Field effect transistor for biosensor for selectively detecting endonucleases, biosensor comprising same, and method for manufacturing same

Abstract

The present invention relates to a field effect transistor for a biosensor for selectively detecting endonucleases, a biosensor comprising same, and a method for manufacturing same. The field effect transistor for a biosensor can successfully detect the activity of EcoRV, which is one of endonucleases, and has potential applicability for new drug development and disease treatment through EcoRV inhibitor screening.

Description

Field effect transistor for a biosensor for selectively detecting endonuclease, a biosensor including the same, and a method for manufacturing the same

[0001] The present invention relates to a field effect transistor for a biosensor for selectively detecting an endonuclease, a biosensor including the same, and a method for manufacturing the same, and more particularly, to a field effect transistor for a biosensor for selectively detecting an endonuclease that can successfully detect the activity of EcoRV, which is one of the endonucleases, and has potential application in new drug development and disease treatment through screening for EcoRV inhibitors, a biosensor including the same, and a method for manufacturing the same.

[0002]

[0003] Eco321 (EcoRV) is one of the type-II restriction endonucleases that catalyzes the hydrolysis of internal phosphodiester bonds in DNA or RNA. The DNA cleavage process through restriction enzymes is widely used in genetic techniques such as single nucleotide polymorphism, restriction fragment length polymorphism, and post-modification analysis.

[0004] Additionally, these type-II restriction endonucleases play a key role in genetic information transfer and gene expression, including replication, DNA repair, and nucleic acid recombination, and their overexpression results in excessive activation of DNA repair in DNA-damaged cells, which may be used in bacterial drug resistance mechanisms and viral replication processes.

[0005] Therefore, detection of restriction endonucleases is important not only for gene cloning but also for antiviral drug development.

[0006] Meanwhile, nanocarbon-based biosensors are being developed and used as diagnostic tools because they are highly sensitive, do not require labels, and can rapidly detect chemical and biological substances.

[0007] In particular, single-walled carbon nanotubes (swCNTs) are promising candidates as channel materials for field-effect transistor (FET) biosensors due to their excellent environmentally sensitive electronic properties.

[0008] However, single-walled carbon nanotube-based field-effect transistor (swCNT-FET) biosensors have a problem of lack of selectivity due to false positive responses to interference.

[0009]

[0010] The present invention has been made to solve the above problems, and the purpose of the present invention is to provide a field effect transistor for a biosensor for selectively detecting an endonuclease, which can confirm the reaction of DNA cleavage by the enzyme as an electrical signal by fixing DNA tagged with methylene blue (MB) as a probe to a field effect transistor biosensor and adding EcoRV, an endonuclease enzyme, thereto, and a biosensor including the same and a method for manufacturing the same.

[0011]

[0012] In order to solve the above-described problem, the field effect transistor for a biosensor for selectively detecting an endonuclease of the present invention may include a substrate, a source electrode and a drain electrode formed to be spaced apart from each other on one surface of the substrate, and a channel region formed between the source electrode and the drain electrode and in electrical contact therebetween.

[0013] In addition, the field effect transistor for a biosensor for selectively detecting the endonuclease of the present invention may include a substrate, a first self-assembled monolayer and a second self-assembled monolayer formed spaced apart from each other on one surface of the substrate, a source electrode formed on one surface of the first self-assembled monolayer, a drain electrode formed on one surface of the second self-assembled monolayer, and a channel region formed between the source electrode and the drain electrode, and in electrical contact with them.

[0014] In a preferred embodiment of the present invention, the channel region may include a single-walled carbon nanotube layer having a DNA substrate for endonuclease detection immobilized on the surface.

[0015] In a preferred embodiment of the present invention, the DNA substrate for endonuclease detection may be a DNA substrate tagged with methylene blue.

[0016] In a preferred embodiment of the present invention, the methylene blue-tagged DNA substrate may have a base sequence of 5' MB-AGTATGATATCCA-3'.

[0017] In a preferred embodiment of the present invention, the endonuclease may be an EcoRV enzyme.

[0018] In a preferred embodiment of the present invention, the average distance between the DNA substrate for endonuclease detection and the single-walled carbon nanotube layer may be 0.15 to 0.5 nm.

[0019] In a preferred embodiment of the present invention, a field effect transistor for a biosensor for selectively detecting the endonuclease of the present invention has a size of 5.8Х10 -4 ~ 9.8Х10 -4 Detection may be possible at EcoRV concentrations of U / mL.

[0020] Meanwhile, the biosensor for selectively detecting the endonuclease of the present invention may include a field effect transistor for a biosensor for selectively detecting the endonuclease of the present invention.

[0021] Furthermore, the method for manufacturing a field effect transistor for a biosensor for selectively detecting an endonuclease of the present invention may include a first step of preparing a field effect transistor including a duplex DNA substrate for EcoRV detection and a single-walled carbon nanotube layer, a second step of drop-casting the duplex DNA substrate for EcoRV detection onto the single-walled carbon nanotube layer of the field effect transistor to fix the duplex DNA substrate for EcoRV detection onto the surface of the single-walled carbon nanotube layer of the field effect transistor, and a third step of manufacturing a field effect transistor for a biosensor by coating a blocking agent onto the surface of the single-walled carbon nanotube layer on which the duplex DNA substrate for EcoRV detection is fixed.

[0022] In a preferred embodiment of the present invention, a duplex DNA substrate for EcoRV detection can be prepared by including a step 1-1 of preparing a DNA solution containing upper strand DNA and an MB-tagged DNA solution containing DNA tagged with methylene blue (MB), a step 1-2 of preparing a mixture by mixing the DNA solution and the MB-tagged DNA solution, and a step 1-3 of heating the mixture to anneal it and then leaving it to stand, thereby preparing a duplex DNA substrate for EcoRV detection.

[0023]

[0024] The field effect transistor for a biosensor for selectively detecting an endonuclease of the present invention, the biosensor including the same, and the method for manufacturing the same enable more sensitive and accurate genetic analysis, thereby enabling a better understanding of variation and diversity of individual genomes and being utilized to study genetic factors related to diseases.

[0025] In addition, the field effect transistor for a biosensor for selectively detecting the endonuclease of the present invention, the biosensor including the same, and the method for manufacturing the same can be utilized for disease diagnosis and prevention by accurately measuring the activity of EcoRV endonuclease, and can particularly contribute to the early diagnosis and risk factor assessment of diseases related to DNA damage.

[0026]

[0027] FIG. 1 is a cross-sectional view of a field effect transistor for a biosensor for selectively detecting the endonuclease of the present invention, according to a preferred embodiment of the present invention.

[0028] FIG. 2 is a cross-sectional view of a field effect transistor for a biosensor for selectively detecting the endonuclease of the present invention, according to another preferred embodiment of the present invention.

[0029] FIG. 3(A) is an AFM image for a field effect transistor including a MB-tagged DNA substrate manufactured in Example 1, FIG. 3(B) is an AFM image for a field effect transistor including a DNA substrate in which MB is not tagged in Comparative Example 1, FIG. 3(C) is a graph of height profiles before and after immobilization of a duplex DNA substrate for EcoRV detection in Example 1, FIG. 3(D) is a graph of height profiles before and after immobilization of a duplex DNA substrate for EcoRV detection in Comparative Example 1, FIG. 3(E) is a graph of the position at the center of mass z in Example 1, and FIG. 3(F) is a graph of the position at the center of mass z in Comparative Example 1.

[0030] Fig. 4(A) is a graph showing the electrical response of a field effect transistor (=MB-tagged DNA / swCNT-FET) including the MB-tagged DNA substrate manufactured in Example 1 when EcoRV was continuously added, and Fig. 4(B) is a graph showing the calibration curve of a field effect transistor including the MB-tagged DNA substrate manufactured in Example 1 as a function of the EcoRV concentration.

[0031] Figure 5(A) is a graph measuring real-time conductivity after injecting 50 U / mL of EcoRV when ATA (aurintricarboxylic acid), a representative inhibitor of EcoRV, was added at various concentrations.

[0032] Figure 5(B) is a graph showing the inhibitory effect of various concentrations of ATA on the reaction of a field effect transistor including the MB-tagged DNA substrate manufactured in Example 1.

[0033] Figure 6(A) is a graph showing the MD simulation results of the MB-tagged DNA substrate of the field effect transistor including the MB-tagged DNA substrate manufactured in Example 1 interacting with the single-walled carbon nanotube layer and the non-MB-tagged DNA substrate of the field effect transistor including the non-MB-tagged DNA substrate of Comparative Example 1.

[0034] Figure 6(B) is a graph calculating the average minimum distance between each nucleic acid in the double-stranded portion and the single-walled carbon nanotube layer.

[0035] FIG. 6(C) is a graph showing the average electrostatic potential generated by the double-stranded portion of the MB-free DNA substrate in the single-walled carbon nanotube layer of the field-effect transistor including the MB-untagged DNA substrate in Comparative Example 1 using Poisson-Boltzmann electrostatic potential simulation, and the graph showing the average electrostatic potential generated by the double-stranded portion of the MB-tagged DNA substrate in the single-walled carbon nanotube layer of the field-effect transistor including the MB-tagged DNA substrate manufactured in Example 1 on the right.

[0036] FIG. 6(D) is a graph showing the global electrostatic potential generated by the MB-free DNA substrate in the single-walled carbon nanotube layer of the field-effect transistor including the MB-free DNA substrate in Comparative Example 1 and the global electrostatic potential generated by the MB-tagged DNA substrate (MB-tagged DNA) in the single-walled carbon nanotube layer of the field-effect transistor including the MB-tagged DNA substrate manufactured in Example 1, using Poisson-Boltzmann electrostatic potential simulation.

[0037] Figure 7 is a graph showing the conductivity measured in a field effect transistor containing the MB-tagged DNA substrate prepared in Example 1 after adding HaeIII, EcoRI, PvuII, and BamHI as interference substances in addition to the EcoRV enzyme.

[0038] Figure 8 is a schematic diagram showing the detection principle of a field effect transistor including an MB-tagged DNA substrate (=MB-tagged DNA) manufactured in Example 1 and an MB-free DNA substrate (=MB-free DNA) manufactured in Comparative Example 1 for measuring EcoRV endonuclease activity.

[0039]

[0040] Hereinafter, with reference to the attached drawings, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. The present invention may be implemented in various different forms and is not limited to the embodiments described herein. In the drawings, parts irrelevant to the description have been omitted for clarity of description, and the same reference numerals are assigned to identical or similar components throughout the specification.

[0041]

[0042] Referring to FIG. 1, a field effect transistor for a biosensor for selectively detecting an endonuclease of the present invention may include a substrate (10), a source electrode (21) and a drain electrode (22) formed spaced apart from each other on one surface of the substrate (10). In addition, the field effect transistor may include a channel region (30) formed between the source electrode (21) and the drain electrode (22) and in electrical contact therewith.

[0043] In addition, referring to FIG. 2, a field effect transistor for a biosensor for selectively detecting an endonuclease of the present invention may include a substrate (10), a first self-assembled monolayer (23) and a second self-assembled monolayer (24) formed to be spaced apart from each other on one surface of the substrate (10). In addition, it may include a source electrode (21) formed on one surface of the first self-assembled monolayer (23) and a drain electrode (22) formed on one surface of the second self-assembled monolayer (24). In addition, it may include a channel region (30) formed between the source electrode (21) and the drain electrode (22) and in electrical contact therewith.

[0044] The substrate (10) can be any material commonly used in the field, and for example, one or more materials selected from the group consisting of silicon, silicon dioxide, glass, quartz, metal, plastic, and oxide can be used.

[0045] The source electrode (21) and the drain electrode (22) can be any metal commonly used in the field, and for example, can be formed of one or more metals selected from the group consisting of platinum, gold, chromium, copper, aluminum, nickel, palladium, and titanium.

[0046] The first self-assembled monolayer (23) and the second self-assembled monolayer (24) can be formed by patterning self-assembled monolayers (SAMs) composed of one or more molecules selected from the group consisting of hydrophobic molecules, particularly octadecyltrichlorosilane (OTS), octadecyltrimethoxysilane (OTMS), octadecyltriethoxysilane (OTE), and octadecanethiols (ODT), on one surface of the substrate (10). In addition, the first self-assembled monolayer (23) and the second self-assembled monolayer (24) each refer to a regularly aligned organic molecule film generated on the substrate surface, and preferably have either a carboxyl group (-COOH) and / or a hydroxyl group (-OH). Preferably, the material for forming each of the first self-assembled monolayer (23) and the second self-assembled monolayer (24) can be OTS having a nonpolar terminal group. In a preferred embodiment of the present invention, the central portion of the substrate may be formed by patterning so as to be exposed in an approximately rectangular shape.

[0047]

[0048] Meanwhile, the channel region (30) may include a single-walled carbon nanotube layer having a DNA substrate for endonuclease detection fixed to the surface. At this time, the single-walled carbon nanotube layer may be composed of carbon nanotubes having a diameter of 5 nm or less, preferably 3 to 4.5 nm.

[0049] Specifically, the DNA substrate for endonuclease detection may be a methylene blue-tagged DNA substrate. Furthermore, the methylene blue-tagged DNA substrate may have the base sequence 5' MB-AGTATGATATCCA-3'. Furthermore, the endonuclease may be an EcoRV enzyme.

[0050] Furthermore, the average distance between the DNA substrate for endonuclease detection and the single-walled carbon nanotube layer may be 0.15 to 0.5 nm, preferably 0.2 to 0.3 nm.

[0051] In addition, the field effect transistor for a biosensor for selectively detecting the endonuclease of the present invention is 5.8 Х 10 -4 ~ 9.8 Х 10 -4 U / mL, preferably 6.8 Х 10 -4 ~ 8.8 Х 10 -4 Detection may be possible at EcoRV concentrations of U / mL.

[0052]

[0053] Meanwhile, the surface of the single-walled carbon nanotube layer immobilized with the DNA substrate for endonuclease detection may be coated with a blocking agent to prevent non-specific binding. In this case, bovine serum albumin (BSA) may be used as the blocking agent, but is not limited thereto.

[0054]

[0055] Furthermore, the biosensor for selectively detecting the endonuclease of the present invention may include the field effect transistor for a biosensor for selectively detecting the endonuclease of the present invention described above.

[0056]

[0057] Meanwhile, the method for manufacturing a field effect transistor for a biosensor for selectively detecting the endonuclease of the present invention may include steps 1 to 3.

[0058] First, the first step of the method for manufacturing a field effect transistor for a biosensor for selectively detecting the endonuclease of the present invention can prepare a field effect transistor including a duplex DNA substrate for detecting EcoRV and a single-walled carbon nanotube layer, respectively.

[0059] At this time, a duplex DNA substrate for EcoRV detection can be prepared including steps 1-1 to 1-3.

[0060] Step 1-1 of the method for preparing a duplex DNA substrate for EcoRV detection can prepare a DNA solution containing upper strand DNA and an MB-tagged DNA solution containing DNA tagged with methylene blue (MB).

[0061] Specifically, the DNA solution may include upper strand DNA and Tris-HCl buffered saline (pH 7.0 to 8.0), and the MB-tagged DNA solution may include methylene blue (MB)-tagged DNA and Tris-HCl buffered saline (pH 7.0 to 8.0). At this time, the upper strand DNA may have a base sequence of 5'-CTA GCT ATG TGC CGA ATT TCA AGG ACA GTT GTA TGG ATA TCA TAC T-3', and the methylene blue-tagged DNA may have a base sequence of 5' MB-AGTATGATATCCA-3'. In addition, the prepared DNA solution and the prepared MB-tagged DNA solution may be stirred and heated at a temperature of 85 to 105°C and a speed of 300 to 400 rpm for 3 to 7 minutes, respectively.

[0062] Steps 1 and 2 of the method for preparing a duplex DNA substrate for EcoRV detection can prepare a mixture by mixing a DNA solution and an MB-tagged DNA solution.

[0063] Steps 1-3 of the method for preparing a duplex DNA substrate for EcoRV detection can prepare a duplex DNA substrate for EcoRV detection by heating and annealing the mixture prepared in Steps 1-2, and then allowing it to stand. At this time, heating can be performed at a temperature of 85 to 105°C for 3 to 7 minutes. Additionally, allowing it to stand can be performed at a temperature of 15 to 25°C for 45 to 75 minutes.

[0064] A field effect transistor including a single-walled carbon nanotube layer comprises the steps of: introducing single-walled carbon nanotubes into 1,2-dichlorobenzene and ultrasonicating the mixture for 15 to 25 minutes to prepare a single-walled carbon nanotube suspension having a concentration of 0.05 to 0.15 mg / mL; preparing a substrate, patterning one surface of the substrate prepared with octadecyltrichlorosilane (OTS) having a non-polar terminal group through a photolithography process, and then immersing the substrate in an OTS solution (OTS: hexane = 1: 450 to 550 v / v) at 20 to 25°C for 5 to 10 minutes to form a patterned self-assembled monolayer (SAM) on one surface of the substrate, thereby preparing a substrate having a self-assembled monolayer formed thereon; A method for manufacturing a self-assembled monolayer may include: a step of immersing a substrate on which a self-assembled monolayer is formed in a single-walled carbon nanotube suspension for 5 to 15 minutes, and then washing the substrate to form a single-walled carbon nanotube layer in a channel region where a self-assembled monolayer is not formed on one surface of the substrate; a step of forming source electrodes and drain electrodes spaced apart from each other (separation distance: 3 to 5 μm) on one surface of the self-assembled monolayer using a thermal evaporation process and a lift-off process;

[0065]

[0066] Next, the second step of the method for manufacturing a field effect transistor for a biosensor for selectively detecting an endonuclease of the present invention is to drop cast the duplex DNA substrate for EcoRV detection onto a single-walled carbon nanotube layer of the field effect transistor, thereby fixing the duplex DNA substrate for EcoRV detection onto the surface of the single-walled carbon nanotube layer of the field effect transistor. At this time, the drop casting can be performed at 18 to 25°C for 2 to 4 hours.

[0067] In addition, after drop casting, a washing and drying process can be performed to remove the duplex DNA substrate for EcoRV detection that is not fixed on the surface of the single-walled carbon nanotube layer. Washing can be performed using Tris-HCl buffered saline, and drying can be performed using nitrogen (N2) gas, but is not limited thereto.

[0068]

[0069] Finally, the third step of the method for manufacturing a field-effect transistor for a biosensor for selectively detecting an endonuclease of the present invention is to coat a blocking agent on the surface of a single-walled carbon nanotube layer on which a duplex DNA substrate for EcoRV detection is fixed, thereby manufacturing a field-effect transistor for a biosensor. Specifically, a BSA solution containing 0.3 to 0.7 wt% of a blocking agent, bovine serum albumin (BSA), and Tris-HCl buffered saline is drop-casted onto the surface of a single-walled carbon nanotube layer on which a duplex DNA substrate for EcoRV detection is fixed, and then washed with Tris-HCl buffered saline, followed by drying using nitrogen (N2) gas, thereby manufacturing a field-effect transistor including an MB-tagged DNA substrate.

[0070]

[0071] Hereinafter, the present invention will be described in more detail through examples, but the following examples do not limit the scope of the present invention, and should be interpreted as helping to understand the present invention.

[0072]

[0073] Preparation Example 1: Preparation of duplex DNA substrate for EcoRV detection

[0074] (1) 500 μL of a DNA solution containing 20 μM upper strand DNA and 10 mM Tris-HCl buffered saline (pH 7.6) and 500 μL of a MB-tagged DNA solution containing 20 μM methylene blue (MB; methylene blue)-tagged DNA and 10 mM Tris-HCl buffered saline (pH 7.6) were prepared, respectively, and each solution was stirred and heated at 95°C and 350 rpm for 5 minutes. At this time, the upper strand DNA had the base sequence 5'-CTA GCT ATG TGC CGA ATT TCA AGG ACA GTT GTA TGG ATA TCA TAC T-3', and the methylene blue-tagged DNA had the base sequence 5' MB-AGTATGATATCCA-3'.

[0075] (2) A mixture was prepared by mixing the heated DNA solution and the heated MB-tagged DNA solution, and the prepared mixture was annealed by heating at 95°C for 5 minutes, and then left at room temperature (20°C) for 1 hour to prepare a duplex DNA substrate for EcoRV detection.

[0076]

[0077] Comparative Preparation Example 1: Preparation of duplex DNA substrate for EcoRV detection

[0078] (1) 500 μL of a DNA solution containing 20 μM upper strand DNA and 10 mM Tris-HCl buffered saline (pH 7.6) and 500 μL of a MB-free DNA solution containing 20 μM MB-free DNA and 10 mM Tris-HCl buffered saline (pH 7.6) were prepared, respectively, and each solution was stirred and heated at 95°C and 350 rpm for 5 minutes. At this time, the upper strand DNA used had a base sequence of 5'-CTA GCT ATG TGC CGA ATT TCA AGG ACA GTT GTA TGG ATA TCA TAC T-3', and the MB-free DNA used had a base sequence of 5'-AGTATGATATCCA-3'.

[0079] (2) A mixture was prepared by mixing the heated DNA solution and the heated MB-free DNA solution, and the prepared mixture was annealed by heating at 95°C for 5 minutes, and then left at room temperature (20°C) for 1 hour to prepare a duplex DNA substrate for EcoRV detection.

[0080]

[0081] Preparation Example 2: Manufacturing of a field effect transistor (FET)

[0082] (1) A purified single-walled carbon nanotube (SWCNT) having a diameter of 4 nm was added to 1,2-dichlorobenzene and sonicated for 20 minutes to prepare a single-walled carbon nanotube suspension having a concentration of 0.1 mg / mL.

[0083] (2) A silicon dioxide (SiO2) substrate with a thickness of 1000 Å was prepared, and octadecyltrichlorosilane (OTS) with a non-polar terminal group was patterned on one side of the silicon dioxide substrate through a photolithography process using AZ5214 photoresist. Then, the substrate was immersed in an OTS solution (OTS: hexane = 1: 500 v / v) at room temperature (23°C) for 10 minutes to form a patterned self-assembled monolayer (SAM) on one side of the silicon dioxide substrate, thereby manufacturing a substrate on which a self-assembled monolayer was formed.

[0084] (3) The substrate on which the self-assembled monolayer was formed was immersed in the single-walled carbon nanotube suspension for 10 minutes and washed using 1,2-dichlorobenzene, thereby forming a single-walled carbon nanotube layer in the channel region on one side of the substrate where the self-assembled monolayer was not formed.

[0085] (4) Using a thermal evaporation process and a lift-off process, a source electrode and a drain electrode spaced apart from each other (spaced apart: 4 μm) were formed on one side of the self-assembled monolayer. At this time, gold (Au) and palladium (Pd) were used to form the source electrode and the drain electrode, and the structure was such that gold (Au) was formed with a thickness of 30 nm on one side of the self-assembled monolayer, and palladium (Pd) was formed with a thickness of 10 nm on one side of the formed gold.

[0086] (5) A field effect transistor was manufactured by passivating the source electrode and drain electrode with photoresist through a photolithography process to expose the single-wall carbon nanotube layer in the channel region formed between the source electrode and the drain electrode.

[0087]

[0088] Example 1: Fabrication of a field-effect transistor comprising an MB-tagged DNA substrate

[0089] (1) 10 μL of the duplex DNA substrate for EcoRV detection manufactured in Preparation Example 1 was drop-casted onto the single-walled carbon nanotube layer of the field effect transistor manufactured in Preparation Example 2 at room temperature (20°C) for 3 hours, thereby fixing the duplex DNA substrate for EcoRV detection manufactured in Preparation Example 1 to the surface of the single-walled carbon nanotube layer of the field effect transistor manufactured in Preparation Example 2.

[0090] (2) After drop casting, to remove the duplex DNA substrate for EcoRV detection that was not fixed on the surface of the single-walled carbon nanotube layer, the layer was washed with Tris-HCl buffered saline (pH 7.6) and dried using nitrogen (N2) gas.

[0091] (3) Then, a field effect transistor including an MB-tagged DNA substrate was manufactured by coating a blocking agent, bovine serum albumin (BSA), on the surface of a single-walled carbon nanotube layer on which a duplex DNA substrate for EcoRV detection was immobilized to prevent non-specific binding. Specifically, a BSA solution containing 0.5 wt% BSA and 10 mM Tris-HCl buffered saline (pH 7.6) was drop-casted on the surface of a single-walled carbon nanotube layer on which a duplex DNA substrate for EcoRV detection was immobilized, and then washed with Tris-HCl buffered saline (pH 7.6) and dried using nitrogen (N2) gas, thereby manufacturing a field effect transistor including an MB-tagged DNA substrate.

[0092]

[0093] Comparative Example 1: Fabrication of a field-effect transistor containing an untagged DNA substrate by MB

[0094] (1) 10 μL of the duplex DNA substrate for EcoRV detection manufactured in Comparative Preparation Example 1 was drop-casted onto the single-walled carbon nanotube layer of the field effect transistor manufactured in Preparation Example 2 at room temperature (20°C) for 3 hours, thereby fixing the duplex DNA substrate for EcoRV detection manufactured in Comparative Preparation Example 1 to the surface of the single-walled carbon nanotube layer of the field effect transistor manufactured in Preparation Example 2.

[0095] (2) After drop casting, to remove the duplex DNA substrate for EcoRV detection that was not fixed on the surface of the single-walled carbon nanotube layer, the layer was washed with Tris-HCl buffered saline (pH 7.6) and dried using nitrogen (N2) gas.

[0096] (3) Then, to prevent non-specific binding, a field-effect transistor including a DNA substrate that is not tagged with MB was manufactured by coating the surface of the single-walled carbon nanotube layer on which the duplex DNA substrate for EcoRV detection was immobilized with a blocking agent, bovine serum albumin (BSA). Specifically, a BSA solution containing 0.5 wt% BSA and 10 mM Tris-HCl buffered saline (pH 7.6) was drop-casted onto the surface of the single-walled carbon nanotube layer on which the duplex DNA substrate for EcoRV detection was immobilized, and then washed with Tris-HCl buffered saline (pH 7.6) and dried using nitrogen (N2) gas, thereby manufacturing a field-effect transistor including a DNA substrate that is not tagged with MB.

[0097]

[0098] Experimental Example 1: Atomic Force Microscope (AFM) Analysis

[0099] The results of scanning probe microscopy analysis are shown in Fig. 3. Specifically, an AFM image for a field effect transistor including a MB-tagged DNA substrate manufactured in Example 1 is shown in Fig. 3(A), a graph for the height profile before and after immobilization of a duplex DNA substrate for EcoRV detection is shown in Fig. 3(C), and a graph for the position at the center of mass z is shown in Fig. 3(E). In addition, an AFM image for a field effect transistor including a DNA substrate without MB tagged in Comparative Example 1 is shown in Fig. 3(B), a graph for the height profile before and after immobilization of a duplex DNA substrate for EcoRV detection is shown in Fig. 3(D), and a graph for the position at the center of mass z is shown in Fig. 3(F). The shaded dilution regions in Figs. 3(E) and 3(F) represent the Debye length (0.70 nm).

[0100] As can be seen in FIGS. 3(C) and 3(E), the field effect transistor including the MB-tagged DNA substrate manufactured in Example 1 was able to confirm that the duplex DNA substrate for EcoRV detection was more closely fixed to the single-walled carbon nanotube layer than the field effect transistor including the non-MB-tagged DNA substrate in Comparative Example 1. In addition, as can be seen in FIGS. 3(D) and 3(F), the distance between the duplex DNA substrate for EcoRV detection and the single-walled carbon nanotube layer was precisely measured, and it was confirmed that the field effect transistor including the MB-tagged DNA substrate manufactured in Example 1 was fixed at a very close distance of 0.25 nm, while the field effect transistor including the non-MB-tagged DNA substrate in Comparative Example 1 showed a distance of 2.5 nm. From these results, it was confirmed that the field effect transistor including the MB-tagged DNA substrate manufactured in Example 1 had the duplex DNA substrate for EcoRV detection well positioned within the Debye length (0.70 nm).

[0101]

[0102] Experimental Example 2: EcoRV Activity Detection Principle

[0103] Figure 8 is a schematic diagram showing the detection principle of a field effect transistor including an MB-tagged DNA substrate (=MB-tagged DNA) manufactured in Example 1 and an MB-free DNA substrate (=MB-free DNA) manufactured in Comparative Example 1 for measuring EcoRV endonuclease activity.

[0104] Referring to Fig. 8, the DNA substrate of EcoRV has a single-stranded region that is fixed to the surface of a single-walled carbon nanotube layer and a double-stranded segment for sequence-specific recognition of EcoRV. The DNA substrate without MB can be fixed to a fixed location on the surface of the single-walled carbon nanotube layer through π-π interactions between the single-stranded region of the substrate DNA and the surface of the single-walled carbon nanotube layer.

[0105] The MB-tagged DNA substrate (=MB-tagged DNA) can be fixed in a lying position by additionally π-π stacking of MB tags, which serve as orientation guides, on a single-walled carbon nanotube layer. After the DNA substrate is cleaved by EcoRV, the electrostatic distribution near the surface of the single-walled carbon nanotube layer of the field-effect transistor is changed, which affects the conductivity of the field-effect transistor. At this time, both the fixation of the MB-tagged DNA substrate and the cleavage by EcoRV on the surface of the single-walled carbon nanotube layer occur within the Debye length. In contrast, the cleavage of the DNA substrate that is not tagged with MB occurs beyond the Debye length. Therefore, it can be expected that a significant change in the electrostatic distribution will occur in the field-effect transistor including the MB-tagged DNA substrate (=MB-tagged DNA) manufactured in Example 1, and it can also be expected that the detection function will be improved. Using computer modeling, we were able to estimate the overall proximity of the anchored substrate to the surface of the single-walled carbon nanotube layer and the changes in the electrostatic potential distribution in the field-effect transistor at the molecular level before and after EcoRV-mediated cleavage.

[0106]

[0107] Experimental Example 3: Real-time Conductivity Measurement for EcoRV Activation and Inhibition

[0108] Fig. 4(A) is a graph showing the electrical response of a field effect transistor (=MB-tagged DNA / swCNT-FET) including the MB-tagged DNA substrate manufactured in Example 1 when EcoRV was continuously added, and Fig. 4(B) is a graph showing a calibration curve of a field effect transistor including the MB-tagged DNA substrate manufactured in Example 1 as a function of the EcoRV concentration. As a control, the electrical response of a field effect transistor (=Bare swCNT-FET) not including the MB-tagged DNA substrate was shown.

[0109] As can be seen in Fig. 4(A), the conductivity of the field effect transistor including the MB-tagged DNA substrate manufactured in Example 1 was confirmed to decrease significantly with each additional addition of EcoRV. In comparison, the field effect transistor not including the MB-tagged DNA substrate was confirmed to show minimal change.

[0110] In addition, as can be seen in Fig. 4(B), the field effect transistor including the MB-tagged DNA substrate manufactured in Example 1 has a dielectric constant of 7.8 Х 10 -4 It was confirmed that detection was possible even at low EcoRV concentrations of U / mL.

[0111] Figure 5(A) is a graph measuring real-time conductivity after injection of 50 U / mL of EcoRV when ATA (aurintricarboxylic acid), a representative inhibitor of EcoRV, was added at various concentrations. As can be seen in Figure 5(A), conductivity decreased as the ATA concentration increased from 5 nM to 50 nM, confirming that EcoRV was successfully inhibited by ATA.

[0112] Figure 5(B) is a graph showing the inhibitory effect of various concentrations of ATA on the reaction of a field effect transistor containing an MB-tagged DNA substrate manufactured in Example 1. As can be seen in Figure 5(B), the inhibition ratio increased correspondingly as the ATA concentration increased, and it was confirmed that it reached saturation at about 20 nM. The half-maximum inhibition value (IC) calculated from these results 50 ; the half-maximal inhibitory value) was calculated to be approximately 8.37 nM. In conclusion, the field-effect transistor comprising the MB-tagged DNA substrate prepared in Example 1 was shown to be suitable for screening EcoRV inhibitors, and it was confirmed that it has potential applications in new drug development and disease treatment.

[0113]

[0114] Experimental Example 4: Molecular Dynamics Simulation and Electrostatic Potential Calculation

[0115] Using MD simulation and Poisson-Boltzmann electrostatic potential simulation, the cause of the change in conductivity of the field effect transistor including the MB-tagged DNA substrate prepared in Example 1 and the field effect transistor including the non-MB-tagged DNA substrate prepared in Comparative Example 1 was investigated.

[0116] FIG. 6(A) is a graph showing the MD simulation results of the MB-tagged DNA substrate of the field effect transistor including the MB-tagged DNA substrate manufactured in Example 1 interacting with the single-walled carbon nanotube layer and the non-MB-tagged DNA substrate of the field effect transistor including the non-MB-tagged DNA substrate of Comparative Example 1. As can be seen in FIG. 6(A), it was found that the MB-tag plays an important role in controlling the interaction between the DNA substrate and the single-walled carbon nanotube layer. Specifically, starting from the same initial structure, the double-stranded region rapidly uncouples from the single-walled carbon nanotube layer and points upward relative to the DNA substrate without methylene blue (MB) (=MB-free DNA). In contrast, it was confirmed that the MB-tagged DNA substrate remains parallel because the methylene blue (MB) interacts with the single-walled carbon nanotube layer.

[0117] Figure 6(B) is a graph calculating the average minimum distance between each nucleic acid in the double-stranded portion and the single-walled carbon nanotube layer, where the shaded gray area represents the Debye length (0.70 nm), the vertical dotted line separates the two strands, and methylene blue (MB) is attached to the electron 1 of DNA-MB. As can be confirmed in Figure 6(A), the nucleic acid was confirmed to maintain a closer distance to the single-walled carbon nanotube layer in the case of MB-tagged DNA than in the case of DNA substrate without methylene blue (MB) (=MB-free DNA).

[0118] Fig. 6(C) is a graph showing the average electrostatic potential generated by the double-stranded portion of the MB-free DNA substrate in the single-walled carbon nanotube layer of the field-effect transistor including the MB-free DNA substrate in Comparative Example 1 on the left, and the average electrostatic potential generated by the double-stranded portion of the MB-tagged DNA substrate (MB-tagged DNA) in the single-walled carbon nanotube layer of the field-effect transistor including the MB-tagged DNA substrate manufactured in Example 1 on the right, using Poisson-Boltzmann electrostatic potential simulation. The black dot in Fig. 6(C) corresponds to the center of mass of the double-stranded portion and indicates the top of the single-walled carbon nanotube layer. As can be seen in Fig. 6(C), the electrostatic potential generated in the double-stranded region of the single-walled carbon nanotube layer is lost when cleaved by the EcoRV enzyme. Since each nucleic acid has a net charge of -1e, the electrostatic potential generated in both the MB-free DNA substrate and the MB-tagged DNA substrate was observed to be negative. Therefore, it was confirmed that the cleavage of the MB-tagged DNA substrate resulted in a small negative electrostatic potential in the single-walled carbon nanotube layer.

[0119] FIG. 6(D) is a graph showing the global electrostatic potential generated by the MB-free DNA substrate in the single-walled carbon nanotube layer of the field-effect transistor including the MB-free DNA substrate in Comparative Example 1 and the global electrostatic potential generated by the MB-tagged DNA substrate (MB-tagged DNA) in the single-walled carbon nanotube layer of the field-effect transistor including the MB-tagged DNA substrate manufactured in Example 1, using Poisson-Boltzmann electrostatic potential simulation. The global electrostatic potential value corresponds to the Riemann sum of the electrostatic potential values ​​of the single-walled carbon nanotube layer for a given configuration. As can be seen in FIG. 6(D), it was confirmed that the electrostatic potential of the MB-tagged DNA substrate showed a large change in the global electrostatic potential value compared to the MB-free DNA substrate. Therefore, it was confirmed that the field effect transistor including the MB-tagged DNA substrate manufactured in Example 1 provided excellent sensitivity for detecting EcoRV activity because the dsDNA was placed closer to the single-walled carbon nanotube layer, thereby increasing the electrostatic potential generated in the single-walled carbon nanotube layer.

[0120]

[0121] Experimental Example 5: Selectivity of EcoRV Activity Detection

[0122] Figure 7 is a graph showing the conductivity measured in a field effect transistor including the MB-tagged DNA substrate manufactured in Example 1 after adding HaeIII, EcoRI, PvuII, and BamHI as interference substances in addition to the EcoRV enzyme. As can be seen in Figure 7, it was confirmed that a slight change in conductivity was observed for the interference substances HaeIII, EcoRI, PvuII, and BamHI, but a significant change in conductivity was observed for EcoRV. From these results, it was confirmed that the field effect transistor including the MB-tagged DNA substrate manufactured in Example 1 had excellent selectivity for detecting the activity of EcoRV.

[0123]

[0124] Simple modifications or changes of the present invention can be easily implemented by a person having ordinary skill in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.

[0125]

[0126] [National Research and Development Project Supporting This Invention]

[0127] [Project ID] 1485019375

[0128] [Assignment Number] ARQ201902181005

[0129] [Ministry Name] Ministry of Environment

[0130] [Name of Project Management (Specialist) Institution] Korea Environmental Industry & Technology Institute

[0131] [Research Project Name] Development of Eco-inspired Environmental Pollution Management Technology

[0132] [Research Project Title] Biomimetic Technology-Based Synthesis of Peptide Receptors for Environmental Hormone Detection and Development of a Portable Measurement Device

[0133] [Contribution rate] 30 / 100

[0134] [Name of the project performing organization] G&C Bio Co., Ltd.

[0135] Research Period: April 17, 2019 - December 31, 2023

[0136] [Project ID] 1711181666

[0137] [Assignment Number] 2020R1A2C1014918

[0138] [Ministry Name] Ministry of Science and ICT

[0139] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0140] [Research Project Name] Individual Basic Research (Ministry of Science and ICT)

[0141] [Research Project Title] Development of a MoS2 transistor-based myocardial infarction diagnostic biosensor using biomimetic technology.

[0142] [Contribution rate] 30 / 100

[0143] [Name of the project performing organization] Soonchunhyang University

[0144] Research Period: March 1, 2020 - February 28, 2025

Claims

1. Substrate; A source electrode and a drain electrode formed spaced apart from each other on one surface of the substrate; and Including a channel region formed between the source electrode and the drain electrode and in electrical contact therewith; A field effect transistor for a biosensor for selectively detecting endonuclease, characterized in that the channel region comprises a single-walled carbon nanotube layer having a DNA substrate for endonuclease detection fixed to the surface.

2. Substrate; A first self-assembled layer and a second self-assembled layer formed spaced apart from each other on one surface of the substrate; A source electrode formed on one surface of the first self-assembled layer and a drain electrode formed on one surface of the second self-assembled layer; and Including a channel region formed between the source electrode and the drain electrode and in electrical contact therewith; A field effect transistor for a biosensor for selectively detecting endonuclease, characterized in that the channel region comprises a single-walled carbon nanotube layer having a DNA substrate for endonuclease detection fixed to the surface.

3. In paragraph 1 or 2, A field effect transistor for a biosensor for selectively detecting endonuclease, characterized in that the DNA substrate for detecting the endonuclease is a DNA substrate tagged with methylene blue.

4. In paragraph 3, A field effect transistor for a biosensor for selectively detecting an endonuclease, wherein the methylene blue-tagged DNA substrate has a base sequence of 5' MB-AGTATGATATCCA-3'.

5. In paragraph 1 or 2, A field effect transistor for a biosensor for selectively detecting an endonuclease, wherein the endonuclease is an EcoRV enzyme.

6. In paragraph 1 or 2, A field effect transistor for a biosensor for selectively detecting endonuclease, characterized in that the average distance between the DNA substrate for detecting the endonuclease and the single-walled carbon nanotube layer is 0.15 to 0.5 nm.

7. In paragraph 1 or 2, The above field effect transistor is 5.8 Х 10 -4 ~ 9.8 Х 10 -4 A field-effect transistor for a biosensor for selectively detecting an endonuclease, characterized in that it can be detected at an EcoRV concentration of U / mL.

8. A biosensor for selectively detecting an endonuclease comprising a field effect transistor for a biosensor according to claim 1 or 2.

9. A first step of preparing a field effect transistor including a duplex DNA substrate and a single-walled carbon nanotube layer for EcoRV detection; A second step of drop casting the duplex DNA substrate for EcoRV detection onto the single-walled carbon nanotube layer of the field effect transistor, thereby fixing the duplex DNA substrate for EcoRV detection onto the surface of the single-walled carbon nanotube layer of the field effect transistor; and A third step of manufacturing a field effect transistor for a biosensor by coating a blocking agent on the surface of a single-walled carbon nanotube layer on which the duplex DNA substrate for EcoRV detection is fixed; A method for manufacturing a field effect transistor for a biosensor for selectively detecting an endonuclease, characterized by including a .

10. In the 9th paragraph, the duplex DNA substrate for detecting EcoRV is Step 1-1 of preparing a DNA solution containing upper strand DNA and an MB-tagged DNA solution containing DNA tagged with methylene blue (MB), respectively; Step 1-2 of preparing a mixture by mixing the above DNA solution and MB-tagged DNA solution; and Step 1-3 of producing a duplex DNA substrate for EcoRV detection by heating and annealing the above mixture and then leaving it alone; A method for manufacturing a field effect transistor for a biosensor for selectively detecting an endonuclease, characterized in that it is manufactured including.

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