Biosensor comprising Anti-nuclear antibody, talen, or nanobody bound to channel

Abstract

The technical idea of the present invention provides a biosensor including: a substrate extending in a first direction and a second direction crossing the first direction; a source electrode and a drain electrode disposed apart from each other on the substrate; a channel disposed between the source electrode and the drain electrode; a probe disposed on the channel and composed of a transcription activator-like effector nuclease (TALEN), which is capable of chemically binding to low DNA; a linker molecule coupling the probe to the channel therethrough; a gate electrode for electrically controlling the channel; and a gate dielectric layer between the channel and the gate electrode.

Description

A biosensor comprising an antinuclear antibody, TALEN or nanobody bound to a channel

[0001] The technical idea of ​​the present invention relates to a biosensor comprising an antinuclear antibody, a transcription activator-like effector nuclease (TALEN), or a nanobody bound to a channel.

[0002] The recent COVID-19 pandemic has dramatically increased the need for rapid and accurate sensors for detecting biological substances such as DNA and RNA. While various optical, mechanical, and electrical sensors have been developed to detect biological substances, most sensors require extensive preprocessing to increase the concentration of the target substance in solution. In particular, for patient samples such as cancer cells, viruses, and antibodies, the number of extractions from patients is limited, resulting in significantly smaller sample volumes. This necessitates lengthy and complex preprocessing to detect target substances. Typically, detecting DNA within the nucleus of somatic cells requires unwinding the double helix of thousands of base pairs (bps) of DNA and then slicing it to the desired length. Therefore, the development of a method that simplifies the preprocessing process and enables rapid DNA detection has been urgently needed.

[0003] Programmable nucleases (CRISPRs), such as ZFNs (zinc finger nucleases), TALENs, and type 2 CRISPR / Cas (clustered regularly interspaced repeat / CRISPR-associated), and RGENs (RNA-guided engineered nucleases) derived from the prokaryotic adaptive immune system, are widely used for genome editing in cultured cells and organisms. Genome editing technology using CRISPRs is a highly useful technology that can be utilized for various purposes in life sciences, biotechnology, and medicine. For example, targeted genetic modification in stem cells or somatic cells has enabled gene / cell therapy for various genetic or acquired diseases. Various technologies utilizing CRISPRs are being developed based on the ability of CRISPRs to specifically bind to target sites.

[0004] Aberrant DNA methylation is a key epigenetic mechanism. Hypermethylation of CpG islands of tumor suppressor genes and the resulting transcriptional silencing are associated with malignant transformation in cancer. Other diseases with well-known epigenetic defects include ICF (Imunodeficiency, Centromeric region instability, and Facial anomalies) syndrome, Prader-Willi and Beckwith-Wiedemann syndromes, and Rett syndrome. Indeed, epigenetic architecture may explain several characteristics of many diseases, including age-dependent and quantitative patterns of disease, and mechanisms mediating genetic predisposition to disease. Therefore, numerous technologies for detecting aberrant DNA methylation are being investigated.

[0005] The technical idea of ​​the present invention is to provide a biosensor comprising an antinuclear antibody, TALEN or nanobody bound to a channel.

[0006] In order to solve the above-described problem, the technical idea of ​​the present invention provides a biosensor including a substrate extending in a first direction and a second direction intersecting the first direction; a source electrode and a drain electrode spaced apart from each other on the substrate; a channel disposed between the source electrode and the drain electrode; a probe disposed on the channel and comprising a transcription activator-like effector nuclease (TALEN) capable of chemically bonding to raw DNA; a linker molecule connecting the probe and the channel; a gate electrode electrically controlling the channel; and a gate dielectric film between the channel and the gate electrode.

[0007] According to one embodiment, the channel includes graphene having a two-dimensional hexagonal carbon (C) structure, and includes peaks and valleys having a height difference in a direction perpendicular to the upper surface of the substrate, and the biosensor is provided in which the peaks and valleys are alternately arranged in a zigzag shape along the first direction or a diagonal direction between the first direction and the second direction.

[0008] According to one embodiment, a biosensor is provided, characterized in that the peak portions are arranged at regular intervals within a range of 1 nanometer to 10 nanometers along the first direction.

[0009] According to one embodiment, a biosensor is provided, characterized in that the height difference between the peak portion and the valley portion is in the range of 0.2 nanometers to 20 nanometers.

[0010] According to one embodiment, a biosensor is provided, characterized in that the peak portion and the valley portion are alternately arranged in a zigzag shape along the first direction, and the peak portion and the valley portion extend in a line shape.

[0011] According to one embodiment, the biosensor is provided, wherein the probe is characterized by binding to DNA methylated DNA.

[0012] In order to solve the above-described problem, the technical idea of ​​the present invention provides a biosensor including a substrate extending in a first direction and a second direction intersecting the first direction; a source electrode and a drain electrode spaced apart from each other on the substrate; a channel disposed between the source electrode and the drain electrode; a probe disposed on the channel and comprising an antinuclear antibody capable of specifically binding to dsDNA included in raw DNA; a linker molecule connecting the probe and the channel; a gate electrode electrically controlling the channel; and a gate dielectric film between the channel and the gate electrode.

[0013] According to one embodiment, the channel includes graphene having a two-dimensional hexagonal carbon (C) structure, and includes peaks and valleys having a height difference in a direction perpendicular to the upper surface of the substrate, and the biosensor is provided in which the peaks and valleys are alternately arranged in a zigzag shape along the first direction or a diagonal direction between the first direction and the second direction.

[0014] According to one embodiment, a biosensor is provided, characterized in that the peak portions are arranged at regular intervals within a range of 1 nanometer to 10 nanometers along the first direction.

[0015] According to one embodiment, a biosensor is provided, characterized in that the height difference between the peak portion and the valley portion is in the range of 0.2 nanometers to 20 nanometers.

[0016] According to one embodiment, a biosensor is provided, characterized in that the peak portion and the valley portion are alternately arranged in a zigzag shape along the first direction, and the peak portion and the valley portion extend in a line shape.

[0017] In order to solve the above-described problem, the technical idea of ​​the present invention provides a biosensor including a substrate extending in a first direction and a second direction intersecting the first direction; a source electrode and a drain electrode spaced apart from each other on the substrate; a channel disposed between the source electrode and the drain electrode; a probe disposed on the channel and comprising a nanobody; a linker molecule connecting the probe and the channel; a gate electrode electrically controlling the channel; and a gate dielectric film between the channel and the gate electrode.

[0018] According to one embodiment, the channel includes graphene having a two-dimensional hexagonal carbon (C) structure, and includes peaks and valleys having a height difference in a direction perpendicular to the upper surface of the substrate, and the biosensor is provided in which the peaks and valleys are alternately arranged in a zigzag shape along the first direction or a diagonal direction between the first direction and the second direction.

[0019] According to one embodiment, a biosensor is provided, characterized in that the peak portions are arranged at regular intervals within a range of 1 nanometer to 10 nanometers along the first direction.

[0020] According to one embodiment, a biosensor is provided, characterized in that the height difference between the peak portion and the valley portion is in the range of 0.2 nanometers to 20 nanometers.

[0021] According to one embodiment, a biosensor is provided, characterized in that the peak portion and the valley portion are alternately arranged in a zigzag shape along the first direction, and the peak portion and the valley portion extend in a line shape.

[0022] According to exemplary embodiments of the present invention, by using a biosensor comprising an antinuclear antibody, TALEN or nanobody bound to a channel, the DNA pretreatment process of a sample required for use of the biosensor can be shortened.

[0023] FIG. 1 is a drawing for explaining the structure of a biosensor according to one embodiment of the present invention.

[0024] FIG. 2 is a drawing illustrating a wrinkled graphene channel included in a biosensor according to one embodiment of the present invention.

[0025] Figure 3 is an enlarged view of the R1 region of the wrinkled graphene channel of Figure 2.

[0026] FIG. 4 is a drawing for explaining a method for manufacturing a flat graphene channel included in a biosensor according to one embodiment of the present invention.

[0027] FIG. 5 is a drawing for explaining a method for manufacturing a wrinkled graphene channel included in a biosensor according to one embodiment of the present invention.

[0028] Hereinafter, embodiments of the technical concept of the present invention will be described in detail with reference to the attached drawings. Identical components in the drawings are designated by the same reference numerals, and redundant descriptions thereof will be omitted.

[0029] The biosensor according to embodiments of the present invention is used for analyzing biomolecules contained in a bio sample, thereby performing gene expression profiling, genotyping, detection of mutations and polymorphisms such as SNPs (Single Nucleotide Polymorphisms), protein and peptide analysis, screening of potential drugs, development and manufacturing of new drugs, etc. In addition, a biosensor based on an FET (Field Effect Transistor) that detects a target substance with an electrical signal is formed by combining a target substance with a very small wire-shaped or thin-film-shaped semiconductor structure, changing the electrical conductivity of the semiconductor structure by the target substance, and detecting the target substance through the change in electrical conductivity.

[0030] Biosensors employ appropriate probes depending on the target biological sample to be analyzed. Examples of probes that can be employed in biosensors include DNA probes, enzymes or antibodies / antigens, protein probes such as bacteriorhodopsin, microbial probes, and neuronal probes. Biosensors manufactured in the form of chips are also referred to as biochips. For example, they can be referred to as DNA chips, protein chips, cell chips, neuron chips, etc. depending on the type of probe employed. Biosensors according to some embodiments of the present invention may include oligomeric probes as probes. The oligomeric probe implies that the number of monomers of the employed probe is at the oligomeric level. Here, the term "oligomer" may be used to mean a polymer composed of two or more covalently bonded monomers having a molecular weight of about 1000 or less. Specifically, it may include about 2 to 500 monomers, and preferably 5 to 30 monomers. However, the meaning of the oligomer probe is not limited to the above figures. The monomers constituting the oligomer probe can be modified depending on the type of biological sample to be analyzed, and can be, for example, nucleosides, nucleotides, amino acids, peptides, etc. Nucleosides and nucleotides can include not only known purine and pyrimidine bases, but also methylated purines or pyrimidines, acylated purines or pyrimidines, etc. In addition, nucleosides and nucleotides can include not only conventional ribose and deoxyribose sugars, but also modified sugars in which one or more hydroxyl groups are substituted with halogen atoms or aliphatics, or functional groups such as ethers and amines are bonded. Amino acids can be L-, D-, and achiral 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 specified, the probes exemplified in the examples below are DNA probes, which are oligomeric probes in which monomers of about 5 to 30 nucleotides are covalently bonded. However, the present invention is not limited thereto, and it is apparent that various probes described above can be applied.

[0031] The term "TALEN" used herein refers to a protein composed of a DNA-binding domain and a DNA cleavage domain (nuclease domain). Specifically, the DNA-binding domain located at the N-terminus of TALEN includes a TALE (transcription activator-like effector) protein derived from a plant pathogenicity gene, which is a domain that recognizes a specific target sequence in the genome. TALE proteins have a structure in which modules composed of 33 to 35 amino acids are repeatedly arranged, and one module can recognize one base pair. The base pair recognized by the module is determined by the types of the 12th and 13th amino acids of the module, and these two amino acids are each referred to as repeat variable residues (RVDs). The relationship between an RVD and its corresponding base is as follows. Asn-Asn corresponds to guanine, Asn-Ile corresponds to adenine, His-Asp corresponds to cytosine, and Asn-Gly corresponds to thymine, respectively. Within the TALE protein, the RVD consists of 20 units, each of which can bind to one DNA base in the major groove of the DNA chain. In addition, the DNA cleavage domain located at the C-terminus of TALEN can include the cleavage region of the FokI restriction enzyme. Two FokI restriction enzymes can work together to cleave the double-stranded DNA structure.

[0032] As used herein, the term "antinuclear antibody" refers to not only autoantibodies directed against nuclear components such as deoxyribonucleic acid (DNA) and small nuclear ribonucleoprotein, but also, in a broader sense, autoantibodies directed against cellular components other than the nucleus, such as the mitotic spindle, cytosol, cytoplasmic organelles, and cell membrane.

[0033] As used herein, the term "nanobody (single domain antibody, sdAb)" refers to a single domain of an antinuclear antibody. For example, a nanobody may include the antigen recognition variable region (variable region 5) of a heavy chain-only antibody. Compared to typical antibodies, the nanobody has a simple single-domain structure and small size, resulting in high structural stability and ease of purification and production.

[0034] The term "gene scissors" used herein refers to a technology used to destroy genes, insert genes, or correct genes (genome editing) through DNA double-strand breaks, and refers to an artificial enzyme that binds to a specific gene and cuts the DNA of the gene, thereby removing an incorrect portion of the gene to solve a problem. For the purpose of the present invention, the gene scissors may bind to a target gene to be detected by a biosensor.

[0035] As used herein, "DNA methylation" refers to the attachment of a methyl group to a base that constitutes DNA. Specifically, methylation refers to methylation occurring at a cytosine at a specific CpG site of a specific gene. In one embodiment of the present invention, "methylation status" may refer to the presence or absence of 5-methyl-cytosine at one or more CpG dinucleotides within a DNA base sequence. In other words, it may include adding a methyl group (methyl) to the 5th position of the pyrimidine ring of cytosine or to the nitrogen at the 6th position of the purine ring of adenine. In one embodiment of the present invention, "methylation level" refers to the amount of methylation present in the DNA base sequence of a target gene methylation gene, for example, within all genomic regions and some non-genomic regions. The above methylation level can be performed by one or more methods selected from the group consisting of, but not limited to, PCR, methylation specific PCR, real time methylation specific PCR, MethyLight PCR, MehtyLight digital PCR, EpiTYPER, PCR using methylated DNA specific binding protein, quantitative PCR, DNA chip, pyrosequencing, and bisulfite sequencing.

[0036] As used herein, "DNA pretreatment" refers to a process of fragmenting a nucleic acid molecule to match the NGS read length, and specifically, may include 1) a step of fragmenting a nucleic acid molecule to match the NGS read length, 2) a step of making the ends of the fragmented nucleic acid molecules blunt ends through end-repair, 3) a step of adding A to the 3' end through A-tailing, 4) a step of attaching an adapter through a ligation reaction, and 5) a step of performing PCR to increase the ratio of molecules in which a ligation reaction has occurred. In the present specification, DNA that has not undergone DNA pretreatment may be referred to as raw DNA.

[0037] FIG. 1 is a drawing for explaining the structure of a biosensor according to one embodiment of the present invention.

[0038] Referring to FIG. 1, a biosensor (100) may include a substrate (110), a gate electrode (112), a wiring structure (114), a gate dielectric film (116), a source electrode (120), a drain electrode (122), and a channel (124). The substrate (110) may extend in a first direction (X) and a second direction (Y). The first direction (X) and the second direction (Y) may intersect each other. A direction perpendicular to the upper surface of the substrate (110) may be defined as a third direction (Z).

[0039] The substrate (110) may be formed of a non-conductive material such as ceramic, glass plate, or polymer resin, and may also be formed of organic polymer materials such as polyester, polyvinyle chloride, and polycarbonate. However, the material of the substrate (110) is not limited thereto, and may be changed or modified in any way considering cost or the configuration of the channel (124) provided on the substrate (110).

[0040] In one embodiment, the substrate (110) may include a material capable of causing shrinkage deformation such that the channels (124) disposed on the substrate (110) are wrinkled. The substrate (110) may include a material that causes shrinkage deformation, particularly at high temperatures. For example, the substrate (110) may include a thermoplastic polymer that causes shrinkage deformation at about 100° C. to 200° C. Examples of polymers suitable for use in the present invention include one or a combination of polystyrene, polyetherimide, polyethersulfone, polyetheretherketone, polytetrafluoroethylene, and liquid crystal polymer. For example, when a substrate (110) including polystyrene (PS) is annealed at about 110°C to 115°C for about 10 to 120 minutes, wrinkles may be formed in the channel (124) in a range of several micrometers to several hundred nanometers. The substrate (110) may have a different degree of shrinkage deformation depending on the annealing temperature. For example, the substrate (110) may have a greater degree of shrinkage deformation as the annealing temperature increases. In addition, the substrate (110) may have a different degree of shrinkage deformation depending on the annealing time. For example, the substrate (110) may have a greater degree of shrinkage deformation as the annealing time increases.

[0041] The source electrode (120) and the drain electrode (122) may be disposed on the substrate (110). The drain electrode (122) may be disposed spaced apart from the source electrode (120) in the first direction (X). The source electrode (120) and the drain electrode (122) may be formed of various materials and may include a metal or a metal alloy. For example, the source electrode (120) and the drain electrode (122) 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 an alloy thereof. However, when the channel (124) is made of graphene, it is preferable that the source electrode (120) and the drain electrode (122) be made of gold (Au) or a chromium / gold (Cr / Au) alloy in terms of contact with the graphene and ease of etching.

[0042] A channel (124) may be interposed between the source electrode (120) and the drain electrode (122), and the channel (124) may extend in a first direction (X) on the substrate (110). The channel (124) may be formed of a material whose electrical properties change by an external electric field. For example, the channel (124) may include crystalline silicon, amorphous silicon, a doped layer doped with impurities, a semiconductor layer, an oxide layer, a compound layer, a carbon nanotube (CNT), or a semiconductor nanowire. The channel (124) may include graphene having a two-dimensional hexagonal carbon (C) structure. In the present specification, a channel (124) including graphene may be referred to as a “graphene channel.” However, in some embodiments, the graphene channel may not have a flat structure extending along the upper surface of the substrate (110), but may include peaks and valleys having a height difference in a direction perpendicular to the upper surface of the substrate (110) (e.g., the third direction (Z)). In the present specification, a graphene channel having peaks and valleys may be referred to as a “wrinkled graphene channel.” Conversely, a graphene channel not including peaks and valleys may be referred to as a “flat graphene channel.” A specific description of the structure of the wrinkled graphene channel will be described later in the description of FIG. 2. The degree of wrinkles in the graphene channel may vary depending on the temperature and time of the annealing process described above. At this time, considering the yield and sensitivity of the graphene channel, the degree of wrinkles in the graphene channel is preferably about 55%.

[0043] The sample (126) in contact with the channel (124) may include a target substance. The target substance may include a protein, DNA, RNA, or any base sequence, and the sample (126) may include fragments containing the target substance, a biological solution such as blood, saliva, etc. In addition, the sample (126) may include raw DNA. The sample (126) may be placed in a sample storage container (130). The sample storage container (130) may be a container for containing the sample (126) and may be formed of a polymer or non-polymer material. For example, silicone rubber may be used for the sample storage container (130). The shape and size of the sample storage container (130) are not limited to those illustrated, and any container capable of containing the sample (126) may be used as the sample storage container (130) of the present invention.

[0044] The channel (124) can be electrically controlled by the gate electrode (112). The gate electrode (112) can include a conductive metal material. In one embodiment, the gate electrode (112) can be made of silver (Ag). The gate electrode (112) can be connected to a power source through a wiring structure (114) to supply a gate voltage to the channel (124). A gate dielectric film (116) can be positioned between the channel (124) and the gate electrode (112). At this time, by observing the electrical parameters according to the gate voltage of the channel (124), it is possible to determine whether a target material exists in the sample (126). At this time, a change in the electrical parameters can correspond to the presence of the target material. For example, the electrical parameters can include capacitance, voltage, average current, etc.

[0045] The biosensor (100) may further include a probe (128) capable of chemically bonding with a target substance molecule. The probe (128) may be disposed on a channel (124). When the probe (128) on the channel (124) chemically bonds with the target substance, an electrical redox reaction occurs, thereby changing the electrical parameter. This may determine whether the target substance is present in the sample (126). The biosensor (100) may further include a linker molecule (129) to facilitate chemical bonding between the probe (128) and the channel (124). The linker molecule (129) may be disposed on the channel (124). In one embodiment, the channel (124) may be bonded to the linker molecule (129), and the probe (128) may be bonded to the channel (124) via the linker molecule (129).

[0046] As an example, a PASE (Pyrenebutanoic acid succinimidyl ester) molecule of the following chemical formula 1 may be selected as the linker molecule (129).

[0047] [Chemical Formula 1]

[0048]

[0049] The pyrene group in the PASE molecule can bind to the graphene channel (124), and the succinimidyl ester group in the PASE molecule can bind to the probe (128). The binding of the pyrene group in the PASE molecule to the graphene channel (124) may be through a π-π bond among van der Waals bonds.

[0050] In some embodiments, the probe (128) may include a TALEN. As described above, a TALEN is a protein composed of a DNA-binding domain and a DNA cleavage domain (nuclease domain). As described above, the DNA-binding domain includes a TALE protein derived from a plant pathogenicity gene, which is a domain that recognizes a specific target sequence on the genome. The TALE protein has a structure in which modules composed of 33 to 35 amino acids are repeatedly arranged, and one module can recognize one base pair. The base pair recognized by the module is determined by the types of the 12th and 13th amino acids of the module, and the two amino acids are each referred to as RVD. Since one RVD unit of the TALE protein of a TALEN corresponds one-to-one to one DNA base, the RVD units of the TALE protein can be arranged to correspond to the target base sequence of the target gene, which is a target substance. As an example, when the target base sequence is guanine-adenine-adenine (GAA), the TALE protein may include Asn-Asn, Asn-Ile, and Asn-Ile as RVD units. Using the above-described characteristics of TALENs, the probe (128) may be formed of a TALE protein that includes RVD units arranged to correspond to the target base sequence of the target gene for detection of the target gene. As an example, when the probe (128) is formed of a TALE protein having an RVD unit corresponding to a methylated cytosine base, it is possible to determine whether methylated DNA exists in the sample (126) by observing whether methylated DNA binds to the probe (128).

[0051] In some embodiments, the probe (128) may include an anti-nuclear antibody. The anti-nuclear antibody may include an anti-DNA antibody, and the anti-DNA antibody may include an anti-dsDNA antibody. The anti-dsDNA antibody is an antibody against dsDNA (double-stranded DNA) and may be an antibody against a substance within the cell nucleus. In addition, the anti-DNA antibody may include an anti-ssDNA antibody, and the anti-ssDNA antibody is an antibody against ssDNA (single-stranded DNA) and may be an antibody against a substance within the cell nucleus. The anti-DNA antibody may include an antibody that reacts only with dsDNA, an antibody that reacts with both dsDNA and ssDNA, an antibody that reacts only with ssDNA, and an antibody that reacts with ZDNA. The probe (128) including the anti-dsDNA antibody can detect dsDNA. A probe (128) containing an anti-ssDNA antibody can detect ssDNA. When the probe (128) is composed of anti-dsDNA, it is possible to detect whether the target substance dsDNA binds to the probe (128), thereby detecting whether the target substance dsDNA exists in the sample (126).

[0052] In some embodiments, the probe (128) may comprise a nanobody. A nanobody, as described above, refers to a single domain of an antinuclear antibody. For example, the nanobody may comprise the antigen recognition variable region (variable region 5) of a heavy chain-only antibody. The size of the nanobody may be small compared to the size of the antinuclear antibody. For example, the nanobody may be a substance that is composed of 10% of the size of the antinuclear antibody. When the probe (128) is composed of a nanobody, the presence of a target substance in the sample (126) can be detected by detecting whether the target substance binds to the probe (128). When the probe (128) is composed of a nanobody, the target substance may be a protein.

[0053] When detecting a target substance using a biosensor (100) according to embodiments of the technical idea of ​​the present invention, since it includes a probe (128) made of TALEN or an antinuclear antibody, the target substance, i.e., target DNA, can be detected without DNA pretreatment of the sample (126). As a comparative example, in the case of a biosensor that does not include a probe (128) made of TALEN or an antinuclear antibody, the sample must undergo DNA pretreatment in order to detect the target substance, i.e., target DNA. At this time, DNA pretreatment refers to a process of fragmenting a nucleic acid molecule according to the NGS read length. The ends of the fragmented nucleic acids undergo end-repair and A-tailing to change into a form suitable for ligation, and then an adapter is attached by ligation. This process often involves selectively selecting only those molecules that have undergone a ligation reaction. Typically, PCR (polymerase chain reaction) is performed to increase the proportion of molecules that have undergone a ligation reaction. Because DNA pretreatment requires various enzymes and experimental procedures, even if the costs associated with specific enzymes or experiments are significantly reduced, the overall cost effectiveness is limited. Furthermore, the time required for DNA pretreatment makes it difficult to rapidly detect target genes.

[0054] In the case of detecting a target substance using a biosensor (100) according to embodiments of the technical idea of ​​the present invention, since it includes a probe (128) made of TALEN or an antinuclear antibody, the target DNA can be detected using raw DNA that has not undergone DNA pretreatment for the sample (126), and in terms of cost, it is economical because no DNA pretreatment cost is required, and in terms of time, the time required for DNA pretreatment can be reduced, enabling rapid target gene detection.

[0055] In addition, when detecting a target substance using a biosensor (100) according to embodiments of the technical idea of ​​the present invention, since it includes a probe (128) made of a nanobody, it is easier for the target substance to be located within the device length, so the sensitivity of the biosensor (100) can be further improved.

[0056] Hereinafter, a method for binding a PASE molecule as a linker molecule to a graphene channel will be described in detail with reference to an experimental example of the present invention. This experimental example is only intended to more clearly understand the present invention and is not intended to limit the scope of the present invention. Referring to an experimental example of the present invention, a PASE solution was prepared from DMSO (Dimethyl Sulfoxide). The PASE solution was prepared at a concentration of 30 μM. The prepared PASE solution was dropped onto the surface of the graphene channel so that the graphene channel and the prepared PASE solution were in contact with each other. The graphene channel treated with the PASE solution was washed three times with a DMSO solution and three times with ethanol. The washed graphene channel was dried for approximately 1 hour or more. The dried graphene channel was washed with DI water. As a result, a graphene channel to which a PASE molecule is bound can be obtained.

[0057] Hereinafter, a method for binding DNA as a probe to a graphene channel will be described in detail with reference to an experimental example of the present invention. When binding TALEN and antinuclear antibodies as probes to a graphene channel, a method similar to the method for binding DNA as a probe to a graphene channel can be used. In the present specification, DNA bound as a probe is referred to as probe DNA. Referring to an experimental example of the present invention, a graphene channel that had been treated with a PASE solution was incubated in 1X PBS (1X Phosphate-Buffered Saline) for more than one day. After the incubation, a diluted solution containing the probe DNA was dropped onto the graphene channel, and after about 3 hours, the channel was washed with a 1X PBS solution. The probe DNA was diluted to a concentration of about 264 μM. The washed graphene channel was dried at about 25°C for about 1 hour or more. As a result, a graphene channel to which the probe DNA was bound via a PASE molecule can be obtained.

[0058] Next, we describe two experimental examples of a method for detecting target substances through graphene channels bound to probe DNA using PASE molecules. A similar method for detecting target substances through graphene channels bound to probe DNA can also be used when detecting target substances using TALEN and antinuclear antibodies as probes.

[0059] [Experimental Example 1]

[0060] After the PASE solution treatment was completed, the graphene channel was incubated in 1XPBS, washed with 1XPBS, and then incubated with 50 μL of 100 μM pDNA-NH2 for about 3 hours or more. After the incubation with 100 μM pDNA-NH2 was completed, it was washed with 1XPBS three or more times. A 50 μL tDNA solution containing x μM (where x is a number greater than or equal to 0) tDNA was brought into contact with the second end of the graphene channel, and then left to stand for about 20 minutes. After the waiting period, the electrode was washed with 1XPBS, and then brought into contact with a sample containing a mixture of 5 mM methylene blue, 5 mM [Fe(CN)6]-4, and the target substance. Afterwards, the CV (Cyclic voltammetry) graph was observed at a scan frequency of 50 mV, and the current was observed while applying the gate voltage from approximately 0 V to -0.6 V. The above-described experimental process was repeated while gradually increasing the concentration of tDNA in the tDNA solution.

[0061]

[0062] [Experimental Example 2]

[0063] After PASE solution treatment, the graphene channel was incubated in 1XPBS, washed with 1XPBS, and then 50 μL of 100 μM MB-pDNA-NH2 was applied onto the graphene channel and incubated for more than 3 hours. After incubation with 50 μL of 100 μM MB-pDNA-NH2, the channel was washed with 1XPBS three or more times. The subsequent process is similar to the first experimental example.

[0064] However, there is a difference in that the first experimental example incubates the graphene channel with pDNA-NH2 after incubation in 1XPBS, and the second experimental example incubates with MB-pDNA-NH2 containing methylene blue.

[0065]

[0066] FIG. 2 is a drawing illustrating a wrinkled graphene channel included in a biosensor according to one embodiment of the present invention.

[0067] Referring to FIG. 2, the channel (124) may include a peak portion (P) and a valley portion (V) having a height difference in the third direction (Z). At this time, the channel (124) may be a graphene channel. The wrinkled shape of the channel (124) may vary. As an example, referring to (a) of FIG. 2, the peak portions (P) and valley portions (V) of the wrinkled graphene channel may be alternately arranged in a zigzag shape along the first direction (X). As another example, referring to (b) of FIG. 2, the peak portions (P) and valley portions (V) of the wrinkled graphene channel may be alternately arranged in a zigzag shape in a diagonal direction between the first direction (X) and the second direction (Y). The wrinkled shape of the channel (124) is not limited to that illustrated, and may be wrinkled in a shape other than that illustrated. Additionally, for some embodiments, the channel (124) includes a peak portion (P) and a valley portion (V) having a height difference in the third direction (Z), and the channel (124) may be configured to be flat in the first direction (X) and the second direction (Y).

[0068] In a channel (124) having a peak portion (P) and a valley portion (V), the spacing between the peak portions (P) and the valley portions (V) may vary. In one embodiment, the peak portions (P) may be arranged to be spaced apart at a constant first spacing (W1) in a range of about 1 nanometer to 10 nanometers along the first direction (X). The valley portions (V) may be arranged to be spaced apart at a constant second spacing (W2) in a range of about 1 nanometer to 10 nanometers along the first direction (X). In another embodiment, the peak portions (P) and the valley portions (V) may be arranged to be spaced apart at a non-constant spacing along the first direction (X). As the degree of wrinkles in the channel (124) increases, the spacing between the peak portions (P) and the valley portions (V) along the first direction (X) may decrease.

[0069] In one embodiment, the height difference in the third direction (Z) between adjacent peaks (P) and valleys (V) of the channel (124) may be constant in a range of about 0.2 nanometers to 20 nanometers. In another embodiment, the height difference in the third direction (Z) between adjacent peaks (P) and valleys (V) of the channel (124) may not be constant.

[0070] A valley portion (V) may be arranged between adjacent peak portions (P) of the channel (124), and a peak portion (P) may be arranged between adjacent valley portions (V). The shapes of the peak portions (P) and the valley portions (V) of the channel (124) are not limited to those illustrated, and may be formed in various shapes. In one embodiment, for the first valley portion (V1) and the second valley portion (V2), which are valley portions (V) adjacent to the peak portion (P) of the channel (124), the slope of the first intermediate region (COL1) from the first valley portion (V1) to the peak portion (P) and the slope of the second intermediate region (COL2) from the second valley portion (V2) to the peak portion (P) may be different from each other. In another embodiment, for the first valley portion (V1) and the second valley portion (V2), which are valley portions (V) adjacent to the peak portion (P) of the channel (124), the slope of the first intermediate region (COL1) from the first valley portion (V1) to the peak portion (P) and the slope of the second intermediate region (COL2) from the second valley portion (V2) to the peak portion (P) may be the same. In this case, the first intermediate region (COL1) from the first valley portion (V1) to the peak portion (P) and the second intermediate region (COL2) from the second valley portion (V2) to the peak portion (P) may be symmetrical with respect to the peak portion (P).

[0071] The channel (124) may have a thickness of about 1 nanometer to 2 nanometers and a surface area of ​​about 1 square millimeter to 100 square millimeters.

[0072] In addition, the number of graphene layers constituting the channel (124) is not limited to that illustrated. The channel (124) may be composed of a plurality of graphene layers. The channel (124) may be composed of first graphene and second graphene disposed on the first graphene. The first graphene and the second graphene may have the same or different shapes. For example, the first graphene may have a smaller separation distance between adjacent peaks (P) than the second graphene. In addition, the plurality of carbon (C) atoms included in the plurality of graphene layers constituting the channel (124) may be arranged in a line in the third direction (Z) or may be arranged in an offset manner. In one embodiment, the carbon (C) atoms constituting the first graphene and the carbon (C) atoms constituting the second graphene may be arranged in a line in the third direction (Z). As another example, the carbon (C) atoms constituting the first graphene and the carbon (C) atoms constituting the second graphene may be arranged to be misaligned in the third direction (Z).

[0073] A target material can be directly or indirectly attached to the channel (124), and the presence or absence of the target material in the reaction solution can be determined by changing the electrical parameters when the target material is attached to the channel (124).

[0074] Figure 3 is an enlarged view of the R1 region of the wrinkled graphene channel of Figure 2.

[0075] Referring to FIG. 3, an intermediate region (COL) between a peak portion (P) and a valley portion (V) of a channel (124) is illustrated. A distance (d1) from a line (A-A') crossing the intermediate region (COL) of the channel (124) to the peak portion (P) in a third direction (Z) may be about 0.54 nanometers. A distance (d2) from a line (A-A') crossing the intermediate region (COL) of the channel (124) to the valley portion (V) in a third direction (Z) may be about 0.54 nanometers.

[0076] FIG. 4 is a diagram illustrating a method for manufacturing a flat graphene channel included in a biosensor according to one embodiment of the present invention. The present invention is not limited to what is illustrated, and a flat graphene channel can be formed through various processes.

[0077] Referring to FIG. 4, the channel (124) can be formed by depositing a carbon compound containing carbon (C) on a preliminary substrate (110') using a chemical vapor deposition (CVD) process.

[0078] The preliminary substrate (110') may include a metal such as copper (Cu), nickel (Ni), or platinum (Pt). When the preliminary substrate (110') includes copper (Cu), it may be preferable for forming a single layer of graphene. A raw material gas containing a carbon (C) compound may be injected onto the preliminary substrate (110') to form a channel (124). For example, the carbon compound may be natural graphite, synthetic graphite, highly ordered pyrolytic graphite, activated graphite, carbon monoxide, carbon dioxide, methane, ethane, ethylene, methanol, ethanol, acetylene, propane, propylene, butane, butadiene, pentane, pentene, cyclopentadiene, hexane, cyclohexane, benzene, pyridine, hexaphenylborazine, poly(4-vinylpyridine) (P4VP), 1,3,5-triazine, piperidine, toluene, polymethyl methacrylate, polystyrene, acrylonitrile, polyacrylonitrile, Melamine, polycyclic aromatic hydrocarbon structure methylnaphthalene, hexabromobenzene, naphthalene, pentachloropyridine, terphenyl, tetrabromothiophene, benzopyrene, azulene, trimethylnaphthalene, acenaphthene, acenaphthylene, anthracene, fluorene, phenalene, phenanthrene, benz(a)anthracene,Benzofluorene, Benzo(c)phenanthrene, Chrysene, Fluoranthene, Pyrene, Tetracene, Triphenylene, Benz(e)acephenanthrylene, Benzofluoranthene, Dibenzanthracene, Olympicene, Pentacene, Perylene, Picene, Tetraphenylene, Zethrene, Ovalene, Kekulene, Hexacene, Heptacene, Diindenoperylene, Dicoronylene, It may include Coronene, Corannulene, Benzo(ghi)perylene, Anthanthrene, Hexamethyldihydro-4H-benzoquinolizinoacridine, 4Hbenzoquinolizinoacridinetrione or Hexaazatriphenylenehexacarbonitrile.

[0079] Next, a protective film (134) can be formed on the channel (124). In one embodiment, the protective film (134) can include a polymer such as an acrylic resin (PMMA; Polymethyl methacrylate). In one embodiment, the protective film (134) can be formed by a spin coating method. Specifically, a small amount of liquid for forming the protective film (134) can be dropped on the channel (124), and then the preliminary substrate (110') can be rotated. The rotation of the preliminary substrate (110') causes the small amount of liquid to spread to the edge of the channel (124), thereby forming a protective film (134) that coats the channel (124).

[0080] Next, the preliminary substrate (110') under the channel (124) can be removed. In one embodiment, the preliminary substrate (110') can be removed using a chemical etching method. The etchant of the chemical etching method can include a substance that can dissolve metal. In one embodiment, the etchant can include sodium persulfate (Na2S2O8). Thereafter, the substrate (110) can be formed under the channel (124) using a direct transfer method. A plasma treatment can be performed on the substrate (110) to combine the substrate (110) and the channel (124) on which the protective film (134) is formed. In one embodiment, the plasma treatment can include oxygen (O) as a reaction gas. After combining the channel (124) with the substrate (110), the protective film (134) on the channel (124) can be removed. As an example, when the protective film (134) includes an acrylic resin (PMMA), the protective film (134) can be removed using acetic acid (CH3COOH) and DI water. By removing the protective film (134), a channel (124) bonded to the substrate (110) can be obtained.

[0081]

[0082] FIG. 5 is a diagram illustrating a method for manufacturing a wrinkled graphene channel included in a biosensor according to one embodiment of the present invention. The present invention is not limited to the illustrated process, and wrinkled graphene channels can be formed through various processes. Since the method for manufacturing a wrinkled graphene channel is generally similar to the method for manufacturing a flat graphene channel, the following description will focus on the differences from the method for manufacturing a flat graphene channel.

[0083] Referring to FIG. 5, a preliminary channel (124') can be formed on a preliminary substrate (110'). The preliminary channel (124') can be formed by depositing carbon (C) atoms on the preliminary substrate (110') using a chemical vapor deposition (CVD) process.

[0084] Next, a protective film (134) can be formed on the preliminary channel (124'). As an example, the protective film (134) can be formed by a rotational coating method. Specifically, a small amount of liquid for forming the protective film (134) can be dropped on the preliminary channel (124'), and then the preliminary substrate (110') can be rotated. The rotation of the preliminary substrate (110') causes the small amount of liquid to spread to the edge of the preliminary channel (124'), thereby forming a protective film (134) that coats the preliminary channel (124').

[0085] Next, the preliminary substrate (110') under the preliminary channel (124') can be removed. In one embodiment, the preliminary substrate (110') can be removed using a chemical etching method. The etchant for the chemical etching method can include a material that can melt metal. Thereafter, the substrate (110) can be formed under the preliminary channel (124') using a direct transfer method. The substrate (110) and the preliminary channel (124') on which the protective film (134) is formed can be combined by performing a plasma treatment on the upper portion of the substrate (110). After the preliminary channel (124') is combined with the substrate (110), the protective film (134) on the preliminary channel (124') can be removed.

[0086] Next, the substrate (110) to which the preliminary channel (124') from which the protective film (134) has been removed can be annealed. For example, the annealing process can be performed at a temperature of about 110°C to 120°C and can be performed for about 10 to 120 minutes. At this time, the length in the direction in which the channel (124) extends can be formed to be 30% to 90% of the length in the direction in which the preliminary channel (124') extends. Through shrinkage deformation of the substrate (110), the preliminary channel (124') can be formed into a wrinkled channel (124).

[0087] The degree of wrinkles in the channel (124) may vary depending on the time or temperature of the annealing process. In one embodiment, when the length of the preliminary channel (124') in the first direction (X) is defined as the first length and the length of the channel (124) in the first direction (X) is defined as the second length, when the substrate (110) to which the preliminary channel (124') is bonded is annealed at about 120° C. for about 60 minutes, the second length may be 1 / 4 of the first length. Since the greater the degree of wrinkles in the wrinkled channel (124), the longer the time consumed in the process for bonding the channel (124) and the linker molecule, it is preferable that the degree of wrinkles in the wrinkled channel (124) from the preliminary channel (124') be about 55% when considering yield and sensitivity. That is, it is preferable that the length in the direction in which the channel (124) extends is about 45% of the length in the direction in which the spare channel (124') extends.

[0088]

[0089] As described above, exemplary embodiments have been disclosed in the drawings and specifications. While specific terminology has been used to describe embodiments herein, it is intended solely to illustrate the technical concept of the present disclosure and is not intended to limit the scope of the present disclosure as defined in the claims. Therefore, those skilled in the art will appreciate that various modifications and equivalent embodiments are possible. Therefore, the true technical protection scope of the present disclosure should be determined by the technical concept of the appended claims.

Claims

1. A substrate extending in a first direction and a second direction intersecting the first direction; A source electrode and a drain electrode spaced apart from each other on the substrate; A channel disposed between the source electrode and the drain electrode; A probe disposed on the above channel and composed of a transcription activator-like effector nuclease (TALEN) capable of chemically binding to raw DNA; A linker molecule that connects the probe and the channel; a gate electrode electrically controlling the above channel; and A biosensor comprising a gate dielectric film between the channel and the gate electrode.

2. In paragraph 1, The above channel includes graphene having a two-dimensional hexagonal carbon (C) structure, and includes a peak portion and a valley portion having a height difference in the vertical direction on the upper surface of the substrate, A biosensor in which the peak portion and the valley portion are alternately arranged in a zigzag shape along the first direction or the diagonal direction between the first direction and the second direction.

3. In paragraph 2, A biosensor characterized in that the peak portions are arranged at regular intervals within a range of 1 nanometer to 10 nanometers along the first direction.

4. In paragraph 2, A biosensor characterized in that the height difference between the peak and the valley is in the range of 0.2 nanometers to 20 nanometers.

5. In paragraph 2, A biosensor characterized in that the peak portion and the valley portion are alternately arranged in a zigzag shape along the first direction, and the peak portion and the valley portion extend in a line shape.

6. In paragraph 1, A biosensor characterized in that the above probe binds to DNA methylated DNA.

7. A substrate extending in a first direction and a second direction intersecting the first direction; A source electrode and a drain electrode spaced apart from each other on the substrate; A channel disposed between the source electrode and the drain electrode; A probe comprising an antinuclear antibody capable of specifically binding to dsDNA contained in raw DNA and positioned on the above channel; A linker molecule that connects the probe and the channel; a gate electrode electrically controlling the above channel; and A biosensor comprising a gate dielectric film between the channel and the gate electrode.

8. In paragraph 7, The above channel includes graphene having a two-dimensional hexagonal carbon (C) structure, and includes a peak portion and a valley portion having a height difference in the vertical direction on the upper surface of the substrate, A biosensor in which the peak portion and the valley portion are alternately arranged in a zigzag shape along the first direction or the diagonal direction between the first direction and the second direction.

9. In paragraph 8, A biosensor characterized in that the peak portions are arranged at regular intervals within a range of 1 nanometer to 10 nanometers along the first direction.

10. In paragraph 8, A biosensor characterized in that the height difference between the peak and the valley is in the range of 0.2 nanometers to 20 nanometers.

11. In paragraph 8, A biosensor characterized in that the peak portion and the valley portion are alternately arranged in a zigzag shape along the first direction, and the peak portion and the valley portion extend in a line shape.

12. A substrate extending in a first direction and a second direction intersecting the first direction; A source electrode and a drain electrode spaced apart from each other on the substrate; A channel disposed between the source electrode and the drain electrode; A probe disposed on the above channel and composed of a nanobody; A linker molecule that connects the probe and the channel; a gate electrode electrically controlling the above channel; and A biosensor comprising a gate dielectric film between the channel and the gate electrode.

13. In paragraph 12, The above channel includes graphene having a two-dimensional hexagonal carbon (C) structure, and includes a peak portion and a valley portion having a height difference in the vertical direction on the upper surface of the substrate, A biosensor in which the peak portion and the valley portion are alternately arranged in a zigzag shape along the first direction or the diagonal direction between the first direction and the second direction.

14. In paragraph 13, A biosensor characterized in that the peak portions are arranged at regular intervals within a range of 1 nanometer to 10 nanometers along the first direction.

15. In paragraph 13, A biosensor characterized in that the height difference between the peak and the valley is in the range of 0.2 nanometers to 20 nanometers.

16. In paragraph 13, A biosensor characterized in that the peak portion and the valley portion are alternately arranged in a zigzag shape along the first direction, and the peak portion and the valley portion extend in a line shape.

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