Micro-fluidic chip and use method, and cell sorting apparatus

Abstract

Disclosed in the present application are a micro-fluidic chip and a use method, and a cell sorting apparatus. The micro-fluidic chip comprises: a sample pool, which is provided with a sample injection channel at one end and is provided with an optical-tweezer sorting channel at the other end; a sheath liquid channel, which is provided with a sheath liquid inlet at one end and is in communication with a sheath liquid sorting channel at the other end, the sheath liquid channel intersecting the end of the optical-tweezer sorting channel; and a sample injection switch element, which is configured to cut off or open the sample injection channel. In a cell sorting state, the sample injection switch element cuts off the sample switch channel to keep samples in the sample pool in a stationary state, a target sample in the sample pool is observed and determined by means of an imaging device, and the target sample is dragged to the end of the optical-tweezer sorting channel by means of optical tweezers, so that the target sample is delivered to the sheath liquid sorting channel by means of sheath liquid in the sheath liquid channel; and in a sample injection state, the sample injection switch element opens the sample injection channel, and samples are fed into the sample pool through the sample injection channel.

Description

Microfluidic chip and usage method, cell sorting equipment

[0001] This application claims priority to a Chinese patent application filed with the Patent Office of China on June 27, 2024, with application number 202410843368.8 and application name “A continuous sampling microfluidic chip and method of use, cell sorting device”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application belongs to the field of cell sorting technology, and specifically relates to a microfluidic chip and a method of use, and a cell sorting device. Background Art

[0003] As the most abundant species on Earth, microorganisms play a vital role in the ecosystem and are an indispensable part of biomass synthesis, degradation, and circulation. At the same time, microorganisms are also closely related to human health. The number of microorganisms in the human body is equivalent to ten times that of the human body's own cells. However, to date, more than 90% of microorganisms cannot be cultured under laboratory conditions. The phenotypic identification, sorting, and genotypic analysis of single living cells (i.e., "single-cell technology") can avoid the lengthy incubation process of microorganisms and analyze the "deepest" heterogeneity and operating mechanisms of living systems. Single-cell technology for microorganisms has always faced this technical challenge: how to non-destructively and accurately isolate single microbial cells.

[0004] Currently, the main technologies capable of isolating single-cell microorganisms include fluorescence flow cytometry (FACS) and micromanipulation (Eppendorf). However, each has its own technical limitations. For example, FACS requires fluorescent labeling of cells, which often leads to labeling difficulties or degradation of cell viability. Furthermore, FACS for microbial isolation is expensive. Micromanipulation techniques are complex in single operations (requiring precise control of the capillary tip position, including needle insertion and withdrawal) and have low throughput.

[0005] Research groups both domestically and internationally have reported methods for isolating single microbial cells using microfluidics or laser ejection strategies. For example, in F. Teng et al.'s "Nondestructive Identification and Accurate Isolation of Single Cells through a Chip with Raman Optical Tweezers," published in Anal. Chem., optical tweezers were used to drag cells from a cell pool to a sorting pool, where they were then sorted using a pipette. However, the dragging distance required for single cells is several millimeters, requiring a long manipulation time. Furthermore, the wide separation channel structure employed prevents imaging of the entire channel within the field of view, making it easy to overlook non-target cells.

[0006] Y. Wang, et al., Raman Activated Cell Ejection for Isolation of Single Cells, Anal. Chem., proposes a pulsed laser ejection method. However, the cells must be dried naturally on the ejection substrate before ejection. Furthermore, the pulsed laser generates intense photothermal activity on the dried substrate, which can severely affect the physiological activity of the isolated cells.

[0007] 202011027416.4 discloses a microfluidic chip for particle screening and separation, and discloses an oil-droplet cell sorting chip. Due to the characteristics of its structure, it is impossible to achieve secondary sampling, and the chip cannot be reused, which makes the cost higher. It is also impossible to achieve effective sorting for low-abundance samples. Summary of the Invention

[0008] In response to at least one shortcoming of the prior art, the present application provides a microfluidic chip and a method of use, and a cell sorting device.

[0009] The first aspect of the present application provides a microfluidic chip, comprising:

[0010] A sample pool, one end of which is provided with a sample injection channel and the other end is provided with an optical tweezers sorting channel;

[0011] a sheath liquid channel, one end of which is a sheath liquid inlet and the other end of which is connected to a sheath liquid sorting channel, and the sheath liquid channel intersects with the end of the optical tweezers sorting channel;

[0012] An injection switch element, used to cut off or open the injection channel;

[0013] In the cell sorting state, the injection switch element cuts off the injection channel to keep the sample in the sample pool in a static state, observes and identifies the target sample in the sample pool through an imaging device, and uses optical tweezers to drag the target sample to the end of the optical tweezers sorting channel, so that the target sample is transported to the sheath fluid sorting channel through the sheath fluid in the sheath fluid channel;

[0014] In the injection state, the injection switch element switches on the injection channel, and the sample is injected into the sample pool through the injection channel.

[0015] In some embodiments, the microfluidic chip further comprises: a guide assembly for intercepting a liquid flow formed by the sheath fluid containing the target sample to form droplets containing the target sample.

[0016] In some embodiments, the outlet component is an air flow channel; the air flow channel intersects with the sheath liquid sorting channel, and by inputting gas into the air flow channel, the air flow shears the liquid flow to form droplets containing target samples.

[0017] In some embodiments, the outlet component is a piezoelectric element; the piezoelectric element is arranged at the sheath liquid sorting channel, and a rapidly changing voltage signal is applied to the piezoelectric element to deform the piezoelectric element, thereby cutting off the liquid flow through vibration to form droplets containing the target sample.

[0018] In some embodiments, the injection channel is a truncated structure to form a first guide port and a second guide port; the injection switch element includes a conduction cavity, which is connected to the first guide port and the second guide port, and the outer end of the conduction cavity is an elastic layer; the first guide port and the second guide port are truncated and connected by squeezing or loosening the elastic layer.

[0019] In some embodiments, the injection channel, the optical tweezers sorting channel, and the sheath fluid sorting channel are located on the symmetry axis of the sample cell.

[0020] In some embodiments, two sheath fluid channels are provided and are symmetrically distributed on both sides of the sheath fluid sorting channel to form a Y-shaped structure.

[0021] In some embodiments, the gas flow channel includes a first gas flow channel and a second gas channel symmetrically arranged on both sides of the sheath liquid sorting channel; the end portions of the first gas flow channel and the second gas channel are arranged in a collinear structure with the outlet of the sheath liquid sorting channel.

[0022] A second aspect of the present application provides a method for using a microfluidic chip, which is applied to the microfluidic chip according to any one of the first aspects. The method for using the microfluidic chip comprises:

[0023] The cell sorting method includes opening an injection channel and injecting a sample into a sample pool. After the sample is injected, the injection channel is cut off and cell sorting is performed in the sample pool. The target sample to be captured is dragged to the intersection of the optical tweezers sorting channel and the sheath fluid channel by optical tweezers, and the target sample is transferred to the sheath fluid sorting channel using the sheath fluid in the sheath fluid channel.

[0024] The injection method is to open the injection channel to inject the sample into the sample pool. After the sample injection is completed, the injection channel is cut off and sheath liquid is continuously injected into the sheath liquid channel to flush the sheath liquid sorting channel. After the flushing is completed, the cells in the sample pool are sorted.

[0025] In some embodiments, the microfluidic chip further comprises a guide assembly configured to intercept a liquid flow formed by the sheath fluid containing the target sample to form droplets containing the target sample;

[0026] The method of use also includes a target sample derivation method: calculating the time when the liquid flow containing the target sample reaches the position of the derivation component, and then sending a signal to the derivation component to cut off the liquid flow containing the target sample to form droplets containing the target sample.

[0027] A third aspect of the present application provides a cell sorting device, comprising the microfluidic chip as described in the first aspect, and further comprising:

[0028] A microfluidic chip placement platform is used to place the microfluidic chip and move it in the horizontal direction;

[0029] Imaging module, used for real-time imaging and identification analysis of samples in the sample pool and sheath fluid sorting channel;

[0030] The automatic optical tweezers capture module captures and fixes the identified target sample through optical tweezers; by adjusting the horizontal position of the microfluidic chip placement platform, the target sample is dragged to the intersection of the sheath fluid channel and the end of the optical tweezers sorting channel;

[0031] a sheath liquid channel pumping device, used for pumping sheath liquid into the sheath liquid channel, so that the target sample is transported to the sheath liquid sorting channel along with the flow of the sheath liquid;

[0032] The export component control unit calculates the time when the liquid flow containing the target sample reaches the position of the export component, and then sends a signal to the export component to intercept the liquid flow containing the target sample and form droplets containing the target sample;

[0033] A collection well plate placement platform is used to place the collection well plate and receive droplets containing target samples at the outlet of the sheath liquid sorting channel through displacement;

[0034] The injection control module is used to control the injection switch element to realize the cutoff or conduction of the injection channel to perform cell sorting or injection.

[0035] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0036] This application suspends cells in an aqueous phase (sheath fluid) and utilizes low-damage near-infrared optical tweezers technology to ensure the activity of sorted cells, and can perform single-cell culture, sequencing and other analyses. With the help of the high resolution of optical tweezers, single cells and particles of 0.5 microns and above can be accurately separated. At the same time, due to the adoption of the structural form of aqueous phase sorting, the functions of secondary injection and stable static sorting can be achieved by controlling the injection switch element, which solves the problem that the current single-cell sorting cannot be secondary injected and is susceptible to laminar flow during the sorting process, and the structural mode of secondary injection can greatly solve the sorting problem of low-abundance samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The drawings described herein are used to provide further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute improper limitations on the present application.

[0038] FIG1 is a schematic structural diagram of a microfluidic chip according to an embodiment of the present application;

[0039] FIG2 is a partial enlarged view of the sample pool portion of the microfluidic chip according to an embodiment of the present application;

[0040] FIG3 is a partial enlarged view of the cutoff portion of the injection channel in the microfluidic chip according to an embodiment of the present application;

[0041] FIG4 a is a cross-sectional view of the sample injection switch element in the embodiment of the present application in a conducting state;

[0042] FIG4 b is a cross-sectional view of the sample injection switch element in the embodiment of the present application in a cut-off state;

[0043] FIG5 is an exploded view of a microfluidic chip in one embodiment of the present application;

[0044] FIG6 is a top view of each layer of the microfluidic chip in the embodiment shown in FIG5 ;

[0045] FIG7 is a partial enlarged view of portion A in FIG6 ;

[0046] FIG8 is a partial enlarged view of portion B in FIG6 ;

[0047] FIG9 is an exploded view of a microfluidic chip in another embodiment of the present application;

[0048] FIG10 is a top view of the second layer and the fourth layer in the embodiment shown in FIG9 ;

[0049] FIG11 is a top view of the fourth layer provided with an air flow channel in one embodiment of the present application.

[0050] FIG12 is a schematic diagram of the CFD simulation analysis of the sample pool of the present application.

[0051] FIG13 is a schematic structural diagram of a cell sorting device in an embodiment of the present application.

[0052] 1. Sample pool; 2. Injection channel; 21. First injection section; 211. First guide port; 22. Second injection section; 221. Second guide port; 23. Partitioning part; 24. Sample inlet; 3. Optical tweezers sorting channel; 4. Sheath liquid channel; 41. Sheath liquid inlet; 5. Sheath liquid sorting channel; 51. Sheath liquid sorting channel outlet; 6. Injection switch element; 61. Valve body; 611. First valve port; 612. Second valve port; 62. Conducting cavity; 621. First conducting flow channel; 622. Second conducting flow channel; 623. Third conducting flow channel; 631. First on-off control terminal; 632. Second on-off control terminal; 64. Elastic layer; 7. Derivative assembly; 71. Cut-off medium channel; 711. Cut-off medium channel inlet; 712. Cut-off medium channel outlet; 713. First Cut-off medium channel; 714, second cut-off medium channel; 715, air flow channel; 716, first gas channel; 717, second gas channel; 811, first through hole; 812, second through hole; 813, third through hole; 821, first connecting hole; 822, second connecting hole; 91, first receiving hole; 92, second receiving hole; 100, microfluidic chip; 101, first layer; 102, second layer; 103, third layer; 104, fourth layer; 105, fifth layer; 200, cell sorting equipment; 201, microfluidic chip placement platform; 202, imaging module; 203, automatic optical tweezers capture module; 204, sheath liquid channel pumping device; 205, export component control unit; 206, collection well plate placement platform; 207, injection control component module. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical solutions and advantages of this application more clearly understood, the present application is described and illustrated below in conjunction with the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely used to explain this application and are not intended to limit this application. Based on the embodiments provided in this application, all other embodiments obtained by those of ordinary skill in the art without making any creative efforts are within the scope of protection of this application.

[0054] Obviously, the drawings described below are merely examples or embodiments of the present application. Those skilled in the art can, without inventive effort, apply the present application to other similar scenarios based on these drawings. Furthermore, it is also understood that, although the effort involved in such a development process may be complex and lengthy, for those skilled in the art related to the content disclosed in this application, changes in design, manufacturing, or production based on the technical content disclosed in this application are merely conventional technical means and should not be construed as an insufficiency of the content disclosed in this application.

[0055] References to "embodiments" in this application mean that a particular feature, structure, or characteristic described in connection with the embodiment may be included in at least one embodiment of the application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it refer to independent or alternative embodiments that are mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described in this application may be combined with other embodiments unless there is a conflict.

[0056] As shown in FIG1 and FIG2 , an embodiment of the present application provides a microfluidic chip 100, comprising:

[0057] The sample pool 1 has a sample injection channel 2 at one end and an optical tweezers sorting channel 3 at the other end; the end of the sample injection channel 2 away from the sample pool 1 is a sample inlet 24;

[0058] A sheath liquid channel 4, one end of which is a sheath liquid inlet 41, and the other end of which is connected to a sheath liquid sorting channel 5, and the sheath liquid channel 4 intersects with the end of the optical tweezers sorting channel 3, so that the starting end of the sheath liquid sorting channel 5 intersects with the end of the optical tweezers sorting channel 3;

[0059] The sampling switch element 6 is used to cut off or conduct the sampling channel 2; among them, the sampling switch element 6 has various structural forms that can ensure that the sampling channel 2 is cut off or conducted, such as through channel deformation caused by external force, through air pressure, mechanical force, magnetism, temperature-sensitive materials or biological materials, etc. to achieve on-off; or use the vertical movement of the valve or the rotation of the intermediate on-off mechanism to control the opening and closing of the channel; or the pipeline connected to the sampling channel 2 is controlled by external force, such as the rapid start and stop of the injection pump, or the closing of the hose, to achieve on-off, etc.

[0060] In the cell sorting state, the injection switch element 6 cuts off the injection channel 2 to keep the sample in the sample pool 1 in a static state. The target sample in the sample pool is observed and identified by the imaging device, and the target sample is dragged to the end of the optical tweezers sorting channel 3 by the optical tweezers, and then the sheath fluid in the sheath fluid channel 4 is used to transport the target sample to the sheath fluid sorting channel 5.

[0061] In the injection state, the injection switch element 6 turns on the injection channel 2 , and the sample is injected into the sample cell 1 through the injection channel 2 .

[0062] The microfluidic chip 100 provided in the above embodiment controls the on-off of the injection channel 2 through the injection switch element 6; when performing a cell sorting operation, the injection switch element 6 cuts off the injection channel 2 to ensure a static flow field in the sample pool 1, so that the sample cells remain in a static state, which is conducive to the smooth progress of the sorting operation; after the current sorting is completed, the injection switch element 6 is controlled to turn on the injection channel 2, and at the same time, the injection pump, peristaltic pump and other devices connected to the sample inlet 24 are turned on, and the unsorted sample is again input into the sample pool 1 through the injection channel 2 to perform the next sorting operation.

[0063] Through the above process, the cell sample in the sample pool 1 can be replaced and rinsed, realizing the secondary sampling function. With the help of the same microfluidic chip, multiple sorting operations of the cell sample can be performed, which increases the probability of capturing target cells and reduces the cost of cell sorting.

[0064] In the description of this application, the “starting end” and “ending end” of a channel are defined according to the flow direction of the fluid in the channel.

[0065] In some embodiments, as shown in Figures 3 and 4 , the injection switch element 6 utilizes an elastic valve. The injection channel 2 has a partitioned, truncation-type structure, with a first guide port 211 and a second guide port 221 formed on either side of the partition. The injection switch element 6 includes a conducting cavity 62, which communicates with the first and second guide ports 211, 221. The outer end of the conducting cavity 62 is formed by an elastic layer 64. The first and second guide ports 211, 221 are separated and connected by squeezing or releasing the elastic layer 64.

[0066] In some embodiments, as shown in Figures 3, 4, and 5-8, the injection channel 2 includes a first injection section 21 and a second injection section 22 extending along the same axis, and the first injection section 21 and the second injection section 22 are separated by a partition 23; the first injection section 21 forms a first guide port 211 at one end close to the second injection section 22, and the second injection section 22 forms a second guide port 221 at one end close to the first injection section 21; the injection switch element 6 adopts an elastic valve, including a valve body 61, which is made of an elastic material and defines a guide port 221 therein. The valve body 61 is provided with a first valve port 611 and a second valve port 612, which are communicated through the conduction cavity 62, and the first valve port 611 and the second valve port 612 are respectively connected to the first guide port 211 and the second guide port 221, so that the first injection section 21 and the second injection section 22 are communicated through the conduction cavity 62; the valve body 61 is configured to: elastically deform under the action of external force and make the inner walls of the conduction cavity 62 fit together, so as to block the communication between the first injection section 21 and the second injection section 22.

[0067] In the solution provided in the above embodiment, the partition design of the injection channel 2 is combined with the elastic valve body 61 to realize the cutoff or conduction of the injection channel 2. The injection switch element 6 has a simple structure and is easy to operate. By squeezing or releasing the valve body 61, the cutoff or conduction of the injection channel 2 can be controlled, and high-precision start and stop of the fluid transportation in the channel can be achieved. When performing cell sorting operations, the injection channel 2 is controlled to be closed by squeezing the valve body 61; when injection is required, the external force is removed, the valve body 61 returns to its original shape, the injection channel 2 is opened, and at the same time, the injection pump, peristaltic pump and other devices connected to the sample inlet 24 are turned on to inject the cell sample into the chip. In some embodiments, as shown in FIG4 , the conducting cavity 62 includes a first conducting channel 621 connected to the first valve port 611 and a second conducting channel 622 connected to the second valve port 612. The ends of the first and second conducting channels 621 and 622 located within the valve body 61 extend in the same direction and respectively form a first on-off control end 631 and a second on-off control end 632. The first on-off control end 631 and the second on-off control end 632 are connected via a third conducting channel 623. By squeezing the valve body 61, the inner wall of the third conducting channel 623 is deformed and the first on-off control end 631 and / or the second on-off control end 632 are blocked, thereby blocking the connection between the first and second valve ports 611 and 612, thereby shutting off the injection channel 2.

[0068] In the above embodiment, the structure of the conducting cavity 62 inside the valve body 61 and the direction of the flow channel are defined, the on-off control position of the valve body 61 is clarified, and then the external force extrusion position is clarified, thereby ensuring the reliability of the on-off control of the injection switch element on the injection channel 2.

[0069] In some embodiments, the microfluidic chip 100 further includes: a guide assembly 7 for intercepting a liquid flow formed by the sheath fluid containing the target sample to form droplets containing the target sample.

[0070] In some embodiments, as shown in Figures 9 and 10, the outlet component 7 includes a cutoff medium channel 71, and the outlet 712 of the cutoff medium channel 71 intersects with the sheath liquid sorting channel outlet 51. The cutoff medium channel 71 is configured to transport the cutoff medium to the sheath liquid sorting channel outlet 51, and the cutoff medium is used to shear the liquid flow containing the target sample to form droplets containing the target sample.

[0071] In the solution provided in the above embodiment, by delivering the truncation medium to the outlet 51 of the sheath fluid sorting channel, the export of small-volume single-cell samples is achieved through the generation of air-in-water or oil-in-water droplets. The volume of the exported droplets can be less than 0.5 μL, which is about 10 times smaller than the volume of the droplets exported before the truncation process (about 5 μL). The droplet volume is reduced from the microliter level to the nanoliter level, which is more conducive to the subsequent genome amplification reaction.

[0072] The cut-off medium is an oil-phase fluid or gas. Optionally, the gas used as the cut-off medium can be a relatively inert gas such as air, nitrogen or carbon dioxide; the oil-phase liquid used as the cut-off medium can be mineral oil, fluorocarbon oil or dimethyl silicone oil.

[0073] In some embodiments, as shown in FIG10 , the cutting medium channel 71 includes a first cutting medium channel 713 and a second cutting medium channel 714 symmetrically disposed on either side of the sheath liquid sorting channel 5. The distal ends of the first cutting medium channel 713 and the distal ends of the second cutting medium channel 714 are aligned and perpendicular to the sheath liquid sorting channel 5. The first cutting medium channel 713 and the second cutting medium channel 714 simultaneously deliver cutting medium to the sheath liquid sorting channel outlet 51 from both sides, allowing the cutting medium to squeeze the liquid flow from both sides simultaneously, forming droplets containing the target sample, thereby ensuring stable droplet interception.

[0074] In some embodiments, as shown in FIG10 , the cut-off medium channel 71 has a curved shape, which can extend the flow channel and increase the flow resistance of the cut-off medium, thereby preventing the cut-off medium (such as oil phase liquid) from entering the upper aqueous phase channel and causing contamination.

[0075] Specifically, the truncation method and approach may be an air jet droplet printing approach or a piezoelectric droplet printing approach.

[0076] Air jet droplet printing method: The rapid rise and fall of air pressure can lead to the formation and ejection of droplets. By controlling the change of air pressure, the size and release speed of the droplets can be adjusted. When the air pressure acts on the liquid in the chip, the liquid will be ejected from the nozzle to form droplets. The size of the droplets can be controlled by adjusting the size and action time of the air pressure.

[0077] In some embodiments, as shown in FIG11 , the outlet component 7 is an air flow channel 715 ; the air flow channel 715 intersects with the sheath liquid sorting channel 5 , and by inputting gas into the air flow channel 715 , the air flow shears the liquid flow to form droplets containing the target sample.

[0078] In some embodiments, the gas flow channel 715 includes a first gas channel 716 and a second gas channel 717 symmetrically arranged on either side of the sheath liquid sorting channel. The distal ends of the first and second gas channels 716, 717 are collinear with the sheath liquid sorting channel outlet 51. This collinear arrangement ensures stable droplet truncation during pneumatic droplet shearing. In this collinear configuration, the distal ends of the first and second gas channels 716, 717 lie on the same straight line. The outlet of the sheath liquid sorting channel 51 is located on this straight line and coincides with the outlet 712 of the truncation medium channel.

[0079] Piezoelectric droplet printing method: By applying a rapidly changing voltage signal to the piezoelectric element, the piezoelectric element will rapidly expand and contract, generating vibrations; the vibration of the piezoelectric element will cause pressure fluctuations in the liquid inside the chip, thereby forming droplets at the nozzle; by precisely controlling the frequency and amplitude of the voltage signal, the size of the droplets and the injection speed can be precisely controlled.

[0080] In some embodiments, the outlet component is a piezoelectric element (not shown in the figure); the piezoelectric element is arranged in the sheath liquid sorting channel 5, and a rapidly changing voltage signal is applied to the piezoelectric element to deform the piezoelectric element, thereby cutting off the liquid flow through vibration to form droplets containing the target sample.

[0081] In some embodiments, as shown in FIG2 , the sample cell 1 is shaped like an axisymmetric figure, such as an axisymmetric hexagon. The injection channel 2 and the optical tweezers sorting channel 3 are respectively located at two end points on the axis of symmetry of the sample cell 1 .

[0082] In some embodiments, the injection channel 2 , the optical tweezers sorting channel 3 , and the sheath liquid sorting channel 5 are located on the symmetry axis of the sample cell 2 .

[0083] In some embodiments, two sheath liquid channels 4 are provided and symmetrically distributed on both sides of the sheath liquid sorting channel 5 .

[0084] In some embodiments, the angle between the two sheath liquid channels 4 and the axis of symmetry of the sample cell 1 is less than 90°, thereby forming a Y-shaped structure with the sheath liquid separation channel 5. This angle design allows the sheath liquid transported by the sheath liquid channels 4 to flow toward the sheath liquid separation channel 5 without flowing back into the sample cell, thereby reducing interference with the flow field within the sample cell 1 and improving the stability of the flow field within the sample cell 1.

[0085] In some embodiments, the microfluidic chip 100 is provided with a sheath liquid inlet 41 , and the two sheath liquid channels 4 intersect at the sheath liquid inlet 41 and extend from both sides of the symmetry axis of the sample cell 1 to the end of the optical tweezers sorting channel 3 .

[0086] A microfluidic chip for particle screening and separation, disclosed in 202011027416.4, requires a T-shaped channel width of approximately 30 microns to function as a static flow field cell storage device. Because the double-sided tape hollowing process cannot achieve the micron-level precision of traditional PDMS chip soft lithography, the resulting channel width is at least 100 microns. Therefore, it can only be produced using soft lithography, which has high process requirements and high costs.

[0087] By optimizing the sheath fluid channel structure and chip assembly, this application achieves consistent flow field stability even with a sheath fluid channel width of 200 microns. This allows for large-scale production of the product using existing double-sided adhesive tape processes for microfluidic chips, reducing the difficulty and cost of chip fabrication. Furthermore, the sample reservoir 1 has only one outlet connected to the optical tweezers sorting channel 3; the sample channel 2 at the other end is shut off by the sample switch element 6, further ensuring flow channel stability during the sorting process.

[0088] Computational Fluid Dynamics (CFD) fluid simulation was used to analyze flow field stability. In Figure 12, darker colors represent slower flow rates. The simulation results show that the flow rate within sample pool 1 is approximately zero. This indicates that the high flow rate of sheath fluid channel 4 does not disturb sample pool 1. Consequently, the flow rate within sample pool 1 is unaffected by external channels. During the sorting process, the cells in sample pool 1 remain stationary, facilitating a smoother sorting process.

[0089] In the embodiment shown in Figures 5 and 6, the microfluidic chip 100 includes a first layer 101, a second layer 102 and a third layer 103 stacked in sequence from top to bottom, wherein the sample pool 1, the injection channel 2, the optical tweezers sorting channel 3, the sheath liquid channel 4 and the sheath liquid sorting channel 5 are hollowed out in the second layer 102.

[0090] Optionally, the first layer 101 and the third layer 103 are made of quartz or glass, and the second layer 102 is made of pressure sensitive adhesive (PSA) and is bonded to the first layer 101 and the third layer 103 .

[0091] First through holes 811 are provided in the third layer 103 corresponding to the sheath liquid inlet 41 , the sample inlet 24 , and the sheath liquid sorting channel outlet 51 , thereby enabling input and output of corresponding fluids in each channel in the second layer 102 .

[0092] In some embodiments, as shown in Figures 6-8, the third layer 103 is provided with a first connecting hole 821 and a second connecting hole 822 corresponding to the first guide port 211 and the second guide port 221 in the second layer 102, respectively. The first guide port 211 and the second guide port 221 are connected to the first valve port 611 and the second valve port 612 of the injection switch element 6 through the first connecting hole 821 and the second connecting hole 822, respectively.

[0093] In the embodiment shown in Figures 9 and 10, the microfluidic chip 100 further includes a fourth layer 104 and a fifth layer 105 stacked sequentially below the third layer 103, with the cutoff medium channel 71 hollowed out in the fourth layer 104. The third layer 103 and the fifth layer 105 cover the upper and lower sides of the fourth layer 104, thereby sealing the cutoff medium channel 71.

[0094] The fifth layer 105 is provided with second through holes 812 corresponding to the inlet 711 and outlet 712 (i.e., the sheath liquid sorting channel outlet 51) of the cut-off medium channel 71, respectively, for transporting the cut-off medium to the sheath liquid sorting channel outlet 51 and for the cut-off droplets to be discharged; the fourth layer 104 and the fifth layer 105 are also provided with third through holes 813 corresponding to the sheath liquid inlet 41 and the sample inlet 24, respectively, for inputting the sheath liquid and the cell sample into the corresponding channels of the second layer 102 from the bottom through the fifth layer 105 and the fourth layer 104 in sequence.

[0095] In some embodiments, the through holes of the third layer 103 and / or the fifth layer 105 corresponding to the outlet 51 of the sheath liquid sorting channel are subjected to superhydrophobic treatment, such as femtosecond laser etching and superhydrophobic coating treatment, which is conducive to the formation of small-sized droplets, thereby achieving contactless single-cell separation.

[0096] In addition, the fourth layer 104 is provided with a first accommodating hole 91, and the fifth layer 105 is provided with a second accommodating hole 92. The first accommodating hole 91 and the second accommodating hole 92 are correspondingly arranged to form a accommodating portion in the microfluidic chip 100. The size of the accommodating portion is adapted to the size of the sampling switch element 6, so that the sampling switch element 6 is accommodated in the accommodating portion.

[0097] The sample injection switch element 6 is made of polydimethylsiloxane (PDMS for short).

[0098] In the above embodiment, the microfluidic chip 100 is formed by stacking five layers. Each microchannel of the microfluidic chip 100 is formed by bonding upper and lower layers of quartz glass to a pressure-sensitive adhesive with a hollow microstructure. The aqueous buffer (sheath fluid) and cell sample suspension are located in the second layer 102, while the oily or gaseous cutoff medium is located in the fourth layer 104. The injection channel 2 in the second layer 102 is in a cutoff state, controlled by the injection switch element 6 located below.

[0099] The present application also discloses a method for using a microfluidic chip, which is applicable to the microfluidic chip of any of the above embodiments. The method for using the microfluidic chip includes:

[0100] Cell sorting method: The sample injection channel 2 is opened and the sample is injected into the sample pool 1. After the sample injection is completed, the sample injection channel 2 is cut off and sheath liquid is continuously injected into the sheath liquid channel 4. The intersection between the optical tweezers sorting channel 3 and the sheath liquid channel 4 is set to keep the sample in the sample pool 1 in a stable state, and the target sample to be captured is dragged to the intersection of the optical tweezers sorting channel 3 and the sheath liquid channel 4 by the optical tweezers. The target sample is then transferred to the sheath liquid sorting channel 5 by the sheath liquid in the sheath liquid channel 4;

[0101] Injection method: Open the injection channel 2 to inject the sample into the sample pool 1. After the sample injection is completed, cut off the injection channel 2 and continue to inject sheath liquid into the sheath liquid channel 4 to flush the sheath liquid sorting channel 5. After the flushing is completed, prepare to sort the cells in the sample pool 1.

[0102] The method for using the microfluidic chip provided in the above embodiment is to cut off the sample injection channel during cell sorting to ensure a static flow field in the sample pool, keeping the sample cells in a static state and facilitating the smooth progress of the sorting operation. When the cell sample in the sample pool needs to be replaced, the sample injection channel is opened, and the syringe pump, peristaltic pump, or other device connected to the sample inlet is simultaneously turned on to inject a new cell sample into the chip. Through this process, the cell sample in the sample pool can be replaced and flushed, achieving secondary sampling, thereby allowing the microfluidic chip to be reused and reducing the cost of cell sorting.

[0103] In some embodiments, the method of using the microfluidic chip also includes a target sample export method: calculating the time it takes for the liquid flow containing the target sample to reach the position of the export component 7 (specifically the outlet of the export component 7, that is, the outlet 712 of the cut-off medium channel 71), and then sending a signal to the export component 7 to cut off the liquid flow containing the target sample to form droplets containing the target sample.

[0104] The microfluidic chip 100 includes a derivation component 7, which includes a cutoff medium channel 71. The outlet 712 of the cutoff medium channel 71 overlaps with the outlet 51 of the sheath fluid sorting channel. In the target sample derivation method provided in the above embodiment, the time for the target sample-containing liquid flow to reach the outlet 712 of the cutoff medium channel 71 (i.e., the sheath fluid sorting channel outlet 51) is calculated. The delivery and flow rate of the cutoff medium in the derivation component 7 are controlled to ensure that the cutoff medium arrives at the outlet 712 of the cutoff medium channel 71 at the same time as the target sample-containing liquid flow arrives at that location. This cutoff effect of the derivation component 7 allows for the derivation of small single-cell sample droplets. The volume of the derivate droplets is less than 0.5 ml, a tenfold reduction compared to the pre-cutoff droplet volume, reducing the droplet volume from the microliter level to the nanoliter level, which is more conducive to subsequent genome amplification reactions.

[0105] In the method for using the microfluidic chip provided in the embodiments of the present application, one of the cell sorting method, the injection method, and the target sample derivation method can be executed separately, or two or three of them can be executed according to the operation sequence requirements. The order can be changed according to actual needs, and this application does not impose any restrictions.

[0106] The present application also discloses a cell sorting device 200, as shown in FIG13 , comprising the microfluidic chip 100 according to any one of the above embodiments, and further comprising:

[0107] The microfluidic chip placement platform 201 is used to place the microfluidic chip and can be adjusted in the horizontal direction; it can be designed as a two-axis movement platform or a three-axis movement platform according to needs;

[0108] Imaging module 202, for performing real-time bright field / fluorescence imaging of samples in the sample pool and sheath fluid sorting channel; the imaging module includes an imaging optical path formed by various optical elements such as prisms, reflectors, and lenses. Those skilled in the art can implement it based on existing technologies and will not be described in detail in this application;

[0109] The automatic optical tweezers capture module 203 is used to analyze and identify samples in the sample pool and capture and fix the identified target samples using optical tweezers. For example, the absolute position of the laser is fixed, and the horizontal position of the microfluidic chip placement platform is adjusted to drag the target sample to the intersection of the sheath fluid channel and the end of the optical tweezers sorting channel.

[0110] The sheath liquid channel pumping device 204 is used to pump the sheath liquid into the sheath liquid channel, so that the target sample is transported to the sheath liquid sorting channel through the sheath liquid; the sheath liquid channel pumping device includes a pump and a pipeline for pumping the sheath liquid;

[0111] The derivation component control unit 205 calculates the time when the liquid flow containing the target sample reaches the position of the derivation component, and then sends a signal to the derivation component to intercept the liquid flow containing the target sample and form droplets containing the target sample; the derivation component control unit can be implemented with the help of a computer device;

[0112] The collection well plate placement platform 206 is used to place the collection well plate and receive the target sample at the outlet of the sheath liquid sorting channel through displacement;

[0113] The injection control module 207 is used to control the injection switch element to realize the cutoff or conduction of the injection channel; the injection control module can be realized with the help of external mechanical components, or controlled by computer equipment.

[0114] The modules or devices in the above-mentioned cell sorting equipment are not the focus of this application and can be implemented by using existing technologies. Therefore, they will not be described in detail in this application.

[0115] In the embodiment of the cell sorting device shown in FIG13 , the microfluidic chip 100 is placed on a three-dimensional moving platform, and cells are captured and sorted by a micro-Raman system coupled with 1064 nm optical tweezers.

[0116] Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A microfluidic chip, characterized in that: include: A sample pool, one end of which is provided with a sample injection channel and the other end is provided with an optical tweezers sorting channel; a sheath liquid channel, one end of which is a sheath liquid inlet and the other end of which is connected to a sheath liquid sorting channel, and the sheath liquid channel intersects with the end of the optical tweezers sorting channel; An injection switch element, used to cut off or open the injection channel; In the cell sorting state, the injection switch element cuts off the injection channel to keep the sample in the sample pool in a static state, observes and identifies the target sample in the sample pool through an imaging device, and uses optical tweezers to drag the target sample to the end of the optical tweezers sorting channel, so that the target sample is transported to the sheath fluid sorting channel through the sheath fluid in the sheath fluid channel; In the injection state, the injection switch element switches on the injection channel, and the sample is injected into the sample pool through the injection channel.

2. The microfluidic chip according to claim 1, characterized in that Also includes: The outlet component is used to intercept the liquid flow formed by the sheath liquid containing the target sample to form droplets containing the target sample.

3. The microfluidic chip according to claim 2, characterized in that: The derivation component is an air flow channel; the air flow channel intersects with the sheath liquid sorting channel, and by inputting gas into the air flow channel, the air flow shears the liquid flow to form droplets containing target samples.

4. The microfluidic chip according to claim 2, characterized in that The outlet component is a piezoelectric element; the piezoelectric element is arranged at the sheath liquid sorting channel, and a rapidly changing voltage signal is applied to the piezoelectric element to deform the piezoelectric element, thereby cutting off the liquid flow through vibration to form droplets containing the target sample.

5. The microfluidic chip according to claim 1, characterized in that The injection channel is a truncated structure to form a first guide port and a second guide port; the injection switch element includes a conduction cavity, the conduction cavity is connected to the first guide port and the second guide port, and the outer end of the conduction cavity is an elastic layer; The first guide opening and the second guide opening are cut off and connected by squeezing or releasing the elastic layer.

6. The microfluidic chip according to claim 1, characterized in that The injection channel, the optical tweezers sorting channel and the sheath liquid sorting channel are located on the symmetry axis of the sample pool.

7. The microfluidic chip according to claim 1, characterized in that The sheath fluid channels are provided with two channels and are symmetrically distributed on both sides of the sheath fluid sorting channel to form a Y-shaped structure.

8. The microfluidic chip according to claim 3, characterized in that: The gas flow channel includes a first gas flow channel and a second gas channel symmetrically arranged on both sides of the sheath liquid sorting channel; the end portions of the first gas flow channel and the second gas channel are arranged in a collinear structure with the outlet of the sheath liquid sorting channel.

9. A method for using a microfluidic chip, characterized in that: Applied to the microfluidic chip according to any one of claims 1 to 8, the method for using the microfluidic chip comprises: The cell sorting method includes opening an injection channel and injecting a sample into a sample pool. After the sample is injected, the injection channel is cut off and cell sorting is performed in the sample pool. The target sample to be captured is dragged to the intersection of the optical tweezers sorting channel and the sheath fluid channel by optical tweezers, and the target sample is transferred to the sheath fluid sorting channel using the sheath fluid in the sheath fluid channel. The injection method is to open the injection channel to inject the sample into the sample pool. After the sample injection is completed, the injection channel is cut off and sheath liquid is continuously injected into the sheath liquid channel to flush the sheath liquid sorting channel. After the flushing is completed, the cells in the sample pool are sorted.

10. The method for using the microfluidic chip according to claim 9, characterized in that: The microfluidic chip further includes a guide assembly for intercepting a liquid flow formed by the sheath fluid containing the target sample to form droplets containing the target sample; The method of use also includes a target sample derivation method: calculating the time when the liquid flow containing the target sample reaches the position of the derivation component, and then sending a signal to the derivation component to cut off the liquid flow containing the target sample to form droplets containing the target sample.

11. A cell sorting device, characterized in that: The microfluidic chip according to any one of claims 2 to 8, further comprising: A microfluidic chip placement platform is used to place the microfluidic chip and move it in the horizontal direction; Imaging module, used for real-time imaging and identification analysis of samples in the sample pool and sheath fluid sorting channel; The automatic optical tweezers capture module captures and fixes the identified target sample through optical tweezers; by adjusting the horizontal position of the microfluidic chip placement platform, the target sample is dragged to the intersection of the sheath fluid channel and the end of the optical tweezers sorting channel; a sheath liquid channel pumping device, used for pumping sheath liquid into the sheath liquid channel, so that the target sample is transported to the sheath liquid sorting channel along with the flow of the sheath liquid; The export component control unit calculates the time when the liquid flow containing the target sample reaches the position of the export component, and then sends a signal to the export component to intercept the liquid flow containing the target sample and form droplets containing the target sample; A collection well plate placement platform is used to place the collection well plate and receive droplets containing target samples at the outlet of the sheath liquid sorting channel through displacement; The injection control module is used to control the injection switch element to realize the cutoff or conduction of the injection channel to perform cell sorting or injection.

Images