CN103966090B - A kind of dismountable electroporation orifice fitting - Google Patents

Abstract

The invention discloses a kind of based on the dismountable electroporation device of biochip, comprising: independently electroporation chip module, carrier board, PCB; Described electroporation chip module comprises the carrying substrate (1) of electrode, electrode (2), chip wiring contact (3), cell culture chamber (4), described electrode is annular, every two is a pair, comprise the anode (5) and negative electrode (6) that are oppositely arranged, and mutually nested between negative electrode and anode.Independently electroporation chip module electroporation of the present invention is effective, without chemical pollution, damage, efficiency will be caused high, simple in actually operating, convenient to cell.Use device of the present invention in once testing, the cell culture chamber of differing capacities can be selected flexibly, process while achieving mass disposal cell and single adherent cell, and treated cell is without the need to transfer, directly carries out follow-up cultivation at cell culture chamber.

Description

A kind of dismountable electroporation orifice fitting

Technical field

The present invention relates to a kind of novel dismountable electroporation chip, more specifically, the present invention relates to a kind of device process cell utilizing cell electroporation, and directly carry out the method for cell cultures.

Background technology

Cytolemma is enclosed in pericellular thin film, is cell and the extraneous permeability barrier carrying out selective substances exchange.Cytolemma makes cell become an independently living unit, and has a metastable environment.Some materials in surrounding environment can pass through cytolemma, and other material is then not all right.Cell can absorb nutriment by cytolemma from surrounding environment, discharges meta-bolites, makes the transhipment of material reach equilibrium state.So the basic function of cytolemma is exactly the relatively stable of microenvironment in maintenance cell and carries out exchange of substance with external environment selectively.

Research finds, if apply the electricity irritation of some strength to cell and continue for some time, just inducing cell film can produce some micropores, the permeability of cell is strengthened, so-called cell electroporation (Electroporation) is exactly that phalangeal cell is under the effect of additional pulsed electrical field, cell membrane lipid bilayer is formed the biophysics process (WaverJ.C. " Electroporation:Adramatic, nothermalelectricfieldphenomenon " 1992) of instantaneous micropore.When cytolemma generation electroporation, its permeability and membrane conductance can instantaneous increases, make hydrophilic molecules, DNA, protein, virion, drug particles etc. not entered cell by the molecule of cytolemma under normal circumstances.After removing electricity irritation at short notice, cytolemma can self-recovery, again becomes selective permeation barrier.Compare with viral perforation with traditional chemical poration, because electroporation has without chemical pollution, can not cause permanent damage, efficiency comparatively 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 on film, form temporary micropore, allows exogenous molecule by diffusing into cell.

In prior art, mainly contain three class methods to complete the process of cell electroporation:

1, cell is positioned over a pair between the parallel pole of several millimeters to several centimetres.Make to be subject to electricity irritation in cell electric field in-between the electrodes, to realize the object of electroporation.(such as, United States Patent (USP) U.S.Pat.5389069)

2, use needle electrode to penetrate in tissue or cell to shock by electricity to cell, reach the object of electroporation.(such as, United States Patent (USP) U.S.Pat.5389069; Chinese patent application, publication number CN101020892A)

3, a chamber is placed between pair of parallel electrode, is shocked by electricity while the aaerosol solution of cell is flowed in the chamber.(such as, United States Patent (USP) U.S.Pat.6773669; Chinese patent application, publication number: CN1195997A)

In the prior art in disclosed system, the distance between electrode, usually in 10 millimeter, is far longer than the typical size (20 microns) of cell, so be difficult to accurately control the actual electric field be applied on cell.Simultaneously, in the electric field that two plate electrodes produce, (V is the voltage be applied between two-plate to electric field E=V/D, D is the distance between two-plate), in the conventional electroporation system of the distance D between pole plate comparatively large (being greater than 10 millimeters), required voltage is usually up to several thousand volts.This adds difficulty to design power supply system, is also unfavorable for energy-conserving and environment-protective.

And disclosed electroporation device and method are not all suitable for a large amount of sample of process in the prior art, be not suitable for continuous processing sample yet.That is, the electroporation chamber obtained in prior art all " static state " works, that is: after electroporation chamber processes a batch sample, need to clean electroporation chamber, the process such as culturing cell again, could continue next batch sample preparation.In disclosed technology, electroporation usually carries out in disposable single chamber test tube, and its maximum capacity for electroporation is generally 1 milliliter.Needing to process in the occasion of a large amount of sample, this technology tedium, labour intensity is large.

In order to overcome above-mentioned defect, the instrument of cell electroporation disclosed in prior art comprises:

The biochip electroporation apparatus that EP2004000789 relates to and applying in comprehensive single cell electroporation.This biochip is for the treatment of other electroporation of cell adhering to any kind of matrix, different unicellular to realize processing in same culture environment.

A kind of electroporation chip that CN101870949A relates to and the porous plate device based on electroporation chip.Adopt the structure that porous plate is separated up and down with chip, porous plate adopts the transparent material such as glass or polymkeric substance to make, the change of microscope Real Time Observation cell easy to use in electroporation process.

Above-mentioned patent concentrates on the application using electroporation chip process cell, but the size of the porous plate of electroporation is all fixing, the cell concentration adjustment that can not process as required, the cultivation of cell and electroporation separately carry out, in the process of cultivate the cell transfer through electroporation, the interference of other factors can reduce the working efficiency of electroporation, and porous plate is an entirety, sometimes do not need holes all on porous plate to process in practical application, waste the space of porous plate.And electrode is also an overall alignment, after forming electrical connection, electrode integral all can form electric field, and when not needing to process corresponding porous plate, base wastes the electric field that porous plate is formed, also loss work-ing life of electrode.

The present invention is in order to overcome above-mentioned defect, the technical problem mainly solved is to provide and a kind of provides customized type demand for cell electroporation, general row is strong, electroporation is effective, without chemical pollution, damage, efficiency will be caused high to cell, independently, dismountable, the electroporation device based on micro-electrode chip that need not shift cultivation, can adjust cell cultures chamber vol flexibly.

Summary of the invention

Instant invention overcomes above-mentioned deficiency of the prior art, an object of the present invention there is provided a kind of dismountable electroporation device, and it has independently dismountable electroporation chip module; Another object of the present invention is to provide a kind of direct cultivation and observes the cell after electroporation process.

To achieve the above object, the technical scheme of dismountable electroporation device provided by the present invention is summarized as follows:

A kind of based on the dismountable electroporation device of biochip, comprising: independently electroporation chip module, carrier board, PCB; It is characterized in that,

Described electroporation chip module comprises the carrying substrate of electrode, electrode, chip wiring contact, cell culture chamber, and described electrode be annular, and every two is a pair, comprises the anode and negative electrode that are oppositely arranged, and mutually nested between negative electrode and anode;

Described carrier board comprises multiple lower deep gouge, the size of each lower deep gouge is slightly larger than the size of described chip substrate, to make, described electroporation chip module is dismountable to be fixed in lower deep gouge, described lower deep gouge has the rim openings of side at least, electrical connector is connected to form, with pcb board compact siro spinning technology bottom carrier board for the contact in chip wiring contact and PCB;

Described PCB arranges multiple contact and circuit, and contact is led to outside connecting terminal by circuit, and electricimpulse, as the parts be connected with external electrical, is incorporated in chip by described outside connecting terminal.

Described substrate is made up of insulating material.

Described substrate is the metal covering insulating material.

Described insulating material is glass or silicon.

Described electrode width is 1 micron to 1 millimeter.

Described electrode is made up of metal or conductive polymers.

Described electrode shape is annular or square.

Described electrode is arranged in the surface of substrate or embeds substrate.

Described electrode, often pair of electrode connects the zonal control realizing electric field respectively.

All anodes of described electrode link together, and all negative electrodes link together simultaneously, control while realizing electric field.

Described cell culture chamber is connected with substrates into intimate.

The size of described cell culture chamber is suitable with the size of chip electrode, can process the size of the amount of capacity adjust size of cell as required.

Only cultivate a kind of cell in described cell culture chamber, or cultivate various kinds of cell in a cell culture chamber.

The chamber vol of described cell culture chamber is between 20 microlitres to 10 milliliter, preferably between 10 microlitres to 1 milliliter, preferably between 1 milliliter to 10 milliliters.

Under described carrier board, the height of deep gouge is not less than the thickness of chip substrate, and preferably, the height of lower deep gouge equals the thickness of chip substrate and the thickness sum of pcb board.

Described carrier board comprises 6,8,16,24,48,96 lower deep gouges.

The direction of the sinking channel opening of described carrier board is towards side, carrier board edge.

The lower deep gouge of described carrier board can be any shape, as being square or circle, is arranged to multiple row and column.

Described carrier board is transparent, one or more compositions in preferred carrier board material comprises glass, plastics, pottery and metal.

The preferred technical scheme of described electroporation device is:

A kind of based on the dismountable electroporation device of biochip, comprising: independently electroporation chip module, PCB; It is characterized in that,

Described electroporation chip module comprises the carrying substrate of electrode, electrode, chip wiring contact, cell culture chamber, and described electrode be annular, and every two is a pair, comprises the anode and negative electrode that are oppositely arranged, and mutually nested between negative electrode and anode; Described PCB comprises multiple lower deep gouge, the size of each lower deep gouge is slightly larger than the size of described chip substrate, to make, described electroporation chip module is dismountable to be fixed in lower deep gouge, the height of lower deep gouge is suitable with the height of chip substrate, described lower deep gouge has at least the edge of side to arrange multiple contact, make to form electrical connector with chip wiring contact, contact is led to outside connecting terminal by circuit, electricimpulse, as the parts be connected with external electrical, is incorporated in chip by described outside connecting terminal.

In order to reach another above-mentioned object, a kind of electroporation of cells method and technology scheme provided by the present invention is summarized as follows:

A kind of electroporation of cells method, is characterized in that, use above-mentioned based on biochip dismountable electroporation device process cell.

Described cell comprises human body and zooblast or bacterium.

When carrying out electroporation to cell, the pulsed voltage of setting is 10-2000 volt, pulse width 0.05-20 millisecond, pulse number 1-100 time, recurrent interval 0.1-60 second.

Electroporation device described in the invention can be common with this area electroporation apparatus support the use.

Compared with prior art, useful effect is in the present invention:

1, in electroporation chip module of the present invention, the design of the capacity of adjustable cell culture chamber brings a significant advantage, user can according to the amount oneself using cell, the cell culture chamber of flexible selection differing capacities, achieve the mode that different gradient concentration processes simultaneously, easy to use, flexible, efficient.

2, according to dismountable electroporation chip module, cell after electroporation process can together be cultivated together with electroporation chip module by researchist, without the need to cell is shifted, reduce the interference of other factors in cell transfer process, for stechiology research, there is the advantage of highly significant.

3, the general row of electroporation device of the present invention is strong, and electroporation is effective, without chemical pollution, damage, efficiency will be caused high to cell.

4, PCB edge of the present invention can arrange screw thread, electroporation device of the present invention and common electroporation apparatus can be made to support the use, apply extensively easy.

5, in certain embodiments, adopt the transparent material such as glass or polymkeric substance to make substrate, now, just can carry out the change of Real Time Observation cell in electroporation process by microscope.For stechiology research, this is significant advantage.In the middle of preferred embodiment, adopt material that the bio-compatibility such as glass is good as substrate, can make cell attachment on substrate, carry out electroporation more afterwards.In other words, cell culture chamber of the present invention, both can cell under electroporation adhered state, also can cell under electroporation suspended state.This elasticity, for stechiology research, is also significant advantage.

Accompanying drawing explanation

In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing used required in describing embodiment is briefly described, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings, wherein:

Fig. 1 is independently electroporation chip modular structure schematic diagram;

Fig. 2 is the schematic diagram of single electroporation chip electrode;

Fig. 3 is the Facad structure schematic diagram of device;

Fig. 4 is the bottom construction schematic diagram of device;

Fig. 5 is the schematic diagram installing electroporation chip;

Fig. 6 is device electrical connection schematic diagram.

Wherein, substrate 1; Electrode 2; Chip wiring contact 3; Cell culture chamber 4; ; 5-anode; 6-negative electrode; 7-carrier board; Deep gouge under 8-; 9-sinking channel opening edge; 10-PCB circuit card; 11-PCB circuit card wiring contact; 12-chip wiring contact slot; 13-chip wiring contact is advancing to the position anterior diastema; 14-chip wiring contact is advancing to the position post gap; The outside connecting terminal of 15-; 16-electrical connector; 17-voltage source.

Embodiment

Be clearly and completely described to the technical scheme in inventive embodiments below, obviously, described embodiment is only a part of embodiment of the present invention, instead of whole embodiments.Based on embodiments of the invention, those of ordinary skill in the art, not making other embodiments all obtained under creative work prerequisite, belong to the scope of protection of the invention.

Embodiment:

Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail:

1 substrate:

Because substrate plays support effect and plays insulation buffer action in-between the electrodes, so substrate needs to be made up of insulating material, or be made up of non-insulating material covering insulation layer.Can be made up of any solid substrate being applicable to above condition according to substrate of the present invention, preferred substrate is that those can be cut into by mold or machine the substrate requiring specification.Particularly preferably be transparent glass or polymkeric substance, because in this case, can by the state of microscope Real Time Observation cell.As shown in Figure 1, independently dismountable bio-chip module comprises: substrate 1; Electrode 2; Chip wiring contact 3; Cell culture chamber 4.

In the preferred embodiment shown in Fig. 1, substrate adopts simple glass to make, and the size of this substrate can be determined by real needs, and obtains the substrate of desired size by methods such as cuttings.

In other embodiments, baseplate material also can adopt silicon, pottery or cover the metal of insulation layer.Processing technology due to these materials is known in the art, and thus those skilled in the art can adopt these materials easily in enforcement of the present invention.

2 electrodes

Electrode can be made up of the mixture of suitable electro-conductive material or these materials.Preferred material is the material that bio-compatibility is good, such as gold, titanium and be mixed with the PDMS (polydimethylsiloxane, polymkeric substance conventional in a kind of bio-science) of silver ions.When adopting multiple material (such as a kind of electro-conductive material (such as gold) is plated on another kind of electro-conductive material (such as copper)), outermost preferred material is the material that bio-compatibility is good.

In the embodiment shown in fig. 1, electrode materials is chromium and gold.First on substrate, sputter the chromium that (this is the method for known metal refining in a kind of semiconducter process) a layer thickness is 0.1 micron, then sputter the gold that a layer thickness is 0.5 micron.Then the electrode shape that photoetching (being the method for known making figure in semiconducter process equally) obtains needs is carried out to whole substrate.Then unwanted part in layer gold and layers of chrome is eroded.Finally obtain required electrode.In the middle of other embodiments, also can open corresponding groove on substrate, electrode is inlayed in groove.Certainly, the method for the processing metal well known in the art such as plating also can be used for manufacturing electrode.

The present embodiment adopts chromium and Jin Lai to make electrode, be because gold does not have bio-toxicity, and chromium is the adhesion in order to increase between gold and glass.Under different demands, other electro-conductive material, such as aluminium, copper or conductive polymers can be used for manufacturing electrode.

In embodiment as shown in Figure 1, the height of electrode is 0.6 micron, this considers that the cell culture chamber on electrode needs and substrate realization sealing, in the present embodiment, this sealing is realized by bonding (in semiconducter process the known method be closely linked by two kinds of materials), so the height of electrode should not be excessive to hinder sealing.Be experimentally determined: when adopting bonded seal, electrode height is all suitable from 0.1 micron to 10 microns.When adopting other sealing means such as hot pressing, tackiness agent 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.

As shown in Figure 2, the layout that in figure, visible electrode anode 5 is mutually nested with negative electrode 6.Every two electrodes are a pair, and often pair of electrode comprises the anode and negative electrode that are oppositely arranged, and all anodes link together, and all negative electrodes link together simultaneously, control while can realizing electric field.In other application scenario, those skilled in the art are easy to realize the zonal control that often pair of electrode connects to realize electric field respectively.

In embodiment as shown in Figure 2, the width of electrode is 100 microns, and the distance between 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, suitable electrode width and interelectrode distance can be selected voluntarily by those skilled in the art.

3 cell culture chambers

Can being made up of any insulating material being applicable to shaping of cell culture chamber, or at surface coverage one layer insulating of non-insulating material.Preferably, the material making cell culture chamber should be the material that bio-compatibility is good.

In embodiment as shown in Figure 1, adopt PDMS (polydimethylsiloxane) to make cell culture chamber, this is a kind of known biocompatible polymer material being easy to machine-shaping being usually used in biological devices field.Described PDMS cell culture chamber is obtained by the method for mold, uses silicon chip to serve as mould.First on common stainless steel mould embryo, utilize machine tooling to go out required groove, then by the groove on the PDMS solution-cast progressive die tool of liquid state, after it solidifies, its demoulding is taken out, just obtain shaping cell culture chamber.Certainly, cell culture chamber also can adopt the materials'use working methods more known in the field such as glass, plastics, other polymkeric substance to process, such as, use and carry out punching to plastics.

As shown in Figure 3, the shape of each integral part of visible electroporation device in figure, cell culture chamber is linked together by the method for bonding and substrate.This considers very easily to be formed by bonding between PDMS and glass firmly to seal, and bonding process between PDMS and glass is also very simple, only needing the oxygen plasma treatment about 10 seconds (this is the dangling bonds in order to excite PDMS and glass surface) excited by the surface high pressure of PDMS and glass, then applying certain pressure and being compressed.Leave standstill the sealing namely forming robust after 24 hours.For the application that other are different, method of attachment well known in the art is also all applicable, such as, use tackiness agent or hot pressing cell culture chamber and base plate seals to be linked together.

In the embodiment shown in fig. 3, on cell culture chamber, bore dia is respectively 7,15,21 millimeters, is highly 12 millimeters.The same with the size design of electrode, in preferred cell culturing room, bore dia is respectively 7 millimeters, and this packet size is also the most preferably condition arranged for human embryonic kidney cell's electroporation.In other applications, this area researchist can arrange size voluntarily to meet the different needs.

In the embodiment shown in fig. 3, because PDMS and glass all have good bio-compatibility, so the hole of each electroporation device can be used as the cavity of cell cultures.By this method, the process of cell cultures and electroporation can be combined.

4 carrier boards and PCB

Carrier board can be made up of insulating material, or cover insulation layer by non-insulating material to make, support according to the present invention plate can be made up of any solid substrate being applicable to above condition, one or more compositions in preferred carrier board material comprises glass, plastics, pottery and metal.Preferred carrier board plate is that those can be cut into by mold or machine the carrier board requiring specification.

PCB (Printedcircuitboard) circuit card is base material with insulcrete, is furnished with circuitry lines and wiring contact, both can form electrical connector with chip module, the cable connection that can be connected with voltage source again.Described voltage source and cable connection are all the disclosed equipment easily obtained with those skilled in the art.

As shown in Figure 4, visible carrier board and PCB compact siro spinning technology in figure, under carrier board, deep gouge outward flange side is provided with PCB, is linked together by the method for bonding and carrier board.

Carrier board 7 plays the effect of support biochip, need to be fixedly connected with PCB 10 simultaneously, under carrier board, deep gouge 8 is near carrier board edge one side opening 9, in embodiment as shown in Figure 4, the edge of PCB deep gouge under each carrier board arranges two PCB wiring contacts 11, the chip wiring contact slot 12 that under carrier board, the height of the bottom of deep gouge and the contact of PCB lower surface is formed can make electroporation chip module chip wiring contact 3 insert, and forms electrical connector.

5 electroporation devices and electroporation method

In embodiment as shown in Figure 5, electroporation chip module is vertically put into deep gouge 8 under carrier board, the size of the substrate 1 of preferred chip module is slightly less than the size of lower deep gouge 8, in figure, visible chip wiring contact is advancing to the position anterior diastema 13, in figure, the direction of arrow represents the direction of chip module translation in lower deep gouge, chip wiring contact is advancing to the position post gap 14 and represents that chip wiring contact forms some web members with the contact in PCB.

As shown in Figure 6, figure comprises the outside connecting terminal 15 of electroporation device, the cable connection 16 be connected with electroporation device and voltage source 17.Described voltage source and cable connection are all the disclosed equipment easily obtained with those skilled in the art.As shown in the figure 6 system in, adopt voltage source to export as the voltage source of positive and negative 100 volts.This is also the most preferably condition arranged for human embryonic kidney cell's electroporation.

Those skilled in the art can select like device voluntarily.This electroporation device can electroporation human body and zooblast and bacterium, in an embodiment as illustrated in figure 2, 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 wants 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.

Electroporation device of the present invention can make multiple different macromolecular compound enter cell by the method for electroporation, comprises nucleic acid (plasmid DNA, linear DNA, siRNA, antisense nucleic acid), protein (peptide section, antibody).In the embodiment shown in fig. 3, preferred plasmid is carrier for expression of eukaryon (pEGFP-C3), and those skilled in the art can select various eucaryon voluntarily, and prokaryotic expression carrier uses.

Electroporation device of the present invention can become various size by designing and making, the cell culture vessel of these sizes and known in this field and conventional various size matches, thus the electroporation experiment of different flux can be carried out, and the cell after electroporation is cultivated and follow-up test analysis.In an embodiment as illustrated in figure 3, preferably 6 orifice plates, those skilled in the art can select 8,16,24,48,96,192,288,384,576,672,768,1536 orifice plates as required.This device both to attached cell, also can carry out electroporation to suspension cell.By realizing the Real Time Observation to cell to the electroporation of attached cell, thus the change of research cell physiology and morphology function after being subject to electricity irritation.In the embodiment shown in fig. 3, preferably adopt the conventional Laser Scanning Confocal Microscope in this area to observe, those skilled in the art can select other high speed microscopic examination equipment.

The electroporation of suspension cell is combined with the porous plate device in the present invention, the sample preparation speed of the existing electroporation technology in this area can be significantly improved, thus realize different flux electroporation.

Voltage source in system can provide the voltage of different wave to different cell, in the embodiment shown in fig. 3, preferably adopt square-wave pulse.Those skilled in the art can select suitable voltage source to export according to different cells.

The damping fluid used in electroporation experiment is taken from by KCl (Repone K), KH2PO4 (potassium primary phosphate), K2HPO4 (dipotassium hydrogen phosphate), the group of carbohydrate and water composition.For the embodiment shown in Fig. 2, preferred buffer formulation is: containing KCl (15-50mM mmole) in 1000ml (milliliter) damping fluid, KH2PO4 (0.1-2mM), K2HPO4 (0.1-2mM), inositol (20-60mM).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

Device of the present invention has successfully been produced by the different manufacture craft of following two cover.Providing concrete making method is to help skilled in the art to understand manufacture method of the present invention, and is not the material to device of the present invention, and size and manufacture method make restriction.

Making method A:

Adopt 2 inch glass sheets that semiconductor fabrication process is conventional.Sputter the chromium metal level of 0.1 micron thickness on the glass sheet, then on chromium metal level, sputter the layer gold of 0.5 micron thickness.Photoetching is carried out to sheet glass, produces the shape of required electrode, and then use liquor kalii iodide to corrode layer gold, use ceric ammonium nitrate solution corrosion layers of chrome.So just obtain substrate and on electrode.Then sheet glass is pressed long 33 millimeters, the shape emery wheel of wide 32 millimeters cuts.Adopt common stainless steel mold base, use machine tooling to go out the groove of corresponding 12 millimeters deep.Pour in groove by liquid PDMS, after it solidifies, the demoulding is taken out.Pressed long 23 millimeters, the shape blade cuts of wide 22 millimeters, so just obtains cell culture chamber.After the surface of glass substrate and glass roof and PDMS cell cultures chamber surface oxygen plasma treatment, be close together and apply pressure, leave standstill 24 hours, namely obtain porous plate electroporation device, wherein electroporation chip is arranged at the bottom in each hole.

Making method B:

Adopt 2 inches of N-type silicon chip that semiconductor fabrication process is conventional.Silicon chip sputters the chromium metal level of 0.1 micron thickness, then on chromium metal level, electroplates the layer gold of 5 micron thickness.Photoetching is carried out to silicon chip, produces the shape of required electrode, and then use liquor kalii iodide to corrode layer gold, use ceric ammonium nitrate corrosion layers of chrome.So just obtain substrate and on electrode.Then by this silicon chip by long 33 millimeters, the size that the shape emery wheel cutting of wide 32 millimeters thus obtain is applicable to.Adopt common plastics, thickness 12 millimeters, use process for stamping to process the shape of porous plate.By plastic sheet by long 13 millimeters, the shape emery wheel of wide 12 millimeters cuts, and so just obtains cell culture chamber.Silicon substrate and plastic multi hole plate are bonded together with ultraviolet curing adhesive, namely obtain porous plate electroporation device, wherein electroporation chip is arranged at the bottom in each hole.

7 concrete electroporation methods

Successful electroporation experiment is carried out to device of the present invention and system by the different electroporation method of following three cover.Providing concrete electroporation method is using method in order to help skilled in the art to understand electroporation device, and is not make restriction to the scope of application of device of the present invention.

Electroporation method A: the electroporation of differing capacities suspension cell

Collect the MDCK (dog renal epithelial cell) being in logarithmic phase, centrifugal 5 minutes of rotating speed 800g, abandons supernatant, with electroporation buffer (Repone K 15mM, potassium primary phosphate 0.3mM, dipotassium hydrogen phosphate 0.85mM, inositol 56mM) re-suspended cell, make the density of cell be 2X10 ³individual/ul (microlitre), adds the plasmid pEGFP-C3 needing electroporation to proceed to cell, makes the concentration of plasmid be 20ug (microgram)/ml (milliliter), softly mixes.Mixed cell suspension 20ul, 10ul, 5ul are evenly dropped to cell culture chamber, adopts following condition to carry out electricity irritation: voltage 100V (volt), pulse width 0.2ms (millisecond), pulse number 3 times, 2 seconds recurrent intervals.

After electroporation terminates, each chip drips the DMEM substratum of 200ul containing 10% serum, connects and add 0.2-1ml substratum, corresponding porous plate chip is cultivated, culture condition: temperature 37 DEG C, gas concentration lwevel 5%.Fluorescence microscopy Microscopic observation after 24 hours, the cell successfully can observing more than 90% has green fluorescence to express, and shows that the transfection efficiency of electroporation reaches more than 90%.

Electroporation method B: adherent electroporation

Collect the cell HEK293 (human embryonic kidney cell) being in logarithmic phase, centrifugal 5 minutes of rotating speed 800g, abandons supernatant, resuspended to 1X10 with the DMEM substratum containing 10% serum ⁵/ ml, mixes.Then in each hole of electroporation device, drip 500ul cell suspension, put into incubator and cultivate, culture condition is: temperature 37 DEG C, CO2 concentration 5%.After 24 hours, start electroporation when the density of cell reaches more than 80%.The Anti-IaminA/CSiRNA of cell to be proceeded to (siRNA for lamin) electroporation buffer (Repone K 15mM (mmole), potassium primary phosphate 0.3mM, dipotassium hydrogen phosphate 0.85mM, inositol 56mM) is diluted to 40ug/ml, the damping fluid of the mixed siRNA of 10ul is added in every hole, following condition is adopted to carry out electricity irritation: voltage 60V, pulse width 0.1ms, pulse number 3 times, 2 seconds recurrent intervals.

After electroporation terminates, then on each chip, drip the DMEM substratum of 500ul containing 10% serum, put into incubator and cultivate, culture condition is the same.24 h before harvest cells, carry out the expression level that Real-timePCR (real-time fluorescence quantitative PCR) detects lamin, can detect that expression level reduces more than 80%.

Use the direct culturing cell of culturing room on independently electroporation chip of the present invention, pass through the cell cultures of the process shifted, without the better effects if of the cell cultures of transfer with the cell through electroporation.

Specific embodiment

For HEK293A, contrast the present invention's detachable electroporation chip device and traditional time required for non-removable electroporation chip device process cell and electroporation efficiency.

Table 1 electroporation chip of the present invention device and 6 traditional orifice plate electroporation device testing sequence and test effects

Dismountable electroporation chip device of the present invention has saved 3 steps than traditional non-removable electroporation chip device in step, eliminates much loaded down with trivial details process, and the time can save about 1/4 in operating process.The more important thing is, use identical electrical parameter, observe contrast and experiment, the non-removable electroporation chip plant efficiency of dismountable electroporation chip plant efficiency average specific of the present invention can be obtained and exceed 10%-15%.

The above is only the preferred embodiment of the present invention, but does not limit the present invention, can not assert that embodiments of the present invention are confined to these embodiments.It should be pointed out that for those skilled in the art, under the prerequisite not departing from the technology of the present invention principle and design, some improvement or retouching can also be made, all should be considered as protection scope of the present invention.

Claims (13)

1. based on the dismountable electroporation device of biochip, comprising: independently electroporation chip module, carrier board,

PCB; It is characterized in that,

Described electroporation chip module comprises the carrying substrate (1) of electrode, electrode (2), chip wiring contact (3), cell culture chamber (4), described electrode is annular, every two is a pair, comprise the anode (5) and negative electrode (6) that are oppositely arranged, and mutually nested between negative electrode and anode, described cell culture chamber is connected with substrates into intimate;

Described carrier board (7) comprises multiple lower deep gouge (8), the size of each lower deep gouge is slightly larger than the size of described chip substrate, to make, described electroporation chip module is dismountable to be fixed in lower deep gouge, described lower deep gouge has the rim openings (9) of side at least, electrical connector is connected to form, with pcb board compact siro spinning technology bottom carrier board for chip wiring contact (3) and the contact (11) in PCB (10);

Described PCB arranges multiple contact and circuit, and contact is led to outside connecting terminal (15) by circuit, described in

Electricimpulse, as the parts be connected with external electrical, is incorporated in chip by outside connecting terminal.

2. electroporation device according to claim 1, is characterized in that, only cultivates one in described cell culture chamber

Cell, or various kinds of cell is cultivated in a cell culture chamber.

3. the electroporation device according to any one of claim 1-2, is characterized in that, the room of described cell culture chamber

Capacity is between 20 microlitres to 10 milliliter.

4. electroporation device according to claim 1, is characterized in that, under described carrier board, the height of deep gouge equals the thickness of chip substrate and the thickness sum of pcb board.

5. electroporation device according to claim 1, is characterized in that, described carrier board comprises 6,8,16 or 24 lower deep gouges.

6. the electroporation device according to claim 1 or 4, is characterized in that, the lower deep gouge of described carrier board is any shape.

7. electroporation device according to claim 6, is characterized in that, the lower deep gouge of described carrier board is square or circular.

8. the electroporation device according to claim 4 or 5, is characterized in that, the lower deep gouge of described carrier board is arranged to many

Individual row and column.

9. electroporation device according to claim 1, is characterized in that, described carrier board is transparent.

10. the electroporation device according to claim 1 or 9, is characterized in that, described carrier board material comprises glass, moulds

One or more compositions in material, pottery and metal.

The method of 11. 1 kinds of electroporation of cells, is characterized in that using according to described device arbitrary in claim 1-10.

The method of 12. electroporation of cells according to claim 11, is characterized in that, described cell comprise human body and

Zooblast or bacterium.

The method of 13. electroporation of cells according to claim 11, is characterized in that, when electroporation is carried out to cell, if

Fixed pulsed voltage is 10-2000 volt, pulse width 0.05-20 millisecond, pulse number 1-100 time, recurrent interval 0.1-60 second.