MICROFLUIDIC CHIP, BOX DEVICE, MICROFLUIDIC DEVICE

Abstract

The present disclosure provides a microfluidic chip, a box device adapted to the microfluidic chip, and a microfluidic device including the microfluidic chip and the box device. The microfluidic chip includes a first container for accommodating a first fluid, a second container for accommodating a second fluid, a delivery channel, a sorting channel and a collector. The delivery channel is shaped such that the first fluid and the second fluid merge at a confluence. The sorting channel includes a first sorting channel and a second sorting channel. The collector includes a first collector and a second collector.

Description

TECHNICAL FIELD

The present disclosure relates to the field of biomedical detection, and in particular, to a microfluidic chip, a box device used with the microfluidic chip, and a microfluidic device comprising the microfluidic chip and the box device.

BACKGROUND

Cells are the basic structural and functional units of living organisms. Because there is usually a high degree of heterogeneity among the cells, the average value of data obtained by analyzing the cell population essentially conceals the differences between individual cells, so it cannot characterize the random nature of gene expression and cannot reflect the real situation. With the continuous development of life sciences and precision medicine, cell population analysis is gradually developing towards single-cell analysis. A key technology in single-cell analysis is how to isolate a single cell from a highly heterogeneous biological sample comprising a plurality of cells. Single cell sorting technology provides new options for hot medical fields such as single cell analysis, early diagnosis of cancer and companion diagnostics.

SUMMARY

According to an aspect of the present disclosure, a microfluidic chip is provided. The microfluidic chip comprises: a first container configured to accommodate a first fluid; a second container configured to accommodate a second fluid comprising a cell suspension; a delivery channel comprising a first delivery channel and a second delivery channel, the first delivery channel communicating with the first container and the second delivery channel communicating with the second container, the first delivery channel intersecting and communicating with the second delivery channel at a confluence, a shape of the delivery channel being designed so that the first fluid and the second fluid merge at the confluence; a sorting channel downstream of the delivery channel and comprising a first sorting channel and a second sorting channel; and a collector downstream of the sorting channel and comprising a first collector and a second collector, the first collector communicating with the first sorting channel, and the second collector communicating with the second sorting channel.

In some embodiments, a portion of the first delivery channel is divided by the confluence into a first section and a second section, in each section of the first section and the second section, an area of a first cross-section of the section gradually increases along a first direction away from the confluence, the first cross-section is perpendicular to the first direction, and, the second delivery channel is divided by the confluence into a third section and a fourth section, in each section of the third section and the fourth section, an area of a second cross-section of the section gradually increases along a second direction away from the confluence, and the second cross-section is perpendicular to the second direction.

In some embodiments, both a beginning of the first sorting channel and a beginning of the second sorting channel communicate with an end of the delivery channel, an end of the first sorting channel communicates with the first collector and an end of the second sorting channel communicates with the second collector, the first sorting channel and the second sorting channel bend from the end of the delivery channel toward the confluence, and the first collector and the second collector are between the confluence and the end of the delivery channel.

In some embodiments, the sorting channel further comprises at least two connecting channels. The second sorting channel comprises at least two branches which are cascaded, a connecting channel is provided between any two adjacent branches of the at least two branches which are cascaded, and the any two adjacent branches communicate via the connecting channel; a beginning of the first sorting channel communicates with an end of the delivery channel, an end of the first sorting channel communicates with the first collector, the first sorting channel is adjacent to a first branch of the at least two branches which are cascaded, a connecting channel is provided between the first sorting channel and the first branch, and the first sorting channel communicates with the first branch via the connecting channel; and the second collector comprises at least two sub-collectors, the branches which are cascaded correspond to the sub-collectors one by one, and one of the branches which are cascaded communicates with a corresponding one of the sub-collectors.

In some embodiments, the second sorting channel comprises a first branch, a second branch and a third branch which are cascaded, the at least two connecting channel comprises a first connecting channel, a second connecting channel, and a third connecting channel, the second collector comprises a first sub-collector, a second sub-collector, and a third sub-collector. The first sorting channel communicates with the first branch via the first connecting channel, the first branch communicates with the second branch via the second connecting channel, and the second branch communicates with the third branch via the third connecting channel; and an end of the first branch communicates with the first sub-collector, an end of the second branch communicates with the second sub-collector, and an end of the third branch communicates with the third sub-collector.

In some embodiments, the second connecting channel is closer to the collector in a second direction than the first connecting channel, and the third connecting channel is closer to the collector in the second direction than the second connecting channel.

In some embodiments, the microfluidic chip further comprises two third containers, a beginning of the first branch and a beginning of the second branch respectively communicate with one of the two third containers, and the third container is configured to accommodate the first fluid.

In some embodiments, the sorting channel further comprises at least two connecting channels. The first sorting channel comprises at least two branches which are cascaded, a connecting channel is provided between any adjacent two branches of the at least two branches which are cascaded, and the any two adjacent branches communicate via the connecting channel, ends of the at least two branches which are cascaded communicate with the first collector; and a beginning of the second sorting channel communicates with a last branch of the first sorting channel via a connecting channel, and an end of the second sorting channel communicates with the second collector.

In some embodiments, the sorting channel further comprises a main channel, the main channel is spiral in a plane where the microfluidic chip is located, an end of the main channel communicates with the first sorting channel and the second sorting channel, the first sorting channel is configured to sort first droplets, the second sorting channel is configured to sort second droplets, and the first droplets sorted by the first sorting channel and the second droplets sorted by the second sorting channel have different particle sizes.

In some embodiments, the portion of the first delivery channel comprises a first sub-portion, a second sub-portion comprising the confluence, and a third sub-portion, the first sub-portion belongs to the first section, the third sub-portion belongs to the second section, the second sub-portion spans the first section and the second section and is between the first sub-portion and the third sub-portion, areas of the first cross-section of the first sub-portion and the third sub-portion are larger than an area of the first cross-section of the second sub-portion.

In some embodiments, a size of the first cross-section of the second sub-portion of the first delivery channel at the confluence is configured to allow the first fluid having a specific particle size to flow therein, the specific particle size of the first fluid is larger than a particle size of a single cell in the cell suspension.

In some embodiments, the second delivery channel comprises a first sub-channel, a second sub-channel and a third sub-channel, the first sub-channel and the second sub-channel belong to the third section, and the third sub-channel belongs to the fourth section. The first end of the first sub-channel communicates with the second container, a second end of the first sub-channel communicates with a first end of the second sub-channel, a second end of the second sub-channel communicates with a first end of the third sub-channel, and both the second end of the second sub-channel and the first end of the third sub-channel are at the confluence. The areas of the second cross-section of the first sub-channel and the third sub-channel are larger than an area of the second cross-section of the second sub-channel.

In some embodiments, a size of the second cross-section of the second sub-channel is configured to allow the second fluid having a specific particle size to flow therein, the specific particle size of the second fluid is larger than 1 time of a particle size of a single cell in the cell suspension and smaller than 2 times of the particle size of the single cell.

In some embodiments, the area of the second cross-section of the third sub-channel gradually increases along a direction from the first end to a second end of the third sub-channel.

In some embodiments, an area of the first cross-section of the second sub-portion of the first delivery channel at the confluence is greater than or equal to an area of the second cross-section of each of the second sub-channel and the third sub-channel of the second delivery channel at the confluence.

In some embodiments, a surface of an inner wall of the delivery channel is hydrophobic.

In some embodiments, contours of the first container and the second container comprise four chamfers, and a shape of the chamfers comprises an arc shape.

In some embodiments, both the first container and the second container are provided with a filter structure, the filter structure comprises a plurality of microstructures, a gap between adjacent two of the plurality of microstructures is larger than 1 time of a particle size of a single cell in the cell suspension and smaller than 2 times of the particle size of the single cell.

In some embodiments, the microfluidic chip further comprises an inlet and an outlet. The inlet is arranged in the first container and the second container, and the outlet is arranged in the collector.

According to another aspect of the present disclosure, a box device is provided. The box device is configured to be used with the microfluidic chip described in any one of the previous embodiments, and the microfluidic chip comprises an inlet and an outlet, the box device comprises: an accommodating cavity configured to accommodate the microfluidic chip described in any one of the previous embodiments; an inlet unit communicated with the inlet of the microfluidic chip, the inlet unit being configured to store a first reagent and release the first reagent to the inlet of the microfluidic chip; and an outlet unit communicated with the outlet of the microfluidic chip, the outlet unit being configured to receive and store a second reagent processed by the microfluidic chip and flowing into the outlet unit from the outlet of the microfluidic chip. The inlet unit comprises an inlet hole and a first storage cavity, the inlet hole is a through hole and communicates with the first storage cavity, the inlet hole is recessed from a surface of the box device to an inside of the box device, and the first storage cavity is on a side of the inlet hole away from the surface of the box device.

In some embodiments, the first storage cavity is inside the box device, and an orthographic projection of the inlet hole on the box device falls within an orthographic projection of the first storage cavity on the box device.

In some embodiments, the inlet unit further comprises a second storage cavity, the second storage cavity is on a side of the first storage cavity away from the inlet hole and communicates with the first storage cavity, the second storage cavity comprises a first opening communicating with the first storage cavity and a second opening facing to the first opening, an orthographic projection of the second opening on the box device falls within an orthographic projection of the first opening on the box device.

In some embodiments, the orthographic projection of the second opening of the second storage cavity on the box device falls within an orthographic projection of the inlet hole on the box device.

In some embodiments, the outlet unit comprises an outlet hole and a third storage cavity, the outlet hole is a through hole and communicates with the third storage cavity, the outlet hole is recessed from the surface of the box device to the inside of the box device, and the third storage cavity is on a side of the outlet hole away from the surface of the box device.

In some embodiments, the third storage cavity is inside the box device, and an orthographic projection of the outlet hole on the box device falls within an orthographic projection of the third storage cavity on the box device.

In some embodiments, the outlet unit further comprises a fourth storage cavity, and the fourth storage cavity is on a side of the third storage cavity away from the outlet hole and communicates with the third storage cavity.

In some embodiments, an orthographic projection of the fourth storage cavity on the box device overlaps at most a portion of an orthographic projection of the outlet hole on the box device.

In some embodiments, an orthographic projection of the fourth storage cavity on the box device falls within an orthographic projection of the outlet hole on the box device.

In some embodiments, the inlet unit comprises a first inlet unit, a second inlet unit, and a third inlet unit, the inlet of the microfluidic chip comprises a first inlet, a second inlet, and a third inlet, and the first reagent comprises the first fluid, the cell suspension, and a biochemical reagent. The first inlet unit communicates with the first inlet of the microfluidic chip, the first inlet unit is configured to store the first fluid and release the first fluid to the first inlet of the microfluidic chip; the second inlet unit communicates with the second inlet of the microfluidic chip, the second inlet unit is configured to store the cell suspension and release the cell suspension to the second inlet of the microfluidic chip; the third inlet unit communicates with the third inlet of the microfluidic chip, and the third inlet unit is configured to store the biochemical reagent and release the biochemical reagent to the third inlet of the microfluidic chip.

In some embodiments, the box device further comprises a first installation region and a second installation region, the first installation region is configured to install an optical identification device, and the second installation region is configured to install a driving electrode device.

In some embodiments, the inlet unit comprises a first inlet unit and a second inlet unit, the inlet of the microfluidic chip comprises a first inlet and a second inlet, the first reagent comprises the first fluid and a droplet comprising a single cell. The first inlet unit communicates with the first inlet of the microfluidic chip, the first inlet unit is configured to store the first fluid and release the first fluid to the first inlet of the microfluidic chip; the second inlet unit communicates with the second inlet of the microfluidic chip, and the second inlet unit is configured to store the droplet comprising the single cell and release the droplet comprising the single cell to the second inlet of the microfluidic chip. The outlet unit comprises a first outlet unit, a second outlet unit, and a third outlet unit between the first outlet unit and the second outlet unit, the second reagent comprises a first droplet and a second droplet, the third outlet unit is configured to receive and store the first droplet, the first outlet unit and the second outlet unit are configured to receive and store the second droplet.

In some embodiments, the inlet unit comprises a first inlet unit, a second inlet unit, and a third inlet unit, the inlet of the microfluidic chip comprises a first inlet, a second inlet, and a third inlet, the first reagent comprises the first fluid, the cell suspension, and a biochemical reagent. The first inlet unit communicates with the first inlet of the microfluidic chip, the first inlet unit is configured to store the first fluid and release the first fluid to the first inlet of the microfluidic chip; the second inlet unit communicates with the second inlet of the microfluidic chip, the second inlet unit is configured to store the cell suspension and release the cell suspension to the second inlet of the microfluidic chip; the third inlet unit communicates with the third inlet of the microfluidic chip, and the third inlet unit is configured to store the biochemical reagent and release the biochemical reagent to the third inlet of the microfluidic chip. The outlet unit comprises a first outlet unit and a second outlet unit, the second reagent comprises a first droplet and a second droplet, the first outlet unit is configured to receive and store the first droplet, and the second outlet unit is configured to receive and store the second droplet.

In some embodiments, the first outlet unit and the second outlet unit are between the inlet unit and the first installation region and the second installation region.

In some embodiments, the first installation region and the second installation region are between the inlet unit and the outlet unit, the first installation region comprises a first sub-installation unit, a second sub-installation unit, and a third sub-installation unit, the second installation region comprises a fourth sub-installation unit, a fifth sub-installation unit, and a sixth sub-installation unit, the first sub-installation unit is associated with the fourth sub-installation unit, the second sub-installation unit is associated with the fifth sub-installation unit, and the third sub-installation unit is associated with the sixth sub-installation unit.

In some embodiments, the inlet unit comprises a first inlet unit, a second inlet unit, and a third inlet unit, the inlet of the microfluidic chip comprises a first inlet, a second inlet, and a third inlet, the first reagent comprises the first fluid and a droplet comprising a single cell. The first inlet unit communicates with the first inlet of the microfluidic chip, the first inlet unit is configured to store the first fluid and release the first fluid to the first inlet of the microfluidic chip; the second inlet unit communicates with the second inlet of the microfluidic chip, the second inlet unit is configured to store the first fluid and release the first fluid to the second inlet of the microfluidic chip; the third inlet unit communicates with the third inlet of the microfluidic chip, and the third inlet unit is configured to store the droplet comprising the single cell and release the droplet comprising the single cell to the third inlet of the microfluidic chip. The outlet unit comprises a first outlet unit and a second outlet unit, the second reagent comprises a first droplet and a second droplet, the first outlet unit is configured to receive and store the first droplet, and the second outlet unit is configured to receive and store the second droplet.

In some embodiments, a number of the first outlet unit is one, and a number of the second outlet unit is three.

In some embodiments, a number of the first outlet unit is one, and a number of the second outlet unit is one.

In some embodiments, the box device comprises one inlet unit and two outlet units, the second reagent comprises a first droplet and a second droplet, the first droplet and the second droplet have different particle sizes, one of the two outlet units is configured to receive and store the first droplet, and the other of the two outlet units is configured to receive and store the second droplet.

According to yet another aspect of the present disclosure, a microfluidic device is provided, the microfluidic device comprises the microfluidic chip described in any of the preceding embodiments and the box device described in any of the preceding embodiments. The microfluidic chip is assembled with the box device.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly describe the technical solutions in the embodiments of the present disclosure, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present disclosure, and for those ordinary skill in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1A shows a schematic structural diagram of a microfluidic chip according to an embodiment of the present disclosure;

FIG. 1B shows an enlarged view of region I of the microfluidic chip of FIG. 1A;

FIG. 1C shows an enlarged view of the container of the microfluidic chip of FIG. 1A;

FIG. 2 shows a schematic structural diagram of a microfluidic chip according to another embodiment of the present disclosure;

FIG. 3 shows a schematic structural diagram of a variant of the microfluidic chip of FIG. 2;

FIG. 4 shows a schematic structural diagram of a microfluidic chip according to yet another embodiment of the present disclosure;

FIG. 5A shows a schematic structural diagram of a box device according to an embodiment of the present disclosure;

FIG. 5B shows a schematic structural diagram of a microfluidic chip adapted to the box device of FIG. 5A;

FIG. 6A shows a schematic structural diagram of a box device according to another embodiment of the present disclosure;

FIG. 6B shows a schematic structural diagram of a microfluidic chip adapted to the box device of FIG. 6A;

FIG. 7 shows a schematic structural diagram of a box device according to yet another embodiment of the present disclosure;

FIG. 8 shows a schematic structural diagram of a box device according to still another embodiment of the present disclosure;

FIG. 9 shows a schematic structural diagram of a box device according to still another embodiment of the present disclosure; and

FIG. 10 shows a block diagram of a microfluidic device according to an embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE DISCLOSURE

The technical solutions in the embodiments of the present disclosure will be described clearly and completely in the following with reference to the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only a part, not all, of the embodiments of the present disclosure. Based on the embodiments in the present disclosure, all other embodiments obtained by those of ordinary skill in the art without paying creative work are within the scope of protection of the present disclosure.

Before formally describing the technical solutions in the embodiments of the present disclosure, the following explanations and definitions are made for the terms used in the embodiments, so as to help those skilled in the art to understand the technical solutions in the embodiments of the present disclosure more clearly.

As used herein, the term “fluid” refers to all substances that can flow, and is a general term for liquids and gases. Fluid is a substance that can be continuously deformed under the action of tiny shearing force. Fluid can be composed of a single substance or a mixture of many different substances. Fluid can be a continuous phase (such as an oil phase), a dispersed phase (such as an aqueous phase), or a mixture of continuous and dispersed phases. Fluid has characteristics such as fluidity, compressibility and viscosity.

As used herein, the term “oil phase” means that substances that are not soluble in water belong to the oil phase according to the principle of similar compatibility. For example, when a substance is mixed with water, if the mixed liquid shows stratification or turbidity, the substance belongs to the oil phase. Oil may have a density higher or lower than that of water and/or a viscosity higher or lower than that of water. For example, liquid paraffin, silicone oil, vaseline, mineral oil and perfluorinated oil all belong to the oil phase.

As used herein, the term “aqueous phase” means that substances that are easily soluble in water belong to the aqueous phase according to the principle of similar compatibility. For example, when a substance is mixed with water, if the mixed liquid presents a transparent and uniform solution, the substance belongs to the aqueous phase. For example, water, glycerin, alcohol and acetone all belong to the aqueous phase.

As used herein, the term “cell suspension” refers to a cell solution obtained by separating cells from tissues by mechanical or chemical method and diluted uniformly with cell culture solution. A large number of cells can be included in the cell suspension, such as hundreds, thousands, tens of thousands, millions, tens of millions or more cells. The cells in the cell suspension can be any type of cells, including but not limited to prokaryotic cells, eukaryotic cells, bacteria, fungi, plants, mammals or other animal cell types, mycoplasma, normal tissue cells, tumor cells or any other type of cells, whether or not they are derived from single-cell or multicellular organisms. The cells in the cell suspension may include DNA, RNA, organelles, protein or any combination thereof.

As used herein, the term “A communicates with B” means that element A and element B are connected and communicated with each other, which allows fluid to flow between element A and element B, that is, fluid can flow from element A to element B or from element B to element A according to product design requirements. Element A and element B can communicate directly, that is, fluid can flow directly from element A to element B or from element B to element A without passing through other intermediate elements (such as pipes). Alternatively, element A and element B can communicate indirectly, that is, fluid can flow from element A to element B or from element B to element A via one or more intermediate elements (such as pipes).

As used herein, the term “polymerase chain reaction (PCR)” is a molecular biology technique for amplifying specific deoxyribonucleic acid (DNA) fragments. It can be regarded as a special DNA replication in vitro, which can replicate trace DNA into a large amount and greatly increase its quantity. The basic principle of PCR is that DNA can denature and unwind into a single strand at high temperature (for example, about 95° C.), and when the temperature drops to low temperature (for example, about 60° C.), the primer(s) and the single strand combine according to the principle of base complementary pairing to become a double strand. Therefore, the denaturation and renaturation of DNA can be controlled by temperature change, and a large number of DNA replication can be achieved by adding the primer(s). PCR reactions include but are not limited to digital PCR (dPCR), quantitative PCR and real-time PCR. DPCR technology can provide quantitative analysis technology of digitized DNA quantitative information, which can provide higher sensitivity and accuracy when combined with microfluidic technology.

As used herein, the term “microfluidic chip” refers to a chip with micro-scale microchannels, which can integrate basic operation units such as sample preparation, reaction, separation and detection involved in the fields of biology, chemistry and medicine into the micro-scale chip to automatically complete the whole process of reaction and analysis. The analysis and detection device based on microfluidic chip may have the advantages such as controllable liquid flow, less sample consumption, fast detection speed, simple operation, multi-functional integration, small volume and portability, etc.

As used herein, the term “particle size of XX” refers to the size of the substance XX, that is, the length of the substance XX in a certain direction. The substance XX may be a single cell or a single droplet. For example, when the shape of a cell or a droplet is spherical, the term “particle size of a single cell” refers to the diameter of a single cell, and “particle size of a single droplet” refers to the diameter of a single droplet. When the shape of a cell or a droplet is a rod, the term “particle size of a single cell” refers to the length of a single cell in the direction of the shorter side, and “particle size of a single droplet” refers to the length of a single droplet in the direction of the shorter side.

The inventor(s) of this application found that in the conventional technology, the methods for sorting single cells are mainly divided into two categories: one is to use fluorescence activated cell sorting (FACS) to automatically sort single cell, but the fluorescence activated cell sorting is expensive and costly to maintain; the other is to sort single cell manually by professional operators, but this manual sorting method not only depends on the skills and proficiency of operators, but also needs large and medium-sized instruments such as micro-pipetting platform and optical tweezers. In addition, the single cell sorting process is easily polluted by aerosols and microorganisms floating in the environment, which is usually difficult to remove in the subsequent detection. Therefore, the existing single cell sorting methods have shortcomings such as high cost, high requirements for operators' skills, limited instruments required by the site, easy to be polluted by the environment and so on.

In view of this, embodiments of the present disclosure provide a microfluidic chip. The microfluidic chip can be used to prepare a droplet comprising a single cell derived from cell suspension and to sort target droplets from the prepared droplets. The preparation and sorting of a single cell can be realized with the microfluidic chip, which can effectively improve the automatic operation while reducing the use cost, eliminate cross-contamination and improve the survival rate of cells.

FIG. 1A shows a schematic structural diagram of a microfluidic chip 300, wherein (a) is a front view of the microfluidic chip 300, (b) is a rear view of the microfluidic chip 300, (c) is a left view of the microfluidic chip 300, and (d) is a trimetric view of the microfluidic chip 300. As shown in FIG. 1A, the microfluidic chip 300 comprises: a first container 301, a second container 302, a delivery channel 303, a sorting channel 305 and a collector 306. The first container 301 is configured to accommodate a first fluid, and the second container 302 is configured to accommodate a second fluid comprising the cell suspension. The delivery channel 303 comprises a first delivery channel 3031 and a second delivery channel 3032. The first delivery channel 3031 communicates with the first container 301 and the second delivery channel 3032 communicates with the second container 302. The first delivery channel 3031 intersects and communicates with the second delivery channel 3032 at a confluence 304. The shape of the delivery channel 303 is designed to make the first fluid and the second fluid merge at the confluence 304. The sorting channel 305 is downstream of the delivery channel 303, and the sorting channel 305 comprises a first sorting channel 3051 and a second sorting channel 3052. The collector 306 is downstream of the sorting channel 305 and comprises a first collector 3061 and a second collector 3062, the first collector 3061 communicates with the first sorting channel 3051, and the second collector 3062 communicates with the second sorting channel 3052.

In some embodiments, the first sorting channel 3051 can be configured to sort a first droplet and the second sorting channel 3052 can be configured to sort a second droplet. In such a case, the first collector 3061 is configured to collect the first droplet, and the second collector 3062 is configured to collect the second droplet.

It should be noted that, herein, the term “first droplet” may refer to a non-target droplet, and the term “second droplet” may refer to a target droplet. The non-target droplet means that the droplet comprises non-target cells from the cell suspension, while the target droplet means that the droplet comprises a single target cell from the cell suspension. The cell suspension comprises a large number of cells, most of which are non-target cells and a small fraction of which are target cells (e.g., circulating tumor cells, rare cells, cancer cells, etc. in peripheral blood samples). Herein, the terms “first droplet” and “non-target droplet” are used interchangeably, and the terms “second droplet” and “target droplet” are used interchangeably.

The microfluidic chip 300 can not only prepare a droplet comprising a single cell (a single target cell or a single non-target cell) from the cell suspension, but also can sort out the target droplet comprising a single target cell from the droplet. Therefore, the microfluidic chip 300 has a high degree of integration, and can automatically complete the preparation of a droplet comprising a single cell and the sorting of a droplet comprising a single target cell without manual operation by an operator, thereby effectively improving the degree of automation of the operation. In addition, since the first fluid and the second fluid only flow in the delivery channel 303 and are completely isolated from the external environment, contamination by aerosols, microorganisms, etc. floating in the environment can be avoided. Moreover, since the single cell isolated from the cell suspension is wrapped and protected by the droplet, the whole preparation process is relatively mild, and hence the cell viability can be effectively improved.

The following will specifically describe how to prepare the droplet comprising a single cell through the microfluidic chip 300.

FIG. 1B is an enlarged view of region I of the microfluidic chip 300 in FIG. 1A. Referring to FIG. 1A and FIG. 1B, the delivery channel 303 of the microfluidic chip 300 comprises the first delivery channel 3031 and the second delivery channel 3032. The first delivery channel 3031 communicates with the first container 301 and allows the first fluid to flow therein. The first fluid is a continuous phase (e.g., oil phase) liquid, which may be, for example, mineral oil, perfluorinated oil, or any other suitable fluid. Optionally, a surfactant may be mixed in the first fluid, which facilitates stabilization of the resulting droplets, e.g., inhibits subsequent coalescence of the resulting droplets. When the first fluid is a perfluorinated oil, the surfactant may be a perfluorinated surfactant. The second delivery channel 3032 communicates with the second container 302 and allows the second fluid to flow therein. The second fluid is an aqueous phase liquid. In the embodiment in the figure, the second container 302 comprises a first sub-container 3021 and a second sub-container 3022, the first sub-container 3021 is configured to accommodate cell suspension, and the second sub-container 3022 is configured to accommodate biochemical reagent. Different biochemical reagents can be used according to different biochemical reactions, and the embodiments of the present disclosure do not specifically limit the chemical composition of the biochemical reagents. It should be noted that although it is shown in FIG. 1A that the cell suspension is accommodated in the first sub-container 3021, the biochemical reagent is accommodated in the second sub-container 3022 separated from the first sub-container 3021, but this is just an example, and embodiments of the present disclosure are not limited thereto. In alternative embodiments, the cell suspension and biochemical reagent may be pre-mixed and accommodated within the same container. The first delivery channel 3031 and the second delivery channel 3032 intersect and communicate at the confluence 304.

A portion of the first delivery channel 3031 is divided by the confluence 304 into a first section and a second section, in each section of the first section and the second section, the area of the first cross-section of the section gradually increases along a first direction away from the confluence 304, the first cross-section is perpendicular to the first direction, and the first direction is the vertical direction in the figure. The second delivery channel 3032 is divided by the confluence 304 into a third section and a fourth section, in each section of the third section and the fourth section, the area of the second cross-section of the section gradually increases along a second direction away from the confluence 304, the second cross-section is perpendicular to the second direction, and the second direction refers to the flow direction of the second fluid in the second delivery channel 3032.

Specifically, the first delivery channel 3031 comprises a first sub-portion 3031-1, a second sub-portion 3031-2, and a third sub-portion 3031-3 arranged in sequence along the first direction, and the second sub-portion 3031-2 is between the first sub-portion 3031-1 and the third sub-portion 3031-3 and comprises the confluence 304. The first sub-portion 3031-1 belongs to the first section described above, the third sub-portion 3031-3 belongs to the second section described above, and the second sub-portion 3031-2 spans the first section and the second section. The area of the first cross-section of the first sub-portion 3031-1 and the third sub-portion 3031-3 is larger than the area of the first cross-section of the second sub-portion 3031-2, that is, along the direction from the first sub-portion 3031-1 to the third sub-portion 3031-3, the first delivery channel 3031 gradually becomes thinner and then gradually thicker, so that, the first delivery channel 3031 is thick at its upper and lower parts (the first sub-portion 3031-1 and the third sub-portion 3031-3) and thin at its middle part (the second sub-portion 3031-2). With such a shape design, when the first fluid in the first delivery channel 3031 flows from the first sub-portion 3031-1 to the second sub-portion 3031-2 or flows from the third sub-portion 3031-3 to the second sub-portion 3031-2, since the channel becomes thinner and thinner, the flow velocity of the first fluid in the first delivery channel 3031 becomes larger, so that, the pressure of the first fluid can be increased to promote the flow of the first fluid in the first sub-portion 3031-1 and the third sub-portion 3031-3 to the confluence 304 of the second sub-portion 3031-2, to merge at the confluence 304. This provides sufficient first fluid for subsequent droplet formation. The shape of the first cross-section of the first sub-portion 3031-1, the second sub-portion 3031-2 and the third sub-portion 3031-3 of the first delivery channel 3031 may be a circle, a square, a rectangle, a regular polygon, an irregular shape, etc., which are not limited in the embodiments of the present disclosure.

The size of the first cross-section of the second sub-portion 3031-2 of the first delivery channel 3031 at the confluence 304 is configured to allow the first fluid having a specific particle size to flow therein. The specific particle size of the first fluid is larger than the particle size of a single cell (e.g., a single target cell). That is, the width of the first cross-section of the second sub-portion 3031-2 of the first delivery channel 3031 at the confluence 304 is larger than the particle size of a single cell. In an example, the particle size of each cell in the cell suspension is about 10 μm, and the width of the cross-section of the second sub-portion 3031-2 of the first delivery channel 3031 at the confluence 304 is greater than 10 μm, for example slightly larger than 10 μm. Here, “slightly greater than 10 μm” means that the width of the first cross-section of the second sub-portion 3031-2 of the first delivery channel 3031 at the confluence 304 is greater than 10 μm but less than 20 μm, that is, the width is greater than the particle size of a single cell but smaller than the sum of the particle sizes of two cells. It should be noted that the phrase “the width of the first cross-section of the second sub-portion 3031-2 of the first delivery channel 3031 at the confluence 304” can be understood as, when the shape of the first cross-section of the second sub-portion 3031-2 of the first delivery channel 3031 at the confluence 304 is a circle, the width of the first cross-section is the diameter of the circle; when the shape of the first cross-section of the second sub-portion 3031-2 of the first delivery channel 3031 at the confluence 304 is a square, the width of the first cross-section is the length of a side of the square; when the shape of the first cross-section of the second sub-portion 3031-2 of the first delivery channel 3031 at the confluence 304 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-portion 3031-2 of the first delivery channel 3031 at the confluence 304 is a regular polygon, the width of the first cross-section is the distance between two farthest vertices of the regular polygon. In an example, when the first cross-section of the second sub-portion 3031-2 of the first delivery channel 3031 at the confluence 304 is circular and the shape of a single cell is spherical, the width of the first cross-section of the second sub-portion 3031-2 of the first delivery channel 3031 at the confluence 304 is greater than the particle size of a single cell, which should be understood that, the diameter of the second sub-portion 3031-2 of the first delivery channel 3031 at the confluence 304 is larger than the diameter of the single cell. By this design, when the first fluid in the first delivery channel 3031 flows from the first sub-portion 3031-1 to the second sub-portion 3031-2 or from the third sub-portion 3031-3 to the second sub-portion 3031-2, the first fluid can be made to form a single row of fluid particles arranged in sequence near the confluence 304, and the particle size of each particle in the single row of fluid particles is greater than 1 time of the particle size of a single cell and smaller than 2 times of the particle size of a single cell. In this way, the particle size of each particle formed by the first fluid can be slightly larger than the particle size of a single cell, so that a single cell can be better wrapped and a better encapsulation effect can be achieved. Moreover, such a design can also increase the flow velocity of the first fluid at the confluence 304, which is beneficial to the formation of droplets.

The second delivery channel 3032 comprises a first sub-channel 3032-1, a second sub-channel 3032-2 and a third sub-channel 3032-3. The first sub-channel 3032-1 and the second sub-channel 3032-2 belong to the third section described above, the third sub-channel 3032-3 belongs to the fourth section described above. The first end of the first sub-channel 3032-1 communicates with the second container 302, the second end of the first sub-channel 3032-1 communicates with the first end of the second sub-channel 3032-2; the second end of the second sub-channel 3032-2 communicates with the first end of the third sub-channel 3032-3, and both the second end of the second sub-channel 3032-2 and the first end of the third sub-channel 3032-3 are at the confluence 304; the second end of the third sub-channel 3032-3 communicates with the beginning of the sorting channel 305. The first sub-channel 3032-1 comprises a first branch and a second branch, the first branch communicates with the first sub-container 3021 of the second container 302 and is configured to allow the cell suspension to flow therein, the second branch communicates with the second sub-container 3022 of the second container 302 and is configured to allow biochemical reagents to flow therein. As shown in FIG. 1B, the first branch intersects and communicates with the second branch at a point, and the angle between the first branch and the second branch at this point is an acute angle. In an example, the angle between the first branch and the second branch at this point is about 60 degrees. On the one hand, the design of the angle between the first branch and the second branch can ensure that the cell suspension in the first branch and the biochemical reagents in the second branch have a sufficient forward flow velocity (towards the direction of the confluence 304), so as to buffer the pressure; on the other hand, this design can also ensure that the cell suspension and the biochemical reagents can be fully mixed at this point; on yet the other hand, the dead volume of the mixed solution in the flow channel can be reduced, and the liquid storage accuracy of the first branch and the second branch can be improved.

The area of the second cross-section of each of the first sub-channel 3032-1 and the third sub-channel 3032-3 of the second delivery channel 3032 is larger than the area of the second cross-section of the second sub-channel 3032-2 of the second delivery channel 3032. That is, the area of the second cross-section of each of the first branch and the second branch of the first sub-channel 3032-1 is larger than the area of the second cross-section of the second sub-channel 3032-2, and the area of the second cross-section of the third sub-channel 3032-3 is greater than the area of the second cross-section of the second sub-channel 3032-2. Along the direction from the first sub-channel 3032-1 to the third sub-channel 3032-3, the size of the second delivery channel 3032 becomes thick, and then thin, and then thick. Similar to the first delivery channel 3031, the shape of the second cross-section of the first sub-channel 3032-1, the second sub-channel 3032-2 and the third sub-channel 3032-3 of the second delivery channel 3032 can be circle, square, rectangle, regular polygon, irregular shapes, etc., which are not limited in the embodiments of the present disclosure.

The size of the second cross-section of the second sub-channel 3032-2 of the second delivery channel 3032 is configured to allow the second fluid having a specific particle size to flow therein. The specific particle size of the second fluid is larger than 1 time of the particle size of a single cell and smaller than 2 times of the particle size of a single cell. That is to say, the width of the second cross-section of the second sub-channel 3032-2 is larger than 1 time of the particle size of a single cell and smaller than 2 times of the particle size of a single cell. In an example, when the second cross-section of the second sub-channel 3032-2 is circular and the shape of a single cell is spherical, the width of the second cross-section of the second sub-channel 3032-2 is larger than 1 time of the particle size of a single cell and smaller than 2 times of the particle size of a single cell, which should be understood that the diameter of the second sub-channel 3032-2 is larger than 1 time of the diameter of a single cell and smaller than 2 times of the diameter of a single cell. In this case, the diameter of the second sub-channel 3032-2 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, etc. of the diameter of a single cell. When the cell suspension and the biochemical reagent are mixed and flow forward (toward the confluence 304), by making the width of the second cross-section of the second sub-channel 3032-2 larger than 1 time of the particle size of a single cell and smaller than 2 times of the particle size of a single cell, the mixed solution is arranged in a single row of string which is composed of a plurality of single cells in the second sub-channel 3032-2, as shown in FIG. 1B. That is to say, the width of the second cross-section of the second sub-channel 3032-2 can only accommodate a single cell in its width direction, but cannot accommodate two cells which are arranged side-by-side. When the single row of string which is composed of a plurality of single cells moves to the confluence 304, under the pressure of the first fluid in the first delivery channel 3031, a cell closest to the confluence 304 in the cell string (that is, the frontmost cell in the cell string) is separated from the cell string. The separated one cell combines with a single particle in the first fluid at the confluence 304, thereby forming a droplet comprising the single cell. As mentioned above, the first fluid is the oil phase, the second fluid (that is, the mixed solution of cell suspension and biochemical reagents) is the aqueous phase. Therefore, the droplet is water-in-oil, that is, the first fluid in the oil phase wraps the second fluid in the aqueous phase.

As shown in the figure, the area of the second cross-section of the third sub-channel 3032-3 of the second delivery channel 3032 gradually increases along the direction from the first end to the second end thereof. That is, the third sub-channel 3032-3 gradually becomes thicker along the direction from the first end to the second end. The purpose of this design is to make the droplet gradually becomes larger when moving forward along the third sub-channel 3032-3, so as to facilitate the phase stabilization of the droplets. The area of the first cross-section of the second sub-portion 3031-2 of the first delivery channel 3031 at the confluence 304 is greater than or equal to the area of the second cross-section of the second sub-channel 3032-2 and the third sub-channel 3032-3 of the second delivery channel 3032 at the confluence 304. In an example, the area of the first cross-section of the second sub-portion 3031-2 of the first delivery channel 3031 at the confluence 304 is equal to the area of the second cross-section of the second sub-channel 3032-2 and the third sub-channel 3032-3 of the second delivery channel 3032 at the confluence 304. Through such a design, at the confluence 304, the particle size of a single oil phase particle in the first fluid is approximately equal to the particle size of a single cell in the second fluid, so that the size of the droplet can be precisely controlled.

The above describes in detail how to use the microfluidic chip 300 to prepare the droplet comprising a single cell. Next, how to use the microfluidic chip 300 to sort out the target droplets from the prepared droplets, that is, the droplet comprising a single target cell, will be described in detail.

The microfluidic chip 300 may further comprise an optical recognition device and a driving electrode device (not shown in the figure), and the optical recognition device and the driving electrode device may be disposed near the third sub-channel 3032-3 of the second delivery channel 3032. The droplets generated at the confluence 304 flow forward along the third sub-channel 3032-3, which communicates with the downstream sorting channel 305. As mentioned above, the cell suspension comprises a large number of cells, among which a large number of non-target cells and a small number of target cells (e.g. circulating tumor cells, rare cells, cancer cells, etc. in peripheral blood samples) are included. The cell suspension has been dyed before being provided into the first sub-container 3021. Since the target cells and non-target cells in the cell suspension have different antibodies, after fluorescent dye, these target cells and non-target cells will show different colors under the optical recognition device. Therefore, the droplet can be roughly divided into the following three categories: (a) a droplet comprising a single target cell with a target color; (b) a droplet comprising non-target cell(s) (including two cases where a droplet comprising one or more non-target cells and a droplet comprising multiple target cells); or (c) a droplet comprising no cells. When the droplet moves forward along the third sub-channel 3032-3, the optical recognition device performs real-time detection on the optical signal (e.g., color) of the droplet in the third sub-channel 3032-3. When the optical recognition device detects that the droplet is in the above situation (b) or (c), it will not notify the circuit system, so the circuit system does not apply a voltage to the driving electrode, and the non-target droplet flows into the first sorting channel 3051 under the action of inertial force, and then flows into the first collector 3061. In an alternative embodiment, when the optical recognition device detects that the droplet is in the above situation (b) or (c), it can also notify the circuit system, and the circuit system applies a certain voltage to the driving electrode after receiving the notification, and the non-target droplet flows into the first sorting channel 3051 under the driving of dielectric force, and then flows into the first collector 3061. When the optical recognition device detects that the droplet is in the above situation (a), it immediately informs the circuit system to apply an appropriate voltage (e.g. 800-1000V) to the driving electrode, and the target droplet comprising the single target cell is polarized. Under the action of the electric field, the target droplet is deflected upward and flows into the second sorting channel 3052, and then flows into the second collector 3062. Therefore, the microfluidic chip 300 realizes the sorting of droplets, the non-target droplets are collected into the first collector 3061, and the target droplets are collected into the second collector 3062.

It should be noted that the dyeing 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 can be distinguished from the non-target cells in the cell suspension, the treatment methods are all within the protection scope of the present disclosure.

It should be noted that although FIG. 1A shows that the sorting channel 305 comprises two channels 3051 and 3052, the embodiments of the present disclosure are not limited thereto. In alternative embodiments, the sorting channel 305 may also comprise more channels (e.g., three channels, four channels, or even more), one channel of the plurality of channels is configured to sort out the non-target droplets from the droplets, and the remaining channels of the plurality of channels are configured to sort out target droplets from the droplets. Correspondingly, the collector 306 may comprise a plurality of collectors, the plurality of collectors are in one-to-one correspondence with the plurality of channels of the sorting channel 305, one of the plurality of collectors communicates with one of the plurality of channels of the sorting channel 305 and is configured to collect non-target droplets, and the remaining collectors of the plurality of collectors respectively communicate with the remaining channels of the plurality of channels of the sorting channel 305 and are configured to collect target droplets.

With continued reference to FIG. 1A, the beginning of the first sorting channel 3051 and the beginning of the second sorting channel 3052 are both communicated with the end of the delivery channel 303 (i.e., the end of the third sub-channel 3032-3 of the delivery channel 303), the end of the first sorting channel 3051 communicates with the first collector 3061, and the end of the second sorting channel 3052 communicates with the second collector 3062. The first sorting channel 3051 and the second sorting channel 3052 are bent from the end of the delivery channel 303 toward the confluence 304, so that the first collector 3061 and the second collector 3062 are located between the confluence 304 and the end of the delivery channel 303. In contrast to a situation where the first sorting channel 3051 and the second sorting channel 3052 extend straight in the horizontal direction (that is, the first sorting channel 3051 and the second sorting channel 3052 extend straight toward the right direction in the figure) and hence the first collector 3061 and the second collector 3062 are connected to their ends respectively, by making the first sorting channel 3051 and the second sorting channel 3052 fold back from the end of the delivery channel 303 toward the confluence 304, the volume occupied by the microfluidic chip 300 can be reduced, and the microfluidic chip 300 can be more miniaturized, and the production costs can be saved.

It should be noted that, herein, the term “the end of the delivery channel 303” refers to the end of the third sub-channel 3032-3 of the delivery channel 303, that is, the terminal of the third sub-channel 3032-3 of the delivery channel 303, which is directly connected to the downstream sorting channel 305. The term “the beginning of the first sorting channel 3051 and the beginning of the second sorting channel 3052” refers to the first end of the first sorting channel 3051 and the first end of the second sorting channel 3052. The first ends of the first sorting channel 3051 and the second sorting channel 3052 are directly connected to the terminal of the third sub-channel 3032-3 of the upstream delivery channel 303, and the droplets flow from the terminal of the third sub-channel 3032-3 into the first end of the first sorting channel 3051 and the first end of the second sorting channel 3052, respectively. The term “the end of the first sorting channel 3051 and the end of the second sorting channel 3052” refers to the second end of the first sorting channel 3051 and the second end of the second sorting channel 3052. The second end of the first sorting channel 3051 is connected to the first collector 3061, and the second end of the second sorting channel 3052 is connected to the second collector 3062. The non-target droplets flow from the first end of the first sorting channel 3051 to the second end of the first sorting channel 3051, and then flow into the first collector 3061. The target droplets flow from the first end of the second sorting channel 3052 to the second end of the second sorting channel 3052, and then flow into the second collector 3062.

In some embodiments, the inner wall surface of the delivery channel 303 is hydrophobic treated and thus has hydrophobicity. As previously described, the delivery channel 303 comprises the first delivery channel 3031 configured to allow the first fluid to flow therein and the second delivery channel 3032 configured to allow the second fluid to flow therein. The hydrophobically-treated first delivery channel 3031 may facilitate the flow of the first fluid therein. The hydrophobically-treated second delivery channel 3032 may promote the cell suspension to flow smoothly in the first branch of the first sub-channel 3032-1 of the second delivery channel 3032 without sticking to the inner wall surface, and may promote the mixed solution of cell suspension and biochemical reagent to flow smoothly in the second sub-channel 3032-2 and the third sub-channel 3032-3 of the second delivery channel 3032 without sticking to the inner wall surface. In this way, the dosage of the cell suspension can be precisely controlled, which is conducive to the uniform mixing of the cell suspension and the biochemical reagent, thereby promoting the uniform generation of droplets. At the same time, this can also improve the utilization of the cell suspension and avoid the waste of the cell suspension.

In the microfluidic chip 300 shown in FIG. 1A, the first container 301 further comprises an inlet to which an external device (such as a box device described below) is connected and through which the first fluid is injected into the first container 301. The first sub-container 3021 of the second container 302 comprises an inlet to which an external device (such as a box device described below) is connected and through which the cell suspension is injected into the first sub-container 3021. The second sub-container 3022 of the second container 302 comprises an inlet to which an external device (such as a box device described below) is connected and through which the biochemical reaction reagent is injected into the second sub-container 3022. The first collector 3061 and the second collector 3062 respectively comprise an outlet, and the outlets are used to connect with an external device (such as a box device described below) to deliver the droplets in the first collector 3061 and the second collector 3062 into the external device.

FIG. 1C is an enlarged view of any one of the first container 301, the first sub-container 3021 and the second sub-container 3022 of the second container 302 in FIG. 1A. As shown in FIG. 1C, the outline of each of the first container 301, the first sub-container 3021 and the second sub-container 3022 of the second container 302 comprises four chamfers. The shape of the four chamfers can be any appropriate shape, for example, it can be arc. It should be understood that the specific dimensions of the chamfer are not limited in the embodiments of the present disclosure. Taking the first sub-container 3021 of the second container 302 as an example, as shown in FIG. 1C, the outline of the first sub-container 3021 comprises four chamfers 313, and the shape of the four chamfers 313 is arc. The arc-shaped chamfer can reduce the dead volume of the cell suspension in the first sub-container 3021 and improve the liquid storage accuracy of the first sub-container 3021. “Dead volume” here refers to the uncontrollable volume during reagent injection. Specifically, if the four corners of the first sub-container 3021 are right angles instead of arc-shaped chamfers, due to the existence of the surface tension of the droplet, the cell suspension is not in a right-angle shape at the four right-angle positions of the first sub-container 3021. That is, the cell suspension cannot perfectly match the shape of the first sub-container 3021 and cannot fill the space occupied by the four right angles of the first sub-container 3021. Thus, the shape and volume of the cell suspension will change, and there is a certain randomness in this change in shape and volume, thereby introducing dead volume. This may cause the first sub-container 3021 of the microfluidic chip 300 to accommodate a different volume of cell suspension in each operation than in the previous operation, resulting in the inability to accurately control the amount of cell suspension. In the embodiment of the present disclosure, the four corners 313 of the first sub-container 3021 are designed as arc-shaped chamfers, which can make the cell suspension perfectly match the shape of the first sub-container 3021, in particular, the cell suspension can be filled with the space occupied by the four arc-shaped chamfers of the first sub-container 3021. Therefore, the difference in the accommodation volume of the first sub-container 3021 can be effectively reduced or even avoided, and the control accuracy of the cell suspension can be improved.

Similarly, the four arc-shaped chamfers of the first container 301 can reduce the dead volume of the first fluid in the first container 301 and improve the liquid storage accuracy of the first container 301. The four arc-shaped chamfers of the second sub-container 3022 of the second container 302 can reduce the dead volume of the biochemical reagent in the second sub-container 3022 and improve the liquid storage accuracy of the second sub-container 3022.

Continuing to refer to FIG. 1C, any one of the first container 301, the first sub-container 3021 and the second sub-container 3022 of the second container 302 of the microfluidic chip 300 is provided with a filter structure 312. Since the filter structures 312 of the first container 301, the first sub-container 3021, and the second sub-container 3022 have the same structure, the structure and function of the filter structure 312 will be described in detail below by taking the filter structure 312 in the first sub-container 3021 as an example.

As shown in FIG. 1C, the filter structure 312 comprises a plurality of microstructures spaced apart from each other, and the gap d between two adjacent microstructures 312-1 and 312-2 is larger than 1 time of the particle size of a single cell and smaller than 2 times of the particle size of a single cell. In some embodiments, the particle size of a single cell derived from the cell suspension is about 10 μm, and correspondingly, the gap d between two adjacent microstructures 312-1 and 312-2 is greater than 10 μm and less than 20 μm. The heights of a plurality of microstructures of the filter structure 312 may be completely the same, may be completely different, or may be only partly the same. The specific height can be flexibly designed according to product requirements, which is not specifically limited in the embodiments of the present disclosure. In some embodiments, the height of each micropillar is about 100-200 μm. In a direction parallel to the plane where the first sub-container 3021 is located, the shape of the cross-section of each micropillar can be any appropriate shape, such as rhombus, square, rectangle, circle, ellipse, regular polygon, irregular shape, etc., which are not specifically limited in the embodiments of the present disclosure.

During the operation of the microfluidic chip 300, the cell suspension in the first sub-container 3021 flows through the gap between the adjacent microstructures of the filter structure 312, then flows into the first branch of the first sub-channel 3032-1 of the second delivery channel 3032. Since the gap d between two adjacent microstructures is greater than 1 time of the particle size of a single cell and less than 2 times of the particle size of a single cell, when the cell suspension flows through the gap between adjacent microstructures, on the one hand, it can prevent excessively large impurities in the cell suspension (for example, impurities with a particle size larger than 2 times of the particle size of a single cell, such as dust, salt-out substances, etc.) from flowing into the subsequent channel, so as to avoid excessively large impurities from blocking the channel and affecting the normal flow of the cell suspension. On the other hand, under the force of the adjacent microstructures on the cell suspension and the sorting of the size of the cell suspension by the gap between adjacent microstructures, multiple cells adhered to each other in the cell suspension (for example, two cells, three cells or more cells adhered to each other) can be separated into a plurality of single cells separated from each other, so as to facilitate the preparation of droplet comprising a single cell and reduce the probability that a single droplet comprises two or more cells.

For the structure of the filter structure 312 in the first container 301 and the second sub-container 3022, reference may be made to the above description of the filter structure in the first sub-container 3021, and details are omitted here for the sake of brevity. During the operation of the microfluidic chip 300, the first fluid in the first container 301 flows through the gap between the adjacent microstructures of the filter structure 312, and then flows into the first delivery channel 3031 of the delivery channel 303. When the first fluid flows through the gap between adjacent microstructures of the filter structure 312, oversized impurities in the first fluid (for example, impurities with a particle size larger than 2 times of the particle size of a single cell, such as dust, salt-out substances, etc.) can be prevented from flowing into the first delivery channel 3031, so as to prevent oversized impurities from blocking the first delivery channel 3031 and affecting the normal flow of the first fluid. During the operation of the microfluidic chip 300, the biochemical reagent in the second sub-container 3022 flows through the gap between the adjacent microstructures of the filter structure 312, then flows into the second branch of the first sub-channel 3032-1 of the second delivery channel 3032. When the biochemical reagent flows through the gap between the adjacent microstructures of the filter structure 312, the oversized impurities in the biochemical reagent (for example, impurities with a particle size larger than 2 times of the particle size of a single cell, such as dust, salt-out substances, etc.) can be prevented from flowing into the second branch of the first sub-channel 3032-1, so as to prevent the second branch from being blocked by the oversized impurities and affecting the normal flow of biochemical reagent.

FIG. 2 shows a schematic structural diagram of a microfluidic chip 400, wherein (a) is a front view of the microfluidic chip 400, (b) is a left view of the microfluidic chip 400, (c) is a rear view of the microfluidic chip 400, and (d) is a trimetric view of the microfluidic chip 400. The microfluidic chip 400 can be used to sort out a target droplet comprising a single target cell from the droplets. The microfluidic chip 400 can be used alone as an independent component to realize the sorting of target droplets, or can also be used to replace the sorting channel 305 and the collector 306 of the microfluidic chip 300, so as to realize the preparation of a droplet comprising a single cell and the sorting of the target droplets.

As shown in FIG. 2, the microfluidic chip 400 comprises a sorting channel 403, a connecting channel 404, and collectors 405 and 406. The sorting channel 403 comprises a first sorting channel 4031 and a second sorting channel 4032, and the second sorting channel 4032 comprises a first branch 4032A, a second branch 4032B, and a third branch 4032C which are cascaded. The connecting channel 404 comprises a first connecting channel 4041, a second connecting channel 4042 and a third connecting channel 4043. The collector comprises a first collector 405 and a second collector 406, and the second collector 406 comprises a first sub-collector 4061, a second sub-collector 4062, and a third sub-collector 4063. Optionally, the microfluidic chip 400 may further comprise two third containers 401 and one fourth container 402, each third container 401 is configured to accommodate the first fluid of the oil phase, and the fourth container 402 is configured to accommodate a large number of droplets, which comprise target droplets and non-target droplets, wherein each target droplet comprises a single target cell. The droplets can be prepared by other devices. As shown in the figure, the beginning of the first sorting channel 4031 communicates with the fourth container 402, the end of the first sorting channel 4031 communicates with the first collector 405, and the first sorting channel 4031 communicates with the first branch 4032A of the second sorting channel 4032 via the first connecting channel 4041. The beginning of the first branch 4032A of the second sorting channel 4032 communicates with the third container 401, the end of the first branch 4032A of the second sorting channel 4032 communicates with the first sub-collector 4061, and the first branch 4032A of the second sorting channel 4032 communicates with the second branch 4032B of the second sorting channel 4032 via the second connecting channel 4042. The beginning of the second branch 4032B of the second sorting channel 4032 communicates with the third container 401, the end of the second branch 4032B of the second sorting channel 4032 communicates with the second sub-collector 4062, and the second branch 4032B of the second sorting channel 4032 communicates with the third branch 4032C of the second sorting channel 4032 via the third connecting channel 4043. The beginning of the third branch 4032C of the second sorting channel 4032 communicates with the third connecting channel 4043, and the end of the third branch 4032C of the second sorting channel 4032 communicates with the third sub-collector 4063. The microfluidic chip 400 may further comprise a plurality of optical recognition devices and a plurality of driving electrode devices (not shown in the figure), so that the microfluidic chip 400 can realize cascaded sorting of target cells.

In the cell suspension, there may be only one type target cell, or there may be many different types of target cells. When there are multiple different types of target cells, these different types of target cells need to be sorted out and collected into different collectors for subsequent detection.

The process of sorting target droplets by using the microfluidic chip 400 is as follows: adding the first fluid into the third container 401, and adding the droplets prepared by using other devices (such as other microfluidic chips) into the fourth container 402. The droplets comprise target droplets and non-target droplets, wherein the target droplet comprises a single target cell. Suppose that a droplet may comprise four different types of cells, A, B, C, and D, where type A, B, and C cells are target cells, and type D cell is non-target cell. Thus, the target droplet comprises: (a) a droplet comprising a single type A target cell, (b) a droplet comprising a single type B target cell, and (c) a droplet comprising a single type C target cell; the non-target droplet comprises: (d) a droplet comprising one or more type D non-target cells. The above droplets have been dyed in the early stage.

The droplets in the fourth container 402 flow into the first sorting channel 4031. At the connection position of the first sorting channel 4031 and the first connecting channel 4041, the first optical recognition device detects the optical signal (e.g., color) of the droplet in real-time. When the first optical recognition device detects that the droplet is in the above-mentioned situation (d), it will not notify the circuit system, and the circuit system will not apply a voltage to the first driving electrode device associated with the first optical recognition device. Therefore, the non-target droplet moves along the first sorting channel 4031 until it flows into the first collector 405. When the first optical recognition device detects that the droplet is in any of the above-mentioned situations (a)-(c), it immediately informs the circuit system to apply an appropriate voltage to the first driving electrode device, and the target droplet is polarized. Under the action of the electric field, the target droplet is deflected upward and flows into the first connecting channel 4041, and then flows into the first branch 4032A of the second sorting channel 4032 via the first connecting channel 4041. At the connecting position of the first branch 4032A and the second connecting channel 4042, the second optical recognition device performs real-time detection on the optical signal of the target droplet. When the second optical recognition device detects that the target droplet is in the above-mentioned situation (a), it will not notify the circuit system, and the circuit system will not apply a voltage to the second driving electrode device associated with the second optical recognition device. Therefore, the target droplet (a) continues to move along the first branch 4032A until it flows into the first sub-collector 4061, so that the target droplet comprising a single type A target cell can be sorted from the droplets. When the second optical recognition device detects that the target droplet is in the above situation (b) or (c), it immediately informs the circuit system to apply an appropriate voltage to the second driving electrode device, and the target droplet (b) or (c) is polarized. Under the action of the electric field, the target droplet (b) or (c) is deflected upward and flows into the second connecting channel 4042, and then flows into the second branch 4032B via the second connecting channel 4042. At the connecting position of the second branch 4032B and the third connecting channel 4043, the third optical recognition device performs real-time detection on the optical signal of the target droplet (b) or (c). When the third optical recognition device detects that the target droplet is in the above-mentioned situation (b), it will not notify the circuit system, and the circuit system will not apply a voltage to the third driving electrode device associated with the third optical recognition device. Therefore, the target droplet (b) continues to move along the second branch 4032B until it flows into the second sub-collector 4062, so that the target droplet comprising a single type B target cell can be sorted from the droplets. When the third optical recognition device detects that the target droplet is in the above-mentioned situation (c), it immediately informs the circuit system to apply an appropriate voltage to the third driving electrode device, and the target droplet (c) is polarized. Under the action of the electric field, the target droplet (c) is deflected upward and flows into the third connecting channel 4043, and then flows into the third branch 4032C via the third connecting channel 4043, and finally flows into the third sub-collector 4063, so that the target droplet comprising a single type C target cell can be sorted from the droplets.

With the microfluidic chip 400, three different types of target cells can be sorted out through a single sorting process, which greatly improves the speed and efficiency for sorting cells. Moreover, compared to using three different microfluidic chips to sort three different types of target cells, in the embodiment of the present disclosure, only one microfluidic chip 400 is used to realize the sorting of three different types of target cells, which greatly saves the number of required microfluidic chips, thereby saving production costs.

When the microfluidic chip 400 is used to replace the sorting channel 305 and the collector 306 of the microfluidic chip 300, the fourth container 402 can be omitted. Alternatively, the beginning of the first sorting channel 4031 is connected to the end of the third sub-channel 3032-3 of the microfluidic chip 300, and other arrangements of the microfluidic chip 400 may remain unchanged. In this way, the droplets generated at the confluence 304 flow into the first sorting channel 4031 along the third sub-channel 3032-3, and the droplets are then subjected to cascaded sorting as described above. With this design, a microfluidic chip can not only prepare a droplet comprising a single cell, but also sort the droplets in a cascaded manner to sort many different types of target cells.

In actual operation, the oil-phase first fluid in the third container 401 can be pre-filled with the microfluidic chip 400, so that the droplets in the sorting channel 403 can flow more smoothly.

As shown in FIG. 2, one end of the first connecting channel 4041 is located between the beginning and the end of the first sorting channel 4031, and the other end of the first connecting channel 4041 is located between the beginning and the end of the first branch 4032A; one end of the second connecting channel 4042 is located between the beginning and the end of the first branch 4032A, the other end of the second connecting channel 4042 is located between the beginning and the end of the second branch 4032B, and the second connecting channel 4042 is closer to the collector in the second direction (i.e., the lateral direction in FIG. 2) than the first connecting channel 4041 (i.e., in the figure, the second connecting channel 4042 is offset to the right by a distance compared to the first connecting channel 4041). One end of the third connecting channel 4043 is located between the beginning and the end of the second branch 4032B, the other end of the third connecting channel 4043 communicates with the beginning of the third branch 4032C, and the third connecting channel 4043 is closer to the collector in the lateral direction than the second connecting channel 4042 (that is, in the figure, the third connecting channel 4043 is offset to the right by a distance compared to the second connecting channel 4042). In other words, in the second direction, the first connecting channel 4041 is located on the left side of the second connecting channel 4042, and the second connecting channel 4042 is located on the left side of the third connecting channel 4043. With this arrangement, the droplets can smoothly flow from the first sorting channel 4031 to the first branch 4032A, the second branch 4032B, and the third branch 4032C of the second sorting channel 4032 sequentially, thereby achieving cascaded sorting as described above. Further, the sorting channel 403 and the connecting channel 404 are configured so that the droplets flow from the first sorting channel 4031 through the connecting channel 404 into the first branch 4032A, the second branch 4032B, and the third branch 4032C of the second sorting channel 4032 in sequence, and the direction of flow of the droplets is irreversible. With such arrangement, the droplets flowing into the next branch are prevented from flowing back to the previous last branch, thereby avoiding the cross flow of different types of target cells.

It should be noted that although the second sorting channel 4032 of the microfluidic chip 400 shown in FIG. 2 comprises three branches 4032A, 4032B, and 4032C, this is only an example. The number of branches of the second sorting channel 4032 depends on the types of target cells to be sorted, which is not specifically limited in the embodiment of the present disclosure. For example, when N (N≥2) different types of target cells need to be sorted from the droplets, the microfluidic chip 400 may comprise N connecting channels, and the second sorting channel 4032 may comprise N cascaded branches, a connecting channel is provided between any two adjacent branches in the N cascaded branches, and the any two adjacent branches are communicated via the connecting channel. Correspondingly, the second collector 406 includes N sub-collectors, and the N cascaded branches of the second sorting channel 4032 are in one-to-one correspondence with the N sub-collectors, and one of the N cascaded branches communicates with a corresponding one of the N sub-collectors.

FIG. 3 shows a variant 400′ of the microfluidic chip 400, wherein (a) is a front view of the microfluidic chip 400′, (b) is a left view of the microfluidic chip 400′, (c) is a rear view of the microfluidic chip 400′, and (d) is a trimetric view of the microfluidic chip 400′. Compared with the microfluidic chip 400 shown in FIG. 2, the microfluidic chip 400′ shown in FIG. 3 has a similar structure as the microfluidic chip 400 except for the sorting channel 403 and the collectors 405′ and 406. The same reference numerals refer to the same components. Therefore, for the sake of brevity, the functions of these same components will not be described, reference may be made to the description of the microfluidic chip 400, and only different components will be described below.

The microfluidic chip 400′ can be used to sort out a target droplet comprising a single target cell from the droplets. The microfluidic chip 400′ can be used alone as an independent component to realize the sorting of target droplets, or can also be used to replace the sorting channel 305 and the collector 306 of the microfluidic chip 300, so as to realize the preparation of a droplet comprising a single cell and the sorting of target droplets.

As shown in FIG. 3, the microfluidic chip 400′ comprises a sorting channel 403, a connecting channel 404, and collectors 405′ and 406. The sorting channel 403 comprises a first sorting channel 4031 and a second sorting channel 4032, and the first sorting channel 4031 comprises a first branch 4031A, a second branch 4031B, and a third branch 4031C which are cascaded. The connecting channel 404 comprises a first connecting channel 4041, a second connecting channel 4042 and a third connecting channel 4043. The collector comprises a first collector 405′ and a second collector 406. Optionally, the microfluidic chip 400′ may further comprise two third containers 401 and one fourth container 402, each third container 401 is configured to accommodate the first fluid of the oil phase, and the fourth container 402 is configured to accommodate a plurality of droplets comprising target droplets and non-target droplets, wherein each target droplet comprises a single target cell. As shown in FIG. 3, the beginning of the first branch 4031A of the first sorting channel 4031 communicates with the fourth container 402, the end of the first branch 4031A of the first sorting channel 4031 communicates with the first collector 405′, and the first branch 4031A and the second branch 4031B of the first sorting channel 4031 communicate via the first connecting channel 4041. The beginning of the second branch 4031B of the first sorting channel 4031 communicates with the third container 401, the end of the second branch 4031B of the first sorting channel 4031 communicates with the first collector 405′, and the second branch 4031B and the third branch 4031C of the first sorting channel 4031 communicate via the second connecting channel 4042. The beginning of the third branch 4031C of the first sorting channel 4031 communicates with the third container 401, the end of the third branch 4031C of the first sorting channel 4031 communicates with the first collector 405′, and the third branch 4031C of the first sorting channel 4031 communicates with the second sorting channel 4032 via the third connecting channel 4043. The beginning of the second sorting channel 4032 communicates with the third connecting channel 4043, and the end of the second sorting channel 4032 communicates with the second collector 406. The microfluidic chip 400′ may further comprise a plurality of optical recognition devices and a plurality of driving electrode devices (not shown in the figure), so that the microfluidic chip 400′ can realize cascaded sorting of target cells.

When one type of target cells is present in the cell suspension, there may be a situation where this type of target cells is so similar to non-target cells in the cell suspension that they are indistinguishable. Therefore, it is difficult to sort out the desired target cells from the cell suspension by only one sorting process, or the possibility of success of sorting out the desired target cells from the cell suspension by one sorting process is low. Therefore, unlike the microfluidic chip 400, the microfluidic chip 400′ is not used to simultaneously sort out multiple different types of target cells, but is used to improve the purity of one type of target cells.

The process of sorting the target droplets by using the microfluidic chip 400′ is as follows: adding the first fluid into the third container 401, and adding droplets prepared by other devices (for example, other microfluidic chips) into the fourth container 402. The droplets comprise target droplets and non-target droplets, wherein the target droplet comprises a single target cell. Suppose that the droplet comprises two different types of cells, E and F, where type E cells are target cells, type F cells are non-target cells, and the type E target cells are indistinguishable from the type F non-target cells. Thus, a target droplet comprises: (e) a droplet comprising a single type E target cell. A non-target droplet comprises: (f) a droplet comprising one or more type F non-target cells. The above droplets have been dyed in the early stage. The droplets in the fourth container 402 flow into the first branch 4031A of the first sorting channel 4031. At the connection position of the first branch 4031A and the first connecting channel 4041, the first optical recognition device detects the optical signal (e.g., color) of the droplet in real time. When the first optical recognition device detects that the droplet is in the above-mentioned situation (f), it will not notify the circuit system, and the circuit system will therefore not apply a voltage to the first driving electrode device associated with the first optical recognition device. Therefore, the non-target droplet continues to move along the first branch 4031A until it flows into the first collector 405′. When the first optical recognition device determines that the droplets are in the above-mentioned situation (e), it immediately informs the circuit system to apply an appropriate voltage to the first driving electrode device, and the above-mentioned droplets (actually they still comprise some non-target droplets) are polarized. Under the action of the electric field, the above-mentioned droplets are deflected upward and flow into the first connecting channel 4041, and then flow into the second branch 4031B through the first connecting channel 4041. At the connecting position of the second branch 4031B and the second connecting channel 4042, the second optical recognition device performs real-time detection on the optical signal of the droplets. When the second optical recognition device detects that some of the droplets are still in the above-mentioned situation (f), the circuit system will not be notified, and the circuit system will therefore not apply a voltage to the second driving electrode device associated with the second optical recognition device. Therefore, the sorted non-target droplets (f) continue to move along the second branch 4031B, and finally flow into the first collector 405′. When the second optical recognition device determines that the droplets are in the above-mentioned situation (e), it immediately informs the circuit system to apply an appropriate voltage to the second driving electrode device, and the droplets are polarized. Under the action of the electric field, the droplets are deflected upward and flow into the second connecting channel 4042, and then flow into the third branch 4031C through the second connecting channel 4042. At the connecting position of the third branch 4031C and the third connecting channel 4043, the third optical recognition device performs real-time detection on the optical signal of the droplets (which actually still comprise a small amount of non-target droplets). When the third optical recognition device detects that some of the droplets are still in the above-mentioned situation (f), the circuit system will not be notified, and the circuit system will not apply a voltage to the third driving electrode device associated with the third optical recognition device. Therefore, the non-target droplets continue to move along the third branch 4031C and then flow into the first collector 405′. When the third optical recognition device detects that the droplet is in the above-mentioned situation (e), it immediately informs the circuit system to apply an appropriate voltage to the third driving electrode device, and the target droplet (e) is polarized. Under the action of the electric field, the target droplet (e) is deflected upward and flows into the third connecting channel 4043, and then flows into the second sorting channel 4032 through the third connecting channel 4043, and finally flows into the second collector 406, the target droplet comprising a single type E target cell is sorted from the droplets.

Using the microfluidic chip 400′, through multiple cascaded sorting of droplets, indistinguishable target droplets and non-target droplets can be distinguished from each other, which greatly improves the purity of the final collected target droplets. The possibility of non-target droplets being comprised in the collected target droplets is reduced or even excluded.

It should be noted that although the first sorting channel 4031 of the microfluidic chip 400′ shown in FIG. 3 comprises three branches 4031A, 4031B and 4031C, this is only an example. The specific number of branches of the first sorting channel 4031 may be determined according to the difficulty of distinguishing target cells from non-target cells, which is not specifically limited in the embodiment of the present disclosure.

When the microfluidic chip 400′ is used to replace the sorting channel 305 and the collector 306 of the microfluidic chip 300, the fourth container 402 may be omitted, and alternatively, the beginning of the first branch 4031A of the first sorting channel 4031 is connected to the end of the third sub-channel 3032-3 of the microfluidic chip 300, and other arrangements of the microfluidic chip 400′ can remain unchanged. In this way, the droplets generated at the confluence 304 flow along the third sub-channel 3032-3 into the first branch 4031A of the first sorting channel 4031, and then the droplets are subjected to cascaded sorting as described above. With such design, a microfluidic chip can not only prepare a droplet comprising a single cell, but also perform a cascaded sorting on such droplets, so that indistinguishable target droplets and non-target droplets can be distinguished from each other, which greatly improves the purity of the final collected target droplets.

FIG. 4 shows a schematic structural diagram of a microfluidic chip 500, wherein (a) is a front view of the microfluidic chip 500, (b) is a left view of the microfluidic chip 500, (c) is a rear view of the microfluidic chip 500, and (d) is a trimetric view of the microfluidic chip 500. The microfluidic chip 500 can be used to sort two types of droplets with different particle sizes from the droplets. The microfluidic chip 500 can be used alone as an independent component, or can be used to replace the sorting channel 305 and the collector 306 of the microfluidic chip 300, so that the preparation of a droplet comprising a single cell and the sorting of the target droplets can be realized.

As shown in FIG. 4, the microfluidic chip 500 comprises a sorting channel 502 and a collector 506, and the sorting channel 502 comprises a main channel 503, a first sorting channel 504, and a second sorting channel 505. The collector 506 comprises a first collector 507 and a second collector 508. The main channel 503 is helical in the plane where the microfluidic chip 500 is located. The end of the main channel 503 communicates with the first sorting channel 504 and the second sorting channel 505. The end of the first sorting channel 504 communicates with the first collector 507, and the end of the second sorting channel 505 communicates with the second collector 508. Optionally, the microfluidic chip 500 may further comprise a third container 501, the third container 501 is configured to accommodate droplets which comprise first type droplets and second type droplets with different particle sizes.

The cell suspension comprises cells with a smaller particle size and cells with a larger particle size, and when such a cell suspension is mixed with the first fluid and forms a plurality of droplets each comprising a single cell through the above-mentioned process, the droplets thus have different particle sizes. Here, droplets comprising cells with smaller particle size are referred to as the first type of droplets, and the first type of droplets have smaller particle size; droplets comprising cells with larger particle size are referred to as the second type of droplets, and the second type of droplets have a larger particle size. When the microfluidic chip 500 is used to sort droplets, the droplets in the third container 501 flow into the helical main channel 503, due to the difference in particle size of the droplets, the inertial force is different. At the end bifurcation of the main channel 503, the first type of droplets with smaller particle size are subject to less inertial force, so they follow along the extension direction of the main channel 503 into the first sorting channel 504 and then flow into the first collector 507. The second type of droplets with larger particle size are subjected to larger inertial force, and are thrown out of the main channel 503 under the action of the inertial force and enter the second sorting channel 505, and finally flow into the second collector 508.

FIG. 4 only shows one possible shape of the main channel 503 as an example, but the shape of the main channel 503 is not limited to this, as long as the shape of the main channel 503 can enable droplets with different particle sizes to enter different sorting channels under the action of different inertial forces.

The microfluidic chip 500 does not need to be provided with an optical recognition device and a driving electrode device, and only depends on the shape of the main channel 503 to distinguish droplets of different particle sizes. Since the optical recognition device and the driving electrode device are not required, not only the volume of the microfluidic chip 500 can be reduced, but also the production cost can be saved.

When the microfluidic chip 500 is used to replace the sorting channel 305 and the collector 306 of the microfluidic chip 300, the third container 501 can be omitted. Alternatively, the beginning of the main channel 503 is connected to the end of the third sub-channel 3032-3 of the microfluidic chip 300, and other arrangements of the microfluidic chip 500 may remain unchanged. In this way, the droplets generated at the confluence 304 flow into the main channel 503 along the third sub-channel 3032-3, and then the sorting operation as described above is performed on the droplets. Through this design, a microfluidic chip can not only prepare a droplet comprising a single cell, but also distinguish droplets of different particle sizes.

The inventors of the present application found that, in conventional techniques, the first fluid and the second fluid (comprising cell suspension and biochemical reagent) described in the above embodiments need to be respectively stored in an external device independent of the microfluidic chip. During the operation of the microfluidic chip, manual operation is required each time to connect the external device with the inlet of the microfluidic chip by using a flexible pipe, so as to inject the first fluid and the second fluid into the microfluidic chip in real time, and then through the corresponding processing of the microfluidic chip, the droplets are prepared and/or the target droplets are sorted from the droplets. Therefore, the preparation of droplets and/or the sorting of target droplets requires at least the external device for storing fluids, the flexible pipe, and the microfluidic chip. This makes the system bulky and not easy to carry. In addition, when the microfluidic chip is replaced to prepare different reagents, the external device needs to be cleaned to accommodate the new reagents adapted to the replaced microfluidic chip, but it is usually impossible to guarantee that the external device can be thoroughly cleaned, so the reagents remaining before are likely to remain in the external device, thereby causing contamination of the replaced new reagents.

In view of this, the embodiments of the present disclosure provide a box device adapted to a microfluidic chip, each microfluidic chip has a corresponding box device, and the box device can be combined with the microfluidic chip by an appropriate bonding method. The box device can store reagents and release the reagents to the inlet of the microfluidic chip, and can receive and store the reagents flowing into the box device from the outlet of the microfluidic chip. Such a box device can provide a sterile environment as the cell suspension can be completely contained within the sealed box device before and after cell sorting.

FIG. 5A shows a schematic structural diagram of a box device 1000 according to an embodiment of the present disclosure, wherein (a) is a front view of the box device 1000, (b) is a right view of the box device 1000, (c) is a top view of the box device 1000, and (d) is a trimetric view of the box device 1000. FIG. 5B shows a schematic structural diagram of a microfluidic chip 100, which is described in the priority application (No. 202180000922.0). The box device 1000 is adapted to the microfluidic chip 100, and the combination of the two can be used to prepare the droplet comprising a single cell. For the specific preparation process of the droplets, reference can be made to the description of the priority application.

Referring to FIG. 5A and FIG. 5B, the box device 1000 is configured to be used with the microfluidic chip 100, and the microfluidic chip 100 comprises inlets 1, 2, 3 and an outlet 4. The box device 1000 comprises: an accommodating cavity configured to accommodate the microfluidic chip 100; an inlet unit 1001 communicated with the inlets 1, 2, and 3 of the microfluidic chip 100, and the inlet unit 1001 being configured to store a first reagent and release the first reagent to the inlets 1, 2, and 3 of the microfluidic chip 100; and an outlet unit 1002 communicated with the outlet 4 of the microfluidic chip 100. The outlet unit 1002 is configured to receive and store the second reagent that is processed by the microfluidic chip 100 and flows into the outlet unit 1002 from the outlet 4 of the microfluidic chip 100. The second reagent comprises target droplets, each target droplet comprises a single target cell. The inlet unit 1001 comprises inlet holes 1003A/1004A/1005A and first storage cavities 1003B/1004B/1005B, each inlet hole is a through hole and communicates with the corresponding first storage cavity, each inlet hole is recessed from the surface of the box device 1000 to the inside of the box device 1000, and the first storage cavity corresponding to the inlet hole is located on a side of the inlet hole away from the surface of the box device 1000.

By providing the box device 1000, each microfluidic chip 100 can be provided with a separate box device 1000, and the box device 1000 can store the injection reagent (i.e., the first reagent) required by the microfluidic chip 100 and the output reagent (i.e., the second reagent) processed by the microfluidic chip 100. Therefore, there is no need to provide an external storage device, which can greatly reduce the size of the device and make it easy to carry. In addition, since each microfluidic chip 100 is provided with a separate box device 1000, the box device 1000 stores the first reagent required by the microfluidic chip 100 and the second reagent produced by the microfluidic chip 100. Therefore, there is no risk of cross-contamination of the reagents in the external storage device due to the replacement of the microfluidic chip in the conventional technology. Further, the inlet unit 1001 comprises the inlet holes and the first storage cavities. Such a design can better guide the first reagent to flow from the inlet hole to the first storage cavity, and then flow into the inlet of the microfluidic chip 100 through the first storage cavity.

Continuing to refer to FIG. 5A and FIG. 5B, the inlet unit 1001 of the box device 1000 comprises a first inlet unit 1003, a second inlet unit 1004, and a third inlet unit 1005, the inlets of the microfluidic chip 100 comprise a first inlet 1, a second inlet 2, and a third inlet 3, and the first reagent comprises a first sub-reagent (i.e., the first fluid), a second sub-reagent (i.e., the cell suspension), and a third sub-reagent (i.e., the biochemical reagent).The first inlet unit 1003 of the box device 1000 communicates with the first inlet 1 of the microfluidic chip 100, and the first inlet unit 1003 is configured to store the first sub-reagent and release the first sub-reagent to the first inlet 1 of the microfluidic chip 100. The second inlet unit 1004 of the box device 1000 communicates with the second inlet 2 of the microfluidic chip 100, and the second inlet unit 1004 is configured to store the second sub-reagent and release the second sub-reagent to the second inlet 2 of the microfluidic chip 100. The third inlet unit 1005 of the box device 1000 communicates with the third inlet 3 of the microfluidic chip 100, and the third inlet unit 1005 is configured to store the third sub-reagent and release the third sub-reagent to the third inlet 3 of the microfluidic chip 100. The outlet unit 1002 of the box device 1000 comprises an outlet unit 1006, and the second reagent received and stored by the outlet unit 1006 comprises target droplets and non-target droplets.

As shown in the figure, the first inlet unit 1003 comprises the inlet hole 1003A and the first storage cavity 1003B, the second inlet unit 1004 comprises the inlet hole 1004A and the first storage cavity 1004B, and the third inlet unit 1005 comprises the inlet hole 1005A and the first storage cavity 1005B. The first inlet unit 1003, the second inlet unit 1004, and the third inlet unit 1005 have the same structures, and the first inlet unit 1003 is taken as an example to describe the structure of each inlet unit below. Since the first inlet unit 1003, the second inlet unit 1004, and the third inlet unit 1005 have the same structure, the following description about the structure of the first inlet unit 1003 is also applicable to the second inlet unit 1004 and the third inlet unit 1005.

The first storage cavity 1003B of the first inlet unit 1003 is located inside of the box device 1000, and the orthographic projection of the inlet hole 1003A on the box device 1000 falls within the orthographic projection of the first storage cavity 1003B on the box device 1000. For example, as shown in FIG. 5A, the width of the inlet hole 1003A in the lateral direction is smaller than the width of the first storage cavity 1003B in the lateral direction. With such an arrangement, the flow rate of the first sub-reagent in the inlet hole 1003A can be increased, and the first sub-reagent is promoted to flow from the inlet hole 1003A into the first storage cavity 1003B, and finally the first sub-reagent flows into the first inlet 1 of the microfluidic chip 100.

In some embodiments, the first inlet unit 1003 may further comprise a second storage cavity 1003C (similarly, the second inlet unit 1004 may further comprise a second storage cavity 1004C, and the third inlet unit 1005 may further comprise a second storage cavity 1005C), the second storage cavity 1003C is located on the side of the first storage cavity 1003B away from the inlet hole 1003A and communicates with the first storage cavity 1003B. The second storage cavity 1003C comprises a first opening communicated with the first storage cavity 1003B and a second opening opposite to the first opening. The orthographic projection of the second opening of the second storage cavity 1003C on the box device 1000 falls within the orthographic projection of the first opening on the box device 1000. In an example, as shown in FIG. 5A, the second storage cavity 1003C has a bowl-like shape, that is, the second storage cavity 1003C has a shape that is wide at the top and narrow at the bottom. With such an arrangement, the second storage cavity 1003C can well collect the first sub-reagent flowing into it from the first storage cavity 1003B, and guide the first sub-reagent to the first inlet 1 of the microfluidic chip 100. In some embodiments, the orthographic projection of the second opening of the second storage cavity 1003C on the box device 1000 falls within the orthographic projection of the inlet hole 1003A on the box device 1000.

Continuing to refer to FIG. 5A, the outlet unit 1006 of the box device 1000 comprises the outlet hole 1006A and the third storage cavity 1006B. The outlet hole 1006A is a through hole and communicates with the third storage cavity 1006B, the outlet hole 1006A is recessed from the surface of the box device 1000 to the inside of the box device 1000, and the third storage cavity 1006B is located on a side of the outlet hole 1006A away from the surface of the box device 1000. In some embodiments, the third storage cavity 1006B is located inside of the box device 1000, and the orthographic projection of the outlet hole 1006A on the box device 1000 falls within the orthographic projection of the third storage cavity 1006B on the box device 1000. For example, as shown in FIG. 5A, the width of the outlet hole 1006A in the lateral direction is smaller than the width of the third storage cavity 1006B in the lateral direction. With this arrangement, the third storage cavity 1006B mainly plays the role of storing the second reagent, and the outlet hole 1006A can better facilitate the transfer of the second reagent in the third storage cavity 1006B to the external device (if necessary).

In some embodiments, the outlet unit 1006 may further comprise a fourth storage cavity 1006C, which is located on a side of the third storage cavity 1006B away from the outlet hole 1006A and communicates with the third storage cavity 1006B. The fourth storage cavity 1006C can be used to connect the outlet 4 of the microfluidic chip 100 with the outlet unit 1006 of the box device 1000, and can guide the second reagent flowing out from the outlet 4 of the microfluidic chip 100 to the third storage cavity 1006B of the box device 1000. In some embodiments, the orthographic projection of the fourth storage cavity 1006C on the box device 1000 overlaps at most a part with the orthographic projection of the outlet hole 1006A on the box device 1000.

The general process of preparing a droplet comprising a single cell using the box device 1000 and the microfluidic chip 100 can be described as follows:

• • (1) Pre-adding the first fluid, the cell suspension and the biochemical reagent to the first inlet unit 1003, the second inlet unit 1004 and the third inlet unit 1005 respectively. The first fluid is the oil phase, which may be mixed with surfactants.
  • (2) Connecting the inlet holes of the first inlet unit 1003, the second inlet unit 1004 and the third inlet unit 1005 of the box device 1000 to a flow pump through flexible pipes, and controlling the flow rate of fluid injected into the inlet units by adjusting the pressure of the flow pump.
  • (3) The first fluid in the first inlet unit 1003 flows into the first inlet 1 of the microfluidic chip 100 through the inlet hole 1003A, the first storage cavity 1003B and the second storage cavity 1003C; the cell suspension in the second inlet unit 1004 flows into the second inlet 2 of the microfluidic chip 100 through the inlet hole 1004A, the first storage cavity 1004B and the second storage cavity 1004C; the biochemical reagent in the third inlet unit 1005 flows into the third inlet 3 of the microfluidic chip 100 through the inlet hole 1005A, the first storage cavity 1005B and the second storage cavity 1005C. Note that the first fluid of the oil phase can be filled with the microfluidic chip 100 first, and then the cell suspension and biochemical reagent can be injected.
  • (4) The above-mentioned first fluid, cell suspension, and biochemical reagent merge at the confluence 105 of the microfluidic chip 100 and generate droplets (i.e., the above-mentioned second reagent), and the droplets comprise target droplets and non-target droplets, where the target droplet includes a single target cell. The droplets flow into the first collector 104 through the delivery channel 103 of the microfluidic chip 100, and then flow into the outlet unit 1006 of the box device through the outlet 4 of the first collector 104. The outlet unit 1006 can store the droplets or can transfer the droplets to other equipment as needed.

FIG. 6A shows a schematic structural diagram of a box device 2000 according to another embodiment of the present disclosure, wherein (a) is a front view of the box device 2000, (b) is a right view of the box device 2000, (c) is a top view of the box device 2000, and (d) is a trimetric view of the box device 2000. FIG. 6B shows a schematic structural diagram of a microfluidic chip 200, which is described in the priority application (No. 202180000922.0). The box device 2000 is adapted to the microfluidic chip 200, and the combination of the two can be used to sort droplets to obtain target droplets. For the specific sorting process of droplets, reference can be made to the description of the priority application.

The box device 2000 comprises an inlet unit 2001 and an outlet unit 2002. The inlet unit 2001 communicates with the inlets of the microfluidic chip 200, and is configured to store a first reagent and release the first reagent to the inlets of the microfluidic chip 200, the first reagent is a plurality of droplets, each droplet of at least a portion of the plurality of droplets comprises a single cell. The outlet unit 2002 communicates with the outlets of the microfluidic chip 200 and is configured to receive and store a second reagent which is processed by the microfluidic chip 200 and flows from the outlets of the microfluidic chip 200 into the outlet unit 2002, the second reagent comprises target droplets and non-target droplets, wherein the target droplet comprises a single target cell. The inlet unit 2001 comprises a first inlet unit 2003 and a second inlet unit 2004, and the outlet unit 2002 comprises a first outlet unit 2005, a second outlet unit 2006, and a third outlet unit 2007 located between the first outlet unit 2005 and the second outlet unit 2006. The inlets of the microfluidic chip 200 comprise a first inlet 5 and a second inlet 6. The first inlet unit 2003 communicates with the first inlet 5 of the microfluidic chip 200, and the first inlet unit 2003 is configured to store a first sub-reagent (i.e., the first fluid) and release the first sub-reagent to the first inlet 5 of the microfluidic chip 200. The second inlet unit 2004 communicates with the second inlet 6 of the microfluidic chip 200, and the second inlet unit 2004 is configured to store a second sub-reagent (that is, a droplet comprising a single cell) and release the second sub-reagent to the second inlet 6 of the microfluidic chip 200. The third outlet unit 2007 of the outlet unit 2002 is configured to receive and store non-target droplets, and the first outlet unit 2005 and the second outlet unit 2006 of the outlet unit 2002 are configured to receive and store target droplets.

The first inlet unit 2003 of the box device 2000 comprises an inlet hole 2003A, a first storage cavity 2003B, and a second storage cavity 2003C; the second inlet unit 2004 comprises an inlet hole 2004A, a first storage cavity 2004B, and a second storage cavity 2004C. The structures of the first inlet unit 2003 and the second inlet unit 2004 of the box device 2000 are exactly the same as that of the first inlet unit 1003 of the box device 1000. Therefore, the first inlet unit 2003 and the second inlet unit 2004 of the box device 2000 have the same technical effects as the first inlet unit 1003 of the box device 1000. For the sake of brevity, their structures and technical effects are not repeated here. The first outlet unit 2005 of the box device 2000 comprises an outlet hole 2005A, a third storage cavity 2005B and a fourth storage cavity 2005C, and the second outlet unit 2006 of the box device 2000 comprises an outlet hole 2006A, a third storage cavity 2006B and a fourth storage cavity 2006C, the third outlet unit 2007 of the box device 2000 comprises an outlet hole 2007A, a third storage cavity 2007B and a fourth storage cavity 2007C. The first outlet unit 2005, the second outlet unit 2006 and the third outlet unit 2007 have exactly the same structures. Except for the relative position of the fourth storage cavity and the outlet hole, the structures of the first outlet unit 2005, the second outlet unit 2006 and the third outlet unit 2007 of the box device 2000 are the same as the structure of the outlet unit 1006 of the box device 1000, so the structure and technical effect of each outlet unit of the box device 2000 can refer to the structure and technical effect of the outlet unit 1006 of the box device 1000. In the box device 2000, taking the first outlet unit 2005 as an example, the orthographic projection of the fourth storage cavity 2005C on the box device 2000 falls within the orthographic projection of the outlet hole 2005A on the box device 2000.

The box device 2000 further comprises a first installation region 2008 and a second installation region 2009, the first installation region 2008 is configured to install the optical recognition device, and the second installation region 2009 is configured to install the driving electrode device. The optical recognition device and the driving electrode device are used to cooperate with the microfluidic chip 200 to realize the sorting of target droplets.

The general process of sorting target droplets using the box device 2000 and the microfluidic chip 200 can be described as follows:

• • (1) Pre-adding the first fluid and droplets each comprising a single cell to the first inlet unit 2003 and the second inlet unit 2004 respectively, and the droplets can be prepared by the above-mentioned box device 1000 and the microfluidic chip 100. The first fluid is the oil phase, which may be mixed with surfactants.
  • (2) Connecting the inlet hole 2003A of the first inlet unit 2003 and the inlet hole 2004A of the second inlet unit 2004 of the box device 2000 to the corresponding flow pumps through flexible pipes, and controlling the flow rate of fluid injected into the inlet units by adjusting the pressure of the flow pumps.
  • (3) The first fluid in the first inlet unit 2003 flows into the first inlet 5 of the microfluidic chip 200 through the inlet hole 2003A, the first storage cavity 2003B and the second storage cavity 2003C. The droplets in the second inlet unit 2004 flow into the second inlet 6 of the microfluidic chip 200 through the inlet hole 2004A, the first storage cavity 2004B and the second storage cavity 2004C. Note that the microfluidic chip 200 can be filled with the first fluid of the oil phase first, and then the droplets are injected.
  • (4) The above-mentioned droplets are sorted at the sorting channel 203 of the microfluidic chip 200 and enter the corresponding sub-collectors, the target droplets (each target droplet comprises a single target cell) are collected into the first sub-collector 2041 and the second sub-collector 2042, and the non-target droplets are collected into the third sub-collector 2043. The target droplets in the first sub-collector 2041 flow into the first outlet unit 2005 of the box device 2000 through the outlet 7A, the target droplets in the second sub-collector 2042 flow into the second outlet unit 2006 of the box device 2000 through the outlet 7B, and the non-target droplets in the third sub-collector 2043 flow into the third outlet unit 2007 of the box device 2000 through the outlet 7C. The first outlet unit 2005, the second outlet unit 2006, and the third outlet unit 2007 may store corresponding droplets or may transfer these droplets to other devices as required.

The box device 2000 is adapted to the microfluidic chip 200 to jointly realize the sorting of target droplets. Such a box device 2000 provides an environment for aseptic operation since the droplets are completely confined within the sealed box device 2000 and microfluidic chip 200 before and after sorting. Moreover, the existence of the box device 2000 makes the system composed of the box device 2000 and the microfluidic chip 200 more concise and convenient, and easy to carry.

FIG. 7 shows a schematic structural diagram of a box device 3000 according to yet another embodiment of the present disclosure, wherein (a) is a front view of the box device 3000, (b) is a right view of the box device 3000, (c) is a top view of the box device 3000, and (d) is a trimetric view of the box device 3000. The box device 3000 is adapted to the microfluidic chip 300 shown in FIG. 1A of the present application, and the combination of the two can be used to prepare droplets each comprising a single cell and sort the droplets to obtain target droplets. For the preparation and sorting process of droplets, reference may be made to the description of the microfluidic chip 300.

The box device 3000 comprises an inlet unit 3001 and an outlet unit 3002. The inlet unit 3001 communicates with the inlets of the microfluidic chip 300 and is configured to store a first reagent and release the first reagent to the inlets of the microfluidic chip 300. The outlet unit 3002 communicates with the outlets of the microfluidic chip 300 and is configured to receive and store a second reagent which is processed by the microfluidic chip 300 and flows into the outlet unit 3002 from the outlets of the microfluidic chip 300, the second reagent comprises target droplets and non-target droplets, wherein the target droplet comprises a single target cell. The inlet unit 3001 comprises a first inlet unit 3003, a second inlet unit 3004 and a third inlet unit 3005. The outlet unit 3002 comprises a first outlet unit 3006 and a second outlet unit 3007. The inlets of the microfluidic chip 300 comprise a first inlet located at the first container 301, a second inlet located at the first sub-container 3021, and a third inlet located at the second sub-container 3022. The first inlet unit 3003 communicates with the first inlet of the microfluidic chip 300, and the first inlet unit 3003 is configured to store a first sub-reagent (i.e., the first fluid) and release the first sub-reagent to the first inlet of the microfluidic chip 300. The second inlet unit 3004 communicates with the second inlet of the microfluidic chip 300, and the second inlet unit 3004 is configured to store a second sub-reagent (i.e., the cell suspension) and release the second sub-reagent to the second inlet of the microfluidic chip 300. The third inlet unit 3005 communicates with the third inlet of the microfluidic chip 300, and the third inlet unit 3005 is configured to store a third sub-reagent (i.e., the biochemical reagent) and release the third sub-reagent to the third inlet of the microfluidic chip 300. The first outlet unit 3006 of the outlet unit 3002 is configured to receive and store non-target droplets, and the second outlet unit 3007 of the outlet unit 3002 is configured to receive and store target droplets.

The first inlet unit 3003 of the box device 3000 comprises an inlet hole 3003A, a first storage cavity 3003B, and a second storage cavity 3003C; the second inlet unit 3004 comprises an inlet hole 3004A, a first storage cavity 3004B, and a second storage cavity 3004C; the third inlet unit 3005 comprises an inlet hole 3005A, a first storage cavity 3005B, and a second storage cavity 3005C. The structures of the first inlet unit 3003, the second inlet unit 3004 and the third inlet unit 3005 of the box device 3000 are exactly the same as that of the first inlet unit 1003 of the box device 1000. Therefore, the first inlet unit 3003, the second inlet unit 3004 and the third inlet unit 3005 of the box device 3000 have the same technical effects as the first inlet unit 1003 of the box device 1000. For the sake of brevity, their structures and technical effects are not repeated here. The first outlet unit 3006 of the box device 3000 comprises an outlet hole 3006A, a third storage cavity 3006B and a fourth storage cavity 3006C, and the second outlet unit 3007 of the box device 3000 comprises an outlet hole 3007A, a third storage cavity 3007B and a fourth storage cavity 3007C. Both the first outlet unit 3006 and the second outlet unit 3007 have exactly the same structures. Except for the relative position of the fourth storage cavity and the outlet, the structures of the first outlet unit 3006 and the second outlet unit 3007 of the box device 3000 are basically the same as that of the outlet unit 1006 of the box device 1000. Therefore, the structure and technical effect of each outlet unit of the box device 3000 may refer to the structure and technical effect of the outlet unit 1006 of the box device 1000. In the box device 3000, taking the first outlet unit 3006 as an example, the orthographic projection of the fourth storage cavity 3006C on the box device 3000 falls within the orthographic projection of the outlet hole 3006A on the box device 3000.

The box device 3000 further comprises a first installation region 3008 and a second installation region 3009, the first installation region 3008 is configured to install the optical recognition device, and the second installation region 3009 is configured to install the driving electrode device. The optical recognition device and the driving electrode device are used to cooperate with the microfluidic chip 300 to realize the sorting of target droplets. The first outlet unit 3006 and the second outlet unit 3007 are located between the inlet unit 3001 and the first installation region 3008 and the second installation region 3009. Similar to the microfluidic chip 300, through such an arrangement, the size of the box device 3000 can be reduced, the box device 3000 can be more miniaturized, and the cost can be saved.

The general process of preparing droplets and sorting the target droplets by using the box device 3000 and the microfluidic chip 300 can be described as follows:

• • (1) Pre-adding the first fluid, the cell suspension and the biochemical reagent to the first inlet unit 3003, the second inlet unit 3004 and the third inlet unit 3005 respectively. The first fluid is the oil phase, which may be mixed with surfactants.
  • (2) Connecting the inlet hole 3003A of the first inlet unit 3003, the inlet hole 3004A of the second inlet unit 3004, and the inlet hole 3005A of the third inlet unit 3005 of the box device 3000 to the corresponding flow pumps via the flexible pipes, and controlling the flow rate of the fluid injected into the inlet units by adjusting the pressure of the flow pumps.
  • (3) The first fluid in the first inlet unit 3003 flows into the first inlet of the microfluidic chip 300 through the inlet hole 3003A, the first storage cavity 3003B and the second storage cavity 3003C; the cell suspension in the second inlet unit 3004 flows into the second inlet of the microfluidic chip 300 through the inlet hole 3004A, the first storage cavity 3004B and the second storage cavity 3004C; the biochemical reagent in the third inlet unit 3005 flows into the third inlet of the microfluidic chip 300 through the inlet hole 3005A, the first storage cavity 3005B and the second storage cavity 3005C. Note that the microfluidic chip 300 can be filled with the first fluid of the oil phase first, and then the cell suspension and biochemical reagent can be injected.
  • (4) The first fluid, the cell suspension, and the biochemical reagent merge at the confluence 304 of the microfluidic chip 300 to generate droplets each comprising a single cell, and then the droplets are sorted at the sorting channel 305 and enter the corresponding sub-collector. Non-target droplets are collected into the first sub-collector 3051, and target droplets (each target droplet comprises a single target cell) are collected into the second sub-collector 3052. The non-target droplets in the first sub-collector 3051 flow into the first outlet unit 3006 of the box device 3000 through the outlet, and the target droplets in the second sub-collector 3052 flow into the second outlet 3007 of the box device 3000 through the outlet. The first outlet unit 3006 and the second outlet unit 3007 can store corresponding droplets or can transfer these droplets to other devices as needed.

The box device 3000 is adapted to the microfluidic chip 300 to jointly realize the preparation of the droplet comprising a single cell and the sorting of target droplets. Such a box device 3000 provides an environment for aseptic operation since the droplets are completely confined within the sealed box device 3000 and microfluidic chip 300 before and after sorting. In addition, the existence of the box device 3000 makes the system composed of the box device 3000 and the microfluidic chip 300 more concise and convenient, and easy to carry.

FIG. 8 shows a schematic structural diagram of a box device 4000 according to still another embodiment of the present disclosure, wherein (a) is a front view of the box device 4000, (b) is a right view of the box device 4000, (c) is a top view of the box device 4000, and (d) is a trimetric view of the box device 4000. The box device 4000 is adapted to the microfluidic chip 400 shown in FIG. 2 of the present application, and the combination of the two can be used for cascaded sorting of target droplets to obtain target droplets comprising different types of target cells. For the cascaded sorting process of droplets, reference may be made to the description about the microfluidic chip 400.

The box device 4000 comprises an inlet unit 4001 and an outlet unit 4002. The inlet unit 4001 communicates with the inlets of the microfluidic chip 400 and is configured to store a first reagent and release the first reagent to the inlets of the microfluidic chip 400. The outlet unit 4002 communicates with the outlets of the microfluidic chip 400 and is configured to receive and store a second reagent which is processed by the microfluidic chip 400 and flows into the outlet unit 4002 from the outlets of the microfluidic chip 400, the second reagent comprises target droplets and non-target droplets, wherein the target droplets comprise: a target droplet comprising a single type A target cell, a target droplet comprising a single type B target cell, and a target droplet comprising a single type C target cell. The non-target droplet is a droplet comprising a type D non-target cell. The inlet unit 4001 comprises a first inlet unit 4003, a second inlet unit 4004 and a third inlet unit 4005. The outlet unit 4002 comprises a first outlet unit 4006 as well as second outlet units 4007, 4008 and 4009. The inlets of the microfluidic chip 400 comprise a first inlet and a second inlet located at the two third containers 401 and a third inlet located at the fourth container 402. The first inlet unit 4003 communicates with the first inlet of the microfluidic chip 400, and the first inlet unit 4003 is configured to store a first sub-reagent (i.e., the first fluid) and release the first sub-reagent to the first inlet of the microfluidic chip 400; the second inlet unit 4004 communicates with the second inlet of the microfluidic chip 400, and the second inlet unit 4004 is configured to store the first sub-reagent (i.e., the first fluid) and release the first sub-reagent to the second inlet of the microfluidic chip 400; the third inlet unit 4005 communicates with the third inlet of the microfluidic chip 400, and the third inlet unit 4005 is configured to store a second sub-reagent (i.e., a droplet comprising a single cell) and release the second sub-reagent to the third inlet of the microfluidic chip 400. The first outlet unit 4006 of the outlet unit 4002 is configured to receive and store non-target droplets, and the second outlet units 4007-4009 of the outlet unit 4002 are respectively configured to receive and store the target droplet comprising a single type A cell, the target droplet comprising a single type B cell, and the target droplet comprising a single type C cell.

The first inlet unit 4003 of the box device 4000 comprises an inlet hole 4003A, a first storage cavity 4003B, and a second storage cavity 4003C; the second inlet unit 4004 comprises an inlet hole 4004A, a first storage cavity 4004B, and a second storage cavity 4004C; the third inlet unit 4005 comprises an inlet hole 4005A, a first storage cavity 4005B, and a second storage cavity 4005C. The structures of the first inlet unit 4003, the second inlet unit 4004 and the third inlet unit 4005 of the box device 4000 are exactly the same as that of the first inlet unit 1003 of the box device 1000, thus, the first inlet unit 4003, the second inlet unit 4004 and the third inlet unit 4005 of the box device 4000 have the same technical effect as the first inlet unit 1003 of the box device 1000. For the sake of brevity, their structures and technical effects are not repeated here. The first outlet unit 4006 of the box device 4000 comprises an outlet hole 4006A, a third storage cavity 4006B and a fourth storage cavity 4006C; the second outlet unit 4007 of the box device 4000 comprises an outlet hole 4007A, a third storage cavity 4007B and a fourth storage cavity 4007C; the second outlet unit 4008 of the box device 4000 comprises an outlet hole 4008A, a third storage cavity 4008B and a fourth storage cavity 4008C; the second outlet unit 4009 of the box device 4000 comprises an outlet hole 4009A, a third storage cavity 4009B, and a fourth storage cavity 4009C. The first outlet unit 4006 and the second outlet units 4007-4009 have exactly the same structures. Except for the relative position of the fourth storage cavity and the outlet hole, the structures of the first outlet unit 4006 and the second outlet units 4007-4009 of the box device 4000 are basically the same as the structure of the outlet unit 1006 of the box device 1000. Therefore, the structure and technical effect of each outlet unit of the box device 4000 may refer to the structure and technical effect of the outlet unit 1006 of the box device 1000. In the box device 4000, taking the first outlet unit 4006 as an example, the orthographic projection of the fourth storage cavity 4006C on the box device 4000 falls within the orthographic projection of the outlet hole 4006A on the box device 4000.

The box device 4000 further comprises a first installation region and a second installation region located between the inlet unit 4001 and the outlet unit 4002. The first installation region is configured to mount a plurality of optical recognition devices, and the second installation region is configured to mount a plurality of driving electrode devices. The optical recognition devices and the driving electrode devices are used to cooperate with the microfluidic chip 400 to realize cascaded sorting of target droplets. Specifically, the first installation region comprises a first sub-installation unit 4010, a second sub-installation unit 4011, and a third sub-installation unit 4012, the second installation region comprises a fourth sub-installation unit 4013, a fifth sub-installation unit 4014, and a sixth sub-installation unit 4015. The first sub-installation unit 4010 is associated with the fourth sub-installation unit 4013, the second sub-installation 4011 is associated with the fifth sub-installation unit 4014, and the third sub-installation unit 4012 is associated with the sixth sub-installation unit 4015.

The general process of cascaded sorting of target droplets by using the box device 4000 and the microfluidic chip 400 can be described as follows:

• • (1) Pre-adding the first fluid to the first inlet unit 4003 and the second inlet unit 4004 respectively and pre-adding the droplet comprising a single cell to the third inlet unit 4005. The droplets can be prepared by the above-mentioned box device 1000 and the microfluidic chip 100. The first fluid is the oil phase, which may be mixed with surfactants.
  • (2) Connecting the inlet hole 4003A of the first inlet unit 4003, the inlet hole 4004A of the second inlet unit 4004, and the inlet hole 4005A of the third inlet unit 4005 of the box device 4000 to the corresponding flow pumps through flexible pipes, respectively. The flow rate of fluid injected into the inlet unit is controlled by adjusting the pressure of the flow pump.
  • (3) The first fluid in the first inlet unit 4003 flows into the first inlet of the microfluidic chip 400 through the inlet hole 4003A, the first storage cavity 4003B and the second storage cavity 4003C; the first fluid in the second inlet unit 4004 flows into the second inlet of the microfluidic chip 400 through the inlet hole 4004A, the first storage cavity 4004B and the second storage cavity 4004C; the droplets in the third inlet unit 4005 flow into the third inlet of the microfluidic chip 400 through the inlet hole 4005A, the first storage cavity 4005B and the second storage cavity 4005C. Note that the microfluidic chip 400 may be filled with the first fluid of the oil phase first, and then the droplets are injected.
  • (4) The above-mentioned droplets are sorted at the sorting channel 403 of the microfluidic chip 400 and enter the corresponding sub-collector, the non-target droplets comprising type D non-target cells are collected into the first collector 405, the target droplet comprising a single type A target cell is collected into the first sub-collector 4061, the target droplet comprising a single type B target cell is collected into the second sub-collector 4062, and the target droplet comprising a single type C target cell is collected into the third sub-collector 4063. The non-target droplets in the first collector 405 flow into the first outlet unit 4006 of the box device 4000 through the outlet, the target droplets in the first sub-collector 4061 flow into the second outlet unit 4007 of the box device 4000 through the outlet, the target droplets in the second sub-collector 4062 flow into the second outlet unit 4008 of the box device 4000 through the outlet, and the target droplets in the third sub-collector 4063 flow into the second outlet unit 4009 of the box device 4000 through the outlet. The first outlet unit 4006 and the second outlet units 4007-4009 can store the corresponding droplets or can transfer these droplets to other devices as needed.

The box device 4000 is adapted to the microfluidic chip 400 to jointly realize cascaded sorting of target droplets. Using the box device 4000 and the microfluidic chip 400, three different types of target cells can be sorted out through a single sorting process, which greatly improves the speed and efficiency of sorting cells. Moreover, compared to using three different microfluidic chips to sort three different types of target cells, the embodiment of the present disclosure can realize the sorting of three different types of target cells by using only one box device 4000 and microfluidic chip 400, which greatly saves the number of microfluidic chips and box devices required, thereby saving production costs.

The box device 4000 can be slightly modified to obtain a variant box device, which can be adapted to the microfluidic chip 400′ shown in FIG. 3 of the present application. Compared with the box device 4000, the variant box device only needs to change the number of outlet units, and other components do not need to be changed. In the box device 4000, the number of the first outlet unit 4006 is one, and the number of the second outlet unit is three. In the variant box device, the number of the first outlet unit is one, and the number of the second outlet unit is one.

The first three steps for the cascaded sorting of target droplets using the variant box device and the microfluidic chip 400′ are the same as the above-mentioned first three steps (1)-(3) for the cascaded sorting of target droplets using the box device 4000 and the microfluidic chip 400. For the sake of brevity, the description is not repeated here. Next, the description will be started from the fourth step.

(4) The droplets are sorted at the sorting channel 403 of the microfluidic chip 400′ and enter the corresponding sub-collector, the non-target droplets each comprising type F non-target cells are collected into the first collector 405′ via the first sorting channels 4031A, 4031B, 4031C, and the target droplets each comprising a single type E target cell are collected into the second collector 406. The non-target droplets in the first collector 405′ flow into the first outlet unit of the variant box device through the outlet, and the target droplets in the second collector 406 flow into the second outlet of the variant box device through the outlet. The first outlet unit and the second outlet unit can store the corresponding droplets or can transfer these droplets to other equipment as needed.

The variant box device is adapted to the microfluidic chip 400′ to jointly realize the cascaded sorting of target droplets. Using the variant box device and the microfluidic chip 400′, indistinguishable target droplets can be distinguished from non-target droplets through multiple cascaded sorting of droplets, which greatly improves the purity of the collected target droplets, and reduces or even eliminates the possibility that the collected target droplets comprise non-target droplets.

FIG. 9 shows a schematic structural diagram of a box device 5000 according to yet another embodiment of the present disclosure, wherein (a) is a front view of the box device 5000, (b) is a right view of the box device 5000, (c) is a top view of the box device 5000, and (d) is a trimetric view of the box device 5000. The box device 5000 is adapted to the microfluidic chip 500 shown in FIG. 4 of the present application, and the combination of the two can be used to sort droplets with different particle sizes. For the specific process of sorting droplets, reference may be made to the description about the microfluidic chip 500.

The box device 5000 comprises an inlet unit 5001 and an outlet unit 5002. The inlet unit 5001 communicates with the inlet of the microfluidic chip 500 and is configured to store a first reagent and release the first reagent to the inlet of the microfluidic chip 500. The first reagent is a plurality of droplets, each droplet of at least a portion of the plurality of droplets comprises a single cell. The outlet unit 5002 communicates with the outlets of the microfluidic chip 500 and is configured to receive and store a second reagent which is processed by the microfluidic chip 500 and flows into the outlet unit 5002 from the outlets of the microfluidic chip 500, the second reagent comprises two types of droplets with different particle sizes. The inlet unit 5001 comprises an inlet unit 5003, and the outlet unit 5002 comprises a first outlet unit 5004 and a second outlet unit 5005. The inlet unit 5003 communicates with the inlet of the microfluidic chip 500, and the inlet unit 5003 is configured to store droplets and release the droplets to the inlet of the microfluidic chip 500. The first outlet unit 5004 of the outlet unit 5002 is configured to receive and store droplets with smaller particle size, and the second outlet unit 5005 of the outlet unit 5002 is configured to receive and store droplets with larger particle size.

The inlet unit 5003 of the box device 5000 comprises an inlet hole 5003A, a first storage cavity 5003B, and a second storage cavity 5003C. The structure of the inlet unit 5003 of the box device 5000 is exactly the same as that of the first inlet unit 1003 of the box device 1000. Therefore, the inlet unit 5003 of the box device 5000 has the same technical effect as the first inlet unit 1003 of the box device 1000. For the sake of brevity, its structure and technical effects are not repeated here. The first outlet unit 5004 of the box device 5000 comprises an outlet hole 5004A, a third storage cavity 5004B and a fourth storage cavity 5004C; the second outlet unit 5005 of the box device 5000 comprises an outlet hole 5005A, a third storage cavity 5005B and a fourth storage cavity 5005C. The first outlet unit 5004 and the second outlet unit 5005 have exactly the same structures. Except for the relative position of the fourth storage cavity and the outlet hole, the structures of the first outlet unit 5004 and the second output unit 5005 of the box device 5000 are basically the same as the structure of the outlet unit 1006 of the box device 1000. Therefore, the structure and technical effect of each outlet unit of the box device 5000 may refer to the structure and technical effect of the outlet unit 1006 of the box device 1000. In the box device 5000, taking the first outlet unit 5004 as an example, the orthographic projection of the fourth storage cavity 5004C on the box device 5000 falls within the orthographic projection of the outlet hole 5004A on the box device 5000.

The general process of sorting target droplets by using the box device 5000 and the microfluidic chip 500 can be described as follows:

• • (1) Pre-adding the droplets each comprising a single cell to the inlet unit 5003. The droplets can be prepared by the above-mentioned box device 1000 and the microfluidic chip 100. The first fluid is the oil phase, which may be mixed with surfactants.
  • (2) Connecting the inlet hole 5003A of the inlet unit 5003 of the box device 5000 to the flow pump through a flexible pipe, and controlling the flow rate of the fluid injected into the inlet unit by adjusting the pressure of the flow pump.
  • (3) The droplets in the inlet unit 5003 flow into the inlet of the microfluidic chip 500 through the inlet hole 5003A, the first storage cavity 5003B and the second storage cavity 5003C.
  • (4) The above-mentioned droplets flow in the main channel 503 of the microfluidic chip 500 and are sorted under the action of inertial force and then enter the corresponding collector. At the end bifurcation of the main channel 503, the first type of droplets with smaller particle size are subjected to less inertial force, so they enter the first sorting channel 504 along the extending direction of the main channel 503, and then flow into the first collector 507. The second type of droplets with larger particle size are subjected to larger inertial force, and are thrown out of the main channel 503 under the inertial force to enter the second sorting channel 505, and finally flow into the second collector 508. The first type of droplets in the first collector 507 flow into the first outlet unit 5004 of the box device 5000 through the outlet, and the second type of droplets in the second collector 508 flow into the second outlet unit 5005 of the box device 5000 through the outlet. The first outlet unit 5004 and the second outlet unit 5005 can store the corresponding droplets or can transfer the droplets to other devices as needed.

The box device 5000 is adapted to the microfluidic chip 500, and can sort droplets with different particle sizes. The box device 5000 does not need to leave an area for installing the optical recognition device and an area for installing the driving electrode device. The microfluidic chip 500 also does not need to be provided with an optical recognition device and a driving electrode device, but distinguishes droplets of different particle sizes only depending on the shape of the main channel 503. Since the optical recognition device and the driving electrode device are not required, not only the size of the box device 5000 and the microfluidic chip 500 can be reduced, but also the production cost can be saved.

According to yet another aspect of the present disclosure, a microfluidic device is provided. FIG. 10 shows a block diagram of the microfluidic device. The microfluidic device comprises the microfluidic chip described in any of the preceding embodiments and the box device described in any of the preceding embodiments, the microfluidic chip being assembled with the corresponding box device. Since the microfluidic device has basically the same technical effect as the microfluidic chip and box device described in the previous embodiments, for the sake of brevity, the description of the technical effects of the microfluidic device will not be described here.

It will be understood that although the terms first, second, third, etc. may be used herein to describe various elements, components, regions, layers and/or sections, these elements, components, regions, layers and/or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer or section from another region, layer or section. Thus, a first element, component, region, layer or section discussed above could be termed a second element, component, region, layer or section without departing from the teachings of the present disclosure.

Spatially relative terms such as “row,” “column,” “below,” “above,” “left,” “right,” etc. may be used herein for ease of description to describe the relationship of one element or feature to another element or feature(s) illustrated in the figures. It will be understood that these spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, elements described as “below” other elements or features would then be oriented “above” other elements or features. Thus, the exemplary term “below” can encompass both an orientation of above and below. The device may be oriented otherwise (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. In addition, it will also be understood that when a layer is referred to as being “between” two layers, it can be the only layer between the two layers, or one or more intervening layers may also be present.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present disclosure. As used herein, the singular forms “a”, “an” and “the” are intended to comprise the plural forms as well, unless the context clearly dictates otherwise. It will be further understood that the terms “comprising” and/or “including” when used in this specification specify the presence of stated features, integers, steps, operations, elements and/or parts, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, parts and/or groups thereof. As used herein, the term “and/or” comprises any and all combinations of one or more of the associated listed items. In the description of this specification, descriptions with reference to the terms “an embodiment”, “another embodiment” and the like mean that a particular feature, structure, material, or characteristic described in connection with the embodiment is comprised in at least one embodiment of the present disclosure. In this specification, the schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the described specific features, structures, materials or characteristics may be combined in any suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine different embodiments or examples and features of different embodiments or examples described in this specification without conflicting with each other.

It will be understood that when an element or layer is referred to as being “on”, “connected to”, “coupled to” or “adjacent to” another element or layer, it may be directly on, connected to, coupled to, or adjacent to another element or layer, or intervening elements or layers may be present. In contrast, when an element is referred to as being “directly on”, “directly connected to”, “directly coupled to” or “directly adjacent to” another element or layer, no intermediate elements or layers present. In no event, however, “on” or “directly on” should be construed as requiring that one layer completely cover the underlying layer.

Embodiments of the disclosure are described herein with reference to schematic illustrations of idealized embodiments (and intermediate structures) of the disclosure. As such, variations from the shapes of the illustrations, for example, as a result of manufacturing techniques and/or tolerances, should be expected. Thus, embodiments of the present disclosure should not be construed as limited to the particular shapes of regions illustrated herein but are to comprise deviations in shapes that result, for example, from manufacturing. Thus, the regions illustrated in the figures are schematic in nature and their shapes are not intended to illustrate the actual shape of a region of a device and are not intended to limit the scope of the present disclosure.

Unless otherwise defined, all terms (comprising technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms such as those defined in commonly used dictionaries should be interpreted to have meanings consistent with their meanings in the relevant art and/or in the context of this specification, and will not be interpreted in an idealized or overly formal sense unless explicitly so defined herein.

The above description is only a specific implementation manner of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Anyone skilled in the art within the technical scope disclosed in the present disclosure can easily think of changes or substitutions, which should be covered by the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be determined by the protection scope of the claims.

Claims

1. A microfluidic chip comprising: a first container configured to accommodate a first fluid;a second container configured to accommodate a second fluid comprising a cell suspension;a delivery channel comprising a first delivery channel and a second delivery channel, the first delivery channel communicating with the first container and the second delivery channel communicating with the second container, the first delivery channel intersecting and communicating with the second delivery channel at a confluence, a shape of the delivery channel being designed so that the first fluid and the second fluid merge at the confluence;a sorting channel downstream of the delivery channel and comprising a first sorting channel and a second sorting channel; anda collector downstream of the sorting channel and comprising a first collector and a second collector, the first collector communicating with the first sorting channel, and the second collector communicating with the second sorting channel.

2. The microfluidic chip according to claim 1, wherein a portion of the first delivery channel is divided by the confluence into a first section and a second section, in each section of the first section and the second section, an area of a first cross-section of the section gradually increases along a first direction away from the confluence, the first cross-section is perpendicular to the first direction, andwherein the second delivery channel is divided by the confluence into a third section and a fourth section, in each section of the third section and the fourth section, an area of a second cross-section of the section gradually increases along a second direction away from the confluence, and the second cross-section is perpendicular to the second direction.

3. The microfluidic chip according to claim 1, wherein both a beginning of the first sorting channel and a beginning of the second sorting channel communicate with an end of the delivery channel, an end of the first sorting channel communicates with the first collector and an end of the second sorting channel communicates with the second collector, the first sorting channel and the second sorting channel bend from the end of the delivery channel toward the confluence, and the first collector and the second collector are between the confluence and the end of the delivery channel.

4. The microfluidic chip according to claim 1, wherein the sorting channel further comprises at least two connecting channels, wherein the second sorting channel comprises at least two branches which are cascaded, a connecting channel is provided between any two adjacent branches of the at least two branches which are cascaded, and the any two adjacent branches communicate via the connecting channel,wherein a beginning of the first sorting channel communicates with an end of the delivery channel, an end of the first sorting channel communicates with the first collector, the first sorting channel is adjacent to a first branch of the at least two branches which are cascaded, a connecting channel is provided between the first sorting channel and the first branch, and the first sorting channel communicates with the first branch via the connecting channel, andwherein the second collector comprises at least two sub-collectors, the branches which are cascaded correspond to the sub-collectors one by one, and one of the branches which are cascaded communicates with a corresponding one of the sub-collectors.

5. The microfluidic chip according to claim 4, wherein the second sorting channel comprises a first branch, a second branch and a third branch which are cascaded, the at least two connecting channels comprises a first connecting channel, a second connecting channel, and a third connecting channel, and the second collector comprises a first sub-collector, a second sub-collector, and a third sub-collector, wherein the first sorting channel communicates with the first branch via the first connecting channel, the first branch communicates with the second branch via the second connecting channel, and the second branch communicates with the third branch via the third connecting channel,wherein an end of the first branch communicates with the first sub-collector, an end of the second branch communicates with the second sub-collector, and an end of the third branch communicates with the third sub-collector,wherein the second connecting channel is closer to the collector in a second direction than the first connecting channel, and the third connecting channel is closer to the collector in the second direction than the second connecting channel, andwherein the microfluidic chip further comprises two third containers, each of a beginning of the first branch and a beginning of the second branch communicates with a corresponding one of the two third containers, and the third container is configured to accommodate the first fluid.

6. (canceled)

7. (canceled)

8. The microfluidic chip according to claim 1, wherein the sorting channel further comprises at least two connecting channels, wherein the first sorting channel comprises at least two branches which are cascaded, a connecting channel is provided between any two adjacent branches of the at least two branches which are cascaded, and the any two adjacent branches communicate via the connecting channel, ends of the at least two branches which are cascaded communicate with the first collector, andwherein a beginning of the second sorting channel communicates with a last branch of the first sorting channel via a connecting channel, and an end of the second sorting channel communicates with the second collector.

9. The microfluidic chip according to claim 1, wherein the sorting channel further comprises a main channel, the main channel is spiral in a plane where the microfluidic chip is located, an end of the main channel communicates with the first sorting channel and the second sorting channel, the first sorting channel is configured to sort first droplets, the second sorting channel is configured to sort second droplets, and the first droplets sorted by the first sorting channel and the second droplets sorted by the second sorting channel have different particle sizes.

10. The microfluidic chip according to claim 2, wherein the portion of the first delivery channel comprises a first sub-portion, a second sub-portion comprising the confluence, and a third sub-portion, the first sub-portion belongs to the first section, the third sub-portion belongs to the second section, the second sub-portion spans the first section and the second section and is between the first sub-portion and the third sub-portion, areas of the first cross-section of the first sub-portion and the third sub-portion are larger than an area of the first cross-section of the second sub-portion, and wherein a size of the first cross-section of the second sub-portion of the first delivery channel at the confluence is configured to allow the first fluid having a specific particle size to flow therein, the specific particle size of the first fluid is larger than a particle size of a single cell in the cell suspension.

11. (canceled)

12. The microfluidic chip according to claim 10, wherein the second delivery channel comprises a first sub-channel, a second sub-channel and a third sub-channel, the first sub-channel and the second sub-channel belong to the third section, and the third sub-channel belongs to the fourth section,wherein a first end of the first sub-channel communicates with the second container, a second end of the first sub-channel communicates with a first end of the second sub-channel, a second end of the second sub-channel communicates with a first end of the third sub-channel, and both the second end of the second sub-channel and the first end of the third sub-channel are at the confluence,wherein areas of the second cross-section of the first sub-channel and the third sub-channel are larger than an area of the second cross-section of the second sub-channel,wherein a size of the second cross-section of the second sub-channel is configured to allow the second fluid having a specific particle size to flow therein, the specific particle size of the second fluid is larger than 1 time of a particle size of a single cell in the cell suspension and smaller than 2 times of the particle size of the single cell,wherein the area of the second cross-section of the third sub-channel gradually increases along a direction from the first end to a second end of the third sub-channel, andwherein an area of the first cross-section of the second sub-portion of the first delivery channel at the confluence is greater than or equal to an area of the second cross-section of each of the second sub-channel and the third sub-channel of the second delivery channel at the confluence.

13. (canceled)

14. (canceled)

15. (canceled)

16. (canceled)

17. (canceled)

18. The microfluidic chip according to claim 1, wherein both the first container and the second container are provided with a filter structure, the filter structure comprises a plurality of microstructures, a gap between adjacent two of the plurality of microstructures is larger than 1 time of a particle size of a single cell in the cell suspension and smaller than 2 times of the particle size of the single cell.

19. (canceled)

20. A box device configured to be used with the microfluidic chip according to claim 1, the microfluidic chip comprising an inlet and an outlet, wherein the box device comprises: an accommodating cavity configured to accommodate the microfluidic chip;an inlet unit communicated with the inlet of the microfluidic chip, the inlet unit being configured to store a first reagent and release the first reagent to the inlet of the microfluidic chip; andan outlet unit communicated with the outlet of the microfluidic chip, the outlet unit being configured to receive and store a second reagent processed by the microfluidic chip and flowing into the outlet unit from the outlet of the microfluidic chip,wherein the inlet unit comprises an inlet hole and a first storage cavity, the inlet hole is a through hole and communicates with the first storage cavity, the inlet hole is recessed from a surface of the box device to an inside of the box device, and the first storage cavity is on a side of the inlet hole away from the surface of the box device, andwherein the first storage cavity is inside the box device, and an orthographic projection of the inlet hole on the box device falls within an orthographic projection of the first storage cavity on the box device.

21. (canceled)

22. The box device according to claim 20, wherein the inlet unit further comprises a second storage cavity, the second storage cavity is on a side of the first storage cavity away from the inlet hole and communicates with the first storage cavity, the second storage cavity comprises a first opening communicating with the first storage cavity and a second opening facing to the first opening, an orthographic projection of the second opening on the box device falls within an orthographic projection of the first opening on the box device, andwherein the orthographic projection of the second opening of the second storage cavity on the box device falls within an orthographic projection of the inlet hole on the box device.

23. (canceled)

24. The box device according to claim 20, wherein the outlet unit comprises an outlet hole and a third storage cavity, the outlet hole is a through hole and communicates with the third storage cavity, the outlet hole is recessed from the surface of the box device to the inside of the box device, and the third storage cavity is on a side of the outlet hole away from the surface of the box device, andwherein the third storage cavity is inside the box device, and an orthographic projection of the outlet hole on the box device falls within an orthographic projection of the third storage cavity on the box device.

25. (canceled)

26. The box device according to claim 24, wherein the outlet unit further comprises a fourth storage cavity, and the fourth storage cavity is on a side of the third storage cavity away from the outlet hole and communicates with the third storage cavity,wherein an orthographic projection of the fourth storage cavity on the box device overlaps at most a portion of an orthographic projection of the outlet hole on the box device, andwherein an orthographic projection of the fourth storage cavity on the box device falls within an orthographic projection of the outlet hole on the box device.

27. (canceled)

28. (canceled)

29. The box device according to claim 20, wherein the inlet unit comprises a first inlet unit, a second inlet unit, and a third inlet unit, the inlet of the microfluidic chip comprises a first inlet, a second inlet, and a third inlet, and the first reagent comprises the first fluid, the cell suspension, and a biochemical reagent,wherein the first inlet unit communicates with the first inlet of the microfluidic chip, the first inlet unit is configured to store the first fluid and release the first fluid to the first inlet of the microfluidic chip,wherein the second inlet unit communicates with the second inlet of the microfluidic chip, the second inlet unit is configured to store the cell suspension and release the cell suspension to the second inlet of the microfluidic chip, andwherein the third inlet unit communicates with the third inlet of the microfluidic chip, and the third inlet unit is configured to store the biochemical reagent and release the biochemical reagent to the third inlet of the microfluidic chip.

30. (canceled)

31. The box device according to claim 20, wherein the inlet unit comprises a first inlet unit and a second inlet unit, the inlet of the microfluidic chip comprises a first inlet and a second inlet, the first reagent comprises the first fluid and a droplet comprising a single cell, wherein the first inlet unit communicates with the first inlet of the microfluidic chip, the first inlet unit is configured to store the first fluid and release the first fluid to the first inlet of the microfluidic chip; the second inlet unit communicates with the second inlet of the microfluidic chip, and the second inlet unit is configured to store the droplet comprising the single cell and release the droplet comprising the single cell to the second inlet of the microfluidic chip, andwherein the outlet unit comprises a first outlet unit, a second outlet unit, and a third outlet unit between the first outlet unit and the second outlet unit, the second reagent comprises a first droplet and a second droplet, the third outlet unit is configured to receive and store the first droplet, the first outlet unit and the second outlet unit are configured to receive and store the second droplet.

32. The box device according to claim 20, wherein the inlet unit comprises a first inlet unit, a second inlet unit, and a third inlet unit, the inlet of the microfluidic chip comprises a first inlet, a second inlet, and a third inlet, the first reagent comprises the first fluid, the cell suspension, and a biochemical reagent. wherein the first inlet unit communicates with the first inlet of the microfluidic chip, the first inlet unit is configured to store the first fluid and release the first fluid to the first inlet of the microfluidic chip,wherein the second inlet unit communicates with the second inlet of the microfluidic chip, the second inlet unit is configured to store the cell suspension and release the cell suspension to the second inlet of the microfluidic chip; the third inlet unit communicates with the third inlet of the microfluidic chip, and the third inlet unit is configured to store the biochemical reagent and release the biochemical reagent to the third inlet of the microfluidic chip,wherein the outlet unit comprises a first outlet unit and a second outlet unit, the second reagent comprises a first droplet and a second droplet, the first outlet unit is configured to receive and store the first droplet, and the second outlet unit is configured to receive and store the second droplet, andwherein the box device further comprises a first installation region and a second installation region, the first installation region is configured to install an optical identification device, and the second installation region is configured to install a driving electrode device, the first outlet unit and the second outlet unit are between the inlet unit and the first installation region and the second installation region.

33. (canceled)

34. The box device according to claim 30, wherein the box device further comprises a first installation region and a second installation region, the first installation region is configured to install an optical identification device, and the second installation region is configured to install a driving electrode device, the first installation region and the second installation region are between the inlet unit and the outlet unit, the first installation region comprises a first sub-installation unit, a second sub-installation unit, and a third sub-installation unit, the second installation region comprises a fourth sub-installation unit, a fifth sub-installation unit, and a sixth sub-installation unit, the first sub-installation unit is associated with the fourth sub-installation unit, the second sub-installation unit is associated with the fifth sub-installation unit, and the third sub-installation unit is associated with the sixth sub-installation unit,wherein the inlet unit comprises a first inlet unit, a second inlet unit, and a third inlet unit, the inlet of the microfluidic chip comprises a first inlet, a second inlet, and a third inlet, the first reagent comprises the first fluid and a droplet comprising a single cell,wherein the first inlet unit communicates with the first inlet of the microfluidic chip, the first inlet unit is configured to store the first fluid and release the first fluid to the first inlet of the microfluidic chip; the second inlet unit communicates with the second inlet of the microfluidic chip, the second inlet unit is configured to store the first fluid and release the first fluid to the second inlet of the microfluidic chip; the third inlet unit communicates with the third inlet of the microfluidic chip, and the third inlet unit is configured to store the droplet comprising the single cell and release the droplet comprising the single cell to the third inlet of the microfluidic chip, andwherein the outlet unit comprises a first outlet unit and a second outlet unit, the second reagent comprises a first droplet and a second droplet, the first outlet unit is configured to receive and store the first droplet, and the second outlet unit is configured to receive and store the second droplet.

35. (canceled)

36. (canceled)

37. (canceled)

38. The box device according to claim 20, wherein the box device comprises one inlet unit and two outlet units, the second reagent comprises a first droplet and a second droplet, the first droplet and the second droplet have different particle sizes, one of the two outlet units is configured to receive and store the first droplet, and the other of the two outlet units is configured to receive and store the second droplet.

39. A microfluidic device comprising a microfluidic chip and a box device, wherein the microfluidic chip being assembled with the box device, wherein the microfluidic chip comprises;a first container configured to accommodate a first fluid;a second container configured to accommodate a second fluid comprising a cell suspension;a delivery channel comprising a first delivery channel and a second delivery channel, the first delivery channel communicating with the first container and the second delivery channel communicating with the second container, the first delivery channel intersecting and communicating with the second delivery channel at a confluence, a shape of the delivery channel being designed so that the first fluid and the second fluid merge at the confluence;a sorting channel downstream of the delivery channel and comprising a first sorting channel and a second sorting channel; anda collector downstream of the sorting channel and comprising a first collector and a second collector, the first collector communicating with the first sorting channel, and the second collector communicating with the second sorting channel,wherein the box device is configured to be used with the microfluidic chip, the microfluidic chip further comprises an inlet and an outlet, the box device comprises:an accommodating cavity configured to accommodate the microfluidic chip;an inlet unit communicated with the inlet of the microfluidic chip, the inlet unit being configured to store a first reagent and release the first reagent to the inlet of the microfluidic chip; andan outlet unit communicated with the outlet of the microfluidic chip, the outlet unit being configured to receive and store a second reagent processed by the microfluidic chip and flowing into the outlet unit from the outlet of the microfluidic chip,wherein the inlet unit comprises an inlet hole and a first storage cavity, the inlet hole is a through hole and communicates with the first storage cavity, the inlet hole is recessed from a surface of the box device to an inside of the box device, and the first storage cavity is on a side of the inlet hole away from the surface of the box device.