CN101643701A - Cell sorter micro-fluidic chip based on immunomagnetic separation technology and application thereof in aspect of enrichment of rare cells - Google Patents
Cell sorter micro-fluidic chip based on immunomagnetic separation technology and application thereof in aspect of enrichment of rare cells Download PDFInfo
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Abstract
本发明公开了一种属于微流控芯片技术领域的基于免疫磁性分离技术的细胞分选微流控芯片及其在富集稀少细胞方面的应用。该芯片是在现有Y型微流控芯片基础上的改造，包括键合在一起的基片和盖片，所述基片上设有Y型微通道以及分别位于Y型微通道上部两个端点的样品池(1)和缓冲液池(2)、还有位于Y型微通道下部端点的废液池(3)，所述基片上还设有抗原抗体免疫反应室(4)，所述抗原抗体免疫反应室(4)位于Y型微通道交叉点(5)和废液池(3)之间的微通道上，且抗原抗体免疫反应室(4)的宽度为微通道宽度的2－5倍。本发明的芯片可使磁珠高效、快速的被固定，增大细胞的进样量，提高了分析通量，减少进样死体积、减少滞留和交叉感染。
The invention discloses a cell sorting microfluidic chip based on immunomagnetic separation technology and its application in enriching rare cells, belonging to the technical field of microfluidic chips. The chip is a transformation based on the existing Y-shaped microfluidic chip, including a substrate and a cover sheet bonded together. The substrate is provided with a Y-shaped microchannel and two endpoints on the upper part of the Y-shaped microchannel. The sample pool (1) and the buffer pool (2), and the waste liquid pool (3) located at the lower end of the Y-shaped microchannel, the substrate is also provided with an antigen-antibody immune reaction chamber (4), and the antigen The antibody immune reaction chamber (4) is located on the microchannel between the intersection of the Y-shaped microchannel (5) and the waste liquid pool (3), and the width of the antigen-antibody immune reaction chamber (4) is 2-5 times the width of the microchannel. times. The chip of the invention can fix the magnetic beads efficiently and quickly, increase the sample injection volume of cells, improve the analysis flux, reduce the dead volume of sample injection, and reduce retention and cross-infection.
Description
Technical field
The invention belongs to the micro-fluidic chip technical field, be specifically related to a kind of cell sorting micro-fluidic chip and application thereof based on the immunomagnetic isolation technology.
Background technology
Early 1990s micro-total analysis system (Miniaturized Total Analysis Systems, μ-TAS) proposition of notion has produced great effect in analytical instrument and life science, and the guiding chemical analysis apparatus is towards the trend development of microminiaturization, automatization, rapid, integrated and portability.With micro electronmechanical processing technology (MicroElectromechanical Systems, MEMS) be the basis, on chip, make functional units such as Micropump, little valve, microchannel, microelectrode, microreactor, microflow sensor, little detector and constitute the very small chemical system, the function in whole laboratory, comprise sampling, dilute, add on the chip that full chemical process such as reagent, reaction, separation, detection is integrated in the heart size and realize, can be referred to as the meaning " laboratory on the chip " (Lab on a Chip, LOC).It is fast that micro-fluidic chip has analysis speed, and the separation efficiency height has reduced the consumption of sample and reagent, can realize the high throughput analysis of cell, and easily and other test set couplings.
Along with the raising of the development of micro-fluidic chip technology, particularly detection sensitivity, micro-fluidic chip comes into one's own with detection day by day to the analysis of the micro substance in individual cells and the born of the same parents.It mainly is because have following key character that micro-fluidic chip is suitable for cell analysis: the first, and the channel size of micro-fluidic chip and the diameter of typical cells of mamma animals adapt, and help manipulation, the analysis of cell; Secondly, the multidimensional network structure of chip forms the environment of relative closure, and is approaching with the space environment of cell under the physiological status; The 3rd, conduct heat under the chip channel microscale, mass transfer is very fast, and favourable cell research environment can be provided; The 4th, chip can satisfy the needs that high-flux cell is analyzed, and can obtain a large amount of biological informations simultaneously; The 5th, the flexible combination of multiple monotechnics makes integrated cell research become possibility on the chip, can be integrated on the chip piece such as processes such as cell sample introduction, cultivation, sorting, cracking and separation detection and finish.
The common technology of cell sorting is based on flow cytometer (flow cytometry), but its apparatus expensive, bulky, need the professional person to operate, and the cell consumption is big, is difficult to be used widely in laboratory and hospital.The micro-fluidic chip technology has overcome above-mentioned limitation to a certain extent, can realize the miniaturization of analogous instrument, integrated, automatization and portability.At present, the cell sorting method that with the micro-fluidic chip is platform mainly can be divided into two classes, the first has or not or strong and weak the variation carries out sorting according to signal collected, and it two is to carry out sorting according to the changing features of some differences under different motivating force effects of cell self.
(fluorescence activated cell sorter is a kind of method relatively more commonly used in the micro-fluidic chip cell sorting FACS) to the fluorescence excitation cell sorting, and its principle and flow cytometer are similar.At first pair cell carries out fluorescent mark, when cell when the detector, the having or not or the strong and weak flow direction of controlling cell of the fluorescent signal that goes out according to laser excitation.Wolff etc. have designed the micro-fluidic chip that is integrated with functional structure, adopt the method pair cell of fluorescence-activation sorting to screen, when detecting fluorescent signal, the valve closes of waste fluid channel, thereby cell flows into purpose (WolffA. etc., Lab Chip, 2003 that collecting tank has reached cellular segregation, 3,22-27).Schwille etc. carry out fluorescence-activated cell sorting according to the difference of cell optical characteristics.The micro-fluidic affluent channel system that use is made up of the silicone rubber glass-chip of hybridization mixes fluid, coaxially then focuses on a thin fluidised bed, carries out the fluorescence activated cell detection at this thin fluidised bed.(Dittrich P.S. etc., Anal.Chem.2003,75,5767-5774.)
In addition, by induced polarization, because differences such as dielectric characteristics, specific conductivity, shape or sizes, cell that degree of polarization is different or particle are subjected to different dielectric power and separate in electric field in the heterogeneity alternating electric field for integrated microelectrode on micro-fluidic chip, cell.Cheng etc. by the cell that is fixed in the microfluidic channel surface discharge variation that ion causes the surrounding medium conductivity come to cell counting (Cheng X. etc., Lab Chip, 2007,7,746-755).Utilize that low electricity is led, hypotonic medium makes fixed cytolysis, with the picture on surface electrode detection changing conditions of impedance, thereby the number of quantization cell.The method of cytolysis impedance spectra is relatively more responsive, and detectability can reach 20cells μ L
-1Li etc. have designed a microfluidic device that is integrated with injection, separated region and two outlets, adopt the method for continuous dielectrophoresis separate and purifying the biological cell sample of suspension.At sheath stream and sample injection port, with the waterpower method of focusing cell sample is expelled in the passage, in uneven electric field, formed the dielectrophoresis zone come isolated cell and collect in two sample pools (Li Y. etc., Lab Chip, 2007,7,239-248).
Except that aforesaid method, also have by micromachining technology, micron-sized network of etching or passage are realized the sorting of cell as cell filter on chip.Wheeler etc. have made a ultra micro insulated chamber in T shape channel bottom, focus between two buffer stream of cell introducing by waterpower, after a cell falls into the ultra micro chamber, because other cells of space constraint can not enter, thereby in cell suspending liquid, isolate individual cells (Wheeler A.R. etc., Anal.Chem., 2003 rapidly, 75,3581-3586).Fang Zhaolun study group handles and analyzes individual cells in conjunction with fluid gravity and electric drive method on the cruciform channel chip.The applicating fluid segregation drive is introduced sample intake passage with single blood erythrocyte by mocro, and electricity consumption drives cell introducing split tunnel.On split tunnel, apply high pressure, and use gsh in the laser induced fluorescence(LIF) pair cell carried out separation detection (Gao J. etc., Lab Chip, 2004,4,47-52).
Micro-fluidic chip binding immunoassay paramagnetic particle method sorting cells also has been subjected to paying close attention to widely.Its principle is that specific cell-surface antigens can carry out immune response with the magnetic bead that is coated with antibody, under the effect of externally-applied magnetic field, the cell that contains specific antigens is adsorbed and is trapped in the magnetic field, and do not have this kind surface antigen cell can not with antibodies, there is not magnetic, do not rest in the magnetic field, thereby make different cells obtain separating.Successive in micro-fluidic magnetic resolution equipment such as Pamme separated the cell that is loaded with magnetic nano-particle (Pamme N. etc., Lab Chip, 2006,6,974-980), the separation thereby cell deflects under the effect in magnetic field when flowing through micro-fluidic chamber.The micro-fluidic chip of employing continuous flows such as Inglis successfully from people's whole blood, separated by the white corpuscle of magnetic corpuscular mark (Lnglis D.W. etc., Appl.Phys.Lett., 2004,85,5093-509).Make the little magnetic stripe of a row in chip channel, when the cell successive flow through magnetic stripe, the high magnetic field gradients that magnetic stripe produced was caught by the cell of magnetic mark and is changed the flow direction of cell, thereby reaches isolating purpose.Harrison research group in 2003 adopt immunomagnetic beads method with antibody sandwich outside magnetic bead, success caught cell rare in the cell (Furdui V.I. etc., J.Micromech.Microeng., 2003,13, S164-S170).This research group in 2004 adopts Y shape micro-fluidic chip passage, and the T lymphocyte in the blood is separated and concentrates.They have finishing the albumin A magnetic bead of CD3 antibody to introduce chip, by CD3 antibody protein and the lymphocytic specific combination of T, the T lymphocyte is attracted to both sides and has in the Y shape passage in magnetic field, make only to contain ten thousand/ T cell enrichment (the Furdui V.I. etc. that are hunted down, Lab Chip, 2004,4,614-618).Chip of the present invention is the transformation to above-mentioned Y shape micro-fluidic chip, the present invention compares with the Y shape micro-fluidic chip of Furdui design, on Y shape passage, design one cleverly and be used for the immunoreactive reaction chamber of antigen-antibody, long-pending by the partial cross section that enlarges Y shape passage, reduced fluid in this regional linear velocity, target cell is easier like this is hunted down, thereby has improved the capture rate of cell, can increase the flux of cell sorting in addition.
Summary of the invention
The object of the present invention is to provide a kind of cell sorting micro-fluidic chip based on the immunomagnetic isolation technology.
Another object of the present invention is to provide the application of said chip aspect enriching rare cells.
A kind of cell sorting micro-fluidic chip based on the immunomagnetic isolation technology, this chip is the transformation on existing Y type micro-fluidic chip basis, comprise the substrate and the cover plate that are bonded together, sample pool (1) and buffering liquid pool (2) that described substrate is provided with Y type microchannel and lays respectively at two end points in top, Y type microchannel, be positioned at the waste liquid pool (3) of bottom, Y type microchannel end points in addition, it is characterized in that, also be provided with antigen-antibody immune response chamber (4) on the described substrate, described antigen-antibody immune response chamber (4) is positioned on the microchannel between point of crossing, Y type microchannel (5) and the waste liquid pool (3), and the width of antigen-antibody immune response chamber (4) be the microchannel width 2-5 doubly.
Described antigen-antibody immune response chamber (4) is a round rectangle groove, and rectangle length is 1.8-2mm, and width is 0.2-1.0mm, and the degree of depth of groove is identical with the microchannel degree of depth.
The fillet of described round rectangle groove is that the quadrntnt arc of the circle of 0.9-1mm constitutes by radius.
The point of crossing, width between centers Y type microchannel (5) of described antigen-antibody immune response chamber (4) is 10-15mm.
The material of described micro-fluidic chip is one or both in glass, quartz and the superpolymer.
Described superpolymer is one or both in polydimethylsiloxane, polystyrene and the polyoxyethylene glycol.
Described sample pool (1), buffer pool (2), waste liquid pool (3) are cylindrical.
The application of chip of the present invention aspect enriching rare cells, its operation steps is as follows: the zygomorphy up and down in antigen-antibody immune response chamber (4) is put permanent magnet, the magneticstrength scope of permanent magnet is at 280-310mT, the magnetic bead that surface bonding is had a specific antibodies with the speed of 6-10 μ L/min from sample pool (1) sample introduction, speed with 6-10 μ L/min flows into damping fluid from buffering liquid pool (2), the magnetic bead that remains on the microchannel, nonimmune reaction chamber is flushed in the antigen-antibody immune response chamber (4), then the flow velocity of sample to be separated with 0.1-0.5 μ L/min is passed into the chip microchannel from sample pool (1), wash 3-5 time from buffering liquid pool (2) inflow microchannel with the flow velocity of damping fluid at last with 6-10 μ L/min, the cell that contains corresponding antigens is attracted to antigen-antibody immune response chamber (4), thereby target cell obtains separating.
The magneticstrength of both sides has determined to be combined with the sample introduction speed of the magnetic bead of specific antibodies up and down in antigen-antibody immune response chamber (4), when magneticstrength is big, the sample introduction speed of magnetic bead can be big, magneticstrength hour, the sample introduction speed of magnetic bead is little, otherwise magnetic bead can not 100% be hunted down in antigen-antibody immune response chamber (4).
The detection principle of chip of the present invention: the both sides up and down that earlier two block permanent magnets are placed on symmetrically the micro-fluidic chip reaction chamber, then surface bonding there is the magnetic micro-beads of monoclonal antibody to be passed in the passage with big slightly flow velocity, the magnetic micro-beads that is combined with monoclonal antibody is fixed in the reaction chamber under the effect of externally-applied magnetic field, then cell suspension is passed in the passage with less flow velocity, specific immune response between the antigen by cell surface and the antibody of magnetic bead surfaces, the cell that contains specific antigens is adsorbed and is trapped in the magnetic field, and do not have this kind surface antigen cell can not with antibodies, there is not magnetic, do not rest in the magnetic field, thereby make different cells obtain separating, adopt the inverted microscope be equipped with CCD to the cell of the separating counting of taking pictures, as shown in Figure 4 at last.
The making method of chip of the present invention:
1) chip design: designed mask on substrate, such as, with glass is that substrate is made chip employing wet etching technique, with polydimethylsiloxane (PDMS) is that substrate is made chip employing photolithography, because the glass isotropy can make passage expand to both sides, and the size of PDMS passage is compared variation not quite with the mask of design, so the former the mask size of design is slightly littler than the latter.
2) making method of chip:
A) be that substrate, PDMS are the chip manufacture method of cover plate with glass:
A. the micro-fluidic chip figure of design is made mask, graph area is the clear area, and light-transmissive, non-graph area are black region, extinction and can not transmitted light.
B. mask is covered even glue chromium plate (the chromium type: LRC of 63mm * 63mm * 1.5mm; The thick T:145nm glue of chromium class: positive-working photoresist; Glue is thick: 570nm), expose photoresists generation photochemical reaction under uviolizing.
C. the chromium plate after the exposure develops in 0.5% NaOH solution, and to remove the positive-working photoresist that is exposed, the figure on the mask is copied on the optical cement layer.
D. the glue chromium plate after will exposing under the room temperature is put into chromium film etching liquid, and (cerous sulfate: perchloric acid: water=50g: 15mL: 300mL) the exposed chromium film of corrosion guarantees that simultaneously the optical cement layer of non-graph area and chromium film are not destroyed.After high purity water is rinsed well, oven dry.Micrographics structure on the optical cement layer has been transferred on the glass substrate.
E. wet etching microchannel.With 0.5M HF/0.5M NH4F is etching agent etching microchannel, and speed is about 10 μ m h
-1, getting cross section is the recessed microchannel of trapezoid.Remove remaining optical cement layer and chromium film with acetone and chromium film etching liquid successively again, rinse well with high purity water and promptly get totally transparent substrate.
F. punch with miniature bench drill place in the injection port position of substrate, as the chip sample pond.
G. with substrate behind acetone and deionized water for ultrasonic 5-10min, put into H
2SO
4/ H
2O
2(3/1, heated and boiled half an hour in mixing solutions V/V).After cooling, be neutral with the substrate taking-up and with deionized water rinsing to glass sheet surface.
H. the intercepting and the PDMS cover plate that is cured of the same size of substrate, and with glass substrate being put into the oxygen plasma valve tube, vacuumized 3 minutes, open high frequency electric source, take out substrate and cover plate, bonding immediately after 90 seconds.
B) with PDMS be substrate, be the making method of cover glass
A. design and produce the substrate of force plate mask: the black graphics district is microfluidic channel on the mask, and is light tight.
B. get rid of on clean silicon chip and be coated with skim SU-8 photoresist material, adopt SU-8 dissimilar, viscosity, bondline thickness can be controlled between 1～300 μ m.Ultraviolet ray makes resist exposure by photomask, the unexposed area developing solution dissolution.Remaining protruding SU-8 structure is gone up as the formpiston of making the PDMS substrate in silicon chip and surface.
The making of c.PDMS substrate: PDMS monomer (dimethyl siloxane) is mixed with the part by weight of initiator with 10: 1, waters after the degassing and cast from the substrate force plate,, be cooled to room temperature 80 ℃ of polymerizations 1.5 hours, lift-off stencil, water white transparency PDMS substrate.
D. substrate and clean cover glass are cut into identical size (error: 0.2 μ m).In the punching of the liquid storage tank position of substrate, as the chip liquid storage tank.
E. glass substrate is put into the oxygen plasma valve tube together, vacuumized 3 minutes, open high frequency electric source, take out substrate and cover plate, bonding immediately after 90 seconds.
The advantage of chip of the present invention: the present invention utilizes the technology of immunomagnetic isolation, and design antigen-antibody immune response chamber (4) on Y type micro-fluidic chip realizes that easy, quick, the high-throughput of target cell separates and detection.According to the hydromeehanics principle of continuity as can be known, the long-pending general goal flow velocity in stream pipe middle section is little, the little place of sectional area flow velocity is big, reaction chamber (4) sectional area is designed to greater than channel cross-sectional area, thereby make magnetic bead efficiently, be fixed fast, the sample introduction speed of cell increases simultaneously, has improved analysis throughput, reduces the probability of dead volume, minimizing delay and the cross infection of sample introduction.Target cell by the antigen-antibody immune response in the reaction chamber enrichment that is hunted down, thereby separate with other non-target cells, do not need to use expensive instrument in the whole separation detection process, and very little of the consumption of reagent, this chip is not only applicable to the cellular segregation that enriching rare cells reaches fast high-flux, carry out the clinical detection of cancer cells but also can utilize immunocytochemical assay to combine with the magnetic cell sorting, this micro-fluidic chip low cost of manufacture, be easy to produce in batches, realized microminiaturization, integrated and simplification that cellular segregation detects.
Description of drawings
What Fig. 1 made for embodiment 2 is substrate with glass, and PDMS is the size design figure of microchannel of the micro-fluidic chip of cover plate;
What Fig. 2 made for embodiment 3 is substrate with PDMS, and glass is the size design figure of microchannel of the micro-fluidic chip of cover plate;
Fig. 3 is a Y type PDMS micro-fluidic chip synoptic diagram;
" A " is chip PDMS substrate, and " B " is the chip glass cover plate;
1: sample pool, 2: buffer pool, 3: waste liquid pool, 4: antigen-antibody immune response chamber, point of crossing, 5:Y type microchannel
Fig. 4 is the step and the principle of immunomagnetic isolation on the chip;
Fig. 5 is the size design of microchannel on the mask film of prior art Y type micro-fluidic chip.
Embodiment
The structure of embodiment 1 chip of the present invention
Chip of the present invention comprises substrate and the cover plate that is bonded together, substrate is provided with Y type microchannel and lays respectively at the sample pool (1) of two end points in top, Y type microchannel and buffering liquid pool (2), be positioned at the waste liquid pool (3) of bottom, Y type microchannel end points in addition, design has antigen-antibody immune response chamber (4) on the microchannel of point of crossing, distance Y type microchannel (5) 10mm, reaction chamber (4) is a round rectangle groove, width is 4 times of microchannel width, and length is 2mm.Zygomorphy up and down in antigen-antibody immune response chamber (4) is put permanent magnet, the magnetic bead that surface bonding is had specific antibodies is from sample pool (1) sample introduction, damping fluid flows into from buffering liquid pool (2) then, the magnetic bead that remains on the microchannel, nonimmune reaction chamber is flushed in the antigen-antibody immune response chamber (4), then sample to be separated is passed into the chip microchannel from sample pool (1), flowing into the microchannel with damping fluid from buffering liquid pool (2) at last washes, the cell that contains corresponding antigens is attracted to antigen-antibody immune response chamber (4), waste liquid flows into waste liquid pool (3), thereby target cell obtains separating.
(1) making of glass substrate: the size design of microchannel is seen Fig. 1 on the mask film.The mask film is placed on the even glue chromium plate of 63mm * 63mm * 1.5mm, ultraviolet exposure 7 minutes (wavelength 365nm) after developing 100 seconds in the developing solution, is dried half an hour down for 100 ℃.At room temperature use chromium film etching liquid (cerous sulfate: perchloric acid: water=50 grams: 15 milliliters: 300 milliliters) corrode the chromium film, rinse oven dry then with high purity water well.By the digit microscope shooting, the channel size that records on the chromium plate has all increased about 30 μ m.With 0.5M HF/0.5M NH
4The exposed borosilicate glass of F etching agent corrosion, speed is about 10 μ m min
-1, etching is removed residual light glue-line and chromium film with acetone, chromium film etching liquid after 10 minutes more successively, promptly gets substrate.With miniature bench drill punching, drill bit is the diamond bit of 2mm, and the diameter in hole is liquid pool diameter 2mm.Microscopically is measured the microchannel size: microfluidic channel, the wide 110 μ m in top, the wide 60 μ m in bottom, the degree of depth 20 μ m, the size of antigen-antibody immune response chamber: the wide 440 μ m in top, the wide 240 μ m in bottom, the degree of depth 20 μ m.
(2) making of cover plate: the PDMS of intercepting and the same size of substrate is as cover plate.
(3) with substrate and cover plate successively acetone and deionized water for ultrasonic 5 minutes, at H
2SO
4/ H
2O
2(3/1) solution boils little boiling 30 minutes, is electric neutrality with washing in the high purity water to glass surface after cooling, dries up in super clean bench.Then glass substrate and PDMS cover plate are put into the oxygen plasma valve tube, vacuumized 3 minutes, open high frequency electric source, take out substrate and cover plate, bonding immediately after 90 seconds.
Embodiment 3 is a substrate with PDMS, and glass is the chip manufacturing of the present invention of cover plate
(1) making of PDMS substrate: the size design of microchannel is seen Fig. 2 on the mask film.Silicon chip is put into dense H
2SO
4/ H
2O
2(3/1) solution boils little boiling 30 minutes, takes out after cooling with deionized water rinsing to neutral, and acetone is heating 30 minutes on hot-plate after ultrasonic 5 minutes.
(2) on silicon chip, get rid of and be coated with skim SU-8 photoresist material, make bondline thickness be approximately 50 μ m by control whirl coating speed.Ultraviolet ray makes resist exposure by photomask, unexposed part developing solution dissolution.The SU-8 structure of projection is as the formpiston of PDMS substrate on the silicon chip surface.Then the silicon formpiston was carried out silanization 1 hour.
(3) PDMS performed polymer (mix monomer/solidifying agent=10/1) is cast on the silicon formpiston and vacuum outgas, puts into 70 ℃ of baking ovens then and solidifies about 1 hour, then PDMS is peeled off from formpiston, as the substrate of microchannel.
(4) the PDMS substrate is put in the oxygen plasma valve tube with clean cover glass, vacuumized 3 minutes, open high frequency electric source, take out substrate and cover plate, bonding immediately after 90 seconds.
The chip that embodiment 4 utilizes embodiment 2 to make separates and detects HIV-positive CD4
+The T lymphocyte
(1) with magneticstrength is the both sides up and down that two block permanent magnets of 290mT are placed on micro-fluidic chip immune response chamber symmetrically.
(2) be 1.5 * 10 with 2 μ L, concentration
8The anti-CD4 of beads/mL
+Immunomagnetic beads (available from American I nvitrogen company) with the speed of 6 μ L/min from sample pool (1) sample introduction.
(3) contain the PBS buffered soln of 0.1%BSA from the speed feeding of buffering liquid pool (2), with the anti-CD4 that remains on the microchannel, nonimmune reaction chamber with 5 μ L then with 6 μ L/min
+It is indoor that immunomagnetic beads is flushed to immune response.
(4) cell suspension of the 1 μ L flow velocity with 0.2 μ L/min is passed into the chip microchannel from sample pool (1).
(5) wash 3 times from buffering liquid pool (2) inflow microchannel with the flow velocity of the PBS buffered soln that contains 0.1%BSA, contain CD4 with 6 μ L/min
+The T lymphocyte be adsorbed and be trapped in the magnetic field, thereby make target cell obtain separating.
(6) adopt the inverted microscope be equipped with CCD to the cell of the separating counting of taking pictures.
The preparation method of above-mentioned cell suspension: aseptic taking-up mouse spleen puts it in the plate of PBS buffered soln, grinding spleen to spleen with piston is white in color cotton-shaped, place 300 orders not have the mycetocyte screen filtration then, flushing, with the centrifugal 5min of 1000rpm, abandon its supernatant, with the RPMI-1640 re-suspended cell.
The chip and the comparison test of prior art Y type micro-fluidic chip aspect cell sorting of 2 preparations of embodiment 5 embodiment of the invention
The making of Y type micro-fluidic chip in the prior art: the method according to embodiment 2 is made, and wherein, the size design of microchannel is seen Fig. 5 on the mask film.
The chip and the above-mentioned prior art Y type micro-fluidic chip that use embodiment 2 to make respectively according to the method for embodiment 4 carry out HIV-positive CD4
+The lymphocytic separation of T, prior art Y type micro-fluidic chip is 20% to the capture rate of target cell, and chip of the present invention is 90% to the capture rate of target cell.
Reference
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Claims (8)
1, a kind of cell sorting micro-fluidic chip based on the immunomagnetic isolation technology, comprise the substrate and the cover plate that are bonded together, sample pool (1) and buffering liquid pool (2) that described substrate is provided with Y type microchannel and lays respectively at two end points in top, Y type microchannel, be positioned at the waste liquid pool (3) of bottom, Y type microchannel end points in addition, it is characterized in that, also be provided with antigen-antibody immune response chamber (4) on the described substrate, described antigen-antibody immune response chamber (4) is positioned on the microchannel between point of crossing, Y type microchannel (5) and the waste liquid pool (3), and the width of antigen-antibody immune response chamber (4) be the microchannel width 2-5 doubly.
2, micro-fluidic chip according to claim 1 is characterized in that, described antigen-antibody immune response chamber (4) is a round rectangle groove, and rectangle length is 1.8-2mm, and width is 0.2-1.0mm, and the degree of depth of groove is identical with the microchannel degree of depth.
3, micro-fluidic chip according to claim 2 is characterized in that, the fillet of described round rectangle groove is that the quadrntnt arc of the circle of 0.9-1mm constitutes by radius.
4, micro-fluidic chip according to claim 1 is characterized in that, the point of crossing, width between centers Y type microchannel (5) of described antigen-antibody immune response chamber (4) is 10-15mm.
5, micro-fluidic chip according to claim 1 is characterized in that, the material of described micro-fluidic chip is one or both in glass, quartz and the superpolymer.
6, micro-fluidic chip according to claim 5 is characterized in that, described superpolymer is one or both in polydimethylsiloxane, polystyrene and the polyoxyethylene glycol.
7, the application of the described chip of claim 1 aspect enriching rare cells.
8, application according to claim 7, it is characterized in that, zygomorphy up and down in antigen-antibody immune response chamber (4) is put permanent magnet, the magneticstrength scope of permanent magnet is at 280-310mT, the magnetic bead that surface bonding is had a specific antibodies with the speed of 6-10 μ L/min from sample pool (1) sample introduction, speed with 6-10 μ L/min flows into damping fluid from buffering liquid pool (2), the magnetic bead that remains on the microchannel, nonimmune reaction chamber is flushed in the antigen-antibody immune response chamber (4), then the flow velocity of sample to be separated with 0.1-0.5 μ L/min is passed into the chip microchannel from sample pool (1), wash 3-5 time from buffering liquid pool (2) inflow microchannel with the flow velocity of damping fluid at last with 6-10 μ L/min, the cell that contains corresponding antigens is attracted to antigen-antibody immune response chamber (4), thereby target cell obtains separating.
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Application publication date: 20100210 |