1 in preparing a product for detecting cMyBP-C.

7. A colorimetric-fluorescence dual-modality microfluidic chip for detecting cMyBP-C, characterized in that: The microfluidic chip comprises the colorimetric-fluorescent dual-modal nanoprobe described in claim 1.

8. The microfluidic chip according to claim 7, characterized in that: The main structure of the microfluidic chip includes a bottom plate and an upper cover; according to the sample flow direction, the bottom plate structure includes a sample addition area, a sample filtration area, a reaction area, a time-controlled valve, a detection area and a waste liquid pool connected in sequence, wherein the reaction area is fixed by spotting the colorimetric-fluorescence dual-modal nanoprobe according to claim 1, and the spotting volume is 1μL-2μL / piece; the detection area includes a detection line and a quality control line, and the detection line area is fixed by spotting the capture antibody, the capture antibody is an anti-cMyBP-C antibody, and the spotting volume is 0.2μL-1μL / piece; the quality control line area is fixed by spotting sheep anti-mouse IgG.

9. The microfluidic chip according to claim 8, characterized in that: The reaction zone, time-controlled valve and detection zone are composed of flow channels and micron-scale micro-column structures. According to the sample flow direction, the reaction zone is sequentially provided with three array structures. First, there is a high capillary action zone with a relatively dense array structure, and its array structure is arranged obliquely; followed by a short section of low capillary action zone without array structure; and then there is a medium capillary action zone with a relatively sparse array structure, and its array structure is arranged horizontally.

10. The microfluidic chip according to claim 9, characterized in that: The timing valve is provided with dense transverse grooves for blocking the fluid, reducing the liquid flow rate, and prolonging the reaction time of the sample and reagent to be tested; there is a triangular high capillary action area at the end of the timing valve for guiding the fluid to always enter the microfluidic detection area at the same position.

11. The microfluidic chip according to claim 8, characterized in that: The microfluidic chip is sprayed with a nano-scale hydrophilic coating on the surface of the chip bottom plate by ultrasonic atomization spraying technology. The nano-scale hydrophilic coating is prepared by dissolving 4, 3 or 2 of 0.1%-5% PVP, 0.01%-1% sodium alginate, 0.05%-0.3% polyethylene glycol diamine, 0.01%-0.5% methyl carbamate and 0.1%-6% PVA in ethanol, wherein the "%" refers to the mass percentage of each component relative to ethanol, and the mass concentration of the ethanol is 50%-100%.

12. The microfluidic chip according to claim 8, characterized in that: The detection lines and the accusation lines in the detection zone of the microfluidic chip are cross-linked with a cross-linking agent to cross-link the embedded antibodies and the nano-scale hydrophilic coating of the bottom plate. The cross-linking agent is prepared by dissolving 3, 2 or 1 of 0.05%-5% aminosilane, 0.05%-0.3% polyethylene glycol diamine and 0.1%-6% PVA (polyvinyl alcohol) in ethanol, wherein the "%" refers to the mass percentage of each component relative to ethanol, and the mass concentration of ethanol is 75%-100%.

13. The microfluidic chip according to claim 12, characterized in that: The cross-linking agent is prepared by dissolving 0.05%-5% aminosilane, 0.05%-0.3% polyethylene glycol diamine and 0.1%-6% PVA in anhydrous ethanol.

14. A cMyBP-C dual-modality detection method based on a microfluidic chip, wherein the detection method is not for the purpose of disease diagnosis and treatment, and is characterized in that: The method comprises the following steps: S1: Using the microfluidic chip according to any one of claims 7 to 13, establishing standard curves of colorimetric intensity and fluorescence intensity versus cMyBP-C concentration; S2: Dilute the biological sample with a diluent and load it onto the sample loading area of ​​the microfluidic chip according to any one of claims 7 to 13. Load the AEC staining reagent 5 to 8 minutes later. Measure the colorimetric intensity and fluorescence intensity of the test line and the quality control line 5 to 7 minutes later, and compare them with the standard curve. Calculate the dilution multiple to obtain the cMyBP-C concentration in the biological sample.

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