Biosensor including corrugated graphene layer and method for manufacturing same

Abstract

The technical idea of the present invention provides a biosensor and a method for manufacturing same, the biosensor comprising: a lower substrate extending in a first horizontal direction and a second horizontal direction intersecting the first horizontal direction; a working electrode disposed on the lower substrate; a probe disposed on the working electrode and capable of chemically bonding to a target substance to be detected; a linker bonding the probe and the working electrode; and a reference electrode disposed on the lower substrate so as to be spaced apart from the working electrode, wherein the working electrode includes at least one graphene layer, and the at least one graphene layer includes a peak portion and a valley portion having a height difference in a vertical direction on an upper surface of the lower substrate.

Description

Biosensor comprising a wrinkled graphene layer and method for manufacturing the same

[0001] The technical idea of ​​the present invention relates to a biosensor comprising a corrugated graphene layer.

[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 methods have been developed to detect biological substances, most sensors require extensive preprocessing to increase the concentration of the target substance in the solution. In particular, for patient samples such as cancer cells, viruses, and antibodies, the number of samples extracted from patients is limited, resulting in a limited amount of sample extraction. This necessitates lengthy and complex preprocessing to detect the target substance. Among these, electrical sensors offer high sensitivity without the need for preprocessing to increase the concentration of the target substance in the solution, and thus, extensive research is currently underway. Electrochemical biosensors are well-known in the art and have been used to determine the concentration of various analytes in biological samples, particularly blood.

[0003] The above biosensor comprises an electrode system comprising multiple electrodes formed by screen printing or other methods on an insulating lower substrate. Here, by applying a certain voltage after a sample is introduced and measuring the resulting current, the presence or concentration of a target substance contained in the sample can be determined.

[0004] Graphene, a two-dimensional hexagonal carbon (C) structure, is a new material that has been actively researched worldwide as a replacement for semiconductors. Furthermore, graphene is known to have electrically semi-metallic properties, and because charges within it act as zero-effective mass particles, it has extremely high electrical conductivity (intrinsic electron mobility of 20,000 cm2 / Vs). In particular, after it was reported that a graphene layer composed of two-dimensional hexagonal carbon (C) atoms mechanically exfoliated from graphite exhibited field effect characteristics when used in a transistor, graphene has been attracting attention as a material that can replace conventional semiconductor materials such as silicon. Accordingly, much research is being conducted on technologies that can improve the selectivity and sensitivity of biosensors containing graphene layers while maintaining the excellent electrical conductivity inherent in the graphene layer, and can rapidly modify the surface of the graphene layer without additional chemical reaction steps and stably immobilize bioreceptors.

[0005] The technical idea of ​​the present invention is to provide a biosensor including a wrinkled graphene layer and a method for manufacturing the same.

[0006] In order to solve the above-described problem, according to exemplary embodiments according to the technical idea of ​​the present invention, a biosensor is provided, including: a lower substrate extending in a first horizontal direction and a second horizontal direction intersecting the first horizontal direction; a working electrode disposed on the lower substrate; a probe disposed on the working electrode and capable of chemically bonding with a target substance to be detected; a linker connecting the probe and the working electrode; and a reference electrode disposed on the lower substrate and spaced apart from the working electrode, wherein the working electrode includes at least one graphene layer, and the at least one graphene layer includes a peak portion and a valley portion having a height difference in a vertical direction on an upper surface of the lower substrate.

[0007] In order to solve the above-described problem, according to exemplary embodiments according to the technical idea of ​​the present invention, a method for manufacturing a biosensor is provided, comprising the steps of: preparing a lower substrate extending in a first horizontal direction and a second horizontal direction intersecting the first horizontal direction; forming a working electrode including at least one graphene layer on the lower substrate; and connecting a linker to the at least one graphene layer and connecting a probe chemically bondable to a target substance to be detected to the linker, wherein the step of forming the working electrode including the at least one graphene layer comprises: forming a preliminary graphene layer on the lower substrate; and annealing the lower substrate to cause the preliminary graphene layer to undergo shrinkage deformation.

[0008] According to exemplary embodiments of the present invention, the working electrode may include at least one wrinkled graphene layer, and the sensitivity to a target material may be enhanced using the working electrode.

[0009] Additionally, since the surface area of ​​the wrinkled graphene layer is larger than that of the flat graphene layer, the wrinkled graphene layer can bind to more probes, thereby providing a biosensor with improved sensitivity to target substances.

[0010] Furthermore, since the bonding strength between the linker and the working electrode is greater than the bonding strength between the flat graphene layer and the linker, a biosensor with improved structural stability can be provided.

[0011] FIG. 1 is a drawing for explaining the structure of a biosensor according to exemplary embodiments of the present invention.

[0012] FIG. 2 is a drawing for explaining a working electrode included in a biosensor according to exemplary embodiments of the present invention.

[0013] Figure 3 is an enlarged view of the R1 region of the working electrode of Figure 2.

[0014] FIG. 4A is a drawing for explaining a process for manufacturing a biosensor according to exemplary embodiments of the present invention.

[0015] FIG. 4b is a drawing for explaining a step of forming a working electrode included in a biosensor according to exemplary embodiments of the present invention.

[0016] FIGS. 5A to 5C are graphs illustrating the number of linkers coupled to a working electrode included in a biosensor according to exemplary embodiments of the present invention.

[0017] Figures 6a to 6d are graphs illustrating the results of detecting tDNA using a biosensor according to exemplary embodiments of the present invention.

[0018] 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.

[0019] The biosensor according to embodiments of the present invention is used to analyze biomolecules contained in a biosample, thereby performing gene expression profiling, genotyping, detecting mutations and polymorphisms such as SNPs (Single Nucleotide Polymorphisms), analyzing proteins and peptides, screening for potential drugs, developing and manufacturing new drugs, etc.

[0020] Biosensors employ appropriate probes depending on the target of the 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 biosample 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.

[0021] To measure electrode potential in a solution containing a target substance, two electrodes must be used to measure the potential difference between two points. When measuring the potential difference, the electrode to be measured is called the working electrode, and another electrode is connected to it to measure the potential difference. For example, the electrode system for detecting the target substance may be a two-electrode system including a working electrode and a reference electrode, or a three-electrode system additionally including a counter electrode. In cases where the resistance of the electrolyte is high or the flowing current is large, a three-electrode system may be introduced to minimize errors due to resistance.

[0022] In this specification, the term "probe" refers to a material that is connected to a working electrode and can chemically bond with a target material in a sample, and "linker" refers to a material that plays a role in connecting the probe and the working electrode.

[0023] In this specification, the term "working electrode" refers to an electrode where a reaction of interest occurs in an electrochemical experiment, and may be referred to as a cathode or an anode depending on whether the reaction occurring at the electrode is an oxidation reaction or a reduction reaction, and may also be used interchangeably with "working electrode."

[0024] The term "reference electrode" as used herein refers to an electrode that provides a reference potential, for example, a potential difference, i.e., a voltage, can be established between the reference electrode and the working electrode.

[0025] In this specification, the term "counter electrode" refers to an electrode in an electrochemical circuit that acts as a current source or sink to complete the electrochemical circuit, and may also be used as a substitute for "counter electrode."

[0026] The term "ion" in this specification includes cations and anions, for example, Na + , Ka + , Li + , Ag + Monovalent cations such as Mg 2+ , Zn 2 + Divalent cations such as Cl - , OH - , Br - A monovalent anion such as SO4 2- It contains divalent anions such as .

[0027] FIG. 1 is a drawing for explaining the structure of a biosensor according to exemplary embodiments of the present invention.

[0028] Referring to FIG. 1, the biosensor (100) may include a lower substrate (110), an intermediate substrate (120), and an upper substrate (130) including a working electrode (112), a reference electrode (114), and a counter electrode (116).

[0029] The lower substrate (110) may extend in a first horizontal direction (X) and a second horizontal direction (Y). The first horizontal direction (X) and the second horizontal direction (Y) may intersect each other. A direction perpendicular to the upper surface of the lower substrate (110) may be defined as a vertical direction (Z).

[0030] The lower substrate (110) may include a material capable of causing shrinkage deformation. The lower substrate (110) may particularly include a material capable of causing shrinkage deformation at high temperatures. For example, the lower substrate (110) may include a thermoplastic polymer capable of causing 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 (PS), polyetherimide (PEI), polyethersulfone (PES), polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), and liquid crystal polymer.

[0031] The working electrode (112) may include a graphene layer (GL) having at least one two-dimensional hexagonal carbon structure. However, the graphene layer (GL) may not have a flat structure extending along the upper surface of the lower substrate (110), but may include peaks and valleys having a height difference in a direction perpendicular to the upper surface of the lower substrate (110) (for example, the vertical direction (Z)). A specific description of the structure of the working electrode (112) will be described later in the description of FIG. 2. The degree to which the graphene layer (GL) is wrinkled may vary depending on the temperature and time of the above-described annealing process.

[0032] The sample in contact with the working electrode (112) may include a target substance. The target substance may include a protein, DNA, RNA, or any base sequence, and the sample may include fragments containing the target substance, blood, saliva, or other biological solutions. The sample may be placed in a microchannel (not shown). The microchannel (not shown) may be a container for containing the sample and may be formed of a polymeric or non-polymeric material. As an exemplary embodiment, silicone rubber may be used. The shape and size of the microchannel (not shown) are not limited to those shown, and any container capable of containing a sample may be used as the microchannel (not shown) of the present invention.

[0033] The biosensor (100) may further include a probe (118) capable of chemically binding to a target substance molecule on the working electrode (112). For example, the probe (118) may be one of a polynucleotide, a peptide nucleic acid (PNA) probe, an aptamer (DNA having a specific binding ability to a specific substance), a protein, an antibody, or a capture agent. For example, when the target substance is DNA, the probe (118) may have a nucleotide sequence complementary to the nucleotide sequence of the target substance, DNA, for chemical binding to the target substance.

[0034] The biosensor (100) may further include a linker (119) between the probe (118) and the working electrode (112) to facilitate chemical bonding between the probe (118) and the working electrode (112). As an exemplary embodiment, when the target material is DNA, a PASE (Pyrenebutanoic acid succinimidyl ester) molecule of the following chemical formula 1 may be selected as the linker (119).

[0035]

[0036] The pyrene group within the PASE molecule can bind to the working electrode (112), and the succinimidyl ester group within the PASE molecule can bind to the probe (118). In this case, the probe (118) to which the succinimidyl ester group binds can be DNA having a nucleotide sequence complementary to the nucleotide sequence of the target substance, DNA.

[0037] The reference electrode (114) may include one of gold, silver, aluminum, copper, platinum, tin oxide, or indium tin oxide (ITO). For example, the reference electrode (114) may include an Ag / AgCl electrode (111).

[0038] The counter electrode (116) may be formed of one of gold, silver, aluminum, copper, platinum, tin oxide, or indium tin oxide (ITO).

[0039] The presence of a target substance within a sample can be determined by observing the electrical parameters of the working electrode (112), reference electrode (114), and counter electrode (116). At this time, changes in the electrical parameters may correspond to the presence of the target substance. For example, the electrical parameters may include capacitance, voltage, average current, and the like.

[0040] The intermediate substrate (120) may be placed on the lower substrate (110) so as to electrically insulate the working electrode (112) and the reference electrode (114). The intermediate substrate (120) may be composed of a similar material to the lower substrate (110), but is not limited thereto. The intermediate substrate (120) may include a sample inlet (122). A sample including a target material may be introduced into the lower substrate (110) through the sample inlet (122). The sample inlet (122) may be placed so as to overlap the working electrode (112) and the reference electrode (114) of the lower substrate (110) in the vertical direction (Z). The sample inlet (122) may extend in the direction in which the intermediate substrate (120) extends. The shape of the sample inlet (122) is not limited to that illustrated, and may also be formed in a “T-shape” or an “L-shape.” Additionally, the sample inlet (122) may be configured in multiple numbers, for example, the intermediate substrate (120) may include two or more sample inlets (122).

[0041] The upper substrate (130) may be placed on the upper portion of the intermediate substrate (120). The upper substrate (130) may be composed of a material similar to that of the lower substrate (110), but is not limited thereto. The upper substrate (130) may include an air exhaust hole (132) through which air is exhausted when a sample is introduced, so as to allow the sample to be moved. The air exhaust hole (132) of the upper substrate (130) may be arranged to overlap the sample inlet (122) of the intermediate substrate (120) in the vertical direction (Z). The shape of the air exhaust hole (132) is not limited to that illustrated, and may be formed in various shapes. In addition, the air exhaust hole (132) may be composed of a plurality of air exhaust holes, and for example, the upper substrate (130) may include two or more air exhaust holes (132).

[0042] FIG. 2 is a drawing for explaining a working electrode included in a biosensor according to exemplary embodiments of the present invention.

[0043] Referring to FIG. 2, the working electrode (112) may include at least one graphene layer (GL), and the graphene layer (GL) may include a peak portion (P) and a valley portion (V) having a height difference in the vertical direction (Z). The shape in which the graphene layer (GL) is wrinkled may vary. As exemplary embodiments, referring to FIG. 2 (a), the peak portions (P) and the valley portions (V) of the graphene layer (GL) may be alternately arranged in a zigzag shape along the first horizontal direction (X). As another embodiment, referring to FIG. 2 (b), the peak portions (P) and the valley portions (V) of the graphene layer (GL) may be alternately arranged in a zigzag shape in a diagonal direction between the first horizontal direction (X) and the second horizontal direction (Y). The shape in which the graphene layer (GL) is wrinkled is not limited to that illustrated, and the graphene layer (GL) may be wrinkled in a shape other than that illustrated.

[0044] The spacing between the peaks (P) and the valleys (V) in the graphene layer (GL) may vary. As exemplary embodiments, the peaks (P) may be arranged to be spaced apart at a first constant interval (W1) in a range of about 1 nanometer to 10 nanometers along the first horizontal direction (X). The valleys (V) may be arranged to be spaced apart at a second constant interval (W2) in a range of about 1 nanometer to 10 nanometers along the first horizontal direction (X). As another embodiment, the peaks (P) and the valleys (V) may be arranged to be spaced apart at non-constant intervals along the first horizontal direction (X). As the degree of wrinkles in the graphene layer (GL) increases, the spacing between the peaks (P) and the valleys (V) along the first horizontal direction (X) may decrease.

[0045] As exemplary embodiments, the height difference in the vertical direction (Z) between adjacent peaks (P) and valleys (V) of the graphene layer (GL) may be constant within a range of about 0.2 nanometers to 20 nanometers. In another embodiment, the height difference in the vertical direction (Z) between adjacent peaks (P) and valleys (V) of the graphene layer (GL) may not be constant.

[0046] A valley portion (V) may be arranged between adjacent peak portions (P) of the graphene layer (GL), 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 graphene layer (GL) are not limited to those illustrated, and may be formed in various shapes. As exemplary embodiments, 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 graphene layer (GL), 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 graphene layer (GL), 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).

[0047] The graphene layer (GL) may have a thickness of about 1 nanometer to 2 nanometers and a surface area of ​​about 1 square millimeter to 100 square millimeters.

[0048] The number of graphene layers (GL) constituting the working electrode (112) is not limited to that illustrated. The working electrode (112) may be composed of a plurality of graphene layers (GL). For example, the working electrode (112) may be composed of a first graphene layer and a second graphene layer disposed on the first graphene layer. The first graphene layer and the second graphene layer may have the same or different shapes. For example, the first graphene layer may have a smaller separation distance between adjacent peak portions (P) than the second graphene layer.

[0049] In addition, the plurality of carbon (C) atoms included in the plurality of graphene layers (GL) constituting the working electrode (112) may be arranged in a row in the vertical direction (Z) or may be arranged in an offset manner. As exemplary embodiments, the carbon (C) atoms constituting the first graphene layer and the carbon (C) atoms constituting the second graphene layer may be arranged in a row in the vertical direction (Z). As other exemplary embodiments, the carbon (C) atoms constituting the first graphene layer and the carbon (C) atoms constituting the second graphene layer may be arranged in an offset manner in the vertical direction (Z).

[0050] The presence or absence of a target substance or its concentration in the reaction solution can be determined by observing the electrical parameters of the working electrode (112).

[0051] When detecting a target substance using a biosensor (100) according to the technical concept of the present invention, sensitivity to the target substance can be improved compared to using a working electrode composed of a flat graphene layer. Accordingly, a biosensor (100) with improved sensitivity to the target substance can be provided.

[0052] Figure 3 is an enlarged view of the R1 region of the working electrode of Figure 2.

[0053] Referring to FIG. 3, an intermediate region (COL) between a peak portion (P) and a valley portion (V) of a graphene layer (GL) is illustrated. A distance (d1) in the vertical direction (Z) from a line (A-A') crossing the intermediate region (COL) of the graphene layer (GL) to the peak portion (P) may be about 0.54 nanometers. A distance (d2) in the vertical direction (Z) from a line (A-A') crossing the intermediate region (COL) of the graphene layer (GL) to the valley portion (V) may be about 0.54 nanometers.

[0054]

[0055] FIG. 4A is a diagram illustrating a process for manufacturing a biosensor according to exemplary embodiments of the present invention. In particular, FIG. 4A is a diagram illustrating a process for manufacturing a biosensor using a substrate comprising a thermoplastic polymer according to exemplary embodiments of the present invention.

[0056] Referring to FIG. 4a, the manufacturing process of the biosensor (100) may include a step (S110) of preparing a lower substrate (110) including a thermoplastic polymer and extending in a first horizontal direction (X) and a second horizontal direction (Y) intersecting the first horizontal direction (X), a step (S120) of forming a working electrode (112) including at least one graphene layer (GL) on the lower substrate (110), a step (S130) of connecting a linker (119) to the at least one graphene layer (GL) and connecting a probe (118) capable of chemically bonding to a target substance to be detected to a molecule of the linker (119), and a step (S140) of bonding an intermediate substrate (120) and an upper substrate (130) to the lower substrate (110).

[0057] The step (S120) of forming a working electrode including at least one graphene layer may include the step (S121) of forming a preliminary graphene layer on the lower substrate; and the step (S123) of annealing the lower substrate to cause the preliminary graphene layer to undergo shrinkage deformation.

[0058]

[0059] FIG. 4b is a diagram illustrating a step (S120) of forming a working electrode included in a biosensor according to exemplary embodiments of the present invention. The present invention is not limited to what is illustrated, and the working electrode can be formed through various processes.

[0060] Referring to FIG. 4b, a preliminary graphene layer (PGL) having a flat structure can be formed on a sacrificial substrate (110'). The preliminary graphene layer (PGL) can be formed by depositing carbon (C) atoms on the sacrificial substrate (110') using a chemical vapor deposition (CVD) process. The sacrificial substrate (110') can include a metal such as copper (Cu), nickel (Ni), or platinum (Pt). When the sacrificial substrate (110') includes copper (Cu), it may be preferable for forming a single-layer graphene layer. The preliminary graphene layer (PGL) can be formed by injecting a raw material gas (e.g., methane (CH4)) containing carbon (C) onto the sacrificial substrate (110').

[0061] Next, a protective film (PL) can be formed on the preliminary graphene layer (PGL). As exemplary embodiments, the protective film (PL) can include a polymer such as an acrylic resin (PMMA; Polymethyl methacrylate). As exemplary embodiments, the protective film (PL) can be formed by a spin coating method. Specifically, a small amount of liquid for forming the protective film (PL) can be dropped on the preliminary graphene layer (PGL), and then the sacrificial substrate (110') can be rotated. The rotation of the sacrificial substrate (110') causes the small amount of liquid to spread to the edge of the preliminary graphene layer (PGL), thereby forming a protective film (PL) that coats the preliminary graphene layer (PGL).

[0062] Next, the sacrificial substrate (110') under the preliminary graphene layer (PGL) can be removed. As exemplary embodiments, the sacrificial substrate (110') can be removed using a chemical etching method. The etchant of the chemical etching method can include a material that can dissolve metal. As exemplary embodiments, the etchant can include sodium persulfate (Na2S2O8). Thereafter, a lower substrate (110) can be formed under the preliminary graphene layer (PGL) using a direct transfer method. A plasma treatment can be performed on the lower substrate (110) to bond the lower substrate (110) and the preliminary graphene layer (PGL) on which the protective film (PL) is formed. As exemplary embodiments, oxygen (O) can be included as a reaction gas during the plasma treatment. After bonding the preliminary graphene layer (PGL) to the lower substrate (110), the protective film (PL) on the preliminary graphene layer (PGL) can be removed. As exemplary embodiments, when the protective film (PL) includes an acrylic resin (PMMA), the protective film (PL) can be removed using acetic acid (CH3COOH) and DI water.

[0063] Next, the lower substrate (110) to which the preliminary graphene layer (PGL) from which the protective film (PL) has been removed is bonded can be annealed. For example, when the lower substrate (110) including polystyrene (PS) is annealed at about 110°C to 115°C for about 10 to 120 minutes, wrinkles in the range of several micrometers to several hundred nanometers can be formed in the graphene layer (GL). At this time, the length in the direction in which the graphene layer (GL) extends can be formed to be 30% to 90% of the length in the direction in which the preliminary graphene layer (PGL) of the flat structure extends. In addition, the degree of shrinkage deformation of the lower substrate (110) can vary depending on the annealing time. The degree of shrinkage deformation of the lower substrate (110) can increase as the annealing time increases. Through shrinkage deformation of the lower substrate (110), a working electrode (112) including a wrinkled graphene layer (GL) can be formed.

[0064] The degree to which the graphene layer (GL) is wrinkled may vary depending on the time or temperature of the annealing process. As exemplary embodiments, when the length of the preliminary graphene layer (PGL) in the first horizontal direction (X) is defined as the first length and the length of the graphene layer (GL) in the first horizontal direction (X) is defined as the second length, when the lower substrate (110) to which the preliminary graphene layer (PGL) is bonded is annealed at about 120° C. for about 60 minutes, the second length may be 1 / 4 of the first length.

[0065] The greater the degree of wrinkles in the graphene layer (GL), the longer the time required for the process of bonding the graphene layer (GL) with the linker (119) molecule. Therefore, the degree of wrinkles in the graphene layer (GL) is preferably about 55% when considering yield and sensitivity. In other words, it is preferable that the length of the graphene layer (GL) in the extending direction is about 45% of the length of the preliminary graphene layer (PGL) in the extending direction.

[0066]

[0067] Hereinafter, a specific experimental example will be described for a step (S130) of connecting a linker (119) to at least one graphene layer (GL) and connecting a probe (118) that is chemically bondable to a target substance to be detected to the linker (119) molecule.

[0068] A method for binding a PASE molecule to a working electrode as a linker (119) is specifically described 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 working electrode so that the working electrode and the prepared PASE solution were in contact with each other. The working electrode treated with the PASE solution was washed three times with a DMSO solution and three times with ethanol. The working electrode that had undergone the washing process was dried for about an hour or more. The working electrode that had undergone the drying process was washed using DI water. As a result, a working electrode to which a PASE molecule was bound could be obtained.

[0069] A method for binding DNA to a working electrode as a probe (118) will be described in detail with reference to an experimental example of the present invention. In the present specification, DNA bound as a probe (118) is referred to as probe DNA. Referring to an experimental example of the present invention, a working electrode that had been treated with a PASE solution was incubated in 1X PBS (1X Phosphate-Buffered Saline) for more than one day. A diluted solution containing probe DNA was dropped on the incubated working electrode, and after about 3 hours, it was washed using a 1X PBS solution. The probe DNA was diluted to a concentration of about 264 μM. The washed working electrode was dried at about 25°C for about 1 hour or more. As a result, a working electrode to which probe DNA was bound via PASE molecules can be obtained.

[0070]

[0071] Next, we describe two experimental examples of a method for detecting a target substance through a working electrode bound to a probe DNA via a PASE molecule.

[0072] Experimental Example 1

[0073] The working electrode, which had been treated with the PASE solution, 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 working electrode, and then left to stand for about 20 minutes. After the waiting period, the electrode was washed with 1XPBS, and then 5 mM methylene blue and 5 mM [Fe(CN)6] -4The electrode was brought into contact with the sample containing the target material. 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 in the tDNA solution, gradually increasing the concentration of tDNA.

[0074]

[0075] Experimental Example 2

[0076] The working electrode, which had been treated with PASE solution, was incubated in 1XPBS, washed with 1XPBS, and then 50 μL of 100 μM MB-pDNA-NH2 was applied onto the working electrode and incubated for more than 3 hours. After incubation with 50 μL of 100 μM MB-pDNA-NH2, the electrode was washed three or more times with 1XPBS. The subsequent process is similar to Experimental Example 1.

[0077] However, there is a difference in that Experimental Example 1 incubates the working electrode incubated in 1XPBS with pDNA-NH2, and Experimental Example 2 incubates it with MB-pDNA-NH2 containing methylene blue.

[0078] FIGS. 5A to 5C are graphs illustrating the number of linkers coupled to a working electrode included in a biosensor according to exemplary embodiments of the present invention.

[0079] Figures 5a to 5c are CV graphs that observe current density using a biosensor manufactured through the experimental example of the present invention described above. Figure 5c is a graph that records the anodic current peak (Ipa) of Figures 5a and 5b. Line (a) of Figure 5a shows the change in current density when voltage is applied to a flat graphene layer, and line (b) shows the change in current density when voltage is applied to a flat graphene layer that has undergone PASE treatment. Line (a) of Figure 5b shows the change in current density when voltage is applied to a wrinkled graphene layer, and line (b) shows the change in current density when voltage is applied to a wrinkled graphene layer that has undergone PASE treatment.

[0080] Referring to Fig. 5c, the cathode current peak is -0.907 Acm when voltage is applied to the wrinkled graphene layer. -2 When a voltage of -8.185 Acm- was applied to the wrinkled graphene layer after PASE processing, 2 , while when voltage was applied to the flat graphene layer, it was -2.671 Acm -2 When a voltage was applied to the flat graphene layer after PASE processing, -5.503 Acm -2 The increase in the cathode current peak can be attributed to the PASE electron-mediated transport reaction, which confirms that the PASE electron-mediated transport reaction occurs more actively on the wrinkled graphene layer.

[0081] The reason why the change in the cathode current peak from the cathode current peak of the wrinkled graphene layer to the PASE-treated wrinkled graphene layer is greater than the change in the cathode current peak from the cathode current peak of the flat graphene layer to the PASE-treated flat graphene layer is that the wrinkled graphene layer has a larger surface area than the flat graphene layer, so that more PASE molecules can be adsorbed on the surface of the graphene layer. Since more PASE molecules are adsorbed on the surface of the wrinkled graphene layer, more electron transfer is induced, and the change in the cathode current peak can be greater than that of the flat graphene layer. Therefore, when the working electrode according to the present invention is used, since the working electrode is coupled to a large number of linkers, a biosensor with improved sensitivity to a target substance can be provided.

[0082] Figures 6a to 6d are graphs illustrating the results of detecting tDNA using a biosensor according to exemplary embodiments of the present invention.

[0083] Figures 6a and 6b are data comparing the results of detecting tDNA inside a bulk solution (e.g., electrolyte) containing methylene blue using a wrinkled graphene layer and a flat graphene layer.

[0084] Figure 6a shows the average current density (I) according to the molar concentration of tDNA in the flat graphene channel and the deformed graphene channel for tDNA detection. ave, Acm -2) spectrum, and Fig. 6b is an enlarged graph of the second region (R2) illustrated in Fig. 6a. In general, when a target material is combined with a probe, the charge distribution on the surface of the working electrode can change along with the electrostatic surface potential induced by the gate voltage and the change in the current flowing between the source and drain. Therefore, the presence of the target material can be detected through the change in the charge distribution on the surface of the working electrode.

[0085] Referring to FIGS. 6A and 6B, it can be confirmed that the average current density spectrum according to the molar concentration of tDNA in the wrinkled graphene layer is well separated from the average current density spectrum according to the molar concentration of tDNA in the flat graphene layer. For example, it can be seen that the degree to which the average current density graph for 0 aM and the average current density graph for 2 aM in the wrinkled graphene layer are separated is greater than the degree to which the average current density graph for 0 aM and the average current density graph for 2 aM in the flat graphene layer are separated. The fact that the average current density spectra according to the molar concentration of each tDNA are well separated means that the current according to the change in the concentration of tDNA can be detected delicately. Therefore, the sensitivity to a target substance can be improved by using a biosensor using a working electrode according to the present invention.

[0086] Figures 6c and 6d are graphs showing the change in current density of a flat graphene layer and the current density (Current Density, μAcm) of a wrinkled graphene layer, respectively, when the concentration of tDNA is changed from 0 aM to 2 μM. -2) is a graph for the change in tDNA concentration. Similar to the data in FIGS. 6a and 6b, it can be confirmed that the current density spectrum according to the molar concentration of tDNA in the wrinkled graphene layer is well separated from the current density spectrum according to the molar concentration of tDNA in the flat graphene layer. Therefore, by using a biosensor using a working electrode according to the present invention, the sensitivity to a target substance can be further improved.

[0087] 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 lower substrate extending in a first horizontal direction and a second horizontal direction intersecting the first horizontal direction; A working electrode disposed on the lower substrate; A probe disposed on the working electrode and capable of chemically bonding with a target substance to be detected; a linker connecting the probe and the working electrode; and On the lower substrate, a reference electrode is disposed spaced apart from the working electrode, The working electrode comprises at least one graphene layer, A biosensor characterized in that the at least one graphene layer includes peaks and valleys having a height difference in a vertical direction on the upper surface of the lower substrate.

2. In paragraph 1, A biosensor characterized in that the peak portion and the valley portion are arranged alternately spaced apart in the first horizontal direction.

3. In paragraph 2, A biosensor characterized in that the peak portion and the valley portion extend in a line shape along the second horizontal direction.

4. In paragraph 1, A biosensor characterized in that the peak portion and the valley portion are arranged alternately spaced apart in the diagonal direction of the first horizontal direction and the second horizontal direction.

5. In paragraph 1, A biosensor characterized in that the peak portion and the valley portion are arranged alternately at regular intervals in the range of 1 nanometer to 10 nanometers along the first horizontal direction.

6. In paragraph 1, 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.

7. In paragraph 1, The above probe is a DNA containing a nucleotide sequence that can complementarily bind to the nucleotide sequence of the target material, A biosensor characterized in that the linker comprises a PASE (Pyrenebutanoic acid succinimidyl ester) molecule.

8. In paragraph 1, An intermediate substrate disposed on the working electrode and the reference electrode; A sample inlet penetrating the intermediate substrate and vertically overlapping an end of the working electrode and an end of the reference electrode; an upper substrate disposed on the intermediate substrate; and A biosensor characterized in that it further includes an air exhaust hole that overlaps the sample inlet in the vertical direction on the upper substrate.

9. A step of preparing a lower substrate extending in a first horizontal direction and a second horizontal direction intersecting the first horizontal direction; forming a working electrode including at least one graphene layer on the lower substrate; and comprising the step of connecting a linker to at least one graphene layer and connecting a probe chemically bondable to a target substance to be detected to the linker; The step of forming the working electrode including at least one graphene layer comprises: a step of forming a preliminary graphene layer on the lower substrate; and A method for manufacturing a biosensor, characterized in that it comprises a step of annealing the lower substrate so that the preliminary graphene layer undergoes shrinkage deformation.

10. In paragraph 9, In the step of forming the working electrode including at least one graphene layer, A method for manufacturing a biosensor, characterized in that the length in the direction in which at least one graphene layer extends is formed to be 45% of the length in the direction in which the preliminary graphene layer extends.

11. In paragraph 9, In the step of forming the working electrode including at least one graphene layer, the preliminary graphene layer is heated within 110°C to 120°C for 10 to 120 minutes, A method for manufacturing a biosensor, characterized in that the length in the direction in which at least one graphene layer extends is formed to be 30% to 90% of the length in the direction in which the preliminary graphene layer extends.

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