Biotin-linker-coupled graphene and use thereof

Abstract

The present invention relates to a biotin-linker-coupled graphene and, specifically, to a biotin-linker-coupled graphene obtained by using an oligo(ethylene glycol) as a linker to bind an amine group and biotin, thereby forming a biotin-linker, and then binding same to a carboxyl group of graphene oxide. The biotin-linker-coupled graphene has fluorescence labeling efficiency superior to that of conventional graphene, exhibits excellent binding to a functional group and has improved dispersibility and cohesiveness, and thus can be effectively used in drug delivery systems, biosensors, tissue engineering and the like in the biomedical field.

Description

Biotin-linker-bound graphene and its uses

[0001] The present invention relates to graphene bonded with a biotin-linker having improved dispersibility and cohesion, and uses thereof.

[0002] Graphene is an allotrope of carbon, a two-dimensional plane of carbon atoms. Oxidized graphene is a material obtained by oxidizing graphene. Graphene is thin and flexible, and boasts excellent mechanical, thermal, electrical, and optical properties, making it a promising candidate for future electronic materials. Due to these properties, graphene holds greater value and promise for practical applications than other carbon nanomaterials, such as fullerenes and carbon nanotubes. However, despite its excellent physical and chemical properties, graphene presents several challenges within a matrix. Its hydrophobic surface renders it insoluble in most solvents and matrices, and the electrical repulsion between graphene particles makes stable dispersion difficult. Furthermore, ππ stacking interactions between graphene layers tend to cause graphene sheets to clump together. These factors, combined, significantly reduce graphene dispersibility, resulting in irreversible agglomeration and sedimentation of graphene particles.

[0003] Various studies have been conducted to improve graphene aggregate formation, including mechanical exfoliation, graphene surface functionalization, and solvent modification. However, mechanical exfoliation presents limitations, including the difficulty of large-scale production, the high cost of exfoliation equipment, and the risk of defects in graphene during the exfoliation process. Surface functionalization, on the other hand, presents limitations, such as the complexity of chemical reactions and the potential for impurities. Furthermore, changing the specific solvent can affect the electrical and mechanical properties of graphene.

[0004] Biotin, also known as vitamin B7 or vitamin H, is a water-soluble vitamin that plays an important role in the body's metabolic processes, including the metabolism of fats, carbohydrates, and amino acids. Biotin is known to form very strong non-covalent bonds with avidin, streptavidin, and other proteins. It has a heterocyclic structure consisting of a ureido ring and a tetrahydrothiophene ring, with a valeric acid terminal group.

[0005] Against this backdrop, in the present invention, a biotin linker was attached to graphene oxide to increase the bioavailability of graphene and reduce interactions on the graphene surface, and as a result, the dispersibility and cohesion of graphene oxide were improved not only in deionized water (DI water) but also in physiological conditions and cell culture environments with high ionic strength and the presence of proteins, thereby completing the present invention.

[0006] [Previous literature]

[0007] [Patent Document]

[0008] (Patent Document 0001) Republic of Korea Publication Patent Document No. 10-2017-0126292 (November 17, 2017)

[0009] [Non-patent literature]

[0010] (Non-patent document 0001) Sabih Qamar et al, "Graphene dispersion, functionalization techniques and applications: A review", Synthetic Metals 307 (2024) 117697

[0011] (Non-patent document 0002) Vasilios Georgakilas et al, “Functionalization of Graphene: Covalent and Non-Covalent Approaches, Derivatives and Applications”, Chem Rev. 2012 Nov 14;112(11):6156-214.

[0012] An object of the present invention is to provide graphene having a biotin-linker bonded thereto.

[0013] Another object of the present invention is to provide a method for producing graphene with a biotin-linker bond, comprising the steps of: (a) covalently bonding biotin and a linker to produce a biotin-linker; and (b) covalently bonding the biotin-linker and graphene.

[0014] To achieve the above purpose, the present invention provides a graphene having a biotin-linker bonded thereto, comprising a structure represented by the following chemical formula 1.

[0015] [Chemical Formula 1]

[0016]

[0017] In the above chemical formula 1,

[0018] The above GO is graphene,

[0019] The above X is at least one selected from the group consisting of -NH, -H, -OH, -NH2, -COOH, -SH, -COOR, -CONH2, -PEG, -N3, -C≡ and -C6H5CH2,

[0020] The above L is a linker,

[0021] The above n is an integer between 1 and 10.

[0022] The present invention also provides a method for producing graphene with a biotin-linker bond, comprising the steps of: (a) covalently bonding biotin and a linker to produce a biotin-linker; and (b) covalently bonding the biotin-linker and graphene.

[0023] The graphene with biotin-linker bonded according to the present invention, unlike conventional graphene, maintains excellent dispersibility even in a physiological environment such as a cell culture medium and exhibits a significantly reduced aggregation phenomenon. In addition, compared to graphene to which functional groups are directly bonded without a linker, the graphene has excellent binding affinity with functional groups and improved fluorescent labeling efficiency, so it can be utilized as graphene with high potential for in vivo applications.

[0024] Figure 1 shows the results of analyzing nano-graphene oxide using X-ray diffractometer (XRD).

[0025] Figure 2 shows the results of analyzing nano-oxide graphene using X-ray photoelectron spectroscopy (XPS).

[0026] Figure 3 shows a reaction scheme for synthesizing a biotin complex (Biotin-DG-NH2) having an amine group at the terminal end using biotin and bis(3-aminopropyl)diethylene glycol and carbonyldiimidazole.

[0027] Figure 4 shows the results of analyzing a biotin complex (Biotin-DG-NH2) produced by synthesizing biotin and bis(3-aminopropyl)diethylene glycol using nuclear magnetic resonance (NMR).

[0028] Figure 5 shows a reaction scheme for synthesizing NXB using a biotin complex (Biotin-DG-NH2) and Nano Graphene Oxide through an EDC / NHS coupling reaction.

[0029] Figure 6 shows the results of analyzing the molecular bonds of NXB produced by synthesizing a biotin complex (Biotin-DG-NH2) and Nano Graphene Oxide using Fourier Transform Infra-red (FT-IR) spectroscopy.

[0030] Figure 7 shows the results of measuring the particle size distribution of NXB synthesized with nano graphene oxide using a centrifuge particle sizer (CPS).

[0031] Figure 8 shows the results of measuring the morphological image of nano graphene oxide using a spherical aberration corrector-transmission electron microscope (Cs-TEM).

[0032] Figure 9 shows the fluorescent labeling images and fluorescence signal intensity measurement results of the control group, nano graphene oxide (NGO), biotinylated NGO (NGO) directly bound to nano graphene oxide without a linker, and NXB as a bar graph.

[0033] Figure 10 shows the results of comparing the aggregation and precipitation formation of nano-oxidized graphene and biotin-linker-bound graphene in various culture media.

[0034] Hereinafter, the invention will be described in detail.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0036] When the present invention is said to “include” a certain component or a certain step, this does not mean that other components or other steps are excluded, but rather that other components or other steps may be further included, unless specifically stated otherwise.

[0037] The term "graphene" in the present invention refers to a material that is one of the carbon allotropes, in which carbon atoms are present at the vertices of a hexagon (sp2 bonding) and form a widely spread two-dimensional planar crystal structure in the shape of a hexagonal honeycomb. Graphene can exist as a stable structure as a film made of one atom thick.

[0038] In the present invention, the term "graphene oxide" may be abbreviated as "GO" and may include a structure in which a functional group containing an oxygen atom, such as a carboxyl group, a hydroxyl group, or an epoxy group, is bonded to graphene, but is not limited thereto.

[0039] The above graphene oxide may be obtained by a method known to those skilled in the art. The above graphene oxide may be produced from graphite by physical exfoliation, chemical vapor deposition, chemical exfoliation, or epitaxial growth, but is not limited thereto.

[0040] In the present invention, “graphene oxide variant” may mean a complex in which an additional substance / material is combined with graphene oxide to improve the dispersibility and cohesion of the graphene oxide, and specifically, it may mean a complex in which an additional substance / material such as a functional group, a metal particle, a magnetic particle, a nanoparticle, a polymer, a peptide, and / or a nucleic acid is combined / attached to the nano-sized graphene oxide through a physical van der Waals force bond, an ionic bond, a hydrogen bond, a covalent bond, and / or a non-covalent bond.

[0041] The above graphene oxide may have the form of a nanofilm, nanosheet, nanowire, nanorod, nanotube, pulverized nanowire, nanotetrapod, tripod, bipod, nanocrystal, nanodot, quantum wire, or nanoparticle.

[0042] The above graphene oxide may include graphene quantum dots (GQDs). Additionally, it may include graphene particles having a width, length, and height of several nm manufactured through appropriate processing, but is not limited thereto.

[0043] The structure and chemical properties of the above graphene oxide can be verified by X-ray diffraction, X-ray photoelectron spectroscopy, Raman spectroscopy, and Fourier transform infrared spectroscopy. X-ray diffraction is a technique that can confirm the structural information of a material such as chemical composition, crystal structure, crystal size, strain, preferred orientation, and layer thickness in a laboratory, and X-ray photoelectron spectroscopy is a sensitive quantitative spectroscopy technique based on the photoelectric effect that can identify not only elements existing within a material or covering the surface, but also the overall electronic structure and density within a material. In addition, Raman spectroscopy is a technique that can distinguish the number of layers of graphene or confirm the degree of structural defects, and Fourier transform infrared spectroscopy is a technique used to obtain an infrared spectrum absorbed or emitted from a solid, liquid, or gas.

[0044] Various residues such as carboxyl groups, carbonyl groups, epoxy groups, and hydroxyl groups may exist on the surface of the above nano-graphene oxide.

[0045] In the present invention, the nano-oxide graphene may be characterized by an average diameter of 1 to 100 nm, preferably 5 to 75 nm, and more preferably 10 to 50 nm, but is not limited thereto.

[0046] In the present invention, the average diameter refers to the average diameter in the lateral size of graphene oxide having a certain thickness, and the lateral size can be interpreted as the relatively longer length among the horizontal and vertical lengths when the length of graphene oxide having a certain thickness is measured based on a rectangular frame, or the longest distance among the distances connecting any two ends of graphene oxide having a certain thickness.

[0047] In the present invention, the nano-oxide graphene may be characterized by a thickness of 0.01 to 10 nm, preferably 0.1 to 10 nm, and more preferably 0.5 to 5 nm, but is not limited thereto.

[0048]

[0049] Graphene has the disadvantage of not being well dispersed in solvents and tending to aggregate due to its large surface area, van der Waals forces and interactions, electrical and mechanical properties damaged by functional groups present on the surface, and hydrophobic characteristics. Therefore, when applied to the body, there has been a problem that the unique characteristics of existing graphene are lost due to aggregation and toxicity may occur. In the present invention, in order to solve the above problems and minimize interactions on the graphene surface, a biotin-linker combined with an oligo(ethylene glycol) linker was combined with graphene, thereby confirming the effect of improving the dispersibility and aggregation of graphene.

[0050]

[0051] Accordingly, the present invention relates to a graphene having a biotin-linker bond, comprising a structure represented by the following chemical formula 1.

[0052] [Chemical Formula 1]

[0053]

[0054] In the above chemical formula 1,

[0055] The above GO is graphene,

[0056] The above X is at least one selected from the group consisting of -NH, -H, -OH, -NH2, -COOH, -SH, -COOR, -CONH2, -PEG, -N3, -C≡ and -C6H5CH2,

[0057] The above L is a linker,

[0058] The above n is an integer between 1 and 10, preferably an integer between 1 and 5, but is not limited thereto.

[0059] The above linker may be, but is not limited to, oligo(ethylene glycol).

[0060] When n is 11 or greater, the overall length of the molecule increases as the linker chain lengthens, leading to an increase in molecule size. This increase in size reduces intracellular permeability and may reduce efficiency in crossing the cell membrane or transporting the molecule into the cell. Furthermore, excessively high molecular flexibility may reduce binding stability with the target, resulting in reduced binding efficiency.

[0061] If biotin and NGO are directly bound without a linker, the proximity of the two substances limits the inherent physicochemical properties of the body and edge structures of NGO, which may reduce the efficiency of graphene in applications such as biosensing, drug delivery, and catalysis. Furthermore, this direct binding can cause steric hindrance that impedes the access of streptavidin or antibodies, thereby inhibiting the binding between biotin and streptavidin or the specific binding of anti-biotin antibodies. This could reduce the efficiency of biomolecule detection or targeted drug delivery systems utilizing the biotin-streptavidin system.

[0062] Furthermore, graphene inherently emits weak fluorescence, which can lead to fluorescence quenching when directly bound to a fluorescent substance. This quenching phenomenon can make it difficult to quantify the presence of intracellular graphene using fluorescence.

[0063] The above XLX may be at least one selected from the group consisting of 4,7,10-trioxa-1,13-tridecanediamine, bis(3-aminopropyl)diethylene glycol, polyethylene glycol (PEG) diamine, polyoxypropylene amine, bis(3-aminopropyl)-terminated polyethylene glycol, and polypropylene glycol bis(2-aminopropyl ether), and preferably 4,7,10-trioxa-1,13-tridecanediamine, but is not limited thereto. Doesn't.

[0064] When the above X is NH, the graphene to which the biotin-linker is bound can be represented by the following chemical formula 2:

[0065] [Chemical Formula 2]

[0066]

[0067] In the above chemical formula 2, n is an integer from 1 to 10, preferably an integer from 1 to 5, but is not limited thereto.

[0068] The graphene with the biotin-linker bonded according to the present invention was manufactured taking into account binding to streptavidin.

[0069] Streptavidin binding occurs through two main interactions. First, the NH and O atoms of the ureido ring of biotin form hydrogen bonds with amino acids within the streptavidin protein, and second, the nonpolar region of the thiophene ring interacts with the lipophilic pocket of streptavidin. Considering these binding characteristics, these two binding points should be avoided when modifying biotin. Therefore, the modification of biotin according to the present invention may be primarily performed at the end of the thiophene ring or the valeric acid terminal group, and preferably, but not limited to, at the valeric acid terminal group.

[0070] The above graphene may be at least one selected from the group consisting of, but is not limited to, graphene oxide, graphene oxide variants, and graphene quantum dots.

[0071] The above graphene oxide may be, but is not limited to, nano graphene oxide.

[0072] The above nano-graphene oxide may have an average of 1 to 3 layers and a diameter of 10 to 20 mm, but is not limited thereto.

[0073] The above graphene may have improved dispersibility and cohesion.

[0074] The above dispersibility may refer to the ability to uniformly distribute graphene particles within a solvent or matrix.

[0075] The above cohesion may refer to a phenomenon in which graphene particles clump together to form lumps.

[0076] The above-mentioned graphene to which the biotin-linker is bound may be formed by a peptide bond between a carboxyl group (-COOH) present on the graphene surface and an amine group (-NH) of the biotin-linker, but is not limited thereto.

[0077] The above peptide bond may be formed via an EDC / NHS mediated reaction, but is not limited thereto.

[0078] The above graphene may have a diameter of 0.1 to 100 nm and a thickness of 0.1 to 100 nm, preferably a diameter of 1 to 50 nm and a thickness of 0.5 to 50 nm, but is not limited thereto.

[0079] In the above biotin-linker-bound graphene, the ratio of biotin:L:GO may be 1:1:1 to 1:10:3, preferably 1:1:1 to 1:5:3, but is not limited thereto.

[0080] The above graphene to biotin linker may be combined in a ratio of 1:0.1 to 1:1, preferably in a ratio of 1:0.4 to 1:1, but is not limited thereto.

[0081]

[0082] The present invention also provides a method for producing graphene with a biotin-linker bond, comprising the steps of: (a) covalently bonding biotin and a linker to produce a biotin-linker; and (b) covalently bonding the biotin-linker and graphene.

[0083] The covalent bond of the above step (a) and the covalent bond of the above step (b) may be formed by at least one bond selected from the group consisting of a peptide bond, an amide bond, an ester bond, an imide bond, a cytronitrile bond, a thioester bond, an epoxy bond, a urethane bond, a Schulze bond, a disulfide bond, and a click chemistry bond, but is not limited thereto.

[0084] The linker of step (a) above may be oligo(ethylene glycol),

[0085] The above oligo(ethylene glycol) may be at least one selected from the group consisting of ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, heptaethylene glycol, octaethylene glycol, nonaethylene glycol, and decaethylene glycol, but is not limited thereto.

[0086]

[0087] The description of the above manufacturing method, its effects and all related descriptions are the same as those described above, so the description thereof is omitted to avoid excessive complexity of this specification due to duplicate description.

[0088]

[0089] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0090]

[0091] Example 1. Fabrication of nano-graphene oxide

[0092]

[0093] 1-1. Production of graphene oxide

[0094]

[0095] Graphite (Qingdao Kropfmuehl, China) was used as a carbon structure, and nano-graphene oxide was synthesized through the Couette-Tayler flow method, which generates a vortex in a container containing a graphite mixture to allow sulfuric acid to penetrate the graphite layers and promote a chemical reaction. Sulfuric acid (H2SO4), sodium nitrate (NaNO3), and potassium permanganate (KMnO4) below were all purchased from Samchun Pure Chemicals. Graphite (graphite) and sulfuric acid (H2SO4) were mixed in a ratio of 1:40 to 1:70, and sodium nitrate (NaNO3), which lowers the viscosity of the mixed solution and facilitates stirring, was mixed in a ratio of 1:0.2 to 1:0.7 to graphite (graphite). At this time, sulfuric acid (H2SO4) is inserted between the interlayer structures of the graphite, expanding the interlayer spacing.

[0096] The above mixture is mixed at 150 rpm to 500 rpm for 1 to 3 hours. Thereafter, the mixture and potassium permanganate (KMnO4) are mixed at a ratio of 1:5 to 1:15, and a rotational force of 1,200 rpm to 5,000 rpm is applied for 1 to 72 hours to promote interlayer expansion and separation of graphite.

[0097] At this time, appropriately managing the reaction time and rotational force through oxidation reaction control is crucial for controlling the interlayer spacing and producing single-layer graphene oxide. The graphene oxide produced through the above process is nanosized by applying ultrasonic waves of at least 10 W to 900 W. Nano-ization conditions vary depending on the size of the graphene oxide being nanosized.

[0098]

[0099] 1-2. Nano-firing of graphene oxide

[0100]

[0101] To enhance the frictional strength of the obtained graphene oxide, deionized water (DI water) or PBS solvent is added 50 to 100 times the amount of graphene oxide solids. A certain amount of graphene oxide mixed in the solvent is fed into a nano-forming device that uses a physical method to physically reduce the particle size of the graphene oxide. In addition, the ratio of each material applied when producing graphene oxide can act as a ratio that promotes the nano-forming of graphene oxide. In this experiment, nano-formed graphene oxide can be separated through a physical method in the nano-forming device. In addition, it can be separated by size through a centrifugation process. At this time, the centrifugation speed can be used by applying a rotation speed of 8,000 to 20,000 rpm depending on the size.

[0102]

[0103] Example 2. Confirmation of the structural and chemical properties of nano-graphene oxide.

[0104]

[0105] The structural and chemical properties of each nano-graphene oxide were verified using X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and Fourier-transform infrared spectroscopy (FT-IR), and the types of functional groups and bonds were confirmed. Furthermore, the nano-size and precision were confirmed using a particle size analysis method.

[0106] As a result of X-ray diffractometer (XRD) analysis, it was confirmed that the nano-graphene oxide of the present invention was made by splitting graphene oxide into small pieces of 30 nm or less using high energy, and thus a peak was formed at around 10° (10.56°), which is similar to the 2θ value that fundamentally appears in graphene oxide (Fig. 1).

[0107] X-ray photoelectron spectroscopy (XPS) analysis results showed that the XPS wide peaks of nano graphene oxide represented C=0, C-O-C, and C-C bonds, respectively, and that oxygen accounted for approximately 30% of the weight (Fig. 2).

[0108]

[0109] Example 3. Fabrication and characterization of graphene (NXB) bound to a biotin complex.

[0110]

[0111] To minimize the interaction between nano-graphene oxide and biotin, a linker was inserted between the nano-graphene oxide and biotin to bind them (Fig. 3). First, 4,7,10-Trioxa-1,13-tridecanediamine, in which an amine group is bonded to oligo(ethylene glycol), was used as a linker, and the amine group was bonded to the valeric acid terminal group of biotin at a ratio of 1 (linker):5 (biotin), thereby synthesizing Biotin-EG(Ethylene Glycol)n-NH2.

[0112]

[0113] The biotinylation reaction at this time was carried out by mixing biotin: 4,7,10-trioxa-1,13-tridecanediamine: CDI (N,N'-Carbonyl diimidazole): TEA (trimethylamine) = 1:5:1.3:3 in a ratio and stirring at room temperature for 16 hours under anhydrous conditions. After that, it was purified by precipitation using cold ether, and the results were analyzed by NMR (Fig. 4).

[0114] Biotin-NH2-(EG)n-NH2 is combined with the previously synthesized Biotin-NH2-(EG)n-NH2 through a peptide bond between the carboxyl groups formed on the nano-oxidized graphene. Nano-oxidized graphene was synthesized (Fig. 5). The reaction of binding biotin-(EG)n-NH2 to nano-oxidized graphene was carried out using EDC and NHS, which catalyzes amide group formation, at a mixing ratio of NHS:EDC:biotinylated amine-linker = 1:2:20. The molar ratio of biotin:graphene-binding linker was set to 1:5, and the concentration ratio was set to 1:4.48. Nano-oxidized graphene and this mixture were stirred at room temperature for 10 hours, purified using a dialysis membrane, and the results were analyzed using FT-IR (Fig. 6).

[0115] As a result of Fourier-transform infrared spectroscopy (FT-IR) analysis, the FT-IR peak of the nano graphene oxide of the present invention was CO (1045 cm -1 ), C=O(1630cm -1 , 1729cm -1 ), CH(2859cm -1 ) is observed (Fig. 6). In addition, it was confirmed that the OH peak broadened due to damage caused by high energy during the nano-process.

[0116] After classifying nano-graphene oxide and NXB by particle size using CPS (Centrifuge Particle Sizer) analysis, particle size distribution was determined. After preparing sucrose solution and dodecane at different concentrations, they were placed in CPS to create a concentration gradient. After confirming that the drift value was within -0.005 to 0.005, standard samples were placed and measured. Nano-graphene oxide and NXB were each analyzed for particle size using CPS (Fig. 7).

[0117] The particle sizes of nano graphene oxide and NXB were compared by comparing the overall dispersion and average particle size of nano graphene oxide and NXB. In CPS, D50 means the median of the particle, and the D50 of nano graphene oxide is 15.9 nm and the PDI value is 7.39 nm. The D50 of NXB is 17.1 nm and the PDI value is 13.08 nm. These values ​​do not show large significant figures in CPS, confirming that introducing a biotin complex (Biotin-DG-NH2) to nano graphene oxide does not have a critical effect on the particle size.

[0118] The morphological images of nano-graphene oxide and NXB were confirmed through spherical aberration-corrected transmission electron microscope (Cs-TEM) analysis (Fig. 8). The actual size of each particle was analyzed using IMAGEJ from the Cs-TEM images. The average particle size of nano-graphene oxide was measured to be 10–15 nm, and that of NXB was measured to be 8–12 nm.

[0119]

[0120] Example 4. Comparative experiment of fluorescent labeling of nano graphene oxide and NXB

[0121]

[0122] Graphene itself can emit weak fluorescence, and when a specific substance is directly bound to the graphene surface, the fluorescence may be reduced or quenched, which may cause a problem in that the signal of the fluorescent substance is reduced. In order to confirm whether the NXB according to the present invention can solve this problem, a fluorescent labeling experiment was performed to evaluate the effective binding of biotin and graphene in NXB and to confirm an increase in fluorescence labeling intensity compared to the existing nano-graphene oxide (Fig. 9). The experimental samples consisted of a control group (Control), NGO, biotinylated NGO (NGO) in which biotin was directly bound to nano-graphene oxide without a linker, and NXB in which biotin was bound to nano-graphene oxide as a linker. Each sample was observed in three images: nucleus, biotin, and merge. The nucleus image showed the cell nucleus in blue through DAPI staining, and similar nuclear staining was observed in all samples. For biotin imaging, a fluorescent antibody (Biotin antibody - conjugated to FITC) was used to confirm the appearance of a fluorescent signal from biotin bound to NGO. At this time, almost no fluorescence was observed in the control, NGO, and biotinylated NGO samples, whereas a fluorescent signal was detected in the NXB sample. In particular, the strongest fluorescent signal was observed in the NXB sample. The merged image shows the result of combining the nuclear and biotin signals, indicating that NXB has penetrated into the cell (cellular localization). When the fluorescence signal intensity of each sample was measured, the highest fluorescent signal was observed in NXB. This indicates that the binding affinity was enhanced in NXB, where biotin was bound to nano-graphene oxide via a linker, compared to biotinylated NGO, where biotin was directly bound to nano-graphene oxide without a linker, and that fluorescence quenching did not occur.Therefore, it can be confirmed that the biotin binding method using a linker most effectively increased the fluorescence signal intensity.

[0123]

[0124] Example 5. Comparative experiment on aggregation and precipitation of nano graphene oxide and NXB.

[0125]

[0126] To determine whether nano-graphene oxide and NXB formed sedimentation and agglomeration, the following four different solutions were evaluated as experimental groups (Fig. 10): DI water (triple-distilled water), PBS (phosphate buffer saline; Gibco, GIB-70011-069), FBS (Fetal Bovine Serum, Gibco, 10437-028), and DMEM (DMEM supplied with High Glucose; Hyclone, SH30243.FS). Nano-graphene oxide and NXB were added to each solution at a concentration of 10 μg / ml, and then cultured in an incubator (37°C, 5% CO2) for 24 hours. After incubation, the presence or absence of sedimentation and agglomeration was determined, and centrifugation was performed at a speed of 13,000 rpm in a centrifuge. Thereafter, the agglomeration and sedimentation formed at the bottom due to centrifugal force were observed.

[0127] Observations revealed that for daNGO (10 μg / mL), the solution remained clear under all conditions before incubation. After incubation, slight aggregation occurred in triple-distilled water, but the solution was relatively uniformly dispersed. In PBS, a somewhat clear precipitate formed, and the particles settled and appeared to be aggregated. Considerable aggregation and precipitation were observed in FBS, while distinct aggregation and precipitation were observed in DMEM.

[0128] In the case of NXB (10 μg / mL), the solution remained clear and transparent both before and after centrifugation in triple-distilled water, and no aggregation or precipitation was observed. Similarly, under PBS conditions, the solution remained clear before and after centrifugation, and no precipitation or aggregation was observed. Under FBS conditions, although some bubbles formed within the solution, the overall dispersion was stable, and no precipitation or aggregation was observed. Finally, under DMEM conditions, the solution maintained a uniform dispersion with a clear pink color, and no precipitation or aggregation was observed even after centrifugation.

[0129] In conclusion, nano-graphene oxide (daNGO) showed a high tendency for particle aggregation in physiological or nutrient-containing culture media such as PBS, FBS, and DMEM, which may result in poor dispersion stability, whereas NXB maintained stable particle dispersion under various culture conditions.

Claims

1. A graphene having a biotin-linker bond, comprising a structure represented by the following chemical formula 1: [Chemical Formula 1] In the above chemical formula 1, The above GO is graphene, The above X is -NH, -H, -OH, -NH 2 , -COOH, -SH, -COOR, -CONH 2 , -PEG, -N 3 , -C≡and -C 6 H 5 CH 2 At least one selected from the group consisting of, The above L is a linker, The above n is an integer between 1 and 10.

2. Graphene in the first paragraph, wherein the linker is oligo(ethylene glycol).

3. In the first paragraph, the XLX is at least one selected from the group consisting of 4,7,10-trioxa-1,13-tridecanediamine, Bis(3-aminopropyl)diethylene glycol, Polyethylene Glycol (PEG) Diamine, Polyoxypropylene amine, Bis(3-aminopropyl)-terminated Polyethylene Glycol, and Poly(propylene glycol) bis(2-aminopropyl ether). Graphene.

4. In the first paragraph, when X is NH, the graphene to which the biotin-linker is bound is represented by the following chemical formula 2: [Chemical formula 2] In the above chemical formula 2, n is an integer between 1 and 10.

5. In the first paragraph, the graphene is at least one selected from the group consisting of graphene oxide, graphene oxide variants, and graphene quantum dots.

6. In the fifth paragraph, the graphene is nano-oxidized graphene.

7. In the first paragraph, the graphene has improved dispersibility and cohesion.

8. In the first paragraph, when X is NH, the graphene is formed by a peptide bond between a carboxyl group (-COOH) and an amine group (-NH) present on the graphene surface.

9. Graphene according to claim 8, wherein the peptide bond is formed through an EDC / NHS mediated reaction.

10. In the first paragraph, the graphene has a diameter of 0.1 to 100 nm and a thickness of 0.1 to 100 nm.

11. Graphene in the first paragraph, wherein the ratio of biotin:L:GO is 1:1:1 to 1:10:

3. 12.(a) a step of preparing a biotin-linker by covalently bonding biotin and a linker; and (b) a step of covalently bonding the biotin linker and graphene; A method for producing graphene comprising a biotin-linker bond.

13. A manufacturing method in claim 12, wherein the covalent bond of step (a) and the covalent bond of step (b) are formed by at least one bond selected from the group consisting of a peptide bond, an amide bond, an ester bond, an imide bond, a citronitrile bond, a thioester bond, an epoxy bond, a urethane bond, a Schultz bond, a disulfide bond, and a click chemical bond.

14. A manufacturing method in claim 12, wherein the linker in step (a) is oligo(ethylene glycol).

15. A manufacturing method in claim 14, wherein the oligo(ethylene glycol) is at least one selected from the group consisting of ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, heptaethylene glycol, octaethylene glycol, nonaethylene glycol, and decaethylene glycol.

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