CN116656744A - Use of microfluidic devices in cell electroporation or exogenous material introduction into cells - Google Patents

Use of microfluidic devices in cell electroporation or exogenous material introduction into cells Download PDF

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CN116656744A
CN116656744A CN202310676649.4A CN202310676649A CN116656744A CN 116656744 A CN116656744 A CN 116656744A CN 202310676649 A CN202310676649 A CN 202310676649A CN 116656744 A CN116656744 A CN 116656744A
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cells
cell
electrode
sodium
electroporation
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周乐乐
刘一凡
张蓉
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ShanghaiTech University
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ShanghaiTech University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present application relates to microfluidic devices, in particular to the use of microfluidic devices in cell electroporation. The microfluidic device comprises a microfluidic device chip, wherein the microfluidic device chip comprises an insulating substrate, a moving electrode, a conductive electrode and a gap between the moving electrodes. The cell electroporation only needs a few volts of direct current voltage, so that energy is saved; the device is assembled in a modularized way, and the signal generator can adjust the voltage and the voltage acting time by controlling the input voltage signal so as to meet the electroporation demands of different cells; the microfluidic device chip is simple to process and low in cost, and can select proper chip patterns according to different cell electroporation demands, so that cell electroporation can be efficiently realized.

Description

Use of microfluidic devices in cell electroporation or exogenous material introduction into cells
Technical Field
The present application relates to microfluidic devices, and in particular to the use of microfluidic devices in cell electroporation or the introduction of foreign substances into cells.
Background
Electroporation (electric corporation) is a technique that uses electric pulses to create transient pores in the cell membrane and to promote the transfer of molecules into the cell. Electroporation has been widely used in biotechnology, medicine and agriculture, such as gene transfer, cell fusion, tissue regeneration, plant transformation, etc.
The basic principle of electroporation is that when cells are exposed to high-intensity electric fields, the membrane potential changes and the lipid bilayer is permeable to small ions and molecules. The duration and intensity of the electrical pulse determine the degree and reversibility of membrane permeation. The optimal parameters for electroporation depend on the cell type, size, shape and environment, as well as the desired experimental results.
The existing electroporation devices generally require high voltage pulses, which cause a large number of cell death, decrease in electroporation efficiency, and problems of complicated device structure, increased cost, large energy consumption, etc.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, an object of the present application is to provide a use of a microfluidic device in electroporation of cells or introduction of exogenous substances into cells, for solving the problems of reduced electroporation efficiency, high cell death rate, complex device structure, increased cost, and high energy consumption in the prior art.
To achieve the above and other related objects, the present application provides a use of a microfluidic device in cell electroporation, the microfluidic device comprising a microfluidic chip, a power supply and a signal generator, the microfluidic chip comprising an insulating substrate and an electrode pattern formed on the insulating substrate, a dielectric layer being provided between the insulating substrate and the electrode pattern, the electrode pattern comprising a plurality of moving electrodes and a plurality of conductive electrodes, a gap being provided between adjacent moving electrodes; the motion electrode is connected with the conductive electrode through a conductive element, and the conductive electrode is connected with a power supply through a signal generator; the exogenous material is selected from a polypeptide, a protein, or a polynucleotide.
Preferably, the motion electrode is rectangular; preferably, the motion electrode is square; more preferably, the moving electrode is square with a side length of 0.5-4 mm.
The present application provides a method of electroporation of cells, the method comprising the steps of: and adding the liquid drop containing the cells onto the microfluidic device, controlling voltage by a signal generator of the microfluidic device, enabling the liquid drop to pass through a gap between the moving electrodes, and realizing cell electroporation by utilizing the electric field effect formed by the gap.
As described above, the use of the microfluidic device of the present application in cell electroporation has the following beneficial effects:
1. when the cell electroporation is carried out, only a few volts of direct current voltage is needed, so that the energy is greatly saved.
2. The device used in the application is assembled in a modularized way, and the signal generator can adjust the voltage and the voltage acting time by controlling the input voltage signal, thereby meeting the electroporation demands of different cells.
3. The microfluidic device chip used in the application has simple processing and low cost, and can select proper chip patterns according to different cell electroporation demands, thereby efficiently realizing cell electroporation.
4. The application aims to realize the cell electroporation directly on the digital microfluidic chip, not only simplifies the electroporation operation flow, but also can conveniently combine other experimental operations, and further expands the functions of the digital microfluidic.
Drawings
Fig. 1 shows a schematic structural diagram of a microfluidic device chip according to the present application. Wherein reference numeral 1 is a moving electrode, reference numeral 2 is a wire, reference numeral 3 is a conductive electrode, reference numeral 4 is a gap of the moving electrode, reference numeral 5 is a dielectric layer, and reference numeral 6 is an insulating substrate.
Fig. 2 shows a physical diagram of a microfluidic device chip according to the present application.
Fig. 3 shows a graph of the results of the movement of a droplet of the application on a microfluidic device chip.
FIG. 4 is a graph showing the result of electroporation of cells of the present application into a fluorescent protein plasmid.
Detailed Description
The present application provides the use of a microfluidic device in cell electroporation or the introduction of foreign substances into cells.
The microfluidic device comprises a microfluidic chip, a power supply and a signal generator, wherein the microfluidic chip comprises an insulating substrate and electrode patterns formed on the insulating substrate, a dielectric layer is arranged between the insulating substrate and the electrode patterns, the electrode patterns comprise a plurality of moving electrodes and a plurality of conducting electrodes, and gaps are arranged between adjacent moving electrodes; the motion electrode is connected with the conductive electrode through a conductive element, and the conductive electrode is connected with a power supply through a signal generator; the exogenous material is selected from a polypeptide, a protein, or a polynucleotide.
In some embodiments, the signal generator controls the voltage of each moving electrode separately.
In some embodiments, the number of moving electrodes is 2 or more, for example 2 to 5, 5 to 8, 8 to 10 or more.
In some embodiments, the conductive element is a wire.
In some embodiments, the moving electrode is rectangular. More specifically, the moving electrode is square.
Further, the moving electrode is square with the side length of 0.5-4 mm. Specifically, the side length of the motion electrode is 0.5-1mm, 1-1.5mm, 1.5-2mm, 2.5-3mm, 3-3.5mm or 3.5-4mm.
In some embodiments, the width of the gap between the moving electrodes is 10-300 μm. More specifically, the width of the gap between the moving electrodes is 10-20 μm, 20-30 μm, 30-50 μm, 50-70 μm, 70-90 μm, 90-130 μm, 130-170 μm, 170-180 μm, 180-190 μm, 190-200 μm, 210-230 μm, 230-250 μm, 250-270 μm, 270-290 μm or 290-300 μm.
In some embodiments, the moving or conductive electrode has a resistivity of 0.004 Ω -cm or less.
In some embodiments, the insulating substrate is formed of a silicon material. Specifically, the material constituting the insulating substrate is low-doped substrate silicon.
In some embodiments, a top surface of the electrode pattern may be exposed. The device is convenient for scientific researchers to operate, saves materials and is convenient for processing and manufacturing.
In some embodiments, the electrodes and/or power connection elements may be formed by photolithography or etching after forming a conductive layer on an insulating substrate.
In some embodiments, a dielectric layer is disposed between the insulating substrate and the electrode pattern.
In some embodiments, the dielectric layer may have a relative permittivity greater than 3. The relative permittivity is a physical parameter that characterizes the dielectric or polarization properties of a dielectric material. The relative dielectric constants of different materials at different temperatures are different, and capacitors or related elements with different performance can be manufactured by utilizing the characteristics
In some embodiments, the dielectric layer is comprised of silicon dioxide.
In some embodiments, the electrode pattern is processed from conductive silicon.
In some embodiments, the signal generator is connected to the conductive electrode by a conductive element; the power supply is connected with the signal generator through the conductive element.
In the application, the signal generator can be used for generating large and complex analog waveforms in sequence or by using real-time data flow.
In some embodiments, the power source is a dc power source; more specifically, the power supply is selected from the group consisting of an NI PXI power supply; the signal generator is selected from the group consisting of NI PXI waveform generator.
In some embodiments, the microfluidic device is modified on a microfluidic chip as described in application No. 202211124391.9. The reconstruction method comprises the following steps: a signal generator is arranged between the electrode and the power supply so as to control the voltage parameter of the moving electrode in the cell electroporation.
The application also provides a method of electroporation of cells, the method comprising the steps of: and adding the liquid drop containing the cells onto the microfluidic device, controlling voltage by a signal generator of the microfluidic device, enabling the liquid drop to pass through a gap between the moving electrodes, and realizing cell electroporation by utilizing the electric field effect formed by the gap.
In some embodiments, when the droplet is applied to the microfluidic device, the droplet is placed on the electrode of the microfluidic chip so that the droplet contacts at least two electrodes, and the droplet is moved from one electrode to the other electrode along the preset motion path by adjusting the voltage to the motion electrode on the preset motion path.
In some embodiments, the voltage difference between the moving electrodes is 0-30V. More specifically, the voltage difference of the moving electrode is 0-1V, 1-4V, 4-8V, 8-12V, 12-16V, 16-20V, 20-24V, 24-28V or 28-30V.
In some embodiments, the droplet comprises isolated cells and an ionic surfactant. More specifically, the cells are selected from prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. The cells include a number of cell types such as prokaryotic cells like E.coli or Bacillus subtilis, fungal cells like yeast cells or Aspergillus, insect cells like S2 Drosophila cells or Sf9, or animal cells like fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or human cells. The droplets may contain an ionic surfactant therein. In some embodiments, the ionic surfactant may include a cationic surfactant and an anionic surfactant. In some embodiments, the cationic surfactant may include one or more of dodecyl trimethyl ammonium bromide, cetyl trimethyl ammonium bromide, tetradecyl trimethyl ammonium bromide, octadecyl trimethyl ammonium chloride, distearyl hydroxyethyl methyl ammonium methyl sulfate, cetostearyl alcohol ether-21. In some embodiments, the anionic surfactant may include one or more of sodium dodecyl sulfate, sodium dodecyl alcohol polyoxyethylene ether sulfate, ammonium dodecyl sulfate, dodecylbenzenesulfonic acid, triethanolamine lauryl sulfate, sodium secondary alkyl sulfonate, sodium fatty alcohol isethionate, sodium N-lauroyl sarcosinate, sodium cocoyl methyl taurate, sodium N-lauroyl glutamate, magnesium amidopolyoxyethylene ether sulfate, sodium laureth carboxylate, dodecyl phosphate, potassium dodecyl phosphate, dodecyl phosphate triethanolamine, disodium dodecyl polyoxyethylene ether sulfosuccinate, sodium alpha-alkenyl sulfonate.
Further, the concentration of the surfactant in the droplet is 0.08 times or less the critical micelle concentration. Specifically, the concentration of the surfactant in the liquid drop is selected from 0.01-00.3 times, 0.03-0.04 times, 0.04-0.05 times, 0.05-0.06 or 0.06-0.08 critical micelle concentration.
In some embodiments, the droplets further comprise a biological sample, such as whole blood, lymph, serum, plasma, sweat, tears, saliva, sputum, cerebrospinal fluid, amniotic fluid, semen, vaginal secretions, serum, synovial fluid, pericardial effusion, ascites, pleural effusion, exudates, secretion, cyst fluid, bile, urine, gastric juice, intestinal juice, fecal samples, single-cell or multi-cell containing fluids, organelle containing fluids, fluidized tissue, fluidized organisms, multicellular organism containing fluids, biological swabs, and biological washes. In addition, the droplets may include reagents such as water, deionized water, saline solution, acidic solution, alkaline solution, detergent solution, and/or buffer. The droplets may comprise a protein or an enzyme. The droplets may comprise nucleic acids, such as DNA, genome DNA, RNA, mRNA, or analogs thereof; nucleotides such as deoxyribonucleotides, ribonucleotides or analogs thereof such as analogs having a terminator moiety.
In some embodiments, the droplets comprise an enzyme such as a polymerase, ligase, recombinase, or transposase; binding partners such as antibodies, epitopes, streptavidin, avidin, biotin, lectin or carbohydrates; or other biochemically active molecules. In some embodiments, the droplet may include reagents, such as reagents for a biochemical protocol, a nucleic acid amplification protocol, an affinity-based assay protocol, an enzymatic assay protocol, a sequencing protocol, and/or a biological fluid analysis protocol.
The term "critical micelle concentration" refers to the concentration of surfactant on the interface to adsorb a common monolayer, when the surface adsorption reaches saturation, the surfactant molecules cannot be continuously enriched on the surface, but the hydrophobic effect of the hydrophobic group still strives to promote the hydrophobic group molecules to escape from the water environment, so that the surfactant molecules self-aggregate in the solution, i.e. the hydrophobic groups aggregate together to form a core, the hydrophilic groups outwards contact with water to form a shell, the simplest micelle is formed, and the concentration of the surfactant when the micelle begins to form is called the critical micelle concentration.
The term "droplet operations" means any manipulation of one or more droplets in a droplet microfluidic chip. The droplet operations may include, for example: loading the droplets into a droplet microfluidic chip; dispensing one or more droplets from a droplet source, splitting, separating, or dividing the droplets into two or more droplets; transporting the droplets from one location to another in any direction; combining or combining two or more droplets into a single droplet; diluting the droplets; mixing the droplets; agitating the droplets; deforming the droplets; holding the droplets in place; disposing of the droplets; other droplet operations described herein; and/or any combination of the foregoing. The terms "merging," "combining," and the like are used to describe forming one droplet from two or more droplets. It will be understood that when such terms are used with reference to two or more droplets, any combination of droplet operations sufficient to cause two or more droplets to be combined into one droplet may be used. For example, "merging droplet a with droplet B" may be achieved by transporting droplet a into contact with static droplet B, transporting droplet B into contact with static droplet a, or transporting droplet a and droplet B into contact with each other. The terms "split", "separate" and "divide" are not intended to imply any particular result in terms of the volume of the resulting droplets (i.e., the volumes of the resulting droplets may be the same or different) or the number of resulting droplets (the number of resulting droplets may be 2, 3, 4, 5 or more). The term "mixing" refers to droplet operations that result in a more uniform distribution of one or more components within a droplet. Examples of "loading" droplet operations include microdialysis loading, pressure assisted loading, robotic loading, passive loading, and pipette loading. Droplet operations may be electrode-mediated. In some cases, droplet operations are further facilitated by the use of hydrophilic and/or hydrophobic regions on the surface and/or by physical barriers.
The term "resistivity" refers to the ratio of the product of the resistance of an original (20 ℃ C. At ordinary temperature) made of a certain substance and the cross-sectional area to the length, and is a physical quantity used to represent the resistance characteristics of various substances. The resistivity is independent of the length, cross-sectional area, etc. of the conductor, is an electrical property of the conductor material itself, is determined by the material of the conductor, and is temperature dependent.
Other advantages and effects of the present application will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present application with reference to specific examples. The application may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present application.
Before the embodiments of the application are explained in further detail, it is to be understood that the application is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the application is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the application; in the description and claims of the application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present application may be used to practice the present application according to the knowledge of one skilled in the art and the description of the present application.
Example 1 microfluidic device chip preparation
The microfluidic chip can be manufactured by the following steps:
a, cleaning the silicon wafer for 10 minutes at 120 ℃ by using piranha solution;
b, putting the silicon wafer obtained in the step a into hydrofluoric acid with the mass fraction of 50% to clean for 15-30 seconds;
c, washing and spin-drying the silicon wafer obtained in the step b by using deionized water to finish a washing step;
d, processing the conductive silicon on the top layer of the silicon wafer obtained in the step c according to the figure 1 through photoetching and etching. Cutting the processed silicon wafer to a proper size, packaging, and connecting with a power supply to complete the microfluidic device chip, wherein the prepared microfluidic device chip is shown in fig. 2.
Example 2 cell electroporation experiments Using microfluidic device chips
Material preparation:
droplet preparation: collecting bacteria in BL 21-containing bacteria (i.e. escherichia coli) culture solution, cleaning with 10% glycerol by mass fraction, repeating for 2-3 times, adding plasmid with green fluorescent protein GFP or RED fluorescent protein RED and having nucleotide sequence as SEQ ID No.1 or SEQ ID No.2, and making plasmid concentration in the liquid drop reach 200ng/ml.
Preparing a microfluidic device chip: a microfluidic device chip was prepared as in example 1, with a gap of 20 μm between the moving electrodes.
Cell electroporation experiments:
a 1 μl volume of the droplet was placed on a microfluidic device chip, a 10V potential difference was generated by the adjacent moving electrodes, and at this voltage, the droplet movement speed was v=2 mm/s, and the cell moved integrally with the droplet within the droplet at a speed of approximately 2mm/s. A physical diagram of the movement of the droplet through the electric field for time t=d/v=10 ms is shown in fig. 3. Immediately after the movement was completed, the mixture was aspirated by a pipette, added to 1ml of LB medium, shake-cultured at 37℃for 1 hour, taken out, added to 5ml of LB medium, and mixed according to 1:1000 kana antibiotics (corresponding to resistance gene only on plasmid) were added and the shaker was overnight. The cultured system was imaged under a microscope, and as shown in fig. 4, more than 90% of the fluorescent protein was expressed in the cells, so that plasmids carrying green fluorescent protein GFP or RED fluorescent protein RED could be efficiently introduced into the cells through the microfluidic device.
In this experiment, the electric field strength of the gap of the moving electrode, i.e., the electric field strength of cell electroporation, was calculated by the following formula:
E gap of ＝(E + -E - ) D, wherein E Gap of Electric field strength for electroporation of cells, E + For the voltage of the moving electrode in the direction of droplet movement, E - The voltage of the moving electrode at the position of the liquid drop is shown as D, and the distance of the gap of the moving electrode is shown as D.
In this experiment, cells were in a droplet and electroporation occurred as the droplet moved, only when the cells moved through the gap between the electrodes.
The above examples are provided to illustrate the disclosed embodiments of the application and are not to be construed as limiting the application. Further, various modifications of the methods set forth herein, as well as variations of the methods of the application, will be apparent to those skilled in the art without departing from the scope and spirit of the application. While the application has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the application should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the application which are obvious to those skilled in the art are intended to be within the scope of the present application.

Claims (10)

1. The application of the microfluidic device in cell electroporation or exogenous material introduction into cells comprises a microfluidic chip, a power supply and a signal generator, wherein the microfluidic chip comprises an insulating substrate and electrode patterns formed on the insulating substrate, a dielectric layer is arranged between the insulating substrate and the electrode patterns, the electrode patterns comprise a plurality of moving electrodes and a plurality of conducting electrodes, and gaps are arranged between adjacent moving electrodes; the motion electrode is connected with the conductive electrode through a conductive element, and the conductive electrode is connected with a power supply through a signal generator; the exogenous material is selected from a polypeptide, a protein, or a polynucleotide.
2. The use according to claim 1, wherein the moving electrode is rectangular; preferably, the motion electrode is square; more preferably, the moving electrode is square with a side length of 0.5-4 mm.
3. Use according to claim 1, characterized in that the width of the gap between the moving electrodes is 10-300 μm.
4. The use according to claim 1, wherein the cells are selected from prokaryotic or eukaryotic cells; preferably, the cell is selected from the group consisting of E.coli, bacillus subtilis, yeast cells, aspergillus, S2 Drosophila cells, sf9 cells, fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, or HEK 293 cells.
5. A method of electroporation of cells, the method comprising the steps of: a droplet containing cells is applied to a microfluidic device for use according to any one of claims 1-4, the voltage is controlled by a signal generator of the microfluidic device, the droplet is caused to pass through the gap between the moving electrodes, and electroporation of cells is achieved by the action of an electric field formed by the gap.
6. The method of claim 5, wherein the voltage difference between the moving electrodes is 30V or less and not 0.
7. The method of claim 5, wherein the cells in the droplet are selected from prokaryotic cells or eukaryotic cells.
8. The method of claim 7, wherein the cell is selected from the group consisting of E.coli, bacillus subtilis, yeast cell, aspergillus, S2 Drosophila cell, sf9 cell, fibroblast, CHO cell, COS cell, NSO cell, heLa cell, BHK cell, and HEK 293 cell.
9. The method of claim 5, wherein the droplets further comprise an ionic surfactant; the ionic surfactant is selected from one or more of dodecyl trimethyl ammonium bromide, hexadecyl trimethyl ammonium bromide, tetradecyl trimethyl ammonium bromide, octadecyl trimethyl ammonium chloride, distearyl hydroxyethyl methyl ammonium sulfate, cetostearyl alcohol ether-21, sodium dodecyl sulfate, sodium dodecyl alcohol polyoxyethylene ether sulfate, ammonium dodecyl sulfate, dodecyl benzene sulfonic acid, triethanolamine lauryl sulfate, sodium secondary alkyl sulfonate, sodium fatty alcohol hydroxyethyl sulfonate, sodium N-lauroyl sarcosinate, sodium cocoyl methyl taurate, sodium N-lauroyl glutamate, magnesium amidopolyoxyethylene ether sulfate, sodium laureth carboxylate, dodecyl phosphate, potassium dodecyl phosphate, triethanolamine dodecyl phosphate, disodium dodecyl polyoxyethylene ether sulfonate and sodium alpha-alkenyl sulfonate.
10. The method of claim 9, wherein the concentration of the surfactant in the droplet is less than 0.08 times the critical micelle concentration.
CN202310676649.4A 2023-06-08 2023-06-08 Use of microfluidic devices in cell electroporation or exogenous material introduction into cells Pending CN116656744A (en)

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