Genetic material detection apparatus and genetic material detection method using same

Abstract

The technical idea of the present invention provides a genetic material detection 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; a first insulating film disposed on an upper surface of the first electrode and having a first window exposing a portion of the upper surface 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; a power source configured to apply an alternating current 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, and then to increase a peak voltage of the alternating current voltage to rupture membranes of the cells so as to extract genetic material; and a biosensor including a probe capable of binding to the genetic material.

Description

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

[0001] The present invention relates to an apparatus for detecting genetic material that responds to dielectrophoretic force and a method for detecting 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 detection device capable of trapping cells using dielectrophoretic force and detecting genetic material within said cells.

[0005] The problem that the technical concept of the present invention aims to solve is to provide a method for detecting genetic material using a genetic material detection device capable of trapping cells using dielectrophoretic force and detecting 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 detection 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, 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; a power source configured to increase the peak voltage of the alternating voltage to generate an electric field for trapping the cells within the first window or the second window by applying an alternating voltage to the first electrode and the second electrode, and then to rupture the membrane of the cells to extract genetic material within the cells; and a biosensor comprising a probe capable of binding to the genetic material.

[0008] According to one embodiment, the biosensor comprises: a biosensor substrate; a source electrode and a drain electrode spaced apart from each other and disposed on the biosensor substrate; and a PEDOT:PSS layer electrically connected to the source electrode and the drain electrode and comprising PEDOT:PSS (poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate)); wherein the probe is disposed on the PEDOT:PSS layer.

[0009] According to one embodiment, a genetic material detection device is provided, characterized in that the PEDOT:PSS layer comprises a peak portion and a valley portion having a height difference in a direction perpendicular to the upper surface of the biosensor substrate.

[0010] According to one embodiment, the biosensor comprises: a biosensor substrate; a source electrode and a drain electrode spaced apart from each other and disposed on the biosensor substrate; and graphene electrically connected to the source electrode and the drain electrode, wherein high points and low points having a height difference are repeated in a zigzag shape along the length direction; and wherein the probe is disposed on the graphene.

[0011] According to one embodiment, a dielectric material detection device is provided, characterized in that the height difference between the high point and the low point of the graphene is in the range of 0.2 nanometers to 20 nanometers.

[0012] According to one embodiment, a genetic material detection 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.

[0013] To solve the above problem, the technical concept of the present invention provides a method for detecting genetic material comprising: a step of providing a substrate in which 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 are arranged facing each other on a main surface; a step of positioning a sample storage container containing 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; a step of increasing the peak voltage of the alternating voltage through the power source until the membrane of the cells ruptures in order to extract genetic material within the cells trapped in the first window or the second window; and a step of detecting the genetic material extracted from the cells through a probe of a biosensor located within the sample.

[0014] According to one embodiment, a method for detecting genetic material is provided, characterized in that the sample storage container comprises: an inlet which is a passage into which the sample is introduced; a first outlet which is a passage into which the sample is discharged; and a second outlet which is a passage into which the membrane of the ruptured cell is discharged.

[0015] According to one embodiment, a method for detecting 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.

[0016] According to one embodiment, a method for detecting 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.

[0017] A genetic material detection 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 at 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 detected more efficiently.

[0018] In addition, by placing a biosensor that detects the genetic material within the sample, the series of processes for extraction and detection of the genetic material can be carried out efficiently.

[0019] 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.

[0020] FIG. 1 is a perspective view of a genetic material extraction device according to one embodiment of the present invention.

[0021] Figure 2 is a plan view of the dielectric material extraction device shown in Figure 1.

[0022] 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.

[0023] FIG. 5a is a cross-sectional view of the biosensor shown in FIG. 1 to 3, and FIG. 5b is a diagram illustrating the structure of PEDOT:PSS.

[0024] FIG. 5c is a drawing for explaining the PEDOT:PSS electrode of FIG. 5a, FIG. 5d is an enlarged view of the area marked "EX" in FIG. 5c, and FIG. 5e is a drawing showing the area corresponding to the area marked "EX" in FIG. 5c.

[0025] FIG. 6a is a cross-sectional view of a biosensor according to another embodiment of the present invention.

[0026] FIG. 6b is a diagram illustrating the graphene shown in FIG. 6a, and FIG. 6c is an enlarged view of the Q region of the graphene shown in FIG. 6b.

[0027] FIG. 7 is a flowchart of a method for detecting genetic material according to one embodiment of the present invention.

[0028] FIGS. 8 to 11 are cross-sectional views illustrating a method for detecting genetic material according to an embodiment of the present invention in chronological order.

[0029] Figure 12 is a diagram illustrating the mobility of cells according to the frequency of alternating current voltage.

[0030] Figure 13 is a diagram illustrating the rupture of a cell membrane as the peak voltage of an alternating current voltage increases.

[0031] Figure 14 is a diagram illustrating the process of rupturing the cell membrane to extract genetic material in chronological order.

[0032] Figures 15a to 15d 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 genetic 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), a control unit (60), and a biosensor (80).

[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 (20a), 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 (20a), and the second insulating line (31b) of the second insulating film (30b) may overlap with the second conductive line (21b) of the second electrode (20b). Also, the first insulating pattern (32a) of the first insulating film (30a) may overlap with the first conductive pattern (22a) of the first electrode (20a), 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 (20b), 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 first 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 (33b).

[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 a first outlet (44) into which a sample (41) can be discharged. The 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 first outlet (44). Additionally, the sample storage container (40) may further include a second outlet (46) located between the inlet (42) and the first outlet (44) along a second horizontal direction (Y direction). As will be explained in detail later, among the cells suspended in the sample (41), cells from which genetic material has been extracted due to membrane rupture may be discharged outside the sample storage container (40) through the second outlet (46). The second outlet (46) may be located between the first and second electrodes (20a, 20b) and the biosensor (80) along the second horizontal direction (Y direction). Thus, cells trapped in the first and second electrodes (20a, 20b) and with their membranes ruptured may be discharged through the second outlet (46) before reaching the biosensor (80).

[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 the 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] According to one embodiment, the biosensor (80) may be placed in a sample storage container (40) on the main surface (10a) of the substrate (10). The biosensor (80) may be used to perform gene expression profiling, genotyping, detection of mutations and polymorphisms such as SNPs (Single Nucleotide Polymorphisms), protein and peptide analysis, screening of potential drugs, and new drug development and manufacturing by analyzing the genetic material of cells contained in the sample (41).

[0063] The biosensor (80) employs probes suitable for the target of the genetic material to be analyzed. Examples of probes that can be employed in the biosensor include DNA probes, protein probes such as enzymes, antibodies / antigens, and bacteriorhodopsin, microbial probes, and neuronal probes. A biosensor (80) manufactured in the form of a chip is also referred to as a biochip. For example, depending on the type of probe employed, it may be referred to as a DNA chip, protein chip, cell chip, neuron chip, etc. A biosensor (80) according to some embodiments of the present invention may include an oligomer probe as a probe. The oligomer probe implies that the number of monomers in the probe employed is at the oligomer level. Here, the term oligomer may be used to refer to a polymer composed of two or more covalently bonded monomers with a molecular weight of about 1000 or less. Specifically, it may include 2 to 500 monomers, preferably 5 to 30 monomers. However, the meaning of an oligomer probe is not limited to the above figures. The monomers constituting the oligomer probe can be modified depending on the type of biosample to be analyzed, and may be, for example, nucleosides, nucleotides, amino acids, peptides, etc. Nucleosides and nucleotides may include known purine and pyrimidine bases, as well as methylated purines or pyrimidines, acylated purines or pyrimidines, etc. Furthermore, nucleosides and nucleotides may include conventional ribose and deoxyribose sugars, as well as modified sugars in which one or more hydroxyl groups are substituted with halogen atoms or aliphatic atoms, or functional groups such as ethers or amines are attached.Amino acids may be L-, D-, and nonchiral types of amino acids found in nature, as well as modified amino acids or amino acid analogs. A peptide refers to a compound formed by an amide bond between the carboxyl group of an amino acid and the amino group of another amino acid. Unless otherwise specifically noted, the probe exemplarily conceived in the following examples is a DNA probe, which is an oligomeric probe in which monomers of about 5 to 30 nucleotides are covalently bonded. However, the present invention is not limited thereto, and it goes without saying that various probes described above may be applied. A detailed description of the structure of the biosensor (80) will be provided later.

[0064] FIG. 5a is a cross-sectional view of the biosensor (80) shown in FIG. 1 to 3, and FIG. 5b is a drawing for explaining the structure of PEDOT:PSS.

[0065] Referring to FIG. 5a, the biosensor (80) may include a biosensor substrate (811), a source electrode (812), a drain electrode (813), and a PEDOT:PSS electrode (PS). The biosensor substrate (811) may extend in a first horizontal direction (X direction) and a second horizontal direction (Y direction).

[0066] In exemplary embodiments, the biosensor substrate (811) may comprise a material selected from the group consisting of glass, quartz, SiC, MgO, Si, SiO2, Ge, GaN, AlN, GaP, InP, GaAs, SiC, Al2O3, LiAlO3, MgO, graphite, graphene, plastic, ceramic, rubber, and combinations thereof. However, this is exemplary, and the biosensor substrate (811) of the present invention is not limited to the material exemplified.

[0067] The source electrode (812) and the drain electrode (813) may be disposed on the biosensor substrate (811). The drain electrode (813) may be spaced apart from the source electrode (812) in a second horizontal direction (Y direction). The source electrode (812) and the drain electrode (813) may be formed of various materials and may include metals or metal alloys. For example, the source electrode (812) and the drain electrode (813) may include gold (Au), copper (Cu), ruthenium (Ru), aluminum (Al), cobalt (Co), tungsten (W), molybdenum (Mo), titanium (Ti), tantalum (Ta), nickel (Ni), platinum (Pt), chromium (Cr), or alloys thereof.

[0068] A PEDOT:PSS electrode (PS) may be interposed between the source electrode (812) and the drain electrode (813). However, the PEDOT:PSS electrode (PS) may not have a flat structure extending along the upper surface of the biosensor substrate (811), but may include a peak portion and a valley portion having a height difference in a direction perpendicular to the upper surface of the biosensor substrate (811) (e.g., the vertical direction (Z direction)). A detailed description of the structure of the PEDOT:PSS electrode (PS) will be provided later in the description regarding FIG. 5c and FIG. 5d. The shape of the uneven surface and the degree of unevenness of the PEDOT:PSS electrode (PS) may vary depending on the manufacturing process. Although not illustrated, a gate electrode for supplying voltage to the PEDOT:PSS electrode (PS) may be electrically connected to the PEDOT:PSS electrode (PS).

[0069] The biosensor (80) may further include a probe (815) capable of chemically binding to genetic material on a PEDOT:PSS electrode (PS). For example, the probe (815) may be one of a polynucleotide, a peptide nucleic acid probe, an aptamer (DNA having the ability to specifically bind to a specific substance), a protein, an antibody, or a capture agent. For example, if the genetic material is miRNA, the probe (815) may have a nucleotide sequence complementary to the nucleotide sequence of the miRNA, which is the genetic material, for chemical binding with the genetic material. Additionally, the length of the probe (815) may be adjusted to fit the entire sequence or a specific part of the miRNA. For example, a probe (815) complementary to the entire sequence of the miRNA may include about 20 to 25 bases.

[0070] The biosensor (80) may further include a linker (814) between the probe (815) and the PEDOT:PSS electrode (PS) to facilitate chemical bonding between the probe (815) and the PEDOT:PSS electrode (PS). In one embodiment, when the genetic material is miRNA, nano-sized gold (Au) particles may be selected as the linker (814).

[0071] It is possible to determine whether dielectric material is present in a sample (SP) by observing electrical parameters measured by supplying voltage to the PEDOT:PSS electrode (PS). For example, electrical parameters may include capacitance, voltage, average current, etc. Specifically, regarding a first sample in which dielectric material is absent and a second sample for which observation regarding the presence of dielectric material is required, the presence of dielectric material in the second sample can be confirmed by comparing the electrical parameters measured by supplying voltage to the PEDOT:PSS electrode (PS) when the first sample is introduced with the electrical parameters measured by supplying voltage to the PEDOT:PSS electrode (PS) when the second sample is introduced. A difference in the electrical parameters of the second sample compared to the first sample may indicate the presence of dielectric material.

[0072] FIG. 5c is a drawing for explaining the PEDOT:PSS electrode (PS) of FIG. 5a, FIG. 5d is an enlarged view of the area marked "EX" in FIG. 5c, and FIG. 5e is a drawing showing the area corresponding to the area marked "EX" in FIG. 5c.

[0073] Referring to FIGS. 5c, 5d, and 5e, the surface of the PEDOT:PSS electrode (PS) may have a rough shape. For example, the PEDOT:PSS electrode (PS) may include a peak (P) and a valley (V) having a height difference in the vertical direction (Z direction). In some embodiments, the peak (P) and the valley (V) of the PEDOT:PSS electrode (PS) may be alternately arranged along one direction (e.g., a first horizontal direction (X direction), a second horizontal direction (Y direction), or a diagonal direction between the first horizontal direction (X direction) and the second horizontal direction (Y direction). In some embodiments, the peak (P) and the valley (V) of the PEDOT:PSS electrode (PS) may extend in another direction perpendicular to the one direction in which the peak (P) and the valley (V) are alternately arranged. For example, the peak portion (P) and the valley portion (V) can be formed in a line shape extending in the other direction.

[0074] As illustrated in FIG. 5c, regarding the first valley portion (V1) disposed on one side of the peak portion (P) of the PEDOT:PSS electrode (PS) and the second valley portion (V2) disposed on the other side of the peak portion (P), the first valley portion (V1) and the second valley portion (V2) are spaced apart from each other in the second horizontal direction (Y direction) with the peak portion (P) in between, and the peak portion (P), the first valley portion (V1), and the second valley portion (V2) can be extended in the first horizontal direction (X direction).

[0075] In exemplary embodiments, as illustrated in FIG. 5d, a first inclined surface (SL1) extending from a peak (P) to a first valley (V1) and a second inclined surface (SL2) extending from a peak (P) to a second valley (V2) may face each other in a first horizontal direction (X direction) with the peak (P) in between.

[0076] In exemplary embodiments, the peak portion (P) and the valley portion (V) are shown to have a curved shape, but are not limited thereto and may have a pointed shape.

[0077] In exemplary embodiments, the peak portion (P) may be arranged at a first interval (W1) in a range of about several nanometers to several tens of micrometers along the second horizontal direction (Y direction). The valley portion (V) may be arranged at a second interval (W2) in a range of about several nanometers to several tens of micrometers along the second horizontal direction (Y direction). For example, the first interval (W1) and the second interval (W2) may be within about 0.01 nanometers to 10 micrometers. In one embodiment, the peak portion (P) and the valley portion (V) may each be arranged at a constant interval along the second horizontal direction (Y direction). In another embodiment, the peak portion (P) and the valley portion (V) may each be arranged at an inconsistent interval along the second horizontal direction (Y direction).

[0078] In one embodiment, the height difference in the vertical direction (Z direction) between adjacent peaks (P) and valleys (V) of the PEDOT:PSS electrode (PS) may be within a range of about 0.1 nanometers to 10 micrometers. In one embodiment, the height difference in the vertical direction (Z direction) between adjacent peaks (P) and valleys (V) of the PEDOT:PSS electrode (PS) may be constant. In another embodiment, the height difference in the vertical direction (Z direction) between adjacent peaks (P) and valleys (V) of the PEDOT:PSS electrode (PS) may not be constant and may vary.

[0079] As one embodiment, as shown in FIG. 5d, for a first valley portion (V1) disposed on one side of the peak portion (P) of a PEDOT:PSS electrode (PS) and a second valley portion (V2) disposed on the other side of the peak portion (P), the slope of the first inclined surface (SL1) from the first valley portion (V1) to the peak portion (P) and the slope of the second inclined surface (SL2) from the second valley portion (V2) to the peak portion (P) may be the same. The first inclined surface (SL1) from the first valley portion (V1) to the peak portion (P) and the second inclined surface (SL2) from the second valley portion (V2) to the peak portion (P) may be symmetrical with respect to the peak portion (P). As another embodiment, as shown in FIG. 5e, for the first valley (V1) and the second valley (V2) which are valleys (V) adjacent to the peak (P) of the PEDOT:PSS electrode (PS), the slope of the first sloped surface (SL1) from the first valley (V1) to the peak (P) and the slope of the second sloped surface (SL2) from the second valley (V2) to the peak (P) may be different from each other.

[0080] In exemplary embodiments, the PEDOT:PSS electrode (PS) may have a thickness of about tens of nanometers to hundreds of nanometers, and preferably may have a thickness of about 10 nanometers to 100 nanometers.

[0081] In FIG. 5c, only the front surface (FS) of the PEDOT:PSS electrode (PS) is shown, but the rear surface (BS) of the PEDOT:PSS electrode (PS) may also have a rough surface similar to the front surface (FS). In this specification, the description of the front surface (FS) of the PEDOT:PSS electrode (PS) may be applied in the same or similarly to the rear surface (BS) of the PEDOT:PSS electrode (PS).

[0082] In some embodiments, the rough shape of the front surface (FS) of the PEDOT:PSS electrode (PS) and the rough shape of the rear surface (BS) of the PEDOT:PSS electrode (PS) may correspond to each other. For example, for the location where the peak portion (P) and the valley portion (V) are formed respectively on the front surface (FS) of the PEDOT:PSS electrode (PS), the peak portion (P) and the valley portion (V) may be formed respectively on the rear surface (BS) of the PEDOT:PSS electrode (PS) at a corresponding location. However, the present invention is not limited thereto, and the rough shape of the front surface (FS) of the PEDOT:PSS electrode (PS) and the rough shape of the rear surface (BS) of the PEDOT:PSS electrode (PS) may not correspond to each other.

[0083] The number of PEDOT:PSS layers forming the PEDOT:PSS electrode (PS) is not limited to one as illustrated, but can be composed of multiple layers. For example, the biosensor may include a first PEDOT:PSS layer and a second PEDOT:PSS layer disposed on the first PEDOT:PSS layer. The first PEDOT:PSS layer and the second PEDOT:PSS layer may have the same or different shapes from each other.

[0084] When detecting dielectric material using an electrical method, the charge of the dielectric material may be obscured by the charge of ions other than the dielectric material in the solution, so stable detection is possible only when the dielectric material is close to the sensor surface. In other words, since the dielectric material can be detected only when it is within the Debye length of the sensor, increasing the Debye length of the sensor can increase the sensitivity of the sensor from Debye shielding, which is an important challenge in the industry. When detecting dielectric material using a biosensor (80) that uses a PEDOT:PSS electrode having a rough surface according to the technical concept of the present invention, compared to using a PEDOT:PSS electrode having a flat surface, the Debye length for the dielectric material increases, and sensitivity can be improved. Therefore, a biosensor (80) with improved sensing sensitivity for dielectric material can be provided.

[0085]

[0086] FIG. 6a is a cross-sectional view of a biosensor (80a) according to another embodiment of the present invention.

[0087] Referring to FIG. 6a, the biosensor (80a) may include a biosensor substrate (820), an insulating layer (823), a wiring structure (821), a gate electrode (822), a source electrode (824), a drain electrode (825), and graphene (840).

[0088] The biosensor substrate (820) is substantially the same as the biosensor substrate (811) described with reference to FIG. 5a, so a detailed description will be omitted below.

[0089] According to one embodiment, an insulating layer (823) may be disposed on a biosensor substrate (820). The insulating layer (823) may be formed to have a constant thickness along the upper surface of the biosensor substrate (820). The insulating layer (823) may include, for example, an inorganic insulating material, an organic insulating material, or a combination thereof. The inorganic insulating material may include, for example, silicon oxide, silicon nitride, or a combination thereof. The organic insulating material may include, for example, polyimide, epoxy resin, or a combination thereof.

[0090] According to one embodiment, the gate electrode (822) can be connected to a power source through a wiring structure (821) to supply a gate voltage to the graphene (840). The presence of a target substance in a sample can be determined by observing the electrical parameters of the biosensor according to the gate voltage. In this case, a change in the electrical parameters may correspond to the presence of the target substance. For example, the electrical parameters may include capacitance, voltage, average current, etc. The gate electrode (822) may include a conductive metallic material. As one embodiment, the gate electrode (822) may be composed of silver (Ag).

[0091] According to one embodiment, a source electrode (824) and a drain electrode (825) may be disposed within an insulating layer (823). The drain electrode (825) may be disposed spaced apart horizontally from the source electrode (824). The source electrode (824) and the drain electrode (825) may be formed of various materials and may include metals or metal alloys. For example, the source electrode (824) and the drain electrode (825) may include copper (Cu), ruthenium (Ru), aluminum (Al), cobalt (Co), molybdenum (Mo), titanium (Ti), tantalum (Ta), nickel (Ni), platinum (Pt), chromium (Cr), or alloys thereof.

[0092] Graphene (840) may be disposed on the upper surface of the source electrode (824) and the drain electrode (825). The graphene (840) may have a three-dimensional wrinkled geometry. A detailed description of the structure of the graphene (840) will be provided with reference to FIG. 6b. The graphene (840) may be disposed on the upper surface of the insulating layer (823) and may extend horizontally along the upper surface of the insulating layer (823). The dielectric material in the sample may come into contact with the graphene (840).

[0093] According to one embodiment, the biosensor (80a) may further include a well structure (831) disposed on the upper surface of the insulating layer (823) at the outer edge of the insulating layer (823). The well structure (831) may include first and second boundary members (831a, 831b). The first and second boundary members (831a, 831b) may be disposed spaced apart from each other in a horizontal direction on the upper surface of the insulating layer (823). At least a portion of the upper surface of the graphene (840) may be exposed in the area surrounded by the well structure (831), and the reaction area (832) surrounded by the well structure (831) on the upper surface of the graphene (840) may be defined as an area where the probe of the graphene (840) can react with a target substance. According to one embodiment, the well structure (831) may include an insulating material such as silicon oxide or silicon nitride.

[0094]

[0095] FIG. 6b is a drawing for explaining the graphene (840) shown in FIG. 6a. FIG. 6c is an enlarged view of the Q region of the graphene (840) shown in FIG. 6b.

[0096] Referring to FIGS. 6b and 6c, the graphene (840) may include a body (842) that extends in the longitudinal direction with high points (P_a) and low points (V_a) having height differences arranged repeatedly in a zigzag pattern, and a probe (844) disposed on one side of the high points (P_a) and low points (V_a) and capable of chemically bonding with the dielectric material to be detected. The probe (844) may be disposed not only between the high points (P_a) and low points (V_a) of the body (842), but also between the high points (P_a) and low points (V_a).

[0097] Adjacent high points (P_a) and adjacent low points (V_a) of the graphene (840) may be spaced apart in a second horizontal direction (Y direction) and may each extend in a first horizontal direction (X direction). Low points (V_a) may be placed between adjacent high points (P_a) of the graphene (840), and low points (V_a) may be placed between adjacent high points (P_a). However, the wrinkled shape of the body (842) of the graphene (840) is not limited to that shown in FIG. 6b and may be crumpled into various shapes.

[0098] According to one embodiment, the point having the highest level of the body (842) of the graphene (840) having a three-dimensional wrinkled geometry is defined as a high point (P_a), and the point having the lowest level is defined as a low point (V_a). In one embodiment, adjacent high points (P_a) and adjacent low points (V_a) of the graphene (840) may be spaced apart with a variable spacing (W). The spacing (W) may be in the range of 0.1 micrometers to 10 micrometers. If the spacing (W) is smaller than 0.1 micrometers, the graphene (840) is wrinkled too tightly, so that sufficient reaction space may not be formed for the probes (844) placed on one side of the graphene (840) to chemically bond with the dielectric material (GM). When the gap (W) is greater than 10 micrometers, the body (842) of the graphene (840) can be seen as having a flat geometry rather than a three-dimensional wrinkled geometry.

[0099] In one embodiment, the height difference in the vertical direction (Z direction) between adjacent high points (P_a) and low points (V_a) of graphene (840) may not be constant in the range of 20 nanometers to 30 nanometers. That is, the slope of the first intermediate region (COL1) from the low point (V_a) to the high point (P_a) and the slope of the second intermediate region (COL2) from another low point (V_a) to the high point (P_a) may be different from each other.

[0100] As shown in image (a) of FIG. 6b, the graphene (840) may have high points (P_a) and low points (V_a) repeating along the length direction. In this case, the length direction may be a second horizontal direction (Y direction), and the direction in which the high points (P_a) or low points (V_a) extend may be a first horizontal direction (X direction). However, as shown in image (b) of FIG. 6b, the graphene (840) may have high points (P_a) and low points (V_a) arranged irregularly. In this case, the high points (P_a) and low points (V_a) do not repeat along a specific direction, but rather the high points (P_a) and low points (V_a) may be arranged randomly.

[0101] The number of bodies (842) included in the graphene (840) is not limited to that shown in FIG. 6b. In another embodiment, the graphene (840) may include a first body and a second body disposed on the first body. In a planar view, the carbon atoms constituting the first body and the carbon atoms constituting the second body may be arranged in an overlapping manner. In yet another embodiment, the carbon atoms constituting the first body and the carbon atoms constituting the second body may be arranged in an offset manner. Since the graphene (840) shown in FIG. 6b and 6c includes one body (842), the expression that the graphene (840) has a three-dimensional wrinkled geometry may have substantially the same meaning as the expression that the body (842) of the graphene (840) has a three-dimensional wrinkled geometry.

[0102] In the region between the high point (P_a) and the low point (V_a) of the graphene (840), more probes (844) can be placed compared to the high point (P_a) and the low point (V_a), so it can contain many target materials. However, it is not limited to this, and more probes (844) may be placed in the high point (P_a) and the low point (V_a) than in the region between the high point (P_a) and the low point (V_a).

[0103] In one embodiment, the distance (d1) from a line (A-A') crossing the center of the graphene (840) to a high point (P_a) may be 10 nanometers to 15 nanometers. The distance (d2) from a line (A-A') crossing the center of the graphene (840) to a low point (V_a) may be 10 nanometers to 15 nanometers. Accordingly, the height difference between the high point (P_a) and the low point (V_a) may be 20 nanometers to 30 nanometers. If the distance (d1) from the line (A-A') crossing the center of the graphene (840) to the high point (P_a) or the distance (d2) from the line (A-A') crossing the center of the graphene (840) to the low point (V_a) is less than 10 nanometers, the length in the third direction (Z direction) relative to the length in the first direction (X direction) of the graphene (840) is shortened, so that the graphene (840) can be seen as having a flat geometry rather than a three-dimensional wrinkled geometry. If the distance (d1) from the line (A-A') crossing the center of the graphene (840) to the high point (P_a) or the distance (d2) from the line (A-A') crossing the center of the graphene (840) to the low point (V_a) is greater than 15 nanometers, the length in the third direction (Z direction) relative to the length in the first direction (X direction) of the graphene (840) becomes longer, so that the graphene (840) may have a densely wrinkled shape. In this case, sufficient reaction space may not be formed for the probes (844) placed on one side of the graphene (840) to chemically bond with the target material.

[0104] According to one embodiment, a probe (844) disposed on one side of graphene (840) may be one of a polynucleotide, a peptide nucleic acid (PNA) probe, an aptamer, a protein, an antibody, or a capture agent. The probe (844) may be selected for binding with genetic material (GM). For example, if the genetic material (GM) is miRNA, the probe (844) may have an arrangement complementary to the miRNA to be detected for binding with the miRNA.

[0105] When graphene (840) has a three-dimensional wrinkled geometry, compared to when it has a flat geometry, the Debye length is increased, allowing it to capture more miRNA molecules. Therefore, the sensitivity of the probe (844) to the target substance can be increased despite the Debye shielding effect.

[0106] FIG. 7 is a flowchart of a genetic material detection method according to one embodiment of the present invention, and FIGS. 8 to 11 are cross-sectional views illustrating a genetic material detection method according to one embodiment of the present invention in chronological order.

[0107] Referring to FIG. 8 together with FIG. 7, 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. 8, the second electrode (20b) may be exposed to a second window (33b, see FIG. 2) of a second insulating film (30b).

[0108] 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).

[0109] Additionally, the biosensor (80) may be disposed on the main surface (10a) of the substrate (10). The biosensor (80) may be disposed on the main surface (10a) of the substrate (10) spaced apart from the first electrode (20a) and the second electrode (20b) in the second horizontal direction (Y direction).

[0110]

[0111] Referring to FIG. 9 together with FIG. 7, a genetic material extraction method according to one embodiment may include the step (S120) of placing a sample storage container (40) that receives a sample (41) in which cells (CE) are suspended on a first electrode (20a) and a second electrode (20b).

[0112] 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 first outlet (44) of the sample storage container (40).

[0113] 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. 9, 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). The second outlet (46) of the sample storage container (40) can be positioned between the first and second electrodes (20a, 20b) and the biosensor (80) along the second horizontal direction (Y direction).

[0114]

[0115] Referring to FIG. 10 together with FIG. 7, 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).

[0116] 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).

[0117]

[0118] Referring to FIG. 10 together with FIG. 7, 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.

[0119] 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.

[0120] 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. 12.

[0121]

[0122] Referring to FIG. 11 together with FIG. 7, 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 ruptures in order to extract genetic material within a cell trapped in a first window (33a) or a second window (33b, see FIG. 2).

[0123] 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.

[0124] 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. 13.

[0125] 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).

[0126] 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).

[0127]

[0128] Referring to FIG. 11 together with FIG. 7, a genetic material extraction method according to one embodiment may include the step (S160) of detecting genetic material (GM) extracted from a cell (CE_R) through a probe of a biosensor (80) located in a sample (41).

[0129] As the membrane of the cell (CE_R) ruptures, the extracted genetic material (GM) flows along the solution of the sample (41) toward the first outlet (44). At this time, the biosensor (80) is positioned between the first and second electrodes (20a, 20b) and the first outlet (44), so that the genetic material (GM) floating toward the first outlet (44) can be bound to the probe of the biosensor (80). The probe of the biosensor (80) can chemically bind to the genetic material (GM) to detect the genetic material (GM). The probe of the biosensor (80) may be the probe (815) shown in FIG. 5a or the probe (844) shown in FIG. 6c.

[0130] Figure 12 is a diagram illustrating the mobility of a cell (CE) according to the frequency of the alternating current voltage.

[0131] 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).

[0132] Referring to FIG. 12, 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).

[0133] 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).

[0134] 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).

[0135] 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).

[0136] FIG. 12 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.

[0137] 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.

[0138] 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.

[0139] Figure 13 is a diagram illustrating membrane rupture of a cell (CE) as the peak voltage of the alternating current voltage increases.

[0140] For convenience of explanation, FIG. 13 will be described with reference to FIG. 12. As with FIG. 12, 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. 12).

[0141] Specifically, Photo 13-a of FIG. 13 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. 12. 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.

[0142] Photograph 13-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. 12). 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.

[0143] Figure 14 is a diagram illustrating the process of rupturing the membrane of a cell (CE) to extract genetic material in chronological order.

[0144] For convenience of explanation, the following description will be made with reference to FIG. 14 in conjunction with FIG. 12. The configurations shown in the photograph in FIG. 14 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. 12. As with FIG. 12, 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).

[0145] 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.

[0146] In graph 14-a located at the top of FIG. 14, 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 14-b located at the bottom of FIG. 14 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).

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152]

[0153] 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 isp-p ) shall be referred to as rupture voltage.

[0154] Figures 15a to 15d are experimental data on various factors affecting the rupture of the membrane of a cell (CE).

[0155] The following description will be explained with reference to FIGS. 15a to 15d. 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 the constituent materials of the membrane.

[0156] 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.

[0157] 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.

[0158] Referring to the graph shown in FIG. 15a, the horizontal axis represents the methyl-beta-cyclodextrin concentration of the solution of sample (41), and the vertical axis represents the measured value of the critical energy required to form a perforation in the membrane of the cell (CE).

[0159] Referring to the graph shown in FIG. 15b, 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).

[0160] Figure 15c is a drawing showing the results of the graph in Figure 15a and the graph in Figure 15b as an image.

[0161] As illustrated in the graph of FIG. 15a, 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. 15c, 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.

[0162] In addition, as shown in the graph of Fig. 15b and Fig. 15c, 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.

[0163] Referring to FIG. 15d, 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.

[0164] As shown in FIG. 15d, 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.

[0165] 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.

[0166] 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; A power source configured to apply 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, and then increase the peak voltage of the alternating voltage to rupture the membrane of the cell and extract genetic material within the cell; and A genetic material detection device comprising a biosensor including a probe capable of binding to the genetic material.

2. In Paragraph 1, The above biosensor is, Biosensor substrate; Source electrodes and drain electrodes spaced apart from each other and disposed on the biosensor substrate; and A PEDOT:PSS layer electrically connected to the source electrode and the drain electrode and comprising PEDOT:PSS (poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate)); A dielectric material detection device characterized by the above probe being disposed on the above PEDOT:PSS layer.

3. In Paragraph 2, A genetic material detection device characterized in that the above-mentioned PEDOT:PSS layer includes a peak portion and a valley portion having a height difference in a direction perpendicular to the upper surface of the biosensor substrate.

4. In Paragraph 1, The above biosensor is, Biosensor substrate; Source electrodes and drain electrodes spaced apart from each other and disposed on the biosensor substrate; and Graphene electrically connected to the source electrode and drain electrode, with high points and low points having a height difference repeating in a zigzag shape along the length direction; comprising A dielectric material detection device characterized by the probe being disposed on the graphene.

5. In Paragraph 4, A dielectric material detection device characterized in that the height difference between the high point and the low point of the graphene is in the range of 0.2 nanometers to 20 nanometers.

6. In Paragraph 1, The above power source is, A genetic material detection 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.

7. A step of providing a substrate in which 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 are arranged facing each other on a main surface; A step of positioning a sample storage container for receiving a sample in which cells are suspended on the first electrode and the second electrode on the main surface of the substrate; 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; To extract genetic material within the cell trapped in the first window or the second window, the power source increases the peak voltage of the alternating voltage until the membrane of the cell ruptures; and A method for detecting genetic material comprising the step of detecting the genetic material extracted from the cell through a probe of a biosensor located within the sample.

8. In Paragraph 7, The above sample storage container is, An inlet, which is a passage into which the above sample is introduced; A first outlet, which is a passage through which the above sample is discharged; and A method for detecting genetic material characterized by including a second outlet, which is a passage through which the membrane of the ruptured cell is expelled.

9. In Paragraph 7, In the step of trapping the cell within the first window or the second window, A method for detecting genetic material, characterized in that the power source increases the frequency of the alternating voltage until the cell is trapped within the first window or the second window.

10. In Paragraph 7, 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 detecting dielectric material characterized by the above power source maintaining the frequency of the above alternating voltage constant.

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