Biosensor for analyzing nucleic acid-protein interaction and method for analyzing same

Abstract

The present invention relates to a biosensor for analyzing nucleic acid-protein interactions and a method for same, and more specifically to a device that can analyze, by means of electrical signals, the interaction between proteins and nucleic acids by using nucleic acid bound to an electrode array comprising electrodes, and a method for analyzing interactions between proteins and nucleic acids by means of the device.

Description

Biosensors for Nucleic Acid-Protein Interaction Analysis and Nucleic Acid-Protein Interaction Analysis Methods

[0001] The present invention was made under the support of the Ministry of Environment under the project identification number 1485019339 and project number 2024010283. The research management specialized institution of the project is the Korea Environmental Industry & Technology Institute, the research project name is “Development of a field-type and real-time detection system for microbial metabolites”, the main institution is the Daegu Gyeongbuk Institute of Science and Technology, and the research period is from 2024-01-01 to 2024-12-31.

[0002] The present invention was made under the support of the Ministry of Science and ICT under the research identification number 1711188408 and research number 2024030074. The research management organization of the said project is the National Research Foundation of Korea, the research project name is “Electronic DNA Curtain: Development of a novel biosensor for detecting protein-nucleic acid interactions at the single molecule level”, the main organization is the Daegu Gyeongbuk Institute of Science and Technology, and the research period is from 2024-03-01 to 2025-02-28.

[0003] The present invention relates to a biosensor for analyzing nucleic acid-protein interactions and a method for analyzing nucleic acid-protein interactions, and more particularly, to a device capable of analyzing interactions between proteins and nucleic acids through electrical signals via nucleic acids bound to an electrode array including electrodes, and a method for analyzing interactions between proteins and nucleic acids using the same.

[0004] Nucleic acid-protein interactions are fundamental phenomena of life, playing a crucial role in diverse biological processes, including gene expression regulation, DNA replication, repair, transcription, and translation. These interactions are essential for understanding and controlling the complex processes occurring within cells. In particular, the process by which specific proteins bind to specific DNA or RNA sequences to regulate gene expression is a crucial research topic for disease development and treatment.

[0005] Existing nucleic acid-protein interaction analysis methods are often time-consuming and expensive, and often require complex equipment and procedures. These limitations have led to a growing demand for simpler, faster, and more sensitive analytical methods. Biosensors, a technology developed to meet this demand, are devices capable of detecting the presence and concentration of specific biological molecules in real time. Nucleic acid-protein interaction analysis using biosensors offers high accuracy and reproducibility, and can be applied in a variety of fields.

[0006] Existing nucleic acid-protein interaction analysis techniques include EMSA (Electrophoretic Mobility Shift Assay), ChIP (Chromatin Immunoprecipitation), SPR (Surface Plasmon Resonance), and ITC (Isothermal Titration Calorimetry). EMSA analyzes the mobility of protein-bound DNA through electrophoresis. While relatively simple, it has limitations in quantitative analysis. ChIP identifies DNA sequences bound to specific proteins. While highly accurate, it requires complex experimental procedures. SPR can monitor interactions in real time, but it requires expensive equipment and complex analytical techniques. ITC analyzes interactions by measuring the thermodynamic changes of binding reactions. While highly sensitive, it is subject to demanding experimental conditions.

[0007] Furthermore, attempts have been made to analyze interactions between nucleic acids and proteins using optical detection methods, including DNA curtain technology. However, these methods require skilled technicians, expensive fluorescent probes, and long analysis times, limiting their application in general laboratories and clinical applications. Furthermore, they require expensive equipment and their large size hinders field application. Furthermore, observation is limited in certain molecular systems that cannot utilize fluorescent probes, and analysis results are significantly affected by the resolution and optical instability of the equipment.

[0008] The present invention aims to address the above issues, and provides a biosensor and an analysis method utilizing the same that enables simpler and more accurate analysis of nucleic acid-protein interactions. This biosensor can monitor nucleic acid-protein interactions in real time and boasts high sensitivity and reproducibility. Furthermore, the analysis method of the present invention is simpler and more cost-effective than existing technologies, making it widely applicable in research and diagnostic fields.

[0009] Furthermore, the biosensor and analysis method according to the present invention can be applied not only in the laboratory but also in various fields, including clinical diagnosis, environmental monitoring, and food safety testing. For example, it can be used to study gene expression regulation mechanisms, detect biomarkers for specific diseases, and monitor environmental hazards. This is expected to significantly improve the efficiency of life science research.

[0010] The present inventors fabricated a biosensor having an electrode array including electrodes and nucleic acids connected to each electrode, and confirmed that the biosensor according to the present invention can analyze the interaction between proteins and nucleic acids through electrical signals and can simultaneously analyze a large number of protein-nucleic acid interactions.

[0011] Accordingly, the purpose of the present invention is to provide a biosensor for analyzing nucleic acid-protein interactions.

[0012] Another object of the present invention is to provide a method for analyzing nucleic acid-protein interactions.

[0013] One aspect of the present invention is a biosensor for analyzing nucleic acid-protein interactions, comprising: an electrode array including a plurality of source electrodes spaced at regular intervals and a plurality of drain electrodes corresponding to the source electrodes, wherein the source electrodes and the drain electrodes corresponding to the source electrodes are arranged in parallel; and a nucleic acid having one end connected to the source electrode via a first linker and the other end connected to the drain electrode via a second linker or not connected to the drain electrode.

[0014] The term "nucleic acid" as used herein refers to a polymer of nucleotides, including DNA (deoxyribonucleic acid), RNA (ribonucleic acid), or nucleic acid analogues. Nucleic acids store and transmit genetic information in living organisms, and nucleotides are composed of purine or pyrimidine bases, sugars (ribose or deoxyribose), and phosphate groups. DNA has a double helix structure and is responsible for storing and replicating genetic information. RNA exists as a single strand and plays a crucial role in protein synthesis. Nucleic acids are key molecules for the development, function, and maintenance of living organisms, and are involved in various biochemical and genetic processes. In the present invention, nucleic acids can be connected between source and drain electrodes, and when voltage is applied to the electrode array, current can flow along the nucleic acids, and the changes in the measured current signal can be used to analyze the interaction between nucleic acids and proteins.

[0015] The term "nucleic acid analogue" used herein refers to a compound that mimics or modifies the structure of nucleic acids (deoxyribonucleic acid, ribonucleic acid). Nucleic acid analogues can be used in various genetic and molecular biology studies and are typically created by modifying the bases, sugars, or phosphate groups of nucleic acids. Nucleic acid analogues have biochemical properties similar to those of natural nucleic acids, but may enhance or suppress specific functions.

[0016] The term “nucleic acid-protein interaction” in this specification refers to a process in which DNA or RNA and a protein bind to perform a biological function, and specifically may refer to the binding or diffusion activity of a nucleic acid (e.g., DNA) and a protein. This interaction plays a key role in important cellular mechanisms such as gene expression regulation, replication, repair, recombination, and translation, and the nucleic acid-protein interaction occurs through recognition of a specific structure or sequence.

[0017] In one embodiment of the present invention, a fluorescent protein may be bound to the nucleic acid, and the interaction between the nucleic acid and DNA may be confirmed through the fluorescent protein. At this time, the fluorescent protein is green fluorescent protein (GFP), modified green fluorescent protein (mGFP), enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), modified red fluorescent protein (mRFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), cyan green fluorescent protein (CGFP), enhanced cyan fluorescent protein (ECFP), AzG (Azami Green), HcR (Heteractis crispa red fluorescent protein; HcRed), or Discosoma red fluorescent protein (DsRed). However, the present invention is not limited thereto. Meanwhile, a motif capable of binding to a nucleic acid may be combined with a fluorescent protein, and the motif may be a peptide, and specifically, may include a sequence capable of binding to a nucleic acid molecule and may be composed of 5 to 20 amino acids.

[0018] In one embodiment of the present invention, the source electrode and / or the drain electrode may be formed of various materials capable of transmitting current to and binding to nucleic acids, and various materials known to those skilled in the art may be used without limitation. For example, the source electrode and / or the drain electrode may be formed of one or more materials selected from the group consisting of carbon nanotubes (CNTs), graphene, silicon, silver, platinum, gold, polypyrrole, and polyaniline, and specifically, may be an electrode containing gold.

[0019] In the present invention, a linker can connect a source electrode, a drain electrode, and a nucleic acid. Specifically, when a nucleic acid is singly connected to an electrode, the nucleic acid can be connected to the source electrode via the linker. When a nucleic acid is doubly connected to an electrode, the nucleic acid can be connected to the source electrode via the first linker and to the drain electrode via the second linker.

[0020] At this time, the linker can be used without limitation from various materials known to those skilled in the art that can firmly bind the electrode and nucleic acid, and an appropriate linker can be used depending on the material of the electrode. For example, when a metal electrode is used, the linker can be polypyrrole, organometallic complexes, maleimide, biotin-streptavidin, when a gold (Au) electrode is used, thiols and carbamoyl can be used, and when a silicon or glass electrode is used, a silane group can be used. Additionally, when other electrodes are used, N-hydroxysuccinimide (NHS) ester may be used as a linker for an electrode into which an amine group is introduced, an azide or alkyne group may be used as a linker for an electrode into which an alkyne or azide group is introduced, and an amino group may be used as a linker for an electrode into which a carboxyl group or ester is introduced.

[0021] In one embodiment of the present invention, at least one of the first linker and the second linker may be a thiol group.

[0022] In one embodiment of the present invention, the nucleic acid may have biotin linked to the terminal end.

[0023] In one embodiment of the present invention, at least one of the first linker and the second linker,

[0024] It may be biotin-PEG-thiol and streptavidin conjugated with biotin-PEG-thiol.

[0025] In the present invention, a biosensor for analyzing nucleic acid-protein interactions may include a microchannel disposed adjacent to an electrode array.

[0026] In one embodiment of the present invention, the microchannel is selected from the group consisting of polyvinylsiloxane (PVS), poly(methylhydrosiloxane) (PMHS), fluorosilicone (FVMQ), polydiphenylsiloxane (PDPS), polymethylphenylsiloxane (PMPS), poly(dimethyl-co-methylphenylsiloxane), polytrifluoropropylmethylsiloxane, polyethylmethylsiloxane (PEMS), polyphenylsiloxane (PPS), polyphenylmethylsiloxane (PPMS), polybiphenylsiloxane (Poly(biphenylsiloxane)), polyhydromethylsiloxane (Poly(hydromethylsiloxane)), It may include at least one selected from the group consisting of polyoctamethylcyclotetrasiloxane (Poly(octamethylcyclotetrasiloxane; D4), polydimethylcosiloxane (Poly(dimethyl-co-siloxane)), poly(dimethylsiloxane-co-phenylsiloxane)), polysiloxane urethane (Poly(siloxane-urethane)), and poly(ethylmethylsiloxane) (Poly(ethylmethylsiloxane)).

[0027] In one embodiment of the present invention, the electrode may include a protrusion to which a nucleic acid is connected.

[0028] In one embodiment of the present invention, the protrusion may have a thin thickness relative to the remainder of the source electrode.

[0029] In one embodiment of the present invention, the protrusion may have a width of 0.2 to 5.0 μm, 0.3 to 5.0 μm, 0.4 to 5.0 μm, 0.5 to 5 μm, 0.5 to 3.5 μm, 0.75 to 3.25 μm, 1.0 to 3.0 μm, 1.25 to 2.75 μm, 1.5 to 2.5 μm, 1.6 to 2.4 μm, 1.7 to 2.3 μm, 1.8 to 2.2 μm, 1.85 to 2.15 μm or 1.9 to 2.1 μm.

[0030] In one embodiment of the present invention, the source electrode and the drain electrode are 10.0 to 30.0 μm, 9.0 to 29.0 μm, 8.0 to 28.0 μm, 7.0 to 27.0 μm, 6.0 to 26.0 μm, 5.0 to 25.0 μm, 4.0 to 24.0 μm, 3.0 to 23.0 μm, 2.0 to 22.0 μm, 1.0 to 21.0 μm, 9.5 to 20.5 μm, 9.0 to 20.0 μm, 8.5 to 19.5 μm, 8.0 to 19.0 μm, 7.5 to 18.5 μm, 7.0 to 18.0 μm, 6.5 to 17.5 μm, They may be spaced apart from each other at intervals of 6.0 to 17.0 μm, 5.5 to 16.5 μm, 5.0 to 16.0 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm.

[0031] In one embodiment of the present invention, the microchannel may be arranged in a direction perpendicular to the source electrode and the drain electrode.

[0032] In one embodiment of the present invention, the nucleic acids may be arranged in a direction perpendicular to the source electrode and the drain electrode according to the flow of fluid within the microchannel.

[0033] Another aspect of the present invention is a method for analyzing nucleic acid-protein interactions, comprising: an application step of applying voltage to a nucleic acid, one end of which is connected to a source electrode and the other end of which is connected or not connected to a drain electrode corresponding to the source electrode; and an analysis step of analyzing a nucleic acid-protein interaction by measuring an electric signal according to the voltage application.

[0034] The method according to the present invention may include a stretching step of stretching the nucleic acid by applying a flow of fluid in a direction perpendicular to the source electrode and the drain electrode.

[0035] The biosensor for analyzing nucleic acid-protein interactions according to the present invention is a high-throughput electronic DNA curtain (e-curtain) platform that innovatively improves the existing nano-fluorescence technology-based DNA curtain assay technology and applies it to electronic devices, miniaturizing it. It features large-capacity data processing, low-cost implementation, and field usability, and can analyze nucleic acid-protein interactions in a way that does not require the photostability of fluorescent labels and complex labeling processes.

[0036] FIG. 1 is a drawing showing a biosensor for analyzing nucleic acid-protein interactions according to one embodiment of the present invention.

[0037] FIG. 2 is a drawing showing the arrangement between a nucleic acid and an electrode in a biosensor device according to one embodiment of the present invention.

[0038] FIG. 3 is a drawing showing a binding process between a nucleic acid and an electrode in a biosensor device according to one embodiment of the present invention.

[0039] FIG. 4 is a diagram illustrating a process of analyzing a nucleic acid-protein binding as an electrical signal using a biosensor according to one embodiment of the present invention.

[0040] FIG. 5 is a drawing for showing the electrode array pattern conduction manufactured as a 6-inch wafer in a biosensor according to one embodiment of the present invention, the appearance of a 4-part array pattern divided according to the size of the source / drain gap, and the shape of the pattern according to the source / drain gap.

[0041] FIG. 6 is a drawing showing a process of preparing Thiol-λ in a biosensor according to one embodiment of the present invention (a process of linking a thiol-functionalized oligomer using a ligase).

[0042] Figure 7 is a drawing showing the results of an experiment in which nucleic acids were single-linked and treated with DNase in a biosensor according to one embodiment of the present invention.

[0043] Figure 8 is a graph showing changes in current signals in double-linked nucleic acids in a biosensor according to one embodiment of the present invention.

[0044] FIG. 9 is a graph showing the change in current signal in double-tethered nucleic acids in a biosensor according to one embodiment of the present invention [(A) Blank measurement in 1x PBS buffer after biotin reaction (flow rate OFF) (B) Blank measurement in 1x PBS buffer after streptavidin reaction (flow rate OFF) (C) Reaction measurement before DNA stretching (flow rate OFF) (D) Double-tethered reaction measurement after DNA stretching (flow rate OFF)]

[0045] A biosensor for analyzing nucleic acid-protein interactions, comprising: an electrode array comprising a plurality of source electrodes spaced at regular intervals and a plurality of drain electrodes corresponding to the source electrodes, wherein the source electrodes and the drain electrodes corresponding to the source electrodes are arranged in parallel; and a nucleic acid having one end connected to the source electrodes via a first linker and the other end connected to the drain electrode via a second linker or the other end not connected to the drain electrode.

[0046] Hereinafter, the present invention will be described with reference to the attached drawings. However, the present invention can be implemented in various different forms and is therefore not limited to the embodiments described herein. In the drawings, irrelevant parts have been omitted for clarity of description, and similar parts have been designated with similar reference numerals throughout the specification.

[0047] Throughout the specification, when a part is said to be "connected (connected, contacted, joined)" to another part, this includes not only cases where it is "directly connected," but also cases where it is "indirectly connected" with another part in between. Furthermore, when a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0048] The terminology used herein is merely used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this specification, it should be understood that the terms "comprises" or "has" indicate the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0049]

[0050] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings.

[0051]

[0052] FIG. 1 is a drawing showing a biosensor for analyzing nucleic acid-protein interactions according to one embodiment of the present invention, and FIG. 2 is a drawing showing the arrangement between a nucleic acid and an electrode in a biosensor device according to one embodiment of the present invention.

[0053] Referring to FIGS. 1 and 2, a biosensor (1000) for analyzing nucleic acid-protein interactions according to one embodiment of the present invention may include an electrode array (100), nucleic acids, and microchannels (300).

[0054] The electrode array (100) may include a source electrode (110) and a drain electrode (120). The source electrodes (110) and the drain electrodes (120) may be included in plurality within the electrode array (100), and the source electrodes (110) and the drain electrodes (120) may be arranged in pairs, including the drain electrodes (120) or the source electrodes (110) corresponding to each other.

[0055] The source electrode (110) and the drain electrode (120) may be spaced apart from each other by a certain distance. At this time, the distance may be appropriately adjusted to be suitable for measuring the length of the nucleic acid and the electrical signal of the connected nucleic acid. For example, the source electrode (110) and the drain electrode (120) may have a thickness of 10.0 to 30.0 μm, 9.0 to 29.0 μm, 8.0 to 28.0 μm, 7.0 to 27.0 μm, 6.0 to 26.0 μm, 5.0 to 25.0 μm, 4.0 to 24.0 μm, 3.0 to 23.0 μm, 2.0 to 22.0 μm, 1.0 to 21.0 μm, 9.5 to 20.5 μm, 9.0 to 20.0 μm, 8.5 to 19.5 μm, 8.0 to 19.0 μm, 7.5 to 18.5 μm, 7.0 to 18.0 μm, 6.5 to 17.5 μm, They may be spaced apart from each other at intervals of 6.0 to 17.0 μm, 5.5 to 16.5 μm, 5.0 to 16.0 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm.

[0056] The source electrode (110) and the drain electrode (120) corresponding to the source electrode can be arranged parallel to each other. Accordingly, the source electrode (110) and the drain electrode (120) can be spaced apart with the same interval maintained regardless of the lengths of the source electrode (110) and the drain electrode (120).

[0057] The electrode may include a metallic or non-metallic material, or may be made of a single or composite metallic or non-metallic material. When the electrode includes a metallic material, the electrode may include silver, platinum, or gold, or may be made of silver, platinum, or gold. When the electrode includes a non-metallic material, the electrode may include or be made of carbon nanotubes (CNTs), graphene, silicon, polypyrrole, or polyaniline.

[0058] At this time, the source electrode (110) and the drain electrode (120) may include the same material or be manufactured from the same material, but they do not necessarily use the same material and may include different materials or be manufactured from different materials. For example, the source electrode (110) may include gold while the drain electrode (120) may include silicon, and conversely, the source electrode (110) may include graphene while the drain electrode (120) may include silver.

[0059] A source electrode of a biosensor for nucleic acid-protein interaction analysis according to one embodiment may be bound to a nucleic acid (200). The nucleic acid (200) may be DNA (deoxyribonucleic acid), RNA (ribonucleic acid), or nucleic acid analogues.

[0060] The nucleic acid (200) may be single-connected to the electrode array (100) with one end of the nucleic acid connected to the source electrode (110). Alternatively, the nucleic acid (200) may be double-connected with one end of the nucleic acid connected to the source electrode (110) and the other end of the nucleic acid connected to the drain electrode (120).

[0061] A linker may be used to connect the nucleic acid (200) and the electrode (110, 120). The linker may be connected to one or both ends of the nucleic acid, and the connection between the nucleic acid and the electrode may be easily and firmly supported through the linker. When one end of the nucleic acid (200) is connected to the source electrode (110), one end of the nucleic acid (200) may be connected to the source electrode (110) via a first linker. When the nucleic acid (200) is doubly connected to the electrode array (100), one end of the nucleic acid (100) may be connected to the source electrode (110) via a first linker, and the other end of the nucleic acid (200) may be connected to the drain electrode (120) via a second linker. Alternatively, even if one end of the nucleic acid (200) is connected to the source electrode (110), a second linker may be connected to the other end of the nucleic acid (200), and the other end may be connected to the drain electrode (120) by receiving an external force such as a fluid through the second linker.

[0062] A suitable linker can be used depending on the type of electrode used in the electrode array (100).

[0063] When a metal is used as the electrode material, the electrode and the nucleic acid can be connected through a suitable linker capable of binding the metal electrode and the nucleic acid.

[0064] For example, when gold (Au) is used as the electrode material, a thiol group can be used as a linker. At this time, the thiol group can be directly introduced into the nucleic acid to connect the nucleic acid and the gold electrode, or the thiol group can be connected to the gold electrode and the nucleic acid and the gold can be connected via biotin, etc. If direct connection via the thiol group is not possible, a chemical linker such as maleimide can be used to bind the nucleic acid or electrode to which the thiol group is connected. Similarly, when gold (Au) is used as the electrode material, biotin and streptavidin can be used as linkers. At this time, a thiol group or a compound containing a thiol group can be connected to the gold electrode and connected to streptavidin via biotin. Specifically, biotin-PEG-thiol can be connected to the electrode side and connected to a nucleic acid having biotin attached to the terminal through streptavidin.

[0065] When gold is used as the electrode material, the nucleic acid and the electrode can be connected using a carbamoyl group as a linker. Specifically, a nucleic acid with a carbamoyl group introduced can be directly connected to the gold electrode, or a nucleic acid with a compound containing a carbamoyl group attached to its terminal can be connected to the gold electrode.

[0066] The nucleic acid (200) and the electrode (110, 120) can be linked using an amino group and a carboxyl or ester group as a link. An amino group can be introduced into the nucleic acid, or a compound containing an amino group can be linked to the nucleic acid. A carboxyl group or ester can be introduced into the electrode, and the nucleic acid and the electrode can be linked through a bond between the amino group and the carboxyl or ester group. For example, the nucleic acid and the electrode can be linked through a bond between an N-hydroxysuccinimide (NHS) ester and an amine group.

[0067] Nucleic acid (200) and electrode (110, 120) can be linked using azide and alkyne as linkers. An azide or alkyne group can be introduced into the nucleic acid, and an alkyne or azide group can be attached to the electrode, so that the electrode and the nucleic acid can be linked through a click chemical reaction between the azide group and the alkyne group.

[0068] Nucleic acid (200) and electrode (110, 120) can be combined using polypyrrole as a linker, and an electrode coated with polypyrrole as a conductive polymer can be connected to a nucleic acid having a chemical group capable of reacting with polypyrrole.

[0069] When a non-metal is used as the electrode (110, 120), a linker capable of connecting the non-metal electrode and the nucleic acid may be utilized. For example, when a silicon or glass electrode is utilized, a nucleic acid to which a silane group has been introduced may be connected to the electrode. Silane can stably connect the nucleic acid and the electrode through chemical bonding with the silicon or glass surface.

[0070] A biosensor (1000) for analyzing nucleic acid-protein interactions according to one embodiment may include a microchannel (300). The microchannel (300) may be positioned adjacent to an electrode array (100) and may include a microchannel.

[0071] The microchannel (300) may include an inlet (310) and an outlet (320), and a fluid may be injected into the inlet via a pump (400) to cause the fluid to flow within the microchannel, and the fluid may be discharged to the outside through the outlet. Any pump capable of providing pressure for injecting a fluid may be used as the pump (400) without limitation, and for example, a pump such as a syringe pump as illustrated in FIG. 1 may be used.

[0072] The microchannel (300) may be positioned on the upper side of the electrode array (100). Accordingly, a fluid flowing within the microchannel may flow on the upper side of the electrode array (100), and a laminar flow may occur on the upper side of the electrode array (100). When a laminar flow occurs, the nucleic acids (200) attached to the electrode array (100) also receive an external force due to the fluid, and through this, the nucleic acids (200) may be aligned in the direction in which the fluid flows.

[0073] The microchannel (300) may be formed of various materials known in the art that can form microchannels. For example, the microchannel (300) may be formed of polyvinylsiloxane (PVS), poly(methylhydrosiloxane) (PMHS), fluorosilicone (FVMQ), polydiphenylsiloxane (PDPS), polymethylphenylsiloxane (PMPS), poly(dimethyl-co-methylphenylsiloxane), polytrifluoropropylmethylsiloxane, polyethylmethylsiloxane (PEMS), polyphenylsiloxane (PPS), polyphenylmethylsiloxane (PPMS), polybiphenylsiloxane. It may include at least one selected from the group consisting of poly(biphenylsiloxane)), poly(hydromethylsiloxane)), poly(octamethylcyclotetrasiloxane; D4), poly(dimethyl-co-siloxane)), poly(dimethylsiloxane-co-phenylsiloxane)), poly(siloxane-urethane) and poly(ethylmethylsiloxane).

[0074] FIG. 2 is a drawing showing the arrangement between a nucleic acid and an electrode in a biosensor device according to one embodiment of the present invention.

[0075] Referring to FIG. 2, as described above, the nucleic acid (200) may be attached to the side of the source electrode (110). At this time, the source electrode (110) may include a protrusion (115) to which the nucleic acid (200) is connected. The protrusion (115) may have a narrow width relative to the remainder of the source electrode.

[0076] For example, the protrusion (115) may have a width of 0.2 to 5.0 μm, 0.3 to 5.0 μm, 0.4 to 5.0 μm, 0.5 to 5 μm, 0.5 to 3.5 μm, 0.75 to 3.25 μm, 1.0 to 3.0 μm, 1.25 to 2.75 μm, 1.5 to 2.5 μm, 1.6 to 2.4 μm, 1.7 to 2.3 μm, 1.8 to 2.2 μm, 1.85 to 2.15 μm or 1.9 to 2.1 μm, and a portion of the source electrode (110) other than the protrusion (115) may be designed to have a width wider than the above width.

[0077] In addition, the protrusion (115) of the source electrode (110) may be positioned corresponding to the drain electrode (120). Specifically, when the nucleic acid (200) is coupled to the protrusion (115) of the source electrode (120) as shown in (A) of FIG. 2, the connecting portion (116) connecting the protrusion (115) and the source electrode body (111) may be positioned corresponding to the end of the drain electrode (120) in a direction corresponding to the direction perpendicular to the source electrode (110) and the drain electrode (120). In this way, when the connecting portion (116) is positioned at a position corresponding to the end of the drain electrode (120), the nucleic acid (200) whose both ends are connected to the drain electrode (120) and the source electrode (110) can be positioned at the protrusion (115), and since the width of the protrusion (115) is narrower than the rest of the source electrode (110), the nucleic acid (200) can be positioned while maintaining a uniform gap between the source electrode (110) and the drain electrode (120) (see (B) of FIG. 2).

[0078] The length of the protrusion (115) of the source electrode (110) may be appropriately set depending on the distance from the drain electrode (120) and the experimental purpose, for example, the length of the protrusion may be 1.0 to 90.0 μm, 2.0 to 86.0 μm, 3.0 to 85.0 μm, 4.0 to 84.0 μm, 5.0 to 83.0 μm, 6.0 to 82.0 μm, 7.0 to 81.0 μm, 8.0 to 80.0 μm, 9.0 to 79.0 μm, 10.0 to 78.0 μm, 11.0 to 77.0 μm, 12.0 to 76.0 μm, 13.0 to 75.0 μm, 14.0 to 74.0 μm, 15.0 to It may be 73.0 μm, 16.0 to 72.0 μm, 17.0 to 71.0 μm, 18.0 to 70.0 μm, 19.0 to 69.0 μm, or 20.0 to 68.0 μm, and may have a length of, for example, 44 μm.

[0079] As described above, the attachment of nucleic acids to electrodes can be achieved by reacting exposed nucleic acids with a fluid flowing through a microchannel arranged above the electrode array, thereby attaching the nucleic acids to the electrodes. However, this is not limited to this method, and various methods for attaching nucleic acids to electrodes used in the art can be utilized. For example, an inkjet printer can be used to precisely drop λ drops in parallel to the electrode array trench locations using picoliter-volume inkjet printing. This can be advantageous in experiments requiring high precision and precise positioning.

[0080] FIG. 3 is a drawing for showing a binding process between a nucleic acid and an electrode in a biosensor device according to one embodiment of the present invention, and FIG. 4 is a drawing for explaining a process for analyzing a binding between a nucleic acid and a protein into an electrical signal through a biosensor according to one embodiment of the present invention.

[0081] Referring to FIGS. 3 and 4, the presence or absence of binding between a nucleic acid and a protein or the binding affinity between a nucleic acid and a protein can be confirmed through an electrical signal through a biosensor device (1000) according to one embodiment.

[0082] Specifically, as shown in Fig. 3, one end of the nucleic acid can be attached to the source electrode of the biosensor, and the other end can be aligned in a specific direction by receiving an external force through a fluid flowing in a microchannel and attached to the drain electrode.

[0083] Therefore, the source electrode and the drain electrode are electrically connected to each other through the nucleic acid, and it is possible to measure the current according to the applied voltage, and it is possible to determine whether the nucleic acid is bound or not through the measured current.

[0084] For reference, at this time, the nucleic acid may be attached only at one end via a linker (biotin-PEF-thiol, streptavidin, and biotin at the nucleic acid end in Figure 3), but instead, both ends may be attached to the source and drain electrodes, respectively, via linkers. Therefore, when both ends are attached to the electrode array, the nucleic acid can be electrically connected to the electrode array without alignment and attachment of the nucleic acid through a fluid.

[0085] When nucleic acids are electrically connected to an electrode array, the attachment and detachment of nucleic acids can be detected through changes in the electrical signal. Specifically, as shown in the upper graph of Figure 4, when one end of the nucleic acid detaches from the electrode, a low-level current signal is measured. However, when both ends are firmly attached to the source and drain electrodes, a high-level current signal can be measured. Therefore, users can detect the level of nucleic acid attachment and detachment through current signal measurement.

[0086] Furthermore, as nucleic acids are electrically connected to the electrode array, the level of interaction between nucleic acids and proteins can be measured by observing electrical signals. Specifically, as shown in the lower graph of Figure 4, proteins attach to nucleic acids, and changes in current signals are observed depending on the binding affinity of the attached proteins. This allows for analysis of factors such as the binding affinity between nucleic acids and specific proteins.

[0087] Meanwhile, in addition to analyzing interactions through electrical signals, a biosensor device according to one embodiment also allows for visual analysis of binding between nucleic acids and proteins or their conformational changes using fluorescent proteins. Specifically, by attaching a fluorescent protein to the protein being analyzed, the interaction between nucleic acids and proteins can be measured by measuring the fluorescence signal. At this time, the fluorescent protein is green fluorescent protein (GFP), modified green fluorescent protein (mGFP), enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), modified red fluorescent protein (mRFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), cyan green fluorescent protein (CGFP), enhanced cyan fluorescent protein (ECFP), AzG (Azami Green), HcR (Heteractis crispa red fluorescent protein; HcRed), or Discosoma red fluorescent protein (DsRed). You can use the back.

[0088] However, as described above, the biosensor device according to one embodiment can analyze the interaction between proteins and nucleic acids through electrical signal measurements without the use of fluorescent proteins. Therefore, it is possible to observe nucleic acid structure and dynamics with high sensitivity through electronic signals. Furthermore, since separate fluorescence-based methods are not required, fluorescence analysis equipment and other equipment are no longer required, thereby overcoming cost and observation limitations.

[0089]

[0090] Example 1: Fabrication of an electrode array

[0091] Considering the length of λ DNA (approximately 12 μm when stretched), as shown in Fig. 5, a split chip electrode array was designed to effectively introduce and precisely manipulate DNA curtains. Using a 6-inch quartz wafer, a source / drain (S / D) array with a spacing of 9, 12, 15, or 18 μm was fabricated by a photolithography process. At this time, the trench electrode (source electrode) where the curtain is formed was fabricated with a width of 44 μm x a height of 2 μm to induce the DNA curtain to be regularly arranged on the source electrode, and was fabricated with a transparent quartz substrate to enable simultaneous measurement of electronic signals and DNA fluorescence images.

[0092] The electrode array is designed to be arranged in a single line in the direction of flow with the center of the trench as shown in Fig. 5, so that multiple DNA curtain sensors can be effectively created, and multiple DNA curtain sensors can be created simultaneously by considering the laminar flow applied to the microfluidic channel.

[0093] Gold (Au) was used as the electrode array material to effectively introduce DNA into the electrode through strong bonding with thiol-DNA or thiol-biotin.

[0094]

[0095] Example 2: Trench functionalization

[0096] Preparation of Thiol-λ-DNA (Single-tethering)

[0097] As shown in Fig. 6, a thiol-oligomer having a complementary base sequence to λ-DNA was complementarily bound, and the thiol-oligomer was covalently bound to one end of the DNA through ligase treatment, thereby introducing a thiol to one end of the DNA to form a strong bond with the Au electrode. Accordingly, the fabricated DNA was fixed to the Au electrode in a single-tethered manner, and only one end of the DNA was connected to the electrode, allowing the remaining portion to move freely.

[0098]

[0099] Biotinylated λ preparation (double-tethering)

[0100] Along with the single linkage of DNA previously fabricated, a biosensor was fabricated in which DNA was doubly linked to an electrode.

[0101] Specifically, biotin-PEG-thiol was introduced to a gold electrode, followed by conjugation with streptavidin. Subsequently, λ, with biotin attached to both ends, was flowed through the PDMS channel to initiate a reaction, inducing DNA to form bridges on both electrodes. The fabricated doubly linked structure allows stable immobilization of DNA on the electrode without stretching due to fluid flow, enabling long-term observation of nucleosomes.

[0102]

[0103] Example 3: Measurement of DNA current signal

[0104] Single connection current measurement

[0105] The fabricated DNA solution was flowed using a syringe pump and reacted overnight across the entire exposed area through the PDMS microfluidic channel. 0.1x PBS solution was flowed at a rate of 2000 μl / min through the PDMS channel tube. With a voltage of 1.0 V applied between the source and drain electrodes, the flow was adjusted at regular time intervals (e.g., flow ON for 30 seconds, flow OFF for 1 minute 30 seconds, flow ON for 2 minutes 30 seconds, flow OFF for 3 minutes 30 seconds) and the current signal responding to the flow rate was measured. The curtain formation reaction of DNA was captured by this periodic flow change, and the results are shown in Fig. 7.

[0106] As a result of the experiment, as can be confirmed in Fig. 7, when the flow was turned on in a single-connected biosensor, the current value was relatively high (more than 1 nA). Afterwards, the current change was measured by inactivating the DNA by treating it with DNase in real time. In the single-connected biosensor, when the flow was turned off after DNA introduction, the current value was confirmed to be low because the DNA was not connecting between the source and drain electrodes (less than 1 nA, Fig. 7 (A)). When the flow was turned on around 30-40 seconds during the real-time current measurement, the DNA unfolded and connected between the source and drain electrodes, and the current value was confirmed to increase significantly and then be maintained (more than 1 nA, Fig. 7 (B)). When DNase was flowed into the microfluidic channel around 60 seconds with the flow turned on, the current immediately decreased significantly and then was maintained (less than 1 nA, Fig. 7 (C)). This indicates that the current values ​​before and after DNase treatment directly reflect the DNA linking the source and drain electrodes, which is then severed by DNase. This confirms that the high current values ​​observed when the flow rate is turned on are due to DNA tethering.

[0107]

[0108] Dual connection current measurement

[0109] After introducing biotin and streptavidin to the electrode array, biotinylated λ was introduced to double-link the nucleic acids to the electrodes through flow control. To confirm double linkage, the flow was turned on to form a curtain, and the current signal was continuously measured due to DNA linkage even when the fluid flow was stopped. The results are shown in Figures 8 and 9.

[0110] After the DNA reaction, real-time measurements were initiated without turning on the flow. As shown in Figures 8 and 9, the current was relatively low at approximately 1 nA before the flow was turned on, but when the flow was turned on after 30 seconds, the current increased by approximately twofold to approximately 2 nA. Even when the flow was turned off after approximately 50 seconds, the increased current value was maintained. This confirmed that, unlike single connection, the current value was maintained even without fluid (buffer) flow.

[0111]

[0112] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. For example, each component described as a single component may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0113] The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

[0114] The present invention relates to a biosensor for analyzing nucleic acid-protein interactions and a method for analyzing nucleic acid-protein interactions, and more particularly, to a device capable of analyzing interactions between proteins and nucleic acids through electrical signals via nucleic acids bound to an electrode array including electrodes, and a method for analyzing interactions between proteins and nucleic acids using the same.

Claims

1. An electrode array including a plurality of source electrodes spaced at regular intervals and a plurality of drain electrodes corresponding to the source electrodes, wherein the source electrodes and the drain electrodes corresponding to the source electrodes are arranged in parallel; and A nucleic acid having one end connected to the source electrode via a first linker and the other end connected to the drain electrode via a second linker or the other end not connected to the drain electrode; A biosensor for nucleic acid-protein interaction analysis, comprising:

2. In the first paragraph, the biosensor for analyzing nucleic acid-protein interaction is A biosensor for nucleic acid-protein interaction analysis, further comprising a microchannel disposed adjacent to the electrode array.

3. In paragraph 1, A biosensor for analyzing nucleic acid-protein interactions, wherein the nucleic acid is DNA.

4. A biosensor for analyzing nucleic acid-protein interactions, wherein the source electrode and the drain electrode comprise gold in the first paragraph.

5. A biosensor for analyzing nucleic acid-protein interactions, wherein at least one of the first linker and the second linker is a thiol group.

6. A biosensor for analyzing nucleic acid-protein interactions, wherein the nucleic acid has biotin linked to the terminal end of the nucleic acid in the first paragraph.

7. In the fourth paragraph, at least one of the first linker and the second linker, A biosensor for nucleic acid-protein interaction analysis, comprising biotin-PEG-thiol and streptavidin conjugated to the biotin-PEG-thiol.

8. In the second paragraph, the microchannel is selected from the group consisting of polyvinylsiloxane (PVS), poly(methylhydrosiloxane) (PMHS), fluorosilicone (FVMQ), polydiphenylsiloxane (PDPS), polymethylphenylsiloxane (PMPS), poly(dimethyl-co-methylphenylsiloxane), polytrifluoropropylmethylsiloxane, polyethylmethylsiloxane (PEMS), polyphenylsiloxane (PPS), polyphenylmethylsiloxane (PPMS), polybiphenylsiloxane (Poly(biphenylsiloxane)), polyhydromethylsiloxane (Poly(hydromethylsiloxane)), A biosensor for nucleic acid-protein interaction analysis, comprising at least one selected from the group consisting of polyoctamethylcyclotetrasiloxane (Poly(octamethylcyclotetrasiloxane; D4), polydimethylcosiloxane (Poly(dimethyl-co-siloxane)), poly(dimethylsiloxane-co-phenylsiloxane)), polysiloxane urethane (Poly(siloxane-urethane)), and poly(ethylmethylsiloxane) (Poly(ethylmethylsiloxane)).

9. In the first paragraph, the source electrode includes a protrusion to which the nucleic acid is connected, A biosensor for nucleic acid-protein interaction analysis, wherein the protrusion has a narrow width relative to the rest of the source electrode.

10. A biosensor for analyzing nucleic acid-protein interactions, wherein the protrusion has a width of 0.5 to 5 μm in the 9th paragraph.

11. A biosensor for analyzing nucleic acid-protein interactions, wherein the source electrode and the drain electrode are spaced apart from each other by a distance of 1 to 20 μm in the first paragraph.

12. In the second paragraph, the microchannel is arranged in a direction perpendicular to the source electrode and the drain electrode, A biosensor for analyzing nucleic acid-protein interactions, wherein the nucleic acids are arranged in a direction perpendicular to the source electrode and the drain electrode according to the flow of fluid within the microchannel.

13. An application step of applying voltage to a nucleic acid whose one end is connected to a source electrode and whose other end is connected or not connected to a drain electrode corresponding to the source electrode; An analysis step for analyzing nucleic acid-protein interactions by measuring electrical signals according to voltage application; A method for analyzing nucleic acid-protein interactions, comprising:

14. In the 13th paragraph, the method, A method for analyzing nucleic acid-protein interactions, comprising a stretching step of stretching the nucleic acid by applying a flow of fluid in a direction perpendicular to the source electrode and the drain electrode.

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