CN209098693U - A kind of intermittent streaming electrotransfection device - Google Patents

A kind of intermittent streaming electrotransfection device Download PDF

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Publication number
CN209098693U
CN209098693U CN201821468883.9U CN201821468883U CN209098693U CN 209098693 U CN209098693 U CN 209098693U CN 201821468883 U CN201821468883 U CN 201821468883U CN 209098693 U CN209098693 U CN 209098693U
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China
Prior art keywords
electrode
tube body
liquid
electrotransfection
cell
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Withdrawn - After Issue
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CN201821468883.9U
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Chinese (zh)
Inventor
戴晓兵
沈琦
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SUZHOU ETTA BIOTECH Co Ltd
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SUZHOU ETTA BIOTECH Co Ltd
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Abstract

The utility model discloses a kind of intermittent streaming electrotransfection devices, including tube body, first electrode, second electrode, it is characterized in that, the first electrode, second electrode are in the both ends nozzle of the tube body in parallel, and the inside formation of the tube body and first electrode, second electrode has the cavity for accommodating target liq sample;At least one liquid entrance is arranged in the tube body and at least one liquid passes in and out valve, and the liquid disengaging valve is located at the liquid entrance of the tube body, controls the opening and closing of liquid entrance;The first electrode and/or second electrode can be relatively moved along the tube body.The intermittent streaming electrotransfection device of the utility model avoids electrotransfection process from generating bubble using pressure, improves electrotransfection efficiency, and realization quickly handles a large amount of samples.

Description

A kind of intermittent streaming electrotransfection device
Technical field
The utility model relates to electrotransfection fields, are related to a kind of intermittent streaming electrotransfection device, more specifically, this reality It is related to a kind of intermittent streaming electrotransfection device for inhibiting electrotransfection to generate bubble in such a way that electrode movement is pressurizeed with novel.
Background technique
Cell membrane is the thin film for being enclosed in pericellular, be cell with the external world carry out selective substances exchange it is penetrating Property barrier.Cell membrane makes cell become an independent living unit, and possesses a metastable interior environment.Ambient enviroment In some substances can be by cell membrane, other substances are then not all right.Cell can be absorbed by cell membrane from ambient enviroment Nutriment is discharged metabolite, the transhipment of substance is made to reach equilibrium state.So the basic function of cell membrane is exactly to maintain cell Interior microenvironment relatively stable simultaneously selectively carries out mass exchange with external environment.
The study found that if applying the electro photoluminescence of some strength to cell and continuing for some time, so that it may inducing cell Some micropores are generated on film, enhance the permeability of cell, and so-called cell electroporation (Electroporation) just refers to cell Under the action of adding impulse electric field outside, the biophysics process of instantaneous micropore is formed on cell membrane lipid bilayer.Electrotransfection It (Electrotransfection) is to be imported the large biological molecule of external source, such as DNA, RNA or protein using electroporation technology The technology of cell.When electroporation occurs for cell membrane, permeability and membrane conductance can instantaneously increase, and make hydrophilic molecules, DNA, egg White matter, virion, drug granule etc. cannot be able to enter cell by micropore by the molecule of cell membrane under normal circumstances.? After removing electro photoluminescence in short time, micropore disappears on cell membrane, and cell membrane becomes selective permeation barrier again.With traditional change Learn transfection compared with virus transfection, due to electrotransfection with no chemical contamination, cell will not be caused permanent damage, efficiency compared with The advantages that high, has broad application prospects in fields such as biophysics, molecular biology, clinical medicine.
Although the mechanism of electrotransfection effect is not entirely clear that, cell electrotransfection herein is well known, including thin The movement of after birth lipid bilayer causes to form temporary micropore on film, and exogenous molecule is allowed to enter cell by micropore.
In the prior art, three classes method mainly completes the process of cell electrotransfection:
Cell is placed in a pair of between several millimeters to several centimeters parallel pole.Make the electricity of cell between the electrodes By electro photoluminescence in, to realize the purpose of electrotransfection.For example, United States Patent (USP) US5389069.
It is penetrated in tissue or cell liquid using miniature needle electrode and is shocked by electricity to cell, achieve the purpose that electrotransfection.Example Such as, United States Patent (USP) US5389069.
One chamber is placed between a pair of of parallel pole, while so that the aaerosol solution of cell flows in the chamber It is shocked by electricity.For example, United States Patent (USP) US6773669.
Chinese patent CN201010242144 discloses a kind of streaming electrotransfection apparatus and system, which includes: streaming Electrotransfection device, including: substrate, and the electrode being produced on substrate, the electrode are parallel and place in pairs , each pair of electrode includes the anode and cathode being oppositely arranged;It is placed in the channel of the limitation fluid flowing on electrode;The channel Starting point has multiple inlet branch channels, and pools a main channel, and end has multiple outlet branch channels, described Setting has the top cover of multiple fluid inlets and outlet above channel;Syringe pump is connected to the streaming electrotransfection by pipeline and fills Set the entrance of middle top cover and the flow velocity of control of export fluid;Voltage source is set by electrical connector connection electrode and generates pulse electricity Pressure.Streaming electrotransfection system realizes various suspension in the fluid passage using fluid channel and the syringe pump being connected Continuous flowing, so that cell be enable to be continued to carry out by the process of electrotransfection, realization quickly handles a large amount of samples.
Chinese patent CN201610806987 discloses the disposable product of the electrotransfection for cell comprising: in institute State the fluid compartment of the inside of disposable product;First fluid port is used to provide cell suspension to the fluid compartment;With And second fluid port, be used for will include to by the fluid delivery of electrotransfection at least one of cell compound extremely The fluid compartment;First electrode and second electrode in the fluid compartment are set;At least one outlet port, delivering The fluid from the fluid compartment, wherein the first fluid port and the second fluid port and hybrid channel are flowed Body connection, the hybrid channel and the fluid compartment are in fluid communication.
But streaming electrotransfection device disclosed above can generate a large amount of bubbles during practical electrotransfection, generation Bubble, which is attached on electrode plate, influences field uniformity, unstable so as to cause electrotransfection effect.Inventor passes through retrieval discovery, The technical issues of electrotransfection process generates bubble is solved not yet in streaming electrotransfection field.
Summary of the invention
The utility model is provided a kind of by moveable electricity for above-mentioned existing technology The mode of pole pressurization inhibits the intermittent streaming electrotransfection device of generation bubble during electrotransfection.The utility model it is another Purpose is to improve the stability of electrotransfection device, improves the efficiency of electrotransfection.
In order to achieve the above object, the technical solution of intermittent streaming electrotransfection device provided by the utility model is summarized It is as follows: a kind of intermittent streaming electrotransfection device, including tube body, first electrode, second electrode, which is characterized in that described first Electrode, second electrode are in the both ends nozzle of the tube body in parallel, and the inside of the tube body and first electrode, second electrode is formed With the cavity for accommodating target liq sample;At least one liquid entrance is arranged in the tube body and at least one liquid passes in and out valve Door, the liquid disengaging valve are located at the liquid entrance of the tube body, control the opening and closing of liquid entrance;It is described First electrode and/or second electrode can be relatively moved along the tube body.
It is preferred that snorkel is arranged in the tube body, gas valve, the further preferred snorkel and tube body is arranged in snorkel Gas valve is arranged in junction, and the snorkel is between the first electrode and second electrode, the end of the snorkel Air filter is set.
It is preferred that at least two liquid entrances are arranged in the tube body, the liquid entrance either liquid-inlet, It can be liquid outlet, corresponding at least two liquid that are arranged pass in and out valve;Liquid disengaging valve either liquid into Sample check valve is also possible to liquid discharge sample check valve;At least one liquid-inlet and one is arranged in the further preferred tube body Liquid outlet, at least one Liquid sample introduction check valve and a liquid discharge sample check valve;
It is preferred that the cross section of the first electrode and second electrode and the cross section of the tubular body are identical or close, Further preferably the shape of the cross section of the tubular body is rectangular, and round or other shapes also can be set into;
It is preferred that sealing device is arranged in the first electrode and second electrode and tube body contact position, the sealing device is preferred Seal washer, sealing joint strip or other devices with sealing performance etc.;
It is preferred that when the liquid passes in and out valve and gas valve and closes, the tube body and first electrode, second electrode Inside forms closed cavity;
It is preferred that the first electrode and/or second electrode are opposite when the liquid passes in and out valve and gas valve is closed After tube body is mobile, the pressure value of the closed inside cavity is 1.0-2.5Mpa;The pressure of the further preferred inside cavity Force value is 1.1-2.0Mpa;The pressure value of the further preferred inside cavity is 1.2-1.5Mpa;
It is preferred that described device further includes that conduit is several, it is divided into inlet tube and outlet tube, conduit and tube body sealed set;
It is preferred that described device further includes pump, for letting the liquid out or flow into cavity, or for being discharged or being filled with cavity Interior gas, the type of pump include but is not limited to peristaltic pump, air pump, liquid pump, syringe pump etc.;
It is preferred that described device further includes that the first electrode and/or second electrode is enable to relatively move along the tube body Pressurizing device, the type of the pressurizing device includes but is not limited to air bag, hydraulic stem, atmospheric pressure pole, screw press, electromagnetism Power pressurizing device etc.;
It is preferred that the first electrode and/or second electrode setting operating position and off-position, the electrode is from non-work It is moved to operating position as position, pressure can be provided for inside cavity;The further preferred liquid entrance is located at described Between first electrode and second electrode.The further preferred liquid entrance is located at the first electrode and/or second electrode Between the operating position and off-position of setting.
It is preferred that the second electrode is fixedly installed on one end nozzle of tube body.
The preparation method of device described in the utility model prepares tube body, selection cured plastics work after capable of heating first For the material of tube body, tube body is prepared using the mold for preparing tube body, Liquid sample introduction mouth and liquid discharge sample are respectively set on tube body Mouthful, one relatively parallel electrode is respectively set at tube body both ends, electrode is the sheet metal injection molding of two panels same shape, size in modeling It is first electrode and second electrode in material frame.It is connect in the side that electrode does not contact cavity with pressurizing device, electrode frame of plastic four Soft adhesive tape is arranged in week.Second electrode fixed installation, first electrode and pressurizing device are connected, first electrode is allow to pressurize Displacement is generated under the pressure that device generates;Or connect first electrode and second electrode with pressurizing device respectively, make the first electricity Pole and second electrode can generate displacement under the pressure that pressurizing device generates.
Technical solutions of the utility model bring the utility model has the advantages that
The intermittent streaming electrotransfection device of the utility model is capable of handling a large amount of fluid samples.
The intermittent streaming electrotransfection device of the utility model, to liquid pressing, inhibits bubble to produce during electrotransfection Raw, electrotransfection efficiency fluctuation caused by reducing because of bubble attachment improves the stability of electrotransfection device.
The intermittent streaming electrotransfection apparatus structure of the utility model is novel in design, has initiative.
Detailed description of the invention
The intermittent streaming electrotransfection device exemplary diagram of Fig. 1 the utility model;
The intermittent streaming electrotransfection device exemplary diagram of Fig. 2 the utility model;
Attached drawing annotation: 1- first electrode, 2- second electrode, 3- tube body, 4- inlet tube, 5- outlet tube, 6- pressurizing device, 7- First electrode off-position, 8- first electrode operating position, 9- second electrode off-position, 10- second electrode working position It sets, 11- sample introduction check valve, 12- goes out sample check valve, 13- snorkel, 14- gas valve.
Specific embodiment
Embodiment one
Overall construction design (one)
Intermittent streaming electrotransfection device overall structure provided by the utility model as shown in Figure 1, the tube body (3) two End pipe mouth blocks sealed set, and wherein the sealing end of nozzle one is used as sealed set using second electrode (2), and another sealing end of nozzle uses First electrode (1) is used as sealed set, and snorkel (13) are arranged in the tube body (3), and the snorkel (13) connect with tube body (3) Place's setting gas valve (14).The first electrode (1) setting operating position (8) and off-position (7) in tube body (3), First electrode (1) is connect with pressurizing device (6), pressurizing device (6) preferred hydraulic stem, and pressurizing device (6) can push the first electricity Pole (1) is moved to operating position (8) from off-position (7), and the liquid for carrying inside cavity bears certain pressure.? Applying electric pulse on one electrode (1) and second electrode (2), electric pulse makes to form electric field in cavity, so that cell membrane forms micropore, To make the target substance in liquid sample be able to enter in cell.After the completion of electrotransfection, pressurizing device (6) can be by the first electricity Pole (1) is returned to off-position (7) from operating position (8).It repeats aforesaid operations and carries out intermittent streaming electrotransfection.
Embodiment two
Overall construction design (two)
Intermittent streaming electrotransfection device overall structure provided by the utility model as shown in Fig. 2, the tube body (3) two First electrode (1) and second electrode (2) sealed set is respectively adopted in end pipe mouth sealing end, wherein first electrode (1), second electrode (2) it is connect with pressurizing device (6), pressurizing device (6) preferred hydraulic stem.Snorkel (13) are arranged in the tube body (3), the ventilation Manage (13) and tube body (3) junction setting gas valve (14).First electrode (1) in the tube body (3) setting operating position (8) and Operating position (10) and off-position (9) is arranged in off-position (7), second electrode (2) in tube body.According to the first electricity Following three kinds of working conditions can be set in the relative movement situation of pole (1) and second electrode (2).
The pressurizing device (6) of the first working condition, both ends nozzle pushes first electrode (1), second electrode (2) Cong Feigong Make position (7) (9) and be moved to operating position (8) (10), so that the liquid of inside cavity is born certain pressure, at this time first Applying electric pulse on electrode (1) and second electrode (2), electric pulse makes to form electric field in cavity, so that micropore is formed on cell membrane, To make the target substance in liquid sample be able to enter in cell.After the completion of electrotransfection, pressurizing device (6) can be by the first electricity Pole (1), second electrode (2) are returned to off-position from operating position.
Second of working condition, second electrode (2) is fixed in off-position (9), and pressurizing device (6) pushes first electrode (1) operating position (8) are moved to from off-position (7), so that the liquid of inside cavity is born certain pressure, at this time first Applying electric pulse on electrode (1) and second electrode (2), electric pulse makes to form electric field in cavity, so that micropore is formed on cell membrane, To make the target substance in liquid sample be able to enter in cell.After the completion of electrotransfection, pressurizing device (6) can be by the first electricity Pole (1) is returned to off-position (7) from operating position (8).
The third working condition, first electrode (1) is fixed in off-position (7), and pressurizing device (6) pushes second electrode (2) operating position (10) are moved to from off-position (9), the liquid of inside cavity is made to bear certain pressure, at this time Apply electric pulse on one electrode (1) and second electrode (2), electric pulse makes to form electric field in cavity, so that cell membrane is with penetrating Property, so that the target substance in liquid sample be made to be able to enter in cell.After the completion of electrotransfection, pressurizing device (6) can be by Two electrodes (2) are returned to off-position (9) from operating position (10).
Embodiment three
Liquid batch formula feed liquor mode
When feed liquor, sample introduction check valve (11) is opened, and sample check valve (12) is closed out, is opened gas valve (14), is used air pump By snorkel (13) extraction and tube body (3) isometric gas, target liq sample enters inside cavity from inlet tube (4); Or without using air pump by snorkel (13) extraction and tube body (3) isometric gas, using peristaltic pump by target liq sample This is pressed into inside cavity from inlet tube (4);After cavity is full of, sample introduction check valve (11) and gas valve (14) are closed.
After the completion of electrotransfection, open out sample check valve (12) and gas valve (14), with air pump by snorkel (14) to Tube body is filled with the gas isometric with cavity, and target liq sample is discharged from outlet tube (5);Or pass through ventilation without using air pump Pipe (14) is filled with the gas isometric with tube body to cavity, and target liq sample is discharged from outlet tube (5) by gravity;Target After liquid sample is completely exhausted out, sample check valve (12) is closed out, and sample introduction check valve (11) is opened, and repeats the dynamic of the above feed liquor Make.
Example IV
Intracavity liquid pressuring method
Target liq is entered by sample introduction check valve (11) and full of after cavity, and sample introduction check valve (11) is closed, and is led at this time It is overpressurized device (6) and specific pressure is applied to first electrode (1) and/or second electrode (2), make it from off-position (7) (9) it is moved to operating position (8) (10), completes the pressurization to liquid, the electrotransfection of liquid is carried out in pressurized state.
Embodiment five
Bioassay (one)
The CHO-S cell (Chinese hamster ovary cell) for being in logarithmic growth phase is collected, 1000 revs/min of revolving speed are centrifuged 5 points Clock abandons supernatant, and cell is resuspended with electrotransfection buffer, so that the density of cell is 1X107A/ml, addition need electrotransfection to turn The plasmid pCDNA3.1-GFP for entering cell makes the concentration 20ug/ml of plasmid, soft to be uniformly mixed.The intermittent streaming used Electrotransfection device as shown in Figure 1, the tube body both ends nozzle sealing end first electrode and second electrode sealed set is respectively adopted, First electrode is connect with hydraulic stem.Before liquid enters cavity, sample introduction check valve is opened, out sample closed check valve, opens ventilation Valve, with air pump by snorkel extraction and the isometric gas of tube body, target liq sample enters inside cavity from inlet tube; After cavity is full of, sample introduction check valve and gas valve are closed.First electrode is set to be maintained at off-position, liquid is not pressurized.This When apply electric pulse in first electrode and second electrode, 180 volts of pulse voltage, 6 milliseconds of pulse width, pulse number 2 times, Pulse spacing 1 second, electric pulse made to form electric field in cavity, so that cell membrane forms micropore, to make the target in liquid sample Substance is able to enter in cell.After the completion of electrotransfection, sample check valve and gas valve are opened out, passes through snorkel to pipe with air pump Body is filled with the gas isometric with cavity, and target liq sample is discharged from outlet tube.Cell suspension after transfection is placed in centrifugation In pipe, 1000 revs/min, it is centrifuged 5 minutes.Supernatant is abandoned, CD-OptiCHO culture medium is added, cell, inoculation and triangular pyramidal is resuspended Culture in glassware, culture density 2 × 106/ ml is subsequently placed on shaking table and cultivates, shaking speed 125rpm/min, condition of culture: temperature 37 DEG C of degree, gas concentration lwevel 5%.In fluorescence microscopy microscopic observation after 24 hours, and imitated with flow cytomery electrotransfection Rate and cell survival rate.
Embodiment six
Bioassay (two)
The CHO-S cell (Chinese hamster ovary cell) for being in logarithmic growth phase is collected, 1000 revs/min of revolving speed are centrifuged 5 points Clock abandons supernatant, and electricity consumption turns buffer and cell is resuspended, so that the density of cell is 1X107A/milliliter, addition need electrotransfection to turn The plasmid pCDNA3.1-GFP for entering cell makes the concentration 20ug/ml of plasmid, soft to be uniformly mixed.The intermittent streaming used Electrotransfection device as shown in Figure 1, the tube body both ends nozzle sealing end first electrode and second electrode sealed set is respectively adopted, First electrode is connect with hydraulic stem.Before liquid enters cavity, sample introduction check valve is opened, out sample closed check valve, opens ventilation Valve, with air pump by snorkel extraction and the isometric gas of tube body, target liq sample enters inside cavity from inlet tube; After cavity is full of, sample introduction check valve and gas valve are closed.Hydraulic stem pushes first electrode to be moved to work from off-position Position makes the liquid of inside cavity bear certain pressure.Apply electric pulse, pulse in first electrode and second electrode at this time 180 volts of voltage, 6 milliseconds of pulse width, pulse number 2 times, the pulse spacing 1 second, electric pulse made to form electric field in cavity, so that Cell membrane forms micropore, so that the target substance in liquid sample be made to be able to enter in cell.After the completion of electrotransfection, hydraulic stem First electrode can be returned to off-position from operating position.After the completion of electrotransfection, sample check valve and breather valve are opened out Door is filled with the gas isometric with cavity to tube body by snorkel with air pump, and target liq sample is discharged from outlet tube.It will turn Cell suspension after dye is placed in centrifuge tube, 1000 revs/min, is centrifuged 5 minutes.Supernatant is abandoned, OptiCHO culture medium is added and is resuspended Cell, inoculation and triangular pyramidal culture in glassware, culture density 2 × 106/ ml is subsequently placed on shaking table and cultivates, shaking speed 125rpm/min, condition of culture: 37 DEG C of temperature, gas concentration lwevel 5%.In fluorescence microscopy microscopic observation after 24 hours, it is used in combination Flow cytomery electrotransfection efficiency and cell survival rate.
Experimental result observation:
It can obviously observe after pulse, occur a small amount of bubble in cavity in experiment five, and test in six lumen bodies still It is so hydraulically full, and bubble-free generates.
Detect that experiment five cell dye rate of rotation is 61%-63%, cell survival rate 70%-81% after 24 hours;Experiment Six cell transfection rate 83-%87%, cell survival rate 81%-84%.
To sum up, pressurized cavity provided by the utility model can be improved electrotransfection efficiency, reduce the effect that bubble generates Fruit is obvious.
Although the utility model has been described in detail, the modification in the spirit and scope of the utility model is for ability Field technique personnel will be apparent.It should be understood that sheet described above and/or in the appended claims is practical The part of novel aspect and various embodiments and various features can be combined or completely or partially be interchangeable.Such as this Field technical staff will be realized that, in the description of aforementioned various embodiments, refers to those of another embodiment and implements Scheme can suitably with other combination of embodiment.In addition, it will be recognized by those of ordinary skill in the art that foregoing description is only to make To illustrate, and it is not intended to limit the utility model.

Claims (10)

1. a kind of intermittent streaming electrotransfection device, including tube body, first electrode, second electrode, which is characterized in that described first Electrode, second electrode are in the both ends nozzle of the tube body in parallel, and the inside of the tube body and first electrode, second electrode is formed With the cavity for accommodating target liq sample;At least one liquid entrance is arranged in the tube body and at least one liquid passes in and out valve Door, the liquid disengaging valve are located at the liquid entrance of the tube body, control the opening and closing of liquid entrance;It is described First electrode and/or second electrode can be relatively moved along the tube body.
2. intermittent streaming electrotransfection device according to claim 1, which is characterized in that the tube body setting at least two Liquid entrance.
3. intermittent streaming electrotransfection device according to claim 2, which is characterized in that the liquid entrance is located at institute It states between first electrode and second electrode.
4. intermittent streaming electrotransfection device according to claim 1, which is characterized in that the first electrode and/or Operating position and off-position is arranged in two electrodes.
5. intermittent streaming electrotransfection device according to claim 1, which is characterized in that snorkel is arranged in the tube body, Gas valve is arranged in snorkel.
6. intermittent streaming electrotransfection device according to claim 1, which is characterized in that the first electrode and the second electricity Sealing device is arranged in pole and tube body contact position.
7. according to the described in any item intermittent streaming electrotransfection devices of claim 5-6, which is characterized in that when the liquid into When valve and gas valve are closed out, the inside of the tube body and first electrode, second electrode forms closed cavity.
8. intermittent streaming electrotransfection device according to claim 7, which is characterized in that when the liquid disengaging valve and When gas valve is closed, the first electrode and/or second electrode are after tube body relative movement, in the closed cavity The pressure value in portion is 1.0-2.50Mpa.
9. intermittent streaming electrotransfection device according to claim 1, which is characterized in that described device further include make it is described The pressurizing device that first electrode and/or second electrode can be relatively moved along the tube body.
10. intermittent streaming electrotransfection device according to claim 1-6, which is characterized in that second electricity Pole is fixedly installed on one end nozzle of tube body.
CN201821468883.9U 2018-09-11 2018-09-11 A kind of intermittent streaming electrotransfection device Withdrawn - After Issue CN209098693U (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110885857A (en) * 2018-09-11 2020-03-17 苏州壹达生物科技有限公司 Intermittent flow type formula electrotransfection device
WO2024012261A1 (en) * 2022-07-14 2024-01-18 苏州壹达生物科技有限公司 Flow electroporation device

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110885857A (en) * 2018-09-11 2020-03-17 苏州壹达生物科技有限公司 Intermittent flow type formula electrotransfection device
CN110885857B (en) * 2018-09-11 2024-01-26 苏州壹达生物科技有限公司 Intermittent flow type formula electrotransfection device
WO2024012261A1 (en) * 2022-07-14 2024-01-18 苏州壹达生物科技有限公司 Flow electroporation device

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