Genetic material extraction apparatus and genetic material extraction method using same

Abstract

The technical idea of the present invention provides a genetic material extraction apparatus comprising: a substrate for accommodating, on a main surface thereof, a sample in which cells are suspended; a first electrode disposed on the main surface of the substrate; a second electrode disposed to face the first electrode along a first horizontal direction, and configured to generate an electric field across the first electrode and the second electrode; a first insulating film disposed on an upper surface of the first electrode and having a first window exposing a portion of the first electrode; a second insulating film disposed on an upper surface of the second electrode and having a second window exposing a portion of the upper surface of the second electrode; and a power source for applying an alternating current voltage to the first electrode and the second electrode so as to generate the electric field for trapping the cells within the first window or the second window.

Description

Genetic material extraction device and genetic material extraction method using the same

[0001] The present invention relates to an apparatus for extracting genetic material that responds to dielectrophoretic force and a method for extracting genetic material using the same.

[0002] When a living cell is exposed to an electric field, a dipole is formed within the cell. A force is generated through the interaction between the formed dipole and the electric field, and such a force polarizes the cell. This phenomenon is called dielectrophoresis, and such a force is called the dielectrophoretic force (DEP force).

[0003] Recently, there has been an increasing demand for research that uses electrophoretic forces to precisely control cells.

[0004] The problem that the technical concept of the present invention aims to solve is to provide a genetic material extraction device capable of trapping cells using dielectrophoretic force and extracting genetic material within the cells.

[0005] The problem that the technical concept of the present invention aims to solve is to provide a method for extracting genetic material using a genetic material extraction device capable of trapping cells using dielectrophoretic force and extracting genetic material within said cells.

[0006] The problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0007] To solve the above problem, the technical concept of the present invention provides a genetic material extraction device comprising: a substrate that accommodates a sample in which cells are suspended on a main surface; a first electrode disposed on the main surface of the substrate; a second electrode disposed to face the first electrode along a first horizontal direction, and configured to generate an electric field across the first electrode and the second electrode; a first insulating film disposed on the upper surface of the first electrode and having a first window that exposes a portion of the upper surface of the first electrode; a second insulating film disposed on the upper surface of the second electrode and having a second window that exposes a portion of the upper surface of the second electrode; and a power source that applies an alternating voltage to the first electrode and the second electrode to generate an electric field for trapping the cells within the first window or the second window; wherein the power source is configured to increase the peak voltage of the alternating voltage to rupture the membrane of the cells and extract genetic material after the cells are trapped in the first window or the second window.

[0008] According to one embodiment, the first electrode comprises a plurality of first conductive patterns extending toward the second electrode and having the first window located on the upper surface; and the second electrode comprises a plurality of second conductive patterns extending toward the first electrode and having the second window located on the upper surface; wherein the plurality of first conductive patterns and the plurality of second conductive patterns are arranged to intersect each other along a second horizontal direction perpendicular to the first horizontal direction.

[0009] According to one embodiment, a genetic material extraction device is provided, characterized in that the power source is configured to increase the frequency of the alternating voltage applied to the first electrode and the second electrode until the cell is trapped within the first window or the second window.

[0010] According to one embodiment, a genetic material extraction device is provided, characterized in that the frequency increases within a range of 0.1 kHz to 100 kHz.

[0011] According to one embodiment, a genetic material extraction device is provided, characterized in that the first window and the second window are circular holes in a horizontal view.

[0012] According to one embodiment, a genetic material extraction device is provided, characterized in that the first window and the second window do not overlap each other along a second horizontal direction perpendicular to the first horizontal direction.

[0013] According to one embodiment, a dielectric material extraction device is provided, characterized in that the first electrode and the second electrode do not come into direct contact with each other.

[0014] According to one embodiment, a genetic material extraction apparatus is provided, wherein the sample comprises a fluid containing the cell, and the fluid comprises at least one of an aqueous fluid, an aqueous buffer, an organic solvent, a hydrophobic fluid, and a gas.

[0015] According to one embodiment, a dielectric material extraction device is provided, characterized in that the peak voltage increases within a range of 0.1 V to 100 V.

[0016] According to one embodiment, a dielectric material extraction device is provided, characterized in that each of the first electrode and the second electrode comprises at least one of a metal material or a doped semiconductor material.

[0017] According to one embodiment, a dielectric material extraction device is provided, characterized in that each of the first insulating film and the second insulating film comprises silicon oxide.

[0018] To solve the above problem, the technical concept of the present invention provides a method for extracting genetic material comprising: a step of providing a first electrode exposed to a first window of a first insulating film and a second electrode exposed to a second window of a second insulating film so as to be arranged facing each other on the main surface of a substrate; a step of placing a sample in which cells are suspended on the first electrode and the second electrode; a step of generating an electric field within the first window and the second window by applying an alternating voltage to the first electrode and the second electrode through a power source; a step of trapping the cells within the first window or the second window through a dielectrophoresis force generated by the electric field; and a step of increasing the peak voltage of the alternating voltage by the power source until the membrane of the cells trapped in the first window or the second window is ruptured in order to extract genetic material within the cells trapped in the first window or the second window.

[0019] According to one embodiment, a method for extracting genetic material is provided, wherein in the step of trapping the cell within the first window or the second window, the power source increases the frequency of the alternating voltage until the cell is trapped within the first window or the second window.

[0020] According to one embodiment, a method for extracting genetic material is provided, characterized in that, in the step of trapping the cell within the first window or the second window, the power source maintains the peak voltage of the alternating voltage constant, and in the step of increasing the peak voltage of the alternating voltage until the membrane of the cell ruptures, the power source maintains the frequency of the alternating voltage constant.

[0021] A genetic material extraction device according to the technical concept of the present invention exposes cells suspended in a sample to an electric field. Subsequently, the frequency of an alternating current voltage source that generates the electric field is increased first to trap the cells exposed to the electric field in an electrode. Secondly, the peak voltage of the alternating current voltage source that generates the electric field is increased to rupture the membrane of the cells exposed to the electric field. Since the genetic material within the cells passes through the ruptured membrane and enters the sample, the target genetic material can be extracted more efficiently.

[0022] The effects of the present invention are not limited to the effects described above, and unmentioned effects will be clearly understood by those skilled in the art from this specification and the attached drawings.

[0023] FIG. 1 is a perspective view of a genetic material extraction device according to one embodiment of the present invention.

[0024] Figure 2 is a plan view of the dielectric material extraction device shown in Figure 1.

[0025] Figure 3 is a cross-sectional view along the line I-I' of Figure 2.

[0026] Figure 4 is a cross-sectional view along the line II-II' of Figure 2.

[0027] FIG. 5 is a flowchart of a genetic material extraction method according to one embodiment of the present invention.

[0028] FIGS. 6 to 9 are cross-sectional views illustrating a method for extracting genetic material according to an embodiment of the present invention in chronological order.

[0029] Figure 10 is a diagram illustrating the mobility of cells at different frequencies of alternating current voltage.

[0030] Figure 11 is a diagram illustrating the rupture of a cell membrane as the peak voltage of an alternating current voltage increases.

[0031] Figure 12 is a diagram illustrating the process of rupturing the cell membrane to extract genetic material in chronological order.

[0032] Figures 13a to 13d are experimental data on various factors affecting the rupture of cell membranes.

[0033] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. Identical components in the drawings are denoted by the same reference numerals, and redundant descriptions thereof are omitted.

[0034] FIG. 1 is a perspective view of a genetic material extraction device (1) according to one embodiment of the present invention, and FIG. 2 is a plan view of the genetic material extraction device (1) shown in FIG. 1. FIG. 3 is a cross-sectional view along line I-I' of FIG. 2, and FIG. 4 is a cross-sectional view along line II-II' of FIG. 2.

[0035] Referring to FIG. 1, the dielectric material extraction device (1) may include a substrate (10), a first electrode (20a), a second electrode (20b), a first insulating film (30a), a second insulating film (30b), a sample storage container (40), a power source (50), and a control unit (60).

[0036] According to one embodiment, a sample storage container (40) may be mounted on the main surface (10a) of the substrate (10).

[0037] The substrate (10) may include a base structure composed of a material such as silicon. According to an embodiment, the base structure may include a Complementary Metal-Oxide Semiconductor (CMOS). The substrate (10) may include a semiconductor material, for example, a Group IV semiconductor, a Group III-V compound semiconductor, or a Group II-VI oxide semiconductor. For example, the Group IV semiconductor may include silicon (Si), germanium (Ge), or silicon-germanium. The substrate (10) may be provided as a bulk wafer or an epitaxial layer. In another embodiment, the substrate (10) may include glass or a polymer. In yet another embodiment, the substrate (10) may include a Silicon-On-Insulator (SOI) substrate or a Germanium-On-Insulator (GeOI) substrate.

[0038] According to one embodiment, a first electrode (20a) and a second electrode (20b) may be disposed on a main surface (10a) of a substrate (10), and a first insulating film (30a) and a second insulating film (30b) may be disposed on the first electrode (20a) and the second electrode (20b). The first insulating film (30a) may cover at least a portion of the upper surface of the first electrode (20a), and the second insulating film (30b) may cover at least a portion of the upper surface of the second electrode (20b). As will be described later, the first insulating film (30a) may cover the upper surface of the first electrode (20a), except for a portion where a portion of the first electrode (20a) is exposed through a first window (33a) and a portion where a first conductive connection (23a) to which a power source (50) is connected is exposed. Likewise, the second insulating film (30b) can cover the upper surface of the second electrode (20b), except for the portion where a part of the second electrode (20b) is exposed through the second window (33b) and the portion where a second conductive connection (23b) to which a power source (50) is connected is exposed.

[0039] From a planar perspective, the layout of the first insulating film (30a) may correspond to the layout of the first electrode (30a), and the layout of the second insulating film (30b) may correspond to the layout of the second electrode (20b). Accordingly, from a planar perspective, the first insulating line (31a) of the first insulating film (30a) may overlap with the first conductive line (21a) of the first electrode (30a), and the second insulating line (31b) of the second insulating film (30b) may overlap with the second conductive line (21b) of the second electrode (30b). Also, the first insulating pattern (32a) of the first insulating film (30b) may overlap with the first conductive pattern (22a) of the first electrode (30a), except for the portion where the first window (33a) is formed. The second insulating pattern (32b) of the second insulating film (30b) may overlap with the second conductive pattern (22b) of the second electrode (30b), except for the portion where the second window (33b) is formed.

[0040] The first electrode (20a) and the second electrode (20b) may be arranged to face each other along a first horizontal direction (X direction). The first electrode (20a) may include a first conductive line (21a), a first conductive pattern (22a), and a first conductive connection (23a). The second electrode (20b) may include a second conductive line (21b), a second conductive pattern (22b), and a second conductive connection (23b).

[0041] In the present specification, the direction in which the first electrode (20a) and the second electrode (20b) face each other may be defined as the first horizontal direction (X direction), and the direction parallel to the main surface (10a) of the substrate (10) and perpendicular to the first horizontal direction (X direction) may be defined as the second horizontal direction (Y direction). Also, the direction perpendicular to the main surface (10a) of the substrate (10), the first horizontal direction (X direction), and the second horizontal direction (Y direction) may be defined as the vertical direction (Z direction).

[0042] According to one embodiment, the first electrode (20a) and the second electrode (20b) are spaced apart in the first horizontal direction (X direction) and face each other, so they may not physically come into contact with each other.

[0043] The first conductive line (21a) can be extended along the second horizontal direction (Y direction), and a plurality of first conductive patterns (22a) can be arranged sequentially from one end of the first conductive line (21a). Likewise, the second conductive line (21b) can be extended along the second horizontal direction (Y direction), and a plurality of second conductive patterns (22b) can be arranged sequentially from one end of the second conductive line (21b).

[0044] A plurality of first conductive patterns (22a) may be provided, and the plurality of first conductive patterns (22a) may be arranged spaced apart along a second horizontal direction (Y direction) that extends toward a first horizontal direction (X direction) and is perpendicular to the first horizontal direction (X direction). A plurality of second conductive patterns (22b) may be provided, and the plurality of second conductive patterns (22b) may be arranged spaced apart along a second horizontal direction (Y direction) that extends toward a first horizontal direction (X direction).

[0045] Each of the plurality of first conduction patterns (22a) and each of the plurality of second conduction patterns (22b) can be arranged alternately along the second horizontal direction (Y direction). That is, one second conduction pattern (22b) can be placed between a pair of first conduction patterns (22a) spaced apart along the second horizontal direction (Y direction), and one first conduction pattern (22a) can be placed between a pair of second conduction patterns (22b) spaced apart along the second horizontal direction (Y direction).

[0046] According to one embodiment, the first electrode (20a) and the second electrode (20b) may comprise at least one of a metallic material or a doped semiconductor material. For example, the metallic material may comprise copper (Cu), ruthenium (Ru), aluminum (Al), cobalt (Co), molybdenum (Mo), titanium (Ti), tantalum (Ta), nickel (Ni), platinum (Pt), chromium (Cr), or an alloy thereof. The doped semiconductor material may comprise a material such as doped silicon (Si) or doped germanium (Ge). Examples of the metallic material and the doped semiconductor material are not limited thereto.

[0047] Since the layout of the first insulating film (30a) and the second insulating film (30b) matches the layout of the first electrode (20a) and the second electrode (20b), the first insulating film (30a) and the second insulating film (30b) can also be arranged to face each other along the first horizontal direction (X direction). The first insulating film (30a) may include a first insulating line (31a), a first insulating pattern (32a), and a first window (33a). The second insulating film (30b) may include a second insulating line (31b), a second insulating pattern (32b), and a second window (33b).

[0048] The first insulating line (31a) can cover the upper surface of the first conductive line (21a) so that the first conductive line (21a) is not exposed. Additionally, the second insulating line (31b) can cover the upper surface of the second conductive line (21b) so that the second conductive line (21b) is not exposed. The first insulating pattern (32a) can cover the upper surface of the second conductive pattern (22a) so that the first conductive pattern (22a) of the first electrode (20a) is not exposed, except for a portion of the first electrode (20a) exposed through the first window (33a). The second insulating pattern (32b) can cover the upper surface of the second conductive pattern (22b) so that the second conductive pattern (22b) of the second electrode (20b) is not exposed, except for a portion of the second electrode (20b) exposed through the second window (330b).

[0049] The first insulating film (30a) can define a first window (33a) on the upper surface of the first conductive pattern (22a) to prevent cells in the sample (41) from being trapped in the first electrode (20a), except for a portion of the first electrode (20a) exposed through the first window (33a). As will be explained in detail later, when exposed to an electric field generated across the first electrode (20a) and the second electrode (20b), a dipole can be formed inside the cells, which are organisms in the sample (41). A force is generated by the interaction between the formed dipole and the electric field, and such a force can polarize the cells. This force is called a dielectrophoretic force (DEP force), and the dielectrophoretic force can move the cells in the sample (41) in a specific direction within the area where the electric field is applied. However, since the area of ​​the first electrode (20a) covered by the first insulating film (30a) does not generate an electric field for the cells in the sample (41), the cells cannot move toward the first electrode (20a) covered by the first insulating film (30a).

[0050] In the same principle, the second insulating film (30b) can define a second window (33b) on the upper surface of the second conductive pattern (22b) to prevent cells in the sample (41) from being trapped in the second electrode (20b), except for a portion of the second electrode (20b) exposed through the second window (33b).

[0051] According to one embodiment, as shown in FIG. 3, the first insulating film (30a) may cover not only a part of the upper surface of the first electrode (20a) but also the sidewall of the first electrode (20a). The second insulating film (30b) may also cover not only a part of the upper surface of the second electrode (20b) but also the sidewall of the second electrode (20b). The first insulating film (30a) and the second insulating film (30b) may include, for example, silicon oxide.

[0052] As illustrated in FIG. 3, the sample storage container (40) may be positioned vertically (Z direction) on the main surface (10a) of the substrate (10). The sample storage container (40) may be a container for receiving a sample (41) and may be composed of a polymer or non-polymer material. In one embodiment, the sample storage container (40) may include silicon rubber.

[0053] The first electrode (20a) and the second electrode (20b) may be alternately arranged along the second horizontal direction (Y direction). At this time, a portion of the first electrode (20a) and a portion of the second electrode (20b) that are alternately arranged along the second horizontal direction (Y direction) are a plurality of first conductive patterns (22a) and a plurality of second conductive patterns (22b). The first electrode (20a) and the second electrode (20b) may be placed within the substrate (10) and the sample storage container (40).

[0054] A sample storage container (40) may be configured to contain a sample (41) inside. The sample (41) may include a fluid and cells (CE) suspended in said fluid. The fluid flowing within the sample storage container (40) may include, for example, an aqueous fluid, an aqueous buffer, an organic solvent, a hydrophobic fluid, or a gas, but is not limited thereto.

[0055] In various examples, the fluid may flow within the sample storage container (40) at a flow rate of 10 mL / s or less. The sample storage container (40) may include an inlet (42) into which a sample (41) can be introduced and an outlet (44) into which a sample (41) can be discharged. A sample (41) introduced into the sample storage container (40) through the inlet (42) may flow within the sample storage container (40) at a flow rate within a specific range and then be discharged through the outlet (44).

[0056] According to one embodiment, a first insulating film (30a) covering a portion of the first electrode (20a) and a second insulating film (30b) covering a portion of the second electrode (20b) may be surrounded by a sample (41). The first insulating film (30a) may include a first window (33a) disposed on the upper surface of the first electrode (20a) to expose a portion of the first electrode (20a). Likewise, the second insulating film (30b) may include a second window (33b) disposed on the upper surface of the second electrode (20b) to expose a portion of the second electrode (20b).

[0057] In the first window (33a), the first electrode (20a) can come into contact with the sample (41), and in the second window (33b), the second electrode (20b) can come into contact with the sample (41). Cells in the sample (41) can be trapped in the first window (33a) and the second window (33b) by a dielectrophoretic force resulting from the electric field generated across the first electrode (20a) and the second electrode (20b).

[0058] According to one embodiment, the first window (33a) and the second window (33b) may each have a circular shape in a horizontal view. However, the shapes of the first window (33a) and the second window (33b) in a horizontal view are not necessarily limited to circular shapes, and the first window (33a) and the second window may have any non-circular geometric structure. In various examples, the width (d) of the first window (33a) (also referred to herein as diameter in the case of a circular shape or lateral dimension in the case of any non-circular geometric structure) may be in the range of about 0.1 nm to 1 mm. The same applies to the second window (33b).

[0059] According to one embodiment, a plurality of first windows (33a) may be provided along the first insulation pattern (32a), and a plurality of second windows (33b) may be provided along the second insulation pattern (32b). In this case, the plurality of first windows (33a) and the plurality of second windows (33b) may not overlap each other along the second horizontal direction (Y direction). If the plurality of first windows (33a) and the plurality of second windows (33b) do not overlap each other along the second horizontal direction (Y direction), the windows are evenly distributed, allowing more cells to be efficiently trapped. However, this is merely an example, and depending on the embodiment, the plurality of first windows (33a) and the plurality of second windows (33b) may partially overlap along the second horizontal direction (Y direction).

[0060] According to one embodiment, the power source (50) may be electrically connected to the first electrode (20a) through the first conductive connection (23a) and electrically connected to the second electrode (20b) through the second conductive connection (23b). The power source (50) may be configured to apply an alternating voltage to the first electrode (20a) and the second electrode (20b) by being connected to the first conductive connection (23a) and the second conductive connection (23b).

[0061] The control unit (60) may be configured to transmit / receive electrical signals to apply alternating voltage to the first electrode (20a) and the second electrode (20b) to the power source (50). The control unit (60) may control the power applied by the power source (50). In an exemplary embodiment, the control unit (60) may be implemented as hardware, firmware, software, or any combination thereof. For example, the control unit (60) may be a computing device such as a workstation computer, a desktop computer, a laptop computer, or a tablet computer. The control unit (60) may be a simple controller, a complex processor such as a microprocessor, a CPU, a GPU, etc., a processor configured by software, dedicated hardware, or firmware. The control unit (60) may be implemented by, for example, a general-purpose computer or by application-specific hardware such as a Digital Signal Processor (DSP), a Field Programmable Gate Array (FPGA), and an Application Specific Integrated Circuit (ASIC).

[0062]

[0063] FIG. 5 is a flowchart of a genetic material extraction method according to one embodiment of the present invention, and FIGS. 6 to 9 are drawings illustrating the genetic material extraction method in chronological order.

[0064] Referring to FIG. 6 together with FIG. 5, a method for extracting a dielectric material according to one embodiment may include the step (S110) of providing a first electrode (20a) and a second electrode (20b) arranged to face each other on a main surface (10a) of a substrate (10). At this time, the first electrode (20a) may be exposed to a first window (33a) of a first insulating film (30a), and although not shown in FIG. 6, the second electrode (20b) may be exposed to a second window (33b, see FIG. 2) of a second insulating film (30b).

[0065] The width of the first window (33a) in the lateral direction (X direction and / or Y direction) may be smaller than the width of the first electrode (20a) in the lateral direction (X direction and / or Y direction). Therefore, the entire upper surface of the first electrode (20a) is not exposed through the first window (33a). However, this is merely an example, and according to the embodiment, the width of the first window (33a) in the lateral direction (X direction and / or Y direction) may be greater than or equal to the width of the first electrode (20a) in the lateral direction (X direction and / or Y direction), so that the entire upper surface of the first electrode (20a) is exposed. The same applies to the second electrode (20b) and the second window (33b, see FIG. 2).

[0066]

[0067] Referring to FIG. 7 together with FIG. 5, a genetic material extraction method according to one embodiment may include the step (S120) of placing a sample (41) in which cells (CE) are suspended on a first electrode (20a) and a second electrode (20b).

[0068] First, a sample storage container (40) can be placed on the main surface (10a) of the substrate (10), and a sample (41) can be introduced into the sample storage container (40) through the inlet (42) of the sample storage container (40). The sample (41) may include a solution and a cell (CE) suspended in the solution. The cell (CE) from which genetic material has been extracted can be discharged together with the solution through the outlet (44) of the sample storage container (40).

[0069] The sample (41) can be positioned to completely overlap the first electrode (20a) and the second electrode (20b) in a vertical direction (Z direction). Specifically, the sample (41) can come into contact with the first electrode (20a) through the first window (33a) of the first insulating film (30a). Although not shown in detail in FIG. 7, the sample (41) can come into contact with the second electrode (20b) through the second window (33b, see FIG. 2) of the second insulating film (30b).

[0070]

[0071] Referring to FIG. 8 together with FIG. 5, a method for extracting genetic material according to one embodiment may include the step (S130) of generating an electric field within a first window (33a) and a second window (33b) by applying an alternating voltage to a first electrode (20a) and a second electrode (20b) through a power source (50).

[0072] The control unit (60) can control the power source (50) to apply an alternating voltage to the first electrode (20a) and the second electrode (20b). When the power source (50) applies an alternating voltage to the first electrode (20a) and the second electrode (20b), a non-uniform electric field induced by the alternating signal may be generated across the first electrode (20a) and the second electrode (20b).

[0073]

[0074] Referring to FIG. 8 together with FIG. 5, a genetic material extraction method according to one embodiment may include the step (S140) of trapping a cell (CE) within a first window (33a) or a second window (33b) through a dielectrophoretic force generated by an electric field.

[0075] The control unit (60) can increase the frequency of the alternating voltage applied to the first electrode (20a) and the second electrode (20b) to trap cells (CE) suspended in the solution of the sample (41) within the first window (33a) or the second window (33b). At this time, the control unit (60) can control the power source (50) to maintain the peak voltage of the alternating voltage constant.

[0076] When the frequency of the alternating voltage applied by the power source (50) is lower than the critical frequency, the cell may hardly move. Subsequently, when the frequency of the alternating voltage gradually increases and exceeds a specific critical frequency, the cell may move toward the first window (33a) or the second window (33b, see FIG. 2) and be trapped. The principle of how the cell is trapped within the first window (33a) or the second window (33b, see FIG. 2) will be explained in detail later with reference to FIG. 10.

[0077]

[0078] Referring to FIG. 9 together with FIG. 5, a genetic material extraction method according to one embodiment may include a step (S150) in which a power source (50) increases the peak voltage of an alternating voltage until the cell membrane is ruptured in order to extract genetic material within a cell trapped in a first window (33a) or a second window (33b, see FIG. 2).

[0079] The control unit (60) can increase the peak voltage of the alternating voltage applied by the power source (50) to rupture the membrane of a cell trapped within the first window (33a) or the second window (33b, see FIG. 2). At this time, the control unit (60) can control the power source (50) to maintain the frequency of the alternating voltage constant.

[0080] When the peak voltage of the alternating current voltage applied by the power source (50) increases significantly, the membrane of the cell (CE_R) may rupture. Specifically, when the peak voltage increases significantly, a perforation may be formed in the membrane of the cell (CE_R). At this time, genetic material floating within the membrane of the cell (CE_R) may escape through the perforation of the membrane and flow into the solution of the sample (41). The genetic material within the cell (CE_R) may include DNA, miRNA, plasmid, or fragments having any base sequence. The principle of the rupture of the cell (CE_R) membrane will be explained in detail later with reference to FIG. 11.

[0081] When a perforation is formed in the cell (CE_R), the control unit (60) can control the power source (50) to reduce the frequency of the alternating voltage. When the frequency of the alternating voltage applied by the power source (50) is reduced, the cell (CE_R) under the influence of the electric field generated across the first electrode (20a) and the second electrode (20b) is no longer trapped within the first window (33a) and the second window (33b).

[0082] The perforated cell (CE_R) floats along the solution of the sample (41) flowing at a flow rate within a specific range, and the perforated cell (CE_R) is discharged from the sample storage container (40) through the outlet (44) of the sample storage container (40).

[0083]

[0084] Figure 10 is a diagram illustrating the mobility of a cell (CE) according to the frequency of an alternating current voltage.

[0085] The following description may also apply to the second electrode (20b, see FIG. 2), but for the convenience of explanation, the description will focus on the first electrode (20a).

[0086] Referring to FIG. 10, when a cell (CE) in a sample (41) is under the influence of an electric field generated by the first electrode (20a), charges are induced on the surface of the cell (CE) along the direction of the electric field, causing polarization. If the electric field is uniform, the magnitudes of the electrostatic forces acting on the left and right sides of the polarized cell (CE) are equal, so the net force acting on the cell (CE) is zero and the cell (CE) does not move. However, if the electric field acting on the cell (CE) is non-uniform, the magnitudes of the electrostatic forces acting on the cell (CE) are different, so the cell (CE) undergoes translational motion according to the gradient of the electric field. At this time, if the polarizability of the cell (CE) is greater than the polarizability of the solution of the sample (41) surrounding the cell (CE), the cell (CE) moves to a place where the electric field is strong (positive dielectrophoresis, positive DEP), and conversely, if the polarizability of the cell (CE) is less than the polarizability of the solution, the cell (CE) moves in a direction where the electric field strength is weak (negative dielectrophoresis, negative DEP). Generally, the polarizability of the cell (CE) can be determined by the material constituting the cell (CE), the surface properties of the cell (CE), and the solution surrounding the cell (CE).

[0087] When a power source (50, see FIG. 2) applies an alternating voltage to the first electrode (20a), a non-uniform electric field induced by the alternating voltage may be generated across the first electrode (20a).

[0088] In the case of this embodiment, the polarity of the cell (CE) may be greater than the polarity of the solution of the sample (41) surrounding the cell (CE). Since the electric field strength is stronger the closer it is to the first electrode (20a) exposed to the first window (33a), the cell (CE) approaches the first window (33a) formed in the first insulation pattern (32a).

[0089] When the frequency of the alternating voltage applied by the power source (50) is lower than the critical frequency, the cell (CE) hardly moves. Subsequently, when the frequency of the alternating voltage gradually increases and exceeds a specific critical frequency, the cell (CE) moves toward the first window (33a). At this time, the critical frequency can be referred to as the effective crossover frequency. The effective crossover frequency may depend on independent variables such as the material constituting the cell (CE), the surface properties of the cell (CE), and the solution surrounding the cell (CE).

[0090] FIG. 10 illustrates the process of a cell (CE) approaching the first window (33a) when the frequency increase rate of the AC voltage is 100 Hz / s, 400 Hz / s, and 1600 Hz / s. When the frequency increase rate of the AC voltage is 100 Hz / s, the cell (CE) does not move because the frequency of the AC voltage exceeds the effective crossover frequency over a long period of time. When the frequency increase rate of the AC voltage is 400 Hz / s, the cell (CE) approaches the first window (33a) because the frequency of the AC voltage exceeds the effective crossover frequency. When the frequency increase rate of the AC voltage is 1600 Hz / s, the frequency of the AC voltage exceeds the effective crossover frequency quickly, and because the frequency increases at a rapid speed, the cell (CE) approaches the first window (33a) quickly.

[0091] However, specific values ​​such as 100 Hz / s, 400 Hz / s, and 1600 Hz / s are merely illustrative figures to explain that as the frequency of the alternating voltage increases rapidly, the cell (CE) moves closer to the first window (33a) by the dielectrophoretic force, and the frequency values ​​of the alternating voltage applied by the power source (50, see FIG. 2) are not limited to the above.

[0092] According to one embodiment, the frequency of the alternating voltage applied by the power source (50, see FIG. 2) can be increased or decreased within a range of 0.1 kHz to 100 kHz.

[0093]

[0094] Figure 11 is a diagram illustrating membrane rupture of a cell (CE) as the peak voltage of the alternating current voltage increases.

[0095] For convenience of explanation, FIG. 11 will be described with reference to FIG. 10. As with FIG. 10, the following description may also apply to the second electrode (20b, see FIG. 2), but for convenience of explanation, the description will focus on the first electrode (20a, see FIG. 10).

[0096] Specifically, Photograph 11-a of FIG. 11 is a photograph showing the phenomenon in which a cell (CE) in a sample (41) is trapped within the first window (33a) of the first insulation pattern (32a) by positive dielectrophoresis (positive DEP) described with reference to FIG. 10. In order to trap the cell (CE) within the first window (33a) using positive dielectrophoresis, the peak voltage of the alternating voltage applied by the power source (50, see FIG. 2) is maintained at a relatively low level.

[0097] Photograph 11-b is a photograph showing the phenomenon of the membrane of a cell (CE) in a sample (41) trapped within the first window (33a) of the first insulation pattern (32a) being ruptured. The membrane of the cell (CE_R) trapped within the first window (33a) may be ruptured by electroporation under the influence of an electric field generated at the first electrode (20a; see FIG. 10). As the membrane of the cell (CE_R) is ruptured, genetic material within the cell (CE_R) may escape into the solution of the sample (41). The genetic material may include DNA, miRNA, plasmid, or fragments having any base sequence.

[0098]

[0099] Figure 12 is a diagram illustrating the process of rupturing the membrane of a cell (CE) to extract genetic material in chronological order.

[0100] For convenience of explanation, the following description will be made with reference to FIG. 12 in conjunction with FIG. 10. The configurations shown in the photograph in FIG. 12 correspond to the first electrode (20a), the first insulating pattern (32a), the first window (33a), the sample (41), and the cell (CE) shown in FIG. 10. As with FIG. 10, the following description may also apply to the second electrode (20b, see FIG. 2), but for convenience of explanation, the description will focus on the first electrode (20a).

[0101] The principle of rupturing the membrane of a cell (CE) is based on the pore formation mechanism. Factors such as the strength and duration of the electric field, ambient temperature, and the inherent characteristics of the cell (CE) membrane can determine the energy essential for pore formation.

[0102] In graph 12-a located at the top of FIG. 12, the horizontal axis represents the peak voltage of the alternating voltage applied by the power source (50, see FIG. 2) to the first electrode (20a), and the vertical axis represents the grayscale intensity of the cell (CE), i.e., the measured value of the brightness value of the cell (CE). Photographs 12-b located at the bottom of FIG. 12 are photographs of the cell (CE) observed according to changes in the frequency or peak voltage of the alternating voltage applied by the power source (50, see FIG. 2).

[0103] First, when the polarity of the cell (CE) is smaller than the polarity of the solution in the sample (41), the cell (CE) moves in the direction of the weak electric field strength (negative dielectrophoresis, Negative DEP). When the frequency of the alternating voltage applied by the power source (50, see FIG. 2) is 1 kHz and the maximum power is 2 V, the polarity of the cell (CE) under the influence of the electric field becomes smaller than the polarity of the solution in the sample (41). Therefore, the cell (CE) is positioned on the first insulation pattern (32a) rather than the first window (33a) where the electric field strength is relatively strong and the first electrode (20a) is exposed.

[0104] Subsequently, when the frequency of the alternating voltage applied by the power source (50, see FIG. 2) gradually increases, the polarity of the cell (CE) becomes greater than the polarity of the solution in the sample (41), causing the cell (CE) to move in the direction where the electric field strength is strong (positive dielectrophoresis, Positive DEP). When the frequency of the alternating voltage applied by the power source (50, see FIG. 2) is 41 kHz, the polarity of the cell (CE) under the influence of the electric field becomes greater than the polarity of the solution in the sample (41). Accordingly, the cell (CE) moves to the first window (33a) where the first electrode (20a) is exposed and the electric field strength is relatively strong.

[0105] In order to trap a cell (CE) into the first window (33a) through positive electrophoresis, it is not necessary to increase the peak voltage of the alternating voltage applied by the power source (50, see FIG. 2). According to an embodiment, the cell (CE) can be trapped into the first window (33a) by increasing the frequency of the alternating voltage while maintaining the peak voltage of the alternating voltage applied by the power source (50, see FIG. 2) constant. In another embodiment, the cell (CE) can be trapped into the first window (33a) by increasing the frequency of the alternating voltage applied by the power source (50, see FIG. 2) while simultaneously increasing the peak voltage of the alternating voltage.

[0106] After trapping the cell (CE) in the first window (33a), the peak voltage of the alternating voltage applied by the power source (50, see FIG. 2) is increased. In this example, the peak-to-peak voltage (V p-p The voltage rose from 2 V to 10 V. When the peak-to-peak voltage reached approximately 6.5 V, it was confirmed that the membrane of the cell (CE) had ruptured. When the membrane of the cell (CE) ruptured, the intensity of the grayscale increased rapidly. It can be seen that the rapid change in the grayscale was due to the irreversible electroporation phenomenon. According to one example, when the peak voltage (peak-to-peak voltage in this experiment) of the alternating voltage applied by the power source (50, see FIG. 2) increases, the power source (50, see FIG. 2) can maintain the frequency of the alternating voltage constant.

[0107] According to one embodiment, the peak voltage of the AC voltage applied by the power source (50, see FIG. 2) can be increased in the range of 0.1 V to 100 V. Accordingly, the peak-to-peak voltage of the AC voltage (V p-p ) can be increased in the range of 0.2 V to 200 V, which is twice the peak voltage.

[0108]

[0109] If the peak voltage of the alternating current applied by the power source (50, see FIG. 2) increases significantly, perforations may form in the membrane of the cell (CE). If the peak voltage of the alternating current applied by the power source (50, see FIG. 2) is relatively small, the membrane of the cell (CE) is not permanently damaged, and the perforations formed in the membrane naturally close after a certain period of time. On the other hand, if the peak voltage of the alternating current applied by the power source (50, see FIG. 2) is relatively large, the membrane of the cell (CE) is permanently damaged, the perforations formed in the membrane do not close, and the cell (CE) eventually dies. In this specification, the peak-to-peak voltage (V) at which membrane rupture begins is p-p ) shall be referred to as rupture voltage.

[0110]

[0111] Figures 13a to 13d are experimental data on various factors affecting the rupture of the membrane of a cell (CE).

[0112] The following description will be explained with reference to FIGS. 13a to 13d. The membrane of a cell (CE) is a lipid bilayer composed of phospholipids, cholesterol, and various lipids and proteins. Physical properties such as resistance to membrane rupture are derived from these constituent materials of the membrane.

[0113] The cholesterol content of the cell (CE) membrane and the rate of increase of the alternating voltage applied by the power source (50, see FIG. 2) can affect the resistance to rupture of the membrane.

[0114] Methyl-β-cyclodextrin (MβCD) can be configured to remove cholesterol that makes up the membrane of a cell (CE). Thus, when a certain amount of methyl-β-cyclodextrin is contained in a sample (41) in which a cell (CE) is suspended, cholesterol within the membrane of the cell (CE) can be removed.

[0115] Referring to the graph shown in FIG. 13a, the horizontal axis represents the concentration of methyl-beta-cyclodextrin in the solution of sample (41), and the vertical axis represents the result of measuring the critical energy required to form a perforation in the membrane of the cell (CE).

[0116] Referring to the graph shown in FIG. 13b, the horizontal axis represents the concentration of methyl-beta-cyclodextrin in the solution of sample (41), and the vertical axis represents the radius of the perforation formed in the membrane of the cell (CE).

[0117] Figure 13c is a drawing showing the results of the graph in Figure 13a and the graph in Figure 13b as an image.

[0118] As illustrated in the graph of FIG. 13a, when the concentration of methyl-beta-cyclodextrin in the solution of sample (41) is high, it is observed that cholesterol in the membrane is removed more effectively, and the critical energy required to form a perforation in the membrane is reduced. As illustrated in the image of FIG. 13c, methyl-beta-cyclodextrin destroys cholesterol in the membrane of the cell (CE), allowing a perforation to form in the membrane under a weaker electric field influence. Therefore, when the concentration of methyl-beta-cyclodextrin in the solution of sample (41) is high, the bursting voltage required to rupture the membrane can be reduced. Since the bursting voltage is proportional to the critical energy required to form a perforation in the membrane, the critical energy can also be reduced when the concentration of methyl-beta-cyclodextrin is high.

[0119] In addition, as shown in the graph of Fig. 13b and Fig. 13c, when the concentration of methyl-beta-cyclodextrin is high, the resistance to membrane rupture is reduced, and the radius of the perforation formed in the membrane may be reduced. That is, since the resistance to membrane rupture is reduced, lower critical energy is required for membrane rupture, and thus the radius of the perforation formed may also be reduced. A decrease in the critical energy required to form a perforation within the membrane is correlated with a decrease in the resistance to membrane rupture.

[0120] Referring to FIG. 13d, the horizontal axis represents the rate of increase of the alternating voltage applied by the power source (50, see FIG. 2), and the vertical axis represents the peak-to-peak voltage (V) at which the membrane of the cell (CE) begins to rupture. p-p ...represents ). In addition, the square, circle, and triangle-shaped points represent cases where the concentration of methyl-beta-cyclodextrin in the solution of sample (41) is 0 mM, 2.5 mM, and 5.0 mM, respectively.

[0121] As shown in FIG. 13d, it can be seen that regardless of the concentration of methyl-beta-cyclodextrin in the solution of sample (41), as the rate of increase of the alternating voltage applied by the power source (50) increases, the peak-to-peak voltage at which the membrane of the cell (CE) begins to rupture increases.

[0122] Accordingly, a genetic material extraction device (1, see FIG. 2) according to one embodiment of the present invention may provide methyl-beta-cyclodextrin in the sample (41) or maintain a low rate of increase of the alternating voltage applied by the power source (50, see FIG. 2) in order to lower the rupture voltage of the power source (50, see FIG. 2) required for the rupture of the membrane of a cell (CE) suspended in the sample (41). For example, the concentration of methyl-beta-cyclodextrin in the sample (41) may be 5.0 mM or less, and the rate of increase of the alternating voltage may be 0.1 V / s or less. However, the above figures are merely exemplary and are not limited thereto.

[0123] As described above, exemplary embodiments have been disclosed in the drawings and specification. Although specific terms have been used to describe the embodiments in this specification, they are used only for the purpose of explaining the technical concept of this disclosure and are not intended to limit the meaning or the scope of this disclosure as defined in the claims. Therefore, those skilled in the art will understand that various modifications and equivalent alternative embodiments are possible therefrom. Accordingly, the true technical scope of protection of this disclosure should be determined by the technical concept of the appended claims.

Claims

1. A substrate that accommodates a sample in which cells are suspended on its main surface; A first electrode disposed on the main surface of the above substrate; A second electrode positioned to face the first electrode along a first horizontal direction, configured to generate an electric field across the first electrode and the second electrode; A first insulating film disposed on the upper surface of the first electrode and having a first window that exposes a portion of the upper surface of the first electrode; A second insulating film disposed on the upper surface of the second electrode and having a second window that exposes a portion of the upper surface of the second electrode; and A power source that applies an alternating voltage to the first electrode and the second electrode to generate an electric field for trapping the cell within the first window or the second window; The above power source is a genetic material extraction device configured to increase the peak voltage of the alternating voltage to rupture the membrane of the cell and extract genetic material after the cell is trapped in the first window or the second window.

2. In Paragraph 1, The first electrode above is, A plurality of first conductive patterns extending toward the second electrode and having the first window located on the upper surface; The second electrode above is, A plurality of second conductive patterns extending toward the first electrode and having the second window located on the upper surface; A dielectric material extraction device characterized in that the plurality of first conduction patterns and the plurality of second conduction patterns are arranged to intersect each other along a second horizontal direction perpendicular to the first horizontal direction.

3. In Paragraph 1, The above power source is, A genetic material extraction device characterized by being configured to increase the frequency of the alternating voltage applied to the first electrode and the second electrode until the cell is trapped within the first window or the second window.

4. In Paragraph 3, A genetic material extraction device characterized by the above frequency increasing within a range of 0.1 kHz to 100 kHz.

5. In Paragraph 1, The above first window and the above second window are, A genetic material extraction device characterized by being a circular hole from a horizontal perspective.

6. In Paragraph 1, The above first window and the above second window are, A genetic material extraction device characterized by not overlapping each other along a second horizontal direction perpendicular to the first horizontal direction.

7. In Paragraph 1, The first electrode and the second electrode are, A genetic material extraction device characterized by not coming into direct contact with each other.

8. In Paragraph 1, The above sample includes a fluid that accommodates the cell, and A dielectric material extraction device characterized in that the above fluid comprises at least one of an aqueous fluid, an aqueous buffer, an organic solvent, a hydrophobic fluid, and a gas.

9. In Paragraph 1, A dielectric material extraction device characterized by the above peak voltage increasing within a range of 0.1 V to 100 V.

10. In Paragraph 1, Each of the first electrode and the second electrode is, A dielectric material extraction device characterized by comprising at least one of a metallic material and a doped semiconductor material.

11. In Paragraph 1, Each of the above-mentioned first insulating film and the above-mentioned second insulating film is, A dielectric material extraction device characterized by including silicon oxide.

12. A step of providing a first electrode exposed to a first window of a first insulating film and a second electrode exposed to a second window of a second insulating film so as to be arranged facing each other on the main surface of a substrate; A step of placing a sample in which cells are suspended on the first electrode and the second electrode; A step of generating an electric field within the first window and the second window by applying an alternating voltage to the first electrode and the second electrode through a power source; A step of trapping the cell within the first window or the second window through a dielectrophoresis force generated by the electric field; and A method for extracting genetic material, comprising the step of increasing the peak voltage of the alternating voltage of the power source until the membrane of the cell ruptures, in order to extract genetic material within the cell trapped in the first window or the second window.

13. In Paragraph 12, In the step of trapping the cell within the first window or the second window, A method for extracting genetic material characterized by the above power source increasing the frequency of the alternating voltage until the cell is trapped within the first window or the second window.

14. In Paragraph 12, In the step of trapping the cell within the first window or the second window, The above power source maintains the peak voltage of the above alternating voltage constant, and In the step where the power source increases the peak voltage of the alternating voltage until the membrane of the cell ruptures, A method for extracting dielectric material characterized by the above power source maintaining the frequency of the above alternating voltage constant.

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