CN103320319A - Alternative high-throughput electroporation device - Google Patents
Alternative high-throughput electroporation device Download PDFInfo
- Publication number
- CN103320319A CN103320319A CN201310281908XA CN201310281908A CN103320319A CN 103320319 A CN103320319 A CN 103320319A CN 201310281908X A CN201310281908X A CN 201310281908XA CN 201310281908 A CN201310281908 A CN 201310281908A CN 103320319 A CN103320319 A CN 103320319A
- Authority
- CN
- China
- Prior art keywords
- electroporation
- electroporation device
- pcb
- electrode
- hole
- 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.)
- Granted
Links
Images
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention discloses an alternative high-throughput electroporation device. The electroporation device comprises a substrate, wherein multiple through holes are formed in the substrate; a pair of electrodes is arranged at each through hole; the electrodes are connected with a wire arranged on the substrate; the electrodes can be gated in a single-point mode. The provided electroporation device can be manufactured by using a printed circuit board (PCB) manufacturing method. The electroporation device can be used for performing the electroporation on cells, such as animal cells or bacteria. During use of the device, an electroporation pulse is applied to an electroporation system in the through holes through the electrodes at the through holes. The electroporation device can be manufactured by using a PCB process, is mature in technology and low in cost and can be produced in batches. The electroporation device manufactured by using the PCB process can be seamlessly connected with a PCB-based circuit control system, and the electroporation device contributes to realizing high-throughput automatic gene function screening based on the electroporation.
Description
Technical field
The present invention relates to a kind of alternative apparatus for high-throughput electroporation, belong to electroporation arts.
Background technology
Electroporation is to make cytolemma produce transmembrane voltage by the external pulse electric field, and after cell transmembrane voltage surpassed certain threshold value, cytolemma reversible perforation took place with cell transfecting or the bacterium transformed technology of allogenic material transfered cell.Electroporation has that no cytotoxicity, cost are low, transfection efficiency is than advantages such as height.Electroporation technology has been widely used in biological study at present.Existing a few class electroporation method as cell being placed at a distance of (for example, United States Patent (USP) U.S.Pat.5389069) between several millimeters parallel poles, or makes cell flow through (for example, United States Patent (USP) U.S.Pat.6773669 between parallel pole; Chinese patent application, publication number: CN1195997A), all exist to use comparatively to bother and be difficult to deficiency such as a plurality of samples of parallel processing.High-throughput electroporation allows a plurality of samples are carried out synchronously or automatic continuous electroporation, and this class technology is applicable to many processes such as high-throughput gene functional research and the screening for the treatment of target spot.
Existing apparatus for high-throughput electroporation all is based on the multiaperture-type structure, the electroporation system is added in the multiaperture-type structure, and each hole of this AND DEWATERING FOR ORIFICE STRUCTURE comprises the electrode that a pair of electricimpulse takes place, and applies electricimpulse by certain program and just can make cell generation electroporation in each hole (patent for example: International Patent Application PCT is open: WO2004/050866A1; Chinese patent: CN101384697B).Though this class device has solved the problem of high-throughput electroporation, sample preparation, transfer and device cleaning process are more loaded down with trivial details, and cell and transfected material that its each reaction consumes are many, and this has improved the cost of high flux screening.
The G.Emmanuel of U.S. Stanford university in 2008 etc. has been reported to a kind of hanging drop electroporation device at nature methods magazine.This device is made up of the tinsel of two slotting finger-like, forms 96 pairs of metal electrodes corresponding to 96 orifice plates.The cell suspension of 10 to 20 microlitres is added to the gap that can stick to electrode between the metal electrode because of surface tension.Apply electricimpulse and get final product the cell electroporation that will stick between the electrode pair between two electrodes, the cell after the perforation can be washed into by a large amount of cell culture mediums in the porous plate of electrode below.The sample operation process of this device is very simple, with conventional porous plate compatible and can with the coupling of liquid working station.But the electroporation conditions of the optimum between the combination of different cells and transfection object there are differences, and this device has only two electrodes, thus can't carry out the selectivity electroporation to different samples synchronously, and also tooling cost is higher, and this has limited its application.Therefore how to realize low cost, optionally high-throughput electroporation becomes very important problem in the extensive screening.Printed circuit board (PCB) (PCB) is that a class is applied to the maturation process that electronic circuit is made, and can produce in batches, and tooling cost is lower, has some at present and the PCB technology is applied to the report of cell manipulation.For example, utilize the PCB technology can generate the through hole of diameter about 100 microns, the integrated cell capture based on dielectric power of this class through hole (L.Marta of Italy Bologna university in 2010 etc. once reported at Lab on a Chip magazine that the through hole that utilizes among the PCB caught single celled research) and impedance bioelectrical measurement functions such as (F.Andrea of Italy Bologna university in 2012 etc. use the through hole on the PCB to survey the cell impedance at Lab on a Chip magazine report) has the potentiality that are applied to unicellular operation and analysis.Equally, the PCB technology also has the potentiality that are applied to cell electroporation.
Summary of the invention
The purpose of this invention is to provide a kind of alternative apparatus for high-throughput electroporation easy to use.
A kind of alternative apparatus for high-throughput electroporation provided by the invention, it comprises a substrate;
Described substrate is provided with several through holes; Each described through hole is provided with 1 counter electrode, and described electrode is connected with lead on being located at described substrate;
But described electrode single-point gating.
" single-point gating " described in the present invention refers to: between the every counter electrode on the described substrate for being connected in parallel, thereby can control separately, realize selective control.
In the above-mentioned electroporation device, described substrate can be made by insulating material, as adopting glass or insulation high molecular polymer.
In the above-mentioned electroporation device, described through hole can be circle, ellipse, square or rectangle etc.
In the above-mentioned electroporation device, described electrode is made by metal or conductive polymers.
Cell is in pulsed electrical field, and cytolemma can produce transmembrane voltage, and after transmembrane voltage surpassed certain threshold value, surface of cell membrane produced recoverable micropore, thereby with the allogenic material transfered cell.Producing suitable electric field is the key of efficient electroporation.It is to finish by the conductive electrode that contacts with it with the mixed system for the treatment of the transfection material that the pulse signal importing contains cell.Therefore, for electroporation device, arranging of electrode can directly influence the electroporation effect, and reasonably lead is arranged particularly important to the complexity and the realization selective control that reduce device.According to above-mentioned principle, among the present invention, described electrode can be arranged on the sidewall of described through hole; Perhaps, described electrode can be arranged on the both sides up and down of described substrate, and the inwall of the middle part section of described electrode and described through hole is on the same perpendicular.
In the above-mentioned electroporation device, distance is 0.2～2mm between 2 battery lead plates of described electrode.
In the above-mentioned electroporation device, described substrate can be printed circuit board (PCB) (PCB), and it is compared with disclosed electroporation device, and it can be produced in batches, and tooling cost greatly reduces.In addition, because the pcb board cost of producing in batches is very low, can disposablely use, can reduce the cleaning process of electroporation device behind the electroporation, reduced operation steps, avoided the pollution problem between the different batches experiment.
Electroporation device provided by the invention can be made according to the PCB making method and obtain.
Electroporation device provided by the invention can be used for cell is carried out electroporation, as zooblast or bacterium are carried out electroporation.During use, the electroporation pulse is applied in the electroporation system that is suspended in the through hole by the electrode at described lead to the hole site place.
The present invention is used with the porous plate that is arranged on below the described electroporation device in use, as the standard porous plate of commercialization, can be 6,8,16,24,48,96,384 orifice plates.
Compared with prior art, advantage of the present invention is:
1, application of sample, electroporation and move the liquid process and easily carry out, and can apply corresponding multiple optimization electroporation conditions synchronously to several samples.
2, among the present invention, the system of each electroporation is less, can be at 1 microlitre between 50 microlitres.The cell of each sample consumption and treat that the transfection material is less is conducive to realize extensive screening.
3, the electroporation system is suspended in two distances between 0.2～2 millimeter parallel metal sheet, and its large surface area is conducive to heat radiation, thereby can avoid the damage of rising and bringing to the perforation cell because of solution temperature that electroporation causes.
4, electroporation device of the present invention can be made by PCB technology, technology maturation, and cost is low, can produce in batches.The electroporation device made from PCB technology can with the circuit control system seamless link based on PCB, be conducive to realize the gene function screening based on electroporation of high-throughput, automatization.
Description of drawings
Fig. 1 is the vertical view of apparatus for high-throughput electroporation of the present invention.
Fig. 2 is the structural representation of 1 unit of electroporation device shown in Figure 1.
Fig. 3 is the pictorial diagram based on the apparatus for high-throughput electroporation of printed-board technology (PCB) making.
Fig. 4 is the principle schematic of two electrodes for the metal level with PCB through hole inboard among Fig. 3 interrupts.
Fig. 5 is the use schematic flow sheet of electroporation device proposed by the invention.
Fig. 6 is for using the process of volley of rifle fire application of sample.
Fig. 7 is the principle schematic that mills the metal level of circular PCB through hole inboard.
Each mark is as follows among the figure: 1 substrate, 2 through holes, 3 electrodes, 4 top layer leads, 5 bottom leads.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The vertical view of the electroporation device that Fig. 1 provides for present embodiment, it can mate with 96 orifice plates.
As shown in Figure 1, the electroporation device in the present embodiment comprises a substrate 1, is provided with 96 through holes 2 at this substrate 1, and mates with 96 orifice plates.Be equipped with 1 electrode 3 on the sidewall of each through hole 2, be 1 counter electrode.Distance between two electrode plate is 2mm.
In order to realize the selective control to each through hole with the pad of trying one's best few, all electrodes of upside in 12 holes of delegation are connected in the top layer lead 4 that substrate 1 top distributes along each row jointly, and all electrodes of downside in 8 holes of row are connected in B.B.P jointly along the bottom lead 5 of each column distribution.Two lateral electrodes power up because the electroporation of each through hole 2 position needs up and down, therefore use 8 * 12 pads just can realize the selective control to the single-point gating of all 96 through holes here.Here the top layer lead on the substrate 4 distributes and can exchange with bottom lead 5, and if substrate 1 can the multilayer cabling, lead also can be walked the middle layer of substrate 1.
Fig. 2 is the enlarged view of a unit of the electroporation device among Fig. 1, and as shown in Figure 2, the character of the through hole 2 in the present embodiment is rectangle, and it can also be set to circle, ellipse, different shape such as square.
Fig. 3 is with the pictorial diagram of printed circuit board (PCB) (PCB) as the apparatus for high-throughput electroporation of substrate.
What specifically use is the pcb board of two-layer wiring, comprises 96 ellipse holes.The metal level of the major axis upper and lower of each ellipse hole is connected on the pcb board on 8 * 12 pads by above-mentioned wire laying mode.The via hole inwall that conventional PCB technology is made is the block of metal that links together, and in order to form the required electrode pair of electroporation, need carry out one extra technology.As shown in Figure 4, after via hole covers copper, use to the drill bit of PCB punching the via hole two ends are covered the copper mill off.By this operation, generate a pair of metal electrode that parallels in each via hole on the pcb board.
When using electroporation device of the present invention, can carry out according to method shown in Figure 5, specifically be divided into 3 steps: 1 application of sample, 2 hanging drop electroporations, 3 sample transfer.
Detailed process is as follows: the electroporation device that makes up is placed on the porous plate top, and the through hole of electroporation device is corresponding one by one with the hole of following porous plate.To contain cell on a small quantity and treat that the electroporation system of transfection material is added in the through hole of electroporation device by pipettor.The electroporation system can be suspended in the through hole of electroporation device because of surface tension.The system of electroporation can be controlled by the size design of the through hole of electroporation device.Electroporation system in all through holes is applied synchronously or applies electricimpulse fast successively, finish the cell electroporation process.The cell culture medium of comparatively large vol is added through the through hole of electroporation device, and cell culture medium can pour the cell suspension after the perforation in the porous plate of below, finishes the transfer of perforation cell.Then porous plate is placed on to cultivate in the cell culture incubator in order to follow-up experiment and analysis and uses.
The operation of whole electroporation is very simple, and the application of sample process can finish with the volley of rifle fire (as shown in Figure 6) or automated mechanical hand, is very suitable for high-throughout screening.
Present embodiment is similar to Example 1, all is to adopt PCB to make electroporation device, and difference is: what present embodiment used is that circular PCB through hole carries the electroporation system.Be similar to embodiment 1, PCB through hole inboard is coated with metal level, and in order to generate the needed electrode pair of electroporation, available drill bit is with the metal mill off (as shown in Figure 7) of PCB by inwall, and the metal level of PCB top layer and bottom can become the electroporation electrode like this.A kind of alternative processing mode is: directly punch with drill bit in the position that the upper/lower layer metallic cabling crosses, as long as bit diameter also can obtain to be similar to the structure shown in the right figure of Fig. 7 less than the width of metal routing.Distance between two electrode plate on the horizontal direction is 0.2mm.
The use flow process of the electroporation device that this method generates with embodiment 1, equally also is 3 steps: 1 application of sample, 2 hanging drop electroporations, 3 sample transfer.Concrete operating process is: the electroporation device that builds is placed on the porous plate top, and the through hole of electroporation device is corresponding one by one with the hole of following porous plate.To contain cell and treat that the electroporation system of transfection material is added in the through hole of electroporation device by pipettor.Add system volume should be enough to be full of through hole on the PCB, ie in solution can touch the electrode of PCB upper and lower surface simultaneously.Electroporation system in all through holes is applied synchronously or applies electricimpulse fast successively, finish the cell electroporation process.The cell culture medium of comparatively large vol is added through the through hole of electroporation device, and cell culture medium can pour the cell suspension after the perforation in the porous plate of below, finishes the transfer of perforation cell.Then porous plate is placed on to cultivate in the cell culture incubator in order to follow-up experiment and analysis and uses.
Than embodiment 1, in the present embodiment, the contact area of electrode and solution is less, thereby electrode surface is unfavorable for the factor of cell survival because of the solution temperature that water electrolysis brings raises, pH value change etc. can reduce electroporation the time.In addition, the via hole that this method is made is less, is suitable for small amounts of cells even single celled electroporation.This method is potential mutually integrated with the cell impedance bioelectrical measurement (F.Andrea of Italy Bologna university in 2012 etc. reports at Lab on a Chip magazine) based on PCB, becomes the platform of unicellular transfection and cellular electrophysiologicalsensor monitoring in real time.
Claims (9)
1. alternative apparatus for high-throughput electroporation is characterized in that:
Described electroporation device comprises a substrate;
Described substrate is provided with several through holes; Each described through hole is provided with 1 counter electrode, and described electrode is connected with lead on being located at described substrate;
But described electrode single-point gating.
2. electroporation device according to claim 1, it is characterized in that: described substrate is made by insulating material.
3. electroporation device according to claim 1 and 2, it is characterized in that: described through hole is circular, oval, square or rectangle.
4. according to each described electroporation device among the claim 1-3, it is characterized in that: described electrode is made by metal or conductive polymers.
5. according to each described electroporation device among the claim 1-4, it is characterized in that: described electrode is arranged on the sidewall of described through hole.
6. according to each described electroporation device among the claim 1-4, it is characterized in that: described electrode is arranged on the both sides up and down of described substrate, and the inwall of the middle part section of described electrode and described through hole is on the same perpendicular.
7. according to each described electroporation device among the claim 1-6, it is characterized in that: distance is 0.2～2mm between 2 battery lead plates of described electrode.
8. according to each described electroporation device among the claim 1-7, it is characterized in that: described substrate is printed circuit board (PCB).
9. the making method of each described electroporation device among the claim 1-8 is characterized in that: described electroporation device is made according to the PCB making method and is obtained.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310281908.XA CN103320319B (en) | 2013-07-05 | 2013-07-05 | Alternative high-throughput electroporation device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310281908.XA CN103320319B (en) | 2013-07-05 | 2013-07-05 | Alternative high-throughput electroporation device |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103320319A true CN103320319A (en) | 2013-09-25 |
| CN103320319B CN103320319B (en) | 2015-06-24 |
Family
ID=49189365
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310281908.XA Active CN103320319B (en) | 2013-07-05 | 2013-07-05 | Alternative high-throughput electroporation device |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103320319B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966090A (en) * | 2014-01-15 | 2014-08-06 | 苏州文曲生物微系统有限公司 | Detachable electroporation pore plate device |
| CN104403943A (en) * | 2014-12-02 | 2015-03-11 | 陈剑 | Electroporation cuvette and cell electrotransfection device with same |
| CN106591118A (en) * | 2017-01-05 | 2017-04-26 | 博奥生物集团有限公司 | Cell in-situ electroporation device and use method |
| CN104789468B (en) * | 2014-07-22 | 2017-10-20 | 奥克莱流体公司 | Particle screen selecting device |
| CN107988070A (en) * | 2017-06-13 | 2018-05-04 | 北京呈诺医学科技有限公司 | A kind of few cells electricity turns micro-fluidic chip, electricity turns sorter and application |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030070917A1 (en) * | 2001-10-11 | 2003-04-17 | Symyx Technologies, Inc. | Synthesis and characterization of materials for electrochemical cells |
| CN101343613A (en) * | 2008-08-22 | 2009-01-14 | 重庆大学 | Flexible high-pass cell electric amalgamation microelectrode array chip apparatus |
| CN101384697A (en) * | 2006-02-10 | 2009-03-11 | 生物辐射实验室股份有限公司 | Apparatus for high-throughput electroporation |
| CN101693875A (en) * | 2009-09-30 | 2010-04-14 | 重庆大学 | Cell electrofusion chip device based on columnar microelectrode array and electrofusion method |
| CN101870949A (en) * | 2009-11-10 | 2010-10-27 | 北京大学 | Electroporated chip and porous plate device base on electroporated chip |
| CN102132156A (en) * | 2008-08-01 | 2011-07-20 | 生物风险公司 | Devices and methods for the purification, isolation, desalting or buffer/solvent exchange of substances |
| CN102656260A (en) * | 2009-10-19 | 2012-09-05 | 瑞生生物技术有限公司 | Method, device and apparatus for inducing self-adjusting cell electroporation |
-
2013
- 2013-07-05 CN CN201310281908.XA patent/CN103320319B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030070917A1 (en) * | 2001-10-11 | 2003-04-17 | Symyx Technologies, Inc. | Synthesis and characterization of materials for electrochemical cells |
| CN101384697A (en) * | 2006-02-10 | 2009-03-11 | 生物辐射实验室股份有限公司 | Apparatus for high-throughput electroporation |
| CN102132156A (en) * | 2008-08-01 | 2011-07-20 | 生物风险公司 | Devices and methods for the purification, isolation, desalting or buffer/solvent exchange of substances |
| CN101343613A (en) * | 2008-08-22 | 2009-01-14 | 重庆大学 | Flexible high-pass cell electric amalgamation microelectrode array chip apparatus |
| CN101693875A (en) * | 2009-09-30 | 2010-04-14 | 重庆大学 | Cell electrofusion chip device based on columnar microelectrode array and electrofusion method |
| CN102656260A (en) * | 2009-10-19 | 2012-09-05 | 瑞生生物技术有限公司 | Method, device and apparatus for inducing self-adjusting cell electroporation |
| CN101870949A (en) * | 2009-11-10 | 2010-10-27 | 北京大学 | Electroporated chip and porous plate device base on electroporated chip |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966090A (en) * | 2014-01-15 | 2014-08-06 | 苏州文曲生物微系统有限公司 | Detachable electroporation pore plate device |
| CN104789468B (en) * | 2014-07-22 | 2017-10-20 | 奥克莱流体公司 | Particle screen selecting device |
| CN104403943A (en) * | 2014-12-02 | 2015-03-11 | 陈剑 | Electroporation cuvette and cell electrotransfection device with same |
| CN104403943B (en) * | 2014-12-02 | 2016-06-15 | 陈剑 | A kind of cell electroporation shocking by electricity pipe and there is electric shock pipe |
| CN106591118A (en) * | 2017-01-05 | 2017-04-26 | 博奥生物集团有限公司 | Cell in-situ electroporation device and use method |
| CN107988070A (en) * | 2017-06-13 | 2018-05-04 | 北京呈诺医学科技有限公司 | A kind of few cells electricity turns micro-fluidic chip, electricity turns sorter and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103320319B (en) | 2015-06-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103320319B (en) | Alternative high-throughput electroporation device | |
| CN101343613B (en) | Flexible high-pass cell electric amalgamation microelectrode array chip apparatus | |
| CN106591118B (en) | A kind of cell in-situ electroporation device and application method | |
| CN101693875B (en) | Cell electrofusion chip device based on columnar microelectrode array and electrofusion method | |
| CN101693874A (en) | Cell electrofusion chip device based on micro-chamber array structure | |
| CN101870949B (en) | Electroporated chip and porous plate device base on electroporated chip | |
| CN103903993A (en) | Diode lead wire welding assembly method and device | |
| CN207300964U (en) | A kind of multichannel microarray screen-printing electrochemical sensing device | |
| CN205940516U (en) | Electroplate and plate capability test circuit board deeply | |
| CN108303559A (en) | A kind of method and apparatus of sample-adding tracer and record | |
| Qu et al. | Somatic and stem cell pairing and fusion using a microfluidic array device | |
| CN203523138U (en) | Electroplating capability test module for HDI plate | |
| CN105543940A (en) | Device and method for promoting electroplating uniformity of VCP electroplating wire | |
| CN204434735U (en) | Electrolyzer fault pole plate locating and detecting device | |
| CN201002015Y (en) | Conic tooth shaped medium-barrier discharge plasma chemical reactor | |
| CN202913065U (en) | Novel spraying and copper-dissolving device | |
| CN203065488U (en) | Cell electro-fusion device | |
| CN101580797B (en) | Low-cost cellular positioning arrangement chip and application method | |
| CN102943037B (en) | Multi-hole cell culture dish structure | |
| CN202766645U (en) | Electrolysis phosphorization equipment | |
| CN201713591U (en) | Plating equipment for electrode of silicon solar battery plate | |
| CN210194005U (en) | Electrolysis device and electrolysis system | |
| CN103074212B (en) | Cell electrofusion device and method | |
| CN103966090B (en) | A kind of dismountable electroporation orifice fitting | |
| CN110158116A (en) | Electrolysis unit and electrolysis 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 | ||
| C53 | Correction of patent of invention or patent application | ||
| CB02 | Change of applicant information |
Address after: 102206 Beijing City, Changping District Life Science Park Road No. 18 Applicant after: CAPITALBIO CORPORATION Applicant after: Tsinghua University Address before: 102206 Beijing City, Changping District Life Science Park Road No. 18 Applicant before: Capitalbio Corporation Applicant before: Tsinghua University |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: APPLICANT; FROM: CAPITALBIO CORPORATION TO: CAPITALBIO CORPORATION CO., LTD. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |