WO2019096070A1 - 单个微粒包裹液滴在微流控芯片中形成并分别导出的方法 - Google Patents

单个微粒包裹液滴在微流控芯片中形成并分别导出的方法 Download PDF

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WO2019096070A1
WO2019096070A1 PCT/CN2018/114807 CN2018114807W WO2019096070A1 WO 2019096070 A1 WO2019096070 A1 WO 2019096070A1 CN 2018114807 W CN2018114807 W CN 2018114807W WO 2019096070 A1 WO2019096070 A1 WO 2019096070A1
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sample
microfluidic chip
channel
particle
microfluidic
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PCT/CN2018/114807
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English (en)
French (fr)
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徐腾
马波
徐健
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中国科学院青岛生物能源与过程研究所
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Priority to US16/764,803 priority Critical patent/US20200376490A1/en
Priority to JP2020545423A priority patent/JP7071519B2/ja
Priority to EP18880018.9A priority patent/EP3711855A4/en
Publication of WO2019096070A1 publication Critical patent/WO2019096070A1/zh

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    • 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
    • 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
    • B01L3/502715—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 interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • 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
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    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • 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
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01L2300/00—Additional constructional details
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    • B01L2300/0883—Serpentine channels
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    • B01L2300/16—Surface properties and coatings
    • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
    • B01L2300/165—Specific details about hydrophobic, oleophobic surfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • 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
    • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • 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
    • B01L2400/0454—Moving fluids with specific forces or mechanical means specific forces radiation pressure, optical tweezers
    • 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
    • B01L2400/0457—Moving fluids with specific forces or mechanical means specific forces passive flow or gravitation

Definitions

  • the invention relates to the field of microfluidic technology, in particular to a method for forming a single microparticle-encapsulated droplet by using a microfluidic chip, which can be used for single cell screening, single cell separation, single cell sequencing, single cell morphology analysis, single cell. Training, drug screening and other fields.
  • microparticle refers to particles that are capable of being suspended in a non-organic phase solution (eg, an aqueous phase) and passed within the microfluidic chip of the present invention, including both biomass-derived and non-living-derived particles.
  • a non-organic phase solution eg, an aqueous phase
  • microfluidic chip of the present invention including both biomass-derived and non-living-derived particles.
  • eukaryotic cells eukaryotic cells, prokaryotic cells, unicellular organisms, viral particles, organelles, particles formed by biomacromolecules, drug particles, drug carrier particles, liposomes, polymer particles, and the like.
  • a microfluidic chip comprising a cover sheet layer and a substrate layer; the substrate layer comprising at least one sample channel, the sample channel comprising a microfluidic channel a detection pool (5); the cover sheet layer includes a sample injection hole (1) and an oil storage pool hole (6); the oil storage pool hole (6) forms an oil storage pool with the substrate layer; The inlet end (7) of the sample channel is connected to the injection port (1), and the outlet end (8) of the sample channel is connected to the reservoir.
  • the material of the cover sheet layer is selected from, but not limited to, quartz, PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate), borosilicate glass, calcium fluoride
  • the material of the substrate layer is selected from, but not limited to, quartz, PDMS, PMMA, borosilicate glass, calcium fluoride.
  • the surface of the oil reservoir is a hydrophobic oleophilic surface.
  • the injection port (1) is connected to a sample conduit interface.
  • the microfluidic channel comprises a straight channel (2) and at least one curved channel (3).
  • the curved channel according to the present invention refers to a microfluidic channel that is not linear.
  • the microfluidic channel has a width of 30 to 300 ⁇ m and a height of 15 to 50 ⁇ m.
  • the detection cell (5) is connected to the reservoir hole (6) via a channel (23) which is a straight channel.
  • the sample channel further comprises at least one reservoir (4).
  • the volume of the detection cell (5) is smaller than the reservoir (4) ) the capacity.
  • the detection tank (5) is located between the liquid storage tank (4) and the oil reservoir hole (6), the liquid storage tank (4) and the detection pool ( 5) connected by a channel (22), the reservoir (4) being connected to the curved channel (3) through a channel (21), the channel (21, 22) comprising at least one straight channel or at least one curved channel .
  • the detection pool of the present invention can be used for the collection of particle characteristic signals or the capture of individual particles; the reservoir of the present invention is used for storing the sample liquid to be tested.
  • the width of the channel (22) is smaller than the width of the channel (21);
  • the width of the channel (23) is smaller than the width of the channel (22);
  • the channel (21) has a width of 200 to 300 ⁇ m; preferably 200 ⁇ m;
  • the channel (22) has a width of 45 to 230 ⁇ m; preferably 100 ⁇ m;
  • the channel (23) has a width of 30 to 100 ⁇ m; preferably 45 ⁇ m;
  • the sample channel has a height of 15 to 50 ⁇ m.
  • Another aspect of the present invention provides a method for preparing a microfluidic chip provided by the first aspect of the present invention, comprising: 1 a substrate layer is etched according to a sample channel design, and 2 a cover layer is implanted with a sample hole and a reservoir The oil pool hole, the 3 cover sheet layer is aligned with the substrate layer and is low temperature bonded, and the 4 surface is hydrophobized.
  • the sample channel has a height of 15 to 50 ⁇ m.
  • a microfluidic chip device for forming a single particle-coated droplet, the device comprising the microfluidic chip and the liquid sample introduction device provided by the first aspect of the invention, the liquid The sample introduction device is in communication with the injection port (1), and the liquid sample injection device is selected from the group consisting of, but not limited to, a gravity-driven adjustment sample introduction device, a syringe, a peristaltic pump, and a syringe pump.
  • the gravity-driven adjustment sample introduction device comprises a height-adjustable sample holder, a sample container, and a conduit through which the sample container communicates with the injection hole (1), the sample container can be Moving up and down on the height adjustable sample holder.
  • the manner in which the sample container moves up and down is manually adjusted.
  • the manner in which the sample container moves up and down is an electric adjustment.
  • the height-adjustable sample holder is a slide rail design having an electrically movable slider for fixing the sample container; more preferably, the height-adjustable sample holder further includes a height An adjustment controller that controls the slider to move up and down on the slide rail.
  • a microfluidic operating system for forming a single particle-coated droplet comprising a microfluidic chip or microfluidic chip device provided by the present invention, and a particle capture device,
  • the particle capture device is selected from the group consisting of a diaphragm and a magnetic diaphragm.
  • the aperture and magnetostrictive devices employed in the present invention are prior art in the art.
  • the capturing according to the present invention means that the target particles are fixed by a particle trapping device including a diaphragm and a magnetic yoke, so that when the microfluidic chip of the present invention is moved, the target particles do not move with the chip.
  • the microfluidic operating system further includes a sample detecting device including, but not limited to, a Raman detecting device, an optical microscope, and a fluorescence microscope.
  • the microfluidic operating system further includes means for deriving a single particle-wrapped droplet, the means for deriving a single microparticle-coated droplet selected from the group consisting of a capillary tube and a pipette tip.
  • a method of forming a single particle-wrapped droplet and deriving which utilizes the microfluidic chip or microfluidic chip device or microfluidic operating system provided by the present invention, and includes the steps of: 1 inject the oil phase into the oil reservoir; 2 inject the particulate phase solution into the microfluidic chip sample channel through the inlet, 3 adjust the liquid flow in the sample channel, and make the interface between the particulate phase and the reservoir oil phase at the sample channel outlet Stabilization of the vicinity; 4 capture the target particles with a particle capture device, move the microfluidic chip, drag the target particles to the vicinity of the water phase and the oil phase interface; 5 adjust the liquid flow in the sample channel, and make the target particles enter the oil reservoir and form a droplet encapsulating a single target particle; 6 deriving a droplet encapsulating a single target particle; the manner of adjusting the flow of the liquid in the sample channel is selected from, but not limited to, gravity driven adjustment, syring
  • the particle capture device captures target particles from the detection cell.
  • the oil phase is selected from the group consisting of mineral oil, silicone oil, fluorocarbon oil, vegetable oil, petroleum ether.
  • the particulate phase contains particles and a liquid that is incompatible with the oil phase liquid in the oil reservoir; preferably, the particulate phase is a non-organic phase; more preferably, the particulate The phase is an aqueous phase or a non-organic buffer.
  • the method of forming a single particle-wrapped droplet and deriving further comprises a sample detecting step, the sample detecting step being preceded by step 4, wherein the sample detecting step is performed by a method selected from, but not limited to, Raman Spectral analysis, fluorescence detection, optical microscopy, conductivity detection;
  • the method of deriving droplets encapsulating a single target particle comprises a capillary derivation method, a pipetting method;
  • the method of forming a single microparticle-encapsulated droplet and deriving further comprises performing further manipulations on the derivatized target microparticle, the operation comprising single cell sequencing, single cell morphology analysis, single cell culture.
  • the method of forming a single particle-wrapped droplet and deriving comprises the steps of: 1 injecting an oil phase into the oil reservoir, 2 injecting the particulate phase solution into the sample channel through the inlet, and adjusting the sample container at a height Adjust the height of the sample holder to h0, so that the interface between the particle phase and the oil phase of the sample port is stable and stationary near the exit of the sample channel. 3 Collect the characteristic signal of the single particle in the detection cell (5) and capture the target through the pupil.
  • the method of injecting the particulate phase solution into the sample channel through the inlet is a hydrostatic injection method.
  • the method for collecting the characteristic signals of the individual particles in the sample channel is selected from the group consisting of Raman signal acquisition, fluorescence detection, and optical microscopy; more preferably, the position of the characteristic signal of the individual particles is collected as a detection pool. .
  • the pupil has a laser wavelength of 1064 nm.
  • an application of a microfluidic chip, a microfluidic chip device, or a microfluidic manipulation system including single particle screening, formation of individual particle wrapped droplets, or derivation of individual particle wrapped droplets.
  • the chip can be reused to reduce operating costs.
  • Figure 1 is a schematic diagram of the design of the microfluidic chip.
  • Figure 2 is a physical diagram of the microfluidic chip.
  • Figure 3 is a diagram of a microfluidic chip device forming a single particle-wrapped droplet.
  • A shows a schematic diagram of a microfluidic chip device consisting of a liquid sample introduction device and a microfluidic chip.
  • B shows the placement of a microfluidic chip on a microscope platform, which together with the particle capture device form a physical map of a microfluidic operating system for forming individual microparticle-coated droplets.
  • Figure 4 shows the process of forming a single particle-wrapped droplet.
  • A is the droplet just formed by the package, and then the capillary is used to suck the droplet from the open reservoir. This process does not require external power, depending on the capillary action of the oil phase in the capillary. Place the capillary at the exit B of the channel. The oil phase will flow into the capillary by capillary action. The single cell in the oil phase will wrap the droplet and then flow into the capillary along with the oil phase.
  • C ⁇ F is a video screenshot of this process.
  • G and H are the droplets of the wrapped cells in the field of 50X objective lens. Escherichia coli cells and yeast cells were used as model cells. It was found that two cells of different sizes could be successfully encapsulated into droplets and exported.
  • Figure 6 shows the results of DNA amplification.
  • a and C are MDA amplification results
  • B and D are 16S identification results
  • N is a negative control for MDA (multiple displacement) amplification.
  • the droplets can successfully encapsulate and export the cells, and the exported cells can be successfully used for downstream amplification and sequencing analysis.
  • the microfluidic chip, the microfluidic chip device, the microfluidic operating system and the method for forming a single particle-packed droplet and derived therefrom can be used for separating single particles of biological origin and non-biological origin, for example, In eukaryotic cells (such as animal cells, plant cells, fungal cells, etc.), prokaryotic cells (such as bacterial cells, etc.), single-celled organisms, viral particles, organelles, particles formed by biomacromolecules, drug particles, drug carrier particles, lipids Separation of individual particles of plastids, polymer particles, other natural or synthetic particles.
  • eukaryotic cells such as animal cells, plant cells, fungal cells, etc.
  • prokaryotic cells such as bacterial cells, etc.
  • single-celled organisms viral particles, organelles, particles formed by biomacromolecules, drug particles, drug carrier particles, lipids Separation of individual particles of plastids, polymer particles, other natural or synthetic particles.
  • the injection hole and the reservoir hole are placed on the upper quartz glass, and the hole spacing is in accordance with the channel length.
  • the upper quartz glass is a smooth plane with a thickness of 0.5 to 1 mm and no etched pattern. (The order of the upper and lower quartz glass depends on the optical path of the diaphragm. Since the microscope used in this experiment is an upright microscope, the optical path is passed up and down through the chip, so the upper surface of the chip is smooth and optical, so the upper layer is smooth. Quartz glass, quartz glass engraved with channels in the lower layer)
  • the surface of the lower quartz glass is etched according to the channel design, and the channel height is about 15 to 50 microns.
  • the oil phase is made of mineral oil (containing 2% by weight of surfactant 80). Since the oil phase density is less than water, the generated droplets are located at the outlet of the microchannel at the bottom of the oil reservoir for easy observation and export.
  • the liquid sample introduction device selected here is a gravity-driven adjustment sample introduction device, and the sample container is suspended on the height-adjustable sample holder, and the sample container is connected to the injection hole on the microfluidic chip through the catheter.
  • a schematic diagram of a microfluidic chip device consisting of a liquid sample introduction device and a microfluidic chip is shown in Fig. 3A.
  • the microfluidic chip is placed on a microscope platform, and the microfluidic chip device and the particle capture device together form a microfluidic operating system for forming a single particle-wrapped droplet.
  • the physical map is shown in FIG. 3B.
  • the microparticle phase (the cell phase in this embodiment) solution is injected into the chip channel through the injection port, and the height of the sample holder is adjusted to h0, so that the interface between the cell phase and the oil phase of the sample port is sampled. Stable and stationary near the mouth.
  • the height of the sample holder depends on the internal fluid resistance of the quartz chip.
  • the 1-meter-high sample holder is selected according to the size of the quartz chip channel used and the height of the microscope platform.
  • the sample holder is designed as a slide rail, and the slide rail has an electrically movable slider for fixing the sample container, as shown in FIG. 3B.
  • DNA amplification and electrophoresis detection were performed on the isolated single cells.
  • Example 2 The droplets separated in Example 2 were observed under a 50X objective lens, and it was found that single cells were successfully encapsulated in the droplets.
  • This method is suitable for cells of different sizes, from about one micron to several tens of micrometers, as shown in Fig. 5G.
  • Figure 5H shows the encapsulation of E. coli cells and yeast cells in droplets using this method, respectively. Since the density of the mineral oil used in this method is smaller than that of the cell suspension (water), the droplets are automatically distributed at the bottom of the capillary during the droplet transfer process, and only the capillary containing the droplets is touched on the test tube or the centrifuge tube wall. Droplets can be exported, which makes the transfer process more successful and simpler.
  • the droplets of the encapsulated single cells in the capillary were centrifuged into a separate centrifuge tube, and the amplified substrate was added for DNA amplification, and the amplification was as shown in FIG.
  • Escherichia coli Fig. 6A and Fig. 6B
  • yeast Fig. 6C and Fig. 6D
  • E1 and E2 were E.

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Abstract

一种可用于单个微粒筛选并形成液滴包裹导出的微流控芯片，该微流控芯片与液体样品进样装置相连，可组成一种用于形成单个微粒包裹液滴的微流控芯片装置，该微流控芯片装置可进一步与微粒捕获装置组成可用于形成单个微粒包裹液滴的微流控操作系统。还提供了一种在微流控芯片中形成目标单个微粒包裹液滴并分别导出的方法。

Description

单个微粒包裹液滴在微流控芯片中形成并分别导出的方法 技术领域
本发明涉及微流控技术领域，具体涉及一种利用微流控芯片形成单个微粒包裹液滴并导出的方法，可用于单细胞筛选、单细胞分离、单细胞测序、单细胞形态分析、单细胞培养、药物筛选等领域。
背景技术
众所周知，细胞多样性普遍存在于各细胞种群中，例如细菌、酵母、哺乳动物细胞等。即使是由同一母细胞分裂而来的子细胞，细胞个体间也会存在差异。单细胞分析(single-cell analysis)是指在细胞种群中，针对细胞个体间差异包括细胞尺寸、生长速度、化学组成(磷脂、蛋白、代谢物、DNA/RNA)，等方面的研究，以及细胞间差异产生的原因、机理。其研究内容涉及到肿瘤生物学、干细胞、微生物学、神经系统学和免疫学等领域。
单细胞分析中最大的挑战体现在细胞尺寸微小，以及小尺寸带来的化学组成复杂、组分痕量等问题。单个细胞的尺寸大都分布于微米尺度，在该尺度下对单个细胞的操纵、分析对仪器精度、灵敏度等方面提出了极高要求。因此要实现单细胞分析，首先要解决两个问题：单细胞捕获、分离提取；单细胞信号获取。
借助于荧光显微、单细胞拉曼、电化学技术、质谱以及近两年兴起的qPCR技术的发展，已实现对单细胞组分的多重分析。但是在单细胞捕获、分离提取方面进展缓慢，通过微流控芯片技术，目前大致可以分为单细胞阵列捕获、流式两种方法。阵列捕获的方法可实现单细胞的实时监控，并且方便改变实验条件，研究不同培养条件对单细胞的影响，但是通量较低。流式的方法通常基于单细胞液滴包裹技术，具有检测通量高，方便细胞分离的优势，但是由于检测时间短，一般难以获得单个细胞更详细的信息，目前大都还是以荧光流式筛选为主。然而如何将微流控芯片内分析筛选的细胞单个分离取出，接着进行下游分析，仍然是当前的难点。如单细胞基因谱测序，需要将当个目标细胞从捕获阵列或芯片中转移到试管中，并且避免转移过程中对细胞的损伤以及外界环境的污染。
发明内容
本发明的目的是提供一种将目标单个微粒分离并导出的技术，本发明实现了单个目标微粒在微流控芯片内的检测和捕获，以及从微流控芯片到外试管的转移。
本发明所称的“微粒”，是指能够悬浮在非有机相溶液(例如水相)、并在本发明微流控芯片内通过的颗粒，包括生物体来源的和非生物体来源的颗粒，例如真核细胞、原核细胞、单细胞生物、病毒颗粒、细胞器、生物大分子形成的颗粒、药物颗粒、药物载体颗粒、脂质体、多聚物粒子等。
本发明的第一方面，提供了一种微流控芯片，所述微流控芯片包括盖片层和基片层；所述基片层包括至少一条样品通道，所述样品通道包括微流体通道、检测池(5)；所述盖片层包括进样孔(1)、储油池孔(6)；所述储油池孔(6)与所述基片层形成储油池；所述样品通道的入口端(7)与进样孔(1)相连，所述样品通道的出口端(8)与储油池相连。
在另一优选例中，所述盖片层的材质选自但不限于石英、PDMS(聚二甲基硅氧烷)、PMMA(聚甲基丙烯酸甲酯)、硼硅玻璃、氟化钙，所述基片层的材质选自但不限于石英、PDMS、PMMA、硼硅玻璃、氟化钙。
在另一优选例中，所述储油池的表面为疏水亲油表面。
在另一优选例中，所述进样孔(1)连接有进样导管接口。
在另一优选例中，所述微流体通道包括直通道(2)和至少一段曲通道(3)。
本发明所述的曲通道，是指不是直线型的微流体通道。在一优选例中，所述微流体通道的宽度为30～300μm，高度为15～50μm。
在另一优选例中，所述检测池(5)与所述储油池孔(6)通过通道(23)相连，所述通道(23)为直通道。
在另一优选例中，所述样品通道还包括至少一个储液池(4)，较佳的，在另一优选例中，所述检测池(5)的容量小于所述储液池(4)的容量。
在另一优选例中，所述检测池(5)位于所述储液池(4)和所述储油池孔(6)之间，所述储液池(4)与所述检测池(5)通过通道(22)相连，所述储液池(4)与所述曲通道(3)通过通道(21)相连，所述通道(21，22)包括至少一段直通道或至少一段曲通道。
本发明所述的检测池，可用于微粒特征信号的采集或单个微粒的捕获；本发明所述的储液池，用于储存待测样品液体。
在另一优选例中，所述通道(22)的宽度小于所述通道(21)的宽度；
在另一优选例中，所述通道(23)的宽度小于所述通道(22)的宽度；
在另一优选例中，所述通道(21)的宽度为200～300μm；优选200μm；
在另一优选例中，所述通道(22)的宽度为45～230μm；优选100μm；
在另一优选例中，所述通道(23)的宽度为30～100μm；优选45μm；
在另一优选例中，所述样品通道高度为15～50μm。
本发明的另一方面，提供了一种制备本发明第一方面提供的微流控芯片的方法，包括：①基片层依据样品通道设计做刻蚀，②盖片层打上进样孔与储油池孔，③盖片层与基片层对齐并低温键合，④表面疏水化处理。在一优选例中，所述样品通道高度为15～50μm。
本发明的另一方面，提供了一种用于形成单个微粒包裹液滴的微流控芯片装置，该装置包括本发明第一方面提供的微流控芯片和液体样品进样装置，所述液体样品进样装置与所述进样孔(1)连通，所述液体样品进样装置选自但不限于：重力驱动调节进样装置、注射器、蠕动泵、注射泵。
在一优选例中，所述重力驱动调节进样装置包含高度可调样品架、样品容器、导管，所述样品容器通过所述导管与所述进样孔(1)连通，所述样品容器可在所述高度可调样品架上上下移动。
在另一优选例中，所述样品容器上下移动的方式为手动调节。
在另一优选例中，所述样品容器上下移动的方式为电动调节。
在另一优选例中，所述高度可调样品架为滑轨设计，滑轨上具有可电动移动的滑块，用于固定样品容器；更佳的，所述高度可调样品架还包括高度调节控制器，所述高度调节控制器控制所述滑块在所述滑轨上进行上下移动。
本发明的另一方面，提供了一种用于形成单个微粒包裹液滴的微流控操作系统，包含本发明所提供的微流控芯片或微流控芯片装置，以及微粒捕获装置，所述微粒捕获装置选自光镊、磁镊。
本发明采用的光镊和磁镊装置，为本领域的现有技术。
本发明所述的捕获，是指利用包括光镊和磁镊在内的微粒捕获装置固定住目标微粒，从而实现在移动本发明微流控芯片时、目标微粒不随芯片移动。
在另一优选例中，所述微流控操作系统进一步包括样品检测装置，所述样品检测装置包括但不限于拉曼检测装置、光学显微镜、荧光显微镜。
在另一优选例中，所述微流控操作系统进一步包括将单个微粒包裹液滴导出的装置，所述将单个微粒包裹液滴导出的装置选自毛细管、移液枪枪头。
本发明的另一方面，提供了一种形成单个微粒包裹液滴并导出的方法，该方法利用本发明提供的微流控芯片或微流控芯片装置或微流控操作系统，并包括步骤：①在储油池内注入油相；②将微粒相溶液通过进样口注入微流控芯片样品通道内，③调节样品通道内液体流动、使微粒相与储油池油相的界面在样品通道出口附近稳定静止；④用微粒捕获装置捕获目标微粒，移动微流控芯片、将目标微粒拖拽到水相与油相界面附近；⑤调节样品通道内液体流动、使目标微粒进入储油池并形成包裹有单个目标微粒的液滴；⑥将包裹有单个目标微粒的液滴导出；所述调节样品通道内液体流动的方式选自但不限于重力驱动调节法、注射泵驱动调节法、蠕动泵驱动调节法。
在一优选例中，所述微粒捕获装置从所述检测池中捕获目标微粒。
在另一优选例中，所述油相选自矿物油、硅油、氟碳油、植物油、石油醚。
在另一优选例中，所述微粒相含有微粒和与储油池中的油相液体互不相溶的液体；较佳的，所述微粒相为非有机相；更佳的，所述微粒相为水相或非有机缓冲液。
在另一优选例中，所述形成单个微粒包裹液滴并导出的方法还包括样品检测步骤，所述样品检测步骤位于步骤④之前，所述样品检测步骤采用的方法选自但不限于拉曼光谱分析、荧光检测、光学显微镜检测、电导检测；
在另一优选例中，所述将包裹有单个目标微粒的液滴导出的方法包括毛细管导出法、移液枪导出法；
在另一优选例中，所述形成单个微粒包裹液滴并导出的方法还包括对导出的目标微粒进行进一步操作，所述操作包含单细胞测序、单细胞形态分析、单细胞培养。
在另一优选例中，所述形成单个微粒包裹液滴并导出的方法包括步骤：①在储油池内注入油相，②将微粒相溶液通过进样口注入样品通道内，调节样品容器在高度可调样品架上的高度至h0，使微粒相与出样口油相的界面在 样品通道出口附近稳定静止，③在检测池(5)内采集单个微粒的特征信号，并通过光镊捕获目标微粒，移动微流控芯片、将想要的目标微粒拖拽到水相与油相界面附近，④调节样品容器在高度可调样品架上的高度至h，将微粒与一部分水依靠重力作用推进油相，形成油包水液滴，之后将样品容器高度重新调回h0，使微粒相与油相之间的界面在样品通道出口附近再度达到稳定，⑤用毛细管接触微粒包裹液滴，由于油相对毛细管良好的浸润作用，依据毛细力，液滴随部分油自动进入毛细管内。
在另一优选例中，所述将微粒相溶液通过进样口注入样品通道内的方法为静压注射法。
在另一优选例中，所述在样品通道内采集单个微粒的特征信号的方法选自拉曼信号采集、荧光检测、光学显微镜检测；更佳的，采集单个微粒的特征信号的位置为检测池。
在另一优选例中，所述光镊的激光波长为1064nm。
本发明的另一方面，提供了微流控芯片、微流控芯片装置或微流控操控系统的应用，包括单个微粒筛选、单个微粒包裹液滴的形成或单个微粒包裹液滴的导出。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
本发明具有以下技术优势：
1.适用于各尺寸微粒，如数十微米的酵母细胞和1微米左右细菌细胞的拉曼检测并导出。
2.实现了单个细胞的挑选并分离导出，该过程对细胞活性影响低，能成功与下游单细胞测序对接。
3.单个微粒筛选移取速度快，单个微粒从芯片内到试管中移取操作时间为1～2分钟。
4.芯片可重复利用，降低操作成本。
5.操作简便。
附图说明
图1微流控芯片设计示意图。
图2微流控芯片实物图。
图3形成单个微粒包裹液滴的微流控芯片装置图。A显示了由液体样品进样装置和微流控芯片组成的微流控芯片装置的示意图。B显示了将微流控芯片置于显微镜平台上，微流控芯片装置与微粒捕获装置共同组成用于形成单个微粒包裹液滴的微流控操作系统的实物图。
图4形成单个微粒包裹液滴的过程。
图5微流控芯片装置分选单个细胞的过程对细胞活性影响低。A为刚刚包裹生成的液滴，之后用毛细管将液滴从开放的储油池中吸出，这个过程无需外源动力，依据油相在毛细管中的毛细作用。将毛细管置于通道出口处B，油相会靠毛细作用流入毛细管中，油相中的单细胞包裹液滴进而随着油相一同流入毛细管，C～F为这一过程的视频截图。G、H为50X物镜视野下包裹细胞的液滴，分别以大肠杆菌细胞和酵母细胞为模式细胞，发现两种不同大小的细胞都能成功的被包裹入液滴中并导出。
图6DNA扩增结果。A和C为MDA扩增结果，B和D为16S鉴定结果，其中N为MDA(多重置换)扩增的阴性对照。实验证明液滴能成功将细胞包裹并导出，导出后的细胞能成功用于下游扩增、测序分析。
具体实施方式
本发明提供的微流控芯片、微流控芯片装置、微流控操作系统和形成单个微粒包裹液滴并导出的方法，可用于分离生物体来源的和非生物体来源的单个微粒，例如用于真核细胞(如动物细胞、植物细胞、真菌细胞等)、原核细胞(如细菌细胞等)、单细胞生物、病毒颗粒、细胞器、生物大分子形成的颗粒、药物颗粒、药物载体颗粒、脂质体、多聚物粒子、其他天然或人工合成颗粒的单个微粒的分离。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1
1、微流控芯片的制备：
①用超声打孔方法在上层石英玻璃打上进样孔与储液池孔，孔间距依照通道长度。上层石英玻璃为光滑平面，厚0.5～1毫米，无刻蚀图案。(上下层石英玻璃的顺序依据光镊光路而定，由于本实验采用的显微镜为正置显微镜，光镊光路由上而下通过芯片，所以要保证芯片上表面为光滑光学表面，故上层为光滑石英玻璃，刻有通道的石英玻璃在下层)
②下层石英玻璃表面依据通道设计做刻蚀，通道高度约15～50微米。
③两层玻璃对齐并低温键和。
④表面疏水化处理(用硅烷化试剂)，在储油池位置形成疏水亲油表面。
⑤单个微粒液滴包裹时，油相采用矿物油(含2％wt的表面活性剂司盘80)。由于该油相密度小于水，生成的液滴位于储油池底部微通道出口处，方便观察并导出。
微流控芯片的设计示意图和实物图分别如图1和图2所示。
2、用于形成单个微粒包裹液滴的微流控芯片装置和系统的制备
此处选用的液体样品进样装置为重力驱动调节进样装置，将样品容器悬挂在高度可调样品架上，样品容器通过导管与微流控芯片上的进样孔相连。由液体样品进样装置和微流控芯片组成的微流控芯片装置的示意图见图3A。将微流控芯片置于显微镜平台上，微流控芯片装置与微粒捕获装置共同组成用于形成单个微粒包裹液滴的微流控操作系统，实物图见图3B。
实施例2
目标单个微粒包裹液滴的形成和导出，步骤示意图见图4。
①在样品通道出口端的储油池内注入油相(一般采用包含表面活性剂的矿物油)。
②采用静压注射的方法，将微粒相(本实施例采用细胞相)溶液通过进样口注入芯片通道内，调节样品架高度至h0，使细胞相与出样口油相的界面在出样口附近稳定静止。
③在检测池内采集单个细胞的特征图谱(如拉曼光谱，532nm激光)，并通过光镊(例如1064nm激光)捕获想要的目标细胞，移动微流控芯片、将目标细胞拖拽到水相与油相界面附近。
④调节样品架高度至h，将细胞与一部分水依靠重力作用推进油相，形成 油包水液滴，之后将样品架高度重新调回h0，使细胞液与油相之间的界面在通道开口处再度达到稳定。
⑤用毛细管接触细胞包裹液滴。由于油相对毛细管良好的浸润作用，依据毛细力，液滴随部分油自动进入毛细管内。图5A-F显示单细胞包裹液滴可被毛细管成功导出。
样品架高度依据石英芯片内部流体阻力而定，本实验依据所用石英芯片通道尺寸以及显微镜平台高度，选用1米高样品架。样品架为滑轨设计，滑轨上具有可电动移动的滑块，用于固定样品容器，如图3B所示
实施例3
对分离的单细胞进行DNA扩增和电泳检测。
实施例2分离得到的液滴在50X物镜下观察，能看到单细胞被成功包裹在液滴当中，此方法适用于不同尺寸的细胞，从一微米左右到数十微米左右，如图5G和图5H分别为大肠杆菌细胞和酵母细胞应用此方法在液滴中的包裹情况。由于此方法选用的矿物油密度小于细胞悬液(水)，所以液滴转移过程中，液滴会自动分布于毛细管的底部，仅需将装有液滴的毛细管触碰试管或离心管壁即可导出液滴，这使得转移过程成功率更高、方法更简单。
将毛细管中的包裹单细胞的液滴离心导入单独离心管中，加入扩增底物进行DNA扩增，扩增情况如图6所示。此次试验分别以大肠杆菌(图6A和图6B)和酵母(图6C和图6D)为模式细胞，另外移取了部分空液滴(E1和E2为大肠杆菌样本对照空液滴，E3E4E5为酵母对照空液滴)进行对照，发现空液滴中没有扩增产物出现，单细胞包裹液滴成功扩增，证明此方法分离的细胞能成功用于单细胞测序等下游分析，另外在单细胞筛选过程中能够成功避免污染发生。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种微流控芯片，其特征在于，所述微流控芯片包括盖片层和基片层；所述基片层包括至少一条样品通道，所述样品通道包括微流体通道、检测池(5)；所述盖片层包括进样孔(1)、储油池孔(6)；所述储油池孔(6)与所述基片层形成储油池；所述样品通道的入口端(7)与进样孔(1)相连，所述样品通道的出口端(8)与储油池相连。
  2. 如权利要求1所述的微流控芯片，其特征在于，所述微流体通道包括直通道(2)和至少一段曲通道(3)。
  3. 如权利要求1或2所述的微流控芯片，所述检测池(5)与所述储油池孔(6)通过通道(23)相连，所述通道(23)为直通道。
  4. 如权利要求1-3中任一所述的微流控芯片，其特征在于，所述样品通道还包括至少一个储液池(4)，所述检测池(5)位于所述储液池(4)和所述储油池孔(6)之间，所述储液池(4)与所述检测池(5)通过通道(22)相连，所述储液池(4)与所述曲通道(3)通过通道(21)相连，所述通道(21，22)包括至少一段直通道或至少一段曲通道。
  5. 如权利要求1-4中任一所述的微流控芯片的制备方法，包括步骤：①基片层依据样品通道设计做刻蚀，②盖片层打上进样孔与储油池孔，③盖片层与基片层对齐并低温键合，④表面疏水化处理。
  6. 一种用于形成单个微粒包裹液滴的微流控芯片装置，其特征在于，所述微流控芯片装置包括如权利要求1-4中任一所述的微流控芯片和液体样品进样装置，所述液体样品进样装置与所述进样孔(1)连通，所述液体样品进样装置选自下组：重力驱动调节进样装置、注射器、蠕动泵、注射泵、或其组合。
  7. 如权利要求6所述的微流控芯片装置，其特征在于，所述重力驱动调节进样装置包含高度可调样品架、样品容器、导管，所述样品容器通过所述导管与所述进样孔(1)连通，所述样品容器可在所述高度可调样品架上上下移动。
  8. 一种用于形成单个微粒包裹液滴的微流控操作系统，包含如权利要求1-4中任一所述的微流控芯片或权利要求6或7所述的微流控芯片装置，以及微粒捕获装置，所述微粒捕获装置选自光镊、磁镊。
  9. 一种形成单个微粒包裹液滴并导出的方法，其特征在于，利用如权利要求1-4中任一所述的微流控芯片、权利要求6或7所述的微流控芯片装置或权利要求 8所述的微流控操作系统，并包括步骤：①在储油池内注入油相；②将微粒相溶液通过进样口注入微流控芯片样品通道内；③调节样品通道内液体流动、使微粒相与储油池油相的界面在样品通道出口附近稳定静止；④用微粒捕获装置捕获目标微粒，移动微流控芯片、将目标微粒拖拽到水相与油相界面附近；⑤调节样品通道内液体流动、使目标微粒进入储油池并形成包裹有单个目标微粒的液滴；⑥将包裹有单个目标微粒的液滴导出；所述调节样品通道内液体流动的方式选自下组：重力驱动调节法、注射泵驱动调节法、蠕动泵驱动调节法、或其组合；所述将包裹有单个目标微粒的液滴导出的方法选自下组：毛细管导出法、移液枪导出法、或其组合。
  10. 如权利要求1-4中任一所述的微流控芯片、权利要求6或7所述的微流控芯片装置或权利要求8所述的微流控操作系统的应用，其特征在于，用于单个微粒筛选、单个微粒包裹液滴的形成或单个微粒包裹液滴的导出。
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