A three-dimensional digital immunoassay microfluidic chip based on microchannel plates

Abstract

The present invention discloses a three-dimensional digital immunoassay detection microfluidic chip based on a microchannel plate. The MCP microchannel plate of the microfluidic chip is embedded in the middle of the PDMS middle cavity plate, and two layers of PDMS outer cavity plates are stacked and bonded on the upper and lower sides of the PDMS middle cavity plate. The preparation method comprises: mixing a polydimethylsiloxane (PDMS) solution and a curing agent, evacuating the mixture to remove bubbles, pouring the mixture into a mold, heating and curing, ultrasonically cleaning the mixture, and then drying the mixture with nitrogen to obtain a PDMS cavity plate; performing fluorescence enhancement and external modification treatment on the MCP microchannel plate, and then embedding the plate into the PDMS middle cavity plate; performing plasma cleaning and bonding to obtain a microfluidic chip. The microfluidic chip of the present invention has a 3D structure, fast sample flow and detection speed, extremely low detection concentration, relatively high sensitivity, wide detection range, simple production, and is adaptable to a variety of usages.

Description

Technical Field

[0001] The present invention relates to a microfluidic chip, in particular to a three-dimensional digital immunoassay detection microfluidic chip based on a microchannel plate. Background Art

[0002] Common immunoassay methods include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), and chemiluminescence. Although immunoassays have become a common analytical method, traditional immunoassays require complex procedures and bulky laboratory equipment, hindering their widespread adoption in point-of-care (POCT) diagnostics. However, combining immunoassays with microfluidics can significantly address these shortcomings. Microfluidic immunoassay systems are used for disease diagnosis, environmental monitoring, and food and drug screening. Immunofluorescence technology, among other techniques, uses fluorescein-labeled antibodies to detect corresponding antigens in tissues, cells, or serum. Due to their safety and sensitivity, fluorescent antibodies have been widely used in immunofluorescence assays and flow cytometry. Enzyme-linked immunosorbent assay (ELISA) is currently the most widely used immunoassay method. This method combines the specificity of the antigen-antibody reaction with the action of an enzyme-catalyzed substrate. The test result is determined by the color change produced by the enzyme's action on the substrate, with sensitivity reaching nanograms (ng).

[0003] The development and innovation of microfluidic immunoassay technology is rapidly evolving. For example, lateral flow immunochromatographic testing, a major form of point-of-care diagnostics (POCT), is essentially an antibody-antigen immune reaction. A liquid sample is added through a sample pad and flows toward the side of absorbent paper. As it passes through the conjugate pad, the immunoprobes on the pad are released and react with the analyte in the sample during the flow. These probes are then captured and aggregated by the detection line antibodies immobilized on the NC membrane, generating an optical signal. This method requires the analyte in the sample to diffuse onto the antibody surface, resulting in low capture efficiency and poor accuracy. It also requires large sample volumes and long reaction times. Compared to lateral flow immunoassays, immunomagnetic bead technology is primarily used in the field of microbial detection. It can also be combined with other detection mechanisms to perform immunoassays. It offers high efficiency, selective isolation of microorganisms, and effective reduction of background interference. However, due to the need for improvement in domestic magnetic bead technology, it still needs to be imported, which is expensive. Furthermore, magnetic bead immunoassays almost always require additional methods, such as separation plates, PCR, and fermentation experiments, to avoid cross-reaction with contaminants. Another example is the centrifugal microfluidic immunoassay platform, which uses centrifuge tubes and optical discs, which are expected to develop towards automation, integration, and miniaturization. However, the complex operation steps, large equipment volume, and complex external equipment are all obstacles to the further promotion of such platforms. Summary of the Invention

[0004] In order to solve the problems existing in the background technology, the present invention provides a three-dimensional digital immunoassay detection microfluidic chip based on a microchannel plate.

[0005] The technical solution adopted in the present invention is:

[0006] 1. A three-dimensional digital immunoassay microfluidic chip based on a microchannel plate:

[0007] The microfluidic chip consists of two PDMS outer cavity plates, a PDMS middle cavity plate, and an MCP microchannel plate. The MCP microchannel plate is embedded in the middle of the PDMS middle cavity plate, and the two PDMS outer cavity plates are laminated and bonded to the upper and lower surfaces of the PDMS middle cavity plate. The MCP microchannel plate is specifically a single-sided gold nanopillar microchannel plate.

[0008] The center of one side surface of the PDMS outer cavity plate is provided with an axially symmetrical groove that is wide in the middle and narrow on both sides in the vertical direction. The middle part of the groove is a rectangular groove, and through holes that pass through the PDMS outer cavity plate are symmetrically provided at both ends of the groove; the groove of the PDMS outer cavity plate located on the upper side of the PDMS middle cavity plate faces downward, and the groove of the PDMS outer cavity plate located on the lower side of the PDMS middle cavity plate faces upward, and the groove centers of the two layers of PDMS middle cavity plates are symmetrical to the PDMS middle cavity plate.

[0009] A rectangular through groove running through the PDMS middle cavity plate is opened in the center of the plate surface in the vertical direction. The rectangular through groove faces the rectangular groove of the PDMS outer cavity plate. The MCP microchannel plate is embedded in the rectangular through groove in the middle of the PDMS middle cavity plate.

[0010] The depth of the groove of the PDMS outer cavity plate is slightly less than the thickness of the PDMS outer cavity plate. The MCP microchannel plate is rectangular and has the same dimensions as the rectangular through-groove of the PDMS middle cavity plate. The rectangular surfaces of the rectangular groove of the PDMS outer cavity plate and the rectangular through-groove of the PDMS middle cavity plate have the same or slightly different dimensions.

[0011] 2. Preparation method of a three-dimensional digital immunoassay microfluidic chip:

[0012] The method comprises the following steps:

[0013] Step 1) preparing two layers of PDMS outer cavity plates and a PDMS middle cavity plate: the steps for preparing each layer of the PDMS outer cavity plate and the PDMS middle cavity plate are the same, specifically, mixing the polydimethylsiloxane PDMS solution and the curing agent and stirring them evenly, then vacuuming to remove bubbles to obtain a PDMS curing mixed solution, pouring the PDMS curing mixed solution into the respective pre-prepared molds, heating and curing on a hot plate to obtain a PDMS cured plate, removing the PDMS cured plate from the mold, and ultrasonically cleaning with acetone, isopropyl alcohol, and DI water in sequence, and then drying with nitrogen to obtain a PDMS outer cavity plate or a PDMS middle cavity plate.

[0014] Step 2) The MCP microchannel plate is sequentially subjected to fluorescence enhancement treatment and external modification treatment, and then embedded in the rectangular through-groove in the middle of the PDMS middle cavity plate.

[0015] Step 3) Plasma cleaning and bonding of the two PDMS outer cavity plates and the PDMS middle cavity plate embedded in the MCP microchannel plate are performed to obtain a microfluidic chip.

[0016] In the step 1), 20 g to 30 g of polydimethylsiloxane (PDMS) solution and curing agent are mixed and stirred evenly, and then vacuumed to remove bubbles to obtain a PDMS curing mixed solution. The mass ratio of the polydimethylsiloxane (PDMS) solution to the curing agent is 1 / 10.

[0017] In the step 1), the PDMS curing mixed solution is poured into a pre-prepared mold, heated on a hot plate at 75°C-85°C for 1.5-2.5 hours to obtain a PDMS cured plate, and the PDMS cured plate is removed from the mold and ultrasonically cleaned with acetone for 5-10 minutes, isopropyl alcohol for 5-10 minutes, and DI water for 5-10 minutes, followed by drying with nitrogen.

[0018] In the step 2), the fluorescence enhancement treatment specifically includes first placing metal particles in a glass dish, then placing the MCP microchannel plate in the glass dish, sealing the top of the glass dish with a glass sheet, and placing the MCP microchannel plate tightly against the bottom of the glass sheet. The glass dish is heated until the metal particles melt, and then the MCP microchannel plate is reversely evaporated, so that the collimated vapor atoms generated by the melted metal particles are uniformly vacuum-evaporated and etched on the inner wall of the MCP microchannel plate, thereby completing the fluorescence enhancement treatment.

[0019] Since the holes in the MCP microchannel plate have a certain tilt angle, a large incident angle (>70°) is provided between the collimated vapor atoms and the local surface normal, thereby inducing a shadow effect, thereby achieving local oblique angle deposition, and selectively forming nanorod structures on the steep sidewalls of the surface microcavity through standard photolithography and silicon dry etching.

[0020] In the step 2), the external modification treatment is specifically to immerse the MCP microchannel plate in the cross-linker DSP for 2-2.5 hours, then wash it with phosphate buffered saline (PBST), and then immerse it in 0.1 ng / ml goat immunoglobulin GOAT-IgG or 0.1 ng / ml BGAL-Streptavidinβ-galactosidase labeled streptavidin SβG for 2 hours, followed by washing with phosphate buffered saline (PBST), and then immersing it in bovine serum albumin (BSA) for 1 hour to fill the blanks, and finally using phosphate buffered saline (PBST) to wash away excess bovine serum albumin (BSA) to complete the external modification treatment.

[0021] In the step 3), the two PDMS outer cavity plates and the PDMS middle cavity plate embedded with the MCP microchannel plate are plasma cleaned for 50-60 seconds in a 60W plasma cleaner and then bonded in sequence to obtain a microfluidic chip.

[0022] 3. Application of a three-dimensional digital immunoassay microfluidic chip:

[0023] Application of microfluidic chips in fluorescence immunoassay and fluorescence enzyme-linked immunosorbent assay.

[0024] The beneficial effects of the present invention are:

[0025] 1. High-speed detection, high sensitivity, and easy observation: The invention utilizes millions of pores as capture channels, breaking away from the traditional 2D approach to immunoassays and adopting a 3D approach. Furthermore, the Poisson distribution formula allows for the detection of extremely low concentrations of antigenic proteins, resulting in low detection concentrations, high sensitivity, and rapid sample flow. The gold nanopillars on the channel walls enhance fluorescence amplification for both fluorescent enzyme-linked immunosorbent assays and direct fluorescent immunoassays, facilitating observation.

[0026] 2. Wide detection range: In addition to detecting antigens and antibodies used in experiments, it can also detect samples based on other ELISA reactions. Because the volume of the microchannel is in the pL-nL range, the pore density is high, which is conducive to the detection of high concentrations, resulting in a wide detection range.

[0027] 3. Easy to manufacture: The labeled MCP used in the main structure can be manufactured on a large scale. The gold nanocolumns on the inner wall of the channel can also be easily vacuum-deposited. All the materials and equipment required for the device only require polydimethylsiloxane (PDMS) and a plasma cleaner. Finally, the three are combined to form the final chip.

[0028] 4. Multiple usage modes: It can not only be used as a two-way channel, but also can be turned into a reaction vessel by using high-density heavy oil to seal the bottom.

[0029] The microfluidic chip of the present invention has a 3D structure, fast sample flow and detection speed, extremely low detection concentration, relatively high sensitivity, wide detection range, simple production, and is adaptable to various usage modes. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 (a) is a schematic diagram of the cross-sectional structure of the microchannel plate MCP of the present invention;

[0031] Figure 1 (b) is a schematic diagram of the three-dimensional structure of the microchannel plate MCP of the present invention;

[0032] Figure 2 This is a scanning electron microscope (SEM) image of the unilateral gold nanocolumn microchannel plate structure of the present invention;

[0033] Figure 3 (a) is a cross-sectional view of the microfluidic chip of the present invention;

[0034] Figure 3 (b) is a top view of the microfluidic chip of the present invention;

[0035] Figure 3 (c) is a 3D view of the microfluidic chip of the present invention;

[0036] Figure 4 (a) is a schematic diagram of the process of the protein capture fluorescence reaction in the pores of the microchannel plate MCP of the microfluidic chip of the present invention;

[0037] Figure 4 (b) is a schematic diagram of the process of the microfluidic chip of the present invention being used as a container for reaction after the bottom is sealed;

[0038] Figure 5 Schematic diagram of the through-holes of the microfluidic chip of the present invention;

[0039] Figure 6 Schematic diagram of the manual gas valve operation mode of the microfluidic chip of the present invention;

[0040] Figure 7 (a) is a schematic diagram of the upper and lower structures of the microfluidic chip of the present invention;

[0041] Figure 7 (b) is a schematic diagram of the middle layer structure of the present invention. DETAILED DESCRIPTION

[0042] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0043] like Figure 1 (a) and Figure 1As shown in (b), the MCP microchannel plate is based on a glass sheet. On the substrate, cylindrical microchannels with an aperture slightly smaller than the spatial period are arranged in a hexagonal pattern with a spatial period of several microns to more than ten microns. They are usually used in the field of photon amplification. There are about one million microchannels on an MCP. The volume of the channels on the same MCP is the same. The volume of the microchannels varies from 10pL to 10nL due to different models. At the same time, due to the extremely small spacing between the microchannel plates (about 1um), there are a large number of reaction holes in one field of view. Specifically, Figure 1 (b) A rectangular plate is cut on the MCP microchannel rod as the MCP microchannel plate.

[0044] The microchannel plate used in this microfluidic chip is a "single-sided gold nanopillar microchannel plate". Figure 2 Shown is the SEM image of the structure.

[0045] Under specific excitation conditions, periodically distributed metal nanostructures can generate surface plasmon polaritons (SPPs), creating a localized enhanced electric field on the substrate surface. This localized field effectively modulates the electronic transitions of fluorescent molecules near the metal substrate surface, thereby controlling fluorescence. Nanometal structures enhance fluorescence, enabling fluorescence enhancement to become visible to the naked eye in reactions such as fluorescent immunoassays and fluorescent enzyme immunoassays (ELISAs).

[0046] Figure 3 (a) is a cross-sectional view of the microfluidic chip of the present invention, which is composed of upper, middle and lower layers formed by combining PDMS polydimethylsiloxane and MCP microchannel plates, four upper and lower functional multiplexed liquid inlet / discharge cavities, and the middle layer is inlaid with MCP microchannel plates. Figure 3 (b) is a top view. Figure 3 (c) is a rough schematic diagram of the 3D structure. The upper cavity of the upper layer is the reaction site, and the lower layer also has a lower cavity. The cavity is filled with liquid with a volume of about 30ul. Then, according to the needs of the reaction experiment, the holes are appropriately blocked and the reaction liquid is added. The reaction area is in the hole of MCP. Since PDMS polydimethylsiloxane is transparent, the reaction fluorescence effect in the hole can be directly observed. Figure 5 As shown, the upper through-holes a and b as well as the lower through-holes c and d are all drainage ports / inlet ports.

[0047] In specific implementation, plugs can be used to block the corresponding holes according to the required liquid flow direction. Figure 4 (a) and Figure 5As shown in the figure, the microfluidic chip captures the fluorescence reaction of proteins directly in the pores of MCP. In order to allow liquid 1 to pass through the MCP to the bottom, the three holes b, c, and d are blocked. At this time, the flow resistance is minimal and the liquid can fill the entire chamber. When the liquid needs to be released, the two plugs below are removed to release the immersed reaction liquid. Figure 4 (b) and Figure 5 As shown, the bottom of the microfluidic chip is sealed to serve as a container reaction. First, three liquids of different densities are used. The upper end is not blocked, and the liquid 2 with the highest density is added from the c hole and then the bottom is sealed. Subsequently, the b port is blocked, and the sample liquid (liquid 3) is added from the a port until the sample liquid enters the pores. Finally, a mineral oil with a low density (liquid 4) is used to seal the pores. The sample liquid is added from the a port, and the b port is used to discharge the excess sample liquid. After the reaction in the pores is complete, the final observable state is reached. Mineral oil can be used to discharge the excess reaction liquid and separate adjacent microchannels into independent reaction chambers. Liquid 1 is an antigen liquid, an antibody liquid, a buffer solution, a chain enzyme or avidin, etc., liquid 2 is a high-visibility liquid such as heavy oil, liquid 3 is an enzyme substrate, and liquid 4 is mineral oil.

[0048] like Figure 6 As shown, the microfluidic chip is used as a passive pressure microfluidic system. When liquid is poured in, the air inside the chip needs to be drawn out by retracting and releasing the plug, so as to achieve liquid immersion and sealing. The two holes in the lower chamber are sealed with one of the holes in the upper chamber. At this time, if the solution is poured from the upper chamber, air resistance will be generated. One of the openings in the lower chamber is opened and the solution is poured in. The solution will expel part of the air in the lower chamber. At this time, the entire lower chamber is sealed again, the plug in the upper chamber is removed, and the liquid is squeezed in again to discharge the air in the upper chamber. Finally, the plug removed from the upper chamber is plugged back in, the plug on the other side of the lower chamber is removed, and another part of the air in the lower chamber is discharged by squeezing the liquid in the upper chamber. This step is used to control the liquid immersion and sealing of the entire device.

[0049] The specific embodiments of the present invention are as follows:

[0050] Example 1:

[0051] The preparation method of the three-dimensional digital immunoassay detection microfluidic chip comprises the following steps:

[0052] Step 1) Prepare two layers of PDMS outer cavity plates and PDMS middle cavity plates: The steps for preparing each layer of PDMS outer cavity plate and PDMS middle cavity plate are the same, specifically, 20g of polydimethylsiloxane PDMS solution and curing agent are mixed and stirred evenly, and then vacuumed to remove bubbles to obtain a PDMS curing mixed solution, and the mass ratio of polydimethylsiloxane PDMS solution and curing agent is 1 / 10; the PDMS curing mixed solution is poured into a pre-prepared mold, heated on an 80°C hot plate for 2h to obtain a PDMS cured plate, and the PDMS cured plate is removed from the mold, and ultrasonically cleaned with acetone for 5min, isopropyl alcohol for 10min, and DI water for 10min, and then blown dry with nitrogen to obtain a PDMS outer cavity plate or a PDMS middle cavity plate.

[0053] Step 2) The MCP microchannel plate is sequentially subjected to fluorescence enhancement treatment and external modification treatment, and then embedded in the rectangular through-groove in the middle of the PDMS middle cavity plate. The fluorescence enhancement treatment specifically involves first placing metal particles in a glass dish, then placing the MCP microchannel plate in the glass dish, sealing the top of the glass dish with a glass sheet, and placing the MCP microchannel plate close to the bottom of the glass sheet. The glass dish is heated until the metal particles melt, and then the MCP microchannel plate is reverse-evaporated, so that the aligned vapor atoms generated by the melted metal particles are evenly vacuum-evaporated and etched on the inner wall of the MCP microchannel plate to complete the fluorescence enhancement treatment.

[0054] Since the holes in the MCP microchannel plate have a certain tilt angle, a large incident angle (>70°) is provided between the collimated vapor atoms and the local surface normal, thereby inducing a shadow effect, thereby achieving local oblique angle deposition, and selectively forming nanorod structures on the steep sidewalls of the surface microcavity through standard photolithography and silicon dry etching.

[0055] In step 2), the external modification treatment is specifically to immerse the MCP microchannel plate in the crosslinker DSP for 2 hours, followed by washing with phosphate buffered saline (PBST), and then immersed in 0.1 ng / ml goat immunoglobulin GOAT-IgG or 0.1 ng / ml BGAL-Streptavidinβ-galactosidase labeled streptavidin SβG for 2 hours, followed by washing with phosphate buffered saline (PBST), and then immersed in bovine serum albumin (BSA) for 1 hour to fill the blanks, and finally use phosphate buffered saline (PBST) to wash away excess bovine serum albumin (BSA) to complete the external modification treatment.

[0056] Step 3) The two-layer PDMS outer cavity plate and the PDMS middle cavity plate embedded in the MCP microchannel plate were plasma cleaned for 60 seconds in a 60W plasma cleaner and bonded in sequence to obtain a microfluidic chip.

[0057] like Figure 7 As shown in (a), 1mm holes are punched on both sides of the torn PDMS curing plate. Two such PDMS curing plates are made in total. Then, a hollow PDMS mold with a microchannel plate MCP in the middle is used to make a hollow PDMS membrane with a microchannel plate MCP in the middle. Figure 7 As shown in (b), the size of the PDMS cured film can be specifically 4*4*1mm, and the size of the microchannel plate MCP can be specifically 4mm*4mm*1mm. According to the different cutting sizes of the microchannel plate, the size of the hollow in the middle of the PDMS film can be changed accordingly.

[0058] The prepared microfluidic chip can be used for the following experiments:

[0059] a) Enzyme-linked immunosorbent assay (ELISA) with streptavidin:

[0060] First, the c, d, and b holes (1 mm) were blocked with solid cylinders (1.5 mm), and the cross-linking agent DSP was added from the a port. After flowing through the microchannel plate MCP, it entered the lower cavity and was soaked for 2 h. The c and d ports were opened to discharge the cross-linking agent DSP. Subsequently, the c and d ports were blocked, and PBST was added from the a port to clean the excess DSP. After that, the c and d ports were opened to discharge the PBST.

[0061] Repeat the above steps and add BGAL-Streptavidinβ-galactosidase labeled streptavidin (SβG) at a concentration of 0.1ng / ml. Soak at room temperature for 2h - drain - wash three times with PBS containing 0.1wt% Tween-20 and 0.1wt% BSA (PBST) - drain PBST - add bovine serum albumin (BSA) and soak for 1h - drain - wash with PBST - drain PBST. Then, plug holes a, b, and d. Use a syringe to add a liquid with a density greater than water, such as FC40, from the unblocked holes at the bottom until the bottom is filled. Seal the bottom to make the holes a container. After ensuring that the two lower holes and one of the upper holes are sealed, add fluorescein substrate di(β-d-galactoside) (FDG) under pressure to make fluorescein substrate di(β-d-galactoside) FDG in the holes of the microchannel plate (MCP). Finally, open the two upper holes and add a layer of mineral oil with a lower density than water to drain. For details, please refer to Figure 4 (b) process.

[0062] b) Fluorescence immunoassay experiment:

[0063] First, the c, d, and b holes (1 mm) were blocked with solid cylinders (1.5 mm), and the cross-linking agent disulfide bis (succinimidyl propionate) DSP was added from port a. After flowing through the microchannel plate MCP, it entered the lower cavity and was soaked for 2-2.5 hours. The c and d ports were opened to discharge the cross-linking agent DSP. Subsequently, the c and d ports were blocked, and PBST was added from port a to clean the excess cross-linking agent DSP. After that, the c and d ports were opened to discharge the PBST.

[0064] Repeat the above steps and add Goat-IgG antigen at a concentration of 0.1ng / ml. Soak at room temperature for 2h - drain - wash 3 times with PBS containing 0.1wt% Tween-20 and 0.1wt% BSA (PBST) - drain PBST - add bovine serum albumin BSA and soak for 1h - drain - wash with PBST - drain PBST. Then block ports b, c, and d and pressurize the anti-goat-IgG antibody into the device from port a, allowing it to flow through the holes and soak for 1h. Then open the bottom hole, drain the excess antibody solution, and finally wash 3 times with PBST buffer. When observing, fill the chamber with buffer PBS or pure water for observation.

[0065] In a fluorescent immunoassay, ports b, c, and d are blocked, and anti-goat IgG antibody is introduced into the device through port a under pressure. The antibody is then passed through the holes and allowed to soak for 1 hour. The bottom hole is then opened to drain the excess antibody solution, followed by two washes with PBST buffer. For observation, the chamber can be filled with PBS or purified water.

[0066] In the fluorescent enzyme-linked immunosorbent assay, specifically, holes a, b, and d are blocked, and a liquid with a density greater than water, such as FC40, is added through a syringe from the unblocked holes at the bottom until the bottom is filled. The bottom is sealed to make the holes a container. After ensuring that the two holes below and one of the holes above are sealed, the fluorescein substrate di(β-d-galactoside) (FDG) is added under pressure to ensure that FDG is present in the holes of the microchannel plate MCP. The FDG is filled in the upper chamber for observation.

[0067] The present invention's 3D digital immunoassay device based on a microchannel plate is compact and portable. It utilizes the passive pressurization mode of microfluidics, allowing the test liquid to flow rapidly through the holes and be captured at the wall edges without the need for auxiliary materials such as magnetic beads. Because the microchannel plate has a large number of reaction chambers, it can also accurately and quantify the test substance, resulting in a high throughput and ultra-low detection concentration. For a macromolecular protein test substance of 1 pg / ml, the light-to-dark ratio is approximately 75%, calculated using the Poisson distribution formula. Therefore, it has higher sensitivity and efficiency. The present invention utilizes precise micro-nanofabrication to produce a large number of millions of micron- and nanometer-scale holes, a number that is proportional to the ultra-low concentration of the test sample liquid. Each channel wall of the microchannel plate (MCP) can serve as a separate capture zone. Furthermore, the interior of the channel is covered with nanogold pillars using a simple vapor deposition technique. During fluorescence immunoassay, the surface plasmon resonance of the nanogold pillar structure can enhance fluorescence, enabling clearer observation of the results of antigen-antibody capture. The present invention mainly involves a microfluidic immunoassay chip with a microchannel plate, which performs protein immunoassay in a 3D manner. Compared with 2D microchannel capture, 3D magic can more effectively improve capture efficiency in terms of speed and accuracy. Finally, it can also digitally quantify the sample to be tested to detect concentration.

Claims

1. A three-dimensional digital immunoassay detection microfluidic chip based on a microchannel plate, characterized by: It includes two layers of PDMS outer cavity plates, a PDMS middle cavity plate and an MCP microchannel plate. The MCP microchannel plate is embedded in the middle of the PDMS middle cavity plate. The two layers of PDMS outer cavity plates are laminated and bonded on the upper and lower sides of the PDMS middle cavity plate. The center of one side of the PDMS outer cavity plate is provided with an axially symmetrical groove that is wide in the middle and narrow on both sides in the vertical direction. The middle part of the groove is a rectangular groove, and through holes that pass through the PDMS outer cavity plate are symmetrically provided at both ends of the groove; the groove of the PDMS outer cavity plate located on the upper side of the PDMS middle cavity plate faces downward, and the groove of the PDMS outer cavity plate located on the lower side of the PDMS middle cavity plate faces upward. The grooves of the two layers of PDMS middle cavity plates are symmetrical to the PDMS middle cavity plate. A rectangular through groove running through the PDMS middle cavity plate is opened in the center of the plate surface in the vertical direction. The rectangular through groove faces the rectangular groove of the PDMS outer cavity plate. The MCP microchannel plate is embedded in the rectangular through groove in the middle of the PDMS middle cavity plate.

2. The three-dimensional digital immunoassay detection microfluidic chip based on a microchannel plate according to claim 1, characterized in that: The depth of the groove of the PDMS outer cavity plate is less than the thickness of the PDMS outer cavity plate; the MCP microchannel plate is a rectangular plate, and the size of the MCP microchannel plate is the same as the rectangular through groove of the PDMS middle cavity plate.

3. The method for preparing a three-dimensional digital immunoassay microfluidic chip according to any one of claims 1-2, characterized in that: The method comprises the following steps: Step 1) Preparing two layers of PDMS outer cavity plates and a PDMS middle cavity plate: The steps for preparing each layer of the PDMS outer cavity plate and the PDMS middle cavity plate are the same, specifically, mixing and stirring a polydimethylsiloxane (PDMS) solution and a curing agent, and then vacuuming to remove bubbles to obtain a PDMS curing mixed solution, pouring the PDMS curing mixed solution into a pre-prepared mold, heating and curing on a hot plate to obtain a PDMS cured plate, removing the PDMS cured plate from the mold, ultrasonically cleaning with acetone, isopropyl alcohol, and DI water in sequence, and then drying with nitrogen to obtain a PDMS outer cavity plate or a PDMS middle cavity plate; Step 2) The MCP microchannel plate is sequentially subjected to fluorescence enhancement treatment and external modification treatment, and then embedded into the rectangular through-groove in the middle of the PDMS middle cavity plate; Step 3) The two-layer PDMS outer cavity plate and the PDMS middle cavity plate embedded in the MCP microchannel plate are plasma cleaned and bonded to obtain a microfluidic chip.

4. The method for preparing a three-dimensional digital immunoassay detection microfluidic chip according to claim 3, characterized in that: In the step 1), 20 g to 30 g of polydimethylsiloxane (PDMS) solution and curing agent are mixed and stirred evenly, and then vacuumed to remove bubbles to obtain a PDMS curing mixed solution. The mass ratio of the polydimethylsiloxane (PDMS) solution to the curing agent is 1 / 10.

5. The method for preparing a three-dimensional digital immunoassay detection microfluidic chip according to claim 3, characterized in that: In the step 1), the PDMS curing mixed solution is poured into a pre-prepared mold, heated on a hot plate at 75°C-85°C for 1.5-2.5 hours to obtain a PDMS cured plate, and the PDMS cured plate is removed from the mold and ultrasonically cleaned with acetone for 5-10 minutes, isopropyl alcohol for 5-10 minutes, and DI water for 5-10 minutes, followed by drying with nitrogen.

6. The method for preparing a three-dimensional digital immunoassay detection microfluidic chip according to claim 3, characterized in that: In the step 2), the fluorescence enhancement treatment specifically includes first placing metal particles in a glass dish, then placing the MCP microchannel plate in the glass dish, sealing the top of the glass dish with a glass sheet, and placing the MCP microchannel plate tightly against the bottom of the glass sheet. The glass dish is heated until the metal particles melt, and then the MCP microchannel plate is reversely evaporated, so that the collimated vapor atoms generated by the melted metal particles are uniformly vacuum-evaporated and etched on the inner wall of the MCP microchannel plate, thereby completing the fluorescence enhancement treatment.

7. The method for preparing a three-dimensional digital immunoassay detection microfluidic chip according to claim 3, characterized in that: In the step 2), the external modification treatment is specifically to immerse the MCP microchannel plate in the cross-linker DSP for 2-2.5 hours, then wash it with phosphate buffered saline (PBST), and then immerse it in 0.1 ng / ml goat immunoglobulin GOAT-IgG or 0.1 ng / ml BGAL-Streptavidinβ-galactosidase labeled streptavidin SβG for 2 hours, followed by washing with phosphate buffered saline (PBST), and then immersed in bovine serum albumin (BSA) for 1 hour, and finally using phosphate buffered saline (PBST) to wash away excess bovine serum albumin (BSA) to complete the external modification treatment.

8. The method for preparing a three-dimensional digital immunoassay detection microfluidic chip according to claim 3, characterized in that: In the step 3), the two PDMS outer cavity plates and the PDMS middle cavity plate embedded with the MCP microchannel plate are plasma cleaned for 50-60 seconds in a 60W plasma cleaner and then bonded in sequence to obtain a microfluidic chip.

9. Use of the three-dimensional digital immunoassay detection microfluidic chip according to any one of claims 1-2 or the three-dimensional digital immunoassay detection microfluidic chip prepared by the preparation method according to any one of claims 3-8, characterized in that: The microfluidic chip is used in fluorescence immunoassay and fluorescence enzyme-linked immunosorbent assay.

Images