CN111378556A - Micro-fluidic chip and preparation method thereof, and preparation method of single-cell micro-droplets - Google Patents
Micro-fluidic chip and preparation method thereof, and preparation method of single-cell micro-droplets Download PDFInfo
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Abstract
A micro-fluidic chip comprises a chip body and a confluence channel, wherein the chip body is provided with an oil phase inlet, a micro-bead solution inlet, a cell suspension inlet and a micro-droplet outlet; the oil phase inlet, the microbead solution inlet and the cell suspension inlet are inlets for the liquid drop generating oil, the microbead solution and the cell suspension to enter the chip body in sequence; the cell suspension comprises a plurality of cells, and the microbead solution comprises a plurality of microbeads; the confluence channel is communicated with the micro-droplet outlet and the oil phase inlet; the micro-fluidic chip also comprises a first fluid control unit communicated with the microbead solution inlet and the confluence channel and a second fluid control unit communicated with the cell suspension inlet and the confluence channel, wherein the first and second fluid control units are used for arranging microbeads or cells according to a certain sequence; the micro-bead solution is firstly converged with the cell suspension to form an immiscible laminar flow, and the converged laminar flow is then converged with the droplet generating oil to form micro-droplets, and the micro-droplets are output at a micro-droplet outlet. The microfluidic chip provided by the invention has high single wrapping rate.
Description
Technical Field
The invention relates to the field of microfluidic technology and related devices, in particular to a microfluidic chip, a preparation method of the microfluidic chip and a preparation method of single-cell micro-droplets.
Background
Microfluidic chips, also known as lab-on-a-chip, can integrate conventional biochemical reactions into a few square centimeters chip. Since the microfluidic chip has micron-sized channels, which are equivalent to the size of cells, the microfluidic chip has attracted more and more attention in the research of cells.
However, the microfluidic chip on the market has a complex structure, a complex manufacturing process and high manufacturing cost. Some droplets generated by microfluidic chips are easy to wrap multiple particles (such as microbeads or cells) or do not contain particles, and these phenomena will increase the experiment cost, reduce the experiment efficiency and cause the problem of low wrapping rate. In addition, the flux of the microfluidic chip on the market is low.
Disclosure of Invention
In view of this, the invention provides a microfluidic chip with high single-encapsulation rate, high flux, low experimental cost and high experimental efficiency, a preparation method of the microfluidic chip and a preparation method of single-cell micro-droplets.
A micro-fluidic chip comprises a chip body and a confluence channel, wherein the chip body is provided with an oil phase inlet, a micro-bead solution inlet, a cell suspension inlet and a micro-droplet outlet; the oil phase inlet, the microbead solution inlet and the cell suspension inlet are inlets for liquid drop generating oil, microbead solution and cell suspension to enter the chip body in sequence; the cell suspension comprises a plurality of cells, and the microbead solution comprises a plurality of microbeads; the confluence channel is respectively communicated with the micro-droplet outlet and the oil phase inlet; the microfluidic chip further comprises: the first fluid manipulation unit is communicated with the microbead solution inlet and one end of the confluence channel and is used for arranging microbeads in the microbead solution according to a certain sequence; the second fluid manipulation unit is communicated with the cell suspension inlet and one end of the confluence channel and is used for arranging the cells in the cell suspension according to a certain sequence; the micro-bead solution is firstly converged with the cell suspension to form an immiscible laminar flow, and the converged laminar flow is then converged with the droplet generating oil to form micro-droplets, and the micro-droplets are output at the micro-droplet outlet.
Further, the first fluid control unit and the second fluid control unit are any one of a centrifugal channel, a displacement channel and a flow resistance channel, or a combination of any two or more of the centrifugal channel, the displacement channel and the flow resistance channel.
Further, the centrifugal channel comprises a centrifugal channel main body, a fluid inlet and a fluid outlet, the centrifugal channel main body is spirally distributed, the fluid inlet is positioned at the inner side of the centrifugal channel main body, the fluid outlet is positioned at the outer side of the centrifugal channel main body, the fluid inlet is communicated with the microbead solution inlet or the cell suspension inlet, and the fluid outlet is communicated with the confluence channel.
Further, the displacement channel comprises a displacement channel main body and at least one flow guide structure formed on the displacement channel main body, the flow guide structure is located on the inner side wall of one side of the displacement channel main body, each flow guide structure is composed of a plurality of protrusions, an inclined surface is formed on each protrusion, the inclination direction of each inclined surface is consistent with the flowing direction of liquid in the displacement channel main body, and the height of each protrusion gradually increases along the flowing direction of the liquid in the displacement channel main body.
Further, the flow resistance channel comprises a plurality of curved channels and a plurality of straight channels, and at least one end of each straight channel is connected with one curved channel.
Furthermore, the straight channel is provided with at least one flow guide structure, the flow guide structure is positioned on the inner side wall of one side of the straight channel, each flow guide structure is composed of a plurality of protrusions, each protrusion is provided with an inclined surface, the inclination direction of each inclined surface is consistent with the flowing direction of liquid in the straight channel, and the height of each protrusion gradually increases along the flowing direction of the liquid in the straight channel.
Furthermore, the micro-fluidic chip also comprises a micro-bead solution channel, a cell solution channel and an oil phase channel which are positioned on the chip body, wherein two ends of the micro-bead solution channel are respectively communicated with the micro-bead solution inlet and the confluence channel, and two ends of the cell suspension channel are respectively communicated with the cell suspension inlet and the confluence channel; the oil phase channel is communicated with the oil phase inlet and the confluence channel; preferably, the microbead solution channel, the cell suspension channel and the confluence channel are intersected in a Y-shaped structure, and the oil phase channel and the confluence channel are intersected in a cross-shaped structure.
Further, the oil phase inlet faces the confluence channel and is communicated with the confluence channel, and preferably, a T-shaped structure is formed between the oil phase inlet and the confluence channel.
A method for preparing single-cell micro-droplets comprises the following steps: preparing a microbead solution, a cell suspension and droplet generating oil; and adding the micro-bead solution, the cell suspension and the droplet-generating oil into the micro-bead solution inlet, the cell suspension inlet and the oil phase inlet of the micro-fluidic chip respectively at a certain flow rate, and collecting the generated micro-droplets at the micro-droplet outlet.
A method for preparing a microfluidic chip comprises the following steps: manufacturing a structural mould of a microfluidic chip channel; performing low adhesion treatment on a structure mold of a microfluidic chip channel by using trimethylsilane, placing polydimethylsiloxane on the chip channel structure mold, solidifying and forming, and taking down to form an upper chip, wherein the lower surface of the upper chip forms a microfluidic chip channel structure; punching holes at preset positions of the upper layer chip to form the oil phase inlet, the microbead solution inlet, the cell suspension inlet and the micro-droplet outlet of the micro-fluidic chip; and sealing the upper chip on the lower chip to obtain the microfluidic chip.
According to the microfluidic chip provided by the invention, the first fluid manipulation unit is connected between the microbead solution inlet and the microbead solution channel, the second fluid manipulation unit is connected between the cell suspension inlet and the cell suspension channel, the microbeads and the cells can be arranged in a certain sequence by the first fluid manipulation unit and the second fluid manipulation unit, the phenomenon of conglomeration and stacking between the microbeads in the microbead solution and the cells in the cell suspension is less, the probability that a plurality of microbeads and cells are wrapped in one water-in-oil droplet is reduced, and thus more droplets only contain one microbead and one cell is ensured. Therefore, the microfluidic chip provided by the invention has the advantages of high single-wrapping rate, high flux, high experiment efficiency and low experiment cost. In addition, the microfluidic chip provided by the invention has the advantages of simple structure, simple manufacturing process and low production cost. The invention can be used for the pretreatment of single cell sequencing samples.
Drawings
Fig. 1 is a schematic diagram of a microfluidic chip provided by the present invention.
Fig. 2 is a schematic view of a fluid manipulation unit in the microfluidic chip shown in fig. 1.
Fig. 3 is a schematic view of another fluid manipulation unit in the microfluidic chip shown in fig. 1.
Fig. 4 is a schematic view of another fluid manipulation unit in the microfluidic chip shown in fig. 1.
Fig. 5 is a schematic view of the flow of cells or microbeads within the channels of the fluid manipulation unit shown in fig. 2.
Fig. 6 is a schematic view of the flow of cells or microbeads within the channels of the fluid manipulation unit shown in fig. 3.
Fig. 7 is a schematic view of the flow of cells or microbeads through the channels of the fluid manipulation unit shown in fig. 4.
Description of the main elements
The following detailed description will further illustrate the invention in conjunction with the above-described figures.
Detailed Description
In order to further explain the technical means and effects of the present invention adopted to achieve the predetermined objects, the following detailed description will be made on the specific embodiments, structures, features and effects of the microfluidic chip, the method for preparing the microfluidic chip and the method for preparing the single-cell micro-droplets provided by the present invention with reference to the accompanying drawings 1-7 and the preferred embodiments.
Referring to fig. 1-4, the present invention provides a microfluidic chip 100, wherein the microfluidic chip 100 includes a chip body 10, an oil phase inlet 20, a bead solution inlet 30, a cell suspension inlet 40, a first fluid control unit 50, a second fluid control unit 60, a droplet outlet 70, a bead solution channel 81, a cell suspension channel 82, an oil phase channel 83, and a confluence channel 84.
Wherein, the oil phase inlet 20, the microbead solution inlet 30, the cell suspension inlet 40 and the micro-droplet outlet 70 are located on the surface of the chip body 10. The first fluid manipulation unit 50, the second fluid manipulation unit 60, the bead solution channel 81, the cell suspension channel 82, the oil phase channel 83 and the confluence channel 84 are located in the chip body 10.
Wherein the bead solution inlet 30 is communicated with the first fluid handling unit 50. The cell suspension inlet 40 communicates with the second fluid handling unit 60. The bead solution passage 81 communicates with the first fluid handling unit 50 and the confluence passage 84. The cell suspension channel 82 communicates with the second fluid handling unit 60 and the confluence channel 84. The oil phase passage 83 communicates the oil phase inlet 20 and the confluence passage 84. The confluence channel 84 communicates with the micro-droplet outlet 70.
Wherein, the micro-bead solution channel 81, the cell suspension channel 82 and the confluence channel 84 are intersected in a Y-shaped structure. The oil phase passage 83 communicates the oil phase inlet 20 and the confluence passage 84. The oil phase passage 83 and the confluence passage 84 are intersected in a cross-shaped structure. Namely, the microbead solution channel 81 and the cell suspension channel 82 converge to form a laminar flow (water phase), and then converge with the oil phase channel 83 to the converging channel 84 to form a water-in-oil droplet.
In other embodiments, the oil phase passage 83 may be omitted, and in this case, the oil phase inlet 20 may be disposed directly above the confluence passage 84 and communicate with the confluence passage 84, and in this case, a T-shaped structure is formed between the oil phase inlet 20 and the confluence passage 84.
Wherein the oil phase inlet 20 is used for connecting an external droplet generating oil (oil phase, a liquid that is not easily soluble in water) and as an inlet of the droplet generating oil into the chip body 10.
The bead solution inlet 30 is used for connecting an external bead solution (water phase, water-soluble liquid) and serving as an inlet for the bead solution to enter the chip body 10. Wherein, the micro-bead solution contains a plurality of micro-beads 200.
Wherein, the cell suspension inlet 40 is used for connecting an external cell suspension (aqueous phase, liquid easily dissolved in water) and serving as an inlet for the cell suspension to enter the chip body 10. Wherein the cell suspension comprises cells 300.
The droplet generating oil, the microbead solution and the cell suspension respectively enter the chip body 10 from the oil phase inlet 20, the microbead solution inlet 30 and the cell suspension inlet 40. The bead solution flows through the first fluid handling unit 50 and the bead solution passage 81 in this order. The cell suspension flows through the second fluid manipulation unit 60 and the cell suspension channel 82 in sequence, and is joined with the bead solution at one end of the confluence channel 84 to form a non-intermixed laminar flow, and at this time, the beads and the cells have a certain probability to appear on both sides of the laminar flow. The oil phase (droplet forming oil) flows through the oil phase channel 83 and meets the laminar flow in the confluence channel 84, and at this time, a plurality of micro droplets containing one micro bead and one cell are formed under the action of surface tension. After the droplet-forming oil meets the laminar flow, water-in-oil droplets are immediately formed in the cross-shaped structure, and then discharged from the droplet outlet 70.
In the present embodiment, the oil phase inlet 20, the microbead solution inlet 30, the cell suspension inlet 40 and the micro-droplet outlet 70 are located on the same surface of the chip body 10.
In other embodiments, the oil phase inlet 20, the microbead solution inlet 30, the cell suspension inlet 40, and the microdroplet outlet 70 may be located on different surfaces of the chip body 10.
The first fluid handling unit 50 is configured to handle the distribution of microbeads in the microbead solution flowing through the first fluid handling unit 50, so that the microbeads can be arranged in a certain order without being stacked.
The first fluid control unit 50 may be any one of a centrifugal channel 51, a displacement channel 52 and a flow resistance channel 53, or a combination of any two or more of the centrifugal channel 51, the displacement channel 52 and the flow resistance channel 53. Of course, the number of the centrifugal channels 51, the displacement channels 52 and the flow resistance channels 53 can be determined according to actual conditions.
When the centrifugal channel 51, the displacement channel 52 and the flow resistance channel 53 are arbitrarily combined, the centrifugal channel 51, the displacement channel 52 and the flow resistance channel 53 may be connected end to end, or the structure of the displacement channel 52 may be directly applied to the flow resistance channel 53 or the structure of the centrifugal channel 51, for example, the displacement channel 52 is disposed on a straight channel 532 (see fig. 4) of the flow resistance channel 53.
Specifically, referring to fig. 2, the centrifugal channel 51 includes a centrifugal channel main body 511, a fluid inlet 512 and a fluid outlet 513. The centrifugal channel body 511 is spirally distributed, the fluid inlet 512 is located at one end (inside) of the centrifugal channel body 511, and the fluid outlet 513 is located at the other end (outside) of the centrifugal channel body 511.
Wherein, when the first fluid handling unit 50 is a centrifugal channel 51, the fluid inlet 512 is communicated with the bead solution inlet 30, and the fluid outlet 513 is communicated with the bead solution channel 81.
Preferably, the number of turns of the spiral of the centrifugal channel body 511 is 3-6. The centrifuge channel body 511 has a maximum profile diameter of about 1.5 to 3 mm, a channel cross-sectional width of about 60 to 150 microns, and a height of about 60 to 150 microns.
Referring to fig. 5, when the bead solution flows through the centrifugal channel 51, the bead solution flows into the centrifugal channel body 511 from the fluid inlet 512 of the centrifugal channel 51. When the bead solution is injected into the centrifugal channel body 511 at a certain initial velocity, the beads contained in the bead solution generate a centrifugal force along a tangential direction of the spiral centrifugal channel body 511 in the centrifugal channel body 511, and the beads gradually move to the outer side wall of the centrifugal channel body 511 under the action of the centrifugal force to form a single row. The microbeads in the microbead solution will flow in a single row from the fluid outlet 513 into the microbead solution channel 81.
Specifically, referring to fig. 3, the displacement channel 52 includes a displacement channel body 521 and at least one flow guide structure 522 formed on the displacement channel body 521. Wherein the flow guiding structure 522 is located on an inner sidewall of one side of the displacement channel body 521. Each of the flow guide structures 522 is composed of a plurality of protrusions 5221. Each protrusion 5221 is formed with a sloped surface 5222. The inclined surface 5222 is inclined in the same direction as the direction in which the bead solution flows in the displacement channel body 521. The height of each of the protrusions 5221 is gradually increased along the flow direction of the microbead solution in the displacement channel body 521.
When the first fluid handling unit 50 is a displacement channel 52, one end of the displacement channel 52 is communicated with the bead solution inlet 30, and the other end is communicated with the bead solution channel 81.
Preferably, each of the displacement channels 52 includes at least six of the projections 5221. The displacement channel body 521 has a cross-sectional width of about 30-75 microns and a cross-sectional height of about 60-120 microns.
In the present embodiment, the protrusions 5221 are triangular saw-toothed. In other embodiments, the projections 5221 may also have a trapezoidal saw-tooth shape or other shapes, so long as the projections have an inclined surface whose inclined direction coincides with the flowing direction of the bead solution in the displacement channel body 521.
Referring to fig. 6, when the bead solution flows through the flow guide structure 522, due to the flow guide effect of the inclined surface 5222 of the flow guide structure 522, the bead solution flows along the inclined surface 5222 of the flow guide structure 522 to the side of the displacement channel main body 521 opposite to the flow guide structure 522, and due to the increasing height of the flow guide structure 522, the bead solution is accelerated to flow to the side opposite to the flow guide structure 522. Meanwhile, the microbeads 200 carried in the microbead solution will also flow toward the side opposite to the flow guide structure 522 along with the microbead solution, thereby passing through the flow guide structure 522 in a single row.
Specifically, referring to fig. 4, the flow resistance channel 53 includes a plurality of curved channels 531 and a plurality of straight channels 532. At least one end of each of the straight roads 532 is connected to one of the curved roads 531.
In the present embodiment, the flow resistance passage 53 has a serpentine shape.
In other embodiments, the flow-resistant channel 53 may also have a flow-guiding structure 522 disposed on the straight channel 532.
Preferably, the flow resistance channel 53 has a cross-sectional width of 60 to 150 microns and a cross-sectional height of about 60 to 120 microns. The length of one straight road 532 plus one curve 531 is 1-1.5 mm.
The flow resistance channel 53 continuously changes the flow direction of the microbead solution, so that microbeads carried in the microbead solution flow orderly and singly under the action of inertia to reduce aggregation and conglomeration. Fig. 7 shows the flow trajectory of microbeads in the flow resistance channel 53. Assuming that the bead solution flows into the flow resistance channel 53 from the left side, the beads 200 flow closely to the side of the maximum radius when they encounter the first turn, flow along the side of the minimum radius when they go to the second turn, flow along the side of the maximum radius when they go to the third turn, and so on. The beads flow only along the same side and do not flow along the other side when encountering a curve, which is determined by the microfluidic principle and is different from the macroscopic phenomenon. The straight channel 532 is used for drawing the distance between the front and the rear microbeads, and the effect of orderly and single-row flowing of the microbeads is enhanced.
The second fluid handling unit 60 is configured to handle the distribution of cells in the cell suspension flowing through the second fluid handling unit 60, so that the cells can be arranged in a certain order without being packed.
The second fluid manipulation unit 60 may be any one of a centrifugal channel 61, a displacement channel 62 and a flow resistance channel 63, or a combination of any two or more of the centrifugal channel 61, the displacement channel 62 and the flow resistance channel 63. Of course, the number of the centrifugal channels 61, the displacement channels 62 and the flow resistance channels 63 can be determined according to actual conditions.
The centrifugal channel 61, the displacement channel 62 and the flow resistance channel 63 have the same structure as the centrifugal channel 51, the displacement channel 52 and the flow resistance channel 53. The trajectory of the cells flowing in these channels can be referenced to the flow trajectory of microbeads in these channels.
A serpentine channel may also be disposed on the oil phase channel 83 to increase the length of the oil phase channel 83, so that the oil phase can be intersected after the intersection of the microbead solution and the cell suspension, and thus the oil phase can wrap the cells and the microbeads in time.
It should be noted that all the channels in the present invention are disposed inside the microfluidic chip.
The invention also provides a preparation method of the single-cell micro-droplet, which adopts the micro-fluidic chip 100 to prepare the single-cell micro-droplet. The preparation method comprises the following steps:
firstly, preparing a microbead solution, a cell suspension and an oil phase.
Wherein the concentration of the bead solution is 100-. In this embodiment, the concentration of the bead solution is 400 beads/μ l. Wherein the microbead is a microbead with a diameter of about 30 micrometers and contains a barcode sequence (DNA sequence label).
Wherein the concentration of the cell suspension is 50-1000 cells/microliter. In this embodiment, the concentration of the cell suspension is 300 cells/microliter.
And secondly, adding the microbead solution, the cell suspension and the oil phase into the microbead solution inlet 30, the cell suspension inlet 40 and the oil phase inlet 20 respectively at a certain flow rate, and collecting the generated water-in-oil microdroplets at the microdroplet outlet 70.
Wherein the flow rate of the micro-bead solution and the cell suspension is 0.5-4 ml/h. The flow rate of the oil phase is 3-10 ml/h.
In this embodiment, the flow rate of the bead solution and the cell suspension is 1 ml/h. The flow rate of the oil phase was 6 ml/h.
When the microbead solution or the cell suspension flows through the first fluid handling unit 50 or the second fluid handling unit 60, the microbeads or the cells in the microbead solution or the cell suspension are reduced in conglomerate in the channel of the first fluid handling unit 50 or the second fluid handling unit 60 and flow from the first fluid handling unit 50 or the second fluid handling unit 60 into the microbead solution channel 81 or the cell suspension channel 82 in a single row. Beads and cells in a single row meet at one end of the confluent channel 84 and form a laminar flow, where there is some chance that one bead will mate with one cell. The paired cells and microbeads meet the oil phase (droplet forming oil) in the confluence channel 84, are encapsulated by the oil phase to form microdroplets, and are output at the microdroplet outlet 70. After the cells are lysed within the droplets, the microbeads capture their nucleic acids. The beads of the present invention may be magnetic beads or beads made of other materials (such as hydrogel beads, silica beads, glass beads, etc.).
The above method can produce droplets that encapsulate only one cell (single cell) and one microbead. The cells are subjected to processes such as lysis in the droplets, and the nucleic acids are captured by the microbeads for subsequent sequencing.
The invention also provides a preparation method of the microfluidic chip 100, which comprises the following steps:
firstly, a structural mould of a microfluidic chip channel is manufactured.
The method comprises the following specific steps of manufacturing a structural mould of the microfluidic chip channel: firstly, providing a first silicon wafer or a first glass substrate, and forming a photoresist layer on the surface of the first silicon wafer or the first glass substrate; and secondly, forming the structure of the microfluidic chip channel on the photoresist layer by an image transfer process so as to manufacture the structural mould of the microfluidic chip channel.
Specifically, the photoresist layer may be formed by a spin coating method and then baked at 95 ℃ for 15-60 minutes.
Preferably, the thickness of the photoresist layer is 60 micrometers, and the photoresist is SU-8 photoresist. The baking time was 45 minutes.
The specific steps of forming the fluidic chip channel structure on the photoresist layer by an image transfer process include: firstly, fixing a mask containing a structure of a microfluidic chip channel on the photoresist layer, and carrying out ultraviolet exposure to polymerize SU-8 according to the structure of the mask; secondly, baking for 10-30 minutes at 95 ℃, naturally cooling, and then removing unexposed SU-8 glue by using a developer; and hardening at the temperature of 180 ℃ for 2 hours, and cooling to form a chip channel structure.
And secondly, performing low adhesion treatment (adhesion prevention) on a structural mold of the microfluidic chip channel by using trimethylsilane, placing Polydimethylsiloxane (PDMS) on the structural mold of the chip channel, solidifying and forming, and then taking down to form an upper chip, wherein the lower surface of the upper chip forms the channel structure of the microfluidic chip, and holes are formed in preset positions of the upper chip to form an oil phase inlet 20, a microbead solution inlet 30, a cell suspension inlet 40 and a micro-droplet outlet 70.
Preferably, the heating temperature required for solidification is 80 ℃ and the heating time is 0.5 to 2 hours.
And thirdly, sealing the upper chip on a second silicon chip or a second glass substrate (lower chip) to obtain the microfluidic chip 100.
Wherein the second silicon wafer may be a silicon wafer of Polydimethylsiloxane (PDMS) material.
Before the step of the third step, the method further comprises the steps of: and processing the upper layer chip and the lower layer chip by using a plasma cleaning machine.
According to the microfluidic chip 100 provided by the invention, the first fluid manipulation unit 50 is connected between the bead solution inlet 30 and the bead solution channel 81, the second fluid manipulation unit 60 is connected between the cell suspension inlet 40 and the cell suspension channel 82, the beads and the cells can be arranged in a certain sequence (in a single-row form) by the first fluid manipulation unit 50 and the second fluid manipulation unit 60, the aggregation and stacking phenomena between the beads in the bead solution and the cells in the cell suspension are less, the probability that a plurality of beads and cells are wrapped in one water-in-oil droplet is reduced, and thus more droplets only contain one bead and one cell. Therefore, the microfluidic chip provided by the invention has the advantages of high single-wrapping rate, high flux, high experiment efficiency and low experiment cost. In addition, the microfluidic chip provided by the invention has the advantages of simple structure, simple manufacturing process and low production cost.
Although the present invention has been described with reference to the above preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A micro-fluidic chip comprises a chip body and a confluence channel, wherein the chip body is provided with an oil phase inlet, a micro-bead solution inlet, a cell suspension inlet and a micro-droplet outlet; the oil phase inlet, the microbead solution inlet and the cell suspension inlet are inlets for liquid drop generating oil, microbead solution and cell suspension to enter the chip body in sequence; the cell suspension comprises a plurality of cells, and the microbead solution comprises a plurality of microbeads; the confluence channel is respectively communicated with the micro-droplet outlet and the oil phase inlet; characterized in that, the micro-fluidic chip also comprises:
the first fluid manipulation unit is communicated with the microbead solution inlet and one end of the confluence channel and is used for arranging microbeads in the microbead solution according to a certain sequence; and
the second fluid manipulation unit is communicated with the cell suspension inlet and one end of the confluence channel and is used for arranging the cells in the cell suspension according to a certain sequence; the micro-bead solution is firstly converged with the cell suspension to form an immiscible laminar flow, and the converged laminar flow is then converged with the droplet generating oil to form micro-droplets, and the micro-droplets are output at the micro-droplet outlet.
2. The microfluidic chip according to claim 1, wherein the first fluid manipulation unit and the second fluid manipulation unit are any one of a centrifugal channel, a displacement channel, and a flow resistance channel, or a combination of any two or more of the centrifugal channel, the displacement channel, and the flow resistance channel.
3. The microfluidic chip according to claim 2, wherein the centrifugal channel comprises a centrifugal channel body, a fluid inlet and a fluid outlet, the centrifugal channel body is spirally disposed, the fluid inlet is located at an inner side of the centrifugal channel body, the fluid outlet is located at an outer side of the centrifugal channel body, the fluid inlet is connected to the bead solution inlet or the cell suspension inlet, and the fluid outlet is connected to the confluent channel.
4. The microfluidic chip according to claim 2, wherein the displacement channel comprises a displacement channel body and at least one flow guiding structure formed on the displacement channel body, the flow guiding structure is located on an inner side wall of one side of the displacement channel body, each flow guiding structure is composed of a plurality of protrusions, each protrusion is formed with an inclined surface, an inclination direction of each inclined surface is consistent with a flowing direction of the liquid in the displacement channel body, and a height of each protrusion gradually increases along the flowing direction of the liquid in the displacement channel body.
5. The microfluidic chip of claim 2, wherein the flow resistance channel comprises a plurality of curved channels and a plurality of straight channels, and at least one end of each straight channel is connected with one curved channel.
6. The microfluidic chip according to claim 5, wherein the straight channel is provided with at least one flow guiding structure, the flow guiding structure is located on an inner side wall of one side of the straight channel, each flow guiding structure is composed of a plurality of protrusions, each protrusion is formed with an inclined surface, an inclination direction of each inclined surface is consistent with a flowing direction of the liquid in the straight channel, and a height of each protrusion gradually increases along the flowing direction of the liquid in the straight channel.
7. The microfluidic chip according to claim 1, further comprising a bead solution channel, a cell suspension channel, and an oil phase channel on the chip body, wherein two ends of the bead solution channel are respectively connected to the bead solution inlet and the confluent channel, and two ends of the cell suspension channel are respectively connected to the cell suspension inlet and the confluent channel; the oil phase channel is communicated with the oil phase inlet and the confluence channel; preferably, the microbead solution channel, the cell suspension channel and the confluence channel are intersected in a Y-shaped structure, and the oil phase channel and the confluence channel are intersected in a cross-shaped structure.
8. The microfluidic chip according to claim 1, wherein the oil phase inlet faces the bus channel and is in communication with the bus channel, and preferably, a T-shaped structure is formed between the oil phase inlet and the bus channel.
9. A method for preparing single-cell micro-droplets comprises the following steps:
preparing a microbead solution, a cell suspension and droplet generating oil; and
adding the micro-bead solution, the cell suspension and the droplet-forming oil into the micro-bead solution inlet, the cell suspension inlet and the oil phase inlet of the micro-fluidic chip according to any one of claims 1 to 8 at a certain flow rate, and collecting the generated micro-droplets at the micro-droplet outlet.
10. A method for preparing a microfluidic chip comprises the following steps:
manufacturing a structural mould of a microfluidic chip channel;
performing low adhesion treatment on a structure mold of a microfluidic chip channel by using trimethylsilane, placing polydimethylsiloxane on the chip channel structure mold, solidifying and forming, and taking down to form an upper chip, wherein the lower surface of the upper chip forms a microfluidic chip channel structure; and perforating the upper chip at predetermined positions to form an oil phase inlet, a microbead solution inlet, a cell suspension inlet and a micro-droplet outlet of the microfluidic chip as claimed in any one of claims 1 to 8; and
and sealing the upper chip on the lower chip to obtain the microfluidic chip.
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