CN101928666B - Flow type electroporation device and system - Google Patents
Flow type electroporation device and system Download PDFInfo
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- CN101928666B CN101928666B CN 201010242144 CN201010242144A CN101928666B CN 101928666 B CN101928666 B CN 101928666B CN 201010242144 CN201010242144 CN 201010242144 CN 201010242144 A CN201010242144 A CN 201010242144A CN 101928666 B CN101928666 B CN 101928666B
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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 flow type electroporation device and a system, and the system comprises the flow type electroporation device, a channel, an injection pump and a voltage source, wherein the flow type electroporation device comprises a substrate and electrodes which are made on the substrate and are arranged in parallel and in pairs, and each pair of the electrodes comprise an anode and a cathode which are oppositely arranged; the channel is arranged above the electrodes and limits fluid to flow; an initial end of the channel is provided with a plurality of inlet branch channels which converge into a main channel, a terminating end is provided with a plurality of outlet branch channels, and a plurality of head covers for fluid inlets and outlets are arranged above the channel; the injection pump is connected to inlets and outlets of the head covers in the flow type electroporation device and limits the flow speed of the fluid; and the voltage source is connected with the electrodes through electrical connectors and sets and produces pulse voltage. The flow type electroporation system utilizes the fluid channel and the connected injection pump to realize continuous flow of various suspensions in the fluid channel, thereby enabling the process that cells are subjected to electroporation to be continuous and realizing rapid treatment of a large number of samples.
Description
Technical field
The present invention relates to a kind of flow electroporation device, more specifically, the present invention relates to a kind of system that utilizes efficiently flow electroporation device cell to be carried out electroporation.
Background technology
Cytolemma is to be enclosed in pericellular thin film, is cell and the extraneous permeability barrier that carries out the selective substances exchange.Cytolemma makes cell become an independently living unit, and has a metastable interior environment.Some materials in the surrounding environment can pass through cytolemma, and other material is then not all right.Cell can absorb nutriment from surrounding environment by cytolemma, discharges meta-bolites, makes the transhipment of material reach equilibrium state.So the basic function of cytolemma is exactly to keep the relatively stable of the interior microenvironment of cell and carry out exchange of substance with external environment selectively.
Research is found, if cell is applied the electricity irritation of some strength and continues for some time, just can produce some micropores on the inducing cell film, the permeability of cell is strengthened, so-called cell electroporation (Electroporation) is exactly that phalangeal cell is under the effect that adds pulsed electrical field, form the biophysics process (Waver J.C. " Electroporation:A dramatic, nothermal electric field phenomenon " 1992) of instantaneous micropore on the cell membrane lipid bilayer.When cytolemma generation electroporation, its permeability and the instantaneous increase of membrane conductance meeting make hydrophilic molecules, DNA, protein, virion, drug particles etc. can not be entered cell by the molecule of cytolemma under normal circumstances.After removing electricity irritation at short notice, cytolemma can self-recovery, again becomes the selective permeation barrier.Compare with viral perforation with traditional chemistry perforation, because electroporation has without chemical pollution, can not cause permanent damage, efficient than advantages of higher to cell, have broad application prospects in fields such as biophysics, molecular biology, clinical medicine.
Although the mechanism of electroporation effect also imperfectly understands, cell electroporation is known in this article, comprises breaking of cell membrane lipid bilayer, causes forming temporary micropore at film, allows exogenous molecule by diffusing into cell.
In the prior art, mainly contain the process that three class methods are finished cell electroporation:
1 cell is positioned over a pair of between several millimeters to several centimetres the parallel pole.Make in the electric field of cell between electrode and be subject to electricity irritation, to realize the purpose of electroporation.(for example, United States Patent (USP) U.S.Pat.5389069)
2 use miniature needle electrode to penetrate in tissue or the cell shocks by electricity to cell, reaches the purpose of electroporation.(for example, United States Patent (USP) U.S.Pat.5389069; Chinese patent application, publication number CN 101020892A)
3 are placed on a chamber between the pair of parallel electrode, so that the aaerosol solution of cell is shocked by electricity when flowing in chamber.
(for example, United States Patent (USP) U.S.Pat.6773669; Chinese patent application, publication number: CN 1195997A) disclosed electroporation device and method major part are not suitable for a large amount of sample of processing in the prior art, are not suitable for continuous processing sample yet.That is to say that the electroporation chamber that obtains in the prior art all is that " static state " works, and after electroporation chamber handles a batch sample, need to clean to electroporation chamber, again add the processing such as cell that is:, could continue the next batch sample preparation.In disclosed technology, electroporation usually carries out in disposable single chamber test tube, and its maximum capacity that is used for electroporation is generally 1 milliliter.Process in the occasion of a large amount of samples at needs, this technology tedium, labour intensity is large.The researchist has invented the form of a plurality of electroporations chamber parallel connection on this basis, and this technology has its advantage, but can not solve the difficulty that is difficult to realize a large amount of samples of fast processing at all.
When the needs electroporation is processed a large amount of sample, also can adopt a kind of semicontinuous streaming system (Flow through system) (for example, United States Patent (USP) U.S.Pat.5676646), in this system, need the sample of electroporation to be injected into electroporation chamber, and apply Electroporation.Then, this electroporation chamber is found time and refill as required repeatedly so that a large amount of cells are carried out electroporation.In this system, because the distance between the electrode is far longer than the typical size (10 microns) of cell, so be difficult to accurately control the actual electric field that is applied on the cell usually in 10 millimeter.Simultaneously, in the electric field that two plate electrodes produce, (V is the voltage that is applied between the two-plate to electric field E=V/D, D is the distance between the two-plate), in traditional streaming electric perforating system of the distance B between pole plate large (10 millimeters), required voltage is usually up to thousands of volts.This has increased difficulty for the design power supply system, also is unfavorable for energy-conserving and environment-protective.Simultaneously, this system is not complete non-stop run, need to annotate to electroporation chamber-electroporation-find time-the again operation of filling-electroporation.This mode of operation has lowered efficient, can not realize real high-throughput operation.
By placing precision machined electrode in the inside of miniature fluid passage, can realize the accurate control to electric field distribution, thereby realize relatively high piercing efficiency and processing speed.Yet in this type systematic, electrode directly contacts with cell suspending liquid, cell is being carried out in the process of electroporation, and the Electrolysis of water can occur inevitably.What follow that the electrolysis of water produces is the violent change of a large amount of heat, bubble and cell suspending liquid pH value, thus more than these phenomenons homogeneity that can significantly reduce the survival rate of cell and destroy electric field in the runner reduce the piercing efficiency of cell.
It is present a kind of multi-layered fluid control techniques of widespread use in the equipment such as flow cytometer (Flow Cytometer) that hydrokinetics focuses on (Hydrodynamic Focusing) technology.According to principle of hydrodynamics, under typical low reynolds number (Reynolds Number) states such as miniature fluid passage, fluid presentation layer stream mode, can't mutually mix (J.B.Knight, " Hydrodynamic Focusing on a Silicon Chip:Mixing Nano liters in Microseconds ", 1998).Utilize this principle, can have a plurality of access roades and pool the miniature fluid passage of a main channel by design, different fluids is injected by different runners, thereby control the thickness of any layer fluid.Although the effect that hydrokinetics focuses on and implementation method are different and different with fluid channel design, but the hydrokinetics focusing technology is known in this article, comprises low reynolds number fluid in the miniature fluid passage, has the miniature fluid passage of a plurality of fluid intakes, controlled fluid layering thickness.
Summary of the invention
The present invention has overcome above-mentioned deficiency of the prior art, and one of purpose of the present invention has provided a kind of electroporation device, and this device has based on the miniature fluid passage of hydrokinetics focusing technology design and is placed on electrode in this passage; Another object of the present invention provides the complete flow electroporation system of a cover.
In order to reach first above-mentioned purpose, the technical scheme of flow electroporation device provided by the present invention is summarized as follows:
A kind of flow electroporation device comprises:
Substrate, and be produced on electrode on the substrate, described electrode is parallel and paired placement, every pair of electrode comprises anode and the negative electrode that is oppositely arranged;
The passage that places the limit fluid on the electrode to flow; Described passage initiating terminal has a plurality of entrance branch passages, and pools a main channel, finishes end and has a plurality of outlet branched bottoms, and described passage top arranges the top cover with a plurality of fluid intakes and outlet.
Described passage is made by insulating material.
Described insulating material is glass or silicon.
Described passage is made by PDMS or organic polymer.
Described passage width is 5 microns to 10 millimeters.
Described passage has 1 to 10 entrance branch passage at initiating terminal, has 1 to 10 outlet branched bottom at the end end.
Described substrate is made by insulating material or electro-conductive material covering surfaces insulating material.
Described insulating material is glass or silicon or plastics.
Described electrode is made by electro-conductive material.
Described electro-conductive material is gold.
Described electrode is parallel to the fluid channel and places in pairs.
Described top cover is made by insulating material or electro-conductive material covering surfaces insulating material.
Described insulating material is glass or silicon or plastics.
In order to reach second above-mentioned purpose, the technical scheme of flow electroporation provided by the present invention system is summarized as follows:
A kind of flow electroporation system comprises:
Flow electroporation device, comprising:
Substrate, and be produced on electrode on the substrate, described electrode is parallel and paired placement, every pair of electrode comprises anode and the negative electrode that is oppositely arranged;
The passage that places the limit fluid on the electrode to flow; Described passage initiating terminal has a plurality of entrance branch passages, and pools a main channel, finishes end and has a plurality of outlet branched bottoms, and described passage top arranges the top cover with a plurality of fluid intakes and outlet;
Syringe pump is connected to entrance and the outlet of top cover in the described flow electroporation device by pipeline, is used for the flow velocity of control fluid;
Voltage source by the electrical connector connection electrode, is used for setting and producing pulsed voltage.
The cell that this electric perforating system is used for convection cell carries out electroporation, described cell comprises zooblast or bacterium, and described zooblast comprises: primary cell, passage cell, neurocyte, endotheliocyte, epithelial cell, inoblast and myocyte.
When this electric perforating system was used for that cell carried out electroporation, the pulsed voltage of setting was 1～2000V, 0.05～20 millisecond of pulse width, 0.1～60 second recurrent interval, flow velocity 0～10 ml/min of fluid.
Compared with prior art, the invention has the beneficial effects as follows:
The flow electroporation system utilizes the fluid channel and the syringe pump that is connected is realized the continuous flow of cell suspending liquid in the fluid channel, thereby makes cell can be continued to carry out by the process of electroporation.So just can realize a large amount of samples of fast processing.Simultaneously, can significantly improve processing speed by the mode of integrated a plurality of flow electroporation devices in a flow electroporation system.
Under the condition that adopts the fine machining methods such as semiconductor etching, than published electroporation device and system, size and the distance between the electrode of electrode all can be dwindled a lot, simultaneously, the size of fluid channel also can narrow down to the degree that matches with electrode size.Like this, want much smaller voltage to realize the purpose of cell electroporation with regard to using with respect to published technology.Thereby reduce equipment cost and energy-conservation.
Utilize the hydrokinetics focusing technology, the present invention has realized the laminar flow of different liqs in the fluid channel.Use the electroporation damping fluid of conduction, cell suspending liquid and electrode are separated.In the present invention, the not direct and electrode contact of cell, thus the deleterious effect (comprising the change of bubble, heating and pH value etc.) that is produced by brine electrolysis of having avoided occurring in electrode region to the injury of cell, has improved the survival rate of cell greatly.Simultaneously, by improving the specific conductivity of electroporation damping fluid, can form the required strength of electric field of electroporation in the zone that stream of cells is crossed with the voltage of less
The interelectrode distance of dwindling has also been brought a significant advantage, is exactly higher electroporation efficiency.Because cell suspending liquid is inhomogeneous, interelectrode distance is less, just means that the inhomogeneous solution between the electrode is fewer, also with regard to the consistence of easier control electroporation conditions, reaches high electroporation efficiency.
The electrode that is parallel to the runner placement can produce a uniform electric field distribution in electric field, cell all flows through in this uniform electric field region, thereby obtains the controlled high efficiency cell electroporation of homogeneous.
In certain embodiments, adopt the transparent materials such as glass or polymkeric substance to make substrate, at this moment, just can come the variation of Real Time Observation cell in electroporation process by microscope.For stechiology research, this is significant advantage.
Description of drawings
Fig. 1 is the structural representation of flow electroporation device;
Fig. 2 is the phantom view along AA ' among Fig. 1 and BB ';
Fig. 3 is the decomposition view of flow electroporation device;
Fig. 4 is the synoptic diagram of fluid channel 4;
Fig. 5 is the synoptic diagram of electrode 6;
Fig. 6 is the each several part connection diagram of flow electroporation system;
Among the figure, the 1-top cover; 2-fluid channel entrance; The outlet of 3-fluid channel; The 4-fluid channel; The 5-substrate; The 6-electrode; Main channel in the 7-fluid channel; Branch's access road of 8-fluid channel initiating terminal; Another branch's entrance channel of 9-fluid channel initiating terminal; The cell branch access road of 10-fluid channel initiating terminal; A branch outlet passage of end is finished in the 11-fluid channel; Another branch outlet passage of end is finished in the 12-fluid channel; The cell branch outlet passage of end is finished in the 13-fluid channel; An electrode of 14-paired electrode centering; Another electrode of 15-paired electrode centering; A flow electroporation device in the 16-flow electroporation system; Another flow electroporation device in the 17-flow electroporation system; Voltage generator in the 18-flow electroporation system; Syringe pump in the 19-flow electroporation system; The 20-cable connection; 21-electroporation damping fluid inlet seal pipeline; 22-cell suspending liquid inlet seal pipeline.
Embodiment
Below in conjunction with the drawings and specific embodiments the present invention is described in further detail:
1 substrate:
Because substrate plays the support effect and play the insulation buffer action between electrode, so substrate need to be made by insulating material, perhaps cover insulation layer by non-insulating material and make.Can have any solid substrate that is fit to above condition to make according to substrate of the present invention, preferred substrate is that those can be cut into the substrate that requires specification by mold or machine.Particularly preferably be transparent glass or polymkeric substance, because in this case, can be by the state of microscope Real Time Observation cell.
In preferred embodiment shown in Figure 2, substrate adopts simple glass to make, long 8 centimetres, wide 2.5 centimetres, high 1 millimeter.The size of this substrate can be determined by real needs, and obtain the substrate of desired size by methods such as cuttings.
In other embodiments, baseplate material also can adopt silicon, pottery or covered the metal of insulation layer.Because the processing technology of these materials is known in the art, thereby those skilled in the art can adopt these materials easily in enforcement of the present invention.
2 electrodes
Electrode can be made by the mixture of suitable electro-conductive material or these materials.Preferred material is the good material of bio-compatibility, for example gold, titanium and be mixed with the PDMS (polydimethylsiloxane, a kind of polymkeric substance commonly used) of silver ions.When adopting multiple material (for example a kind of electro-conductive material (for example gold) is plated on the another kind of electro-conductive material (for example copper)), outermost preferred material is the good material of bio-compatibility.
In embodiment as shown in Figure 2, electrode materials is chromium and gold.At first sputter on substrate (this is the method for known metal refining in a kind of semiconducter process) a layer thickness is 0.1 micron chromium, and again sputter a layer thickness is 0.5 micron gold.Then whole substrate is carried out the electrode shape that photoetching (being the method for known making figure in the semiconducter process equally) obtains needs.Then erode unwanted part on gold layer and the chromium layer.Finally obtain required electrode.Certainly, the method for the processing metal well known in the art such as plating also can be used for making electrode.
Present embodiment adopts chromium and Jin Lai to make electrode, be because gold does not have bio-toxicity, and chromium is in order to increase the adhesion between gold and the glass.Under different demands, other electro-conductive material, for example aluminium, copper or conductive polymers can be used for making electrode.
Among the embodiment as shown in Figure 2, the height of electrode is 0.6 micron, this is to consider that the fluid channel on the electrode needs and substrate realization sealing, in the present embodiment, this sealing is to realize by bonding (the known method that two kinds of materials are closely linked in the semiconducter process), so the height of electrode is unsuitable excessive to hinder sealing.By what experiment was determined be: when adopting bonded seal, electrode height all is suitable from 0.1 micron to 10 microns.When adopting other sealing means such as hot pressing, tackiness agent is bonding, then there is not the restriction of electrode height, so electrode height can be determined according to different demands and working method by those skilled in the art.
Among the embodiment as shown in Figure 2, the width of electrode is 100 microns, and the distance between the electrode is 500 microns, and the electric field that the electrode of this size produces is the most preferably electric field of human embryonic kidney cell's electroporation conditions.When needs carry out electroporation to other cell, can those skilled in the art select voluntarily suitable electrode width and interelectrode distance.
3 fluid channels
The effect of fluid channel is flowing of constrain fluids, makes cell suspending liquid by the specific track electrode region of flowing through, and is subject to the effect of electric field, thereby finishes the process of cell electroporation.The fluid channel can be made by any insulating material that is fit to shape, or at surface coverage one layer insulating of non-insulating material.Preferably, the material of fluid channel should be the good material of bio-compatibility.
Among the embodiment as shown in Figure 2, adopt PDMS (polydimethylsiloxane) to make the fluid channel, this is a kind of known bio-compatible polymer materials that is easy to machine-shaping that is usually used in biological devices field.Described PDMS fluid channel is that the method by mold obtains, and uses silicon chip to serve as mould.At first utilize photoetching in general semiconductor processing with silicon chip, the techniques such as corrosion are produced corresponding groove, then the PDMS solution-cast of liquid state are advanced the groove on the silicon chip, with its demoulding taking-up, have just obtained the fluid channel of moulding after it solidifies.Certainly, the fluid channel also can be adopted the material such as glass, plastics, other polymkeric substance and be used working methods more known in the field to process, and for example uses laser ablation glass to form the fluid channel.
Among the embodiment as shown in Figure 2, the fluid channel links together by method and the substrate of bonding.This is to consider between PDMS and the glass very easily to form firmly sealing by bonding, and the bonding process between PDMS and the glass is also very simple, then the oxygen plasma treatment that only surface of PDMS and glass need to be excited with high pressure applies certain pressure it is compressed about 10 seconds (this is in order to excite the dangling bonds of PDMS and glass surface).Leave standstill and namely form reliably firmly sealing after 24 hours.For other different application, method of attachment well known in the art also all is suitable for, and for example uses tackiness agent that fluid channel and base plate seals are linked together.
As shown in Figure 4, a plurality of entrance and exits in the fluid channel, have been designed to realize the hydrokinetics focusing to cell suspending liquid.The initiating terminal of fluid channel has 3 entrance branch passages and is used for passing into different fluids, and the end end has 3 outlet branched bottoms and flows out for different fluids.All branched bottoms pool a main channel.The width of entrance branch passage is respectively 1.2 millimeters, 0.6 millimeter, 1.2 millimeters; The width of exit passageway is respectively 0.5 millimeter, 0.2 millimeter, 0.5 millimeter; The width of sprue is 1.2 millimeters, and length is 30 millimeters.The height of all runners all is 80 microns.The same with the size design of electrode, this packet size also is the most preferably condition that arranges for human embryonic kidney cell's electroporation.In other is used, this area researchist can arrange size voluntarily to meet the different needs, such as in the preferred embodiment aspect this, main fluid passageway is arranged to linear, so that the flow direction of cell suspending liquid is perpendicular to direction of an electric field, thereby make cell be subject to the electricity irritation of appropriate time.According to the requirement of different cells, in further embodiments, main fluid passageway is arranged to the coil shape of multiturn, prolongs the time that cell passes through in runner, so that cell is subject to the electric field action of longer time.In other some embodiment, main fluid passageway can be arranged as annular, to adapt to the special requirement such as cell electrophoresis separation.In miniature fluid passage described herein, it all is feasible that the main fluid passageway width is 5 microns to 10 millimeters, and simultaneously, 1 to 10 entrance branch passage and 1 to 10 outlet branched bottom also all are feasible.
4 top covers
The effect of top cover is by jointly realizing constraint to cell suspending liquid with being tightly connected of fluid channel, and provides access and export for passage.Any insulating material or the solid material that has covered insulating material can be used for making top cover.Top cover can use the material identical with the fluid channel and and make together the fluid channel and simultaneously once shaped.
The structure of flow electroporation device comprises: top cover 1 as shown in Figure 1; Fluid channel entrance 2 and outlet 3; The fluid channel 4 of top cover below (invisible concrete runner is arranged among this figure); The electrode 6 (invisible concrete arrangement of electrodes among this figure) of runner below; The substrate 5 of electrode below.In order more clearly to find out the structure of top cover, sectional view as shown in Figure 2 can be found out the structure of top cover, and the glass that can adopt laser to beat via hole is made top cover.Identical with aforementioned reason, this be for utilize glass to cut easily, punch and be easy to and fluid channel that the PDMS material is made between form and firmly be tightly connected.In other is used, the insulating material that can adopt plastics, polymkeric substance etc. to be easy to process, the metals such as stainless steel of insulating material that also can adopt surface coverage.Sectional view as shown in Figure 2 can be found out the structure of fluid channel and the part-structure of electrode.
The all parts of whole fluid electroporation device and installation, as shown in Figure 3, substrate 5 is positioned at the lowest layer, electrode 6 is put in its top, fluid channel 4 is placed on above the electrode 6, and is bonded together with substrate 5, be manufactured with top cover 1 above the fluid channel 4, and leave fluid channel entrance 2 and outlet 3.
The design of fluid channel as shown in Figure 4, the cell branch access road 10 of the electroporation damping fluid branch access road 8,9 of both sides and central authorities converges and forms main fluid passageway 7.The main fluid passageway fork becomes the electroporation damping fluid branch outlet passage 11,12 of both sides and the cell branch outlet passage 13 of central authorities.
The set-up mode of electrode as shown in Figure 5, electrode 14 and 15 is placed in pairs and is drawn outside the runner in the fluid channel parallel beneath.5 flow electroporation system and experimental techniques
As shown in Figure 6, the flow electroporation system is except two flow electroporation devices 16,17, and whole system also needs a voltage source 18 and the cable connection 20 that matches with it, a syringe pump 19 and the sealing-duct 21 that matches with it, 22 to realize the controlled flowing of cell suspending liquid.Described voltage source, cable connection, syringe pump and sealing-duct all are equipment disclosed and that those skilled in the art obtain easily.
In system as shown in Figure 6, adopt voltage source to be output as positive and negative 100 volts voltage source, and the controlled flow velocity of syringe pump that adopts is 0 to 10 milliliter of per minute.This also is the most preferably condition that arranges for human embryonic kidney cell's electroporation.Those skilled in the art can select like device voluntarily.As required, voltage is that 1 to 2000 volt, pulse width are that 0.05 to 20 millisecond, recurrent interval are that the electricimpulse of 0.4-60 second all is feasible.
In the native system parallel join two flow electroporation devices, those skilled in the art can select 1 to 1000 flow electroporation device parallel join to realize higher processing speed as required.
This electroporation device can electroporation zooblast and bacterium, preferred clone has: HEK293 (human embryonic kidney cell), Hela (human cervical carcinoma cell), HepG2 (human liver cancer cell), Neuro-2A (mouse brain neuroma cell), Jurkat (human lymphoma cell), HL60 (the former myelocyte of people) and MDCK (dog renal epithelial cell); Preferred primary cell has: HUVEC (Human umbilical vein endothelial cells), DRG (the rat back of the body is wanted ganglion cell), T lymphocyte and human embryo stem cell; Preferred bacterium has intestinal bacteria, pasteurellosis bacillus.Those skilled in the art can select different cells as required.
This electric perforating system can make by the method for electroporation multiple different macromolecular compound enter cell, comprises nucleic acid (plasmid DNA, linear DNA, siRNA, antisense nucleic acid), protein (peptide section, antibody).Preferred plasmid is carrier for expression of eukaryon (pEGFP-C3), and those skilled in the art can select various eucaryons voluntarily, and prokaryotic expression carrier uses.
Native system can significantly improve the sample preparation speed of the existing electroporation technology in this area, realizes high-throughput electroporation.
Voltage source in the system can provide to different cells the voltage of different wave, in system shown in Figure 6, preferably adopts square-wave pulse.Those skilled in the art can select suitable voltage source output according to different cells.
The damping fluid of using in the electroporation experiment is taken from by KCl (Repone K) KH
2PO
4(potassium primary phosphate), K
2HPO
4(dipotassium hydrogen phosphate), the group that carbohydrate and water form.For embodiment shown in Figure 2, preferred buffer formulation is: contain KCl (15-50 mmole) in 1000 milliliters of damping fluids, KH
2PO
4(0.1-2 mmole), K
2HPO
4(0.1-2 mmole), inositol (20-60 mmole).Those skilled in the art can adjust the concentration of each component to obtain the highest piercing efficiency according to the difference of electroporation of cells.
6 concrete manufacturing steps
Successfully produced Apparatus and system of the present invention by the different manufacture craft of following two covers.Provide concrete making method and be in order to help those skilled in the art to understand manufacture method of the present invention, and be not the material to device of the present invention, size and manufacture method are made restriction.
Making method A:
Adopt semiconductor fabrication process 4 inches sheet glass commonly used.The chromium metal level of sputter 0.1 micron thickness on sheet glass, again the gold layer of sputter 0.5 micron thickness on the chromium metal level.Sheet glass is carried out photoetching, produce the shape of required electrode, and then use liquor kalii iodide acid gilding layer, use ceric ammonium nitrate solution corrosion chromium layer.Then sheet glass is pressed long 8 centimetres, wide 2.5 centimetres shape is cut with emery wheel.So just obtained substrate and on electrode.Adopt 4 inches silicon chips of N-type, after making the planeform of runner by lithography, use semi-conductor ICP dry etching (induction plasma etching, namely the high energy plasma with sulfur hexafluoride and tetrafluoro-methane comes etch silicon) commonly used to go out 80 microns dark grooves.Liquid PDMS is poured in the groove, solidify the rear demoulding until it and take out.By long 7.5 centimetres, wide 2 centimetres shape has so just obtained fluid channel and corresponding top cover with blade cuts and in place punching with it.With the surface of glass substrate and surface, PDMS fluid channel with oxygen plasma treatment after, be close together and exert pressure, left standstill 24 hours, namely obtain flow electroporation device.
Use two flow electroporation devices, use common plastics tube to connect the fluid intake of vacuum pump and two flow electroporation devices, between use the ultraviolet curing adhesive sealing.Carry out drawing of cable by ultrasonic welding on the gold electrode that device substrate exposes, the cable that two flow electroporation devices are drawn is connected on the voltage source simultaneously, can finish building of a whole set of flow electroporation system.
Making method B:
Adopt semiconductor fabrication process 4 inches N-type silicon chips commonly used.The chromium metal level of sputter 0.1 micron thickness on silicon chip is electroplated the gold layer of 5 micron thickness again at the chromium metal level.Silicon chip is carried out photoetching, produce the shape of required electrode, and then use liquor kalii iodide acid gilding layer, use ceric ammonium nitrate solution corrosion chromium layer.Then silicon chip is pressed long 8 centimetres, wide 2.5 centimetres shape is cut with emery wheel.So just obtained substrate and on electrode.Adopt common glass sheet, 1 millimeter of thickness uses the laser fluid channel of sizing out thereon.By long 7.5 centimetres, wide 2 centimetres shape is cut with emery wheel, has so just obtained the fluid channel with sheet glass.Adopt common polytetrafluoroethylplastic plastic, use laser boring as fluid inlet and outlet.By long 7.5 centimetres, wide 2 centimetres shape is cut with emery wheel with plastic sheet.With ultraviolet curing adhesive silicon substrate, glass flow passage and plastic roof are bonded together, namely obtain flow electroporation device.
Use two flow electroporation devices, use common plastics tube to connect the fluid intake of vacuum pump and two flow electroporation devices, between use the ultraviolet curing adhesive sealing.Carry out drawing of cable by ultrasonic welding on the gold electrode that device substrate exposes, the cable that two flow electroporation devices are drawn is connected on the voltage source simultaneously, can finish building of a whole set of flow electroporation system.
7 concrete electroporation methods
Device of the present invention and system have been carried out successful electroporation experiment by following electroporation method.Provide concrete electroporation method and be in order to help those skilled in the art to understand the using method of electroporation device, and be not that the scope of application of device of the present invention is made restriction.
Collection is in the HEK293 (human embryonic kidney cell) of logarithmic phase, centrifugal 5 minutes with 800 rev/mins of rotating speeds, abandoning supernatant, with electroporation damping fluid (chloride containing potassium 15 mmoles in per 100 milliliters, potassium primary phosphate 0.3 mmole, dipotassium hydrogen phosphate 0.85 mmole, inositol 56 mmoles) re-suspended cell, so that the density of cell is 2X10
3Individual/microlitre, adding need to enter by electroporation the plasmid pEGFP-C3 of cell, and making the concentration of plasmid is 20 ug/ml, softly mixes.Cell suspension 100 microlitres that mix are added in the microsyringe (the high dove board in Shanghai, capacity is 250 microlitres), and microsyringe is by hose connection on the cell branch access road of flow electroporation device fluid channel central authorities.Microsyringe is installed on the syringe pump, regulates syringe pump, so that flow velocity is 4 mul/min.Electroporation damping fluid 250 microlitres that do not contain cell are added (the high dove board in Shanghai in another microsyringe, capacity is 250 microlitres), this microsyringe is by hose connection on the electroporation damping fluid branch access road of both sides, flow electroporation device fluid channel.This microsyringe is installed on another syringe pump, regulates syringe pump, so that flow velocity is 18 mul/min.Can in main fluid passageway, observe cell suspending liquid by the about 2.2 millimeters layer fluid of successful boil down to width, and be under the stable laminar flow regime.When cell suspension begins to flow, apply electric pulse stimulation in passage.The condition of electricity irritation is: voltage 80V, pulse width 0.2ms, 2 seconds recurrent intervals.
After electroporation finishes, by flexible pipe cell suspending liquid is introduced in 96 orifice plates and to be cultivated.Culture condition: 37 ℃ of temperature, gas concentration lwevel 5%.At the fluorescence microscopy Microscopic observation, the cell that can successfully observe more than 90% has green fluorescence to express, and shows that the transfection efficiency of electroporation reaches more than 90% after 24 hours.After this use PI (Propidium Iodide, propidium iodide) that cell is dyeed, observe and be less than 20% cell and present redness, show that cell survival rate reaches more than 80%.
Claims (16)
1. flow electroporation device comprises:
Substrate, and be produced on electrode on the substrate, described electrode is parallel and paired placement, every pair of electrode comprises anode and the negative electrode that is oppositely arranged;
The passage that places the limit fluid on the electrode to flow; Described passage initiating terminal is provided with cell passage and is positioned at the damping fluid passage of cell passage both sides, and pools a main channel, finishes end and has a plurality of outlet branched bottoms, and described passage top arranges the top cover with a plurality of fluid intakes and outlet;
The width of described main channel is 5 microns to 10 millimeters; Described electrode is positioned at the damping fluid passage.
2. electroporation device as claimed in claim 1 is characterized in that, described passage is made by insulating material.
3. electroporation device as claimed in claim 2 is characterized in that, described insulating material is glass or silicon.
4. electroporation device as claimed in claim 1 is characterized in that, described passage is made by organic polymer.
5. electroporation device as claimed in claim 4 is characterized in that, described passage is made by PDMS.
6. electroporation device as claimed in claim 1 is characterized in that, described passage has 1 to 10 entrance branch passage at initiating terminal, has 1 to 10 outlet branched bottom at the end end.
7. electroporation device as claimed in claim 1 is characterized in that, described substrate is made by insulating material or electro-conductive material covering surfaces insulating material.
8. electroporation device as claimed in claim 7 is characterized in that, described insulating material is glass or silicon or plastics.
9. electroporation device as claimed in claim 1 is characterized in that, described electrode is made by electro-conductive material.
10. electroporation device as claimed in claim 9 is characterized in that, described electro-conductive material is gold.
11. electroporation device as claimed in claim 1 is characterized in that, described electrode is parallel to the fluid channel and places in pairs.
12. electroporation device as claimed in claim 1 is characterized in that, described top cover has 1 to 10 entrance, 1 to 10 outlet.
13. electroporation device as claimed in claim 1 is characterized in that, described top cover is made by insulating material or electro-conductive material covering surfaces insulating material, and adopts identical material and simultaneously once shaped with the fluid channel.
14. a flow electroporation system is characterized in that, comprising:
Flow electroporation device comprises:
Substrate, and be produced on electrode on the substrate, described electrode is parallel and paired placement, every pair of electrode comprises anode and the negative electrode that is oppositely arranged;
The passage that places the limit fluid on the electrode to flow; Described passage initiating terminal is provided with cell passage and is positioned at the damping fluid passage of cell passage both sides, and pools a main channel, finishes end and has a plurality of outlet branched bottoms, and described passage top arranges the top cover with a plurality of fluid intakes and outlet;
The width of described main channel is 5 microns to 10 millimeters; Described electrode is positioned at the damping fluid passage;
Syringe pump is connected to entrance and the outlet of top cover in the described flow electroporation device by pipeline, is used for the flow velocity of control fluid;
Voltage source by the electrical connector connection electrode, is used for setting and producing pulsed voltage.
15. electric perforating system as claimed in claim 14 is characterized in that, this system's parallel join 1 to 1000 flow electroporation device.
16. electric perforating system as claimed in claim 14, it is characterized in that, when this electric perforating system is used for that cell carried out electroporation, the pulsed voltage of setting is 1～2000 volt, 0.05～20 millisecond of pulse width, 0.1～60 second recurrent interval, rate of flow of fluid 0～10 ml/min of each fluid channel entrance.
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| EP3440186A4 (en) * | 2016-04-04 | 2019-12-11 | Cytequest, Inc. | System, device and method for electroporation of cells |
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| CN102367414B (en) * | 2011-06-04 | 2014-04-09 | 苏州文曲生物微系统有限公司 | Method for conveying nucleic acid to vivo tissue cells by flexible electrode chip |
| KR20130061831A (en) * | 2011-12-02 | 2013-06-12 | (주)엘지하우시스 | Sliding system window for higher stories having drain hole cap |
| KR102036623B1 (en) * | 2013-02-20 | 2019-11-26 | 지엔 첸 | Methods and Devices for Electroporation |
| CN103966090B (en) * | 2014-01-15 | 2016-04-06 | 苏州壹达生物科技有限公司 | A kind of dismountable electroporation orifice fitting |
| KR102428463B1 (en) * | 2014-12-28 | 2022-08-03 | 주식회사 펨토바이오메드 | Device for Putting Material into Cell |
| CN116987589A (en) * | 2017-10-19 | 2023-11-03 | 苏州壹达生物科技有限公司 | Flow electroporation device |
| WO2019194799A1 (en) | 2018-04-04 | 2019-10-10 | Hewlett-Packard Development Company, L.P. | Microfluidic cellular membrane modification devices |
| DE112019003498T5 (en) * | 2018-07-09 | 2021-04-08 | NanoCav, LLC | MICROFLOW ELECTROPORATION DEVICES AND METHODS OF CELL TRANSFECTION |
| USD1016324S1 (en) | 2020-07-08 | 2024-02-27 | NanoCav, LLC | Biological cell processing chip |
| CN114686371A (en) * | 2020-12-29 | 2022-07-01 | 苏州壹达生物科技有限公司 | Flow type electrotransfection device |
| TWI806062B (en) * | 2021-06-07 | 2023-06-21 | 新加坡商克雷多生醫有限公司 | Method and device for opening the external layer structure of cells using laser |
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