CN101434904A - Cell electrofusion chip and method for manufacturing the same - Google Patents
Cell electrofusion chip and method for manufacturing the same Download PDFInfo
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- CN101434904A CN101434904A CN 200810233274 CN200810233274A CN101434904A CN 101434904 A CN101434904 A CN 101434904A CN 200810233274 CN200810233274 CN 200810233274 CN 200810233274 A CN200810233274 A CN 200810233274A CN 101434904 A CN101434904 A CN 101434904A
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- microelectrode
- chip
- cell
- cell electrofusion
- silicon chip
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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 cell electrofusion chip which is formed by bonding a silicon wafer and a glass sheet, wherein microelectrode units are arranged on the silicon wafer, a plurality of microelectrodes are arranged on the microelectrode units, and the microelectrode units are provided with signal leading-out wires; the microelectrodes are arranged on one side or two sides of each microelectrode unit in a jagged shape, the microelectrodes arranged on the adjacent microelectrode units are opposite in a staggered way, and a continuous microchannel is formed among the microelectrode units; the cell electrofusion chip not only can observe and record the cell fusion process more easily during the fusion process, but also can improve the anti-chemical corrosion ability, the high-temperature and high-pressure resistant ability and the biocompatibility of the cell electrofusion chip; meanwhile, the invention also discloses a manufacturing method of the cell electrofusion chip.
Description
Technical field
The present invention relates to biomass cells integration technology field, particularly a kind of cell electrofusion chip and manufacture method.
Background technology
Biomass cells can form new cell by merging, and has great significance in modern biomedical engineering foundation field, is widely used in research fields such as health care, modern agriculture, comprises Monoclonal Antibody, new species generation or improvement etc.Cytogamy has chemical fusion method (as the PEG method), physics fusion method, and wherein in the physics fusion method, the electric field fusion method is convenient to accurate control, good reproducibility, fusion rate advantages of higher because of it and has been obtained using widely.
The process that the cell electricity merges by several successive realizes: cell is subjected to dielectrophoresis force in electric field, is in contact with one another to form the pearl string, and this process is called the cell queuing; Apply high electric field pulse and make cytolemma produce reversible puncture on the cell of bunchiness, form hole, two intracellular fluids are by perforation exchange material, and last cytolemma, nucleus merge and form a new cell.
In order to realize that cell electricity merges, most critical be that cell is placed inhomogeneous field, make cell be subjected to dielectrophoresis force and highfield piercing force.According to electric field basic theories electric field strength E=V/d, in order to obtain stronger strength of electric field, must be by increasing voltage or shortening interelectrode distance and realize, in the design of cell electrofusion chip, realize high strength of electric field to reduce interelectrode distance, so, interelectrode distance can be carried out design alternative according to the cell size, in cell size order of magnitude scope, generation is enough to make the membrane perforation required voltage to be reduced to tens volts to tens volts to get final product, greatly reduce cell electricity fusion signal generator and manufacture and design difficulty, improved the surviving rate of cell after the cell electricity merges.
Chip for cell fusion is as the core integral part of a complete cell electricity emerging system, requirement to its structure and manufacture craft is also more and more higher, but on the whole, at present domestic less in the cell electrofusion chip research and development, mainly have following defective: strength of electric field that microelectrode produces and electric-force gradient a little less than, can not realize controlling comparatively accurately; Very poor aspect the visual degree of enhancing, be not easy (or effect is bad) and observe chip internal cell electricity fusion process, be unfavorable for that real-time pair cell carries out observed and recorded in the cell electricity fusion process, be not easy to manipulation cell.And aspect the work material selection, also there is the problem that material is anticorrosive, resistance of oxidation is more weak.
Summary of the invention
In view of this, at the prior art above shortcomings, the present invention proposes a kind of cell electrofusion chip, arrange microelectrode by special spination, strength of electric field and electric-force gradient that microelectrode is produced not only can satisfy requirement of strength, and electric field distribution is even, can realize control comparatively accurately, simultaneously owing to adopt silicon glass structure, in fusion process, cell fusion process be can observe and write down more easily, and resistance to chemical attack, high temperature high voltage resistant ability, the physiologically acceptable performance of cell electrofusion chip improved.
Visual cells electrofusion chip of the present invention, described cell electrofusion chip is formed by silicon chip and sheet glass bonding, and described silicon chip is provided with the microelectrode group, and described microelectrode group is provided with a plurality of microelectrodes, and described microelectrode group is provided with the signal outlet line;
Described microelectrode indentation is arranged in the one or both sides of microelectrode group, and the microelectrode that is arranged on the adjacent microelectrode group is relatively staggered, forms the successive microchannel between the microelectrode group;
Further, described microelectrode is the fleam-tooth shape or the rectangle spination is arranged on the microelectrode group;
Further, described silicon chip is provided with a plurality of microelectrode groups and many microchannels;
Further, described silicon wafer thickness is 40～60 μ m, and sheet glass thickness is 500～1000 μ m;
Further, the signal outlet line of described microelectrode group adopts spun gold;
Further, the signal outlet line of described microelectrode group connection connects independently control signal separately;
Further, described microelectrode group from outward appearance to inner essence is made up of golden electrolytic coating, golden sputtering layer, silicon wafer layer successively;
Further, the thickness of described golden sputtering layer is 150～200nm, and described golden thickness of plating layer is 500～1000nm;
Further, the invention also discloses a kind of working method of cell electrofusion chip, its step is as follows:
1) silicon chip and sheet glass bonding;
2) adopt ion implantation mode to mix the trivalent or the pentad that can improve the semiconductor silicon conductive capability to silicon chip, doping content is greater than 1 * 10
19Cm
-3, to form P type or N type Si;
3) at silicon chip surface sputter 150～200nm gold, form golden sputtering layer;
4) electrogilding sputtering layer to 500～1000nm forms golden electrolytic coating;
5) etching by the golden sputtering layer of step 3 and step 4 gained and golden electrolytic coating to silicon chip;
6) etching silicon chip, etching depth is a silicon wafer thickness, obtains the substrate of glass cell electrofusion chip.
Further, described trivalent element is boron or indium, and described pentad is arsenic or antimony.
The invention has the beneficial effects as follows:
1, adopts the unique design of microelectrode group, wherein the arrangement mode of microelectrode can be the rectangle spination, also can be fleam-tooth shape or other shape, this unique design can improve around the microelectrode and the strength of electric field and the electric-force gradient of inside microchannels greatly, improve the electric field inductive capacity in the fusion process, thereby improved fusion efficiencies.
2, adopt silicon chip and sheet glass bonding, improve the stopping property of microelectrode and silicon contact surface, fluid seepage can not take place.The silicon glass has very strong resistance to corrosion, improves the resistance to chemical attack of chip pair cell damping fluid.Silicon glass heat-resisting ability is strong, is convenient to chip and carries out high-temperature sterilization, sterilization.The silicon glass has lower thermal expansivity, makes chip structure deformation can not take place under hot conditions.The silicon glass has good biological fitness (consistency), and by the cytogamy environment that the silicon glass constitutes, the cell after being convenient to merge is directly cultivated in chip, improves cell survival rate.
3, silicon chip has higher conductive capability by the microelectrode that mixes formation, and carry out sputter coating process on its surface and handle, its conductive capability further strengthens, what make the microelectrode that is connected on the same electric power polarity has equal Potential Distributing everywhere, thereby guarantees that same microelectrode electric field distribution everywhere is identical.
4, glass has constituted the microchannel that the cell electricity merges as chip base jointly with microelectrode, and glass has the good optical visuality, is convenient to observe, writes down chip inner cell fusion process.Glass also has high insulating property, has enlarged the voltage range that chip adapts to, and lays a good foundation for chip adapts to various cytogamy required voltages, has enlarged the range of application of chip.
5, select the microelectrode precision height of silicon doping coating process etching, conductivity is good, the good uniformity that voltage distributes, and tooling cost is low, is easy to the commercialization marketing.
Other advantages of the present invention, target, to set forth in the following description to a certain extent with feature, and to a certain extent,, perhaps can obtain instruction from the practice of the present invention based on being conspicuous to those skilled in the art to investigating hereinafter.Target of the present invention and other advantages can realize and obtain by following specification sheets and claims.
Description of drawings
In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing, wherein:
Fig. 1 is the cell electrofusion chip structural representation;
Fig. 2 is a cell electrofusion chip manufacture craft schematic flow sheet;
Fig. 3 is the structural representation of a kind of cell electricity emerging system of the present invention's composition;
Fig. 4 is the electric-field intensity distribution situation contrast synoptic diagram on intersection microelectrode of the present invention and the traditional symmetrical microelectrode diagonal lines;
Fig. 5 is the electric field intensity gradient distribution situation contrast synoptic diagram of intersection microelectrode of the present invention and traditional symmetrical microelectrode.
Embodiment
Fig. 1 is a kind of cell electrofusion chip structural representation; As shown in Figure 1: cell electrofusion chip is formed by silicon chip 1 and sheet glass 2 bondings, silicon chip 1 is provided with microelectrode group 3, microelectrode group 3 is provided with a plurality of microelectrodes 5, described microelectrode group 3 is provided with the signal outlet line and the external electric control signal links, microelectrode 5 indentations are arranged in a side of microelectrode group 3, the microelectrode that is arranged on the adjacent microelectrode group is relatively staggered, forms successive microchannel 4 between the microelectrode group 3; Wherein silicon chip 1 thickness is 40～60 μ m, and sheet glass 2 thickness are 500～1000 μ m; The signal outlet line of microelectrode 5 adopts spun gold.
Microelectrode 5 also can be the fleam-tooth shape or other shapes are arranged on the microelectrode group 3.
Fig. 2 is a cell electrofusion chip manufacture craft schematic flow sheet, as shown in Figure 2, may further comprise the steps: 1) silicon glass bonding: select for use silicon chip 1 thickness about 300 μ m, sheet glass 2 thickness are about 500 μ m, can be behind the bonding according to the planner need carry out attenuate to silicon chip 1, cell electrofusion chip in the present embodiment, silicon chip 1 reduced thickness to 40 μ m; 2) silicon chip mixes: adopt ion implantation mode to mix boron and indium to silicon chip, doping content is greater than 1 * 10
19Cm
-3, form P type Si; 3) sputter:, form golden sputtering layer 7 at silicon chip 1 surface sputtering 100nm gold; 4) electroplate: electrogilding sputtering layer 7 forms golden electrolytic coating 6 to 500～1000nm; 5) etching gold: etching by the golden sputtering layer 7 of step 3 and step 4 gained and golden electrolytic coating 6 to silicon chip 1; 6) etch silicon: etching depth is silicon chip 1 thickness, obtains cell electrofusion chip.
The microelectrode group of the cell electrofusion chip of making from outward appearance to inner essence is made up of golden electrolytic coating, golden sputtering layer, silicon wafer layer and sheet glass successively, should be noted that in the etching technics of the present invention, glass can not be carried out etching, otherwise influences the chip optical property.
For the advantage of cell electrofusion chip of the present invention is described, wave-front method in the employing ANSYS10.0 analysis software carries out electromagnetic-field simulation to the symmetrical microelectrode arrangement mode and the intersection microelectrode arrangement mode of the present invention of traditional way, wave-front method is the direct method for solving of system of equations, find the solution the unit and adopt the PLANE230 cell type, it is two-dimentional eight node electroanalysis unit, simulation result such as Fig. 4, shown in Figure 5, in comparative analysis, the interelectrode distance of being got, electrode materials, the specific conductivity of cell damping fluid in the microchannel, institute's making alive direction and size parameter thereof are identical on the microelectrode, Fig. 4 intersects electric-field intensity distribution situation contrast synoptic diagram on microelectrode and the symmetrical microelectrode diagonal lines (wherein a intersects microelectrode, b is symmetrical microelectrode), therefrom as can be seen, under identical condition, the maximum electric field intensity that intersection microelectrode of the present invention can produce is greater than symmetrical microelectrode, this shows, in cell fusion process, puncture same cell, the needed pulse control of crossed electrode voltage is lower, help the design of signal generator, different with electroporation conditions is, the queuing of cell mainly is the size that relies on electric-force gradient, Fig. 5 is that (wherein a is for intersecting microelectrode for the electric field intensity gradient distribution situation of two kinds of different electrode array designs, b is symmetrical microelectrode), the result shows, the electric field intensity gradient that in most positions all is interdigitated electrode design is bigger, particularly the zone of the zone of its maximum and strength of electric field maximum is basic overlaps, show that most cells can be positioned in these positions and carry out electroporation and fusion, these results show, the traditional relatively symmetric design of intersection microelectrode design of the present invention, all bigger on strength of electric field and electric-force gradient, the pair cell electricity merges more favourable, can improve cell electricity fusion efficiencies.
Need to prove, chip for cell fusion of the present invention can be formed the cytogamy system by the outer package parts, the silicon glass structure of this chip for cell fusion can guarantee that in the process of test the trier can be by the clear observation of substrate of glass and the record chip inner cell fusion process of chip.A kind of structure as shown in Figure 3, wherein have the hole slot that chip for cell fusion 8 of the present invention is installed in the middle of the test tank 9, the set-up mode of chip for cell fusion 8 is that substrate of glass is provided with outwardly, the inner chamber test solution of microelectrode and test tank 9 keeps in touch, and test tank 9 also is provided with input duct 10 and fluid pipeline 11 in addition.
Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can make amendment or be equal to replacement technical scheme of the present invention, and not breaking away from the aim and the scope of the technical program, it all should be encompassed in the middle of the claim scope of the present invention.
Claims (10)
1. cell electrofusion chip, it is characterized in that: described cell electrofusion chip is formed by silicon chip (1) and sheet glass (2) bonding, described silicon chip (1) is provided with microelectrode group (3), described microelectrode group (3) is provided with a plurality of microelectrodes (5), and described microelectrode group (3) is provided with the signal outlet line;
Described microelectrode (5) indentation is arranged in the one or both sides of microelectrode group (3), and the microelectrode (5) that is arranged on the adjacent microelectrode group (3) is relatively staggered, forms successive microchannel (4) between the microelectrode group (3).
2. cell electrofusion chip according to claim 1 is characterized in that: described microelectrode (5) is the fleam-tooth shape or the rectangle spination is arranged on the microelectrode group (3).
3. cell electrofusion chip according to claim 1 and 2 is characterized in that: described silicon chip (1) is provided with a plurality of microelectrode groups (5) and many microchannels (4).
4. cell electrofusion chip according to claim 3 is characterized in that: described silicon chip (1) thickness is 40～60 μ m, and sheet glass (2) thickness is 500～1000 μ m.
5. cell electrofusion chip according to claim 1 is characterized in that: the signal outlet line of described microelectrode group (5) adopts spun gold.
6. cell electrofusion chip system according to claim 1 or 5, it is characterized in that: the signal outlet line that described microelectrode group (5) connects connects independently control signal separately.
7. cell electrofusion chip according to claim 4 is characterized in that: described microelectrode group (5) from outward appearance to inner essence is made up of golden electrolytic coating (6), golden sputtering layer (7), silicon chip (1) layer successively.
8. cell electrofusion chip according to claim 7 is characterized in that: the thickness of described golden sputtering layer (7) is 150～200nm, and the thickness of described golden electrolytic coating (6) is 500～1000nm.
9. the manufacture method of the described cell electrofusion chip of claim 8, its step is as follows:
1) with silicon chip (1) and sheet glass (2) bonding;
2) adopt ion implantation mode to mix the trivalent element or the pentad that can improve the semiconductor silicon conductive capability to silicon chip (1), doping content is greater than 1 * 10
19Cm
-3, to form P type or N type Si;
3) at silicon chip (1) surface sputtering 150～200nm gold, form golden sputtering layer (7);
4) electrogilding sputtering layer (7) forms golden electrolytic coating (6) to 500～1000nm;
5) etching by the golden sputtering layer (7) of step 3 and step 4 gained and golden electrolytic coating (6) to silicon chip (1);
6) etching silicon chip (1), etching depth are silicon chip (1) thickness, obtain the substrate of glass cell electrofusion chip.
10. the working method of the described cell electrofusion chip of claim 9, its step is as follows: described trivalent element is boron or indium, described pentad is arsenic or antimony.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN 200810233274 CN101434904A (en) | 2008-12-08 | 2008-12-08 | Cell electrofusion chip and method for manufacturing the same |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810233274 CN101434904A (en) | 2008-12-08 | 2008-12-08 | Cell electrofusion chip and method for manufacturing the same |
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| CN101434904A true CN101434904A (en) | 2009-05-20 |
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| CN 200810233274 Pending CN101434904A (en) | 2008-12-08 | 2008-12-08 | Cell electrofusion chip and method for manufacturing the same |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103396944A (en) * | 2013-07-22 | 2013-11-20 | 博奥生物有限公司 | Electroporation chip for cell transfection and making method thereof |
| CN115895876A (en) * | 2022-11-30 | 2023-04-04 | 重庆大学 | Cell electrofusion chip device based on bilateral flow field pairing structure array and preparation method |
-
2008
- 2008-12-08 CN CN 200810233274 patent/CN101434904A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103396944A (en) * | 2013-07-22 | 2013-11-20 | 博奥生物有限公司 | Electroporation chip for cell transfection and making method thereof |
| CN103396944B (en) * | 2013-07-22 | 2015-08-26 | 博奥生物有限公司 | A kind of electroporation chip for cell transfecting and preparation method thereof |
| CN115895876A (en) * | 2022-11-30 | 2023-04-04 | 重庆大学 | Cell electrofusion chip device based on bilateral flow field pairing structure array and preparation method |
| CN115895876B (en) * | 2022-11-30 | 2024-04-02 | 重庆大学 | Cell electrofusion chip device based on double-side flow field pairing structure array and preparation method |
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Application publication date: 20090520 |