CN206906211U - A kind of circulating tumor cell separating micro-fluidic chip device - Google Patents
A kind of circulating tumor cell separating micro-fluidic chip device Download PDFInfo
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- CN206906211U CN206906211U CN201720315262.6U CN201720315262U CN206906211U CN 206906211 U CN206906211 U CN 206906211U CN 201720315262 U CN201720315262 U CN 201720315262U CN 206906211 U CN206906211 U CN 206906211U
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Abstract
It the utility model is related to a kind of circulating tumor cell separating micro-fluidic chip device, the device includes micro-fluidic chip (1) and chip fixture (2), micro-fluidic chip (1) includes chip entrance (11), single-screw chip (12), rare cell collection conduit (13,14) and leucocyte outlet (15), chip entrance (11) of the single-screw type passage from round screw thread channel center, passage is originated through semicircle, into spiral shape microfluidic channel, end includes 3 outlets：Different-grain diameter size rare cell is collected respectively；Chip fixture (2) automatic butt micro-fluidic chip (1) gateway.Compared with prior art, the utility model micro flow control chip device can automatically to after processing sample carry out automating the tumour cell in the liquid samples such as the separation of high flux tumour cell, enrichment tumour patient peripheral blood/pleural effusion/cerebrospinal fluid, significantly improve the disconnected accurate and personalized treatment specificity of tumor patient and accuracy.
Description
Technical field
Cell sorting field is the utility model is related to, particularly relates to one kind on micro-fluidic chip from biofluid
The device of automatic sorting circulating tumor cell is carried out in sample.
Background technology
In fields such as biomedicines, it is often necessary to from complex sample, sub-elect specific target cell.Such as inspection
Survey, sort and count the circulating tumor cell in cancer patient's blood (CTC, circulating tumor cell) number
In cancer diagnosis and the ever more important that seems in treatment.The good and bad jumbled together for CTC detection techniques at present, be generally exactly using and it is unfavorable
With the major class of label two, the positive concentration method generally included based on antibody coating magnetic bead and feminine gender based on label capture are rich
Collection method.It is such to belong to first generation CTC capture techniques, it is represented as the Cell search systems of Johnson ＆ Johnson.But its technological deficiency is
Antibody capture is limited only to the capture of the epithelial origin CTC to specifically expressing EpCAM epithelium antigens, and can not realize living cells
Capture carries out subsequent gene detection.Negative concentration method reversely removes leucocyte in peripheral blood by CD45 magnetic beads, and this method can not
Enrichment detection is carried out to the micro CTC in substantially product sample, and the non-specific adsorption of magnetic bead be present.First generation technique
Filtering embrane method can only capture big particle diameter tumour cell, missing inspection small particle tumour cell, and the cell captured does not have cytoactive,
It is only used for cell count, morphology and FISH detections.
Micro-fluidic chip is called the support technology of 21 century life science, is the technology core of portable biochemical analytical instrument
The heart.The technology is the passage by building minute yardstick, by the sample preparation involved by the fields such as biological and chemical, biology and chemistry
Reaction Separation is integrated on one piece several square centimeters of chip with basic operation units such as detections, to complete different biologies or
Chemical reaction process, substantial amounts of biomolecule can be analyzed in a short time, the accurate bulk information obtained in sample, information content
It is hundreds and thousands of times of traditional detection means.
To the circulating tumor cell in blood sample sort the report of some existing researchs on micro-fluidic chip, such as
Using dielectrophoresis, fluid dynamic, based on immunomagnetic beads and fluorescence separating method etc..But different degrees of deficiency all be present in this method,
Method such as direct current dielectrophoresis needs to apply higher electric-field intensity, is easy to cause cell to crack；Based on immuno magnetic cell separation
Although method improves expresses tumour cell separative efficiency to specific antigen, but still is confined to tumor-cell antigen epitope
Identification, and circulating tumor cell heterogeneity determines and carries out the capture side based on antigen recognizing using monospecific antibody or several antibody
Method, the missing inspection to non-antigen presentation CTC all be present.And the method for fluorescence sorting needs to carry out cell fluorescence labeling, and
And need to build the Systems for optical inspection of complexity.
Patent 201410336948.4 discloses a kind of circulating tumor cell separation PDMS micro-fluidic chips, including liquid storage hole
A to liquid storage hole F, sense channel, main channel, first, second focus channel, sample export passage, target cell collection passage, electricity
Magnetic separation passage；When circulating tumor cell passes through sense channel, caused voltage signal is detected and is sent to NI collections
Card and processing terminal, processing terminal send voltage signal by NI capture cards, close electromagnetic relay, then make electromagnetic micro valve
Structure oppresses PDMS layer below, brings it about deformation, so as to discharge a part of liquid from liquid storage hole E, promotes circulating tumor thin
Born of the same parents are flowed into target cell collection passage.The chip apparatus needs electric field and magnetic field device to realize the separation of cell.The chip is whole
Body prepares complicated, operating process more and cost is high.
Patent 201410345305.6 discloses a kind of double helix micro-fluidic chip, and it includes red blood cell exit, leucocyte
Outlet, fluid issuing and cell filter membrane, fundamentally also it is to rely on 8 micron pore size filter membranes and realizes to tumour cell filtering point
From such method still can missing inspection small particle tumour cell.
The content of the invention
The purpose of this utility model is exactly to provide a kind of circulating tumor the defects of overcoming above-mentioned prior art to exist
Cell separating micro-fluidic chip device.The utility model is based on circulating tumor cell and haemocyte nucleocytoplasmic ratio and surface charge
Difference, realize that the separation relied on the non-antibody of circulating tumor cell in sample is rich using hydrodynamics on micro-fluidic chip
Collection.
The purpose of this utility model can be achieved through the following technical solutions：A kind of circulating tumor cell separation is micro-fluidic
Chip apparatus, it is characterised in that the device includes micro-fluidic chip and chip fixture, and described micro-fluidic chip enters including chip
Mouth, single-screw chip, rare cell collection conduit and leucocyte outlet, wherein, the rare cell collection conduit includes two,
Inside outlet (13) and outlet (14) are connected respectively；Described single-screw chip is made up of single-screw type passage, the single-screw
Type passage originates passage, into spiral shape microfluidic channel, end from the chip entrance of round screw thread channel center through semicircle
Include 3 outlets：The outlet (13) and outlet (14) of different-grain diameter size rare cell are collected respectively, and collect leucocyte
Waste liquid outlet (15)；
Chip entrance, rare cell collection conduit and the waste liquid of described chip fixture automatic butt micro-fluidic chip go out
Mouthful.
The width of described inside outlet is 110 ± 50 μm, and the width of outlet is 110 ± 50 μm, the width of leucocyte outlet
Spend for 700 ± 100 μm.
Described CTC collection conduits outlet (13) and outlet (14) is carried out at face coat using positive charge polymer
Reason.
Described positive charge polymer is cationic polymer, including polyethyleneimine, acrylamide after modified two
Parent's property polymer.
Described single-screw type passage is main channel, and its width is 500 ± 100 μm, is highly 120 ± 50 μm, Mei Gedan
One spiral spacing is 500 ± 100 μm, and complete helix number is more than or equal to 3.
Described chip fixture includes sample pipe joint, CTC collects pipe joint and waste collection pipe joint；Close chip gripper
Tool, described sample pipe joint dock with chip entrance, described different-grain diameter CTC collect pipe joint and it is described export (13),
(14) dock, the leukocyte collection pipe joint docks with export (15), makes each gateway of micro-fluidic chip and external
Conduit connects, wherein the sample cell joint other end connects biological sample, by syringe pump or pressure pump device by biology
Sample constant speed injects chip apparatus, and its flow velocity is 50-300ml/h.
Flow velocity by biological sample constant speed injection chip apparatus is preferably 60-200ml/h.
Described flow velocity is more preferably 100ml/h, 120ml/h or 150ml/h.
The biological sample is peripheral blood, peripheral blood, pleural effusion, seroperitoneum, cerebrospinal fluid, bone marrow fluid and/or urine
Liquid.
A kind of application method of circulating tumor cell separating micro-fluidic chip device, it is characterised in that comprise the following steps：
(1) blood sample to be measured is used into erythrocyte cracked liquid (erythrocyte cracked liquid for commercially available prod, such as using difficult to understand without sijna
The lysate that biological medicine Co., Ltd produces and sold) cracking processing after, add PBS, be diluted to sample introduction sample；
(2) by supporting syringe pump or pressure pump device, by sample introduction sample in step (1) by conduit from chip fixture
Sample pipe joint injects the chip entrance of micro-fluidic chip, according to the difference of cell nucleocytoplasmic ratio and surface charge in single-screw chip
Inside realize the concentration and separation of circulating tumor cell；
(3) the different-grain diameter CTC cells that concentration and separation goes out collect pipe joint and attached from outlet (13), (14) outflow through CTC
Carrying pipe is exported and accessed in sterile centrifugation tube, and leucocyte exports outflow through leukocyte collection pipe joint and attached from outside leucocyte
Carrying pipe exports.
The volume ratio of PBS and blood sample to be measured described in step (1) is 10~30:1.
Compared with prior art, the utility model biological specimen is when passing through micro-fluidic chip, using fluid in spiral flow
The inertia effect and the effect of Dean streams in road, tumour cell and other cells are focused on apart from the different position of entrance center origin
Put, the tumour cell and leucocyte of different nucleocytoplasmic ratios and surface charge, under hydrodynamic interaction, pool the flat of diverse location
Line, in the flow rates that the utility model limits, cell can bear different fluids because of its nucleocytoplasmic ratio and the difference of electric charge
Power, different cells form difference arrangement spatially in pipeline, finally flowed out from different outlets.Wherein, it is most white
Cell enters blood cells flow road after line is pooled, and most of tumour cell then passes through the charge adsorption effect point in structure runner
Jin Ru not CTC runners.Using the micro-fluidic chip of above-mentioned helical flow path inertia sorting technology structure and charge adsorption Integration ofTechnology,
The defects such as the integrated level that overcomes existing circulating tumor cell separation method is low, capture rate is low, are realized to tumour cell non-antibody
The circulating tumor cell fast high-flux separation of dependence；In addition, the chip structure of the system is simple, easy to process, there is operation
Simply, automaticity height etc. is excellent, available for fields such as rare cell biological study, disease early diagnosis and treatments.
Brief description of the drawings
Fig. 1 is microfluidic chip structure figure of the present utility model；
Fig. 2 is chip fixture opening structure figure of the present utility model；
Fig. 3 is the utility model chip fixture closing structure figure；
Fig. 4 is the simulation schematic diagram that micro-fluidic chip of the present utility model separates to cell；
Fig. 5 is the MGC803 cells transfected using the utility model micro fluidic device to the GFP being added in normal blood
Concentration effect figure (adds 300 MGC803 cells, the MGC803 cells of recovery are counted after sorting) in per sample；
Fig. 6 is circulating tumor cell immunofluorescence dyeing figure in the peripheral blood that specific embodiment 1 is isolated；
Fig. 7 is that the circulating tumor cell Giemsa isolated in the chest hydrops that specific embodiment 2 is isolated dyes aspect graph.
Embodiment
The utility model is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment
A kind of circulating tumor cell separating micro-fluidic chip device, the device include micro-fluidic chip 1 and chip fixture 2.
As described in Figure 1, described micro-fluidic chip 1 includes chip entrance 11, single-screw chip 12, rare cell collecting pipe
Road and leucocyte outlet 15, wherein, the rare cell collection conduit includes two, connects inside outlet 13 and outlet respectively
14；Described single-screw chip 12 is made up of single-screw type passage, and the single-screw type passage is from round screw thread channel center
Chip entrance 11, passage is originated through semicircle, into spiral shape microfluidic channel, end includes 3 outlets：Collect respectively different
The inside outlet 13 of particle size rare cell and outlet 14, and collect the outside leucocyte outlet 15 of leucocyte；Wherein, institute
The width for the inside outlet 13 stated is 110 ± 50 μm, and the width for exporting 14 is 110 ± 50 μm, and the width of leucocyte outlet 15 is
700±100μm.Described single-screw type passage is main channel, and its width is 500 ± 100 μm, is highly 120 ± 50 μm, each
Single spiral spacing is 500 ± 100 μm, and complete helix number is more than or equal to 3, is in the present embodiment 5 complete helix.
Described inside outlet 13 and outlet 14 carries out surface coated treatment using positive charge polymer polyethylenimine.
The processing method is conventional liquid cladding process, i.e., injecting outlet by syringe using the polymer solution of certain volume is carried out
Natural coating method.
As shown in figures 2-3, described chip fixture 2 includes sample pipe joint 21, CTC collects pipe joint 22,23 and white
Cell collects pipe joint 24；Chip fixture 2 is closed, described sample pipe joint 21 docks with chip entrance 11, described CTC
Collect pipe joint 22,23 to dock with the outlet 13,14 respectively, described leukocyte collection pipe joint 24 goes out with the leucocyte
Mouth 15 docks, and makes each gateway and the external catheter automatic butt of micro-fluidic chip 1, wherein the other end of sample cell joint 21
Biological sample is connected, biological sample constant speed is injected by chip apparatus by syringe pump or pressure pump device, its flow velocity can be
50-300ml/h。
The biological sample is peripheral blood, peripheral blood, pleural effusion, seroperitoneum, cerebrospinal fluid, bone marrow fluid and/or urine
Liquid.
It is biological sample to choose peripheral blood below, and blood is separated using above-mentioned circulating tumor cell separating micro-fluidic chip device
Circulating tumor cell in liquid, comprises the following steps：
(1) after blood sample to be measured is handled using erythrocyte cracked liquid (Wuxi Naao Bio-Pharm Co., Ltd.) cracking, add
Enter 15ml PBSs, be diluted to sample introduction sample；
(2) after micro-fluidic chip 1 and chip fixture 2 being assembled, with external sample liquid pipe and the outer tube of enriched product, in
Pipe, inner tube, as shown in figure 4, and supporting conduit, syringe pump or pressure pump device fit together；
(3) start instrument, by sample introduction sample in step (1) by conduit from the sample pipe joint 21 of chip fixture 2 according to
Flow velocity 120ml/h injects the chip entrance 11 of micro-fluidic chip 1, according to the difference of cell nucleocytoplasmic ratio and surface charge in single-screw
The concentration and separation of circulating tumor cell is realized in chip 12；The CTC cells that concentration and separation goes out are received from the outflow of outlet 13,14 through CTC
Collection pipe joint 22,23 and subsidiary conduit are exported and accessed in 5ml sterile centrifugation tubes (inner tube), and leucocyte exports from outside leucocyte
15 outflows are collected in outer tube (as shown in Figure 4) through leukocyte collection pipe joint 24 and subsidiary conduit export.
(4) circulating tumor cell through microfluidic separation is subjected to rejection tablet, makes sample slide.4% paraformaldehyde is consolidated
It is fixed, cell permeabilization is handled 10 minutes with 0.25%triton X-100 (PBS preparations), PBS is rinsed 3 times, 5min/ times；2%
BSA closes 30min, and PBS is rinsed 3 times, 5min/ times；Add EpCAM (1：200, PBS prepare), darkroom is incubated 2 hours, PBS drifts
Wash 3 times, 5min/ times；Add CK-18 (1：400, PBS prepare), darkroom is incubated 2 hours；PBS is rinsed three times, each 5min；Add
Enter CD45 (1：200, PBS prepare), darkroom is incubated 30min, and PBS is rinsed 3 times, 5min/ times；Add 0.5ug/mlDAPI (PBS
Prepare) dye 10 minutes, PBS is rinsed 3 times, 5min/ times；Add 20ul mountant mountings；Pass through fluorescence microscope.
The tumour cell sorted through micro fluidic device of the present utility model, can be observed complete cell shape under the microscope
State, such as Fig. 6 immunofluorescence dyeings display EPCAM, positive cell quantity is 5, and cell nucleocytoplasmic ratio is more than 0.8.
As shown in figure 5, different sample introduction speed separate tumour cell capture rate result for micro-fluidic chip, (GFP is positive
MGC803 cells starting addition is 300), the results showed that：In 1.0ml/min, 1.5ml/min, 2.0ml/min sample introduction speed
The average accumulation rate of the lower nanometer micro-fluidic chip of degree is respectively 57.7%, 66.3%, 75.2%.
Embodiment 2
Circulating tumor cell in micro fluidic device separation pleural effusion, device is same as Example 1, and method is as follows：
(1) pleural effusion sample is allowed to mix by gentle inversion back and forth；Pleural effusion is diluted 100 times with PBS, used
30ml syringe is drawn；Then syringe is integrally fastened on the supporting constant flow pump of instrument, syringe outlet and micro-fluidic chip
The interface of device is connected, and the flow velocity that pleural effusion is pumped into is 130ml/h.
(2) concentration and separation liquid exports from concentration and separation and flows out and access in 5ml sterile centrifugation tubes.
(3) circulating tumor cell through microfluidic separation is subjected to rejection tablet, makes sample slide.4% paraformaldehyde is consolidated
It is fixed, carry out Giemsa dyeing, micro- Microscopic observation is taken pictures, as shown in fig. 7, the tumour cell isolated show typical core greatly, core
The matter cellular prion protein bigger than high, particle diameter.
Embodiment 3
Described inside outlet 13 and outlet 14 carries out surface coated treatment using positive charge polymer propylene acid amides.It is logical
Cross syringe pump or pressure pump device and biological sample constant speed is injected into chip apparatus, its flow velocity is 50ml/h.Described PBS bufferings
The volume ratio of liquid and blood sample to be measured is 10:1.Remaining is the same as embodiment 1.
Embodiment 4
Described inside outlet 13 and outlet 14 carries out surface coated treatment using positive charge polymer propylene acid amides.It is logical
Cross syringe pump or pressure pump device and biological sample constant speed is injected into chip apparatus, its flow velocity is 300ml/h.Described PBS delays
Fliud flushing and the volume ratio of blood sample to be measured are 30:1.Remaining is the same as embodiment 1.
Embodiment described above is only that preferred embodiment of the present utility model is described, not to this practicality
New scope is defined, and on the premise of the utility model design spirit is not departed from, those of ordinary skill in the art are to this
The various modifications and improvement that the technical scheme of utility model is made, the guarantor that claims of the present utility model determine all should be fallen into
In the range of shield.
Claims (8)
- A kind of 1. circulating tumor cell separating micro-fluidic chip device, it is characterised in that the device include micro-fluidic chip (1) and Chip fixture (2), described micro-fluidic chip (1) include chip entrance (11), single-screw chip (12), rare cell collecting pipe Road and leucocyte outlet (15), wherein, the rare cell collection conduit includes two, connects inside outlet (13) respectively and goes out Mouth (14)；Described single-screw chip (12) is made up of single-screw type passage, and the single-screw type passage is from round screw thread passage The chip entrance (11) at center, passage is originated through semicircle, into spiral shape microfluidic channel, end includes 3 outlets：Respectively Inside outlet (13) and the outlet (14) of different-grain diameter size rare cell are collected, and the outside leucocyte of collection leucocyte goes out Mouth (15)；The chip entrance (11) of described chip fixture (2) automatic butt micro-fluidic chip (1), rare cell collection conduit (13, 14) and leucocyte exports (15).
- 2. a kind of circulating tumor cell separating micro-fluidic chip device according to claim 1, it is characterised in that described The width of inside outlet (13) is 110 ± 50 μm, and the width of outlet (14) is 110 ± 50 μm, the width of leucocyte outlet (15) For 700 ± 100 μm.
- 3. a kind of circulating tumor cell separating micro-fluidic chip device according to claim 1, it is characterised in that described Inside outlet (13) and outlet (14) carry out surface coated treatment using positive charge polymer.
- 4. a kind of circulating tumor cell separating micro-fluidic chip device according to claim 3, it is characterised in that described Positive charge polymer is cationic polymer, including polyethyleneimine, acrylamide amphipathic nature polyalcohol after modified.
- 5. a kind of circulating tumor cell separating micro-fluidic chip device according to claim 1, it is characterised in that described Single-screw type passage is main channel, and its width is 500 ± 100 μm, is highly 120 ± 50 μm, and each single spiral spacing is 500 ± 100 μm, complete helix number is more than or equal to 3.
- 6. a kind of circulating tumor cell separating micro-fluidic chip device according to claim 1, it is characterised in that described Chip fixture (2) includes sample pipe joint (21), CTC collects pipe joint (22,23) and waste collection pipe joint (24)；Close core Plate clamp (2), described sample pipe joint (21) dock with chip entrance (11), and described CTC collects pipe joint (22) and institute State inside outlet (13) docking, described different-grain diameter CTC collects pipe joint (23), and with the different-grain diameter CTC to export (14) right Connect, the waste collection pipe joint (24) is docked with the outlet (15), is made each gateway of micro-fluidic chip (1) and external is led Pipe connects, wherein the sample cell joint (21) other end connects biological sample, will be raw by syringe pump or pressure pump device Thing sample constant speed injects chip apparatus, and its flow velocity is 50-300ml/h.
- 7. a kind of circulating tumor cell separating micro-fluidic chip device according to claim 6, it is characterised in that by biology The flow velocity of sample constant speed injection chip apparatus is preferably 60-200ml/h.
- 8. a kind of circulating tumor cell separating micro-fluidic chip device according to claim 6, it is characterised in that described Flow velocity is more preferably 100ml/h, 120ml/h or 150ml/h；The biological sample is peripheral blood, peripheral blood, pleural effusion, seroperitoneum, cerebrospinal fluid, bone marrow fluid and/or urine.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107084916A (en) * | 2017-03-29 | 2017-08-22 | 无锡纳奥生物医药有限公司 | A kind of circulating tumor cell separating micro-fluidic chip device and its application method |
| CN109486653A (en) * | 2018-11-27 | 2019-03-19 | 上海昆道生物技术有限公司 | Trace cell capture system based on micro-fluidic and immune Magneto separate dual strategy |
| CN111733056A (en) * | 2020-06-18 | 2020-10-02 | 上海交通大学 | Micro-fluidic chip integrating circulating tumor cell separation and single-cell immunoblotting |
| CN111971378A (en) * | 2018-05-21 | 2020-11-20 | 深圳华大生命科学研究院 | High-flux organ chip and preparation method and application thereof |
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2017
- 2017-03-29 CN CN201720315262.6U patent/CN206906211U/en active Active
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107084916A (en) * | 2017-03-29 | 2017-08-22 | 无锡纳奥生物医药有限公司 | A kind of circulating tumor cell separating micro-fluidic chip device and its application method |
| CN107084916B (en) * | 2017-03-29 | 2023-12-01 | 上海纳奥生物科技有限公司 | Circulating tumor cell separation micro-fluidic chip device and application method thereof |
| CN111971378A (en) * | 2018-05-21 | 2020-11-20 | 深圳华大生命科学研究院 | High-flux organ chip and preparation method and application thereof |
| CN109486653A (en) * | 2018-11-27 | 2019-03-19 | 上海昆道生物技术有限公司 | Trace cell capture system based on micro-fluidic and immune Magneto separate dual strategy |
| CN109486653B (en) * | 2018-11-27 | 2022-03-22 | 上海昆道生物技术有限公司 | Trace cell capture system based on micro-fluidic and immunomagnetic separation dual strategies |
| CN111733056A (en) * | 2020-06-18 | 2020-10-02 | 上海交通大学 | Micro-fluidic chip integrating circulating tumor cell separation and single-cell immunoblotting |
| CN111733056B (en) * | 2020-06-18 | 2022-09-27 | 水熊健康科技(南通)有限公司 | Micro-fluidic chip integrating circulating tumor cell separation and single-cell immunoblotting |
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Effective date of registration: 20210709 Address after: Room 102, building 2, 947 Jinle Road, Baoshan District, Shanghai 201900 Patentee after: Shanghai Nao Biotechnology Co.,Ltd. Address before: No. 88, Meiliang West Road, Mashan, Wuxi, Jiangsu, 214060 Patentee before: WUXI FOXGENE Co.,Ltd. |