CN103642671A - Micro-fluid biochip for enriching and extracting cells - Google Patents
Micro-fluid biochip for enriching and extracting cells Download PDFInfo
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- CN103642671A CN103642671A CN201310602808.2A CN201310602808A CN103642671A CN 103642671 A CN103642671 A CN 103642671A CN 201310602808 A CN201310602808 A CN 201310602808A CN 103642671 A CN103642671 A CN 103642671A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
Abstract
A micro-fluid biochip for enriching and extracting cells is composed of a cover plate layer and a slide plate arranged below the cover plate layer, the cover plate layer is provided with a fluid feeding area, an enriching channel, which communicates with the fluid feeding area, a target cell collecting area and a residual fluid discharging area, wherein the target cell collecting area and the residual fluid discharging area communicate with the enriching channel; one side of the enriching channel is a flat and vertical wall surface, and the other side of the enriching channel is provided with internally-protruded sharp corner structures or other internally-protruded structures; the tail part of the enriching channel is a Y forked structure, and is used to communicate the target cell collecting area and the residual fluid discharging area; the fluid feeding area, the target cell collecting area and the residual fluid discharging area are through holes that are arranged in the cover plate layer, the enriching channel is a typhlosole that is arranged in the cover plate layer, and the bottom communicates with the surface of the slide glass. The biochip can high efficiently achieve enriching and extracting of target cells, has the advantages of simple structure, convenient operation, low energy consumption, no pollution, low cost, portability, and easy promotion.
Description
Technical field
The present invention relates to a kind of biochip, be specifically related to a kind of microfluid biochip for cell enrichment and extraction.
Background technology
Microfluid biochip was born in for 20 end of the centurys, and development in recent years is rapid, became a kind of effective detection means, and was widely used in the fields such as medical science, biology.It embodies the function of Liao Jiang assay laboratory and transfers to the thought on microchip, has portable and height integration.In view of developing to the fruitful property of microfluid biochip correlation technique for a long time, AAAS is chosen as microfluid biochip one of ten large technological breakthroughs in 1998, thinks that microfluid biochip technology has again far-reaching scientific and technological revolution by being after large-scale integrated circuit.Microfluid biochip is to utilize micro-processing technology, on the substrates such as silicon, glass, plastics, etch microchannel and other functional unit of design in advance, then with cover plate, sealed, by design and the layout of the building blocks such as different channel networks, reactor, detecting unit, to realize, integrate micro-example the functions such as preparation, sample introduction, reaction, separation, detection fast, the microscale chemical experiment apparatus of efficient, less energy-consumption.Microfluid biochip is noticeable in the potentiality of the design of efficient separated, handiness fast and the aspect such as multifunctional single unit is integrated, becomes one of current study hotspot.
Recent years, large quantities of scholars attempt utilizing microfluid biochip to carry out enrichment and extraction to micro-nano biomolecules and cell.The enrichment of biomolecules (as protein, nucleic acid, antigen and antibody) or cell be extracted in the fields such as biomedicine, food and environmental monitoring and play an important role, as medical diagnosis on disease, food safety detection and water quality monitoring (WQM) etc.In biomedical research, first need by target cell enrichment extracting from whole blood, to get rid of the interference of other cells or particulate.For example, in the diagnosis of cancer and treatment, malignant cell (being commonly called as cancer cells) need to be carried out to effective enrichment and extraction from blood.Cancer cells can produce transfer in morbidity late period, by initial focal zone, depart from and enter blood circulation, then depend on another organ or tissue, form new pathogenesis of cancer point, but at cancer cells diffusion period, cancer cells content in blood only accounts for 1/100000000th of blood cell total amount, be equivalent in one milliliter of blood, to only have at most a cancer cells, therefore the cancer cells that shifts extremely low concentration in phase blood is carried out to effective enrichment and extraction, estimating of the monitor therapy of cancer and the state of an illness had great significance.On the other hand, the enrichment of cancer cells and extraction, can be for the concentration level of cancer cells in blood before and after medication is detected and contrasted, great to the evaluating significance of anticancer drug effect and result for the treatment of.
Traditional equipment for cell enrichment and extraction, different according to principle, be mainly divided into centrifugal force enrichment, dielectric StrongmenGroup collection, velocity sedimentation enrichment, the enrichment of Solid-Phase Extraction harmony wave surface.What be widely adopted at present is centrifugal force enrichment, and as stem cell treatment system, it is the density variation based on different sorts cell, under the effect of centrifugal force, target cell is carried out to effective enrichment and extraction.Its cell extract comprises hemopoietic stem cell, red corpuscle, blood plasma, thrombocyte etc., for next step clinical diagnosis or scientific research lays the foundation.Dielectric power cell enrichment, is the difference in dielectric constant of utilizing different cells, the enrichment of realize target cell and extraction under DC Electric Field, and the more centrifugal cell enrichment extraction equipment of its structure is simple, but cannot get rid of the impact of polarization phenomena on cell.In addition, electric field strong and weak, fluid viscosity, cell size and electrical characteristic all can exert an influence to dielectric power cell enrichment and extraction effect, thereby are difficult to wide popularization and application.The difference of velocity sedimentation enrichment based on cell size, makes different cells in the medium with very mild density gradient, with different velocity sedimentations, reach the object of enrichment and extraction.While it is pointed out that this kind of method enrichment different target cell, thereby need the sedimentation medium of different densities to guarantee that effective sedimentation occurs target cell in sedimentation medium.In addition, velocity sedimentation cell extraction is very slow, and inconvenience is applied to cell real-time analysis occasion.All the other are such as Solid-Phase Extraction, surface acoustic wave enrichment etc. still in theoretical research stage, and rarely seen have practical application and a popularization.Because conventional cell extraction equipment generally all needs as motor, flowrate control valve, special precision elements such as mobile loop line, manufacture difficulty is larger, mechanism's complex.And expensive, be unfavorable for the quick progress of medical skill, run counter to the advanced health idea such as domestic medicine and individual health care.
Mainly there is following problem in traditional cell enrichment and extraction equipment:
1) bulky, Portable belt not;
2) need additionaling power, consume energy high;
3) flow needs accurately to control, and affected by various factors;
4) assembly is many, and complex structure, as precision components such as needs motor, flow rate control devices, is manufactured comparatively difficulty;
5) single device is expensive, is unfavorable for popularization and use in basic medical unit.
Summary of the invention
For solving above-mentioned problems of the prior art, the object of the present invention is to provide a kind of microfluid biochip for cell enrichment and extraction, can carry out efficiently concentrating and extraction to cell, also can be used for the extraction of blood plasma, there is the advantages such as easy to operate, simple in structure, cost is low, precision is high, be easy to carry, there is good application prospect.
In order to achieve the above object, the present invention adopts following technical scheme:
For a microfluid biochip for cell enrichment and extraction, described microfluid biochip is comprised of cover plate layer 5 and the slide glass 6 being placed under cover plate layer 5; On described cover plate layer 5, offer and add liquid zone 1 and add enrichment passage 2 and the target cell collecting region 4 being communicated with enrichment passage 2 and the remaining liq drainage region 3 that liquid zone 1 is communicated with; One side of described enrichment passage 2 is straight wall, the horn structure that the oriented enrichment passage 2 of opposite side band is projecting inward or other interior convex structure, and described enrichment passage 2 afterbodys are bifurcated structure, for being communicated with target cell collecting region 4 and remaining liq drainage region 3; Described liquid zone 1, target cell collecting region 4 and remaining liq drainage region 3 through hole for offering on cover plate layer 5 of adding, the sidewalk for visually impaired people of described enrichment passage 2 for offering on cover plate layer 5, bottom and slide glass 6 surfaces communicate.
Described cover plate layer 5 and slide glass 6 combine by the irreversible processing of plasma.
Described enrichment passage 2 is positioned at the combine middle position at place of cover plate layer 5 and slide glass 6.
Described liquid zone 1, target cell collecting region 4 and the remaining liq drainage region 3 of adding is cylindrical hole.
The projecting inward horn structure of described enrichment passage 2 is 80.
Also comprise for making cell produce the manual squeezing plug 7 of mobilization dynamic, described manual squeezing plug 7 comprises and adds the suitable cylinder 7-1 of liquid zone 1 shape, be arranged on the extrusion head 7-2 of cylinder 7-1 one end, on described cylinder 7-1, have sealing-ring 7-3 with extrusion head 7-2 contact position cover.
Described cylinder 7-1 adopts PDMS or other soft material.
Described cylinder 7-1 bottom is inclined-plane.
The material of described cover plate layer 5 is polymetylmethacrylate or polydimethylsiloxane.
The material of described slide glass 6 is glass or silicon.
Compared to the prior art, tool has the following advantages in the present invention:
1), volume is little, simple in structure, is easy to carry, easy to operate.Adopt the modes such as simple manual squeezing or power wheel drive to carry out sample filling, carry out easily the collection of target cell, be adapted at middle-size and small-size medical institutions and scientific research institutions and use, there are great using value and market outlook.The manual squeezing plug of configuration, can squeeze into biological specimen in microchannel efficiently and easily.
2), precision is high, suitability is wide.The inertial centrifugal effect that in the present invention, a series of horn structure causes, can carry out accurately enrichment and extraction by all kinds of target cells.Compare with extractive technique with other microfluid cell enrichment, the cell enrichment in biochip of the present invention is insensitive to flow, has increased greatly the suitability of this passage.
3), target extract produce is not polluted, the microchannel for cell enrichment in the present invention does not directly contact with external environment, has avoided the pollution of biological specimen in enrichment process.
4), efficiency is high, consuming time few.Clinical application is convenient in the present invention, and fast and easy is made diagnosis.
5), cost is low, make easily.Compare with extractive technique with other tradition and microfluid cell enrichment, without complicated structure and expensive utility appliance, thereby the present invention is suitable for scale operation and marketing.
Accompanying drawing explanation
Fig. 1 is the microfluid biochip vertical view for cell enrichment and extraction.
Fig. 2 be Fig. 1 along A-A to sectional view.
Fig. 3 be Fig. 1 along B-B to sectional view.
Fig. 4 is the schematic three dimensional views for the microfluid biochip of cell enrichment and extraction.
Fig. 5 is the schematic three dimensional views of manual squeezing plug.
Fig. 6 be the present invention to the enrichment of particle with extract experimental result picture, wherein Fig. 6 a is different flow lower channel outlet particle motion track, Fig. 6 b is the statistics of particle position under different flow.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation:
As shown in Figures 1 to 4, a kind of microfluid biochip for cell enrichment and extraction of the present invention, this biochip is processed by standard soft lithography, cover plate layer 5 and the slide glass 6 being placed under cover plate layer 5, consists of; On cover plate layer 5, offer and add liquid zone 1 and add enrichment passage 2 and the target cell collecting region 4 being communicated with enrichment passage 2 and the remaining liq drainage region 3 that liquid zone 1 is communicated with; One side of enrichment passage 2 is straight wall, the horn structure that the oriented enrichment passage 2 of opposite side band is projecting inward or other interior convex structure, and enrichment passage 2 afterbodys are bifurcated structure, for being communicated with target cell collecting region 4 and remaining liq drainage region 3; Add liquid zone 1, target cell collecting region 4 and remaining liq drainage region 3 through hole for offering on cover plate layer 5, the sidewalk for visually impaired people of enrichment passage 2 for offering on cover plate layer 5, bottom and slide glass 6 surfaces communicate.It is 10mm that the present embodiment adds liquid zone 1 cavity diameter, and height is highly consistent with cover plate layer 5.The microchannel that adds liquid zone 1, enrichment passage 2, target cell collecting region 4 and 3 formation of remaining liq drainage region is positioned at the middle position that cover plate layer 5 combines with slide glass 6, and width is 80 μ m, and the degree of depth is 50 μ m, and design overall length is about 16.6mm.Comprise 80 horn structure that are positioned at passage one side, wedge angle is 45 degree, and horn structure width and length are about 40-45 μ m, and adjacent wedge angle distance is about 120 μ m.
As the preferred embodiment of the present invention, in the present invention, microfluid biochip is used the processing of standard soft lithography, and described cover plate layer 5 and slide glass 6 combine by the irreversible processing of plasma.Also can use other materials or method to process microchannel of the present invention.
As the preferred embodiment of the present invention, described in add liquid zone 1, target cell collecting region 4 and remaining liq drainage region 3 and be cylindrical hole.Corresponding, the cylinder 7-1 of described manual squeezing plug 7 is also right cylinder.
As the preferred embodiment of the present invention, the projecting inward horn structure of described enrichment passage 2 is 80.
As the preferred embodiment of the present invention, as shown in Figure 5, also comprise for making cell produce the manual squeezing plug 7 of mobilization dynamic, described manual squeezing plug 7 comprises and adds the suitable cylinder 7-1 of liquid zone 1 shape, be arranged on the extrusion head 7-2 of cylinder 7-1 one end, on described cylinder 7-1, have sealing-ring 7-3 with extrusion head 7-2 contact position cover.Manual squeezing plug 7 applies in adding sap cavity top, and and add cylindrical surface, liquid zone inner tight and coordinate, prevent that mating surface from existing gap and the sealing problem that causes.Manual squeezing plug 7 design heights need guarantee that testing liquid is squeezed in microchannel completely, thereby make target cell by fully enrichment and extraction, the present embodiment cylinder 7-1 diameter is 10mm, the grommet type seals 7-3 that cock body upper end is 10mm with an internal diameter, sealing circle is prepared by the high material of elasticity, can definitely avoid biological specimen to occur in the process of being squeezed excessive.The syringe that also can drive by manpower or other equipment (as syringe pump) injects biological specimen.
As the further preferred implementation of the present invention, described cylinder 7-1 adopts PDMS or other soft material.
As the further preferred implementation of the present invention, described cylinder 7-1 bottom is inclined-plane, while making liquid feeding, in biochip, forms comparatively stably and flows, and prevents that sample liquid is excessive simultaneously.
Described cylinder 7-1 adopts PDMS or other soft material.
Described cylinder 7-1 bottom is inclined-plane.
As the preferred embodiment of the present invention, the material of described cover plate layer 5 is polymetylmethacrylate or polydimethylsiloxane.
As the preferred embodiment of the present invention, the material of described slide glass 6 is glass or silicon.
With embodiment, implementation process of the present invention is described below:
The enrichment of cancer cells is as follows with extraction concrete operations, first gathers patient blood, then pending blood is filled in liquid zone 1, and manual squeezing plug 7 is placed in and adds on liquid zone 1, and guarantees sealing-ring 7-3 effect, and anti-Hemostatic Oral Liquid is excessive.Thereby pressing extrusion head 7-2 applies pressure to and adds the blood in liquid zone 1, make blood flow into enrichment passage 2, when the cancer cells in blood is flowed through the horn structure of enrichment passage 2, can be subject to the centrifugal inertial force that horn structure causes, this power and flow positive correlation, make cell trend towards straight wall one side.Every through a horn structure, cancer cells can produce a displacement in the direction away from wedge angle, and after 80 identical horn structure, due to storage effect, all cancer cells will be compressed in straight wall one side, and enter cell harvesting district 4.By means such as liquid-transfering guns, the cancer cells of high density is extracted from cell harvesting district 4, for follow-up diagnosis and detection.As enrichment concentration of cancer cells does not meet the needs of subsequent detection, can make it Multiple through then out biochip of the present invention, constantly to realize the enrichment of cancer cells, thereby improve the enrichment concentration of cancer cells.In addition, also can use syringe manual or that other driving arrangements (as syringe pump) drive that patient's blood is injected to microchannel, to realize enrichment and the extraction of cancer cells.
Insensitive to flow for verifying enrichment process of the present invention, thus the feasibility that proof manual squeezing plug is used is used the fluorescence granules of polystyrene (size and hemocyte are suitable) that the present invention is 9.94 microns to diameter to carry out enrichment experiment checking.Particle is dissolved in deionized water, and adopts the syringe pump that can accurately control flow to inject particle liquid, utilize fluorescent microscope and CCD camera track up particle swarm track.Experimental results show that the present invention flow be 300 microlitre per minutes in the scope of 700 microlitre per minutes, particle fully can be enriched in to straight wall one side.In Fig. 6: (a) the particle enrichment phenomenon figure under different flow; (b) under different flow, the fluorescence intensity distribution curve of the particle of channel exit.Experimental verification the present invention insensitive to flow in target compound enrichment process, there is suitability widely.Therefore the application of manual squeezing plug can meet completely to the requirement to flow in target compound enrichment process.
Claims (10)
1. for a microfluid biochip for cell enrichment and extraction, it is characterized in that: by cover plate layer (5) and the slide glass (6) that is placed under cover plate layer (5), formed; On described cover plate layer (5), offer and add liquid zone (1) and add enrichment passage (2) and the target cell collecting region (4) being communicated with enrichment passage (2) and remaining liq drainage region (3) that liquid zone (1) is communicated with; One side of described enrichment passage (2) is straight wall, the horn structure that the oriented enrichment passage of opposite side band (2) is projecting inward or other interior convex structure, described enrichment passage (2) afterbody is bifurcated structure, for being communicated with target cell collecting region (4) and remaining liq drainage region (3); Described liquid zone (1), target cell collecting region (4) and remaining liq drainage region (3) through hole for offering on cover plate layer (5) of adding, the sidewalk for visually impaired people of described enrichment passage (2) for offering on cover plate layer (5), bottom and slide glass (6) surface communicates.
2. a kind of microfluid biochip for cell enrichment and extraction according to claim 1, is characterized in that: described cover plate layer (5) and slide glass (6) combine by the irreversible processing of plasma.
3. a kind of biochip for cell high-efficient separation according to claim 1, is characterized in that: described enrichment passage (2) is positioned at the combine middle position at place of cover plate layer (5) and slide glass (6).
4. a kind of microfluid biochip for cell enrichment and extraction according to claim 1, is characterized in that: described in add liquid zone (1), target cell collecting region (4) and remaining liq drainage region (3) are cylindrical hole.
5. a kind of microfluid biochip for cell enrichment and extraction according to claim 1, is characterized in that: the projecting inward horn structure of described enrichment passage (2) is 80.
6. a kind of microfluid biochip for cell enrichment and extraction according to claim 1, it is characterized in that: also comprise for making cell produce the manual squeezing plug (7) of mobilization dynamic, described manual squeezing plug (7) comprises and adds the suitable cylinder (7-1) of liquid zone (1) shape, be arranged on the extrusion head (7-2) of cylinder (7-1) one end, at described cylinder (7-1), upper and extrusion head (7-2) contact position is overlapped and is had sealing-ring (7-3).
7. a kind of microfluid biochip for cell enrichment and extraction according to claim 6, is characterized in that: described cylinder (7-1) adopts PDMS or other soft material.
8. a kind of microfluid biochip for cell enrichment and extraction according to claim 6, is characterized in that: described cylinder (7-1) bottom is inclined-plane.
9. a kind of microfluid biochip for cell enrichment and extraction according to claim 1, is characterized in that: the material of described cover plate layer (5) is polymetylmethacrylate or polydimethylsiloxane.
10. a kind of microfluid biochip for cell enrichment and extraction according to claim 1, is characterized in that: the material of described slide glass (6) is glass or silicon.
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| CN106190829A (en) * | 2016-07-26 | 2016-12-07 | 西安交通大学 | A kind of for cell high accuracy arrangement and the microflow controlled biochip of detection |
| CN108709985A (en) * | 2018-09-06 | 2018-10-26 | 湖南乐准智芯生物科技有限公司 | A kind of biochip reaction device topples over drainage structure |
| CN109482249A (en) * | 2018-12-12 | 2019-03-19 | 西安交通大学 | Micro fluidic device and method for macromolecule content detection in non-newtonian fluid |
| CN110935238A (en) * | 2019-12-05 | 2020-03-31 | 西安交通大学 | Micro-nano particle enrichment device based on coupling of variable cross-section microchannel and viscoelastic fluid |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110935238A (en) * | 2019-12-05 | 2020-03-31 | 西安交通大学 | Micro-nano particle enrichment device based on coupling of variable cross-section microchannel and viscoelastic fluid |
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