An electrochemiluminescence chip, its fabrication method and application

Abstract

This invention discloses an electrochemiluminescence chip, its preparation method, and its application, belonging to the field of biochips. The electrochemiluminescence chip provided by this invention includes a PDMS sheet and a glass substrate from top to bottom. The PDMS sheet has an inlet and an outlet that penetrate the PDMS sheet. Between the inlet and outlet are a first detection chamber, a second detection chamber, a third detection chamber, and a fourth detection chamber. The glass substrate has a first working electrode, a second working electrode, a third working electrode, a fourth working electrode, a first pair of electrodes, a second pair of electrodes, a third pair of electrodes, a fourth pair of electrodes, a first solder joint, a second solder joint, a third solder joint, and a fourth solder joint. The electrochemiluminescence chip provided by this invention combines electrochemiluminescence and microfluidic technology to simultaneously detect three biomarkers related to myocardial infarction: cTnI, Myo, and CK-MB. It has advantages such as short detection time, high application efficiency, good detection accuracy, small supporting instruments, simple operation, small sample volume, and small reagent volume required.

Description

Technical Field

[0001] This invention relates to the intersection of biochips and in vitro diagnostics, and more specifically, to an electrochemiluminescence chip, its fabrication method, and its applications. Background Technology

[0002] Myocardial infarction is one of the most significant cardiovascular diseases threatening human health. Early detection of myocardial cell damage and heart failure caused by myocardial infarction is of great clinical importance for monitoring treatment efficacy and prognosis, and reducing disease mortality. Myocardial infarction biomarkers, which are generated during myocardial infarction, possess specificity and sensitivity and are widely used for screening, diagnosing, evaluating prognosis, and monitoring treatment efficacy for myocardial injury or abnormalities. Troponin I (cTnI), myoglobin (Myo), and serum creatine kinase isoenzyme (CK-MB) are the most common myocardial infarction biomarkers.

[0003] Currently, routine detection techniques for myocardial infarction biomarkers include radioimmunoassay, high-performance liquid chromatography (HPLC), electrochemical analysis, protein analyzers, various diagnostic reagent kits, and immunochromatographic strips. These techniques have various drawbacks, such as radioactive contamination, the need for large instruments, long processing times, inability to perform quantitative detection, or low sensitivity and accuracy, which limit their clinical application.

[0004] Currently available clinical diagnostic chips of this type mostly consist of colloidal gold test strips with an external slotted structure or observation chamber. For example, patent document CN1363839A describes a biochip for early myocardial infarction diagnosis, composed of a carrier box and a protein test strip. Another example is patent CN201707336U, which discloses a test strip device for rapid quantitative detection of cTnI. However, this patent can only detect one myocardial infarction marker, cTnI, and cannot provide a more accurate assessment of the severity of myocardial infarction. Chinese invention patent CN102305866A describes a rapid diagnostic device for acute myocardial infarction that can simultaneously detect three myocardial infarction markers. However, using fluorescent labeling to detect antigens cannot eliminate the influence of excitation light on signal detection sensitivity and accuracy. Furthermore, due to the presence of a sample application system and an excitation detection system, the size of the accompanying instrument cannot be miniaturized. Therefore, there is a need to develop a completely new clinically applicable detection method. Summary of the Invention

[0005] The problem to be solved by this invention is to provide a novel method for detecting myocardial infarction indicators with high sensitivity, high accuracy and miniaturization.

[0006] To address the aforementioned problems, the first aspect of this invention provides an electrochemiluminescence chip, comprising, from top to bottom, a PDMS wafer and a glass substrate. The area of ​​the PDMS wafer is smaller than that of the glass substrate. The glass substrate and the PDMS wafer are bonded together. The PDMS wafer has an inlet and an outlet that penetrate the PDMS wafer. A first detection cavity, a second detection cavity, a third detection cavity, and a fourth detection cavity are sequentially formed on the lower surface of the PDMS wafer from the inlet to the outlet. The inlet and the outlet are connected by a flow channel, on which the first detection cavity, the second detection cavity, the third detection cavity, and the fourth detection cavity are sequentially arranged from the inlet to the outlet. The upper surface of the glass substrate has a first... The system comprises a working electrode, a second working electrode, a third working electrode, and a fourth working electrode. The first working electrode is surrounded by a first pair of electrodes, the second working electrode by a second pair of electrodes, the third working electrode by a third pair of electrodes, and the fourth working electrode by a fourth pair of electrodes. Each of the first, second, third, and fourth pairs of electrodes has an opening allowing a metal sputtered layer to pass through. The first working electrode and the first pair of electrodes are completely fitted and covered by a first detection cavity. The second working electrode and the second pair of electrodes are completely fitted and covered by the second detection cavity. The third working electrode and the third pair of electrodes are completely fitted and covered by the third detection cavity. The fourth working electrode... The electrodes and the fourth pair of electrodes are completely adhered to and covered by the fourth detection cavity. The glass substrate also includes a first solder joint, a second solder joint, a third solder joint, a fourth solder joint, a fifth solder joint, a first metal sputtering layer, a second metal sputtering layer, a third metal sputtering layer, a fourth metal sputtering layer, and a fifth metal sputtering layer. The first solder joint, the second solder joint, the third solder joint, the fourth solder joint, and the fifth solder joint are all located outside the overlap area between the PDMS sheet and the glass substrate. The first solder joint is electrically connected to the first working electrode through the first metal sputtering layer, the second solder joint is electrically connected to the second working electrode through the second metal sputtering layer, the third solder joint is electrically connected to the third working electrode through the third metal sputtering layer, and the fourth solder joint is electrically connected to the fourth metal sputtering layer. The metal sputtering layer is electrically connected to the fourth working electrode. The first pair of electrodes is electrically connected to the fifth solder joint through the fifth metal sputtering layer. The second pair of electrodes is electrically connected to the fifth solder joint through the fifth metal sputtering layer. The third pair of electrodes is electrically connected to the fifth solder joint through the fifth metal sputtering layer. The fourth pair of electrodes is electrically connected to the fifth solder joint through the fifth metal sputtering layer. The first working electrode is modified with troponin I monoclonal antibody for detecting troponin I. The second working electrode is modified with myoglobin primary antibody for detecting myoglobin. The third working electrode is modified with serum creatine kinase isoenzyme primary antibody for detecting serum creatine kinase isoenzyme. The fourth working electrode is unmodified and is used for background rendering.The electrochemiluminescence chip provided by this invention combines microfluidic technology and electrochemiluminescence technology, requiring very little detection liquid during use, and exhibiting high sensitivity and accuracy.

[0007] Preferably, the materials of the first metal sputtering layer, the second metal sputtering layer, the third metal sputtering layer, the fourth metal sputtering layer, and the fifth metal sputtering layer are all selected from one or more of Cr / Au layer, platinum, silver, graphite, ITO, and glassy carbon.

[0008] Preferably, the thickness of the first metal sputtering layer, the second metal sputtering layer, the third metal sputtering layer, the fourth metal sputtering layer, and the fifth metal sputtering layer are all 50 to 500 nm.

[0009] Preferably, the thickness of the PDMS sheet is 2-10 mm, the inlet is circular with a diameter of 1-5 mm, and the outlet is circular with a diameter of 1-5 mm.

[0010] Furthermore, a second aspect of the present invention provides a method for preparing the aforementioned electrochemiluminescence chip, comprising the following steps:

[0011] Preparation of S1 and PDMS sheets:

[0012] S11: Using single-crystal silicon as the substrate, photoresist is spin-coated onto the silicon wafer using a spin coater. After spin coating, pre-drying is performed at a temperature of 60–68°C for 25–35 minutes. After pre-drying, photolithography is performed according to the design layout. After photolithography, post-drying is performed at a temperature of 90–98°C for 25–35 minutes. After post-drying, development is performed using a developer to remove the photoresist and clean the wafer, forming a microstructure of the designed height. The wafer is then hard-baked at 95°C for 30 minutes to obtain a silicon wafer mold with microstructures of different heights.

[0013] S12: Place the silicon wafer mold obtained in step S11 into a fluorosilane atmosphere and incubate for 200-300 min. Mix the PDMS prepolymer and curing agent evenly, then vacuum dry and remove the PDMS mixture. Cast the PDMS mixture into the silicon wafer mold, solidify the mold, peel off the PDMS sheet, and punch holes in the PDMS sheet to form an inlet and outlet. Place the punched PDMS sheet into a plasma cleaner, maintain a vacuum environment for 50-70 min, and irradiate with glow discharge for 100-120 s. Remove the PDMS sheet and drop a PEG-siloxane and acetone mixture (V:V = 1:1) at the inlet and outlet positions. Incubate at room temperature for 40-80 min and then rinse with ultrapure water.

[0014] S2. Preparation of glass substrate: A single-polished glass substrate is used as the substrate. Plasma cleaning is performed under the following conditions: vacuum environment for 50-70 min, glow discharge for 100-120 s. Photoresist is applied to the polished surface of the glass substrate using a coating machine. Photolithography is then performed under the following conditions: exposure for 15 s followed by development to obtain the desired pattern. A metal sputtering layer is obtained by sputtering. Excess photoresist and metal are removed, while the Au electrode is retained.

[0015] S3. Antibody Modification: Protein modification of the four different working electrodes of the chip. The first working electrode is modified with a monoclonal antibody against troponin I, the second working electrode is modified with a primary antibody against myoglobin, the third working electrode is modified with a primary antibody against a serum creatine kinase isoenzyme, and the fourth working electrode is left unmodified. After modification, plasma cleaning is performed under vacuum conditions for 50-70 minutes followed by glow discharge for 100-120 seconds. After removal, the glass substrate and PDMS sheet are rapidly bonded under a microscope with the structural planes facing each other to obtain the electrochemical chip.

[0016] Preferably, in step S12, the mass ratio of the PDMS prepolymer to the curing agent is (13-17):1.

[0017] Preferably, in step S12, the vacuum drying environment is a vacuum of 13 psi, and the drying is allowed to stand for 25 to 40 minutes.

[0018] A third aspect of the present invention provides an application of the aforementioned electrochemiluminescence chip, the application including using the aforementioned electrochemiluminescence chip to prepare drugs and kits for treating and / or preventing myocardial infarction.

[0019] The beneficial effects of this invention are as follows: This invention provides a rapid clinical diagnostic chip for myocardial infarction based on electrochemiluminescence technology. This chip combines electrochemiluminescence and microfluidic technology to simultaneously detect three biomarkers related to myocardial infarction: troponin I (cTnI), myoglobin (Myo), and serum creatine kinase isoenzyme (CK-MB). It has the advantages of short detection time, high application efficiency, good detection accuracy, small supporting instruments, simple operation, small sample volume, and small reagent volume. Attached Figure Description

[0020] Figure 1 An overall top view of the electrochemiluminescence chip provided for a specific embodiment of the present invention;

[0021] Figure 2 A diagram showing the PDMS underlay structure of an electrochemiluminescence chip provided for a specific embodiment of the present invention;

[0022] Figure 3 This is a diagram showing the surface bonding structure of an electrochemiluminescence chip on a glass substrate, provided for a specific embodiment of the present invention.

[0023] Explanation of reference numerals in the attached figures: 1. PDMS sheet; 2. Inlet; 3. Outlet; 4. First detection chamber; 5. Second detection chamber; 6. Third detection chamber; 7. Fourth detection chamber; 8. Flow channel; 9. Glass substrate; 10. First solder joint; 11. Second solder joint; 12. Third solder joint; 13. Fourth solder joint; 14. Fifth solder joint; 15. First working electrode; 16. Second working electrode; 17. Third working electrode; 18. Fourth working electrode; 19. First pair of electrodes; 20. Second pair of electrodes; 21. Third pair of electrodes; 22. Fourth pair of electrodes; 231. First metal sputtering layer; 232. Second metal sputtering layer; 233. Third metal sputtering layer; 234. Fourth metal sputtering layer; 235. Fifth metal sputtering layer. Detailed Implementation

[0024] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the claims of the present invention.

[0025] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0026] See Figure 1 To address the shortcomings of existing myocardial infarction detection kits, such as low sensitivity, low accuracy, and long processing time, this invention provides an electrochemiluminescence chip. The chip comprises a PDMS sheet 1 and a glass substrate 9 from top to bottom, with the PDMS sheet 1 having a smaller area than the glass substrate 9. The two are bonded together. In this embodiment, the PDMS sheet 1 has two through-holes, designated as an inlet 2 and an outlet 3. In this embodiment, the inlet 2 and outlet 3 are circular with a diameter of 3 mm. In other embodiments, the inlet 2 and outlet 3 can be square or other regular shapes. The inlet 2 is mainly used for the entry of samples, cleaning solutions, and reaction reagents, while the outlet 3 is mainly used for the outflow of samples, cleaning solutions, or the reaction solution after the reaction.

[0027] See Figure 1 and Figure 2In the above embodiment, the lower surface of the PDMS sheet 1 is provided with a first detection cavity 4, a second detection cavity 5, a third detection cavity 6, and a fourth detection cavity 7. The first detection cavity 4 is connected to the injection port 2 through a flow channel 8. The first detection cavity 4 is connected to the second detection cavity 5 through a flow channel 8. The second detection cavity 5 is connected to the third detection cavity 6 through a flow channel 8. The third detection cavity 6 is connected to the fourth detection cavity 7 through a flow channel 8. The fourth detection cavity 7 is connected to the outlet port 3 through a flow channel 8. In this embodiment, the width of the flow channel 8 between the injection port 2 and the first detection cavity 4 is 1 mm, the length is 1 cm, and the height is 50 μm. The length of the flow channel 8 between the first detection cavity 4 and the second detection cavity 5, between the second detection cavity 5 and the third detection cavity 6, and between the third detection cavity 6 and the fourth detection cavity 7 is 4 mm, the width is 200 μm, and the height is 50 μm.

[0028] See Figure 1 and Figure 3 In the above embodiment, the upper surface of the glass substrate 9 of the electrochemiluminescence chip has four working electrodes, four counter electrodes, and five solder joints. The four working electrodes are respectively a first working electrode 15 attached to the first detection cavity 4, a second working electrode 16 attached to the second detection cavity 5, a third working electrode 17 attached to the third detection cavity 6, and a fourth working electrode 18 attached to the fourth detection cavity 7. The first working electrode 15 is surrounded by a corresponding first counter electrode 19, the second working electrode 16 is surrounded by a corresponding second counter electrode 20, the third working electrode 17 is surrounded by a corresponding third counter electrode 21, and the fourth working electrode 18 is surrounded by a corresponding fourth counter electrode 22. In this embodiment, all detection cavities should completely cover the working electrodes and counter electrodes to ensure the smooth progress of electrochemiluminescence. In this embodiment, the first working electrode 15 is connected to the first solder joint 10 through the first metal sputtering layer 231, the second working electrode 16 is connected to the second solder joint 11 through the second metal sputtering layer 232, the third working electrode 17 is connected to the third solder joint 12 through the third metal sputtering layer 233, the fourth working electrode 18 is connected to the fourth solder joint 13 through the fourth metal sputtering layer 234, and the first pair of electrodes 19, the second pair of electrodes 20, the third pair of electrodes 21, and the fourth pair of electrodes 22 are all connected to the fifth solder joint 14 through the fifth metal sputtering layer 235. In this embodiment, to ensure that all detection chambers function normally, all solder joints should be externally voltaged and exposed, and should not be covered by the PDMS sheet 1.

[0029] See Figure 3 In this embodiment, the first working electrode 15 of the electrochemiluminescence chip is modified with troponin I, the second working electrode 16 is modified with myoglobin primary antibody, the third working electrode 17 is modified with primary antibody of serum creatine kinase isoenzyme, and the fourth working electrode 18 is not modified as a background parameter.

[0030] The present invention also provides a method for using the aforementioned electrochemiluminescence chip, including an electrochemiluminescence detection instrument based on a silicon photomultiplier tube or a photomultiplier tube, and a detection method for using the instrument and the chip together. That is, based on this chip, rapid clinical diagnosis of myocardial infarction can be achieved. The silicon photomultiplier tube instrument is a detection instrument that uses a silicon photomultiplier tube as a photosensitive detection element. It can provide the dark environment, the required voltage, and the electrochemiluminescence detection chip required by the chip. The dark environment refers to the light signal emitted by the reaction within the chip when the electrochemiluminescence reaction occurs; therefore, a dark environment is required to minimize signal interference. The required voltage means that the electrochemiluminescence chip needs to be connected to or supplied with a working voltage to undergo the electrochemiluminescence reaction. Therefore, the instrument needs to provide a working voltage to ensure the normal operation of the diagnostic chip. Since the luminescence signal is a response indicator for myocardial infarction markers, the instrument needs to provide reliable luminescence detection.

[0031] When using the electrochemical chip provided in the specific embodiments of the present invention, a pipette or an automatic sampler can be used to inject the sample to be tested into the injection port 2. After the sample to be tested is injected into the injection port 2, it flows sequentially through the flow channel into the first detection chamber 4, the second detection chamber 5, the third detection chamber 6, and the fourth detection chamber 7 for incubation. This allows the troponin I primary antibody on the first working electrode 15, the myoglobin primary antibody on the second working electrode 16, the serum creatine kinase isoenzyme primary antibody on the third working electrode 17, and the fourth working electrode 18 within the chip to fully react with the sample to be tested. Once the reaction is complete... Afterwards, the cleaning solution is injected into the inlet 2, and the unreacted sample is washed out from the outlet 3. After cleaning, the secondary antibodies containing the electrochemiluminescent substrates of myocardial infarction markers are added to the first working electrode 15, the second working electrode 16, and the third working electrode 17. After incubation at room temperature, the cleaning solution is added to wash out the unreacted secondary antibodies. The chip is placed in the detection instrument. After applying voltage in the dark, an electrochemiluminescent reaction will occur in the four detection chambers. After the detection instrument collects the luminescence signal, the concentration index of the relevant analyte can be directly determined according to the standard curve.

[0032] Example 1

[0033] Fabrication of electrochemiluminescence chips

[0034] 1. Incubate the silicon wafer mold in a fluorosilane atmosphere for 4 hours to facilitate the subsequent peeling of the PDMS chip from the mold;

[0035] 2. Weigh the PDMS prepolymer and curing agent in a weight ratio of 15:1, place them in a container, stir them evenly with a glass rod, put the container in a vacuum desiccator, evacuate to a vacuum degree of 13psi, and let stand for 30 minutes to remove air bubbles.

[0036] 3. Take out the PDMS mixture, place the silicon wafer mold on a horizontal platform, pour in PDMS, and let it stand for 30 minutes to fill the mold with PDMS; put the mold in an oven and heat at 80°C for 1 hour until the PDMS is completely cured, then carefully peel the PDMS off the silicon wafer.

[0037] 4. Using a needle with a specific aperture, make holes at the inlet 2 and outlet 3 on the PDMS chip; place the PDMS chip in a plasma cleaner, keep it under vacuum for 1 hour, and irradiate it with glow discharge for 2 minutes.

[0038] 5. Remove the chip and add a mixture of PEG(6-9)-siloxane and acetone (V:V = 1:1) to the sample inlet 2. Use the negative pressure inside the PDMS chip as the driving force to fill the entire inner cavity and channels of the chip with the mixture and incubate at room temperature for 1 hour. Then rinse with ultrapure water to make the surfaces of all inner cavities and channels of the PDMS chip hydrophilic, so as to reduce the non-specific adhesion of irrelevant proteins in the sample to each inner cavity and channel.

[0039] The fabrication process of the glass substrate 9 containing the electrode is as follows: A four-inch single-polished glass substrate 9 is used as the substrate. First, plasma treatment and cleaning are performed. The glass substrate is placed in a plasma cleaner and kept under vacuum for 1 hour, followed by glow discharge irradiation for 2 minutes. LC100A photoresist is evenly coated onto the polished surface of the glass substrate 9 using a coating machine. Photolithography: After exposure for 15 seconds, development is performed to obtain the desired pattern. Sputtering: A Cr / Au layer with a thickness of 100 / 1000 Å is sputtered onto the entire glass substrate.

[0040] Photoresist removal: Removes the photoresist that does not need to form patterns, and can also remove excess metal, leaving only the Au electrode block and electrode leads.

[0041] Modification of antibodies required for clinical diagnosis: Protein modification of four different working electrodes of the chip. Working electrode 1 is modified with a primary antibody against troponin I, working electrode 2 is modified with a primary antibody against myoglobin, working electrode 3 is modified with a primary antibody against serum creatine kinase isoenzyme, and working electrode 4 is not modified and is used as a background parameter.

[0042] After the PDMS sheet 1 and the electrode glass substrate 9 modified with the primary antibody are prepared, they are placed in a plasma cleaner and irradiated with glow discharge under vacuum for two minutes. After being taken out, they are quickly aligned and bonded under a microscope with their structural surfaces facing each other to ensure a firm bond.

[0043] Example 2

[0044] After bonding, the electrochemiluminescence chip for myocardial infarction was filled with BSA solution through injection port 2. The chip was then incubated at room temperature for 5 minutes to prevent nonspecific reactions. Unreacted BSA was washed with washing buffer. After washing, different standard concentrations of troponin I (cTnI) were added to injection port 2. Once the chip was filled with this antigen, it was incubated at room temperature for 5 minutes to allow the primary antibody to fully react with the analyte (antigen) in the sample. After the reaction was complete, the chip was removed from the incubator, and washing buffer was added through injection port 2 to remove any unreacted sample. After washing, secondary antibody containing cTnI containing the electrochemiluminescence substrate was added. The chip was then incubated at room temperature for 5 minutes to allow the cTnI and secondary antibody to fully react. After the reaction was complete, washing buffer was added again through injection port 2 to remove any unreacted secondary antibody.

[0045] After cleaning, the chip is placed in the detection instrument in a dark environment. The chip's solder joints are connected to the instrument. The instrument can control the application of working voltage to the detection chamber through the connection with the solder joints. The reaction chamber undergoes an electrochemiluminescence reaction. The detection instrument collects the electrochemiluminescence signal and can detect the signal at the calcium concentration of the antigen. By sequentially detecting the electrochemiluminescence signals corresponding to different standard concentrations of cTnI, the standard curve of cTnI can be obtained.

[0046] Following the aforementioned method, standard curves for myoglobin (Myo) and serum creatine kinase isoenzyme (CK-MB) can be obtained sequentially.

[0047] Example 3

[0048] After bonding, the electrochemiluminescence chip for myocardial infarction is filled with BSA solution through injection port 2. The chip is then incubated at room temperature for 5 minutes to prevent nonspecific reactions. Unreacted BSA is washed away with washing buffer. Human serum sample is then added through injection port 2. Once the chip is filled with serum sample, it is incubated at room temperature for 5 minutes to allow the primary antibody to fully react with the analyte (antigen) in the sample. After the reaction is complete, the chip is removed from the incubator, and washing buffer is added through injection port 2 to remove any unreacted sample. Finally, secondary antibodies containing myocardial infarction biomarkers, including troponin I (cTnI), myoglobin (Myo), and serum creatine kinase isoenzyme (CK-MB), are added. Then incubate at room temperature for 5 minutes to allow the analyte (antigen) and secondary antibody in the sample bound to the primary antibody in the chip to react fully. After the reaction is complete, add washing solution again through injection port 2 to wash out the unreacted secondary antibody.

[0049] After cleaning, the chip is placed in the detection instrument in a dark environment, and the chip's solder joints are connected to the instrument. The instrument can control whether to apply a working voltage to the reaction chamber where a certain electrode is located through the connection with the solder joints. When a working voltage is applied to a certain reaction chamber, the specific reaction chamber undergoes an electrochemiluminescence reaction. The detection instrument detects the electrochemiluminescence signal. The working voltage is applied to different electrodes at different times, and then the electrochemiluminescence signal is collected. The signals of different reaction chambers can be detected. According to the standard curve in Example 2, the concentration of the target analyte can be obtained.

[0050] Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, terms having a conventional meaning are defined herein for clarification or ease of reference, and such definitions should not be construed as indicating a significant difference from conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and employed by conventional methods. Unless otherwise stated, the use of commercially available instruments is performed according to the manufacturer's specifications and parameters.

[0051] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. An electrochemiluminescence chip, characterized in that, The electrochemiluminescence chip comprises, from top to bottom, a PDMS sheet (1) and a glass substrate (9). The area of ​​the PDMS sheet (1) is smaller than that of the glass substrate (9). The glass substrate (9) and the PDMS sheet (1) are bonded together. The PDMS sheet (1) has an inlet (2) and an outlet (3) that penetrate the PDMS sheet (1). The lower surface of the PDMS sheet (1) has a first detection cavity (4), a second detection cavity (5), a third detection cavity (6), and a fourth detection cavity (7) sequentially formed from the inlet (2) to the outlet (3). The sample inlet (2) and the sample outlet (3) are connected by a flow channel (8). The first detection chamber (4) is connected to the sample inlet (2) by a flow channel (8). The first detection chamber (4) and the second detection chamber (5) are connected by a flow channel (8). The second detection chamber (5) and the third detection chamber (6) are connected by a flow channel (8). The third detection chamber (6) and the fourth detection chamber (7) are connected by a flow channel (8). The fourth detection chamber (7) and the sample outlet (3) are connected by a flow channel (8). The width of the flow channel (8) between the injection port (2) and the first detection chamber (4) is 1 mm, the length is 1 cm, and the height is 50 μm. The length of the flow channel (8) between the first detection chamber (4) and the second detection chamber (5), between the second detection chamber (5) and the third detection chamber (6), and between the third detection chamber (6) and the fourth detection chamber (7) is 4 mm, the width is 200 μm, and the height is 50 μm. The inner surfaces of the flow channel (8) and each detection chamber are hydrophilic surfaces formed after treatment with a mixed solution of PEG(6-9)-siloxane and acetone in a volume ratio of 1:

1. The upper surface of the glass substrate (9) has a first working electrode (15), a second working electrode (16), a third working electrode (17), and a fourth working electrode (18). The first working electrode (15) is surrounded by a first pair of electrodes (19), the second working electrode (16) is surrounded by a second pair of electrodes (20), the third working electrode (17) is surrounded by a third pair of electrodes (21), and the fourth working electrode (18) is surrounded by a fourth pair of electrodes (22). The first pair of electrodes (19), the second pair of electrodes (20), the third pair of electrodes (21), and the fourth pair of electrodes (22) all have openings that allow the corresponding metal sputtered layers to pass through. The first working electrode (15) and the first pair of electrodes (19) are completely covered by the first detection cavity (4), the second working electrode (16) and the second pair of electrodes (20) are completely covered by the second detection cavity (5), the third working electrode (17) and the third pair of electrodes (21) are completely covered by the third detection cavity (6), and the fourth working electrode (18) and the fourth pair of electrodes (22) are completely covered by the fourth detection cavity (7). The glass substrate (9) further includes a first solder joint (10), a second solder joint (11), a third solder joint (12), a fourth solder joint (13), a fifth solder joint (14), a first metal sputtering layer (231), a second metal sputtering layer (232), a third metal sputtering layer (233), a fourth metal sputtering layer (234), and a fifth metal sputtering layer (235). The first solder joint (10), the second solder joint (11), the third solder joint (12), the fourth solder joint (13), and the fifth solder joint (14) are all located outside the overlap area between the PDMS sheet (1) and the glass substrate (9). The first solder joint (10) is connected to the first working electrode. (15) The second solder joint (11) is electrically connected to the second working electrode (16) through the first metal sputtering layer (231), the third solder joint (12) is electrically connected to the third working electrode (17) through the third metal sputtering layer (233), the fourth solder joint (13) is electrically connected to the fourth working electrode (18) through the fourth metal sputtering layer (234), and the first pair of electrodes (19), the second pair of electrodes (20), the third pair of electrodes (21), and the fourth pair of electrodes (22) are all electrically connected to the fifth solder joint (14) through the fifth metal sputtering layer (235). The first metal sputtering layer (231), the second metal sputtering layer (232), the third metal sputtering layer (233), the fourth metal sputtering layer (234), and the fifth metal sputtering layer (235) are all Cr / Au layers, and the thickness of the Cr / Au layer is 100 / 1000 Å. The first working electrode (15) is modified with troponin I monoclonal antibody for detecting troponin I, the second working electrode (16) is modified with myoglobin primary antibody for detecting myoglobin, the third working electrode (17) is modified with serum creatine kinase isoenzyme primary antibody for detecting serum creatine kinase isoenzyme, and the fourth working electrode (18) is unmodified and is used as a background parameter.

2. The electrochemiluminescence chip as described in claim 1, characterized in that, The thickness of the PDMS sheet (1) is 2-10 mm, the inlet (2) is circular and the diameter of the inlet (2) is 1-5 mm, the outlet (3) is circular and the diameter of the outlet (3) is 1-5 mm.

3. The electrochemiluminescence chip as described in claim 2, characterized in that, The diameter of both the inlet (2) and outlet (3) is 3 mm.

4. A method for preparing an electrochemiluminescence chip according to any one of claims 1 to 3, characterized in that, Includes the following steps: Preparation of S1 and PDMS tablets (1): S11: Using single-crystal silicon as the substrate, photoresist is spin-coated onto the silicon wafer using a spin coater. After spin coating, pre-drying is performed at a temperature of 60–68°C for 25–35 minutes. After pre-drying, photolithography is performed according to the design layout. After photolithography, post-drying is performed at a temperature of 90–98°C for 25–35 minutes. After post-drying, development is performed using a developer to remove the photoresist and clean the wafer, forming a microstructure of the designed height. The wafer is then hard-baked at 95°C for 30 minutes to obtain a silicon wafer mold with microstructures of different heights. S12: Place the silicon wafer mold obtained in step S11 into a fluorosilane atmosphere and incubate for 200-300 min. Mix the PDMS prepolymer and curing agent evenly and then vacuum dry the mixture. Remove the PDMS mixture and cast it into the silicon wafer mold. Cure the cast mold and peel off the PDMS sheet (1). Drill holes in the PDMS sheet (1) to form an inlet (2) and an outlet (3). Place the drilled PDMS sheet (1) into a plasma cleaner and maintain the vacuum environment for 50-70 min. Irradiate with glow discharge for 100-120 s. Remove the PDMS sheet (1) and add a PEG(6-9)-siloxane and acetone mixed solution with a volume ratio of 1:1 at the inlet (2) and outlet (3). Incubate at room temperature for 40-80 min and then rinse with ultrapure water. S2. Preparation of glass substrate (9): A single-polished glass substrate (9) is used as the substrate. Plasma cleaning is performed. The cleaning conditions are vacuum environment for 50-70 min and glow discharge for 100-120 s. Photoresist is applied to the polished surface of the glass substrate (9) using a coating machine. Photolithography is performed. The photolithography conditions are exposure for 15 s followed by development to obtain the desired pattern. A Cr / Au layer with a thickness of 100 / 1000 Å is obtained by sputtering. Excess photoresist and metal are removed, while Au electrode blocks and electrode leads are retained. S3. Antibody modification: Protein modification was performed on the four different working electrodes of the chip. The first working electrode (15) was modified with a monoclonal antibody of troponin I, the second working electrode (16) was modified with a primary antibody of myoglobin, the third working electrode (17) was modified with a primary antibody of serum creatine kinase isoenzyme, and the fourth working electrode (18) was not modified and was used as a background parameter. After modification, plasma cleaning was performed under vacuum conditions for 50-70 min and glow discharge for 100-120 s. After removal, the glass substrate (9) and PDMS sheet (1) were quickly bonded under a microscope in a way that the structural planes met, and an electrochemiluminescence chip was obtained.

5. The method for preparing the electrochemiluminescence chip as described in claim 4, characterized in that, In step S11, the spin coating thickness of the photoresist is 28–32 μm.

6. The method for preparing the electrochemiluminescence chip as described in claim 4, characterized in that, In step S12, the mass ratio of the PDMS prepolymer to the curing agent is (13-17):

1.

7. The method for preparing the electrochemiluminescence chip as described in claim 4, characterized in that, In step S12, the vacuum drying environment is a vacuum of 13 psi, and the drying time is 25-40 minutes.

8. The application of the electrochemiluminescence chip according to any one of claims 1 to 3 in the preparation of a myocardial infarction biomarker detection kit, characterized in that, The myocardial infarction biomarkers include troponin I, myoglobin, and serum creatine kinase isoenzyme. The kit includes a secondary antibody containing a myocardial infarction biomarker with an electrochemiluminescent substrate. The secondary antibody contains three biomarkers: troponin I, myoglobin, and serum creatine kinase isoenzyme.

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