A microfluidic system for biomolecule enrichment detection

Abstract

The present application relates to a kind of microfluidic systems for biomolecule enrichment detection, the microfluidic system is provided with first reservoir (1), second reservoir (2), waste pool (3);The first reservoir (1), second reservoir (2) are connected by three-way electromagnetic valve (4) and first diaphragm pump (5);The first diaphragm pump (5) is connected with magnetic bead capture PDMS chip (6) and is further connected second diaphragm pump (7);The waste liquid discharged by the second diaphragm pump (7) enters the waste pool (3);The flow direction of the first diaphragm pump (5) and the second diaphragm pump (7) is opposite.By optimizing the design of capture structure, the efficient capture of magnetic beads is realized, the reciprocating motion of microfluid is used, the distribution of magnetic beads is more uniform, and the reaction efficiency between magnetic beads and target molecules is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microfluidics, and particularly relates to a microfluidic system for biomolecule enrichment detection. BACKGROUND

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

[0003] Microfluidic technology is a scientific and technological technique for manipulating fluids at the micron scale, and is an emerging interdisciplinary subject involving chemistry, fluid physics, microelectronics, new materials, biology, and biomedical engineering. Microfluidic chips can precisely manipulate fluids by designing microchannels, reaction chambers, and other functional components, and can integrate basic operation units such as sample preparation, reaction, separation, and detection in biological, chemical, and medical analysis processes. This technology has the advantages of controllable liquid flow, integration, low consumption, high throughput, and fast analysis. Microfluidic technology has been widely applied in the fields of biomedical and environmental science, 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 kind of excellent magnetic separation carrier. 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 is its efficient separation and enrichment, which makes it widely used in nucleic acid extraction, immunoassay, cell sorting, and protein purification, with the advantages of high sensitivity, rapid reaction, and multiple detection.

[0005] However, the current magnetic microspheres still have limited capture efficiency for biomolecules, which limits the further application of magnetic bead detection technology. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a microfluidic system for biomolecule enrichment detection, which optimizes the design of the capture structure, realizes efficient capture of magnetic beads, and improves the reaction efficiency between magnetic beads and target molecules by means of the reciprocating motion of microfluids, making the distribution of magnetic beads more uniform.

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

[0008] Preferably, the first liquid pool is provided with probe molecule modified magnetic beads; and the second liquid pool is provided with a sample to be detected.

[0009] The application further provides a use method of the microfluidic system for biomolecule enrichment detection, which comprises the following steps:

[0010] Firstly, the first diaphragm pump is connected with the first liquid pool through the three-way valve, the second liquid pool is disconnected, the first diaphragm pump is controlled to be opened by an upper computer and the magnetic beads in the first liquid pool are continuously pumped into the magnetic bead capture PDMS chip; when the pumping is completed, the first diaphragm pump is closed, the second diaphragm pump is opened, then the second diaphragm pump is closed and the first diaphragm pump is opened, and the cycle is repeated so that the magnetic beads are uniformly distributed at the designed capture sites in the chip; subsequently, the second liquid pool is connected with the first diaphragm pump by adjusting the three-way valve, the first liquid pool is disconnected; the first diaphragm pump is opened and the second diaphragm pump is closed, the sample to be detected is pumped into the magnetic bead capture PDMS chip, so that the sample is fully contacted with the modified magnetic beads; after the reaction is completed, the magnetic bead capture PDMS chip is taken out, the fluorescence intensity or the dyeing effect on the magnetic beads is observed and analyzed by a microscope, so that the detection of the sample is realized.

[0011] The application further provides another microfluidic system for biomolecule enrichment detection, which is provided with a first liquid pool, a first waste liquid pool, a second liquid pool and a second waste liquid pool; the first liquid pool is connected with the inlet of a first microfluidic chip through a conveying pump; the outlet of the first microfluidic chip is connected with a three-way valve; the first road of the three-way valve is connected with the first liquid pool through a first double-way valve, the second road of the three-way valve is connected with the first waste liquid pool through a second double-way valve, and the third road of the three-way valve is connected with the second liquid pool; the second liquid pool is connected with the first inlet of a second microfluidic chip through a first peristaltic pump; the second inlet of the second microfluidic chip is connected with a second peristaltic pump, and simultaneously the second inlet is connected with the second waste liquid pool; the flow directions of the first peristaltic pump and the second peristaltic pump are opposite.

[0012] The second microfluidic system is also called a multi-chip cascade microfluidic system, and the upper half thereof mainly transports the liquid required by the first microfluidic chip, such as culture medium and drugs, by means of a delivery pump. The lower half thereof transports the liquid by means of two peristaltic pumps, and the uniform distribution of magnetic beads in the chip is realized by means of the first peristaltic pump and the second peristaltic pump. The liquid containing target biomolecules generated by the upper half is transported into the second microfluidic chip by means of the first peristaltic pump for detection. The process is switched by means of the intermediate three-way valve.

[0013] Preferably, the first liquid pool is provided with a biological reaction liquid; and the second liquid pool is provided with a magnetic bead suspension.

[0014] The application further provides a use method of another microfluidic system for biomolecule enrichment detection, comprising the following steps:

[0015] First, the three-way valve is switched to only communicate the upper half, the first double-way valve is opened, and the second double-way valve is closed; the delivery pump is opened to pump the biological reaction liquid into the first microfluidic chip, so as to provide a perfusion culture environment for the cell sample in the first microfluidic chip; when the culture is mature, the delivery pump and the first double-way valve are closed, the second double-way valve is opened, the target reagent is added into the first liquid pool, and then the delivery pump is opened to discharge the liquid in the first microfluidic chip to the first waste liquid pool, and introduce the target reagent to participate in the reaction in the chip; then the first peristaltic pump is opened to pump the magnetic beads into the first peristaltic pump, and the second peristaltic pump is kept closed at this time; when the magnetic beads are completely pumped into the second microfluidic chip, the first peristaltic pump is closed and the second peristaltic pump is opened, then the second peristaltic pump is closed and the first peristaltic pump is opened, and the circulation is repeated to make the magnetic beads uniformly distributed at the designed capture sites in the chip; when the reaction of the upper half is completed, the three-way valve 14 is switched to only communicate the lower half, the liquid containing target biomolecules is pumped into the second liquid pool by means of the delivery pump, and then the liquid containing target biomolecules is pumped into the second microfluidic chip by means of the first peristaltic pump to react with the magnetic beads, so as to finally realize the enrichment and detection of target molecules.

[0016] Advantages

[0017] The application realizes the efficient capture of magnetic beads by optimizing the design of the capture structure, and makes the distribution of magnetic beads more uniform by means of the reciprocating motion of microfluids, so as to improve the reaction efficiency between the magnetic beads and target molecules. The application combines the advantages of microfluidic technology and magnetic bead detection technology, and provides a new solution for the rapid and sensitive detection of biomolecules, and has important application prospects in the early diagnosis and prevention and control of diseases, and is expected to play a greater role in the field of biomedicine in the future. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a structural schematic diagram of the first microfluidic system of the application.

[0019] Figure 2 This is a schematic diagram of the structure of the second microfluidic system of the present invention.

[0020] Figure 3 The results show the test results of the magnetic bead capture efficiency of the microfluidic system used in this invention.

[0021] Figure 4 The fluorescence of the magnetic beads on the surface of the microfluidic system used in this invention is shown.

[0022] Figure 5 This is the quantitative result of fluorescent antibody binding on the surface of magnetic beads in the microfluidic system used in this invention.

[0023] Figure 6 The results of NTA testing are for the microfluidic system used in this invention. Detailed Implementation

[0024] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0025] Example 1

[0026] like Figure 1 As shown, this embodiment provides a microfluidic system for biomolecule enrichment detection. The microfluidic system includes a first reservoir 1, a second reservoir 2, and a waste liquid reservoir 3. The first reservoir 1 and the second reservoir 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-captured PDMS chip 6 and further connected to a second diaphragm pump 7. The waste liquid discharged from 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 storage tank 1 is filled with magnetic beads modified with probe molecules; the second liquid storage tank 2 is filled with the sample to be tested.

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

[0029] First, the first diaphragm pump 5 and the first reservoir 1 are connected via a three-way valve 4, while the second reservoir 2 is disconnected. The host computer controls the first diaphragm pump 5 to continuously pump the magnetic beads from the first reservoir 1 into the magnetic bead capture PDMS chip 6 (this process takes about 1 minute). After pumping is complete, the first diaphragm pump 5 is turned off and the second diaphragm pump 7 is turned on for 5 seconds. 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 ensure that the magnetic beads are evenly distributed at the designed capture sites in the chip. Then, the three-way valve 4 is adjusted to connect the second reservoir 2 and the first diaphragm pump 5, while the first 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 tested is pumped into the magnetic bead capture PDMS chip 6 to ensure full contact with the modified magnetic beads. After the reaction is complete, 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 includes a first reservoir 8, a first waste reservoir 9, a second reservoir 10, and a second waste reservoir 11. The first 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 reservoir 8 via a first two-way valve 12, the second path of the three-way valve 14 is connected to the first waste reservoir 9 via a second two-way valve 13, and the third path of the three-way valve 14 is connected to the second reservoir 10. The second reservoir 10 is connected to the first inlet of a second microfluidic chip 19 via a first peristaltic pump 17. The second inlet of the second microfluidic chip 19 is connected to a second peristaltic pump 18, which is also connected to the second waste reservoir 11. The flow directions of the first peristaltic pump 17 and the second peristaltic pump 18 are opposite.

[0032] Preferably, the first storage tank 8 contains a biological reaction solution; the second storage tank 10 contains a magnetic bead suspension.

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

[0034] First, ensure that the three-way valve 14 is switched to only the upper half of the circuit (taking the three-way valve in the diagram as an example, the right path is 1, the lower path is 2, and the left path is 3; the upper half of the circuit means that 1 and 3 are connected, and 2 is disconnected). Open the first double-way valve 12 and close the second double-way valve 13. Turn on the delivery pump 15 to pump the bioreactor solution into the first microfluidic chip 16, providing a perfusion culture environment for the cell samples in the first microfluidic chip 16. When the culture time is ripe, close the delivery pump 15 and the first double-way valve 12, open the second double-way valve 13, add the target reagent to the first reservoir 8, and then turn on the delivery pump 15 to 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, turn on the first peristaltic pump 17 to pump the magnetic beads... The magnetic beads are pumped into the first peristaltic pump 17, while the second peristaltic pump 18 remains closed. Once all the magnetic beads have been 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. This cycle is repeated for 4 minutes to ensure that the magnetic beads are evenly distributed at the designed capture sites in the chip. When the upper part of the reaction is complete, the three-way valve 14 is switched so that only the lower part is connected (meaning 1 and 2 are connected, and 3 is disconnected). The liquid containing the target biomolecules is pumped into the second reservoir 10 by the delivery pump 15, and then pumped into the second microfluidic chip 19 by the first peristaltic pump 17 to react with the magnetic beads, ultimately achieving the enrichment and detection of the target molecules.

[0035] Example 3

[0036] Capture of ferrite beads in chips:

[0037] In the initial stage of operation of the microfluidic system for biomolecule enrichment detection as described in Example 1, a magnetic bead suspension is added to the reservoir via a micropump. The magnetic beads are then pumped from the first reservoir 1 into the microfluidic chip 6 by the forward fluid in the magnetic bead-capturing PDMS chip 6. The continuous switching between forward and reverse fluids achieves uniform distribution of the magnetic beads within the chip. Statistical calculations were performed on the magnetic bead capture efficiency in the microfluidic system for biomolecule enrichment detection. After the initial introduction of magnetic beads, the capture efficiency within the PDMS chip 6 was only 58.94%. After repeated forward and reverse fluid driving, the capture and utilization efficiency of the magnetic beads reached 95.18%. Figure 3 As shown. Goat anti-rabbit antibody with 488 fluorescence was introduced into the chip as a secondary antibody. After incubation for 0.5 hours, the secondary antibody was removed, and the chips were washed with PBS. Finally, the fluorescence on the surface of the magnetic beads was observed under a confocal microscope, as shown. Figure 4 As shown. Figure 5 The quantitative results of fluorescent antibody binding on the surface of magnetic beads indicate that the enriched antibody has a good binding effect with the magnetic beads after this step.

[0038] Enrichment and detection of magnetic beads in the chip:

[0039] When the magnetic beads are uniformly distributed in the magnetic bead capture PDMS chip 6, the sample to be tested is pumped into the magnetic bead capture PDMS chip 6 through the microfluidic system for biomolecule enrichment and detection. The test sample solution repeatedly flows through the enrichment chip, enhancing the enrichment capacity of the chip for trace solutions. The enriched sample solution is pumped into the collection pool. Subsequently, the original sample solution that has not flowed through the enrichment chip and the enriched solution in the collection pool are taken as two batches of samples for NTA test analysis to detect the content of target exosomes in the two liquids before and after enrichment, as shown in Figure 6 The microfluidic system can achieve enrichment of 93.29% of 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 pool (8), a first waste liquid pool (9), a second liquid pool (10) and a second waste liquid pool (11); the first liquid pool (8) is connected with the inlet of the first microfluidic chip (16) through a delivery pump (15); the outlet of the first microfluidic chip (16) is connected with a three-way valve (14); the first path of the three-way valve (14) is connected with the first liquid pool (8) through a first double-way valve (12), the second path of the three-way valve (14) is connected with the first waste liquid pool (9) through a second double-way valve (13), and the third path of the three-way valve (14) is connected with the second liquid pool (10); the second liquid pool (10) is connected with 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 with a second peristaltic pump (18), and simultaneously the second inlet is connected with the second waste liquid pool (11); the flow directions of the first peristaltic pump (17) and the second peristaltic pump (18) are opposite.

2. The microfluidic system for biomolecule enrichment detection according to claim 1, wherein: The first liquid pool (8) is filled with biological reaction liquid; and the second liquid pool (10) is filled with magnetic bead suspension.

3. A method for using the microfluidic system for biomolecule enrichment detection according to claim 1, comprising the following steps: firstly, ensuring that the three-way valve (14) is switched to only the upper half communication, opening the first double-way valve (12) and closing the second double-way valve (13); opening the delivery pump (15) to pump the biological reaction liquid into the first microfluidic chip (16), so as to provide a perfusion culture environment for the cell sample in the first microfluidic chip (16); when the culture time is ripe, closing the delivery pump (15) and the first double-way valve (12), opening the second double-way valve (13), adding target reagent into the first liquid pool (8), then opening the delivery pump (15) to discharge the liquid in the first microfluidic chip (16) into the first waste liquid pool (9) and introduce the target reagent to participate in the reaction in the chip; then opening the first peristaltic pump (17) to pump the magnetic beads into the first peristaltic pump (17), at this time the second peristaltic pump (18) remains closed; when the magnetic beads are completely pumped into the second microfluidic chip (19), closing the first peristaltic pump (17) and opening the second peristaltic pump (18), then closing the second peristaltic pump (18) and opening the first peristaltic pump (17), so as to circulate to make the magnetic beads uniformly distributed at the designed capture sites in the chip; when the upper half reaction is completed, switching the three-way valve (14) to only the lower half communication, pumping the liquid containing the target biomolecules into the second liquid pool (10) through the delivery pump (15), then pumping the liquid containing the target biomolecules into the second microfluidic chip (19) through the first peristaltic pump (17) to react with the magnetic beads, so as to finally realize the enrichment and detection of the target molecules.

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