Colorimetric-fluorescent dual-mode nanoprobe and microfluidic chip for detecting cmybp-c

Abstract

The present invention belongs to the technical field of biomedical detection and in-vitro diagnosis. Disclosed are a colorimetric-fluorescent dual-mode nanoprobe and microfluidic chip for detecting cMyBP-C. In the present invention, a colorimetric-fluorescent dual-mode nanoprobe, which comprises UCNPs as a core and MOF as a shell and is loaded with horseradish peroxidase and a labeled antibody, is prepared by means of a "layer-by-layer self-assembly" technique; and then a microfluidic chip is prepared on the basis of the nanoprobe. The microfluidic chip realizes dual-mode detection of colorimetric and fluorescent signals of cMyBP-C, thereby solving technical problems such as limited sensitivity, susceptibility to background interference, limited linear range, photobleaching and signal instability in single-signal detection.

Description

Colorimetric-fluorescence dual-modal nanoprobe and microfluidic chip for detecting cMyBP-C Technical Field

[0001] This invention belongs to the field of biomedical detection and in vitro diagnostic technology, and particularly relates to a colorimetric-fluorescence dual-modal nanoprobe and microfluidic chip for detecting cMyBP-C. Background Technology

[0002] Cardiovascular disease is a serious threat to human health 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 showing a high incidence and affecting younger people, making it a significant factor impacting national health. Furthermore, cardiovascular disease can trigger various other illnesses, especially serious conditions such as myocardial damage. The causes of myocardial damage are complex, potentially including insufficient blood or oxygen supply to myocardial cells under various conditions, other diseases, drug toxicity, strenuous exercise, harsh living environments, and unhealthy lifestyle habits. These factors lead to myocardial cell necrosis, apoptosis, or pyroptosis. Since mature myocardial cells cannot regenerate, the condition worsens, eventually leading to heart failure.

[0003] When myocardial injury occurs, the left ventricle accumulates specific cardiac proteins and releases them into the bloodstream. These specific proteins combine with inflammatory markers and other blood indicators to form a specific set of biomarkers. Depending on the timing and concentration of their release into the blood, they have different clinical significance. Monitoring the release time and concentration of these specific biomarkers can reveal the degree of myocardial injury.

[0004] Cardiac myosin binding protein-C (cMyBP-C) is a thick myofibril structural protein specific to cardiomyocytes. It exists in cardiomyocytes in a phosphorylated form. In the very early stage of acute myocardial infarction, intact cMyBP-C and its hydrolysis products are continuously and massively released into the blood, and its blood concentration rises sharply. Due to its myocardial specificity and large molecular weight, detecting serum cMyBP-C concentration can help in the diagnosis of myocardial injury.

[0005] Currently, the main methods for detecting cardiac biomarkers include chemiluminescence immunoassay, first-generation colloidal gold immunoassay, and second-generation immunofluorescence immunoassay. Chemiluminescence immunoassay offers advantages such as high sensitivity and a wide detection range, but its equipment is expensive, large in size, requires sophisticated laboratory procedures, and demands specialized operation, making it unsuitable for point-of-care testing. First-generation colloidal gold immunoassay, based on a chromatography platform, enables qualitative and visual detection of targets through colorimetric signals. Second-generation immunofluorescence immunoassay enables quantitative detection of targets through fluorescence signals, but it is limited by the inherent physical properties of the chromatography membrane, such as inconsistent pore sizes and the inability to guarantee consistent liquid flow, resulting in poor detection accuracy (CV ≥ 15%), low sensitivity, and significant fluctuations in results depending on incubation time.

[0006] Furthermore, single colorimetric or fluorescence signal detection faces several challenges, such as: ① Sensitivity limitations: Single colorimetric detection cannot accurately detect low concentrations or weak signal changes, limiting 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, leading to inaccurate results; ③ Linear range limitations: Colorimetric and fluorescence detection each have specific linear ranges. Beyond these ranges, the relationship between signal intensity and concentration may no longer be linear, affecting the accuracy of quantitative analysis; ④ Photobleaching: 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, and ionic strength, leading to signal instability and variability.

[0007] Lanthanide-doped upconversion nanoparticles (UCNPs) have shown 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 interference from autofluorescence in biological tissues. The chemical stability and low toxicity of UCNPs make them suitable for biomedical applications, while their high photostability ensures signal reliability. UCNPs have broad application prospects in biosensing, disease diagnosis, and environmental monitoring.

[0008] Currently, there are no reports on colorimetric-fluorescence dual-modal probes based on UCNPs for the detection of cMyBP-C. Therefore, this invention provides a colorimetric-fluorescence dual-modal nanoprobe based on UCNPs that can detect cMyBP-C, realizing dual-modal detection of cMyBP-C colorimetric and fluorescence signals, and solving technical problems such as sensitivity limitation and susceptibility to background interference in single signal detection. Summary of the Invention

[0009] One objective of this invention is to provide a colorimetric-fluorescence bimodal nanoprobe for detecting cMyBP-C and its preparation method. This invention utilizes a layer-by-layer self-assembly process to form a core-shell complex with UCNPs as the core and an MOF shell, followed by loading horseradish peroxidase and antibody. This enables bimodal detection of cMyBP-C through both colorimetric and fluorescence signals, solving technical problems such as sensitivity limitations, susceptibility to background interference, linear range limitations, photobleaching, and signal instability inherent in single-signal detection. A second objective of this invention is to provide the application of the aforementioned colorimetric-fluorescence bimodal nanoprobe in the preparation of products for detecting cMyBP-C. A third objective of this invention is to provide a microfluidic chip based on the aforementioned colorimetric-fluorescence bimodal nanoprobe. A fourth objective of this invention is to provide a method for bimodal detection of cMyBP-C.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] In a first aspect, the present invention provides a colorimetric-fluorescence bimodal nanoprobe for detecting cMyBP-C, characterized in that the bimodal nanoprobe uses upconversion nanoparticles (UCNPs) as the core, metal-organic frameworks (MOFs) as the shell, the shell is loaded with horseradish peroxidase (HRP), and the surface of the shell is coupled with labeled antibodies (Abs) to form a colorimetric-fluorescence bimodal nanoprobe, 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 commercially.

[0014] The upconversion 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+ One or a combination of two of them, with a molar content of sensitizing ions ranging from 0.01% to 60%.

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

[0017] The activating ions are selected from Pr 3+ 、Nd 3+ 、Sm 3+ 、Tb 3+ Ho3+ Er 3+ Tm 3+ One or more combinations of the active ions, with a molar content of 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, wherein 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 PEG-phosphate ligand-stabilized upconversion nanoparticles, specifically, the PEG-phosphate ester is PEG(1000)-phosphate ester.

[0021] The metal-organic framework described in this invention is a porous material formed by the coordination of metal nodes with multidentate organic ligands.

[0022] The metal nodes are selected from Zn. 2+ Cu 2+ Fe 3+ Al 3+ Zr 4+ One or more of them, with the molar content of metal nodes ranging from 0.01% to 60%.

[0023] The multidentate organic ligand is selected from one or more of terephthalic acid, dimethyl terephthalate, and 2-imidazolium 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] Secondly, the present invention provides a method for preparing a colorimetric-fluorescence dual-modal nanoprobe for detecting cMyBP-C, characterized in that the preparation method includes the following steps:

[0027] (1) Preparation of UCNPs

[0028] Rare earth raw materials were dissolved in deionized water, and a certain amount of high-temperature solvent was added. The water was evaporated to dryness. The reaction was carried out at 150-170℃ for 1-1.5h under an argon atmosphere 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 to dryness. The temperature was raised to 290-320℃ under a nitrogen atmosphere and kept at that temperature for 1-2h, then cooled to room temperature. The solution was precipitated with ethanol, centrifuged, washed, and the upconversion nanoparticles were obtained and stored in cyclohexane for later use.

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

[0030] The above-prepared upconversion nanoparticles were dispersed in anhydrous ethanol containing PEG-phosphate ligands, slowly heated to 60-70℃, reacted for 8-10 h, 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 reaction was carried out at 60-65℃ for 30-40 min, followed by the dropwise addition of BDC-NH2 solution. The reaction was carried out at 100-120℃ for 1-1.5 h to grow a thin UIO-66-NH2 shell on the surface of UCNPs. The UIO-66-NH2 growth method was repeated to obtain the ideal shell thickness, and 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, and HRP was added to a final concentration of 1.0-1.5 mg / mL. The mixture was incubated at room temperature for 8-24 hours, centrifuged, washed, and the HRP-loaded nanoparticles were redispersed. NHS and EDC were added for activation, and labeled antibody was added for reaction for 3-4 hours. Blocking buffer was added, centrifuged, and labeled antibody protective buffer was added for resuspending. UCNP@MOF@Ab / HRP was thus prepared.

[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 mentioned in step (1) is selected from one or more combinations 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 ester is PEG(1000)-phosphate ester, and the mass ratio of the upconversion nanoparticles to the PEG(1000)-phosphate ester is (30-40mg):1g.

[0040] In step (3), the molar concentrations of the ZrOCl2·8H2O solution and the BDC-NH2 solution are 5-6 mM, preferably freshly prepared solutions.

[0041] Thirdly, the present invention provides the use of a colorimetric-fluorescence dual-modal nanoprobe in the preparation of products for detecting cMyBP-C.

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

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

[0044] Fourthly, the present invention provides a colorimetric-fluorescence dual-modal microfluidic chip for detecting cMyBP-C, characterized in that the microfluidic chip contains the colorimetric-fluorescence 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 a top cover. The base plate is precision-formed by one-time injection molding. The base plate is provided with welding lines of 0.05-1.00mm. The surface of the top cover is flat and smooth. After welding, the base plate and the top cover can form a microchannel with capillary driving force.

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

[0047] The detection area includes a detection line and a control line. The detection line area is used to fix and capture antibodies by spotting, with a spotting volume of 0.2 μL-1 μL / piece. The control line area is used to fix 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 inlet and an air hole. The elliptical sample inlet is located in the front half of the sample inlet 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 micropillar structures.

[0051] In a specific embodiment of the present invention, the reaction zone is provided with three array structures in sequence 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), then there is a short section of low capillary action zone without an array structure, and then there is a medium capillary action zone with a relatively sparse array structure (the array structure is arranged laterally).

[0052] In a specific embodiment of the present invention, the time-controlled valve is provided 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 end of the time-controlled valve, there is a triangular high capillary action area, which guides the fluid to always enter the microchannel detection area at the same position.

[0053] In a specific embodiment of the present invention, the area of ​​the microchannel detection region is 100-105 mm². 2 The region includes 11,000-12,000 microstructures with a diameter of 50±5 μm, which can significantly increase the specific surface area of ​​the flow channel, increase antigen capture efficiency, and improve the dynamic detection range, sensitivity, and repeatability of the microfluidic chip.

[0054] The advantages of the microfluidic chip reaction zone provided by this invention are as follows: the sample first enters and fills the high capillary action zone of the reaction zone, the faster-moving fluid is slightly blocked by the low capillary action zone, waiting for the liquid behind to catch up, and then the liquid enters the medium capillary action zone in a relatively uniform distribution state and comes into contact with and mixes with the reagent (the dual-modal nanoprobe described in the first aspect of this invention) pre-embedded in the zone, and is integrated through the laterally arranged microarray structure. Finally, the fluid enters the time-controlled valve through a relatively flat gas-liquid interface to guide the fluid to always enter the microchannel detection zone at the same position, thereby ensuring the consistency of sample flow rate and direction and the stability of the reaction system.

[0055] The microfluidic chip is coated with a nanoscale hydrophilic coating of controllable thickness on the chip substrate surface using ultrasonic atomization spraying technology. The nanoscale hydrophilic coating is used to change the hydrophilic angle of the chip substrate to regulate the magnitude of capillary driving force and avoid non-specific adsorption of biological samples on the chip surface.

[0056] The nanoscale hydrophilic coating is prepared by dissolving 0.1%-5% PVP (polyvinylpyrrolidone), 0.01%-1% sodium alginate, 0.05%-0.3% polyethylene glycol diamine, 0.01%-0.5% methyl urethane, and 0.1%-6% PVA (polyvinyl alcohol) in ethanol. 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 control line areas of the microfluidic detection zone use a cross-linking agent to cross-link the embedded antibody and the substrate with a nanoscale hydrophilic coating. The cross-linking agent is a mixture of silane coupling agent and high molecular weight organic solvent, and the embedded antibody is a capture antibody and goat anti-mouse IgG.

[0058] The crosslinking 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, comprising three, two, or one of these components. The percentage refers to the mass percentage of each component relative to ethanol, and the mass concentration of ethanol is 75%-100%. The crosslinking agent provided by this invention is rich in amino groups, which can stably crosslink the carboxyl terminus of the capture antibody, preventing the detection line and control line from dissolving and detaching as the blood sample flows.

[0059] Preferably, the crosslinking 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 this invention for detecting cMyBP-C is as follows: using immunomicrofluidic technology, the colorimetric-fluorescence dual-signal quantitative detection of cMyBP-C in the test sample is realized. The detection process is as follows: The sample to be tested is added to the sample application area of ​​the microfluidic chip, and the sample is filtered through a blood filtration membrane in the sample filtration area. When the sample contains cMyBP-C, cMyBP-C reaches the reaction area through capillary action and binds to the anti-cMyBP-C antibody on the colorimetric-fluorescent bimodal nanoprobe provided by this invention, which is fixed in this area, to form an antigen-antibody complex. Under capillary action, the antigen-antibody complex continues to move forward. When it encounters the detection line (T line) with the fixed 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 by this area and forms a fluorescent band under near-infrared light irradiation. A color signal will appear simultaneously after the addition of a chromogenic agent. Some of the colorimetric-fluorescent bimodal nanoprobes that have not bound will continue to move. When they move to the 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, which will fluoresce in the control area. A color signal will appear simultaneously after the addition of a chromogenic agent. When the sample does not contain cMyBP-C, the colorimetric-fluorescent bimodal nanoprobe fixed in the reaction zone will not bind to the capture antibody on the detection line (T line), and the detection line will not show any fluorescence or color signal. It will move directly to the control line (C line) and form a primary-secondary antibody conjugate with the goat anti-mouse IgG antibody, showing fluorescence in the control zone. After the chromogenic agent is added, a color signal will appear at the same time.

[0061] Fifthly, 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 by comprising 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 diluent and load it onto the sample application area of ​​the microfluidic chip described in the fourth aspect of this invention. After 5-8 minutes, load the AEC staining reagent. After 5-7 minutes, measure the colorimetric intensity and fluorescence intensity of the detection line and control line. Compare 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 known concentration of cMyBP-C standard is mixed and diluted with a diluent to form different concentration gradients. 280 μL of the test sample of different concentration gradients is loaded onto the sample loading area of ​​the microfluidic chip. After 5-8 min, 3-amino-9-ethylcarbazole (AEC) staining reagent is loaded again. After 5-7 min, the colorimetric intensity and fluorescence intensity of the detection line and control line are measured. 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 prior art, the technical solution provided by the present invention has the following technical advantages:

[0067] 1) This invention employs a layer-by-layer self-assembly technique to coat the surface of a UCNP with a MOF (UIO-66-NH2) shell, achieving efficient loading of HRP and efficient antibody labeling. The UCNP can output a fluorescence signal upon near-infrared light excitation, avoiding interference from the sample matrix; the MOF-loaded HRP catalyzes the corresponding substrate to output a color signal, enabling the chip to simultaneously perform colorimetric and fluorescence dual-modal detection. The combination of colorimetric and fluorescence signals can effectively distinguish between target signals and background interference signals, and mutual verification between the two helps reduce false positive and false negative results, improving detection accuracy; the dual-modality can extend the linear range of detection. In the low concentration range, the fluorescence 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) This invention uses self-driven microfluidic technology to solve some of the inherent technical problems of existing chromatography platforms, such as poor detection accuracy (CV≥15%), low sensitivity, and large fluctuations in detection results with different incubation times.

[0069] 3) This invention employs technologies such as nanoscale hydrophilic coatings and silane coupling agents to ensure the stability of the chip substrate surface treatment and the capture antibody, thereby reducing the impact of environmental changes on the fluorescence signal and ensuring the stability and reproducibility of the signal. Attached Figure Description

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

[0071] [Corrected according to Rule 91 25.07.2025] Figure 2 Characterization of the core-shell structure of UCNP@UIO-66-NH2; (A) OA-encapsulated UCNPs, (B) PEG-phosphate modified UCNPs, (C) TEM image of UCNP@UIO-66-NH2 (UIO-66-NH2 shell is indicated by red arrow); (D) Elemental mapping analysis of UCNP@UIO-66-NH2; (E) OA-encapsulated UCNPs, (F) PEG-phosphate modified UCNPs, (G) Particle size distribution of UCNP@UIO-66-NH2.

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

[0073] Figure 4. Photographs and fluorescence images of cMyBP-C detected by the colorimetric-fluorescence dual-modal microfluidic chip.

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

[0075] Figure 6. Standard curve of cMyBP-C concentration versus fluorescence signal intensity. Detailed Implementation

[0076] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

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

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

[0079] Step 1: Preparation of PEG-phosphate ligand-stabilized UCNPs

[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 atmosphere for 1 hour to remove oxygen. The solution was then slowly heated to 120 °C to remove water and reacted at 156 °C for about 1 hour until a homogeneous, transparent yellow solution was obtained. After cooling the solution to room temperature under argon atmosphere, 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 held for 20 minutes to evaporate the methanol. Subsequently, the solution was heated to 290 °C and reacted for 2 hours, after which it was cooled to room temperature. Add 20 mL of anhydrous ethanol to the above reaction solution, shake thoroughly, and centrifuge at 9000 rpm for 10 minutes. Collect the centrifuged product, i.e., UCNPs. After repeated washing with cyclohexane and ethanol, redisperse the final product in 20 mL of cyclohexane, freeze-dry, and prepare for further use.

[0081] The synthesized 30 mg 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 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. The product was washed twice with cyclohexane (2 mL) and then four times with anhydrous ethanol (2 mL) to remove free PEG(1000)-phosphate ligand. 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 the UCNP@UIO-66-NH2 core-shell complex

[0083] This experiment employed a "layer-by-layer self-assembly" method to coat UCNPs with a UIO-66-NH2 shell. The specific steps were as follows: 2 mL of the PEG(1000)-phosphate ligand-stabilized UCNPs dispersion was added to 8 mL of DMF, followed by dropwise addition of 5 mL of freshly prepared ZrOCl2·8H2O solution (5 mM). The reaction was carried out at 60 °C with stirring for 40 minutes to allow for efficient adsorption of Zr on the UCNPs surface. 4+Ions. Subsequently, 5 mL of freshly prepared BDC-NH2 solution (5 mM) was added dropwise to the solution, which was then heated to 120 °C and reacted for 1 hour. A thin 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 above method of growing the UIO-66-NH2 shell can be repeated using a "layer-by-layer self-assembly" method to form MOF shells of different thicknesses on the surface of UCNPs. Finally, the final product was washed six times alternately with ethanol and deionized water and redispersed in 10 mL of DMF for short-term stable storage.

[0084] This invention involves coating the surface of UCNPs once, twice, three times, and four times according to the above method, forming MOF shells of different thicknesses on the UCNPs surface. The inventors found that the core-shell composites with one and two coatings exhibited poor nanoparticle stability and dispersion in aqueous solution due to the thin MOF shell, showing a certain degree of aggregation. The four-coating MOF shell had a thickness of approximately 14.5 nm and better dispersion; however, TEM revealed uneven MOF coating thickness on the UCNPs surface, with less uniform thickness than the three-coating method. Ultimately, this invention preferably uses the above method to coat the UCNPs surface three times.

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

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

[0087] UCNP@UIO-66-NH2 was dispersed in Tris-HCl buffer, and HRP was added to a final concentration of 1.0 mg / mL. The mixture was incubated on a shaker for 8 hours to obtain an HRP-loaded core-shell complex, named UCNP@MOF / HRP. Subsequently, UCNP@MOF / HRP was dispersed in 100 μL of MES buffer, and 1.5 μL of NHS solution was added. The mixture was shaken for 1 min, followed by the addition of 1.5 μL of EDC solution. The mixture was incubated on a shaker at 37°C for 30 min, 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 shaker at 37°C for 120 min. Then, 30 μL of blocking buffer was added, and the mixture was sonicated for 1 min, continuing the reaction for 60–120 min. After the reaction, the constructed probe was collected by centrifugation at 9000 rpm for 15 min. Finally, the collected product was washed three times with HEPES buffer to remove free antibody, centrifuged to remove supernatant, and resuspended in 500 μL of labeled antibody protection solution to obtain UCNP@MOF@Ab / HRP.

[0088] Example 2: Fabrication of a colorimetric-fluorescence dual-modal microfluidic chip

[0089] The colorimetric-fluorescence dual-modal microfluidic chip consists of a top cover and a chip substrate. The top cover has an elliptical sample inlet and vents. The elliptical sample inlet is located in the front half of the sample application area of ​​the substrate, and the vents are located at the front end of the detection area of ​​the substrate. The substrate is precision-machined by a single injection molding process. A 0.05-1.00 mm welding line is provided on the substrate. A nano-scale hydrophilic coating is sprayed onto the substrate surface using ultrasonic atomization spraying technology. The nano-scale hydrophilic coating material is prepared by the following method: 2% PVP and 2% PVA (by mass of anhydrous ethanol) are added sequentially to anhydrous ethanol and stirred for 10-15 minutes; then 0.3% polyethylene glycol diamine and 0.3% methyl carbamate (by mass of anhydrous ethanol) are added and stirred for 10-15 minutes; finally, 1% sodium alginate (by mass of anhydrous ethanol) is added and stirred for 10-15 minutes to obtain the nano-scale hydrophilic coating material.

[0090] The base plate is equipped with a sample dispensing area, a sample filtration area, a reaction area, a timed valve, a detection area, and a waste liquid pool, which are connected in sequence. The sample dispensing area is used to add the sample to be tested; a blood filtration membrane is installed in the sample filtration area to filter the sample.

[0091] According to the sample flow direction, the reaction zone is arranged with three array structures in sequence: first, a high capillary action zone with a relatively dense array structure (the array structure is arranged obliquely); then, 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 laterally). 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 / piece, and then air-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. A triangular high capillary action area is set at the end of the time-controlled valve to guide the fluid to always enter the microchannel detection area at the same position.

[0093] The area of ​​the microchannel detection region is 105 mm². 2 The region contains 11,000-12,000 microstructures with a diameter of 50±5 μm, which can significantly increase the specific surface area of ​​the flow channel and increase the antigen capture efficiency. A detection line and a control line are set in the detection area. A cross-linking agent is first sprayed onto the detection line and control line areas and then air-dried at room temperature. cMyBP-C capture antibody is fixed in the detection line area by spotting, with a spotting volume of 0.42 μL / piece. Goat anti-mouse IgG is fixed in the control line area by spotting, with a spotting volume of 0.42 μL / piece, and then air-dried at room temperature. In this embodiment of the invention, two cross-linking agents are prepared in the experimental stage, namely cross-linking agent 1 and cross-linking agent 2. Cross-linking agent 1 is prepared by the following method: 4% aminosilane and 2% PVA by mass of anhydrous ethanol are added sequentially to anhydrous ethanol, stirred for 10-15 minutes, and then 0.3% polyethylene glycol diamine by mass of anhydrous ethanol is added, stirred for 10-15 minutes to prepare the cross-linking agent. The preparation method of crosslinking agent 2 is the same as that of crosslinking agent 1, except that it does not contain polyethylene glycol diamine. Crosslinking agent 1 and crosslinking agent 2 were sprayed onto two substrates respectively, and the effect of the crosslinking agents on the stability of the detection lines was verified. The final verification results showed that crosslinking agent 1 had a better crosslinking effect between the antibody and the hydrophilic layer of the substrate, resulting in better stability of the detection and control lines. The chip coated with crosslinking agent 2 experienced deviation of the detection and control lines with sample flow when the sample volume was large, affecting the interpretation of the final signal intensity. Therefore, this invention preferably uses crosslinking agent 1 as the crosslinking agent for preparing the microfluidic chip. Technical personnel believe that polyethylene glycol diamine, as a linear molecule containing amino groups, increases the compatibility of the crosslinking agent with the hydrophilic coating and also enhances the antibody capture effect of the crosslinking agent.

[0094] The waste liquid tank is used to absorb the sample liquid from the end of the chromatography process. Finally, the top cover is installed, the ultrasonic welding program is run, and the welding detection card is welded to complete the fabrication of the microfluidic chip.

[0095] The microfluidic chip prepared in this embodiment is shown in Figure 3. As can be seen from the figure, the chip surface is fabricated with multiple functional units, which can 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 microchannel detection area. The sample loading area (a) is designed with transverse and longitudinal guide channels to facilitate sample flow. The reaction area (b) features three array structures, as described above. The sample first enters and fills the high capillary action zone of the reaction area. Faster-moving fluids are slightly blocked by the low capillary action zone, allowing subsequent liquids to catch up. The liquid then enters the medium capillary action zone in a relatively uniform distribution and mixes with the pre-embedded reagents within this zone. Further integration is achieved through a transversely arranged microarray structure, and finally, a relatively flat gas-liquid interface is established before entering the time-controlled valve (c). The bottom of the time-controlled valve is designed with dense transverse grooves to impede fluid flow, reduce liquid velocity, and extend the reaction time between the sample and reagents. Simultaneously, a triangular high capillary action zone is designed at the end of the time-controlled valve area, guiding the fluid to always enter the microchannel detection area (d) from the same position. At 105 mm... 2 The microfluidic detection region is fabricated with more than 11,000 microstructures, each with a diameter of approximately 50 μm. This significantly increases the specific surface area of ​​the flow channel, enhances antigen capture efficiency, and thereby improves the dynamic detection range, sensitivity, and repeatability of the microfluidic chip.

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

[0097] Take 20 μL of 5 ng / mL cMyBP-C corporate reference and mix it with 480 μL of diluent. Take 280 μL of the sample and load it onto the sample application area of ​​the microfluidic chip. After 8 min, load 100 μL of AEC staining reagent again. After 5 min, observe the color change of the detection line and control line in the detection area of ​​the microfluidic chip and observe the upconversion fluorescence of the detection line and control line using a fluorescence microscope.

[0098] As shown in Figure 4, both color and fluorescence signals indicate that the T-line has a detection signal output, proving that the colorimetric-fluorescence dual-modal microfluidic chip prepared in this invention can be used to detect cMyBP-C and is feasible.

[0099] Example 4: Detection Performance Verification of a Colorimetric-Fluorescence Dual-Modal 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 them with 480 μL of diluent to prepare sample solutions containing cMyBP-C with a series of concentration gradients; 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, use the colorimetric and fluorescence detection instrument supporting the microfluidic chip to measure the colorimetric intensity and fluorescence intensity of the test line and the quality control line. Respectively, use the cMyBP-C concentration as the abscissa and the colorimetric intensity and fluorescence intensity as the ordinate to establish a standard curve, and analyze performance indicators such as the linear equation, correlation coefficient, linear range, and detection limit of the corresponding standard curve under the two signals of colorimetry and fluorescence. The results are shown in Table 1 and Figures 5 - 6.

[0101] Table 1 Linear equations, correlation coefficients, linear ranges, and detection limit indicators corresponding to the two signals of colorimetry and fluorescence

[0102] From the standard curves, linear equations, correlation coefficients, and linear range data shown in Table 1 and Figures 5 - 6, it can be seen that there is a significant linear relationship between the colorimetric and fluorescence signals and the cMyBP-C concentration, and the correlation coefficient > 0.99. The cMyBP-C concentration in the sample can be quantitatively detected by measuring the colorimetric and fluorescence signal intensities of the test sample and then according to the provided standard curve. In addition, from the detection limit data, it can be seen that the colorimetric-fluorescence dual-modal signal can expand the linear range of detection. In the low-concentration range, the fluorescence signal provides high-sensitivity detection; in the high-concentration range, the colorimetric signal provides stable quantitative analysis, thus achieving a wider quantitative linear range.

[0103] Preparation of fluorescent nanoprobe UCNP@MOF@Ab in Comparative Example 1

[0104] The preparation method is the same as in Example 1, except that in step three, HRP is not loaded onto the surface of UCNP@UIO-66-NH2, and the antibody is directly conjugated. Specifically, UCNP@UIO-66-NH2 is dispersed in 100 μL of MES buffer solution, 1.5 μL of NHS solution is added, and the mixture is shaken for 1 min. Then, 1.5 μL of EDC solution is added, and the mixture is placed on a 37°C shaker and reacted for 30 min. After centrifugation, the mixture is redispersed in 200 μL of HEPES buffer. 100 μg of labeled antibody (anti-cMyBP-C antibody) is added to the above solution, and the mixture is placed on a 37°C shaker and reacted for 120 min. Then, 30 μL of blocking buffer is added, and the mixture is sonicated for 1 min, and the reaction continues for 60-120 min. After the reaction, the constructed probe is collected by centrifugation at 9000 rpm for 15 min. Finally, the collected product was washed three times with HEPES buffer to remove free antibody, centrifuged to remove supernatant, and resuspended in 500 μL of labeled antibody protection solution to obtain UCNP@MOF@Ab.

[0105] Comparative Example 2: Fabrication of a microfluidic chip without color signals

[0106] The preparation method is the same as in Example 2, except that the reaction area of ​​the chip substrate is fixed by spotting the UCNP@MOF@Ab prepared in Comparative Example 1, with a spotting amount of 2.1 μL / chip. The settings of other structural regions are exactly the same as in Example 2.

[0107] 20 μL of 0.01 ng / mL cMyBP-C enterprise reference standard was mixed with 480 μL of diluent to prepare a low-concentration cMyBP-C sample solution, totaling 100 aliquots, along with 100 blank controls. These 200 samples were randomly arranged and qualitatively detected using the microfluidic chips prepared in Example 2 and Comparative Example 2, respectively, according to the method provided in Example 3. For the microfluidic chip prepared in Comparative Example 2, no AEC staining reagent was used, and the detection result only showed a fluorescence signal. The final positive rates were: 50% using the microfluidic chip prepared in Example 2 and 53% using the microfluidic chip prepared in Comparative Example 2. The results demonstrate that the microfluidic chip prepared in Example 2 has higher detection accuracy. This is because for low-concentration samples, where only the fluorescence signal is used for interpretation, background signals in the system may interfere with the target signal, leading to false positive results. Adding a colorimetric signal can correct for false positive data. Therefore, the combination of colorimetric and fluorescence signals can effectively distinguish between target signals and background interference signals. The mutual verification between the two helps to reduce false positive and false negative results and improve the accuracy of detection.

[0108] Comparative Example 3: Fabrication of microfluidic chips with different reaction region array structures

[0109] The preparation method is the same as in Example 2, except that the array structure of the reaction area on the chip substrate is as follows: first, there is a high capillary action area with a relatively dense array structure (the array structure is arranged obliquely), then there is a medium capillary action area with a relatively sparse array structure (the array structure is arranged laterally), and there is no low capillary action area. The arrangement of other structural regions is exactly the same as in Example 2.

[0110] Comparative Example 4: Fabrication of a microfluidic chip without a time-controlled valve

[0111] The preparation method is the same as in Example 2, except that the chip substrate does not include a time-controlled valve in its structure. The chip substrate includes a sample application area, a sample filtration area, a reaction area, a detection area, and a waste liquid pool connected in sequence. The specific structure of the area is the same as in Example 2.

[0112] The microfluidic chips prepared in Comparative Examples 3 and 4 were used to detect cMyBP-C samples (>100 samples). The results showed that the microfluidic chips prepared in Comparative Examples 3 and 4 had no impact on the qualitative detection results of the samples, and the qualitative detection results were accurate. However, when using the microfluidic chips prepared in Comparative Examples 3 and 4 to construct standard curves, the technicians found that the correlation coefficients of the obtained standard curves were significantly lower than those of the microfluidic chips prepared in Example 2. 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. Technical personnel analyzed that the three array structures in the reaction zone and the array structure in the time-control valve of this invention effectively control the chromatography speed, ensuring that the antigen in the sample to be tested fully binds to 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-fluorescence signal. If the array structure in the reaction zone is arbitrarily changed, or the time-control valve structure is removed, the antigen in the sample to be tested cannot fully bind to UCNP@MOF@Ab / HRP, the formed antigen-antibody complex structure is unstable, and it binds to the capture antibody at an uneven rate, affecting the stability of the colorimetric-fluorescence signal.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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-fluorescence dual-modal nanoprobe for detecting cMyBP-C, characterized in that, The bimodal nanoprobe uses 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 bimodal nanoprobe, denoted as UCNP@MOF@Ab / HRP. 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 antibodies, polyclonal antibodies, or antigen-specific binding fragments.

3. The dual-modal nanoprobe according to claim 1, characterized in that, The upconversion 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 them; the activating ion is selected from Pr 3+ 、Nd 3+ 、Sm 3+ 、Tb 3+ Ho 3+ Er 3+ Tm 3+ One or more of the following; the metal-organic framework is a porous material formed by the coordination of metal nodes with multidentate organic ligands.

4. A method for preparing the colorimetric-fluorescence dual-modal nanoprobe for detecting cMyBP-C as described in claim 1, characterized in that, The preparation method includes the following steps: (1) Preparation of UCNPs Rare earth raw materials were dissolved in deionized water, and a certain amount of high-temperature solvent was added. The water was evaporated to dryness. The reaction was carried out at 150-170℃ for 1-1.5h under an argon atmosphere 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 to dryness. The temperature was raised to 290-320℃ under a nitrogen atmosphere and kept at that temperature for 1-2h, then cooled to room temperature. The solution was precipitated with ethanol, centrifuged, washed, and the upconversion nanoparticles were obtained and stored in cyclohexane for later use. (2) Preparation of PEG-phosphate ligand-stabilized UCNPs The above-prepared upconversion nanoparticles were dispersed in anhydrous ethanol containing PEG-phosphate ligands, slowly heated to 60-70℃, reacted for 8-10 h, 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 reaction was carried out at 60-65℃ for 30-40 min, followed by the dropwise addition of BDC-NH2 solution. The reaction was carried out at 100-120℃ for 1-1.5 h to grow a thin UIO-66-NH2 shell on the surface of UCNPs. The UIO-66-NH2 growth method was repeated to obtain the ideal shell thickness, and 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, and HRP was added to a final concentration of 1.0-1.5 mg / mL. The mixture was incubated at room temperature for 8-24 hours, centrifuged, washed, and the HRP-loaded nanoparticles were redispersed. NHS and EDC were added for activation, and labeled antibody was added for reaction for 3-4 hours. Blocking buffer was added, centrifuged, and antibody protection buffer was added for resuspending. UCNP@MOF@Ab / HRP was thus prepared.

5. The preparation method according to claim 4, characterized in that, The high-temperature solvent mentioned 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-fluorescence dual-modal nanoprobe of claim 1 in the preparation of a product for detecting cMyBP-C.

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

8. The microfluidic chip according to claim 7, characterized in that, The main structure of the microfluidic chip includes a base plate and a top cover. Following the sample flow direction, the base plate structure includes a sample application area, a sample filtration area, a reaction area, a timing valve, a detection area, and a waste liquid reservoir connected sequentially. The reaction area uses a spotting method to fix the colorimetric-fluorescence dual-modal nanoprobe described in claim 1, with a spotting volume of 1 μL-2 μL / piece. The detection area includes a detection line and a control line. The detection line area uses a spotting method to fix a capture antibody, specifically an anti-cMyBP-C antibody, with a spotting volume of 0.2 μL-1 μL / piece. The control line area uses a spotting method to fix goat 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 micropillar structures. According to the sample flow direction, the reaction zone is arranged with three array structures in sequence. First, there is a high capillary action zone with a relatively dense array structure, whose array structure is arranged obliquely; then there is a short section of low capillary action zone without an array structure; and then there is a medium capillary action zone with a relatively sparse array structure, whose array structure is arranged laterally.

10. The microfluidic chip according to claim 9, characterized in that, The time-controlled valve is equipped with dense transverse grooves to block the fluid, reduce the liquid flow rate, and prolong the reaction time of the sample and reagent. At the end of the time-controlled valve, there is a triangular high capillary action area to guide the fluid to always enter the microchannel detection area at the same position.

11. The microfluidic chip according to claim 8, characterized in that, The microfluidic chip is coated with a nano-scale hydrophilic coating on the chip substrate surface using ultrasonic atomization spraying technology. The nano-scale hydrophilic coating is prepared by dissolving 4, 3, or 2 of the following components in ethanol: 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. The "%" refers to the mass percentage of each component relative to ethanol, and the mass concentration of ethanol is 50%-100%.

12. The microfluidic chip according to claim 8, characterized in that, The detection lines and control lines of the detection area of ​​the microfluidic chip are cross-linked with a cross-linking agent to cross-link the nanoscale hydrophilic coating of the embedded antibody and the substrate. 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, or by dissolving 2, 3, or 4 of these components. 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 crosslinking 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-modal detection method based on a microfluidic chip, wherein the detection method is not intended for disease diagnosis and treatment, characterized in that, The method includes the following steps: S1: Using the microfluidic chip according to any one of claims 7-13, establish standard curves for colorimetric intensity and fluorescence intensity versus cMyBP-C concentration, respectively; S2: Dilute the biological sample with diluent and load it onto the sample application area of ​​the microfluidic chip according to any one of claims 7-13. After 5-8 minutes, load the AEC staining reagent. After 5-7 minutes, measure the colorimetric intensity and fluorescence intensity of the detection line and control line. Compare with the standard curve and calculate the dilution factor to obtain the cMyBP-C concentration in the biological sample.

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