CN104513787B - For unicellular capture, the integrated microfluidic chip cultivating and be administered and system - Google Patents
For unicellular capture, the integrated microfluidic chip cultivating and be administered and system Download PDFInfo
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- CN104513787B CN104513787B CN201510005699.5A CN201510005699A CN104513787B CN 104513787 B CN104513787 B CN 104513787B CN 201510005699 A CN201510005699 A CN 201510005699A CN 104513787 B CN104513787 B CN 104513787B
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Abstract
The integrated microfluidic chip that single celled capture, cultivation and mass difference are administered can be realized for prior art there is no simultaneously, the invention provides a kind of for unicellular capture, the integrated microfluidic chip cultivating and be administered and system, belong to the microfluidic chip technology application at cell pharmacology analysis field.Micro-fluidic chip is made up of with cultivating unit, drug diffusion unit and medicine feed unit the cell capture of sealing-in the most successively.The system of micro-fluidic chip includes micro-fluidic chip, fluorescence microscope, image processing equipment and numeral syringe pump.Its preparation method is, three unit makes by lithography on silicon plate base plate plane figure respectively and loses groove, then pour in groove by the chip material of liquid, obtain unit after solidification；The punching of sealing-in bonding, obtains micro-fluidic chip again.The present invention achieves on one piece of integrated microfluidic chip and single celled capture, cultivation and mass difference is administered three kinds of functions, and efficiently avoid sample contamination.
Description
Technical field
The invention belongs to the microfluidic chip technology application at cell pharmacology analysis field, especially relate to a kind of for list
Cell capture, the integrated microfluidic chip cultivated and be administered and system.
Background technology
Society, the health of the mankind in the especially cancer serious threat of various diseases.And the research of unicellular level for
The research of the major diseases such as cancer, hematopathy, Parkinson's disease, senile dementia is particularly significant, therefore, it is achieved single celled capture,
Cultivate, mass difference is administered, and for drugs high flux screening, the isocellular growth of cancerous cell, shifts, spreads and art
Rear judgement waits and all has great importance.
In recent years, along with the fast development of micro-electromechanical technology Yu the subject such as life sciences, analytical chemistry, Bio-MEMS technology
And micro-total analysis system (u-TAS) of based on Bio-MEMS technology is being increasingly becoming the focus of scientist's research.Micro-fluidic
Involved sample in chemistry and the field such as biological is prepared, reacts, separates, is detected by chip, the sorting of cell, capture, cultivate,
Be administered, merge, the basic operation unit such as cracking is integrated on a little chip block, in order to realize conventional chemical or biology laboratory
Various functions, have that volume is little, consume that reagent is few, result accurately and reliably, reaction quickly, light and fast, with after be easily handled
Advantage.
Dino Di Carlo etc., at Lab Chip, in 2006,6,1445-1449, utilize soft lithography and PDMS material
Material, has designed and produced a kind of unicellular acquisition equipment of many arrays with the micro-well structure of U-shaped in one direction, the arrangement achieves
Single celled fast Acquisition；But this researcher grinds the aspects such as the long-term cultivation after cell capture, mass difference administration
Study carefully and do not indicate.
Alison M Skelley etc., at Nature Methods, in 2009,6 (2), 147-152, utilize soft lithography
And PDMS material, designed and produced former and later two directions a kind of respectively with many arrays cell capture of a micro-trap of U-shaped, pairing,
Fusing device, this device and fluorescent tracer technique combine, and the cell that can observe capture matches inside the micro-trap of U and melts
The dynamic overall process closed；But this researcher is for the research of the aspects such as the follow-up long-term cultivation of cell and mass difference administration
Do not give to indicate.
Jing Zhu etc. are in Sensors and Actuators A.215 (2014), in 197-203, by micro-to PDMS
Micro-trap geometry and the adjustment of stress distribution in fluidic chip, to micro-trap adjustable in unicellular or many cells capture chip
Joint property is studied；But this researcher does not carry out the research after unicellular capture in terms of mass difference administration.
The patent of Application No. CN201410141283.1: a kind of single cell analysis micro-fluidic chip and system and slender
Born of the same parents analyze method, devise and a kind of realize unicellular capture, cracking, the chip system of intracellular component analysis；But this integrated core
Sheet is the capture hole by arranging vertical direction for the realization of unicellular capture, and lean on is weight-driven, and process is the most steerable
And easily cell is caused damage；This chip functionally can not realize the cultivation after unicellular capture and mass difference to
Medicine.
The patent of Application No. CN201110224663.8: a kind of integrated microfluidic chip for capture of cancer cells in whole blood, design
A kind of can realize whole blood cancerous cell separate, the micro-fluidic chip of capture；This micro-fluidic chip is a layer unit structure, permissible
Realize cellulous separating trap, it is impossible to enough realize single celled capture, cultivation and mass difference and be administered.
The patent of Application No. CN201180072343: the micro-current controlled cell for high throughput analysis captures and analysis sets
Standby, devise a kind of for many cells capture and the micro fluidic chip unit of analysis, but have the disadvantage that this chip is at design chi
Cannot ensure to realize single celled capture on very little and structural principle.
The patent of Publication No. US8475730B2: Apparatus for single cell seperation and
Position fixing, devises the integrated microfluidic chip of a kind of three-layer cell structure, but the function of this chip is mainly
Separating individual cells and location positioning, the defect of this chip is can not to arrange diffusion micropore and administration in this structure again
Chamber, is unable to realize being administered single celled mass difference.
The patent of Publication No. US005821073A: Method and apparatus for single step
Assays of whole blood, devises a kind of three-layer cell structure integrated microfluidic chip, but the master of this integrated chip
Wanting function is to carry out the cell mass in blood and other materials separating analyzing, and separation is not unicellular, but many cells
The large scale dough such as group and some protein molecule groups；Drawingdimension can not realize single celled with arresting structure
Capture.
The patent of Publication No. WO2014066888 (A1): High-Throughput Mutagenized Cell
Screening Systerm For Selective Single Cell Extraction, devises the collection of a kind of multilevel-cell
Become micro-fluidic chip, but the major function of this integrated chip is for screening individual cells, carries out the extraction of cellular material, and
And the design conditions of this integrated chip internal passage makes it cannot be used for single celled cultivation and mass difference is administered.
In the disclosedest technology, relate to the documents and materials to the unicellular follow-up cultivation of capture, administration few,
At present the most still not over designing and producing a kind of integrated microfluidic chip, to realize single celled capture, cultivation and mass poor simultaneously
The report of different administration.
Summary of the invention
The invention aims to the defect overcoming above-mentioned technology to exist, it is provided that a kind of for unicellular capture, cultivation
With the integrated microfluidic chip being administered and system, this integrated chip degree is high, simple to operate, processing technology is ripe, it is possible to fast
Speed accurately realizes being administered single celled capture, cultivation and mass difference.
For realizing the object of the invention, the technical scheme used is as follows:
One of technical scheme is, a kind of for unicellular capture, the integrated microfluidic chip cultivating and be administered, by from upper and
Under successively sealing-in cell capture with cultivate unit, drug diffusion unit and medicine feed unit composition；
Wherein, described cell capture includes cell culture chamber, unicellular capture trap with cultivating unit, and microfluid flows into logical
Road, microfluid flow pass, ostium and tap hole, it is also possible to liquid storage tank is set；Cell culture chamber is for being arranged at cell capture
With cultivate unit bottom surface cavity, unicellular capture trap is positioned at cell culture chamber, cell culture chamber two ends respectively with microfluid
Flow channel is connected with microfluid flow pass, ostium and tap hole respectively by cell capture with cultivate unit upper surface or
Side surface penetrates unit, and ostium is connected with microfluid flow channel, and tap hole is connected with microfluid flow pass；Liquid storage tank can
To be arranged on ostium and microfluid flow channel junction and tap hole and microfluid flow pass junction；
Described unicellular capture trap is cylinder, has groove at cylinder facing fluid inflow direction, and groove is U-shaped or square
Shape groove, the size of cell body, for being only capable of one cell of receiving, has the gap running through cylinder on cylinder, and gap is that 1-3 is individual relatively
Good；Multiple unicellular capture traps arrange along X and Y-direction respectively and form unicellular seizure trap array, preferable line number be 100～
200, preferable columns is 100～200；Microfluid flow channel and microfluid flow pass can be horizontally disposed leading directly to
Road, it is also possible to be the serpentine channel of bending；Preferably design is to set near cell culture chamber one end in microfluid flow channel
Have the array that multiple vertical cylinder is formed as pre-mix zone, its role is to, the cell mass of inflow is dispersed as single carefully
Born of the same parents；Liquid storage tank is preferably cylindrical；
Multiple micro channel is vertically distributed on described drug diffusion unit, arranges along X and Y-direction respectively and form micropore
Array, preferably, micro channel is positioned at immediately below the level of cell capture trap groove；Micro channel can be arranged to identical or not
Same diameter；
Described medicine feed unit includes to drug slot, medicine flow channel, medicine flow pass, medicine ostium and medicine
Logistics is portalled；Be to be arranged at the groove of medicine feed unit upper surface to drug slot, be positioned at the lower section of microwell array, two ends respectively with
Medicine flow channel is connected with medicine flow pass；Medicine flow channel and medicine flow pass respectively with medicine ostium and medicine
Logistics is portalled connected；Medicine ostium and medicine tap hole can penetrate with cultivation unit and drug diffusion unit through cell capture
Medicine feed unit or the side surface by medicine feed unit penetrate；Can also be connected with medicine ostium at medicine flow channel
Place and medicine flow pass arrange medicine storage pool with medicine tap hole junction；
Can to drug slot, medicine storage pool, medicine flow channel and medicine flow pass and medicine ostium and medicine tap hole
Being one or more；During administration, the medicine of variable concentrations can be carried by multiple medicine ostiums and multiple medicine flow channel
Thing to multiple to drug slot, it is also possible to be delivered to multiple to drug slot by an ostium and multiple medicine flow channel, it is also possible to one
Individual ostium and medicine flow channel be delivered to one to drug slot, or other combining form；Medicine flows in and out
Passage can be horizontally disposed straight channel, it is also possible to be the serpentine channel of bending；Liquid storage tank is preferably cylindrical.
When being administered by above-mentioned integrated microfluidic chip, within the identical time, the micro channel of different-diameter is input to slender
It is different that born of the same parents catch the medicine liquid volume in trap, it is achieved thereby that mass difference is administered；Or micro channel is arranged to identical directly
Footpath, by arranging different medicinal liquid service times, it is achieved differentiation over time is administered；Or micro channel is arranged to phase
Same diameter, is supplied respectively to different medicinal liquids by different to drug slot, it is achieved the diversity of different medicinal liquids is administered；Or other combinations
Form.
The material of described integrated microfluidic chip is conventional transparent organic polymer material, the intensity that disclosure satisfy that perforate
Require and the character of material itself does not interferes with single celled physiological property, such as, polymethyl methacrylate (PMMA), poly-two
Methylsiloxane (PDMS), Merlon (PC) and hydrogel etc., preferably polydimethylsiloxane (PDMS).
The two of technical scheme are, a kind of for unicellular capture, the integrated micro-fluidic chip system cultivating and be administered, bag
Include,
Above-mentioned for unicellular capture, the integrated microfluidic chip cultivating and be administered；
Fluorescence microscope, in observing chip, whether the cell in unicellular capture trap array has fluorescence acquisition figure
Picture；
Image processing system, such as two dimension, three dimensional particles image speed measurement system, be used for analyzing described fluorescence microscope and obtain
Image；
Digital injection pump, connects the ostium of described micro-fluidic chip, to drive cell suspending liquid, cell culture fluid, medicine
The flowing of the microfluids such as liquid.
The three of technical scheme are, a kind of for unicellular capture, the preparation side of integrated microfluidic chip cultivating and be administered
Method, comprises the steps:
1) cell capture is with cultivation unit: use N-type silicon chip, and Wafer Cleaning is clean, uses wet oxidation, at silicon chip table
Face generates layer of oxide layer, coats SU-8 photoresist uniformly on its surface, is placed on above uniform photoresist by mask plate, adopts
With ultraviolet exposure technology make cell culture chamber by lithography, unicellular capture array, microfluid flow in and out passage, pre-mix zone circle
The flat shape such as cylinder, liquid storage tank；Re-use the conventional ICP dry method (induction plasma etching) of semiconductor applications or wet method (is carved
Erosion liquid etching) etch groove；Pouring in groove by the chip material of liquid, after it solidifies, the demoulding takes out that to be cut into rectangle little
Sheet, obtains cell capture and cultivates unit；
2) drug diffusion unit: use N-type silicon chip, Wafer Cleaning is clean, use wet oxidation, generate at silicon chip surface
One oxide layer, coats SU-8 photoresist uniformly on its surface, is placed on by mask plate above uniform photoresist, uses ultraviolet
Exposure technique makes the flat shape of diffusion micro channel array by lithography；Re-use the ICP dry method (sensing etc. that semiconductor applications is conventional
Ion etching) or wet method (etching liquid etching) etch groove；The chip material of liquid is poured in groove, the demoulding after it solidifies
Rectangular-shaped pieces is cut in taking-up, obtains drug diffusion unit；
3) medicine feed unit: use N-type silicon chip, Wafer Cleaning is clean, use wet oxidation, generate at silicon chip surface
Layer of oxide layer, is uniformly coated with SU-8 photoresist on its surface, is placed on by mask plate above uniform photoresist, uses ultraviolet
Exposure technique makes the planar graph flowing in and out passage and liquid storage tank to drug slot and medicine by lithography；Re-use semiconductor applications
Conventional ICP dry method (induction plasma etching) or wet method (etching liquid etching) etch groove, are fallen by the chip material of liquid
Entering in groove, after it solidifies, the demoulding is taken out and is cut into rectangular-shaped pieces, obtains medicine feed unit；
4) sealing-in bonding: respectively process cell capture with cultivate the lower surface of unit, the upper and lower surface of drug diffusion unit,
They are passed sequentially through oxygen plasma or solidification glue bond etc. in ultraviolet irradiation, vacuum by the upper surface of medicine feed unit
Method is bonded, the micro-fluidic integrated chip that final acquisition is complete；
5) punching: use card punch to get ostium and stream with cultivating on unit and medicine feed unit at cell capture
Portal and medicine ostium and tap hole；
Above-mentioned a kind of chip material in unicellular capture, the preparation method of integrated microfluidic chip cultivating and be administered
Material does not interferes with list for the character of conventional transparent organic polymer material, the requirement of strength that disclosure satisfy that perforate and material itself
The physiological property of cell, such as, polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), Merlon (PC)
And hydrogel etc., preferably polydimethylsiloxane (PDMS).
Compared with prior art, the invention have the advantages that
1, the present invention achieve on one piece of integrated microfluidic chip to single celled capture, cultivation and mass difference to
Medicine, not only integrates three kinds of functions, and efficiently avoid the pollution in off-chip operation and sample transfer process.
2, present invention achieves the high flux to sample quickly to process: the present invention, based on micro-fabrication technology, utilizes
The materials such as PDMS, have made the capture trap array that can accommodate individual cells, it is possible to realize the capture of single celled high flux；This
Outward, the present invention is integrated micro-fluidic chip, and sample usage amount required in experiment is few, response speed is fast, easy and simple to handle.
3, the present invention uses three-layer cell structure in design, and under the conditions of existing micro-fabrication technology, processing technology compares
Simply, it is easy to processing；It addition, the present invention using cell capture with cultivate unit as ground floor, individually made by drug diffusion unit
For the second layer, using medicine feed unit as third layer, each layer of division of labor clearly, has good compatibility between each layer.
4, the micro-fluidic chip of the present invention is applied widely, and the size capturing trap in cell capture and cultivation unit can root
It is adjusted according to different cells, if needing for the biggest cell of size difference, it is only necessary to process various sizes of capture
Trap.
5, the unicellular capture of the present invention is that the streaming capture trap opening double slit by arranging horizontal direction realizes, and leans on
It is that the horizontal shear force of fluid drives, has and be easily achieved unicellular fast Acquisition, controllability less to unicellular damage relatively
High, easy to operate, the advantage of precise and high efficiency.
In sum, the present invention, by micro-manufacture process technology and microflow control technique, develops a kind of multi-layer micro-fluidic collection
Become chip, it is achieved that capture single celled in cell suspending liquid, cultivation and diversity are administered；Carry for single celled administration research
Supplied a kind of effective realization means, it is possible to more efficient, more accurately, more high-throughout realize single celled diversity and be administered
Journey.
Accompanying drawing explanation
Fig. 1 is the population structure schematic diagram of micro-fluidic chip of the present invention；
Wherein, 1, cell capture and cultivation unit, 2, drug diffusion unit, 3, medicine feed unit；
Fig. 2 is the cell capture of micro-fluidic chip of the present invention and the upward view cultivating unit；
Wherein, H is the enlarged drawing of the microcosmic local of unicellular capture trap array, and unicellular capture trap is along X-Y direction shape
Become array；101, cell culture chamber, 102, unicellular capture trap, 103, microfluid flow channel, 104, microfluid flow pass,
105, ostium, 106, tap hole, 107, liquid storage tank, 110, unicellular seizure trap array, 112, pre-mix zone, 34, medicine flows into
Hole, 35, medicine tap hole；
Fig. 3 is the top view of the drug diffusion unit of micro-fluidic chip of the present invention；
Wherein, I is the enlarged drawing of the microcosmic local of microwell array, and micro channel forms array along X-Y direction；21, micro-
Hole path, 22, microwell array；
Fig. 4 is the top view of the medicine feed unit of micro-fluidic chip of the present invention；
Wherein, 31, to drug slot, 32, medicine flow channel, 33, medicine flow pass, 36, medicine storage pool；
Fig. 5 is cell capture and the pre-mix zone schematic diagram cultivating unit；
Wherein, Fig. 5 A is the pre-mix zone sectional view along the A-A ' direction of Fig. 1, and Fig. 5 B is Fig. 5 A section view along C-C ' direction
Figure；111, cylinder；
Fig. 6 is the enlarged diagram of cell culture chamber and drug diffusion unit faying face microcosmic local；
Wherein, Fig. 6 A is cell culture chamber with drug diffusion unit faying face along the sectional view in the B-B ' direction of Fig. 1；Fig. 6 B
For Fig. 6 A along the sectional view in D-D ' direction；108, groove, 109, gap.
Detailed description of the invention
Embodiment 1
A kind of for unicellular capture, the integrated microfluidic chip cultivating and be administered, thin by sealing-in the most successively
Born of the same parents' capture forms with cultivating unit 1, drug diffusion unit 2 and medicine feed unit 3；
Wherein, described cell capture includes cell culture chamber 101, unicellular capture trap 102, miniflow with cultivating unit
Body flow channel 103, microfluid flow pass 104, ostium 105 and tap hole 106, it is also possible to liquid storage tank 107 is set；Cell
Culture chamber 101 is the cavity being arranged at cell capture with cultivating unit 1 bottom surface, and unicellular capture trap 102 is positioned at cell culture chamber
In 101, cell culture chamber 101 two ends are connected with microfluid flow channel 103 and microfluid flow pass 104 respectively, ostium
105 and tap hole 106 respectively by cell capture with cultivate the upper surface of unit 1 or side surface penetrates in unit, ostium 105 with
Microfluid flow channel 103 is connected, and tap hole 106 is connected with microfluid flow pass 104；Liquid storage tank 107 can be arranged on stream
Hand-hole 105 and microfluid flow channel 103 junction and tap hole 106 and microfluid flow pass 104 junction；
Cell capture and cultivation unit 1 high 5-10 millimeter, be preferably cuboid, long 20-30 millimeter, wide 7.5-15 milli
Rice；Described cell culture chamber 101 is preferably rectangular recess, and the degree of depth is 10～100 microns, is preferably 10～30 microns；
Described unicellular capture trap 102 is cylinder, and height is identical with the degree of depth of cell culture chamber 101, is 10～100 microns, preferably
Being 10～30 microns, a length of 50～100 microns, width is 50～100 microns, has recessed at cylinder facing fluid inflow direction
Groove 108, groove is U-type groove or rectangular channel, and the size of cell body is for being only capable of one cell of receiving, preferably, U-type groove mouth opening width
Degree 10-20 micron, degree of depth 10-20 micron, rectangular channel is the rectangle of length of side 10-20 micron, has and run through cylinder on cylinder
Gap 109, gap is that 1-3 is individual preferably；Multiple unicellular capture traps 102 arrange along X and Y-direction respectively and form unicellular seizure trap
Array 110, line number is 100～200, and columns is 100～200, and line space is 20～100 microns, and column pitch is 20～100 micro-
Rice；Microfluid flow channel 103 and flow pass 104 can be horizontally disposed straight channel, it is also possible to be the snakelike logical of bending
Road, the width of passage is 10～100 microns, and height is 10～100 microns；Near cell culture chamber 101 in flow channel 103
One end is provided with array that multiple vertical cylinder 111 formed as pre-mix zone 112, and cylindrical radius is 2～10 microns, and height is
10～100 microns, line number is 3～8, and columns is 8～20, and line space and column pitch are 20～80 microns, its role is to, will
The cell mass flowed into is dispersed as individual cells；Liquid storage tank 107 is cylindrical, and radius is 500～1000 microns, height be 10～
100 microns；Ostium 105 is 100～500 microns with the aperture of tap hole 106；
Multiple micro channel 21 is vertically distributed on described drug diffusion unit 2, arranges along X and Y-direction respectively and formed
Microwell array 22, preferably, micro channel 21 is positioned at immediately below groove 108 level of cell capture trap 102；Drug diffusion unit
2 height are 10～100 microns, are preferably cuboid, and length and width are with cell capture and cultivation unit 1；The aperture of micro channel 21
Must be smaller than the diameter of individual cells, this like cell just will not decline in medicine cavity from micropore, preferably, and micro channel
21 for diameter 0.5～the circular hole of 6 microns or are respectively the rectangular opening of 0.5～6 micron for length, width；Can be by micro channel
21 are arranged to identical or different diameter；
Described medicine feed unit 3 includes to drug slot 31, medicine flow channel 32, medicine flow pass 33, medicine stream
Hand-hole 34 and medicine tap hole 35；It is the groove being arranged at medicine feed unit 3 upper surface to drug slot 31, is positioned at microwell array
Lower section, two ends are connected with medicine flow channel 32 and medicine flow pass 33 respectively；Medicine flow channel 32 and medicine flow out logical
Road 33 is connected with medicine ostium 34 and medicine tap hole 35 respectively；Medicine ostium 34 and tap hole 35 can be through cell captures
With cultivate unit 1 and drug diffusion unit 2 penetrates medicine feed unit 3 or the side surface by medicine feed unit 3 penetrates；Also may be used
To arrange drug storage with medicine tap hole junction and medicine flow channel with medicine ostium junction at medicine flow pass
Pond 36；
Medicine feed unit 3 a height of 5-10 millimeter, can be cuboid, and length and width are with cell capture and cultivation unit 1；It is administered
Groove 31, medicine flow channel 32, medicine flow pass 33, medicine ostium 34, medicine tap hole 35 and medicine storage pool 36 are permissible
It is one or more；During administration, variable concentrations can be carried by multiple medicine ostiums 34 and multiple medicine flow channel 32
Medicine to multiple to drug slot 31, it is also possible to be delivered to multiple to drug slot by an ostium 34 and multiple medicine flow channel 32
31, it is also possible to an ostium 34 and medicine flow channel 32 be delivered to one to drug slot 31, or other combination shape
Formula；Medicine storage pool 36 can on-demand arrange multiple；
It is respectively greater than the length and width equal to cell culture chamber 101 to drug slot 31 total length and overall width；Medicine flows into
Can be horizontally disposed straight channel with flow pass, it is also possible to be the serpentine channel of bending, the width of passage be 10～100 micro-
Rice, the degree of depth is 10～100 microns；Medicine ostium is 100～500 microns with the circular aperture of tap hole；Medicine storage pool 36 is circle
Cylindricality, radius is 500～1000 microns, and height is 10～100 microns.
When using integrated microfluidic chip to carry out unicellular capture, pre-set cell capture and cultivate unit 1 inside
Pressure be slightly less than the pressure within medicine feed unit 3, put before this, ostium 105 and tap hole 106 are respectively cell
Suspension or the inflow entrance of cell culture fluid and flow export, cell suspending liquid is flowed into by ostium 105, through liquid storage tank 107 He
Microfluid flow channel 103, is dispersed as individual cells by the cell mass in cell suspending liquid in pre-mix zone 112, subsequently into
Cell culture chamber 101, owing to capture trap is the streaming capture trap that horizontal direction is cracked, lean on is that the horizontal shear force of fluid drives
Dynamic, make the unicellular seizure trap battle array that the cell in cell suspending liquid is formed by the unicellular capture trap 102 in cell culture chamber 101
Row 110 capture, and unicellular have stayed in groove 108, and liquid flows to miniflow by the passage between gap 109 and unicellular capture trap
Body flow pass 104, then discharged by tap hole 106, complete cell capture.
When using integrated microfluidic chip to carry out single cell culture, pre-set cell capture and cultivate unit 1 inside
Pressure be slightly less than the pressure within medicine feed unit 3, put before this, ostium 105 stop conveying cell suspending liquid, change
For conveying cell culture fluid, make cell culture chamber 101 is full of culture fluid, regularly replace culture fluid, can realize single celled
Long-term cultivation；
When using integrated microfluidic chip to carry out unicellular administration experiment, administering mode has multiple, can select
One in following manner:
1) micro channel 21 is arranged to different-diameter, micro channel 21 and unicellular seizure trap 102 one_to_one corresponding；By medicine
Liquid flows into from medicine ostium 34, enters to drug slot 31 through medicine flow channel 33, pings a period of time and treats that flow reaches stable
After state, block tap hole 35；Due to pressure effect, medicinal liquid is entered the slender of its correspondence by drug slot 31 by micro channel 21
Born of the same parents catch trap 102, and the micro channel 21 of different-diameter is input to the unicellular seizure trap 102 of its correspondence within the identical time
Interior medicine liquid volume is different, it is achieved thereby that mass concentration difference is administered；
2) micro channel 21 is arranged to identical or different diameter, micro channel 21 and unicellular seizure trap 102 1 a pair
Should；Medicine ostium 34, medicine flow channel 33 and to give drug slot 31 be multiple, and medicine ostium 34 one medicine of correspondence
Flow channel 33 and one give drug slot 31；Medicinal liquid is flowed into, through medicine from different medicine ostiums 34 at different time respectively
Flow channel 33 enters to drug slot 31, pings a period of time after flow reaches steady statue, blocks tap hole 35；Or by medicine
Liquid flows into from different medicine ostiums 34 simultaneously, enters to drug slot 31 through medicine flow channel 33, pings a period of time and waits to flow
After amount reaches steady statue, block tap hole 35 at different time respectively；Due to pressure effect, medicinal liquid is by passing through micro-to drug slot 31
Hole path 21 enters the unicellular seizure trap 102 of its correspondence, because the difference of administration time or block tap hole 35 time
Successively, the medicinal liquid time in micro channel 21 is input to unicellular seizure trap 102 is variant, creates different medicinal liquid supplies
Time, thus realize the mass time difference opposite sex and be administered；
3) micro channel 21 is arranged to identical or different diameter, micro channel 21 and unicellular seizure trap 102 1 a pair
Should；Medicine ostium 34, medicine flow channel 33 and to give drug slot 31 be multiple, and medicine ostium 34 one medicine of correspondence
Flow channel 33 and one give drug slot 31；It is supplied respectively to different medicines, by medicinal liquid from medicine by different medicine ostiums 34
Ostium 34 flows into, and enters to drug slot 31 through medicine flow channel 33, pings a period of time after flow reaches steady statue, resistance
Plug flow portals 35；Owing to the medicinal liquid kind in being input to unicellular seizure trap 102 through micro channel 21 is different, thus realize batch
Chemical medicine species diversity is administered；
4) micro channel 21 is arranged to same diameter, some micro channels 21 and unicellular seizure trap 102 one_to_one corresponding,
Some micro channels 21 are also had to be positioned at the lower section of several unicellular seizure trap 102 centrage；During administration, medicinal liquid is flowed into from medicine
Hole 34 flows into, and enters to drug slot 31 through medicine flow channel 33, pings a period of time after flow reaches steady statue, blocks stream
Portal 35；Due to pressure effect, micro channel 21 with unicellular seizure trap 102 one to one, can directly by medicinal liquid by giving
Drug slot 31 is entered by micro channel 21 in the unicellular seizure trap 102 of its correspondence, but some micro channels 21 are positioned at several
The lower section of unicellular seizure trap 102 centrage, medicinal liquid is needed by after entering cell culture tank to drug slot 31 by micro channel 21
Entered in these unicellular seizure traps 102 by diffusion, so make the medicine in each unicellular seizure trap 102 dense
Degree difference, thus realize the mass concentration difference opposite sex and be administered；
5) other compound modes.
It is achieved thereby that single celled mass difference is administered, and single celled capture, cultivation and mass difference are given
Medicine has been integrated on one piece of micro-fluidic chip.
Embodiment 2
A kind of for unicellular capture, the integrated microfluidic chip cultivating and be administered, thin by sealing-in the most successively
Born of the same parents' capture forms with cultivating unit 1, drug diffusion unit 2 and medicine feed unit 3；
Cell capture is cuboid with cultivation unit 1, long 30 millimeters, wide 10 millimeters, high 10 millimeters；Cell culture chamber 101
For length 12 millimeters, width 8 millimeters, the rectangle cavity of the degree of depth 16 microns；Unicellular capture trap 102 is cylinder, highly 16 microns,
A length of 50 microns, width is 50 microns, has groove 108 at cylinder facing fluid inflow direction, and groove is U-type groove, U-type groove
Mouth A/F 16 microns, the degree of depth 16 microns, the gap 109 running through cylinder is had at groove 108 bottom land, gap is 2；
10000 unicellular capture traps 12 arrange along X and Y-direction respectively and form unicellular seizure trap array 110, and line number is 100, columns
Being 100, line space is 20 microns, and column pitch is 50 microns；Microfluid flow channel 103 and flow pass 104 are arranged horizontally
Straight channel, the width of passage is 50 microns, height be 16 microns；Near cell culture chamber 101 one end in flow channel 103
Being provided with multiple vertical cylinder 111 and form array as pre-mix zone 112, cylindrical radius is 2 microns, and height is 16 microns, OK
Number is 3, and columns is 10, and line space and column pitch are 80 microns, its role is to, the cell mass of inflow is dispersed as single carefully
Born of the same parents；Liquid storage tank 107 is cylindrical, and radius is 600 microns, and height is 16 microns；Ostium 105 with the aperture of tap hole 106 is
500 microns；
Described drug diffusion unit 2 is cuboid, and length and width are with cell capture and cultivation unit 1, and height is 40 microns；Medicine
10000 micro channels 21 are vertically distributed on thing diffusion unit 2, arrange along X and Y-direction respectively and form 100 row × 100 row
Microwell array 22, each micro channel 21 is all located at immediately below groove 108 level of each cell capture trap 102；Micro channel
The aperture of 21 must be smaller than the diameter of individual cells, and this like cell just will not decline in medicine cavity from micropore；For reality
Existing mass difference is administered, and 100 row micro channels 21 are in turn divided into 5 regions, and each region comprises 20 row, corresponding expansion
Scattered micro-pore diameter is respectively 0.5 micron, 1 micron, 2 microns, 3 microns and 4 microns, and the height of micro channel 21 is 40 microns；
Medicine feed unit 3 is cuboid, and length and width are with cell capture and cultivate unit 1, a height of 5 millimeters；To drug slot 31, medicine
Thing flow channel 32 and medicine flow pass 33 are 5, and medicine ostium 34 and medicine tap hole 35 are 1；Medicine flows into
Passage 32 and medicine flow pass 33 are straight channel, and the width of passage is 50 microns, and height is 40 microns；Each to drug slot 31
Length is 1.6 millimeters equal to the length 12 millimeters of cell culture chamber 101, width equal to the 1/5 of cell culture chamber 101 width；
Other parts are with embodiment 1.
Embodiment 3
The diameter of micro channel 21 is identical, and to drug slot 31, medicine flow pass 32, medicine flow channel 33, medicine flows into
Hole 34 and medicine tap hole 35 are multiple；Different types of medicine is carried by different pharmaceutical ostium 34 and medicine flow channel 33
To multiple to drug slot 31, other are with embodiment 1.
Embodiment 4
A kind of for unicellular capture, the integrated micro-fluidic chip system cultivating and be administered, including,
Described in embodiment 1 for unicellular capture, the integrated microfluidic chip cultivating and be administered；
Fluorescence microscope, for Japanese Nikon Ti-s type bioluminescence inverted microscope, catches for observing the unicellular of chip
Obtain whether the cell in trap array has fluorescence and obtain image；
Image processing system, the DM3-16M500 type three-dimensional PIV system produced for the world, Beijing cube Science and Technology Ltd.,
For analyzing the image that described fluorescence microscope obtains；
And digital injection pump, for auspicious wound RSP04-B four-way push-pull mode syringe pump, connect described micro-fluidic chip
Ostium, to drive the flowing of the microfluids such as Cell sap, cell culture fluid, medicinal liquid.
When system uses, in chip, inject cell suspending liquid first with digital injection pump, be stably passed through a period of time,
After chip captures cell, stop injecting cell suspending liquid；Then digital injection pump is utilized to inject cell culture fluid in chip,
Inculcating continuously, unicellular by capture is cultivated after 1～48h in micro-fluidic chip, then is noted in chip by digital injection pump
Enter the cell culture fluid containing fluorescent probe and with labelling and show cell surface molecule, add finally by digital injection pump and be used as medicine
Thing, and by cell surface fluorescence intensity before and after fluorescence microscope and image processing equipment observation detection dosing, determine analysis
The medicine of the variable concentrations drug effect to cell.
Embodiment 5
Prepare chip:
1) cell capture is with cultivation unit: use N-type 4 inch silicon wafer, and Wafer Cleaning is clean, uses wet oxidation,
Silicon chip surface generates the oxide layer that one layer of 0.5-0.8 micron is thick, is coated with last layer SU-8 photoresist uniformly on its surface, by mask
Plate is placed on above uniform photoresist, uses ultraviolet exposure technology to make cell culture chamber, unicellular capture array, miniflow by lithography
Body flows in and out the flat shapes such as passage, pre-mix zone cylinder, liquid storage tank；Re-use the ICP dry method that semiconductor applications is conventional
(induction plasma etching) or wet method (etching liquid etching) etch the groove that 10-100 micron is deep, by the chip material of liquid
PDMS pours in groove, and after it solidifies, the demoulding is taken out according to long 20-30 millimeter, wide 7.5-15 millimeter, the profile of high 5-10 millimeter
Size is cut into rectangular-shaped pieces, obtains cell capture and cultivates unit；
2) drug diffusion unit: use N-type 4 inch silicon wafer, Wafer Cleaning is clean, use wet oxidation, at silicon chip table
Face generates the oxide layer that one layer of 0.4-1.0 micron is thick, is coated with last layer SU-8 photoresist on its surface uniformly, is placed on by mask plate
Above uniform photoresist, ultraviolet exposure technology is used to make the flat shape of diffusion micro channel array by lithography；Re-use half
ICP dry method (induction plasma etching) that conductor field is conventional or wet method (etching liquid etching) etch deep recessed of 10-100 micron
Groove, pours in groove by the chip material of liquid, and after it solidifies, the demoulding is taken out according to long 20-30 millimeter, wide 7.5-15 millimeter, height
Degree is cut into rectangular-shaped pieces for the overall dimensions of 40-100 micron, obtains drug diffusion unit；
3) medicine feed unit: use N-type 4 inch silicon wafer, Wafer Cleaning is clean, use wet oxidation, at silicon chip table
Face generates one layer of 0.5 micron of thick oxide layer, is uniformly coated with one layer of SU-8 photoresist on its surface, is placed on uniformly by mask plate
Above photoresist, ultraviolet exposure technology is used to make the plane flowing in and out passage and liquid storage tank to drug slot and medicine by lithography
Figure；Re-use the conventional ICP dry method (induction plasma etching) of semiconductor applications or wet method (etching liquid etching) etches 10-
100 microns of deep grooves, pour into liquid PDMS in groove, and after it solidifies, the demoulding is taken out according to long 20-30 millimeter, wide 7.5-15
Millimeter, the overall dimensions of high 5-10 millimeter is cut into rectangular-shaped pieces, obtains medicine feed unit；
4) sealing-in bonding: the bonding of micro-fluidic chip can use ultraviolet to irradiate, oxygen plasma and adopting in vacuum
By the solidification method such as glue bond, process cell capture respectively with cultivate the lower surface of unit, the upper and lower surface of drug diffusion unit,
They are bonded by the upper surface of medicine feed unit successively, the micro-fluidic integrated chip that final acquisition is complete；
5) punching: cell capture with cultivate cell surface use card punch get respectively cell capture and cultivation unit with
And the ostium of medicine feed unit and tap hole and medicine ostium and tap hole.
Claims (9)
1. the integrated microfluidic chip being used for unicellular capture, cultivating and be administered, it is characterised in that by the most successively
The cell capture of sealing-in forms with cultivating unit, drug diffusion unit and medicine feed unit；
Wherein, described cell capture includes cell culture chamber with cultivating unit, unicellular capture trap, microfluid flow channel,
Microfluid flow pass, ostium and tap hole；Cell culture chamber is the cavity being arranged at cell capture with cultivating unit bottom surface,
Unicellular capture trap is positioned at cell culture chamber, and cell culture chamber two ends are flowed out logical with microfluid flow channel and microfluid respectively
Road is connected, and ostium and tap hole are penetrated unit, ostium by cell capture with the upper surface or side surface cultivating unit respectively
Being connected with microfluid flow channel, tap hole is connected with microfluid flow pass；
Multiple micro channel is vertically distributed on described drug diffusion unit, arranges along X and Y-direction respectively and form micropore battle array
Row；
Described medicine feed unit includes to drug slot, medicine flow channel, medicine flow pass, medicine ostium and medicine stream
Portal；Be to be arranged at the groove of medicine feed unit upper surface to drug slot, be positioned at the lower section of microwell array, two ends respectively with medicine
Flow channel is connected with medicine flow pass；Medicine flow channel and medicine flow pass respectively with medicine ostium and medicine stream
Portal connected；Medicine ostium and medicine tap hole penetrate medicine supply through cell capture and cultivation unit and drug diffusion unit
Unit or the side surface by medicine feed unit penetrate.
The most according to claim 1 a kind of for unicellular capture, the integrated microfluidic chip cultivating and be administered, its feature
Being, described cell capture also includes being arranged on ostium and microfluid flow channel junction and outflow with cultivating unit
Hole and the liquid storage tank of microfluid flow pass junction；Described medicine feed unit also includes being arranged on medicine flow pass and medicine
Logistics is portalled junction and the medicine flow channel medicine storage pool with medicine ostium junction.
The most according to claim 1 a kind of for unicellular capture, the integrated microfluidic chip cultivating and be administered, its feature
Being, described unicellular capture trap is cylinder, has groove at cylinder facing fluid inflow direction, has and run through on cylinder
The gap of cylinder；Multiple unicellular capture traps arrange along X and Y-direction respectively and form unicellular capture trap array.
The most according to claim 1 a kind of for unicellular capture, the integrated microfluidic chip cultivating and be administered, its feature
It is, in described microfluid flow channel, is provided with the array that multiple vertical cylinder is formed near cell culture chamber one end
As pre-mix zone.
The most according to claim 3 a kind of for unicellular capture, the integrated microfluidic chip cultivating and be administered, its feature
Being, described micro channel is positioned at immediately below the level of cell capture trap groove；Micro channel diameter is identical or different.
Integrated microfluidic chip the most according to claim 1, it is characterised in that described chip material is polymethylacrylic acid
Methyl ester, polydimethylsiloxane, Merlon or hydrogel.
The most according to claim 3 a kind of for unicellular capture, the integrated microfluidic chip cultivating and be administered, its feature
Being, the groove of described unicellular capture trap is U-shaped or rectangular channel, and the size of cell body is for being only capable of one cell of receiving；Described
The gap running through cylinder is 1～3.
8. one kind for unicellular capture, the system of integrated microfluidic chip cultivating and be administered, it is characterised in that include basis
Integrated microfluidic chip described in claim 1, fluorescence microscope, image processing system and digital injection pump.
The most according to claim 1 a kind of for unicellular capture, the preparation of integrated microfluidic chip cultivating and be administered
Method, it is characterised in that comprise the steps:
1) prepare cell capture and cultivate unit: using N-type silicon chip, Wafer Cleaning is clean, use wet oxidation, at silicon chip table
Face generates layer of oxide layer, coats SU-8 photoresist uniformly on its surface, is placed on above uniform photoresist by mask plate, adopts
With ultraviolet exposure technology make cell culture chamber by lithography, unicellular capture array, microfluid flow in and out passage, pre-mix zone circle
Cylinder, liquid storage tank flat shape；The ICP dry or wet etch re-using semiconductor applications conventional goes out groove；By the core of liquid
Sheet material is poured in groove, and after it solidifies, the demoulding is taken out and is cut into rectangular-shaped pieces, obtains cell capture and cultivates unit；
2) drug diffusion unit is prepared: use N-type silicon chip, Wafer Cleaning is clean, use wet oxidation, generate at silicon chip surface
Layer of oxide layer, coats SU-8 photoresist uniformly on its surface, is placed on by mask plate above uniform photoresist, uses ultraviolet
Line exposing technology makes the flat shape of diffusion micro channel array by lithography；Re-use the ICP dry method or wet that semiconductor applications is conventional
Method etches groove；Pouring in groove by the chip material of liquid, after it solidifies, the demoulding is taken out and is cut into rectangular-shaped pieces, obtains medicine
Thing diffusion unit；
3) medicine feed unit is prepared: use N-type silicon chip, Wafer Cleaning is clean, use wet oxidation, generate at silicon chip surface
Layer of oxide layer, coats SU-8 photoresist uniformly on its surface, is placed on by mask plate above uniform photoresist, uses ultraviolet
Line exposing technology makes the planar graph flowing in and out passage and liquid storage tank to drug slot, medicine by lithography；Re-use quasiconductor neck
The ICP dry or wet etch that territory is commonly used goes out groove, pours in groove by the chip material of liquid, and after it solidifies, the demoulding is taken out and cut
Rectangularity small pieces, obtain medicine feed unit；
4) sealing-in bonding: process cell capture respectively and cultivate the lower surface of unit, the upper and lower surface of drug diffusion unit, medicine
They are passed sequentially through oxygen plasma or solidification glue bond method key in ultraviolet irradiation, vacuum by the upper surface of feed unit
Close, the micro-fluidic integrated chip that final acquisition is complete；
5) punching: use card punch to get ostium and tap hole with cultivating on unit and medicine feed unit at cell capture
And medicine flows in and out hole.
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