Colorimetric-fluorescent dual-modal nanoprobe and microfluidic chip for detecting cMyBP-C

Abstract

The present invention discloses a colorimetric-fluorescent dual-modal nanoprobe and microfluidic chip for detecting cMyBP-C, belonging to the fields of biomedical testing and in vitro diagnostics. The present invention utilizes a layer-by-layer self-assembly technique to prepare a colorimetric-fluorescent dual-modal nanoprobe composed of UCNPs as a core, a MOF as a shell, and loaded with horseradish peroxidase and a labeled antibody. This probe is then used to prepare a microfluidic chip. This microfluidic chip enables dual-modal detection of both colorimetric and fluorescent cMyBP-C signals, addressing technical issues such as limited sensitivity, susceptibility to background interference, limited linear range, photobleaching, and signal instability in single-signal detection.

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 serious health hazard and is the leading cause of death in my country. It is estimated that there are approximately 330 million cardiovascular patients in my country. Cardiovascular disease is prevalent and is affecting younger people, making it a significant health concern for the nation. It can also trigger a variety of other diseases, particularly major illnesses such as myocardial injury. The causes of myocardial injury are complex and can be caused by insufficient blood or oxygen supply to myocardial cells in various situations, other diseases, drug toxicity, strenuous exercise, a harsh living environment, and unhealthy lifestyle habits, leading to myocardial cell necrosis, apoptosis, or pyroptosis. As mature myocardial cells cannot regenerate, the condition worsens, resulting in heart failure.

[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 myofilament structural protein unique to cardiomyocytes and exists in cardiomyocytes in a phosphorylated form. In the ultra-early stages of acute myocardial infarction, intact cMyBP-C and its hydrolysis products are continuously and massively released into the blood, causing a sharp increase in their blood concentration. Due to its myocardial specificity and large molecular weight, measuring serum cMyBP-C concentration can aid in the diagnosis of 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, require high laboratory conditions, 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 achieve qualitative visual detection of the target through colorimetric signals. The second-generation immunofluorescence method can achieve 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 the 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 fluorescence 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 fluorescence detection each have a specific linear range. 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 signal stability and repeatability; ⑤ signal stability and variability: fluorescence signals 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 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, enhancing the penetration depth of light into biological tissues while effectively reducing interference from tissue autofluorescence. The chemical stability and low toxicity of UCNPs make them suitable for biomedical applications, and their high photostability ensures signal reliability. UCNPs hold broad application prospects in fields such as biosensing, disease diagnosis, and environmental monitoring.

[0008] Currently, there are no reports on UCNPs-based colorimetric-fluorescent dual-modal probes for detecting cMyBP-C. Therefore, 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 objective of the present invention is to provide a colorimetric-fluorescent dual-modal nanoprobe for detecting cMyBP-C and a method for preparing the same. This invention utilizes layer-by-layer self-assembly to form a core-shell complex composed of UCNPs as a core and a MOF shell. After loading with horseradish peroxidase and antibodies, the complex achieves dual-modal detection of cMyBP-C colorimetric and fluorescent signals, addressing technical issues such as sensitivity limitations, susceptibility to background interference, linear range limitations, photobleaching, and signal instability in single-signal detection. A second objective is to provide the use of the colorimetric-fluorescent dual-modal nanoprobe in the preparation of products for detecting cMyBP-C. A third objective is to provide a microfluidic chip based on the colorimetric-fluorescent dual-modal nanoprobe. A fourth objective is to provide a method for dual-modal detection of cMyBP-C.

[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 recorded 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 an anti-cMyBP-C monoclonal antibody, a polyclonal antibody or an 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 of the present invention is a porous material formed by the coordination of metal nodes and multidentate organic ligands.

[0022] The metal node is selected from Zn 2+ 、Cu 2+ 、Fe 3+ 、Al 3+ 、Zr 4+ 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-imidazolecarboxylic 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-modal 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 was dissolved in deionized water, a certain amount of high-temperature solvent was added, and the water was evaporated; the reaction was carried out at 150-170°C under an argon atmosphere for 1-1.5 hours to obtain a transparent yellow solution, which was then cooled to room temperature; the solution was added dropwise to a methanol solution containing NH4F and NaOH, and the methanol solvent was evaporated; the temperature was raised to 290-320°C under a nitrogen atmosphere, and the reaction was maintained for 1-2 hours, and the solution was cooled to room temperature; the solution was ethanol precipitated, centrifuged, and washed to obtain upconversion nanoparticles, which were 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 PEG-phosphate ligand-stabilized upconversion nanoparticles;

[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. The mixture was reacted at 60-65°C for 30-40 minutes. Subsequently, BDC-NH2 solution was added dropwise, and the mixture was heated to 100-120°C and reacted for 1-1.5 hours. A thin layer of UIO-66-NH2 shell was grown on the surface of the UCNPs. The above UIO-66-NH2 growth method was repeated to obtain the desired shell thickness. UCNP@MOF was prepared and 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 and activated by adding NHS and EDC. The labeled antibody was added and reacted for 3-4 hours. The blocking solution was added, centrifuged, and the labeled antibody protection solution was added to resuspend for later 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 the 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, the PEG-phosphate in step (2) 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 concentrations of the ZrOCl2·8H2O solution and the BDC-NH2 solution in step (3) are 5-6 mM, and they are preferably freshly prepared solutions.

[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 direction of sample flow, 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 sample filtration area is installed with a blood filter membrane, and the sample is filtered through the blood filter membrane, and the reaction area is fixed by spotting the colorimetric-fluorescence dual-modal nanoprobe described in the first aspect of the present invention, 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 by spotting the capture antibody, and the spotting volume is 0.2μL-1μL / piece; the quality control line area is fixed by spotting the goat anti-mouse IgG.

[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 sample loading area of ​​the bottom plate, and the air hole is located at the front end of the detection area of ​​the bottom plate.

[0050] Preferably, the reaction zone, time-controlled valve and 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. First, there is a high capillary action zone with a relatively dense array structure (the array structure is arranged obliquely), followed by a small section of low capillary action zone without an array structure, and then 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, each with a diameter of 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 area of ​​the reaction zone, 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 evenly distributed state and contact and mix with the reagent pre-buried in the area (the dual-modal nanoprobe described in the first aspect of the present invention), and is integrated through the horizontally arranged microarray structure. Finally, the time-controlled valve guides the fluid to enter the microfluidic detection area at the same position at a relatively flat gas-liquid interface, 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 base plate through ultrasonic atomization spraying technology. The nano-scale hydrophilic coating is used to change the hydrophilic angle of the chip base plate to regulate the magnitude of the capillary driving force and avoid nonspecific adsorption of biological samples on the chip surface.

[0056] The nanoscale 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 crosslinker is prepared by dissolving 0.05%-5% aminosilane, 0.05%-0.3% polyethylene glycol diamine, and 0.1%-6% PVA (polyvinyl alcohol) in ethanol, where the "%" represents the mass percentage of each component relative to the ethanol, and the mass concentration of the ethanol is 75%-100%. The crosslinker provided by the present invention is rich in amino groups and can stably crosslink the carboxyl termini of the capture antibody, preventing the test line and quality control line from dissolving and falling 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 microfluidic chip provided by the present invention is used to detect cMyBP-C by using immunomicrofluidic technology to achieve colorimetric and fluorescence dual signal quantitative detection of cMyBP-C in the test sample. The detection process is as follows: a sample to be tested is added to the sample addition area of ​​the microfluidic chip, and the sample is filtered through a blood filter membrane in the sample filtration area; when the sample contains cMyBP-C, the cMyBP-C reaches the reaction area through capillary action and binds to the anti-cMyBP-C antibody on the colorimetric-fluorescent dual-modal nanoprobe provided by the present invention fixed in the area, forming an antigen-antibody complex; under the capillary action, the antigen-antibody complex continues to move forward, and when it encounters the detection line (T line) fixed with the capture antibody, the antigen in the antigen-antibody complex binds to the capture antibody to form a double sandwich structure. The antigen-antibody complex is captured in the area, forming a fluorescent band under near-infrared light irradiation, and a color signal appears when a color developer is added; some unbound colorimetric-fluorescent dual-modal nanoprobes continue to move. When they move to the quality control line (C line) coated with goat anti-mouse IgG, they form a primary antibody-secondary antibody conjugate with the goat anti-mouse IgG antibody, fluoresce in the quality control area, and a color signal appears when 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 has no fluorescence and color signal and moves directly to the quality control line (C line). It forms a primary antibody-secondary antibody conjugate with the goat anti-mouse IgG antibody, and shows fluorescence in the quality control area. After the color developer is added, a color signal will appear at the same time.

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

[0062] S1: Using the colorimetric-fluorescence dual-modal microfluidic chip described in the fourth aspect of the present invention, standard curves of colorimetric intensity and fluorescence intensity versus cMyBP-C concentration are established respectively;

[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. After 5-8 minutes, load the AEC staining reagent. After 5-7 minutes, measure the colorimetric intensity and fluorescence intensity of the test line and the quality control line, compare them with the standard curve, and calculate the dilution factor 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 test sample of different concentration gradients is 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. Standard curve 1 is established based on the colorimetric intensity and cMyBP-C concentration, and standard curve 2 is established based on the fluorescence intensity and cMyBP-C concentration.

[0066] Compared with the existing technology, 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, achieving efficient loading of HRP and efficient labeling of antibodies. UCNP can output a fluorescent signal through near-infrared light excitation, avoiding interference from the sample matrix; the MOF-loaded HRP catalyzes the corresponding substrate to output a color signal, allowing the chip to simultaneously perform both colorimetric and fluorescent dual-modal detection. The combination of colorimetric and fluorescent signals can effectively distinguish target signals from background interference signals. The mutual verification of the two helps 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 uses self-driven microfluidic technology to solve some technical difficulties 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 uses technologies such as nano-scale hydrophilic coating and silane coupling agent to ensure the surface treatment of the chip base plate and the stability of the capture antibody, thereby reducing the impact of changes in environmental conditions on the fluorescence signal and ensuring signal stability and repeatability. 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) Schematic diagram of the overall 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 following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts 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 with 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 solution was stirred at room temperature under argon for 1 hour to remove oxygen from the solution. The solution was then slowly heated to 120°C to remove water from the solution and reacted at 156°C for approximately 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. 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 reaction mixture, shaken thoroughly, and 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] 30 mg of the synthesized UCNPs powder was dispersed in 2 mL of anhydrous ethanol containing 1 g of PEG(1000)-phosphate ligand. The solution was slowly heated to 70°C and allowed to react 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. The reaction product was washed twice with cyclohexane (2 mL) and then four times with anhydrous ethanol (2 mL) to remove free PEG(1000)-phosphate ligands from the reaction system. Finally, the final product was redispersed in 2 mL of N,N-dimethylformamide (DMF) for subsequent coating with the UIO-66-NH2 shell.

[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 using the layer-by-layer self-assembly method. The specific operation steps were 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 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. The intermediate product was collected by centrifugation at 9000 rpm for 10 minutes. To further increase the thickness of the MOF shell, the "layer-by-layer self-assembly" method can be repeated to form MOF shells of varying thicknesses on the surface of UCNPs. Finally, the final product was washed alternately with ethanol and deionized water six 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, forming MOF shells of varying thicknesses on the surface of the UCNPs. The inventors found that the core-shell complexes coated once and twice, respectively, had poor nanoparticle stability and poor dispersibility in aqueous solution due to the thin thickness of the MOF shell, and both exhibited a certain degree of agglomeration. The thickness of the MOF shell after four coatings was approximately 14.5 nm, indicating good dispersibility. However, under a TEM microscope, the thickness of the MOF coating layer on the surface of the UCNPs was uneven, and the thickness uniformity was not as good as that after three coatings. Ultimately, 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-mentioned 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 the ideal thickness; element mapping, elements Y, F, Tm and Zr are evenly distributed on the nanoparticles ( Figure 2 D), showing 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 Antibody Modification on 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 incubated on a shaker for 8 hours to obtain an HRP-loaded core-shell complex, designated UCNP@MOF / HRP. Subsequently, the UCNP@MOF / HRP complex was dispersed in 100 μL of MES buffer, 1.5 μL of NHS solution was added, and the mixture was mixed on a shaker for 1 minute. Then, 1.5 μL of EDC solution was added, and the mixture was incubated on a 37°C shaker for 30 minutes. The mixture was then centrifuged and redispersed in 200 μL of HEPES buffer. 100 μg of labeled antibody (anti-cMyBP-C antibody) was added to the above solution, and the mixture was incubated on a 37°C shaker for 120 minutes. Then, 30 μL of blocking buffer was added, ultrasonically mixed for 1 minute, and the reaction continued for another 60-120 minutes. After the reaction, the probe was collected by centrifugation at 9000 rpm for 15 minutes. 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 a colorimetric-fluorescence dual-modal microfluidic chip

[0089] The colorimetric-fluorescence dual-modal microfluidic chip consists of a top cover and a chip base. The top cover has an elliptical sample injection port and an air hole. The elliptical sample injection port is located in the front half of the base sample injection area, and the air hole is located at the front end of the base detection area. The base structure is finely processed by injection molding in one step. A 0.05-1.00mm welding line is set on the base. A nano-scale hydrophilic coating is sprayed on the base surface using ultrasonic atomization spraying technology. The nano-scale hydrophilic coating material is prepared by the following method: PVP and PVA are added to anhydrous ethanol in a certain amount (2% by weight of the anhydrous ethanol) and stirred for 10-15 minutes; polyethylene glycol diamine is added in a certain amount (0.3% by weight of the anhydrous ethanol) and methyl carbamate is added in a certain amount (0.3% by weight of the anhydrous ethanol) and stirred for 10-15 minutes; finally, sodium alginate is added in a certain amount (1% by weight of the anhydrous ethanol) and stirred for 10-15 minutes to prepare the 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, which are 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.

[0091] According to the direction of sample flow, 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 diagonally), followed by a small 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 transversely); the UCNP@MOF@Ab / HRP prepared in Example 1 was fixed in the reaction zone by spotting, with a spotting volume of 2.1 μL / sheet and dried at room temperature.

[0092] Dense transverse grooves are set in the time-controlled 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 time-controlled 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 11,000-12,000 microstructures, each with a diameter of 50±5 μm, which can significantly increase the specific surface area of ​​the flow channel and enhance antigen capture efficiency. A test line and a quality control line are set up in the detection area. A crosslinker is first sprayed on the test line and quality control line areas and allowed to dry at room temperature. The cMyBP-C capture antibody is fixed in the test line area by spotting, with a spotting volume of 0.42 μL / sheet. The goat anti-mouse IgG is fixed in the quality control line area with a spotting volume of 0.42 μL / sheet, and allowed to dry at room temperature. In the experimental phase of the present invention, two crosslinkers, namely crosslinker 1 and crosslinker 2, are configured. Crosslinker 1 is prepared by the following method: aminosilane (4% by weight of the anhydrous ethanol) and PVA (2% by weight of the anhydrous ethanol) are sequentially added to anhydrous ethanol, stirred for 10-15 minutes, and polyethylene glycol diamine (0.3% by weight of the anhydrous ethanol) is continuously added and stirred for 10-15 minutes to prepare the 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 were sprayed on two base plates respectively, and the effects of the cross-linkers on the stability of the detection line were verified respectively. The final verification results showed that cross-linker 1 had 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 had good stability. When the chip sprayed with cross-linker 2 had a large sample volume, the detection line and the quality control line would be offset as the sample flowed, affecting the interpretation of the final signal intensity. The present invention preferably uses cross-linker 1 as a cross-linker for preparing microfluidic chips. 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 test 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, the chip surface is processed with multiple 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 addition area (a) is designed with horizontal and vertical guide groove structures, which can realize 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. 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 pre-buried in the area, and further integrate through the horizontally 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 horizontal grooves to block the fluid, reduce the liquid flow rate, and extend 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-modality microfluidic chip

[0097] Take 20 μL of 5 ng / mL cMyBP-C corporate reference product, 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 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] 20 μL of cMyBP-C corporate reference material 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 were taken and mixed with 480 μL of diluent to prepare a series of cMyBP-C sample solutions with a concentration gradient; 280 μL was loaded onto the sample loading area of ​​the microfluidic chip, and 100 μL of AEC staining reagent was loaded again 8 minutes later. After 5 minutes, the colorimetric and fluorescence detection instruments supporting the microfluidic chip were used to measure the colorimetric intensity and fluorescence intensity of the test line and the quality control line. The standard curves were established with cMyBP-C concentration as the horizontal axis and colorimetric intensity and fluorescence intensity as the vertical axis, and the performance indicators such as linear equation, correlation coefficient, linear range and detection limit of the standard curves corresponding to the colorimetric and fluorescence signals were analyzed. 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 demonstrate a significant linear relationship between the colorimetric and fluorescent signals and cMyBP-C concentration, with a correlation coefficient >0.99. The cMyBP-C concentration in the sample can be quantitatively determined by measuring the colorimetric and fluorescent signal intensities of the sample, and then using the provided standard curve. Furthermore, the detection limit data demonstrates that the colorimetric-fluorescence dual-modality signal can extend the linear range of detection. In the low concentration range, the fluorescent signal provides highly sensitive detection, while in the high concentration range, the colorimetric signal provides stable quantitative analysis, thus 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 and mixed for 1 minute, then 1.5 μL EDC solution is added, and the mixture is placed on a 37°C constant temperature shaker for 30 minutes, and then centrifuged and redispersed in 200 μL HEPES buffer. 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 minutes, then add 30 μL of blocking solution, ultrasonically mix for 1 minute, and continue the reaction for 60-120 minutes. 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, centrifuged to remove the supernatant, and resuspended in 500 μL labeled antibody protection solution to obtain UCNP@MOF@Ab.

[0106] Comparative Example 2 Preparation of a Microfluidic Chip Without Colorless Signal

[0107] The preparation method is the same as that in Example 2, with the only difference being the reaction area of ​​the chip bottom plate, specifically the UCNP@MOF@Ab prepared in Comparative Example 1 is fixed by spotting, with a spotting volume of 2.1 μL / chip. The settings of other structural areas are exactly the same as in Example 2.

[0108] Take 20 μL of the cMyBP-C enterprise reference product with a concentration of 0.01 ng / mL and mix it with 480 μL of diluent to prepare a low-concentration cMyBP-C sample solution. A total of 100 copies are prepared, and 100 blank controls are set up at the same time. The 200 samples are randomly arranged and qualitatively tested using the microfluidic chips prepared in Example 2 and Comparative Example 2 according to the method provided in Example 3. Among them, the microfluidic chip prepared in Comparative Example 2 is used for detection without loading the AEC staining reagent, and the detection result is only the fluorescence 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 fluorescence signal is used as the basis for interpretation. The background signal in the system may interfere with the target signal, resulting in false positive results. The addition of colorimetric signals can correct false positive data. Therefore, the combination of colorimetric and fluorescent signals can effectively distinguish target signals from background interference signals. The mutual verification between the two helps to reduce false positive and false negative results and improve the accuracy of detection.

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

[0110] The preparation method is the same as that of Example 2, with the only difference being the array structure of the reaction area of ​​the chip base plate. The array structure is: first, a high capillary action area with a relatively dense array structure (the array structure is arranged obliquely), followed by a medium capillary action area with a relatively sparse array structure (the array structure is arranged horizontally), and no low capillary action area. The arrangement of other structural areas is exactly the same as that of 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, except 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. 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 samples). 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 using the microfluidic chips prepared in Comparative Examples 3 and 4 to generate standard curves, technicians found that the correlation coefficients of the obtained standard curves were significantly lower than those of the microfluidic chip prepared in Example 2. The correlation coefficients of the colorimetric signal standard curve decreased from 0.9934 to 0.9615 and 0.9462, respectively, and the correlation coefficients of the fluorescence signal standard curve decreased from 0.9996 to 0.9482 and 0.9133, respectively. Technical personnel 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 excellent control over the chromatography speed, ensuring that the antigen in the sample to be tested fully binds to the UCNP@MOF@Ab / HRP, increasing the stability of the formed antigen-antibody complex, and ensuring that the antigen-antibody complex binds to the capture antibody on the detection line at a uniform rate, forming a stable colorimetric-fluorescent signal. If the array structure of the reaction zone is arbitrarily changed, or the time-controlled valve structure is eliminated, the antigen in the sample to be tested will not fully bind to the UCNP@MOF@Ab / HRP, the formed antigen-antibody complex structure will be unstable, and it will bind to the capture antibody at an uneven rate, 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 above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above 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. Use of a colorimetric-fluorescent dual-modal nanoprobe in preparing a product for detecting cMyBP-C, characterized in that: The colorimetric-fluorescent dual-modal nanoprobe is composed of 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. The colorimetric-fluorescence dual-modal nanoprobe is prepared by the following method: (1) Preparation of UCNPs The rare earth raw material was dissolved in deionized water, a certain amount of high-temperature solvent was added, and the water was evaporated. The reaction was carried out at 150-170°C under an argon atmosphere for 1-1.5 hours to obtain a transparent yellow solution, which was then cooled to room temperature. The solution was then added dropwise to a methanol solution containing NH4F and NaOH, and the methanol solvent was evaporated. The temperature was raised to 290-320°C under a nitrogen atmosphere and the reaction was maintained for 1-2 hours, and the solution was cooled to room temperature. The solution was precipitated with ethanol, centrifuged, and washed to obtain upconversion nanoparticles, which were then 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 PEG-phosphate ligand-stabilized upconversion nanoparticles; (3) Preparation of UCNP@MOF The PEG-phosphate ligand-stabilized UCNPs prepared above were dispersed in DMF, and ZrOCl2•8H2O solution was added dropwise. The mixture was reacted at 60-65°C for 30-40 minutes. Subsequently, BDC-NH2 solution was added dropwise, and the mixture was heated to 100-120°C and reacted for 1-1.5 hours. A thin layer of UIO-66-NH2 shell was grown on the surface of the UCNPs. The above UIO-66-NH2 growth method was repeated to obtain the desired shell thickness. UCNP@MOF was prepared and 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 and activated by adding NHS and EDC. The labeled antibody was reacted for 3-4 hours, blocking solution was added, centrifuged, and antibody protection solution was added to resuspend for later use to prepare UCNP@MOF@Ab / HRP.

2. The use 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 use according to claim 1, characterized in that The high-temperature solvent in step (1) is a combination of oleic acid and octadecene; and the rare earth raw material is a combination of YCl3, YbCl3 and TmCl3.

4. A colorimetric-fluorescence dual-modality microfluidic chip for detecting cMyBP-C, 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 / chip; the detection area includes a detection line and a quality control line, and the detection line area is fixed by spotting the capture antibody, and the capture antibody is an anti-cMyBP-C antibody, and the spotting volume is 0.2μL-1μL / chip; the quality control line area is fixed by spotting the goat anti-mouse IgG; The reaction zone, time-controlled valve, and detection zone are composed of flow channels and micron-scale micro-column structures. Three array structures are sequentially arranged in the reaction zone according to the sample flow direction. First, there is a high capillary action zone with a relatively dense array structure, whose array structure is arranged diagonally; followed by a short low capillary action zone without an array structure; and finally, a medium capillary action zone with a relatively sparse array structure, whose array structure is arranged horizontally. 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.

5. The microfluidic chip according to claim 4, characterized in that: The microfluidic chip is sprayed with a nano-scale hydrophilic coating on the surface of the chip base plate using ultrasonic atomization spraying technology. The nano-scale hydrophilic coating is prepared by dissolving 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. The "%" refers to the mass percentage of each component relative to ethanol, and the mass concentration of ethanol is 50%-100%.

6. The microfluidic chip according to claim 4, 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 embed antibodies and the nano-scale hydrophilic coating of the base plate. 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 ethanol. The "%" refers to the mass percentage of each component relative to ethanol, and the mass concentration of ethanol is 75%-100%.

7. A dual-modality detection method for cMyBP-C based on a microfluidic chip, wherein the detection method is not intended for 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 4 to 6, establishing standard curves of colorimetric intensity and fluorescence intensity versus cMyBP-C concentration, respectively; 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 4 to 6. After 5 to 8 minutes, load the AEC staining reagent. After 5 to 7 minutes, measure the colorimetric intensity and fluorescence intensity of the test line and the quality control line, and compare them with the standard curve. By calculating the dilution factor, the cMyBP-C concentration in the biological sample is obtained.

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