CN109900624A - A kind of unicellular separator and method based on micro-fluidic chip - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 19
- 238000001514 detection method Methods 0.000 claims abstract description 47
- 238000000926 separation method Methods 0.000 claims abstract description 14
- 238000005370 electroosmosis Methods 0.000 claims description 41
- 239000006285 cell suspension Substances 0.000 claims description 40
- 210000005266 circulating tumour cell Anatomy 0.000 claims description 33
- 238000010790 dilution Methods 0.000 claims description 16
- 239000012895 dilution Substances 0.000 claims description 16
- 210000004027 cell Anatomy 0.000 claims description 14
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 12
- 238000005183 dynamical system Methods 0.000 claims description 10
- 238000002347 injection Methods 0.000 claims description 10
- 239000007924 injection Substances 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- 230000000694 effects Effects 0.000 claims description 7
- 239000002699 waste material Substances 0.000 claims description 7
- 230000015572 biosynthetic process Effects 0.000 claims description 6
- 229910052697 platinum Inorganic materials 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 239000002861 polymer material Substances 0.000 claims description 3
- 238000003672 processing method Methods 0.000 claims description 3
- 238000001039 wet etching Methods 0.000 claims description 3
- 230000003111 delayed effect Effects 0.000 claims 1
- 238000011010 flushing procedure Methods 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000005259 measurement Methods 0.000 abstract description 2
- 238000011160 research Methods 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 208000005443 Circulating Neoplastic Cells Diseases 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000001746 injection moulding Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000013019 agitation Methods 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000002032 lab-on-a-chip Methods 0.000 description 1
- 238000011528 liquid biopsy Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000003746 surface roughness Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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Abstract
一种基于微流控芯片的单细胞分离装置和方法，装置包括微流控芯片，微流控芯片配备有动力系统，微流控芯片依次设有单细胞排列区、单细胞电阻检测区和单细胞分离区；单细胞电阻检测区设有电阻检测上电极、电阻检测下电极，电阻检测电极和配备有虚拟仪器界面的计算机连接；单细胞分离区采用四路分支结构，并设有电渗驱动右电极和电渗驱动左电极，电渗驱动电极和计算机连接；分离方法通过结合细胞电阻测量和电渗驱动技术，实现单细胞自动分离，大大提升了单细胞生成效率和生成纯度。
A single cell separation device and method based on a microfluidic chip, the device comprises a microfluidic chip, the microfluidic chip is equipped with a power system, and the microfluidic chip is sequentially provided with a single cell arrangement area, a single cell resistance detection area and a single cell resistance detection area. Cell separation area; single cell resistance detection area is equipped with resistance detection upper electrode, resistance detection lower electrode, resistance detection electrode is connected with computer equipped with virtual instrument interface; single cell separation area adopts four-way branch structure and is equipped with electroosmotic drive The right electrode and the electroosmotic drive left electrode, and the electroosmotic drive electrode is connected to the computer; the separation method realizes the automatic separation of single cells by combining cell resistance measurement and electroosmotic drive technology, which greatly improves the generation efficiency and purity of single cells.
Description
Technical field
It is the invention belongs to micro-fluidic single-cell technique field, in particular to a kind of based on the unicellular of micro-fluidic chip
Separator and method.
Background technique
Circulating tumor cell (CTCs) is the tumour cell entered in peripheral blood by primary tumors, is the weight of liquid biopsy
Want marker.The research of CTCs is primarily limited to two aspects, first is that CTCs content is few, every milliliter of blood in tumor patient body
CTCs content is about at 1-10, it is difficult to carry out large sample statistical analysis (Gwak, Hogyeong, et al. " Progress in
Circulating Tumor Cell Research Using Microfluidic Devices."Micromachines 9.7
(2018))；In addition, extremely strong (Kulasinghe, Arutha, et al. " the Capture of of single CTCs specificity
Circulating Tumour Cell Clusters Using Straight Microfluidic Chips."Cancers
11.1(2019)).In view of above two big challenges, the unicellular application of CTCs is of great significance.
Firstly the need of single CTC is separated, can be realized unicellular isolated method at present is mainly the unicellular application of CTCs
Both unicellular sorting may be implemented in flow cytometer and micrurgy, lay a good foundation for unicellular research.But it flows
Formula cell instrument is expensive, bulky, and it is universal to can not achieve large area；Micrurgy requires height to operator, and imitates
Rate is low, it is difficult to meet research needs (Gao, Dan, et al. " Recent advances in single cell
manipulation and biochemical analysis on microfluidics."Analyst 144.3(2019):
766-781；Huang,Qiushi,et al."Single-cell assay on microfluidic devices."
Analyst 144.3(2019):808-823.).In addition to this, obtaining single celled method, there are also limiting dilution assays and drop
Method, though limiting dilution assay operation is simple, efficiency generally only 20%, needs experimenter to carry out observation exclusion, time-consuming and laborious
(Gross,Andre,et al."Technologies for Single-Cell Isolation."International
Journal of Molecular Sciences 16.8(2015):16897-16919.)；Sessile drop method is due to can be generated nanoliter
The even unicellular drop of picoliters magnitude is particularly suitable for micro- reaction, but CTCs research is needed to handle single drop demulsification,
Increase experimental procedure；The unicellular separation method of drop formation method obeys Poisson point since the drop of generation is coated with unicellular number
Cloth, production rate lower than 30% (Collins, D.J., et al. " The Poisson distribution and beyond:
methods for microfluidic droplet production and single cell encapsulation."
Lab on a Chip 15.17(2015):3439-3459.)。
To sum up, the prior art haves the shortcomings that unicellular formation efficiency, generation purity are low.
Summary of the invention
In order to overcome the disadvantages of the above prior art, the purpose of the present invention is to provide a kind of lists based on micro-fluidic chip
Cell separation apparatus and method, by combination cell resistance measurement and driven by electroosmosis technology, it can be achieved that unicellular be automatically separated, greatly
Unicellular formation efficiency is improved greatly and generates purity.
In order to achieve the above object, the technical scheme adopted by the invention is as follows:
A kind of unicellular separator based on micro-fluidic chip, including micro-fluidic chip, micro-fluidic chip is equipped with dynamic
Force system, dynamical system effect be that dilution for many times cell suspension, buffer are injected into micro-fluidic chip, micro-fluidic chip according to
It is secondary to be equipped with unicellular alignment area 100, unicellular resistance detection area 200 and unicellular Disengagement zone 300；
The unicellular alignment area 100 includes cell suspension circulation duct 101,101 entrance of cell suspension circulation duct
External dilution for many times CTCs cell suspension；
The unicellular resistance detection area 200 includes downstream line 201, the entrance and cell suspension of downstream line 201
The outlet of circulation duct 101 connects into straight channel, and downstream line 201 is equipped with resistance detection top electrode 202, downstream line 201 above
Electrode 203 under resistance detection are arranged below, electrode 203 and computer 204 connect under resistance detection top electrode 202, resistance detection；
The unicellular Disengagement zone 300 includes Disengagement zone pipeline, and Disengagement zone pipeline is four road branched structures, Disengagement zone pipe
Road entrance and the outlet of downstream line 201 connect into straight channel, and Disengagement zone pipeline is equipped with buffer injection port 301, unicellular
Outlet 302 and waste liquid outlet 304, the interior electric osmose that is equipped with of Disengagement zone pipeline between buffer injection port 301 and waste liquid outlet 304 are driven
Move right electrode 303 and the left electrode 305 of driven by electroosmosis, the right electrode 303 of driven by electroosmosis, the left electrode 305 of driven by electroosmosis and computer 204
Connection.
The CTCs cell equivalent diameter that 101 diameter of cell suspension circulation duct is 3-6 times.
The CTCs cell equivalent diameter that the diameter of the downstream line 201 is 3-6 times.
203 width of electrode is 50 μm, 100 μm of length under the resistance detection top electrode 202, resistance detection；Resistance inspection
Surveying electrode 203 under top electrode 202, resistance detection is platinum electrode.
The Disengagement zone pipe diameter is 3-6 times of CTCs cell equivalent diameter, and 302 entrances of unicellular outlet are one
Grading structure.
The right electrode 303 of the driven by electroosmosis and left 305 length of electrode of driven by electroosmosis are 60 μm, and 20 μm of width, electric osmose is driven
It moves right electrode 303 and the left electrode 305 of driven by electroosmosis is platinum electrode.
PCR pipe is directly docked in the unicellular outlet 302, loads the single cell suspension of generation.
The micro-fluidic chip area is no more than 10 square centimeters, 4~5cm of fluid channel total length.
The micro-fluidic chip material is glass or high molecular polymer material, and processing method is wet etching, number
Control CNC or die sinking injection molding.
A kind of separation method of the unicellular separator based on micro-fluidic chip, comprising the following steps:
1) dilution for many times CTCs cell suspension injects micro-fluidic chip pipeline by dynamical system, while passing through dynamical system
Buffer is injected into buffer injection port 301；CTCs cell suspension connects in cell suspension circulation duct 101, downstream line 201
Gradually reach equilbrium position in the straight channel connect in flow process under lift effect, unicellular arrangement is stablized in formation；
2) when dilution for many times CTCs cell suspension flows through unicellular resistance detection area 200, resistance detection top electrode 202 adds
It carries electrode 203 under DC voltage, resistance detection and acquires signal, signal is by 204 continuous acquisition of computer and carries out data processing；
3) when unicellular Disengagement zone 300 be not detected it is unicellular by when, the right electrode 303 of driven by electroosmosis, driven by electroosmosis are left
Electrode 305 is not loaded with driven by electroosmosis voltage；When unicellular Disengagement zone 300 detect it is unicellular by when, the right electrode of driven by electroosmosis
303, the left electrode 305 of driven by electroosmosis loads driven by electroosmosis voltage, realizes unicellular separation.
The invention has the benefit that
(1) micro-fluidic chip of the present invention directly detects unicellular resistance variations, and simultaneously real-time control is slender for real-time data analysis
The collection of born of the same parents, from detecting that separation can complete within the ms time.
(2) present invention improves unicellular generation purity, without sky in the single cell suspension generated using micro-fluidic chip
Suspension and be free of many cells.
(3) the present invention is based on the unicellular separator integrated level of micro-fluidic chip height, high degree of automation, device can be with
Micromation, micro-fluidic chip area is only several square centimeters, the CTCs cell equivalent diameter that fluid channel diameter is 3-6 times, always
4~5cm of length.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of micro-fluidic chip of the present invention.
Fig. 2 is cell single-row arrangement schematic diagram in cell suspension circulation duct 101 of the present invention.
Fig. 3 is the flow chart of the method for the present invention.
Specific embodiment
The present invention is described in detail below in conjunction with drawings and examples, it is clear that described embodiment is only
It is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill people
Member's every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
As shown in Figure 1, a kind of unicellular separator based on micro-fluidic chip, including micro-fluidic chip, micro-fluidic core
Piece is used as dynamical system equipped with syringe pump, constant pressure pump or peristaltic pump etc., and dynamical system effect is to hang dilution for many times cell
Liquid, buffer are injected into micro-fluidic chip, and micro-fluidic chip is successively arranged unicellular alignment area 100, unicellular resistance detection
Area 200 and unicellular Disengagement zone 300；
The unicellular alignment area 100 includes cell suspension circulation duct 101,101 entrance of cell suspension circulation duct
External dilution for many times CTCs cell suspension, concentration of cell suspension are 100 cell/mL；It, can in order to keep cell liquid concentration uniform
Using the methods of magnetic agitation and oscillation, to avoid that cell settlement or reunion occurs；
The CTCs cell equivalent diameter that 101 diameter of cell suspension circulation duct is 3-6 times；Hydrodynamics it is found that
Cell suspension flows through straight pipe channel, and under wall surface induced lift and shear-induced lift collective effect, cell can gradually reach flat
Weighing apparatus position, since experiment sample is the CTCs cell suspension of dilution for many times, stable unicellular arrangement can be formed.
The unicellular resistance detection area 200 includes downstream line 201, the entrance and cell suspension of downstream line 201
The outlet of circulation duct 101 connects into straight channel, and downstream line 201 is equipped with resistance detection top electrode 202, downstream line 201 above
Electrode 203 under resistance detection are arranged below, electrode 203 and computer 204 connect under resistance detection top electrode 202, resistance detection；
According to Coulter-counter principle, when thering is cell to pass through between electrode 203 under resistance detection top electrode 202, resistance detection, carefully
Born of the same parents' occupy-place causes resistance between two electrodes to change；Resistance changes can generate impact peak between the electrodes；Computer 204 is responsible for providing
The control voltage signal of resistance detection top electrode 202, and acquire and handle the signal that electrode 203 returns under resistance detection, identification
The unicellular signal impact peak generated when passing through out.
The CTCs cell equivalent diameter that the diameter of the downstream line 201 is 3-6 times.
203 width of electrode is 50 μm, 100 μm of length under the resistance detection top electrode 202, resistance detection；Resistance inspection
Surveying electrode 203 under top electrode 202, resistance detection is platinum electrode.
The unicellular Disengagement zone 300 includes Disengagement zone pipeline, and Disengagement zone pipeline is four road branched structures, Disengagement zone pipe
Road entrance and the outlet of downstream line 201 connect into straight channel, and Disengagement zone pipeline is equipped with buffer injection port 301, unicellular outlet
302 and waste liquid outlet 304, it is right that driven by electroosmosis is equipped in the Disengagement zone pipeline between buffer injection port 301 and waste liquid outlet 304
Electrode 303 and the left electrode 305 of driven by electroosmosis, the right electrode 303 of driven by electroosmosis, the left electrode 305 of driven by electroosmosis and computer 204 connect
It connects；When computer 204 does not detect unicellular flow through, the cell suspension of unicellular Disengagement zone is flowed into buffer injection port
Waste liquid outlet 304 is flowed under the fluid matasomatism of buffer in 301；When computer 204 has detected unicellular pass through, calculate
Machine 204 controls the right electrode 303 of driven by electroosmosis and driven by electroosmosis left electrode 305 and single cell suspension driving is flowed into unicellular outlet
302。
The Disengagement zone pipe diameter is 3-6 times of CTCs cell equivalent diameter, and 302 entrances of unicellular outlet are one
Grading structure.
The right electrode 303 of the driven by electroosmosis and left 305 length of electrode of driven by electroosmosis are 60 μm, and 20 μm of width, electric osmose is driven
It moves right electrode 303 and the left electrode 305 of driven by electroosmosis is platinum electrode.
PCR pipe is directly docked in the unicellular outlet 302, loads the single cell suspension of generation.
As shown in Fig. 2, the function of the unicellular alignment area 100 be dilution for many times cell suspension is formed it is uniform steady
Fixed unicellular stream.
The micro-fluidic chip material is glass or high molecular polymer material, and processing method is wet etching, number
Control the methods of CNC or die sinking injection molding, it is desirable that its internal duct surface roughness is smaller, avoids that cell wall built-up phenomenon occurs；
The problems such as section circle or ellipse of pipeline, the dead volume for avoiding rectangular pipeline from generating.
As shown in figure 3, a kind of separation method of unicellular separator based on micro-fluidic chip, including it is following
Step:
1) dilution for many times CTCs cell suspension injects micro-fluidic chip pipeline by dynamical system, while passing through dynamical system
Buffer is injected into buffer injection port 301；CTCs cell suspension connects in cell suspension circulation duct 101, downstream line 201
Gradually reach equilbrium position in the straight channel connect in flow process under lift effect, unicellular arrangement is stablized in formation；
2) when dilution for many times CTCs cell suspension flows through unicellular resistance detection area 200, resistance detection top electrode 202 adds
It carries electrode 203 under DC voltage, resistance detection and acquires signal, signal is by 204 continuous acquisition of computer and carries out data processing；
3) when unicellular Disengagement zone 300 be not detected it is unicellular by when, the right electrode 303 of driven by electroosmosis, driven by electroosmosis are left
Electrode 305 is not loaded with driven by electroosmosis voltage；When unicellular Disengagement zone 300 detect it is unicellular by when, the right electrode of driven by electroosmosis
303, the left electrode 305 of driven by electroosmosis loads driven by electroosmosis voltage, realizes unicellular separation.
Here, it should also be noted that, in order to avoid having obscured the present invention because of unnecessary details, in the accompanying drawings only
Show with closely related structure and/or processing step according to the solution of the present invention, and be omitted little with relationship of the present invention
Other details.
Claims (10)
1. a kind of unicellular separator based on micro-fluidic chip, including micro-fluidic chip, micro-fluidic chip is equipped with dynamic
System, dynamical system effect is that dilution for many times cell suspension, buffer are injected into micro-fluidic chip, it is characterised in that: micro-
Fluidic chip is successively arranged unicellular alignment area (100), unicellular resistance detection area (200) and unicellular Disengagement zone (300)；
The unicellular alignment area (100) includes cell suspension circulation duct (101), and cell suspension circulation duct (101) enters
The external dilution for many times CTCs cell suspension of mouth；
The unicellular resistance detection area (200) includes downstream line (201), and the entrance and cell of downstream line (201) are outstanding
Liquid stream thread a pipe (101) outlet connection, cell suspension circulation duct (101) and downstream line (201) connect into straight channel, downstream
Pipeline (201) is equipped with resistance detection top electrode (202) above, and electrode under resistance detection is arranged below in downstream line (201)
(203), electrode (203) and computer (204) connection under resistance detection top electrode (202), resistance detection；
The unicellular Disengagement zone (300) includes Disengagement zone pipeline, and Disengagement zone pipeline is four road branched structures, Disengagement zone pipeline
Entrance and downstream line (201) outlet connection, Disengagement zone pipeline and downstream line (201) connect into straight channel, Disengagement zone pipeline
Equipped with buffer injection port (301), unicellular outlet (302) and waste liquid outlet (304), buffer injection port (301) and waste liquid
It exports and is equipped with the right electrode of driven by electroosmosis (303) and the left electrode of driven by electroosmosis (305), electric osmose in the Disengagement zone pipeline between (304)
Drive right electrode (303), the left electrode of driven by electroosmosis (305) and computer (204) connection.
2. a kind of unicellular separator based on micro-fluidic chip according to claim 1, it is characterised in that: described
The CTCs cell equivalent diameter that cell suspension circulation duct (101) diameter is 3-6 times.
3. a kind of unicellular separator based on micro-fluidic chip according to claim 1, it is characterised in that: described
The CTCs cell equivalent diameter that the diameter of downstream line (201) is 3-6 times.
4. a kind of unicellular separator based on micro-fluidic chip according to claim 1, it is characterised in that: described
Electrode (203) width is 50 μm, 100 μm of length under resistance detection top electrode (202), resistance detection；Resistance detection top electrode
(202), electrode (203) is platinum electrode under resistance detection.
5. a kind of unicellular separator based on micro-fluidic chip according to claim 1, it is characterised in that: described
Disengagement zone pipe diameter is 3-6 times of CTCs cell equivalent diameter, and unicellular outlet (302) entrance is a grading structure.
6. a kind of unicellular separator based on micro-fluidic chip according to claim 1, it is characterised in that: described
The right electrode of driven by electroosmosis (303) and driven by electroosmosis left electrode (305) length are 60 μm, 20 μm of width, the right electrode of driven by electroosmosis
(303) and the left electrode of driven by electroosmosis (305) is platinum electrode.
7. a kind of unicellular separator based on micro-fluidic chip according to claim 1, it is characterised in that: described
Unicellular outlet (302) directly docks PCR pipe, loads the single cell suspension of generation.
8. a kind of unicellular separator based on micro-fluidic chip according to claim 1, it is characterised in that: described
Micro-fluidic chip area is no more than 10 square centimeters, 4~5cm of fluid channel total length.
9. a kind of unicellular separator based on micro-fluidic chip according to claim 1, it is characterised in that: described
Micro-fluidic chip material is that perhaps high molecular polymer material processing method is wet etching, numerical control CNC or die sinking note to glass
Modeling.
10. a kind of separation method of unicellular separator based on micro-fluidic chip according to claim 1, feature
It is, comprising the following steps:
1) dilution for many times CTCs cell suspension injects micro-fluidic chip pipeline by dynamical system, while will be delayed by dynamical system
Fliud flushing is injected into buffer injection port (301)；CTCs cell suspension is in cell suspension circulation duct (101), downstream line (201)
Gradually reach equilbrium position in the straight channel of connection in flow process under lift effect, unicellular arrangement is stablized in formation；
2) when dilution for many times CTCs cell suspension flows through unicellular resistance detection area (200), resistance detection top electrode (202) adds
It carries electrode (203) under DC voltage, resistance detection and acquires signal, signal is by computer (204) continuous acquisition and carries out at data
Reason；
3) when unicellular Disengagement zone (300) be not detected it is unicellular by when, the right electrode of driven by electroosmosis (303), driven by electroosmosis are left
Electrode (305) is not loaded with driven by electroosmosis voltage；When unicellular Disengagement zone (300) detect it is unicellular by when, driven by electroosmosis is right
Electrode (303), the left electrode of driven by electroosmosis (305) load driven by electroosmosis voltage, realize unicellular separation.
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