CN115529820A

CN115529820A - Chip, microfluidic device, and method for sorting target droplets

Info

Publication number: CN115529820A
Application number: CN202180000922.0A
Authority: CN
Inventors: 邓林; 杨帆; 李达; 马相国; 丁丁
Original assignee: BOE Technology Group Co Ltd; Beijing BOE Technology Development Co Ltd
Current assignee: BOE Technology Group Co Ltd; Beijing BOE Technology Development Co Ltd
Priority date: 2021-04-27
Filing date: 2021-04-27
Publication date: 2022-12-27
Also published as: WO2022226791A1; CN218281795U; CN115245846A

Abstract

The present disclosure provides a chip, a microfluidic device including the chip, and a method of sorting target droplets. The chip includes: a first container portion containing a first fluid; a second container portion containing a second fluid; a delivery flow passage including a first flow passage and a second flow passage, the first flow passage communicating with the first accommodation portion and the second flow passage communicating with the second accommodation portion, the first flow passage and the second flow passage intersecting and communicating with each other at a junction point; and at least one collecting portion. A portion of the first flow path includes a junction and is divided by the junction into two segments, in each of which the segment becomes progressively thicker along a first direction away from the junction. The second flow path includes a junction and is divided by the junction into two segments, in each of which the segment becomes progressively thicker along a second direction away from the junction.

Description

Chip, microfluidic device, and method for sorting target droplets

Technical Field

The present disclosure relates to the field of biomedical detection, and more particularly, to a chip, a microfluidic device including the chip, and a method of sorting target droplets.

Background

Cells are the basic structural and functional units of an organism. Since there is usually a high degree of heterogeneity between individual cells, the mean of the data obtained by analyzing the cell population substantially masks the variability between individual cells, and thus does not characterize the stochastic nature of gene expression and does not reflect the real situation. With the continuous development of life sciences and precise medicine, cell population analysis gradually develops towards single cell analysis. One key technology for single cell analysis is how single cells can be isolated from highly heterogeneous biological samples containing numerous cells. The single cell sorting technology provides a new choice for the hot medical fields of single cell analysis, early cancer diagnosis, companion diagnosis and the like.

Disclosure of Invention

According to an aspect of the present disclosure, there is provided a chip including: a first containing portion configured to contain a first fluid; a second receptacle configured to receive a second fluid, the second fluid comprising a cell suspension; a delivery flow channel comprising a first flow channel and a second flow channel, the first flow channel in communication with the first receptacle and the second flow channel in communication with the second receptacle, the first flow channel and the second flow channel intersecting and communicating with each other at a junction, the delivery flow channel configured to cause the first fluid and the second fluid to join at the junction and generate at least one droplet, each of at least a portion of the at least one droplet comprising a single cell derived from the cell suspension; and at least one collecting portion configured to collect the at least one droplet. A portion of the first flow passage includes the junction and is divided by the junction into a first section and a second section, and in each 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 junction, the first cross section being perpendicular to the first direction. The second flow path includes the junction and is divided by the junction into a third section and a fourth section, and in each 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 junction, the second cross section being perpendicular to the second direction.

In some embodiments, a portion of the first flow passage includes a first sub-portion belonging to the first section, a second sub-portion including the junction, and a third sub-portion belonging to the second section, the second sub-portion spanning and located between the first and third sections, the first cross-sectional areas of the first and third sub-portions each being greater in area than the first cross-sectional area of the second sub-portion.

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

In some embodiments, the second flow passage comprises a first portion, a second portion, and a third portion, the first and second portions belonging to the third section, the third portion belonging to the fourth section. A first end of the first portion of the second flow passage communicates with the second receiving portion, and a second end of the first portion of the second flow passage communicates with a first end of the second portion of the second flow passage; a second end of the second portion of the second flow passage communicates with a first end of the third portion of the second flow passage, and both the second end of the second portion of the second flow passage and the first end of the third portion of the second flow passage are located at the junction; a second end of the third portion of the second flow passage communicates with the at least one collection portion. The area of the second cross-section of the first and third portions of the second flow passage are each greater than the area of the second cross-section of the second portion of the second flow passage.

In some embodiments, the second cross-section of the second portion of the second flow channel is sized to allow a second fluid having a specific particle size to flow therein, the specific particle size of the second fluid being greater than 1-fold the particle size of the single cell and less than 2-fold the particle size of the single cell.

In some embodiments, the area of the second cross-section of the third portion of the second flow passage gradually increases in a direction from the first end to the second end of the third portion of the second flow passage.

In some embodiments, the area of the first cross-section of the second sub-portion of the first flow passage at the junction is greater than or equal to the area of the second cross-section of the second and third portions of the second flow passage at the junction.

In some embodiments, the second receiving portion includes at least one sub-receiving portion.

In some embodiments, the second fluid comprises a first reagent and a second reagent, the first reagent comprising the cell suspension. The second housing part includes a first sub-housing part and a second sub-housing part separated from each other, the first sub-housing part being configured to house the first reagent, and the second sub-housing part being configured to house the second reagent.

In some embodiments, the first portion of the second flow passage includes a first branch and a second branch, the first branch is in communication with the first sub-receiving portion, the second branch is in communication with the second sub-receiving portion, and the first branch and the second branch intersect and communicate with each other at a first point. The included angle between the first branch and the second branch at the first point is an acute angle.

In some embodiments, the at least one collection portion comprises a first collection portion configured to collect the at least one droplet via the delivery channel.

In some embodiments, the at least one collection portion comprises a second collection portion comprising at least two sub-collection portions configured to collect the at least one droplet via the transport flow channel.

In some embodiments, the at least one collecting portion comprises a first collecting portion and a second collecting portion, the second collecting portion comprising at least two sub-collecting portions. The first collection portion is in communication with the second collection portion, and the first collection portion is located between the junction and the second collection portion.

In some embodiments, the chip further comprises an electrode structure located between the junction and the second collection portion.

In some embodiments, the delivery flow channel further comprises a sorting flow channel located between the junction and the second collection portion. The sorting channel includes at least two branches, one of the at least two branches configured to sort non-target droplets from the at least one droplet, the remaining branches of the at least two branches configured to sort the target droplets from the at least one droplet. At least two sub-collecting portions of the second collecting portion correspond to at least two branches of the sorting flow channel one to one, one of the at least two sub-collecting portions is communicated with one of the at least two branches of the sorting flow channel and is configured to collect the non-target droplets, and the remaining sub-collecting portions of the at least two sub-collecting portions are respectively communicated with the remaining branches of the at least two branches of the sorting flow channel and are configured to collect the target droplets.

In some embodiments, the at least two branches of the sorting flow channel include a first branch and a second branch configured to sort the target droplet from the at least one droplet and a third branch configured to sort the non-target droplet from the at least one droplet. The first branch, the second branch and the third branch intersect at a second point and the third branch is located between the first branch and the second branch, and a first included angle of the first branch and the third branch at the second point and a second included angle of the second branch and the third branch at the second point are both greater than 10 °.

In some embodiments, the space between the first and third branches of the sort flow channel defines a first right triangle, the space between the second and third branches of the sort flow channel defines a second right triangle, the first included angle faces a first right side of the first right triangle, and the second included angle faces a second right side of the second right triangle. The lengths of the first right-angle side of the first right-angle triangle and the second right-angle side of the second right-angle triangle are both larger than or equal to the particle size of the single liquid drop.

In some embodiments, the inner wall surface of the transport flow channel has hydrophobicity.

In some embodiments, the profile of the first and second receptacles includes four chamfers.

In some embodiments, the shape of the chamfer comprises a circular arc.

In some embodiments, each of the first and second receptacles is provided with a filter structure comprising a plurality of microstructures, a gap between two adjacent of the plurality of microstructures being greater than 1-fold the particle size of the single cell and less than 2-fold the particle size of the single cell.

In some embodiments, the chip is a microfluidic chip.

According to another aspect of the present disclosure, there is provided a microfluidic device comprising a chip as described in any of the previous embodiments.

According to yet another aspect of the present disclosure, there is provided a method of sorting target droplets, the method comprising the steps of: providing a first fluid and a second fluid comprising a cell suspension to the first and second receptacles, respectively, of the chip described in any of the previous embodiments, bringing the first and second fluids together at the junction of the delivery flow path and generating at least one droplet, each of at least a portion of the at least one droplet comprising a single cell derived from the cell suspension; and applying a voltage to the chip described in any of the previous embodiments to sort out a target droplet having a target property from the at least one droplet, the target droplet comprising the single cell.

In some embodiments, the chip further comprises an electrode structure located between the junction and the at least one collection portion. The step of applying a voltage to the chip described in any of the previous embodiments to sort out a target droplet having a target property from the at least one droplet comprises: detecting an optical signal of the at least one droplet in real time using an optical device, and applying a transient voltage of 800-1000V to the electrode structure in response to the optical device detecting a droplet having a target optical signal, the target droplet including the single cell, to sort out the target droplet having the target optical signal from the at least one droplet.

In some embodiments, before the step of applying a voltage to the chip described in any of the previous embodiments, the method further comprises: transferring at least one droplet of the chip to another reaction vessel for polymerase chain reaction or fluorescent staining.

In some embodiments, the first fluid is an oil phase, the second fluid is an aqueous phase, and the droplets have a water-in-oil structure.

Drawings

In order to more clearly describe the technical solutions in the embodiments of the present disclosure, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present disclosure, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

Fig. 1A schematically illustrates a front view of a chip according to an embodiment of the disclosure;

fig. 1B schematically illustrates a side view of a chip according to an embodiment of the disclosure;

FIG. 1C schematically shows a back view of a chip according to an embodiment of the disclosure;

FIG. 1D schematically illustrates a positive triaxial view of a chip according to an embodiment of the present disclosure;

FIG. 2 schematically illustrates a partial enlarged schematic view of the chip of FIG. 1A;

FIG. 3 schematically illustrates another partial enlarged schematic view of the chip of FIG. 1A;

FIG. 4 schematically illustrates yet another partial enlarged schematic view of the chip of FIG. 1A;

fig. 5A schematically illustrates a front view of a chip according to another embodiment of the disclosure;

fig. 5B schematically illustrates a side view of a chip according to another embodiment of the disclosure;

FIG. 5C schematically illustrates a back view of a chip according to another embodiment of the disclosure;

FIG. 5D schematically illustrates a positive triaxial view of a chip according to another embodiment of the present disclosure;

FIG. 6 schematically illustrates a partial enlarged schematic view of the chip of FIG. 5A;

FIG. 7 schematically illustrates a front view of a chip according to yet another embodiment of the disclosure;

fig. 8 schematically illustrates a block diagram of a microfluidic device according to yet another embodiment of the present disclosure; and

fig. 9 schematically illustrates a flow diagram of a method of sorting target droplets according to yet another embodiment of the present disclosure.

Detailed Description

The technical solutions in the embodiments of the present disclosure will be clearly and completely described below with reference to the drawings in the embodiments of the present disclosure. It is to be understood that the described embodiments are merely illustrative of some, and not restrictive, of the embodiments of the disclosure. All other embodiments, which can be derived by a person skilled in the art from the embodiments disclosed herein without making any creative effort, shall fall within the protection scope of the present disclosure.

Before formally describing the technical solutions of the embodiments of the present disclosure, the terms used in the embodiments of the present disclosure are explained and defined as follows to help those skilled in the art to more clearly understand the technical solutions of the embodiments of the present disclosure.

As used herein, the term "fluid" refers to all substances capable of flowing, being a generic term for liquids and gases. A fluid is a substance that can be continuously deformed under the action of a slight shear force. The fluid may consist of a single substance or a mixture of a plurality of different substances. The fluid may be a continuous phase (e.g., an oil phase), a dispersed phase (e.g., an aqueous phase), or a mixture of a continuous phase and a dispersed phase. The fluid has the characteristics of easy fluidity, compressibility, viscosity and the like.

As used herein, the term "oil phase" means that a substance that is not readily soluble in water belongs to the oil phase according to the principle of similar phase solubility. For example, a substance is miscible with water and belongs to the oil phase if the mixed liquid exhibits stratification or cloudiness. The 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 perfluorooil are all included in the oil phase.

As used herein, the term "aqueous phase" means that a substance that is readily soluble in water belongs to the aqueous phase according to the principle of similar phase solubility. For example, a substance is miscible with water and belongs to the aqueous phase if the liquid after mixing is a clear and homogeneous solution. For example, water, glycerin, alcohol, acetone, etc. are included in the aqueous phase.

As used herein, the term "cell suspension" refers to a cell solution obtained by mechanically or chemically separating cells from a tissue and diluting the mixed solution with a cell culture solution. A cell suspension can include a large number of cells, e.g., hundreds, thousands, tens of thousands, millions, tens of millions, or more. The cells in the cell suspension may be any type of cell, including but not limited to prokaryotic cells, eukaryotic cells, bacteria, fungi, plant, mammalian or other animal cell types, mycoplasma, normal tissue cells, tumor cells, or any other cell type, whether derived from a single cell or multicellular organism. The cells in the cell suspension may comprise DNA, RNA, organelles, proteins, or any combination thereof.

As used herein, the term "a and B in communication" means that the a and B elements are interconnected and in communication, which allows fluid to flow between the a and B elements, i.e., fluid may flow from the a to the B or from the B to the a element as required by the product design. The a-component and the B-component may be in direct communication, i.e., fluid may flow directly from the a-component to the B-component or from the B-component to the a-component without passing through other intermediate components (e.g., conduits). Alternatively, the a and B elements may be in indirect communication, i.e. fluid may flow from the a element to the B element via one or more intermediate elements (e.g. pipes) or from the B element to the a element via one or more intermediate elements (e.g. pipes).

As used herein, the term "Polymerase Chain Reaction (PCR)" is a molecular biological technique for amplifying a specific deoxyribonucleic acid (DNA) fragment, which can be regarded as a specific DNA replication in vitro, and can largely replicate a minute amount of DNA to largely increase the amount thereof. The basic principle of PCR is that DNA may be denatured and melted at a high temperature (e.g., about 95 ℃) to become single-stranded, and when the temperature is lowered to a low temperature (e.g., about 60 ℃), primers bind to the single strand and become double-stranded again according to the base-complementary pairing principle. Therefore, the denaturation and renaturation of DNA are controlled by the change of temperature, and the large-scale replication of DNA can be realized by adding designed primers. PCR reactions include, but are not limited to, digital PCR (dPCR), quantitative PCR, real-time PCR. The dPCR technique may provide a quantitative analysis technique of quantitative information of digitized DNA, which, in combination with microfluidic techniques, may provide higher sensitivity and accuracy.

As used herein, the term "microfluidic chip" refers to a chip having micro-scale micro-channels, which can integrate basic operation units of sample preparation, reaction, separation, detection, etc. involved in the fields of biology, chemistry, medicine, etc. onto the micro-scale chip, and automatically complete the whole process of reaction and analysis. The analysis and detection device based on the microfluidic chip can have the following advantages: controllable liquid flow, less sample consumption, high detection speed, simple and convenient operation, multifunctional integration, small volume, convenient carrying and the like.

As used herein, the term "particle size of XX" refers to the size of substance XX, i.e. the length of 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 droplet is spherical, then 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 the cell or droplet is a rod, the term "particle diameter of a single cell" refers to the length of a single cell in the direction of the shorter side, and "particle diameter of a single droplet" refers to the length of a single droplet in the direction of the shorter side.

The inventors of the present application found that, in the conventional art, methods for sorting single cells are largely classified into two types: one is to use a Fluorescent Activated Cell Sorting (FACS) to automatically sort single cells, but the fluorescent flow cytometer is expensive and has high maintenance cost; the other is manual sorting of single cells by professional operators, but the manual sorting method not only depends on the skill and proficiency of operators, but also requires large and medium-sized instruments such as micro-pipetting platforms, optical tweezers and the like. In addition, the single cell sorting process is very vulnerable to the pollution of aerosol and microorganism floating in the environment, and the pollution is usually difficult to remove in the subsequent detection process. Therefore, the existing single cell sorting method has the disadvantages of high cost, high requirement on the skill of operators, site limitation on required instruments and equipment, environmental pollution and the like.

In view of the above, embodiments of the present disclosure provide a chip, a microfluidic device including the chip, and a method of sorting target droplets. The chip may be used to prepare droplets containing single cells derived from a cell suspension, or may be used to sort target droplets from the prepared droplets, or may be used to prepare droplets containing single cells derived from a cell suspension and sort target droplets from the prepared droplets. The chip may be a microfluidic chip. The chip can realize the preparation and/or sorting of single cells, can effectively improve the automation operation and reduce the use cost, can eliminate cross contamination and improve the survival rate of the cells.

Fig. 1A-1D illustrate a chip 100 (hereinafter first chip 100) according to some embodiments of the present disclosure, which first chip 100 may be used to prepare droplets comprising single cells. Fig. 1A is a front view of the first chip 100, fig. 1B is a side view of the first chip 100, fig. 1C is a rear view of the first chip 100, and fig. 1D is a front triaxial side view of the first chip 100. As shown in fig. 1A to 1D, the first chip 100 includes: a first containing portion 101 configured to contain a first fluid 107; a second containment portion 102 configured to contain a second fluid 108, the second fluid 108 comprising a suspension of cells; a delivery flow channel 103 (hereinafter referred to as a first delivery flow channel 103) comprising a first flow channel 1031 and a second flow channel 1032, the first flow channel 1031 being in communication with the first housing 101 and the second flow channel 1032 being in communication with the second housing 102, the first flow channel 1031 and the second flow channel 1032 intersecting and communicating with each other at a junction 105, the delivery flow channel 103 being configured such that the first fluid 107 and the second fluid 108 join at the junction 105 and generate at least one droplet 110 (hereinafter referred to as a first droplet 110), each of at least a portion of the at least one first droplet 110 comprising a single cell derived from the cell suspension; and at least one collection portion 104 (hereinafter referred to as a first collection portion 104) configured to collect at least one first droplet 110. A portion 1031B of the first flow passage 1031 includes a junction 105 and is divided by the junction 105 into a first section (e.g., the section of 1031B in fig. 1A that is above the junction 105) and a second section (e.g., the section of 1031B in fig. 1A that is below the junction 105), in each of which the area of a first cross-section of the section gradually increases along a first direction away from the junction 105, the first cross-section being perpendicular to the first direction. The second flow channel 1032 includes the junction 105 and is divided by the junction 105 into a third section (e.g., 1032 of fig. 1A, the section located on the left side of the junction 105) and a fourth section (e.g., 1032 of fig. 1A, the section located on the right side of the junction 105), in each of which the area of the second cross section of the section gradually increases along a second direction away from the junction 105, the second cross section being perpendicular to the second direction. That is, in a part 1031B of the first flow passage 1031, the first flow passage 1031 becomes gradually thicker in an upward direction from the junction 105; the first flow path 1031 also becomes gradually thicker in a downward direction from the merging point 105. In the second flow channel 1032, the second flow channel 1032 becomes gradually thicker in a direction leftward from the merging point 105; the second flow channel 1032 also becomes progressively thicker in a direction to the right from the junction 105.

As can be seen from the above description, the first cross section refers to a cross section of a portion 1031B of the first flow channel 1031 in a direction perpendicular to the flow direction of the first fluid 107; the second cross-section refers to a cross-section of the second flow channel 1032 in a direction perpendicular to the flow direction of the second fluid 108.

By the structural design of the first flow channel 1031 and the second flow channel 1032 of the first chip 100, the first chip 100 is enabled to facilitate the generation of the first liquid droplet 110 containing a single cell. The first chip 100 can prepare the first droplet 110 containing a single cell, and the first chip 100 has a high degree of integration, so that the preparation of the first droplet 110 containing a single cell can be automatically completed without manual operation of an operator, and the degree of automation of the operation can be effectively improved. Since the first fluid 107 and the second fluid 108 completely flow in the first conveying flow channel 103 and are completely isolated from the external environment, the pollution caused by aerosol, microorganisms and the like floating in the environment can be avoided. In addition, the whole preparation process is mild, and the single cell separated from the cell suspension is wrapped and protected by the liquid drop, so that the survival rate of the cell can be effectively improved.

In the first chip 100 shown in fig. 1A, the first container 101 includes a sample inlet 1, and an external device (e.g., a micro flow pump) is connected to the sample inlet 1 and injects the first fluid 107 into the first container 101 through the sample inlet 1. The first fluid 107 is a continuous phase (e.g., oil phase) liquid, which may be any suitable fluid such as, for example, a mineral oil, a perfluorinated oil, and the like. Optionally, a surfactant may be mixed into the first fluid 107, which surfactant facilitates stabilization of the resulting first droplets 110, e.g., inhibits subsequent coalescence of the resulting droplets 110. When the first fluid 107 is a perfluorinated oil, the surfactant may be a perfluorinated surfactant. The second receiving part 102 includes a first sub receiving part 1021 and a second sub receiving part 1022 which are separated from each other. The first sub-housing 1021 includes a sample inlet 2, and an external device (e.g., a micro flow pump) is connected to the sample inlet 2 and injects the cell suspension 109-1 into the first sub-housing 1021 through the sample inlet 2. The second sub-receiving portion 1022 includes a sample inlet 3, and an external device (e.g., a micro flow pump) is connected to the sample inlet 3 and injects the biochemical reaction reagent 109-2 into the second sub-receiving portion 1022 through the sample inlet 3. Different biochemical reagents can be adopted according to different biochemical reactions, and the chemical composition of the biochemical reagent 109-2 is not particularly limited by the embodiment of the disclosure. The first fluid 107 and the second fluid 108 comprising the cell suspension 109-1 and the biochemical reaction reagent 109-2 meet at the meeting point 105 of the first transport channel 103 and generate a plurality of first droplets 110, and the plurality of first droplets 110 flow along the first transport channel 103 into the first collection portion 104. The first collection portion 104 includes a plurality of sample outlets 4, and the sample outlets 4 are used for connecting with an external device to transfer the plurality of first droplets 110 in the first collection portion 104 to other containers for subsequent operations.

It should be noted that, although fig. 1A shows that the cell suspension 109-1 is accommodated in the first sub-accommodation portion 1021 and the biochemical reaction reagent 109-2 is accommodated in the second sub-accommodation portion 1022 separated from the first sub-accommodation portion 1021, this is merely an example and the embodiment of the present disclosure is not limited thereto. In an alternative embodiment, cell suspension 109-1 and biochemical reaction reagent 109-2 may be pre-mixed and contained in the same containment.

With continued reference to fig. 1A-1D, the first delivery flow path 103 includes a first flow path 1031 and a second flow path 1032. The first flow path 1031 communicates with the first accommodating portion 101 and allows the first fluid 107 to flow therein. The second flow channel 1032 communicates with the second receiving portion 102 and allows the second fluid 108 to flow therein. The first and

second flow passages

1031, 1032 intersect and communicate at a junction 105. In a partial section of the first supply channel 103 (for example in the second part 1031B of the first flow channel 1031 and the second flow channel 1032 of the first supply channel 103), the cross-sectional area of the first supply channel 103 first decreases and then increases, i.e. in this section the first supply channel 103 is made thicker and narrower and then thicker. This will be described in detail below.

The first flow path 1031 includes a first portion 1031A and a second portion 1031B, and the first portion 1031A and the second portion 1031B of the first flow path 1031 together form a closed pentagon that is approximately axisymmetric about a horizontal axis in which the junction 105 is located. A part of the first fluid 107 in the first container 101 flows along a part of the first flow passage 1031 above the horizontal axis to the junction 105, and another part of the first fluid 107 in the first container 101 flows along a part of the first flow passage 1031 below the horizontal axis to the junction 105.

Fig. 2 is an enlarged view of the AA area of fig. 1A. As shown in fig. 2, the second part 1031B of the first flow passage 1031 includes a first sub-part 1031B-1, a second sub-part 1031B-2 and a third sub-part 1031B-3 arranged in sequence in the first direction (vertical direction in the figure), and the second sub-part 1031B-2 is located between the first sub-part 1031B-1 and the third sub-part 1031B-3 and includes a junction 105. The first sub-part 1031B-1 belongs to the previously described first section, the third sub-part 1031B-3 belongs to the previously described second section, and the second sub-part 1031B-2 spans the first section and the second section. The first cross-sectional areas of the first and third sub-portions 1031B-1 and 1031B-3 are each larger than the first cross-sectional area of the second sub-portion 1031B-2, i.e., the first flow passage 1031 tapers first and then becomes thicker in a direction from the first sub-portion 1031B-1 to the third sub-portion 1031B-3, such that the first flow passage 1031 takes a shape that is thicker in the upper and lower (first and third sub-portions 1031B-1 and 1031B-3) and thinner in the middle (second sub-portion 1031B-2). By such a shape design, when the first fluid 107 in the first flow passage 1031 flows from the first sub-portion 1031B-1 to the second sub-portion 1031B-2 or flows from the third sub-portion 1031B-3 to the second sub-portion 1031B-2, as the flow passage becomes thinner, the flow velocity of the first fluid 107 in the first flow passage 1031 becomes larger, so that the pressure of the first fluid 107 can be increased, and the first fluid 107 in the first sub-portion 1031B-1 and the third sub-portion 1031B-3 is promoted to flow to the junction 105 of the second sub-portion 1031B-2 and be collected at the junction 105. This may provide sufficient first fluid 107 for subsequent formation of the first droplet 110. The shape of the first cross section of the first, second and third sub-portions 1031B-1, 1031B-2, 1031B-3 of the second portion 1031B of the first flow passage 1031 may be circular, square, rectangular, regular polygonal, irregular, etc., which is not limited by the embodiments of the present disclosure.

A first cross-section of the second sub-portion 1031B-2 of the first flow path 1031 at the junction 105 is sized to allow the first fluid 107 having a particular particle size to flow therein, the particular particle size of the first fluid 107 being greater than the particle size of an individual cell. That is, the width of the first cross-section of the second sub-portion 1031B-2 of the first flow passage 1031 at the junction 105 is greater than the particle size of the individual cells. In one example, each cell in the cell suspension has a particle size of about 10 μm, and the width of the cross-section of the second sub-portion 1031B-2 of the first flow channel 1031 at the junction 105 is greater than 10 μm, for example slightly greater than 10 μm. By "slightly greater than 10 μm" herein is meant that the width of the first cross-section of the second sub-portion 1031B-2 of the first flow passage 1031 at the junction 105 is greater than 10 μm but less than 20 μm, i.e. the width is greater than the particle size of a single cell but less 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 1031B-2 of the first flow path 1031 at the junction 105" can be understood to mean that when the shape of the first cross section of the second sub-portion 1031B-2 of the first flow path 1031 at the junction 105 is a circle, then 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 1031B-2 of the first flow passage 1031 at the junction 105 is square, then the width of the first cross section is the side length of the square; when the shape of the first cross-section of the second sub-portion 1031B-2 of the first flow passage 1031 at the junction 105 is rectangular, then 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 1031B-2 of the first flow path 1031 at the meeting point 105 is a regular polygon, then the width of the first cross-section is the distance between the two farthest vertices of the regular polygon. In one example, when the first cross-section of the second sub-portion 1031B-2 of the first flow passage 1031 at the junction 105 is circular and the shape of the individual cell is spherical, then the width of the first cross-section of the second sub-portion 1031B-2 of the first flow passage 1031 at the junction 105 being greater than the particle size of the individual cell should be understood that the diameter of the second sub-portion 1031B-2 of the first flow passage 1031 at the junction 105 is greater than the diameter of the individual cell. By this design, when the first fluid 107 in the first flow path 1031 flows from the first sub-portion 1031B-1 to the second sub-portion 1031B-2 or from the third sub-portion 1031B-3 to the second sub-portion 1031B-2, the first fluid 107 can be formed into a single row of fluid particles arranged in sequence near the junction 105, each particle in the single row of fluid particles having a particle size larger than a particle size of a single cell and smaller than a sum of particle sizes of two cells. In this way, the particle size of each particle formed by the first fluid 107 can be made slightly larger than the particle size of an individual cell, so that individual cells can be better encapsulated for better encapsulation. Moreover, such a design may also increase the flow rate of the first fluid 107 at the junction 105, facilitating the formation of the first droplet 110.

With continued reference to fig. 1A-1D and 2, the second flow channel 1032 includes a first portion 1032A, a second portion 1032B, and a third portion 1032C arranged in sequence in a second direction (horizontal direction in the figure) different from the first direction. The first and

second portions

1032A, 1032B belong to the previously described third section, and the third portion 1032C belongs to the previously described fourth section. A first end of the first section 1032A of the second flow channel 1032 communicates with the second accommodation portion 102, and a second end of the first section 1032A of the second flow channel 1032 communicates with a first end of the second section 1032B of the second flow channel 1032; a second end of the second portion 1032B of the second channel 1032 communicates with a first end of the third portion 1032C of the second channel 1032, and the second end of the second portion 1032B of the second channel 1032 and the first end of the third portion 1032C of the second channel 1032 are both located at the junction 105; a second end of the third portion 1032C of the second flow channel 1032 communicates with the first collecting portion 104. As shown, the first portion 1032A of the second channel 1032 includes a first branch communicating with the first sub-receiving part 1021 of the second receiving part 102 and configured to flow the cell suspension 109-1 therein, and a second branch communicating with the second sub-receiving part 1022 of the second receiving part 102 and configured to flow the biochemical reaction reagent 109-2 therein. The first and second branches intersect and communicate with each other at a first point 106, and the angle α between the first and second branches at the first point 106 is acute. In one example, the first branch and the second branch include an angle α of about 60 degrees at the first point 106. The angle between the first branch and the second branch is designed to ensure that the cell suspension 109-1 in the first branch and the biochemical reaction reagent 109-2 in the second branch have sufficient forward (towards the junction 105) flow rate and buffer pressure; on the other hand, it can be ensured that the cell suspension 109-1 and the biochemical reaction reagent 109-2 can be mixed sufficiently at the first point 106; on the other hand, the dead volume of the mixed solution in the flow channel can be reduced, and the liquid storage precision of the first branch and the second branch is improved. It should be noted that the phrase "the included angle α between the first branch and the second branch at the first point 106 is about 60 degrees" includes the cases that the included angle α between the first branch and the second branch at the first point 106 is greater than 60 degrees, the included angle α between the first branch and the second branch at the first point 106 is less than 60 degrees, and the included angle α between the first branch and the second branch at the first point 106 is equal to 60 degrees.

The second cross-sectional areas of the first and

third portions

1032A, 1032C of the second flow channel 1032 are each greater than the second cross-sectional area of the second portion 1032B of the second flow channel 1032. That is, the areas of the second cross-sections of the first and second branches of the first portion 1032A of the second flow channel 1032 are each greater than the area of the second cross-section of the second portion 1032B of the second flow channel 1032, and the area of the second cross-section of the third portion 1032C of the second flow channel 1032 is greater than the area of the second cross-section of the second portion 1032B of the second flow channel 1032. The second flow channel 1032 is tapered to be thicker along a direction from the first portion 1032A to the third portion 1032C of the second flow channel 1032. Similar to the first flow channels 1031, the shape of the second cross-section of the first, second, and

third portions

1032A, 1032B, 1032C of the second flow channels 1032 may be circular, square, rectangular, regular polygonal, irregular, etc., which are not limited by the embodiments of the present disclosure.

The second cross-section of the second portion 1032B of the second flow channel 1032 is sized to allow the second fluid 108 having a particular particle size to flow therein, the particular particle size of the second fluid 108 being greater than 1 times the particle size of an individual cell and less than 2 times the particle size of an individual cell. That is, the width of the second cross-section of the second portion 1032B of the second flow channel 1032 is greater than 1 times the particle size of an individual cell and less than 2 times the particle size of an individual cell. In one example, when the second cross-section of the second portion 1032B of the second flow channel 1032 is circular and the shape of the single cell is round and spherical, then a width of the second cross-section of the second portion 1032B of the second flow channel 1032 is greater than 1 times a particle size of a single cell and less than 2 times a particle size of a single cell should be understood as a diameter of the second portion 1032B of the second flow channel 1032 that is greater than 1 times a diameter of a single cell and less than 2 times a diameter of a single cell. In this case, the diameter of the second portion 1032B of the second flow channel 1032 may be 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, etc. the diameter of a single cell. When cell suspension 109-1 and biochemical reactant 109-2 are mixed at first point 106 and flow forward (toward junction 105), the mixed solution is arranged in a single row of single cell clusters within second portion 1032B by having the width of the second cross section of second portion 1032B greater than 1 times the particle size of a single cell and less than 2 times the particle size, as shown in FIG. 2. That is, the width of the second cross-section of the second portion 1032B of the second flow channel 1032 only allows for accommodation of a single cell in its width direction, and cannot accommodate two side-by-side cells. When the single-row single-cell string moves to the junction 105, under the pressure of the first fluid 107 in the second portion 1031B of the first flow passage 1031, one cell of the cell string closest to the junction 105 (i.e., the first cell of the cell string) is separated from the cell string, and the separated one cell is combined with a single particle in the first fluid 107 at the junction 105 to form a first droplet 110 containing a single cell 111. The upper right hand corner of fig. 2 shows an enlarged view of a first droplet 110 containing a single cell 111 generated at the junction 105. As mentioned above, the first fluid 107 is an oil phase and the second fluid 108 (i.e., the mixed solution of the cell suspension 109-1 and the biochemical reaction reagent 109-2) is an aqueous phase, so that the first droplets 110 formed have a water-in-oil structure, i.e., the first fluid 107 of the oil phase encloses the second fluid 108 of the aqueous phase.

As shown, the third portion 1032C of the second flow channel 1032 has a second cross-sectional area that gradually increases in a direction from the first end to the second end thereof, i.e., the third portion 1032C of the second flow channel 1032 gradually becomes thicker in the direction from the first end to the second end thereof. The purpose of this design is to make the first droplet 110 gradually larger as it moves forward along the third portion 1032C of the second channel 1032, thereby facilitating droplet phase stabilization. The area of the first cross section of the second sub-portion 1031B-2 of the first flow passage 1031 at the junction 105 is greater than or equal to the area of the second cross section of the second and

third portions

1032B, 1032C of the second flow passage 1032 at the junction 105. In one example, the area of a first cross section of the second sub-portion 1031B-2 of the first flow passage 1031 at the junction 105 is equal to the area of a second cross section of the second and

third portions

1032B, 1032C of the second flow passage 1032 at the junction 105. By such a design, the particle size of the single particle in the first fluid 107 can be approximately equal to the particle size of the mixed solution containing the single cell 111 (the mixed solution of the cell suspension 109-1 and the biochemical reaction reagent 109-2) at the merging point 105, so that the size of the formed first droplet 110 can be precisely controlled.

Fig. 3 is an enlarged schematic view of the first collection portion 104 of the first chip 100 in fig. 1A. The formed first droplets 110 finally flow into the first collection portion 104 along the third portion 1032C of the second flow channel 1032. As described above, each of the plurality of first droplets 110 is desirably formed to surround a single cell 111. However, in practice, due to the adhesion between cells in the cell suspension, the flow rate of the fluid, the surface design of the flow channel, etc., it is not completely guaranteed that each first droplet 110 includes a single cell 111. As shown in FIG. 3, in the formed first droplets 110, a single cell 111 derived from cell suspension 109-1 may be included in one first droplet 110, a single cell 111 derived from cell suspension 109-1 may not be included (i.e., an "empty" droplet), and two cells 111 derived from cell suspension 109-1 may be included. The structural design of the first chip 100 in the embodiments of the present disclosure (e.g., the structures of the first and

second flow passages

1031, 1032, the design of a filtering structure to be described later, etc.) is advantageous in facilitating the inclusion of only a single cell 111 within each first droplet 110.

In some embodiments, the inner wall surface of the first conveying flow channel 103 is subjected to hydrophobic treatment, and thus has hydrophobicity. As previously described, the first delivery flow path 103 includes the first flow path 1031 configured to flow the first fluid 107 therein and the second flow path 1032 configured to flow the second fluid 108 therein. The hydrophobic-treated first flow path 1031 may facilitate the flow of the first fluid 107 therein. The hydrophobically treated second channel 1032 can promote smooth flow of the cell suspension 109-1 in the first branch of the first portion 1032A of the second channel 1032 without adhesion to the inner wall surface, and can promote smooth flow of the mixed solution of the cell suspension 109-1 and the biochemical reaction reagent 109-2 in the second portion 1032B and the third portion 1032C of the second channel 1032 without adhesion to the inner wall surface. This allows precise control of the amount of cell suspension 109-1, which facilitates uniform mixing of cell suspension 109-1 with biochemical reaction reagent 109-2, thereby facilitating uniform formation of first droplet 110. Meanwhile, the utilization rate of the cell suspension 109-1 can be improved, and the waste of the cell suspension 109-1 is avoided.

With continued reference back to fig. 1A-1D, the profiles of the first receiving portion 101, the first sub-receiving portion 1021, and the second sub-receiving portion 1022 of the second receiving portion 102 each include four chamfers. The shape of the four chamfers may be any suitable shape, and may be, for example, an arc shape. It should be understood that the embodiments of the present disclosure do not limit the specific dimensions of the chamfer. Fig. 4 is an enlarged schematic view of the first sub-receiving portion 1021 of the second receiving portion 102. Taking the first sub-receiving portion 1021 of the second receiving portion 102 as an example, as shown in fig. 4, the contour of the first sub-receiving portion 1021 of the second receiving portion 102 includes four chamfers 113, and the four chamfers 113 have an arc shape. The arc-shaped chamfer design can reduce the dead volume of the cell suspension 109-1 in the first sub-containing part 1021, and improve the liquid storage precision of the first sub-containing part 1021. The "dead volume" here refers to the volume that is not controlled during the introduction of the reagent. Specifically, if the four corners of the first sub-housing 1021 are right angles rather than rounded chamfers, the cell suspension 109-1 is not right-angled at the four right-angled positions of the first sub-housing 1021 due to the presence of droplet surface tension, i.e., the cell suspension 109-1 cannot perfectly match the shape of the first sub-housing 1021, and cannot fill the space occupied by the four right angles of the first sub-housing 1021. Thus, the shape and volume of the cell suspension 109-1 will change, and there will be some randomness in this change in shape and volume, thereby introducing dead volume. This may cause the first sub-receiving part 1021 of the first chip 100 to receive a different volume of the cell suspension 109-1 in each operation than in the previous operation, thereby causing an inaccurate control of the amount of the cell suspension 109-1. In the embodiment of the disclosure, the four corners 113 of the first sub-receiving portion 1021 are designed to be arc-shaped chamfers, so that the shape of the cell suspension 109-1 can be perfectly matched with that of the first sub-receiving portion 1021, and especially, the cell suspension 109-1 can fill the space occupied by the four arc-shaped chamfers of the first sub-receiving portion 1021, thereby effectively reducing or even avoiding the difference in receiving volume of the first sub-receiving portion 1021, and improving the control precision of the cell suspension 109-1.

Similarly, the four rounded chamfers of the first container 101 can reduce the dead volume of the first fluid 107 in the first container 101, and improve the liquid storage accuracy of the first container 101. The four arc-shaped chamfers of the second sub-receiving portion 1022 of the second receiving portion 102 can reduce the dead volume of the biochemical reaction reagent 109-2 in the second sub-receiving portion 1022 and improve the liquid storage accuracy of the second sub-receiving portion 1022.

With continued reference to fig. 1A to 1D and fig. 4, the first receiving portion 101 and the second receiving portion 102 of the first chip 100 are each provided with the filtering structure 112, that is, the first receiving portion 101 of the first chip 100, the first sub-receiving portion 1021 of the second receiving portion 102, and the second sub-receiving portion 1022 are each provided with the filtering structure 112. Since the filter structures 112 of the first accommodating part 101, the first sub-accommodating part 1021, and the second sub-accommodating part 1022 are all configured identically, the structure and function of the filter structure 112 will be described in detail below by taking the filter structure 112 in the first sub-accommodating part 1021 shown in fig. 4 as an example.

As shown in FIG. 4, the filter structure 112 includes a plurality of microstructures spaced apart from each other, and a gap d between two adjacent microstructures 112-1 and 112-2 is larger than 1 times a particle size of a single cell and smaller than 2 times the particle size of the single cell. In some embodiments, the size of the individual cells derived from cell suspension 109-1 is about 10 μm, and accordingly, the gap d between two adjacent microstructures 112-1 and 112-2 is greater than 10 μm and less than 20 μm. The heights of the plurality of microstructures of the filtering structure 112 may be completely the same, or completely different, or may be only partially the same, and the specific heights may be flexibly designed according to product requirements, which is not specifically limited by the embodiments of the present disclosure. In some embodiments, the height of each microcolumn is about 100-200 μm. The shape of the cross section of each micro-pillar in a direction parallel to the plane of the first sub-receiving part 1021 may be any suitable shape, such as a diamond shape, a square shape, a rectangular shape, a circular shape, an oval shape, a regular polygon shape, an irregular shape, and the like, and embodiments of the present disclosure are not particularly limited thereto.

During operation of the first chip 100, the cell suspension 109-1 in the first sub-volume 1021 flows through the gaps between adjacent microstructures of the filter structure 112 and then into the first branch of the first portion 1032A of the second flow channel 1032. Because the gap d between two adjacent microstructures is larger than 1-time particle size of a single cell and smaller than 2-time particle size of the single cell, when the cell suspension 109-1 flows through the gap between the two adjacent microstructures, on one hand, overlarge impurities (for example, impurities with particle size larger than 2-time particle size of a single cell, such as dust, salting-out substances and the like) in the cell suspension 109-1 can be prevented from flowing into a subsequent flow channel, so that the flow channel is prevented from being blocked by the overlarge impurities, and normal flow of the cell suspension 109-1 is prevented from being influenced; on the other hand, under the action of the adjacent microstructures on the cell suspension 109-1 and the screening of the size of the cell suspension 109-1 by the gap between the adjacent microstructures, a plurality of cells (e.g., two cells, three cells or more cells adhered to each other) adhered to each other in the cell suspension 109-1 can be separated into a plurality of single cells separated from each other, so as to facilitate the generation of the first droplet 110 containing single cells, and reduce the probability of containing two or more cells in the single first droplet 110.

The structure of the filter structure 112 in the first receiving portion 101 and the second sub-receiving portion 1022 may refer to the above description of the filter structure in the first sub-receiving portion 1021, and for brevity, will not be described again. During operation of the first chip 100, the first fluid 107 in the first container 101 flows through the gaps between adjacent microstructures of the filter structure 112 and then flows into the first flow path 1031 of the first delivery flow path 103. When the first fluid 107 flows through the gaps between the adjacent microstructures of the filter structure 112, the flow of the oversized impurities (e.g. impurities with a particle size 2 times larger than that of a single cell, such as dust, salting-out substances, etc.) in the first fluid 107 into the first flow channel 1031 can be blocked, thereby avoiding the oversized impurities from blocking the first flow channel 1031 and affecting the normal flow of the first fluid 107. During operation of the first chip 100, the biochemical reaction reagent 109-2 in the second sub-accommodation part 1022 flows through the gap between the adjacent microstructures of the filter structure 112 and then flows into the second branch of the first portion 1032A of the second flow channel 1032 of the first transporting flow channel 103. When the biochemical reaction reagent 109-2 flows through the gap between the adjacent microstructures of the filtering structure 112, the excessive impurities (for example, impurities having a particle size 2 times larger than that of a single cell, such as dust, salting-out substances, etc.) in the biochemical reaction reagent 109-2 can be blocked from flowing into the second branch of the first portion 1032A of the second flow channel 1032, thereby preventing the excessive impurities from blocking the second flow channel 1032 and affecting the normal flow of the biochemical reaction reagent 109-2.

The first chip 100 described in any of the previous embodiments may be a microfluidic chip. The microfluidic chip has the advantages of controllable liquid flow, less sample consumption, high detection speed, simple and convenient operation, multifunctional integration, small volume, portability and the like. In addition to the above advantages, in the embodiment of the present disclosure, through the optimized design of the first conveying flow channel 103 of the first chip 100, the separation of single cells from the cell suspension 109-1 can be promoted, so that each generated first droplet 110 can be promoted to contain a single cell, and the probability of containing two or more cells in the single first droplet 110 can be reduced; by optimizing the spacing between adjacent microstructures of the filter structure 112, the inclusion of individual cells within each first droplet 110 generated may be further facilitated; four corners of the first accommodating part 101 and the second accommodating part 102 are designed to be arc-shaped chamfers, so that dead volume in the accommodating parts can be avoided, and liquid storage precision of the accommodating parts is improved; by performing hydrophobic treatment on the inner wall surface of the first conveying flow channel 103, the adhesion of the fluid in the flow channel to the inner wall surface of the flow channel can be reduced or even avoided, so that the amount of the fluid can be accurately controlled, the waste of the reagent can be avoided, and the uniform generation of the first liquid drops 110 can be promoted. The first droplet 110 may constitute a microreactor for biochemical reactions of individual cells and may also constitute a carrier for droplets for subsequent single-cell sorting. In addition, since the first chip 100 is highly integrated, the preparation of the first droplet 110 including a single cell can be automatically completed without manual operation by an operator, and thus the degree of automation of the operation can be effectively improved. Since the first fluid 107 and the second fluid 108 completely flow in the first conveying flow channel 103 and are completely isolated from the external environment, the pollution caused by aerosol, microorganisms and the like floating in the environment can be avoided. In addition, the whole preparation process is mild, and the single cell separated from the cell suspension is wrapped and protected by the liquid drop, so that the survival rate of the cell can be effectively improved.

Other embodiments of the present disclosure provide a chip 200 (hereinafter referred to as a second chip 200), which second chip 200 may be used to sort target droplets. Fig. 5A-5D illustrate the second chip 200, wherein fig. 5A is a front view of the second chip 200, fig. 5B is a side view of the second chip 200, fig. 5C is a rear view of the second chip 200, and fig. 5D is a front triaxial side view of the second chip 200. As shown in fig. 5A to 5D, the second chip 200 includes: a third housing portion 201 configured to house a third fluid 205; a fourth container 202 configured to contain a fourth fluid, the fourth fluid comprising a cell suspension; a delivery flow path 208 (hereinafter referred to as a second delivery flow path 208) comprising a third flow passage 2081 and a fourth flow passage 2082, the third flow passage 2081 being in communication with the third receptacle 201 and the fourth flow passage 2082 being in communication with the fourth receptacle 202, the third flow passage 2081 and the fourth flow passage 2082 intersecting and communicating with each other at a junction 209, the second delivery flow path 208 being configured such that the third fluid 205 and the fourth fluid meet at the junction 209 and generate at least one droplet 206 (hereinafter referred to as a second droplet 206), each of at least a portion of the at least one second droplet 206 comprising a single cell derived from the cell suspension; and at least one collection portion 204 (hereinafter referred to as second collection portion 204) configured to collect at least one second droplet 206. A portion of the third flow passage 2081 (i.e., the vertical portion of the third flow passage 2081 in fig. 5A) includes the junction 209 and is divided by the junction 209 into a first section (e.g., the section of the third flow passage 2081 in fig. 5A that is above the junction 209) and a second section (e.g., the section of the third flow passage 2081 in fig. 5A that is below the junction 209), in each of which the area of a first cross-section of the section gradually increases along a first direction away from the junction 209, the first cross-section being perpendicular to the first direction. The fourth flow passage 2082 includes a junction 209 and is divided by the junction 209 into a third section (e.g., the section of 2082 in fig. 5A that is located on the left side of the junction 209) and a fourth section (e.g., the section of 2082 in fig. 5A that is located on the right side of the junction 209), and in each 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 junction 209, and the second cross-section is perpendicular to the second direction. That is, in the third flow passage 2081, the third flow passage 2081 becomes gradually thicker in the upward direction from the junction 209; the third flow passage 2081 also becomes progressively thicker in a direction downward from the junction 209. In the fourth flow passage 2082, the fourth flow passage 2082 becomes progressively thicker in the leftward direction from the junction 209; the fourth flow passage 2082 also becomes progressively thicker in the rightward direction from the junction 209.

It should be noted that the third fluid 205 and the fourth fluid are only used for describing the second chip 200, and in fact, the third fluid 205 may be identical to the first fluid 107, and the fourth fluid may be identical to the second fluid 108 (including the cell suspension 109-1 and the biochemical reaction reagent 109-2). Of course, the fourth fluid may not be identical to the second fluid 108. In one example, the fourth fluid is the first droplet 110 described previously.

As can be seen from the above description, the first cross-section of a portion of the third flow passage 2081 (i.e., the vertical portion of the third flow passage 2081 in fig. 5A) refers to a cross-section of a portion of the third flow passage 2081 in a direction perpendicular to the flow direction of the third fluid 205; the second cross section of the fourth flow passage 2082 refers to a cross section of the fourth flow passage 2082 in a direction perpendicular to the flow of the fourth fluid.

The second delivery channel 208 further comprises a sorting channel 203, the sorting channel 203 being configured to sort a droplet of interest, which comprises a single cell, from the at least one second droplet 206.

As shown in fig. 5A to 5D, the third container 201 includes a sample inlet 5, and an external device (e.g., a micro flow pump) is connected to the sample inlet 5 and injects a third fluid 205 into the third container 201 through the sample inlet 5. Third fluid 205 is a continuous phase (e.g., oil phase) liquid that may have the same chemical composition as first fluid 107. Third fluid 205 may be any suitable fluid such as, for example, mineral oil, perfluorinated oil, and the like. Optionally, a surfactant may be mixed with third fluid 205, which surfactant facilitates stabilization of subsequently formed second droplets 206, e.g., inhibits subsequent coalescence of second droplets 206. When the third fluid 205 is a perfluorinated oil, the surfactant may be a perfluorinated surfactant.

The fourth container 202 includes a sample inlet 6, and an external device (e.g., a micro flow pump) is connected to the sample inlet 6 and injects a fourth fluid (e.g., the first droplet 110 described above) into the fourth container 202 through the sample inlet 6. Before injecting the first droplet 110 into the second chip 200, the first droplet 110 may be transferred to another device for performing a corresponding biochemical process (e.g., PCR amplification, staining, etc.), and then the processed first droplet 110 may be injected into the second receiving portion 202 of the second chip 200.

Similar to the first and second receiving

portions

101 and 102 of the first chip 100, the profiles of the third and fourth receiving

portions

201 and 202 of the second chip 200 also each include four chamfers, the shape of which may be a circular arc or any other suitable shape. The chamfered design of the third container 201 and the fourth container 202 can reduce the dead volume of the third fluid 205 and the first droplet 110 in the third container 201 and the fourth container 202, respectively, so that the liquid storage accuracy of the third container 201 and the fourth container 202 can be improved.

Like the first and second receiving

parts

101 and 102 of the first chip 100, the third and fourth receiving

parts

201 and 202 are also provided with a filtering structure (not shown in the drawings). The filter structures in the third and

fourth receptacles

201 and 202 include a plurality of microstructures spaced apart from each other, and a gap between two adjacent microstructures is greater than 1 times a particle size of a single first droplet 110 and less than 2 times a particle size of the single first droplet 110. The heights of the plurality of microstructures of the filtering structure may be completely the same or different, or may be only partially the same, and the specific height may 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 microcolumn is about 100-200 μm. The shape of the cross section of each micro-column in a direction parallel to the plane of the third receiving part 201 and the fourth receiving part 202 may be any suitable shape, such as a diamond shape, a square shape, a rectangular shape, a circular shape, an oval shape, a regular polygon shape, an irregular shape, and the like, and embodiments of the present disclosure are not particularly limited thereto.

During operation of the second chip 200, the third fluid 205 in the third receptacle 201 flows through the gaps between adjacent microstructures of the filter structure, the filtered third fluid 205 then flows into the third flow passage 2081 of the second transfer flow passage 208. When the third fluid 205 flows through the gaps between the adjacent microstructures of the filter structure, the excessive impurities (for example, impurities having a particle diameter 2 times larger than that of the single first droplet 110, such as dust, salting substances, etc.) in the third fluid 205 can be blocked from flowing into the third flow passage 2081, so that the excessive impurities are prevented from blocking the third flow passage 2081 and affecting the normal flow of the third fluid 205. The first droplet 110 in the fourth receptacle 202 flows through the gap between adjacent microstructures of the filter structure, and the filtered first droplet 110 then flows into the fourth flow passage 2082 of the second transport flow passage 208. Since the gap between two adjacent microstructures is larger than 1 times the particle diameter of the single first liquid droplet 110 and smaller than 2 times the particle diameter of the single first liquid droplet 110, when the first liquid droplet 110 flows through the gap between the adjacent microstructures, on one hand, potential oversized impurities (for example, impurities with a particle diameter larger than 2 times the particle diameter of the single first liquid droplet 110, such as dust, salting-out substances, etc.) in the first liquid droplet 110 can be blocked from flowing into the fourth flow passage 2082, thereby preventing the oversized impurities from blocking the fourth flow passage 2082 and affecting the normal flow of the first liquid droplet 110; on the other hand, under the action of the first droplet 110 by the adjacent microstructures and the screening of the size of the first droplets 110 by the gap between the adjacent microstructures, a plurality of droplet particles (e.g., two droplet particles, three droplet particles, or more droplet particles that are adhered to each other) that may exist in the first droplets 110 may be separated to become a plurality of individual droplets separated from each other, so that when the individual first droplets 110 are merged with the third fluid 205, the individual second droplets 206 may be generated.

The second transportation flow passage 208 includes a third flow passage 2081 and a fourth flow passage 2082, and the third flow passage 2081 and the fourth flow passage 2082 intersect at a junction 209 and communicate with each other at the junction 209. The third flow passage 2081 communicates with the third housing portion 201 and allows the third fluid 205 to flow therein, and the fourth flow passage 2082 communicates with the fourth housing portion 202 and allows the first liquid droplet 110 to flow therein. The third fluid 205 flows along the third flow passage 2081 to a junction 209 between the third flow passage 2081 and the fourth flow passage 2082, the first droplet 110 flows along the fourth flow passage 2082 to a junction 209 between the third flow passage 2081 and the fourth flow passage 2082, and the third fluid 205 joins the first droplet 110 at the junction 209 and generates the second droplet 206.

Fig. 6 is an enlarged view of a region BB of the sorting flow channel 203 shown in fig. 5A. Referring to fig. 5A and 6, the sorting flow channel 203 includes a first branch 2031, a second branch 2032, and a third branch 2033, the third branch 2033 being located between the first branch 2031 and the second branch 2032. The second collecting portion 204 includes a first sub-collecting portion 2041, a second sub-collecting portion 2042 and a third sub-collecting portion 2043, the first sub-collecting portion 2041 includes a sample outlet 7A, the second sub-collecting portion 2042 includes a sample outlet 7B, and the third sub-collecting portion 2043 includes a sample outlet 7C. The first sub-collecting portion 2041 communicates with the first branch 2031 of the sorting channel 203, the second sub-collecting portion 2042 communicates with the second branch 2032 of the sorting channel 203, and the third sub-collecting portion 2043 communicates with the third branch 2033 of the sorting channel 203. An electrode structure (not shown) is provided at the sorting flow path 203, which may for example comprise positive and negative electrodes for applying a voltage to drive the deflection of the second droplet 206 into the respective branch of the sorting flow path 203, thereby sorting out the targeted droplet in the second droplet 206. The second chip 200 may further comprise an optical device (not shown in the figure, such as a fluorescence microscope, etc.) for identifying the target droplet in the second droplet 206.

The sorting process of the target droplets is roughly as follows: cell suspension 109-1 contains a plurality of cells. Among the plurality of cells, a small number of target cells to be analyzed and detected, i.e., target cells that the present application desires to sort out (e.g., circulating tumor cells, rare cells, cancer cells, etc. in a peripheral blood sample), and other non-target cells, are contained. Since these target cells and non-target cells in the cell suspension contain different antibodies, they will show different colors under a fluorescent microscope after fluorescent staining. The cell suspension 109-1 may be stained, and the first liquid drop 110 may also be stained, and the staining sequence is not particularly limited by the embodiment of the disclosure. After the dyeing process, the first droplet 110 is injected into the fourth containing portion 202 of the second chip 200, and the third fluid 205 and the first droplet 110 are merged in the second transport flow channel 208 to generate a second droplet 206. The second droplets 206 can be roughly classified into the following two types: (a) The second droplet 206 contains a single target cell with a target color; (b) The second droplet 206 contains non-target cells or no cells. The second droplet 206 moves along the second transporting flow path 208 toward the second collecting portion 204, and the optical device detects an optical signal (e.g., color) of the second droplet 206 in the second transporting flow path 208 in real time. When the optical device detects that the second droplet 206 is in the condition (b), the second droplet 206 flows along the second transporting channel 208 directly into the third branch 2033 of the sorting channel 203 and then into the third sub-collecting portion 2043 of the second collecting portion 204 without triggering the circuitry to apply a voltage to the electrode structure. When the optical device detects that the second droplet 206 is in the condition (a), the optical device immediately triggers the circuitry to apply a voltage (e.g., 800-1000V) to the electrode structure at the sorting channel 203, so that the second droplet 206 containing a single target cell is polarized, and under the action of the electric field, the second droplet 206 containing a single target cell is deflected upward into the first branch 2031 of the sorting channel 203 or deflected downward into the second branch 2032 of the sorting channel 203, and then flows into the first sub-collection portion 2041 or the second sub-collection portion 2042 of the second collection portion 204, respectively. Thus, the second chip 200 realizes sorting of target droplets.

It should be noted that the fluorescent staining process for the cell suspension is only an example of the embodiment of the present disclosure, and the processing manner for the cell suspension is not limited thereto, as long as the processing manner capable of distinguishing the target cells from the non-target cells in the cell suspension is within the protection scope of the present disclosure.

It should be noted that, although fig. 5A shows that the sorting channel 203 includes three branches and the second collecting portion 204 includes three sub-collecting portions, respectively, embodiments of the present disclosure are not limited thereto. In alternative embodiments, the sorting channel 203 may include at least two branches (e.g., two branches, four branches, or more branches), one branch of the at least two branches configured to sort non-target droplets from the plurality of second droplets 206, and the remaining branches of the at least two branches configured to sort target droplets from the plurality of second droplets 206. Accordingly, the second collecting part 204 may include at least two sub-collecting parts corresponding to the at least two branches of the sorting flow channel 203 one to one, one of the at least two sub-collecting parts being in communication with one of the at least two branches of the sorting flow channel 203 and configured to collect the non-target droplets, and the remaining sub-collecting parts of the at least two sub-collecting parts being in communication with the remaining branches of the at least two branches of the sorting flow channel 203 and configured to collect the target droplets, respectively.

With continued reference to fig. 6, the first branch 2031, the second branch 2032, and the third branch 2033 of the sorting flow channel 203 intersect at the second point 207 and the third branch 2033 is located between the first branch 2031 and the second branch 2032. The first angle θ 1 between the first branch 2031 and the third branch 2033 at the second point 207 and the second angle θ 2 between the second branch 2032 and the third branch 2033 at the second point 207 are both greater than 10 °. The space between the first branch 2031 and the third branch 2033 of the sorting flow channel 203 defines a first right triangle and the space between the second branch 2032 and the third branch 2033 of the sorting flow channel 203 defines a second right triangle. The first included angle theta 1 faces a first right-angle side of the first right-angle triangle, and the second included angle theta 2 faces a second right-angle side of the second right-angle triangle. The length L1 of the first leg of the first right triangle and the length L2 of the second leg of the second right triangle are both greater than or equal to the diameter of a single second droplet 206.

It should be noted that the phrase "defined" in the phrase "the space between the first branch 2031 and the third branch 2033 of the sorting flow channel 203 defines a first right triangle, and the space between the second branch 2032 and the third branch 2033 of the sorting flow channel 203 defines a second right triangle" means that, as shown in fig. 5A, the third branch 2033 of the sorting flow channel 203 extends in the second direction (i.e., the horizontal direction in the drawing); the first branch 2031 of the sort flow channel diverges from the third branch 2033 at the second point 207 and extends upwardly at an inflection point; the second branch 2032 of the sort flow channel diverges from the third branch 2033 at the second point 207 and extends downward at another inflection point. The inflection point of the first branch 2031 and the inflection point of the second branch 2032 are connected by a straight line which intersects the third branch 2033 at a point. The inflection point of the first branch 2031, the second point 207, and the intersection point of the straight line and the third branch 2033 are connected to form a first right-angled triangle, the first included angle θ 1 faces the first right-angled side of the first right-angled triangle, the length of the first right-angled side is L1, and the length L1 is greater than or equal to the particle size of a single second droplet 206. An inflection point of the second branch 2032, the second point 207, and an intersection point of the straight line and the third branch 2033 are connected to form a second right-angled triangle, the second included angle θ 2 faces a second right-angled side of the second right-angled triangle, the length of the second right-angled side is L2, and the length L2 is greater than or equal to the particle size of the single second droplet 206.

By designing the angle between the branches of the sorting flow channel 203 to be greater than 10 ° and the length of the right-angled side of the right-angled triangle to be greater than or equal to the particle size of one second droplet 206, sorting of the target droplets into the corresponding sub-collecting sections is facilitated. This is because: if the slope of the branch between the branches of the sorting flow path 203 is too small, the target droplet may not be deflected normally upward or downward into the corresponding sub-collecting portion, and may be erroneously entered into the intermediate sub-collecting portion 2043; if the slope of the branch between the branches of the sorting flow path 203 is too large, a larger voltage is applied to cause the target droplets to flow into the corresponding sub-collecting sections in order to sort the target droplets, but the second droplet 206 is broken or damaged by the excessive voltage, and thus sorting of the target droplets cannot be achieved. Therefore, the appropriate fork slope of the sorting flow path 203 facilitates the sorting of the target droplet from the second droplet 206.

The second chip 200 described in any of the previous embodiments may be a microfluidic chip. In the embodiments of the present disclosure, by using a microfluidic chip as the first chip 100 and the second chip 200, a liquid flow path and a pump and valve system thereof may be effectively simplified. By controlling the flow of liquid into the chip, it is achieved that the size of the formed second droplet 206 is controllable in the range of a few micrometers to a few tens of micrometers.

Further embodiments of the present disclosure provide a chip 300 (hereinafter referred to as a third chip 300), which third chip 300 can be used to simultaneously achieve the preparation of droplets containing a single cell and the sorting of target droplets. Fig. 7 shows the third chip 300. The third chip 300 may be regarded as a combination of the first chip 100 and the second chip 200, but in the third chip 300, the third receiving portion 201, the fourth receiving portion 202, and the second conveying flow channel 208 of the second chip 200 are removed. In fig. 7, the same reference numerals as in fig. 1A and 5A denote the same structures, and thus, for the sake of brevity, the same structures as the first chip 100 and the second chip 200 in the third chip 300 are not described again, and different portions are described below.

During operation of the third chip 300, the first fluid 107 and the second fluid 108 (including the cell suspension 109-1 and the biochemical reaction reagent 109-2) are combined in the first transport channel 103 to form the first droplet 110, and the first droplet 110 flows into the first collection portion 104 along the third portion 1032C of the second channel 1032, as shown in FIG. 7. The first droplets 110 have a water-in-oil structure. The first collecting portion 104 is in direct communication with the sorting channel 203, that is, the first droplets 110 within the first collecting portion 104 may flow into the sorting channel 203. Since the first droplet 110 has been subjected to fluorescent staining in the previous stage, the first droplet 110 can be roughly classified into the following two types: (a) The first droplet 110 contains a single target cell with a target color; (b) The first droplet 110 contains non-target cells or no cells. The plurality of first droplets 110 move along the sorting flow path 203 toward the second collecting portion 204, and the optical device detects an optical signal (e.g., color) of the first droplets 110 in real time. When the optical device detects that the first droplet 110 is in the condition (b), the electronic circuit system is not triggered to apply the voltage to the electrode structure, and the first droplet 110 flows straight forward into the third branch 2033 of the sorting channel 203 and then flows into the third sub-collecting portion 2043 of the second collecting portion 204. When the optical device detects that the first droplet 110 is in the above-mentioned condition (a), the optical device immediately triggers the circuitry to apply a voltage (e.g., 800-1000V) to the electrode structure at the sorting channel 203, the first droplet 110 containing a single target cell is polarized, and under the action of the electric field, the first droplet 110 containing a single target cell is deflected upward to flow into the first branch 2031 of the sorting channel 203 or deflected downward to flow into the second branch 2032 of the sorting channel 203, and then flows into the first sub-collecting portion 2041 or the second sub-collecting portion 2042 of the second collecting portion 204, respectively. Therefore, the third chip 300 can simultaneously achieve the two purposes of preparing the first droplet 110 containing a single cell and sorting the target droplet from the first droplet 110. The target droplets contain individual target cells desired to be obtained by the present application, such as circulating tumor cells, rare cells, cancer cells, etc. in a peripheral blood sample.

As shown in fig. 7, the third chip 300 is provided with an electrode structure at the sorting flow channel 203. Above the sorting flow channel 203, electrode structures E1, E2, E3, and E4 are provided; below the sorting flow channel 203, electrode structures E5, E6, E7, and E8 are provided. Each electrode structure is connected with a lead. As shown, one end of wire W1 is electrically connected to electrode structure E1, one end of wire W2 is electrically connected to electrode structure E2, one end of wire W3 is electrically connected to electrode structure E3, one end of wire W4 is electrically connected to electrode structure E4, and the other ends of wires W1, W2, W3, and W4 intersect at a point. The wires W1, W2, W4 are substantially polygonal lines, and the wire W3 is substantially straight. One end of the wire W5 is electrically connected to the electrode structure E5, one end of the wire W6 is electrically connected to the electrode structure E6, one end of the wire W7 is electrically connected to the electrode structure E7, one end of the wire W8 is electrically connected to the electrode structure E8, and the other ends of the wires W5, W6, W7, and W8 intersect at one point. The wires W5, W6, W8 are substantially polygonal lines, and the wire W7 is substantially straight. During operation of the third chip 300, when the optical device detects that the first droplet 110 is in the above condition (a), then the triggering circuitry applies a voltage to the electrode structures at the sorting flow channel 203, for example, a positive voltage (or a negative voltage) to the electrode structures E1-E4, and a negative voltage (or a positive voltage) to the electrode structures E5-E8, and the voltage difference between the two may be, for example, 800-1000V. The first droplet 110 containing the single target cell is polarized, and under the action of the electric field, the first droplet 110 containing the single target cell is deflected upward to flow into the first branch 2031 of the sorting channel 203 or deflected downward to flow into the second branch 2032 of the sorting channel 203 according to the direction of the electric field, and then flows into the first sub-collecting portion 2041 or the second sub-collecting portion 2042 of the second collecting portion 204, respectively.

The third chip 300 combines the first chip 100 with the second chip 200, and removes the third container 201, the fourth container 202, and the second transfer channel 208 of the second chip 200, thereby achieving the two purposes of preparing the first droplet 110 containing a single cell and sorting the target droplet from the first droplet 110. Therefore, the third chip 300 has the respective technical effects of the first chip 100 and the second chip 200, and can simplify the structure, further reduce the volume occupied by the chip, further improve the integration degree of the chip, and make the chip lighter and easier to carry on the premise of realizing the preparation of the droplets and the sorting of the target droplets.

According to yet another aspect of the present disclosure, a microfluidic device is provided. Fig. 8 shows a block diagram of the microfluidic device 400. The microfluidic device 400 includes a chip as described in any of the previous embodiments. Since the microfluidic device 400 may have substantially the same technical effects as the first chip 100, the second chip 200, and the third chip 300 described in the previous embodiments, for the sake of brevity, the technical effects of the microfluidic device 400 will not be described repeatedly herein.

According to yet another aspect of the present disclosure, a method of sorting target droplets is provided, and a flow chart of the method 800 is shown in fig. 9. The method 800 is described below by taking the third chip 300 in fig. 7 as an example. The method 800 includes the steps of:

step S801: providing a first fluid 107 and a second fluid 108 comprising a cell suspension 109-1 to the first container 101 and the second container 102, respectively, of the third chip 300 described in any of the previous embodiments, bringing the first fluid 107 and the second fluid 108 together at the junction 105 of the transfer flow channel 103 and generating at least one droplet 110, each of at least a portion of the at least one droplet 110 comprising a single cell derived from the cell suspension 109-1; and

step S802: a voltage is applied to the third chip 300 described in any of the previous embodiments to sort out a target droplet having a target property, which includes a single cell, from the at least one droplet 110.

In some embodiments, step S802 includes the sub-steps of: an optical signal of the plurality of droplets 110 is detected in real time by an optical device, and in response to the optical device detecting the droplet 110 having the target optical signal, an instantaneous voltage of 800 to 1000V is applied to the electrode structure of the third chip 300 described in any of the foregoing embodiments to sort out the target droplet having the target optical signal, which includes the above-described single cell, from the plurality of droplets 110.

In some embodiments, before step S802, the method further includes: the plurality of first droplets 110 are transferred to another reaction vessel to perform a polymerase chain reaction or a fluorescent staining process.

In some embodiments, the first fluid 107 is an oil phase, such as any suitable oil, such as mineral oil, perfluorinated oil, etc., and the second fluid 108 is an aqueous phase, and the droplets 110 formed have a water-in-oil structure.

The method of sorting the droplets of the object is described in more detail below with a specific example, taking the first chip 100 and the second chip 200 as an example.

Step S901: the input channels of the micro flow pump are respectively connected to the sample inlet 1 of the first receiving part 101 of the first chip 100, the sample inlet 2 of the first sub-receiving part 1021 of the second receiving part 102, and the sample inlet 3 of the second sub-receiving part 1022 of the second receiving part 102, so as to respectively inject the first fluid 107, the cell suspension 109-1, and the biochemical reaction reagent 109-2 into the first receiving part 101, the first sub-receiving part 1021, and the second sub-receiving part 1022. The first fluid 107 is an oil phase, which may be mixed with a surfactant.

Step S902: the sample injection speed of the first containing part 101 and the second containing part 102 to the first conveying flow channel 103 is adjusted to control the flow rate of the oil water and the droplet generation effect. Typically, the flow rate of first fluid 107 is greater than the flow rates of cell suspension 109-1 and biochemical reagents 109-2.

Step S903: the first receiving portion 101 is controlled such that the first fluid 107 fills most of the first chip 100, and then the first sub-receiving portion 1021 and the second sub-receiving portion 1022 are controlled such that the cell suspension 109-1 and the biochemical reaction reagent 109-2 flow into the first transfer channel 103. Controlling the first container 101 to fill the first fluid 107 first in most areas of the first chip 100 means controlling the first container 101 to fill the first fluid 107 first in the first flow channels 1031, the second portions 1032B and the third portions 1032C of the first flow channels 103, and optionally the first collection portion 104 of the first chip 100. Since the amount of cell suspension 109-1 is usually very small and precious, it is possible to fill a partial region of first chip 100 with first fluid 107 first to achieve a better encapsulation effect.

Step S904: the first fluid 107 in the first container 101 flows through gaps between the plurality of microstructures of the filtering structure 112 in the first container 101 to perform filtering, and then flows into the first flow passage 1031, thereby preventing the flow passage from being clogged with excessive foreign materials. The cell suspension 109-1 in the first sub-housing part 1021 flows through the gaps between the microstructures of the filtering structure 112 in the first sub-housing part 1021 to realize filtering, and then flows into the second flow channel 1032, so that on one hand, the blockage of the flow channel by the overlarge impurities can be avoided, and on the other hand, the cells adhered together in the cell suspension 109-1 can be separated into a plurality of single cells separated from each other. The biochemical reaction reagent 109-2 in the second sub-accommodation part 1022 flows through the gaps between the plurality of microstructures of the filtering structure 112 in the second sub-accommodation part 1022 to perform filtering, and then flows into the second flow channel 1032, so that it is possible to prevent the flow channel from being blocked by the excessive impurities.

Step S905: the first fluid 107, the cell suspension 109-1, and the biochemical reactant 109-2 meet at the junction 105 of the first transport channel 103 and form a plurality of first droplets 110, and the plurality of first droplets 110 move along the third portion 1032C of the second channel 1032 and stabilize as the diameter of the third portion 1032C of the second channel 1032 gradually widens. Finally, the first droplets 110 flow along the third portion 1032C of the second flow channel 1032 into the first collection portion 104.

Step S906: the first droplets 110 are collected at the outlet 4 of the first collection portion 104 for a period of time (e.g., 30 seconds, 1 minute, 2 minutes, etc.) and discarded as waste. This is because, at an initial operation stage of the first chip 100, initial states of the respective components within the first chip 100 may be less stable (e.g., pressure instability), which may affect an encapsulation effect of the first chip 100, thereby being disadvantageous to forming the first liquid droplets 110 of good quality. Therefore, the first droplet 110 formed in the initial stage is not generally used. When the parameters indicated by the first chip 100 reach a steady state, the first droplet 110 can be collected for subsequent operations.

Step S907: the first droplet 110 collected by the first collection portion 104 is taken out and transferred to another reaction vessel, such as a 96-well cell culture plate, a PCR instrument, or the like, to perform a desired biochemical reaction (e.g., PCR amplification, incubation reaction, fluorescent staining of droplets, or the like).

Step S908: the input channels of the micro flow pumps are respectively connected to the sample inlet 5 of the third containing part 201 and the sample inlet 6 of the fourth containing part 202 of the second chip 200, so as to respectively inject the third fluid 205 and the first droplet 110 prepared by the first chip 100 into the third containing part 201 and the fourth containing part 202. Third fluid 205 may be the same oil phase as first fluid 107 and may be mixed with a surfactant.

Step S909: the sample injection speed of the third accommodating part 201 and the fourth accommodating part 202 to the second conveying flow channel 208 is adjusted to control the droplet flow speed. Typically, the flow rate of third fluid 205 is greater than the flow rate of first droplets 110.

Step S910: similar to step S903, the third container 201 is controlled to fill most of the second chip 200 with the third fluid 205, and then the fourth container 202 is controlled to flow the first droplets 110 into the second transport channel 208.

Step S911: the third fluid 205 in the third container 201 flows through the gaps between the microstructures of the filtering structure 112 in the third container 201 to be filtered, and then flows into the third flow passage 2081 of the second conveying flow passage 208, so as to prevent the flow passage from being blocked by the excessive impurities. The first liquid droplet 110 in the fourth container 202 flows through the gaps between the microstructures of the filtering structure 112 in the fourth container 202 to achieve filtering, and then flows into the fourth flow passage 2082 of the second conveying flow passage 208, thereby preventing the flow passage from being blocked by the excessive foreign matters.

Step S912: third fluid 205 and first droplet 110 meet at a junction of second delivery flow channel 208 and generate a plurality of second droplets 206. The second droplets 206 can be roughly classified into the following two types: (a) The second droplet 206 contains a single target cell with a target color; (b) The second droplet 206 contains non-target cells or no cells.

Step S913: the second droplet 206 moves along the second transport channel 208 toward the second collection portion 204, and the optical device detects an optical signal (e.g., color) of the second droplet 206 in the second transport channel 208 in real time. When the optical device detects that the second droplet 206 is in the condition (b), the second droplet 206 flows along the second transporting channel 208 directly into the third branch 2033 of the sorting channel 203 and then into the third sub-collecting portion 2043 of the second collecting portion 204 without triggering the circuitry to apply a voltage to the electrode structure. When the optical device detects that the second droplet 206 is in the condition (a), the optical device immediately triggers the circuitry to apply a voltage (e.g., 800-1000V) to the electrode structure at the sorting channel 203, so that the second droplet 206 containing a single target cell is polarized, and under the action of the electric field, the second droplet 206 containing a single target cell is deflected upward into the first branch 2031 of the sorting channel 203 or deflected downward into the second branch 2032 of the sorting channel 203, and then flows into the first sub-collection portion 2041 or the second sub-collection portion 2042 of the second collection portion 204, respectively. In this way, the separation of the target droplet from the second droplet 206 may be achieved.

The method of sorting the object droplets is implemented based on the structures of the first chip 100 and the second chip 200 described in the foregoing embodiments, and thus, the method may have substantially the same technical effects as the first chip 100 and the second chip 200 described in the foregoing embodiments. For the sake of brevity, the technical effects of the method of sorting target droplets are not repeated here.

In the description of the present disclosure, the terms "upper", "lower", "left", "right", and the like indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of describing the present disclosure but do not require that the present disclosure must be constructed and operated in a specific orientation, and thus, cannot be construed as limiting the present disclosure.

In the description herein, references to the description of "one embodiment," "another embodiment," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction. In addition, it should be noted that the terms "first" and "second" in this specification are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or to imply that the number of indicated technical features is high.

As one of ordinary skill in the art will appreciate, although the various steps of the methods of the present disclosure are depicted in the drawings in a particular order, this does not require or imply that the steps must be performed in this particular order, unless the context clearly dictates otherwise. Additionally or alternatively, multiple steps may be combined into one step execution and/or one step may be broken down into multiple step executions. In addition, other method steps may be inserted between the steps. The intervening steps may represent modifications to the methods, such as those described herein, or may be unrelated to the methods. Furthermore, a given step may not have been completely completed before the next step begins.

The above description is only a specific embodiment of the present disclosure, but the scope of the present disclosure is not limited thereto. Any person skilled in the art can easily think of changes or substitutions in the technical scope of the disclosure, and all shall cover the protection scope of the disclosure. Therefore, the protection scope of the present disclosure shall be subject to the protection scope of the claims.

Claims

A chip, comprising:

a first containing portion configured to contain a first fluid;

a second receptacle configured to receive a second fluid, the second fluid comprising a cell suspension;

a delivery flow channel comprising a first flow channel and a second flow channel, the first flow channel in communication with the first receptacle and the second flow channel in communication with the second receptacle, the first flow channel and the second flow channel intersecting and communicating with each other at a junction, the delivery flow channel configured to cause the first fluid and the second fluid to join at the junction and generate at least one droplet, each of at least a portion of the at least one droplet comprising a single cell derived from the cell suspension; and

at least one collecting portion configured to collect the at least one droplet,

wherein a portion of the first flow passage includes the junction and is divided by the junction into a first section and a second section, in each of the first section and the second section, an area of a first cross section of the section gradually increases along a first direction away from the junction, the first cross section is perpendicular to the first direction, and

wherein the second flow path includes the junction and is divided by the junction into a third section and a fourth section, and in each 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 junction, the second cross section being perpendicular to the second direction.
The chip of claim 1, wherein a portion of the first flow channel comprises a first sub-portion belonging to the first section, a second sub-portion comprising the junction, and a third sub-portion belonging to the second section, the second sub-portion spanning the first and second sections and located between the first and third sub-portions, the first cross-sectional area of each of the first and third sub-portions being greater than the first cross-sectional area of the second sub-portion.
The chip of claim 2, wherein said first cross-section of said second sub-portion of said first flow channel at said confluence is sized to allow a first fluid having a specific particle size to flow therein, said first fluid having a specific particle size larger than the particle size of said single cell.
The chip of claim 2, wherein the chip is a chip,

wherein the second flow channel comprises a first portion, a second portion and a third portion, the first portion and the second portion belonging to the third section, the third portion belonging to the fourth section,

wherein a first end of the first portion of the second flow passage communicates with the second receiving portion, a second end of the first portion of the second flow passage communicates with a first end of the second portion of the second flow passage, a second end of the second portion of the second flow passage communicates with a first end of the third portion of the second flow passage, and both the second end of the second portion of the second flow passage and the first end of the third portion of the second flow passage are located at the junction, the second end of the third portion of the second flow passage communicates with the at least one collecting portion, and

wherein the area of the second cross-section of both the first and third portions of the second flow passage is greater than the area of the second cross-section of the second portion of the second flow passage.
The chip of claim 4, wherein the second cross-section of the second portion of the second flow channel is sized to allow a second fluid having a specific particle size to flow inside thereof, the specific particle size of the second fluid being greater than 1-fold particle size of the single cell and less than 2-fold particle size of the single cell.
The chip of claim 4 or 5, wherein the area of the second cross-section of the third portion of the second flow channel gradually increases in a direction from the first end to the second end of the third portion of the second flow channel.
The chip of any one of claims 4-6, wherein the area of the first cross-section of the second sub-portion of the first flow channel at the junction is greater than or equal to the area of the second cross-section of the second and third portions of the second flow channel at the junction.
The chip of any one of claims 1-7, wherein the second pocket comprises at least one sub-pocket.
The chip of claim 8, wherein the chip is a chip,

wherein the second fluid comprises a first reagent and a second reagent, the first reagent comprising the cell suspension; and is

Wherein the second accommodating part includes a first sub-accommodating part and a second sub-accommodating part separated from each other, the first sub-accommodating part is configured to accommodate the first reagent, and the second sub-accommodating part is configured to accommodate the second reagent.
The chip of claim 9, wherein the chip is a chip,

wherein the first portion of the second flow passage includes a first branch communicating with the first sub housing portion and a second branch communicating with the second sub housing portion, and the first branch and the second branch intersect and communicate with each other at a first point, and

wherein the first branch and the second branch form an acute angle at the first point.
The chip of any one of claims 1-10, wherein the at least one collection portion comprises a first collection portion configured to collect the at least one droplet via the delivery flow channel.
The chip of any one of claims 1-10, wherein the at least one collection portion comprises a second collection portion comprising at least two sub-collection portions configured to collect the at least one droplet via the transport flow channel.
The chip of any one of claims 1-10,

wherein the at least one collecting part includes a first collecting part and a second collecting part including at least two sub-collecting parts, and,

wherein the first collection portion is in communication with the second collection portion, and the first collection portion is located between the junction and the second collection portion.
The chip of claim 12 or 13, further comprising an electrode structure, wherein the electrode structure is located between the junction and the second collection portion.
The chip of claim 12 or 13, wherein the delivery flow channel further comprises a sorting flow channel located between the junction and the second collection portion,

wherein the sorting flow channel comprises at least two branches, one branch of the at least two branches being configured to sort non-target droplets from the at least one droplet, the remaining branches of the at least two branches being configured to sort target droplets from the at least one droplet; and is

Wherein the at least two sub-collecting portions of the second collecting portion correspond to the at least two branches of the sorting channel one-to-one, one of the at least two sub-collecting portions is communicated with one of the at least two branches of the sorting channel and is configured to collect the non-target droplets, and the remaining sub-collecting portions of the at least two sub-collecting portions are respectively communicated with the remaining branches of the at least two branches of the sorting channel and are configured to collect the target droplets.
The chip of claim 15, wherein said chip is,

wherein the at least two branches of the sorting flow channel comprise a first branch and a second branch configured to sort the target droplet from the at least one droplet and a third branch configured to sort the non-target droplet from the at least one droplet, and

wherein the first branch, the second branch, and the third branch intersect at a second point and the third branch is located between the first branch and the second branch, a first included angle of the first branch and the third branch at the second point and a second included angle of the second branch and the third branch at the second point are both greater than 10 °.
The chip of claim 16, wherein said chip is,

wherein a first right-angled triangle is defined by a space between the first branch and the third branch of the sorting flow channel, a second right-angled triangle is defined by a space between the second branch and the third branch of the sorting flow channel, the first included angle faces a first right-angled side of the first right-angled triangle, the second included angle faces a second right-angled side of the second right-angled triangle, and

wherein the lengths of the first right-angle side of the first right-angle triangle and the second right-angle side of the second right-angle triangle are both larger than or equal to the particle size of the single liquid drop.
The chip of any one of claims 1 to 17, wherein the inner wall surface of the transport flow channel has hydrophobicity.
The chip of any one of claims 1-18, wherein the contours of the first and second receptacles comprise four chamfers.
The chip of claim 19, wherein the shape of the chamfer comprises a circular arc.
The chip of any one of claims 1-20, wherein the first and second receptacles are each provided with a filter structure comprising a plurality of microstructures, a gap between two adjacent of the plurality of microstructures being greater than 1 particle size and less than 2 particle sizes of the single cell.
The chip of any one of claims 1-21, wherein the chip is a microfluidic chip.
A microfluidic device comprising a chip according to any one of claims 1-22.
A method of sorting target droplets, comprising the steps of:

providing a first fluid and a second fluid comprising a cell suspension to the first and second receptacles of the chip of any one of claims 1-22, respectively, bringing the first and second fluids together at the confluence of the delivery flow channel and generating at least one droplet, each of at least a portion of the at least one droplet comprising a single cell derived from the cell suspension; and

applying a voltage to the chip according to any one of claims 1-22 to sort out a target droplet having a target property from the at least one droplet, the target droplet comprising the single cell.
The method of claim 24, wherein the chip further comprises an electrode structure located between the junction and the at least one collection portion,

wherein the step of applying a voltage to the chip according to any one of claims 1-22 to sort out a target droplet having a target property from the at least one droplet comprises: detecting an optical signal of the at least one droplet in real time using an optical device, and applying a transient voltage of 800-1000V to the electrode structure in response to the optical device detecting a droplet having a target optical signal, the target droplet including the single cell, to sort out the target droplet having the target optical signal from the at least one droplet.
The method of claim 24 or 25, further comprising, prior to the step of applying a voltage to the chip of any of claims 1-22:

transferring the at least one droplet in the chip to another reaction vessel for polymerase chain reaction or fluorescent staining process.
The method of any one of claims 24-26, wherein the first fluid is an oil phase, the second fluid is an aqueous phase, and the droplets have a water-in-oil structure.