CN104513787A - Integrated micro-fluidic chip and system for capture, culture and administration of single cells - Google Patents

Abstract

Aiming at the lack of an integrated micro-fluidic chip capable of realizing capture, culture and batch differential administration of single cells simultaneously in the prior, the invention provides an integrated micro-fluidic chip and system for capture, culture and administration of single cells and belongs to application of micro-fluidic chip technologies to the field of cell pharmacology analysis. The integrated micro-fluidic chip comprises a cell capture and culture unit, a drug diffusion unit and a drug supply unit, which are sealed in sequence from top to bottom; the system employing the integrated micro-fluidic chip comprises the integrated micro-fluidic chip, a fluorescence microscope, image processing equipment and a digital injection pump. A preparation method of the integrated micro-fluidic chip comprises the following steps: photo-etching baseplate plane figures on the silicon plates of the three units to etch grooves, pouring a liquid chip material into the grooves, and obtaining the three units after the liquid chip material is solidified; conducting sealing bonding and punching to obtain the integrated micro-fluidic chip. The integrated micro-fluidic chip provided by the invention integrates the three functions of capture, culture and batch differential administration of single cells and effectively avoids sample contamination.

Description

For unicellularly catching, cultivating and the integrated microfluidic chip of administration and system

Technical field

The invention belongs to the application of microfluidic chip technology at cell pharmacology analysis field, especially relate to a kind of for unicellularly catching, cultivating and the integrated microfluidic chip of administration and system.

Background technology

Society, various disease especially cancer serious threat the health of the mankind.And the research of unicellular level is very important for the research of the major diseases such as cancer, hemopathy, parkinsonism, senile dementia, therefore, realize single celledly catching, cultivate, the administration of mass difference, for drugs high flux screening, the isocellular growth of cancer cells, transfer, diffusion and postoperative judgement etc. all have great importance.

In recent years, along with the fast development of micro-electromechanical technology and the subject such as life science, analytical chemistry, Bio-MEMS technology and the micro-total analysis system (u-TAS) based on Bio-MEMS technology are becoming the focus of scientist's research gradually.Micro-fluidic chip by sample preparation, the reaction involved by chemistry and the field such as biological, be separated, detection, the sorting of cell, catch, cultivate, administration, fusion, the basic operation unit such as cracking be integrated on a little chip block, in order to realize the various functions of conventional chemical or biology laboratory, have volume little, consume few, the result of reagent accurately and reliably, reaction fast, light and fast, with after be easy to the advantages such as process.

Dino Di Carlo etc. at Lab Chip, 2006,6, in 1445 – 1449, utilize soft lithography and PDMS material, design and produce a kind of in one direction with the unicellular acquisition equipment of many arrays of the micro-well structure of U-shaped, the arrangement achieves single celled fast Acquisition; But the research of this investigator to aspects such as the long-term cultivation after cell capture, the administrations of mass difference does not indicate.

Alison M Skelley etc. are at Nature Methods, 2009,6 (2), in 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 can observe the dynamic whole process that the cell of catching carries out in U micro-trap inside matching and merging; But this investigator does not give to indicate for the research of the aspect such as follow-up long-term cultivation and the administration of mass difference of cell.

Jing Zhu etc. at Sensors and Actuators A.215 (2014), in 197-203, by the adjustment to trap geometry micro-in PDMS micro-fluidic chip and stress distribution, the controllability of unicellular or many cells being caught to micro-trap in chip is studied; But this investigator does not carry out unicellular research of catching rear mass difference administration aspect.

Application number is the patent of CN201410141283.1: a kind of single cell analysis micro-fluidic chip and system and single cell analysis method, devise a kind ofly realize unicellularly catching, cracking, composition analysis in born of the same parents chip system; But this integrated chip is catch hole by what arrange vertical direction for unicellular realization of catching, and what lean on is segregation drive, and process is not easily handled and easily caused damage to cell; This chip functionally can not realize the cultivation after unicellular catching and the administration of mass difference.

Application number is the patent of CN201110224663.8: a kind of integrated microfluidic chip for capture of cancer cells in whole blood, devises a kind of micro-fluidic chip that can realize whole blood cancer cells and be separated, catch; This micro-fluidic chip is one deck modular construction, can realize cellulous separating trap, can not realize single celledly catching, cultivating and the administration of mass difference.

Application number is the patent of CN201180072343: the micro-current controlled cell for high throughput analysis is caught and Analytical equipment, devise a kind of micro fluidic chip unit of catching for many cells and analyzing, but shortcoming is that this chip can not be guaranteed to realize single celled catching in size and structural principle.

Publication number is the patent of US8475730B2: Apparatus for single cell seperation and positionfixing, devise a kind of integrated microfluidic chip of three-layer cell structure, but the function of this chip is mainly separated individual cells and location, position, the defect of this chip to arrange diffusion micropore and administration chamber in this structure again, therefore can not realize the administration of single celled mass difference.

Publication number is the patent of US005821073A: Method and apparatus for single step assays ofwhole blood, devise a kind of three-layer cell structure integrated microfluidic chip, but the major function of this integrated chip carries out compartment analysis to the cell mass in blood and other materials, what be separated is not unicellular, but the large size dough such as many cells group and some protein molecule groups; Drawing dimension can not realize catching single celled with arresting structure.

Publication number is the patent of WO2014066888 (A1): High-Throughput Mutagenized CellScreening Systerm For Selective Single Cell Extraction, devise a kind of integrated microfluidic chip of multilevel-cell, but the major function of this integrated chip is for screening individual cells, carry out the extraction of cellular material, and the design conditions of this integrated chip internal passages makes it can not be used for single celled cultivation and the administration of mass difference.

In current disclosed technology, relate to catch unicellular follow-up cultivation, administration documents and materials few, still not realizing single celledly catching by designing and producing a kind of integrated microfluidic chip at present simultaneously, cultivating and the report of mass difference administration.

Summary of the invention

The object of the invention is the defect existed to overcome above-mentioned technology, there is provided a kind of for unicellularly catching, cultivating and the integrated microfluidic chip of administration and system, this integrated chip degree is high, simple to operate, manufacture craft is ripe, can accurately realize fast single celledly catching, cultivating and the administration of mass difference.

For realizing the object of the invention, the technical scheme adopted is as follows:

One of technical scheme is, a kind of for unicellularly catching, cultivating and the integrated microfluidic chip of administration, by sealing-in successively from top to bottom cell capture with cultivate unit, drug diffusion unit and medicine feed unit and form;

Wherein, described cell capture with cultivate unit and comprise cell culture chamber, unicellularly catch trap, microfluid flow channel, microfluid flow pass, ostium and taphole, can also liquid storage tank be set; Cell culture chamber is be arranged at cell capture and the cavity cultivating unit bottom surface, unicellular trap of catching is positioned at cell culture chamber, cell culture chamber two ends are connected with microfluid flow pass with microfluid flow channel respectively, ostium and taphole penetrate unit by cell capture and the upper surface or side surface cultivating unit respectively, ostium is connected with microfluid flow channel, and taphole is connected with microfluid flow pass; Liquid storage tank can be arranged on ostium and microfluid flow channel junction and taphole and microfluid flow pass junction;

Described unicellular trap of catching is cylinder, and have groove at cylinder towards fluid inflow direction, groove is U-shaped or rectangular tank, and cell body is of a size of and only can holds a cell, and cylinder has the gap running through cylinder, and gap is that 1-3 is better individual; Multiple unicellular trap of catching forms unicellular seizure trap array along X and Y-direction arrangement respectively, and good line number is 100 ~ 200, and good columns is 100 ~ 200; Microfluid flow channel and microfluid flow pass can be horizontally disposed straight channel, also can be bending serpentine channels; Good design is, the array being provided with the formation of multiple vertical right cylinder in microfluid flow channel near cell culture chamber one end, as pre-confounding, its role is to, the cell mass of inflow is dispersed as individual cells; Liquid storage tank is cylindrical preferably;

Described drug diffusion unit is vertically distributed with multiple micro channel, and form microwell array along X and Y-direction arrangement respectively, preferably, micro channel is positioned at immediately below the level of cell capture trap groove; Micro channel can be arranged to identical or different diameter;

Described medicine feed unit comprises administration groove, medicine flow channel, medicine flow pass, medicine ostium and medicine taphole; Administration groove is the groove being arranged at medicine feed unit upper surface, is positioned at the below of microwell array, and two ends are connected with medicine flow pass with medicine flow channel respectively; Medicine flow channel is connected with medicine taphole with medicine ostium respectively with medicine flow pass; Medicine ostium and medicine taphole can penetrate drug diffusion unit through cell capture and cultivation unit and drug diffusion unit or be penetrated by the side surface of medicine feed unit; Medicine storage pool can also be set at medicine flow channel and medicine ostium junction and medicine flow pass and medicine taphole junction;

Administration groove, medicine storage pool, medicine flow channel and medicine flow pass and medicine ostium and medicine taphole can be one or more; During administration, the medicine of different concns can be carried to multiple administration groove by multiple medicine ostium and multiple medicine flow channel, also multiple administration groove can be delivered to by an ostium and multiple medicine flow channel, an ostium and a medicine flow channel can also be delivered to an administration groove, or other array configuration; Medicine flows into and flow pass can be horizontally disposed straight channel, also can be bending serpentine channel; Liquid storage tank is cylindrical preferably.

During by above-mentioned integrated microfluidic chip administration, the medicine liquid volume that the micro channel of different diameter is input in unicellular seizure trap in same time is different, thus achieves the administration of mass difference; Or micro channel being arranged to same diameter, by arranging different liquid service times, realizing differentiation over time administration; Or micro channel is arranged to same diameter, supplies different liquids respectively by different administration grooves, realize the otherness administration of different liquid; Or other array configurations.

The requirement of strength that the material of described integrated microfluidic chip is conventional transparent organic polymer material, can meet perforate and the character of material itself can not affect single celled physiological property, such as, polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate (PC) and hydrogel etc., preferred polydimethylsiloxane (PDMS).

Two of technical scheme is, a kind ofly comprises for unicellularly catching, cultivating and the integrated micro-fluidic chip system of administration,

Above-mentioned for unicellularly catching, cultivating and the integrated microfluidic chip of administration;

Whether fluorescent microscope, have fluorescence for observing unicellular cell of catching in trap array in chip and obtain image;

Image processing system, such as two dimension, three dimensional particles image speed measurement system, for analyzing the image that described fluorescent microscope obtains;

Digital injection pump, connects the ostium of described micro-fluidic chip, to drive the flowing of the microfluids such as cell suspending liquid, cell culture fluid, liquid.

Three of technical scheme is, a kind of for unicellularly catching, cultivating and the preparation method of integrated microfluidic chip of administration, comprises the steps:

1) cell capture and cultivation unit: adopt N-type silicon chip, Wafer Cleaning is clean, adopt wet oxidation, layer of oxide layer is generated at silicon chip surface, SU-8 photoresist material is coated at its surface uniform, mask plate is placed on above uniform photoresist material, adopts ultraviolet exposure technology to make cell culture chamber, unicellular capture array, microfluid inflow and the planeform such as flow pass, pre-confounding right cylinder, liquid storage tank by lithography; Re-use the conventional ICP dry method (induction plasma etching) of semiconductor applications or wet method (etching liquid etching) etches groove; Pour in groove by the chip material of liquid state, 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: adopt N-type silicon chip, Wafer Cleaning is clean, adopt wet oxidation, a zone of oxidation is generated at silicon chip surface, SU-8 photoresist material is coated at its surface uniform, mask plate is placed on above uniform photoresist material, adopts ultraviolet exposure technology to make the planeform of diffusion micro channel array by lithography; Re-use the conventional ICP dry method (induction plasma etching) of semiconductor applications or wet method (etching liquid etching) etches groove; Pour in groove by the chip material of liquid state, after it solidifies, the demoulding is taken out and is cut into rectangular-shaped pieces, obtains drug diffusion unit;

3) medicine feed unit: adopt N-type silicon chip, Wafer Cleaning is clean, adopt wet oxidation, layer of oxide layer is generated at silicon chip surface, SU-8 photoresist material is coated at its surface uniform, mask plate is placed on above uniform photoresist material, adopts ultraviolet exposure technology to make the planar graph of administration groove and medicine inflow and flow pass and liquid storage tank by lithography; Re-use the conventional ICP dry method (induction plasma etching) of semiconductor applications or wet method (etching liquid etching) etches groove, the chip material of liquid state is poured 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: process cell capture and lower surface, the upper and lower surface of drug diffusion unit, the upper surface of medicine feed unit of cultivating unit respectively, by them successively by method bondings such as oxygen plasma in uviolizing, vacuum or solidification glue bonds, the micro-fluidic integrated chip that final acquisition is complete;

5) punch: use punch tool unit and medicine feed unit to get ostium and taphole and medicine ostium and taphole at cell capture with cultivating;

Above-mentioned a kind of for unicellularly to catch, cultivate and administration integrated microfluidic chip preparation method in chip material be conventional transparent organic polymer material, the requirement of strength that can meet perforate and the character of material itself can not affect single celled physiological property, such as, polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate (PC) and hydrogel etc., preferred polydimethylsiloxane (PDMS).

Compared with prior art, the present invention has the following advantages:

1, the present invention achieves single celledly catching, cultivating and the administration of mass difference on one piece of integrated microfluidic chip, not only integrates three kinds of functions, and efficiently avoid sheet and operate outward and the pollution in sample transfer process.

2, present invention achieves the high-throughput fast processing to sample: the present invention, based on micro-fabrication technology, utilizes the materials such as PDMS, made can hold individual cells catch trap array, single celled high-throughput can be realized and catch; In addition, the present invention is integrated micro-fluidic chip, and sample usage quantity required in experiment is few, speed of response is fast, easy and simple to handle.

3, the present invention adopts three-layer cell structure in design, and under existing micro-fabrication technology condition, manufacture craft is fairly simple, is easy to processing; In addition, the present invention using cell capture with cultivate unit as the first layer, using drug diffusion unit separately as the second layer, using medicine feed unit as third layer, the division of labor of every one deck is clear and definite, has good compatibility between each layer.

4, micro-fluidic chip of the present invention is applied widely, and cell capture can adjust according to different cells from the size of catching trap in cultivation unit, if need for the very large cell of size difference, what only need processing different size catches trap.

5, unicellular catching of the present invention is that the streaming opening double slit by arranging horizontal direction catches trap to realize, what lean on is that the horizontal shear force of fluid drives, have be easy to realize unicellular fast Acquisition, less to unicellular damage, controllability is higher, the advantage of easy to operate, precise and high efficiency.

In sum, the present invention, by micro-manufacture processing technology and microflow control technique, develops a kind of multi-layer micro-fluidic integrated chip, achieves single celledly in cell suspending liquid catching, cultivating and otherness administration; For single celled administration research provides a kind of effective realization means, can more efficiently, more accurately, more high-throughoutly realize single celled otherness administration process.

Accompanying drawing explanation

Fig. 1 is the overall 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 unicellular enlarged view of catching the microcosmic local of trap array, and unicellular trap of catching forms array along X-Y direction; 101, cell culture chamber, 102, unicellularly catch trap, 103, microfluid flow channel, 104, microfluid flow pass, 105, ostium, 106, taphole, 107, liquid storage tank, 110, unicellular seizure trap array, 112, pre-confounding, 34, medicine ostium, 35, medicine taphole;

Fig. 3 is the vertical view of the drug diffusion unit of micro-fluidic chip of the present invention;

Wherein, I is the enlarged view of the microcosmic local of microwell array, and micro channel forms array along X-Y direction; 21, micro channel, 22, microwell array;

Fig. 4 is the vertical view of the medicine feed unit of micro-fluidic chip of the present invention;

Wherein, 31, administration groove, 32, medicine flow channel, 33, medicine flow pass, 36, medicine storage pool;

Fig. 5 is cell capture and the pre-confounding schematic diagram cultivating unit;

Wherein, Fig. 5 A is the sectional view of pre-confounding along the A-A ' direction of Fig. 1, and Fig. 5 B is the sectional view of Fig. 5 A along C-C ' direction; 111, right cylinder;

Fig. 6 is the enlarged diagram of cell culture chamber and drug diffusion unit bonding surface microcosmic local;

Wherein, Fig. 6 A is cell culture chamber and the drug diffusion unit bonding surface sectional view along the B-B ' direction of Fig. 1; Fig. 6 B is the sectional view of Fig. 6 A along D-D ' direction; 108, groove, 109, gap.

Embodiment

Embodiment 1

For unicellularly catching, cultivating and the integrated microfluidic chip of administration, by sealing-in successively from top to bottom cell capture with cultivate unit 1, drug diffusion unit 2 and medicine feed unit 3 and form;

Wherein, described cell capture with cultivate unit and include cell culture chamber 101, unicellularly catch trap 102, microfluid flow channel 103, microfluid flow pass 104, ostium 105 and taphole 106, can also arrange liquid storage tank 107; Cell culture chamber 101 is for being arranged at cell capture and the cavity cultivating unit 1 bottom surface, unicellular trap 102 of catching is positioned at cell culture chamber 101, cell culture chamber 101 two ends are connected with microfluid flow pass 104 with microfluid flow channel 103 respectively, ostium 105 and taphole 106 penetrate in unit by cell capture and the upper surface or side surface cultivating unit 1 respectively, ostium 105 is connected with microfluid flow channel 103, and taphole 106 is connected with microfluid flow pass 104; Liquid storage tank 107 can be arranged on ostium 105 and microfluid flow channel 103 junction and taphole 106 and microfluid flow pass 104 junction;

Cell capture and the high 5-10 millimeter of cultivation unit 1 are rectangular parallelepiped preferably, long 20-30 millimeter, wide 7.5-15 millimeter; Described cell culture chamber 101 is rectangular recess preferably, and the degree of depth is 10 ~ 100 microns, is 10 ~ 30 microns preferably; Described unicellular trap 102 of catching is for cylinder, height is identical with the degree of depth of cell culture chamber 101, it is 10 ~ 100 microns, be 10 ~ 30 microns preferably, length is 50 ~ 100 microns, width is 50 ~ 100 microns, have groove 108 at cylinder towards fluid inflow direction, groove is U-type groove or rectangular tank, and cell body is of a size of and only can holds a cell, preferably, U-type groove mouth A/F 10-20 micron, degree of depth 10-20 micron, rectangular tank is the rectangle of length of side 10-20 micron, cylinder has the gap 109 running through cylinder, and gap is that 1-3 is better individual; Multiple unicellular trap 102 of catching forms unicellular seizure trap array 110 along X and Y-direction arrangement respectively, and line number is 100 ~ 200, and columns is 100 ~ 200, and line space is 20 ~ 100 microns, and column pitch is 20 ~ 100 microns; Microfluid flow channel 103 and flow pass 104 can be horizontally disposed straight channel, also can be bending serpentine channels, and the width of passage is 10 ~ 100 microns, is highly 10 ~ 100 microns; The array of multiple vertical right cylinder 111 formation is provided with near cell culture chamber 101 one end as pre-confounding 112 in flow channel 103, cylindrical radius is 2 ~ 10 microns, be highly 10 ~ 100 microns, line number is 3 ~ 8, columns is 8 ~ 20, line space and column pitch are 20 ~ 80 microns, its role is to, and the cell mass of inflow is dispersed as individual cells; Liquid storage tank 107 is cylindrical, and radius is 500 ~ 1000 microns, is highly 10 ~ 100 microns; Ostium 105 is 100 ~ 500 microns with the aperture of taphole 106;

Described drug diffusion unit 2 is vertically distributed with multiple micro channel 21, and form microwell array 22 along X and Y-direction arrangement respectively, preferably, micro channel 21 is positioned at immediately below groove 108 level of cell capture trap 102; Drug diffusion unit 2 is highly 10 ~ 100 microns, is rectangular parallelepiped preferably, and length and width are with cell capture and cultivation unit 1; The aperture of micro channel 21 must be less than the diameter of individual cells, and this like cell just can not decline in medicine cavity from micropore, and preferably, micro channel 21 is the circular hole of diameter 0.5 ~ 6 micron or is respectively the rectangular opening of 0.5 ~ 6 micron for length, width; Micro channel 21 can be arranged to identical or different diameter;

Described medicine feed unit 3 comprises administration groove 31, medicine flow channel 32, medicine flow pass 33, medicine ostium 34 and medicine taphole 35; Administration groove 31, for being arranged at the groove of medicine feed unit 3 upper surface, is positioned at the below of microwell array, and two ends are connected with medicine flow pass 33 with medicine flow channel 32 respectively; Medicine flow channel 32 is connected with medicine taphole 35 with medicine ostium 34 respectively with medicine flow pass 33; Medicine ostium 34 and taphole 35 can penetrate drug diffusion unit 3 through cell capture and cultivation unit 1 and drug diffusion unit 2 or be penetrated by the side surface of medicine feed unit 3; Medicine storage pool 36 can also be set at medicine flow pass and medicine taphole junction and medicine flow channel and medicine ostium junction;

Medicine feed unit 3 height is 5-10 millimeter, can be rectangular parallelepiped, and length and width are with cell capture and cultivation unit 1; Administration groove 31, medicine flow channel 32, medicine flow pass 33, medicine ostium 34, medicine taphole 35 and medicine storage pool 36 can be one or more; During administration, the medicine of different concns can be carried to multiple administration groove 31 by multiple medicine ostium 34 and multiple medicine flow channel 32, also multiple administration groove 31 can be delivered to by an ostium 34 and multiple medicine flow channel 32, an ostium 34 and a medicine flow channel 32 can also be delivered to an administration groove 31, or other array configuration; Medicine storage pool 36 can arrange multiple as required;

Administration groove 31 total length and total width are more than or equal to length and the width of cell culture chamber 101 respectively; Medicine flows into and flow pass can be horizontally disposed straight channel, and also can be bending serpentine channel, the width of passage be 10 ~ 100 microns, and the degree of depth is 10 ~ 100 microns; The circular aperture of medicine ostium and taphole is 100 ~ 500 microns; Medicine storage pool 36 is cylindrical, and radius is 500 ~ 1000 microns, is highly 10 ~ 100 microns.

When using integrated microfluidic chip to carry out unicellular catching, the pressure pre-setting cell capture and cultivation unit 1 inside is slightly less than the pressure of medicine feed unit 3 inside, putting before this, ostium 105 and taphole 106 are respectively influx and the spout of cell suspending liquid or cell culture fluid, cell suspending liquid is flowed into by ostium 105, through liquid storage tank 107 and microfluid flow channel 103, in pre-confounding 112, the cell mass in cell suspending liquid is dispersed as individual cells, then cell culture chamber 101 is entered, be that the streaming that horizontal direction is cracked catches trap owing to catching trap, lean on be fluid horizontal shear force drive, make the cell in cell suspending liquid be caught by unicellular in cell culture chamber 101 the unicellular seizure trap array 110 that trap 102 formed to catch, unicellularly stay in groove 108, liquid flows to microfluid flow pass 104 by gap 109 and unicellular passage of catching between trap, discharged by taphole 106 again, complete cell capture.

When using integrated microfluidic chip to carry out single cell culture, the pressure pre-setting cell capture and cultivation unit 1 inside is slightly less than the pressure of medicine feed unit 3 inside, putting before this, ostium 105 stops conveying cell suspending liquid, change conveying cell culture fluid into, make to be full of nutrient solution in cell culture chamber 101, periodic replacement nutrient solution, can realize single celled long-term cultivation;

When using integrated microfluidic chip to carry out unicellular administration experiment, administering mode has multiple, can select as the one under type:

1) micro channel 21 is arranged to different diameter, micro channel 21 and unicellular seizure trap 102 one_to_one corresponding; Flowed into from medicine ostium 34 by liquid, enter administration groove 31 through medicine flow channel 33, examination logical for some time reaches after steady state until flow, and blocked flow portals 35; Due to pressure effect, liquid is entered the unicellular seizure trap 102 of its correspondence by micro channel 21 by administration groove 31, and the medicine liquid volume that the micro channel 21 of different diameter is input in the unicellular seizure trap 102 of its correspondence in same time is different, thus achieves the administration of mass concentration difference;

2) micro channel 21 is arranged to identical or different diameter, micro channel 21 and unicellular seizure trap 102 one_to_one corresponding; Medicine ostium 34, medicine flow channel 33 and administration groove 31 are multiple, and a corresponding medicine flow channel 33 of medicine ostium 34 and an administration groove 31; Flowed into from different medicine ostiums 34 at different time respectively by liquid, enter administration groove 31 through medicine flow channel 33, examination logical for some time reaches after steady state until flow, and blocked flow portals 35; Or flowed into from different medicine ostiums 34 by liquid, enter administration groove 31 through medicine flow channel 33, examination logical for some time reaches after steady state until flow, portals 35 respectively at different time blocked flow simultaneously; Due to pressure effect, liquid is entered the unicellular seizure trap 102 of its correspondence by micro channel 21 by administration groove 31, the priority of 35 times because the difference of administration time or blocked flow portal, the time that liquid is input in unicellular seizure trap 102 through micro channel 21 is variant, create different liquid service times, thus realize the opposite sex administration of mass time difference;

3) micro channel 21 is arranged to identical or different diameter, micro channel 21 and unicellular seizure trap 102 one_to_one corresponding; Medicine ostium 34, medicine flow channel 33 and administration groove 31 are multiple, and a corresponding medicine flow channel 33 of medicine ostium 34 and an administration groove 31; Supply different medicines respectively by different medicine ostiums 34, flowed into by liquid from medicine ostium 34, enter administration groove 31 through medicine flow channel 33, examination logical for some time reaches after steady state until flow, and blocked flow portals 35; Because the liquid kind be input to through micro channel 21 in unicellular seizure trap 102 is different, thus realize the administration of mass medicament categories otherness;

4) micro channel 21 is arranged to same diameter, some micro channels 21 and unicellular seizure trap 102 one_to_one corresponding, also has some micro channels 21 to be positioned at the below of several unicellular seizure trap 102 medullary ray; During administration, flowed into by liquid from medicine ostium 34, enter administration groove 31 through medicine flow channel 33, examination logical for some time reaches after steady state until flow, and blocked flow portals 35; Due to pressure effect, micro channel 21 and unicellular seizure trap 102 are one to one, can directly liquid be entered in the unicellular seizure trap 102 of its correspondence by administration groove 31 by micro channel 21, but some micro channels 21 are positioned at the below of several unicellular seizure trap 102 medullary ray, liquid needs after entering cell cultures groove by administration groove 31 by micro channel 21 to enter into these unicellular seizure traps 102 by diffusion, make the drug level in each unicellular seizure trap 102 different like this, thus realize the administration of the mass concentration difference opposite sex;

5) other array modes.

Thus achieve the administration of single celled mass difference, and will single celledly to catch, to cultivate and the administration of mass difference has been integrated on one piece of micro-fluidic chip.

Embodiment 2

For unicellularly catching, cultivating and the integrated microfluidic chip of administration, by sealing-in successively from top to bottom cell capture with cultivate unit 1, drug diffusion unit 2 and medicine feed unit 3 and form;

Cell capture is rectangular parallelepiped with cultivating unit 1, long 30 millimeters, wide 10 millimeters, high 10 millimeters; Cell culture chamber 101 is length 12 millimeters, width 8 millimeters, the rectangle cavity of the degree of depth 16 microns; Unicellular trap 102 of catching is for cylinder, height 16 microns, length is 50 microns, width is 50 microns, has groove 108 at cylinder towards fluid inflow direction, and groove is U-type groove, U-type groove mouth A/F 16 microns, the degree of depth 16 microns, has the gap 109 running through cylinder at groove 108 bottom land, gap is 2; 10000 unicellular traps 12 of catching form unicellular seizure trap array 110 along X and Y-direction arrangement respectively, and line number is 100, and columns is 100, and line space is 20 microns, and column pitch is 50 microns; The straight channel that microfluid flow channel 103 and flow pass 104 are arranged horizontally, the width of passage is 50 microns, is highly 16 microns; In flow channel 103, be provided with multiple vertical right cylinder 111 near cell culture chamber 101 one end form array as pre-confounding 112, cylindrical radius is 2 microns, be highly 16 microns, line number is 3, columns is 10, line space and column pitch are 80 microns, its role is to, and the cell mass of inflow is dispersed as individual cells; Liquid storage tank 107 is cylindrical, and radius is 600 microns, is highly 16 microns; Ostium 105 is 500 microns with the aperture of taphole 106;

Described drug diffusion unit 2 is rectangular parallelepiped, and length and width, with cell capture and cultivation unit 1, are highly 40 microns; Drug diffusion unit 2 is vertically distributed with 10000 micro channels 21, respectively along the microwell array 22 that X and Y-direction arrangement formation 100 row × 100 arrange, each micro channel 21 is positioned at immediately below groove 108 level of each cell capture trap 102; The aperture of micro channel 21 must be less than the diameter of individual cells, and this like cell just can not decline in medicine cavity from micropore; In order to realize the administration of mass difference, 100 row micro channels 21 are divided into 5 regions successively, each region comprises 20 row, and corresponding diffusion 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;

Drug diffusion unit 3 is rectangular parallelepiped, and length and width are with cell capture and cultivation unit 1, and height is 5 millimeters; Administration groove 31, medicine flow channel 32 and medicine flow pass 33 are 5, and medicine ostium 34 and medicine taphole 35 are 1; Medicine flow channel 32 and medicine flow pass 33 are straight channel, and the width of passage is 50 microns, is highly 40 microns; The length of each administration groove 31 equals the length 12 millimeters of cell culture chamber 101, and it is 1.6 millimeters that width equals 1/5 of cell culture chamber 101 width;

Other parts are with embodiment 1.

Embodiment 3

The diameter of micro channel 21 is identical, administration groove 31, medicine flow pass 32, medicine flow channel 33, and medicine taphole 34 and medicine ostium 35 are multiple; Carry different types of medicine to multiple administration groove 31 by different pharmaceutical ostium 35 and medicine flow channel 33, other are with embodiment 1.

Embodiment 4

For unicellularly catching, cultivating and the integrated micro-fluidic chip system of administration, comprise,

Described in embodiment 1 for unicellularly catching, cultivating and the integrated microfluidic chip of administration;

Fluorescent microscope, is Japanese Nikon Ti-s type bioluminescence inverted microscope, whether has fluorescence for the unicellular cell of catching in trap array observing chip and obtain image;

Image processing system is the three-dimensional PIV system of DM3-16M500 type that Beijing cube world Science and Technology Ltd. produces, for analyzing the image that described fluorescent microscope obtains;

And digital injection pump, be auspicious wound RSP04-B four-way push-pull mode syringe pump, connect the ostium of described micro-fluidic chip, to drive the flowing of the microfluids such as enchylema, cell culture fluid, liquid.

When system uses, first utilize digital injection pump to inject cell suspending liquid in chip, stablize and pass into for some time, after chip captures cell, stop injecting cell suspending liquid; Then digital injection pump is utilized to inject cell culture fluid in chip, inculcate continuously, by unicellularly cultivating after 1 ~ 48h in micro-fluidic chip of catching, again by digital injection pump injects in chip containing fluorescent probe cell culture fluid with mark and showed cell surface molecular, medicine is added finally by digital injection pump, and observe cell surface fluorescence intensity before and after detection dosing by fluorescent microscope and image processing equipment, determine that the medicine analyzing different concns is to the drug effect of cell.

Embodiment 5

Prepare chip:

1) cell capture and cultivation unit: adopt N-type 4 inch silicon wafer, Wafer Cleaning is clean, adopt wet oxidation, the zone of oxidation of one deck 0.5-0.8 micron thickness is generated at silicon chip surface, at the painting last layer SU-8 photoresist material of its surface uniform, mask plate is placed on above uniform photoresist material, adopts ultraviolet exposure technology to make cell culture chamber, unicellular capture array, microfluid inflow and the planeform such as flow pass, pre-confounding right cylinder, liquid storage tank by lithography; Re-use the conventional ICP dry method (induction plasma etching) of semiconductor applications or wet method (etching liquid etching) etches the dark groove of 10-100 micron, the chip material PDMS of liquid state is poured in groove, after it solidifies, the demoulding is taken out according to long 20-30 millimeter, wide 7.5-15 millimeter, the physical dimension of high 5-10 millimeter is cut into rectangular-shaped pieces, obtains cell capture and cultivates unit;

2) drug diffusion unit: adopt N-type 4 inch silicon wafer, Wafer Cleaning is clean, adopt wet oxidation, the zone of oxidation of one deck 0.4-1.0 micron thickness is generated at silicon chip surface, at the painting last layer SU-8 photoresist material of its surface uniform, mask plate is placed on above uniform photoresist material, adopts ultraviolet exposure technology to make the planeform of diffusion micro channel array by lithography; Re-use the conventional ICP dry method (induction plasma etching) of semiconductor applications or wet method (etching liquid etching) etches the dark groove of 10-100 micron, the chip material of liquid state is poured in groove, after it solidifies, the demoulding is taken out according to long 20-30 millimeter, wide 7.5-15 millimeter, highly for the physical dimension of 40-100 micron is cut into rectangular-shaped pieces, obtain drug diffusion unit;

3) medicine feed unit: adopt N-type 4 inch silicon wafer, Wafer Cleaning is clean, adopt wet oxidation, the zone of oxidation of one deck 0.5 micron thickness is generated at silicon chip surface, last layer SU-8 photoresist material is coated with at its surface uniform, mask plate is placed on above uniform photoresist material, adopts ultraviolet exposure technology to make the planar graph of administration groove and medicine inflow and flow pass and liquid storage tank by lithography; Re-use the conventional ICP dry method (induction plasma etching) of semiconductor applications or wet method (etching liquid etching) etches the dark groove of 10-100 micron, liquid PDMS is poured in groove, after it solidifies, the demoulding is taken out according to long 20-30 millimeter, wide 7.5-15 millimeter, the physical dimension 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 adopt the method such as oxygen plasma and employing solidification glue bond in uviolizing, vacuum, process cell capture and lower surface, the upper and lower surface of drug diffusion unit, the upper surface of medicine feed unit of cultivating unit respectively, by they bondings successively, finally obtain complete micro-fluidic integrated chip;

5) punch: cell capture with cultivate cell surface and use punch tool get cell capture respectively and cultivate the ostium of unit and medicine feed unit and taphole and medicine ostium and taphole.

Claims (9)

1., for unicellularly catching, cultivating and the integrated microfluidic chip of administration, it is characterized in that, by sealing-in successively from top to bottom cell capture with cultivate unit, drug diffusion unit and medicine feed unit and form;

Wherein, described cell capture with cultivate unit and comprise cell culture chamber, unicellularly catch trap, microfluid flow channel, microfluid flow pass, ostium and taphole; Cell culture chamber is be arranged at cell capture and the cavity cultivating unit bottom surface, unicellular trap of catching is positioned at cell culture chamber, cell culture chamber two ends are connected with microfluid flow pass with microfluid flow channel respectively, ostium and taphole penetrate unit by cell capture and the upper surface or side surface cultivating unit respectively, ostium is connected with microfluid flow channel, and taphole is connected with microfluid flow pass;

Described drug diffusion unit is vertically distributed with multiple micro channel, forms microwell array along X and Y-direction arrangement respectively;

Described medicine feed unit comprises administration groove, medicine flow channel, medicine flow pass, medicine ostium and medicine taphole; Administration groove is the groove being arranged at medicine feed unit upper surface, is positioned at the below of microwell array, and two ends are connected with medicine flow pass with medicine flow channel respectively; Medicine flow channel is connected with medicine taphole with medicine ostium respectively with medicine flow pass; Medicine ostium and medicine taphole penetrate drug diffusion unit through cell capture and cultivation unit and drug diffusion unit or are penetrated by the side surface of medicine feed unit.

2. according to claim 1 a kind of for unicellularly catching, cultivating and the integrated microfluidic chip of administration, it is characterized in that, described cell capture also comprises with cultivation unit the liquid storage tank being arranged on ostium and microfluid flow channel junction and taphole and microfluid flow pass junction; Described medicine feed unit also comprises the medicine storage pool being arranged on medicine flow pass and medicine taphole junction and medicine flow channel and medicine ostium junction.

3. according to claim 1 a kind of for unicellularly catching, cultivating and the integrated microfluidic chip of administration, it is characterized in that, described unicellular trap of catching is cylinder, has groove, cylinder has the gap running through cylinder at cylinder towards fluid inflow direction; Multiple unicellular trap of catching is formed along X and Y-direction arrangement respectively and unicellularly catches trap array.

4. according to claim 1 a kind of for unicellularly catching, cultivating and the integrated microfluidic chip of administration, it is characterized in that, in described microfluid flow channel, be provided with the array of multiple vertical right cylinder formation near cell culture chamber one end as pre-confounding.

5. according to claim 1 a kind of for unicellularly catching, cultivating and the integrated microfluidic chip of administration, it is characterized in that, described micro channel is positioned at immediately below the level of cell capture trap groove; Micro channel diameter is identical or different.

6. integrated microfluidic chip according to claim 1, is characterized in that, described chip material is polymethylmethacrylate, polydimethylsiloxane, polycarbonate or hydrogel.

7. according to claim 3 a kind of for unicellularly catching, cultivating and the integrated microfluidic chip of administration, it is characterized in that, described groove is U-shaped or rectangular tank, and cell body is of a size of and only can holds a cell; The described gap running through cylinder is 1 ~ 3.

8., for unicellularly catching, cultivating and the system of integrated microfluidic chip of administration, it is characterized in that, comprise integrated microfluidic chip according to claim 1, fluorescent microscope, image processing system and digital injection pump.

9. according to claim 1 a kind of for unicellularly catching, cultivating and the preparation method of integrated microfluidic chip of administration, it is characterized in that, comprise the steps:

1) prepare cell capture and cultivate unit: adopting N-type silicon chip, Wafer Cleaning is clean, adopt wet oxidation, layer of oxide layer is generated at silicon chip surface, SU-8 photoresist material is coated at its surface uniform, mask plate is placed on above uniform photoresist material, adopts ultraviolet exposure technology to make cell culture chamber, unicellular capture array, microfluid inflow and the planeform such as flow pass, pre-confounding right cylinder, liquid storage tank by lithography; Re-use the conventional ICP dry method of semiconductor applications or wet etching goes out groove; Pour in groove by the chip material of liquid state, 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: adopt N-type silicon chip, Wafer Cleaning is clean, adopt wet oxidation, layer of oxide layer is generated at silicon chip surface, SU-8 photoresist material is coated at its surface uniform, mask plate is placed on above uniform photoresist material, adopts ultraviolet exposure technology to make the planeform of diffusion micro channel array by lithography; Re-use the conventional ICP dry method of semiconductor applications or wet etching goes out groove; Pour in groove by the chip material of liquid state, after it solidifies, the demoulding is taken out and is cut into rectangular-shaped pieces, obtains drug diffusion unit;

3) medicine feed unit is prepared: adopt N-type silicon chip, Wafer Cleaning is clean, adopt wet oxidation, layer of oxide layer is generated at silicon chip surface, SU-8 photoresist material is coated at its surface uniform, be placed on by mask plate above uniform photoresist material, employing ultraviolet exposure technology makes administration groove by lithography, medicine flows into and the planar graph of flow pass and liquid storage tank; Re-use the conventional ICP dry method of semiconductor applications or wet etching goes out groove, pour in groove by the chip material of liquid state, after it solidifies, the demoulding is taken out and is cut into rectangular-shaped pieces, obtains medicine feed unit;

4) sealing-in bonding: process cell capture and lower surface, the upper and lower surface of drug diffusion unit, the upper surface of medicine feed unit of cultivating unit respectively, by them successively by method bondings such as oxygen plasma in uviolizing, vacuum or solidification glue bonds, the micro-fluidic integrated chip that final acquisition is complete;

5) punch: use punch tool to get ostium and taphole and medicine on cell capture with cultivation unit and medicine feed unit and flow into and taphole.