WO2022226791A1 - 芯片、微流控装置以及分选目标液滴的方法 - Google Patents

芯片、微流控装置以及分选目标液滴的方法 Download PDF

Info

Publication number
WO2022226791A1
WO2022226791A1 PCT/CN2021/090291 CN2021090291W WO2022226791A1 WO 2022226791 A1 WO2022226791 A1 WO 2022226791A1 CN 2021090291 W CN2021090291 W CN 2021090291W WO 2022226791 A1 WO2022226791 A1 WO 2022226791A1
Authority
WO
WIPO (PCT)
Prior art keywords
flow channel
droplet
section
chip
branch
Prior art date
Application number
PCT/CN2021/090291
Other languages
English (en)
French (fr)
Inventor
邓林
杨帆
李达
马相国
丁丁
Original Assignee
京东方科技集团股份有限公司
北京京东方技术开发有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 京东方科技集团股份有限公司, 北京京东方技术开发有限公司 filed Critical 京东方科技集团股份有限公司
Priority to PCT/CN2021/090291 priority Critical patent/WO2022226791A1/zh
Priority to CN202180000922.0A priority patent/CN115529820A/zh
Priority to CN202220244666.1U priority patent/CN218281795U/zh
Priority to CN202210112214.2A priority patent/CN115245846A/zh
Priority to GB2317811.4A priority patent/GB2621756A/en
Priority to US18/262,223 priority patent/US20230405587A1/en
Priority to PCT/CN2022/078956 priority patent/WO2022227853A1/zh
Priority to US17/731,746 priority patent/US20220339630A1/en
Publication of WO2022226791A1 publication Critical patent/WO2022226791A1/zh

Links

Images

Classifications

    • 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
    • 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
    • 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
    • B01L3/50273—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 means or forces applied to move the fluids
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • 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
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • 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
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single 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
    • B01L2400/02—Drop detachment mechanisms of single droplets from nozzles or pins
    • B01L2400/021—Drop detachment mechanisms of single droplets from nozzles or pins non contact spotting by inertia, i.e. abrupt deceleration of the nozzle or pin
    • 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
    • B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces
    • 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
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure

Definitions

  • the present disclosure relates to the field of biomedical detection, and in particular, to a chip, a microfluidic device including the chip, and a method for sorting target droplets.
  • Cells are the basic structural and functional units of living organisms. Since there is usually a high degree of heterogeneity among individual cells, the mean of the data obtained by analyzing the cell population essentially masks the differences between individual cells, and thus cannot characterize the stochastic nature of gene expression and cannot reflect the real situation. With the continuous development of life science and precision medicine, cell population analysis is gradually developing towards single cell analysis.
  • a key technique for single-cell analysis is how individual cells can be isolated from highly heterogeneous biological samples containing numerous cells.
  • Single-cell sorting technology provides new options for popular medical fields such as single-cell analysis, early cancer diagnosis, and companion diagnostics.
  • a chip comprising: a first accommodating part configured to accommodate a first fluid; a second accommodating part configured to accommodate a second fluid, the second fluid comprising a cell suspension; delivering a flow channel, including a first flow channel and a second flow channel, the first flow channel communicates with the first accommodating part and the second flow channel communicates with the second accommodating part, the first flow channel communicates with the first accommodating part A second flow channel intersects and communicates with each other at a junction, the delivery channel is configured to merge the first fluid and the second fluid at the junction and generate at least one droplet, the at least one droplet Each of at least a portion of the droplets includes a single cell derived from the cell suspension; and at least one collection portion configured to collect the at least one droplet.
  • a portion of the first flow channel includes the junction and is divided by the junction into a first section and a second section, in each of the first section and the second section , the area of the first cross-section of the segment gradually increases along a first direction away from the meeting point, the first cross-section being perpendicular to the first direction.
  • the second flow channel includes the junction and is divided by the junction into a third section and a fourth section, in each of the third section and the fourth section , the area of the second cross-section of the segment gradually increases along a second direction away from the junction, the second cross-section being perpendicular to the second direction.
  • a portion of the first flow channel includes a first subsection, a second subsection including the junction, and a third subsection, the first subsection belonging to the first section,
  • the third subsection belongs to the second section, the second subsection spans the first section and the second section and is located between the first subsection and the third subsection Meanwhile, the area of the first cross-section of the first sub-section and the third sub-section is larger than the area of the first cross-section of the second sub-section.
  • the first cross-section of the second sub-portion of the first flow channel at the junction is sized to allow a first fluid having a specific particle size to flow therein, the first The specific particle size of a fluid is larger than the particle size of the individual cells.
  • the second flow channel includes a first portion, a second portion, and a third portion, the first portion and the second portion belong to the third section, and the third portion belongs to the fourth section section.
  • the first end of the first part of the second flow channel communicates with the second accommodating part, and the second end of the first part of the second flow channel and the first end of the second part of the second flow channel.
  • the second end of the second part of the second flow channel is in communication with the first end of the third part of the second flow channel, and the second end of the second part of the second flow channel is connected to the first end of the third part of the second flow channel.
  • the first ends of the third part of the second flow channel are all located at the confluence point; the second end of the third part of the second flow channel is communicated with the at least one collecting part.
  • the areas of the second cross-section of the first and third portions of the second flow channel are both larger than the area of the second cross-section of the second portion of the second flow channel.
  • the second cross-section of the second portion of the second flow channel is sized to allow a second fluid having a particular particle size to flow therein, the second fluid having a particular particle size It is larger than 1 times the particle size of the single cell and smaller than 2 times the particle size of the single cell.
  • the area of the second cross-section of the third portion of the second flow channel gradually increases in a direction from the first end to the second end of the third portion of the second flow channel .
  • the area of the first cross-section of the second subsection of the first flow channel at the junction is greater than or equal to the area where the second and third sections of the second flow channel are located. the area of the second cross-section at the junction.
  • the second receiving portion includes at least one sub-receiving portion.
  • the second fluid includes a first reagent and a second reagent, the first reagent including the cell suspension.
  • the second accommodating portion includes a first sub-accommodating portion and a second sub-accommodating portion that are separated from each other, the first sub-accommodating portion is configured to accommodate the first reagent, and the second sub-accommodating portion is configured to accommodate the second reagent.
  • the first portion of the second flow channel includes a first branch and a second branch
  • the first branch communicates with the first sub-accommodating portion
  • the second branch communicates with the second sub-accommodation
  • the receiving portion communicates, and the first branch and the second branch intersect and communicate with each other at a first point.
  • the included angle between the first branch and the second branch at the first point is an acute angle.
  • the at least one collection portion includes a first collection portion configured to collect the at least one droplet via the delivery flow channel.
  • the at least one collecting portion includes a second collecting portion that includes at least two sub-collecting portions configured to collect the at least two sub-collecting portions via the delivery flow channel a droplet.
  • the at least one collection portion includes a first collection portion and a second collection portion
  • the second collection portion includes at least two sub-collection portions.
  • the first collection portion communicates with the second collection portion, and the first collection portion is located between the junction and the second collection portion.
  • the chip further includes an electrode structure located between the junction and the second collection portion.
  • the delivery flow channel further includes a sorting flow channel located between the confluence point and the second collection portion.
  • the sorting flow channel includes at least two branches, one of the at least two branches is configured to sort non-target droplets from the at least one droplet, and the remaining branches of the at least two branches is configured to sort the target droplet from the at least one droplet.
  • At least two sub-collection parts of the second collection part correspond one-to-one with at least two branches of the sorting flow channel, and one sub-collection part of the at least two sub-collection parts is associated with at least two branches of the sorting flow channel.
  • One of the two branches communicates and is configured to collect the non-target droplets, and the remaining sub-collecting parts of the at least two sub-collecting parts are respectively connected to the remaining branches of the at least two branches of the sorting flow channel and is configured to collect the target droplets.
  • the at least two branches of the sorting channel include first and second branches configured to sort the target droplet from the at least one droplet and A third branch of the non-target droplet is sorted out of at least one droplet.
  • the first branch, the second branch and the third branch intersect at a second point and the third branch is located between the first branch and the second branch, the first branch and the The first included angle of the third branch at the second point and the second included angle between the second branch and the third branch at the second point are both greater than 10°.
  • the space between the first branch and the third branch of the sorting flow channel defines a first right-angled triangle
  • the space between the second branch and the third branch of the sorting flow channel defines a first right triangle.
  • Two right-angled triangles the first included angle faces the first right-angled side of the first right-angled triangle
  • the second included angle faces the second right-angled side of the second right-angled triangle.
  • the lengths of the first right-angled side of the first right-angled triangle and the second right-angled side of the second right-angled triangle are both greater than or equal to the particle size of a single droplet.
  • the inner wall surface of the delivery channel is hydrophobic.
  • the contours of the first and second receptacles include four chamfers.
  • the shape of the chamfer includes an arc shape.
  • both the first accommodating part and the second accommodating part are provided with a filter structure
  • the filter structure includes a plurality of microstructures, and the space between adjacent two of the plurality of microstructures is The gap is larger than 1 times the particle size of the single cell and smaller than 2 times the particle size of the single cell.
  • the chip is a microfluidic chip.
  • microfluidic device comprising the chip described in any of the preceding embodiments.
  • a method for sorting target droplets comprising the steps of: providing the first accommodating portion and the second accommodating portion of the chip described in any of the preceding embodiments with a first accommodating portion, respectively. a fluid and a second fluid comprising a cell suspension, the first fluid and the second fluid are brought together at the junction of the delivery channel and generate at least one droplet, at least one droplet of the at least one droplet each of a portion comprises a single cell derived from the cell suspension; and applying a voltage to the chip described in any of the preceding embodiments to sort out a target liquid having a target property from the at least one droplet droplets, the target droplets comprising the single cells.
  • the chip further includes an electrode structure located between the junction and the at least one collection portion.
  • the step of applying a voltage to the chip described in any of the preceding embodiments to sort a target droplet having a target property from the at least one droplet comprises detecting, in real time, an optical signal of the at least one droplet with an optical device , in response to the optical device detecting a droplet with a target optical signal, applying an instantaneous voltage of 800 to 1000V to the electrode structure to sort out a target with the target optical signal from the at least one droplet droplets, the target droplets comprising the single cells.
  • the method further comprises: transferring at least one droplet in the chip to another reaction vessel for polymerase reaction Chain reaction or fluorescent staining treatment.
  • the first fluid is an oil phase
  • the second fluid is an aqueous phase
  • the droplets have a water-in-oil structure.
  • FIG. 1A schematically shows a front view of a chip according to an embodiment of the present disclosure
  • FIG. 1B schematically shows a side view of a chip according to an embodiment of the present disclosure
  • FIG. 1C schematically shows a rear view of a chip according to an embodiment of the present disclosure
  • FIG. 1D schematically shows an isometric view of a chip according to an embodiment of the present disclosure
  • FIG. 2 schematically shows a partially enlarged schematic view of the chip of FIG. 1A;
  • FIG. 3 schematically shows another partially enlarged schematic view of the chip of FIG. 1A;
  • FIG. 4 schematically shows another partially enlarged schematic diagram of the chip of FIG. 1A;
  • FIG. 5A schematically shows a front view of a chip according to another embodiment of the present disclosure.
  • 5B schematically shows a side view of a chip according to another embodiment of the present disclosure.
  • FIG. 5C schematically shows a rear view of a chip according to another embodiment of the present disclosure.
  • FIG. 5D schematically shows an isometric view of a chip according to another embodiment of the present disclosure.
  • FIG. 6 schematically shows a partially enlarged schematic view of the chip of FIG. 5A
  • FIG. 7 schematically shows a front view of a chip according to yet another embodiment of the present disclosure.
  • FIG. 8 schematically shows a block diagram of a microfluidic device according to yet another embodiment of the present disclosure.
  • FIG. 9 schematically shows a flowchart of a method for sorting target droplets according to yet another embodiment of the present disclosure.
  • fluid refers to all substances capable of flowing, and is a general term for liquids and gases.
  • a fluid is a substance that can continuously deform under the action of small shear forces. Fluids can be composed of a single substance or a mixture of many different substances.
  • the fluid can be a continuous phase (eg, an oil phase), a dispersed phase (eg, an aqueous phase), or a mixture of continuous and dispersed phases. Fluids have the characteristics of easy flowability, compressibility and viscosity.
  • oil phase means that, according to the principle of similar compatibility, substances that are not easily soluble in water belong to the oil phase. For example, if a substance is miscible with water, if the mixed liquid exhibits layering or turbidity, the substance is in the oil phase.
  • the oil may have a density higher or lower than water and/or a viscosity higher or lower than water.
  • liquid paraffin, silicone oil, petrolatum, mineral oil, perfluorinated oil, etc. belong to the oil phase.
  • aqueous phase means that substances that are readily soluble in water belong to the aqueous phase according to the principle of similar compatibility.
  • a substance that is miscible with water is in the water phase if the mixed liquid presents a clear and homogeneous solution.
  • water, glycerol, alcohol, acetone, etc. belong to the water phase.
  • cell suspension refers to a cell solution obtained by mechanically or chemically separating cells from tissue and diluting and mixing with a cell culture medium. Numerous numbers of cells can be included in the cell suspension, eg, hundreds, thousands, tens of thousands, millions, tens of millions, or more cells.
  • the cells in the cell suspension can be of any type including but not limited to prokaryotic cells, eukaryotic cells, bacteria, fungi, plants, mammalian or other animal cell types, mycoplasma, normal tissue cells, tumor cells or any other cells type, whether derived from unicellular or multicellular organisms.
  • Cells in a cell suspension can include DNA, RNA, organelles, proteins, or any combination thereof.
  • the term "A and B in communication” means that the A element and the B element are interconnected and in communication that allows fluid to flow between the A element and the B element, ie, fluid can flow from the A element as required by the product design to the B element, or from the B element to the A element.
  • the A element can be in direct communication with the B element, ie fluid can flow directly from the A element to the B element or from the B element to the A element without passing through other intermediate elements (eg pipes).
  • the A element and the B element may be in indirect communication, ie fluid may flow from the A element via one or more intermediate elements (eg, conduits) to the B element or from the B element via one or more intermediate elements (eg, conduits) to the B element A component.
  • PCR Polymerase Chain Reaction
  • DNA deoxyribonucleic acid
  • PCR Polymerase Chain Reaction
  • DNA deoxyribonucleic acid
  • the basic principle of PCR is that DNA can be denatured and melted into single-stranded DNA at high temperature (for example, about 95°C). When the temperature drops to low temperature (for example, about 60°C), the primer and the single-strand are combined according to the principle of base complementary pairing. back to double-stranded.
  • PCR reactions include but are not limited to digital PCR (digital PCR, dPCR), quantitative PCR, real-time PCR.
  • dPCR technology can provide quantitative analysis technology for digitizing DNA quantification information, which combined with microfluidic technology can provide higher sensitivity and precision.
  • microfluidic chip refers to a chip with micrometer-scale microchannels, which can integrate basic operation units such as sample preparation, reaction, separation, detection, etc. involved in the fields of biology, chemistry, and medicine. On the micro-scale chip, the whole process of reaction and analysis is automatically completed. Analysis and detection devices based on microfluidic chips can have the following advantages: controllable liquid flow, less sample consumption, fast detection speed, simple operation, multi-functional integration, small size, and easy portability.
  • the term "particle size of XX” refers to the size of substance XX, ie, the length of substance XX in a certain direction.
  • Substance XX can be a single cell or a single droplet.
  • the term “particle size of a single cell” refers to the diameter of a single cell
  • “particle size of a single droplet” refers to the diameter of a single droplet.
  • particle size of a single cell refers to the length of a single cell in the direction of the shorter side
  • particle size of a single droplet refers to the length of a single droplet in the direction of the shorter side The length in the direction of the edge.
  • the inventors of the present application found that in conventional technology, methods for sorting single cells are mainly divided into two categories: one is to use a fluorescence flow cytometry (Fluorescence Activated Cell Sorting, FACS) to automatically sort single cells,
  • FACS Fluorescence Activated Cell Sorting
  • the fluorescence flow cytometer is expensive and expensive to maintain; the other is to manually sort single cells by professional operators, but this manual sorting method not only depends on the skill and proficiency of the operator, but also requires Micropipette platform, optical tweezers and other large and medium-sized instruments.
  • the single-cell sorting process is highly susceptible to contamination by aerosols and microorganisms floating in the environment, which are usually difficult to remove in subsequent detection links. Therefore, the existing single-cell sorting methods have disadvantages such as high cost, high skill requirements for operators, site restrictions on the required instruments and equipment, and easy environmental pollution.
  • embodiments of the present disclosure provide a chip, a microfluidic device including the chip, and a method for sorting target droplets.
  • the chip can be used to prepare droplets containing single cells derived from cell suspensions, or can be used to sort droplets of interest from prepared droplets, or can be used to prepare single droplets containing single cells derived from cell suspensions droplets of cells and sorting of target droplets from the prepared droplets.
  • the chip may be a microfluidic chip.
  • the chip can realize the preparation and/or sorting of single cells, which can effectively improve the automation operation while reducing the use cost, and can eliminate cross-contamination and improve the cell survival rate.
  • FIGS. 1A-1D illustrate a chip 100 (hereinafter referred to as the first chip 100 ), which may be used to prepare droplets including single cells, according to some embodiments of the present disclosure.
  • 1A is a front view of the first chip 100
  • FIG. 1B is a side view of the first chip 100
  • FIG. 1C is a rear view of the first chip 100
  • FIG. 1D is a front triaxial view of the first chip 100 .
  • the first chip 100 includes: a first accommodating part 101 configured to accommodate a first fluid 107 ; a second accommodating part 102 configured to accommodate a second fluid 108 , the second fluid 108 includes the cell suspension;
  • the delivery channel 103 (hereinafter referred to as the first delivery channel 103) includes a first channel 1031 and a second channel 1032, the first channel 1031 communicates with the first accommodating part 101 and the second channel 1032 communicates with the second accommodating portion 102 , the first flow channel 1031 and the second flow channel 1032 intersect and communicate with each other at the confluence point 105 , and the delivery flow channel 103 is configured so that the first fluid 107 and the second fluid 108 are at the confluence point 105 .
  • a portion 1031B of the first flow channel 1031 includes the junction 105 and is divided by the junction 105 into a first section (eg, the section of 1031B above the junction 105 in FIG. 1A ) and a second section (eg, the section of 1031B in FIG.
  • the second flow channel 1032 includes the junction 105 and is divided by the junction 105 into a third section (eg, the section to the left of the junction 105 of 1032 in FIG. 1A ) and a fourth section (eg, 1032 in FIG. 1A ) of the segment to the right of the junction 105 ), in each of the third and fourth segments, the area of the second cross-section of the segment is along a second direction away from the junction 105 Increasing gradually, the second cross section is perpendicular to the second direction.
  • a third section eg, the section to the left of the junction 105 of 1032 in FIG. 1A
  • a fourth section eg, 1032 in FIG. 1A
  • the first flow channel 1031 gradually becomes thicker along the upward direction from the junction point 105; and the first flow channel 1031 also gradually becomes thicker along the downward direction from the junction point 105. thick.
  • the second flow channel 1032 gradually becomes thicker along the leftward direction from the confluence point 105 ; the second flow channel 1032 also gradually becomes thicker along the rightward direction from the confluence point 105 .
  • the first cross section refers to the cross section of a part 1031B of the first flow channel 1031 perpendicular to the flow direction of the first fluid 107 ;
  • the second cross section refers to the second flow channel 1032 perpendicular to the second flow direction 1032.
  • the cross-section in the direction of flow of the fluid 108 is not limited to the above description.
  • the first chip 100 is beneficial to generate the first droplet 110 containing a single cell.
  • the first droplet 110 containing a single cell can be prepared through the first chip 100. Since the first chip 100 has a high degree of integration, the preparation of the first droplet 110 containing a single cell can be automatically completed without the need for an operator. Manual operation, so it can effectively improve the degree of automation of the operation. Since the first fluid 107 and the second fluid 108 flow completely in the first delivery channel 103 and are completely isolated from the external environment, contamination by aerosols, microorganisms, etc. floating in the environment can be avoided. In addition, since the whole preparation process is relatively mild, and the single cells isolated from the cell suspension are wrapped and protected by droplets, the cell viability can be effectively improved.
  • the first accommodating part 101 includes a sample inlet 1 , and an external device (eg, a micro flow pump) is connected to the sample inlet 1 and passes the first fluid 107 through the sample inlet 1 .
  • the first fluid 107 is a continuous phase (eg, oil phase) liquid, which may be, for example, any suitable fluid such as mineral oil, perfluorinated oil, and the like.
  • the first fluid 107 may be mixed with a surfactant that facilitates stabilization of the resulting first droplets 110 , eg, inhibits subsequent coalescence of the resulting droplets 110 .
  • the second accommodating part 102 includes a first sub-accommodating part 1021 and a second sub-accommodating part 1022 which are separated from each other.
  • the first sub-accommodating part 1021 includes an injection port 2 , and an external device (eg, a micro flow pump) is connected to the injection port 2 and injects the cell suspension 109 - 1 into the first sub-accommodating part 1021 through the injection port 2 .
  • an external device eg, a micro flow pump
  • the second sub-accommodating part 1022 includes an injection port 3 , and an external device (eg, a micro flow pump) is connected to the injection port 3 and injects the biochemical reaction reagent 109 - 2 into the second sub-accommodating part 1022 through the injection port 3 .
  • an external device eg, a micro flow pump
  • Different biochemical reaction reagents can be adopted according to different biochemical reactions, and the chemical composition of the biochemical reaction reagent 109-2 is not specifically limited in the embodiments of the present disclosure.
  • the first fluid 107 and the second fluid 108 including the cell suspension 109-1 and the biochemical reaction reagent 109-2 converge at the confluence point 105 of the first delivery channel 103 and generate a plurality of first droplets 110, a plurality of first droplets 110.
  • a droplet 110 flows into the first collecting part 104 along the first conveying channel 103 .
  • the first collection part 104 includes a plurality of sample outlets 4, and the sample outlets 4 are used for connecting with external equipment to transfer the plurality of first droplets 110 in the first collection part 104 to other containers for use in subsequent operations .
  • FIG. 1A shows that the cell suspension 109-1 is accommodated in the first sub-accommodating part 1021, the biochemical reaction reagent 109-2 is accommodated in the second sub-accommodating part separated from the first sub-accommodating part 1021 1022, but this is only an example, and embodiments of the present disclosure are not limited thereto.
  • the cell suspension 109-1 and the biochemical reaction reagent 109-2 may be premixed and contained in the same container.
  • the first delivery flow channel 103 includes a first flow channel 1031 and a second flow channel 1032 .
  • the first flow channel 1031 communicates with the first accommodating portion 101 and allows the first fluid 107 to flow therein.
  • the second flow channel 1032 communicates with the second accommodating portion 102 and allows the second fluid 108 to flow therein.
  • the first flow channel 1031 and the second flow channel 1032 intersect and communicate at the meeting point 105 .
  • the cross-section of the first delivery flow channel 103 The area first gradually decreases and then gradually increases, that is, in this section, the first conveying flow channel 103 becomes narrower and then thicker. This is described in detail below.
  • the first flow channel 1031 includes a first portion 1031A and a second portion 1031B, and the first portion 1031A and the second portion 1031B of the first flow channel 1031 together form a closed pentagon, and the pentagon is approximately about the horizontal axis where the confluence point 105 is located. Axisymmetric. A part of the first fluid 107 in the first accommodating part 101 flows to the confluence point 105 along the part of the first runner 1031 located above the horizontal axis, and another part of the first fluid 107 in the first accommodating part 101 flows along the The portion of the flow channel 1031 below the horizontal axis also flows to the junction 105 .
  • FIG. 2 is an enlarged view of the AA area of FIG. 1A .
  • the second portion 1031B of the first flow channel 1031 includes a first sub-section 1031B- 1 , a second sub-section 1031B- 2 and a third sub-section 1031B- 1 , which are sequentially arranged along a first direction (the vertical direction in the figure).
  • Section 1031B-3, second subsection 1031B-2 is located between first subsection 1031B-1 and third subsection 1031B-3 and includes junction 105 .
  • the first subsection 1031B-1 belongs to the first section described above
  • the third subsection 1031B-3 belongs to the second section described above
  • the second subsection 1031B-2 spans the first section and the second section.
  • the areas of the first cross-sections of the first subsection 1031B-1 and the third subsection 1031B-3 are both larger than the areas of the first cross-sections of the second subsection 1031B-2, that is, along the direction from the first subsection 1031B -1 to the direction of the third sub-section 1031B-3, the first flow channel 1031 is gradually thinner first and then gradually thicker, so that the first flow channel 1031 appears up and down (the first sub-section 1031B-1 and the third sub-section 1031B-3 ) thick middle (second subsection 1031B-2) thin shape.
  • the first fluid 107 in the first flow channel 1031 flows from the first subsection 1031B-1 to the second subsection 1031B-2 or from the third subsection 1031B-3 to the second subsection 1031B- 2, since the flow channel is getting thinner and thinner, the flow velocity of the first fluid 107 in the first flow channel 1031 becomes larger, so that the pressure of the first fluid 107 can be increased, and the first sub-section 1031B-1 and the third sub-section can be promoted.
  • the first fluid 107 within 1031B-3 flows toward the junction 105 of the second subsection 1031B-2, where it collects. This provides sufficient first fluid 107 for subsequent formation of first droplets 110 .
  • the shapes of the first cross-sections of the first subsection 1031B-1, the second subsection 1031B-2 and the third subsection 1031B-3 of the second section 1031B of the first flow channel 1031 may be circular, square, rectangular, regular Polygons, irregular shapes, etc., are not limited in the embodiments of the present disclosure.
  • the first cross-section of the second sub-portion 1031B-2 of the first flow channel 1031 at the confluence point 105 is sized to allow a first fluid 107 having a specific particle size to flow therein, the specific particle size of the first fluid 107 being larger than The particle size of a single cell. That is, the width of the first cross-section of the second subsection 1031B-2 of the first flow channel 1031 at the confluence point 105 is larger than the particle size of a single cell. In one example, the particle size of each cell in the cell suspension is approximately 10 ⁇ m, and the width of the cross-section of the second subsection 1031B-2 of the first flow channel 1031 at the confluence point 105 is greater than 10 ⁇ m, eg slightly greater than 10 ⁇ m.
  • “Slightly larger than 10 ⁇ m” here means that the width of the first cross-section of the second sub-section 1031B-2 of the first flow channel 1031 at the confluence point 105 is larger than 10 ⁇ m, but smaller than 20 ⁇ m, that is, the width is larger than the particle size of a single cell But less than the sum of the particle sizes of the two cells.
  • the phrase "the width of the first cross-section of the second subsection 1031B-2 of the first flow channel 1031 at the confluence point 105" can be understood as when the second subsection 1031B-2 of the first flow channel 1031 When the shape of the first cross section at the confluence point 105 is a circle, the width of the first cross section is the diameter of the circle; When the shape of the first cross-section is square, the width of the first cross-section is the side length of the square; When the shape is a rectangle, the width of the first cross section is the length of the short side of the rectangle; when the shape of the first cross section of the second sub-section 1031B-2 of the first flow channel 1031 at the meeting point 105 is a regular polygon , the width of the first cross section is the distance between the two furthest vertices of the regular polygon.
  • the second subsection 1031B-2 of the first flow channel 1031 at the confluence point 105 when the first cross-section of the second subsection 1031B-2 of the first flow channel 1031 at the confluence point 105 is circular and the shape of a single cell is spherical, then the second subsection 1031B-2 of the first flow channel 1031
  • the width of the first cross-section of the portion 1031B-2 at the confluence point 105 is greater than the particle size of a single cell should be understood to mean that the diameter of the second sub-section 1031B-2 of the first flow channel 1031 at the confluence point 105 is greater than the diameter of a single cell .
  • the first fluid 107 in the first flow channel 1031 flows from the first subsection 1031B-1 to the second subsection 1031B-2 or from the third subsection 1031B-3 to the second subsection 1031B- 2, the first fluid 107 can form a single row of fluid particles arranged in sequence near the confluence point 105, and the particle size of each particle in the single row of fluid particles is larger than the particle size of a single cell and smaller than the particle size of two cells. and.
  • the particle size of each particle formed by the first fluid 107 can be slightly larger than the particle size of a single cell, so that the single cell can be better encapsulated and a better encapsulation effect can be achieved.
  • such a design can also increase the flow rate of the first fluid 107 at the confluence point 105 , which is beneficial to the formation of the first droplet 110 .
  • the second flow channel 1032 includes a first portion 1032A, a second portion 1032B and a third portion 1032C sequentially arranged in a second direction (horizontal direction in the figure) different from the first direction .
  • the first portion 1032A and the second portion 1032B belong to the third section described above, and the third section 1032C belongs to the fourth section described above.
  • the first end of the first portion 1032A of the second flow channel 1032 communicates with the second receiving portion 102 , and the second end of the first portion 1032A of the second flow channel 1032 communicates with the first end of the second portion 1032B of the second flow channel 1032 ;
  • the second end of the second portion 1032B of the second flow channel 1032 communicates with the first end of the third portion 1032C of the second flow channel 1032, and the second end of the second portion 1032B of the second flow channel 1032 communicates with the second
  • the first ends of the third portion 1032C of the flow channel 1032 are all located at the confluence point 105 ; the second end of the third portion 1032C of the second flow channel 1032 communicates with the first collecting portion 104 .
  • the first portion 1032A of the second flow channel 1032 includes a first branch and a second branch
  • the first branch communicates with the first sub-accommodating portion 1021 of the second receiving portion 102 and is configured to allow the cell suspension 109-1
  • the second branch communicates with the second sub-accommodating part 1022 of the second containing part 102 and is configured to allow the biochemical reaction reagent 109-2 to flow therein.
  • the first branch and the second branch intersect and communicate with each other at the first point 106 , and the included angle ⁇ between the first branch and the second branch at the first point 106 is an acute angle. In one example, the angle ⁇ between the first branch and the second branch at the first point 106 is about 60 degrees.
  • the angle design between the first branch and the second branch can ensure that the cell suspension 109-1 in the first branch and the biochemical reaction reagent 109-2 in the second branch are sufficiently forward (towards the confluence point 105). On the other hand, it can also ensure that the cell suspension 109-1 and the biochemical reaction reagent 109-2 can be fully mixed at the first point 106; on the other hand, it can also reduce the mixed solution in the flow channel.
  • the dead volume improves the storage accuracy of the first branch and the second branch.
  • the phrase "the angle ⁇ between the first branch and the second branch at the first point 106 is about 60 degrees” includes that the angle ⁇ between the first branch and the second branch at the first point 106 is greater than 60 degrees degrees, the angle ⁇ between the first branch and the second branch at the first point 106 is less than 60 degrees, and the angle ⁇ between the first branch and the second branch at the first point 106 is equal to 60 degrees.
  • the areas of the second cross section of the first portion 1032A and the third portion 1032C of the second flow channel 1032 are both larger than the area of the second cross section of the second portion 1032B of the second flow channel 1032 . That is, the areas of the first branch of the first portion 1032A of the second flow channel 1032 and the second cross-sectional area of the second branch are both larger than the area of the second cross-section of the second portion 1032B of the second flow channel 1032 , and the second The area of the second cross-section of the third portion 1032C of the flow channel 1032 is greater than the area of the second cross-section of the second portion 1032B of the second flow channel 1032 .
  • the second flow channel 1032 becomes thicker and thinner. Similar to the first flow channel 1031, the shape of the second cross section of the first portion 1032A, the second portion 1032B and the third portion 1032C of the second flow channel 1032 may be a circle, a square, a rectangle, a regular polygon, an irregular shape, etc., The embodiments of the present disclosure do not limit this.
  • the dimensions of the second cross-section of the second portion 1032B of the second flow channel 1032 are configured to allow a second fluid 108 having a specific particle size to flow therein, the specific particle size of the second fluid 108 being greater than 1 times the particle size of a single cell And less than 2 times the particle size of a single cell. That is, the width of the second cross section of the second portion 1032B of the second flow channel 1032 is greater than 1 times the diameter of a single cell and less than 2 times the diameter of a single cell.
  • the second cross-section of the second portion 1032B of the second flow channel 1032 is circular and the shape of the single cell is spherical
  • the second cross-section of the second portion 1032B of the second flow channel 1032 The width of the width is greater than 1 times the diameter of a single cell and less than 2 times the diameter of a single cell should be understood as the diameter of the second part 1032B of the second flow channel 1032 is greater than 1 times the diameter of a single cell and less than 2 times the diameter of a single cell .
  • the diameter of the second portion 1032B of the second flow channel 1032 may be 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times the diameter of a single cell times equal.
  • the width of the second cross-section of the second portion 1032B of the second flow channel 1032 only allows a single cell to be accommodated in its width direction, but cannot accommodate two side-by-side cells.
  • the single row of the single cell string moves to the junction 105, under the pressure of the first fluid 107 in the second portion 1031B of the first flow channel 1031, one cell (ie, the cell in the string that is closest to the junction 105)
  • the first cell in the string) is separated from the string, and the separated cell combines with a single particle in the first fluid 107 at a confluence point 105, thereby forming a first droplet 110 containing a single cell 111.
  • the first droplet 110 has a water-in-oil structure, that is, the first fluid 107 of the oil phase encapsulates the second fluid 108 of the water phase.
  • the area of the second cross-section of the third portion 1032C of the second flow channel 1032 gradually increases along the direction from the first end to the second end thereof, that is, the third portion 1032C of the second flow channel 1032 is along the The direction from the first end to the second end gradually becomes thicker.
  • the purpose of this design is to make the first droplet 110 progressively larger as it moves forward along the third portion 1032C of the second flow channel 1032, thereby facilitating droplet phase stabilization.
  • the area of the first cross-section of the second subsection 1031B-2 of the first flow channel 1031 at the junction 105 is greater than or equal to the area of the second section 1032B and the third section 1032C of the second flow channel 1032 at the junction 105 The area of the cross section.
  • the area of the first cross-section of the second subsection 1031B-2 of the first flow channel 1031 at the junction 105 is equal to the area of the second section 1032B and the third section 1032C of the second flow channel 1032 at the junction 105 the area of the second cross-section.
  • FIG. 3 is an enlarged schematic view of the first collecting part 104 of the first chip 100 in FIG. 1A .
  • the formed first droplet 110 finally flows into the first collecting part 104 along the third portion 1032C of the second flow channel 1032 .
  • each first droplet 110 is wrapped with a single cell 111 .
  • each first droplet 110 includes a single cell 111 .
  • FIG. 3 shows that due to factors such as the adhesion between cells in the cell suspension, the flow rate of the fluid, the surface design of the flow channel, etc., it is not completely guaranteed that each first droplet 110 includes a single cell 111 . As shown in FIG.
  • a single cell 111 derived from the cell suspension 109-1 may be included in one first droplet 110, and may not include a single cell 111 derived from the cell suspension 109-1
  • a single cell 111 ie, an "empty" droplet
  • possibly two cells 111 derived from the cell suspension 109-1 The structural design of the first chip 100 in the embodiment of the present disclosure (eg, the structures of the first flow channel 1031 and the second flow channel 1032 , the design of the filter structure to be described below, etc.) is beneficial to promote the internalization of each first droplet 110 Only single cells 111 are included.
  • the inner wall surface of the first delivery channel 103 is hydrophobic treated and thus has hydrophobicity.
  • the first delivery flow channel 103 includes a first flow channel 1031 configured to flow the first fluid 107 therein and a second flow channel 1032 configured to flow the second fluid 108 therein.
  • the hydrophobic treatment of the first flow channel 1031 may facilitate the flow of the first fluid 107 therein.
  • the hydrophobically treated second flow channel 1032 can facilitate the smooth flow of the cell suspension 109-1 within the first branch of the first portion 1032A of the second flow channel 1032 without adhering to the inner wall surface, and can promote the cell suspension
  • the mixed solution of 109-1 and the biochemical reaction reagent 109-2 flows smoothly in the second part 1032B and the third part 1032C of the second flow channel 1032 without sticking to the inner wall surface.
  • the amount of the cell suspension 109-1 can be precisely controlled, which is beneficial to the uniform mixing of the cell suspension 109-1 and the biochemical reaction reagent 109-2, thereby promoting the uniform generation of the first droplets 110.
  • this can also improve the utilization rate of the cell suspension 109-1 and avoid waste of the cell suspension 109-1.
  • the outlines of the first accommodating portion 101 , the first sub-accommodating portion 1021 and the second sub-accommodating portion 1022 of the second accommodating portion 102 each include four chamfers.
  • the shape of the four chamfers may be any appropriate shape, for example, a circular arc shape. It should be understood that the embodiment of the present disclosure does not limit the specific size of the chamfer.
  • FIG. 4 is an enlarged schematic view of the first sub-accommodating portion 1021 of the second accommodating portion 102 . Taking the first sub-accommodating portion 1021 of the second accommodating portion 102 as an example, as shown in FIG.
  • the outline of the first sub-accommodating portion 1021 of the second accommodating portion 102 includes four chamfers 113 .
  • arc-shaped The arc-shaped chamfer design can reduce the dead volume of the cell suspension 109 - 1 in the first sub-accommodating part 1021 and improve the liquid storage accuracy of the first sub-accommodating part 1021 .
  • the "dead volume” here refers to the uncontrollable volume during reagent injection.
  • the cell suspension 109-1 will be at the four right angles of the first sub-accommodating part 1021 is not a right-angle shape, that is, the cell suspension 109-1 cannot perfectly match the shape of the first sub-accommodating portion 1021, and cannot fill the space occupied by the four right angles of the first sub-accommodating portion 1021. Therefore, the shape and volume of the cell suspension 109-1 will change, and there is a certain randomness in the change of the shape and volume, thereby introducing dead volume.
  • the first sub-accommodating part 1021 of the first chip 100 may accommodate a different volume of the cell suspension 109-1 in each operation and the previous operation, thereby making it impossible to precisely control the amount of the cell suspension 109-1.
  • the four corners 113 of the first sub-accommodating portion 1021 are designed as arc-shaped chamfers, so that the cell suspension 109-1 can be perfectly matched with the shape of the first sub-accommodating portion 1021, especially It can make the cell suspension 109-1 fill the space occupied by the four arc-shaped chamfers of the first sub-accommodating part 1021, thereby effectively reducing or even avoiding the difference in the accommodation volume of the first sub-accommodating part 1021, and improving the cell Handling accuracy of suspension 109-1.
  • the four arc-shaped chamfers of the first accommodating part 101 can reduce the dead volume of the first fluid 107 in the first accommodating part 101 and improve the liquid storage accuracy of the first accommodating part 101 .
  • the four arc-shaped chamfers of the second sub-accommodating part 1022 of the second containing part 102 can reduce the dead volume of the biochemical reaction reagent 109 - 2 in the second sub-accommodating part 1022 and improve the storage capacity of the second sub-accommodating part 1022 Fluid accuracy.
  • the first accommodating part 101 and the second accommodating part 102 of the first chip 100 are both provided with a filter structure 112 , that is, the first accommodating part 101 and the second accommodating part 101 of the first chip 100
  • the first sub-accommodating part 1021 and the second sub-accommodating part 1022 of the part 102 are both provided with a filter structure 112 . Since the structures of the filter structures 112 of the first accommodating portion 101 , the first sub-accommodating portion 1021 and the second sub-accommodating portion 1022 are completely the same, the filter structures 112 in the first sub-accommodating portion 1021 shown in FIG. 4 are as follows: For example, the structure and function of the filtering structure 112 will be described in detail.
  • the filter structure 112 includes a plurality of microstructures spaced apart from each other, and the gap d between two adjacent microstructures 112-1 and 112-2 is larger than 1 times the diameter of a single cell and smaller than that of a single cell. 2 times the particle size.
  • the particle size of a single cell derived from the cell suspension 109-1 is about 10 ⁇ m, and accordingly, the gap d between two adjacent microstructures 112-1 and 112-2 is greater than 10 ⁇ m and smaller than 20 ⁇ m.
  • the heights of the multiple microstructures of the filtering structure 112 may be the same, completely different, or only partially the same.
  • each micropillar is about 100-200 ⁇ m.
  • the shape of the cross section of each micro-pillar can be any suitable shape, such as diamond, square, rectangle, circle, ellipse, regular polygon, irregular shape etc., the embodiments of the present disclosure do not specifically limit this.
  • the cell suspension 109 - 1 in the first sub-accommodating portion 1021 flows through the gaps between the adjacent microstructures of the filter structure 112 , and then flows into the first portion 1032A of the second flow channel 1032 in the first branch.
  • the gap d between two adjacent microstructures is larger than 1 times the diameter of a single cell and smaller than 2 times the diameter of a single cell, when the cell suspension 109-1 flows through the gap between adjacent microstructures, On the one hand, it can prevent the excessively large impurities in the cell suspension 109-1 (for example, the particles with a particle size larger than 2 times the particle size of a single cell, such as dust, salting-out substances, etc.) from flowing into the subsequent flow channels, thereby avoiding excessively large impurities.
  • the excessively large impurities in the cell suspension 109-1 for example, the particles with a particle size larger than 2 times the particle size of a single cell, such as dust, salting-out substances, etc.
  • the cell suspension 109- Under the screening of the size of 1, a plurality of cells in the cell suspension 109-1 that are adhered to each other (for example, two cells, three cells or more cells that are adhered to each other) can be separated into a plurality of cells.
  • the isolated single cells are beneficial to the subsequent generation of the first droplet 110 containing a single cell, and reduce the probability that a single first droplet 110 contains two or more cells.
  • the filtering structure 112 in the first accommodating part 101 and the second sub-accommodating part 1022 For the structure of the filtering structure 112 in the first accommodating part 101 and the second sub-accommodating part 1022, reference may be made to the above description of the filtering structure in the first sub-accommodating part 1021, which is not repeated here for brevity.
  • the first fluid 107 in the first receiving portion 101 flows through the gaps between adjacent microstructures of the filter structure 112 , and then flows into the first flow channel 1031 of the first delivery flow channel 103 .
  • the first fluid 107 When the first fluid 107 flows through the gaps between the adjacent microstructures of the filter structure 112 , it can block excessive impurities in the first fluid 107 (for example, impurities with a particle size larger than 2 times the particle size of a single cell, such as dust) , salting-out substances, etc.) flow into the first flow channel 1031 , so as to prevent excessive debris from clogging the first flow channel 1031 and affecting the normal flow of the first fluid 107 .
  • the biochemical reaction reagent 109 - 2 in the second sub-accommodating part 1022 flows through the gaps between the adjacent microstructures of the filter structure 112 , and then flows into the second channel of the first delivery channel 103 .
  • the biochemical reaction reagent 109-2 flows through the gaps between the adjacent microstructures of the filter structure 112, it can block the excessively large impurities in the biochemical reaction reagent 109-2 (for example, impurities with a particle size larger than 2 times the particle size of a single cell). impurities, such as dust, salting-out substances, etc., flow into the second branch of the first part 1032A of the second flow channel 1032, so as to prevent excessive foreign matter from clogging the second flow channel 1032 and affecting the normal flow of the biochemical reaction reagent 109-2.
  • impurities such as dust, salting-out substances, etc.
  • the first chip 100 described in any of the foregoing embodiments may be a microfluidic chip.
  • Microfluidic chips have advantages such as controllable liquid flow, low sample consumption, fast detection speed, simple operation, multi-functional integration, small size and portability.
  • the separation of single cells from the cell suspension 109-1 can be promoted, thereby promoting Each generated first droplet 110 contains a single cell, reducing the probability that a single first droplet 110 contains two or more cells; by optimizing the gap between adjacent microstructures of the filter structure 112, this can be further facilitated
  • Each generated first droplet 110 contains a single cell; by designing the four corners of the first accommodating part 101 and the second accommodating part 102 as arc-shaped chamfers, the dead volume in the accommodating part can be avoided, and the The storage accuracy of the accommodating part; by hydrophobic treatment of the inner wall surface of the first delivery channel 103, the fluid in the channel
  • the first droplet 110 can constitute a microreactor for biochemical reaction of single cells, and also constitute a droplet carrier for subsequent single cell sorting.
  • the preparation of the first droplet 110 containing a single cell can be completed automatically without manual operation by an operator, thus effectively improving the degree of automation of the operation. Since the first fluid 107 and the second fluid 108 completely flow in the first delivery channel 103 and are completely isolated from the external environment, contamination by aerosols, microorganisms, etc. floating in the environment can be avoided. In addition, since the whole preparation process is relatively mild, and the single cells isolated from the cell suspension are wrapped and protected by droplets, the cell viability can be effectively improved.
  • FIGS. 5A-5D illustrate the second chip 200 , wherein FIG. 5A is a front view of the second chip 200 , FIG. 5B is a side view of the second chip 200 , and FIG. 5C is a rear view of the second chip 200 , and FIG. 5D is an isometric view of the second chip 200 . As shown in FIGS.
  • the second chip 200 includes: a third accommodating part 201 configured to accommodate a third fluid 205 ; a fourth accommodating part 202 configured to accommodate a fourth fluid, the fourth fluid including Cell suspension;
  • the delivery channel 208 (hereinafter referred to as the second delivery channel 208 ) includes a third channel 2081 and a fourth channel 2082, the third channel 2081 communicates with the third accommodating part 201 and the fourth channel 2082 communicates with the fourth accommodating part 202, the third flow channel 2081 and the fourth flow channel 2082 intersect and communicate with each other at the confluence point 209, and the second delivery flow channel 208 is configured so that the third fluid 205 and the fourth fluid are at the confluence point merge at 209 and generate at least one droplet 206 (hereafter referred to as second droplet 206), each of at least a portion of the at least one second droplet 206 comprising a single cell derived from the cell suspension; and at least one collection A portion 204 (hereinafter referred to as the second collection portion 204 )
  • a portion of the third flow channel 2081 (ie, the vertical portion of the third flow channel 2081 in FIG. 5A ) includes a junction 209 and is divided into a first section by the junction 209 (eg, the third flow channel 2081 in FIG. 5A ) the section above the junction 209) and a second section (eg, the section of the third flow channel 2081 in FIG. 5A below the junction 209), in each of the first and second sections In the section, the area of the first cross-section of the section gradually increases along a first direction away from the junction 209, and the first cross-section is perpendicular to the first direction.
  • the fourth flow channel 2082 includes the junction 209 and is divided by the junction 209 into a third section (eg, the section to the left of the junction 209 of 2082 in FIG. 5A ) and a fourth section (eg, 2082 in FIG. 5A ) of the segment to the right of the junction 209 ), in each of the third and fourth segments, the area of the second cross-section of the segment is along a second direction away from the junction 209 Increasing gradually, the second cross section is perpendicular to the second direction. That is to say, in the third flow channel 2081, the third flow channel 2081 gradually becomes thicker in the upward direction from the confluence point 209; and the third flow channel 2081 also gradually becomes thicker in the downward direction from the confluence point 209. thick. In the fourth flow channel 2082 , the fourth flow channel 2082 gradually becomes thicker along the leftward direction from the junction point 209 ; and the fourth flow channel 2082 also gradually becomes thicker along the rightward direction from the junction point 209 .
  • a third section
  • the third fluid 205 and the fourth fluid are only names used for the convenience of describing the second chip 200.
  • the third fluid 205 may be exactly the same as the first fluid 107
  • the fourth fluid may be It is exactly the same as the second fluid 108 (including the cell suspension 109-1 and the biochemical reaction reagent 109-2).
  • the fourth fluid may not be exactly the same as the second fluid 108 .
  • the fourth fluid is the first droplet 110 previously described.
  • the first cross-section of a part of the third flow channel 2081 (ie, the vertical part of the third flow channel 2081 in FIG. 5A ) means that the part of the third flow channel 2081 is perpendicular to the third fluid 205
  • the second cross section of the fourth flow channel 2082 refers to the cross section of the fourth flow channel 2082 perpendicular to the flow direction of the fourth fluid.
  • the second delivery flow channel 208 also includes a sorting flow channel 203 configured to sort a target droplet including a single cell from the at least one second droplet 206 .
  • the third accommodation part 201 includes an injection port 5 , and an external device (eg, a micro flow pump) is connected to the injection port 5 and injects the third fluid 205 into the first injection port 5 through the injection port 5 Inside the three accommodating parts 201 .
  • the third fluid 205 is a continuous phase (eg, oil phase) liquid, which may have the same chemical composition as the first fluid 107 .
  • the third fluid 205 may be any suitable fluid such as mineral oil, perfluorinated oil, and the like.
  • the third fluid 205 may be mixed with a surfactant, which is beneficial for stabilizing the second droplets 206 that are subsequently generated, eg, inhibiting subsequent coalescence of the second droplets 206 .
  • the surfactant may be a perfluorinated surfactant.
  • the fourth containing part 202 includes an injection port 6, an external device (eg, a micro flow pump) is connected to the injection port 6 and through which a fourth fluid (eg, the first droplet 110 described above) is injected into the injection port 6 . inside the fourth accommodating portion 202 .
  • a fourth fluid eg, the first droplet 110 described above
  • the first droplet 110 can be transferred to another device for corresponding biochemical treatment (eg PCR amplification, staining, etc.), and then the treated first droplet 110 can be The droplet 110 is injected into the second receiving portion 202 of the second chip 200 .
  • the contours of the third accommodating portion 201 and the fourth accommodating portion 202 of the second chip 200 also include four chamfers,
  • the shape can be arc-shaped or any other suitable shape.
  • the chamfered design of the third accommodating part 201 and the fourth accommodating part 202 can reduce the dead volume of the third fluid 205 and the first droplet 110 in the third accommodating part 201 and the fourth accommodating part 202 respectively, thereby improving the Accuracy of liquid storage of the three accommodating parts 201 and the fourth accommodating part 202 .
  • the third accommodating part 201 and the fourth accommodating part 202 are also provided with filter structures (not shown in the figure).
  • the filter structures in the third accommodating part 201 and the fourth accommodating part 202 include a plurality of microstructures spaced apart from each other, and the gap between two adjacent microstructures is larger than 1 times the particle size of a single first droplet 110 and smaller than a single 2 times the particle size of the first droplet 110 .
  • the heights of the multiple microstructures of the filtering structure may be the same, completely different, or only partially the same, and the specific heights may be flexibly designed according to product requirements, which are not specifically limited in the embodiments of the present disclosure.
  • the height of each micropillar is about 100-200 ⁇ m.
  • the shape of the cross-section of each micropillar can be any suitable shape, such as diamond, square, rectangle, circle, ellipse, positive Polygons, irregular shapes, etc., are not specifically limited in the embodiments of the present disclosure.
  • the third fluid 205 in the third accommodating portion 201 flows through the gaps between adjacent microstructures of the filter structure, and the filtered third fluid 205 then flows into the second delivery channel 208 in the third flow channel 2081.
  • the third fluid 205 can block the oversized impurities in the third fluid 205 (for example, impurities with a particle size larger than 2 times the particle size of a single first droplet 110 ) substances, such as dust, salting-out substances, etc.) flow into the third flow channel 2081 , so as to prevent excessive debris from clogging the third flow channel 2081 and affecting the normal flow of the third fluid 205 .
  • the first droplets 110 in the fourth accommodating portion 202 flow through the gaps between adjacent microstructures of the filter structure, and the filtered first droplets 110 then flow into the fourth flow channel 2082 of the second conveying flow channel 208 middle. Since the gap between two adjacent microstructures is larger than 1 times the diameter of a single first droplet 110 and smaller than 2 times the diameter of a single first droplet 110, when the first droplet 110 is separated from the adjacent microstructures When flowing through the gap between the first droplets 110 , on the one hand, it can block the potentially oversized impurities in the first droplet 110 (for example, impurities with a particle size larger than 2 times the particle size of a single first droplet 110 , such as dust, salting-out substances, etc.
  • the second delivery flow channel 208 includes a third flow channel 2081 and a fourth flow channel 2082, the third flow channel 2081 and the fourth flow channel 2082 intersect at a confluence point 209 and communicate with each other at the confluence point 209.
  • the third flow channel 2081 communicates with the third accommodating portion 201 and allows the third fluid 205 to flow therein
  • the fourth flow channel 2082 communicates with the fourth accommodating portion 202 and allows the first droplet 110 to flow therein.
  • the third fluid 205 flows along the third flow channel 2081 to the confluence point 209 of the third flow channel 2081 and the fourth flow channel 2082
  • the first droplet 110 flows along the fourth flow channel 2082 to the third flow channel 2081 and the fourth flow channel 2082 .
  • the third fluid 205 and the first droplet 110 join at the confluence point 209 to generate the second droplet 206 .
  • FIG. 6 is an enlarged view of the region BB of the sorting flow channel 203 shown in FIG. 5A .
  • the sorting flow channel 203 includes a first branch 2031 , a second branch 2032 , and a third branch 2033 , and the third branch 2033 is located between the first branch 2031 and the second branch 2032 .
  • the second collection part 204 includes a first sub-collection part 2041 , a second sub-collection part 2042 and a third sub-collection part 2043 , the first sub-collection part 2041 includes a sample outlet 7A, and the second sub-collection part 2042 includes a sample outlet 7B , the third sub-collecting part 2043 includes a sample outlet 7C.
  • the first sub-collecting part 2041 communicates with the first branch 2031 of the sorting flow channel 203
  • the second sub-collecting part 2042 communicates with the second branch 2032 of the sorting flow channel 203
  • the third sub-collecting part 2043 communicates with the sorting flow channel 203
  • the sorting channel 203 is provided with an electrode structure (not shown in the figure), and the electrode structure may include, for example, a positive electrode and a negative electrode, for applying a voltage to drive the second droplet 206 to deflect into the corresponding branch of the sorting channel 203 , so that the target droplet is sorted out in the second droplet 206 .
  • the second chip 200 may also include an optical device (not shown, such as a fluorescence microscope, etc.) for identifying target droplets in the second droplet 206 .
  • the sorting process of the target droplets is roughly as follows:
  • the cell suspension 109-1 contains a large number of cells. In this large number of cells, there are a small number of target cells to be analyzed and detected, that is, the target cells that the present application wishes to sort (such as circulating tumor cells, rare cells, cancer cells, etc. in peripheral blood samples), and other non-target cells. Since these target cells in the cell suspension contain different antibodies than non-target cells, they will show a different color than non-target cells under a fluorescence microscope after fluorescent staining.
  • the cell suspension 109-1 can be dyed, and the first droplet 110 can also be dyed. The embodiment of the present disclosure does not specifically limit the dyeing sequence.
  • the first droplet 110 is injected into the fourth accommodating part 202 of the second chip 200 , and the third fluid 205 and the first droplet 110 converge in the second delivery channel 208 to generate a second droplet 206.
  • the second droplets 206 can be roughly divided into the following two categories: (a) the second droplet 206 contains a single target cell with the target color; (b) the second droplet 206 contains non-target cells or no cells.
  • the second droplet 206 moves along the second conveying channel 208 toward the second collecting part 204 , and the optical device detects the optical signal (eg, color) of the second droplet 206 in the second conveying channel 208 in real time.
  • the circuit system When the optical device detects that the second droplet 206 is in the above situation (b), the circuit system will not be triggered to apply a voltage to the electrode structure, and the second droplet 206 flows directly into the sorting channel 203 along the second conveying channel 208 The third branch 2033 of the second collecting part 204 then flows into the third sub-collecting part 2043 of the second collecting part 204 .
  • the circuit system When the optical device detects that the second droplet 206 is the above-mentioned situation (a), the circuit system is immediately triggered to apply a voltage (for example, 800-1000V) to the electrode structure at the sorting channel 203, and the second droplet containing a single target cell
  • a voltage for example, 800-1000V
  • the droplet 206 is polarized, and under the action of the electric field, the second droplet 206 containing a single target cell is deflected upward into the first branch 2031 of the sorting channel 203 or deflected downward into the second branch 2032 of the sorting channel 203 , and then flow into the first sub-collection part 2041 or the second sub-collection part 2042 of the second collection part 204 , respectively.
  • the second chip 200 realizes the sorting of target droplets.
  • the fluorescent staining treatment of the cell suspension is only an example of the embodiment of the present disclosure, and the treatment method of the cell suspension is not limited to this, as long as the target cells in the cell suspension can be distinguished from non-target cells
  • the open processing methods are all within the protection scope of the present disclosure.
  • FIG. 5A shows that the sorting flow channel 203 includes three branches, and the second collecting part 204 correspondingly includes three sub-collecting parts, the embodiments of the present disclosure are not limited thereto.
  • the sorting flow channel 203 may include at least two branches (eg, two branches, four branches, or more branches), one branch of the at least two branches being configured from a plurality of second branches Non-target droplets are sorted from droplets 206 , and the remaining branches of the at least two branches are configured to sort target droplets from the plurality of second droplets 206 .
  • the second collection part 204 may include at least two sub-collection parts, the at least two sub-collection parts correspond to the at least two branches of the sorting flow channel 203 one-to-one, and one of the at least two sub-collection parts is associated with the sorting One of the at least two branches of the flow channel 203 is connected and configured to collect non-target droplets, and the remaining sub-collecting parts of the at least two sub-collecting parts are respectively connected to the remaining branches of the at least two branches of the sorting flow channel 203 and is configured to collect target droplets.
  • the first branch 2031 , the second branch 2032 and the third branch 2033 of the sorting flow channel 203 intersect at the second point 207 and the third branch 2033 is located between the first branch 2031 and the second branch 2032 .
  • the first included angle ⁇ 1 between the first branch 2031 and the third branch 2033 at the second point 207 and the second included angle ⁇ 2 between the second branch 2032 and the third branch 2033 at the second point 207 are both greater than 10°.
  • the space between the first branch 2031 and the third branch 2033 of the sorting flow channel 203 defines a first right triangle
  • the space between the second branch 2032 and the third branch 2033 of the sorting flow channel 203 defines a second right triangle.
  • the first included angle ⁇ 1 faces the first right-angled side of the first right-angled triangle
  • the second included angle ⁇ 2 faces the second right-angled side of the second right-angled triangle.
  • the length L1 of the first right-angled side of the first right-angled triangle and the length L2 of the second right-angled side of the second right-angled triangle are both greater than or equal to the particle size of a single second droplet 206 .
  • the phrase "the space between the first branch 2031 and the third branch 2033 of the sorting flow channel 203 defines a first right triangle, and the space between the second branch 2032 and the third branch 2033 of the sorting flow channel 203
  • the “limited” in the space defines the second right-angled triangle” means that, as shown in FIG. 5A, the third branch 2033 of the sorting flow channel 203 extends along the second direction (ie, the horizontal direction in the figure); the sorting flow
  • the first branch 2031 of the channel separates from the third branch 2033 from the second point 207 and extends upward at an inflection point; the second branch 2032 of the sorting channel separates from the third branch 2033 from the second point 207 and extends upward at another point.
  • the inflection point of the first branch 2031 and the inflection point of the second branch 2032 are connected by a straight line, which intersects the third branch 2033 at a point.
  • the inflection point of the first branch 2031, the second point 207, the intersection of the straight line and the third branch 2033, these three points are connected to form a first right-angled triangle, the first angle ⁇ 1 faces the first right-angled side of the first right-angled triangle, and the third
  • the length of the right-angled side is L1 , and the length L1 is greater than or equal to the particle size of a single second droplet 206 .
  • the inflection point of the second branch 2032, the second point 207, the intersection of the straight line and the third branch 2033 are connected to form a second right triangle, and the second included angle ⁇ 2 faces the second right side of the second right triangle.
  • the length of the two right-angled sides is L2, and the length L2 is greater than or equal to the particle size of a single second droplet 206 .
  • the included angle between the branches of the sorting flow channel 203 is greater than 10° and the length of the right-angled side of the right triangle is greater than or equal to the particle size of one second droplet 206 , it is beneficial to sort the target droplets to corresponding in the sub-collection department. This is because: if the gradient of the fork between the branches of the sorting flow channel 203 is too small, the target droplet may not be deflected up or down normally into the corresponding sub-collection part, and may enter the middle sub-collection by mistake.
  • an appropriate fork gradient of the sorting flow channel 203 is beneficial to sorting the target droplets from the second droplets 206 .
  • the second chip 200 described in any of the foregoing embodiments may be a microfluidic chip.
  • the liquid flow path and its pump valve system can be effectively simplified.
  • the size of the formed second droplets 206 can be controlled within the range of several micrometers to several tens of micrometers.
  • FIG. 7 shows the third chip 300 .
  • the third chip 300 can be regarded as a combination of the first chip 100 and the second chip 200 , but in the third chip 300 , the third accommodating part 201 , the fourth accommodating part 202 and the second conveying part of the second chip 200 Runner 208 is removed.
  • the same reference numerals as those in FIGS. 1A and 5A denote the same structures, and thus, for the sake of brevity, the same structures in the third chip 300 as the first and second chips 100 and 200 are no longer used. description, the different parts are described below.
  • the first fluid 107 and the second fluid 108 (including the cell suspension 109 - 1 and the biochemical reaction reagent 109 - 2 ) converge in the first delivery channel 103
  • the first droplets 110 are combined to generate the first droplets 110 , and the first droplets 110 flow into the first collection part 104 along the third portion 1032C of the second flow channel 1032 .
  • the first droplet 110 has a water-in-oil structure.
  • the first collecting part 104 is in direct communication with the sorting flow channel 203 , that is, the first droplets 110 in the first collecting part 104 can flow into the sorting flow channel 203 .
  • the first droplet 110 can be roughly divided into the following two categories: (a) the first droplet 110 contains a single target cell with a target color; (b) ) The first droplet 110 contains non-target cells or no cells.
  • the plurality of first droplets 110 move along the direction of the sorting flow channel 203 toward the second collection part 204 , and the optical device detects the optical signal (eg, color) of the first droplets 110 in real time.
  • the circuit system When the optical device detects that the first droplet 110 is in the above-mentioned situation (b), the circuit system will not be triggered to apply a voltage to the electrode structure, and the first droplet 110 flows straight forward into the third branch 2033 of the sorting channel 203 , and then flows into the third sub-collecting part 2043 of the second collecting part 204 .
  • the circuit system When the optical device detects that the first droplet 110 is the above-mentioned situation (a), the circuit system is immediately triggered to apply a voltage (for example, 800-1000V) to the electrode structure at the sorting channel 203, and the first droplet containing a single target cell
  • a voltage for example, 800-1000V
  • the droplet 110 is polarized, and under the action of the electric field, the first droplet 110 containing a single target cell is deflected upwards into the first branch 2031 of the sorting channel 203 or is deflected downwards into the second branch 2032 of the sorting channel 203 , and then flow into the first sub-collection part 2041 or the second sub-collection part 2042 of the second collection part 204 , respectively.
  • the third chip 300 can achieve the two purposes of preparing the first droplet 110 containing a single cell and sorting the target droplet from the first droplet 110 at the same time.
  • the target droplets contain the single target cells expected to be obtained in the present application, such as circulating tumor cells, rare cells, cancer cells, etc. in peripheral blood samples.
  • the third chip 300 is provided with an electrode structure at the sorting channel 203 .
  • electrode structures E1 , E2 , E3 and E4 are arranged; below the sorting flow channel 203 , electrode structures E5 , E6 , E7 and E8 are arranged.
  • a wire is connected to each electrode structure.
  • one end of the wire W1 is electrically connected to the electrode structure E1
  • one end of the wire W2 is electrically connected to the electrode structure E2
  • one end of the wire W3 is electrically connected to the electrode structure E3
  • one end of the wire W4 is electrically connected to the electrode structure E4.
  • the other ends of W1, W2, W3, and W4 meet at a point.
  • the wires W1, W2, and W4 are basically broken lines, and the wire W3 is basically a straight line.
  • One end of the wire W5 is electrically connected to the electrode structure E5
  • one end of the wire W6 is electrically connected to the electrode structure E6
  • one end of the wire W7 is electrically connected to the electrode structure E7
  • one end of the wire W8 is electrically connected to the electrode structure E8, the wires W5, W6, W7 It intersects with the other end of W8 at one point.
  • the wires W5, W6, and W8 are basically broken lines, and the wire W7 is basically a straight line.
  • the circuit system is immediately triggered to apply a voltage to the electrode structure at the sorting channel 203, for example, to the electrode structure E1
  • a positive voltage or a negative voltage
  • a negative voltage or a positive voltage
  • the electrode structures E5 ⁇ E8 and the voltage difference between the two may be, for example, 800 ⁇ 1000V.
  • the first droplet 110 containing a single target cell is polarized, and under the action of an electric field, the first droplet 110 containing a single target cell is deflected upward and flows into the first branch 2031 of the sorting channel 203 or is deflected downward according to the direction of the electric field It flows into the second branch 2032 of the sorting flow channel 203 , and then flows into the first sub-collection part 2041 or the second sub-collection part 2042 of the second collection part 204 , respectively.
  • the third chip 300 combines the first chip 100 with the second chip 200 , and removes the third accommodating part 201 , the fourth accommodating part 202 and the second conveying channel 208 of the second chip 200 , so as to realize the preparation including
  • the two purposes are the first droplet 110 of a single cell and the sorting of target droplets from the first droplet 110 . Therefore, in addition to the respective technical effects of the first chip 100 and the second chip 200, the third chip 300 can also simplify the structure on the premise of realizing the preparation of droplets and the sorting of target droplets, and further The volume occupied by the chip is reduced, the integration degree of the chip is further improved, and the chip is lighter and easier to carry.
  • FIG. 8 shows a block diagram of the microfluidic device 400 .
  • the microfluidic device 400 includes a chip as described in any of the previous embodiments. Since the microfluidic device 400 can have substantially the same technical effects as the first chip 100 , the second chip 200 and the third chip 300 described in the previous embodiments, for the sake of brevity, the description of the microfluidic device will not be repeated here. The technical effect of the control device 400 is obtained.
  • FIG. 9 shows a flowchart of the method 800 .
  • the method 800 is described below by taking the third chip 300 in FIG. 7 as an example.
  • the method 800 includes the following steps:
  • Step S801 Provide the first fluid 107 and the second fluid 108 including the cell suspension 109-1 to the first accommodating part 101 and the second accommodating part 102 of the third chip 300 described in any of the previous embodiments, so that the The first fluid 107 and the second fluid 108 merge at the junction 105 of the delivery channel 103 and generate at least one droplet 110, each of at least a portion of the at least one droplet 110 comprising a single cell derived from the cell suspension 109-1 cells; and
  • Step S802 applying a voltage to the third chip 300 described in any of the previous embodiments to sort out target droplets with target properties from the at least one droplet 110 , the target droplets including a single cell.
  • step S802 includes the following sub-steps: using the optical device to detect the optical signals of the plurality of droplets 110 in real time, in response to the optical device detecting the droplet 110 with the target optical signal, as described in any one of the preceding embodiments
  • the electrode structure of the third chip 300 applies an instantaneous voltage of 800-1000V to sort out the target droplet with the target optical signal from the plurality of droplets 110, and the target droplet includes the above-mentioned single cell.
  • the method before step S802 , the method further includes: transferring the plurality of first droplets 110 to other reaction vessels to perform polymerase chain reaction or fluorescent dyeing treatment.
  • the first fluid 107 is an oil phase, such as mineral oil, perfluorinated oil and other suitable oils
  • the second fluid 108 is an aqueous phase
  • the formed droplets 110 have a water-in-oil structure .
  • Step S901 Connect the input pipe of the micro flow pump to the injection port 1 of the first accommodating part 101 of the first chip 100 , the injection port 2 of the first sub-accommodating part 1021 of the second accommodating part 102 , and the second accommodating part 102 respectively.
  • the injection port 3 of the second sub-accommodating part 1022 of the part 102 is used to inject the first fluid 107, the cell suspension 109-1 and the first fluid 107, the cell suspension 109-1 and the Biochemical Reagent 109-2.
  • the first fluid 107 is the oil phase, which may be mixed with surfactants.
  • Step S902 adjusting the injection speed of the first accommodating part 101 and the second accommodating part 102 to the first conveying channel 103 to control the oil-water flow rate and the droplet generation effect.
  • the flow rate of the first fluid 107 is greater than the flow rates of the cell suspension 109-1 and the biochemical reaction reagent 109-2.
  • Step S903 control the first accommodating part 101 so that the first fluid 107 fills most of the first chip 100 first, and then control the first sub-accommodating part 1021 and the second sub-accommodating part 1022 to make the cell suspension 109 - 1 and the biochemical reaction reagent 109 - 2 flows into the first delivery channel 103 .
  • Controlling the first accommodating part 101 so that the first fluid 107 fills most of the area of the first chip 100 first refers to controlling the first accommodating part 101 so that the first fluid 107 fills the first delivery channel 103 of the first chip 100 first
  • the first flow channel 1031 , the second portion 1032B and the third portion 1032C of the second flow channel 1032 , and optionally the first collection portion 104 Because the amount of the cell suspension 109-1 is usually very small and very precious, a partial area of the first chip 100 can be filled with the first fluid 107 to achieve a better encapsulation effect.
  • Step S904 the first fluid 107 in the first accommodating part 101 flows through the gaps between the plurality of microstructures of the filtering structure 112 in the first accommodating part 101 to realize filtration, and then flows into the first flow channel 1031 , thereby Avoid excessive debris to block the flow channel.
  • the cell suspension 109-1 in the first sub-accommodating part 1021 flows through the gaps between the plurality of microstructures of the filtering structure 112 in the first sub-accommodating part 1021 to realize filtration, and then flows into the second flow channel 1032 , so that, on the one hand, the flow channel can be prevented from being blocked by excessively large debris, and on the other hand, the multiple cells adhering together in the cell suspension 109-1 can be separated into multiple single cells separated from each other.
  • the biochemical reaction reagent 109 - 2 in the second sub-accommodating part 1022 flows through the gaps between the plurality of microstructures of the filtering structure 112 in the second sub-accommodating part 1022 to realize filtration, and then flows into the second flow channel 1032 , so as to prevent excessive debris from clogging the flow channel.
  • Step S905 the first fluid 107 , the cell suspension 109 - 1 and the biochemical reaction reagent 109 - 2 converge at the confluence point 105 of the first delivery channel 103 to generate a plurality of first droplets 110 .
  • 110 moves along the third portion 1032C of the second flow channel 1032 and stabilizes as the diameter of the third portion 1032C of the second flow channel 1032 gradually widens.
  • the first droplet 110 flows into the first collection part 104 along the third portion 1032C of the second flow channel 1032 .
  • Step S906 collecting the first droplets 110 for a period of time (eg, 30 seconds, 1 minute, 2 minutes, etc.) at the outlet 4 of the first collecting part 104 and discarding them as waste liquid.
  • a period of time eg, 30 seconds, 1 minute, 2 minutes, etc.
  • the initial states of the various components in the first chip 100 may not be stable (for example, the pressure is unstable), which may affect the encapsulation effect of the first chip 100 , which is not conducive to A first droplet 110 of good quality is formed. Therefore, the first droplet 110 formed in the initial stage is generally not used.
  • the parameters indicated by the first chip 100 reach a stable state, the first droplets 110 can be collected for subsequent operations.
  • Step S907 Take out the first droplets 110 collected by the first collection part 104 and transfer them to other reaction vessels, such as a 96-well cell culture plate, a PCR machine, etc., to perform required biochemical reactions (eg, PCR amplification, incubation, etc.). reaction, droplet fluorescent staining, etc.).
  • other reaction vessels such as a 96-well cell culture plate, a PCR machine, etc.
  • biochemical reactions eg, PCR amplification, incubation, etc.
  • reaction droplet fluorescent staining, etc.
  • Step S908 connect the input pipes of the micro flow pump to the injection port 5 of the third accommodation part 201 of the second chip 200 and the injection port 6 of the fourth accommodation part 202
  • the accommodating part 202 is injected with the third fluid 205 and the first droplet 110 prepared by using the first chip 100 , respectively.
  • the third fluid 205 may be the same oil phase as the first fluid 107 and may be mixed with a surfactant.
  • Step S909 adjusting the injection speed of the third accommodating part 201 and the fourth accommodating part 202 to the second conveying channel 208 to control the flow rate of droplets.
  • the flow rate of the third fluid 205 is greater than the flow rate of the first droplet 110 .
  • Step S910 Similar to Step S903, control the third accommodating part 201 so that the third fluid 205 fills most of the second chip 200 first, and then control the fourth accommodating part 202 so that the first droplet 110 flows into the second chip 200. in the delivery channel 208.
  • Step S911 the third fluid 205 in the third accommodating part 201 flows through the gaps between the plurality of microstructures of the filtering structure 112 in the third accommodating part 201 to realize filtration, and then flows into the second delivery channel 208 . in the third flow channel 2081, so as to avoid excessive debris from clogging the flow channel.
  • the first droplets 110 in the fourth accommodating part 202 flow through the gaps between the plurality of microstructures of the filtering structure 112 in the fourth accommodating part 202 to realize filtration, and then flow into the fourth part of the second conveying channel 208 . In the flow channel 2082, so as to avoid excessive debris to block the flow channel.
  • Step S912 the third fluid 205 and the first droplet 110 merge at the merging point of the second delivery channel 208 and generate a plurality of second droplets 206 .
  • the second droplets 206 can be roughly divided into the following two categories: (a) the second droplet 206 contains a single target cell with the target color; (b) the second droplet 206 contains non-target cells or no cells.
  • Step S913 the second droplet 206 moves along the second conveying channel 208 toward the second collecting part 204 , and the optical device performs an optical signal (eg, color) of the second droplet 206 in the second conveying channel 208 .
  • an optical signal eg, color
  • the circuit system will not be triggered to apply a voltage to the electrode structure, and the second droplet 206 flows directly into the sorting channel 203 along the second conveying channel 208
  • the third branch 2033 of the second collecting part 204 then flows into the third sub-collecting part 2043 of the second collecting part 204 .
  • the circuit system When the optical device detects that the second droplet 206 is the above-mentioned situation (a), the circuit system is immediately triggered to apply a voltage (for example, 800-1000V) to the electrode structure at the sorting channel 203, and the second droplet containing a single target cell
  • a voltage for example, 800-1000V
  • the droplet 206 is polarized, and under the action of the electric field, the second droplet 206 containing a single target cell is deflected upward into the first branch 2031 of the sorting channel 203 or deflected downward into the second branch 2032 of the sorting channel 203 , and then flow into the first sub-collection part 2041 or the second sub-collection part 2042 of the second collection part 204 , respectively.
  • the target droplet can be sorted out from the second droplet 206 .
  • the method for sorting target droplets is implemented based on the structures of the first chip 100 and the second chip 200 described in the previous embodiment, therefore, the method can be similar to the first chip 100 and the second chip 200 described in the previous embodiment. Has basically the same technical effect. For the sake of brevity, the description of the technical effects of the method of sorting target droplets is not repeated here.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Hematology (AREA)
  • Analytical Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Dispersion Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Sustainable Development (AREA)
  • Medicinal Chemistry (AREA)
  • Fluid Mechanics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Physics & Mathematics (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

本公开提供了一种芯片、包括该芯片的微流控装置、以及分选目标液滴的方法。该芯片包括：容纳第一流体的第一容纳部；容纳第二流体的第二容纳部；输送流道，包括第一流道和第二流道，第一流道与第一容纳部连通且第二流道与第二容纳部连通，第一流道与第二流道在汇合点处彼此交叉且连通；以及至少一个收集部。第一流道的一部分包括汇合点并被汇合点分割为两个区段，在每一个区段中，该区段沿着远离汇合点的第一方向逐渐变粗。第二流道包括汇合点并被汇合点分割为两个区段，在每一个区段中，该区段沿着远离汇合点的第二方向逐渐变粗。

Description

芯片、微流控装置以及分选目标液滴的方法 技术领域
本公开涉及生物医学检测领域，尤其涉及一种芯片、包括该芯片的微流控装置以及分选目标液滴的方法。
背景技术
细胞是生物体基本的结构和功能单位。由于各个细胞之间通常存在高度异质性，因此通过对细胞群体分析而获得的数据均值实质上掩盖了单个细胞之间的差异性，因而不能表征基因表达的随机性本质，无法反映真实情况。随着生命科学和精准医学的不断发展，细胞群体分析逐渐向单细胞分析发展。单细胞分析的一个关键技术是如何能够从高度异质的包含众多细胞的生物样品中分离出单个细胞。单细胞分选技术为单细胞分析、癌症早期诊断和伴随诊断等热门医学领域提供了新的选择。
发明内容
根据本公开的一方面，提供了一种芯片，包括：第一容纳部，配置为容纳第一流体；第二容纳部，配置为容纳第二流体，所述第二流体包括细胞悬液；输送流道，包括第一流道和第二流道，所述第一流道与所述第一容纳部连通且所述第二流道与所述第二容纳部连通，所述第一流道与所述第二流道在汇合点处彼此交叉且连通，所述输送流道配置为使所述第一流体和所述第二流体在所述汇合点处汇合并生成至少一个液滴，所述至少一个液滴的至少一部分中的每一个包括源自所述细胞悬液的单个细胞；以及至少一个收集部，配置为收集所述至少一个液滴。所述第一流道的一部分包括所述汇合点并被所述汇合点分割为第一区段和第二区段，在所述第一区段和所述第二区段中的每一个区段中，该区段的第一横截面的面积沿着远离所述汇合点的第一方向逐渐增大，所述第一横截面垂直于所述第一方向。所述第二流道包括所述汇合点并被所述汇合点分割为第三区段和第四区段，在所述第三区段和所述第四区段中的每一个区段中，该区段的第二横截面的面积沿着远离所述汇合点的第二方向逐渐增大，所述第二横截面垂直 于所述第二方向。
在一些实施例中，所述第一流道的一部分包括第一子部分、包括所述汇合点的第二子部分、以及第三子部分，所述第一子部分属于所述第一区段，所述第三子部分属于所述第二区段，所述第二子部分跨越所述第一区段和所述第二区段并且位于所述第一子部分和所述第三子部分之间，所述第一子部分和所述第三子部分的所述第一横截面的面积均大于所述第二子部分的所述第一横截面的面积。
在一些实施例中，所述第一流道的第二子部分在所述汇合点处的所述第一横截面的尺寸配置为允许具有特定粒径的第一流体在其内部流动，所述第一流体的特定粒径大于所述单个细胞的粒径。
在一些实施例中，所述第二流道包括第一部分、第二部分以及第三部分，所述第一部分和第二部分属于所述第三区段，所述第三部分属于所述第四区段。所述第二流道的第一部分的第一端与所述第二容纳部连通，所述第二流道的第一部分的第二端与所述第二流道的第二部分的第一端连通；所述第二流道的第二部分的第二端与所述第二流道的第三部分的第一端连通，且所述第二流道的第二部分的第二端与所述第二流道的第三部分的第一端均位于所述汇合点处；所述第二流道的第三部分的第二端与所述至少一个收集部连通。所述第二流道的第一部分和第三部分的所述第二横截面的面积均大于所述第二流道的第二部分的所述第二横截面的面积。
在一些实施例中，所述第二流道的第二部分的所述第二横截面的尺寸配置为允许具有特定粒径的第二流体在其内部流动，所述第二流体的特定粒径大于所述单个细胞的1倍粒径且小于所述单个细胞的2倍粒径。
在一些实施例中，所述第二流道的第三部分的所述第二横截面的面积沿着从所述第二流道的第三部分的第一端到第二端的方向逐渐增大。
在一些实施例中，所述第一流道的第二子部分在所述汇合点处的所述第一横截面的面积大于或等于所述第二流道的第二部分和第三部分在所述汇合点处的所述第二横截面的面积。
在一些实施例中，所述第二容纳部包括至少一个子容纳部。
在一些实施例中，所述第二流体包括第一试剂和第二试剂，所述 第一试剂包括所述细胞悬液。所述第二容纳部包括彼此分离的第一子容纳部和第二子容纳部，所述第一子容纳部配置为容纳所述第一试剂，所述第二子容纳部配置为容纳所述第二试剂。
在一些实施例中，所述第二流道的第一部分包括第一分支和第二分支，所述第一分支与所述第一子容纳部连通，所述第二分支与所述第二子容纳部连通，并且所述第一分支与所述第二分支在第一点处彼此相交且连通。所述第一分支与所述第二分支在所述第一点处的夹角为锐角。
在一些实施例中，所述至少一个收集部包括第一收集部，所述第一收集部配置为经由所述输送流道收集所述至少一个液滴。
在一些实施例中，所述至少一个收集部包括第二收集部，所述第二收集部包括至少两个子收集部，所述至少两个子收集部配置为经由所述输送流道收集所述至少一个液滴。
在一些实施例中，所述至少一个收集部包括第一收集部和第二收集部，所述第二收集部包括至少两个子收集部。所述第一收集部与所述第二收集部连通，并且所述第一收集部位于所述汇合点与所述第二收集部之间。
在一些实施例中，该芯片还包括电极结构，所述电极结构位于所述汇合点和所述第二收集部之间。
在一些实施例中，所述输送流道还包括分选流道，所述分选流道位于所述汇合点和所述第二收集部之间。所述分选流道包括至少两个分支，所述至少两个分支中的一个分支配置为从所述至少一个液滴中分选出非目标液滴，所述至少两个分支中的其余分支配置为从所述至少一个液滴中分选出所述目标液滴。所述第二收集部的至少两个子收集部与所述分选流道的至少两个分支一一对应，所述至少两个子收集部中的一个子收集部与所述分选流道的至少两个分支中的一个分支连通且配置为收集所述非目标液滴，所述至少两个子收集部中的其余子收集部与所述分选流道的至少两个分支中的其余分支分别连通且配置为收集所述目标液滴。
在一些实施例中，所述分选流道的至少两个分支包括配置为从所述至少一个液滴中分选出所述目标液滴的第一分支和第二分支以及配置为从所述至少一个液滴中分选出所述非目标液滴的第三分支。所述 第一分支、所述第二分支以及所述第三分支相交于第二点且所述第三分支位于所述第一分支和所述第二分支之间，所述第一分支与所述第三分支在所述第二点处的第一夹角和所述第二分支与所述第三分支在所述第二点处的第二夹角均大于10°。
在一些实施例中，所述分选流道的第一分支与第三分支之间的空间限定第一直角三角形，所述分选流道的第二分支与第三分支之间的空间限定第二直角三角形，所述第一夹角面向所述第一直角三角形的第一直角边，所述第二夹角面向所述第二直角三角形的第二直角边。所述第一直角三角形的第一直角边和所述第二直角三角形的第二直角边的长度均大于或等于单个所述液滴的粒径。
在一些实施例中，所述输送流道的内壁表面具有疏水性。
在一些实施例中，所述第一容纳部和所述第二容纳部的轮廓包括四个倒角。
在一些实施例中，所述倒角的形状包括圆弧状。
在一些实施例中，所述第一容纳部和所述第二容纳部均设置有过滤结构，所述过滤结构包括多个微结构，所述多个微结构中的相邻两个之间的间隙大于所述单个细胞的1倍粒径且小于所述单个细胞的2倍粒径。
在一些实施例中，所述芯片是微流控芯片。
根据本公开的另一方面，提供了一种微流控装置，该微流控装置包括在前面任一个实施例中描述的芯片。
根据本公开的再一方面，提供了一种分选目标液滴的方法，该方法包括以下步骤：向在前面任一个实施例中描述的芯片的第一容纳部和第二容纳部分别提供第一流体和包括细胞悬液的第二流体，使所述第一流体和所述第二流体在所述输送流道的汇合点处汇合并生成至少一个液滴，所述至少一个液滴的至少一部分中的每一个包括源自所述细胞悬液的单个细胞；以及向在前面任一个实施例中描述的芯片施加电压，以从所述至少一个液滴中分选出具有目标属性的目标液滴，所述目标液滴包括所述单个细胞。
在一些实施例中，所述芯片还包括电极结构，所述电极结构位于所述汇合点和所述至少一个收集部之间。向在前面任一个实施例中描述的芯片施加电压以从所述至少一个液滴中分选出具有目标属性的目 标液滴的步骤包括：利用光学设备实时检测所述至少一个液滴的光学信号，响应于所述光学设备检测到具有目标光学信号的液滴，向所述电极结构施加800～1000V的瞬时电压，以从所述至少一个液滴中分选出具有所述目标光学信号的目标液滴，所述目标液滴包括所述单个细胞。
在一些实施例中，在向前面任一个实施例中描述的芯片施加电压的步骤之前，所述方法还包括：将所述芯片中的至少一个液滴转移到其他反应容器中，以进行聚合酶链式反应或荧光染色处理。
在一些实施例中，所述第一流体为油相，所述第二流体为水相，并且所述液滴具有油包水结构。
附图说明
为了更清楚地描述本公开实施例中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本公开的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1A示意性地示出了根据本公开实施例的芯片的正视图；
图1B示意性地示出了根据本公开实施例的芯片的侧视图；
图1C示意性地示出了根据本公开实施例的芯片的后视图；
图1D示意性地示出了根据本公开实施例的芯片的正三轴测视图；
图2示意性地示出了图1A的芯片的局部放大示意图；
图3示意性地示出了图1A的芯片的另一局部放大示意图；
图4示意性地示出了图1A的芯片的又一局部放大示意图；
图5A示意性地示出了根据本公开另一实施例的芯片的正视图；
图5B示意性地示出了根据本公开另一实施例的芯片的侧视图；
图5C示意性地示出了根据本公开另一实施例的芯片的后视图；
图5D示意性地示出了根据本公开另一实施例的芯片的正三轴测视图；
图6示意性地示出了图5A的芯片的局部放大示意图；
图7示意性地示出了根据本公开又一实施例的芯片的正视图；
图8示意性地示出了根据本公开再一实施例的微流控装置的框图；以及
图9示意性地示出了根据本公开又一实施例的分选目标液滴的方法的流程图。
具体实施方式
下面将结合本公开实施例中的附图，对本公开实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例仅仅是本公开一部分实施例，而不是全部的实施例。基于本公开中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本公开保护的范围。
在正式描述本公开实施例的技术方案之前，对本公开实施例中使用的术语做出如下解释和定义，以帮助本领域技术人员能够更加清楚地理解本公开实施例的技术方案。
如本文所使用的，术语“流体”是指能够流动的所有物质，是液体和气体的总称。流体是一种在微小剪切力的作用下能够连续变形的物质。流体可以由单纯的一种物质组成，也可以由多种不同的物质混合而成。流体可以是连续相(例如油相)，也可以是分散相(例如水相)，还可以是连续相和分散相的混合。流体具有易流动性、可压缩性、黏性等特性。
如本文所使用的，术语“油相”是指，根据相似相溶原理，不易溶于水的物质属于油相。例如，将一种物质与水混溶，如果混合后的液体呈现分层或浑浊现象，则该物质属于油相。油可以具有高于或低于水的密度和/或高于或低于水的粘度。例如，液状石蜡、硅油、凡士林、矿物油、全氟化油等均属于油相。
如本文所使用的，术语“水相”是指，根据相似相溶原理，易溶于水的物质属于水相。例如，将一种物质与水混溶，如果混合后的液体呈现透明且均匀的溶液，则该物质属于水相。例如，水、甘油、酒精、丙酮等均属于水相。
如本文所使用的，术语“细胞悬液”是指通过机械或化学方法将细胞从组织上分离并用细胞培养液稀释混匀得到的细胞溶液。细胞悬液中可以包括众多数量的细胞，例如数百个、数千个、数万个、数百万个、数千万个或更多个细胞。细胞悬液中的细胞可以是任何类型的细胞，包括但不限于原核细胞、真核细胞、细菌、真菌、植物、哺乳 动物或其他动物细胞类型、支原体、正常组织细胞、肿瘤细胞或任何其他细胞类型，无论是否衍生来自单细胞或多细胞生物。细胞悬液中的细胞可以包括DNA、RNA、细胞器、蛋白质或其任何组合。
如本文所使用的，术语“A与B连通”是指A元件与B元件互相连接且相通，其允许流体在A元件与B元件之间流动，即，流体可以按照产品设计要求从A元件流动至B元件，或者从B元件流动至A元件。A元件与B元件可以直接连通，即流体可以从A元件直接流动至B元件或从B元件直接流动至A元件而不经过其他中间元件(例如管道)。替代地，A元件与B元件可以间接连通，即流体可以从A元件经由一个或多个中间元件(例如管道)流动至B元件或从B元件经由一个或多个中间元件(例如管道)流动至A元件。
如本文所使用的，术语“聚合酶链式反应(Polymerase Chain Reaction，PCR)”是一种用于放大扩增特定的脱氧核糖核酸(DNA)片段的分子生物学技术，它可以看作是生物体外的特殊DNA复制，其能将微量的DNA大量复制，使其数量大幅增加。PCR的基本原理是，DNA在高温(例如95°左右)时可以发生变性解链变为单链，当温度降至低温(例如60℃左右)时，引物与单链按碱基互补配对原则结合又变为双链。因此，通过温度变化控制DNA的变性和复性，加入设计引物，就可以实现DNA的大量复制。PCR反应包括但不限于数字PCR(digital PCR，dPCR)、定量PCR、实时PCR。dPCR技术可以提供数字化DNA量化信息的定量分析技术，其与微流控技术相结合，可以提供更高的灵敏度和精确度。
如本文所使用的，术语“微流控芯片”是指具有微米尺度微通道的芯片，其可以将生物、化学和医学等领域中所涉及的样本制备、反应、分离、检测等基本操作单元集成到该微尺度芯片上，自动完成反应和分析的全过程。基于微流控芯片的分析检测装置可以具有下列优点：液体流动可控、样品消耗少、检测速度快、操作简便、多功能集成、体积小和便于携带等。
如本文所使用的，术语“XX的粒径”是指物质XX的大小，即物质XX在某个方向上的长度。物质XX可以是单个细胞或单个液滴。例如，当细胞或液滴的形状为球形时，则术语“单个细胞的粒径”是指单个细胞的直径，“单个液滴的粒径”是指单个液滴的直径。当细胞或 液滴的形状为棒状时，则术语“单个细胞的粒径”是指单个细胞在较短边的方向上的长度，“单个液滴的粒径”是指单个液滴在较短边的方向上的长度。
本申请的发明人发现，在常规技术中，用于分选单细胞的方法主要分为两类：一类是使用荧光流式细胞分选仪(Fluorescence Activated Cell Sorting，FACS)来自动分选单细胞，但是该荧光流式细胞分选仪价格昂贵且维护成本很高；另一类是通过专业操作人员来手动分选单细胞，但是该手动分选方法不仅依赖操作人员的技巧和熟练程度，而且也需要显微移液平台、光镊等大中型仪器。此外，单细胞分选过程极易受到环境中飘浮的气溶胶、微生物的污染，这种污染通常难以在后续的检测环节去除。因此，现有单细胞分选方法存在诸如成本昂贵、对操作人员技能要求高、所需仪器设备受场地限制、易受到环境污染等缺点。
有鉴于此，本公开的实施例提供了一种芯片、包括该芯片的微流控装置以及分选目标液滴的方法。该芯片可以用来制备包含源自细胞悬液的单个细胞的液滴，或者可以用来从所制备的液滴中分选出目标液滴，或者可以用来制备包含源自细胞悬液的单个细胞的液滴以及从所制备的液滴中分选出目标液滴。该芯片可以是微流控芯片。通过该芯片可以实现单细胞的制备和/或分选，可以在有效提高自动化操作的同时降低使用成本，并且可以消除交叉污染，提高细胞存活率。
图1A-图1D示出了根据本公开一些实施例的芯片100(以下称为第一芯片100)，该第一芯片100可以用来制备包括单个细胞的液滴。图1A是该第一芯片100的正视图，图1B是该第一芯片100的侧视图，图1C是该第一芯片100的后视图，图1D是该第一芯片100的正三轴测视图。如图1A-图1D所示，该第一芯片100包括：第一容纳部101，其配置为容纳第一流体107；第二容纳部102，其配置为容纳第二流体108，该第二流体108包括细胞悬液；输送流道103(以下称为第一输送流道103)，包括第一流道1031和第二流道1032，第一流道1031与第一容纳部101连通且第二流道1032与第二容纳部102连通，第一流道1031与第二流道1032在汇合点105处彼此交叉且连通，输送流道103配置为使第一流体107和第二流体108在汇合点105处汇合并生成至少一个液滴110(以下称为第一液滴110)，该至少一个第一液 滴110的至少一部分中的每一个包括源自细胞悬液的单个细胞；以及至少一个收集部104(以下称为第一收集部104)，其配置为收集至少一个第一液滴110。第一流道1031的一部分1031B包括汇合点105并被汇合点105分割为第一区段(例如图1A中的1031B的位于汇合点105上方的区段)和第二区段(例如图1A中的1031B的位于汇合点105下方的区段)，在第一区段和第二区段中的每一个区段中，该区段的第一横截面的面积沿着远离汇合点105的第一方向逐渐增大，第一横截面垂直于第一方向。第二流道1032包括汇合点105并被汇合点105分割为第三区段(例如图1A中的1032的位于汇合点105左侧的区段)和第四区段(例如图1A中的1032的位于汇合点105右侧的区段)，在第三区段和第四区段中的每一个区段中，该区段的第二横截面的面积沿着远离汇合点105的第二方向逐渐增大，第二横截面垂直于第二方向。也就是说，在第一流道1031的一部分1031B中，沿着从汇合点105向上的方向，第一流道1031逐渐变粗；沿着从汇合点105向下的方向，第一流道1031也逐渐变粗。在第二流道1032中，沿着从汇合点105向左的方向，第二流道1032逐渐变粗；沿着从汇合点105向右的方向，第二流道1032也逐渐变粗。
通过上文描述可知，第一横截面是指第一流道1031的一部分1031B在垂直于第一流体107的流动方向上的横截面；第二横截面是指第二流道1032在垂直于第二流体108的流动方向上的横截面。
通过对第一芯片100的第一流道1031和第二流道1032的结构设计，使得该第一芯片100有利于生成包含单个细胞的第一液滴110。通过该第一芯片100可以制备得到包含单个细胞的第一液滴110，由于该第一芯片100集成化程度高，因此可以自动完成包含单个细胞的第一液滴110的制备而无需操作人员的手动操作，因此可以有效提高操作的自动化程度。由于第一流体107和第二流体108完全在第一输送流道103内流动，与外界环境完全隔离，因此可以避免受到环境中飘浮的气溶胶、微生物等的污染。此外，由于整个制备过程比较温和，并且从细胞悬液中分离出的单个细胞被液滴包裹和保护，因此可以有效提高细胞的存活率。
在图1A所示的第一芯片100中，第一容纳部101包括进样口1，外部设备(例如微型流量泵)与该进样口1连接并通过该进样口1将 第一流体107注入到第一容纳部101内。第一流体107为连续相(例如油相)液体，其例如可以是矿物油、全氟化油等任意适当的流体。可选地，第一流体107中可以混合有表面活性剂，表面活性剂有利于稳定所得的第一液滴110，例如，抑制所得液滴110的后续聚结。当第一流体107为全氟化油时，表面活性剂可以是全氟化表面活性剂。第二容纳部102包括彼此分离的第一子容纳部1021和第二子容纳部1022。第一子容纳部1021包括进样口2，外部设备(例如微型流量泵)与该进样口2连接并通过该进样口2将细胞悬液109-1注入到第一子容纳部1021内。第二子容纳部1022包括进样口3，外部设备(例如微型流量泵)与该进样口3连接并通过该进样口3将生化反应试剂109-2注入到第二子容纳部1022内。可以根据不同的生化反应来采取不同的生化反应试剂，本公开的实施例对生化反应试剂109-2的化学成分不作具体限定。第一流体107和包括细胞悬液109-1与生化反应试剂109-2的第二流体108在第一输送流道103的汇合点105处汇合并生成多个第一液滴110，多个第一液滴110沿着第一输送流道103流入到第一收集部104中。第一收集部104包括多个出样口4，出样口4用于与外部设备连接以将第一收集部104中的多个第一液滴110转移至其他容器中，以供后续操作使用。
需要说明的是，虽然图1A中示出了细胞悬液109-1容纳在第一子容纳部1021内，生化反应试剂109-2容纳在与第一子容纳部1021分离的第二子容纳部1022内，但是这仅是一个示例，本公开的实施例并不仅限于此。在替代的实施例中，细胞悬液109-1和生化反应试剂109-2可以预先混合并容纳在同一个容纳部内。
继续参考图1A-图1D，第一输送流道103包括第一流道1031和第二流道1032。第一流道1031与第一容纳部101连通且供第一流体107在其内部流动。第二流道1032与第二容纳部102连通且供第二流体108在其内部流动。第一流道1031和第二流道1032在汇合点105处相交且连通。在第一输送流道103的部分区段中(例如在第一输送流道103的第一流道1031的第二部分1031B和第二流道1032中)，第一输送流道103的横截面的面积先逐渐减小后逐渐增大，也即，在该区段中，第一输送流道103由粗变窄再变粗。这在下文中将详细介绍。
第一流道1031包括第一部分1031A和第二部分1031B，第一流道 1031的第一部分1031A和第二部分1031B共同围成一个闭合的五边形，该五边形关于汇合点105所在的水平轴线近似呈轴对称。第一容纳部101内的第一流体107的一部分沿第一流道1031的位于水平轴线上方的部分流道流到汇合点105处，第一容纳部101内的第一流体107的另一部分沿第一流道1031的位于水平轴线下方的部分流道也流到汇合点105处。
图2是图1A的AA区域的放大图。如图2所示，第一流道1031的第二部分1031B包括沿第一方向(图中的竖直方向)依次布置的第一子部分1031B-1、第二子部分1031B-2以及第三子部分1031B-3，第二子部分1031B-2位于第一子部分1031B-1和第三子部分1031B-3之间并且包括汇合点105。第一子部分1031B-1属于前文描述的第一区段，第三子部分1031B-3属于前文描述的第二区段，第二子部分1031B-2跨越第一区段和第二区段。第一子部分1031B-1和第三子部分1031B-3的第一横截面的面积均大于第二子部分1031B-2的第一横截面的面积，也即，沿着从第一子部分1031B-1到第三子部分1031B-3的方向，第一流道1031先逐渐变细后逐渐变粗，这样使得第一流道1031呈现上下(第一子部分1031B-1和第三子部分1031B-3)粗中间(第二子部分1031B-2)细的形状。通过这样的形状设计，当第一流道1031内的第一流体107从第一子部分1031B-1流向第二子部分1031B-2时或者从第三子部分1031B-3流向第二子部分1031B-2时，由于流道越来越细，因此第一流体107在第一流道1031内的流速变大，从而可以增加第一流体107的压力，促进第一子部分1031B-1和第三子部分1031B-3内的第一流体107向第二子部分1031B-2的汇合点105流动，并在汇合点105处汇集。这样可以提供充足的第一流体107，以便于后续形成第一液滴110。第一流道1031的第二部分1031B的第一子部分1031B-1、第二子部分1031B-2以及第三子部分1031B-3的第一横截面的形状可以是圆形、正方形、矩形、规则多边形、不规则形状等，本公开的实施例对此不做限定。
第一流道1031的第二子部分1031B-2在汇合点105处的第一横截面的尺寸配置为允许具有特定粒径的第一流体107在其内部流动，第一流体107的特定粒径大于单个细胞的粒径。也即，第一流道1031的第二子部分1031B-2在汇合点105处的第一横截面的宽度大于单个细 胞的粒径。在一个示例中，细胞悬液中的每个细胞的粒径大约为10μm，第一流道1031的第二子部分1031B-2在汇合点105处的横截面的宽度大于10μm，例如略微大于10μm。这里的“略微大于10μm”是指第一流道1031的第二子部分1031B-2在汇合点105处的第一横截面的宽度大于10μm，但是小于20μm，也即该宽度大于单个细胞的粒径但是小于两个细胞的粒径的和。需要说明的是，短句“第一流道1031的第二子部分1031B-2在汇合点105处的第一横截面的宽度”可以理解为，当第一流道1031的第二子部分1031B-2在汇合点105处的第一横截面的形状为圆形时，则该第一横截面的宽度为该圆形的直径；当第一流道1031的第二子部分1031B-2在汇合点105处的第一横截面的形状为正方形时，则该第一横截面的宽度为该正方形的边长；当第一流道1031的第二子部分1031B-2在汇合点105处的第一横截面的形状为矩形时，则该第一横截面的宽度为该矩形的短边的长度；当第一流道1031的第二子部分1031B-2在汇合点105处的第一横截面的形状为规则多边形时，则该第一横截面的宽度为该规则多边形的最远两个顶点之间的距离。在一个示例中，当第一流道1031的第二子部分1031B-2在汇合点105处的第一横截面为圆形且单个细胞的形状为圆球状时，则第一流道1031的第二子部分1031B-2在汇合点105处的第一横截面的宽度大于单个细胞的粒径应被理解为第一流道1031的第二子部分1031B-2在汇合点105处的直径大于单个细胞的直径。通过这种设计方式，当第一流道1031内的第一流体107从第一子部分1031B-1流向第二子部分1031B-2时或者从第三子部分1031B-3流向第二子部分1031B-2时，可以使第一流体107在汇合点105附近形成依次排列的单排流体颗粒，该单排流体颗粒中的每个颗粒的粒径大于单个细胞的粒径且小于两个细胞的粒径的和。这样，可以使由第一流体107形成的每个颗粒的粒径略微大于单个细胞的粒径，从而可以更好地包裹单个细胞，起到更好的包封效果。而且这样的设计还可以增大第一流体107在汇合点105处的流速，有利于第一液滴110的形成。
继续参考图1A-图1D和图2，第二流道1032包括沿不同于第一方向的第二方向(图中的水平方向)依次布置的第一部分1032A、第二部分1032B以及第三部分1032C。第一部分1032A和第二部分1032B属于前文描述的第三区段，第三部分1032C属于前文描述的第四区段。 第二流道1032的第一部分1032A的第一端与第二容纳部102连通，第二流道1032的第一部分1032A的第二端与第二流道1032的第二部分1032B的第一端连通；第二流道1032的第二部分1032B的第二端与第二流道1032的第三部分1032C的第一端连通，且第二流道1032的第二部分1032B的第二端与第二流道1032的第三部分1032C的第一端均位于汇合点105处；第二流道1032的第三部分1032C的第二端与第一收集部104连通。如图所示，第二流道1032的第一部分1032A包括第一分支和第二分支，第一分支与第二容纳部102的第一子容纳部1021连通并且配置为使细胞悬液109-1在其内部流动，第二分支与第二容纳部102的第二子容纳部1022连通并且配置为使生化反应试剂109-2在其内部流动。第一分支与第二分支在第一点106处彼此相交且连通，并且第一分支与第二分支在第一点106处的夹角α为锐角。在一个示例中，第一分支与第二分支在第一点106处的夹角α约为60度。第一分支与第二分支这样的夹角设计，一方面可以保证第一分支内的细胞悬液109-1和第二分支内的生化反应试剂109-2有足够向前(朝向汇合点105的方向)的流速，缓冲压力；另一方面还可以保证细胞悬液109-1和生化反应试剂109-2能够在第一点106处充分混合；再一方面还可以减少混合溶液在该流道内的死体积，提高第一分支与第二分支的储液精度。需要说明的是，短语“第一分支与第二分支在第一点106处的夹角α约为60度”包含了第一分支与第二分支在第一点106处的夹角α大于60度、第一分支与第二分支在第一点106处的夹角α小于60度、第一分支与第二分支在第一点106处的夹角α等于60度等几种情况。
第二流道1032的第一部分1032A和第三部分1032C的第二横截面的面积均大于第二流道1032的第二部分1032B的第二横截面的面积。也即，第二流道1032的第一部分1032A的第一分支和第二分支的第二横截面的面积均大于第二流道1032的第二部分1032B的第二横截面的面积，并且第二流道1032的第三部分1032C的第二横截面的面积大于第二流道1032的第二部分1032B的第二横截面的面积。沿着从第二流道1032的第一部分1032A到第三部分1032C的方向，第二流道1032由粗变细再变粗。与第一流道1031相似，第二流道1032的第一部分1032A、第二部分1032B以及第三部分1032C的第二横截面的形状可以是圆形、正方形、矩形、规则多边形、不规则形状等，本公开的实 施例对此不做限定。
第二流道1032的第二部分1032B的第二横截面的尺寸配置为允许具有特定粒径的第二流体108在其内部流动，第二流体108的特定粒径大于单个细胞的1倍粒径且小于单个细胞的2倍粒径。也就是说，第二流道1032的第二部分1032B的第二横截面的宽度大于单个细胞的1倍粒径且小于单个细胞的2倍粒径。在一个示例中，当第二流道1032的第二部分1032B的第二横截面为圆形且单个细胞的形状为圆球形时，则第二流道1032的第二部分1032B的第二横截面的宽度大于单个细胞的1倍粒径且小于单个细胞的2倍粒径应被理解为第二流道1032的第二部分1032B的直径大于单个细胞的1倍直径且小于单个细胞的2倍直径。在这种情况下，第二流道1032的第二部分1032B的直径可以是单个细胞的直径的1.1倍、1.2倍、1.3倍、1.4倍、1.5倍、1.6倍、1.7倍、1.8倍、1.9倍等。当细胞悬液109-1和生化反应试剂109-2在第一点106处混合后并向前(朝向汇合点105)流动时，通过使第二部分1032B的第二横截面的宽度大于单个细胞的1倍粒径且小于2倍粒径，使得该混合溶液在第二部分1032B内排列成单排的单个细胞串，如图2所示。也就是说，第二流道1032的第二部分1032B的第二横截面的宽度仅允许在其宽度方向上容纳单个细胞，而无法容纳两个并排的细胞。当该单排的单个细胞串移动到汇合点105时，在第一流道1031的第二部分1031B内的第一流体107的压力下，细胞串中最靠近汇合点105的一个细胞(也即细胞串中最靠前的一个细胞)与该细胞串分离，该分离的一个细胞与第一流体107中的单个颗粒在汇合点105处结合，进而形成包含单个细胞111的第一液滴110。图2的右上角示出了在汇合点105处生成的包含单个细胞111的第一液滴110的放大图。如前所述，第一流体107是油相，第二流体108(也即细胞悬液109-1和生化反应试剂109-2的混合溶液)是水相，因此，所形成的第一液滴110具有油包水结构，即油相的第一流体107将水相的第二流体108包裹在内。
如图所示，第二流道1032的第三部分1032C的第二横截面的面积沿其第一端到第二端的方向逐渐增大，也即，第二流道1032的第三部分1032C沿其第一端到第二端的方向逐渐变粗。这样设计的目的是使得第一液滴110在沿着第二流道1032的第三部分1032C向前移动时逐 渐变大，从而便于液滴相稳定。第一流道1031的第二子部分1031B-2在汇合点105处的第一横截面的面积大于或等于第二流道1032的第二部分1032B和第三部分1032C在汇合点105处的第二横截面的面积。在一个示例中，第一流道1031的第二子部分1031B-2在汇合点105处的第一横截面的面积等于第二流道1032的第二部分1032B和第三部分1032C在汇合点105处的第二横截面的面积。通过这样的设计方式，可以使得在汇合点105处，第一流体107中的单个颗粒的粒径与包含单个细胞111的混合溶液(细胞悬液109-1和生化反应试剂109-2的混合溶液)的粒径大致相等，从而可以精准控制所形成的第一液滴110的大小。
图3是图1A中第一芯片100的第一收集部104的放大示意图。所形成的第一液滴110沿着第二流道1032的第三部分1032C最终流入到第一收集部104中。根据前面的描述，理想情况下，所形成的多个第一液滴110中，每个第一液滴110均包裹有单个细胞111。但是在实际操作中，由于细胞悬液中细胞之间的彼此粘附力、流体的流速、流道的表面设计等因素，并不能完全保证每个第一液滴110内均包括单个细胞111。如图3所示，在所形成的第一液滴110中，一个第一液滴110内可能包括源自细胞悬液109-1的单个细胞111，可能不包括源自细胞悬液109-1的单个细胞111(即“空”液滴)，也可能包括源自细胞悬液109-1的两个细胞111。本公开实施例中的第一芯片100的结构设计(例如第一流道1031和第二流道1032的结构、下文中将描述的过滤结构的设计等)有利于促进每个第一液滴110内仅包括单个细胞111。
在一些实施例中，第一输送流道103的内壁表面经过疏水处理，因此具有疏水性。如前所述，第一输送流道103包括配置为供第一流体107在其内部流动的第一流道1031和配置为供第二流体108在其内部流动的第二流道1032。经过疏水处理的第一流道1031可以有利于第一流体107在其内部流动。经过疏水处理的第二流道1032可以促进细胞悬液109-1在第二流道1032的第一部分1032A的第一分支内顺畅地流动而不粘附在内壁表面上，并且可以促进细胞悬液109-1与生化反应试剂109-2的混合溶液在第二流道1032的第二部分1032B和第三部分1032C内顺畅地流动而不粘附在内壁表面上。这样可以精准控制细胞悬 液109-1的用量，有利于细胞悬液109-1与生化反应试剂109-2的均匀混合，从而促进第一液滴110的均匀生成。同时，这也可以提高细胞悬液109-1的利用率，避免对细胞悬液109-1的浪费。
返回继续参考图1A-图1D，第一容纳部101、第二容纳部102的第一子容纳部1021和第二子容纳部1022的轮廓均包括四个倒角。四个倒角的形状可以是任意适当的形状，例如可以是圆弧状。应当理解，本公开的实施例对倒角的具体尺寸不做限定。图4是第二容纳部102的第一子容纳部1021的放大示意图。以第二容纳部102的第一子容纳部1021为例，如图4所示，第二容纳部102的第一子容纳部1021的轮廓包括四个倒角113，四个倒角113的形状为圆弧状。圆弧状的倒角设计，可以减少细胞悬液109-1在第一子容纳部1021内的死体积，提高第一子容纳部1021的储液精度。这里的“死体积”指在试剂入样过程中不可控的体积。具体地，如果第一子容纳部1021的四个角为直角而非圆弧状的倒角，由于液滴表面张力的存在，细胞悬液109-1在第一子容纳部1021的四个直角的位置处并不是直角形状，即细胞悬液109-1不能与第一子容纳部1021的形状完美匹配，无法充满第一子容纳部1021的四个直角所占的空间。因而，细胞悬液109-1的形状和体积会发生变化，并且这种形状和体积的变化存在一定的随机性，从而引入死体积。这可能导致第一芯片100的第一子容纳部1021在每次操作时都可能与上次操作容纳不同体积的细胞悬液109-1，从而导致无法精确控制细胞悬液109-1的用量。而在本公开的实施例中，第一子容纳部1021的四个角113设计为圆弧状的倒角，可以使细胞悬液109-1与第一子容纳部1021的形状完美匹配，尤其是可以使细胞悬液109-1充满第一子容纳部1021的四个圆弧状倒角所占的空间，从而可以有效降低甚至避免第一子容纳部1021的容纳体积的差异，提高对细胞悬液109-1的操控精度。
类似地，第一容纳部101的四个圆弧状的倒角可以减少第一流体107在第一容纳部101内的死体积，提高第一容纳部101的储液精度。第二容纳部102的第二子容纳部1022的四个圆弧状的倒角可以减少生化反应试剂109-2在第二子容纳部1022内的死体积，提高第二子容纳部1022的储液精度。
继续参考图1A-图1D以及图4，第一芯片100的第一容纳部101 和第二容纳部102均设置有过滤结构112，即，第一芯片100的第一容纳部101、第二容纳部102的第一子容纳部1021、第二子容纳部1022均设置有过滤结构112。由于第一容纳部101、第一子容纳部1021、第二子容纳部1022的过滤结构112的构造完全相同，因此，下面以图4示出的第一子容纳部1021内的过滤结构112为例，来详细描述过滤结构112的结构和功能。
如图4所示，过滤结构112包括多个彼此间隔开的微结构，相邻两个微结构112-1和112-2之间的间隙d大于单个细胞的1倍粒径且小于单个细胞的2倍粒径。在一些实施例中，源自细胞悬液109-1的单个细胞的粒径大约为10μm左右，相应地，相邻两个微结构112-1和112-2之间的间隙d大于10μm且小于20μm。过滤结构112的多个微结构的高度可以完全相同，也可以完全不同，还可以仅部分地相同，具体的高度可以根据产品需求而灵活设计，本公开的实施例对此不做具体限定。在一些实施例中，每个微柱的高度约为100-200μm。在平行于第一子容纳部1021所在平面的方向上，每个微柱的横截面的形状可以是任意适当的形状，诸如菱形、正方形、矩形、圆形、椭圆形、正多边形、不规则形状等，本公开的实施例对此不做具体限定。
在第一芯片100的操作期间，第一子容纳部1021内的细胞悬液109-1在过滤结构112的相邻微结构之间的间隙流过，然后流入第二流道1032的第一部分1032A的第一分支中。由于相邻两个微结构之间的间隙d大于单个细胞的1倍粒径且小于单个细胞的2倍粒径，因此当细胞悬液109-1从相邻微结构之间的间隙流过时，一方面可以阻挡细胞悬液109-1中的过大杂物(例如粒径大于单个细胞2倍粒径的杂物，诸如灰尘、盐析物质等)流入后续的流道中，从而避免过大杂物堵塞流道，影响细胞悬液109-1的正常流动；另一方面在相邻微结构对细胞悬液109-1的作用力下以及相邻微结构之间的间隙对细胞悬液109-1的尺寸的筛选下，细胞悬液109-1中彼此粘连在一起的多个细胞(例如彼此粘连的两个细胞、三个细胞或更多个细胞)可以被分隔开而成为多个彼此分离的单个细胞，从而有利于后续包含单个细胞的第一液滴110的生成，降低单颗第一液滴110内包含两个或更多个细胞的概率。
第一容纳部101和第二子容纳部1022内的过滤结构112的结构可以参考上面关于第一子容纳部1021内的过滤结构的描述，为了简洁起 见，在此不再赘述。在第一芯片100的操作期间，第一容纳部101内的第一流体107在过滤结构112的相邻微结构之间的间隙流过，然后流入第一输送流道103的第一流道1031中。当第一流体107从过滤结构112的相邻微结构之间的间隙流过时，可以阻挡第一流体107中的过大杂物(例如粒径大于单个细胞2倍粒径的杂物，诸如灰尘、盐析物质等)流入第一流道1031中，从而避免过大杂物堵塞第一流道1031，影响第一流体107的正常流动。在第一芯片100的操作期间，第二子容纳部1022内的生化反应试剂109-2在过滤结构112的相邻微结构之间的间隙流过，然后流入第一输送流道103的第二流道1032的第一部分1032A的第二分支中。当生化反应试剂109-2从过滤结构112的相邻微结构之间的间隙流过时，可以阻挡生化反应试剂109-2中的过大杂物(例如粒径大于单个细胞2倍粒径的杂物，诸如灰尘、盐析物质等)流入第二流道1032的第一部分1032A的第二分支中，从而避免过大杂物堵塞第二流道1032，影响生化反应试剂109-2的正常流动。
在前面任一个实施例中描述的第一芯片100可以是微流控芯片。微流控芯片具有诸如液体流动可控、样品消耗少、检测速度快、操作简便、多功能集成、体积小和便于携带等优点。除了具有上述优点之外，在本公开的实施例中，通过对第一芯片100的第一输送流道103的优化设计，可以促进从细胞悬液109-1中分离出单个细胞，从而可以促进生成的每个第一液滴110包含单个细胞，降低单个第一液滴110内包含两个或更多个细胞的概率；通过优化过滤结构112的相邻微结构之间的间隙，可以进一步促进生成的每个第一液滴110内包含单个细胞；通过将第一容纳部101和第二容纳部102的四个边角设计成圆弧状的倒角，可以避免容纳部内的死体积，提升容纳部的储液精度；通过将第一输送流道103的内壁表面进行疏水处理，可以减少甚至避免流道内的流体粘附到流道的内壁表面上，从而可以精准控制流体的用量，避免试剂的浪费，促进第一液滴110的均匀生成。第一液滴110可以构成对单个细胞生化反应的微反应器，同时也构成了后续单细胞分选的液滴载体。另外，由于该第一芯片100集成化程度高，因此可以自动完成包含单个细胞的第一液滴110的制备而无需操作人员的手动操作，因此可以有效提高操作的自动化程度。由于第一流体107和第二流体108完全在第一输送流道103内流动，与外界环境完全隔离， 因此可以避免受到环境中飘浮的气溶胶、微生物等的污染。此外，由于整个制备过程比较温和，并且从细胞悬液中分离出的单个细胞被液滴包裹和保护，因此可以有效提高细胞的存活率。
本公开的另一些实施例提供了芯片200(以下称为第二芯片200)，该第二芯片200可以用来分选目标液滴。图5A-图5D示出了第二芯片200，其中，图5A是该第二芯片200的正视图，图5B是该第二芯片200的侧视图，图5C是该第二芯片200的后视图，以及图5D是该第二芯片200的正三轴测视图。如图5A-图5D所示，该第二芯片200包括：第三容纳部201，其配置为容纳第三流体205；第四容纳部202，其配置为容纳第四流体，该第四流体包括细胞悬液；输送流道208(以下称为第二输送流道208)，包括第三流道2081和第四流道2082，第三流道2081与第三容纳部201连通且第四流道2082与第四容纳部202连通，第三流道2081与第四流道2082在汇合点209处彼此交叉且连通，第二输送流道208配置为使第三流体205和第四流体在汇合点209处汇合并生成至少一个液滴206(以下称为第二液滴206)，该至少一个第二液滴206的至少一部分中的每一个包括源自细胞悬液的单个细胞；以及至少一个收集部204(以下称为第二收集部204)，其配置为收集至少一个第二液滴206。第三流道2081的一部分(即图5A中的第三流道2081的竖直部分)包括汇合点209并被汇合点209分割为第一区段(例如图5A中的第三流道2081的位于汇合点209上方的区段)和第二区段(例如图5A中的第三流道2081的位于汇合点209下方的区段)，在第一区段和第二区段中的每一个区段中，该区段的第一横截面的面积沿着远离汇合点209的第一方向逐渐增大，第一横截面垂直于第一方向。第四流道2082包括汇合点209并被汇合点209分割为第三区段(例如图5A中的2082的位于汇合点209左侧的区段)和第四区段(例如图5A中的2082的位于汇合点209右侧的区段)，在第三区段和第四区段中的每一个区段中，该区段的第二横截面的面积沿着远离汇合点209的第二方向逐渐增大，第二横截面垂直于第二方向。也就是说，在第三流道2081中，沿着从汇合点209向上的方向，第三流道2081逐渐变粗；沿着从汇合点209向下的方向，第三流道2081也逐渐变粗。在第四流道2082中，沿着从汇合点209向左的方向，第四流道2082逐渐变粗；沿着从汇合点209向右的方向，第四流道2082 也逐渐变粗。
需要说明的是，此处的第三流体205以及第四流体仅是为了便于描述第二芯片200而采用的名称，事实上，第三流体205可以与第一流体107完全相同，第四流体可以与第二流体108(包括细胞悬液109-1和生化反应试剂109-2)完全相同。当然，第四流体也可以与第二流体108不完全相同。在一个示例中，第四流体是前文所述的第一液滴110。
通过上文描述可知，第三流道2081的一部分(即图5A中的第三流道2081的竖直部分)的第一横截面是指第三流道2081的一部分在垂直于第三流体205的流动方向上的横截面；第四流道2082的第二横截面是指第四流道2082在垂直于第四流体的流动方向上的横截面。
第二输送流道208还包括分选流道203，分选流道203配置为从至少一个第二液滴206中分选出目标液滴，该目标液滴包括单个细胞。
如图5A-图5D所示，第三容纳部201包括进样口5，外部设备(例如微型流量泵)与该进样口5连接并通过该进样口5将第三流体205注入到第三容纳部201内。第三流体205为连续相(例如油相)液体，其可以与第一流体107具有相同的化学成分。第三流体205例如可以是矿物油、全氟化油等任意适当的流体。可选地，第三流体205中可以混合有表面活性剂，该表面活性剂有利于稳定后续生成的第二液滴206，例如，抑制第二液滴206的后续聚结。当第三流体205为全氟化油时，表面活性剂可以是全氟化表面活性剂。
第四容纳部202包括进样口6，外部设备(例如微型流量泵)与该进样口6连接并通过该进样口6将第四流体(例如前文描述的第一液滴110)注入到第四容纳部202内。在将第一液滴110注入到第二芯片200之前，可以先将第一液滴110转移到另外的设备进行相应的生化处理(例如PCR扩增、染色等)，然后将处理后的第一液滴110注入到第二芯片200的第二容纳部202内。
与第一芯片100的第一容纳部101和第二容纳部102相似，第二芯片200的第三容纳部201和第四容纳部202的轮廓也均包括四个倒角，该四个倒角的形状可以是圆弧状或任何其他合适的形状。第三容纳部201和第四容纳部202的倒角设计，可以减少第三流体205和第一液滴110分别在第三容纳部201和第四容纳部202内的死体积，从而可以提高第三容纳部201和第四容纳部202的储液精度。
与第一芯片100的第一容纳部101和第二容纳部102相似，第三容纳部201和第四容纳部202也均设置有过滤结构(图中未示出)。第三容纳部201和第四容纳部202内的过滤结构包括多个彼此间隔开的微结构，相邻两个微结构之间的间隙大于单个第一液滴110的1倍粒径且小于单个第一液滴110的2倍粒径。过滤结构的多个微结构的高度可以完全相同，也可以完全不同，还可以仅部分地相同，具体的高度可以根据产品需求而灵活设计，本公开的实施例对此不做具体限定。在一些实施例中，每个微柱的高度约为100-200μm。在平行于第三容纳部201和第四容纳部202所在平面的方向上，每个微柱的横截面的形状可以是任意适当的形状，诸如菱形、正方形、矩形、圆形、椭圆形、正多边形、不规则形状等，本公开的实施例对此不做具体限定。
在第二芯片200的操作期间，第三容纳部201内的第三流体205在过滤结构的相邻微结构之间的间隙流过，经过过滤后的第三流体205然后流入第二输送流道208的第三流道2081中。当第三流体205从过滤结构的相邻微结构之间的间隙流过时，可以阻挡第三流体205中的过大杂物(例如粒径大于单个第一液滴110的2倍粒径的杂物，诸如灰尘、盐析物质等)流入第三流道2081中，从而避免过大杂物堵塞第三流道2081，影响第三流体205的正常流动。第四容纳部202内的第一液滴110在过滤结构的相邻微结构之间的间隙流过，经过过滤后的第一液滴110然后流入第二输送流道208的第四流道2082中。由于相邻两个微结构之间的间隙大于单个第一液滴110的1倍粒径且小于单个第一液滴110的2倍粒径，因此当第一液滴110从相邻微结构之间的间隙流过时，一方面可以阻挡第一液滴110中的潜在的过大杂物(例如粒径大于单个第一液滴110的2倍粒径的杂物，诸如灰尘、盐析物质等)流入第四流道2082中，从而避免过大杂物堵塞第四流道2082，影响第一液滴110的正常流动；另一方面在相邻微结构对第一液滴110的作用力下以及相邻微结构之间的间隙对多个第一液滴110的尺寸的筛选下，多个第一液滴110中可能存在彼此粘连在一起的多个液滴颗粒(例如彼此粘连的两个液滴颗粒、三个液滴颗粒或更多个液滴颗粒)可以被分隔开而成为多个彼此分离的单个液滴，从而当单个第一液滴110与第三流体205汇合后，可以生成单个第二液滴206。
第二输送流道208包括第三流道2081和第四流道2082，第三流道 2081与第四流道2082相交于汇合点209且在该汇合点209处彼此连通。第三流道2081与第三容纳部201连通且供第三流体205在其内部流动，第四流道2082与第四容纳部202连通且供第一液滴110在其内部流动。第三流体205沿着第三流道2081流动至第三流道2081与第四流道2082的汇合点209处，第一液滴110沿着第四流道2082流动至第三流道2081与第四流道2082的汇合点209处，第三流体205与第一液滴110在该汇合点209处汇合并生成第二液滴206。
图6是图5A示出的分选流道203的区域BB的放大图。结合图5A和图6所示，分选流道203包括第一分支2031、第二分支2032、第三分支2033，第三分支2033位于第一分支2031与第二分支2032之间。第二收集部204包括第一子收集部2041、第二子收集部2042以及第三子收集部2043，第一子收集部2041包括出样口7A，第二子收集部2042包括出样口7B，第三子收集部2043包括出样口7C。第一子收集部2041与分选流道203的第一分支2031连通，第二子收集部2042与分选流道203的第二分支2032连通，第三子收集部2043与分选流道203的第三分支2033连通。分选流道203处设置有电极结构(图中未示出)，电极结构例如可以包括正电极和负电极，用来施加电压以驱动第二液滴206偏转进入分选流道203的相应分支，从而在第二液滴206中分选出目标液滴。第二芯片200还可以包括光学设备(图中未示出，例如荧光显微镜等)，用来识别第二液滴206中的目标液滴。
目标液滴的分选过程大致如下：细胞悬液109-1中含有众多数量的细胞。在这众多数量的细胞中，含有少量的待分析和待检测的目标细胞，即本申请希望分选出的目标细胞(例如外周血样本中的循环肿瘤细胞、稀有细胞、癌细胞等)，以及其他的非目标细胞。由于细胞悬液中的这些目标细胞与非目标细胞含有不同的抗体，因此经荧光染色后在荧光显微镜下会显示出与非目标细胞不同的颜色。可以对细胞悬液109-1进行染色处理，也可以对第一液滴110进行染色，本公开的实施例对染色顺序不做具体限定。经过染色处理后，第一液滴110被注入到第二芯片200的第四容纳部202内，第三流体205和第一液滴110在第二输送流道208内汇合并生成第二液滴206。第二液滴206大致可以分为以下两类：(a)第二液滴206内含有带有目标颜色的单个目标细胞；(b)第二液滴206内含有非目标细胞或不含细胞。第二液滴206 沿着第二输送流道208朝向第二收集部204的方向移动，光学设备对第二输送流道208内的第二液滴206的光学信号(例如颜色)进行实时检测。当光学设备检测到第二液滴206为上述情况(b)时，则不会触发电路系统为电极结构施加电压，第二液滴206沿着第二输送流道208径直流入分选流道203的第三分支2033，然后流入第二收集部204的第三子收集部2043。当光学设备检测到第二液滴206为上述情况(a)时，则立即触发电路系统为分选流道203处的电极结构施加电压(例如800～1000V)，含有单个目标细胞的第二液滴206被极化，在电场作用下，含有单个目标细胞的第二液滴206向上偏转流入分选流道203的第一分支2031中或向下偏转流入分选流道203的第二分支2032中，然后分别流入第二收集部204的第一子收集部2041或第二子收集部2042内。于是，该第二芯片200实现了对目标液滴的分选。
需要说明的是，对细胞悬液的荧光染色处理仅是本公开实施例的一个示例，对细胞悬液的处理方式并不仅限于此，只要能够将细胞悬液中的目标细胞与非目标细胞区分开的处理方式均在本公开的保护范围之内。
需要说明的是，虽然图5A示出了分选流道203包括三个分支，第二收集部204对应地包括三个子收集部，但是本公开的实施例并不限于此。在替代的实施例中，分选流道203可以包括至少两个分支(例如两个分支、四个分支或更多个分支)，该至少两个分支中的一个分支配置为从多个第二液滴206中分选出非目标液滴，该至少两个分支中的其余分支配置为从多个第二液滴206中分选出目标液滴。相应地，第二收集部204可以包括至少两个子收集部，至少两个子收集部与分选流道203的至少两个分支一一对应，至少两个子收集部中的一个子收集部与分选流道203的至少两个分支中的一个分支连通且配置为收集非目标液滴，至少两个子收集部中的其余子收集部与分选流道203的至少两个分支中的其余分支分别连通且配置为收集目标液滴。
继续参考图6，分选流道203的第一分支2031、第二分支2032以及第三分支2033相交于第二点207且第三分支2033位于第一分支2031和第二分支2032之间。第一分支2031与第三分支2033在第二点207处的第一夹角θ1和第二分支2032与第三分支2033在第二点207处的第二夹角θ2均大于10°。分选流道203的第一分支2031与第三分支 2033之间的空间限定第一直角三角形，分选流道203的第二分支2032与第三分支2033之间的空间限定第二直角三角形。第一夹角θ1面向第一直角三角形的第一直角边，第二夹角θ2面向第二直角三角形的第二直角边。第一直角三角形的第一直角边的长度L1和第二直角三角形的第二直角边的长度L2均大于或等于单个第二液滴206的粒径。
需要说明的是，短句“分选流道203的第一分支2031与第三分支2033之间的空间限定第一直角三角形，分选流道203的第二分支2032与第三分支2033之间的空间限定第二直角三角形”中的“限定”是指，如图5A所示，分选流道203的第三分支2033沿着第二方向(即图中的水平方向)延伸；分选流道的第一分支2031从第二点207处与第三分支2033分开并在一拐点处向上延伸；分选流道的第二分支2032从第二点207处与第三分支2033分开并在另一拐点处向下延伸。利用直线连接第一分支2031的拐点和第二分支2032的拐点，该直线与第三分支2033相交于一点。第一分支2031的拐点、第二点207、直线与第三分支2033的交点，将这三点相连构成第一直角三角形，第一夹角θ1面向第一直角三角形的第一直角边，该第一直角边的长度为L1，且该长度L1大于或等于单个第二液滴206的粒径。第二分支2032的拐点、第二点207、直线与第三分支2033的交点，将这三点相连构成第二直角三角形，第二夹角θ2面向第二直角三角形的第二直角边，该第二直角边的长度为L2，且该长度L2大于或等于单个第二液滴206的粒径。
通过将分选流道203的分支之间的夹角设计为大于10°且直角三角形的直角边的长度大于或等于一个第二液滴206的粒径，有利于将目标液滴分选到相应的子收集部中。这是因为：如果分选流道203的分支之间的岔口坡度过小，则目标液滴可能无法正常向上或向下偏转到对应的子收集部中，而可能会误入到中间的子收集部2043中；如果分选流道203的分支之间的岔口坡度过大，则为了实现对目标液滴的分选，需要施加更大的电压以使目标液滴流入到对应的子收集部中，但是过大的电压会击碎或损伤第二液滴206，因而也无法实现对目标液滴的分选。因此，分选流道203的适当的岔口坡度才有利于从第二液滴206中分选出目标液滴。
前面任一个实施例中描述的第二芯片200可以是微流控芯片。在本公开的实施例中，通过使用微流控芯片作为第一芯片100和第二芯 片200，可以有效简化液体流路及其泵阀系统。通过控制注入芯片中的液流，可以实现所形成的第二液滴206的尺寸在几微米到几十微米范围内可控。
本公开的另一些实施例提供了芯片300(以下称为第三芯片300)，该第三芯片300可以用来同时实现包含单个细胞的液滴的制备以及目标液滴的分选。图7示出了该第三芯片300。第三芯片300可以看作是第一芯片100与第二芯片200的结合体，但是在该第三芯片300中，第二芯片200的第三容纳部201、第四容纳部202以及第二输送流道208被移除。在图7中，与图1A和图5A中相同的附图标记指示相同的结构，因此，出于简洁的目的，第三芯片300中与第一芯片100和第二芯片200相同的结构不再描述，下面描述不同的部分。
如图7所示，在该第三芯片300的操作期间，第一流体107与第二流体108(包括细胞悬液109-1和生化反应试剂109-2)在第一输送流道103内汇合并生成第一液滴110，第一液滴110沿着第二流道1032的第三部分1032C流入到第一收集部104中。第一液滴110具有油包水结构。第一收集部104与分选流道203直接连通，也就是说，第一收集部104内的第一液滴110可以流入到分选流道203中。由于第一液滴110在前期已被荧光染色处理过，因此第一液滴110大致可以分为以下两类：(a)第一液滴110内含有带有目标颜色的单个目标细胞；(b)第一液滴110内含有非目标细胞或不含细胞。多个第一液滴110沿着分选流道203朝向第二收集部204的方向移动，光学设备对第一液滴110的光学信号(例如颜色)进行实时检测。当光学设备检测到第一液滴110为上述情况(b)时，则不会触发电路系统为电极结构施加电压，第一液滴110径直向前流入分选流道203的第三分支2033中，然后流入第二收集部204的第三子收集部2043。当光学设备检测到第一液滴110为上述情况(a)时，则立即触发电路系统为分选流道203处的电极结构施加电压(例如800～1000V)，含有单个目标细胞的第一液滴110被极化，在电场作用下，含有单个目标细胞的第一液滴110向上偏转流入分选流道203的第一分支2031中或向下偏转流入分选流道203的第二分支2032中，然后分别流入第二收集部204的第一子收集部2041或第二子收集部2042内。因此，该第三芯片300可以同时实现制备包含单个细胞的第一液滴110以及从第一液滴110中分选出 目标液滴这两个目的。目标液滴中含有本申请期望获得的单个目标细胞，例如外周血样本中的循环肿瘤细胞、稀有细胞、癌细胞等。
如图7所示，该第三芯片300在分选流道203处设置有电极结构。在分选流道203的上方，设置有电极结构E1、E2、E3和E4；在分选流道203的下方，设置有电极结构E5、E6、E7和E8。每个电极结构均连接有导线。如图所示，导线W1的一端电连接到电极结构E1，导线W2的一端电连接到电极结构E2，导线W3的一端电连接到电极结构E3，导线W4的一端电连接到电极结构E4，导线W1、W2、W3和W4的另一端相交于一点。导线W1、W2、W4基本上为折线，导线W3基本上为直线。导线W5的一端电连接到电极结构E5，导线W6的一端电连接到电极结构E6，导线W7的一端电连接到电极结构E7，导线W8的一端电连接到电极结构E8，导线W5、W6、W7和W8的另一端相交于一点。导线W5、W6、W8基本上为折线，导线W7基本上为直线。在第三芯片300的操作期间，当光学设备检测到第一液滴110为上述情况(a)时，则立即触发电路系统为分选流道203处的电极结构施加电压，例如向电极结构E1～E4施加正电压(或负电压)，并向电极结构E5～E8施加负电压(或正电压)，两者之间的电压差例如可以是800～1000V。含有单个目标细胞的第一液滴110被极化，在电场作用下，含有单个目标细胞的第一液滴110根据电场方向向上偏转流入分选流道203的第一分支2031中或向下偏转流入分选流道203的第二分支2032中，然后分别流入第二收集部204的第一子收集部2041或第二子收集部2042内。
该第三芯片300将第一芯片100与第二芯片200相结合，并且移除了第二芯片200的第三容纳部201、第四容纳部202以及第二输送流道208，实现了制备包含单个细胞的第一液滴110以及从第一液滴110中分选出目标液滴这两个目的。因此，该第三芯片300除了具有第一芯片100和第二芯片200各自的技术效果之外，还可以在实现液滴的制备以及目标液滴的分选的前提下，使结构更加简化，进一步减小了芯片所占用的体积，进一步提高了芯片的集成程度，使芯片更加轻便且易于携带。
根据本公开的又一方面，提供了一种微流控装置。图8示出了该微流控装置400的框图。该微流控装置400包括在前面任一个实施例 中描述的芯片。由于微流控装置400可以与前面实施例描述的第一芯片100、第二芯片200和第三芯片300具有基本相同的技术效果，因此，出于简洁的目的，此处不再重复描述微流控装置400的技术效果。
根据本公开的再一方面，提供了一种分选目标液滴的方法，图9示出了该方法800的流程图。下面以图7中的第三芯片300为例来描述方法800。该方法800包括以下步骤：
步骤S801：向前面任一个实施例中描述的第三芯片300的第一容纳部101和第二容纳部102分别提供第一流体107和包括细胞悬液109-1的第二流体108，使第一流体107和第二流体108在输送流道103的汇合点105处汇合并生成至少一个液滴110，至少一个液滴110的至少一部分中的每一个包括源自细胞悬液109-1的单个细胞；以及
步骤S802：向前面任一个实施例中描述的第三芯片300施加电压，以从至少一个液滴110中分选出具有目标属性的目标液滴，目标液滴包括单个细胞。
在一些实施例中，步骤S802包括以下子步骤：利用光学设备实时检测多个液滴110的光学信号，响应于光学设备检测到具有目标光学信号的液滴110，向前面任一个实施例中描述的第三芯片300的电极结构施加800～1000V的瞬时电压，以从多个液滴110中分选出具有目标光学信号的目标液滴，该目标液滴包括上述单个细胞。
在一些实施例中，在步骤S802之前，还包括：将多个第一液滴110转移其他反应容器中，以进行聚合酶链式反应或荧光染色处理。
在一些实施例中，第一流体107为油相，例如可以为矿物油、全氟化油等任意适当的油类，第二流体108为水相，所形成的液滴110具有油包水结构。
下面以第一芯片100和第二芯片200为例，以一个具体的示例来更加详细地描述分选目标液滴的方法。
步骤S901：将微型流量泵的输入管道分别接入第一芯片100的第一容纳部101的进样口1、第二容纳部102的第一子容纳部1021的进样口2以及第二容纳部102的第二子容纳部1022的进样口3，以向第一容纳部101、第一子容纳部1021和第二子容纳部1022分别注入第一流体107、细胞悬液109-1以及生化反应试剂109-2。第一流体107为油相，其可以混合有表面活性剂。
步骤S902：调节第一容纳部101和第二容纳部102向第一输送流道103的进样速度，以控制油水流速和液滴生成效果。通常情况下，第一流体107的流速大于细胞悬液109-1和生化反应试剂109-2的流速。
步骤S903：控制第一容纳部101以使第一流体107先充满第一芯片100的大部分区域，然后再控制第一子容纳部1021和第二子容纳部1022以使细胞悬液109-1和生化反应试剂109-2流入到第一输送流道103中。控制第一容纳部101以使第一流体107先充满第一芯片100的大部分区域是指，控制第一容纳部101以使第一流体107先充满第一芯片100的第一输送流道103的第一流道1031、第二流道1032的第二部分1032B和第三部分1032C、以及可选地第一收集部104中。因为通常情况下细胞悬液109-1的量非常少且非常珍贵，因此可以先用第一流体107充满第一芯片100的部分区域，以实现更好的包封效果。
步骤S904：第一容纳部101内的第一流体107从第一容纳部101内的过滤结构112的多个微结构之间的间隙流过，以实现过滤，然后流入第一流道1031中，从而避免过大杂物堵塞流道。第一子容纳部1021内的细胞悬液109-1从第一子容纳部1021内的过滤结构112的多个微结构之间的间隙流过，以实现过滤，然后流入第二流道1032中，从而一方面可以避免过大杂物堵塞流道，另一方面可以将细胞悬液109-1中粘连在一起的多个细胞分隔成多个彼此分离的单个细胞。第二子容纳部1022内的生化反应试剂109-2从第二子容纳部1022内的过滤结构112的多个微结构之间的间隙流过，以实现过滤，然后流入第二流道1032中，从而可以避免过大杂物堵塞流道。
步骤S905：第一流体107、细胞悬液109-1以及生化反应试剂109-2在第一输送流道103的汇合点105处汇合并生成多个第一液滴110，多个第一液滴110沿着第二流道1032的第三部分1032C移动，并随着第二流道1032的第三部分1032C的直径逐渐变宽而趋于稳定。最终，第一液滴110沿着第二流道1032的第三部分1032C流入到第一收集部104中。
步骤S906：在第一收集部104的出口4处收集一段时间(例如30秒、1分钟、2分钟等)的第一液滴110，作为废液抛弃。这是因为，在第一芯片100的初始操作阶段，第一芯片100内的各个部件的初始状态可能不太稳定(例如压力不稳定)，可能影响第一芯片100的包封 效果，从而不利于形成良好质量的第一液滴110。因此，通常并不采用初始阶段形成的第一液滴110。当第一芯片100指示的各项参数达到稳定状态后，可以开始收集第一液滴110以供后续操作使用。
步骤S907：将第一收集部104收集的第一液滴110取出，并转移至其他反应容器，例如96孔细胞培养板、PCR仪等，以进行所需的生化反应(例如PCR扩增、孵育反应、液滴荧光染色等)。
步骤S908：将微型流量泵的输入管道分别接入第二芯片200的第三容纳部201的进样口5以及第四容纳部202的进样口6，以向第三容纳部201和第四容纳部202分别注入第三流体205和利用第一芯片100制备的第一液滴110。第三流体205可以与第一流体107是相同的油相，并且可以混合有表面活性剂。
步骤S909：调节第三容纳部201和第四容纳部202向第二输送流道208的进样速度，以控制液滴流动速度。通常情况下，第三流体205的流速大于第一液滴110的流速。
步骤S910：与步骤S903相似，控制第三容纳部201以使第三流体205先充满第二芯片200的大部分区域，然后再控制第四容纳部202以使第一液滴110流入到第二输送流道208中。
步骤S911：第三容纳部201内的第三流体205从第三容纳部201内的过滤结构112的多个微结构之间的间隙流过，以实现过滤，然后流入第二输送流道208的第三流道2081中，从而避免过大杂物堵塞流道。第四容纳部202内的第一液滴110从第四容纳部202内的过滤结构112的多个微结构之间的间隙流过，以实现过滤，然后流入第二输送流道208的第四流道2082中，从而避免过大杂物堵塞流道。
步骤S912：第三流体205和第一液滴110在第二输送流道208的汇合点处汇合并生成多个第二液滴206。第二液滴206大致可以分为以下两类：(a)第二液滴206内含有带有目标颜色的单个目标细胞；(b)第二液滴206内含有非目标细胞或不含细胞。
步骤S913：第二液滴206沿着第二输送流道208朝向第二收集部204的方向移动，光学设备对第二输送流道208内的第二液滴206的光学信号(例如颜色)进行实时检测。当光学设备检测到第二液滴206为上述情况(b)时，则不会触发电路系统为电极结构施加电压，第二液滴206沿着第二输送流道208径直流入分选流道203的第三分支2033， 然后流入第二收集部204的第三子收集部2043中。当光学设备检测到第二液滴206为上述情况(a)时，则立即触发电路系统为分选流道203处的电极结构施加电压(例如800～1000V)，含有单个目标细胞的第二液滴206被极化，在电场作用下，含有单个目标细胞的第二液滴206向上偏转流入分选流道203的第一分支2031中或向下偏转流入分选流道203的第二分支2032中，然后分别流入第二收集部204的第一子收集部2041或第二子收集部2042中。通过这样的方式，可以实现从第二液滴206中分选出目标液滴。
分选目标液滴的方法是基于前面实施例中描述的第一芯片100和第二芯片200的结构来实现的，因此，该方法可以与前面实施例描述的第一芯片100和第二芯片200具有基本相同的技术效果。出于简洁的目的，此处不再重复描述分选目标液滴的方法的技术效果。
在本公开的描述中，术语“上”、“下”、“左”、“右”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本公开而不是要求本公开必须以特定的方位构造和操作，因此不能理解为对本公开的限制。
在本说明书的描述中，参考术语“一个实施例”、“另一个实施例”等的描述意指结合该实施例描述的具体特征、结构、材料或者特点包含于本公开的至少一个实施例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。另外，需要说明的是，本说明书中，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。
如本领域技术人员将理解的，尽管在附图中以特定顺序描述了本公开中方法的各个步骤，但是这并非要求或者暗示必须按照该特定顺序来执行这些步骤，除非上下文另有明确说明。附加的或可替换的，可以将多个步骤合并为一个步骤执行，以及/或者将一个步骤分解为多个步骤执行。此外，在步骤之间可以插入其他方法步骤。插入的步骤可以表示诸如本文所描述的方法的改进，或者可以与该方法无关。此 外，在下一步骤开始之前，给定步骤可能尚未完全完成。
以上所述，仅为本公开的具体实施方式，但本公开的保护范围并不局限于此。任何熟悉本技术领域的技术人员在本公开揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本公开的保护范围之内。因此，本公开的保护范围应以所述权利要求的保护范围为准。

Claims (27)

  1. 一种芯片，包括：
    第一容纳部，配置为容纳第一流体；
    第二容纳部，配置为容纳第二流体，所述第二流体包括细胞悬液；
    输送流道，包括第一流道和第二流道，所述第一流道与所述第一容纳部连通且所述第二流道与所述第二容纳部连通，所述第一流道与所述第二流道在汇合点处彼此交叉且连通，所述输送流道配置为使所述第一流体和所述第二流体在所述汇合点处汇合并生成至少一个液滴，所述至少一个液滴的至少一部分中的每一个包括源自所述细胞悬液的单个细胞；以及
    至少一个收集部，配置为收集所述至少一个液滴，
    其中，所述第一流道的一部分包括所述汇合点并被所述汇合点分割为第一区段和第二区段，在所述第一区段和所述第二区段中的每一个区段中，该区段的第一横截面的面积沿着远离所述汇合点的第一方向逐渐增大，所述第一横截面垂直于所述第一方向，并且
    其中，所述第二流道包括所述汇合点并被所述汇合点分割为第三区段和第四区段，在所述第三区段和所述第四区段中的每一个区段中，该区段的第二横截面的面积沿着远离所述汇合点的第二方向逐渐增大，所述第二横截面垂直于所述第二方向。
  2. 根据权利要求1所述的芯片，其中，所述第一流道的一部分包括第一子部分、包括所述汇合点的第二子部分、以及第三子部分，所述第一子部分属于所述第一区段，所述第三子部分属于所述第二区段，所述第二子部分跨越所述第一区段和所述第二区段并且位于所述第一子部分和所述第三子部分之间，所述第一子部分和所述第三子部分的所述第一横截面的面积均大于所述第二子部分的所述第一横截面的面积。
  3. 根据权利要求2所述的芯片，其中，所述第一流道的第二子部分在所述汇合点处的所述第一横截面的尺寸配置为允许具有特定粒径的第一流体在其内部流动，所述第一流体的特定粒径大于所述单个细胞的粒径。
  4. 根据权利要求2所述的芯片，
    其中，所述第二流道包括第一部分、第二部分以及第三部分，所述第一部分和第二部分属于所述第三区段，所述第三部分属于所述第四区段，
    其中，所述第二流道的第一部分的第一端与所述第二容纳部连通，所述第二流道的第一部分的第二端与所述第二流道的第二部分的第一端连通，所述第二流道的第二部分的第二端与所述第二流道的第三部分的第一端连通，且所述第二流道的第二部分的第二端与所述第二流道的第三部分的第一端均位于所述汇合点处，所述第二流道的第三部分的第二端与所述至少一个收集部连通，并且
    其中，所述第二流道的第一部分和第三部分的所述第二横截面的面积均大于所述第二流道的第二部分的所述第二横截面的面积。
  5. 根据权利要求4所述的芯片，其中，所述第二流道的第二部分的所述第二横截面的尺寸配置为允许具有特定粒径的第二流体在其内部流动，所述第二流体的特定粒径大于所述单个细胞的1倍粒径且小于所述单个细胞的2倍粒径。
  6. 根据权利要求4或5所述的芯片，其中，所述第二流道的第三部分的所述第二横截面的面积沿着从所述第二流道的第三部分的第一端到第二端的方向逐渐增大。
  7. 根据权利要求4-6中任一项所述的芯片，其中，所述第一流道的第二子部分在所述汇合点处的所述第一横截面的面积大于或等于所述第二流道的第二部分和第三部分在所述汇合点处的所述第二横截面的面积。
  8. 根据权利要求1-7中任一项所述的芯片，其中，所述第二容纳部包括至少一个子容纳部。
  9. 根据权利要求8所述的芯片，
    其中，所述第二流体包括第一试剂和第二试剂，所述第一试剂包括所述细胞悬液；并且
    其中，所述第二容纳部包括彼此分离的第一子容纳部和第二子容纳部，所述第一子容纳部配置为容纳所述第一试剂，所述第二子容纳部配置为容纳所述第二试剂。
  10. 根据权利要求9所述的芯片，
    其中，所述第二流道的第一部分包括第一分支和第二分支，所述 第一分支与所述第一子容纳部连通，所述第二分支与所述第二子容纳部连通，并且所述第一分支与所述第二分支在第一点处彼此相交且连通，并且
    其中，所述第一分支与所述第二分支在所述第一点处的夹角为锐角。
  11. 根据权利要求1-10中任一项所述的芯片，其中，所述至少一个收集部包括第一收集部，所述第一收集部配置为经由所述输送流道收集所述至少一个液滴。
  12. 根据权利要求1-10中任一项所述的芯片，其中，所述至少一个收集部包括第二收集部，所述第二收集部包括至少两个子收集部，所述至少两个子收集部配置为经由所述输送流道收集所述至少一个液滴。
  13. 根据权利要求1-10中任一项所述的芯片，
    其中，所述至少一个收集部包括第一收集部和第二收集部，所述第二收集部包括至少两个子收集部，并且，
    其中，所述第一收集部与所述第二收集部连通，并且所述第一收集部位于所述汇合点与所述第二收集部之间。
  14. 根据权利要求12或13所述的芯片，还包括电极结构，其中，所述电极结构位于所述汇合点和所述第二收集部之间。
  15. 根据权利要求12或13所述的芯片，其中，所述输送流道还包括分选流道，所述分选流道位于所述汇合点和所述第二收集部之间，
    其中，所述分选流道包括至少两个分支，所述至少两个分支中的一个分支配置为从所述至少一个液滴中分选出非目标液滴，所述至少两个分支中的其余分支配置为从所述至少一个液滴中分选出目标液滴；并且
    其中，所述第二收集部的至少两个子收集部与所述分选流道的至少两个分支一一对应，所述至少两个子收集部中的一个子收集部与所述分选流道的至少两个分支中的一个分支连通且配置为收集所述非目标液滴，所述至少两个子收集部中的其余子收集部与所述分选流道的至少两个分支中的其余分支分别连通且配置为收集所述目标液滴。
  16. 根据权利要求15所述的芯片，
    其中，所述分选流道的至少两个分支包括配置为从所述至少一个 液滴中分选出所述目标液滴的第一分支和第二分支以及配置为从所述至少一个液滴中分选出所述非目标液滴的第三分支，并且
    其中，所述第一分支、所述第二分支以及所述第三分支相交于第二点且所述第三分支位于所述第一分支和所述第二分支之间，所述第一分支与所述第三分支在所述第二点处的第一夹角和所述第二分支与所述第三分支在所述第二点处的第二夹角均大于10°。
  17. 根据权利要求16所述的芯片，
    其中，所述分选流道的第一分支与第三分支之间的空间限定第一直角三角形，所述分选流道的第二分支与第三分支之间的空间限定第二直角三角形，所述第一夹角面向所述第一直角三角形的第一直角边，所述第二夹角面向所述第二直角三角形的第二直角边，并且
    其中，所述第一直角三角形的第一直角边和所述第二直角三角形的第二直角边的长度均大于或等于单个所述液滴的粒径。
  18. 根据权利要求1-17中任一项所述的芯片，其中，所述输送流道的内壁表面具有疏水性。
  19. 根据权利要求1-18中任一项所述的芯片，其中，所述第一容纳部和所述第二容纳部的轮廓包括四个倒角。
  20. 根据权利要求19所述的芯片，其中，所述倒角的形状包括圆弧状。
  21. 根据权利要求1-20中任一项所述的芯片，其中，所述第一容纳部和所述第二容纳部均设置有过滤结构，所述过滤结构包括多个微结构，所述多个微结构中的相邻两个之间的间隙大于所述单个细胞的1倍粒径且小于所述单个细胞的2倍粒径。
  22. 根据权利要求1-21中任一项所述的芯片，其中，所述芯片是微流控芯片。
  23. 一种微流控装置，包括根据权利要求1-22中任一项所述的芯片。
  24. 一种分选目标液滴的方法，包括以下步骤：
    向根据权利要求1-22中任一项所述的芯片的第一容纳部和第二容纳部分别提供第一流体和包括细胞悬液的第二流体，使所述第一流体和所述第二流体在所述输送流道的汇合点处汇合并生成至少一个液滴，所述至少一个液滴的至少一部分中的每一个包括源自所述细胞悬液的 单个细胞；以及
    向根据权利要求1-22中任一项所述的芯片施加电压，以从所述至少一个液滴中分选出具有目标属性的目标液滴，所述目标液滴包括所述单个细胞。
  25. 根据权利要求24所述的方法，其中，所述芯片还包括电极结构，所述电极结构位于所述汇合点和所述至少一个收集部之间，
    其中，向根据权利要求1-22中任一项所述的芯片施加电压以从所述至少一个液滴中分选出具有目标属性的目标液滴的步骤包括：利用光学设备实时检测所述至少一个液滴的光学信号，响应于所述光学设备检测到具有目标光学信号的液滴，向所述电极结构施加800～1000V的瞬时电压，以从所述至少一个液滴中分选出具有所述目标光学信号的目标液滴，所述目标液滴包括所述单个细胞。
  26. 根据权利要求24或25所述的方法，在向根据权利要求1-22中任一项所述的芯片施加电压的步骤之前，还包括：
    将所述芯片中的所述至少一个液滴转移到其他反应容器中，以进行聚合酶链式反应或荧光染色处理。
  27. 根据权利要求24-26中任一项所述的方法，其中，所述第一流体为油相，所述第二流体为水相，并且所述液滴具有油包水结构。
PCT/CN2021/090291 2021-04-27 2021-04-27 芯片、微流控装置以及分选目标液滴的方法 WO2022226791A1 (zh)

Priority Applications (8)

Application Number Priority Date Filing Date Title
PCT/CN2021/090291 WO2022226791A1 (zh) 2021-04-27 2021-04-27 芯片、微流控装置以及分选目标液滴的方法
CN202180000922.0A CN115529820A (zh) 2021-04-27 2021-04-27 芯片、微流控装置以及分选目标液滴的方法
CN202220244666.1U CN218281795U (zh) 2021-04-27 2022-01-29 微流控芯片、盒体装置、微流控装置
CN202210112214.2A CN115245846A (zh) 2021-04-27 2022-01-29 微流控芯片、盒体装置、微流控装置
GB2317811.4A GB2621756A (en) 2021-04-27 2022-03-03 Microfluidic chip, box body device, and microfluidic device
US18/262,223 US20230405587A1 (en) 2021-04-27 2022-03-03 Microfluidic chip, box device, microfluidic device
PCT/CN2022/078956 WO2022227853A1 (zh) 2021-04-27 2022-03-03 微流控芯片、盒体装置、微流控装置
US17/731,746 US20220339630A1 (en) 2021-04-27 2022-04-28 Chip, microfluidic device, and method for sorting target droplets

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/090291 WO2022226791A1 (zh) 2021-04-27 2021-04-27 芯片、微流控装置以及分选目标液滴的方法

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US17/731,746 Continuation-In-Part US20220339630A1 (en) 2021-04-27 2022-04-28 Chip, microfluidic device, and method for sorting target droplets

Publications (1)

Publication Number Publication Date
WO2022226791A1 true WO2022226791A1 (zh) 2022-11-03

Family

ID=83697913

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/090291 WO2022226791A1 (zh) 2021-04-27 2021-04-27 芯片、微流控装置以及分选目标液滴的方法

Country Status (2)

Country Link
CN (3) CN115529820A (zh)
WO (1) WO2022226791A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009004533A1 (en) * 2007-07-03 2009-01-08 Nxp B.V. A microfluidic chip for and a method of handling fluidic droplets
CN105944775A (zh) * 2016-06-22 2016-09-21 苏州汶颢芯片科技有限公司 单细胞分离微流控芯片
CN107008517A (zh) * 2017-05-22 2017-08-04 广东顺德工业设计研究院(广东顺德创新设计研究院) 微流控芯片及其微滴生成装置
CN109603932A (zh) * 2018-12-12 2019-04-12 深圳大学 一种双聚焦微流体芯片
CN111378556A (zh) * 2018-12-29 2020-07-07 青岛华大智造普惠科技有限公司 微流控芯片及其制备方法、单细胞微液滴的制备方法

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8765455B2 (en) * 2011-01-27 2014-07-01 Lawrence Livermore National Security, Llc Chip-based droplet sorting
CN102242055B (zh) * 2011-06-03 2013-08-14 博奥生物有限公司 一种精子活力评价及筛选的方法及其专用微流控芯片装置
CN104549587B (zh) * 2015-01-20 2016-02-03 重庆科技学院 一种三通道微球筛选芯片及使用方法
CN108949496A (zh) * 2017-05-18 2018-12-07 中国科学院大连化学物理研究所 一种基于液滴微流控芯片的单细胞分离方法
US10969350B2 (en) * 2017-05-22 2021-04-06 Arizona Board Of Regents On Behalf Of Arizona Stat Metal electrode based 3D printed device for tuning microfluidic droplet generation frequency and synchronizing phase for serial femtosecond crystallography
CN109735429B (zh) * 2019-01-28 2024-05-10 珠海银方舟生物芯片有限公司 微流控芯片及分离多种细胞的系统及其分离方法
CN112657565B (zh) * 2020-12-17 2022-08-19 京东方科技集团股份有限公司 微流控通道及其控制方法、微流控芯片和分析装置
CN112553063B (zh) * 2020-12-22 2024-03-01 苏州缔因安生物科技有限公司 一种基于微液滴的集成化数字核酸扩增芯片及其使用方法和应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009004533A1 (en) * 2007-07-03 2009-01-08 Nxp B.V. A microfluidic chip for and a method of handling fluidic droplets
CN105944775A (zh) * 2016-06-22 2016-09-21 苏州汶颢芯片科技有限公司 单细胞分离微流控芯片
CN107008517A (zh) * 2017-05-22 2017-08-04 广东顺德工业设计研究院(广东顺德创新设计研究院) 微流控芯片及其微滴生成装置
CN109603932A (zh) * 2018-12-12 2019-04-12 深圳大学 一种双聚焦微流体芯片
CN111378556A (zh) * 2018-12-29 2020-07-07 青岛华大智造普惠科技有限公司 微流控芯片及其制备方法、单细胞微液滴的制备方法

Also Published As

Publication number Publication date
CN115245846A (zh) 2022-10-28
CN115529820A (zh) 2022-12-27
CN218281795U (zh) 2023-01-13

Similar Documents

Publication Publication Date Title
ES2669870T3 (es) Células de líquido compuesto
JP5241678B2 (ja) 微小流体粒子分析システム
EP2820394B1 (en) Methods, systems, and devices for multiple single-cell capturing and processing using microfluidics
EP2970849B1 (en) Methods and devices for analysis of defined multicellular combinations
CN104513787A (zh) 用于单细胞捕获、培养和给药的集成微流控芯片及系统
CN105944775A (zh) 单细胞分离微流控芯片
EP4066942A1 (en) Method for using microfluidic chip, and device thereof
WO2018075577A1 (en) Methods of integrated microfluidic processing for preventing sample loss
WO2022226791A1 (zh) 芯片、微流控装置以及分选目标液滴的方法
WO2022227853A1 (zh) 微流控芯片、盒体装置、微流控装置
US10041931B2 (en) Microfluidic device for separating liquid from the same liquid containing deformable particles without external sources of energy
Lan et al. Microfluidic based single cell or droplet manipulation: Methods and applications
WO2023065174A1 (zh) 筛选药物的方法
EP4370247A1 (en) Microfluidic device for manipulating a discrete element
Qiu Microfluidic Devices for Digestion, Dissociation, and Filtration of Tissues into Single Cell Suspensions
CN112871226A (zh) 一种微流控芯片和微流控芯片的使用方法
Ke Microfluidic-based cell assay for biomedical application
CN111019805A (zh) 用于单细胞固定并原位进行医学分析的微流控芯片装置及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21938278

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE