CN110639628A - Micro-fluidic chip and method for cell sorting and positioning - Google Patents

Micro-fluidic chip and method for cell sorting and positioning Download PDF

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CN110639628A
CN110639628A CN201910861908.4A CN201910861908A CN110639628A CN 110639628 A CN110639628 A CN 110639628A CN 201910861908 A CN201910861908 A CN 201910861908A CN 110639628 A CN110639628 A CN 110639628A
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flow channel
pressure control
sorting
outlet
channel
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CN110639628B (en
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靳欣
李歧强
韩琳
王兴元
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Shandong University
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502776—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for focusing or laminating flows
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0636—Focussing flows, e.g. to laminate flows
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0883—Serpentine channels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0893—Geometry, shape and general structure having a very large number of wells, microfabricated wells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/14—Means for pressure control
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1028—Sorting particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
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Abstract

The invention discloses a micro-fluidic chip for cell sorting and positioning and a method thereof, wherein the chip comprises a main flow channel, a sorting flow channel and a pressure control flow channel, wherein one end of the main flow channel is provided with a sample injection port and a sheath fluid injection port, and the other end of the main flow channel is provided with a main flow channel outlet; one end of the separation flow channel and one end of the pressure control flow channel are sealed, and the other end of the separation flow channel and the other end of the pressure control flow channel are respectively a separation flow channel outlet and a pressure control flow channel outlet; the main flow channel is connected with the separation flow channel through a plurality of connecting flow channels, and the separation flow channel is connected with the pressure control flow channel through a plurality of micro flow channels; the joint of the sorting flow channel and the micro flow channel is of a bowl-shaped structure; the chip disclosed by the invention has the advantages of high focusing precision, low cost and simple experimental steps, and is very significant for marine ecological environment research and animal tissue or blood sample detection pretreatment.

Description

Micro-fluidic chip and method for cell sorting and positioning
Technical Field
The invention relates to a microfluidic technology, in particular to a microfluidic chip and a method for cell sorting and positioning.
Background
In the research of marine ecological environment, microalgae is used as a main energy source of coastal water areas and has important influence on offshore environment. Through the identification of fluorescence and images, the species, the propagation and the biomass of the microalgae can be obtained, so that the water body environment is evaluated and predicted. The separation and observation of the biological tissue and the blood sample also have great significance in scientific research and disease diagnosis and treatment.
The focusing of the microscope and the overlapping of the cells are major challenges for image acquisition and fluorescence identification. By reducing the sample concentration, the problem of cell image overlap can be alleviated to some extent. However, a large number of blank pictures are brought, and the detection efficiency and the post-image processing are adversely affected. The best method for solving the problem at present is to use a flow cytometer, but the flow cytometer is expensive, bulky, inconvenient to carry, complex in detection steps and high in detection cost.
Disclosure of Invention
In order to solve the technical problems, the invention provides a micro-fluidic chip for cell sorting and positioning and a method thereof, so as to achieve the purposes of effectively improving the problems of low focusing precision, overlapping of cell images and the like, low cost and simple experimental steps.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a micro-fluidic chip for cell sorting and positioning comprises a main flow channel, a sorting flow channel and a pressure control flow channel, wherein one end of the main flow channel is provided with a sample injection port and a sheath fluid injection port, and the other end of the main flow channel is provided with a main flow channel outlet; one end of the separation flow channel and one end of the pressure control flow channel are sealed, and the other end of the separation flow channel and the other end of the pressure control flow channel are respectively a separation flow channel outlet and a pressure control flow channel outlet; the main flow channel is connected with the separation flow channel through a plurality of connecting flow channels, and the separation flow channel is connected with the pressure control flow channel through a plurality of micro flow channels; the joint of the sorting flow channel and the micro flow channel is of a bowl-shaped structure; select separately the runner and the pressure control runner sets up the multiunit, distributes in the different positions of sprue in proper order along the sample flow direction of sprue, and the diameter that every group selects separately the connection runner between runner and the sprue increases step by step.
In the above-mentioned scheme, the S type runner that the sprue comprises horizontal segment and crooked section, select separately the runner and the pressure control runner is located the horizontal segment outside of S type, and is parallel with the horizontal segment.
In the above embodiment, the diameter of the micro flow channel is much smaller than the diameter of the cell.
In the above scheme, the sorting flow channel outlet and the pressure control flow channel outlet are located at two ends relatively far away.
In a further technical scheme, the chip is made of PDMS, PMMA, silicon or glass.
A method for sorting and positioning cells adopts the microfluidic chip for sorting and positioning cells, and comprises the following processes:
respectively injecting a sample solution containing cells and a sheath liquid from a sample injection port and a sheath liquid injection port, extracting the liquid from an outlet of a first group of pressure control flow channels, enabling a batch of cells with the smallest size in the sample solution to enter a sorting flow channel through a connecting flow channel and enter a bowl-shaped structure, and then sequentially opening an outlet of a pressure control flow channel of a rear group and extracting the liquid, so that the cells in the sample solution are sorted out in a small-to-large order and are positioned in the bowl-shaped structure; and then carrying out image detection, finally pressurizing through a pressure control flow passage outlet, extracting liquid from a sorting flow passage outlet, and extracting cells positioned in the bowl-shaped structure from different sorting flow passages so as to obtain the cells in a certain size interval, thereby facilitating the detection and analysis of the next step.
According to the technical scheme, the micro-fluidic chip for cell sorting and positioning is connected with the main flow channel and the sorting flow channel through connecting flow channels with different diameters, so that cells with different sizes sequentially enter different sorting flow channels and are positioned in the bowl-shaped structure, the pressure in the bowl-shaped structure is controlled by the pressure control flow channel, the cells can be positioned in the bowl-shaped structure, image detection is facilitated, the focusing precision of a microscope can be effectively improved, the problem of cell superposition is thoroughly solved, the cells to be identified are arranged in order, and the accuracy of pattern identification can be greatly improved.
Meanwhile, the S-shaped main flow channel is adopted, the flow channel area is small, and the photoresist stress can be reduced compared with a large-area region to be sorted, so that the manufacturing yield is improved. Adopt two liquid inlets and S type sprue that sample injection port and sheath liquid injection port constitute, utilize laminar flow and centrifugal force, can make most of cells along the sprue outside motion, and then make most of cells within the biggest size all can be screened the location.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a schematic diagram of an internal structure of a microfluidic chip for cell sorting and positioning according to an embodiment of the present invention;
fig. 2 is a partially enlarged schematic view of the embodiment of the present invention.
In the figure, 1, a main flow channel; 2. a sorting flow channel; 3. a pressure control flow path; 4. a sample injection port; 5. a sheath fluid injection port; 6. an outlet of the main runner; 7. an outlet of the sorting flow channel; 8. a pressure control flow passage outlet; 9. connecting the flow channel; 10. a micro flow channel; 11. a bowl-shaped structure; 12. a cell.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
The invention provides a micro-fluidic chip for cell sorting and positioning and a method thereof, and the chip can effectively improve the cell image acquisition quality and has great significance for marine ecological environment research and detection pretreatment of animal samples such as animal tissues or blood samples and the like, such as a structure shown in figure 1.
The micro-fluidic chip for cell sorting and positioning as shown in fig. 1 comprises a main flow channel 1, a sorting flow channel 2 and a pressure control flow channel 3, wherein one end of the main flow channel 1 is provided with a sample injection port 4 and a sheath fluid injection port 5, and the other end is provided with a main flow channel outlet 6; sorting flow channel 2 and pressure control flow channel 3 one end are sealed, and the other end is sorting flow channel export 7 and pressure control flow channel export 8 respectively, and sorting flow channel export 7 and pressure control flow channel export 8 are located the both ends of keeping away from relatively, can guarantee any action like this, and pressure all can pass whole runner, rather than forming short-lived return circuit.
The main flow channel 1 is connected with the sorting flow channel 2 through a plurality of connecting flow channels 9, and the sorting flow channel 2 is connected with the pressure control flow channel 3 through a plurality of micro flow channels 10; the connection part of the sorting flow channel 2 and the micro flow channel 10 is a bowl-shaped structure 11. The diameter of the micro flow channel 10 is much smaller than the diameter of the cell 12, so that the cell can be prevented from entering the pressure control flow channel 3 through the micro flow channel 10.
The sprue 1 is the S type runner that comprises horizontal segment and curved segment, selects separately runner 2 and pressure control runner 3 and sets up the multiunit, distributes in the horizontal segment outside of S type runner along the sample flow direction of sprue 1 in proper order, and is parallel with the horizontal segment, and the diameter of the connecting channel 9 between runner 2 and the sprue 1 is selected separately to every group increases step by step. As can be seen from FIG. 1, the connecting flow channel 9 located at the uppermost layer has the smallest diameter, and the connecting flow channel 9 located at the lowermost layer has the largest diameter, so that cells 12 of different sizes can be sorted and positioned in batches.
The S-shaped flow channel designed by the invention can make the cell solution deflect to the outer side of the S-shaped flow channel under the action of laminar flow, and is convenient to enter the sorting flow channel 2.
The micro-fluidic chip designed by the invention has high structural strength, so that the micro-fluidic chip has no excessive requirements on materials, and can be made of various materials, such as PDMS, PMMA, silicon or glass.
A method for sorting and positioning cells adopts the microfluidic chip for sorting and positioning cells, and comprises the following processes:
firstly, respectively injecting a sample solution containing cells and a sheath solution from a sample injection port 4 and a sheath solution injection port 5, extracting the solution from an outlet of a first group of pressure control flow channels 3 by using an external pressure control device, enabling a batch of cells with the smallest size in the sample solution to enter a sorting flow channel 2 from a connecting flow channel and enter a bowl-shaped structure 11, and then sequentially opening an outlet 8 of a pressure control flow channel of a later group and extracting the solution, so that the cells in the sample solution are sorted out in a descending order and are positioned in the bowl-shaped structure 11; then, image detection is carried out, and finally, the pressure is controlled by the pressure control flow channel outlet 8 to pump liquid from the sorting flow channel 2 outlet, so that the cells in the bowl-shaped structure 11 are pumped from different sorting flow channels 2, and cells in a certain size interval are obtained for the next detection and analysis.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (6)

1. A micro-fluidic chip for cell sorting and positioning is characterized by comprising a main flow channel, a sorting flow channel and a pressure control flow channel, wherein one end of the main flow channel is provided with a sample injection port and a sheath fluid injection port, and the other end of the main flow channel is provided with a main flow channel outlet; one end of the separation flow channel and one end of the pressure control flow channel are sealed, and the other end of the separation flow channel and the other end of the pressure control flow channel are respectively a separation flow channel outlet and a pressure control flow channel outlet; the main flow channel is connected with the separation flow channel through a plurality of connecting flow channels, and the separation flow channel is connected with the pressure control flow channel through a plurality of micro flow channels; the joint of the sorting flow channel and the micro flow channel is of a bowl-shaped structure; select separately the runner and the pressure control runner sets up the multiunit, distributes in the different positions of sprue in proper order along the sample flow direction of sprue, and the diameter that every group selects separately the connection runner between runner and the sprue increases step by step.
2. The microfluidic chip for cell sorting and positioning according to claim 1, wherein the main channel is an S-shaped channel comprising a horizontal section and a curved section, and the sorting channel and the pressure control channel are located outside the horizontal section of the S-shape and are parallel to the horizontal section.
3. The microfluidic chip for cell sorting and positioning according to claim 1, wherein the diameter of the micro flow channel is much smaller than the diameter of the cell.
4. The microfluidic chip for cell sorting and positioning according to claim 1, wherein the sorting channel outlet and the pressure control channel outlet are located at two opposite ends.
5. The microfluidic chip for cell sorting and positioning according to any one of claims 1-4, wherein the chip is made of PDMS, PMMA, silicon or glass.
6. A method for cell sorting and positioning using the microfluidic chip for cell sorting and positioning according to claim 1, comprising the steps of:
respectively injecting a sample solution containing cells and a sheath liquid from a sample injection port and a sheath liquid injection port, extracting the liquid from an outlet of a first group of pressure control flow channels, enabling a batch of cells with the smallest size in the sample solution to enter a sorting flow channel through a connecting flow channel and enter a bowl-shaped structure, and then sequentially opening an outlet of a pressure control flow channel of a rear group and extracting the liquid, so that the cells in the sample solution are sorted out in a small-to-large order and are positioned in the bowl-shaped structure; and then carrying out image detection, finally pressurizing through a pressure control flow passage outlet, extracting liquid from a sorting flow passage outlet, and extracting cells positioned in the bowl-shaped structure from different sorting flow passages so as to obtain the cells in a certain size interval, thereby facilitating the detection and analysis of the next step.
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