CN102174369B - Micro-slit-structure-based full PDMS (polydimethylsiloxane) micro-fluidic cell capturing chip and manufacturing method thereof - Google Patents
Micro-slit-structure-based full PDMS (polydimethylsiloxane) micro-fluidic cell capturing chip and manufacturing method thereof Download PDFInfo
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- CN102174369B CN102174369B CN 201110031106 CN201110031106A CN102174369B CN 102174369 B CN102174369 B CN 102174369B CN 201110031106 CN201110031106 CN 201110031106 CN 201110031106 A CN201110031106 A CN 201110031106A CN 102174369 B CN102174369 B CN 102174369B
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Abstract
The invention discloses a double-layer structure cell capturing chip, which is a micro-slit-structure-based full PDMS (polydimethylsiloxane) micro-fluidic cell capturing chip. The cell capturing chip consists of an upper solidified polydimethylsiloxane thin sheet and a lower solidified polydimethylsiloxane thin sheet, wherein micro-channels with different sizes and shapes are formed in the polydimethylsiloxane thin sheets respectively; the upper layer channel and the lower layer channel are staggered in parallel along edges; and 10+/-5 mu m slits are formed by the channels in an aligning and bonding method, and the length and the quantity of the slits can be adjusted and controlled according to different channel combination and alignment methods so that the amount of captured cells can be controlled. Based on a hydrodynamics principle, the chip blocks cells by using a slit structure so as to capture a limited quantity of cells, and simultaneously, the cell release can be controlled by adjusting the fluid pressure to realize repeated cell capturing. The chip is made by a conventional chip manufacturing method and can capture and release a single cell and a plurality of cells; moreover, the chip can be used for integrating a microelectrode for monitoring captured cells in real time. The invention also discloses a manufacturing method and process of the chip.
Description
Technical field
The present invention relates to that the PDMS micro-current controlled cell is caught chip and micro-current controlled cell is caught control.
Background technology
Cell is the very small elementary cell with unique vital movement, and traditional cell research is usually take a large amount of cell samples as object, and expectation obtains the general properties of similar cell.Yet even also more or less there are differences between the cell of same type, therefore this analysis in fact only can provide the average response of whole cell sample, and has ignored the details between single cell and the cell.The heterogeneity of cell behavior is for bacterium or animal eukaryotic cell all is its inherent nature, also is ubiquitous in all complex biological events such as Growth of Cells, differentiation and infection.Therefore, in biology and chemical research, to controlledization of experiment condition, especially for single celled control, to help scientist to the understanding of the basic vital processes such as cell regulate and control mechanism and molecular level interaction, also be the very strong driving force that promotes scientific and technological development always.At present, the most successful unicellular patch clamp and the laser optical tweezer technology of surely belonging to of catching and handle.The former unicellular is captured in single glass microelectrode suction pipe port by negative pressure with one, and is used for the single cell analysis technology of record ion channel in cell membrane molecule activity.The laser optical tweezer technology is that the three-dimensional potential well that utilizes laser that a branch of height converges to form is captured and most of biological particles such as manipulation cell, organoid even biomacromolecule.But these two kinds of technology all need complex instrument, and efficient is too low.
The microflow control technique that grew up in recent years or lab-on-chip technology control and analyze the thinking of development that provides new for cell.Based on physical and chemical principles different under the microscale, scientist has developed the micro-current controlled cell chip with various functions and different application.As the basic premise of cell subsequent analysis and research, the separation of cell and catching on the especially unicellular yardstick, almost all is an important ring in all micro-current controlled cell analytical equipments.At present, be used for cellular segregation on the chip and the means of catching relate to the various fields such as light, electricity, sound, magnetic, hydromeehanics, mechanical workout and chemical process, mainly be divided into contact and contactless two kinds.The effect such as light, electricity, sound, magnetic all is contactless.Optical instrument is the combination of laser optical tweezer technology and chip technology, has utilized the transparent characteristic of most chips such as glass, PDMS and other macromolecular materials, and it can realize unicellularly catching and controlling, but it relates to the instrument complexity, controls singlely, limits more.Electrical method is the engagement means the most successful with micro-fluidic chip, not only is conducive to instrument integration, and the multiple mode of action is arranged, such as electric osmose, electrophoresis, dielectrophoresis (nDEP, the mode such as pDEP) can carry out not only that the group is somatic to be caught and separate, and also can realize unicellular catching.But this mode often need to be on chip integrated fine electrode, need complicated power supply control; In addition, voltage has adverse influence to cytoactive and solution system.Acoustics and magnetics are emerging contactless cell control devices, but its control effect is limited, and very difficult realization is unicellular to be caught and control.Chemical process is strong as a kind of selectivity, and the application of method on micro-fluidic chip that bio-compatibility is good is increasingly extensive.It is often in conjunction with cell adhesion and the combination of micromachined means by the chemically modified feasible region, can reach unicellular selectivity and catch, be generally used for cell cultures, immunoassay etc., but its collection efficiency be relatively poor, control a little less than, and need complicated chemically modified to process.Also having a kind of chemical process is to utilize hydrogel parcel cell to form to catch, this method action temperature and, but its operability is relatively poor.It is present the most effective cell fixed form that micromachining technology is used for catching of integral body and individual cells sample in conjunction with hydromeehanics control.The geometry traps such as little well that this technology often is complementary by finish size and cell, micropore, Wei Ba, little slit and microchannel or obstacle are caught cell, not only can form open array system, can also realize the control to cell in the microchannel.This method need not other control devices and instrument, to the cell not damaged, can compatible other analyses or control method, can in physiological processs such as unicellular its stimuli responsive of yardstick research, interaction and migration, differentiation, apoptosis, be particularly useful for suspension cell is studied.Its shortcoming is that micro-processing technology is had relatively high expectations, and is difficult to realize in the Experiment of General Chemistry chamber.
Therefore, seek a kind of simple general-purpose, adopting the conventional machining means is attainable chip manufacture method, and is attainable high-level efficiency, high-resolution cell capture method are significant in common lab.
Summary of the invention
The object of the invention is to avoid the use of expensive device and meticulous undressed means, and a kind of micro-current controlled cell catching method simple, efficient general of making is provided, be used for expanding the effective means that common lab is studied cell.
Purpose of the present invention reaches by following measure:
A kind of double-deck cell capture chip, it is based on full polydimethylsiloxane (PDMS) micro-current controlled cell of little narrow slit structure and catches chip, it is made of the polydimethylsiloxane thin slice of up and down two curing, on the polydimethylsiloxane thin slice, be manufactured with respectively the miniature recessed channel of different size and shape, when upper and lower two polydimethylsiloxanes are superimposed, make upper and lower two-layer passage be parallel to each other staggered along the edge, form the single or multiple slits of 10 ± 5 μ m, this slit is caught the controlled of limited quantity cell for stopping that cell forms.
Above-mentioned bilayer structure cell capture chip, described passage slit can make by the method for channel alignment bonding.
Above-mentioned bilayer structure cell capture chip, described passage slit length and quantity can be regulated and control by different combination of channels and alignment methods.
Above-mentioned bilayer structure cell capture chip can be controlled the quantity of catching cell by length and the size of adjusting slit, can form effectively catching from individual cells to tens cell.
Above-mentioned bilayer structure cell capture chip can utilize principle of hydrodynamics that cell is caught, and can pass through the pressure-controlling release cells of gangway after catching, thereby can repeat the operation of catching of cell.
A kind of method of making above-mentioned bilayer structure cell capture chip, it comprises the following steps:
Step 3. slit is made and chip bonding: will make up and down two PDMS chips of respective channel simultaneously through Cement Composite Treated by Plasma, drip rapidly several deionized waters thereon, then cover upper slice on lower, this moment is because the existence of water, two PDMS can slide mutually smoothly, with the microchannel parallel alignment on two-layer, make it form the slit of whole piece or partial-length, adjust slit width at microscopically and make it to be no more than 15 μ m (less than corresponding cell size), suck most of excessive moisture with filter paper, keep up and down the layers of chips position and place under the infrared lamp drying, be bonding firm and obtain slit and catch chip behind about 30min, slit length can partly be adjusted according to align with tube, and the gangway of two-layer passage was to carry out sample introduction operation and pressure adjustment about upper slice need were reserved.
The method of above-mentioned making bilayer structure cell capture chip, described step 3 can be carried out the regulation and control of combination of channels and slit: can adopt the microchannel of isomorphism type not to be combined to form the slit of different lengths, be used for the quantity that cell is caught in regulation and control; Can adopt beeline channel to be combined to form longer slit, thereby form the linear arrangement of cell; Also can adopt point-like combination of channels or straight line and point-like combination of channels to form a plurality of slits of catching, realize catching a small amount of cell; Simultaneously, owing to can pass through the up and down aligned position of the flexible control channel of slip of two PDMS, can adopt suitable point-like combination of channels, the aligning that intersects forms small slit, and individual cells is caught.
Double-layer cell of the present invention is caught chip and can easily single and a plurality of cells be caught and discharge, and the steps include:
Inject cell suspension at upper strata feeder connection place, because the difference of hydrostatic pressure, cell are followed solution stream and are formed shunting to outlet and to lower floor's pipeline, stopped by slit, the cell streamwise constantly on slit ordered aggregation form and to catch, unnecessary cell is discharged from outlet with suspension; Keep pressure-acting, also can be to catching the operations such as cell cleans.
Sample solution from the entrance sucking-off, is injected buffered soln in lower floor entrance and exit of the passage, make liquid pressure reverse, even the solution reverse direction flow is crossed slit, can discharge the cell of catching, and discharge from the upper strata channel outlet, thereby can repeat cell is caught and discharged.
Concrete effect of the present invention is as follows: the inventive method is utilized the microchannel to aim at bonding method and is made the narrow slit structure that can be used for catching a small amount of cell by simple chip manufacture method.Compare with cell capture technology on the traditional chip that utilizes hydrodynamic force principle and microstructure, this invention has the following advantages: 1, making method is simple, reliable, with low cost, adopts full PDMS material and water lubricating bonding method, does not need too much modification step; 2, need not clean room, need not accurate little processing instrument and means, only utilize traditional lithographic method can form the narrow slit structure that is applicable to cell size, can realize in the Experiment of General Chemistry chamber, be convenient to popularize; 3, the method makes structure and can conveniently adjust, and realizes catching and arranging from individual cells to the dozens of cell, and capture rate is high, controls easy; 4, this chip is soft chip, simultaneously the PDMS material so that follow-up modification and integrated operation is simple and feasible also can make integrated microelectrode and be used for cell is carried out Real Time Monitoring and analyzing and testing.
Description of drawings
Fig. 1.The PDMS micro-channel chip is made synoptic diagram;
Fig. 2Slit is made and the integrated chip synoptic diagram.(a) PDMS of lower floor chip microchannel; (b) upper strata PDMS chip microchannel; (1) PBS buffered soln entrance; (2) PBS buffered soln outlet; (1 ') cell solution entrance; The outlet of (2 ') cell solution;
Fig. 3.Cell capture and slit regulation and control.The combination of (a, e) beeline channel; (b, f) point-like passage, beeline channel combination; (c, g) beeline channel, point-like combination of channels; (d, h) point-like passage, point-like combination of channels;
Fig. 4.A small amount of cell capture and unicellular catching.(a) the corresponding slit of point-like-a plurality of cell captures; (b) the staggered slit of point-like-a plurality of cell captures; (c) the staggered slit-individual cells of point-like is caught.
Embodiment
The invention will be further described below in conjunction with accompanying drawing and example.
The water that uses in the experimentation is redistilled water, and the experiment agents useful for same comprises Sodium phosphate dibasic, potassium primary phosphate, and Repone K, sodium-chlor, polyvinyl alcohol etc. are analytical pure.Used cell is human stomach cancer cell line (BGC-823).
The making of embodiment 1. bilayer structure cell capture chips:
1. adopt mode as shown in Figure 1 to prepare the PDMS micro-channel chip.
2. adopt as shown in Figure 2 mode to prepare slit and integrating cell is caught chip
Step 3. slit is made and chip bonding: will make up and down two PDMS chips of respective channel simultaneously through Cement Composite Treated by Plasma, drip rapidly several deionized waters thereon, then cover upper slice on lower, this moment is because the existence of water, two PDMS can slide mutually smoothly, with the microchannel parallel alignment on two-layer, make it form the slit of whole piece or partial-length, adjust slit width at microscopically and make it to be no more than 15 μ m (less than corresponding cell size), suck most of excessive moisture with filter paper, keeping up and down the layers of chips position and place under the infrared lamp drying, is that bonding firmly obtaining slit catches chip behind about 30min.Slit length can partly be adjusted according to align with tube, and the gangway of two-layer passage was to carry out sample introduction operation and pressure adjustment about upper slice need were reserved.
Step 4. combination of channels and slit regulation and control: can adopt the microchannel of isomorphism type not to be combined to form the slit of different lengths, be used for the quantity that cell is caught in regulation and control.Can adopt beeline channel to be combined to form longer slit, thereby form the linear arrangement of cell; Also can adopt point-like combination of channels or straight line and point-like combination of channels to form a plurality of slits of catching, realize catching a small amount of cell.Simultaneously, owing to can pass through the up and down aligned position of the flexible control channel of slip of two PDMS, can adopt suitable point-like combination of channels, the aligning that intersects forms small slit, and individual cells is caught.
In order to characterize this chip to the capture effect of cell, we adopt different combination of channels to make little slit, and suspension BGC-823 cell sample is caught, and the result as shown in Figure 3.The steps include:
Embodiment 3. adopts bilayer structure cell capture chip, catches several cells or individual cells:
In order to characterize this chip to the acquisition performance of a small amount of cell, we adopt and reduce slit length and adopt the mode of staggered bonding that several and individual cells are caught, and the result as shown in Figure 4.
Claims (6)
1. the making method of a bilayer structure cell capture chip, it comprises the following steps:
The step 1) glazing channel is made: the glass-chip passage adopts standard photoetching and the preparation of wet chemical lithographic technique;
Adopt the commercialization sol evenning chromium plate, carry out photoetching according to the channels designs pattern, etch depth is 30-50 μ m;
Step 2) in the making, lower two-layer polydimethylsiloxane (PDMS) micro-channel chip: with the negative template of above-mentioned glass-chip, a certain amount of PDMS thereon casts, be heating and curing, namely get the PDMS micro-channel chip with respective male structure after peeling off, with this PDMS micro-channel chip with respective male structure after air plasma is processed, it is rapidly 0.1 ~ 0.5% polyvinyl alcohol water solution in surperficial spin coating mass percentage concentration, and under infrared lamp, dry, has the positive template of PDMS micro-channel chip of respective male structure with this, PDMS thereon casts, can easily peel off after the curing, can obtain the PDMS micro-channel chip consistent with the etching glass chip channel;
The step 3) slit is made and chip bonding: will make the upper of respective channel, lower two PDMS micro-channel chips are simultaneously through Cement Composite Treated by Plasma, drip rapidly several deionized waters thereon, then upper PDMS micro-channel chip is covered on lower PDMS micro-channel chip, this moment is because the existence of water, two PDMS micro-channel chips can slide mutually smoothly, with the microchannel parallel alignment on the two-layer PDMS micro-channel chip, make it form the slit of whole piece or partial-length, adjust slit width at microscopically and make it to be no more than 15 μ m, suck most of excessive moisture with filter paper, in the maintenance, descend two-layer PDMS micro-channel chip position and place and dry under the infrared lamp, be bonding firm and obtain slit and catch chip behind about 30min, slit length is partly adjusted according to align with tube, and the gangway of two-layer PDMS micro-channel chip passage was to carry out sample introduction operation and pressure adjustment about upper PDMS micro-channel chip was reserved.
2. the making method of bilayer structure cell capture chip according to claim 1, it is characterized in that, the regulation and control of step 4) combination of channels and slit have also been comprised: adopt the microchannel of isomorphism type not to be combined to form the slit of different lengths, be used for the quantity that cell is caught in regulation and control.
3. the making method of bilayer structure cell capture chip according to claim 2 is characterized in that, in described step 4) combination of channels and the slit regulation and control, adopts beeline channel to be combined to form longer slit, thereby forms the linear arrangement of cell.
4. the making method of bilayer structure cell capture chip according to claim 2, it is characterized in that, in described step 4) combination of channels and the slit regulation and control, adopt point-like combination of channels or straight line and point-like combination of channels to form a plurality of slits of catching, realize catching a small amount of cell.
5. the making method of bilayer structure cell capture chip according to claim 2, it is characterized in that, in described step 4) combination of channels and the slit regulation and control, the aligned position of the flexible control channel of slip by upper and lower two PDMS micro-channel chips, adopt the point-like combination of channels, intersect and aim at the small slit of formation, individual cells is caught.
6. the bilayer structure cell capture chip of arbitrary described making method preparation according to claim 1-5.
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| CN103387935A (en) * | 2012-05-09 | 2013-11-13 | 中国人民解放军军械工程学院 | Microfluidic array chip for cell capture |
| CN102787364B (en) * | 2012-08-07 | 2013-12-11 | 中国科学院大连化学物理研究所 | Manufacturing method and application of PDMS (Polydimethylsiloxane) polymer chip with arc sunk holes |
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| CN101827931A (en) * | 2008-08-29 | 2010-09-08 | 北京大学 | A microfluidic chip for accurately controllable cell culture |
| CN101629143B (en) * | 2008-12-02 | 2011-09-21 | 中国科学院上海微系统与信息技术研究所 | Microfluidic cell array chip for high-throughput medicament screening, method and use |
| CN101748060B (en) * | 2008-12-15 | 2012-04-18 | 国家纳米科学中心 | Device and method for arranging a plurality of cells at same plane and controlling cells |
| CN101451105B (en) * | 2008-12-26 | 2011-06-08 | 中国科学院上海微系统与信息技术研究所 | Construction method of blood capillary model and microsystem chip thereof |
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