Carbon nitride-modified gold transducer-based electrochemical sensor for detection of 5-hydroxymethylcytosine and its manufacturing method

Abstract

The present invention relates to an electrochemical sensor based on a carbon nitride-modified gold converter for detecting 5-hydroxymethylcytosine and a method for manufacturing the same. More specifically, the invention relates to an electrochemical sensor based on a carbon nitride-modified gold converter for detecting 5-hydroxymethylcytosine and a method for manufacturing the same, which can selectively detect 5-hydroxymethylcytosine (5hmC) in genomic DNA, which is an important biomarker in various types of cancer, in real-time by an electrochemical method using a carbon nitride-modified gold converter. The electrochemical sensor based on a carbon nitride-modified gold converter for detecting 5-hydroxymethylcytosine according to the present invention is characterized by selectively detecting 5-hydroxymethylcytosine (5hmC) in genomic DNA in real time by an electrochemical method using a graphite carbon nitride (g-C3N4) modified gold converter.

Description

Technology Field

[0001] The present invention relates to an electrochemical sensor based on a carbon nitride-modified gold converter for detecting 5-hydroxymethylcytosine and a method for manufacturing the same. More specifically, the invention relates to an electrochemical sensor based on a carbon nitride-modified gold converter for detecting 5-hydroxymethylcytosine and a method for manufacturing the same, which can selectively detect 5-hydroxymethylcytosine (5hmC) in genomic DNA, which is an important biomarker in various types of cancer, in real-time by an electrochemical method using a carbon nitride-modified gold converter. Background Technology

[0002] Cancer biomarkers are important indicators of cancer status and progression that aid in the early detection and more effective treatment of cancer.

[0003] The loss of 5-hydroxymethylcytosine (hereinafter referred to as '5hmC'), an oxidation product of 5-methylcytosine (hereinafter referred to as '5mC'), is an epigenetic biomarker that occurs repeatedly in various types of cancer.

[0004] There are various cases of research and development related to 5hmC in cancer. According to studies investigating the association between 5hmC and hepatocellular carcinoma and neural progenitor cell phenotypes, a decrease in 5hmC was found to be associated with the progression of hepatocellular carcinoma, and it was also revealed to be associated with the neural progenitor cell phenotype of the normal brain and a shortened survival period in malignant glioma.

[0005] In addition, it was found that TET gene expression and a decrease in 5-methylcytosine hydroxylation are associated with tumor development, and it was confirmed that downregulated 5hmC expression in renal cell carcinoma has prognostic value.

[0006] Recently, the risk of acute myeloid leukemia in healthy people was predicted based on 5hmC levels, and it was discovered that TET2 mutations predict the response to hypomethylating agents in patients with myelodysplastic syndrome.

[0007] These studies comprehensively highlight the importance of 5hmC and TET proteins in cancer progression and the potential of 5hmC as a biomarker for early cancer detection.

[0008] Therefore, accurately quantifying 5hmC has great potential for various clinical applications.

[0009] Traditional methods for selectively detecting 5hmC include the dot blot method.

[0010] The above dot blot method is a technique that involves fixing a DNA or RNA fragment to be detected, detecting 5hmC using a specific antibody or probe specific to 5hmC, and then analyzing the dot blot using a dark room or a photosensitive plate reader.

[0011] However, the above Dot Blot method had the disadvantage of taking a long time to detect 5hmC and being cumbersome to use.

[0012] Meanwhile, recently, sensing strategies utilizing the unique physicochemical properties of functional nanomaterials or electrochemical biosensing technology are being developed for faster and easier 5hmC sensing.

[0013] However, despite their excellent performance, sensing strategies utilizing physicochemical properties have had limitations in actual application in clinical settings because most require complex chemical pretreatment, time-consuming procedures, surface functionalization, and expensive equipment, and have had the disadvantage that incomplete chemical or enzymatic reactions and the short half-life of recombinant enzymes often lead to inaccurate results.

[0014] On the other hand, electrochemical biosensing technology has many advantages over other detection methods, such as rapid response, ease of operation, high sensitivity and selectivity, low cost, excellent reproducibility, and ease of miniaturization.

[0015] As prior art regarding such electrochemical biosensing technology, Korean Published Patent Application No. 10-2017-0000437 (published Jan. 3, 2017) proposes a 'carbon nanotube-polymer composite electrode and a method for electrochemical detection of DNA using the same'.

[0016] However, the above-mentioned conventional technology is a technology that directly detects DNA without surface modification using an electrochemical method, and it had limitations in highly selective real-time detection of 5-hydroxymethylcytosine (5hmC) in genomic DNA, and therefore, there was a problem in that it was not easy to distinguish between 5hmC and 5mC using the existing electrochemical method. The problem to be solved

[0017] The present invention was devised to solve the problems of the prior art as described above. The objective of the present invention is to provide a rapid and highly selective electrochemical sensor based on a carbon nitride modified gold converter for detecting 5-hydroxymethylcytosine (5hmC) without labeling, using a graphite carbon nitride (g-C3N4) modified gold converter, and a method for manufacturing the same. means of solving the problem

[0018] To achieve the above objectives, the electrochemical sensor based on a carbon nitride-modified gold converter for detecting 5-hydroxymethylcytosine according to the present invention is characterized by selectively detecting 5-hydroxymethylcytosine (5hmC) in genomic DNA in real time by an electrochemical method using a graphite carbon nitride (g-C3N4) modified gold converter.

[0019] In addition, the method for manufacturing an electrochemical sensor based on a carbon nitride-modified gold converter for detecting 5-hydroxymethylcytosine according to the present invention is a method for manufacturing an electrochemical sensor capable of selectively detecting 5-hydroxymethylcytosine (5hmC) in genomic DNA in real time by an electrochemical method, comprising: (a) a step of preparing a nitrogen precursor material comprising one or more of urea, thiourea, cyanamide, melamine, or dicyandiamide; (b) a step of synthesizing a two-dimensional graphite carbon nitride (g-C3N4) sheet by thermally decomposing the nitrogen precursor material prepared in step (a) in an atmosphere or nitrogen atmosphere; and (c) a step of individually dispersing the graphite carbon nitride (g-C3N4) sheet synthesized in step (b) in double distilled water and drop-casting the graphite carbon nitride (g-C3N4) dispersion onto the surface of a gold electrode (AuE) to produce a graphite carbon nitride-modified gold converter (AuE-g-C3N4).

[0020] Here, the above step (b) is characterized by melting the nitrogen precursor material while stirring at a certain temperature, stirring further for a certain period of time, pouring the melted nitrogen precursor material into a crucible and cooling it to room temperature to solidify it, and synthesizing a two-dimensional graphite carbon nitride (g-C3N4) sheet by thermally decomposing the solidified nitrogen precursor material at a certain temperature for a certain period of time at a predetermined heating rate in an atmosphere or nitrogen atmosphere.

[0021] Here, step (b) is characterized by using urea as the nitrogen precursor material, melting the urea while stirring at a temperature of 135°C and stirring for 10 minutes, pouring the melted urea into a crucible and cooling it to room temperature to solidify it, and synthesizing a two-dimensional graphite carbon nitride (g-C3N4) sheet by pyrolyzing the solidified urea at 500°C for 3 hours at a heating rate of 3°C / min in an atmosphere of air or nitrogen.

[0022] Here, step (b) is characterized by synthesizing a two-dimensional graphite carbon nitride (g-C3N4) sheet by thermally decomposing the nitrogen precursor material prepared in step (a) in a nitrogen atmosphere.

[0023] Here, step (c) is characterized by producing a graphite carbon nitride modified gold converter (AuE-g-C3N4) by individually dispersing the graphite carbon nitride (g-C3N4) sheet in double distilled water and then ultrasonically treating it for a certain period of time, and then drop-casting the graphite carbon nitride (g-C3N4) dispersion onto the surface of a gold electrode (AuE) and drying it in air, wherein the gold electrode (AuE) is washed with a predetermined solution and rinsed with distilled water before drop-casting, polished using a predetermined powder, and then ultrasonically treated in distilled water. Effects of the invention

[0024] With the above configuration, the carbon nitride-modified gold converter-based electrochemical sensor for detecting 5-hydroxymethylcytosine according to the present invention has the advantage of having high sensitivity, selectivity, excellent reproducibility, and stability in detecting 5hmC in genomic DNA by the method.

[0025] In addition, the method for manufacturing a carbon nitride-modified gold converter-based electrochemical sensor for detecting 5-hydroxymethylcytosine according to the present invention has the advantage that anyone can easily manufacture a carbon nitride-modified gold converter-based electrochemical sensor having the above advantages. Brief explanation of the drawing

[0026] FIG. 1 is a conceptual diagram illustrating the fabrication and 5hmC sensing mechanism of an electrochemical sensor based on a carbon nitride-modified gold converter according to an embodiment of the present invention. FIG. 2 is a state diagram for detecting 5hmC in primary liver samples separated from two mouse models using a carbon nitride-modified gold converter-based electrochemical sensor according to an embodiment of the present invention. FIG. 3 is a flowchart of a method for manufacturing an electrochemical sensor based on a carbon nitride-modified gold converter according to an embodiment of the present invention. FIG. 4 is a spectral graph of a two-dimensional graphite carbon nitride (g-C3N4) sheet synthesized in an air or nitrogen atmosphere according to an embodiment of the present invention. FIG. 5 shows SEM, TEM, HR-TEM, and SAED images of a two-dimensional graphite carbon nitride (g-C3N4) sheet synthesized in an air or nitrogen atmosphere according to an embodiment of the present invention. FIG. 6 is a graph of the current response versus voltage of an electrochemical sensor based on a carbon nitride-modified gold converter according to an embodiment of the present invention. Figure 7 is a graph summarizing the peak current values ​​shown in Figure 6. FIG. 8 is a graph comparing the 5hmC quantification results using a carbon nitride-modified gold converter-based electrochemical sensor according to an embodiment of the present invention and a traditional CMS dot blot. Specific details for implementing the invention

[0027] Hereinafter, a carbon nitride-modified gold converter-based electrochemical sensor for detecting 5-hydroxymethylcytosine according to the present invention will be described in more detail with reference to the embodiments illustrated in the drawings.

[0028] FIG. 1 is a conceptual diagram illustrating the fabrication and 5hmC detection mechanism of an electrochemical sensor based on a carbon nitride-modified gold converter according to an embodiment of the present invention; FIG. 2 is a state diagram of detecting 5hmC in primary liver samples separated from two mouse models using an electrochemical sensor based on a carbon nitride-modified gold converter according to an embodiment of the present invention; FIG. 3 is a flowchart of a method for manufacturing an electrochemical sensor based on a carbon nitride-modified gold converter according to an embodiment of the present invention; FIG. 4 is a spectrum graph of a two-dimensional graphite carbon nitride (g-C3N4) sheet synthesized in an atmospheric or nitrogen atmosphere according to an embodiment of the present invention; FIG. 5 is SEM, TEM, HR-TEM, and SAED images of a two-dimensional graphite carbon nitride (g-C3N4) sheet synthesized in an atmospheric or nitrogen atmosphere according to an embodiment of the present invention; FIG. 6 is a current response versus voltage graph of an electrochemical sensor based on a carbon nitride-modified gold converter according to an embodiment of the present invention. FIG. 7 is a graph summarizing the peak current values ​​shown in FIG. 6, and FIG. 8 is a graph comparing the 5hmC quantitative results using a carbon nitride modified gold converter-based electrochemical sensor according to an embodiment of the present invention and a traditional CMS dot blot.

[0029] Referring to FIG. 1, the electrochemical sensor based on a carbon nitride-modified gold converter for detecting 5-hydroxymethylcytosine according to one embodiment of the present invention is an electrochemical sensor capable of selectively detecting 5-hydroxymethylcytosine (5hmC) in genomic DNA in real time by an electrochemical method using a graphite carbon nitride-modified gold converter (AuE-g-C3N4) fabricated by drop-casting a graphite carbon nitride (g-C3N4) dispersion onto the surface of a gold electrode (AuE).

[0030] The 5hmC sensing mechanism of the carbon nitride-modified gold converter-based electrochemical sensor according to one embodiment of the present invention is as follows.

[0031] 5hmC is a special type of molecule found in DNA, and when this molecule is present in DNA, it can increase the current of a device called a nanopore sequencer used to read DNA base sequences.

[0032] This occurs because, as shown in Fig. 1, 5hmC has an extra atomic group (hydroxymethyl group) that can form additional connections (hydrogen bonding) with the nanopores, which allows DNA to remain more stable and stay longer inside the nanopores (this is referred to as "residence time").

[0033] That is, in the present invention, 5hmC-containing DNA is fixed to graphite nitride (g-C3N4) through hydrogen bonding between the -OH of 5hmC and the -NH2 of graphite nitride (g-C3N4).

[0034] In this case, because DNA stays in the nanopores longer, more charged particles (ions) can pass through the pores, increasing the current.

[0035] In other words, 5hmC causes DNA to stay in the nanopores longer, allowing more electricity to flow and consequently increasing the current, and it becomes possible to selectively detect 5hmC in real time by measuring the current value of a carbon nitride modified gold converter-based electrochemical sensor (AuE-g-C3N4-5hmC) that is distinguished by the amount of 5hmC.

[0036] In addition, the graphite carbon nitride (g-C3N4) based sensor according to the present invention can exhibit higher selectivity for 5hmC compared to other cytosine analogs because it does not form interactions such as those with 5hmC in the case of C or 5mC.

[0037] FIG. 2 illustrates the electrochemical detection of 5 hmC through a current versus potential graph of the sensor according to the present invention in primary liver samples separated from two mouse models, namely the TET knockout or tumor (TKO or T) model and the wild-type or non-tumor (WT or NT) model, as an example of use of the carbon nitride modified gold converter-based electrochemical sensor according to one embodiment of the present invention.

[0038] The experimental results and confirmed performance according to the experimental example of the electrochemical sensor based on a carbon nitride-modified gold converter for detecting 5-hydroxymethylcytosine according to the present invention will be described later.

[0039] FIG. 3 shows a flowchart of a method for manufacturing a carbon nitride-modified gold converter-based electrochemical sensor for detecting 5-hydroxymethylcytosine according to an embodiment of the present invention.

[0040] Referring to FIG. 3, a method for manufacturing an electrochemical sensor based on a carbon nitride-modified gold converter for detecting 5-hydroxymethylcytosine according to one embodiment of the present invention relates to a method for manufacturing an electrochemical sensor capable of selectively detecting 5-hydroxymethylcytosine (5hmC) in genomic DNA in real time by an electrochemical method, comprising (a) a step of preparing a nitrogen precursor material, (b) a step of synthesizing a two-dimensional graphite carbon nitride sheet, and (c) a step of manufacturing a graphite carbon nitride-modified gold converter.

[0041] The above step (a) is a step of preparing a nitrogen precursor material comprising one or more of (a) urea, thiourea, cyanamide, melamine, or dicyandiamide.

[0042] Graphite carbon nitride (g-C3N4) is a promising two-dimensional (2D) polymer due to its easy and cost-effective synthesis, non-toxicity, high thermal and chemical stability, and favorable energy band position.

[0043] Among the five phases of carbon nitride (α-C3N4, β-C3N4, cubic C3N4, pseudocubic C3N4, and g-C3N4), graphitic carbon nitride (g-C3N4) is the most stable allotrope under atmospheric conditions and has a layered structure similar to graphite.

[0044] The above-mentioned graphite carbon nitride (g-C3N4) can generally be produced directly by the thermal decomposition of various nitrogen-rich precursors such as urea, thiourea, cyanamide, melamine, or dicyandiamide, and in step (a) above, a nitrogen precursor material such as the above is prepared for the production of graphite carbon nitride (g-C3N4).

[0045] In one embodiment of the present invention, the nitrogen precursor material may be urea (ACS reagent, 99.0-100.5%) purchased from Sigma-Aldrich (USA).

[0046] Step (b) above is a step of synthesizing two-dimensional graphite carbon nitride (g-C3N4) sheets by thermally decomposing the nitrogen precursor material prepared in Step (a) above in an atmosphere of air or nitrogen.

[0047] In one embodiment of the present invention, step (b) may be configured to synthesize a two-dimensional graphite carbon nitride (g-C3N4) sheet by using urea as the nitrogen precursor material, melting the urea while stirring at a temperature of 135°C, stirring for 10 minutes, pouring the melted urea into a crucible and cooling it to room temperature to solidify it, and pyrolyzing the solidified urea at 500°C for 3 hours at a heating rate of 3°C / min in an atmosphere or nitrogen atmosphere.

[0048] In addition, after the pyrolysis process, it is desirable to continuously wash with 0.1 M nitric acid, water, and acetone to remove impurities and to dry the product.

[0049] When synthesizing two-dimensional graphite carbon nitride (g-C3N4) sheets by pyrolysis in an atmospheric environment (g-C3N4-A), the end of the tube in the furnace can be opened to allow air to flow.

[0050] Meanwhile, when synthesizing two-dimensional graphite carbon nitride (g-C3N4) sheets by pyrolysis in a nitrogen atmosphere (g-C3N4-N2), a closed tubular furnace can be used to apply a constant nitrogen gas flow (0.5 bp / min).

[0051] FIG. 4 is a spectral graph of a two-dimensional graphite carbon nitride (g-C3N4) sheet synthesized in an atmosphere of air or nitrogen according to an embodiment of the present invention.

[0052] Figure 4(a) shows the FTIR spectrum graphs of an atmospheric atmosphere (g-C3N4-A) and a nitrogen atmosphere (g-C3N4-N2), obtained by performing infrared spectroscopy to investigate the molecular structure and chemical bonds present in the synthesized two-dimensional graphite carbon nitride (g-C3N4) sheet.

[0053] Looking at Figure 4(a), compared to the atmospheric atmosphere (g-C3N4-A), the nitrogen atmosphere (g-C3N4-N2) shows an overall decrease in the absorption peak intensity for aromatic CN, C=N, triazine ring, heptazine ring, and -NH2 or =NH stretching vibrations, and this absorption band pattern indicates the successful formation of the carbon nitride structure.

[0054] Figure 4(b) is a graph of the Raman spectra of an atmospheric atmosphere (g-C3N4-A) and a nitrogen atmosphere (g-C3N4-N2).

[0055] Referring to Fig. 4(b), when comparing the Raman peaks in the atmospheric atmosphere (g-C3N4-A) and the nitrogen atmosphere (g-C3N4-N2), at 7 cm⁻¹ in the nitrogen atmosphere (g-C3N4-N2) -1 A blue shift appears, which indicates the phonon limiting effect of the peeled sheet and provides insight into the thickness.

[0056] Figure 4 (c) is a graph of the UV-vis absorption spectra of an atmospheric atmosphere (g-C3N4-A) and a nitrogen atmosphere (g-C3N4-N2).

[0057] Looking at Figure 4(c), a comparison of the UV-vis absorption spectra revealed that the atmospheric atmosphere (g-C3N4-A) and the nitrogen atmosphere (g-C3N4-N2) had maximum absorption at 356 and 301 nm, respectively, and the nitrogen atmosphere (g-C3N4-N2) showed a lower absorption peak intensity than the atmospheric atmosphere (g-C3N4-A) and a 55 nm blue shift.

[0058] Meanwhile, (a), (b), (c), and (d) of Fig. 5 are SEM, TEM, HR-TEM, and SAED (selected area electron diffraction) images of a two-dimensional graphite carbon nitride (g-C3N4) sheet synthesized in an atmospheric atmosphere (g-C3N4-A), and (e), (f), and (g) of Fig. 4 are SEM, TEM, HR-TEM, and SAED images of a two-dimensional graphite carbon nitride (g-C3N4) sheet synthesized in a nitrogen atmosphere (g-C3N4-N2).

[0059] SEM and TEM images showed that the atmospheric atmosphere (g-C3N4-A) had an amorphous and crumpled structure, whereas the nitrogen atmosphere (g-C3N4-N2) showed a 2D smooth sheet-like structure, which could verify the X-ray diffraction (XRD) results.

[0060] The detailed microstructures of the atmospheric atmosphere (g-C3N4-A) and nitrogen atmosphere (g-C3N4-N2) were further observed by HR-TEM, and HR-TEM analysis revealed the existence of lattice patterns with a distance of 0.32 nm, which perfectly matched the crystal planes of the carbon studied, thereby verifying the XRD results.

[0061] In addition, the polycrystalline characteristics of the material could be confirmed through the SAED patterns in an atmospheric (g-C3N4-A) and nitrogen (g-C3N4-N2) atmosphere.

[0062] Based on these results, it can be said that it is more desirable to synthesize two-dimensional graphite carbon nitride (g-C3N4) sheets by pyrolysis in a nitrogen atmosphere (g-C3N4-N2) rather than in an air atmosphere (g-C3N4-A).

[0063] Step (c) above is a step of fabricating a graphite carbon nitride modified gold converter (AuE-g-C3N4) by individually dispersing the graphite carbon nitride (g-C3N4) sheets synthesized in Step (b) above in double distilled water and drop-casting the graphite carbon nitride (g-C3N4) dispersion onto the surface of a gold electrode (AuE).

[0064] In one embodiment of the present invention, step (c) can be performed by individually dispersing the graphite carbon nitride (g-C3N4) sheet in double distilled water and then ultrasonically treating it for 15 minutes, and then drop-casting the graphite carbon nitride (g-C3N4) dispersion onto the surface of a gold electrode (AuE) and drying it in air to produce the graphite carbon nitride modified gold converter (AuE-g-C3N4), wherein the gold electrode (AuE) is washed with a piranha solution for 30 minutes before drop-casting, rinsed with distilled water, polished using Al2O3 powder with a diameter of 0.5 micron, and ultrasonically treated in distilled water to complete the process.

[0066] Example:

[0067] (a) materials

[0068] Urea (ACS reagent, 99.0-100.5%), sodium chloride (ACS reagent, ≥99.0%), potassium ferrocyanide (ACS reagent, 98.5-102.0%), potassium ferrocyanide (ACS reagent, ≥99.0%), potassium chloride (for molecular biology, ≥99.0%), sulfuric acid (ACS reagent, 95.0-98.0%), hydrogen peroxide (35%), Al2O3 powder and sodium perchlorate (ACS reagent, ≥98.0%) were purchased from Sigma-Aldrich (USA).

[0069] All materials were analytical grade and used as received without further purification, and only deionized water (DI) was used for all aqueous solutions and rinsing procedures, and all solutions were prepared with ultrapure water (18 MΩ cm) obtained from an ultrapure HIQ water purification system.

[0071] (b) Synthesis of two-dimensional graphite carbon nitride (g-C3N4) sheets

[0072] Graphite nitride was prepared by simple thermal decomposition of urea in an atmospheric or nitrogen atmosphere. First, urea (20 g) was heated and melted at 135°C while stirring, then stirred for an additional 10 minutes, after which the melted urea was poured into a quartz crucible and cooled to room temperature.

[0073] The urea was solidified and annealed at 500°C for 3 hours at a heating rate of 3°C / min in an atmosphere of air or nitrogen (N2) gas.

[0074] To prepare graphite carbon nitride (g-C3N4) in ambient air (called g-C3N4-A), the end of the tube in the furnace was opened to allow air to flow.

[0075] During the production of graphite carbon nitride (g-C3N4) by pyrolysis in a nitrogen (N2) gas environment (referred to as g-C3N4-N2), a constant nitrogen gas flow (0.5 bp / min) was applied using a closed tubular furnace.

[0076] After the pyrolysis process, the product was washed continuously with 0.1 M nitric acid, water, and acetone to remove impurities and dried.

[0078] (c) Fabrication of carbon nitride-modified gold electrode for 5hmC detection

[0079] Graphite nitride (5 mg) was individually dispersed in double distilled water (DI, 1 mL) and sonicated for 15 minutes.

[0080] A homogeneous dispersion of graphite carbon nitride (g-C3N4) (6 μL) was drop-cast onto the surface of a gold electrode (AuE) and dried in air.

[0081] Before drop casting, the gold electrode (AuE) was washed with piranha solution for 30 minutes and thoroughly rinsed with distilled water.

[0082] The manufactured electrode was polished using Al2O3 powder with a diameter of 0.5 micron and ultrasonically treated in distilled water to complete the process.

[0084] Experimental Example: Measurement of Performance of Carbon Nitride Modified Gold Converter-Based Electrochemical Sensor

[0085] Cyclic voltammetry (CV) measurements were performed on a carbon nitride-modified gold converter-based electrochemical sensor manufactured by the manufacturing method according to one embodiment of the present invention at a specified scan rate in 1M H2SO4 between -400mV and 1400mV until constant current behavior was observed.

[0086] A measurement sample containing 5hmC DNA (5μL) was dropped onto a graphite carbon nitride (g-C3N4) modified gold electrode (AuE), incubated for 1 hour under humid conditions, washed with KCl to remove unreacted 5hmC, and then CV and linear sweep voltammetry (LSV) measurements were performed.

[0087] FIG. 6 is a graph of the current response versus voltage of an electrochemical sensor based on a carbon nitride-modified gold converter according to an embodiment of the present invention.

[0088] The graph in Fig. 6 was obtained through experiments on primary liver samples isolated from two mouse models as in Fig. 2, namely the TET knockout or tumor (TKO or T) model and the wild or non-tumor (WT or NT) model.

[0089] Looking at Figure 6, it can be seen that tumor (T) genomic DNA with a higher level of 5hmC consistently produces a stronger current response than non-tumor (NT) genomic DNA with a significantly reduced 5hmC content.

[0090] Figure 7 is a graph summarizing the peak current values ​​shown in Figure 6.

[0091] Looking at Figure 7, when comparing the maximum current response of the non-tumor (NT) sample and the tumor (T) sample, it can be seen that the peak current value is reduced by about 3 times in the tumor (T) sample compared to the non-tumor (NT) sample.

[0092] This is because the genomic DNA isolated from mice has the same nucleotide composition and sequence but differs only at the 5hmC level, so the difference observed in the current reaction can be attributed mainly to the difference in 5hmC content.

[0093] Figure 8 is a graph comparing the quantification results of 5hmC using a carbon nitride-modified gold converter-based electrochemical sensor (AuE-g-C3N4) according to an embodiment of the present invention and a traditional CMS dot blot.

[0094] Looking at Fig. 8, it can be seen that the quantification results of 5hmC using a carbon nitride modified gold converter (AuE-g-C3N4)-based electrochemical sensor according to one embodiment of the present invention and a traditional CMS dot blot are in great agreement with each other.

[0095] These results indicate that the carbon nitride-modified gold converter (AuE-g-C3N4)-based electrochemical sensor according to one embodiment of the present invention can be used to detect 5hmC in real time more simply and rapidly compared to the dot blot method, while having high accuracy in the selective detection of 5hmC.

[0096] The carbon nitride-modified gold converter-based electrochemical sensor for detecting 5-hydroxymethylcytosine according to one embodiment of the present invention showed high sensitivity, selectivity, excellent reproducibility and stability, and a low oxidation potential (0.23 V) and a very low detection limit (0.316 pM) for 5 hmC.

[0098] In addition, the sensor according to one embodiment of the present invention was tested to see if it could be applied to actual samples using primary liver samples from a mouse model in which 5hmC levels were reduced due to Tet gene knockout or hepatocellular carcinogenesis, and the results showed that the sensor effectively detected reduced genomic 5hmC in TET-deficient liver and hepatocellular carcinoma compared to the control group.

[0099] Therefore, this new sensing strategy has the potential to rapidly quantify genomic 5hmC and develop a clinically applicable sensor for early cancer diagnosis and prognosis evaluation.

[0100] The electrochemical sensor based on a carbon nitride-modified gold converter for detecting 5-hydroxymethylcytosine and the method for manufacturing the same, described above and illustrated in the drawings, are merely one embodiment for carrying out the present invention and should not be interpreted as limiting the technical scope of the present invention. The scope of protection of the present invention is determined solely by the matters described in the following claims, and embodiments that are improved and modified without departing from the gist of the present invention shall be deemed to fall within the scope of protection of the present invention insofar as they are obvious to those skilled in the art to which the present invention belongs. Explanation of the symbols

[0101] 5hmC: 5-hydroxymethylcytosine g-C3N4: Graphite carbon nitride AuE: Gold electrode AuE-g-C3N4: Graphite Carbon Nitride Modified Gold Converter g-C3N4-A: Atmospheric atmosphere g-C3N4-N2: Nitrogen atmosphere

Claims

Claim 1 A carbon nitride modified gold converter-based electrochemical sensor for detecting 5-hydroxymethylcytosine, characterized by selectively detecting 5-hydroxymethylcytosine (5hmC) in genomic DNA in real time by an electrochemical method using a graphite carbon nitride (g-C3N4) modified gold converter. Claim 2 A method for manufacturing an electrochemical sensor capable of selectively detecting 5-hydroxymethylcytosine (5hmC) in genomic DNA in real time by an electrochemical method, comprising: (a) a step of preparing an urea as a nitrogen precursor material; (b) a step of synthesizing a two-dimensional graphite carbon nitride (g-C3N4) sheet by thermally decomposing the nitrogen precursor material prepared in step (a) in an atmosphere of air or nitrogen; and (c) a step of individually dispersing the graphite carbon nitride (g-C3N4) sheet synthesized in step (b) in double distilled water and drop-casting the graphite carbon nitride (g-C3N4) dispersion onto the surface of a gold electrode (AuE) to produce a graphite carbon nitride modified gold converter (AuE-g-C3N4); wherein step (b) is characterized by using urea as the nitrogen precursor material, melting the urea while stirring at a temperature of 135°C and stirring for 10 minutes, pouring the melted urea into a crucible and cooling it to room temperature to solidify it, and pyrolyzing the solidified urea at a heating rate of 3°C / min at 500°C for 3 hours in an atmosphere or nitrogen atmosphere to synthesize a two-dimensional graphite carbon nitride (g-C3N4) sheet. Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 A method for manufacturing a carbon nitride-modified gold converter-based electrochemical sensor for detecting 5-hydroxymethylcytosine, wherein in step (c), the graphite carbon nitride (g-C3N4) sheet is individually dispersed in double distilled water and then ultrasonically treated for a certain period of time, and the graphite carbon nitride (g-C3N4) dispersion is drop-casted onto the surface of a gold electrode (AuE) and then dried in air to produce the graphite carbon nitride-modified gold converter (AuE-g-C3N4), wherein the gold electrode (AuE) is washed with a predetermined solution and rinsed with distilled water before drop-casting, polished using a predetermined powder, and then ultrasonically treated in distilled water.

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