CN218308015U

CN218308015U - Micro-fluidic chip for single cell sorting

Info

Publication number: CN218308015U
Application number: CN202222355681.6U
Authority: CN
Inventors: 陈艳伟; 何炜; 陶建昌; 张传伦
Original assignee: Southwest University of Science and Technology
Current assignee: Southwest University of Science and Technology
Priority date: 2022-09-05
Filing date: 2022-09-05
Publication date: 2023-01-17
Anticipated expiration: 2032-09-05

Abstract

The utility model provides a micro-fluidic chip for single cell sorting, which relates to the technical field of cell sorting and comprises a substrate, wherein the upper surface of the substrate is provided with a micro-flow channel which comprises a capturing channel; the upper surface of the base is also provided with a slot which is communicated with the capturing channel, a baffle is movably inserted in the slot and is used for closing or opening the capturing channel. The utility model discloses a promote the baffle and slide along the slot and can the closing and switching on of effective control miniflow passageway, under the circumstances that the function is not weakened in guaranteeing unicellular separation, greatly reduced the holistic thickness of micro-fluidic chip, make it directly arrange in and observe under the upright microscope, improved suitability and practicality. Moreover, the matched equipment is simplified, and only the upright microscope and the micro-injection pump are involved, so that the operation of the micro-fluidic chip is simpler and more convenient, and the use cost is obviously reduced.

Description

Micro-fluidic chip for single cell sorting

Technical Field

The utility model relates to a cell sorting technology field particularly, relates to a micro-fluidic chip for unicellular separation.

Background

The micro-fluidic chip is based on a micro-electro-mechanical processing technology, and can realize the precise control of a trace fluid by constructing a complex micro-channel on the chip and using a technology that a controllable micro-fluid penetrates through the whole system and completes various biological and chemical processes, and because the size of most of cells is in a micron scale and is just adapted to the size of the micro-channel, a very convenient condition is provided for controlling a few or single cells. Miniaturized, integrated, high-throughput microfluidic chips are known as Lab-on-a-chips.

In the fields of biology, medicine and the like, the required cells are often required to be separated from a more complex sample for directional research. The micro-fluidic chip technology has good advantages in cell sorting, reduces the work which must be finished in a comprehensive laboratory originally on one chip, not only reduces the consumption of detection and reagents, greatly reduces the cost, but also improves the analysis speed by tens of times. The existing micro-fluidic chip is mainly used for separating single cells by properly diluting a cell sample and then controlling the connection or disconnection of a channel to realize the separation or guidance of the single cells. At present, the chip of this type is mainly based on the gas circuit control to control the on/off of the channel, that is, open the micro valve, control the gas supply to supply gas, make the Polydimethylsiloxane (PDMS) film deform towards the fluid channel to block the channel, and the film recovers after exhausting, the channel is on. The single cell can be collected or guided by the connection and the disconnection of the channel, and the single cell can be guided by the combination of a plurality of micro valves. Although the microfluidic chip is complete in function, the thickness of the chip is increased by a complex air path control structure and is usually more than 7mm, and the distance between a lens and an objective table of a common upright microscope is only 1-2mm, so that the chip cannot be applied, and therefore the chip is usually required to be provided with complex observation equipment. In addition, because the micro-valve air inlet/outlet structure is included, a plurality of micro-injection pumps and a plurality of air sources need to be equipped, and the matched equipment is complicated.

SUMMERY OF THE UTILITY MODEL

To micro-fluidic chip thickness among the prior art be not suitable for just putting the loaded down with trivial details problem of microscope and corollary equipment, the utility model provides a micro-fluidic chip for unicellular is selected separately.

The utility model discloses specifically realize through following technical scheme:

a microfluidic chip for single cell sorting comprises a substrate and a baffle, wherein a microfluidic channel is formed in the upper surface of the substrate and comprises a capture channel;

the upper surface of the base is also provided with a slot which is communicated with the capturing channel, the baffle is movably inserted in the slot, and the baffle is used for closing or opening the capturing channel.

Further, the slot is disposed perpendicular to the capture channel.

Further, the part of the baffle plate, which is positioned outside the slot, is provided with a bulge for forming a handle structure.

Further, the outer side wall of the baffle is provided with a waterproof layer.

Further, the microfluidic channel further comprises a collection channel, and an outlet of the capture channel is communicated with an inlet of the collection channel.

Further, the microfluidic channel further comprises a washing channel in communication with the collection channel and the capture channel at a junction.

Further, the microfluidic channel further comprises a flow channel, an outlet of the flow channel is communicated with an inlet of the capture channel through a pipeline, and the pipeline is detachably connected with the outlet of the flow channel and/or the inlet of the capture channel.

Further, at least a segment of the flow channel is serpentine.

Furthermore, the upper surface of the base is also provided with a dilution pool, and the dilution pool is communicated with the inlet of the flow channel.

Further, the base includes flow path layer and substrate layer, the flow path layer is located on the substrate layer, the flow path layer with the substrate layer cooperation forms the miniflow passageway, just the slot is located on the flow path layer.

The beneficial effects of the utility model are that:

the utility model discloses a promoting the baffle and sliding from top to bottom along the slot and can closing and switching on of effective control miniflow passageway, under the circumstances that the function is not weakened in guaranteeing unicellular separation, greatly reduced the holistic thickness of micro-fluidic chip, thickness can reduce to about 1.5mm, combines living cell dyeing technique, can directly arrange in and observe under just putting the microscope, improved suitability and practicality. Moreover, the control of the microfluidic channel can be realized only by additionally arranging the slot and the baffle on the substrate, and the acquisition and collection of single cells are realized, so that the structure of the substrate can be greatly simplified, the difficulty in manufacturing the chip is reduced, and the matched equipment is simplified and only relates to an upright microscope and a micro-injection pump, so that the operation of the microfluidic chip is simpler and more convenient, and the mature application is favorable for obviously reducing the use cost.

Drawings

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

Fig. 1 is a schematic structural diagram of a microfluidic chip for single cell sorting according to an embodiment of the present invention;

description of the reference numerals:

1. a substrate; 11. a flow channel layer; 12. a substrate layer; 2. a microfluidic channel; 21. a flow channel; 22. a capture channel; 23. a collection channel; 24. a flushing channel; 3. inserting slots; 4. a baffle plate; 5. a protrusion; 6. a dilution tank; 7. a sample liquid pool; 71. a cell sample liquid pool; 72. a diluent sample reservoir; 8. a cell culture device; 9. a pipeline.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, embodiments accompanied with figures are described in detail below. It is to be understood that the disclosed embodiments are merely exemplary of the invention, and are not intended to limit the invention to the precise embodiments disclosed. Based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative efforts belong to the protection scope of the present invention.

It should be noted that the technical terms used in the specification and the claims of the present invention should have the ordinary meanings that those having ordinary skill in the art to which the present invention belongs understand. The use of "including" or "comprising" and similar referents in the context of describing the invention and the following description is to be construed to cover all the elements or items listed in the list of "including" or "comprising" and their equivalents, but not to exclude other elements or items. The terms "connected" and "coupled" and the like are not restricted to physical or mechanical connections, nor are they restricted to direct or indirect connections.

It should be understood that the description of the invention in the specification and claims and the above drawings, with respect to the orientation, such as the orientation or positional relationship indicated by the front, back, upper, lower, etc., is based on the orientation or positional relationship shown in the drawings, and is only for convenience of description and simplicity of description, and does not indicate or imply that the device or element so referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore should not be considered as limiting the invention.

The embodiment of the utility model provides a micro-fluidic chip for unicell is selected separately, refer to fig. 1, including base 1, micro-flow channel 2 has been seted up on base 1, micro-flow channel 2 includes flow channel 21, catches passageway 22 and collects passageway 23, flow channel 21 catch passageway 22 and collect passageway 23 along fluid (the utility model discloses the fluid in the context refers to cell suspension) flow direction communicate in proper order, namely, the export of flow channel 21 communicates catches the entry of passageway 22, the export of catching passageway 22 communicates the entry of collecting passageway 23, flow channel 21 is used for separating cell suspension gradually into the unicell liquid drop and flows, realizes that the cell is in flow channel 21 especially latter half section is orderly arranged to the cell sorting of single candidate, catch passageway 22 is used for separating the cell of candidate, collect passageway 23 is used for collecting the aforesaid cell of selecting of separating, from this, forms fluid flow's micro-flow channel 2 to constitute liquid drop generating area and unicell collecting area on base 1.

The upper surface of the base 1 is provided with a slot 3, the slot 3 is communicated with the capturing passage 22, so that the intersection of the slot 3 and the capturing passage 22 is positioned in front of the intersection of the capturing passage 22 and the collecting passage 23, a baffle 4 is movably inserted in the slot 3, and the baffle 4 is used for closing or opening the capturing passage 22.

In actual use, the inlet of the flow channel 21 is connected to the sample solution reservoir 7 through a conduit, and the outlet of the collection channel 23 is connected to the cell culture apparatus 8 such as a test tube, a culture dish, or a cell culture flask through a conduit. It should be noted that each inlet and outlet of the microfluidic chip are connected to the microfluidic channel 2, and each inlet and outlet are connected to an external fluid-driven sample injection device such as a micro syringe pump through a conduit to provide a driving force for fluid flow. Before advancing the appearance, will the utility model discloses a micro-fluidic chip arranges in on just putting microscope like fluorescence microscope's objective table, lets catch passageway 22 and collection channel 23 intersection and be located just putting under microscope's the camera lens, makes the field of vision can observe this region. Then, the inlet of the flow channel 21 of the microfluidic chip is connected with the sample injection pool, the cell suspension (which is subjected to living cell fluorescent staining) in the sample liquid pool 7 enters from the inlet of the flow channel 21 and then sequentially flows along the flow channel 21, the cells in the cell suspension are gradually separated under the extrusion action of the flow channel 21 to form single-row ordered arrangement, the cells are prevented from being aggregated and blocking the subsequent microfluidic channel 2 to form single-cell droplets, then the single-cell droplets enter the capture channel 22 and pass through the intersection of the capture channel 22 and the collection channel 23, the cell concentration and the fluid flow rate just meet the condition that only one cell exists within 3-5 seconds and passes through the lens of the normal microscope, at the moment, the baffle 4 is pulled to move downwards to close the capture channel 22 and block the fluid flow, then the single cell located in the collection channel 23 is collected and cultured, after the collection is finished, the baffle 4 is pulled out to move upwards to open the capture channel 22, the fluid returns to flow, and the next single cell sorting and collection are performed.

The utility model discloses a promoting baffle 4 and sliding from top to bottom along slot 3 and can effectively controlling closing and switching on of miniflow passageway 2, need not to set up complicated gas circuit control structure, under the circumstances that the single cell separation function is not weakened guaranteeing, greatly reduced the holistic thickness of micro-fluidic chip, thickness can fall about 1.5mm, combines living cell dyeing technique, can directly arrange upright microscope in and observe down, has improved suitability and practicality. Moreover, the control of the microfluidic channel 2 can be realized only by additionally arranging the slot 3 and the baffle 4 on the substrate 1, and the acquisition and collection of single cells are realized, so that the structure of the substrate 1 can be greatly simplified, the difficulty in manufacturing the chip is reduced, and the corollary equipment is simplified, only the upright microscope and the micro-injection pump are involved, so that the operation of the microfluidic chip is simpler and more convenient, and the use cost is obviously reduced after the application is mature. Additionally, the utility model discloses at the in-process of separation unicellular, do not carry out other any special treatment except that living cell dyes to the cell, greatly reduced to the injury of cell, improve the survival rate of the unicellular that the separation obtained, be favorable to follow-up cultivation work.

Aforementioned baffle 4 and slot 3 cooperation break-make miniflow channel 2's structure setting is favorable to simplifying basement 1 structure, if the utility model discloses a basement 1 can only adopt 2 layer structure, specifically, basement 1 includes flow channel layer 11 and substrate layer 12, substrate layer 12 can be made by glass, flow channel layer 11 can be made by the PDMS material, sets up groove or pore structure at the PDMS material, will again flow channel layer 11 with glass substrate layer 12 from top to bottom laminates in proper order, encapsulates, can form the miniflow control chip that has the miniflow channel 2 that supplies the cell suspension to flow, and can locate slot 3 on flow channel layer 11. Therefore, the utility model discloses a chip preparation flow is very simple convenient, is favorable to carrying out industrialization mass production.

Optionally, the slot 3 is disposed perpendicular to the capture channel 22, that is, the slot 3 extends along the width direction of the substrate 1, so that the baffle 4 can slide in the slot 3 to open and close the capture channel 22.

Optionally, the one end of baffle 4 extends to in the slot 3 and slidable mounting in the slot 3, the other end extends to outside the slot 3, baffle 4 is located the part outside the slot 3 is equipped with arch 5, arch 5 can be formed by 4 self bending of baffle to be used for forming handle structure, do benefit to the activity of staff promotion baffle 4.

Optionally, the outer side wall of the baffle 4 is provided with a waterproof layer (not shown in the figure). The waterproof layer is made of hydrophobic materials or super-hydrophobic materials, such as polyolefin resin, polyacrylonitrile resin, paraffin and the like. The waterproof layer possesses hydrophobic, waterproof characteristic, can make baffle 4 and the adhesion of liquid in the cell suspension little, is favorable to reducing the corruption to baffle 4, prolongs the life of baffle 4.

In order to facilitate the single cell collection, the microfluidic channel 2 may optionally further comprise a washing channel 24, and the washing channel 24 is communicated with the collection channel 23. Preferably, the flushing channel 24 communicates with the intersection of the collecting channel 23 and the capturing channel 22, so that the outlet of the flushing channel 24, the inlet of the collecting channel 23 and the outlet of the microfluidic channel 2 are connected to form a three-way structure.

The flushing channel 24 is used to flow a flushing liquid, typically a sterile medium, suitable for culturing the collected single cells directly. When a single cell is observed in the field of the upright microscope, the supply of the cell suspension to the flow channel 21 is immediately stopped, while the shutter 4 is pushed to close the capturing channel 22, and then a washing liquid is injected, and the observed single cell is carried out through the collection channel 23 and collected into the culture apparatus. After the baffle 4 closes the capturing channel 22, the single cell liquid drops in the capturing channel 22 are driven to flow when the washing liquid is carried out from the single cell, and the introduction of more cells into the culture device is avoided.

Optionally, the outlet of the flow channel 21 communicates with the inlet of the capture channel 22 via a conduit 9, and the conduit 9 is detachably connected to the outlet of the flow channel 21 and/or the inlet of the capture channel 22. In some embodiments, one end of the conduit 9 is fixedly connected to the outlet of the flow channel 21, and the other end is detachably connected to the inlet of the capture channel 22. In other embodiments, one end of the conduit 9 is detachably connected to the outlet of the flow channel 21, and the other end is fixedly connected to the inlet of the capture channel 22. It will be appreciated that both ends of the conduit 9 may also be removably connected to both the outlet of the flow channel 21 and the inlet of the capture channel 22.

The flow channel 21 and the capturing channel 22 are separated by the pipe 9, and the pipe 9 is detachably connected with one of them, thereby the single-cell droplet generating region and the single-cell collecting region can be mutually independent, namely, can respectively and independently function, and also provides the convenience of operation for adjusting the cell concentration. Specifically, the cell suspension needs to be adjusted to a proper concentration before entering the flow channel 21, but in general, in order to improve the sorting efficiency, the cell concentration is adjusted in real time by injecting a diluent while injecting cells, which results in a higher cell concentration of the cell suspension injected earlier, by the above arrangement, the cell suspension that is not adjusted to a proper concentration can be discharged and discarded via the conduit 9, and after the concentration is proper, the conduit 9 is communicated with the flow channel 21 and the capturing channel 22, so that the microfluidic chip works normally.

It should be noted that, with the above arrangement, the substrate 1 may be arranged as one, that is, the flow channel 21 and the capture channel 22 are respectively arranged at different positions of the substrate 1 to form a large chip; it is also possible to provide 2, that is, the flow channel 21 is disposed on one of the substrates 1, and the capture channel 22 is disposed on the other substrate 1, so that 2 sub-chips are formed, and in use, the 2 sub-chips are connected and combined into one large chip through the pipeline 9.

When observing and adjusting the cell density and the moving speed of the microorganism in the microfluidic channel 2, the pipeline 9 and the joint connecting the flow channel 21 and the capturing channel 22 may block the objective lens of the microscope, which causes inconvenience for single cell collection. Therefore, the substrate 1 preferably adopts a 2-piece mode, so that the single-cell droplet generation region and the single-cell collection region are completely independent and can separately perform respective functions, and the cell suspension diluted in the single-cell droplet generation region is injected into the single-cell collection region for single-cell collection after being connected through the pipeline 9, so that the whole function is not influenced.

Optionally, at least a section of the flow channel 21 is serpentine. As shown in fig. 1, the middle of the flow channel 21 has a serpentine shape, thereby dividing the flow channel 21 into a "straight-curved-straight" structure, and the curved structure, i.e., the serpentine flow channel, has at least one straight channel and at least one curved channel, the straight channel and the curved channel are alternately arranged, and the flow direction of the cell suspension after passing through the curved channel is turned by 180 °, thereby prolonging the flow path and the time. The diluted cell suspension enters the flow channel 21 at a lower cell concentration, and the cells are gradually dispersed and uniformly arranged under the action of continuous straight channels and curved channels to form single cell liquid drops through separation, so that the single cell is accurately sorted. The serpentine-shaped flow channel 21 ensures rapid dispersion and separation of cells under a shorter flow path.

It should be noted that the serpentine flow channel 21 has a plurality of curves, and the number of the curves is set according to the nature and kind of the cell suspension, the required cell interval time, and the like.

In order to adjust the concentration of the diluted cells, optionally, the microfluidic chip further includes a dilution pool 6, the dilution pool 6 is disposed on the upper surface of the substrate 1, and the dilution pool 6 is communicated with the inlet of the flow channel 21.

In this embodiment, the dilution pool 6 is used for connecting the sample pool 7, the sample pool 7 generally comprises a cell sample pool 71 and a dilution liquid sample pool 72, and the dilution liquid can be a buffer liquid or a sterile culture medium. The stained cells and the diluent are respectively injected into a diluting pool 6 through a micro-injection pump to provide space for cell dilution; the cell and the diluent are mixed in the diluting pool 6, the cell concentration is reduced, the dilution times of the cell are adjusted by adjusting the diluent or the injection speed of the cell, the dilution while the injection and the dilution while the adjustment are realized, the convenience in adjusting the cell concentration to a proper range is facilitated, and the loss of the diluent and the cell is reduced.

It should be noted that the size of the dilution pool 6 and the size of the microfluidic channel 2, such as height, width, length, etc., are adapted to the size of the cells and the cell concentration to be studied, so as to ensure the functional transport of the cell suspension in the channel. The utility model discloses do not do special restriction. Generally, the height and width of the dilution pool 6 are 5-10 times the height and width of the microfluidic channel 2, and illustratively, the height and width of the dilution pool 6 are 50 μm, respectively, to perform a cell dilution function, and the height and width of the microfluidic channel 2 are 8 μm, respectively, to perform a single cell sorting and collecting function. The length of each section of the microfluidic channel 2, such as the length of the flow channel 21 (in terms of the linear distance from the inlet to the outlet of the flow channel 21), is 17mm, the length of the capture channel 22 is 12mm, and the length of the collection channel 23 is 12mm.

In addition, it should be noted that the whole size of the microfluidic chip is set according to the experimental requirements, and the utility model discloses do not specially limit. In general, the microfluidic chip may be configured to: the width is 20-30mm, the length is 80-150mm, and the thickness is 1-1.5mm. Illustratively, the microfluidic chip has a width of 25mm, a length of 130mm, and a thickness of 1.5mm.

Although the present disclosure has been described above, the scope of the present disclosure is not limited thereto. Without departing from the spirit and scope of the present disclosure, those skilled in the art can make various changes and modifications, which will fall into the scope of the present disclosure.

Claims

1. The microfluidic chip for single cell sorting is characterized by comprising a substrate (1) and a baffle (4), wherein a microfluidic channel (2) is formed in the upper surface of the substrate (1), and the microfluidic channel (2) comprises a capture channel (22);

the upper surface of the base (1) is further provided with a slot (3), the slot (3) is communicated with the capturing channel (22), the baffle (4) is movably inserted into the slot (3), and the baffle (4) is used for closing or opening the capturing channel (22).

2. The microfluidic chip for single-cell sorting according to claim 1, wherein the slot (3) is arranged perpendicular to the capture channel (22).

3. Microfluidic chip for single-cell sorting according to claim 1, characterized in that the part of the baffle (4) outside the slot (3) is provided with protrusions (5) for forming a handle structure.

4. The microfluidic chip for single-cell sorting according to claim 1, wherein the outer side wall of the baffle (4) is provided with a water-proof layer.

5. The microfluidic chip for single-cell sorting according to any of claims 1-4, wherein the microfluidic channel (2) further comprises a collection channel (23), and the outlet of the capture channel (22) is communicated with the inlet of the collection channel (23).

6. The microfluidic chip for single-cell sorting according to claim 5, wherein the microfluidic channel (2) further comprises a washing channel (24), the washing channel (24) communicating with the collection channel (23) and the capture channel (22) at the intersection.

7. The microfluidic chip for single-cell sorting according to any of claims 1-4, wherein the microfluidic channel (2) further comprises a flow channel (21), an outlet of the flow channel (21) is in communication with an inlet of the capture channel (22) via a conduit (9), and the conduit (9) is detachably connected with the outlet of the flow channel (21) and/or the inlet of the capture channel (22).

8. The microfluidic chip for single-cell sorting according to claim 7, wherein at least a section of the flow channel (21) is serpentine.

9. The microfluidic chip for single-cell sorting according to claim 7, wherein the substrate (1) further comprises a dilution pool (6) formed on the upper surface thereof, and the dilution pool (6) is communicated with the inlet of the flow channel (21).

10. The microfluidic chip for single-cell sorting according to claim 1, wherein the substrate (1) comprises a channel layer (11) and a substrate layer (12), the channel layer (11) is disposed on the substrate layer (12), the channel layer (11) and the substrate layer (12) cooperate to form the microfluidic channel (2), and the insertion groove (3) is disposed on the channel layer (11).