CN208250334U - A kind of flow electroporation device - Google Patents

Abstract

The utility model relates to a kind of flow electroporation devices, plane electrode including bracket and restriction fluid flowing passage, two panels plane electrode is embedded in bracket in parallel, the plane electrode includes that setting and high-voltage pulse power source have the device of electrical communication, so that the fluid moved along the fluid flowing passage be made to undergo impulse electric field；By optimizing the position of fluid drive apparatus, enable cell-bioactive substance suspension evenly into electroporation chamber, while other substances that cell after shock treatment and electricity are changed the line of production raw also can successfully be discharged from electroporation chamber.Flow electroporation device described in the utility model can be used in the processing of ultra-large volume cell liquid, and the survival rate of cell is significantly improved while significantly improving electrotransfection efficiency.

Description

A kind of flow electroporation device

Technical field

The utility model relates to electroporation arts, more particularly to flow electroporation device, by electric field by the life of flowing The method and apparatus that active substances are delivered into the living cells of flowing.

Background technique

Electroporation phenomenon is to find the 1970s, using electric field treatment cell, forms micropore, but not to cell Permanent damage is caused, micropore can self-resetting.This discovery makes it possible that bioactive substance enters cell.It is existing The study found that may work during electroporation there are four phenomenon.First phenomenon is insulation breakdown.Insulation breakdown refers to Be ability that high electric field generates small micropore or hole on cell membrane.Once generating micropore, cell can the work of load biology Property substance.Second phenomenon is insulation bunching effect, it refers to generating in uniform electric field by the stacking of vesicle mutual Self-gravitation.Third phenomenon is vesicle fusion.Vesicle fusion refers to the film that the biological vesicle of micropore is generated by insulation breakdown In closely interior and its combined trend at insulation breakdown site.4th phenomenon is thin in the presence of high-frequency electric field Born of the same parents arrange trend in alignment along their axis.Therefore, in order to load and unload cell vesicle, electroporation is related to It rotates and is pre-positioned in vesicle, the application in the dielectric constant of vesicle perforation and vesicle.

Available electroporation chamber is designed to only use in static state mostly in the prior art, these static electroporations Room is limited by volume, is unsuitable for handling a large amount of sample, can not use high pressure or duplicate charging.In order to carry out large volume Cell is handled, flow electroporation chamber has been developed.

Flow electroporation refers to such process comprising: by the bioactive substance of the suspension of cell and external source Suspension be transferred in the equipment being made of fluid chamber or fluid course；The fluid chamber or fluid course are by along fluid chamber Or the side of fluid course is arranged and is configured as, is suitable for the mixed liquor in the fluid chamber or fluid course The electrode of the electric field action of electroporation is constituted；And it will be transferred out of from the device by the cell suspending liquid of electroporation.This method It is especially effective for the cell of extensive volume.

In general, include two plane electrodes for the device of flow electroporation, to carry out the cell suspension of electroporation Continue and steadily flow through between two electrodes, until entire cell suspension has all carried out electroporation.Flow electroporation device packet Electroporation chamber containing belt electrode and interface, cell suspension can be injected by interface.Electrode with can provide high-voltage pulse to electrode Circuit is connected.High-voltage pulse is controlled by program-con-trolled computer.Flow electroporation system is had developed now, which can Electroporation is carried out to large volume of cell, obtains the living cells for importing molecules of interest.But the streaming of successful commercialization at present Electroporation apparatus is extremely rare, on the one hand has direct association with the demand in market, is on the other hand the efficiency due to electroporation It is related with the stability of product.

The condition of flow electroporation is different due to the difference of cell type, also because expectation imported into the class of intracellular molecule Type is different and different.For any one certain types of cell, all there are an optimal treatment conditions, including Optimum voltage, pulse width, optimal time interval, pulse number carry out pulse.For the electric pulse being applied on cell Number reaches best, when using flow electroporation device described in this field, cell suspension by electroporation chamber, electrode it Between flow velocity and the frequency of pulse be all alternatively, to carry out the arteries and veins of best number to the cell suspension of certain volume Punching.For example, if it is known that the optimum pulse number applied on each cell is 2, passing through electroporation for a specific cells The volume of room is 1ml, then, flow velocity is just set as per unit time 1ml, and the pulse number applied in the per unit time is 2.If carrying out electroporation in this way, each cell just will receive 2 subpulses in this example.But cell suspension It will lead to cell by the hydrodynamics flowing of electroporation chamber to flow with different rates by room and between the electrodes.Due to The place remote from locular wall is higher than the local flow velocity of close locular wall, therefore, passes through between the electrodes to the cell of liquid stream center flow Time required for region between electrode can be lower than the unit time, and the pulse number being subject to may be less than 2 times, and close The cell of locular wall flowing can be higher than the unit time by the time required for the region between electrode, and the pulse number being subject to can It can be higher than 2 times.Since the optimum pulse number that each cell is subject in this example is 2 times, however, it is clear that being not every A cell all receives the pulse of best number, and therefore, the whole electroporation effect of cell suspension is not just optimal.

The optimum condition of electroporation is according to the particular cell types of electroporation and wants to imported into cell by electroporation The difference of interior molecule type and it is different.Benefit experimentally solve this problem be it is possible, have group using static room Change the condition of electroporation with knitting, then the optimum condition for being determined at these tests is applied in flow electroporation system.This There are two disadvantages for a method.First, the static room electroporation needs due to carrying out a large amount of samples take a substantial amount of time, Cell for each static electroporation can also change in test.Second, in this way for no other reason than that people recognize It is the same for the optimum condition and flow electroporation of static room electroporation, but is not aware that whether this is true.

It is desirable that be that just can determine to carry out all electroporation examinations using the same device using a cell sample The optimum condition tested, the extensive electroporation of cell needed for this condition can be used for treatment or other purposes.

Electric revolving cup is mainly the colorimetric cup to be inserted into metal electrode, is used for electroporation or electro' asion.For this use The container on way is that small container, bottom closing, top opening, and inner space pass through two pairs of parallel side walls positioned opposite mostly It constitutes.The effect of inner space is the cell suspending liquid for receiving the cell to be processed that wherein suspended, i.e. usually buffered aqueous solution Or cell culture medium.This cuvette include mostly be arranged near the sidewall bottom of a pair of of arranged relative apply it is alive A pair of electrodes.During electric discharge, electric current flows through the cell suspending liquid between two electrodes, and nucleic acid or other molecules can be made to enter cell Or cell fusion is led to according to selected condition.Electrode is made of metal mostly, and wherein aluminium is the most frequently used.But known in these , commercially available cuvette has one disadvantage in that, be exactly that metal ion can be emitted in buffer solution during electric discharge, cell produced Raw undesirable stimulation, makes cytotoxic.For example, can be illustrated using aluminum cuvette due to Al³⁺What the release of ion generated Side effect.In addition, using have metal made of electrode cuvette, it may occur that undesirable precipitating, this be also due to from What electrode release metal ions generated.This precipitating can be the biology in metal hydroxides or metal ion and buffer solution The conjugate of macromolecular.Finally, aluminum cuvette has another disadvantage that the resistance of cuvette reduces during electric discharge, probably It is by electric current due to the alumina layer with high electrical resistance from electrode delamination.

Tentative improvement is done to electrode material in the prior art, for example, patent CN03805811.1 discloses one kind Electrode made of the electrically conductive synthetic material of the plastics of at least one conductive materials is adulterated, and the total concentration of the dopant in plastics For 20-80%w/w, dopant is made of carbon fiber, graphite, carbon black and/or carbon nanotube, can be effectively treated with electric current carefully Born of the same parents, cell derivative.Patent CN201610044870.8 discloses a kind of cell electrotransfection device, and electrode surface has nano wire Structure, nano wire are selected from cupric oxide nano line, titanium dioxide nano thread, nanowires of gold, silver nanowires, copper nano-wire, titanium nanometer Line, Fe nanowire, Pt nanowires, titanium platinum alloy nano wire, iron oxide nano-wire, stannic oxide nano wire, metallic cover silicon Any one of nano wire, the transfection voltage used are less than or equal to 10V, increase local field strength using electrode surface microstructure The 3-4 order of magnitude, but the electrotransfection device is only used for the static electroporation of attached cell.

According to the research of researcher, existing technical problem underlying is that electrolysis water causes during streaming electrotransfection at present Cathode surrounding medium pH variation, anode electrode surface oxidation generates harmful substance, the Joule effect of electric current leads to solution temperature Raising and the electric foreign matters such as raw bubble of changing the line of production cause the variation of electric field strength and its distribution.

In the R&D process of the flow electroporation chamber of the utility model, electrode research and development are used as core content, wherein electrode material Material is even more key, and electrode material directly contacts with cell solution, electrochemical reaction occurs in electroporation process, electric field is caused to become Change, influence as a result, anode material unstable simultaneously have metal ion precipitation, cause cell death or contamination of cells solution, Cause bigger harm.Therefore, the stabilization electrode material with bio-compatibility of the utility model research and development is to solve streaming electricity The matter of utmost importance for commercial applications of perforating.

The flow electroporation chamber of the utility model is contacted in electroporation process, electrode with cell solution, easy in electrode surface Electrolysis water reaction occurs, generates bubble, ideally, bubble can be flowed with liquid, be quickly discharged from electrode interior.

But due to the influence of electrode structure and electrode material surface roughness, if bubble is stagnant inside electroporation chamber It stays, since bubble is non-conductive, the distribution of electrode electric field intensity inside high changes；Bubble occupies chamber simultaneously, can change solution flow velocity, Cause cell to flow through the change of the electrode zone current pulse signal number received and intensity, electroporation result is caused to become Change.

Therefore, the utility model needs to solve the bubble generated when electroporation and how to ask the technology of bubble discharge Topic.

Summary of the invention

The purpose of this utility model is to provide a kind of modified electrodes of device for flow electroporation, can substitute The use of aluminium electrode, and there is higher electrotransfection efficiency and cell survival rate than aluminium electrode.

The another object of the utility model is to provide a kind of device of flow electroporation, and fluid can be made with relatively permanent Fixed speed flows through flow electroporation chamber.

The another object of the utility model is to provide a kind of device of flow electroporation, by the optimization of electrode structure and Controlling to optimize the generation and discharge of bubble for electric field, avoids bubble from turning result in electrode interior retention effects electricity.

The another object of the utility model is to provide a kind of preparation method of the device of flow electroporation, this method step Simply, easy to operate, product is easy to standardization.

For the above-mentioned purpose for realizing the utility model, the following technical solution is employed:

The flow electroporation device of the utility model, the main plane electrode including bracket and restriction fluid flowing passage, Two panels plane electrode is embedded in bracket in parallel, forms an electroporation chamber, and the plane electrode includes for placement and high-tension pulse The device that power supply has electrical communication is rushed, two panels plane electrode is respectively connected to the positive and negative electrode of high-voltage pulse power source output electric signal, connects After electric signal, the electroporation chamber between two panels plane electrode forms uniform electric field, to make along the fluid stream The mobile fluid experience impulse electric field for flowing through the electroporation chamber in dynamic channel.

The flow electroporation electrode of the utility model selects the good metal material of electric conductivity, while needing stable electricity Chemical property cannot chemically react in electroporation process with cell solution, avoid generating harmful substance.

Alternative electrode material has aluminium, stainless steel, gold, silver, titanium, platinum, iridium, niobium, ruthenium, rhodium, molybdenum, tungsten, conductivity ceramics Deng.

It is preferred that the electrode material is gold, titanium, platinum, iridium, niobium, ruthenium, molybdenum, tungsten, rhodium；

It is preferred that the plane electrode material surface can add coating or coating, the material of coating or coating material and substrate It is different；

It is preferred that the coating or coating material of the plane electrode be gold, silver, titanium, yttrium oxide, iridium, platinum, niobium, ruthenium, molybdenum, rhodium, One or more of tungsten, conducting ceramic material.

It is preferred that the material of the plane electrode is that platinum is gold-plated, platinum is silver-plated, platinum plates iridium, platinum plates niobium, platinum plates ruthenium, platinum plates molybdenum, platinum Rhodanizing, platinum plate conductivity ceramics.

It is preferred that the material of the plane electrode is that titanium is gold-plated, titanium is silver-plated, titanium plates iridium, titanium platinum plating, titanium plates niobium, titanium plates ruthenium, titanium Plate molybdenum, titanium rhodanizing, titanium plating conductivity ceramics.

It is preferred that the material of the plane electrode is that iridium is gold-plated, iridium is silver-plated, iridium platinum plating, iridium plates niobium, iridium plates ruthenium, iridium plates molybdenum, iridium Rhodanizing, iridium plate conductivity ceramics.

It is preferred that the material of the plane electrode is rhodium plating, rhodium is silver-plated, rhodium plating iridium, rhodium platinum plating, rhodium plates niobium, rhodium plates ruthenium, rhodium Plate molybdenum, rhodium plates conductivity ceramics.

It is preferred that the coating of the plane electrode with a thickness of 0.1 μm -10 μm.

It is preferred that the coating of the plane electrode or the preparation method of coating are to smear or be electroplated, preparation process: including preceding place The step of reason, pretreatment, smearing or plating, drying.

Pre-treatment includes choosing suitable substrate to carry out sharp processing, surface sand-blasting process, is chosen according to thickness of coating different Sandblasting mesh number, use sour clean the surface oxide layer.

Pretreatment includes being cleaned by ultrasonic using cleaning agent, and deionized water washes off cleaning agent residual.

Smearing includes that substrate surface is coated uniform coating using drying after immersion or hairbrush or spray gun spray/smearing, electricity Plating includes plating uniform coating in substrate surface using electrolysis method, smears or is rinsed after being electroplated using deionized water, removal applies Liquid or electroplate liquid are smeared, is dried for standby.

It is preferred that the shape of the plane electrode is one or more of rectangular, round, oval.

It is preferred that the surface area of the plane electrode is 10-2500mm², preferably 50-1000mm²。

It is preferred that the plane electrode with a thickness of 0.5-5mm, preferably 1-3mm.

It is preferred that the surface of the plane electrode is smooth, contacted with bracket gently without offset and sealing.

It is preferred that the two panels plane electrode is made of identical or dissimilar material respectively.

It is preferred that the distance between described two panels plane electrode is 0.5-8mm, preferably 1-5mm.

The bracket of the utility model flow electroporation device is used for fixed pan electrode, and the timbering material selects medical grade Engineering plastics, the bracket is made by way of injection molding or CNC or 3D printing.

The flow electroporation device of the utility model further includes conduit, and the conduit is connect with bracket, and the conduit includes Inlet tube and outlet tube.

It is preferred that bracket setting inlet and liquid outlet, the inlet tube connects with inlet, the outlet tube and out The connection of liquid mouth.Fluid flows into chamber by inlet, under the action of electric field, completes electroporation, flows out finally by liquid outlet.

It is preferred that the diameter of the outlet tube is not less than the diameter of the inlet tube.

It is preferred that the diameter of the conduit is between 2-10mm.

It is preferred that the conduit selects medical engineering plastics or medical silica-gel.

It is preferred that the conduit includes but is not limited to be threadedly coupled with the both ends connection type of bracket, snap connection, cladding connects It connects, ultrasonic welding etc..

The flow electroporation device of the utility model further includes fluid drive apparatus, is provided just using peristaltic pump or magnetic drive pump The mode of pressure and/or negative pressure drives fluid to flow through restriction fluid flowing passage.

It is preferred that fluid is driven to flow through restriction fluid flowing passage by the way of negative pressure.

Using the treated electroporation of cells of flow electroporation device described in the utility model, cell electrotransfection rate is at least 60% or more, cell survival rate at least 60% or more.

The preparation method of the flow electroporation device of the utility model, described device pass through injection molding or CNC or 3D printing Manufacture, first the bracket there are two recessed window is stayed in production, then plane electrode is installed in the recessed window, then again by plane Electrode package.

The application method of the flow electroporation device of the utility model applies voltage to the plane electrode, generates electric current, The electric current reaches highest 120A/cm², bioactive substance is transferred to by cell by electroporation principle.

Cell and biological active matter metallic substance are placed in solution by the application method of the flow electroporation device of the utility model In, and transfer the solution into flow electroporation device, the bioactive substance is shifted into cell, by connecting the electricity The current density for pressing pulse is 2-14A/cm², pulse width be 0.01-100ms electric current realize.

The application method of the flow electroporation device of the utility model passes through field strength 200V-10KV/cm, pulse width The voltage pulse of 0.01-100ms realizes transfer of the bioactive substance into living cells.

The flow electroporation device of the utility model is used to bioactive substance transfecting the purposes into cell, and feature exists In, the bioactive substance include but is not limited to medicament, gene DNA, RNA, antibody or other protein, cell factor, Nano particle etc..

It is preferred that the type of the cell includes but is not limited to prokaryotic cell, eukaryocyte, plant protoplast etc..

It is preferred that prokaryotic cell includes bacterium, fungi, saccharomycete.

It is preferred that eukaryocyte includes animal cell line cell, primary cell, egg cell etc..

Compared with prior art, the flow electroporation device of the utility model achieves significant progress and unexpected Technical effect:

Plane electrode made of the preferred plane electrode material of the utility model, is capable of providing continual and steady electric current, In the presence of buffer solution, stable uniform electric field can be provided, electroporation efficiency can be significantly improved, while guarantee that cell height is survived Rate；

The preferred plane electrode of the utility model with after Buffer fluid contacts, even if higher pulse voltage, unexpected Discovery generate cell reaction it is very small, while generate bubble it is considerably less, to can persistently to plane electrode press pulse Provide advantageous condition；

The OH that the plane electrode of the utility model is generated in working condition^-(cathode effect) is few, does not change the pH of cell liquid Value, small to cell damage, cell survival rate is high；

The flow electroporation device simple production process of the utility model, preparation cost is low, and production can standardize Production, batch processed cell liquid volume is bigger, and transfection efficiency and cell survival rate are higher, and stability is more preferable, application method operation It is convenient.

The flow electroporation device of the utility model is current state-of-the-art flow electroporation product, be can be used in actual Laboratory and production link carry out electrotransfection, have huge commercialization space.

Detailed description of the invention

It, below will be to use required in embodiment for the clearer technical solution for illustrating the utility model embodiment Attached drawing do simple introduction.

The electric current fringe of Fig. 1 difference size aluminium electrode is qualitative；

Fig. 2 adds the current stability of the Ti electrode of conductivity ceramics coating, aluminium electrode and stainless steel gold-plated electrode；

Solution colour variation after Fig. 3 adds the titanium plane electrode electricity of conductivity ceramics coating to turn；

Solution colour variation after Fig. 4 aluminium electrode electricity turns；

Solution colour variation after Fig. 5 stainless steel gold-plated electrode electricity turns；

Solution colour after the electricity of the pure Ti electrode of Fig. 6 turns；

Solution colour after the electricity of Fig. 7 titanium platinized electrode turns；

(left side is anode, the right is yin for electrode surface variation after Fig. 8 adds the titanium plane electrode electricity of conductivity ceramics coating to turn Pole)；

Electrode surface variation after Fig. 9 aluminium electrode electricity turns (left side is anode, the right is cathode)；

Electrode surface variation after Figure 10 stainless steel gold-plated electrode electricity turns (left side is anode, the right is cathode)；

Electrode surface variation after the pure Ti electrode electricity of Figure 11 turns (left side is anode, the right is cathode)；

Electrode surface variation after Figure 12 titanium platinized electrode electricity turns (left side is anode, the right is cathode)；

Figure 13 device overall structure decomposition diagram；

Annotation: 1- conduit (outlet tube)；2- bracket liquid outlet；3- first buffering area domain；4- electroporation chamber；The first plane of 5- Electrode；6- the first plane electrode cooling device；7- conduit (inlet tube)；8- conduit (inlet tube)；9- second buffering area domain；10- Bracket；11- the second plane electrode cooling device；The second plane electrode of 12-；

Specific embodiment

The embodiments of the present invention are described in detail below in conjunction with specific embodiment, but art technology Personnel will be understood that the following example is merely to illustrate the utility model, and be not construed as limitation the scope of the utility model.It is real The person that is not specified actual conditions in example is applied, is carried out according to the condition that this field normal condition or manufacturer suggest.

Embodiment one

The selection of the shape and size of electrode

The flow electroporation device of the utility model uses plane electrode, when the size of plane electrode determines unit The speed of interior processing cell, length, width and the spacing of electroporation chamber have different influences respectively.In the feelings of constant flow rate Under condition, the time of the effect length electrode interior solution flowing of plane electrode, increase electrode length will increase bubble and flow through electrode When region with electrode surface time of contact, increase the probability of bubble residence.The widths affect electrode interior solution stream of plane electrode Dynamic speed, fluid intermediate region flow velocity is fast, and fringe region flow velocity is slow, and electrode width is bigger, and deviation is more obvious.Plane electrode The intensity of effect of distance electric field, according to formula E=U/d, under identical voltage, spacing is bigger, and electric field is smaller.Increase electrode spacing, If reaching optimal electricity turns intensity, it is necessary to improve voltage, increase the output of instrument.

Experimental designs: it is filled using the plane electrode production flow electroporation of the proportionate relationship of different length and width Set, with the test current data variation of empty buffer liquid, while observing bubble and generating and discharge situation, optimize the length of plane electrode with Width ratio relationship.The shape selection rectangle or square of plane electrode.Plane electrode test material selects aluminium, test sample Long generous size is set separately are as follows: 15 × 10 × 2mm, 25 × 4 × 2mm, 25 × 2 × 2mm, 12.5 × 4 × 2mm.With CNC or 3D For printing and making band there are two the bracket of recess window, the size of the recess window is consistent with the size of above-mentioned plane electrode.It is flat Electrode insertion in face is recessed in window, then bracket is encapsulated, and forms the electroporation chamber for having fluid entry port and outflux.It will Two plate electrodes of above-mentioned apparatus are separately connected the positive and negative anodes of pulse power output end.Electricity turns condition 150V/11ms/1 times, and phase is added The EL buffer solution (Suzhou one reaches company and its agent's product on sale, article No.: H10305) for answering volume, during record shocks by electricity Current data situation of change (referring to attached drawing 1), whole process observation bubble generate with discharge situation.

Electric shock process testing result: the electrode of length 25mm, the minute bubbles that electric shock generates in the process are easy to tire out in electrode Product, is attached to electrode surface, gradually grows up and be then exhausted from for air pocket, causes current cycle to fluctuate, causes under electric field stability Drop.The electrode of length 12.5mm, the minute bubbles that electric shock generates in the process are flowed with solution to be discharged, and is seldom accumulated in electrode, electricity Stream stability is good, but plane electrode is that electroporation chamber's total volume of 2 × 4mm only has the half of 2 × 8mm, chooses 2 under the same terms × 8mm specification, to improve the treating capacity in the unit time.In the case that length and electrode spacing are constant, it is wide individually to change electrode Degree, width is bigger, and unit time treating capacity is bigger, otherwise width is smaller, and unit time treating capacity is smaller.But larger width Afterwards, electrode two side areas can also become larger with solution flow velocity difference in intermediate region, and comprehensively considering 15 × 10 × 2mm is preferred electricity Pole size.

Embodiment two

The selection of plane electrode material

The flow electroporation device of the utility model, plane electrode material development is as core content, planar electrode surface Answer it is smooth, contacted with bracket gently without segment difference and formed sealed chamber, reduce bubble residence possibility to the greatest extent.Pass through electrode The selection of material, the optimization of electrode structure and the control of electric field strength, come cut down electric shock during electrochemical reaction product to electricity The adverse effect of transfection.

Experimental designs: the type of the alternative plane electrode material of the present embodiment be aluminium, 304 stainless steels, proof gold, Stainless steel is gold-plated, the titanium of pure titanium plus conductivity ceramics coating, pure platinum, titanium platinum plating, pure iridium；

Aluminium: electrotransfection field is widely used in as electroporation material；

304 stainless steels: bioaffinity is good, at low cost, most common medical metal material；

Stainless steel is gold-plated: fitting has stable chemistry and electric conductivity, has good bio-compatibility；

Pure titanium: the metal material with good biological compatibility is widely used in biologic medical field；

Add the titanium of conductivity ceramics coating: University Of Suzhou's Material Physics laboratory provides, and coating material is conductivity ceramics, is had Good antioxygenic property；

Titanium platinum plating: stable anode material is widely used in electrolytic industry, and has good bio-compatibility；

Proof gold: there is high erosion-resisting stability, good electric conductivity and thermal conductivity, easily shape, and very It is easy on the surface for being plated to other metals and pottery and glass

Pure platinum: chemical property is extremely stable, does not dissolve in strong acid and strong base solution, has catalytic activity, can cook anticancer drug.

Pure iridium: chemical property is highly stable, does not dissolve in chloroazotic acid, and iridium is widely used in high-tech sector, such as navigates Its technology, pharmacy and automobile industry.

The plane electrode that length × width x thickness is 15 × 10 × 2mm is made in above-mentioned material.As in the first embodiment, with CNC or 3D For printing and making band there are two the bracket of recess window, the size of the recess window is consistent with the size of above-mentioned plane electrode.It will The insertion of plane electrode made of above-mentioned material is recessed in window, then bracket is encapsulated, and forms one and has fluid entry port and stream The electroporation chamber of outlet.Above-mentioned two plate electrodes having been inserted into bracket are separately connected to the positive and negative anodes of pulse power output end, are made At an electroporation device.

Test one, under the conditions of empty buffer liquid (referring to does not have cell and bioactive substance in buffer), selection, which adds, to be led The titanium plane electrode of electroceramics coating, aluminium electrode, stainless steel gold-plated electrode, pure Ti electrode, titanium platinized electrode use 150V electricity Pressure, 11ms pulsewidth, number of shocks are 1 time, are divided into 2352ms electricity and turn condition, the electricity swivel product electric shock test group loading electrode of 30ml Stability records the current data during electric shock, and experimental result referring to fig. 2, while being observed solution after electricity turns and being changed, referring to figure 3-7 and planar electrode surface variation, referring to Fig. 8-12.

From Figure 2 it can be seen that the electric current of stainless steel gold-plated electrode is gradually increased to 5A from 4A；Aluminium electrode electric current rises rapidly from 4.5A Height arrives 5A, and rear electric current fluctuates above or below 5A always；Add the titanium plane electrode electric current of conductivity ceramics coating all non-from start to end Often stablize, is maintained at 4.8A.Add Ti electrode, aluminium electrode and the titanium platinized electrode electric current of conductivity ceramics coating relatively stable, it is stainless Steel gold-plated electrode electric current is gradually increasing, and pure Ti electrode initial stage electric current can decline, then slowly rise, behind gradually tend to be steady, And current waveform changes.

A variety of materials processing after solution variation by Fig. 3-7 as it can be seen that plus conductivity ceramics coating titanium plane electrode can with it is molten Chloride ion in liquid reacts, solution turned yellow (Fig. 3) after electricity turns, and cell survival rate is made after the ion component of precipitation can turn electricity At influence, without obvious sediment after centrifugation.

During electric shock, vigorous oxidation reaction occurs for aluminium and stainless steel gilding, anode.The Al of aluminium electrode³⁺Can with it is molten OH in liquid^-Equal reactions generate white precipitate (Fig. 4), and therefore, generating oxide can fall in cell solution, are not available as anode Material.And the gold of the gold-plated planar electrode surface of stainless steel falls off (Figure 10), solution becomes micro- yellow (Fig. 5).

Above-mentioned three kinds of materials, which have, generates white foam group, and wherein aluminium electrode generates at most, and has white depositions analysis (Fig. 4) out.

After pure titanium plane electrode electric shock, solution does not change colour, but has obvious particle to precipitate after being centrifuged, and all processes all exist It precipitates (Fig. 6).

After titanium platinum plating plane electrode electric shock, solution does not change colour, has no obvious sediment, also deposit-free (Fig. 7) after centrifugation.

After electricity turns, electrode is dismantled, by Fig. 8-12 as it can be seen that electricity adds the titanium planar electrode surface of conductivity ceramics coating not after turning See obvious oxidation (Fig. 8), and aluminium (Fig. 9) and the gold-plated plane electrode anode of stainless steel have oxidation, the especially gold-plated plane of stainless steel The Gold plated Layer of electrode has fallen off (Figure 10).Pure Ti electrode anodic oxidation forms oxide layer (Figure 11), and titanium platinized electrode surface has no It aoxidizes (Figure 12).

Test two, will containing pMAX plasmid (be purchased from Lonza Human T cell Nucleofector Kit, article No.: ) and CHO-S cell (Medical School of Peking University Zhou professor give, and professor Zhou is purchased from Life, article No. R80007) and EL VAPA-1002 The fluid that Buffer electricity turns buffer is gradually injected in electroporation chamber from above-mentioned electroporation device bottom, selects different electrode materials Material is divided into 2352ms respectively by 150V voltage, 11ms pulsewidth, number of shocks is 1 time, and in 150V voltage, 5ms Pulsewidth, number of shocks are 3 times, are divided into electric field treatment under the conditions of 784ms, then flow out from top open part, carry out to cell Culture measures electrotransfection rate, the indexs such as cell survival rate.

Ti electrode, aluminium electrode, stainless steel gold-plated electrode and the pure Ti electrode of table 1 plus conductivity ceramics coating are in 150V 11ms Cell experiment result is handled under the conditions of 1 time

Annotation: TE: electric transfer efficient；VE: cell survival rate；MIX: overall efficiency；MFI: average fluorescent strength；

Above-mentioned four kinds of electrode material electricity turn the result shows that, 30ml electricity swivel product, transfection efficiency remains unchanged substantially, there is 5% The fluctuation of left and right, electric transfer efficient: pure titanium > adds the titanium > aluminium > stainless steel of conductivity ceramics coating gold-plated；Cell survival rate: pure titanium > adds the gold-plated > aluminium of titanium > stainless steel of conductivity ceramics coating；Overall efficiency: pure titanium > add the titanium > aluminium of conductivity ceramics coating= Gold-plated stainless steel；Fluorescence intensity: pure titanium > aluminium > adds the titanium > stainless steel of conductivity ceramics coating gold-plated.

Ti electrode and titanium platinized electrode electricity under the conditions of 150V 5ms 3 times of table 2 plus conductivity ceramics coating turn cell reality Test result

Titanium platinized electrode and plus conductivity ceramics coating Ti electrode on transfection efficiency inter-individual difference it is smaller, but titanium platinum plating The survival rate of electrode is higher than coated electrode by 15%, and the fluorescence intensity of the two is almost the same, therefore titanium platinized electrode overall efficiency It is higher than the Ti electrode for adding conductivity ceramics coating.

Although pure Ti electrode electricity turns result better than aluminium, stainless steel gold-plated electrode, the anode of pure Ti electrode can be aoxidized, Electric field stability changes, and observes that black object falls off in solution after electricity turns, therefore pure titanium is not suitable as anode Material；And adding the Ti electrode electricity of conductivity ceramics coating to turn solution can turn to be yellow.Titanium platinized electrode and the titanium electricity for adding conductivity ceramics coating Extremely electric transfer efficient is close, and the cell survival rate of titanium platinized electrode is higher, therefore titanium platinized electrode overall efficiency is higher than plus conduction The Ti electrode of ceramic coating.

Embodiment three

Positive pressure or negative pressure drive fluid

The flow electroporation device of the utility model drives fluid to flow in a manner of positive pressure and/or negative pressure, preferably with negative The mode of pressure drives fluid to flow.The power mainly flowed using the rotation of peristaltic pump as fluid, promotes fluid to enter electroporation Room receives electric shock, and by after shock treatment cell and electricity other substances for changing the line of production raw take electric field out of, it is ensured that electricity turn it is accurate orderly Progress.According to the position that peristaltic pump is placed, entire hydrodynamic form can be divided into positive pressure and negative pressure both of which.Just press Force mode is the front side that peristaltic pump is placed on to electroporation chamber, pushes liquid in pipeline system by the thrust that rotation peristaltic pump generates One-way flow enters electroporation chamber in uniting.Negative pressure dynamic mode is the rear side that peristaltic pump is placed on to electroporation chamber, passes through rotation The suction that peristaltic pump generates extracts the sample in sample cell and its one-way flow in pipe-line system is made to pass through electroporation chamber.

Experimental designs: using just into Zheng Chu arc buffering area electroporation device, monitoring two kinds of positive pressure, negative pressure power moulds Under formula, foam is rolled into a ball during current stability and observation during driving fluid to carry out electricity turn shock by electricity generation and feelings are discharged Condition compares the flow stability of the stability and fluid of electric field strength under two kinds of dynamic modes in pipeline.Detect two kinds of work CHO-S cell electricity turns pMAX under mode, and in 150V voltage, 5ms pulsewidth, number of shocks is 3 times, be divided under the conditions of 784ms into Row electricity turns, selection titanium platinum plating plane electrode, resulting cell electrotransfection efficiency, survival rate, overall efficiency and luciferase expression amount, than Electricity compared with cell under two kinds of dynamic modes turns effect and stability.

Cell electricity turns effect under 3 positive pressure of table and negative pressure dynamic mode

Interpretation of result: can speculate from actual average size of current and standard deviation: under two kinds of dynamic modes of positive pressure and negative pressure Electric field strength size is more consistent, and CHO-S cell electricity turns in DNA tests, under positive pressure dynamic mode measured current be 5.20 ± 0.39A, measured current is 4.95 ± 0.55A under negative pressure dynamic mode, and the two difference is smaller, and stability is preferable.

It can be seen that the generation and row that electrode surface foam is rolled into a ball from the moving situation of fluid and foam group in pipe-line system Artificial situation is essentially identical, shows as smaller size smaller foam group before this and generates, and most of is without the continuous quickly discharge of delay, in electricity Pole piece and the deceleration of bracket seam crossing are accumulated compared with macrofoam group, then occur slowing down again at the closing in of electrode outlet end accumulating Bigger foam group, finally enters collection conduit system.But discharge situation of the foam group in collection conduit differs greatly: positive pressure Under the conditions of, foam group is easily in pipeline corner stagnation accumulation, sectional sampling, and leading portion is collected less than foam group, and back segment is collected into A large amount of foam groups, flow velocity is uneven during causing electricity to turn, and the cellular liquid accumulated amount that different sections are collected into is inhomogenous.Condition of negative pressure Under, foam group is lighter in pipeline corner stagnation levels of accumulation；When each section of sampling, the distribution of foam group and cellular liquid in collecting pipe Accumulated amount is more consistent.Relative to positive pressure, under negative pressure dynamic mode, transport of the fluid suspension in pipeline is more stable, and foam The discharge of group is more evenly smooth.

Turn every end value of effect from cell electricity it follows that under negative pressure dynamic mode, cell electrotransfection efficiency is survived Rate, overall efficiency and MFI are higher than acquired results under positive pressure dynamic mode.And electric transfer efficient and survival rate, negative pressure dynamic mode It is more stable.Negative pressure dynamic mode can effectively promote flowing and discharge of the foam group in pipe-line system, and sample cell can at this time With upright placement, caused by avoiding because of reasons such as sample cell poor sealings phenomena such as leakage, while being convenient for later period sample channel The transformation of system.

Although illustrate and describing the utility model with specific embodiment, it will be appreciated that without departing substantially from this In the case where the spirit and scope of utility model, many other change and modification can be made.It is, therefore, intended that appended It include all such changes and modifications belonged in the scope of the utility model in claim.

Claims (7)

1. a kind of flow electroporation device, which is characterized in that the plane electrode including bracket and restriction fluid flowing passage, two panels Plane electrode is embedded in bracket in parallel, and the plane electrode includes that setting and high-voltage pulse power source have the device of electrical communication, to make The fluid moved along the fluid flowing passage undergoes impulse electric field；It further include fluid drive apparatus, the material of the plane electrode Material be selected from gold, silver, titanium, platinum, iridium, niobium, ruthenium, rhodium, molybdenum, tungsten, conductivity ceramics, or containing gold, silver, titanium, platinum, iridium, niobium, ruthenium, rhodium, As one or more of coating or the conductive material of coating, the fluid drive apparatus is peristaltic pump for molybdenum, tungsten, conductivity ceramics Or magnetic drive pump, the fluid drive apparatus drive fluid to flow through restriction fluid flowing passage by the way of positive pressure and/or negative pressure.

2. flow electroporation device according to claim 1, which is characterized in that drive fluid to flow through by the way of negative pressure Limit fluid flowing passage.

3. -2 described in any item flow electroporation devices according to claim 1, which is characterized in that the shape of the plane electrode It is one or more of rectangular, round, oval.

4. -2 described in any item flow electroporation devices according to claim 1, which is characterized in that the surface of the plane electrode Product is 10-2500mm²。

5. -2 described in any item flow electroporation devices according to claim 1, which is characterized in that the thickness of the plane electrode For 0.1-5mm.

6. -2 described in any item flow electroporation devices according to claim 1, which is characterized in that the two panels plane electrode point It is not made of identical or dissimilar material.

7. -2 described in any item flow electroporation devices according to claim 1, which is characterized in that the two panels plane electrode it Between distance be 0.5-8mm.