CN108949496A - A kind of unicellular separation method based on drop micro-fluidic chip - Google Patents
A kind of unicellular separation method based on drop micro-fluidic chip Download PDFInfo
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Abstract
The present invention provides a kind of unicellular separation methods based on drop micro-fluidic chip, the specific steps are as follows: single cell suspension is flowed into dispersed phase feed pathway from dispersed phase entrance by A.；B. oil phase liquid is flowed into continuous phase feed pathway from continuous phase entrance；C. two-phase is converged to form the single celled drop of package, and with drop formations a large amount of in liquid storage tank, drop is flowed through above droplet capture unit, stands 2~5min, and when drop is slowly settled into droplet capture unit, residual droplets are sucked out；D. single celled drop chip will be captured and be placed in 37 DEG C of incubator cultures, DAPI is then added and carries out core dye, detects unicellular capture rate.The present invention has the advantages that simple and convenient for operation；Cell is few with reagent dosage, and experimental cost is cheap；Highgrade integration；It has wide range of applications.
Description
Technical field
The invention belongs to microflow control techniques and cell biology, and in particular to a kind of based on drop micro-fluidic chip
Unicellular separation method.
Background technique
Microfluid based Lab on a chip is sample preparation, reaction, separation, detection involved in the fields such as chemistry and biology
And the basic operation units such as cell culture, separation, division are integrated on the chip of one piece several square centimeters (even more small), by micro-
Channel forms network, runs through whole system with controlled fluid, to replace the various functions of conventional chemical and biology laboratory
A kind of technology platform.The essential characteristic and sharpest edges of micro-fluidic chip are a variety of monotechnicses in whole controllable small platform
Upper flexible combination, scale are integrated.High throughput is a kind of integrated form of scale, and the operating unit or one-element group being only integrated are
It is identical.
Biology and medical research discovery in recent years, have important meaning for the RESEARCH ON CELL-BIOLOGY of individual cells
Justice.Same cell may also have very big difference between different cell individuals, and the most significant cell that makes a variation tends to take off
Show important biomolecule phenomenon or prompt the generation of major disease, disclose pathogenesis of cancer mechanism, understands cell differentiation and group hair-weaving
Principle is educated, identifies gene expression characteristics and cell characteristic.Therefore, it accurately captures the single cell population of significant difference and carries out accurate
Identification is the key technology being badly in need of in biomedical research.
With the rapid development of drop microflow control technique, a kind of method for manipulating micro-meter scale fluid is provided for people,
Its application in single cell analysis causes more and more concerns.Drop can be used as independent unicellular microreactor, energy
Enough effectively control diffusions, improve detection sensitivity, have been successfully applied in a variety of single cell analysis.In addition, from a large amount of sample
Specific cells are accurately captured in this (such as blood) and identification identification and traditional technology (such as flow cytometer) hardly possible are carried out to it
With realization.
The present invention realizes single celled high-throughput isolation, whole device knot using polymer chip and drop microflow control technique
Structure is simple, without any complicated and expensive equipment, it can be achieved that high throughput, easy to operate, can quick separating suspend the list cultivated
Cell reduces cellular damage and is convenient for the cell experiment in later period.
Summary of the invention
The object of the present invention is to provide a kind of unicellular separation methods based on drop micro-fluidic chip, previous single to solve
Complicated, limitation, the present invention such as be easy to cause cellular damage, capture rate low complex for operation step present in cell separation process
Preparation process is stablized, easy to operate, and integrated level is high.
A kind of drop micro-fluidic chip, is formed by two layers, and upper layer is drop formation unit, and lower layer is droplet capture unit；
The chip liquid drop generation unit is specifically provided with following structures: dispersed phase entrance, continuous phase entrance, dispersed phase into
Liquid channel, continuous phase feed pathway, drop formation cross aisle, liquid outflow channel, liquid outlet, liquid storage tank；The chip
Dispersed phase entrance connects dispersed phase feed pathway；
The continuous phase entrance of the chip connects continuous phase feed pathway；
The Liang Ge feed pathway intersection of the chip intersects in " ten " word, forms drop formation cross aisle；
The liquid outflow channel connection drop formation cross aisle and liquid outlet of the chip；
The chip includes an open liquid storage tank, and liquid storage tank connects liquid outlet, is located above droplet capture unit.
The droplet capture unit of the chip is cylindrical recess pitting structure, shares 5000~10000；
The diameter of the droplet capture unit is 100~300 μm, is highly 100~200 μm, and each cell spacing is 50
~100 μm；
A kind of unicellular separation method based on drop micro-fluidic chip, the specific steps are as follows:
A. single cell suspension is flowed into dispersed phase feed pathway from dispersed phase entrance；The chip dispersed phase flow velocity is 0.5
~10 μ L/min；
B. oil phase liquid is flowed into continuous phase feed pathway from continuous phase entrance；Continuous phase flow velocity is 0.5~20 μ L/min；
C. two-phase is converged to form the single celled drop of package, and with drop formations a large amount of in liquid storage tank, drop flows through drop
Above capturing unit, 2~5min is stood, when drop is slowly settled into droplet capture unit, residual droplets are sucked out；
D. single celled drop chip will be captured and be placed in 37 DEG C of incubator cultures, DAPI is then added and carries out core dye, detection
Unicellular capture rate.
The dispersion phase constituent of the chip is single cell suspension, and single cell suspension diameter is 10~20 μm, single cell suspension
Density is 103Cells/mL~108cells/mL。
Span 80 in the mineral oil of the continuous phase of the chip containing 0.01~0.1g/mL；
The single celled drop of the package that the chip is prepared is water-in-oil emulsion drop, and liquid-drop diameter is 80~300 μm；
The droplet capture unit size of the chip only should be slightly bigger than drop size, it is ensured that each droplet capture unit only has
One drop exists.
It is described it is unicellular be wrapped in drop, subsequently enter in droplet capture unit, the ruler of drop and droplet capture unit
It is very little to meet cell long-period growth, proliferation；
The unicellular separative efficiency of the chip is by single cell suspension flow velocity and single cell suspension density synergy；
The chip can be used as high-throughput unicellular capture, can carry out unicellular Culture in situ, administration, proliferation, induction point
The researchs such as change, unicellular sequencing.
The present invention has the advantages that
1, simple and convenient for operation；
2, cell and reagent dosage are few, and experimental cost is cheap；
3, Highgrade integration；
4, it has wide range of applications；
Detailed description of the invention
Fig. 1 is chip structure schematic diagram overall structure figure,
Fig. 2 is micro-fluidic chip superstructure figure；
Fig. 3 is micro-fluidic chip understructure figure；
Fig. 4 be partial enlarged view at cross aisle；
Fig. 5 is the front view of micro-fluidic chip；
Wherein: 1 be dispersed phase entrance, 2 be continuous phase entrance, 3 be dispersed phase feed pathway, 4 be continuous phase feed pathway,
5 it is drop formation cross aisle, 6 be liquid outflow channel, 7 be liquid outlet, 8 be liquid storage tank, 9 is droplet capture unit.
Fig. 6 is micro-fluidic chip pictorial diagram.
Fig. 7 is that micro-fluidic chip captures unicellular flow chart,
Fig. 8 is the drop picture of micro-fluidic chip capture,
Fig. 9 is that drop micro-fluidic chip captures single celled localized fluorescence picture,
Figure 10 is the statistical chart of unicellular capture rate.
Specific embodiment
A kind of drop micro-fluidic chip, as shown in figs. 1 to 6, the chip is formed by two layers, and upper layer is drop formation list
Member, lower layer are droplet capture unit；
The drop formation unit is specifically provided with following structures: dispersed phase entrance 1, continuous phase entrance 2, dispersed phase feed liquor
Channel 3, continuous phase feed pathway 4, drop formation cross aisle 5, liquid outflow channel 6, liquid outlet 7, liquid storage tank 8；
The dispersed phase entrance 1 of the chip connects dispersed phase feed pathway 3；
The continuous phase entrance 2 of the chip connects continuous phase feed pathway 4；
The Liang Ge feed pathway intersection of the chip intersects in " ten " word, forms drop formation cross aisle 5；
The liquid outflow channel 6 of the chip connects drop formation cross aisle 5 and liquid outlet 7；
The chip includes an open liquid storage tank 8, and liquid storage tank connects liquid outlet 7, is located on droplet capture unit 9
Side.
The chip is formed by two layers, and upper layer is drop formation unit, and lower layer is droplet capture unit；The chip includes
Following structures: dispersed phase entrance 1, continuous phase entrance 2, dispersed phase feed pathway 3, continuous phase feed pathway 4, drop formation cross
Channel 5, liquid outflow channel 6, liquid outlet 7, liquid storage tank 8, droplet capture unit 9.
Embodiment 1
A kind of preparation for unicellular isolated drop micro-fluidic chip
The preparation of the superstructure SU-8 template of chip: micro-fluidic chip prepares channel part using photoetching and caustic solution
Divide the SU-8 template of protrusion；Firstly, getting rid of one layer of SU-8 glue on silicon wafer, with a thickness of 100 μm, 95 DEG C of front baking 20min, drop naturally
Exposure mask is placed in SU-8 glue washer by temperature, uv-exposure 30s, dries 20min, Temperature fall after 95 DEG C；It will using ethyl lactate
Above-mentioned SU-8 glue development 10min, 180 DEG C of post bake 2h, Temperature fall are spare.
The preparation of the understructure SU-8 template of chip: firstly, getting rid of one layer of SU-8 glue, on silicon wafer with a thickness of 150 μm, 95
Exposure mask is placed in SU-8 glue washer by DEG C front baking 30min, Temperature fall, uv-exposure 45s, dries 30min after 95 DEG C, natural
Cooling；Above-mentioned SU-8 glue is developed 10min, 180 DEG C of post bake 2h using ethyl lactate, Temperature fall is spare.
Embodiment 2:
Preparation for unicellular isolated PDMS chip
PDMS is uniformly mixed with initiator with volume ratio 10:1, be cast in early period preparation two SU-8 templates, 80 DEG C
PDMS and SU-8 template are removed, are obtained with structured upper layer chip and lower layer chip by curing oven 30min；By upper layer
PDMS chip is cut into penetrating square hollow place along liquid storage tank edge, with punch in dispersed phase entrance and continuous phase entrance
Punching；By the chip upper and lower level band structure plasma treated 120s in side, it is spare to be bonded sealing-in.
Embodiment 3
Single cell suspension density is 104When cells/mL, the unicellular experiment of chip automatic capture
The micro-fluidic chip of above-mentioned preparation is impregnated through 75% ethyl alcohol, after ultraviolet irradiation 1h sterilization treatment, then, by people's colloid
Oncocyte (U87) single cell suspension is with 104The cell density of cells/mL flows through dispersed phase feed pathway 3 from dispersed phase entrance 1,
Flow velocity is 1 μ L/min；Mineral oil containing 3% (w/w) span80 is flowed through into continuous phase feed pathway 4 from continuous phase entrance 2, is flowed
Speed is 3.5 μ L/min, and two-phase liquid converges in 5 infall of cross aisle, and single cell suspension is cut into Water-In-Oil drop by mineral oil；
Unicellular to be retained in drop, drop passes through liquid outflow channel 6, is flowed into liquid storage tank 8 from liquid outlet 7；With a large amount of packages
Single celled drop formation, drop are flowed through 9 top of droplet capture unit, stand 3min and slowly settled due to gravity into liquid
Extra drop is sucked out when whole drops are filled with droplet capture unit in drop capturing unit, and chip is placed in 37 DEG C of incubators
Culture is then added DAPI and carries out core dye, detects unicellular capture rate.
Embodiment 4
Single cell suspension density is 105When cells/mL, the unicellular experiment of chip automatic capture
The micro-fluidic chip of above-mentioned preparation is impregnated through 75% ethyl alcohol, after ultraviolet irradiation 1h sterilization treatment, then, by people's colloid
Oncocyte (U87) single cell suspension is with 105The cell density of cells/mL flows through dispersed phase feed pathway 3 from dispersed phase entrance 1,
Flow velocity is 0.5 μ L/min；Mineral oil containing 3% (w/w) span80 is flowed through into continuous phase feed pathway 4 from continuous phase entrance 2,
Flow velocity is 2 μ L/min, and two-phase liquid converges in 5 infall of cross aisle, and single cell suspension is cut into Water-In-Oil drop by mineral oil；
Unicellular to be retained in drop, drop passes through liquid outflow channel 6, is flowed into liquid storage tank 8 from liquid outlet 7；With a large amount of packages
Single celled drop formation, drop are flowed through 9 top of droplet capture unit, stand 3min and slowly settled due to gravity into liquid
Extra drop is sucked out when whole drops are filled with droplet capture unit in drop capturing unit, and chip is placed in 37 DEG C of incubators
Culture is then added DAPI and carries out core dye, detects unicellular capture rate.
Fig. 7 is that micro-fluidic chip captures unicellular flow chart, and Fig. 8 is the drop picture of micro-fluidic chip capture.Fig. 9 is micro-
Fluidic chip captures single celled localized fluorescence picture, and Figure 10 is the statistical chart of unicellular capture rate.According to 2000 counted
For a single celled fluorescence data of droplet capture it is found that prepared drop size is fixed, diameter is 150 μm；Cell density is
104When cells/mL, the unicellular capture rate of chip is 1.00%；Cell density is 105When cells/mL, chip it is slender
Born of the same parents' capture rate is 9.10%；It follows that single cell suspension density is in a certain range when drop formation size constancy
When, cell density is bigger, and it is higher to capture single celled efficiency.This unicellular capture chip can realize the unicellular capture of high efficiency,
Simultaneously, it can be achieved that unicellular long-term cultivation in situ in the chip and the assay in later period.
Claims (11)
1. a kind of drop micro-fluidic chip, it is characterised in that: the chip is formed by two layers, and upper layer is drop formation unit, under
Layer is droplet capture unit；
The drop formation unit is specifically provided with following structures: dispersed phase entrance (1), continuous phase entrance (2), dispersed phase feed liquor
Channel (3), continuous phase feed pathway (4), drop formation cross aisle (5), liquid outflow channel (6), liquid outlet (7), liquid storage
Pond (8)；
The dispersed phase entrance (1) of the chip connects dispersed phase feed pathway (3)；
The continuous phase entrance (2) of the chip connects continuous phase feed pathway (4)；
The Liang Ge feed pathway intersection of the chip intersects in " ten " word, is formed drop formation cross aisle (5)；
Liquid outflow channel (6) connection drop formation cross aisle (5) of the chip and liquid outlet (7)；
The chip includes an open liquid storage tank (8), and liquid storage tank connects liquid outlet (7), is located on droplet capture unit (9)
Side.
2. according to drop micro-fluidic chip described in claim 1, it is characterised in that: the droplet capture unit of the chip is cylinder
The recess pitting structure of shape, shares 5000~10000.
3. according to drop micro-fluidic chip described in claim 1, it is characterised in that: the diameter of the droplet capture unit is 100
~300 μm, be highly 100~200 μm, and each cell spacing is 50~100 μm.
4. according to a kind of unicellular separation method based on drop micro-fluidic chip described in claim 1, it is characterised in that according to
Following steps carry out:
A. dispersed phase being flowed into dispersed phase feed pathway (3) from dispersed phase entrance (1), the dispersed phase is single cell suspension, point
Dephasing flow velocity is 0.5~10 μ L/min；
B. continuous phase is flowed into continuous phase feed pathway (4) from continuous phase entrance (2), the continuous phase is mineral oil, continuous phase
Flow velocity is 0.5~20 μ L/min；
C. two-phase is converged to form the single celled drop of package, and with a large amount of drop formations in liquid storage tank (8), drop flows through drop and catches
It obtains above unit (9), stands 2~5min, when drop is slowly settled into droplet capture unit, residual droplets are sucked out；
D. single celled drop chip will be captured and be placed in 37 DEG C of incubator cultures, DAPI is then added and carries out core dye, detects slender
Born of the same parents' capture rate.
5. according to a kind of unicellular separation method based on drop micro-fluidic chip described in claim 4, it is characterised in that: described
The single cell suspension diameter of the dispersed phase of chip is 10~20 μm, and single cell suspension density is 103Cells/mL~108cells/
mL。
6. according to a kind of unicellular separation method based on drop micro-fluidic chip described in claim 4, it is characterised in that: described
Span 80 in the mineral oil of the continuous phase of chip containing 0.01~0.1g/mL.
7. according to a kind of unicellular separation method based on drop micro-fluidic chip described in claim 4, it is characterised in that: described
The single celled drop of the package that chip is prepared is water-in-oil emulsion drop, and liquid-drop diameter is 80~300 μm.
8. according to a kind of unicellular separation method based on drop micro-fluidic chip described in claim 4, it is characterised in that: described
The droplet capture unit size of chip only should be slightly bigger than drop size, it is ensured that each only one drop of droplet capture unit is deposited
?.
9. according to a kind of unicellular separation method based on drop micro-fluidic chip described in claim 4, it is characterised in that: described
It is unicellular to be wrapped in drop, it subsequently enters in droplet capture unit, the size of drop and droplet capture unit can meet cell
Long term growth, proliferation.
10. according to a kind of unicellular separation method based on drop micro-fluidic chip described in claim 4, it is characterised in that: institute
The unicellular separative efficiency of chip is stated by single cell suspension flow velocity and single cell suspension density synergy.
11. according to a kind of unicellular separation method based on drop micro-fluidic chip described in claim 4, it is characterised in that: institute
The unicellular separation method for stating chip can be used as high-throughput unicellular capture, can carry out unicellular Culture in situ, administration, proliferation,
The researchs such as induction differentiation, unicellular sequencing.
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|---|---|---|---|---|
| CN108940387A (en) * | 2017-05-18 | 2018-12-07 | 中国科学院大连化学物理研究所 | It is a kind of for unicellular isolated drop micro-fluidic chip and preparation method thereof |
| CN109569752A (en) * | 2018-12-22 | 2019-04-05 | 北京工业大学 | A kind of micro-fluidic chip bearing flow for improving the limit after droplet capture |
| CN109609339A (en) * | 2018-12-14 | 2019-04-12 | 华中科技大学同济医学院附属协和医院 | A kind of micro-fluidic chip and its preparation method and application of real-time observation and processing suspension cell |
| CN109718874A (en) * | 2018-12-22 | 2019-05-07 | 北京工业大学 | A kind of separation influences the micro-fluidic chip of drop internal flow behavior variable |
| CN110004043A (en) * | 2019-04-09 | 2019-07-12 | 东北大学 | A kind of unicellular capture micro-fluidic chip |
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| CN110711608A (en) * | 2019-09-23 | 2020-01-21 | 中国科学院上海微系统与信息技术研究所 | Microfluidic chip for cell detection and preparation method thereof |
| CN110747102A (en) * | 2019-10-09 | 2020-02-04 | 山东大学 | Single cell separation device and method based on micro-fluidic chip |
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| CN112410165A (en) * | 2019-08-23 | 2021-02-26 | 北京致雨生物科技有限公司 | Micro-drop type single cell capturing device, capturing system and single cell micro-drop preparation method |
| CN112675935A (en) * | 2021-01-22 | 2021-04-20 | 中国科学院上海微系统与信息技术研究所 | Droplet array chip for single cell freezing and droplet generation method and application |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101570776A (en) * | 2008-04-29 | 2009-11-04 | 中国科学院大连化学物理研究所 | High-throughput nano-litre micro-droplet forming and fixing method based on microfluidic chip and special chip and application thereof |
| CN101947124A (en) * | 2010-06-25 | 2011-01-19 | 博奥生物有限公司 | Integrated microfluidic chip device and using method thereof |
| US20130065795A1 (en) * | 2010-02-16 | 2013-03-14 | Nancy Allbritton | Array of micromolded structures for sorting adherent cells |
| CN104826673A (en) * | 2015-03-16 | 2015-08-12 | 中国科学院微生物研究所 | Writting-type two-dimensional microfluidic drop arraying device, application and use method thereof |
| WO2015200832A1 (en) * | 2014-06-26 | 2015-12-30 | Northeastern University | Microfluidic device and method for analysis of tumor cell microenvironments |
| CN106148187A (en) * | 2016-07-20 | 2016-11-23 | 国家纳米科学中心 | For expressing unicellular sorting and the micro-fluidic chip of polygenic locus detection of EGFR |
| CN108940387A (en) * | 2017-05-18 | 2018-12-07 | 中国科学院大连化学物理研究所 | It is a kind of for unicellular isolated drop micro-fluidic chip and preparation method thereof |
-
2017
- 2017-05-18 CN CN201710351137.5A patent/CN108949496A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101570776A (en) * | 2008-04-29 | 2009-11-04 | 中国科学院大连化学物理研究所 | High-throughput nano-litre micro-droplet forming and fixing method based on microfluidic chip and special chip and application thereof |
| US20130065795A1 (en) * | 2010-02-16 | 2013-03-14 | Nancy Allbritton | Array of micromolded structures for sorting adherent cells |
| CN101947124A (en) * | 2010-06-25 | 2011-01-19 | 博奥生物有限公司 | Integrated microfluidic chip device and using method thereof |
| WO2015200832A1 (en) * | 2014-06-26 | 2015-12-30 | Northeastern University | Microfluidic device and method for analysis of tumor cell microenvironments |
| CN104826673A (en) * | 2015-03-16 | 2015-08-12 | 中国科学院微生物研究所 | Writting-type two-dimensional microfluidic drop arraying device, application and use method thereof |
| CN106148187A (en) * | 2016-07-20 | 2016-11-23 | 国家纳米科学中心 | For expressing unicellular sorting and the micro-fluidic chip of polygenic locus detection of EGFR |
| CN108940387A (en) * | 2017-05-18 | 2018-12-07 | 中国科学院大连化学物理研究所 | It is a kind of for unicellular isolated drop micro-fluidic chip and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| S. SARKAR等: "Phenotypic drug profiling in droplet microfluidics for better targeting of drug-resistant tumors" * |
| SAHELI SARKAR等: "T Cell Dynamic Activation and Functional Analysis in Nanoliter Droplet Microarray" * |
| 赵述芳等: "液滴流微反应器的基础研究及其应用" * |
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