WO2021088936A1 - 一种分离捕获单细胞的微流控芯片及其制备方法和应用 - Google Patents

一种分离捕获单细胞的微流控芯片及其制备方法和应用 Download PDF

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Publication number
WO2021088936A1
WO2021088936A1 PCT/CN2020/126831 CN2020126831W WO2021088936A1 WO 2021088936 A1 WO2021088936 A1 WO 2021088936A1 CN 2020126831 W CN2020126831 W CN 2020126831W WO 2021088936 A1 WO2021088936 A1 WO 2021088936A1
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branch flow
flow channel
microfluidic chip
branch
flow channels
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PCT/CN2020/126831
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English (en)
French (fr)
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黄斌
陈艳
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深圳先进技术研究院
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Publication of WO2021088936A1 publication Critical patent/WO2021088936A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/12—Specific details about manufacturing devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces

Definitions

  • the invention relates to the technical field of microfluidics, in particular to a microfluidic chip for separating and capturing single cells, and a preparation method and application thereof.
  • the embodiments of the present invention provide a microfluidic chip for separating and capturing single cells, and a preparation method and application thereof.
  • the microfluidic chip for separating and capturing single cells can be fast, simple, low-cost and low-threshold Separate and obtain single cells.
  • the present invention provides a microfluidic chip for separating and capturing single cells, comprising a chip substrate, the chip substrate is provided with a sample hole and a plurality of sampling holes, the sample hole is located on the chip substrate In the center, wherein the injection hole is connected with at least two sets of multi-stage flow channels, and each set of the multi-stage flow channels includes a main flow channel, a first branch flow channel and a plurality of second branch flow channels; One end of the main flow channel is in communication with the injection hole, and the other end is in communication with the first branch flow channel; the plurality of second branch flow channels are symmetrically arranged on both sides of the first branch flow channel, and One end of a plurality of second branch flow channels communicates with the first branch flow channel, the other end of each second branch flow channel is connected with at least one sampling hole, and each sampling hole is connected to one The second branch flow channel is connected.
  • each of the second branch flow channels is directly connected with the sampling hole to achieve communication.
  • each of the second branch flow channels is connected to at least two of the sampling holes through at least two third branch flow channels, wherein each of the third branch flow channels is connected to one of the sampling holes correspondingly .
  • a part of the second branch flow passage is directly connected with the sampling hole to achieve communication; another part of the second branch flow passage passes through at least two third branch flow passages.
  • the channel is connected with at least two of the sampling holes, and each of the third branch flow channels is correspondingly connected with one of the sampling holes.
  • the plurality of second branch flow channels are connected to the sampling hole in a first manner and a second manner, and the second branch flow channels in the first manner are the same as those in the second manner.
  • the second branch flow channels are arranged at intervals; wherein, the first mode is that each of the second branch flow channels is directly connected to the sampling hole; the second mode is that each of the second branch flow channels is directly connected to the sampling hole.
  • the channel is connected to at least two sampling holes through at least two third branch flow channels, and each of the third branch flow channels is correspondingly connected to one sampling hole.
  • the at least two sets of multi-stage flow channels are regularly arranged around the injection hole.
  • the two sets of multi-stage flow channels may be symmetrically arranged on both sides of the injection hole, and the main flow channel in each group of the multi-stage flow channels communicates with the injection hole.
  • the cross-sectional widths of the main flow channel, the first branch flow channel and the second branch flow channel gradually decrease; wherein the cross-sectional width of the second branch flow channel is 50-55 ⁇ m.
  • the main flow channel, the first branch flow channel and the second branch flow channel have the same height.
  • the height of the main flow channel, the first branch flow channel and the second branch flow channel is 30-35 ⁇ m.
  • the main flow channel and the first branch flow channel are T-shaped intersection; the second branch flow channel and the first branch flow channel are T-shaped intersection.
  • the chip substrate is formed by sealing a flow channel layer and a sealing layer, wherein the sample injection hole, the sampling hole and the multi-stage flow channel are arranged on the flow channel layer.
  • the microfluidic chip for separating and capturing single cells includes a sampling hole, a plurality of sampling holes, and at least two sets of multi-stage flow channels communicating with the sampling hole;
  • the microfluidic chip can efficiently and simply obtain single cells from a sample solution containing a large number of cells;
  • the microfluidic chip for separating and capturing single cells according to the first aspect of the present invention has a simplified structure and can be used for integration, with strong initiative and flexibility It has a high degree of accuracy and has a wide range of application prospects, especially suitable for cell-related research and analysis.
  • the microfluidic chip for separating and capturing single cells of the present invention also has the characteristics of small size, low reagent consumption, high throughput, low cost and the like.
  • the present invention also provides a method for preparing a microfluidic chip for separating and capturing single cells as described in the first aspect of the present invention, including:
  • a patterned hard template and runner layer substrate combined with imprinting technology, after embossing, perforating and cleaning the runner layer substrate to form injection holes and multiple samples on the runner layer substrate Pattern structure of holes and at least two sets of multi-stage runners;
  • a sealing layer substrate is provided, and after the sealing layer substrate and the flow channel layer substrate after imprinting, perforating and cleaning are aligned and bonded, and the thermocompression sealing process is performed, the separated and trapped single cells are obtained Microfluidic Chip.
  • the material of the runner layer substrate and the sealing layer substrate includes polydimethylsiloxane, cycloolefin copolymer, polystyrene, polymethacrylate, polyethylene terephthalate One or more of ester, polytetrafluoroethylene, polypropionate, polycarbonate, polyethylene, polypropylene and polyvinyl chloride.
  • the sealing layer substrate forms the sealing layer of the microfluidic chip for separating and capturing single cells; the flow channel layer substrate after imprinting, perforating and washing forms the separating and capturing single cells The runner layer of the microfluidic chip.
  • the preparation method described in the second aspect of the present invention has a simple manufacturing process and a low manufacturing cost, and is suitable for large-scale industrial production.
  • the microfluidic chip for separating and capturing single cells prepared by the preparation method can realize rapid acquisition of single cells, and can obtain multiple single cells at the same time.
  • the method for obtaining the entire single cell is simple and low in cost.
  • the present invention also provides a device for separating and capturing single cells, comprising the microfluidic chip for separating and capturing single cells according to the first aspect of the present invention.
  • the device for separating and capturing single cells of the present invention is one of the applications of the microfluidic chip for separating and capturing single cells.
  • the device for separating and capturing single cells may include, but is not limited to, an automatic sample addition device.
  • the automatic sample addition device can set the sample hole of the microfluidic chip for separating and capturing single cells according to a preset time interval. A sample containing cells is added inside.
  • the single cell separation and capture device can be used in many fields such as biomedicine and stem cell research. Based on the separation and capture single cell device, in cell-related research and analysis work, by using the separated and captured single cell device, single cells can be obtained more conveniently and quickly from a system containing multiple cells; low cost and method Simple, it has huge advantages and prospects in biomedical analysis.
  • FIG. 1 is a schematic structural diagram of a microfluidic chip 100 for separating and capturing single cells according to an embodiment of the present invention
  • FIG. 2 is a schematic cross-sectional view along the A-A direction of the microfluidic chip 100 for separating and capturing single cells according to an embodiment of the present invention in FIG. 1;
  • FIG. 3 is an exploded view of the microfluidic chip 100 for separating and capturing single cells according to an embodiment of the present invention
  • FIG. 4 is a schematic cross-sectional view of a microfluidic chip 200 for separating and capturing single cells according to another embodiment of the present invention
  • FIG. 5 is a schematic cross-sectional view of a microfluidic chip 300 for separating and capturing single cells according to another embodiment of the present invention.
  • the raw materials and other chemical reagents used in the embodiments of the present invention are all commercially available products.
  • an embodiment of the present invention provides a microfluidic chip 100 for separating and capturing single cells, which includes a chip substrate 10 with a sample injection hole 20 in the center of the chip substrate 10.
  • a plurality of sampling holes 30 are provided around the hole 20, wherein the injection hole 10 is connected with two sets of multi-stage flow channels 40, and each set of the multi-stage flow channels 40 includes a main flow channel 41 and a first branch flow.
  • each second branch flow channel 43 is directly connected to the sampling hole 30 to achieve communication.
  • the sample injection hole 10 communicates with two sets of multi-stage flow channels 40.
  • the two sets of multi-stage flow channels 40 can be, but not limited to, symmetrically arranged.
  • the sample injection hole 10 may also communicate with multiple sets of multi-stage flow channels 40.
  • the groups of multi-stage flow channels are regularly arranged around the injection hole.
  • the cross-sectional widths of the main flow channel 41, the first branch flow channel 42 and the second branch flow channel 43 gradually decrease.
  • the cross-sectional width of the main flow channel 41 is greater than the cross-sectional width of the first branch flow channel 42; the cross-sectional width of the first branch flow channel 42 is greater than that of the second branch flow channel 43.
  • the cross-sectional width of the main runner is 150-300 ⁇ m.
  • the cross-sectional width of the main flow channel is 200-300 ⁇ m.
  • the cross-sectional width of the main runner is 150 ⁇ m, or 180 ⁇ m, or 200 ⁇ m, or 230 ⁇ m, or 250 ⁇ m, or 280 ⁇ m, or 300 ⁇ m.
  • the cross-sectional width of the first branch flow channel is 100-150 ⁇ m.
  • the cross-sectional width of the first branch flow channel is 120-150 ⁇ m.
  • the cross-sectional width of the first branch flow channel is 100-130 ⁇ m.
  • the cross-sectional width of the first branch flow channel is 120 ⁇ m, or 130 ⁇ m, or 135 ⁇ m, or 140 ⁇ m, or 150 ⁇ m.
  • the cross-sectional width of the second branch flow channel is 50-55 ⁇ m.
  • the cross-sectional width of the second branch flow channel is 50 ⁇ m, or 51 ⁇ m, or 52 ⁇ m, or 53 ⁇ m, or 54 ⁇ m, or 55 ⁇ m.
  • the sample injection hole 20 is used to add a sample solution containing cells.
  • the sampling hole 30 is used to obtain and separate single cells.
  • the cross-sectional widths of the main flow channel 41, the first branch flow channel 42 and the second branch flow channel 43 are all in the order of micrometers, and have a certain capillary force effect, which is conducive to the flow of the sample solution in the injection hole to all levels. Micro flow channel.
  • the gradually reduced cross-sectional widths of the main flow channel 41, the first branch flow channel 42 and the second branch flow channel 43 are beneficial to the separation of single cells; while the micro flow channel with a reduced cross-sectional width can be fixed. Enhancing the capillary force to a certain extent is beneficial to the step-by-step flow of the sample solution, so as to maintain the same liquid volume in the sampling holes 30 connected to each of the second branch flow channels.
  • the height of each group of the multi-stage runners is the same.
  • the main flow channel, the first branch flow channel and the second branch flow channel have the same height.
  • multi-level flow channels with the same flow channel height are beneficial to maintaining good fluency of each channel.
  • the heights of the main flow channel, the first branch flow channel and the second branch flow channel are all 30-35 ⁇ m.
  • the height of the third branch flow channel is 30-35 ⁇ m.
  • the sampling holes and the openings of the sampling holes are located on the surface of the chip substrate. Therefore, the sampling holes and the openings of the sampling holes are both located on the surface of the chip substrate. The depth of the sampling hole is greater than the height of the multi-stage flow channel.
  • the depth of the sampling hole and the sampling hole are the same.
  • the depth of the sampling hole refers to the distance between the bottom surface of the sampling hole and the opening of the sampling hole in the thickness direction of the chip substrate.
  • the depth of the sampling hole refers to the distance between the bottom surface of the sampling hole and the opening of the sampling hole in the thickness direction of the chip substrate.
  • the depth of the sampling hole and the sampling hole can be adjusted based on the thickness of the chip substrate.
  • the bottom surface of each micro flow channel, the bottom surface of the sampling hole, and the bottom surface of the sampling hole in the multi-stage flow channel are on the same level, which can facilitate the flow of liquid samples (such as cell suspension).
  • the main flow channel 41 and the first branch flow channel 42 meet in a T shape; the second branch flow channel 43 and the first branch flow channel 42 meet in a T shape.
  • the main flow channel 41 and the first branch flow channel 42 are made to be mutually perpendicular to the T-shaped intersection; the second branch flow channel 43 and the first branch flow channel 42 are made to be perpendicular to each other.
  • the second branch flow channel 43 is symmetrically arranged on both sides of the first branch flow channel 42; on the one hand, the overall structure of the multi-level flow channel can be made more symmetrical, and the distribution of each flow channel is more regular, thereby prompting each sampling hole 30
  • the probability of single cells separated is more even; on the other hand, the space of the chip substrate can be fully and rationally used under the premise of reducing the difficulty in the production of microfluidic chips, so that more second cells can be arranged on the chip substrate.
  • Branch flow channel 43 and sampling hole 30 are made to be mutually perpendicular to the T-shaped intersection; the second branch flow channel 43 and the first branch flow channel 42 are made to be perpendicular to each other.
  • the second branch flow channel 43 is symmetrically arranged on both sides of
  • the diameter of the sample injection hole 20 is 2-6 mm.
  • the diameter of the sample injection hole is 2 mm, or 3 mm, or 4 mm, or 5 mm, or 6 mm.
  • the diameter of the sampling hole 30 is 1.5-3.0 mm.
  • the diameter of the sampling hole is 1.5 mm, or 1.6 mm, or 1.8 mm, or 2.0 mm, or 2.2 mm, or 2.5 mm, or 2.8 mm, or 3.0 mm.
  • the sampling holes are regularly arranged on the chip substrate.
  • the length of the main flow channel, the first branch flow channel and/or the second branch flow channel may be adjusted based on the size of the microfluidic chip.
  • the size of the microfluidic chip is too large, the length of the main flow channel, the first branch flow channel and/or the second branch flow channel can be relatively increased; when the size of the microfluidic chip If it is too small, the length of the main flow channel, the first branch flow channel and/or the second branch flow channel can be relatively shortened.
  • the chip substrate may be, but not limited to, a two-layer structure.
  • the chip substrate is formed by sealing the flow channel layer 51 and the sealing layer 52, wherein the sampling hole 20, the sampling hole 30 and the multi-stage flow channel 40 are arranged on the flow channel layer 51 , See Figure 3.
  • the multi-stage flow channel 40 can only see the intersection of the main flow channel communicating with the injection hole 20 in the view 3.
  • the chip substrate is made of water- and gas-proof materials.
  • the flow channel layer 51 and the sealing layer 52 in the chip substrate may be, but not limited to, direct thermal compression sealing.
  • the microfluidic chip obtained by direct thermal compression sealing has a stable structure and good sealing performance.
  • the flow channel layer 51 and the sealing layer 52 can also be sealed with an intermediate sealing layer or an adhesive.
  • the thickness of the flow channel layer and the sealing layer can be adjusted based on actual application requirements.
  • the thickness of the flow channel layer can be, but is not limited to, 3-10 mm.
  • an embodiment of the present invention also provides a microfluidic chip 200 for separating and capturing single cells, including a chip substrate 10 provided with a sampling hole 20 and a plurality of sampling holes 30, The sampling hole 20 is arranged in the center of the chip substrate 10, wherein the sampling hole 10 is connected with two sets of multi-stage flow channels 40, and each set of the multi-stage flow channels 40 includes a main flow channel 41 and a first sub-channel.
  • the passages 43 are symmetrically arranged on both sides of the first branch flow passage 42, and one end of the plurality of second branch flow passages 43 is in communication with the first branch flow passage 42, and each of the second branch flow passages At least one of the sampling holes 30 is connected to the other end of the 43, and each of the sampling holes 30 is in communication with one of the second branch flow channels 43.
  • each of the second branch flow passages 43 is connected to at least two of the sampling holes 30 through at least two third branch flow passages 431, wherein each of the third branch flow passages 431 is connected to one of the Sampling hole 30.
  • each of the second branch flow passages 43 passes through the two third branch flow passages 431 to realize the communication with the two sampling holes 30.
  • the second branch flow passage 43 may also pass through a plurality of third branch flow passages 431 to achieve communication with a plurality of sampling holes 30.
  • the length of the third branch flow channel 431 that intersects with the same second branch flow channel 43 may be the same or different.
  • the cross-sectional width of the third branch flow channel 431 is less than or equal to the cross-sectional width of the second branch flow channel 43.
  • the cross-sectional width of the third branch flow channel 431 is 30-42 ⁇ m. In one embodiment, the cross-sectional width of the second branch flow channel is 30-40 ⁇ m.
  • the cross-sectional width of the second branch flow channel is 30 ⁇ m, or 32 ⁇ m, or 35 ⁇ m, or 38 ⁇ m, or 40 ⁇ m, or 42 ⁇ m.
  • microfluidic chip 200 for separating and capturing single cells may be consistent with the specific limitations of the microfluidic chip 100 for separating and capturing single cells described above, and will not be repeated in this embodiment.
  • an embodiment of the present invention also provides a microfluidic chip 300 for separating and capturing single cells, including a chip substrate 10 provided with a sampling hole 20 and a plurality of sampling holes 30, The sampling hole 20 is arranged in the center of the chip substrate 10, wherein the sampling hole 10 is connected with two sets of multi-stage flow channels 40, and each set of the multi-stage flow channels 40 includes a main flow channel 41 and a first sub-channel.
  • the passages 43 are symmetrically arranged on both sides of the first branch flow passage 42, and one end of the plurality of second branch flow passages 43 is in communication with the first branch flow passage 42, and each of the second branch flow passages
  • At least one of the sampling holes 30 is connected to the other end of the 43, and each of the sampling holes 30 is in communication with one of the second branch flow channels 43.
  • the plurality of second branch flow passages 43 are connected to the sampling hole 30 in a first manner and a second manner, and the second branch flow passages 43a in the first manner are connected to the sampling holes 30 in the second manner.
  • the second branch flow passages 43b are arranged at intervals; wherein, the first method is that each second branch flow passage 43a is directly connected to the sampling hole 30a; the second method is that each of the second branch flow passages 43a is directly connected to the sampling hole 30a;
  • the second branch flow channel 43b is connected to at least two sampling holes 30b through at least two third branch flow channels 431, and each of the third branch flow channels 431 is correspondingly connected to one sampling hole 30b.
  • the second branch flow passage 30 includes the second branch flow passage 43a connected to the sampling hole 30a in a first manner, and the second branch flow passage 43a connected to the sampling hole 30b in a second manner.
  • Branch flow channel 43b is the branch flow channel 43a.
  • the main flow channel 41 and the first branch flow channel 42 meet in a T shape; the second branch flow channel 43 and the first branch flow channel 41 meet in a T shape.
  • two second branch flow passages 43a are provided between two adjacent second branch flow passages 43b, as shown in FIG. 5.
  • one second branch flow passage 43a may be provided between two adjacent second branch flow passages 43b, or between two adjacent second branch flow passages 43b. There are two or more of the second branch flow passages 43a.
  • two sets of the multi-stage flow channels 40 are symmetrically arranged on both sides of the sampling hole 20.
  • the plurality of second branch runners 43 are symmetrically arranged on both sides of the first branch runner 42.
  • the multi-stage flow channel 40 can realize the communication between the injection hole 20 and the sampling hole 30, and each group of the multi-stage flow channel 40 includes 1 main flow channel, 1 first branch flow channel, and 36 first branch channels. Two branch runners and 24 third branch runners. In this embodiment, the number of the second branch flow channel and the third branch flow channel in each group of the multi-stage flow channel 40 can be adjusted based on the size of the microfluidic chip.
  • the sampling holes 30 on the microfluidic chip for separating and capturing single cells can be arranged regularly.
  • the length and shape of the second branch flow channel and the second branch flow channel can be changed.
  • the length of the second branch flow passage 43b is greater than the second branch flow passage 43a; and the second branch flow passage 43a is a linear flow passage, and the second branch flow passage 43b is a combination of a straight line and a curve.
  • Type runner the staggered arrangement of the multiple second branch flow channels described above can increase the number of sampling holes in the same area, further improve the separation efficiency of the entire microfluidic chip for separating and capturing single cells, and can separate simultaneously Multiple single cells out.
  • the cross-sectional width of the third branch flow channel 431 is 30-42 ⁇ m. In one embodiment, the cross-sectional width of the second branch flow channel is 30-40 ⁇ m.
  • the cross-sectional width of the second branch flow channel is 30 ⁇ m, or 32 ⁇ m, or 35 ⁇ m, or 38 ⁇ m, or 40 ⁇ m, or 42 ⁇ m.
  • the cross-sectional shape of the micro-channels of each level of the multi-stage flow channel 40 is rectangular, circular or elliptical. Further, the cross-sectional shape of the micro-channels of each level of the multi-stage flow channel 40 is rectangular.
  • microfluidic chip 300 for separating and capturing single cells may be the same as those of the microfluidic chip 100 for separating and capturing single cells or the specific limitations of the microfluidic chip 200 for separating and capturing single cells as described above. It is consistent, and will not be repeated in this embodiment.
  • the microfluidic chip for separating and capturing single cells of the present invention includes a sampling hole, a plurality of sampling holes, and at least two sets of multi-stage flow channels connected to the sampling hole;
  • the chip can efficiently and simply obtain single cells from a sample solution containing a large number of cells;
  • the microfluidic chip for separating and capturing single cells according to the first aspect of the present invention has a simplified structure and can be used for integration, with strong initiative and high flexibility. It has a wide range of application prospects, especially suitable for cell-related research and analysis.
  • the microfluidic chip for separating and capturing single cells of the present invention also has the characteristics of small size, low reagent consumption, high throughput, low cost and the like.
  • the microfluidic chip for separating and capturing single cells of the present invention can be manufactured by using multilayer soft lithography technology.
  • an embodiment of the present invention also provides a method for preparing the microfluidic chip for separating and capturing single cells, including:
  • a patterned hard template and runner layer substrate combined with imprinting technology, after embossing, perforating and cleaning the runner layer substrate to form injection holes and multiple samples on the runner layer substrate Pattern structure of holes and at least two sets of multi-stage runners;
  • a sealing layer substrate is provided, and after the sealing layer substrate and the flow channel layer substrate after imprinting, perforating and cleaning are aligned and bonded, and the thermocompression sealing process is performed, the separated and trapped single cells are obtained Microfluidic Chip.
  • the patterned hard template may be etched by photolithography technology to form a corresponding pattern capable of embossing a sample injection hole, a plurality of sampling holes, and at least two sets of multi-level flow channels.
  • photolithography technology For example, electron beam etching, photolithography, and wet etching.
  • the material of the runner layer substrate and the sealing layer substrate includes polydimethylsiloxane (PDMS), cycloolefin copolymer, polystyrene, polymethacrylate, polyterephthalic acid One or more of ethylene glycol ester, polytetrafluoroethylene, polypropionate, polycarbonate, polyethylene, polypropylene, and polyvinyl chloride.
  • PDMS polydimethylsiloxane
  • cycloolefin copolymer polystyrene
  • polymethacrylate polyterephthalic acid
  • ethylene glycol ester polytetrafluoroethylene
  • polypropionate polycarbonate
  • polyethylene polyethylene
  • polypropylene polypropylene
  • polyvinyl chloride polyvinyl chloride
  • the sealing layer substrate forms the sealing layer of the microfluidic chip for separating and capturing single cells; the flow channel layer substrate after imprinting, perforating and washing forms the separating and capturing single cells The runner layer of the microfluidic chip.
  • a specific embodiment of the present invention provides a method for preparing the microfluidic chip for separating and capturing single cells, including:
  • the hard substrate is processed by photolithography to form the facing micro-nano structure as shown in FIG. 3 as a hard template; the obtained hard template is transferred to the PDMS material to obtain a PDMS chip. Then use a puncher to punch through the PDMS chip one by one according to the design to obtain the PDMS flow channel layer.
  • the PDMS material used includes a silicone resin and a curing agent, wherein the silicone resin and the curing agent are mixed at a mass ratio of 10:1, and the mixture is uniformly stirred.
  • the material liquid is degassed. The degassing process is: place the mixed material in a vacuum box, evacuate until the internal pressure is lower than 110 Pa, and let it stand for 15 minutes.
  • microfluidic chip for separating and capturing single cells provided by the embodiments of the present invention can also be prepared by other preparation methods, and this embodiment does not make too many restrictions.
  • the operating method of the microfluidic chip for separating and capturing single cells of the present invention may be:
  • the remaining cell suspension in the microfluidic chip for separating and capturing single cells can be recovered with a pipette for subsequent use.
  • An embodiment of the present invention also provides a device for separating and capturing single cells, which includes the microfluidic chip 100, 200, or 300 for separating and capturing single cells provided in the foregoing embodiment of the present invention.
  • the device for separating and capturing single cells includes an automatic sample addition device, and the automatic sample addition device can add cells containing cells to the injection hole of the microfluidic chip for separating and capturing single cells according to a preset time interval. Sample solution.
  • the single cell separation and capture device can be used in many fields such as biomedicine and stem cell research. Based on the separation and capture single cell device, in cell-related research and analysis work, by using the separated and captured single cell device, single cells can be obtained more conveniently and quickly from a system containing multiple cells; low cost and method Simple, it has huge advantages and prospects in biomedical analysis.

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Abstract

一种分离捕获单细胞的微流控芯片（100），包括芯片基板（10），所述芯片基板（10）设有一进样孔（20）和多个取样孔（30），所述进样孔（10）位于所述芯片基板的中央，其中，进样孔连通有至少两组多级流道（40），每一组所述多级流道（40）包括一主流道（41）、一第一分支流道（42）和多条第二分支流道（43）；所述主流道（41）的一端与所述进样孔（20）连通，另一端与所述第一分支流道（42）连通；所述多条第二分支流道（43）对称排布在所述第一分支流道（42）的两侧，所述多条第二分支流道（43）的一端与所述第一分支流道（42）连通，每一条所述第二分支流道（42）的另一端连接有至少一个所述取样孔（30），每一所述取样孔（30）与一条所述第二分支流道（43）连通。

Description

一种分离捕获单细胞的微流控芯片及其制备方法和应用 技术领域
本发明涉及微流控技术领域，特别是涉及一种分离捕获单细胞的微流控芯片及其制备方法和应用。
 背景技术
近几年的研究发现，针对单个细胞的细胞生物学研究有着重要的意义，它能够揭示单个细胞的基因结构和基因表达状态，反映细胞间的异质性，在重大疾病早期诊断、肿瘤、发育生物学、微生物学、神经科学等领域发挥重要作用，正成为生命科学研究的焦点。单细胞分离捕获技术是进行单细胞水平研究的基础。目前能够实现单细胞分选的方法主要集中在流式细胞仪或单细胞分选仪等。流式细胞仪可以实现活细胞的多通道分选；单细胞分选仪是通过负压控制毛细管在显微镜下寻找目标细胞。然而，现有的流式细胞仪价格昂贵，体积庞大，需要专人操作，不能实现大面积普及。单细胞分选仪同样价格昂贵，且操作费时费力，对操作者同样要求高技术。
因此，有必要开发一种成本低、操作简便、条件温和、主动性强和灵活度高的分离捕获单细胞的装置。
有鉴于此，本发明实施例提供了一种分离捕获单细胞的微流控芯片及其制备方法和应用，所述分离捕获单细胞的微流控芯片能够快捷、简易、低成本和低门槛的对单细胞实现分离获取。
 技术解决方案
第一方面，本发明提供了一种分离捕获单细胞的微流控芯片，包括芯片基板，所述芯片基板设有一进样孔和多个取样孔，所述进样孔位于所述芯片基板的中央，其中，所述进样孔连通有至少两组多级流道，每一组所述多级流道包括一主流道、一第一分支流道和多条第二分支流道；所述主流道的一端与所述进样孔连通，另一端与所述第一分支流道连通；所述多条第二分支流道对称排布在所述第一分支流道的两侧，所述多条第二分支流道的一端与所述第一分支流道连通，每一条所述第二分支流道的另一端连接有至少一个所述取样孔，每一所述取样孔与一条所述第二分支流道连通。
可选地，每一条所述第二分支流道与所述取样孔直接连接以实现连通。
可选地，每一条所述第二分支流道通过至少两条第三分支流道与至少两个所述取样孔连接，其中，每条所述第三分支流道对应连接一个所述取样孔。
可选地，所述多条第二分支流道中，一部分所述第二分支流道与所述取样孔直接连接以实现连通；另一部分所述第二分支流道通过至少两条第三分支流道与至少两个所述取样孔连接，每条所述第三分支流道对应连接一个所述取样孔。
可选地，所述多条第二分支流道按第一方式和第二方式与所述取样孔连接，且按所述第一方式的所述第二分支流道与按所述第二方式的所述第二分支流道间隔设置；其中，所述第一方式为每一条所述第二分支流道与所述取样孔直接连接；所述第二方式为每一条所述第二分支流道通过至少两条第三分支流道与至少两个所述取样孔连接，每条所述第三分支流道对应连接一个所述取样孔。
可选地，所述至少两组多级流道围绕所述进样孔规则排布。例如，当存在两组多级流道时，所述两组多级流道可以对称设置在进样孔的两旁，每组所述多级流道中的主流道与所述进样孔连通。
可选地，所述主流道、所述第一分支流道和所述第二分支流道的截面宽度逐渐减小；其中，所述第二分支流道的截面宽度为50-55μm。
可选地，所述主流道、所述第一分支流道和所述第二分支流道的高度相同。
可选地，所述主流道、所述第一分支流道和所述第二分支流道的高度为30-35μm。
可选地，所述主流道与所述第一分支流道呈T型交汇；所述第二分支流道与所述第一分支流道呈T型交汇。
可选地，所述芯片基板由流道层和封闭层封接形成，其中，所述进样孔、所述取样孔和所述多级流道设置在所述流道层上。
本发明第一方面所述的分离捕获单细胞的微流控芯片，所述微流控芯片包括进样孔、多个取样孔和与进样孔连通的至少两组多级流道；所述微流控芯片可以高效、简便地从含有大量细胞的样品溶液中获取单细胞；本发明第一方面所述分离捕获单细胞的微流控芯片结构精简，可以用于集成，主动性强、灵活度高，具有广泛的应用前景，尤其适用于细胞相关的研究和分析工作。本发明所述分离捕获单细胞的微流控芯片还具有尺寸小、试剂消耗量少、高通量、成本低等特点。
第二方面，本发明还提供了一种利用如本发明第一方面所述分离捕获单细胞的微流控芯片的制备方法，包括：
提供一图案化的硬质模板和流道层基板，结合压印技术，对流道层基板进行压印、打孔和清洗后，以在所述流道层基板上形成进样孔、多个取样孔和至少两组多级流道的图案结构；
提供封闭层基板，将所述封闭层基板和所述经压印、打孔和清洗后的所述流道层基板对准贴合以及热压封接处理后，得到所述分离捕获单细胞的微流控芯片。
可选地，所述流道层基板和所述封闭层基板的材质包括聚二甲基硅氧烷、环烯烃共聚物、聚苯乙烯、聚甲基丙烯酸酯、聚对苯二甲酸乙二醇酯、聚四氟乙烯、聚丙酸酯、聚碳酸酯、聚乙烯、聚丙烯和聚氯乙烯中的一种或多种。
可选地，所述封闭层基板形成所述分离捕获单细胞的微流控芯片的封闭层；所述经压印、打孔和清洗后的所述流道层基板形成所述分离捕获单细胞的微流控芯片的流道层。
本发明第二方面所述制备方法，制作工艺简单，制作成本低，能适用于大规模的工业化生产的。由所述制备方法制得的分离捕获单细胞的微流控芯片能实现单细胞的快速获取，且可以同时获取多个单细胞，整个单细胞获取的方法简单，成本低廉。
第三方面，本发明还提供了一种分离捕获单细胞装置，包括本发明第一方面所述分离捕获单细胞的微流控芯片。本发明所述分离捕获单细胞装置为所述分离捕获单细胞的微流控芯片的应用之一。
可选地，所述分离捕获单细胞装置可以但不限于包括自动加样装置，所述自动加样装置可以按照预设的时间间隔在所述分离捕获单细胞的微流控芯片的进样孔内添加含有细胞的样品。
可选地，所述分离捕获单细胞装置可以用于生物医学、干细胞研究等众多领域。基于所述分离捕获单细胞装置，在与细胞相关的研究和分析工作，通过利用所分离捕获单细胞装置，实现更加方便、快速地从含有多个细胞的体系中获取单细胞；成本低，方法简单，在生物医学分析方面有着巨大的优势和前景。
本发明的优点将会在下面的说明书中部分阐明，一部分根据说明书是显而易见的，或者可以通过本发明实施例的实施而获知。
 附图说明
为更清楚地阐述本发明的内容，下面结合附图与具体实施例来对其进行详细说明。
图1为本发明一实施例提供的分离捕获单细胞的微流控芯片100的结构示意图；
图2为图1中本发明一实施例提供的分离捕获单细胞的微流控芯片100沿A-A方向的截面示意图；
图3为本发明一实施例提供的分离捕获单细胞的微流控芯片100的分解图；
图4为本发明另一实施例提供的分离捕获单细胞的微流控芯片200的截面示意图；
图5为本发明另一实施例提供的分离捕获单细胞的微流控芯片300的截面示意图。
 本发明的实施方式
以下所述是本发明实施例的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明实施例原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明实施例的保护范围。
本申请说明书、权利要求书和附图中出现的术语“包括”和“具有”以及它们任何变形，意图在于覆盖不排他的包含。例如包含了一系列步骤或单元的过程、方法、系统、产品或设备没有限定于已列出的步骤或单元，而是可选地还包括没有列出的步骤或单元，或可选地还包括对于这些过程、方法、产品或设备固有的其它步骤或单元。本申请中“第一”、“第二”等术语仅用于对象的区分，并不是指特定的顺序。
若无特别说明，本发明实施例所采用的原料及其它化学试剂皆为市售商品。
如图1和图2所示，本发明一实施例提供了一种分离捕获单细胞的微流控芯片100，包括芯片基板10，所述芯片基板10中央设有一进样孔20，在进样孔20周围设有多个取样孔30，其中，所述进样孔10连通有两组多级流道40，每一组所述多级流道40包括一主流道41、一第一分支流道42和多条第二分支流道43；所述主流道41的一端与所述进样孔20连通，另一端与所述第一分支流道42连通；所述多条第二分支流道43对称排布在所述第一分支流道42的两侧，所述多条第二分支流道43的一端与所述第一分支流道42连通，每一条所述第二分支流道43的另一端连接有至少一个所述取样孔30，每一所述取样孔30与一条所述第二分支流道43连通。
本实施方式中，每一条第二分支流道43与所述取样孔30直接连接以实现连通。
本实施方式中，所述进样孔10与两组多级流道40相连通。所述两组多级流道40可以但不限于对称设置。
本一实施方式中，所述进样孔10还可以与多组多级流道40连通。可选地，所述多组多级流道围绕所述进样孔规则排布。
本实施方式中，所述主流道41、所述第一分支流道42和所述第二分支流道43的截面宽度逐渐减小。所述主流道41的截面宽度大于所述第一分支流道42的截面宽度；所述第一分支流道42的截面宽度大于所述第二分支流道43。
可选地，所述主流道的截面宽度为150-300μm。
进一步地，可选地，所述主流道的截面宽度为200-300μm。
例如，所述主流道的截面宽度为150μm，或为180μm，或为200μm，或为230μm，或为250μm，或为280μm，或为300μm。
可选地，所述第一分支流道的截面宽度为100-150μm。
进一步地，所述第一分支流道的截面宽度为120-150μm。
进一步地，所述第一分支流道的截面宽度为100-130μm。
例如，所述第一分支流道的截面宽度为120μm，或为130μm，或为135μm，或为140μm，或为150μm。
可选地，所述第二分支流道的截面宽度为50-55μm。
例如，所述第二分支流道的截面宽度为50μm，或为51μm，或为52μm，或为53μm，或为54μm，或为55μm。
本实施方式中，所述进样孔20用于添加含有细胞的样品溶液。所述取样孔30用于获取并分离的单细胞。所述主流道41、所述第一分支流道42和所述第二分支流道43的截面宽度均为微米级，具有一定的毛细力作用，有利于进样孔中的样品溶液流向各级微流道。同时，逐渐减小的主流道41、所述第一分支流道42和所述第二分支流道43的截面宽度，一方面有利于单细胞的分离；而截面宽度减少的微流道可以一定程度地增强毛细力，有利于样品溶液的逐级流动，从而维持与各条第二分支流道连接的取样孔30的液量一致。
本实施方式中，每一组所述多级流道的高度相同。可选地，所述主流道、所述第一分支流道和所述第二分支流道的高度相同。本实施方式中，相同的流道高度的多级流道有利于各个通道保持良好的流畅性。
可选地，所述主流道、所述第一分支流道和所述第二分支流道的高度均为30-35μm。
可选地，所述第三分支流道的高度均为30-35μm。
本实施方式中，由于所述多级流道是在所述芯片基板的内部，所述进样孔和所述取样孔的开口都位于所述芯片基板的表面，因此，所述进样孔和所述取样孔深度都大于所述多级流道的高度。
可选地，所述进样孔和所述取样孔的深度相同。其中，所述进样孔的深度是指所述进样孔底部表面与所述进样孔开口在所述芯片基板厚度方向上的距离。所述取样孔的深度是指所述取样孔底部表面与所述取样孔开口在所述芯片基板厚度方向上的距离。
本实施方式中，所述进样孔和所述取样孔的深度可以基于芯片基板的厚度进行调节。本实施方式中，所述多级流道中各个微流道的底部表面、进样孔底部表面和取样孔的底部表面处于同一水平面上，这样可以有利于液体样本（例如细胞悬液）的流动。
本实施方式中，所述主流道41与所述第一分支流道42呈T型交汇；所述第二分支流道43与所述第一分支流道42呈T型交汇。这里是指：所述主流道41与所述第一分支流道42按T型交汇的方式连通；所述第二分支流道43与所述第一分支流道42按T型交汇的方式连通。
本实施方式中，通过使主流道41与所述第一分支流道42呈相互垂直的T型交汇；使所述第二分支流道43与所述第一分支流道42呈相互垂直的T型交汇，且第二分支流道43对称排布在第一分支流道42的两侧；一方面可以使多级流道的整体结构更加对称，各个流道分布更加规整，从而促使各个取样孔30分离得到的单细胞的几率更加平均；另一方面，可在减少微流控芯片的制作难道的前提下，充分、合理利用芯片基板的空间，使芯片基板上可以设有更多的第二分支流道43和取样孔30。
本实施方式中，所述进样孔20的直径为2-6 mm。例如，所述进样孔的直径为2mm，或为3 mm，或为4 mm，或为5 mm，或为6 mm。
可选地，所述取样孔30的直径为1.5-3.0mm。例如，所述取样孔的直径为1.5mm，或为1.6 mm，或为1.8 mm，或为2.0mm，或为2.2 mm，或为2.5mm，或为2.8 mm，或为3.0mm。本实施方式中，所述取样孔规则排布在所述芯片基板上。
本实施方式中，所述主流道、所述第一分支流道和/或所述第二分支流道的长度可以基于所述微流控芯片的尺寸大小进行调节。当所述微流控芯片的尺寸偏大时，所述主流道、所述第一分支流道和/或所述第二分支流道的长度可以相对增长；当所述微流控芯片的尺寸偏小时，所述主流道、所述第一分支流道和/或所述第二分支流道的长度可以相对缩短。
本实施方式中，所述芯片基板可以但不限于由两层结构组分。例如，所述芯片基板由流道层51和封闭层52封接形成，其中，所述进样孔20、所述取样孔30和所述多级流道40设置在所述流道层51上，参见图3。其中，多级流道40在视图3中仅能看到与进样孔20连通的主流道的交汇口。
本实施方式中，所述芯片基板由隔水、隔气的材料制成。所述芯片基板中的流道层51和封闭层52之间可以但不限于通过直接热压封接。通过直接热压封接的方式得到的所述微流控芯片结构稳定、密封性良好。可选地，流道层51和封闭层52之间也可以使用中间密封层或粘黏剂进行密封。
本实施方式中，所述流道层和封闭层的厚度尺寸可以基于实际应用需求进行调节。
可选地，所述流道层的厚度可以但不限于为3-10mm。
如图4所示，本发明一实施例还提供了一种分离捕获单细胞的微流控芯片200，包括芯片基板10，所述芯片基板10设有一进样孔20和多个取样孔30，进样孔20设置在芯片基板10的中央，其中，所述进样孔10连通有两组多级流道40，每一组所述多级流道40包括一主流道41、一第一分支流道42和多条第二分支流道43；所述主流道41的一端与所述进样孔20连通，另一端与所述第一分支流道42连通；所述多条第二分支流道43对称排布在所述第一分支流道42的两侧，所述多条第二分支流道43的一端与所述第一分支流道42连通，每一条所述第二分支流道43的另一端连接有至少一个所述取样孔30，每一所述取样孔30与一条所述第二分支流道43连通。其中，每一条所述第二分支流道43通过至少两条第三分支流道431与至少两个所述取样孔30连接，其中，每条所述第三分支流道431对应连接一个所述取样孔30。
本实施方式中，一条每一条所述第二分支流道43通过两条第三分支流道431，实现对两个取样孔30的连通。本实施方式中，所述第二分支流道43还可以通过多条第三分支流道431，实现对多个取样孔30的连通。
本实施方式中，与同一条第二分支流道43交汇的第三分支流道431的长度可以相同也可不同。所述第三分支流道431的截面宽度小于或等于第二分支流道43的截面宽度。
可选地，所述第三分支流道431的截面宽度为30-42μm。一实施方式中，所述第二分支流道的截面宽度为30-40μm。
例如，所述第二分支流道的截面宽度为30μm，或为32μm，或为35μm，或为38μm，或为40μm，或为42μm。
本实施方式中，所述分离捕获单细胞的微流控芯片200的其他限定可以与上面所述的分离捕获单细胞的微流控芯片100的具体限定一致，本实施方式中不做重复赘述。
如图5所示，本发明一实施例还提供了一种分离捕获单细胞的微流控芯片300，包括芯片基板10，所述芯片基板10设有一进样孔20和多个取样孔30，进样孔20设置在芯片基板10的中央，其中，所述进样孔10连通有两组多级流道40，每一组所述多级流道40包括一主流道41、一第一分支流道42和多条第二分支流道43；所述主流道41的一端与所述进样孔20连通，另一端与所述第一分支流道42连通；所述多条第二分支流道43对称排布在所述第一分支流道42的两侧，所述多条第二分支流道43的一端与所述第一分支流道42连通，每一条所述第二分支流道43的另一端连接有至少一个所述取样孔30，每一所述取样孔30与一条所述第二分支流道43连通。
其中，所述多条第二分支流道43按第一方式和第二方式与所述取样孔30连接，且按所述第一方式的所述第二分支流道43a与按所述第二方式的所述第二分支流道43b间隔设置；其中，所述第一方式为每一条所述第二分支流道43a与所述取样孔30a直接连接；所述第二方式为每一条所述第二分支流道43b通过至少两条第三分支流道431与至少两个所述取样孔30b连接，每条所述第三分支流道431对应连接一个所述取样孔30b。
可选地，所述第二分支流道30包括按第一方式连接所述取样孔30a的所述第二分支流道43a，和按第二方式连接所述取样孔30b的所述第二分支流道43b。
本实施方式中，所述主流道41与所述第一分支流道42呈T型交汇；所述第二分支流道43与所述第一分支流道41呈T型交汇。
可选地，相邻两条所述第二分支流道43b之间设有两条所述第二分支流道43a，见图5。
本实施方式中，相邻两条所述第二分支流道43b之间还可以设有一条所述第二分支流道43a，或者相邻两条所述第二分支流道43b之间还可以设有两条以上条所述第二分支流道43a。
本实施方式中，所述分离捕获单细胞的微流控芯片上，两组所述多级流道40对称排布在所述取样孔20的两边。每组多级流道40中，所述多条第二分支流道43对称排布在所述第一分支流道42的两侧。
可选地，所述多级流道40能实现进样孔20和取样孔30之间的连通，每组多级流道40包括1个主流道、1个第一分支流道、36个第二分支流道及24个第三分支流道。本实施方式中，所述每组多级流道40中第二分支流道和第三分支流道的数目可以基于微流控芯片的尺寸大小进行调节。
本实施方式中，所述分离捕获单细胞的微流控芯片上的所述取样孔30可以规则排布。为了实现所述取样孔30相对紧密设置在分离捕获单细胞的微流控芯片上，所述第二分支流道和第二分支流道的长度和形状可以进行改变。例如，图5中，第二分支流道43b的长度大于第二分支流道43a；且第二分支流道43a为直线型流道，而第二分支流道43b为直线与曲线相结合的混合型流道。本实施方式中，通过上述所述多条第二分支流道的错开排列能使得相同面积内的取样孔的数量增加，进一步提升整个分离捕获单细胞的微流控芯片的分离效率，可以同时分离出多个单细胞。
可选地，所述第三分支流道431的截面宽度为30-42μm。一实施方式中，所述第二分支流道的截面宽度为30-40μm。
例如，所述第二分支流道的截面宽度为30μm，或为32μm，或为35μm，或为38μm，或为40μm，或为42μm。
本实施方式中，所述多级流道40的各级微流道的截面形状为矩形、圆形或椭圆。进一步地，所述多级流道40的各级微流道的截面形状为矩形。
本实施方式中，所述分离捕获单细胞的微流控芯片300的其他限定可以与上面所述的分离捕获单细胞的微流控芯片100或分离捕获单细胞的微流控芯片200的具体限定一致，本实施方式中不做重复赘述。
本发明所述的分离捕获单细胞的微流控芯片，所述微流控芯片包括进样孔、多个取样孔和与进样孔连通的至少两组多级流道；所述微流控芯片可以高效、简便地从含有大量细胞的样品溶液中获取单细胞；本发明第一方面所述分离捕获单细胞的微流控芯片结构精简，可以用于集成，主动性强、灵活度高，具有广泛的应用前景，尤其适用于细胞相关的研究和分析工作。本发明所述分离捕获单细胞的微流控芯片还具有尺寸小、试剂消耗量少、高通量、成本低等特点。
本发明所述分离捕获单细胞的微流控芯片可以采用多层软光刻技术加工制作而成。可选地，本发明一实施例还提供了所述分离捕获单细胞的微流控芯片的制备方法，包括：
提供一图案化的硬质模板和流道层基板，结合压印技术，对流道层基板进行压印、打孔和清洗后，以在所述流道层基板上形成进样孔、多个取样孔和至少两组多级流道的图案结构；
提供封闭层基板，将所述封闭层基板和所述经压印、打孔和清洗后的所述流道层基板对准贴合以及热压封接处理后，得到所述分离捕获单细胞的微流控芯片。
可选地，所述图案化的硬质模板可以采用光刻技术进行刻蚀以形成能压印得到进样孔、多个取样孔和至少两组多级流道的相对应图案。例如电子束刻蚀法，光刻法，湿法刻蚀法。
可选地，所述流道层基板和所述封闭层基板的材质包括聚二甲基硅氧烷（PDMS）、环烯烃共聚物、聚苯乙烯、聚甲基丙烯酸酯、聚对苯二甲酸乙二醇酯、聚四氟乙烯、聚丙酸酯、聚碳酸酯、聚乙烯、聚丙烯和聚氯乙烯中的一种或多种。
可选地，所述封闭层基板形成所述分离捕获单细胞的微流控芯片的封闭层；所述经压印、打孔和清洗后的所述流道层基板形成所述分离捕获单细胞的微流控芯片的流道层。
例如，本发明一具体实施例提供了所述分离捕获单细胞的微流控芯片的制备方法，包括：
用光刻方法将硬质基底加工形成如图3所示的正对的微纳结构，作为硬质模板；将得到的所述硬质模板转移到PDMS材料上，得到PDMS芯片。然后用打孔器将PDMS芯片按设计逐一打通孔得到PDMS流道层。
将干净载玻片与打孔后的PDMS流道层的键合面朝上放入等离子清洗机中，清洗30秒后拿出键合，得到分离捕获单细胞的微流控芯片。进一步放入80°烘箱烘烤30分钟，使键合更牢固。
所述制备方法中，所用的PDMS材料包括硅树脂和固化剂，其中硅树脂和固化剂按质量比10:1的比例混合，搅拌均匀。混合后将材料液体进行除气处理，除气过程是：将混合后的材料置于真空箱中，抽真空至内部压强低于110Pa，静置15分钟。
本发明所述实施例提供的分离捕获单细胞的微流控芯片还可以采用其他制备方法制备得到，本实施方式中不做过多限定。
本发明所述分离捕获单细胞的微流控芯片的操作方法可以为：
将得到的细胞液以1200r/min离心5分钟，弃上清；然后加入一定量缓冲液（如磷酸盐缓冲液），用移液器吸取重悬。
用移液器吸取一定量的细胞悬液至新管中，加入缓冲液以稀释细胞浓度，得到低浓度细胞悬液；
用移液器吸取低浓度细胞悬液，注入微流控芯片的进样孔内，并将压力维持一定的时间；待每个取样孔都充满液体后，在显微镜下寻找取样孔里的单细胞。
分离单细胞的过程中，所述分离捕获单细胞的微流控芯片内剩余的细胞悬液可以用移液器回收，供后续使用。
本发明一实施例还提供了一种分离捕获单细胞装置，包括本发明上述实施例提供的分离捕获单细胞的微流控芯片100、200或300。
可选地，所述分离捕获单细胞装置包括自动加样装置，所述自动加样装置可以按照预设的时间间隔在所述分离捕获单细胞的微流控芯片的进样孔内添加含有细胞的样品溶液。
可选地，所述分离捕获单细胞装置可以用于生物医学、干细胞研究等众多领域。基于所述分离捕获单细胞装置，在与细胞相关的研究和分析工作，通过利用所分离捕获单细胞装置，实现更加方便、快速地从含有多个细胞的体系中获取单细胞；成本低，方法简单，在生物医学分析方面有着巨大的优势和前景。
需要说明的是，根据上述说明书的揭示和阐述，本发明所属领域的技术人员还可以对上述实施方式进行变更和修改。因此，本发明并不局限于上面揭示和描述的具体实施方式，对本发明的一些等同修改和变更也应当在本发明的权利要求的保护范围之内。此外，尽管本说明书中使用了一些特定的术语，但这些术语只是为了方便说明，并不对本发明构成任何限制。

Claims (10)

  1. 一种分离捕获单细胞的微流控芯片，其特征在于，包括芯片基板，所述芯片基板设有一进样孔和多个取样孔，所述进样孔位于所述芯片基板的中央，其中，所述进样孔连通有至少两组多级流道，每一组所述多级流道包括一主流道、一第一分支流道和多条第二分支流道；所述主流道的一端与所述进样孔连通，另一端与所述第一分支流道连通；所述多条第二分支流道对称排布在所述第一分支流道的两侧，所述多条第二分支流道的一端与所述第一分支流道连通，每一条所述第二分支流道的另一端连接有至少一个所述取样孔，每一所述取样孔与一条所述第二分支流道连通。
  2. 如权利要求1所述的微流控芯片，其特征在于，每一条所述第二分支流道与所述取样孔直接连接以实现连通。
  3. 如权利要求1所述的微流控芯片，其特征在于，每一条所述第二分支流道通过至少两条第三分支流道与至少两个所述取样孔连接，其中，每条所述第三分支流道对应连接一个所述取样孔。
  4. 如权利要求1所述的微流控芯片，其特征在于，所述多条第二分支流道按第一方式和第二方式与所述取样孔连接，且按所述第一方式的所述第二分支流道与按所述第二方式的所述第二分支流道间隔设置；其中，所述第一方式为每一条所述第二分支流道与所述取样孔直接连接；所述第二方式为每一条所述第二分支流道通过至少两条第三分支流道与至少两个所述取样孔连接，每条所述第三分支流道对应连接一个所述取样孔。
  5. 如权利要求4所述的微流控芯片，其特征在于，所述另一部分所述第二分支流道间隔设置。
  6. 如权利要求1所述的微流控芯片，其特征在于，所述主流道、所述第一分支流道和所述第二分支流道的截面宽度逐渐减小；其中，所述第二分支流道的截面宽度为50-55μm。
  7. 如权利要求1所述的微流控芯片，其特征在于，所述主流道与所述第一分支流道呈T型交汇；所述第二分支流道与所述第一分支流道呈T型交汇。
  8. 如权利要求1所述的微流控芯片，其特征在于，所述芯片基板由流道层和封闭层封接形成，其中，所述进样孔、所述取样孔和所述多级流道设置在所述流道层上。
  9. 一种利用如权利要求1-8任意一项所述分离捕获单细胞的微流控芯片的制备方法，其特征在于，包括：
    提供一图案化的硬质模板和流道层基板，结合压印技术，对流道层基板进行压印、打孔和清洗后，以在所述流道层基板上形成进样孔、多个取样孔和至少两组多级流道的图案结构；
    提供封闭层基板，将所述封闭层基板和所述流道层基板对准贴合以及热压封接处理后，得到所述分离捕获单细胞的微流控芯片。
  10. 一种分离捕获单细胞装置，其特征在于，包括如权利要求1-8任意一项所述分离捕获单细胞的微流控芯片。
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US20180071736A1 (en) * 2016-01-26 2018-03-15 Lidong Qin Microfluidic Aliquot Chip For Single-Cell Isolation
CN208200905U (zh) * 2018-01-12 2018-12-07 哈尔滨工业大学深圳研究生院 一种集成单细胞捕获与筛选功能的微流控芯片
CN208449339U (zh) * 2018-05-04 2019-02-01 南京邮电大学 一种星-树型复合结构的mhd微流控芯片
CN110713900A (zh) * 2019-11-05 2020-01-21 深圳先进技术研究院 一种分离捕获单细胞的微流控芯片及其制备方法和应用

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