Microfluidic system for biomolecule enrichment detection

Abstract

The invention relates to a microfluidic system for biomolecule enrichment detection. The microfluidic system is provided with a first liquid storage tank (1), a second liquid storage tank (2) and a waste liquid tank (3), the first liquid storage tank (1) and the second liquid storage tank (2) are connected with the first diaphragm pump (5) through the three-way electromagnetic valve (4); the first diaphragm pump (5) is connected with a magnetic bead capture PDMS chip (6) and is further connected with a second diaphragm pump (7); waste liquid discharged by the second diaphragm pump (7) enters the waste liquid pool (3); and the flowing directions of the first diaphragm pump (5) and the second diaphragm pump (7) are opposite. By optimizing the design of a capturing structure, efficient capturing of the magnetic beads is achieved, the magnetic beads are distributed more uniformly by means of reciprocating motion of microfluid, and the reaction efficiency between the magnetic beads and target molecules is improved.

Description

Technical Field

[0001] The invention belongs to the field of microfluidics technology, and in particular relates to a microfluidics system for biomolecule enrichment detection. Background Art

[0002] Biomolecule detection is of great significance in the biomedical field. Biomolecules (such as exosomes, pathogenic microorganisms, etc.) refer to molecules that play an important role in organisms. These molecules can be proteins, nucleic acids, viruses, bacteria, etc. Rapid detection of biomolecules is of great significance for the early diagnosis and prevention of diseases, and can effectively reduce the spread of diseases and provide timely treatment options. Traditional biomolecule detection methods, such as culture and PCR, although highly accurate, have problems such as long detection time, complex operation, and expensive equipment, which limits their application in rapid diagnosis.

[0003] Microfluidics is a science and technology that manipulates fluids at the micrometer scale. It is an emerging interdisciplinary subject involving chemistry, fluid physics, microelectronics, new materials, biology, and biomedical engineering. Microfluidics chips precisely manipulate fluids by designing microchannels, reaction chambers, and other functional components, and realize the integrated analysis of basic operating units such as sample preparation, reaction, separation, and detection in biological, chemical, and medical analysis processes. This technology has significant advantages such as controllable liquid flow, integration, low consumption, high throughput, and fast analysis. Microfluidics has been widely used in research fields such as biomedicine and environmental sciences, and has shown great potential in disease diagnosis, drug screening, and environmental monitoring.

[0004] Magnetic bead detection technology is a separation and detection method based on magnetic microspheres. Magnetic beads have the advantages of simple operation, high separation efficiency, large specific surface area and good physical stability, and are a magnetic separation carrier with excellent performance. The surface of magnetic beads can be modified with various functional groups, such as antibodies, antigens, nucleic acids, etc., for capturing and detecting target molecules. The core of magnetic bead detection technology lies in its efficient separation and enrichment, which makes it widely used in the fields of nucleic acid extraction, immunoassay, cell sorting and protein purification, with the advantages of high sensitivity, rapid response and multiple detection.

[0005] However, at present, the capture efficiency of magnetic microspheres for biological molecules is still limited, which restricts the further application of magnetic bead detection technology. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a microfluidic system for biomolecule enrichment detection, optimize the capture structure design, achieve efficient capture of magnetic beads, and make the magnetic beads more evenly distributed by means of the reciprocating motion of the microfluidic, thereby improving the reaction efficiency between the magnetic beads and the target molecules.

[0007] The present invention provides a microfluidic system for biomolecule enrichment detection, wherein the microfluidic system is provided with a first liquid storage tank, a second liquid storage tank, and a waste liquid tank; the first liquid storage tank and the second liquid storage tank are connected to a first diaphragm pump through a three-way valve; the first diaphragm pump is connected to a magnetic bead capture PDMS chip and is further connected to a second diaphragm pump; the waste liquid discharged by the second diaphragm pump enters the waste liquid tank; the flow directions of the first diaphragm pump and the second diaphragm pump are opposite.

[0008] Preferably, the first liquid reservoir contains magnetic beads modified with probe molecules; and the second liquid reservoir contains samples to be detected.

[0009] The present invention also provides a method for using a microfluidic system for biomolecule enrichment detection, comprising the following steps:

[0010] First, the first diaphragm pump and the first liquid reservoir are connected through a three-way valve, and the second liquid reservoir is disconnected. The first diaphragm pump is controlled by the upper computer to start and continuously pump the magnetic beads in the first liquid reservoir into the magnetic bead capture PDMS chip; when the pumping is completed, the first diaphragm pump is turned off and the second diaphragm pump is turned on, and then the second diaphragm pump is turned off and the first diaphragm pump is turned on, and this cycle is repeated to make the magnetic beads evenly distributed at the capture sites designed in the chip; then the three-way valve is adjusted to connect the second liquid reservoir and the first diaphragm pump, and the first liquid reservoir is disconnected; the first diaphragm pump is turned on and the second diaphragm pump is turned off, and the sample to be tested is pumped into the magnetic bead capture PDMS chip so that it is in full contact with the modified magnetic beads; after the reaction is completed, the magnetic bead capture PDMS chip is removed, and the fluorescence intensity or staining effect on the magnetic beads is observed and analyzed through a microscope, thereby realizing the detection of the sample.

[0011] The present invention also provides another microfluidic system for biomolecule enrichment detection, the microfluidic system is provided with a first liquid reservoir, a first waste liquid reservoir, a second liquid reservoir and a second waste liquid reservoir; the first liquid reservoir is connected to the inlet of the first microfluidic chip through a delivery pump; the outlet of the first microfluidic chip is connected to a three-way valve; the first path of the three-way valve is connected to the first liquid reservoir through a first two-way valve, the second path of the three-way valve is connected to the first waste liquid reservoir through a second two-way valve, and the third path of the three-way valve is connected to the second liquid reservoir; the second liquid reservoir is connected to the first inlet of the second microfluidic chip through a first peristaltic pump; the second inlet of the second microfluidic chip is connected to the second peristaltic pump, and the second inlet is connected to the second waste liquid reservoir; the flow directions of the first peristaltic pump and the second peristaltic pump are opposite.

[0012] The second type of microfluidic system is also called a multi-chip cascade microfluidic system. The upper part of the system mainly uses a delivery pump to transport the liquid required by the first microfluidic chip, such as culture medium and drugs. The lower part uses two peristaltic pumps to transport liquid, and the first peristaltic pump and the second peristaltic pump are used to achieve uniform distribution of magnetic beads in the chip. The first peristaltic pump transports the liquid containing target biomolecules produced in the upper part to the second microfluidic chip for detection. The process is completed by switching the three-way valve in the middle.

[0013] Preferably, the first liquid reservoir is filled with biological reaction liquid; and the second liquid reservoir is filled with magnetic bead suspension.

[0014] The present invention also provides another method for using a microfluidic system for biomolecule enrichment detection, comprising the following steps:

[0015] First, ensure that the three-way valve is switched to only the upper half connected, open the first two-way valve, and close the second two-way valve; turn on the delivery pump to pump the biological reaction liquid into the first microfluidic chip to provide a perfusion culture environment for the cell samples in the first microfluidic chip; when the culture time is ripe, close the delivery pump and the first two-way valve, open the second two-way valve, add the target reagent to the first liquid reservoir, then turn on the delivery pump to discharge the liquid in the first microfluidic chip into the first waste liquid tank, and introduce the target reagent to participate in the reaction in the chip; then turn on the first peristaltic pump to pump the magnetic beads into the first peristaltic pump. The second peristaltic pump remains closed; when all the magnetic beads are pumped into the second microfluidic chip, the first peristaltic pump is closed and the second peristaltic pump is opened, and then the second peristaltic pump is closed and the first peristaltic pump is opened, and this cycle is repeated so that the magnetic beads are evenly distributed at the capture sites designed in the chip; when the reaction in the upper half is completed, the three-way valve 14 is switched so that only the lower half is connected, and the liquid containing the target biological molecules is pumped into the second liquid storage tank through the delivery pump, and then the liquid containing the target biological molecules is pumped into the second microfluidic chip through the first peristaltic pump to react with the magnetic beads, thereby finally achieving the enrichment and detection of the target molecules.

[0016] Beneficial Effects

[0017] The present invention achieves efficient capture of magnetic beads by optimizing the capture structure design, and makes the magnetic beads more evenly distributed by means of the reciprocating motion of the microfluidic, thereby improving the reaction efficiency between the magnetic beads and the target molecules. The present invention combines the advantages of microfluidics and magnetic bead detection technology, and provides a new solution for the rapid and sensitive detection of biomolecules. This system has important application prospects in the early diagnosis and prevention of diseases, and is expected to play a greater role in the future biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the structure of the first microfluidic system of the present invention.

[0019] Figure 2 Schematic diagram of the structure of the second microfluidic system of the present invention.

[0020] Figure 3 The figure is the test result of the magnetic bead capture efficiency of the microfluidic system used in the present invention.

[0021] Figure 4 This is the fluorescence condition of the magnetic bead surface of the microfluidic system used in the present invention.

[0022] Figure 5 This is the quantitative result of the fluorescent antibody bound to the surface of the magnetic beads in the microfluidic system used in the present invention.

[0023] Figure 6 The results of NTA test of the microfluidic system used in the present invention are shown in FIG. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0025] Example 1

[0026] like Figure 1 As shown, this embodiment provides a microfluidic system for biomolecule enrichment detection, wherein the microfluidic system is provided with a first liquid reservoir 1, a second liquid reservoir 2, and a waste liquid reservoir 3; the first liquid reservoir 1 and the second liquid reservoir 2 are connected to a first diaphragm pump 5 through a three-way valve 4; the first diaphragm pump 5 is connected to a magnetic bead capture PDMS chip 6 and is further connected to a second diaphragm pump 7; the waste liquid discharged by the second diaphragm pump 7 enters the waste liquid reservoir 3; the flow directions of the first diaphragm pump 5 and the second diaphragm pump 7 are opposite.

[0027] Preferably, the first liquid reservoir 1 contains magnetic beads modified with probe molecules; and the second liquid reservoir 2 contains samples to be detected.

[0028] This embodiment also provides a method for using a microfluidic system for biomolecule enrichment detection, comprising the following steps:

[0029] First, the first diaphragm pump 5 is connected to the first liquid reservoir 1 through the three-way valve 4, and the second liquid reservoir 2 is disconnected. The first diaphragm pump 5 is controlled by the upper computer to start and continuously pump the magnetic beads in the first liquid reservoir 1 into the magnetic bead capture PDMS chip 6 (this process takes about 1 minute); when the pumping is completed, the first diaphragm pump 5 is turned off and the second diaphragm pump 7 is turned on for 5 seconds, and then the second diaphragm pump 7 is turned off and the first diaphragm pump 5 is turned on for 3 seconds. This cycle is repeated for 4 minutes to make the magnetic beads evenly distributed at the capture sites designed in the chip; then the three-way valve 4 is adjusted to connect the second liquid reservoir 2 and the first diaphragm pump 5, and the first liquid reservoir 1 is disconnected; the first diaphragm pump 5 is turned on and the second diaphragm pump 7 is turned off, and the sample to be detected is pumped into the magnetic bead capture PDMS chip 6 so that it is in full contact with the modified magnetic beads; after the reaction is completed, the magnetic bead capture PDMS chip 6 is removed, and the fluorescence intensity or staining effect on the magnetic beads is observed and analyzed under a microscope, thereby realizing the detection of the sample.

[0030] Example 2

[0031] like Figure 2 As shown, this embodiment provides a microfluidic system for biomolecule enrichment detection, the microfluidic system is provided with a first liquid reservoir 8, a first waste liquid reservoir 9, a second liquid reservoir 10 and a second waste liquid reservoir 11; the first liquid reservoir 8 is connected to the inlet of the first microfluidic chip 16 through a delivery pump 15; the outlet of the first microfluidic chip 16 is connected to a three-way valve 14; the first way of the three-way valve 14 is connected to the first liquid reservoir 8 through a first two-way valve 12, the second way of the three-way valve 14 is connected to the first waste liquid reservoir 9 through a second two-way valve 13, and the third way of the three-way valve 14 is connected to the second liquid reservoir 10; the second liquid reservoir 10 is connected to the first inlet of the second microfluidic chip 19 through a first peristaltic pump 17; the second inlet of the second microfluidic chip 19 is connected to a second peristaltic pump 18, and the second inlet is connected to the second waste liquid reservoir 11; the flow directions of the first peristaltic pump 17 and the second peristaltic pump 18 are opposite.

[0032] Preferably, the first liquid storage tank 8 is filled with biological reaction liquid; and the second liquid storage tank 10 is filled with magnetic bead suspension.

[0033] This embodiment also provides a method for using a microfluidic system for biomolecule enrichment detection, comprising the following steps:

[0034] First, ensure that the three-way valve 14 is switched to connect only the upper half (taking the three-way valve in the figure as an example, the right path is 1, the lower path is 2, and the left path is 3. The upper half connects means that 1 and 3 are connected, and 2 is disconnected), open the first two-way valve 12, and close the second two-way valve 13; turn on the delivery pump 15 to pump the biological reaction liquid into the first microfluidic chip 16 to provide a perfusion culture environment for the cell sample in the first microfluidic chip 16; when the culture time is ripe, turn off the delivery pump 15 and the first two-way valve 12, open the second two-way valve 13, add the target reagent to the first liquid storage tank 8, and then turn on the delivery pump 15 to discharge the liquid in the first microfluidic chip 16 to the first waste liquid tank 9, and introduce the target reagent to participate in the reaction in the chip; then turn on the first peristaltic pump 17 to pump the magnetic beads The magnetic beads are pumped into the first peristaltic pump 17, and the second peristaltic pump 18 remains closed at this time; when all the magnetic beads are pumped into the second microfluidic chip 19, the first peristaltic pump 17 is turned off and the second peristaltic pump 18 is turned on for 5 seconds, then the second peristaltic pump 18 is turned off and the first peristaltic pump 17 is turned on for 3 seconds, and this cycle is repeated for 4 minutes to make the magnetic beads evenly distributed at the capture sites designed in the chip; when the reaction of the upper half is completed, the three-way valve 14 is switched to connect only the lower half (referring to 1 and 2 connected, 3 disconnected), and the liquid containing the target biological molecules is pumped into the second liquid storage tank 10 through the delivery pump 15, and then the liquid containing the target biological molecules is pumped into the second microfluidic chip 19 through the first peristaltic pump 17 to react with the magnetic beads, thereby finally achieving the enrichment and detection of the target molecules.

[0035] Example 3

[0036] Capture of magnetic beads in the chip:

[0037] In the initial stage of the operation of the microfluidic system for biomolecule enrichment detection as described in Example 1, the magnetic bead suspension is added to the liquid reservoir by a micropump, and the magnetic beads are pumped from the first liquid reservoir 1 to the microfluidic chip by the forward fluid in the magnetic bead capture PDMS chip 6, and the magnetic beads are uniformly distributed in the chip by continuous switching of the forward and reverse fluids. The efficiency of magnetic bead capture in the microfluidic system for biomolecule enrichment detection is statistically calculated. After the magnetic beads are first introduced, the efficiency of magnetic bead capture in the PDMS chip 6 is only 58.94%. After repeated driving of the forward and reverse fluids, the efficiency of magnetic bead capture and utilization can reach 95.18%. Figure 3 As shown. A goat anti-rabbit antibody with 488 fluorescence was introduced into the chip as a secondary antibody. After incubation for 0.5 h, the secondary antibody was removed and washed with PBS. Finally, the two chips were placed under a confocal microscope to observe the fluorescence on the surface of the magnetic beads, as shown. Figure 4 shown. Figure 5 This is the quantitative result of the fluorescent antibody bound to the magnetic beads surface, indicating that the enriched antibody has a good binding effect with the magnetic beads after being treated in this step.

[0038] Enrichment and detection applications of magnetic beads in chips:

[0039] When the magnetic beads are evenly distributed in the magnetic bead capture PDMS chip 6, the exosome sample to be tested is pumped into the magnetic bead capture PDMS chip 6 through the microfluidic system for biomolecule enrichment detection. The test sample solution repeatedly flows through the enrichment chip to enhance the chip's enrichment ability for trace solutions, and the enriched test solution is pumped into the collection pool. Subsequently, the original sample solution that has not flowed through the enrichment chip and the solution placed in the collection pool after enrichment are used as two batches of samples for NTA test analysis to detect the content of target exosomes in the two liquids before and after enrichment, such as Figure 6 As shown, the microfluidic system can achieve enrichment of 93.29% of the biomolecules in the solution.

Claims

1. A microfluidic system for biomolecule enrichment detection, characterized in that: The microfluidic system is provided with a first liquid storage tank (1), a second liquid storage tank (2), and a waste liquid tank (3); the first liquid storage tank (1) and the second liquid storage tank (2) are connected to a first diaphragm pump (5) via a three-way valve (4); the first diaphragm pump (5) is connected to a magnetic bead capture PDMS chip (6) and is further connected to a second diaphragm pump (7); the waste liquid discharged by the second diaphragm pump (7) enters the waste liquid tank (3); the flow directions of the first diaphragm pump (5) and the second diaphragm pump (7) are opposite.

2. A microfluidic system for biomolecule enrichment detection according to claim 1, characterized in that: The first liquid storage tank (1) contains magnetic beads modified with probe molecules; the second liquid storage tank (2) contains samples to be detected.

3. A method for using the microfluidic system for biomolecule enrichment detection as claimed in claim 1, comprising the following steps: First, the first diaphragm pump (5) is connected to the first liquid reservoir (1) through a three-way valve (4), and the second liquid reservoir (2) is disconnected. The first diaphragm pump (5) is controlled by a host computer to be turned on and the magnetic beads in the first liquid reservoir (1) are continuously pumped into the magnetic bead capture PDMS chip (6); after the pumping is completed, the first diaphragm pump (5) is turned off and the second diaphragm pump (7) is turned on, and then the second diaphragm pump (7) is turned off and the first diaphragm pump (5) is turned on, and the cycle is repeated so that the magnetic beads are evenly distributed at the capture sites designed in the chip; then the three-way valve (4) is adjusted to connect the second liquid reservoir (2) and the first diaphragm pump (5), and the first liquid reservoir (1) is disconnected; the first diaphragm pump (5) is turned on and the second diaphragm pump (7) is turned off, and the sample to be detected is pumped into the magnetic bead capture PDMS chip (6) so that it is in full contact with the modified magnetic beads; after the reaction is completed, the magnetic bead capture PDMS chip (6) is removed, and the fluorescence intensity or staining effect on the magnetic beads is observed and analyzed through a microscope, thereby realizing the detection of the sample.

4. A microfluidic system for biomolecule enrichment detection, characterized in that: The microfluidic system is provided with a first liquid reservoir (8), a first waste liquid reservoir (9), a second liquid reservoir (10) and a second waste liquid reservoir (11); the first liquid reservoir (8) is connected to the inlet of a first microfluidic chip (16) via a delivery pump (15); the outlet of the first microfluidic chip (16) is connected to a three-way valve (14); the first path of the three-way valve (14) is connected to the first liquid reservoir (8) via a first two-way valve (12), and the second path of the three-way valve (14) is connected to the first liquid reservoir (8) via a second two-way valve (12). The three-way valve (13) is connected to the first waste liquid tank (9), and the third way of the three-way valve (14) is connected to the second liquid storage tank (10); the second liquid storage tank (10) is connected to the first inlet of the second microfluidic chip (19) through the first peristaltic pump (17); the second inlet of the second microfluidic chip (19) is connected to the second peristaltic pump (18), and the second inlet is connected to the second waste liquid tank (11); the flow directions of the first peristaltic pump (17) and the second peristaltic pump (18) are opposite.

5. A microfluidic system for biomolecule enrichment detection according to claim 4, characterized in that: The first liquid storage tank (8) is filled with biological reaction liquid; the second liquid storage tank (10) is filled with magnetic bead suspension.

6. A method for using the microfluidic system for biomolecule enrichment detection as claimed in claim 4, comprising the following steps: First, ensure that the three-way valve (14) is switched to only the upper half connected, open the first two-way valve (12), and close the second two-way valve (13); open the delivery pump (15) to pump the biological reaction liquid into the first microfluidic chip (16), and provide a perfusion culture environment for the cell sample in the first microfluidic chip (16); when the culture time is ripe, close the delivery pump (15) and the first two-way valve (12), open the second two-way valve (13), add the target reagent to the first liquid storage tank (8), then open the delivery pump (15), discharge the liquid in the first microfluidic chip (16) into the first waste liquid tank (9), and introduce the target reagent to participate in the reaction in the chip; then open the first peristaltic pump (17) to pump the magnetic beads into the first peristaltic pump (17). The first peristaltic pump (17) is in the middle of the pump (17), and the second peristaltic pump (18) remains closed at this time; when all the magnetic beads are pumped into the second microfluidic chip (19), the first peristaltic pump (17) is closed and the second peristaltic pump (18) is opened, and then the second peristaltic pump (18) is closed and the first peristaltic pump (17) is opened, and this cycle is repeated so that the magnetic beads are evenly distributed at the capture sites designed in the chip; when the reaction of the upper half is completed, the three-way valve 14 is switched so that only the lower half is connected, and the liquid containing the target biological molecules is pumped into the second liquid storage tank (10) through the delivery pump (15), and then the liquid containing the target biological molecules is pumped into the second microfluidic chip (19) through the first peristaltic pump (17) to react with the magnetic beads, thereby finally achieving the enrichment and detection of the target molecules.

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