Detection device, chip manufacturing method and protein marker detection method

Abstract

The present application relates to the field of chips, and provides a detection device, a method for manufacturing a chip, and a method for detecting protein markers for detecting protein markers, comprising a chip, a first cover plate, and a second cover plate. The chip comprises a glass substrate and a microporous array layer disposed on the glass substrate. The microporous array layer is stacked with the glass substrate, and the microporous array layer comprises a plurality of micropores. The first cover plate and the second cover plate are attached and enclosed to form a cavity. The chip is disposed in the cavity. A liquid inlet and a liquid outlet are disposed on the first cover plate. The liquid inlet and the liquid outlet are configured so that liquid entering through the liquid inlet can enter the cavity, flow through the micropores in the microporous array layer, and finally be discharged from the liquid outlet. By applying the present application, a chip having a microporous array layer can be used to perform single separation of detection magnetic bead particles, thereby achieving higher-precision detection of protein markers.

Description

Technical Field

[0001] The present invention relates to the field of chip technology, and in particular to a detection device, a chip manufacturing method and a protein marker detection method. Background Art

[0002] In the current IVD (in vitro diagnostic products) field, which refers to medical devices, in vitro diagnostic reagents, and pharmaceuticals, the primary methods used to detect protein markers are ELISA (enzyme-linked immunosorbent assay), fluorescence, and chemiluminescence. Chemiluminescence is currently the leading technology in immunodiagnosis in terms of sensitivity, precision, and accuracy. However, because chemiluminescence cannot detect single-molecule proteins, and equipment automation technology has reached a bottleneck, the sensitivity of existing chemiluminescence technology is approaching its theoretical detection limit.

[0003] Therefore, there is an urgent need for a detection device and / or detection method for detecting protein markers to achieve higher-precision detection of protein markers. Summary of the Invention

[0004] The present application aims to solve at least one of the technical problems existing in the prior art, and proposes a detection device, a chip manufacturing method and a protein marker detection method. The detection device uses a chip with a microporous array layer, which can perform single separation of detection magnetic bead particles to achieve higher-precision detection of protein markers.

[0005] To achieve the purpose of the present application, a first aspect provides a detection device for detecting protein markers, comprising a chip, a first cover plate and a second cover plate, the chip comprising a glass substrate and a micropore array layer arranged on the glass substrate, the micropore array layer being stacked on the glass substrate, the micropore array layer comprising a plurality of micropores, the first cover plate and the second cover plate being adhered to and enclosing a cavity, the chip being arranged in the cavity, the first cover plate being provided with a liquid inlet and a liquid outlet, the liquid inlet and the liquid outlet being arranged so that the liquid entering through the liquid inlet can enter the cavity, flow through the micropores in the micropore array layer, and finally be discharged from the liquid outlet.

[0006] Optionally, the cavity is formed on the second cover plate, and the second cover plate further includes a connection area arranged around the cavity, and the connection area is in contact with the first cover plate.

[0007] Optionally, an adhesive is provided between the connection area and the first cover plate; or

[0008] The connection area and the first cover plate bonding surface are bonded by ionic bonding.

[0009] Optionally, the micropore array layer includes a photoresist layer and a passivation layer, the photoresist layer is arranged on the glass substrate and stacked with the glass substrate, initial micropores are formed on the photoresist layer, and the passivation layer covers the photoresist layer and covers the bottom wall and side wall of the initial micropores to form the micropores.

[0010] Optionally, the thickness of the passivation layer is to between.

[0011] Optionally, the detection device also includes a magnetic field component, which is arranged on the periphery of the chip and is used to form a magnetic field at least in the micropore array layer. The direction of the magnetic field is along the extension direction of the micropore and has a force on the magnetic body falling into the micropore toward the bottom wall of the micropore.

[0012] Optionally, the size of the micropore array layer is one third to one half of the size of the glass substrate in the same direction.

[0013] Optionally, the diameter of the micropore is between 4 μm and 5 μm, and the depth of the micropore is between 3 μm and 5 μm.

[0014] To achieve the purpose of this application, a second aspect provides a method for manufacturing a chip, wherein the chip is used in a detection device for detecting protein markers, and the manufacturing method comprises:

[0015] providing a glass substrate;

[0016] A micropore array layer is formed on the glass substrate, wherein the micropore array layer has a plurality of micropores arranged in an array.

[0017] Optionally, the step of forming a micropore array layer on the glass substrate includes:

[0018] coating a photoresist layer on the base layer;

[0019] exposing and developing the photoresist layer to form initial micropores arranged in an array on the photoresist layer;

[0020] The passivation layer is formed on the exposed and developed photoresist layer, and the passivation layer covers the bottom wall and the side wall of the initial micropores to form the micropores arranged in an array.

[0021] To achieve the purpose of this application, a third aspect provides an immunoassay method applied to a solution to be detected containing a protein marker, the method comprising:

[0022] reacting the magnetic beads coupled with the antibody of the protein marker with the solution to be detected and the solution with the labeled antibody in sequence to obtain the magnetic beads with the label;

[0023] Applying the chip manufacturing method provided in the second aspect to manufacture a chip, and manufacturing the detection device provided in the first aspect based on the chip, the first cover plate, and the second cover plate;

[0024] All the magnetic beads are loaded onto the detection device from the liquid inlet, so that at least part of the magnetic beads fall into the micropores. The content of the protein marker in the solution to be detected is determined by detecting the total amount of the magnetic beads in the micropores and the fractional amount of the magnetic beads containing the marker.

[0025] Optionally, after at least part of the magnetic beads fall into each of the microwells, the method further comprises:

[0026] The magnetic beads that have not fallen into the micropores are removed by an oil phase flushing method.

[0027] Optionally, the method of removing the magnetic beads that have not fallen into the micropores by flushing with an oil phase includes:

[0028] The detection device is flushed with 40 μL-60 μL of electronic fluoride solution at a flow rate of 2 μL / s-4 μL / s to remove the magnetic beads that have not fallen into the micropores. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A schematic diagram of the structure of a chip of a detection device provided in an embodiment of the present application;

[0030] Figure 2 A schematic diagram of the chip manufacturing process provided in the embodiment of the present application;

[0031] Figure 3 Fluorescence microscope images of different times of post-staining washing provided in the embodiments of the present application;

[0032] Figure 4 Fluorescence microscope images after flushing with electronic fluoride solution of different volumes and flow rates provided in the embodiments of the present application;

[0033] Figure 5 Fluorescence microscope images of the test results obtained using different concentrations of the test solutions provided in the examples of the present application. DETAILED DESCRIPTION

[0034] The present application is described in detail below. Examples of embodiments of the present application are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar components or components having the same or similar functions. In addition, if the detailed description of the known technology is not necessary for the features of the present application shown, it will be omitted. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application and are not to be construed as limiting the present application.

[0035] It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs. It should also be understood that terms such as those defined in common dictionaries should be understood to have meanings consistent with their meanings in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless specifically defined as herein.

[0036] It will be understood by those skilled in the art that, unless otherwise stated, the singular forms "a," "an," and "the" used herein may also include the plural forms. It should be understood that when we say an element is "connected" or "coupled" to another element, it can be directly connected or coupled to the other element, or there may be intermediate elements. In addition, "connected" or "coupled" as used herein may include wireless connections or wireless couplings. The term "and / or" as used herein includes all or any units and all combinations of one or more associated listed items.

[0037] The technical solution of the present application and how the technical solution of the present application solves the above-mentioned technical problems are described in detail below with reference to specific embodiments in conjunction with the accompanying drawings.

[0038] This embodiment studies and analyzes the detection technology of protein markers using magnetic microparticles as solid phase carriers. It is found that: in the detection technology using magnetic microparticles as solid phase carriers, although the magnetic microparticles have a larger surface area than a two-dimensional plane, which allows them to fully react during the detection process, the pH value of the buffer solution in which the magnetic microparticles are located and the concentration of various ions therein will affect the charged magnetic microparticles (causing agglomeration, etc.). Therefore, if the magnetic microparticles can be distributed in a single and uniform manner, this problem can be solved, a more accurate signal can be obtained, and then a higher precision detection of protein markers can be achieved. For example, the method of heating PDMS (polydimethylsiloxane) can be used to fix the magnetic microparticles labeled with antibodies in a PMDS positive mold. However, this method requires heating the mold, which is very likely to cause protein denaturation, and then lead to the inactivation of antigens and antibodies, resulting in a significant decrease in detection accuracy. In addition, the use of PDMS as a microarray material is prone to aging and denaturation, has a short service life, and is expensive.

[0039] In view of the above problems, this embodiment provides a detection device that can be used to detect protein markers, such as Figure 1 and Figure 2 As shown, the detection device includes a chip 10, a first cover plate and a second cover plate. The chip 10 includes a glass substrate 11 and a micropore array layer 12 arranged on the glass substrate 11. The glass substrate 11 and the micropore array layer 12 can be stacked, and the micropore array layer 12 can include a plurality of micropores 121. The first cover plate and the second cover plate are attached to form a cavity. The chip 10 is arranged in the cavity. The first cover plate is provided with a liquid inlet and a liquid outlet. The liquid inlet and the liquid outlet are both located in the area where the first cover plate and the chip 10 are attached, and are connected to the micropores 121 on the micropore array. The liquid inlet and the liquid outlet can be arranged so that the liquid entering through the liquid inlet can enter the above-mentioned cavity, flow through the micropores 121 in the micropore array layer 12, and finally be discharged from the liquid outlet.

[0040] The detection device provided in this embodiment includes a chip 10 having a micropore array layer 12 and a glass substrate 11, and also includes a first cover plate and a second cover plate. The chip 10 can be assembled into a cavity surrounded by the first cover plate and the second cover plate, and then a buffer solution containing magnetic beads that react with the solution to be detected is injected from the liquid inlet hole on the first cover plate. The buffer solution flows on the chip 10, so that the magnetic beads fall evenly into each micropore 121 of the micropore array. The size of the magnetic beads can be matched with the size of the micropore 121 so that each micropore 121 can accommodate only one magnetic bead. The total amount of magnetic beads falling into the micropore 121 and the amount of magnetic beads that react can be used to accurately calculate the content of protein markers in the solution to be detected according to the Poisson's ratio. The chip 10 with a glass substrate 11 directly opens the micropores 121 in its micropore array layer 12, and no heating is required, which will not denature the protein, thereby ensuring the accuracy of protein marker detection, thereby achieving pg / mL (10 -12 Furthermore, the glass-based chip 10 has strong corrosion resistance, is not easily aging or denaturing, has a long service life, and is low in cost.

[0041] The micropore array layer 12 of the chip 10 may include a photoresist layer 14 and a passivation layer 13. The photoresist layer 14 may be disposed on the glass substrate 11 and stacked with the glass substrate 11 to form initial micropores 141 on the photoresist layer 14. The passivation layer 13 may cover the photoresist layer 14 and the bottom and side walls of the initial micropores 141, thereby forming the aforementioned micropores 121. The passivation layer 13 is a hydrophilic modified layer made of an organic material. The molecular interaction between the passivation layer 13 and the protein is relatively small, thereby reducing protein adsorption, improving the fluidity of the buffer solution and the magnetic beads therein, and avoiding damage to the structure of the magnetic beads after the reaction, thereby increasing the micropore filling rate of the magnetic beads and improving the accuracy of detection using the detection device.

[0042] Specifically, the process of forming the micropore array layer 12 can be as follows: 1) spin coating a photoresist layer 14 on the glass substrate 11 (process parameters, for example: the spin coating rate is 400 rpm, the time is 30 s, and it can be baked at 110°C for 150 s, and the thickness of the photoresist layer 14 can be 4 microns); 2) using a mask to expose and develop the photoresist layer 14 (process parameters, for example: development 70 s, exposure 4500 ms) to obtain the initial micropores 141 arranged in the array; 3) using PECVD (Plasma Enhanced Chemical Vapor Deposition, plasma enhanced chemical vapor deposition) process to evaporate the passivation layer 13. The thickness of the passivation layer 13 can be greater than or equal to and less than or equal to The passivation layer 13 is used to isolate the buffer from the glass substrate 11 and the photoresist layer 14, thereby reducing the effect of protein adsorption. Preferably, the thickness of the passivation layer 13 can be

[0043] It should be noted that the structure of the above-mentioned chip 10 is only one implementation of this embodiment, and this embodiment is not limited to this. As long as the chip 10 has a glass substrate 11 and a micropore array layer 12, and micropores 121 arranged in an array are formed on the micropore array layer 12, it will be sufficient.

[0044] The first cover plate and the second cover plate can be transparent glass substrates to facilitate observation of the magnetic bead particles. When the first cover plate, the second cover plate and the chip 10 are assembled, the first cover plate and the second cover plate can be arranged opposite each other, or the first cover plate and the second cover plate can be arranged alternately, as long as the chip 10 can be encapsulated between the two cover plates, this embodiment does not specifically limit this. It should be noted that this embodiment does not limit the specific material of the cover plate, and the two can be the same or different. Preferably, two first cover plates and second cover plates of different materials can be used. For the second cover plate located on the side of the chip 10 away from the micropores 121, the fluidity of the buffer solution can be ignored, and a material that is easy to fit and fix with the chip 10 can be used; and for the first cover plate located on the side of the chip 10 close to the micropores 121, the fluidity of the buffer solution must be considered, that is, to ensure that the chip 10 and the first cover plate can fit and fix with the chip 10, and to ensure the fluidity of the buffer solution, the selected material cannot hinder the flow of the buffer solution.

[0045] Specifically, a cavity can be formed on the second cover plate, and the second cover plate can also include a connection area arranged around the cavity, which can be used to fit with the first cover plate. In this way, the chip 10 can be confined in the cavity with closed sides to prevent the position of the chip 10 from moving during the detection process, thereby reducing the accuracy of the detection results.

[0046] like Figure 2 As shown, the chip 10 may include an array area 102 and a bonding area 101. The bonding area 101 is arranged around the array area 102 and is bonded and connected to the first cover plate. The micropore array layer 12 is formed in the array area 102. In this way, the bonding area 101 is arranged around the array area 102 to facilitate the connection of the chip 10 to the first cover plate and the second cover plate, which can ensure the compression of the chip 10, and then ensure that the buffer can flow evenly through the array area 102, thereby ensuring that the magnetic beads can fall into each micropore 121. The size of the array area 102 (micropore array layer 12) can be one-third to one-half of the size of the glass substrate 11 in the same direction to ensure sufficient bonding area 101 so that the chip 10 can be tightly bonded to the two cover plates. For example, the chip 10 can be a 10mm×10mm glass sheet, which can contain a 4mm×4mm array area 102 inside, and the array area 102 can contain 500×500 micropores 121.

[0047] Furthermore, an adhesive, such as ultraviolet glue, can be provided between the connection area of ​​the second cover plate and the first cover plate, and the chip 10 can be bonded and bonded to the two cover plates by the adhesive. Alternatively, the connection area and the bonding surface of the first cover plate can also be bonded and fixed by plasma bonding (plasma is generated by ionizing air through high-voltage discharge, and the plasma is blown out by airflow. The plasma undergoes physical and chemical changes with the surface of the processed material to make the surface clean and flat for further processing). Among them, by adopting the plasma bonding method, the force between each position of the bonding area 101 of the chip 10 (or the entire surface in contact with the cover plate) and the cover plate is equivalent, which can make the connection between the cover plate and the chip 10 more uniform and stable.

[0048] It should be noted that the above-mentioned method of encapsulating the chip 10 between the two cover plates is only two optional implementation methods of this embodiment, and this embodiment is not limited to this. For example, two flat-plate structure cover plates can also be used, so that the upper and lower surfaces of the chip 10 can be respectively attached and bonded to the two cover plates. The bonding can be performed in the chip bonding area 101 to avoid affecting the flow of the buffer solution containing the magnetic bead particles.

[0049] In order to allow the buffer solution to fully flow through each micropore 121 of the micropore array layer 12 so that the magnetic beads can fall into each micropore 121, the liquid inlet and outlet holes can be set at the position where the first cover plate and the bonding area 101 are bonded, and the two can be close to the two opposite top corners of the chip 10 respectively, so that the buffer solution enters the above-mentioned cavity from the liquid inlet and flows through each micropore 121 from one corner of the chip 10, and the excess buffer solution is discharged from the liquid outlet at the opposite corner, thereby ensuring that magnetic beads can fall into almost every micropore 121.

[0050] To ensure that only one magnetic bead can be placed within micropore 121, the size of the magnetic bead can be designed to match the size of micropore 121, so that both the diameter and depth of micropore 121 are greater than the diameter of the magnetic bead and less than twice the diameter of the magnetic bead. For example, considering machining accuracy and ease of observation, the diameter of micropore 121 can be greater than or equal to 4μm and less than or equal to 5μm, preferably 4.5μm; the depth of micropore 121 can be greater than or equal to 3μm and less than or equal to 5μm, preferably 4μm. The diameter of the magnetic bead can then be greater than 2.5μm and less than 3μm.

[0051] In order to facilitate the entry of magnetic beads into the micropores 121, the detection device may further include a magnetic field component, which may be arranged on the periphery of the chip 10 to form a magnetic field at least in the micropore array layer 12 (array area 102). The direction of the magnetic field is along the extension direction of the micropores 121, and has a force on the magnetic body (such as magnetic beads) falling into the micropores 121 toward the bottom wall of the micropores 121.

[0052] Based on the same concept as the above detection device, this embodiment further provides a method for manufacturing a chip 10, which is used to manufacture the above chip 10. The chip 10 can be used in the above detection device for detecting protein markers, and can include the following steps:

[0053] (1) Providing a glass substrate 11, which may be a transparent glass substrate 11, so as to facilitate observation of the magnetic beads;

[0054] (2) A micropore array layer 12 is formed on the glass substrate 11. The micropore array layer 12 has a plurality of micropores 121 arranged in an array for separating magnetic beads during protein marker detection.

[0055] As described above, the step of forming the micropore array layer 12 on the glass substrate 11 may include:

[0056] (21) coating a photoresist layer 14 on the base layer, wherein the thickness of the photoresist layer 14 is sufficient to form arrayed micropores 121;

[0057] (22) exposing and developing the photoresist layer 14 to form initial micropores 141 arranged in an array on the photoresist layer 14;

[0058] (23) A passivation layer 13 is formed on the exposed and developed photoresist layer 14, and the passivation layer 13 covers the bottom wall and side wall of the initial micropores 141, forming an array of micropores 121.

[0059] The manufacturing method of the chip 10 provided in this embodiment can manufacture a chip 10 including a glass substrate 11 and a micropore array layer 12. The chip 10 can be applied to the above-mentioned detection device to facilitate separation of magnetic bead particles and improve detection accuracy.

[0060] Based on the same concept as the above detection device, this embodiment also provides a method for detecting protein markers, which can be applied to a solution to be detected containing protein markers. The method may include:

[0061] Step S1: reacting magnetic beads coupled with antibodies to protein markers with a solution to be detected and a solution containing labeled antibodies in sequence to obtain magnetic beads containing labeled antibodies.

[0062] During the detection process, magnetic beads coupled with antibodies to protein markers are added to the solution to be detected, so that the antibodies coupled to the magnetic beads bind to the protein antigens in the solution to be detected. All magnetic beads (including those bound to antigens) are then washed and added to a solution with labeled antibodies for reaction, so that the antigens bound to the magnetic beads react with the labeled antibodies, thereby obtaining magnetic beads with markers.

[0063] Specifically, before using the magnetic beads for immune reaction, the magnetic beads can be stained to obtain fluorescent magnetic beads to facilitate observation of the magnetic beads. After staining, the stained magnetic beads can be washed to minimize the background fluorescence to avoid the staining solution from affecting subsequent detection. Before use, the magnetic beads can be stored in a preservation solution to ensure their biological activity. Furthermore, this embodiment also tested the number of times the dyed magnetic beads were washed to obtain the number of times that can minimize the background fluorescence and ensure the fluorescent effect of the magnetic beads, such as Figure 3 As shown in the figure, (A), (B), and (C) are images observed using a fluorescence microscope after washing three times, four times, and five times respectively. The bright spots in the figure can represent fluorescent magnetic beads. Figure 3 It can be seen that after five washes, the background fluorescence of the magnetic beads that have not fallen into the wells can be reduced to a lower level while ensuring the fluorescence effect of the magnetic beads.

[0064] In step S2, the chip 10 is manufactured using the manufacturing method of the chip 10, and the detection device is manufactured based on the chip 10, the first cover plate and the second cover plate. For details, please refer to the above-mentioned detection device embodiment and the chip 10 manufacturing method embodiment, which will not be repeated here.

[0065] In step S3, all the magnetic beads are loaded onto the detection device from the liquid inlet, so that at least part of the magnetic beads fall into the micropores 121. The total amount of magnetic beads in the micropores 121 and the total amount of magnetic beads containing markers are detected to determine the content of protein markers in the solution to be detected.

[0066] The loading process can refer to the use process of the above-mentioned detection device, that is, injecting buffer solution containing magnetic beads from the liquid inlet of the first cover plate, and discharging excess buffer solution and magnetic beads from the liquid outlet, so that the magnetic beads can fall into each micropore 121 during the flow of the buffer solution.

[0067] It should be noted that this embodiment does not specifically limit the execution order of the above-mentioned step S1 and step S2, and the two can also be performed simultaneously.

[0068] In order to facilitate the observation and quantity detection of the magnetic bead particles, after at least some of the magnetic bead particles fall into each micropore 121, it can also include: using an oil phase flushing method to remove the magnetic bead particles that have not fallen into the micropore 121, so as to avoid the magnetic bead particles that have not fallen into the micropore 121 affecting the observation and detection of the magnetic bead particles that have fallen into the micropore 121 (for example, it is impossible to tell whether the magnetic bead particles have fallen into the micropore 121).

[0069] Specifically, the above-mentioned method of using oil phase flushing to remove the magnetic beads that have not fallen into the micropores 121 may include the following treatment: using 40 μL-60 μL of electronic fluoride liquid at a flow rate of 2 μL / s-4 μL / s to flush the detection device to remove the magnetic beads that have not fallen into the micropores 121. Before determining the volume and flow rate of the oil phase liquid, this embodiment conducted a comparative test on the cleaning effects of using different volumes and flow rates, such as Figure 4 As shown, after 15 μL of the test solution containing 500,000 magnetic beads is passed into the chip 10 with 200,000 micropores 121, the fluorescence microscope images are taken under a fluorescence microscope with an excitation wavelength of 495 nm, a 10x objective lens, and an exposure time of 700 ms (the bright spots in the image can represent the magnetic beads). In the figure, (A) is the image without oil phase flushing after adding the magnetic beads, (B) is the image with 30 μL of electronic fluoride solution at a flow rate of 10 μL / s, (C) is the image with 50 μL of electronic fluoride solution at a flow rate of 5 μL / s, and (D) is the image with 50 μL of electronic fluoride solution at a flow rate of 3 u / s. Figure 4 It can be seen that using 50 μL of electronic fluoride solution at a flow rate of 3 μL / s to flush the chip 10 can maximize the reduction of noise while retaining more magnetic beads in the wells.

[0070] The immunoassay method is described in detail below with reference to specific examples.

[0071] 1. Production of detection device

[0072] The manufacturing method of the chip 10 and the structure of the detection device can be referred to to manufacture the 10 mm×10 mm chip 10 and to manufacture the detection device based on the chip 10 , which will not be described in detail here.

[0073] 2. Reagent Preparation

[0074] Since digital immunoassay requires applying a solution containing magnetic beads to the chip 10, a single magnetic bead falls into each microwell 121 of the array for separation and observation. The conditions for the magnetic beads to fall into the wells are related to the concentration of the solution containing the magnetic beads and the waiting time after addition. For example, if there are 250,000 microwells on the manufactured chip 10, assuming that the enzyme-catalyzed fluorescence response observed for 100 magnetic beads is within the acceptable Poisson noise (≤10%), and that the magnetic beads with enzyme fluorescence response account for 1% of the proportion of all magnetic beads entering the microwell 121 as the detection limit, then the minimum efficiency of the magnetic beads entering the wells can be calculated according to the Poisson formula to be 4.6%, that is, the minimum concentration of the magnetic beads after the immune reaction should be between 4,000 and 7,000 per μL.

[0075] Specifically, 2.8 μm carboxyl magnetic beads are activated with EDC (an activator) and then coupled with the SPRN-5 capture antibody (antibody against bovine serum albumin (BSA)). After the antibody is coupled to the magnetic beads, they need to be stained for subsequent counting, allowing direct localization of the number of magnetic beads that have entered the well (including both those attached and those not attached to the antigen being tested).

[0076] The dye concentration used for magnetic bead staining can be 10 mg / mL (1 mg of dye dissolved in 100 μL of diluent). 10 million magnetic beads linked to capture antibodies are suspended in 600 μL of 0.1 M NaHCO₃ solution, and 4.4 μL of dye at pH 8.5 is added. Staining can be performed at room temperature with continuous shaking for 2 hours. After staining, the magnetic beads can be washed five times with PBST (0.1% Tween). Finally, the stained antibody-containing magnetic beads can be stored in magnetic bead storage solution, which can include 50 mM buffer, 150 mM NaCl at pH 7.8, 10 mM EDTA, 1% BSA, 1% Triton-100, and 0.15% Proclin-100.

[0077] 3. Immune-specific response:

[0078] The sample to be tested is selected to contain 100 μL of standard solution containing different concentrations of PSA antigen, the composition of which is PBST (0.1% Tween) with 2.5% BSA solution added. The number of magnetic beads required for a single reaction is 500,000, which are added to the diluent of the sample to be tested for immune reaction. The reaction is carried out at room temperature for 2 hours with continuous shaking. After the reaction is completed, the magnetic beads are washed five times with washing solution (PBST). After washing, 50 μL of 1nM concentration of labeled antibody solution is added to the magnetic beads for immune reaction. The reaction conditions are 1 hour at room temperature with continuous shaking. After the reaction is completed, the magnetic beads are washed 3 times with washing solution. 40pM PBST and 1mM MgCl2 enzyme marker reaction is added, and the reaction conditions are room temperature for 30 minutes. After the reaction is completed, the beads are separated and washed 5 times. Finally, the obtained magnetic beads are added to 15μL 100uMol substrate and passed onto the chip 10 of the detection device.

[0079] 4. Fluorescent magnetic beads after washing:

[0080] After the solution containing magnetic beads is loaded onto the chip 10, a magnetic field can be applied to the outside of the chip 10 to cause the magnetic beads to settle into the micropores 121. At the same time, a large number of disordered magnetic beads that have not entered the micropores 121 will be displayed on the chip 10, which will affect signal collection. To solve this problem, 50 μL of electronic fluoride solution can be used to flush the detection device (the location of the chip 10) at a flow rate of 3 μL / s to minimize the noise while retaining more magnetic beads in the micropores 121 to ensure the accuracy of the detection.

[0081] 5. Test results

[0082] Different concentrations of the antigen to be tested were used for testing, namely 0 pg / mL (negative sample), 0.2 pg / mL and 1000 pg / mL. A fluorescence microscope equipped with a 1000W pixel camera was used to observe the test results. First, an image of 520 nm fluorescence emission light was obtained under 495 nm excitation light to locate the positions of all magnetic beads in the microwell 121, such as Figure 5 (A), (B), (C); then, under 577 excitation light, a picture of 620nm fluorescence emission light is obtained to locate the fluorescence response of magnetic beads connected with antigens and labeled antibodies, such as Figure 5 (D), (E), and (F) in the figure. The Poisson equation is then used to calculate the proportion of magnetic beads with antigens to the total number of magnetic beads, enabling more accurate digital detection of protein markers and qualitatively observing the response of the detection technology to different concentrations of the analyte.

[0083] In summary, the immunoassay method provided in this embodiment can ensure the precision and accuracy of protein marker detection, thereby achieving pg / mL (10-12 ) concentration level. The detection method is simple and easy to implement.

[0084] Those skilled in the art will understand that the various operations, methods, steps, measures, and solutions in the process discussed in this application may be alternated, modified, combined, or deleted.

[0085] In the description of this application, it should be understood that the terms "center", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship, are based on the orientation or position relationship shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on this application.

[0086] The above are only some of the implementation methods of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.

Claims

1. A detection device for detecting protein markers, characterized in that: The chip comprises a first cover plate and a second cover plate, wherein the chip comprises a glass substrate and a micropore array layer disposed on the glass substrate, the micropore array layer being stacked on the glass substrate and comprising a plurality of micropores, the first cover plate and the second cover plate being attached to and enclosing a cavity, the chip being disposed in the cavity, the first cover plate being provided with a liquid inlet and a liquid outlet, the liquid inlet and the liquid outlet being configured such that liquid entering through the liquid inlet can enter the cavity, flow through the micropores in the micropore array layer, and finally be discharged from the liquid outlet; The liquid entering the liquid inlet hole includes a plurality of magnetic beads; The diameter of the micropores is larger than the diameter of the magnetic beads and smaller than twice the diameter of the magnetic beads; the micropore array layer is capable of allowing at least some of the magnetic beads to fall into the micropores, and different magnetic beads fall into different micropores; The micropore array layer includes a photoresist layer and a passivation layer. The photoresist layer is arranged on the glass substrate and stacked with the glass substrate. Initial micropores are formed on the photoresist layer. The passivation layer covers the photoresist layer and covers the bottom wall and side wall of the initial micropores to form the micropores.

2. The detection device according to claim 1, characterized in that The cavity is formed on the second cover plate, and the second cover plate further includes a connection area arranged around the cavity, and the connection area is in contact with the first cover plate.

3. The detection device according to claim 2, characterized in that An adhesive is provided between the connection area and the first cover plate; or The connection area and the first cover plate bonding surface are bonded by ionic bonding.

4. The detection device according to claim 1, characterized in that The thickness of the passivation layer is between 2500Å and 3500Å.

5. The detection device according to any one of claims 1 to 4, characterized in that: The detection device also includes a magnetic field component, which is arranged on the periphery of the chip and is used to form a magnetic field at least in the micropore array layer. The direction of the magnetic field is along the extension direction of the micropores, and has a force on the magnetic beads falling into the micropores toward the bottom wall of the micropores.

6. The detection device according to any one of claims 1 to 4, characterized in that: The size of the micropore array layer is one third to one half of the size of the glass substrate in the same direction.

7. The detection device according to any one of claims 1 to 4, characterized in that: The diameter of the micropores is between 4 μm and 5 μm, and the depth of the micropores is between 3 μm and 5 μm.

8. A method for manufacturing a detection device according to any one of claims 1 to 7, wherein the detection device is used to detect protein markers, characterized in that: The manufacturing method comprises: Providing a first cover plate and a second cover plate, and forming a liquid inlet hole and a liquid outlet hole on the first cover plate; Providing a glass substrate and forming a micropore array layer on the glass substrate to form a chip; the micropore array layer has a plurality of micropores arranged in an array; The first cover plate and the second cover plate are bonded together to form a cavity, and the chip is placed in the cavity; wherein the liquid inlet and the liquid outlet are configured so that the liquid entering through the liquid inlet can enter the cavity, flow through the micropores in the micropore array layer, and finally be discharged from the liquid outlet.

9. The manufacturing method according to claim 8, characterized in that The step of forming a micropore array layer on the glass substrate comprises: forming a photoresist layer on the base layer; exposing and developing the photoresist layer to form initial micropores arranged in an array on the photoresist layer; The passivation layer is formed on the exposed and developed photoresist layer, and the passivation layer covers the bottom wall and the side wall of the initial micropores to form the micropores arranged in an array.

10. A method for detecting protein markers, characterized in that: The detection device according to any one of claims 1 to 7 is used; wherein the method comprises: reacting the magnetic beads coupled with the antibody of the protein marker with the solution to be detected and the solution with the labeled antibody in sequence to obtain the magnetic beads with the label; All the magnetic beads are loaded onto the detection device from the liquid inlet hole so that at least part of the magnetic beads fall into the micropores. The content of the protein marker in the solution to be detected is determined by detecting the total amount of the magnetic beads in the micropores and the fractional amount of the magnetic beads containing the marker.

11. The method for detecting protein markers according to claim 10, characterized in that: After at least part of the magnetic beads fall into the micropores, the method further comprises: The magnetic beads that have not fallen into the micropores are removed by an oil phase flushing method.

12. The method for detecting protein markers according to claim 11, characterized in that: The method of using oil phase flushing to remove the magnetic beads that have not fallen into the micropores includes: The detection device is flushed with 40 μL to 60 μL of electronic fluoride solution at a flow rate of 2 μL / s to 4 μL / s to remove the magnetic beads that have not fallen into the micropores.

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