CN101580797B - Low-cost cellular positioning arrangement chip and application method - Google Patents
Low-cost cellular positioning arrangement chip and application method Download PDFInfo
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- CN101580797B CN101580797B CN 200910032740 CN200910032740A CN101580797B CN 101580797 B CN101580797 B CN 101580797B CN 200910032740 CN200910032740 CN 200910032740 CN 200910032740 A CN200910032740 A CN 200910032740A CN 101580797 B CN101580797 B CN 101580797B
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- C—CHEMISTRY; METALLURGY
- 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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/22—Transparent or translucent parts
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- C—CHEMISTRY; METALLURGY
- 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
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
Abstract
The invention discloses a low-cost cellular positioning arrangement chip and an application method, and relates to the field of biological micro-fluidic control. When voltage is applied between an upper conductive film (11) and a lower conductive film (31) of the chip, cells in a micro-fluid cavity (4) in the chip move to a 'solitary island' (5) or a 'solitary trap' (6) under the action of dielectrophoresis force to further form regularized arrangement according to the array arrangements of the 'solitary island' (5) or the 'solitary trap' (6). The electric field strength in the chip depends onthe vertical size of the micro-fluid cavity but not on the horizontal 'solitary island' or 'solitary trap' separation distance, thus the generation of a local strong electric field can be avoided so as to prevent damages to the cells. The chip can be used as a low-cost disposable biochemical analysis tool and has considerable prospect in the filed of high-flux bioanalysis.
Description
Technical field
The present invention is a kind of large-scale cellular positioning arrangement technology, relates to biological micro-fluidic field, particularly cellular localization control techniques field.
Background technology
A large amount of cells arrange for the research cell and intercellular interaction has and important meaning; Such as can two cell masses (or two unicellular) being positioned two zones of certain microspur that are separated by respectively, and then study interaction and exchange of substance situation between them.The meaning that cell is arranged also is embodied in and is easy to observation that a large amount of cells physiological situations are walked abreast, is easy to realize the quick counting to a large amount of cells, and is easy to realize biosensor based on cell etc.In addition; It is a tool technique that has Practical significance that the array of cell is arranged to grouping research and high-flux parallel cell analysis with cell; For example, can carry out parallel parsing to different cells group, also can realize a large amount of parallel single cell analysis simultaneously through array control.At present; The scientific research personnel uses extensive microelectrode array, and arranges by the array of carrying out of consequent dielectrophoretic force pair cell, but usually because of go between too much (a hundreds of line end); The external interface cost of manufacture heightens, and also increases greatly for the requirement of peripheral circuit or control device.In addition because these strength of electric field (horizontal direction) of arranging between the very close planar micro electrode are very big, therefore when cell impaired under the highfield effect probably near the planar micro electrode edge time.
Therefore,, can only use a small amount of lead terminal to connect actuation signal, and make viable cell avoid near the local highfield effect of microelectrode, just can overcome the above problems if propose a kind of cellular localization chip.
Summary of the invention
Technical problem: the purpose of this invention is to provide a kind of low-cost cellular positioning arrangement chip and application method; Can be under the situation of only using two signal terminals; Realize that the location of a large amount of cells arranges; And can avoid cell to suffer the infringement of local highfield, present cellular positioning arrangement pin is too much to solve, complex interfaces, and local highfield pair cell causes the deficiency of damage.
Technical scheme: to the problem that prior art exists, the present invention proposes a kind of technical scheme that only has the mass cell location arrangement chip of two signal terminals.In this chip, the electric field line direction is vertical, so strength of electric field depends on the size of the vertical direction of miniflow body cavity in the chip, and does not depend on the size of any horizontal microstructure.Because the vertical size of the fluid cavity of this chip proposed by the invention does not determine the position control accuracy of cell; Therefore can be than big several times to tens times of the horizontal microelectrode spacing of present existing arrangement chip; So in this chip provided by the present invention, can keep away the damage of local highfield pair cell basically.
Low-cost cellular positioning arrangement chip comprise upper substrate that the last conductive film of substrate and lower surface thereof forms, down substrate with and the hypocoxa formed of the following conductive film of upper surface, the miniflow body cavity of dielectric spacer layer formation between last conductive film and following conductive film; Be provided with the semiconductor material layer of array architecture at the lower surface of the upper surface of hypocoxa or upper substrate, on semiconductor material layer, be provided with passivation layer, and form miniature isolated island or miniature lonely trap or form and have the shape of isolated island and lonely trap simultaneously.
Described substrate down and last substrate can be that transparent material also can be translucent material or opaque material.
The shape of described all miniature isolated islands or lonely trap is all identical, or part is identical or have nothing in common with each other.
Semiconductor material layer is cadmium selenide or other specific conductivity semiconductor material littler than cell suspending liquid that non-crystalline silicon or adulterated Cadmium Sulfide perhaps mix.
The application method of low-cost cellular positioning arrangement chip, at first, the liquid that will contain sample cell injects the miniflow body cavity and covers upper substrate then, perhaps injects liquid then through on the chip upper substrate, making micropore; After treating that liquid evenly scatters; Between last conductive film and following conductive film, apply voltage signal and regulate exciting signal frequency; Make each cell under the effect of dielectrophoretic force, be automatically moved to the position at miniature isolated island or miniature lonely trap place; Through behind the certain hour, realize promptly arranging in the location of cell.
The voltage signal that between last conductive film and following conductive film, applies is the sinusoidal signal or the superposed signal of some sinusoidal signals.
Described exciting signal frequency comprises the frequency in all frequency separations that can encourage cell movement.
Strength of electric field in the chip provided by the invention depends on the vertical size of miniflow body cavity, rather than level " isolated island " or " lonely trap " spacing, thereby can avoid producing local highfield, in order to avoid pair cell causes damage and then causes sample fails.
Beneficial effect: cellular positioning arrangement chip provided by the invention and application method; Can overcome that present cellular positioning arrangement chip pin is various, interface cost is high, complex operation and local highfield pair cell cause defectives such as damage; And only need one road electrical signal; Need not the chip periphery assembly, it is very low to have very high safety and a cost.Secondly; Strength of electric field in the chip provided by the invention is not the spacing that depends between level " isolated island " or " the lonely trap "; Thereby can avoid producing local highfield, in order to avoid pair cell causes damage and then causes sample fails, also improved the efficient of subsequent analysis simultaneously.At last, this cellular positioning arrangement chip provided by the invention can be used as the biochemical analysis instrument of cheap jettisonable, can meet economical and feature of environmental protection requirement.The present invention has very considerable prospect in high-throughout bioanalysis field.
Description of drawings
Fig. 1 is the structural representation of the cellular positioning arrangement chip in the embodiment of the invention;
Fig. 2 is the plane formula in the cellular positioning arrangement chip " isolated island " array pattern synoptic diagram in the embodiment of the invention;
Fig. 3 is the plane formula in the cellular positioning arrangement chip in the embodiment of the invention " lonely trap " array pattern synoptic diagram;
Fig. 4 is the array pattern synoptic diagram when having " isolated island " and " lonely trap " simultaneously in the cellular positioning arrangement chip in the embodiment of the invention.
Have among the above figure:
Upper substrate 1, wall 2, hypocoxa 3, upper strata transparent conductive film 11, upward substrate 12, lower floor's transparent conductive film 31, substrate 32 down, passivation layer 51, semiconductor material layer 52, miniflow body cavity 4, isolated island 5, non-isolated island zone 50, lonely trap 6, non-lonely well area 60.
Embodiment
The embodiment of cellular positioning arrangement chip provided by the invention and application method is referring to Fig. 1, Fig. 2 and Fig. 3.
In the present embodiment, it is as shown in Figure 1 to be used for the arrange substruction of localized chip of cell.In this location arrangement chip, upper substrate 1, wall 2, hypocoxa 3 have surrounded miniflow body cavity 4.Miniature " isolated island " 5 is positioned at the upper surface of hypocoxa 3.Because " isolated island " array and " lonely trap " array all can be represented with Fig. 1, so Fig. 1 has been contained two kinds of situation: one, position " isolated island " array (referring to Fig. 2) required when arranging through the negative dielectrophoretic force pair cell; Two, position " lonely trap " array (referring to Fig. 3) required when arranging through positive dielectrophoretic force pair cell.In a chip, can only there be " isolated island 5 " (Fig. 2) or only to have " lonely trap 6 " (Fig. 3), perhaps to have " isolated island 5 " simultaneously and reach " lonely trap 6 " (Fig. 4).Situation shown in Figure 4 mainly is applicable to the situation of two kinds of cells, and promptly a kind of cell experiences positive dielectrophoresis and arrives " lonely trap ", and the negative dielectrophoresis of another kind of cell experience arrives " isolated island ".
In the present embodiment, the upper surface of chip hypocoxa (or bottom surface of upper substrate) has " isolated island " of a lot of array arrangements.These " isolated islands " size is approaching with the size of the individual cells (or set of some cells) of being arranged, and the material of " isolated island " adopts amorphous silicon material.The shape of " isolated island " is not limited to situation shown in Figure 2, and the shape of all isolated islands can be the same or different according to actual needs (square also have circle or other shapes for example, are arranged).By in the miniflow body cavity 4 of cell between upper and lower base plate of arranging.Viable cell need place the nutrient solution with certain ionic concn usually, so the specific conductivity of the cell suspending liquid in the miniflow body cavity 4 is usually than the big times of specific conductivity of non-crystalline silicon.Generally, this chip does not need any chip periphery accessory and interface, and only needs one tunnel actuation signal, so use cost is very low.Between the two-layer transparent conductive film of upper substrate and hypocoxa, be applied in an ac voltage signal; Then direction of an electric field is vertical; But because the liquid level in the miniflow body cavity 4 is different with the dividing potential drop in " non-isolated island " zone in " isolated island " zone; And vertically distance is basic identical, causes the formation of inhomogeneous field, so produced dielectrophoretic force.Be positioned at the little times of strength of electric field that electric field ratio " non-isolated island " that " isolated island " locate is located in the liquid level; So when cell receives to bear dielectrophoresis (being that cell moves to the minimum place of field intensity); Cell understand oneself move to " isolated island " 5 places (referring to Fig. 2, " isolated island " here be the general reference, be not certain specific " isolated island "); And then arrange according to the formation ruleization of arranging of " isolated island " array, therefore the distance between " isolated island " and " isolated island " has also just determined the distance between cell and the cell.When the size of " isolated island " and individual cells near the time, just can realize single celled array arrangement; When the set area occupied of the size of " isolated island " and some cells near the time, just can realize the array arrangement of cell mass.
On the other hand, " lonely trap " 6 can be constructed at upper surface or (lower surface of upper substrate) of the hypocoxa of chip in the location of arranging when receiving positive dielectrophoresis (being cell greatly moves at the place to field intensity) in order to satisfy cell." lonely trap " 6 is the zone that does not have deposition of amorphous silicon, and other zones are " non-lonely trap " 60 zones that deposited non-crystalline silicon.The shape of " lonely trap " is not limited to situation shown in Figure 3, and the shape of all " lonely traps " can be identical, also can difference (for example, can have square, hexagonal, circular or other shapes).Because liquid level is different with the dividing potential drop in " non-lonely trap " zone in " lonely trap " zone, and vertically distance is basic identical, causes the formation of inhomogeneous field, so produced dielectrophoretic force.Be positioned at the big times of strength of electric field at electric field ratio " non-lonely trap " 60 places at " lonely trap " 6 places; So when cell receives positive dielectrophoresis; Cell is understood and oneself is moved to " lonely trap " 6 places; And then arrange according to the formation ruleization of arranging of " lonely trap " array, therefore the distance between " lonely trap " and " lonely trap " has also just determined the distance between cell and the cell.
In the present embodiment, the material of the transparent conductive film in the chip can be selected ITO (Indium Tin Oxides) film." isolated island " 5 contains passivation layer 51 and semiconductor material layer 52.Passivation layer 51 can be silit or silicon nitride film; Semiconductor material layer 52 can be amorphous silicon hydride or adulterated Cadmium Sulfide (CdS) or the cadmium selenide (CdSe) that mixes or the combination of Cadmium Sulfide and cadmium selenide, or other specific conductivity are than the semiconductor material of the little times of cell culture fluid (that is the cell suspending liquid in the miniflow body cavity).The thickness of passivation layer 51 is much smaller than the thickness of semiconductor material layer 52.The substrate up and down of chip can be transparent (for example glass), also can be translucent or opaque material.Transparent material is beneficial to observation most, almost in all are used, can use; Translucent material can use in the time only need seeing through the light of certain limit wavelength; Opaque material in situation (for example, micro sensor being installed on the chip) that light requirement is not learned observation down or allow to open in the application of substrate (for example, need open upper substrate and add reagent, observation etc.) and also can use.
Claims (2)
1. low-cost cellular positioning arrangement chip; It is characterized in that, this chip comprise upper substrate (1) that the last conductive film (11) of substrate (12) and lower surface thereof forms, down substrate (32) with and the hypocoxa (3) formed of the following conductive film (31) of upper surface, the miniflow body cavity (4) of dielectric spacer layer (2) formation between last conductive film (11) and following conductive film (31); Be provided with the semiconductor material layer (52) of array architecture at the lower surface of the upper surface of hypocoxa (3) or upper substrate (1); On semiconductor material layer (52), be provided with passivation layer (51), and form miniature isolated island (5) or miniature lonely trap (6) or form and have the shape of isolated island and lonely trap simultaneously;
Described substrate (32) down and last substrate (12) are transparent material or translucent material or opaque material;
The shape of described all miniature isolated islands or lonely trap is all identical, or part is identical or have nothing in common with each other.
2. low-cost cellular positioning arrangement chip as claimed in claim 1 is characterized in that, semiconductor material layer (52) is cadmium selenide or other specific conductivity semiconductor material littler than cell suspending liquid that non-crystalline silicon or adulterated Cadmium Sulfide perhaps mix.
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| CN 200910032740 CN101580797B (en) | 2009-06-19 | 2009-06-19 | Low-cost cellular positioning arrangement chip and application method |
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| CN 200910032740 CN101580797B (en) | 2009-06-19 | 2009-06-19 | Low-cost cellular positioning arrangement chip and application method |
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| CN101580797B true CN101580797B (en) | 2012-02-22 |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109142717B (en) * | 2013-10-22 | 2021-11-16 | 伯克利之光生命科技公司 | Microfluidic device for determining biological activity |
| US9889445B2 (en) | 2013-10-22 | 2018-02-13 | Berkeley Lights, Inc. | Micro-fluidic devices for assaying biological activity |
| CN104789443B (en) * | 2014-06-23 | 2020-01-17 | 格子生物科技(上海)有限公司 | Cell positioning unit, cell positioning array, cell positioning device and forming method of cell positioning device |
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| US5034486A (en) * | 1990-08-09 | 1991-07-23 | Gaf Chemicals Corporation | Terpolymers of maleic anhydride, C1 -C5 alkyl vinyl ether and a C12 -C14 alpha-olefin, and crosslinked products thereof |
| US7014747B2 (en) * | 2001-06-20 | 2006-03-21 | Sandia Corporation | Dielectrophoretic systems without embedded electrodes |
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| CN101368155A (en) * | 2008-09-19 | 2009-02-18 | 重庆大学 | Continuous flow cell electric amalgamation chip based on silicon structure on insulator |
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2009
- 2009-06-19 CN CN 200910032740 patent/CN101580797B/en not_active Expired - Fee Related
Patent Citations (6)
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| US5034486A (en) * | 1990-08-09 | 1991-07-23 | Gaf Chemicals Corporation | Terpolymers of maleic anhydride, C1 -C5 alkyl vinyl ether and a C12 -C14 alpha-olefin, and crosslinked products thereof |
| US7014747B2 (en) * | 2001-06-20 | 2006-03-21 | Sandia Corporation | Dielectrophoretic systems without embedded electrodes |
| CN101250482A (en) * | 2008-03-28 | 2008-08-27 | 重庆大学 | Microelectrode array chip for cell electrofusion |
| CN101275944A (en) * | 2008-05-13 | 2008-10-01 | 东南大学 | High flux test chip of single cell traveling wave dielectric spectrum and test method |
| CN201188104Y (en) * | 2008-05-13 | 2009-01-28 | 东南大学 | High flux test chip |
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Non-Patent Citations (2)
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| D.F. Chen等.A 3-D Microelectrode System for Dielectrophoretic Manipulation of Microparticles.《Journal of Physics》.2006 IOP Publishing Ltd,2006,第34卷图2. * |
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