CN114717100A - Microfluidic chip for single cell sequencing and application - Google Patents
Microfluidic chip for single cell sequencing and application Download PDFInfo
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Abstract
The invention provides a micro-fluidic chip for single cell sequencing and a method for processing a single cell sequencing sample by using the micro-fluidic chip. According to the microfluidic chip, a cell solution, a cell marker solution (such as microspheres with nucleic acid sequences) and a reaction reagent solution (such as cell lysate) are respectively stored in different storage cavities, so that a cell solution inlet and a reaction reagent solution inlet are separated, and the survival rate of cells can be effectively improved; meanwhile, the mixed solution is cut into water-in-oil droplets at the cross structure of the chip flow channel by the oil phase solution, so that high wrapping rate of microspheres and cells can be realized.
Description
Technical Field
The invention relates to a micro-fluidic chip for single cell sequencing, and belongs to the technical field of micro-droplets, particle packaging and cell packaging.
Background
Microfluidics is a scientific technology which precisely controls and controls microscale fluids and is mainly characterized by controlling fluids in a microscale space. The common technologies of the current microfluidic chip include separation technology, detection technology, micro-droplet technology and microfluidic control and drive technology. Compared with the traditional separation mode, the microfluidic chip separation technology has the advantages of rich carrier materials, quartz, glass, silicon and various polymers, easy separation realization, simple process control, flexible combination with other operation units and wider application range. Compared with the traditional detection technology, the detection technology has higher requirements, such as high sensitivity, high response speed, parallel analysis function, portable characteristic and the like. At present, many detection technologies based on different principles are applied to the research of microfluidic chips, and mainly include methods such as optical detection, electrochemical detection, mass spectrometry and the like.
Microfluidics (Microfluidics) has important applications in the disciplines of single cell sequencing, life sciences, clinical medicine and the like. Single cell sequencing is a new technology which is started in recent years, can obtain a complete transcriptome expression profile from a single cell level, and can carry out high-throughput sequencing after amplification, thereby efficiently detecting the gene expression level in the single cell, and having important application values in the fields of diagnosis and treatment of tumors, design of targeted drugs, development and differentiation of stem cells and the like.
Micro-fluidic technology generally uses micro-analysis devices as carriers for technical implementation, and micro-fluidic chips are the most rapidly developed among various types of micro-analysis devices. The micro-fluidic chip is a micro total analysis system which processes various microstructures on a silicon, quartz, glass or high molecular polymer substrate by using an MEMS technology, then communicates components with fluid conveying, controlling and detecting monitoring functions such as a micro pump, a micro valve, a micro liquid storage device, a micro detection element and the like by using a micro pipeline, and integrates the processes of diluting, adding a reagent, sampling, reacting, separating and dispersing, detecting, monitoring and the like on the chip to the maximum extent.
At present, a common microfluidic detection system usually mixes a cell solution and a reaction reagent solution (such as a lysate which has a destructive effect on cells) and stores the mixture in a same droplet storage cavity with a smaller volume for droplet sample preparation pretreatment, so that the defects of low cell survival rate, low test flux and the like are caused. Manual intervention is often required to move or operate the chip multiple times, which not only has low automation degree, but also has high cost.
Therefore, it is necessary to design a microfluidic chip for single cell sequencing, in which droplets of a cell solution, a cell marker solution (e.g., microspheres with a nucleic acid sequence), and a reaction reagent solution (e.g., cell lysate) are packaged in the same chip.
Disclosure of Invention
The invention provides a micro-fluidic chip for single cell sequencing and a method for processing a single cell sequencing sample by using the micro-fluidic chip. According to the microfluidic chip, a cell solution, a cell marker solution (such as microspheres with nucleic acid sequences) and a reaction reagent solution (such as cell lysate) are respectively stored in different storage cavities, so that a cell solution inlet and a reaction reagent solution (such as cell lysate) inlet are separated, and the survival rate of cells can be effectively improved; meanwhile, the mixed solution is cut into water-in-oil droplets at the intersection structure of the chip flow channels by the oil phase solution, so that high wrapping rate of microspheres and cells can be realized, and the survival rate of the cells is improved.
The invention provides a micro-fluidic chip for single cell sequencing, which comprises an upper chip part and a lower chip part, wherein one side of the upper chip part is provided with an oil phase solution injection port 1, a reaction reagent solution (such as cell lysate) injection port 2, a cell solution injection port 3, a cell marker solution (such as microspheres with nucleic acid sequences) injection port 4 and a droplet outlet 5, and one side of the lower chip part is provided with a micro-channel 6 for generating droplets to wrap cells, cell markers (such as microspheres with nucleic acid sequences) and reaction reagents (such as cell lysate).
Optionally, the micro flow channel 6 comprises:
(1) a first channel, one end of which is connected with the cell solution injection port and the other end of which is connected with the droplet generation intersection part;
(2) one end of the second channel is connected with the oil phase solution injection port, and the other end of the second channel is connected with the droplet generation crossing part;
(3) a third channel having one end connected to the droplet generation intersection and the other end connected to the droplet outlet;
(4) the fourth channel is positioned between the first channel and the second channel, one end of the fourth channel is connected with the cell marker solution injection port, and the other end of the fourth channel is connected with the first channel;
(5) and the fifth channel is positioned between the first channel and the second channel, one end of the fifth channel is connected with the reaction reagent solution injection port, and the other end of the fifth channel is connected with the first channel.
Optionally, a cross section of at least one of the first channel, the second channel, the fourth channel and the fifth channel is narrowed from wide toward a liquid flow direction. The upstream wide region 8 is designed to facilitate easier use of gas pressure control to push the liquid phase fluid, and the downstream narrow region 9 is designed to facilitate easier flow of cells and cell markers (e.g., microspheres with nucleic acid sequences) in a single row through the cross-over structure to maximize single encapsulation.
Optionally, the width of the cross section of the channel is between 20 μm and 300 μm, preferably the width of the cross section of the channel is between 40 μm and 80 μm.
Optionally, a plurality of support micro-columns 7 are arranged in the chip channel to prevent the chip channel from collapsing and promote the uniform mixing of the solution. Preferably, a plurality of support microcolumns 7 are arranged in order within the channel.
Optionally, the support micro-column 7 is a cylindrical structure.
Optionally, each sample inlet is provided with a sample storage chamber, and the droplet outlet is provided with a storage droplet storage chamber.
Optionally, the volume of the droplet storage chamber is between 30 μ L and 700 μ L.
Optionally, at least one of the upper sheet portion and the lower sheet portion is a transparent portion, and the upper sheet portion and/or the lower sheet portion is made of a polymer material such as PMMA, PC, COP, COC, or PS.
Alternatively, the upper sheet portion and the lower sheet portion are bonded by heat pressing, film sticking, or the like.
Meanwhile, the invention also provides a method for processing the single cell sequencing sample by using the microfluidic chip, which is characterized by comprising the following steps:
(1) transferring the oil phase solution, the reaction reagent solution (such as cell lysate), the cell solution and the cell marker solution (such as microspheres with nucleic acid sequences) into each storage cavity on the chip respectively;
(2) controlling the flow rate of each solution by using an air pressure control device, and mixing the cell marker solution, the cell solution and the reaction reagent solution in the chip flow channel;
(3) the oil phase solution cuts the mixed solution at the cross structure of the chip flow channel to generate water-in-oil liquid drops, so that the liquid drops are generated, cells, cell markers and reaction reagents are wrapped, and the sample treatment before the single cell sequencing and library building is completed.
Preferably, the step (2) includes:
(a) controlling the flow rate of each solution by using an air pressure control device, and mixing the cell marker solution and the cell solution into a mixed solution A at one position of a chip flow channel;
(b) the mixed solution A and the reaction reagent solution are mixed into a mixed solution B at the other position of the chip flow channel.
The microfluidic chip disclosed by the invention has the following beneficial effects:
(1) the microfluidic chip of the invention respectively adds a reaction reagent solution (such as cell lysate), a cell solution and a cell marker solution (such as microspheres with nucleic acid sequences) into corresponding sample inlets, so that the cell solution inlet and the reaction reagent solution (such as cell lysate) inlet are separated, thereby obviously improving the cell survival rate (the reading number of free RNA in final data is reduced to within 15 percent from 20-40 percent of common single cell sequencing, wherein the free RNA is usually from dead cells);
(2) the flow channel of the microfluidic chip is internally provided with the plurality of supporting microcolumns, so that the collapse of the flow channel of the chip can be prevented, and the uniform mixing of a solution can be promoted;
(3) the micro-fluidic chip disclosed by the invention adopts a high-capacity liquid drop storage cavity (the volume is 30-700 mu L), each independently-operated micro-fluidic chip in a single experiment can process up to 20000 cell samples, and the processing effect is good, so that high-throughput single-cell sequencing library building is realized;
(4) the microfluidic chip can be combined on one chip, so that a plurality of groups of cell samples can be conveniently processed simultaneously;
(5) the upper plate part and the lower plate part of the microfluidic chip are made of high polymer materials such as PMMA, PC, COP, COC or PS and the like, so that the mass injection production is easy, the cost is low, and the biocompatibility is good;
(6) the microfluidic chip has the advantages of simple and small integral structure, good stability and good repeatability;
(7) the microfluidic chip can realize high wrapping rate of microspheres and cells.
Drawings
Fig. 1 is a schematic view of a microfluidic chip.
Fig. 2 is a schematic chip diagram containing 4 independently operated microfluidic chips.
Fig. 3 is a schematic perspective view of a chip containing 4 independently operated microfluidic chips.
Fig. 4 is an isometric view of a chip housing 4 independently operated microfluidic chips.
FIG. 5 quality control results of single cell sequencing library.
Detailed Description
The technical solution of the present invention is further described and illustrated by the following specific examples, but the present invention is not limited to the following examples.
Unless otherwise specified, the raw materials and other chemicals used in the examples of the present invention are commercially available.
Example 1
As shown in fig. 1, an embodiment of the present invention provides a microfluidic chip, which includes an upper chip portion and a lower chip portion, and:
one side of the upper plate part is provided with an oil phase solution injection port 1, a reaction reagent solution (such as cell lysate) injection port 2, a cell solution injection port 3, a cell marker solution (such as microspheres with nucleic acid sequences) injection port 4 and a droplet outlet 5;
the other side of the upper piece part is provided with a sample storage cavity corresponding to the sample inlet and used for storing a corresponding sample; a droplet storage chamber corresponding to the droplet outlet for storing the generated droplets;
a flow channel 6 for connecting the sample inlet and the droplet outlet is arranged on one side of the lower sheet part, and the flow channel consists of five channels and a droplet generation crossing part, wherein the first channel is connected with the cell solution sample inlet 3 and the droplet generation crossing part; the second channel is connected with the oil phase solution injection port 1 and the droplet generation intersection part; a third channel connects the droplet generation intersection and the droplet outlet 5; the fourth channel is positioned between the first channel and the second channel, one end of the fourth channel is connected with the cell marker solution sample inlet, and the other end of the fourth channel is in cross connection with the first channel; the fifth channel is positioned between the fourth channel and the second channel, one end of the fifth channel is connected with the reaction reagent solution injection port, and the other end of the fifth channel is in cross connection with the first channel; the cross sections of the first channel, the second channel, the fourth channel and the fifth channel are changed from wide to narrow towards the flowing direction of the liquid; cylindrical supporting micro-columns which are arranged in order are arranged in each channel;
at least one of the first channel, the second channel, the fourth channel and the fifth channel has a cross section that is narrowed from wide toward a liquid flow direction.
The upstream wide region 8 is designed to facilitate easier use of gas pressure control to push the liquid phase fluid, and the downstream narrow region 9 is designed to facilitate easier flow of cells and cell markers (e.g., microspheres with nucleic acid sequences) in a single row through the cross-over structure to maximize single encapsulation.
Be provided with the support microcolumn in the runner, support the microcolumn orderly arrangement, can support the runner and prevent that the chip from collapsing and lead to the velocity of flow uneven, simultaneously, the function of liquid stirring can be realized in the setting up of support microcolumn, helps the effective mixture of liquid.
The inner diameter of the flow channel in the embodiment of the invention is controlled between 20 μm and 300 μm, the preferred range is 40 μm to 80 μm, the diameter of the formed liquid drop can be controlled, the single cell encapsulation can be realized, and the encapsulation rate can be improved.
The micro-fluidic chip in the embodiment of the invention adopts a high-capacity liquid drop storage cavity, the capacity of the high-capacity liquid drop storage cavity is 30-700 mu L, each micro-fluidic chip which operates independently in a single experiment can process up to 20000 cell samples, the processing effect is good, and the high-throughput single-cell sequencing library building is realized.
As shown in fig. 2-4, in practical applications, 4 independently operated microfluidic chips can be accommodated on one chip, so as to process 4 groups of cell samples simultaneously, thereby improving the efficiency of single cell sequencing and library building.
The embodiment of the invention also provides a method for processing sequencing samples by utilizing the microfluidic chip, which comprises the following steps:
(1) preparing 125 mu L of reaction reagent solution (such as cell lysate), 125 mu L of cell solution and 65 mu L of cell marker solution (such as microspheres with nucleic acid sequences);
(2) respectively transferring 120 mu L of oil phase solution, 120 mu L of reaction reagent solution (such as cell lysate), 120 mu L of cell solution and 60 mu L of cell marker solution (such as microspheres with nucleic acid sequences) into each sample storage cavity on the chip by using a pipette gun;
(3) controlling the pressure of an oil phase solution, a reaction reagent solution (such as cell lysate), a cell solution and a cell marker solution (such as microspheres with nucleic acid sequences) to be 120mBar, 100mBar and 250mBar respectively by using an air pressure control device, and mixing the cell marker solution (such as microspheres with nucleic acid sequences) and the cell solution at one position of a chip flow channel to form a mixed solution A;
(4) the mixed solution A and the reaction reagent solution are mixed into a mixed solution B at the other position of the chip flow channel;
(5) the oil phase solution cuts the mixed solution B at the cross structure of the chip flow channel to generate water-in-oil droplets, generates droplets, wraps cells, cell markers (such as microspheres with nucleic acid sequences) and reaction reagents (such as cell lysate), flows into a droplet storage cavity, and finishes sample processing before single cell sequencing and library building.
As shown in FIG. 5, the sample obtained in the above example was analyzed by the bioanalyzer 2100, and the sequencing library quality control data, the length and the concentration of the sample were all expected.
The embodiments described above were chosen and described in order to best explain the principles of the invention, but are not intended to be exhaustive or to limit the invention to the precise forms disclosed, and many modifications and variations are possible to those skilled in the art to best utilize the invention, the scope of which is defined by the appended claims.
Claims (11)
1. The utility model provides a micro-fluidic chip, its characterized in that, includes upper segment part and lower segment part, one side of upper segment part is equipped with oil phase solution introduction port 1, reaction reagent solution introduction port 2, cell solution introduction port 3, cell marker solution introduction port 4, droplet export 5, one side of lower segment part is equipped with microchannel 6 for accomplish and generate droplet parcel cell marker, cell and reaction reagent.
2. The microfluidic chip according to claim 1, wherein the micro flow channel 6 comprises:
(1) a first channel having one end connected to the cell solution injection port 3 and the other end connected to the droplet generation intersection;
(2) a second channel, one end of which is connected with the oil phase solution injection port 1 and the other end of which is connected with the droplet generation intersection part;
(3) a third channel having one end connected to the droplet generation intersection and the other end connected to the droplet outlet;
(4) a fourth channel, which is positioned between the first channel and the second channel, and one end of which is connected with the cell marker solution injection port 4 and the other end of which is connected with the first channel;
(5) and the fifth channel is positioned between the first channel and the second channel, one end of the fifth channel is connected with the reaction reagent solution injection port 2, and the other end of the fifth channel is connected with the first channel.
3. The microfluidic chip according to claim 2, wherein at least one of the first channel, the second channel, the fourth channel and the fifth channel has a cross section that is narrowed toward a liquid flowing direction.
4. The microfluidic chip of claim 2, wherein the channel has a cross-sectional width of between 20 μm and 300 μm.
5. The microfluidic chip according to claim 4, wherein the width of the cross section of the channel is between 40 μm and 80 μm.
6. The microfluidic chip according to claim 2, wherein a plurality of support micro-pillars 7 are disposed in said channel.
7. The microfluidic chip according to claim 6, wherein the supporting micro-pillars 7 are cylindrical structures.
8. The microfluidic chip according to claim 1, wherein each sample inlet is provided with a sample storage chamber, and the droplet outlet is provided with a droplet storage chamber.
9. The microfluidic chip of claim 8, wherein the volume of the droplet storage chamber is between 30 μ L and 700 μ L.
10. A method for single cell sequencing sample processing using the microfluidic chip of any of claims 1-9, comprising:
(1) respectively transferring the oil phase solution, the reaction reagent solution, the cell solution and the cell marker solution into each storage cavity on the chip;
(2) controlling the flow rate of each solution by using an air pressure control device, and mixing the cell marker solution, the cell solution and the reaction reagent solution in the chip flow channel;
(3) the oil phase solution cuts the mixed solution at the cross structure of the chip flow channel to generate water-in-oil liquid drops, so that the liquid drops are generated, cells, cell markers and reaction reagents are wrapped, and the sample treatment before the single cell sequencing and library building is completed.
11. The method of claim 10, wherein step (2) comprises:
(a) controlling the flow rate of each solution by using an air pressure control device, and mixing the cell marker solution and the cell solution into a mixed solution A at one position of a chip flow channel;
(b) the mixed solution A and the reaction reagent solution are mixed at the other position of the chip flow channel to form mixed solution B.
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| PCT/CN2021/129696 WO2023284191A1 (en) | 2021-07-16 | 2021-11-10 | Microfluidic chip for single cell sequencing and application thereof |
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| CN115920991A (en) * | 2022-12-23 | 2023-04-07 | 中国科学院基础医学与肿瘤研究所(筹) | Micro-fluidic chip and method for sorting microorganisms |
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| CN114717100B (en) * | 2021-07-16 | 2024-03-19 | 墨卓生物科技(浙江)有限公司 | Microfluidic chip for single-cell sequencing and application |
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| CN114717100B (en) | 2024-03-19 |
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