KR20070022212A - Biochip electroporator and its use in multi-site, single-cell electroporation - Google Patents

Abstract

The introduction of genetic material or biologically interesting molecules into cells is an interesting process for experimentation and use. For this purpose, electroporation is a widely used method, but electroporation of single adherent cells is still insufficient. The present invention discloses a device capable of electroporating any kind of cell attached to a substrate at a stage of development, where an electrical signal is driven and applied to a single adherent cell in culture to electroporate the cell. The method also discloses a method of electroporating single adherent cells using the device of the present invention.

Electroporation, Biochip, Microelectrode

Description

BIOCHIP ELECTROPORATOR AND ITS USE IN MULTI-SITE, SINGLE-CELL ELECTROPORATION}

The present invention relates to a method and an apparatus that can each electroporate any cell that adheres to a substrate at a development stage.

Electroporation is widely used to introduce genetic material or biologically interesting molecules into cells. By applying an electrical signal to the cell to create openings of pores in the cell membrane, molecules in the extracellular solution enter the cell.

Many electroporation methods have been established. These methods can be divided into two categories:

(i) electroporation of cell populations

(ii) single cell electroporation

Electroporation of Cell Population

Most commonly used and representative of the first electroporation method, cell group electroporation in suspension is usually performed after removal of cells from the culture substrate using an enzyme such as trypsin. Thereafter, the cells are suspended in electroporation medium and a high voltage (more than 1000 V) is applied between two large metal electrodes (typically 2 or 4 mm apart). The cell suspension is transferred to a suitable incubator where the cells are free to settle and attach to the bottom of the substrate to be cultured.

This method has some problems.

Cannot control the active voltage drop across each cell During the application of high pressure many cells close to the electrode are destroyed, and other cells are electroporated to different degrees according to their position in the electric field between the electrodes and the change in the position of the non-uniform applied electric field.

The method can only be used for cell populations.

-Cells must be separated from the attached substrate, causing damage to the cells during the process.

Single Cell Electroporation

Methods for electroporating single cells are disclosed in US Pat. No. 6,300,108 to Rubinsky et al.

The method utilizes a cell sized electroporation incubator integrated into a silicon microchip. First, cells are introduced into the incubator and placed on openings in the silicon substrate, followed by electroporation and penetration of the dielectric construct.

This method has several problems.

Not only is the system installation complex, the integration of a series of many "single cell electroporators" into one microchip has not yet been achieved and will be costly as is the state of the art.

Since the cell membrane cannot be extended to attach to the substrate, it is impossible to develop and develop all kinds of cells (most of the cells) that need to be attached.

Cells should be placed in a microchamber, which is a difficult process and is far from the protocol commonly used for cell culture.

As mentioned above, it is obvious that an electroporation apparatus is needed to overcome the above problem.

The Applicant has found an apparatus for each electroporation of any cell adhering to a substrate in order to solve all the above problems.

It is therefore an object of the present invention to provide an electroporation apparatus comprising a waveform generator and a control system for applying a signal transfer to a series of microelectrodes and a single microelectrode of a preselected biochip.

Another object of the invention is a series of microelectrodes included on a suitable insulating layer mounted on a solid substrate; Means for electrically connecting the microelectrode with a switch system; It provides a biochip 13 including a cell incubator that can develop and attach cells in contact with the series of microelectrodes on a surface formed by the insulating layer including a series of microelectrodes on the solid substrate. It is.

Another object of the invention is a method of electroporating a cell using the device, the method comprising further electroporation of the electroporated cells obtained with the same device as the at least one adherent cell.

In addition to the features and advantages of the present invention, the above object will be better understood from the following detailed description of the invention.

The nature of the invention will be apparent from the following detailed description and the preferred examples which are presented to illustrate it and which do not limit the scope of the invention.

The present invention can overcome the problems of the current method through an apparatus comprising an electrical waveform generator and a biochip comprising a series of microelectrodes and a control system for transferring a signal to a preselected microelectrode of the biochip. .

Preferably, the control system consists of a personal computer having a software program capable of designing various waveforms in a waveform generator and selecting a microelectrode using a switch system.

1 diagrammatically shows an apparatus according to the invention listing a personal computer 10, a switch system 11, a signal generator 12 and a biochip 13.

The personal computer 10 is provided with a software program that allows the operator to fully control the electroporation process and programs the shape and timing of the signal waveform to be delivered to the biochip.

The electrical waveform generator 12 used in the device according to the present invention is a conventional device which is readily available on the market, and the switch system 11 can be easily designed and implemented by those skilled in the art.

By a series of microelectrodes, a suitable insulating layer on a solid substrate, means for electrically connecting said series of microelectrodes to a switch system, and said insulating layer, wherein said cells are developed and comprise a series of microelectrodes on said solid substrate. A biochip 13 comprising a cell incubator that can be attached in contact with the series of microelectrodes on the surface to be formed will be described in detail in accordance with the following preferred embodiments.

2A and 2B show a structure of a biochip according to the present invention as a preferred embodiment. The structure will be described with reference to two figures in which reference numerals refer to the same or corresponding elements in both directions.

Figure 2a shows that the biochip is mounted on the dielectric support 21, which in turn, is mounted on the dielectric support 21 is equipped with a cell incubator 24 including an opening 26 located at its center A base surface formed by an insulating layer comprising a series of microelectrodes having a size corresponding to a cell to be electroporated on a substrate 27 and mounted in a cell incubator 24 which imparts the opening 26. The series of microelectrodes 20 integrated on an insulating layer mounted on the solid substrate 27 is mounted. The bottom of the opening 26 is an upper surface of the biochip 13 in which cells are developed by contacting and attaching to a series of microelectrodes. The series of microelectrodes is electrically connected to the conductive pads 29 through the conductive labeling material 28, and in turn, the conductive pads 29 surround the openings 26 through the wire connection 23. It is connected to a pair of external parallel connectors 22 which extends to the outside of 24.

As shown in FIG. 1, the biochip 13 is electrically connected to the switch system 11 through connecting means such as an electric wire connected to the connector 22 by a plug.

In a preferred embodiment of the present invention, the dielectric support 21 is composed of vetronite. Equivalent materials such as glass or ceramics are not excluded.

In a preferred embodiment of the present invention, the solid substrate of the biochip 13 has a suitable shape, for example a square having a suitable side dimension, preferably about 10 mm, and is covered with a dielectric layer of SiO ₂ . It consists of semiconductor substrates, such as a board | substrate. The only material available for such devices is silicon. For example, glass or another transparent substrate can also be used. However, silicon has the advantage of integrated and repeatable manufacturing techniques derived from microelectronics that precisely control the parameters of each device.

In some cases, a transparent substrate is preferred, which uses a reversed phase microscope for cell observation.

Two electrodes 25 of large dimension (about 1 square mm) integrated into a solid substrate covered by an insulating layer 27 serve as ground reference. On the other hand, it serves as a cold terminal for the electroporation current and the wiring, and may comprise the electrode consisting of Ag / AgCl as a ground potential reference point.

In the series of microelectrodes 20 having a size corresponding to the cells to be electroporated and included in the insulating layer covering the solid substrate, since each microelectrode is connected to the switch system 11 respectively, the microelectrodes are individually separated and operated. The electroporation process can be controlled very accurately and timely. The microelectrodes can use either conductive or capacitive forms.

2B shows the arrangement of twenty microelectrodes 20 (no scale). Here, each microelectrode including the active region (the region carrying the signal to the cell) is 20 mu m in length and 20 mu m in width. This arrangement gives a relatively good balance of the microelectrodes, which are many stimulating positions, and the interconnection of the microelectrodes of the biochip required for an external source of microelectrodes. Depending on the type of cells to be electroporated, more and more microelectrodes can be obtained.

To aid cell adhesion on the biochip, adhesion molecules (eg, poly-L-lysine) may be applied to the surface of the opening 26 of the cell incubator 24 before starting cell culture. Many adhesion molecules can be used to coat the substrate, and excellent cell adhesion is possible at a distance of several tens of nanometers between the cell membrane and the semiconductor substrate covered with the insulating layer 27 included in the opening of the incubator.

 As long as they have a size corresponding to the cell size, the microelectrodes can have other shapes and dimensions. Microelectrodes are usually planar or generally designed to ensure good adhesion of the cell membrane on the surface (usually at least 10 percent of the total cell membrane should be in contact with the microelectrode). The dimensions of the microelectrodes range from 1 μm to 50 μm. The microelectrode is made of a conductive material suitable for a living body capable of sending the necessary current / voltage AC and / or DC signals to the cell.

The conductive microelectrode may be composed of a material selected from several biocompatible metals and alloys, and may be a single layer or multiple layers.

3 shows a preferred embodiment of the preferred conductive microelectrode used in the biochip of the device according to the present invention. The microelectrode is SiO ₂ It is embodied on the silicon substrate 31 which is protected by the insulating layer 32. The microelectrode and the labeling material 38 connected thereto are “sandwiched” with an aluminum layer 34 inserted between two titanium nitride (TiN) layers 33, covered with a gold layer 37. Is done. This solution combines the excellent thermal cost savings and biocompatibility of titanium nitride with the low electrical resistance of aluminum and has several advantages. The microelectrode is SiO ₂ Separated by an insulating layer 35. The portion of the chip that is not directly exposed to the cells (including the labeling material 38 connecting the microelectrodes) is covered with a silicon nitride (Si ₃ N ₄ ) layer 36 to expose only the active region of the microelectrode. . Other materials such as SiO ₂ or organic polymers can be used for this purpose.

In another embodiment of the present invention, the microelectrode of the conductive type can introduce a thin insulating material to protect the active region of the microelectrode itself. With reference to FIG. 6, the microelectrode comprises a conductive layer 61 (which may be composed of a metal or semiconductor or sandwich form of TiN / Al / TiN described above, equivalent material and mixture of materials), and a cell through conductive coupling. Is formed on the insulating substrate 60 using a thin insulating layer 64 (generally thinner than 25 nm thick) for electroporation. The microelectrode is separated by the insulating material 62 and protected by the unexposed regions by the passivation layer 63.

 According to another embodiment of a microelectrode of a biochip according to the present invention, a microelectrode may be implemented by using a metal oxide semiconductor (MOS) technique to obtain a high electrode density, and each electrode may be a semiconductor memory. It may be addressed in a manner similar to a cell.

Referring to FIG. 4, a series of metal oxide semiconductor field effect transistors (MOSFETs) include a p-type substrate 40 and two layers consisting of a drain and a source of each transistor. It is embodied on silicon substrate 40 by conventional microelectrode techniques implanting into n-doped regions 41, 42. The gate 43 of the MOSFETs runs on n + doped polysilicon and is common to all devices in the row direction (word line). The drains 41 of all the above devices are connected in a column direction using a metal contact plug (typically realized by tungsten) and a metal wire 44 together (bit line). The metal is surrounded by isolation oxide 45. The source 42 of the transistor is connected via a metal (usually tungsten) plug 46 to a thin gold layer 47 which acts as an active microelectrode. The unexposed areas of the chip are protected by the passivation layer 48.

Switching the p-type region to the n-type region and n doping to p doping can implement a pMOSFET instead of the nMOSFET described above with similar results in terms of integration.

In this way, the minimum distance between the two microelectrodes can be as low as 1 μm, so that they can reach the single cell and be electroporated with high spatial resolution.

5 shows a schematic of the series of microelectrodes described above. The microelectrode 50 has a drain 52 connected to a word line 53 and a gate connected to a source 51 of MOSFETs connected to a bit line 55. If the gate of the MOSFET maintains a voltage V _G higher than the threshold voltage V _TH , the drain remains V _D , the source is open, and the later device's voltage is V _D -V _TH . This mechanism can be used to control the voltage of the microelectrode 50 connected to the source 51 of the selected MOSFET by regulating the voltages of the word line 53 and the bit line 55 defining the arrangement position. .

The electroporation device of the present invention is independent of the process for cell coating , batching and culturing without the need for specific deadlock molecules.

With the device according to the invention electroporation is carried out with the following advantages.

Electroporation on adherent cells

Electroporation on multiple different single cells in the same medium

-Randomly selected electroporation timing for each target cell

The amount of electroporation controlled (ie pore count, pore size, duration) for each target cell by regulating the voltage increasing across the membrane through electrical coupling of each cell / microelectrode

In the case of cells large enough to cover different microelectrodes, electroporation at different locations of the same cell using different microelectrodes

Because of this property, the method according to the invention is very useful for high throughput screening of molecules that cannot penetrate the cell membrane. Pharmaceuticals, genetic constructs and proteins can be tested individually in many single cells on the same microchip. The timing of delivery and combination of different molecules can be tailored to each target cell.

The method according to the invention can be used not only at the "discovery" stage of a new drug, but also at basic examination (eg screening of gene function). Substantial increases in experimental results and reductions in experimental costs are expected. Because of the good efficacy and control of cell electroporation, the method will be useful in the "production" phase of pharmaceuticals and gene therapy from transfected cells.

The electroporation method obtainable by the above-mentioned electroporation substantially includes the following steps.

Culturing the cells until the attachment step;

Adding at least one compound to the medium to be electroporated in at least one single cell of said cell;

Selecting at least one single cell and selecting at least one microelectrode to which the selected single cell is attached; And

Generating at least one electrical signal suitable for electroporation of the at least one single cell with at least one compound and sending the generated electrical signal to the microelectrode to which the selected single cell is attached

1 diagrammatically shows the apparatus.

Figure 2 shows a biochip according to the present invention, where 2a is a sectional view, 2b is a plan view.

3 is a cross-sectional view of a conductive microelectrode according to the present invention.

4 shows a plan view and a cross-sectional view of a series of MOSFET conductive microelectrodes according to the present invention.

5 diagrammatically shows a series of MOSFET conductive microelectrodes according to the present invention.

6 shows a cross-sectional view of a capacitive microelectrode according to the invention.

7 and 8 show two specific waveforms of the electroporation drive signal. Variables such as time and amplitude are described in the examples below.

Now, four experiments performed by the method using the apparatus according to the present invention will be described in the following examples.

Example  One : Cos -7 cells transfected with oligonucleotides ( transfection )

The goal of this experiment is to transfect each target cell with double-stranded DNA oligonucleotides.

Using oligonucleotides labeled with a fluorescent material, their penetration into target cells after electroporation is detected by the presence of intracellular fluorescence.

Cos-7 cells continue to be cultured using conventional media. After incubation for 2 days, trypsinized, resuspended in medium and plated on a biochip cell incubator according to the invention at a density of about 35,000 cells / cm ² of the chip surface.

Prior to plate incubation, the chip surface was carefully washed, rinsed, dried and sterilized by UV light. The biochips were then coated with poly-L-lysine and dried by adsorption from 20 μg / ml aqueous solution for 2 hours. Cells were incubated with 37 ° C., 5% carbon dioxide.

Double-stranded DNA oligonucleotides 87-113 pairs were synthesized with one residue each labeled with a fluorescent dye (NED or FAM). The oligonucleotide was dissolved in water during synthesis and the final concentration was about 40 ng / μl. The oligonucleotide solution was diluted 1: 1 in HBS 2 × before electroporation. After removing the medium, about 60 μl of the oligonucleotide solution was applied to the incubator just before electroporation.

Each cell developing in contact with one microelectrode was confirmed by observing under a microscope. After selecting one of the microelectrodes in contact with the cells as the control system, the same run was repeated for each target cell by sending the appropriate electroporation signal.

A waveform generator driven by a personal computer generates an electrical signal carried to a biochip in accordance with the present invention. The signal is sent via a 50 ohm coaxial cable to a switch system that applies the transfer of the signal to a single preselected microelectrode of the biochip. The external parallel connector is connected to the biochip through a printed circuit. The reference ground is made by connecting the previous switch ground with the Ag / AgCl electrode in the electrolyte solution in which the cultured cells are present.

7 shows the waveform of an electroporation signal. Five rows of 25 spherical pulses repeated in a 500 ms cycle (duration: 1 ms, rise / fall time: 10 ms) are known to be suitable for successful transfection of Cos-7 cells.

Electroporated cells were washed with standard physiological solution and observed by fluorescence microscopy. Successful transfection of each target cell was confirmed by the presence of cell fluorescent. After transfection, cell survival was monitored for 24 hours by observing the morphology of the cells.

About 80% of the target cells were successfully transfected and high survival rates (50-80%) were obtained with pulses of 6-10 V amplitude and 1-10 Hz rise / fall time.

The degree of transfection determined from intracellular fluorescence intensity is directly related to amplitude and inversely related to rise time. In contrast, cell viability decreased with voltage amplitude and increased with rise time. The cycle between rows is important. This is because no transfection occurs when the cycle is larger than 500 ms, and the cell viability is substantially reduced when the cycle is smaller than 500 ms. As a compromise of the highest rate of transfection and cell survival, we typically used pulses with 9 V amplitude and 10 Hz rise time.

Example  2 : Cos -7 cells transfected with DNA plasmid vector

The goal of another experiment carried out by the method according to the invention is to transfect each target cell with a DNA plasmid vector.

Each Cos-7 target cell was transfected with a plasmid vector for green fluorescent protein (GFP) using the device according to the invention. Synthesis of GFP in the cytoplasm was observed under fluorescence microscopy, indicating successful transfection.

Transfection with plasmid vectors is a commonly used method in laboratories to investigate gene function.

The Cos-7 target cells were the same as in the previous experiment.

5 kB of Cos-7 cells containing GFP coding genes were used. After amplifying and clarifying the DNA, it was dissolved at 50 μg / ml in water. DNA solution was diluted 1: 1 in HBS 2 × before electroporation. After removing the medium, about 60 μl (25 μg / ml) of DNA solution was applied to the incubator just before electroporation.

Target cells were selected as in the previous example. The waveform of the electroporation signal used was the same as the previous experiment except that the number of pulses per column rose to 50. After 48 hours, the transfected target cells underwent replication cycle splitting in two cells expressing GFP. Typical cytoplasmic patterns of GFP expression are seen in both cells. Because of replication and intrinsic cell motility, the two cells migrated somewhat from the microelectrode, which was covered only with the parental cells.

Example  3: Fluorescein (fluoroscein) of  Electroporation of Rat Hippocampal Neurons

In a third experiment, mouse hippocampal neurons containing fluoroscein were electroporated using a device performed by the method according to the invention.

Neurons were isolated from the hippocampus of 18-day-old Wistar rats (Banker and Cowan, 1977). Neurons were plated twice in advance to remove glial cells, and glutamax I supplemented with 10% (volume) fetal bovine serum (10106078, Gibco) and 1% (volume) penicillin (15140114, Gibco). In DMEM (no. 61965026, Gibco, Eggenstein, Germany) containing (Brewer et al., 1993; Vassanelli and Fromherz, 1998). The final concentration was 350,000 cell number / mm.

The chip surface was carefully washed with a 1% solution of liquid food detergent, rinsed with milli-Q water (Millipore, Bedford, Mass.), Dried and UV sterilized. The chip was adsorbed from a 20 ug / ml aqueous solution for 1 hour, coated with poly-L-lysine (molecular weight: 300,000 or more; Sigma, Heidelberg, Germany) and dried. We applied 350 μl of cell suspension to the incubator. LeibovitzL-15 medium (100 ui) (31415029, Gibco) containing Glutamax I supplemented with 5% fetal bovine serum was added. Cell density was 100,000 cells / cm ² . The chip was kept at 37 ° C., 10% carbon dioxide for 2 hours. The medium was then removed and neurobasal medium (Gibco, 21103049) supplemented with 450 μl of 2% (volume) B27- medium (17504036, Gibco) and 1% (volume) Glutamax I (35050038, Gibco) Cells were cultured in medium without serum for 4-7 days (Brewer et al., 1993; Evans et al., 1998; Vassanelli and Fromherz, 1998). Electroporation was performed with neurons that survived in the medium for a minimum of 6 days and a maximum of 12 days.

Fluorescein was dissolved in standard physiological solution (10 uM) and applied to the reactor just before electroporation. After washing twice with standard physiological solution, fluorescence microscopy was observed.

One hippocampal neuron was electroporated by applying a triangular column voltage to the microelectrode. Here, each triangular waveform was an amplitude equal to 4 V, and one triangular duration was 1 ms equal to the rise / fall time (see FIG. 8).

In contrast to Cos-7 cells and numerous cell lines, neurons are excitatory cells and unwanted stimulation of electrical activity during electroporation should be limited. The inventors have found that i) decrease the number of pulses per column (10 pulses instead of 25 or 50 used in Cos-7 cells), ii) increase the spacing between rows (5 s instead of 500 ms) and iii) triangles. The voltage was used to reduce unwanted stimuli.

Example  4: Electrophysiological activity during perforation ( Cos -7 cells and rat hippocampal neurons)

Target cells were contacted with a patch-clamp electrode. After defining the total cell layout, the intracellular potential of the cells was monitored. Appropriate electroporation signals were applied to the corresponding microelectrodes to induce intracellular transient voltages up to 0 mV (cell potential is negative, about −70 mV). Due to the electroporation pore formation and continued electrical interaction with the grounded external electrolyte, the transient voltage slowly decreased and the cells fully recovered to rest potential within 1-2 minutes. This is believed to be the time required to reseal the pores. Pore formation was also confirmed by penetration of fluorescein (small fluorescent dye) into the cell from the external electrolyte.

The electroporation and suitable electroporation signals performed with the device according to the invention can be repeated without any electrophysiological signal for cell damage during the entire fragment recording session (typically 20-30 minutes).

As expected, the amplitude of the intracellular transient voltage induced by electroporation and the recovery time to rest potential vary with the applied electroporation signal.

The above-described results demonstrate that the apparatus and method according to the present invention can effectively electroporate many kinds of cells with various compounds, thereby accomplishing the object of the present invention. It will be apparent to those skilled in the art that, within the scope of the present invention, not only embodiments of the present invention, but additional implementations or applications may be considered.

Claims (25)

An electroporation device comprising a waveform generator and a control system for applying a signal transfer to a series of microelectrodes and a single microelectrode preselected in the biochip. 2. The electroporation device of claim 1, wherein the control system comprises a personal computer having a software program for designing various waveform signals and a switch system for controlling the waveform generator output. The biochip of claim 1 or 2, wherein the biochip includes a series of microelectrodes having a size corresponding to that of the cells to be electroporated, and each of the microelectrodes is driven separately from the other electrodes to perform the electroporation process. Electroporation device, characterized in that accurate and timely control. A series of microelectrodes included on a suitable insulating layer mounted on the solid substrate; Means for electrically connecting the microelectrode to a switch system; And And a cell incubator capable of developing and attaching cells in contact with said series of microelectrodes on a surface formed by said insulating layer comprising said series of microelectrodes on said solid substrate. The method of claim 4, wherein A semiconductor substrate as a solid substrate covered with an insulating layer 27 comprising a driven series of microelectrodes 20 having a size corresponding to an electroporated cell, A cell incubator 24 comprising an opening 26 is mounted on a support 21 made of a dielectric material, The microelectrode is electrically connected to the conductive pad 29 through the conductive labeling material 28, The conductive pad 29 is connected via a wire connection 23 to a pair of external parallel connectors 22 external to the cell incubator 24 surrounding the opening 26, The cell incubator 24 including the opening 26 is mounted on top of the semiconductor substrate covered with the insulating layer 27, A biochip, characterized in that the semiconductor substrate and the insulating layer (27) is attached to the insulator support (21). 6. A biochip according to claim 5, further comprising two electrodes integrated into the semiconductor substrate covered with an insulating layer (27), said electrodes serving as reference grounds. 6. The biochip according to claim 5, wherein the semiconductor substrate covered with the insulating layer (27) is a silicon substrate which is preferentially covered with an insulating layer composed of SiO ₂ . 6. The biochip according to claim 4 or 5, wherein the solid substrate is transparent. 6. The biochip of claim 5, wherein the insulator support is betronite, glass, or ceramic. 6. The biochip according to claim 5, wherein the size of the series of microelectrodes (20) is at least 10% or more of the whole cell membrane, and the diameter is preferably in the range of 1 µm to 5 µm. The biochip according to claim 4, wherein the microelectrode (20) is conductive or capacitive. 12. A conductive microelectrode according to claim 11, consisting of a conductive microelectrode obtained on a silicon substrate (31) covered with an insulating layer (27) consisting primarily of SiO ₂ , The microelectrode and the labeling material 38 connected thereto have an aluminum layer 34 interposed between two titanium nitride (TiN) layers 33, covered with a gold layer 37, on the active surface thereof. Microelectrode, characterized in that consisting of "sandwich form". 12. The microelectrode of claim 11, wherein the microelectrode is implemented using a metal oxide semiconductor (MOS) technique. The method of claim 13, A drain 41 and a source 42, which is a silicon p-type substrate 40 and two n-doped regions implanted with conventional microelectrode techniques, The gate 43 of the microelectrode runs in n + doped polysilicon and is in the row direction common to all devices (word lines), The drain 41 of all the devices is connected in the column direction by using a metal contact plug and a metal wire 44 together, Wherein said source (42) of the transistor is connected via a metal (usually tungsten) plug (46) to a gold layer (47) acting as an active electrode. 12. The conductive microelectrode according to claim 11, comprising a conductive microelectrode obtained by an insulating substrate 60, a metal 61, and a thin insulating layer 64, A microelectrode, characterized by being separated by an insulating material (62) and protected by an unexposed region by a passivation layer (63). Electroporation method characterized by using the apparatus according to claim 1. The electroporation method of claim 16, wherein the electroporation is carried out to at least one adherent cell. 18. The electroporation method of claim 17, wherein the device comprises a biochip according to claims 4-10. The electroporation method of claim 16, wherein the biochip comprises the microelectrode according to claim 11. 20. An electroporation method according to any of claims 16 to 19, wherein the waveform generator sends a string of pulses of various amplitudes and durations to the microelectrode. 21. The electroporation method according to any of claims 16 to 20, wherein the waveform generator sends five rows of 25 pulses (1 ms duration) to the microelectrodes by repeating the cycle of 500 ms. 21. The electroporation method according to any one of claims 16 to 20, wherein a voltage of a triangular column consisting of ten pulses is applied to the microelectrodes at intervals of 5 s. The method according to any of the preceding claims, Culturing the cells until the attachment step; Adding at least one compound to the medium to be electroporated in at least one single cell of said cell; Selecting at least one single cell and selecting at least one microelectrode to which the selected single cell is attached; And Generating at least one electrical signal suitable for electroporating the at least one single cell with the at least one compound that is electroporated, and sending the generated electrical signal to the microelectrode to which the selected single cell is attached. How to. An electroporated cell, obtained by the method according to claim 16. The electroporated cell of claim 24, wherein the electroporated individual is a pharmaceutical, genetic construct, and protein.

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