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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English (en)
French (fr)
Chinese (zh)
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徐腾
马波
徐健
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Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
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Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
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Priority to EP18880018.9A priority Critical patent/EP3711855B1/en
Priority to US16/764,803 priority patent/US12403472B2/en
Priority to JP2020545423A priority patent/JP7071519B2/ja
Publication of WO2019096070A1 publication Critical patent/WO2019096070A1/zh
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    • 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 or 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
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • 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
    • 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
    • B01L2200/0647—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
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • 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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    • B01L2300/0883—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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    • B01L2400/04—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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PCT/CN2018/114807 2017-11-15 2018-11-09 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 Ceased WO2019096070A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP18880018.9A EP3711855B1 (en) 2017-11-15 2018-11-09 Method for forming and respectively exporting droplet wrapping single particle in micro-fluidic chip
US16/764,803 US12403472B2 (en) 2017-11-15 2018-11-09 Method for forming and respectively exporting droplet wrapping single particle in micro-fluidic chip
JP2020545423A JP7071519B2 (ja) 2017-11-15 2018-11-09 単一粒子を包んだ液滴のマイクロ流体チップでの形成およびそれぞれ導出される方法

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