KR101362076B1

KR101362076B1 - Method of the localized electroporation using scanning probe microscopy and device for electroporation

Info

Publication number: KR101362076B1
Application number: KR1020100076080A
Authority: KR
Inventors: 이남주; 강치중; 유재우; 김유진; 최영진
Original assignee: 명지대학교 산학협력단
Priority date: 2010-08-06
Filing date: 2010-08-06
Publication date: 2014-02-12
Also published as: KR20120021549A

Abstract

The present invention provides a method of preparing a scanning probe microscope, comprising: placing a scanning probe microscope on a cell in a buffer solution containing a substance to be introduced, maintaining a constant distance between the scanning probe and the cell by monitoring an ion current of the scanning probe, and scanning The present invention provides an electroporation method using a scanning probe microscope and an electroporation apparatus according to the present invention, which includes introducing a substance into a cell by applying a low voltage pulse between an upper electrode which is a probe and a lower electrode which is an indium tin oxide existing under a cell. The method according to the present invention can maintain a constant interval by monitoring the change in the ion current according to the change of the distance between the probe and the cell, and by using this, it is possible to apply a constant electric field to each cell, thereby increasing the efficiency and reliability of the electric drilling. In addition, since it can be directly applied to cultured cells, efficiency and cell survival rate can be increased at the same time.

Description

FIELD OF THE LOCALIZED ELECTROPORATION USING SCANNING PROBE MICROSCOPY AND DEVICE FOR ELECTROPORATION

The present invention relates to electroporation using Scanning Probe Microscope (SPM). More specifically, it relates to an electroporation method in which a uniform electric field is applied to individual cells, including the step of maintaining a constant distance between the scanning probe of the scanning probe microscope and the cells by measuring the ion current.

Recently, in the field of molecular biology, various techniques for injecting exogenous molecules such as DNA, RNA, proteins or genes into cells have been used. For example, biological or chemical pathways of cells in the system are being studied through the insertion of proteins or secondary messenger materials, and even in stem cell studies, cell differentiation is initiated by injection of specific proteins.

Among these technologies, electroporation is a technique for temporarily injecting genetic material such as DNA or protein into cells by making holes in the cell membrane by using electric shock, and more specifically, a combination of genes that cannot penetrate the cell membrane. It is a gene introduction method that allows relatively large substances such as proteins and proteins to be introduced into cells through cell membrane perforation using electric pulses. Since such electroporation is available not only for cell research through gene function analysis but also for new drug development process, it is a field that requires continuous industrialization efforts to create high added value in the future.

The schematic diagram of the conventional electroporation method using the parallel plate electrode which is a prior art is shown in FIG. The conventional cuvette method, which performs electroporation by placing cells between parallel plate electrodes and applying a high voltage, is more efficient than other gene introduction methods, but the cells around the electrodes are damaged and uneven due to several kilovolts (kV) pulses. One electric field has the disadvantage of randomly introducing genes. In particular, since the cells are made in a liquid suspended state, there is a disadvantage that the cells must be cultured afterwards. In addition, it could not be applied to individual cells due to characteristics such as electrode size.

In contrast, electroporation using a probe can be used directly under culture conditions and the probe can be approached to cells within a few micrometers, so electroporation can be performed even at a voltage as low as several volts (V). Therefore, it is possible to prevent damage to the cell membrane that can occur when using the conventional cuvette method. However, there is a problem that it is difficult to apply a uniform electric field because it is very difficult to maintain a constant distance between the cell and the probe.

The present inventors have diligently studied the electroporation method. As a result, when performing electroporation using a scanning probe microscope, a constant electric field is applied to the electroporation target cell by maintaining a constant distance between the probe and the cell by measuring the ion current. The present invention has been achieved.

It is an object of the present invention to perform electroporation on individual cells using a scanning probe microscope (SPM), whereby adjusting the distance between the cells and the probe by applying a constant electric field through ion current measurement rather than an optical method increases work reliability. I'm trying to.

It is another object of the present invention to provide an electroporation device comprising a scanning probe microscope, characterized in that a constant electric field is applied to individual cells through ion current measurements.

In order to achieve the above object, the present invention is characterized by maintaining a constant distance between the scanning probe and the cell by monitoring the ion current between the scanning probe of the scanning probe microscope (SPM) and the indium tin oxide present under the cell An electroporation method using a scanning probe microscope is provided.

More specifically, the electroporation method using a scanning probe microscope according to the present invention,

Positioning the scanning probe of the scanning probe microscope over the cell of electroporation;

Maintaining the distance between the scanning probe and the cell by monitoring the ion current between both electrodes, using the scanning probe as an upper electrode and an indium tin oxide present under the cell as a lower electrode; And

Electroporating the cell by applying a low voltage pulse between the upper electrode and the lower electrode.

To achieve these and other advantages and in accordance with the purpose of the present invention,

An upper electrode which is a scanning probe of a scanning probe microscope;

A lower electrode which is indium tin oxide; And

Electroporation target cell located between the upper electrode and the lower electrode

And a scanning probe microscope, wherein the distance between the scanning probe and the cell is kept constant by monitoring an ion current between the upper electrode and the lower electrode, and a low voltage pulse is applied between the upper electrode and the lower electrode. It further provides an electroporation device.

The present invention uses a scanning probe as an upper electrode and an indium tin oxide as a lower electrode during electroporation using a scanning probe microscope (SPM) to monitor a change in ion current according to a change in distance between a probe and a cell. It is possible to maintain and use this to apply a constant electric field for each cell has the advantage of increasing the efficiency and reliability of the electroporation.

In addition, since the present invention can be directly applied to cultured cells, efficiency and cell survival rate can be increased at the same time. In addition, the prior art has the advantage that the present invention can be applied to any type of cells compared to the limited scope of the application is a wide range of applications.

1 is a schematic diagram of a conventional electroporation method (cuvette method) using a parallel plate electrode.
2 is a schematic view of a scanning probe microscope with an electrode inserted therein according to the present invention and an electrode with an electrode for forming an electric field at the bottom of the sample.
Figure 3 shows a glass pipette probe image with an electrode inserted therein.
Figure 4 shows a metal coated probe image coated with an insulating layer except for the flat end.
Figure 5 illustrates in detail the method of adjusting the distance between the probe and the sample by the ion current measurement method. (a) is an image of the probe approaching the sample, (b) is a graph of the ion current measured at this time.
Figure 6 is an optical microscope to observe the fluorescent protein expressed by the DNA injected into the cell through the electroporation according to the embodiment. (a) is a phase image, (b) is a fluorescence image, (c) is an image which superimposed (a) and (b) of the same position.

Hereinafter, the present invention will be described more specifically.

Electroporation method using the scanning probe microscope of the present invention,

According to the electroporation method using the scanning probe microscope of the present invention, a sample filled with the surroundings with a buffer solution containing the material to be introduced is placed in the cultured electroporation cells. This sample is placed on the scanner of the scanning probe microscope, and the scanning probe of the scanning probe microscope is placed on the cells to be electroporated. In this case, the substance to be introduced into the cell may be a biomolecule or a drug. Examples of biomolecules include, but are not limited to, DNA, RNA, proteins or genes. The cells may use any kind of cells. One of ordinary skill in the art can select appropriately according to the purpose of use.

According to the electroporation method using the scanning probe microscope of this invention, the scanning probe of a scanning probe microscope is used as an upper electrode. Such a scanning probe may be any one so long as no current flows outside and no current flows inside.

According to one embodiment of the invention, the scanning probe according to the invention may use a glass pipette probe with an electrode inserted therein. 3 shows an image of the glass pipette probe. The glass pipette probe is preferably further coated with a metal on the inner wall of the glass pipette probe. If more metal is coated on the inner wall of the glass pipette probe, it is possible to increase the electric field sensitivity of the upper electrode and to remove the ambient noise. Examples of the metal to be coated include gold, silver, aluminum, and copper, but are not limited thereto.

According to another embodiment of the invention, the scanning probe according to the invention may be a metal coated probe coated with an insulating layer except for the flat end of the probe. Such a probe may be manufactured by coating an insulating layer such as an oxide film or a nitride film on a straight probe coated with metal such as platinum, and cutting off the tip of the probe by using a focused ion beam (FIB). . The metal and insulating layer may be appropriately selected from those illustrated as well as those known to those skilled in the art. An enlarged image of the tip of the scanning probe of the present invention prepared in this manner is shown in FIG. 4.

The injection probe according to the invention is nanoscale. Preferably, the tip curvature radius is 10 nm to 100 nm. Since the scanning probe is nanoscale, an electric field for electroporation can be applied locally to individual cells, and more accurately electroporation.

According to the electroporation method using the scanning probe microscope of the present invention, the lower electrode should be under the cell to be electroporated, and indium tin oxide is preferred. Indium tin oxide is easy to manufacture thin films and has excellent linkage with subsequent processes such as PDMS process. Above all, when used as a lower electrode, there is an advantage that can be cultured biological samples such as cells on the thin film.

According to one embodiment of the present invention, the lower electrode may be used that is separated from the medium containing the cells to be electroporated, and may be used patterned at the bottom of the cell culture medium. In the absence of the pattern, the electric field is applied to the front of the lower electrode, whereas when indium tin oxide patterned at the bottom of the cell culture medium is used as the lower electrode, the electric field is applied only to the patterned electrode and thus to the cells of the pattern and the cells without the electrode. The effect of electric field on water is clearly distinguished. Therefore, it is advantageous to measure the presence and efficiency of electroporation for adjacent cells in culture in the medium.

According to the electroporation method using the scanning probe microscope of the present invention, the scanning probe by monitoring the ion current between both electrodes using the scanning probe of the scanning probe microscope as the upper electrode and the indium tin oxide existing under the cell as the lower electrode. The distance between and cells can be kept constant. Specific methods of measuring the ion current are known to those skilled in the art, and thus detailed discussions are omitted.

As shown in (a) and (b) of FIG. 5, the sample is approached while the scanning probe moves, and the ion current between the scanning probe and the indium tin oxide is measured. While monitoring the measured ion current, the moment when the current changes rapidly is taken as the reference height, and electroporation is performed at the reference height. This method has the advantage that the distance adjustment is accurate compared to the prior art because the position of the probe from the cell surface can be adjusted to maintain a constant distance.

According to the electroporation method using the scanning probe microscope of the present invention, electroporation can be performed by applying a low voltage pulse between both electrodes. The low voltage pulse applied is only a voltage of several volts (V), and preferably a voltage of 5 V or less, more preferably 2 V to 5 V can be applied.

When electroporation is performed by applying a low voltage pulse to the cell to be electroporated, a substance present around the cell is introduced into the cell.

As described above, since the present invention performs electroporation with a scanning probe microscope, a series of processes for performing electroporation can be observed, and it is also easy to observe cells after electroporation.

The present invention includes an upper electrode which is a scanning probe of a scanning probe microscope, a lower electrode which is indium tin oxide, and an electroporation target cell located between the upper electrode and the lower electrode, and includes an ion current between the upper electrode and the lower electrode. By monitoring the distance between the scanning probe and the cell is kept constant, it can be performed using an electroporation device, characterized in that a low voltage pulse is applied between the upper electrode and the lower electrode. The present invention includes existing equipment, such as an atomic force microscope (AFM) and a scanning ion conductance microscope (SCIM), which can be modified slightly to be used as an electroporation device. As shown in FIG. 2, the electroporation device of the present invention further includes components characterizing all scanning probe microscopes, such as scanning probes, adjusting devices, lock-in amplifiers, light detectors, laser light sources, scanners, and the like. can do.

Hereinafter, preferred embodiments of the present invention will be described.

Example

Breast cancer cell lines (MCF7) were obtained from US NIH and incubated in DMEM medium for about 24 hours. In order to use the cultured cells in the electroporation experiment, the culture solution was removed, and the culture mixture solution containing DNA and antibiotics was injected. To observe the electroporation in more detail, the buffer solution containing the DNA to be introduced into the cells was labeled with fluorescent material.

In order to perform the electroporation method according to the present invention, a commercial probe of a scanning probe microscope (XE-100) obtained from Park systems was equipped with a probe manufactured through its own semiconductor process such as coating and etching. Cells were cultured in a medium in which indium tin oxide was patterned at the bottom, and the surroundings were filled with DNA labeled with fluorescent material. The ion current between the probe and the lower electrode at the bottom of the sample was monitored to maintain a constant distance between the probe and the sample. At this time, the distance between the probe and the sample was 10 μm. Electroporation was performed by applying a voltage of about 3 V between the scanning probe and the indium tin oxide. An image of the cells into which the fluorescent DNA is introduced is shown in FIG. 6. From the image shown in Figure 6, it can be seen that the electroporation for individual cells without cell membrane damage.

The present invention is not limited to the above-described specific embodiments, and those skilled in the art can make various modifications without departing from the technical spirit. Accordingly, the scope of the present invention should be construed as being determined not by the specific embodiments but by the appended claims.

Claims

Positioning a scanning probe of a scanning probe microscope (SPM) over the cell of electroporation;
Maintaining the distance between the scanning probe and the cell by monitoring the ion current between both electrodes, using the scanning probe as an upper electrode and an indium tin oxide present under the cell as a lower electrode; And
Electroporation of cells by applying a low voltage pulse between the upper and lower electrodes
Electroporation method using a scanning probe microscope (SPM) comprising a.

The electroporation method according to claim 1, wherein the scanning probe is a glass pipette probe having an electrode inserted therein.

The electroporation method using a scanning probe microscope according to claim 2, wherein a metal is coated on an inner wall of the glass pipette probe.

The electroporation method of claim 1, wherein the scanning probe is a metal coated probe coated with an insulating layer except for a flat end of the probe.

The method of claim 1, wherein the lower electrode is electroporation method using a scanning probe microscope, characterized in that the patterned on the bottom of the cell culture medium.

The electroporation method according to claim 1, wherein the low voltage pulse is a voltage of 5 volts (V) or less.

Electroporation using a scanning probe microscope (SPM) characterized by keeping the distance between the scanning probe and the cell constant by monitoring the ion current between the scanning probe of the scanning probe microscope (SPM) and the indium tin oxide present under the cell .

An electroporation device comprising a scanning probe microscope,
An upper electrode which is a scanning probe of a scanning probe microscope;
A lower electrode which is indium tin oxide; And
Electroporation target cell located between the upper electrode and the lower electrode
Wherein the distance between the scanning probe and the cell is kept constant by monitoring the ion current between the upper electrode and the lower electrode, and a low voltage pulse is applied between the upper electrode and the lower electrode. .

9. The electroporation device of claim 8, wherein the scanning probe is a glass pipette probe with an electrode inserted therein.

The electroporation apparatus according to claim 9, wherein a metal is coated on an inner wall of the glass pipette probe.

9. The electroporation device of claim 8, wherein the scan probe is a metal coated probe coated with an insulating layer except for the flat end of the probe.

The electroporation apparatus of claim 8, wherein the lower electrode is patterned at the bottom of the cell culture medium.

9. The electroporation device of claim 8, wherein the low voltage pulse is at or below 5 volts.