KR20070014370A - Patch clamp system and method for giga sealing using the same - Google Patents

Abstract

A patch clamp system and method for sealing of a cell by using the same system are provided to increase reliability of patch clamping by applying cell culture process to the cell absorbed in the area where the patch clamping process is performed, and improve sealing properties of the adsorbed cell. The patch clamp system comprises a substrate structure and a cell attachment-inhibiting membrane(115), wherein the substrate structure contains a substrate consisting of a silicon substrate(111) and a silicon nitride layer(112), and a cell-adsorbing layer(114) coated with a cell adsorption-supporting material; the cell attachment-inhibiting membrane defines the cell adsorption area on the substrate structure and contains a poring channel(113) in the center of the cell adsorption area where poring of the cell is performed; the cell adsorption-supporting material is fibronectin, collagen or PLL(poly-L-lysine); the cell attachment-inhibiting membrane contains polyethylene glycol(PEG); and the cell-adsorbing layer consists of silicon oxide.

Description

Patch clamp system and method for giga sealing using the same

1 is a perspective view of a patch clamp system according to the present invention.

2 is a cross-sectional view taken along line AA ′ of FIG. 1.

3 is a graph showing the degree of giga sealing of cells over time.

4A to 4C are cross-sectional views illustrating a manufacturing process of the patch clamping system according to the present invention.

Description of the main parts of the drawing

111 silicon substrate 112 silicon nitride layer

113: pore channel 114: cell seating layer

115: cell adhesion inhibitory membrane 110: unit patch clamp region

100: patch clamp system A: cell seating area

The present invention relates to a patch clamp system and a cell sealing method using the same. More particularly, the present invention relates to a patch clamp system and a cell sealing method using the same, which improves the sealing property of a cell ion channel, thereby increasing the reliability of patch clamping. .

MEMS (Micro electro mechanical system) technology, which uses semiconductor processing technology as an electromechanical system, has continued to expand its field of application with remarkable developments in the information and communication field, micro machines, micro sensors and displays. In particular, the Bio-MEMS field, which incorporates this technology into biotechnology since the late 90s, shows excellent characteristics in various biochemical analytical devices, including capillary electrophoresis, using the useful properties of microfluidics. It is open to various applications. Bio-chip manufactured by micro-processing technology combines micro / nano processing technology and biotechnology technology to produce biological materials such as genes, proteins, cells, and metabolites from practical biological samples such as trace amounts of blood, urine, and saliva. The device extracts and analyzes materials.

Current disease diagnosis involves the extraction and separation of viruses, bacteria, or proteins from body fluid samples using various equipment in the laboratory to detect disease status by detecting various targets at the millimeter or micro level. In the future, the rapid development of biotechnology using biochips such as DNA chips and protein chips is expected to bring about a revolution in disease diagnosis medicine. Recently, the ultimate goal is to develop a lab on a chip (LOC) that uses MEMS technology to perform all steps, such as dilution, mixing, reaction, separation, and quantification, on a single chip. It is becoming.

On the other hand, the researches on the existing micro biochips have been mainly focused on DNA or protein, and have been focused on chips for DNA analysis, protein analysis or disease diagnosis, and high throughput screening (HTS). Recently, research on biochips using cells, which are the smallest units of a biological system, has been carried out in an attempt to approach. Since cells react sensitively to external stimulants, cell-based biochip sensors that detect toxicity by using cells have been developed, and technology for separating cells precisely using microfluidic technology from FACS (Fluorescence Activated Cell Sorter) used in hospitals. This was developed. By using micro-processing technology, it is easy to manipulate a cell having a size of about 10 μm, thus saving a lot of labor and time of a conventional work.

Existing drug development was validated through animal experiments after screening candidate chemicals to be used as drugs, but the new drug development cycle has been reduced from about 12 years to about 8 years and will have a 4 year cycle in the future. As expected, biochips have been developed to automate screening tasks to reduce the time and expense required for screening. Micro-arrays are used to study interactions between biomaterials, and microfluidics-based systems are currently being developed and used. However, the screened candidates should be finally optimized by feedback from the assay, since the efficacy and safety of the drug should be examined with the results of animal and clinical trials. Therefore, prior to animal testing, toxicity experiments are first performed on living living cells to screen about 20-30% of drug candidates. At this stage, biological cell screening is performed to analyze the potential, stability, toxicity and effects of the drug to reduce the feedback time for optimization of the candidate.

Recently, HCS (High Content Screening) has been used to analyze the number and shape of cells, changes in ion concentration of internal cells for drugs, and the like using biochips in the toxicity test step. It is a method of screening candidates before animal experiments by analyzing various live cell responses to candidates using mainly fluorescent proteins.

On the other hand, in the analysis of cell activity and gene expression, the analysis of the ion channel inside the cell is very important and the ion channel analysis method should be used for the drug targeting the cell's ion channel. The most prominent analytical tool to analyze is the patch clamp method developed by Hamil .

The patch clamp method uses a glass pipette driven by a micromanipulator under a microscope to inhale a cell membrane patch to form a high resistance seal of about 1 GΩ, followed by ion channels. Measure the current in the cell wall patch or cell wall of the whole cell through. Although there have been many improvements, this method is only possible by experimenters who are very skilled at placing pipettes in cells and require a lot of labor and time. For this reason, it is not widely used for proteomics research or drug development that requires high throughput, but is mainly used as a research tool for examining physical phenomena in a single cell.

Recently, patch clamp devices have been developed by applying micromachining technology to automate manipulation. Companies that develop such automated patch clamp devices include PatchXpress, Nanion Technologies, and Sophion Bioscience. However, due to incomplete giga sealing in the ion channel, there is a problem in signal noise and a high process cost based on high-precision silicon (Si) or quartz (SiO ₂ , Quartz). There are disadvantages.

The present invention has been made to solve the above problems, and an object of the present invention is to provide a patch clamp system and a cell sealing method using the same by improving the group sealing characteristics of the cell ion channel to increase the reliability of patch clamping.

The patch clamp system according to the present invention for achieving the above object is made of a combination of the substrate structure and the cell adhesion inhibitory film provided on the substrate structure, the predetermined area on the substrate structure by the cell adhesion inhibitory membrane cells Is defined as a cell seating area to be seated, the center of the cell seating area is provided with a pore channel for providing a space for the poring to the cell, the substrate structure is a double layer structure of the substrate and the cell seating layer On the cell seating layer, a cell adsorption auxiliary material for promoting the adsorption of cells is coated.

Preferably, the cell adsorption auxiliary material is any one of fibronectin, collagen, Poly-L-Lysine (PLL).

Preferably, the cell adhesion inhibitory membrane comprises Poly Ethylene Glycol (PEG).

Preferably, the cell seating layer is composed of silicon oxide.

Preferably, the substrate consists of a double layer of a silicon substrate and a silicon nitride layer.

Cell sealing method using a patch clamp system according to the present invention is a cell sealing method using a patch clamp system having a cell seating area on which cells are seated to perform a series of patch clamping processes such as cell supply, pore, current measurement, etc. In the above, the cell adsorption auxiliary material is applied on the cell seating area, the cell supply process for seating the cell in the cell seating area is carried out, and then the cells are cultured for a predetermined time between the cell and the cell seating area. It is characterized in that the group is sealed.

Preferably, the culture of the cells is carried out for 2-3 hours.

Preferably, the cell supply process, the step of providing a cell on the patch clamp system divided into the cell seating region and the cell adhesion inhibitory membrane region, and located on the cell adhesion inhibitory membrane region using a predetermined wash solution Removing the cells so that the cells exist only in the cell seating region.

Preferably, the cells provided on the patch clamp system are included in a predetermined solution having the same ion concentration as the extracellular fluid.

Preferably, the washing solution is any one of water and media solution.

According to a feature of the present invention, the cells are seated in a region where the patch clamping process is performed, and then a predetermined cell culture process is applied to improve the sealing properties of the seated cells, thereby increasing the reliability of patch clamping.

Hereinafter, a patch clamp system according to the present invention will be described in detail with reference to the accompanying drawings. 1 is a perspective view of a patch clamp system according to the present invention, Figure 2 is a cross-sectional view taken along the line AA 'of FIG.

As shown in FIG. 1, the patch clamp system 100 according to the present invention includes a plurality of unit patch clamp regions 110, and each unit patch clamp region 110 performs patch clamping on predetermined cells. The area of the minimum unit. The patch clamp system 100 according to the present invention may be expressed as the unit patch clamp region 110 is repeatedly arranged. Therefore, the unit patch clamp region 110 may be referred to as a miniature version of the patch clamp system according to the present invention. Hereinafter, the structure of the patch clamp system according to the present invention will be described through the structure of the unit patch clamp region. .

As shown in FIG. 2, the patch clamp system according to the present invention is composed of a combination of a substrate structure and a cell adhesion suppressing layer 115 provided on the substrate structure.

The substrate structure consists of a substrate and a cell seating layer 114. As the substrate, a silicon substrate 111 may be used, and a silicon nitride layer (SiN _x ) 112 may be stacked on the silicon substrate 111 to perform a selective etching process of the silicon substrate 111. On the silicon nitride layer 112 is provided a cell seating layer 114 in direct contact with the cells. The cell seating layer 114 may be a material having excellent cell sealing properties. Specifically, silicon oxide (SiO ₂ ), poly dimethyl siloxane (PDMS), or the like may be used.

Meanwhile, as shown in FIG. 1, a predetermined opening is provided at the center of each unit patch clamp region, which will be referred to as a poring channel 113. As a series of patch clamping processes, such as cell pore and current measurement, are performed through the pore channel 113, the cell seating layer 114 provided on the substrate to improve the sealing property of the cell is provided. It is preferably provided not only on the substrate but also on the inside of the pore channel 113.

Although not shown in the figure, a cell adsorption auxiliary material is applied on the upper surface of the cell seating layer 114 to improve the adsorption of cells. Fibroectin, collagen, Poly-L-Lysine, and the like may be used as the cell adsorption aid.

On the other hand, as described above, one side on the substrate structure consisting of the substrate and the cell seating layer 114 is provided with a cell adhesion inhibiting film 115. The predetermined area on the substrate structure is defined as the cell seating region A by the cell adhesion inhibiting layer 115. The cell seating area (A) is a space that provides a base for a series of patch clamping process to the cell is seated to the cell, the foreing channel 113 is provided in the center of the cell seating area (A) do. The cell adhesion inhibiting layer 115 prevents cells from adsorbing on the cell adhesion inhibiting layer 115 and ultimately induces cell adsorption onto the cell seating region A. Poly Ethylene Glycol) and the like may be used as the cell adhesion inhibiting membrane 115.

Referring to the cell sealing method using a patch clamping system according to the present invention having such a configuration as follows.

First, cells to be analyzed are supplied onto a patch clamping system according to the present invention having a plurality of unit patch clamp regions. At this time, the cells are contained in a predetermined solution having the same ion concentration as the extracellular fluid, so that the cells are supplied by the supply of the solution.

As such, when a predetermined time elapses while cells are supplied on the patch clamp system, the cells may have a difference in degree of adsorption depending on the characteristics of the surface to which the cells are in contact. Specifically, as described above, the patch clamp system according to the present invention may be planarly divided into a cell adhesion inhibiting layer 115 region and a cell seating region A defined by the cell adhesion suppressing membrane 115. The cell seating region A is coated with a cell adsorption auxiliary substance that promotes cell adsorption. Accordingly, the cells located in the cell seating region A are promoted to be adsorbed by the cell adsorption auxiliary material, whereas the cells located on the cell adhesion inhibiting membrane 115 are cell adhesion inhibiting membrane 115. Adsorption strength is relatively weak due to the cell adhesion suppression property of

In this state, the cleaning process is performed on the upper surface of the patch clamp system. The washing process is a process for removing cells located on the cell adhesion inhibiting membrane 115. The washing solution used in the washing process may be water, a media solution, or the like. By the washing process, the cells on the cell adhesion inhibiting layer 115 having a relatively low adsorption strength are removed so that the cells exist only in the cell seating region A.

In general, the patch clamping process includes a cell supply process for supplying a cell to a specific position of a predetermined patch clamping device, a poring process through the cell membrane of the cell, and a current measurement and analysis process for the inner and outer fluids of the cell. In the present invention, by selectively positioning the cells only in the cell seating area (A) by performing the washing process means that the cell supply process is completed.

On the other hand, in order to ensure the reliability of the current measurement and analysis process for the intracellular and external fluids, noise must be minimized in the measured current. In addition, in order to minimize the noise, the electrical properties of the inner liquid and the outer liquid should not be influenced by each other when measuring the current proceeding for each of the intracellular and outer liquids. In order for the intracellular fluid and the extracellular fluid to not be electrically influenced by each other, the intracellular fluid region and the extracellular fluid region must be electrically insulated, that is, a giga sealing state. Here, the intracellular fluid region refers to a region including the intracellular fluid in the cell and the pore channel 113, etc. in the lower part of the cell, and the extracellular fluid region is an upper part based on the cell membrane of the cell. It means an area. The part where the giga sealing is required is between the cell membrane of the cell and the surface of the cell seating area (A). Such a giga sealing must be ensured to improve the reliability of the subsequent pore process and the current measurement process.

Therefore, the group of the supplied cells must be sealed before the pore process, that is, in the cell supply process. In the present invention, as described above, the cells could be selectively positioned only in the cell seating region (A) by performing the washing process.

In addition, the present invention proceeds the cell culture process for the cells seated in the cell seating area (A) to ensure the complete giga sealing properties. The cell culturing process means culturing the cells seated in the cell seating region (A) for a predetermined time. The cell adsorption aid (A) is coated with a cell adsorption aid material, so that the adsorption strength of the seated cells increases, and the adsorption intensity of such cells increases with time as shown in the graph of FIG. 3. . The time required for improving the adsorption strength, that is, the cell culture time is preferably about 2 to 3 hours.

Through such a cell culture process, the cell can improve the sealing property and it is possible to guarantee the reliability of the result during the subsequent pore process and current measurement process.

On the other hand, the manufacturing process of the patch clamping system according to the present invention that provides a basis for the above giga sealing process is described as follows. 4A to 4C are cross-sectional views illustrating a manufacturing process of the patch clamping system according to the present invention.

First, a substrate is prepared as shown in FIG. 4A. As the substrate, a silicon substrate 111 may be used, and a silicon nitride layer 112 may be provided on the silicon substrate 111 to smoothly perform selective etching of the silicon substrate 111. In this state, the pore channel 113 is formed by etching a predetermined portion of the silicon nitride layer 112 through a photolithography process and an etching process. Herein, the width d of the pore channel 113 is preferably about 0.1 to 2 μm, and the thickness of the silicon nitride layer 112 is preferably about 1 to 2 μm.

As shown in FIG. 4 in the state in which the foreing channel 113 is formed, a cell deposition layer (not shown) on the silicon nitride layer 112 may be formed using a plasma enhanced chemical vapor deposition (PECVD) process. 114) are laminated. The cell seating layer 114 is formed to improve the sealing property of the cells, and as the cell seating layer 114, silicon oxide (SiO ₂ ) may be used, and the thickness thereof is preferably about 0.2 μm to 0.7 μm. Do. Meanwhile, the cell seating layer 114 is formed on the inner surface of the pore channel 113 as well as the upper surface of the silicon nitride layer 112.

In this state, although not shown in the figure, a cell adsorption auxiliary material is coated on the entire surface of the cell seating layer 114. As the cell adsorption aid material, as the adsorption aid material, fibronectin, collagen, PLL (Poly-L-Lysine), and the like may be used.

In the state where the cell adsorption auxiliary material is applied, the cell adhesion inhibiting material is deposited on the entire surface of the substrate including the cell seating layer 114 as shown in FIG. 4C. Examples of the cell adhesion inhibitor may include PEG (Poly Ethylene Glycol) and a photo initiator in the PEG. In this state, when the patterned by selectively exposing and developing the laminated cell adhesion inhibitor material, the cells adhere to an area other than the cell seating area (A), which is a space where cells are seated. The suppression film 115 is formed. That is, the predetermined area on the cell seating layer 114 is defined as the cell seating area A by the cell adhesion inhibiting film 115.

Patch clamp system and cell sealing method using the same according to the present invention has the following effects.

After the cells are seated in the area where the patch clamping process is performed, a predetermined cell culture process may be applied to improve the sealing properties of the seated cells, thereby increasing the reliability of patch clamping.

Claims (11)

It consists of a combination of the substrate structure and the cell adhesion inhibiting film provided on the substrate structure, A predetermined region on the substrate structure is defined as a cell seating region in which cells are seated by the cell adhesion inhibiting layer, and a pore channel is provided at the center of the cell seating region to provide a space in which pore for the cells proceeds. , The substrate structure is a double layer structure of the substrate and the cell seating layer, the patch clamp system, characterized in that the cell adsorption auxiliary material is applied on the cell seating layer to promote the adsorption of cells. The patch clamp system of claim 1, wherein the cell adsorption aid is any one of fibronectin, collagen, and poly-L-lysine (PLL). The patch clamp system of claim 1, wherein the cell adhesion inhibiting membrane comprises poly ethylene glycol (PEG). 10. The patch clamp system of claim 1 wherein the cell seating layer is comprised of silicon oxide. 2. The patch clamp system of claim 1 wherein the substrate is comprised of a double layer of a silicon substrate and a silicon nitride layer. In the cell sealing method using a patch clamp system having a cell seating area in which cells are seated to perform a series of patch clamping processes such as cell supply, pore, current measurement, etc. After the cell supply process of seating the cells in the cell seating area in a state in which the cell adsorption auxiliary material is applied on the cell seating area, the cells are cultured for a predetermined time to seal the gas between the cell and the cell seating area. Cell sealing method using a patch clamp system, characterized in that to be done. The cell sealing method of claim 6, wherein the cell sealing method uses the patch clamp system of claim 1. The method of claim 6, wherein the cell culture is carried out for 2 to 3 hours, the cell sealing method using a patch clamp system. The method of claim 6 or 7, wherein the cell supply process, Providing a cell on a patch clamp system that is divided into a cell seating region and a cell adhesion inhibitory membrane region; Removing cells located on the cell adhesion inhibiting membrane region by using a predetermined washing solution so that the cells exist only in the cell seating region. 10. The method of claim 9, wherein the cells provided on the patch clamp system are contained in a predetermined solution having the same ion concentration as the extracellular fluid. The cell sealing method of claim 9, wherein the washing solution is any one of water and a media solution.

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