CN108795693A - A kind of micro-fluidic chip of capture blood rare cell - Google Patents
A kind of micro-fluidic chip of capture blood rare cell Download PDFInfo
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- CN108795693A CN108795693A CN201810667851.XA CN201810667851A CN108795693A CN 108795693 A CN108795693 A CN 108795693A CN 201810667851 A CN201810667851 A CN 201810667851A CN 108795693 A CN108795693 A CN 108795693A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3679—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by absorption
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M2205/00—General characteristics of the apparatus
- A61M2205/02—General characteristics of the apparatus characterised by a particular materials
- A61M2205/0244—Micromachined materials, e.g. made from silicon wafers, microelectromechanical systems [MEMS] or comprising nanotechnology
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M2206/00—Characteristics of a physical parameter; associated device therefor
- A61M2206/10—Flow characteristics
- A61M2206/20—Flow characteristics having means for promoting or enhancing the flow, actively or passively
Abstract
The present invention relates to biologies and pathological body liquids to detect, and is related to a kind of micro-fluidic chip of separating high-purity rare cell in the group from whole blood cells.
Description
Technical field
The present invention relates to biologies and pathological body liquids to detect, and it is rare thin to be related to one kind separating high-purity from whole blood cells group
The micro-fluidic chip of born of the same parents.
Background technology
Rare cell refer in biological fluid samples (including blood, hydrothorax, ascites, urine, cerebrospinal fluid etc.) some are non-
Typical cells.Studies have shown that its completion of collection and utilization NGS to rare cell is analyzed, to finding disease potential treatment machine
Reason, pathomechanism and targeted drug exploitation have great importance.Rare cell detection research method is main in blood at present
There are flow sorting techniques, form fractionation method, density-gradient centrifugation method, membrane filter method and immunomagnetic isolation technology, such as BD
FACSAris may be implemented cell high speed and sort, but the transient laser of airflow classification will damage sorted cell, after sorting activity
Cell activity is impaired.Circulating tumor cell (CTCs) refers to the tumour cell into human peripheral blood.Although tumor patient periphery
CTCs numbers are few in blood, and generally about 107CTCs in order of magnitude haemocyte is only units (2-10/ml), but CTCs
It is a kind of a kind of tool of phase follow-up between epochmaking liquid biopsy, judging prognosis and treatment.Due to circulating tumor cell quantity
Extremely rare, the accuracy and specific requirements detected to it is quite high, and just more difficult to its further analysis, therefore
Being badly in need of development being capable of efficient, big flux, quick the circulating tumor cell portable method and work separated from blood sample
Tool.
Circulating tumor cell in human peripheral is that tumor focus sends out tumour cell (CTCs) into Peripheral Circulation
Or cell mass (CTM), the CTCs or CTM of survival leave the local microenvironment that blood circulation enters secondary internal organs, in all kinds of lifes
Proliferation grows and can finally form transfer stove under the action of the long factor.Circulating tumor cell is the important of tumour Blood route metastasis stove
Source, and DISTANT METASTASES IN is to lead to one of the immediate cause of oncotherapy failure, recurrence and death, as long as however, early detection
Recurrence and metastasis rate will likely be greatly reduced with intervening, therefore detect circulating tumor cell from blood and increasingly cause people
Attention；By the capture and analysis to CTCs, tumour metastasis and recurrence prediction can be carried out with adjuvant clinical doctor and early stage is pre-
Alert, the effect of carrying out the assessing of patients overall survival's phase (OS) and progression free survival phase (PFS), clinical chemicotherapy, monitors etc., in turn
With the important clinical significance for instructing Personalized medicine, the survival condition for improving tumor patient.
But to realize clinical diagnosis or to CTC progress lab analysis premise is that obtaining enough CTC
Cell.Since CTCs is every 10 in peripheral blood6~107A monocyte only contains 1 CTCs, therefore to CTCs detection techniques
Sensitivity and specificity propose high requirement.The detection scheme of current various CTCs includes mainly that CTCs detaches and be enriched with system
System and CTCs detection and identification systems.
By the way that microfluidic chip technology to be combined with antibody capture, to have developed a series of circulating tumor thin in recent years
Born of the same parents' chip capture technique.Micro-fluidic chip sorts and the principle of enrichment CTCs is broadly divided into 4 classes：1. affine using antigen-antibody
Property is sorted；2. being sorted using the difference of cell physical features, such as cell size, morphotropism and different size
Mechanical characteristic of the cell in flow field；3. having the function of that magnetic and connection antibody is sorted using immunomagnetic beads；4. profit
It is sorted etc. with the electric property difference of different cells.The method of micro-current controlled cell immuno-chip is to pass through micro fluidic device
(micro-fluid device) detects CTCs, which has the characteristics that high specificity, sensibility, capture
CTCs has cell activity, separates, and is CTCs clinical applications for cell culture and other various downstream technique researchs
A kind of method of new high efficiency of value research.Currently, rely on antibody CTCs chips (On-Q-ity, Waltham, MA), according to
According to the ClearCellTM systems (ClearbridgeBioMedics, Singapore) of size morphotropism principle, according to immune magnetic
The IsoFluxTM systems (Fluxion) of pearl, according to the ApostreamTM (Apocell) of fluid body dynamic characteristic and according to double
The products such as the DEPArrayTM systems to principle of electrophoresis are just being introduced to the market.However, it is considered that micro-fluidic chip is used with sample
The advantages that measuring less, be highly sensitive, but it is few just because of each equipment detection blood sample amount, and samples selection offset can not be kept away
Exempt from, false negative incidence remains high, and is had an important influence on to sample analysis, clinical diagnosis.Further, since chip often body
Product it is smaller, blood by when, efficiency is too low when flow velocity is too small, while increasing blood coagulation possibility, and flow velocity, which crosses conference, to be caused to mesh
The binding time for marking component is short, and capture is insufficient, in addition, the shearing force that haemocyte is subject to is also larger, may cause haemolysis, because
This, there is higher flow rate, the fluidic chip detecting system of more preferable joint efficiency and more Low haemolysis to become current research for exploitation
Emphasis could provide authentic data support for clinic in this way.On the other hand, some special chips, which involve great expense, is not easy to push away
Wide application.The above problem that microfluidic chip technology is encountered when carrying out cell sorting how is improved, it is excellent to give full play to its
Gesture will be the key that micro-fluidic chip (chipset) carries out CTCs captures.
The present inventor passes through creative work, provides the chip for efficiently collecting blood circulation tumour cell.It should
Chip has following advantages：Improved micro-fluidic chip uses specific spoiler and turbulence columns, is ensureing circulating tumor cell
The damage to haemocyte is reduced while efficiently collection, reduces haemolysis risk；It can will be caught by being based on microfluidic chip technology
The lossless taking-up of the circulating tumor cell that receives, carries out analysis or in vitro culture, will more embody circulating tumor cell in tumour
The effect of the fermentation such as recurrence, drug resistance prediction；The chip of the present invention can be not only used for the rare cell in capture blood, may be used also
For the various cells or cellular component in capture other biological fluid.
Invention content
On the one hand, it includes the micro- of rare cell or cellular component in blood that the present invention, which provides a kind of capture biofluid,
Multiple turbulence columns are arranged in inside in fluidic chip, the chip, form turbulence columns array, to increase chip interior surface with
The contact area of fluid increases the probability of capture target components.
On the one hand, the present invention provides a kind of design method of micro-fluidic chip：The method principle is, is meeting one
Determine under blood flow, cell or other components there can be the longer residence time in the chips in blood, so as in phase
In the case of with flow, in conjunction with more target components, while meaning in the case where combining same amount of target components,
Higher flow velocity may be used, thus improve the working efficiency of chip.In order to meet the principle, the design method is included in
Turbulence columns are set in chip swimming lane, it is characterised in that the turbulence columns make fluid in swimming lane by when can not only generate card
Door vortex street.Due to the presence of Karman vortex street, not only extend fluid upper dead time in unit area runner, moreover it is possible to allow stream
Body is detached from vortex street after a certain period of time in stagnation to be continued to flow, so that flow path and fluid stagnation of the fluid in swimming lane
Time further increases, and affects around turbulence columns whole fluid flow rate in part and chip, to increase in fluid
The time of contact of ingredient and swimming lane is conducive to the capture to target components.
On the one hand, the present invention provides a kind of micro-fluidic chip or chipsets, and the micro-fluidic chip inside includes disturbing
Fluidization tower array, the turbulence columns make fluid in swimming lane by when generate Karman vortex street.
According to any preceding aspect, the component is cycle rare cell, preferably circulating tumor cell.
According to any preceding aspect, wherein the micro-fluidic chip body include the substrate layer from bottom to top set gradually and
Cover plate layer, is equipped with component capture chamber between the substrate layer and cover plate layer, the cover plate layer is equipped with and the component capture chamber
The fluid inlet and fluid outlet of connection, the component capture chamber are divided into buffering area and swimming lane part, and edge is flowed in the swimming lane
Direction is provided with turbulence columns array, wherein the inner surface of the component capture chamber and the surface of turbulence columns are to seamlessly transit
Curved surface.
According to any preceding aspect, wherein the buffering area is at close entrance and exit, it is swimming lane meet, before setting
Branching block is held, there is shunting, reduce flow velocity and cushioning effect.
According to any preceding aspect, wherein by branching block to be divided into two or more disposed in parallel for the swimming lane
Swimming lane, it is preferable that include 5-500 turbulence columns in each swimming lane, preferably 10-200, further preferred 15-100 are a, such as
15,16,17,18,19,20,21,22,23,24,25, most preferably 17；Optionally, the turbulence columns are pressed in swimming lane
Arranged in parallel according to specific spacing, the spacing is 0.1-5 on the same direction, and the diameter of preferably 0.5-1 turbulence columns is (long
Diameter or minor axis).
According to any preceding aspect, 100-500 μm of turbulence columns pillar height, column length diameter 1.0-3.25mm, minor axis 0.5-1.0mm,
The independent turbulence columns that every group of turbulence columns are arranged by 3 isosceles triangles form, and flow-disturbing intercolumniation is 0.1-1mm, preferably 0.3- between group
0.7mm, most preferably 0.45mm；Flow-disturbing intercolumniation is 0.1-1.5mm, preferably 0.5-1mm, most preferably 0.7mm in group.
According to any preceding aspect, the component capture chamber inlet flow rate is 0.01-0.05m/s.
According to any preceding aspect, wherein the branching block both ends respectively constitute the part of the inlet and outlet of swimming lane, it is described into
Exit surface is handled through anti-haemolysis as the curved surface that seamlessly transits, optionally, the sections of inlet and outlet can be arc-shaped, rounded,
Round rectangle, fillet trapezoid etc..
According to any preceding aspect, the surface of the turbulence columns in the flowing direction is the curved surface seamlessly transitted, it is specific and
Speech, the turbulence columns cross section at cylinder, round rectangle, ellipse, dumb-bell shape, streamlined, spindle or olive shape, or
Person connects shape, preferably olive shape similar to two water droplet tail tails so that its surface in the flowing direction seamlessly transits
Curved surface, to reduce the damage of cell in mechanical shear stress convection current body.
According to any preceding aspect, wherein the component capture chamber inner surface and turbulence columns area load has and fluid
In the capture ligands that combine of target components, the preferably described capture ligands are that the anti-EPCAM antibody of Streptavidin-biotin-is multiple
Close object.
According to any preceding aspect, the material of the substrate or cover plate be selected from silicon, glass, siliceous glass, PDMS,
PMMA, or in polypropylene, cyclic olefine copolymer, cyclic olefin polymer, polymethyl methacrylate and makrolon
One or more kinds of macromolecule polymer materials.
On the one hand, the production method that the present invention provides the micro-fluidic chip of any preceding aspect：The method includes under
Row step：
1, chip mask is made；
2, photosensitive film production and exposure：The convex skeleton of floor layout shape is built using light-sensitive surface, mask is attached to light-sensitive surface
On, it is irradiated using ultraviolet light；Light-sensitive surface has been dissolved after removal exposure, has been put into baking oven and dries after washing, obtain floor layout shape
Convex skeleton is spare；
3, PDMS molds are made：184 reagents of SLYGARD are stirred at room temperature uniformly, are poured into convex with floor layout shape
Skeleton is in the PDMS chip grinding tools of bottom plate, and after vacuum degassing, baking oven drying waits for PDMS mold solidifications；
4, it is bonded PDMS：Above-mentioned steps are made to the PDMS Chip molds of gained, and with slide carrier to be put into plasma jointly clear
Washing machine (Plasma Cleaner) is bonded, and obtains design chips finished product；
5, chip is coated with：The chip to complete is placed again into plasma cleaner, chip is taken out after effect, then
Target antibody is injected in chip runner and is coated with.
On the one hand, the present invention also provides the purposes of the micro-fluidic chip of the present invention, the purposes includes for capturing
Rare component in the preferred blood of biofluid, preferably rare cell, more preferable circulating tumor cell.
Description of the drawings
Fig. 1 fluid components capture systems schematic diagrames, wherein 11-15 are power plants, and 2 be fluid line, and 3 be cell capture
Equipment, 4 be human body, and 5 be anti-coagulants release device.
Fig. 2 micro-fluidic chips and its connection diagram.
Fig. 3 spectrophotometry haemolysis situations.
Fig. 4 circulating tumor cells acquisition figure.
The pressure simulation figure of Fig. 5 chips of the present invention.By the 1. 2. 3. 4. baffle design at position before buffer pool, make
Uniform flow distribution in each swimming lane, and the retardance of fluid is formed at A/B/C, play the role of controlling flow in runner.
Fig. 6 turbulence columns rear Karman vortex street forms simulation drawing, and left figure shows the flow velocity simulation for not generating Karman vortex street, right figure
To generate the flow velocity simulation of Karman vortex street, the generation of Karman vortex street is shown at right figure arrow.
Fig. 7 circulating tumor cells acquisition figure.
Specific implementation mode
As used herein, " rare cell " rare cell refers to biological fluid samples (including blood, hydrothorax, ascites, urine
Liquid, cerebrospinal fluid etc.) in some atypical cells.The example of rare cell include but not limited to circulating tumor cell (CTC),
Circulating endothelial cells (CEC) and cycle multiple myeloma cells (CMMC).Preferred rare cell is CTC and CEC, especially
Preferred rare cell is CTC." circulating tumor cell " (CTC) refer to detected in the blood circulation of subject it is pernicious
Tumour cell.
" analyte " refers to molecule or component in the fluid as the target of the method for detection, separation, concentration or extraction.
Exemplary analyte includes cell, virus, nucleic acid, protein, carbohydrate and organic molecule.
" blood constitutent " refers to any component of whole blood, including host's red blood cell, leucocyte and blood platelet.Blood constitutent is also
Including plasma component, such as protein, lipid, nucleic acid and carbohydrate, and for example due to current or past pregnancy, device
Official transplants or infects and be likely to be present in any other cell in blood, including rare cell.
" fluid " or " biofluid " is intended to include natural fluid (such as blood, lymph, cerebrospinal fluid, urine, cervix lavation
Liquid, saliva and water sample), a part for these fluids and the fluid of cell is had been incorporated into (for example, culture medium and liquid texture
Sample).The term further includes lysate.
" capturing unit " or " capture ligands " may refer to according to circumstances for the chemical example of binding analysis object or complete
The composition combinations matter on its surface that cell relies on.Capturing unit can be compound or the composition surface for being coupled to surface
Material.Typical capturing unit includes antibody, oligonucleotides or polypeptide, nucleic acid, other protein, synthetic polymer and carbon water
Compound.
" channel " or " swimming lane " refers to the gap that fluid can flow through.Channel can be capillary on hydrophobic surface, lead
Pipe or it is liquid, aqueous can confined hydrophobic surface hydrophilic texture.
" component " of cell is any component for referring to be separated in cytolysate.Cellular component can be cell
Device (for example, nucleus, nearly core compartment, nuclear membrane, mitochondria, chloroplaset or cell membrane), polymer or molecular complex (for example,
Lipid, polysaccharide, protein (film, cross-film or cytoplasm), nucleic acid (natural, therapeutic or pathogenic), virion or
Ribosomes) or other molecules (for example, hormone, ion, co-factor or drug)." component " of cell sample refers to being wrapped in sample
The cellular component subset contained.
" sample of enrichment " refers to the sample containing such analyte：Relative to usual existing sample, located
Reason is to increase the relative amount of analyte.For example, can by by the amount increase at least 10% of target analytes, 25%,
50%, 75%, 100% or at least 10,100,1000,10,000,100,000 or 1,000,000 times of coefficient be enriched with sample
Product.
" section " refers to profile lateral-view image.
The cell of " rare amount " refers to less than 100 cells/ml fluids, is less than 10 cells/ml fluids or even few
In 1 cells/ml fluid.
Other feature and advantage will be apparent with claim from being described below.
In order to realize that goal of the invention, the present inventor devise a kind of for the micro- of rare cell capture by creative work
Fluidic chip equipment；Cell capture equipment is connected in series or in parallel with fluid line so that fluid can be from cell capture equipment
By to capture the cell contained in fluid.Preferably, the fluid is blood.Preferably, the cell is that cycle is dilute
There is cell, it is further preferred that the cell is circulating tumor cell.The cell capture equipment include micro-fluidic chip or
Chipset.
In one embodiment, a kind of capture biofluid of present invention offer includes rare cell or cell in blood
Multiple turbulence columns are arranged in inside in the micro-fluidic chip of component, the chip, form turbulence columns array.
In one embodiment, the design method of micro-fluidic chip of the invention, which is included in chip swimming lane, is arranged flow-disturbing
Column, it is characterised in that the turbulence columns make fluid generate Karman vortex street in swimming lane.
In one embodiment, using 19.0 version software analogue flow rate of Gambit2.4 versions and ANSYS Fluent
And fluid distrbution, to determine whether chip can generate Karman vortex street.
In one embodiment, the chip body includes the substrate layer from bottom to top set gradually and cover plate layer, institute
It states and is equipped with component capture chamber between substrate layer and cover plate layer, the cover plate layer is equipped with the stream being connected to the component capture chamber
Body entrance and fluid outlet, the component capture chamber are divided into buffering area and swimming lane part, and buffering area enters close to component capture chamber
Mouth and exit are swimming lane meet, and having reduces flow velocity and cushioning effect, and swimming lane part includes one or more swimming lanes, excellent
It gated branching block and is divided at least two swimming lanes, streamwise is provided with turbulence columns array in swimming lane.
In one embodiment, the branching block both ends respectively constitute the part of the inlet and outlet of swimming lane, the inlet and outlet
Surface is handled through anti-haemolysis as the curved surface that seamlessly transits, for example, the section of inlet and outlet can be arc-shaped, rounded, fillet square
Shape, fillet trapezoid etc..
In one embodiment, for the branching block by front end T-type deflector, stage casing linear type deflector and rear end are round
Deflector forms, and deflector compression swimming lane width in front-end and back-end makes the inlet and outlet of swimming lane be narrowed compared to swimming lane width,
Swimming lane is divided into parallel multiple swimming lanes by stage casing deflector.
In one embodiment, the front end T-type deflector is in plane set-hammer type, plane set-hammer type major diameter 1.0-2.0mm, minor axis
0.5-1.0mm；The round water conservancy diversion board diameter 1-3mm in rear end, preferably 2.4mm.
In one embodiment, the turbulence columns cross section is at cylinder, round rectangle, ellipse, dumb-bell shape, streamline
Type, spindle or olive shape, or similar two water droplet tail tails connect shape, preferably olive shape so that it is in flow direction
On surface be the curved surface seamlessly transitted, to reduce the damage of cell in mechanical shear stress convection current body.Turbulence columns array heights
Equal to the internal height of chip component capture chamber.
In one embodiment, turbulence columns are set to side of the cover plate close to substrate.
In one embodiment, turbulence columns are set to side of the substrate close to cover plate.
In one embodiment, each swimming lane diameter 3mm-5mm, 50-500 μm of height, flow-disturbing intercolumniation 0.1-5,
It is preferred that the major diameter or minor axis of 0.5-1 turbulence columns, turbulence columns array heights are 10-500 μm, include 5-500 in each swimming lane
Turbulence columns form microarray.
In one embodiment, 100-500 μm of turbulence columns pillar height, column length diameter 1.0-3.25mm, minor axis 0.5-1.0mm,
The independent turbulence columns that every group of turbulence columns are arranged by 3 isosceles triangles form, and flow-disturbing intercolumniation is 0.1-1mm, preferably 0.3- between group
0.7mm, most preferably 0.45mm；Flow-disturbing intercolumniation is 0.1-1.5mm, preferably 0.5-1mm, most preferably 0.7mm in group.
In one embodiment, the component capture chamber inlet flow rate is 0.01-0.05m/s.
In one embodiment, the swimming lane is arranged in parallel.
In one embodiment, the length of the substrate is 5-100mm, preferably 20-80mm, further preferred 30-
60mm, most preferably 50mm；Width is 5-50mm, preferably 10-30mm, most preferably 20mm.
In one embodiment, the quantity of the swimming lane is 1-20 items, preferred 4-15 items, further preferred 6-10 items,
Most preferably 8.
In one embodiment, the length of the swimming lane is 5-100mm, preferably 20-40mm, most preferably 30mm；Width
For 0.1-50mm, preferably 0.5-5mm, further preferred 1-3mm, most preferably 1.5mm；Height is 0.05-0.5mm, preferably
0.05-0.1mm。
In one embodiment, the material of the substrate or cover plate is silicon, glass, siliceous glass, PDMS, PMMA
Deng can also be in polypropylene, cyclic olefine copolymer, cyclic olefin polymer, polymethyl methacrylate and makrolon
One or more kinds of macromolecule polymer materials.
In one embodiment, cell capture equipment includes chipset clutch, and the chipset clutch is medical cascade
Take over road and card slot make multiple chips connect into chipset in parallel or series for placing micro-fluidic chip.
In one embodiment, in the inner surface of the component capture chamber and turbulence columns area load Streptavidin
(Streptavidin), which can be with anti-epithelial cell adhesion molecule (EPCAM) antibody-biotin (Biotin)
Label biotin specific binding in compound, EPCAM antibody in the EPCAM antibody-biotin compounds can with follow
Ring tumor cell surface EPCAM antigentic specificities combine；Wherein chain in Streptavidin-biotin-EPCAM antibody complexes
The link of mould Avidin-Biotin complex exists by the ability of high-concentration biological element competitive elution.It optionally, can also be in group
The inner surface and turbulence columns surface for dividing capture chamber can also be coated with other capture ligands, such as antigen, antibody, albumin A, albumen
G, agglutinin etc..
In one embodiment, the production method that the present invention provides the micro-fluidic chip of the present invention：The method includes
The following steps：
1, chip mask is made；
2, photosensitive film production and exposure：The convex skeleton of floor layout shape is built using light-sensitive surface, mask is attached to light-sensitive surface
On, it is irradiated using ultraviolet light；Light-sensitive surface has been dissolved after removal exposure, has been put into baking oven and dries after washing, obtain floor layout shape
Convex skeleton is spare；
3, PDMS molds are made：184 reagents of SLYGARD are stirred at room temperature uniformly, are poured into convex with floor layout shape
Skeleton is in the PDMS chip grinding tools of bottom plate, and after vacuum degassing, baking oven drying waits for PDMS mold solidifications；
4, it is bonded PDMS：Above-mentioned steps are made to the PDMS Chip molds of gained, and with slide carrier to be put into plasma jointly clear
Washing machine is bonded, and obtains design chips finished product；
5, chip is coated with：The chip to complete is placed again into plasma cleaner (Plasma Cleaner), is made
With rear taking-up chip, then target antibody is injected in chip runner and is coated with.
In one embodiment, the mask size is (5-10) × (2-4) cm.
In one embodiment, the light-sensitive surface is the light-sensitive surface that 3-13 thickness are 35 μm/specifications.
In one embodiment, the photosensitive film production and step of exposure include：The use of thickness is 35 μm/specifications
Light-sensitive surface builds the convex skeleton of floor layout shape, conventional using 3-13 light-sensitive surfaces, and mask is attached on light-sensitive surface, uses
Ultraviolet light is irradiated -120 seconds 30 seconds and is exposed；Using cleaning solution and soft hairbrush, light-sensitive surface is dissolved after careful removal exposure, clearly
Uniformly shaken using oscillator during washing with ensure fully to remove it is remaining dissolved light-sensitive surface, be washed with deionized；So
After be put into baking oven 10-30 minutes and dry, obtain the convex skeleton of floor layout shape；Chip skeleton situation is observed under stereomicroscope,
Ensure that chip skeleton edges are smooth, small turbulence columns are without breakage；Skeleton finished product is sealed with dustless valve bag.
In one embodiment, the making PDMS molds include：By 184 reagents of SLYGARD in mass ratio 1：10
Ratio, which is stirred at room temperature, to be uniformly poured slowly into using the convex skeleton of floor layout shape as in the PDMS chip grinding tools of bottom plate, with true
Sky pump (pressure 0.06pa) vacuum degassing 8-15 minutes.After the completion of degasification, (3 hours) are dried in 60 DEG C of baking ovens, wait for PDMS molds
Solidification.
In one embodiment, it is bonded PDMS：Above-mentioned steps are made to the PDMS Chip molds and slide carrier of gained
It is put into plasma cleaner bonding jointly；PDMS is pressed on slide after vacuumizing 2 minutes；Then on 60 degrees Celsius of tablets,
Heating 30 minutes, obtains design chips finished product (finished product is sealed with dustless valve bag).
In one embodiment, the chip includes that the chip that will be completed is placed again into plasma cleaner
In (Plasma Cleaner), effect takes out chip after 1-5 minutes, preferably 2 minutes；Target antibody is slowly injected into chip runner
In, 1-20 hours, preferably 12 hours are retained in about 4 DEG C of environment；Removal antibody adds 5%BSA in about 37 DEG C of environment
Close 1-5, preferably 2 hours.
Micro-fluidic chip group described in the disclosure is accessed in vitro according to practical blood sampling volume needs using serial or parallel connection mode
The circulatory system (as shown in Figure 1).The Circulated power system acquisition for entering extracorporeal circulation system after the guiding of blood intravascular follows in vitro
Gyration power, and be one or more in parallel conduits in distal end bifurcated, is respectively connected in micro-fluidic chip group and bypass, and into
Flow-limiting valve is set after entering micro-fluidic chip group.Connection type is as shown in Figure 2.Preferably, the flow-limiting valve is threeway flow-limiting valve.
By the way that bypass duct is arranged so that can conveniently adjust the flow velocity and pressure by component capture chamber, it is not easy to be produced to haemocyte
Raw higher shearing force, prevents haemolysis, and cannot be easily caused pipeline blockage.
Micro-fluidic chip or chipset are accessed into blood circulation system, it is micro- logical by micro-fluidic chip after blood circulation mixing
Road is contacted with the antibody of area load, and anti-using the antibody and circulating tumor cell surface specific of microchannel surface load
Former combination is by circulating tumor cell from capturing and be fixed in microchannel surface in blood sample.
In one embodiment, by the way that antibody is realized in Streptavidin-biotin complex cut-out and is caught by it
The circulating tumor cell obtained is discharged from microchannel surface, to obtain the circulating tumor cell of high-purity, and in circulating collection knot
Shu Houyong cell cleaning solutions clean the micro-fluidic chip, to realize the collection to the captured circulating tumor cell.
The cell cleaning solution is the buffer solution containing high-concentration biological fibroin, and preservative can be added according to the needs of preservation.It adopts
With CD45, CK8/18 fluorescence antibody to the CTC cell dyeings of elution, by fluorescence microscope, by CD45 (-), CK8/18
(+) cell mass is determined as CTC cells.
Low hemolytic described in the disclosure passes through disclosure chip by the blood after anti-freezing that " spectrophotometry " detects
The erythrocyte hemolysis situation measured afterwards is verified (Fig. 3).
Compared with prior art, the present invention has the following advantages that and feature：
1, compared to based on target polypeptide circulating tumor cell catching method and micro-fluidic chip, the present invention is without collecting
Erythrocyte splitting is carried out before CTC, opposite is even more to be protected to red blood cell during collecting CTC；2, in the present invention
Streptavidin-biotin composite combination can be interrupted by cell cleaning solution, can will be captured by being based on microfluidic chip technology
To the lossless taking-up of circulating tumor cell be further analyzed or in vitro culture, it is thin will preferably to play circulating tumor
The effect of born of the same parents.3, micro-fluidic chip group swimming lane entrance and connecting pipe entrance are both designed as the curve shape to smoothly transit,
Avoid the latent lesion caused by blood cell of internal system structure；4, turbulence columns microarray had both increased and contacting blood
Surface area prevent the mechanical damage to haemocyte again, compared to existing CTC detection or blood cell remove device, this patent set
The turbulence columns chip of meter is more suitable for online blood collection systems, and blood collection is carried out in the form of chipset and ensures CTC high
Effect acquisition, and reduce the generation of blood haemolysis, ensure the quality of blood after cycle.5, chip of the invention not only may be used
Rare cell is captured with online extracorporal circulatory system, the extracorporal circulatory system of fluid can also be realized offline, to improve capture rate.6,
The chip of the present invention can be not only used for the rare cell in capture blood, can be also used in capture other biological fluid
Various cells or cellular component.
Embodiment
Below by embodiment, the invention will be further described.
Embodiment 1：The making of chip
1, mask fabrication：With high-precision laser printer output chip mask, mask size is 5-10 × 2-4cm.
2, photosensitive film production and exposure：The convex bone of floor layout shape is built using the light-sensitive surface that thickness is 35 μm/specifications
Frame, it is conventional using 3-13 light-sensitive surfaces, and mask is attached on light-sensitive surface, it is irradiated -120 seconds 30 seconds and is exposed using ultraviolet light；Make
With cleaning solution and soft hairbrush, dissolved light-sensitive surface after careful removal exposure, in cleaning process using oscillator uniformly concussion with
Ensure fully to remove it is remaining dissolved light-sensitive surface, washed 3 times with RO clear water；It is then placed in 10-30 minutes in baking oven and dries,
Obtain the convex skeleton of floor layout shape；Chip skeleton situation is observed under stereomicroscope, it is ensured that chip skeleton edges are smooth, small
Turbulence columns are without breakage；Skeleton finished product is sealed with dustless valve bag.
3, PDMS molds are made：By 184 reagents of SLYGARD in mass ratio 1：10 ratios are stirred at room temperature uniformly, delay
Slowly it pours into using the convex skeleton of floor layout shape in the PDMS chip grinding tools of bottom plate, to be removed with vacuum pump (pressure 0.06pa) vacuum
Gas 8 minutes.After the completion of degasification, (3 hours) are dried in 60 DEG C of baking ovens, wait for PDMS mold solidifications.
4, it is bonded PDMS：Above-mentioned steps are made to the PDMS Chip molds of gained, and with slide carrier to be put into plasma jointly clear
Bonding in washing machine (Plasma Cleaner)；PDMS is pressed on slide after vacuumizing 2 minutes；Then in 60 degrees Celsius of tablets
On, it heats 30 minutes, obtains design chips finished product (finished product is sealed with dustless valve bag).Shown chip layout such as Fig. 4 institutes
Show.
5, chip is coated with：
(1) chip is cleaned 3 times with the 200 sterile PBS of μ L, determining does not have gas bubbles left in chip channel.
100ul Streptavidin albumen is injected into chip with syringe, then chip is placed in wet box, 37 is Celsius
Degree coating 2 hours.
(2) coating of antibody is captured
2.1 are added to people source-EpCAM capture antibody (biotin labeling) in 1 × PBS sterile solutions, and mixing is prepared into
5 μ g/ml concentration antibodies capture working solution.
2.2 are injected into 100 μ L antibody capture working solutions in chip with syringe, then chip are placed in wet box, 4 DEG C
It is incubated overnight.During entire be incubated, it is ensured that be covered with antibody capture working solution always in all chip runners.
2.3 clean the chip 3 times for being coated with capture antibody with 1 × PBS sterile solutions of 200 μ L, and removing remains in chip
In capture antibody working solution.
2.4 with 27 DEG C of 200 μ l 5%BSA solution closing chip 2 hours.
2.5 so far, and the coating that antibody is captured on chip is completed, and can be used at any time.
Embodiment 2：The functional parameter of chip detects
The grid model that chip design is created in Gambit2.4 version softwares, is used in combination 19.0 versions of ANSYS Fluent
Software simulates the flow regime of liquid fluid in the chips.By analogue data as it can be seen that 1. fluid flow distribution is equal in each runner
Even, T-type deflector will be in the more uniform water conservancy diversion to each runner of the liquid of buffer pool；2. being 0.01m/ in inlet porting flow velocity
S-0.05m/s simulates overall flow rate, and fluid density is set as " WATER-LIQUID ", calculate in each runner flow velocity in 0.026-
0.199m/s；3. there is Karman vortex street to form (as shown in Figure 5 and Figure 6) after each turbulence columns.Reach design requirement.
Embodiment 3：Breast cancer circulating tumor cell capture ability detects
In the chip access blood vessel of patient with breast cancer containing 10ml that embodiment 1 is made.After blood intravascular is guided out
Into the extracorporeal blood circulation system for including micro-fluidic chip group of the present invention.
The micro-fluidic chip group that parallel/series mode forms is accessed by medical grade conduit, opens the slow control of front end anti-coagulants
Release.
Open power switch, uninterrupted circle collection cell suspension 1 hour.
Device power switch is closed, automatic front end anti-coagulants slow release device is closed, circulating tumor cell acquisition terminates.
Cleaning chip is injected with 200 μ L 1 × PBS sterile solutions 3 times.
The observation of circulating tumor cell and haemolysis
" spectrophotometry " detection erythrocyte hemolysis situation in blood after cycle is utilized, does not find Hemoglobin Value exception
It increases (Fig. 3).
Circulating tumor cell quantity is collected under the microscope；Judge CTC cells (figure according to cellular morphology size and nucleocytoplasmic ratio
7A)。
Embodiment 4：Breast cancer cell arrests Efficiency testing
In the chip access blood vessel of patient with breast cancer containing 10ml that embodiment 1 is made.After blood intravascular is guided out
Into the extracorporeal blood circulation system for including micro-fluidic chip group of the present invention.
The micro-fluidic chip group that parallel/series mode forms is accessed by medical grade conduit, opens the slow control of front end anti-coagulants
Release.
Open power switch, uninterrupted circle collection cell suspension 1 hour.
Device power switch is closed, automatic front end anti-coagulants slow release device is closed, circulating tumor cell acquisition terminates.
Cleaning chip is injected with 200 μ L 1 × PBS sterile solutions 3 times.
Cell cleaning solution (containing high-concentration biological element) 200 μ l syringes are injected in chip, are stored at room temperature 30min, make
Streptavidin-biotin composite separation, releases biotin-EPCAM composites.
Chip is cleaned with 200 μ L 1 × sterile PBS 3 times, collects cell cleaning solution.
95% ethyl alcohol, 200 μ l are taken to fix collected circulating tumor cell 30 minutes with syringe.
It is cleaned 3 times with 200 μ L 1 × sterile PBS solution, removes remaining detection antibody mixed liquor.
CTC cells carry out CK8 and CD45 fluorescent stainings, in fluorescence microscopy microscopic observation.CK8 (+) and CD45 (-) are indicated
The cell is tumour cell, it was demonstrated that device capture rate, as a result as shown in Figure 7 B.
The result shows that the present invention can be used for detecting the circulating tumor cell in Peripheral Blood In Patients With Breast Cancer liquid, by the greatest extent may be used
Scanning blood more than energy improves the circulating tumor cell detection sensitivity of breast cancer, in particular with the micro-fluidic of innovative design
Chipset significantly improves the detection sensitivity of breast cancer circulating tumor cell.Meanwhile in field of scientific study and clinical examination
On the basis of verification, the specific cell keratin CK of immunological identification circulating tumor cell, leukocyte common antigen are utilized
The method that CD45, nucleus add cellular morphology dyeing, effectively detects the cycle of epithelial origin in Peripheral Blood In Patients With Breast Cancer
Tumour cell.
The present invention is not limited to specific embodiments shown and described herein, but can not depart from and be limited by specification
The spirit and scope of the present invention in the case of various changes and modifications can be made.
Claims (10)
1. the micro-fluidic chip of component in a kind of capture biofluid, wherein including turbulence columns battle array inside the micro-fluidic chip
Row, the turbulence columns make fluid in the chips by when generate Karman vortex street.
2. the micro-fluidic chip of component in a kind of capture biofluid, wherein the chip body includes from bottom to top setting gradually
Substrate layer and cover plate layer, between the substrate layer and cover plate layer be equipped with component capture chamber, the cover plate layer be equipped with it is described
The fluid inlet and fluid outlet of component capture chamber connection, the component capture chamber are divided into buffering area and swimming lane part, the swimming
Streamwise is provided with turbulence columns array in road, wherein micro-fluidic chip inner corner surface is the song seamlessly transitted
Face.
3. according to the micro-fluidic chip of any one of preceding claims, wherein the swimming lane is divided into two or two by branching block
A above swimming lane disposed in parallel, it is preferable that include 5-500 turbulence columns in each swimming lane, preferably 10-200, further preferably
15-100, such as 15,16,17,18,19,20,21,22,23,24,25, most preferably 17；Optionally, the flow-disturbing
Column is arranged in parallel according to specific spacing in swimming lane, and the spacing is 0.1-5, the diameter of preferably 0.5-1 turbulence columns.
4. according to the micro-fluidic chip of any one of preceding claims, wherein the branching block both ends respectively constitute the disengaging of swimming lane
The part of mouth, the disengaging discharge surface handle the curved surface to seamlessly transit through anti-haemolysis, and optionally, the section of inlet and outlet can be
Arc-shaped, rounded, round rectangle, fillet trapezoid etc..
5. according to the micro-fluidic chip of any one of preceding claims, the surface of the turbulence columns in the flowing direction is smoothed
The curved surface crossed, it is preferable that the turbulence columns cross section is at cylinder, round rectangle, ellipse, dumb-bell shape, streamlined, spindle
Or olive shape, or similar two water droplet tail tails connect shape, further preferably olive shape.
6. according to the micro-fluidic chip of any one of preceding claims, wherein in the inner surface and turbulence columns of the component capture chamber
It is Streptavidin-biology that area load, which has the capture ligands combined with the target components in fluid, the preferably described capture ligands,
Element-anti-EPCAM antibody complexes.
7. according to the micro-fluidic chip of any one of preceding claims, the material of the substrate or cover plate is selected from silicon, glass, silicon
Change glass, PDMS, PMMA, or selected from polypropylene, cyclic olefine copolymer, cyclic olefin polymer, polymethyl methacrylate and
The macromolecule polymer material of one or more of makrolon.
8. according to the purposes of the micro-fluidic chip of any one of preceding claims, it is characterised in that rare thin for capturing cycle
Born of the same parents, preferably circulating tumor cell.
9. a kind of design method of micro-fluidic chip, the method includes turbulence columns are arranged in chip swimming lane, it is characterised in that
The turbulence columns make fluid in swimming lane by when generate Karman vortex street.
10. the production method of the micro-fluidic chip of any one of preceding claims：The method includes the following steps：
(1), chip mask is made；
(2), photosensitive film production and exposure：The convex skeleton of floor layout shape is built using light-sensitive surface, mask is attached on light-sensitive surface,
It is irradiated using ultraviolet light；Light-sensitive surface has been dissolved after removal exposure, has been put into baking oven and dries after washing, obtain the convex bone of floor layout shape
Frame is spare；
(3), PDMS molds are made：184 reagents of SLYGARD are stirred at room temperature uniformly, are poured into the convex bone of floor layout shape
Frame is in the PDMS chip grinding tools of bottom plate, and after vacuum degassing, baking oven drying waits for PDMS mold solidifications；
(4), it is bonded PDMS：Above-mentioned steps are made into the PDMS Chip molds of gained and slide carrier is put into plasma cleaning jointly
Switch closes, and obtains design chips finished product；
(5), chip is coated with：The chip to complete is placed again into plasma cleaner, chip is taken out after effect, then will
It is coated in target antibody injection chip runner.
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