CN103018224B - Based on rare cells separation detecting system and the method for centrifugal microfluidic control techniques - Google Patents
Based on rare cells separation detecting system and the method for centrifugal microfluidic control techniques Download PDFInfo
- Publication number
- CN103018224B CN103018224B CN201210545205.9A CN201210545205A CN103018224B CN 103018224 B CN103018224 B CN 103018224B CN 201210545205 A CN201210545205 A CN 201210545205A CN 103018224 B CN103018224 B CN 103018224B
- Authority
- CN
- China
- Prior art keywords
- chip
- microtrabeculae
- centrifugal
- elasticity
- micro
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Centrifugal Separators (AREA)
- Automatic Analysis And Handling Materials Therefor (AREA)
Abstract
The invention discloses a kind of rare cells separation detecting system based on centrifugal microfluidic control techniques and method, described system comprises the micro-fluidic chip of a similar CD, a centrifugal driver module and an optical detecting module.Wherein micro-fluidic chip comprises microchannel and the microcavity of many group radiate, and chip one-piece construction is made up of elasticity microtrabeculae guide rail layer, deformable films layer, pipeline/cavity layer, filtration rete and waste collection layer.During use, first the microballoon of sample liquid and immune modification is imported in its liquid storage cylinder by micro-fluidic chip injection port, and be placed in the centrifugal platform of centrifugal driver module, assemble elasticity microtrabeculae, low speed rotation, realize abundant mixing and the reaction of the microballon liquid of sample liquid and immune modification in liquid storage cylinder, then High Rotation Speed chip is separated; Then drip the fluorescent-labeled antibody solution of specific recognition in each isolated cell collecting region, incubation reaction, add damping fluid and centrifugal; Finally, undertaken identifying and analyzing by optical detecting module.
Description
Technical field
The present invention relates to a kind of rare cells separation detecting system based on centrifugal microfluidic control techniques and using method, can be applicable to without wound pre-natal diagnosis, early diagnosis of tumor and the field such as Prognosis scoveillance and stem-cell research.
Background technology
Optionally be separated from complex mixture rare cells cell biology and clinicing aspect all significant, such as isolating fetal red blood cell from maternal peripheral blood, separating cycle tumour cell (Circulating Tumor Cell in peripheral blood in patients, CTC) separate stem cells etc. and in marrow or tissue, the analysis of these cells provides very valuable information often can to the treatment of clinical diagnosis and disease, but this kind of cell content is in the sample to which very rare, be generally only 1 ~ 10/mL, separation and Extraction is very difficult, therefore greatly limit its clinical practice.Traditional rare cells separation and concentration technology mainly contains density－gradient centrifuga－tion method, isolated by filtration method and immunological magnetic bead sorting method etc.Wherein density－gradient centrifuga－tion method and isolated by filtration method are all screen and enrich target cells by the physical characteristics difference (such as density, size) of various cell; but this kind of to carry out the method specificity of separation and concentration based on cell physical characteristics poor; gained sample purity is lower, and its risk is the mistake that often can cause medical diagnosis on disease.Immunological magnetic bead sorting method is rare cells method for separating and concentrating the most conventional at present, its ultimate principle utilizes rare cells surface antigen to be combined with the monoclonal antibody specificity being connected with immunomagnetic beads, realizes the separation and concentration of rare cells under additional magnetic fields.The concentration and separation effect of the high degree of specificity of antigen-antibody reaction and immunomagnetic beads combines by this method, have easy, sensitive, feature fast, and separation purity is high, and productive rate is large, does not affect the activity of separated cell.But this method is applied to the enrichment aspect of rare cells, still has many deficiencies.On the one hand, traditional separation method based on magnetic force, often adopt centrifuge tube operation, due to magnetic force suffered by magnetic bead, its average settlement path longer (several mm ~ 1cm), therefore magnetic bead-cell complexes complete sedimentation required time is longer, separation efficiency is lower, and because of magnetic force suffered by magnetic bead relatively weak, basic and hydrodynamic shear magnitude, easily cause part magnetic bead or magnetic bead-cell complexes to be taken away together by cleansing solution, affect the accuracy of testing result; In addition, in testing process, the cell of separation and concentration often needs to transfer on new container or flat board and detects, and often easily causes part target cell to be missed because of transfer indfficiency.Therefore, the specificity and susceptibility that detect is separated in order to improve and improve rare cells, lot of domestic and international research institution and enterprise are all devoted to the new rare cells method for separating and concentrating of research and equipment, and the rare cells separation method especially based on microflow control technique is developed rapidly in recent years.Wherein the most strikingly Harvard Medical School report the CTC separation method based on microflow control technique, the method utilizes microfluidic chip technology to be successfully separated to detect a small amount of CTC [the Nagrath S in neoplastic disease human body, Sequist LV, Maheswaran S, et al.Isolation of rare circulating tumour cells in cancer patients by microchip technology.Nature.2007,450:1235-1241.], tumour cell capture rate is up to 99%, and separation purity is about 50%.Micro-fluidic chip used take silicon as substrate, the processes such as photoetching, plasma deep etching, bonding are adopted to make, this chip contains 78,000 silica-based microtrabeculae, by wrapping by anti-EpCAM antibody on microtrabeculae, make the CTC wherein contained when blood flows through microtrabeculae by immunocapture, finally use immunofluorescence method labeled in situ CTC again and to its counting, realize the detection of CTC.Because the method is without the need to the pre-service of whole blood sample, simultaneously operating process is simply gentle, smaller to cellular damage, avoid the tedious steps of separation and purification and centrifuge washing repeatedly, and detection sensitivity is high, shows good application prospect.However, still there is larger deficiency in the method: on the one hand, in order to ensure the capture rate of cell, consider the impact of the factor such as hydrodynamic shear, fluid stream shape space distribution, blood sample must maintain slower speed (~ 1mL/h) in the chips, therefore the method testing process longer (5 ~ 7 hours) consuming time, efficiency is lower.On the other hand, the opaque silica-based making of chip used employing, is unfavorable for optical detection, and in chip, the plasma etching of microtrabeculae is processed, and need expensive instrument, cost of manufacture is higher, limits the large-scale application that it is clinical.Although, someone tries hard to improve this method [Wang S recently, Liu K, Liu J, et al.Highly Efficient Capture of Circulating Tumor Cells by Using Nanostructured Silicon Substrates with Integrated Chaotic Micromixers.Angew.Chem.Int.Ed.2011, 50:3084 – 3088.], catch microtrabeculae spacing and arrangement as by immunity moderation or in microchannel, increase twill ridged microstructure and optimize CTC capture rate in micro-fluidic chip, but improvement effect is limited, still cannot meet the requirement of efficient capture and quick separating simultaneously.Therefore, be badly in need of a kind of being easy to of development to operate, the system that rare cells is rapidly and efficiently separated can be realized.Thus become design of the present invention.
Summary of the invention
The object of this invention is to provide a kind of rare cells separation detecting system based on centrifugal microfluidic control techniques and using method, the separation detecting system provided have simple to operate, automaticity is high, can realize the feature that rare cells is rapidly and efficiently separated, be expected to be applied to circulating tumor cell in fetal erythrocyte in maternal peripheral blood, peripheral blood in patients and be separated with the efficient of rare cells such as stem cells in tissue.
A kind of rare cells separation detecting system based on centrifugal microfluidic control techniques provided by the invention, is characterized in that: described system comprises micro-fluidic chip 1, centrifugal driver module 2 of a similar CD and the optical detecting module 3 of a separation detecting system.This system utilizes the rotation of centrifugal platform 4 in centrifugal module 2, rubber film 15 on the deformation chamber 9 elasticity of compression microtrabeculae 6 be fixed on its fixed pedestal 5 periodically being extruded chip communicates with liquid storage cylinder 7, realize abundant mixing and the rapid reaction of sample liquid and immune modification microballoon in liquid storage cylinder, utilize chip high speed to rotate the centrifugal force of generation in conjunction with the filtration rete 17 of chip disengagement chamber 8 regional ensemble simultaneously, realize the efficient separation of immunocapture cell.
Specifically, the micro-fluidic chip outward appearance of separation detecting system is disc, as shown in Figure 4, is made up of multiple structural sheet, comprises elasticity microtrabeculae guide rail layer 14, deformable films layer 15, pipeline/cavity layer 16 successively, filters rete 17 and waste collection layer 18; Pipeline/the cavity layer 16 of its chips comprises a liquid storage cylinder 7 and one group of disengagement chamber 8, and each disengagement chamber is around liquid storage cylinder periodic arrangement radially; Each disengagement chamber is all in fan-shaped, one end away from chip center is punctured into a semicircle microcavity, as isolated cell collecting region 10, this design isolated cell being gathered in some specific regions, avoids the micro-search operation of loaded down with trivial details large area consuming time in subsequent detection process; And all have injection port above each cell harvesting district, for loading fluorescently-labeled specific antibody solution in cellular identification process, in chip rotary course and incubation reaction process, above each cell harvesting district, injection port all seals with adhesive tape 13, prevents the loss of chip rotary course and incubation period sample liquid.And this mode directly loading identification reaction liquid in isolated cell collecting region, required precious fluorescent-labeled antibody liquor capacity in qualification process can be greatly reduced, reduce testing cost, by one group of microchannel connection between liquid storage cylinder and each disengagement chamber, as fluid passage, microchannel ensures that fluid and cell are transferred to disengagement chamber from liquid storage cylinder on the one hand, also in low driving pressure situation, temporarily limits sample liquid in liquid storage cylinder as capillary valves simultaneously, liquid storage cylinder connects one group of through hole and comprises an injection port 11, blow vent 12 and at least one deformation chamber 9, deformation chamber top layer is rubber film 15, and this thin layer is held between chip elasticity microtrabeculae guide rail layer 14 and pipeline/cavity layer 16, elastic deformation can occur under pressure, thus can change deformation cavity volume by the rubber film on mechanical presses deformation chamber, as shown in Figure 5, the elasticity of compression microtrabeculae 6 be installed on centrifugal module fixed pedestal can provide this mechanical pressure, due to the restriction of elasticity microtrabeculae guide rail layer 14 guide-track groove on chip, elasticity of compression microtrabeculae is only subjected to displacement in the vertical direction, namely the pressure of vertical direction is only applied when it contacts with chip, when chip rotates with centrifugal platform, elasticity of compression microtrabeculae periodically contacts with rubber film 15 on each deformation chamber 9, thus periodicity compressional deformation chamber, cause fluid generation vibratory movement in the liquid storage cylinder communicated with deformation chamber, accelerate Reactive fluids mixing process wherein, the filtration rete 17 of micro-fluidic chip is processed with the thin polymer film of microwell array for one deck, and be held between chip pipeline/cavity layer and waste collection layer, pore size is 8 ~ 12 μm, ensures to stop the target cell after in conjunction with immune microsphere, the waste collection layer 18 of micro-fluidic chip comprises the annular micro-cavity that is processed with micro-pillar array, and micro-pillar array is used as supporting construction, prevents subsiding of filtration membrane, in order to ensure that waste collection chamber can receive separation and waste reaction solution completely, microcavity external diameter should be greater than the distance of pipeline/cavity layer cell harvesting offset from chip center of chip, the making material of the elasticity microtrabeculae guide rail layer of above-mentioned micro-fluidic chip, pipeline/cavity layer and waste collection layer can be glass, PDMS(Polydimethylsiloxane, dimethyl silicone polymer), PS(polystyrene, polystyrene), PC(Polycarbonate, polycarbonate), COC(Cyclic olefin copolymer, cyclic olefine copolymer), PMMA(Polymethyl methacrylate, polymethylmethacrylate), any one in SU-8.In addition, the filtering membrane layer material of micro-fluidic chip can be PDMS(Polydimethylsiloxane, dimethyl silicone polymer), PS(polystyrene, polystyrene), PC(Polycarbonate, polycarbonate), COC(Cyclic olefin copolymer, cyclic olefine copolymer), PMMA(Polymethyl methacrylate, polymethylmethacrylate), PI(Polyimide, polyimide), PA(Parylene, Parylene), any one in SU-8.
The centrifugal driver module of separation detecting system is made up of a rotation motor 4, fixed pedestal 5 and one group of elasticity microtrabeculae 6.Wherein rotation motor 4 is program control governor motor; Fixed pedestal 5 provides support for giving one group of elasticity micro-column structure 6; Elasticity microtrabeculae lower end is smooth spherical shape, ensure rotary course Elastic microtrabeculae and guide-track groove with can deformation film contact the rotation that can not hinder chip; Under the effect of power in the vertical direction, elasticity microtrabeculae can move up and down, and by suitably adjusting its height, the pressure that self spring deformation of elasticity microtrabeculae produces can ensure that its lower end directly contacts with chip guide rail layer groove surface, when chip rotates to deformation chamber 9 perpendicular alignmnet elasticity microtrabeculae, the elasticity microtrabeculae of compression can apply pressure by deformation film 15 to deformation chamber top layer, makes it that deformation occur; Elasticity microtrabeculae is fixed on fixed pedestal by a liftable support, when support rises, elasticity microtrabeculae can not apply extruding to the deformation chamber of chip, and when support declines, elasticity microtrabeculae then applies extruding by elastic force to the deformation chamber of chip, the fluid rapid mixing of auxiliary liquid storage cylinder.
The optical detecting module of separation detecting system is made up of the lens 3 that a group could excite and detect fluorescence, these lens lay respectively at chip upper and lower, lens distance centrifugal platform central shaft distance equals chip isolated cell collecting region distance centrifugal platform central shaft distance, by the rotation of centrifugal driver module driving chip, the original position realizing isolated cell detects automatically, to avoid in classic method from Disengagement zone to the loss that qualification district transfer process causes caught rare cells, further increase rare cells and be separated the sensitivity detected.
In use procedure, first the microballoon of sample liquid and immune modification is imported in its liquid storage cylinder by micro-fluidic chip injection port, due to the capillary valves effect of microchannel, the microballoon of sample liquid and immune modification can be confined in liquid storage cylinder, directly can not enter in disengagement chamber by connecting microchannel, after completing sample introduction, chip is placed in centrifugal platform, and falls elasticity microtrabeculae support, make end in contact guide-track groove under microtrabeculae, and make elasticity microtrabeculae spring be in compressive state, after completing the assembling of elasticity microtrabeculae, low speed rotary centrifugal platform, rotary course Elastic microtrabeculae is utilized to extrude the periodicity of chip deformation chamber upper surface deformable membrane, make fluid generation vibratory movement in liquid storage cylinder, realize fully mixing and the reaction fast of sample liquid and immune modification microballoon in liquid storage cylinder, after reaction terminates, High Rotation Speed centrifugal platform, the effect that the fluid driven in liquid storage cylinder and cell mixture overcome microchannel capillary valves is entered disengagement chamber by larger centrifugal force, in disengagement chamber region, due to the effect of centrifugal force, fluid will flow to lower floor's waste liquid chamber through filtering membrane, the cell that particle diameter is less also will enter waste liquid chamber through fenestra with fluid, and general rare cells is (as fetal erythrocyte, CTC and stem cell) diameter is larger, and after binding immunoassay microballoon, the yardstick of its complex is larger, therefore will be filtered film and be blocked in disengagement chamber upper strata, thus realize being separated of rare cells and other most cells.Under the influence of centrifugal force, the cell on disengagement chamber upper strata is finally gathered in chip collecting region at (comprising the cell of immunocapture).After separation completes, by sealant tape removing on corresponding for each collecting region filling opening, adding can the fluorescent-labeled antibody solution of specific recognition cell to be detected, and again sticks sealant tape, incubation reaction.After reaction terminates, damping fluid is added and high speed centrifugation at chip injection port, utilize the washing of damping fluid to remove unreacted fluorescent-labeled antibody molecule, finally by automatic rotation precise assembly centrifugal platform, utilize optical detecting module to carry out situ identification and analysis to each collecting region cell.
As can be seen here, the present invention is characterised in that:
1. each disengagement chamber is punctured into an isolated cell collecting region away from one end of chip center, and the injection port of the corresponding loading identification reaction liquid in each cell harvesting district, when chip rotates, each identification reaction liquid injection port all seals with adhesive tape;
2. the elasticity microtrabeculae guide rail layer of described micro-fluidic chip is concave surface loop configuration, only length travel occurs in order to limited chip rotary course Elastic microtrabeculae; Its corresponding deformation cavity region has through hole, and the elasticity microtrabeculae compressed by this through hole can bring pressure to bear on the deformable films of deformation chamber top layer;
3. the deformable films layer of described micro-fluidic chip is held between chip elasticity microtrabeculae guide rail layer and pipeline/cavity layer, elastic deformation can occur under pressure;
4. described system comprises the optical detecting module of the micro-fluidic chip of a similar CD, a centrifugal driver module and a separation detecting system; Wherein micro-fluidic chip is made up of multiple structural sheet, is followed successively by and comprises elasticity microtrabeculae guide rail layer, deformable films layer, pipeline/cavity layer, filtration rete and waste collection layer; Centrifugal driver module is made up of a rotation motor, a fixed pedestal and one group of elasticity microtrabeculae; The optical detecting module of separation detecting system is made up of the lens that a group could excite and detect fluorescence;
5. first the microballoon of sample liquid and immune modification is imported in its liquid storage cylinder by micro-fluidic chip injection port when system provided by the invention uses, and be placed in the centrifugal platform of centrifugal driver module, assemble elasticity microtrabeculae, low speed rotation, rotary course Elastic microtrabeculae is utilized to extrude the periodicity of chip surface deformable membrane, realize abundant mixing and the reaction of the microballon liquid of sample liquid and immune modification in liquid storage cylinder, then High Rotation Speed chip, realizes the rapidly and efficiently separation of immunocapture cell by centrifugal force combined filtering film; After separation, dripping in each isolated cell collecting region can the fluorescent-labeled antibody solution of specific recognition cell to be detected, incubation reaction, then adds damping fluid and centrifugal, removes unreacted fluorescent-labeled antibody molecule; Finally, undertaken identifying and analyzing by optical detecting module.
Immune microsphere, micro-pore-film filtration and centrifugal platform combine and build rare cells separation detecting system by the present invention, substantially increase the separation purity of rare cells, accelerate separation detection speed and the efficiency of rare cells.Compared with the existing rare cells separation detecting system based on microflow control technique, rare cells separation detecting system provided by the invention has obvious advantage in separation detection speed and cost, realize rare cell specific isolation by binding immunoassay microballoon and centrifugal filtering method on the one hand, avoid the complicated processing process and expensive cost that make integrated immune modification microtrabeculae micro-fluidic chip; On the other hand, from hybrid reaction angle, immune microsphere is suspended in sample liquid, the reaction of approximate homogeneous phase can be considered as, for the existing micro-fluidic rare cells separation detecting system based on micro-pillar array (its immune response is out-phase reaction system), there is reaction rate faster, and the fluid oscillating motion that period mechanical extruding produces can further improve the reaction bonded efficiency of target cell in immune microsphere and sample liquid.In addition, cell aggregation after immunity can be separated by rare cells separation detecting system provided by the invention is in specific microcell, avoid the micro-search operation of loaded down with trivial details large area consuming time in testing process, greatly reduce the labour intensity that rare cells is separated test experience operation, improve rare cells and be separated detection efficiency.
Accompanying drawing explanation
Fig. 1 is the rare cells separation detecting system structural representation that the present invention is based on centrifugal microfluidic control techniques.
Fig. 2 is the structure of separation detecting system shown in Fig. 1 assembling schematic diagram.
Fig. 3 is rare cells separation detecting system microfluidic chip structure schematic top plan view of the present invention.
Fig. 4 is rare cells separation detecting system microfluidic chip structure of the present invention assembling schematic diagram.
Chip deformation cavity region diagrammatic cross-section when Fig. 5 is rare cells separation detecting system of the present invention execution hybrid reaction.
Embodiment
Substantive distinguishing features of the present invention and significant progress is further illustrated below in conjunction with embodiment.
Embodiment 1
Collect about 5mL tumour patient peripheral blood, and in blood sample, add the immune microsphere that the EpCAM that can be combined with tumour cell marks, then inject the liquid storage cylinder of rare cells separation detecting system micro-fluidic chip; Chip after application of sample is placed in centrifugal platform, and assembles elasticity microtrabeculae, with 60 ~ 100 revs/min of rotating centrifugal platforms, realize the reaction of automatic rapid mixing; After 5 minutes, improve rotating centrifugal platform rotating speed to 500 rev/min, realize target cell separation; After 10 minutes, closedown centrifugal platform operates, and tear adding mouth sealant tape above each cell harvesting district, the CK-PE antibody of the CD45-FITC of identifiable design blood cell and specific mark tumour cell is added cell harvesting district successively, after liquid feeding completes, again closes each adding mouth with adhesive tape; Incubation 20 minutes, injects phosphate buffer (PBS) at chip injection port, and with 600 revs/min of rotating centrifugal platforms, the unconjugated fluorescent-labeled antibody molecule in washing scavenger-cell collecting region.Finally, regulate rotation platform, each for chip cell harvesting district is placed in optical detecting module camera lens place successively, identification and analysis is carried out to isolated cell.
Embodiment 2
Collect about 100mL human bone marrow, and in blood sample, add the immune microsphere that the CD71 that can be combined with fetal erythrocyte marks, then inject the liquid storage cylinder of rare cells separation detecting system micro-fluidic chip; Chip after application of sample is placed in centrifugal platform, and assembles elasticity microtrabeculae, with 60 ~ 100 revs/min of rotating centrifugal platforms, realize the reaction of automatic rapid mixing; After 5 minutes, improve rotating centrifugal platform rotating speed to 500 rev/min, realize target cell separation; After 10 minutes, close centrifugal platform running, and tear adding mouth sealant tape above each cell harvesting district, the fluorescent-labeled antibody solution of identifiable design fetal erythrocyte haemoglobin is added cell harvesting district, after liquid feeding completes, again closes each adding mouth with adhesive tape; Incubation 20 minutes, injects phosphate buffer (PBS) at chip injection port, and with 600 revs/min of rotating centrifugal platforms, the unconjugated fluorescent-labeled antibody molecule in washing scavenger-cell collecting region.Finally, regulate rotation platform, each for chip cell harvesting district is placed in optical detecting module camera lens place successively, identification and analysis is carried out to isolated cell.
Embodiment 3
Collect about 1mL human umbilical cord blood, and add in blood sample can with the immune microsphere of candidate stem cell selective binding, then inject the liquid storage cylinder of rare cells separation detecting system micro-fluidic chip; Chip after application of sample is placed in centrifugal platform, and assembles elasticity microtrabeculae, with 60 ~ 100 revs/min of rotating centrifugal platforms, realize the reaction of automatic rapid mixing; After 5 minutes, improve rotating centrifugal platform rotating speed to 500 rev/min, realize target cell separation; After 10 minutes, close centrifugal platform running, and tear adding mouth sealant tape above each cell harvesting district, the CD45-FITC solution of identifiable design candidate stem cell is added cell harvesting district, after liquid feeding completes, again closes each adding mouth with adhesive tape; Incubation 20 minutes, injects phosphate buffer (PBS) at chip injection port, and with 600 revs/min of rotating centrifugal platforms, the unconjugated fluorescent-labeled antibody molecule in washing scavenger-cell collecting region.Finally, regulate rotation platform, each for chip cell harvesting district is placed in optical detecting module camera lens place successively, identification and analysis is carried out to isolated cell.
Claims (10)
1. based on a rare cells separation detecting system for centrifugal microfluidic control techniques, it is characterized in that: described system comprises the optical detecting module of the micro-fluidic chip of a similar CD, a centrifugal driver module and a separation detecting system; Wherein the micro-fluidic chip of similar CD is made up of multiple structural sheet, is followed successively by elasticity microtrabeculae guide rail layer, deformable films layer, pipeline/cavity layer, filters rete and waste collection layer; Centrifugal driver module is made up of a rotation motor, a fixed pedestal and one group of elasticity microtrabeculae; The optical detecting module of separation detecting system is made up of the lens that a group could excite and detect fluorescence;
Pipeline/the cavity layer of described micro-fluidic chip comprises a liquid storage cylinder and one group of disengagement chamber, and each disengagement chamber is around liquid storage cylinder periodic arrangement radially; Be communicated with by one group of microchannel between liquid storage cylinder and each disengagement chamber; Liquid storage cylinder connects one group of through hole, and described liquid storage cylinder comprises an injection port, a blow vent and at least one deformation chamber; Each disengagement chamber is punctured into an isolated cell collecting region away from one end of chip center, and the injection port of the corresponding loading identification reaction liquid in each cell harvesting district, when chip rotates, each identification reaction liquid injection port all seals with adhesive tape;
Described system is the rotation utilizing centrifugal platform on centrifugal driver module, the deformable films on the deformation chamber elasticity of compression microtrabeculae be fixed on its fixed pedestal periodically being extruded chip communicates with liquid storage cylinder.
2. system according to claim 1, is characterized in that the outward appearance of the micro-fluidic chip of described similar CD is disc.
3. system according to claim 1, is characterized in that:
1. the elasticity microtrabeculae guide rail layer of described micro-fluidic chip is concave surface loop configuration, only length travel occurs in order to limited chip rotary course Elastic microtrabeculae; Its corresponding deformation cavity region has through hole, and the elasticity microtrabeculae compressed by this through hole brings pressure to bear on the deformable films of deformation chamber top layer;
2. the deformable films layer of described micro-fluidic chip is held between chip elasticity microtrabeculae guide rail layer and pipeline/cavity layer, elastic deformation can occur under pressure.
4. system according to claim 2, is characterized in that:
1. described disengagement chamber is fan-shaped, and the one end away from chip center is punctured into a semicircle microcavity, as isolated cell collecting region (10); And all have injection port above each cell harvesting district, for loading fluorescently-labeled specific antibody solution in cellular identification process, above each cell harvesting district, injection port is all with adhesive tape (13) sealing, prevents the loss of chip rotary course and incubation period sample liquid;
2. as fluid passage, the microchannel described in ensures that fluid and cell are transferred to disengagement chamber from liquid storage cylinder on the one hand, also in low driving pressure situation, temporarily limit sample liquid in liquid storage cylinder as capillary valves simultaneously;
3. deformation chamber top layer is rubber film (15), and this thin layer is held between chip elasticity microtrabeculae guide rail layer (14) and pipeline/cavity layer (16);
4. the elasticity of compression microtrabeculae (6) be installed on centrifugal driver module fixed pedestal provides mechanical pressure, and elasticity of compression microtrabeculae is only subjected to displacement in the vertical direction;
5. the filtration rete (17) of micro-fluidic chip is processed with the thin polymer film of microwell array for one deck, and be held between chip pipeline/cavity layer and waste collection layer, pore size is 8 ~ 12 μm;
6. the waste collection layer (18) of micro-fluidic chip comprises the annular micro-cavity that is processed with micro-pillar array, and micro-pillar array is used as supporting construction;
7. described annular micro-cavity external diameter should be greater than the distance of pipeline/cavity layer cell harvesting offset from chip center of chip.
5. system according to claim 1, is characterized in that:
1. the making material of the elasticity microtrabeculae guide rail layer of described micro-fluidic chip, pipeline/cavity layer and waste collection layer is any one in glass, dimethyl silicone polymer, polystyrene, polycarbonate, cyclic olefine copolymer, polymethylmethacrylate and SU-8;
2. filter membrane material is any one in dimethyl silicone polymer, polystyrene, polycarbonate, cyclic olefine copolymer, polymethylmethacrylate, polyimide, Parylene and SU-8.
6. system according to claim 1, is characterized in that rotation motor that described centrifugal driver module comprises is program-controlled governor motor; Comprise the effect of elasticity microtrabeculae power in the vertical direction under, can move up and down, elasticity microtrabeculae lower end is smooth spherical shape, and is directly contacted with chip guide rail layer groove surface by its lower end of pressure that self spring produces; Elasticity microtrabeculae is fixed on fixed pedestal by a liftable support.
7. by system according to claim 6, it is characterized in that the optical detecting module of separation detecting system is made up of the lens (3) that a group could excite and detect fluorescence, lens (3) lay respectively at chip upper and lower, and lens distance centrifugal platform central shaft distance equals chip isolated cell collecting region distance centrifugal platform central shaft distance.
8. use the method for the system according to any one of claim 1-7, it is characterized in that first the microballoon of sample liquid and immune modification being imported in its liquid storage cylinder by micro-fluidic chip injection port, and be placed in the centrifugal platform of centrifugal driver module, assemble elasticity microtrabeculae, low speed rotation, rotary course Elastic microtrabeculae is utilized to extrude the periodicity of chip surface deformable films, realize abundant mixing and the reaction of the microballoon liquid of sample liquid and immune modification in liquid storage cylinder, then High Rotation Speed chip, the rapidly and efficiently separation of immunocapture cell is realized by centrifugal force combined filtering film, after separation, dripping in each isolated cell collecting region can the fluorescent-labeled antibody solution of specific recognition cell to be detected, incubation reaction, then adds damping fluid and centrifugal, removes unreacted fluorescent-labeled antibody molecule, finally, undertaken identifying and analyzing by optical detecting module.
9. method according to claim 8, is characterized in that comprising following steps:
A) microballoon of sample liquid and immune modification is imported in its liquid storage cylinder by micro-fluidic chip injection port, and be placed in the centrifugal platform of centrifugal driver module;
B) the elasticity microtrabeculae support declined on centrifugal driver module, make end in contact guide-track groove under microtrabeculae, and make elasticity microtrabeculae spring be in compressive state, low speed rotary centrifugal platform, utilize rotary course Elastic microtrabeculae to extrude the periodicity of chip deformation chamber upper surface deformable films, realize abundant mixing and the reaction of the microballoon of sample liquid and immune modification in liquid storage cylinder;
C), after reaction terminates, High Rotation Speed centrifugal platform, utilizes centrifugal force combined filtering film to realize the rapidly and efficiently separation of immunocapture cell, and by separate targets cell aggregation in chip collecting region;
D) after being separated, by sealant tape removing on corresponding for each collecting region filling opening, adding can the fluorescent-labeled antibody solution of specific recognition cell to be detected, and again sticks sealant tape, incubation reaction;
E) after reaction terminates, add damping fluid and high speed centrifugation at chip injection port, remove unreacted fluorescent-labeled antibody molecule, finally by optical detecting module collecting region cell identified and analyze.
10. method according to claim 9, is characterized in that:
1. the rotating speed of low speed rotary centrifugal platform is 60-300 rev/min;
2. the rotating speed of High Rotation Speed centrifugal platform is 500-5000 rev/min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210545205.9A CN103018224B (en) | 2012-12-14 | 2012-12-14 | Based on rare cells separation detecting system and the method for centrifugal microfluidic control techniques |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210545205.9A CN103018224B (en) | 2012-12-14 | 2012-12-14 | Based on rare cells separation detecting system and the method for centrifugal microfluidic control techniques |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103018224A CN103018224A (en) | 2013-04-03 |
| CN103018224B true CN103018224B (en) | 2015-07-29 |
Family
ID=47967073
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210545205.9A Active CN103018224B (en) | 2012-12-14 | 2012-12-14 | Based on rare cells separation detecting system and the method for centrifugal microfluidic control techniques |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103018224B (en) |
Families Citing this family (30)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103389371A (en) * | 2013-08-07 | 2013-11-13 | 苏州扬清芯片科技有限公司 | Disc-type multi-index analysis chip |
| CN104251810B (en) * | 2013-12-18 | 2016-08-17 | 中国科学院电子学研究所 | Characterize unicellular Young's modulus and the cell membrane system than electric capacity simultaneously |
| NO337444B1 (en) * | 2014-06-19 | 2016-04-11 | Spinchip Diagnostics As | Analysis Method |
| CN104178413B (en) * | 2014-07-04 | 2016-05-25 | 宁波美晶医疗技术有限公司 | A kind of plastic packaging box packaging structure of rare cell separator |
| CN105400679B (en) * | 2014-09-10 | 2017-10-31 | 清华大学 | A kind of micro flow control chip device for cell separation |
| CN105158492B (en) * | 2015-10-22 | 2017-03-08 | 东南大学 | A kind of automatic pipetting device |
| US20170205404A1 (en) * | 2016-01-19 | 2017-07-20 | General Electric Company | Multifunctional beads and methods of use for capturing rare cells |
| CN106124388B (en) * | 2016-06-12 | 2020-02-11 | 中国科学院电子学研究所 | Capillary tube sample injection system, sample injection method and single cell electrical characteristic detection system |
| CN106754344B (en) * | 2016-11-24 | 2017-11-28 | 大连医科大学附属第二医院 | The combined sorting purification devices and sorting detection method of circulating tumour cell |
| GB201700340D0 (en) * | 2017-01-09 | 2017-02-22 | Gsg Tech Ltd | fluid manipulation cartridge and controller mechanism |
| CN108686722A (en) * | 2017-04-07 | 2018-10-23 | 苏州含光微纳科技有限公司 | A kind of centrifugal immunological magnetic bead sorting micro-fluidic chip and device |
| CN107044972A (en) * | 2017-05-25 | 2017-08-15 | 沈阳优宁生物科技有限公司 | A kind of micro-fluidic chip fluorescence immunoassay quick detection kit and its preparation and detection method |
| CN107702973A (en) * | 2017-09-08 | 2018-02-16 | 深圳市太赫兹科技创新研究院有限公司 | A kind of whole blood blood plasma piece-rate system and method |
| US11099202B2 (en) * | 2017-10-20 | 2021-08-24 | Tecan Genomics, Inc. | Reagent delivery system |
| CN107703981B (en) * | 2017-11-03 | 2020-09-25 | 北京工业大学 | Micro-fluidic chip outlet pressure adjusting device |
| CN107802293A (en) * | 2017-11-10 | 2018-03-16 | 湘潭大学 | The flexible microfluidic body equipment and the sweat collection and detection method of the equipment that a kind of human sweat collects |
| CN108660060B (en) * | 2018-08-13 | 2023-12-08 | 苏州绘真医学检验有限公司 | Microfluidic chip for enriching and purifying circulating tumor cells |
| CN109158136B (en) * | 2018-10-19 | 2023-10-13 | 上海快灵生物工程有限公司 | Micro-fluid chip intercepted by microporous membrane and solution flow path control method thereof |
| CN111068798A (en) * | 2018-10-19 | 2020-04-28 | 上海快灵生物工程有限公司 | Micro-fluidic chip with micro-porous membrane for intercepting aggregated microspheres and detection method thereof |
| CN113660961A (en) * | 2018-12-13 | 2021-11-16 | 科力果疗法公司 | Cell separation device and method of use |
| CN110044838B (en) * | 2019-05-09 | 2021-06-25 | 青岛大学附属医院 | Optical detector for secretion |
| CN112076806B (en) * | 2019-06-14 | 2022-12-30 | 中国科学院青岛生物能源与过程研究所 | Centrifugal enrichment microfluidic chip for low-concentration liquid sample |
| CN110982882B (en) * | 2019-12-20 | 2023-06-20 | 南通大学 | Microfluidic chip for single cell immobilization-isolation and in-situ nucleic acid amplification and application thereof |
| CN110951587A (en) * | 2019-12-24 | 2020-04-03 | 南通大学 | Chip and method for cell filtration, fixation and in-situ fluorescence nucleic acid hybridization experiment |
| WO2021163958A1 (en) * | 2020-02-20 | 2021-08-26 | 京东方科技集团股份有限公司 | Mixing device and driving method therefor, and testing assembly |
| CN112694969A (en) * | 2020-09-22 | 2021-04-23 | 深圳汇芯生物医疗科技有限公司 | Separation detection chip, separation detection device and separation detection method |
| CN112538415A (en) * | 2020-12-23 | 2021-03-23 | 深圳市刚竹医疗科技有限公司 | Single-disc type automatic nucleic acid analyzer |
| CN114994346B (en) * | 2022-08-04 | 2022-10-04 | 天津长和生物技术有限公司 | Cell detection device and detection method thereof |
| CN115920987B (en) * | 2022-12-30 | 2024-04-05 | 华中科技大学 | Drug sensitivity detection chip based on bacterial enrichment |
| CN117548163B (en) * | 2024-01-11 | 2024-04-09 | 杭州联川生物技术股份有限公司 | Microfluidic chip |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4692200B2 (en) * | 2005-10-06 | 2011-06-01 | 横河電機株式会社 | Chemical treatment cartridge and method of use thereof |
| US8741136B2 (en) * | 2006-02-10 | 2014-06-03 | Boehringer Ingelheim Microparts Gmbh | Device and method for treating or cleaning sample material, in particular nucleic acids |
| KR101343034B1 (en) * | 2006-09-05 | 2013-12-18 | 삼성전자 주식회사 | Centrifugal microfluidic device for target protein detection and microfluidic system comprising the same |
| CN101850231B (en) * | 2009-07-03 | 2012-08-29 | 中国科学院上海微系统与信息技术研究所 | Micro-fluid reactor, using method and application thereof |
-
2012
- 2012-12-14 CN CN201210545205.9A patent/CN103018224B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN103018224A (en) | 2013-04-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103018224B (en) | Based on rare cells separation detecting system and the method for centrifugal microfluidic control techniques | |
| JP6200419B2 (en) | Microfluidic disc for use in bead-based immunoassays | |
| CN105062866B (en) | For disposable separating chips module and the using method thereof of Peripheral Circulation tumour cell | |
| CN105675857B (en) | Centrifugal magnetic particle control and detection device and operation method thereof | |
| US9101926B2 (en) | Method for separating a sample into density specific fractions | |
| CN109486653B (en) | Trace cell capture system based on micro-fluidic and immunomagnetic separation dual strategies | |
| CN103240187B (en) | Centrifugal microfluidic video disc and the method from sample separation object | |
| CN105647799B (en) | A kind of circulating tumor cell separator of combined type field flow separation | |
| CN102243234B (en) | Automatic bacterial sorting and labeling method based on immune method, and apparatus thereof | |
| CN105233892A (en) | Magnetic particle chemiluminescence double-layer micro-fluidic chip used for whole-blood sample detection | |
| CN105259163A (en) | Magnetic particle direct chemiluminiscence microfluidic chip for whole blood sample testing | |
| CN102162815B (en) | Plasma separating chip and preparation method thereof | |
| CN108795693A (en) | A kind of micro-fluidic chip of capture blood rare cell | |
| CN107400623A (en) | Circulating tumor cell automatic capture micro-fluidic chip and its automatic capture method | |
| CN108795692B (en) | Rare cell capture system and application thereof | |
| US20240102900A1 (en) | Isolation method for isolating target particles from liquid samples | |
| CN202049159U (en) | Automatic bacterium sorting and labeling device based on immune method | |
| CN220590063U (en) | Microfluidic chip with reagent pre-storing function for chemiluminescence detection | |
| CN111893023A (en) | Tumor cell capturing device and preparation and application thereof | |
| CN212748953U (en) | Micro-fluidic reagent card | |
| CN214953164U (en) | Detection chip for magnetic relaxation signals | |
| CN109012207A (en) | A kind of cross-flow filtration system of circulating tumor cell enrichment | |
| CN102967715B (en) | Novel ELISA biochemical optical disk detection system | |
| CN210945545U (en) | High-flux micro-fluidic rapid nucleic acid extraction and detection device | |
| CN210954058U (en) | Magnetic bead releasing mechanism and system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |