High-throughput single-particle arranged and packaged micro-fluidic chip and application thereof

Abstract

The invention discloses a high-throughput single-particle arranged and packaged micro-fluidic chip and application thereof, and belongs to the field of biomedical engineering. The single-particle arranged micro-fluidic chip comprises a substrate and a slide glass, wherein the substrate is used for supporting the slide glass; a particle suspension liquid inflow port, a sheath liquid inflow port, a periodic focusing flow channel and an outflow port are formed in the slide glass; the particle suspension liquid inlet and the sheath liquid inlet are respectively connected with a micro-channel, and the two micro-channels intersect to form a micro-channel and are communicated with the periodic focusing channel; the included angle of the micro-channel intersection of the particle suspension liquid inlet and the sheath liquid inlet is 30-100 degrees; the periodic focusing flow channel is of a spiral structure from inside to outside; the periodic focusing flow channel is connected with the flow outlet; the micro-fluidic chip provided by the invention integrates droplet arrangement and screening functions, and finally realizes single particle encapsulation.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering, and in particular to a high-throughput single-particle arrangement and packaging microfluidic chip and its application. Background Art

[0002] Compared with traditional multi-cell analysis, single-cell analysis avoids averaging signals and retains heterogeneity information between cells, thereby revealing individual differences in cell behavior and function. Quantification and understanding of cell heterogeneity is crucial to advancing precision medicine, and has important applications in areas such as early disease diagnosis, embryonic development research, abnormal detection of genetic signaling networks, and analysis of tumor composition.

[0003] Droplet-based microfluidic systems are becoming tools for a variety of applications, such as single-cell analysis, complex multi-step biological and chemical analysis, diagnostics, sequencing, drug screening, and directed evolution experiments. Single-cell droplet preparation is achieved in a highly reproducible manner through the geometry of microfluidic devices (such as T-type, flow focusing, and co-flow). Droplets can be generated and operated at kilohertz frequencies, and dividing single cells into microliter or nanoliter droplets can perform high-throughput analysis and sorting of millions of single cells. Using microfluidic technology, scientists are able to achieve high-throughput analysis and sorting of single cells, which allows large-scale data sets to be extracted and analyzed from the single cell level to obtain more detailed genetic, transcriptional, protein, and metabolic information, providing unprecedented insights into understanding cellular processes.

[0004] A particular advantage of droplet microfluidics over traditional screening techniques is that droplets provide a unique tool to link genotype to phenotype through compartmentalization. Cells and molecules secreted by cells are trapped in the droplets throughout the analysis and sorting steps. Due to the Poisson distribution, there may be 0, a single, or multiple cells in the droplet, making it impossible to confirm the number of cells encapsulated in the droplet.

[0005] The existing patent CN109735429A discloses a microfluidic chip for improving cell sorting efficiency and reducing costs. In order to improve the success rate of single-cell encapsulation, the chip is designed to use a sheath fluid focusing method to arrange cells in an orderly manner and realize droplet sorting of encapsulated cells. However, the sheath fluid focusing method used in the patent when encapsulating cells does not deviate from the principle of flow cytometer, and the separation method requires a computer, a fluorescence detector, and a programmable DC power supply, which inevitably increases the complexity and cost of the system. Summary of the invention

[0006] The purpose of the present invention is to provide a high-throughput single-particle arrangement and packaging microfluidic chip and its application. The present invention designs a high-throughput single-particle encapsulation platform, which integrates droplet arrangement and screening functions on the microfluidic chip to ultimately achieve single-particle encapsulation.

[0007] The present invention first provides a single-particle array microfluidic chip, which includes a substrate and a carrier, wherein the substrate is used to support the carrier; The carrier is provided with a particle suspension inlet, a sheath liquid inlet, a periodic focusing flow channel and an outlet; The particle suspension inlet and the sheath liquid inlet are respectively connected to a microchannel, and the two microchannels merge into one microchannel and are connected to the periodic focusing channel; The angle at the intersection of the microchannels of the particle suspension inlet and the sheath liquid inlet is 30-100°, preferably 90°; The periodic focusing channel is a spiral structure extending from inside to outside; The periodic focusing channel is connected to the flow outlet.

[0008] The single-particle array microfluidic chip has one or two outlets; When there are two outlets, the outlet end of the periodic focusing channel is divided into two microchannels, which are respectively connected to the two outlets; the angle between the two microchannels is 30° to 90°, preferably 30°.

[0009] The single-particle array microfluidic chip described above, wherein the number of spiral turns of the periodic focusing channel is 3 to 8 turns; Specifically, when the particle concentration is 2×10 5 ~5.9×10 6 / mL, when the sheath liquid flow rate ranges from 5 to 50 μL / h, the number of spiral turns of the periodic focusing flow channel is 3 to 4 turns; When the particle concentration is 6×10 6 ~1×10 7 When the sheath liquid flow rate is in the range of 51 to 100 μL / h, the number of spiral turns of the periodic focusing flow channel is 5 to 6 turns; When the particle concentration is 1.1×10 7 ~5×10 7 In the range of μL / mL and the sheath liquid flow rate range of 101-200 μL / h, the number of spiral turns of the periodic focusing flow channel is 7-8 turns.

[0010] Second, the present invention also provides a single particle array packaged microfluidic chip, which includes a substrate and a carrier, wherein the substrate is used to support the carrier; The carrier is provided with a particle suspension inlet, a sheath liquid inlet, a periodic focusing flow channel, an oil phase inlet and an outlet; The particle suspension inlet and the sheath liquid inlet are respectively connected to a microchannel, and the two microchannels merge into one microchannel and are connected to the periodic focusing channel; The angle at the intersection of the microchannels of the particle suspension inlet and the sheath liquid inlet is 30° to 100°, preferably 90°; The periodic focusing channel is a spiral structure extending from inside to outside; The periodic focusing flow channel is connected to the flow outlet; The oil phase inlet is arranged on the microchannel of the outflow port where the particles are arranged and flow out.

[0011] The above-mentioned single particle array packaged microfluidic chip, the number of the outlets is 1 or 2; When there are two flow outlets, the outlet end of the periodic focusing channel is divided into two microchannels, which are respectively connected to the two flow outlets; the angle between the two microchannels is 30°.

[0012] The single particle array packaged microfluidic chip, wherein the number of spiral turns of the periodic focusing channel is 3 to 8 turns; Specifically, when the particle concentration is 2×10 5 ~5.9×10 6 / mL, when the sheath liquid flow rate ranges from 5 to 50 μL / h, the number of spiral turns of the periodic focusing flow channel is 3 to 4 turns; When the particle concentration is 6×10 6 ~1×10 7 When the sheath liquid flow rate is in the range of 51 to 100 μL / h, the number of spiral turns of the periodic focusing flow channel is 5 to 6 turns; When the particle concentration is 1.1×10 7 ~5×10 7 In the range of μL / mL and the sheath liquid flow rate range of 101-200 μL / h, the number of spiral turns of the periodic focusing flow channel is 7-8 turns.

[0013] In the above-mentioned single particle array packaged microfluidic chip, the angle between the microchannel of the outflow port and the microchannel connected to the oil phase inflow port is 90° to 120°, and can be specifically 90°.

[0014] In the above-mentioned single-particle array microfluidic chip or single-particle array packaged microfluidic chip, the number of spiral turns of the periodic focusing channel is at least 3, the microchannel height in each period is 25-300 μm, and the width is 25-300 μm; the ratio of the microchannel height to the width is 1:1-3, and the interval between spirals in adjacent periods is 100-800 μm; The periodic focusing channel adopts a spiral microchannel structure with equidistantly increasing radius.

[0015] The substrate is used to support a slide, and the surface of the slide with microchannels is bonded to the slide to form the microfluidic chip; the material of the slide is polydimethylsiloxane, polymethyl methacrylate, polytetrafluoroethylene, paper or pure glass; the material of the substrate is glass or silicon wafer.

[0016] Thirdly, the present invention provides the application of the above-mentioned single-particle arrangement microfluidic chip in single-particle sorting.

[0017] Fourthly, the present invention provides the application of the above-mentioned single particle arrangement and packaging microfluidic chip in single particle arrangement and packaging; Specifically, during the process of single particle arrangement and encapsulation, the flow rate of the particle suspension is 5-100 μL / h; the flow rate of the sheath liquid is 5-200 μL / h; the flow rate of the oil phase is 200-2000 μL / h; Preferably, the flow rate of the particle suspension is 20 μL / h; the flow rate of the sheath liquid is 10 μL / h; and the flow rate of the oil phase is 200 μL / h.

[0018] In the above application, the microfluidic chip is used to sort and encapsulate particles with a particle size of 5 to 200 μm; further, the particles can be cells, gel beads, magnetic beads, glass beads or metal beads.

[0019] Finally, the present invention provides a method for sorting and encapsulating single particles, comprising the following steps: using the above-mentioned microfluidic chip to arrange and encapsulate single particles, injecting a particle suspension and a sheath liquid into the microfluidic chip from the particle suspension flow inlet and the sheath liquid flow inlet respectively, and under the action of centrifugal force, the particle group inertially migrates in the periodic focusing flow channel, and the particles begin to be preliminarily arranged; injecting an oil phase from the oil phase inlet to achieve the encapsulation of a single particle.

[0020] In the above method, the flow rate of the particle suspension is 5-100 μL / h, the flow rate of the sheath liquid is 5-200 μL / h, and the flow rate of the oil phase is 200-2000 μL / h.

[0021] Preferably, the flow rate of the particle suspension is 20 μL / h; the flow rate of the sheath liquid is 10 μL / h; and the flow rate of the oil phase is 200 μL / h.

[0022] Flow rate of particle suspension: flow rate of sheath liquid: flow rate of oil phase = 1:0.5~5:5~10.

[0023] The present invention has the following beneficial effects: (1) The single-particle sorting-packaging chip provided by the present invention utilizes the inertial effect of the particles themselves and a specific flow channel structure to exert inertial force on the particles, thereby controlling the lateral migration of the particles in the flow field and arranging the particles in an orderly manner; inertial focusing breaks the Poisson distribution law, improves the sample processing throughput and the accuracy of particle position control, and the controllable packaging rate of single particles reaches more than 80%; (2) The chip of the present invention utilizes the control of the flow rate of droplet re-injection and the flow rate of sheath fluid to perform one-to-one sorting of magnetic beads and cells, achieve single-cell encapsulation, and greatly improve the utilization rate of downstream single-cell sequencing; (3) In addition, the chip of the present invention is simple and convenient to operate, and has the advantages of high efficiency, no pollution, wide application range and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the chip structure used for K562 cell array in Example 1; Figure 2 For the single cell array in the microfluidic channel in implementation 1, the scale bar is 50 μm; Figure 3 Schematic diagram of the chip structure used for single cell arrangement and packaging in Example 2; Figure 4 This is a single cell encapsulation image at the outlet (4) in Example 2, with a scale of 50 μm; Figure 5 Schematic diagram of the structure of the microfluidic chip for high-throughput single-particle arrangement and packaging in Example 3; Among them, 1 is a particle suspension inlet, 2 is a sheath liquid inlet, 3 and 4 are flow outlets, 5 is an oil phase flow inlet, and 6 is a periodic focusing channel. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0026] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0027] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0028] In the following examples, PDMS, glass slides and syringe pumps were purchased from SMIC Qiheng Scientific Instrument Co., Ltd., and K562, PBS and magnetic beads were purchased from Suzhou Alpha Biotech Co., Ltd.; The droplet-generating oil was HFE 7500 fluorocarbon oil containing 2% surfactant, and the surfactant was 008-FluoroSurfactant, RAN Biotechnologies, FS-Kit #2.

[0029] Example 1 See the schematic diagram of the chip structure for K562 cell array. Figure 1 The microfluidic chip provided in Example 1 uses a glass sheet as a substrate and PDMS as a carrier, and the substrate is used to support the carrier; a particle suspension inlet (1), a sheath liquid inlet (2), a periodic focusing channel (6) and two outlets (3, 4) are arranged on the carrier; The particle suspension inlet (1) and the sheath liquid inlet (2) are connected to a microchannel respectively, and the two microchannels merge into one microchannel and are connected to the periodic focusing channel (6); the angle at the intersection of the microchannels of the particle suspension inlet (1) and the sheath liquid inlet (2) is 90°; the periodic focusing channel (6) is a spiral structure from inside to outside, and the number of spiral turns is 4; the interval between spirals in adjacent periods is 200 μm; the height of the periodic focusing channel (6) is 50 μm, the width is 50 μm, and the ratio of height to width is 1:1; the outlet of the periodic focusing channel (6) is divided into two microchannels, which are respectively connected to the outlets (3) and (4); the angle between the two divided microchannels is 30°.

[0030] The collected 5×10 6 Disperse K562 cells in 1 mL PBS buffer to obtain a cell suspension. Add the cell suspension into a syringe. Then, use a syringe pump to inject from the particle suspension inlet (1) at a flow rate of 10 μL / h; at the same time, inject PBS buffer into the sheath fluid inlet (2) as sheath fluid at a flow rate of 20 μL / h. The sheath fluid can disperse the cells gathered at the inlet. Figure 2 When cells pass through the spiral channel, centrifugal force causes the cells to be evenly arranged in a row. Cells with poor activity or broken cell membranes flow out from the outlet (3), while cells with intact structures flow out from the outlet (4).

[0031] Example 2 See the schematic diagram of the chip structure for single cell arrangement and packaging in this embodiment. Figure 3The microfluidic chip provided in this embodiment uses a glass sheet as a substrate and PDMS as a carrier, and the substrate is used to support the carrier; a particle suspension inlet (1), a sheath liquid inlet (2), a periodic focusing channel (6), an oil phase inlet (5) and two outlets (3, 4) are arranged on the carrier; The particle suspension inlet (1) and the sheath liquid inlet (2) are connected to a microchannel respectively, and the two microchannels intersect to form a microchannel and are connected to the periodic focusing channel (6); the angle at the intersection of the microchannels of the particle suspension inlet (1) and the sheath liquid inlet (2) is 90°; the periodic focusing channel (6) is a spiral structure from inside to outside, and the number of spiral turns is 4; the interval between spirals in adjacent periods is 200 μm; the height of the periodic focusing channel (6) is 50 μm, the width is 50 μm, and the ratio of height to width is 1:1; the outlet of the periodic focusing channel (6) is divided into two microchannels, which are respectively connected to the outlets (3) and (4); the angle between the two microchannels is 30°. Another microchannel is separated from the flow channel of the outlet (4) to connect to the oil phase inlet (5); and the angle between the microchannel of the outlet (4) and the microchannel connected to the oil phase inlet (5) is 90°.

[0032] The collected 5×10 6 K562 cells were dispersed in 1 mL PBS buffer to obtain a cell suspension. The cell suspension was added to a syringe. Then, a syringe pump was used to inject at different flow rates (see Table 1) from the particle suspension inlet (1) of the microfluidic chip; at the same time, PBS buffer was injected into the sheath liquid inlet (2) as the sheath liquid, with flow rates of 0 μL / h, 10 μL / h, 20 μL / h, and 50 μL / h, respectively (as shown in Table 1). The sheath liquid can disperse the cells gathered at the inlet. While injecting the cell suspension, the oil phase inlet (5) was injected with a flow rate of 200 μL / h or 1000 μL / h to generate droplet oil, which can wrap the cells arranged in a row to form single cell droplets. The single cell droplets flowed out from the outlet (4). Cells with incomplete structures flowed out from the outlet (3).

[0033] As shown in Table 1, the sheath liquid flow rate to achieve the highest single-cell droplet ratio is 10 μL / h, the particle suspension flow rate is 20 μL / h, and the oil phase flow rate is 200 μL / h; Figure 4 , each of the generated droplets contains a single cell.

[0034] Table 1 Single cell encapsulation at different flow rates in Example 2

[0035] Example 3 Single cell array and encapsulation chips can modify the chip width to the corresponding size according to the required encapsulation particle diameter. Figure 5 When the particles are magnetic beads (diameter 140-200 μm), the number of spiral turns of the periodic focusing channel (6) in the chip is 3, the height of the microchannel is 200 μm, the width is 300 μm, the ratio of height to width is 1:1.5, and the interval between spirals in adjacent periods is 800 μm; the inlet section is provided with two channel inlets (1) and (2), and the two channel inlets are respectively connected to a microchannel, and the two microchannels intersect and are connected to the periodic focusing channel (6); the angle at the intersection of the microchannels of the two channel inlets is 90°; a microchannel is separated from the flow channel of the outlet (4) and connected to the oil phase flow inlet (5), and the angle between the microchannel of the outlet (4) and the microchannel of the oil phase flow inlet (5) is 90°.

[0036] The method for preparing single magnetic bead droplets is as follows: 2×10 5 The magnetic beads were dispersed in 1 mL PBS buffer to obtain a magnetic bead suspension, which was then injected from the flow channel inlet (1) at a flow rate of 25 μL / h using a syringe pump; at the same time, PBS buffer was injected into the flow channel inlet (2) as a sheath fluid at a flow rate of 50 μL / h; while injecting the magnetic bead suspension, droplet generation oil was injected into the oil phase flow inlet (5) at a flow rate of 300 μL / h to form a single magnetic bead droplet.

Claims

1. A single-particle array microfluidic chip, comprising a substrate and a carrier, wherein the substrate is used to support the carrier; The carrier is provided with a particle suspension inlet, a sheath liquid inlet, a periodic focusing flow channel and an outlet; The particle suspension inlet and the sheath liquid inlet are respectively connected to a microchannel, and the two microchannels merge into one microchannel and are connected to the periodic focusing channel; The angle at the intersection of the microchannels of the particle suspension inlet and the sheath liquid inlet is 30° to 100°, preferably 90°; The periodic focusing channel is a spiral structure extending from inside to outside; The periodic focusing flow channel is connected to the flow outlet.

2. The single-particle array microfluidic chip according to claim 1, characterized in that: The number of the outflow ports is 1 or 2; When there are two outlets, the outlet end of the periodic focusing channel is divided into two microchannels, which are respectively connected to the two outlets; the angle between the two microchannels is 30° to 90°, preferably 30°.

3. The single particle array microfluidic chip according to claim 1 or 2, characterized in that: The number of spiral turns of the periodic focusing flow channel is 3 to 8 turns; Specifically, when the particle concentration is 2×10 5 ~5.9×10 6 / mL, when the sheath liquid flow rate ranges from 5 to 50 μL / h, the number of spiral turns of the periodic focusing flow channel is 3 to 4 turns; When the particle concentration is 6×10 6 ~1×10 7 When the sheath liquid flow rate is in the range of 51 to 100 μL / h, the number of spiral turns of the periodic focusing flow channel is 5 to 6 turns; When the particle concentration is 1.1×10 7 ~5×10 7 In the range of μL / mL and the sheath liquid flow rate range of 101-200 μL / h, the number of spiral turns of the periodic focusing flow channel is 7-8 turns.

4. A single particle array encapsulation microfluidic chip, comprising a substrate and a carrier, wherein the substrate is used to support the carrier; The carrier is provided with a particle suspension inlet, a sheath liquid inlet, a periodic focusing flow channel, an oil phase inlet and an outlet; The particle suspension inlet and the sheath liquid inlet are respectively connected to a microchannel, and the two microchannels merge into one microchannel and are connected to the periodic focusing channel; The angle at the intersection of the microchannels of the particle suspension inlet and the sheath liquid inlet is 30° to 100°, preferably 90°; The periodic focusing channel is a spiral structure extending from inside to outside; The periodic focusing flow channel is connected to the flow outlet; The oil phase inlet is arranged on the microchannel of the outflow port where the particles are arranged and flow out.

5. The single particle array packaged microfluidic chip according to claim 4, characterized in that: The number of the outflow ports is 1 or 2; When there are two flow outlets, the outlet end of the periodic focusing channel is divided into two microchannels, which are respectively connected to the two flow outlets; the angle between the two microchannels is 30°.

6. The single particle array packaged microfluidic chip according to claim 4 or 5, characterized in that: The number of spiral turns of the periodic focusing flow channel is 3 to 8 turns; Specifically, when the particle concentration is 2×10 5 ~5.9×10 6 / mL, when the sheath liquid flow rate ranges from 5 to 50 μL / h, the number of spiral turns of the periodic focusing flow channel is 3 to 4 turns; When the particle concentration is 6×10 6 ~1×10 7 When the sheath liquid flow rate is in the range of 51 to 100 μL / h, the number of spiral turns of the periodic focusing flow channel is 5 to 6 turns; When the particle concentration is 1.1×10 7 ~5×10 7 In the range of μL / mL and the sheath liquid flow rate range of 101-200 μL / h, the number of spiral turns of the periodic focusing flow channel is 7-8 turns.

7. The microfluidic chip with single particle array packaging according to any one of claims 4 to 6, characterized in that: The angle between the microchannel of the outlet and the microchannel connected to the oil phase inlet is 90° to 120°.

8. Use of the microfluidic chip according to any one of claims 1 to 3 in single particle sorting.

9. Use of the microfluidic chip according to any one of claims 4 to 8 in single particle arrangement and packaging.

10. The use according to claim 9, characterized in that: During the process of single particle arrangement and encapsulation, the flow rate of the particle suspension is 5~100 μL / h; the flow rate of the sheath liquid is 5~200 μL / h; the flow rate of the oil phase is 200~2000 μL / h; Preferably, the flow rate of the particle suspension is 20 μL / h; the flow rate of the sheath liquid is 10 μL / h; and the flow rate of the oil phase is 200 μL / h.

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