High-sensitivity cardiac troponin rapid detection chip and detection method thereof

Abstract

The invention discloses a high-sensitivity cardiac troponin rapid detection chip and a detection method thereof. The high-sensitivity cardiac troponin rapid detection chip comprises a substrate with a blood collection port penetrating through the top surface and the bottom surface, and the top surface of the substrate is provided with a plurality of micro-channels which take the blood collection port as the center and gradually extend towards the periphery in a spiral mode. Each microchannel is sequentially divided into a red blood cell processing area, an incubation area and a detection area from the inner ring to the outer ring, and immobilized biomolecules or biomolecule films capable of intercepting red blood cells or breaking the red blood cells are arranged in the red blood cell processing areas; performing function regulation and control on each section of the incubation area and the detection area by adopting a mode I or a mode II; the microfluidic technology is adopted, the problems of environment and time efficiency in the blood sample collection and treatment process of existing human body cardiac troponin detection can be solved, and the prepared human body high-sensitivity cardiac troponin microfluidic rapid detection chip is small in size and low in preparation cost.

Description

Technical Field

[0001] The present invention relates to the technical field of cardiac troponin detection, in particular to the technical field of high-sensitivity cardiac troponin detection chip. Background Art

[0002] Acute coronary syndrome (ACS) is a group of clinical syndromes with the pathological basis of rupture or invasion of coronary atherosclerotic plaques and subsequent complete or incomplete occlusive thrombosis, including acute ST-segment elevation myocardial infarction, acute non-ST-segment elevation myocardial infarction and unstable angina (UA); ACS often presents with symptoms such as paroxysmal chest pain and chest tightness, which can lead to arrhythmia and heart failure (even sudden death), seriously affecting the quality of life and life expectancy of its patients; most ACS patients lack sufficient attention to mild symptoms of chest tightness and chest pain, thus delaying the timely diagnosis and treatment of ACS (if ACS patients adopt appropriate treatment in a timely manner, the mortality rate can be greatly reduced, complications can be reduced, and prognosis can be improved).

[0003] At present, the diagnosis, prognosis assessment, treatment and risk stratification of ACS are mainly judged based on the content of cardiac troponin (cTn) in human blood; cTn is a regulatory protein of myocardial muscle contraction, composed of three different gene subunits, including cardiac troponin T (cTnT), cardiac troponin I (cTnI) and troponin C (TnC); among them, cTnI and cTnT are encoded by separate genes and are both biomarkers of myocardial injury; cTnI is mainly involved in the inhibition of myocardial muscle contraction and has strong specificity. It generally increases gradually in peripheral blood 4 to 8 hours after myocardial injury and the highest value appears 12 to 24 hours after myocardial injury (elevated cTnI can still be detected in peripheral blood 7 to 10 days after myocardial injury); cTnT plays an important role in regulating myocardial contraction. It plays a connecting role in muscle contraction and, like cTnI, is mainly expressed in the myocardium and can also be detected in small amounts in skeletal muscle. However, it has a longer half-life and exists in the blood longer (this makes cTnT suitable for observing later stages of the disease). In addition, in addition to being used in the diagnosis and treatment of acute myocardial injury, cTnT testing can also be used for risk stratification of patients with chronic myocardial diseases (such as heart failure). In other words, rapid, sensitive and accurate determination of a single cTnI, cTnT or both in the blood is of great significance for the diagnosis of acute myocardial infarction, risk stratification of acute coronary syndrome, monitoring of myocardial injury caused by various factors, and prognosis of cardiac events.

[0004] At present, the detection methods for cTnI and / or cTnT mainly include enzyme-linked immunosorbent assay (ELISA) and chemiluminescence method; among them, ELISA method, such as the triple antibody method with announcement number CN1271409C for rapid quantitative determination of cardiac troponin I, usually has the problems of long operation time and low degree of automation; chemiluminescence method, such as the flash homogeneous chemiluminescence technology method for detecting cardiac troponin I / T with publication number CN110031635A, although it has the characteristics of high specificity, high sensitivity and high accuracy, it often requires expensive instruments and experienced operators, and can basically only be used in specific medical institutions.

[0005] With the continuous improvement of the sensitivity of cardiac troponin testing and the widespread implementation of high-sensitivity cardiac troponin testing in clinical laboratories, there is an urgent need to solve the problem of how to achieve point-of-care testing (POCT) while avoiding the need to draw venous blood (i.e., no need for professional medical staff to operate) and reducing the testing time (the existing method usually takes more than 10 minutes) to enable individuals to test at home or grassroots medical staff. Summary of the invention

[0006] The purpose of the present invention is to solve the problems in the prior art, and to propose a high-sensitivity cardiac troponin rapid detection chip and a detection method thereof, which adopts integrated microfluidic technology, and can solve the environmental and time-effect problems in the existing blood sample collection and processing process of human cardiac troponin detection. In addition, the prepared human high-sensitivity cardiac troponin microfluidic rapid detection chip has a small size and low preparation cost.

[0007] To achieve the above-mentioned object, the present invention provides a high-sensitivity cardiac troponin rapid detection chip, comprising a substrate having a blood collection port running through the top and bottom surfaces, the substrate being provided with a plurality of microchannels on the top surface, each of which is centered on the blood collection port and gradually expands spirally toward the periphery, each of which is divided into a red blood cell processing area, an incubation area and a detection area from the inner circle to the outer circle, each of which is provided with an immobilized biomolecule or biomolecule film capable of intercepting or rupturing red blood cells, and the functions of the incubation area and the detection area of ​​each section are regulated by method one or method two, wherein method one is to separately regulate the functions of the incubation area and the detection area of ​​each section only in the incubation area of ​​each section. A conjugate formed by coupling a colloidal carrier with a troponin-specific polyclonal antibody is set. The second method is to set a troponin-specific first monoclonal antibody (hereinafter referred to as the first antibody) labeled with a fluorescent substance in each incubation zone, and to set a troponin-specific second monoclonal antibody (hereinafter referred to as the second antibody) in each detection zone that can capture the immune complex formed by the combination of the troponin-specific first monoclonal antibody labeled with a fluorescent substance and troponin. Microfluidics is a new technology that has emerged in recent years. It refers to the use of channels of tens to hundreds of micrometers to process or manipulate extremely small amounts (10 -9 ~10 -18 liters) of liquid, and one of its important characteristics is the super strong surface tension at the micrometer scale, and has unique fluid properties, such as laminar flow, vortex and droplets; the present invention sets each of the microchannels in a gradually expanding spiral shape, so that the blood can present a strong secondary vortex (i.e., Dean effect) during the flow, thereby achieving a series of transmission dynamics of trace blood that are difficult to achieve with conventional methods, effectively improving the interaction between the blood and the substances set in the red blood cell processing area and the incubation area in each section (realizing some unconventional high-speed chemical reactions), while shortening the blood flow time and reducing the chip size; in addition, the number of the microchannels can be designed according to the required detection number of high-sensitivity cardiac troponin (such as only detecting cTnI, only detecting cTnT, or detecting cTnI and cTnT at the same time).

[0008] Preferably, each section of the red blood cell processing zone has 1 to 2 turns, each section of the incubation zone has 2 to 10 turns, and each microchannel forms a relatively large detection cavity at the tail end as a detection zone.

[0009] The Dean number is an important indicator for measuring the size of the secondary vortex in each of the microchannels, and it depends on properties such as the curvature of the bend, the channel size, the fluid inertia effect, and the viscosity effect. As the Dean number increases, the secondary vortex in the channel will become more intense, causing multiple exchange contacts between the blood and the channel surface, and causing a strong surface catalytic reaction. Based on this, in the present invention, the hydraulic diameter of each section of the red blood cell processing area is 10 to 100 μm, the hydraulic diameter of each section of the incubation area is 5 to 100 μm, and the diameter of each detection cavity is 20 to 500 μm and the depth is 10 to 500 μm. When in use, when the blood enters each microchannel from the blood collection port, it will first pass through the red blood cell processing area in each section to rupture or intercept the red blood cells and allow the remaining serum / plasma to continue to move deeper. Since the red blood cell processing areas in each section are located at the innermost side of the microchannel, the channel curvature is The rate is at the maximum point of the entire channel, and the Dean effect is the strongest; for this reason, the present invention increases the hydraulic diameter of the red blood cell processing area in each section to reduce the flow rate of blood, thereby balancing the excessive cell rupture that may be caused by the Dean flow effect; the second part of each microchannel (i.e., each incubation area) is used for the formation of immune complexes (when blood passes through, the cTnI and / or cTnT therein can respectively bind to their respective specific antibodies and form immune complexes); to ensure that the immune complex can fully complete the incubation reaction, the present invention increases the Dean effect by relatively reducing the hydraulic diameter of this part of the channel to obtain a larger flow rate; in addition, since the present invention sets a larger number of spiral turns for each section of the incubation area, it can further ensure that the immune incubation reaction is fully completed; that is, the present invention can balance the difference in the incubation reaction rates of different protein subunits by adjusting the hydraulic diameter of different sections of each microchannel.

[0010] Preferably, the cross-sections of the red blood cell processing area and incubation area in each section are square, circular, trapezoidal or triangular.

[0011] Preferably, the red blood cell processing zone in each section uses a hemolytic agent to rupture the red blood cells or uses an anti-red blood cell antibody to retain the red blood cells.

[0012] Furthermore, the hemolytic agent is one or a combination of cetyltrimethylammonium chloride (CTAB), polysorbate and saponin.

[0013] Furthermore, the anti-erythrocyte antibody is anti-erythrocyte membrane glycoprotein A (anti-CD235a) and / or anti-hemoglobin antibody (Anti-Hb).

[0014] Preferably, the colloid carrier is latex particles.

[0015] Preferably, the fluorescent substance is fluorescein and / or fluorescent microspheres containing fluorescein.

[0016] Preferably, the troponin-specific polyclonal antibody is a polyclonal antibody that can recognize cTnI and / or cTnT, and the troponin-specific first monoclonal antibody and the troponin-specific second monoclonal antibody are both monoclonal antibodies that can recognize cTnI and / or cTnT; in addition, when selecting troponin-specific antibodies, it is necessary to pay attention to their cross-reactivity (i.e., to avoid cross-reactions between the three subtypes of cTn) to ensure high specificity in the detection.

[0017] Furthermore, the monoclonal antibody capable of recognizing cTnI is a specific amino acid sequence at the N-terminus or C-terminus of cTnI (such as cTnI-2, etc.).

[0018] Furthermore, the polyclonal antibody capable of recognizing cTnI is a goat anti-cTnI polyclonal antibody, a rabbit anti-cTnI polyclonal antibody or a specific amino acid sequence (such as residues 1-30, 31-110 or 111-181, etc.) of the N-terminal region, central region or C-terminal region of different target epitopes of cTnI.

[0019] Furthermore, the monoclonal antibody capable of recognizing cTnT is a C-terminal epitope of cTnT (such as cTnL-2, etc.).

[0020] Furthermore, the polyclonal antibody capable of recognizing cTnT is a rabbit polyclonal antibody (such as Ab188879 or Ab64750, etc.), a rabbit anti-cTnT polyclonal antibody (such as PA5-14314, etc.) or a polyclonal antibody SAB1405743.

[0021] The troponin-specific first monoclonal antibody labeled with a fluorescent substance is usually a troponin monoclonal antibody labeled with fluorescein (such as FITC and Alexa Fluor, etc.), fluorescent microspheres of fluorescein, or other probes to amplify the signal or perform fluorescence detection; at the same time, the correct selection and use of the second antibody is crucial for the successful detection of the immune complex formed by the troponin-specific antibody labeled with a fluorescent substance and troponin; specifically, in the detection of cTnI, the first antibody of the N-terminal epitope (such as residues 1-30) is used to recognize the N-terminal epitope and is coupled to fluorescein, and the second antibody is used to recognize the central region or C-terminal epitope of cTnI, such as the monoclonal antibody against cTnI (residues 50-110); the first antibody of the C-terminal epitope (such as residues 150-181) is used to recognize The first antibody for the C-terminal epitope (e.g., residues 1-80) is used to target the N-terminal epitope and is coupled to fluorescent microspheres, and the second antibody is used to recognize the N-terminal or central region of cTnI, such as the monoclonal antibody against cTnI (residues 1-49); in addition, in the detection of cTnT, the first antibody for the N-terminal epitope (e.g., residues 1-80) is used to target the N-terminal epitope and is coupled to fluorescein, and the second antibody is used to recognize the central or C-terminal epitope, such as the monoclonal antibody against cTnT (residues 100-150), and the first antibody for the C-terminal epitope (e.g., residues 160-290) is used to recognize the C-terminal epitope and is coupled to fluorescent microspheres, and the second antibody is used to recognize the N-terminal or central region, such as the monoclonal antibody against cTnT (residues 1-50).

[0022] Preferably, the top surface of the substrate is covered with a transparent cover plate that can seal the blood collection port and each microchannel, and the bottom surface of the substrate is affixed with a sticker that can seal the blood collection port. The cover plate and the sticker together seal the blood collection port and each microchannel and form a vacuum environment isolated from the outside world or a closed environment filled with non-oxygen gas.

[0023] Furthermore, the thickness of the substrate and the cover plate are both 100-1000 μm.

[0024] Furthermore, the substrate is made of polymer material, pure metal material, alloy material or non-metal material.

[0025] Furthermore, the polymer material is PDMS or polystyrene, the pure metal material is aluminum or copper, and the non-metal material is a carbon-based material or a silicon-based material.

[0026] Furthermore, the cover plate is a transparent inorganic glass plate, a transparent organic glass plate or an optical organic film.

[0027] Furthermore, the non-oxygen gas is an inert gas.

[0028] Furthermore, the inert gas is one or a combination of nitrogen and argon.

[0029] Furthermore, the front and back surfaces of the cover are respectively provided with optical anti-reflection coatings, and the incubation area and detection area of ​​each section are respectively provided with optical reflective coatings; wherein, the incubation area and detection area of ​​each section can respectively utilize optical reflective coatings to increase the intensity of light signals, and the cover can reduce the reflection of incident light for detection and improve the transmittance of blood sample light signals through the optical anti-reflection coatings located on its front and back surfaces.

[0030] Furthermore, the optical anti-reflection coating is a metal film (such as silver or aluminum).

[0031] Furthermore, the optical reflective coating is a TiN film; the TiN film has a very high light reflectivity in the infrared region, and in particular, the film not only has high density, low manufacturing cost and biomass compatibility, but also has a mature process in integrated circuit production.

[0032] Furthermore, the surfaces of the incubation area and detection area of ​​each section need to be chemically polished or chemically mechanically polished before the optical reflective coating is provided until the surface roughness is less than 0.5 μm (the specific roughness may depend on the wavelength of the detection light); wherein, the chemical polishing or chemical mechanical polishing treatment can further enhance the light reflectivity of each section of the incubation area and detection area.

[0033] The present invention also provides a detection method, which requires the use of the above-mentioned high-sensitivity cardiac troponin rapid detection chip and the specific operation steps are firstly to send blood from the bottom surface of the substrate into the blood collection port, and then when the blood flows to each detection area (the immune complex after sufficient incubation reaction will eventually enter the terminal located at each of the microchannels), each detection area is quantitatively analyzed by fluorescence intensity (fluorescence method) or solution turbidity (turbidimetry); wherein, known concentrations of cTnI or cTnL can be used for calibration and a standard curve can be established; when in use, the measured data is compared with the standard curve to determine the concentration of cTnI and / or cTnL.

[0034] Preferably, for the fluorescence method, waste liquid areas may be respectively provided at the ends of the microchannels to collect liquid that has not formed fluorescent complexes.

[0035] Preferably, a separate spectrophotometer is used to penetrate the cover plate at a wavelength of 250 to 900 nm and detect the fluorescence intensity or solution turbidity reflected by each detection area; wherein the measurement of reflected light can effectively increase the measurement accuracy; in addition, the specific fluorescence wavelength depends on the excitation and emission characteristics of the fluorescent substance, such as the commonly used organic fluorescent substance FITC (excitation wavelength is about 495 nm and emission wavelength is about 519 nm) or rhodamine fluorescent substance (excitation wavelength is about 540 nm and emission wavelength is about 570 nm) and lanthanide elements (such as Eu2O3) used for time-resolved fluorescence.3+ The excitation wavelength is approximately 395 nm and the emission wavelength is approximately 610 nm).

[0036] Preferably, dot-matrix optical spectroscopy measurement technology is used to perform separate point-by-point scanning measurements on each section of the incubation area along the direction of blood flow to obtain the immediate reaction progress of the immune incubation reaction along the channel flow direction (such a process can further improve the sensitivity of chip measurement on the one hand, and on the other hand, it can also assist in parameter selection during early development to achieve the purpose of optimizing specific antibodies and optimizing channel length).

[0037] Beneficial effects of the present invention: 1) The present invention integrates a blood collection port and a plurality of multi-level micron-level channels with detection cavities into one integrated microfluidic chip, and further coats each integrated microchannel with a biofilm having a non-conducting function in sections along the blood flow direction, so that blood can be directly drawn from the tail end of a human fingertip during testing, and the blood is fully mixed with the corresponding biofilm in each microchannel and reacts before flowing into the detection cavity, and finally the trace concentration of cardiac troponin is determined by quantitative analysis of fluorescence intensity or solution turbidity, and the whole process does not require the assistance of professional medical personnel to complete the extraction and treatment of blood, thus solving the environmental and time-effect problems of existing cardiac troponin detection, providing the possibility of rapid detection for individuals and clinicians in precision medicine, and can be manufactured using the integrated production process and equipment commonly used in the production of micron-level large-scale integrated circuits, with a simple, mature and stable preparation process and low preparation cost, which meets the expenditure requirements of ordinary people for daily use of medical consumables; 2) The present invention can effectively enhance the eddy effect of the fluid in each microchannel by setting each microchannel in a mosquito coil shape, thereby shortening the length of each microchannel. At the same time, its circular structure can also greatly reduce the chip size, thereby correspondingly reducing the production cost of the chip; 3) The present invention provides optical reflective coatings in each incubation zone and detection zone, thereby enabling the integrated microfluidic chip to capture relatively weak spectral signals; 4) The present invention can meet the processing requirements of different functions (red blood cell processing and incubation) by designing different hydraulic diameters for each section of the red blood cell processing area and the incubation area; 5) The present invention adds a transparent packaging cover plate on the substrate, and can use the material selection of the transparent packaging cover plate to achieve optical detection of the substance in the detection cavity. It can also form a vacuum environment isolated from the outside world or a closed environment filled with non-oxygen gas between the substrate and the transparent packaging cover plate, thereby preventing a trace amount of human blood from contacting the atmosphere and ensuring the accuracy of the detection; 6) The present invention uses dot-matrix optical spectroscopy measurement technology to perform separate point scanning measurements on each incubation area along the direction of blood flow, thereby facilitating real-time knowledge of the reaction progress of each incubation area along the channel (which in turn facilitates developers to optimize the channel length).

[0038] The features and advantages of the present invention will be described in detail through embodiments in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is a schematic diagram of the three-dimensional structure of the first embodiment; Figure 2 is a top view of the first embodiment; Figure 3 yes Figure 2 AA section view; Figure 4 It is a physical diagram of the first embodiment; Figure 5 is a schematic diagram of the three-dimensional structure of the second embodiment; Figure 6 is a top view of the second embodiment; Figure 7 yes Figure 6 BB section view; In the figure: 1-substrate, 11-blood collection port, 12-microchannel, 121-detection cavity, 2-cover plate. DETAILED DESCRIPTION

[0040] Embodiment 1: See also Figures 1 to 4 The present embodiment provides a high-sensitivity cardiac troponin rapid detection chip, comprising a substrate 1 having a blood collection port 11 penetrating the top and bottom surfaces, wherein the substrate 1 is provided with two microchannels 12 on the top surface, each of which is centered on the blood collection port 11 and gradually expands spirally toward the periphery, and each of the microchannels 12 is respectively divided into a red blood cell processing area, an incubation area and a detection area from the inner circle to the outer circle, and each section of the red blood cell processing area is provided with an immobilized biomolecule or biomolecule film that can intercept or rupture red blood cells, and the functions of the incubation area and the detection area of ​​each section are regulated by the first method or the second method, wherein the first method is to respectively set a combination formed by coupling a colloidal carrier and a troponin-specific polyclonal antibody in each section of the incubation area, and the second method is to respectively set a troponin-specific first monoclonal antibody labeled with a fluorescent substance in each section of the incubation area, and on the other hand, to respectively set a troponin-specific second monoclonal antibody that can capture the immune complex formed by the combination of the troponin-specific first monoclonal antibody labeled with a fluorescent substance and troponin in each detection area.

[0041] Each section of the red blood cell processing area has one circle, each section of the incubation area has three circles, and each microchannel 12 forms a relatively large detection cavity 121 at the tail end as a detection area.

[0042] The hydraulic diameter of each section of the red blood cell processing area is 10-100 μm, the hydraulic diameter of each section of the incubation area is 5-100 μm, and the diameter of each detection cavity 121 is 20-500 μm and the depth is 10-500 μm.

[0043] The cross-sections of the red blood cell processing area and the incubation area in each section are trapezoidal.

[0044] The red blood cell processing area described in each section uses a hemolytic agent to rupture the red blood cells or uses an anti-red blood cell antibody to retain the red blood cells.

[0045] The troponin-specific polyclonal antibody is a polyclonal antibody that can recognize cTnI and / or cTnT, and the troponin-specific first monoclonal antibody and the troponin-specific second monoclonal antibody are both monoclonal antibodies that can recognize cTnI and / or cTnT.

[0046] The top surface of the substrate 1 is covered with a transparent cover plate 2 that can seal the blood collection port 11 and each microchannel 12, and the bottom surface of the substrate 1 is affixed with a sticker that can seal the blood collection port 11. The cover plate 2 and the sticker together seal the blood collection port 11 and each microchannel 12 and form a vacuum environment isolated from the outside world or a closed environment filled with non-oxygen gas.

[0047] The front and back surfaces of the cover plate 2 are respectively provided with optical anti-reflection coatings, and the incubation area and detection area in each section are respectively provided with optical reflective coatings.

[0048] This embodiment also provides a detection method, which uses the above-mentioned high-sensitivity cardiac troponin rapid detection chip and the specific operation steps are first sending blood from the bottom surface of the substrate 1 into the blood collection port 11, and then when the blood flows to each detection area, quantitative analysis of the fluorescence intensity or solution turbidity of each detection area is performed.

[0049] The dot-matrix optical spectrum measurement technology is used to perform separate point-by-point scanning measurements on each section of the incubation area along the direction of blood flow to obtain the real-time reaction progress of the immune incubation reaction along the channel flow direction.

[0050] Embodiment 2: See also Figures 5 to 7 The high-sensitivity cardiac troponin rapid detection chip of the present invention has four microchannels 12 on the top surface of the substrate 1, each of which is centered on the blood collection port 11 and gradually expands spirally toward the periphery.

[0051] The cross-sections of the red blood cell processing area and the incubation area in each section are circular.

[0052] The rest is the same as in the first embodiment.

[0053] The above embodiments are intended to illustrate the present invention, not to limit the present invention. Any solution that is a simple transformation of the present invention belongs to the protection scope of the present invention.

Claims

1. High-sensitivity cardiac troponin rapid detection chip, characterized by: The invention comprises a substrate (1) having a blood collection port (11) penetrating the top and bottom surfaces, wherein the substrate (1) is provided with a plurality of microchannels (12) on the top surface, each of which is centered on the blood collection port (11) and gradually expands in a spiral manner toward the periphery, wherein each of the microchannels (12) is divided into a red blood cell processing area, an incubation area, and a detection area in sequence from the inner circle to the outer circle, wherein each section of the red blood cell processing area is provided with an immobilized biomolecule or biomolecule film capable of intercepting or rupturing red blood cells, and wherein the functions of the incubation area and the detection area of ​​each section are regulated by the first method or the second method. The first method is to only provide a combination formed by coupling a colloidal carrier and a troponin-specific polyclonal antibody in each section of the incubation area, and the second method is to provide a troponin-specific first monoclonal antibody labeled with a fluorescent substance in each section of the incubation area, and to provide a troponin-specific second monoclonal antibody capable of capturing an immune complex formed by the combination of the troponin-specific first monoclonal antibody labeled with a fluorescent substance and troponin in each detection area.

2. The high-sensitivity cardiac troponin rapid detection chip according to claim 1, characterized in that: The number of turns of each section of the red blood cell processing area is 1 to 2, and the number of turns of each section of the incubation area is 2 to 10. Each of the microchannels (12) forms a relatively large detection cavity (121) at the tail end to serve as a detection area.

3. The high-sensitivity cardiac troponin rapid detection chip as claimed in claim 2, characterized in that: The hydraulic diameter of each section of the red blood cell processing area is 10 to 100 μm, the hydraulic diameter of each section of the incubation area is 5 to 100 μm, and the diameter of each detection cavity (121) is 20 to 500 μm and the depth is 10 to 500 μm.

4. The high-sensitivity cardiac troponin rapid detection chip according to claim 1, characterized in that: The cross-sections of the red blood cell processing area and the incubation area in each section are square, circular, trapezoidal or triangular.

5. The high-sensitivity cardiac troponin rapid detection chip according to claim 1, characterized in that: The red blood cell processing area described in each section uses a hemolytic agent to rupture the red blood cells or uses an anti-red blood cell antibody to retain the red blood cells.

6. The high-sensitivity cardiac troponin rapid detection chip according to claim 1, characterized in that: The troponin-specific polyclonal antibody is a polyclonal antibody that can recognize cTnI and / or cTnT, and the troponin-specific first monoclonal antibody and the troponin-specific second monoclonal antibody are both monoclonal antibodies that can recognize cTnI and / or cTnT.

7. The high-sensitivity cardiac troponin rapid detection chip according to claim 1, characterized in that: The top surface of the substrate (1) is covered with a transparent cover plate (2) capable of sealing the blood collection port (11) and each microchannel (12); the bottom surface of the substrate (1) is affixed with a sticker capable of sealing the blood collection port (11); the cover plate (2) and the sticker together seal the blood collection port (11) and each microchannel (12) and form a vacuum environment isolated from the outside or a closed environment filled with non-oxygen gas.

8. The high-sensitivity cardiac troponin rapid detection chip according to claim 7, characterized in that: The front and back surfaces of the cover plate (2) are respectively provided with optical anti-reflection coatings, and the incubation area and detection area in each section are respectively provided with optical reflective coatings.

9. A detection method, characterized in that: A high-sensitivity cardiac troponin rapid detection chip as described in any one of claims 1 to 8 is used, and the specific operation steps are firstly to send blood from the bottom surface of the substrate (1) into the blood collection port (11), and then when the blood flows to each detection area, quantitative analysis of the fluorescence intensity or solution turbidity of each detection area is performed.

10. The detection method according to claim 9, characterized in that: The dot-matrix optical spectrum measurement technology is used to perform separate point-by-point scanning measurements on each section of the incubation area along the direction of blood flow to obtain the real-time reaction progress of the immune incubation reaction along the channel flow direction.

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