Method for producing carbon nano-particles

Abstract

To provide a method capable of easily and quickly producing a carbon nanoparticle that can be dispersed in an organic solvent, and to provide a carbon nanoparticle that can be dispersed in an organic solvent.SOLUTION: A method of producing a carbon nanoparticle comprises heating an organic solvent in the presence of a strong acid or strong base catalyst to form the carbon nanoparticle. The carbon nanoparticle has a graphite structure, is soluble in the organic solvent, and has an average particle size of 5 to 1000 nm. The strong acid or strong base is preferably sulfuric acid, p-toluenesulfonic acid, or dimethylaminopyridine, and it is preferable to irradiate the organic solvent with microwaves to heat the organic solvent.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing carbon nanoparticles. Method

Background Art

[0002] Graphene quantum dots (GQDs) are expected to be used as fluorescent probes for bioimaging and medical materials because of their low toxicity, water solubility, and stable photochemical properties.

[0003] For example, Non-Patent Document 1 discloses a method for producing graphene quantum dots by thermally decomposing glutamic acid, which is a natural amino acid.

[0004] Non-Patent Document 2 discloses a method for producing graphene quantum dots by irradiating urea or thiourea with microwaves.

[0005] Non-Patent Document 3 discloses a method for producing graphene quantum dots by irradiating citric acid, which is one of the food additives, with microwaves.

[0006] Non-Patent Document 4 discloses a method for producing graphene quantum dots by irradiating aspartic acid with microwaves in an ammonium carbonate solution.

[0007] Patent Document 1 and Non-Patent Document 5 disclose a method for producing graphene quantum dots from an aqueous hydrogen peroxide solution of graphene oxide.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Non-Patent Documents

[0009]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0010] In the production methods of graphene quantum dots (GQDs) disclosed in Non-Patent Documents 1 to 5 and Patent Document 1, in each case, GQDs are synthesized in an aqueous solvent and prepared as an aqueous dispersion.

[0011] On the other hand, graphene quantum dots dispersed in an organic solvent can be mixed with a polymer to form a film or fiber, and can also be used as a dye. However, in order to disperse conventional graphene quantum dots as an aqueous dispersion in an organic solvent, it is necessary to perform surface modification or the like, and the production thereof is costly.

[0012] Therefore, an object of the present invention is to provide a method for producing carbon nanoparticles that can be produced simply and in a short time and can be dispersed in an organic solvent, and carbon nanoparticles that can be dispersed in an organic solvent. In this specification, "carbon nanoparticles" is a concept including "graphene quantum dots" and "carbon quantum dots".

Means for Solving the Problems

[0013] The present invention includes the following aspects.

[0014] [1] A method for producing carbon nanoparticles, comprising heating an organic solvent in the presence of a catalyst of a strong acid or a strong base to form carbon nanoparticles. [2] The method for producing carbon nanoparticles according to [1], wherein the strong acid or strong base is sulfuric acid, p-toluenesulfonic acid, or dimethylaminopyridine. [3] The method for producing carbon nanoparticles according to [1] or [2], wherein heating the organic solvent is heating the organic solvent by irradiating the organic solvent with microwaves. [4] The method for producing carbon nanoparticles according to any one of [1] to [3], wherein the organic solvent is an organic acid anhydride. [5] The method for producing carbon nanoparticles according to [4], wherein the organic acid anhydride is acetic anhydride. [6] The method for producing carbon nanoparticles according to any one of [1] to [3], wherein the organic solvent is acetone, acetonitrile, dimethylacetamide, or dimethylformamide.

[0015] [7] The method for producing carbon nanoparticles according to any one of [1] to [6], wherein the carbon nanoparticles are soluble in an organic solvent. [8] The method for producing carbon nanoparticles according to any one of [1] to [7], wherein the average particle diameter of the carbon nanoparticles is 5 to 1000 nm. [9] When the carbon nanoparticles are measured by Raman spectroscopy, the intensity I of the G band derived from the graphite structure G and the intensity I of the D band caused by defects in the graphite structure D The relative ratio (I D / I G ) is 0.3 to 0.9. The method for producing carbon nanoparticles according to any one of [1] to [8].

[10] When the carbon nanoparticles are measured by X-ray photoelectron spectroscopy (XPS) and quantified by the Shirley method from the obtained XPS C1s spectrum, the total amount of C-O and C=O is 60% or less with respect to the total amount of 100% of the amounts of C-C, C=C, C-O, and C=O. The method for producing carbon nanoparticles according to any one of [1] to [9].

[11] When the carbon nanoparticles are measured by Fourier transform infrared spectroscopy, 2900 cm -1A peak of C-H bond appears in the vicinity, and a peak of O-H bond does not appear in the vicinity of 3300 cm -1 The method for producing carbon nanoparticles according to any one of [1] to

[10] , wherein a peak of O-H bond does not appear in the vicinity.

[12] The method for producing carbon nanoparticles according to any one of [1] to

[11] , wherein the organic solvent does not contain N atoms and S atoms.

[0016]

[13] Carbon nanoparticles having a graphite structure, soluble in an organic solvent, and having an average particle diameter of 5 to 1000 nm.

[14] When the carbon nanoparticles are measured by Raman spectroscopy, the intensity I of the G band derived from the graphite structure G The relative ratio (I D / I D / I G ) of the intensity I of the D band caused by defects in the graphite structure is 0.3 to 0.9. The carbon nanoparticles according to

[13] .

[15] When the carbon nanoparticles are measured by X-ray photoelectron spectroscopy (XPS) and quantified by the Shirley method from the obtained XPS C1s spectrum, the total amount of C-C, C=C, C-O, and C=O amounts is 100%. The carbon nanoparticles according to

[13] or

[14] , wherein the total amount of C-O and C=O amounts is 60% or less.

[16] When the carbon nanoparticles are measured by Fourier transform infrared spectroscopy, a peak of C-H bond appears in the vicinity of 2900 cm -1 and a peak of O-H bond does not appear in the vicinity of 3300 cm -1 The carbon nanoparticles according to any one of

[13] to

[15] , wherein a peak of O-H bond does not appear in the vicinity.

[17] The carbon nanoparticles according to any one of

[13] to

[16] , wherein the carbon nanoparticles do not contain N atoms and S atoms.

[18] An antibacterial agent containing the carbon nanoparticles according to any one of

[13] to

[17] .

[19] An antiviral agent containing the carbon nanoparticles according to any one of

[13] to

[17] .

[20] A composite film containing the carbon nanoparticles according to any one of

[13] to

[17] and a polymer.

[21] Composite nanoparticles containing the carbon nanoparticles according to any one of

[13] to

[17] and a polymer.

Advantages of the Invention

[0017] According to the present invention, it is possible to provide a method for producing carbon nanoparticles dispersible in an organic solvent, which can be produced simply and in a short time, and carbon nanoparticles dispersible in an organic solvent.

Brief Description of the Drawings

[0018]

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Mode for Carrying Out the Invention

[0019] <Method for Producing Carbon Nanoparticles> The method for producing carbon nanoparticles of the present invention includes heating an organic solvent in the presence of a catalyst of strong acid or strong base to form carbon nanoparticles.

[0020] When an organic solvent is heated in the presence of a catalyst of strong acid or strong base, nanoparticles containing carbon in the organic solvent precipitate, and the reaction solution changes from a transparent state to an opaque brown color. Since the method for producing carbon nanoparticles of the present invention uses an organic solvent as a carbon source, carbon nanoparticles dispersible in the organic solvent can be produced simply and in a short time.

[0021] The carbon nanoparticles are not limited as long as they are nanoparticles containing carbon, and may be graphene quantum dots containing a graphene structure or carbon quantum dots.

[0022] Examples of the strong acid include sulfuric acid, p-toluenesulfonic acid, hydrochloric acid, nitric acid, etc., and sulfuric acid or p-toluenesulfonic acid is preferred.

[0023] Examples of the strong base include dimethylaminopyridine and the like.

[0024] The strong acid or strong base is used as a catalyst, and it is sufficient that there is an amount that functions as a catalyst. The catalyst concentration is preferably 0.1 to 1000 mmol / L, more preferably 1 to 500 mmol / L, still more preferably 2 to 200 mmol / L, and particularly preferably 5 to 50 mmol / L.

[0025] The organic solvent refers to an organic compound that is liquid at normal temperature and pressure. As the organic solvent, a polar solvent is preferred. Examples of the polar solvent include protic polar solvents and aprotic polar solvents. As the protic polar solvent, alcohol solvents such as methanol and ethanol are preferred. Examples of the aprotic polar solvent include ketone solvents such as acetone and methyl ethyl ketone, amide solvents such as dimethylacetamide and dimethylformamide, acetonitrile, and organic acid anhydrides that are liquid at normal temperature and pressure. It is preferable that the organic solvent is acetone, acetonitrile, dimethylacetamide, or dimethylformamide. As the organic solvent, an organic acid anhydride that is liquid at normal temperature and pressure is preferred, and an aliphatic carboxylic acid anhydride that is liquid at normal temperature and pressure is more preferred. An organic solvent that does not contain N atoms and S atoms is preferred.

[0026] At normal temperature and pressure, examples of liquid aliphatic carboxylic acid anhydrides include acetic anhydride (melting point: -73°C, boiling point: 140.0°C), propionic anhydride, isobutyric anhydride, butyric anhydride, pivalic anhydride, isovaleric anhydride, valeric anhydride, hexanoic anhydride, heptanoic anhydride, methacrylic anhydride, acrylic anhydride, etc. Among them, acetic anhydride is preferred as the liquid aliphatic carboxylic acid anhydride at normal temperature and pressure.

[0027] In this specification, "normal temperature" means a temperature without particular cooling or heating, that is, the ordinary temperature, for example, a temperature of 15 to 25°C. In this specification, "normal pressure" refers to the pressure in a state without pressurization or depressurization.

[0028] The heating temperature is preferably 80 to 200°C, more preferably 90 to 190°C, further preferably 100 to 180°C, still further preferably 110 to 170°C, and particularly preferably 120 to 160°C.

[0029] Since the heating time depends on the reaction temperature and catalyst concentration, it is not particularly limited. The heating time is preferably 30 to 240 minutes, more preferably 40 to 200 minutes, further preferably 50 to 160 minutes, and particularly preferably 60 to 120 minutes.

[0030] The means for heating the organic solvent is not limited. Heating by microwave irradiation is preferred.

[0031] Since the method for producing carbon nanoparticles of the present invention forms carbon nanoparticles by heating an organic solvent, the obtained carbon nanoparticles can be dispersed in the organic solvent used as the raw material. The carbon nanoparticles obtained from the method for producing carbon nanoparticles of the present invention are preferably soluble in an organic solvent. The fact that the carbon nanoparticles are soluble in an organic solvent means that when the carbon nanoparticles are dispersed in any one of common organic solvents, they are transparently dispersed. When the carbon nanoparticles are soluble in an organic solvent, the organic solvent may be the organic solvent used as a raw material, or may not be the organic solvent used as a raw material. The carbon nanoparticles obtained from the method for producing carbon nanoparticles of the present invention are preferably water-insoluble. By being water-insoluble, the carbon nanoparticles obtained by dispersing in an organic solvent can be easily washed with water.

[0032] The average particle diameter of the carbon nanoparticles obtained from the method for producing carbon nanoparticles of the present invention is preferably 5 to 1000 nm, more preferably 10 to 500 nm, and even more preferably 20 to 200 nm. Here, the average particle diameter of the carbon nanoparticles is determined from the number average of the equivalent diameters of the projected areas of the particles by image processing of the STEM image of the carbon nanoparticles.

[0033] The method for producing carbon nanoparticles of the present invention can produce carbon nanoparticles having a larger average particle diameter than conventional graphene quantum dots simply and in a short time.

[0034] When the carbon nanoparticles obtained from the method for producing carbon nanoparticles of the present invention are measured by Raman spectroscopy, the intensity I G of the D band caused by the defects of the graphite structure with respect to the intensity I D of the G band derived from the graphite structure, the relative ratio (I D / I G ) is preferably 0.3 to 0.9, more preferably 0.4 to 0.8, and even more preferably 0.5 to 0.7.

[0035] Generally, when graphene quantum dots are measured by X-ray photoelectron spectroscopy (XPS), peaks assigned to C=C and C-C around 285 eV, a peak assigned to C-O around 286 eV, and a peak assigned to C=O around 289 eV are observed in the XPS C1s spectrum. When quantified by the Shirley method from the XPS C1s spectrum of conventional graphene quantum dots, the total amount of C-O and C=O is usually about 80% with respect to 100% of the total amount of C-C, C=C, C-O, and C=O amounts.

[0036] When the carbon nanoparticles obtained from the method for producing carbon nanoparticles of the present invention are measured by X-ray photoelectron spectroscopy (XPS) and quantified by the Shirley method from the obtained XPS C1s spectrum, it is preferable that the total amount of C-O and C=O is 60% or less, more preferably 50% or less, and even more preferably 40% or less with respect to 100% of the total amount of C-C, C=C, C-O, and C=O amounts.

[0037] Usually, when graphene quantum dots are measured by Fourier transform infrared spectroscopy (FT-IR), a peak of an O-H bond appears around 3300 cm -1 and a peak of a C-H bond does not appear around 2900 cm -1

[0038] When the carbon nanoparticles obtained from the method for producing carbon nanoparticles of the present invention are measured by Fourier transform infrared spectroscopy (FT-IR), it is preferable that a peak of a C-H bond appears around 2900 cm -1 and a peak of an O-H bond does not appear around 3300 cm -1

[0039] <Carbon Nanoparticles> The carbon nanoparticles of the present invention have a graphite structure and are soluble in organic solvents. The carbon nanoparticles of the present invention can be obtained from the method for producing the carbon nanoparticles.

[0040] ​​The graphite structure of the carbon nanoparticles can be confirmed by detecting the peak of the G band derived from the graphite structure when the carbon nanoparticles are measured by Raman spectroscopy. The explanation for the solubility in organic solvents is the same as described above.

[0041] The carbon nanoparticles of the present invention have an average particle diameter of 5 to 1000 nm. The average particle diameter of the carbon nanoparticles of the present invention is preferably 10 to 500 nm, and more preferably 20 to 200 nm. Here, the average particle diameter of the carbon nanoparticles is determined from the number average of the equivalent diameters of the projected areas of the particles by image processing of the STEM image of the carbon nanoparticles.

[0042] When the carbon nanoparticles of the present invention are measured by Raman spectroscopy, the intensity I of the G band derived from the graphite structure G of the intensity I of the D band caused by the defects of the graphite structure D relative ratio (I D / I G ) is preferably 0.3 to 0.9, more preferably 0.4 to 0.8, and even more preferably 0.5 to 0.7.

[0043] When the carbon nanoparticles of the present invention are measured by X-ray photoelectron spectroscopy (XPS) and quantified by the Shirley method from the obtained XPS C1s spectrum, the total amount of the C-O amount and the C=O amount is preferably 60% or less, more preferably 50% or less, and even more preferably 40% or less with respect to the total amount of 100% of the C-C amount, the C=C amount, the C-O amount, and the C=O amount.

[0044] When the carbon nanoparticles of the present invention are measured by Fourier transform infrared spectroscopy, a peak of the C-H bond appears in the vicinity of 2900 cm -1 and it is preferable that a peak of the O-H bond does not appear in the vicinity of 3300 cm -1 .

[0045] The carbon nanoparticles of the present invention preferably do not contain N atoms and S atoms.

[0046] <Antibacterial agent> The antibacterial agent of the present invention contains the carbon nanoparticles of the present invention described above. The form of the antibacterial agent is not limited, and a form of a dispersion liquid in which carbon nanoparticles are dispersed in a solvent can be adopted, or a form of a composite film or composite nanoparticles described later can also be adopted. The antibacterial agent of the present invention has antibacterial activity against Escherichia coli, Salmonella, Staphylococcus aureus, etc.

[0047] <Antiviral agent> The antiviral agent of the present invention contains the carbon nanoparticles of the present invention described above. The form of the antiviral agent is not limited, and a form of a dispersion liquid in which carbon nanoparticles are dispersed in a solvent can be adopted, or a form of a composite film or composite nanoparticles described later can also be adopted.

[0048] <Composite film> The composite film of the present invention contains the carbon nanoparticles of the present invention described above and a polymer. As the polymer, a biodegradable polymer is preferable. Examples of the biodegradable polymer include polylactic acid (PLLA), polycaprolactone (PCL), polylactic acid-glycolic acid copolymer (PLGA), silk fibroin (SF), and the like. Since the carbon nanoparticles of the present invention are soluble in an organic solvent, the composite film can be easily produced by dispersing the carbon nanoparticles and the polymer in an organic solvent to form a solution, applying this solution to a substrate, and drying it.

[0049] <Composite nanoparticles> The composite nanoparticles of the present invention contain the carbon nanoparticles of the present invention described above and a polymer. As the polymer, it is the same as the polymer described above. Since the carbon nanoparticles of the present invention are soluble in an organic solvent, the composite nanoparticles can be easily produced by dispersing the carbon nanoparticles and the polymer in an organic solvent to form a solution and then dispersing it in water.

[0050] The average particle diameter of the composite nanoparticles of the present invention measured by the dynamic light scattering method can be 50 to 4000 nm, can be 100 to 2000 nm, and can be 200 to 1000 nm. The polydispersity index of the composite nanoparticles of the present invention measured by the dynamic light scattering method can be 0.05 to 0.40, can be 0.08 to 0.30, and can be 0.10 to 0.20.

Example

[0051] Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the examples shown below.

[0052] [Example 1] 10 μL of 98% sulfuric acid was added to 20 mL of acetic anhydride, and the temperature was raised from room temperature to 180°C in 5 minutes while irradiating with microwaves using a microwave synthesizer (Monowave300) manufactured by Anton Paar, and 180°C was maintained for 60 minutes. The catalyst concentration of sulfuric acid in the reaction solution was 9 mmol / L. The reaction solution changed from a transparent state to an opaque brown color.

[0053] Thereafter, the reaction solution was mixed with twice the volume of chloroform to obtain a uniform mixed solution. Further, twice the volume of water was added, and the chloroform phase was washed in the order of 5% aqueous NaHCO3 solution, saturated brine, and magnesium sulfate in a separatory funnel to remove acetic anhydride and sulfuric acid in the mixed solution. Further, water was added to the chloroform phase and thoroughly mixed, but brown color remained in the chloroform phase. That is, the obtained brown dispersed particles were water-insoluble.

[0054] Images of chloroform-extracted brownish dispersed particles taken with a high-angle annular dark-field scanning transmission electron microscope (HAADF-STEM, a transmission electron microscope TECNAI F20 manufactured by Thermo Fisher Scientific), and the results of energy-dispersive X-ray analysis (EDX) are shown in Fig. 1. Fig. 1(A) is a low-magnification STEM image, Fig. 1(B) is a high-magnification STEM image, Fig. 1(C) is a graph of the results of EDX measurement of the particles, and Fig. 1(D) is a graph of the results of EDX measurement of the carbon grid.

[0055] By image-processing the low-magnification STEM image of Fig. 1(A), the number average of the equivalent circle diameters of the projected areas of 50 particles was determined. As a result, the average particle diameter of the dispersed particles was 50 nm. From these results in Fig. 1, the brownish dispersed particles could be said to be carbon nanoparticles.

[0056] The average particle diameter of conventional graphene quantum dots is approximately less than 10 nm. The carbon nanoparticles of Example 1 have a larger average particle diameter than conventional graphene quantum dots.

[0057] In the EDX results of Fig. 1, neither N atoms nor S atoms were detected in the carbon nanoparticles of Example 1. In addition to the fact that acetic anhydride as a raw material is an organic solvent that does not contain N atoms and S atoms, it was confirmed that the catalyst sulfuric acid could be washed and removed.

[0058] The carbon nanoparticles of Example 1 could be prepared as a chloroform dispersion, so they can be mixed with a polymer to form a film or fiber, or can also be easily used as a dye.

[0059] (Fluorescence measurement) When the chloroform extract was diluted 50-fold with chloroform, it became a pale orange transparent solution. When ultraviolet light of 365 nm was irradiated on this chloroform solution of the carbon nanoparticles of Example 1 using a UV transilluminator device (model number 900-1211-02) manufactured by Ultra-Lum, it was visually confirmed that yellow-green fluorescence was emitted. Also, regarding the chloroform solution of the carbon nanoparticles of Example 1, the results of the fluorescence spectrum (excitation wavelength: 360 nm) measured with a spectrofluorophotometer (FP-6600 manufactured by JASCO Corporation) are shown in Fig. 2, and the results of three-dimensional fluorescence mapping are shown in Fig. 3. From these results, it was found that fluorescence with the maximum intensity was emitted in the vicinity of wavelengths of 490 to 510 nm at excitation wavelengths of 410 to 460 nm.

[0060] (Ultraviolet-visible absorption spectrum) The ultraviolet-visible absorption spectrum when measured with a UV / VIS spectrophotometer (V670 manufactured by JASCO Corporation) is shown in Fig. 4. It was found that the carbon nanoparticles of Example 1 absorb light near 300 nm.

[0061] (Raman spectrum) The chloroform extract of the carbon nanoparticles of Example 1 was cast on a glass plate and dried, and the powder of the carbon nanoparticles of Example 1 was collected from the glass plate. The Raman spectrum measured by Raman spectroscopy for the powder of the carbon nanoparticles of Example 1 using a microscopic Raman spectrometer XploRA (registered trademark) manufactured by HORIBA is shown in Fig. 5. The peak of the G band derived from the graphite structure and the peak of the D band caused by the defects of the graphite structure were observed. The intensity I of the G band derived from the graphite structure G with respect to the intensity I of the D band caused by the defects of the graphite structure D of the relative ratio (I D / I G ) indicates the degree of structural defects. The relative ratio (I D / I G ) of the carbon nanoparticles of Example 1 was 0.609. It was found that the carbon nanoparticles of Example 1 mainly have a graphene structure. Note that this Raman spectrum was similar to that of a known water-soluble graphene quantum dot.

[0062] (Fourier transform infrared spectroscopy (FT-IR) spectrum) Regarding the powder of the carbon nanoparticles of Example 1, the FT-IR spectrum measured by Fourier transform infrared (FT-IR) spectroscopy using an FT / IR-4200 apparatus manufactured by JASCO Corporation was shown in Fig. 6. Reference Example 1 is the FT-IR spectrum of graphene quantum dots (GQDs) obtained by a method prepared from citric acid (cited from Non-Patent Document 1).

[0063] In the FT-IR spectrum of Reference Example 1, a peak of the O-H bond appeared near 3300 cm -1 On the other hand, in the FT-IR spectrum of the carbon nanoparticles of Example 1, a peak of the C-H bond appeared near 2900 cm -1 and a peak of the O-H bond did not appear near 3300 cm -1

[0064] (X-ray photoelectron spectroscopy (XPS) C1s spectrum) Regarding the powder of the carbon nanoparticles of Example 1, the XPS C1s spectrum obtained by measurement using an X-ray photoelectron spectroscopy (XPS) apparatus (SigmaProbe) manufactured by Thermo Electron Corporation was shown in Fig. 7.

[0065] In the XPS C1s spectrum of the carbon nanoparticles of Example 1, peaks assigned to C=C and C-C near 285 eV, a peak assigned to C-O near 286 eV, and a peak assigned to C=O near 289 eV were observed.

[0066] When quantified by the Shirley method from the XPS C1s spectrum, the total amount of the C-O amount and the C=O amount was 20% with respect to 100% of the total amount of the C-C amount, the C=C amount, the C-O amount, and the C=O amount.

[0067] ​In Non-Patent Document 5, for the prepared graphene quantum dots obtained from graphene oxide, the amount of C=C was 18.6%, the amount of C-O was 66.7%, and the amount of C=O was 14.8%. With respect to the total amount of 100% of the amount of C-C, C=C, C-O, and C=O, the total amount of C-O and C=O was 81.4%. It was confirmed that the total amount of C-O and C=O of the carbon nanoparticles of Example 1 was less than that of conventional graphene quantum dots.

[0068] The powder of the carbon nanoparticles of Example 1 was dispersed in each organic solvent of tetrahydrofuran (THF), acetone, acetonitrile, methanol, dimethylformamide (DMF), and chloroform. As a result, all the dispersions became thin yellow transparent solutions. When these dispersion solutions were irradiated with ultraviolet light of 365 nm, they all glowed yellow-green.

[0069] [Example 2] 50 μL of 98% sulfuric acid was added to 20 mL of acetic anhydride, and the mixture was heated under reflux for 1 hour. The catalyst concentration of sulfuric acid in the reaction solution was 45 mmol / L. The reaction solution changed from transparent to black opaque.

[0070] Thereafter, water in the same volume was added to the reaction solution and mixed to obtain the mixed solution of Example 2. This mixed solution was put into a dialysis membrane (Spectra / Pro manufactured by REPLIGEN) with a molecular weight cut-off of 3500 and dialyzed for 3 days. During dialysis, the mixed solution became turbid and precipitates were observed. After 3 days of dialysis, the precipitate and the turbid solution were freeze-dried to obtain 1.2 mg of the powder of Example 2 (that is, carbon nanoparticles).

[0071] For the powder of the carbon nanoparticles of Example 2, Raman spectroscopy measurement was performed. The Raman spectrum is shown in FIG. 10. As a result, the relative ratio (I D / I G ) of the carbon nanoparticles of Example 2 was 0.65. It was found that the carbon nanoparticles of Example 2 mainly had a graphene structure.

[0072] In the XPS C1s spectrum of the carbon nanoparticles of Example 2, peaks assigned to C=C and C-C were observed around 285 eV, a peak assigned to C-O around 286 eV, and a peak assigned to C=O around 289 eV.

[0073] When quantified by the Shirley method from the XPS C1s spectrum, the total amount of C-O and C=O was 35.3% with respect to 100% of the total amount of C-C, C=C, C-O, and C=O.

[0074] [Example 3] 50 μL of 98% sulfuric acid was added to 20 mL of acetic anhydride, and microwave irradiation was performed at 180 °C for 1 hour using a microwave synthesizer manufactured by Anton Paar. The catalyst concentration of sulfuric acid in the reaction solution was 45 mmol / L.

[0075] Thereafter, in the same manner as in Example 2, a mixed solution was prepared, and the mixed solution of this Example 3 was dialyzed for 3 days using a dialysis membrane with a molecular weight cut-off of 3500. During dialysis, the mixed solution became turbid and precipitates were observed. After 3 days of dialysis, the precipitate and the turbid solution were freeze-dried to obtain 106 mg of the powder of Example 3 (i.e., carbon nanoparticles).

[0076] Regarding the powder of the carbon nanoparticles of Example 3, Raman spectroscopy was measured. The Raman spectrum is shown in FIG. 10. As a result, the relative ratio (I D / I G ) of Example 3 was 0.75. It was found that the carbon nanoparticles of Example 3 mainly had a graphene structure, but had more defects in the graphite structure than the carbon nanoparticles of Example 2.

[0077] In the XPS C1s spectrum of the carbon nanoparticles of Example 3, peaks assigned to C=C and C-C were observed around 285 eV, a peak assigned to C-O around 286 eV, and a peak assigned to C=O around 289 eV.

[0078] When quantified by the Shirley method from the XPS C1s spectrum, the total amount of C - O and C = O was 42.9% with respect to 100% of the total amount of C - C, C = C, C - O, and C = O amounts.

[0079] [Example 4] 10 μL of 98% sulfuric acid was added to 20 mL of acetic anhydride, and microwave irradiation was carried out at 180 °C for 1 hour using a microwave synthesizer manufactured by Anton Paar. The catalyst concentration of sulfuric acid in the reaction solution was 9 mmol / L. The reaction solution changed from a transparent state to an opaque brown color.

[0080] Thereafter, in the same manner as in Example 2, a mixed solution was prepared, and the mixed solution of this Example 4 was dialyzed with a dialysis membrane having a molecular weight cut - off of 3500 for 3 days. During dialysis, the mixed solution became turbid and precipitates were observed. After 3 days of dialysis, the precipitate and the turbid solution were freeze - dried to obtain 10 mg of the powder of Example 4 (that is, carbon nanoparticles).

[0081] Regarding the powder of the carbon nanoparticles of Example 4, Raman spectroscopy measurement was performed. The Raman spectrum is shown in FIG. 10. The relative ratio (I D / I G ) of the carbon nanoparticles of Example 4 was 0.63. It was found that the carbon nanoparticles of Example 4 mainly had a graphene structure.

[0082] In the XPS C1s spectrum of the carbon nanoparticles of Example 4, peaks assigned to C = C and C - C were observed near 285 eV, a peak assigned to C - O was observed near 286 eV, and a peak assigned to C = O was observed near 289 eV.

[0083] When quantified by the Shirley method from the XPS C1s spectrum, the total amount of C - O and C = O was 38.8% with respect to 100% of the total amount of C - C, C = C, C - O, and C = O amounts.

[0084] From the results of Examples 2 to 4 above, the yield of carbon nanoparticles was in the order of Example 3 > Example 4 > Example 2, and the amounts of C-O and C=O were also larger in the order of Example 3 > Example 4 > Example 2. It was found that the higher the yield of carbon nanoparticles, the more the C=C and C-C bonds decreased and the C-O and C=O bonds increased. It is considered that the smaller the C-O and C=O bonds, the easier it is to disperse in an organic solvent.

[0085] By using a microwave device, it becomes possible to react at a temperature higher than the boiling point, and it is considered that more carbon nanoparticles were synthesized. Also, it was found that the more the amount of the catalyst sulfuric acid, the more carbon nanoparticles were synthesized. Therefore, among the carbon nanoparticles of Examples 2 to 4, it is considered that the reaction rate of the carbon nanoparticles of Example 3 was the highest and the reaction rate of the carbon nanoparticles of Example 2 was the lowest.

[0086] (Fluorescence measurement) When the carbon nanoparticles of Examples 2 to 4 were dispersed in chloroform, all became pale orange solutions. When these solutions were irradiated with ultraviolet light of 365 nm using an Ultra-Lum UV transilluminator device (model number 900-1211-02), all emitted green light. Also, the results of the fluorescence spectrum (excitation wavelength 340 to 520 nm) measured with a spectrofluorometer (FP-6600 manufactured by JASCO Corporation) for the chloroform solution of the carbon nanoparticles of Example 3 are shown in Fig. 8. It was found that when irradiated with excitation light of 440 nm, it emits maximum fluorescence of 506 nm. Similar fluorescence spectra were also confirmed for the chloroform solutions of the carbon nanoparticles of Examples 2 and 4.

[0087] (Absorption spectrum of ultraviolet-visible light) For the chloroform solutions of the carbon nanoparticles of Examples 2 to 4, when measured with a UV / VIS spectrophotometer (V670 manufactured by JASCO Corporation), the absorption spectra of ultraviolet-visible light are shown in Fig. 9. It was found that the light around 270 nm and around 320 nm was absorbed. Also, while the absorption peak around 270 nm was the same, it was found that the higher the reaction rate (Example 3 > Example 4 > Example 2), the larger the absorption peak around 320 nm. Since the absorption peak around 320 nm has been reported to be the n-π * transition of the carbon-oxygen bond, it is considered that the carbon nanoparticles of Example 3 may have more carbon-oxygen bonds.

[0088] [Example 5] 5 μL of 98% sulfuric acid was added to 10 mL of acetone, and microwave irradiation was performed at 140 °C for 1 hour using a microwave synthesizer manufactured by Anton Paar. The catalyst concentration of sulfuric acid in the reaction solution was 9 mmol / L. It was confirmed that carbon nanoparticles were generated when the reaction solution changed from transparent to opaque brown.

[0089] [Comparative Example 1] For 10 mL of acetone, microwave irradiation was performed at 140 °C for 1 hour using a microwave synthesizer manufactured by Anton Paar. The reaction solution remained transparent and did not emit fluorescence even when irradiated with ultraviolet light of 365 nm.

[0090] [Example 6] 5 μL of 98% sulfuric acid was added to 10 mL of dimethylacetamide, and microwave irradiation was performed at 180 °C for 1 hour using a microwave synthesizer manufactured by Anton Paar. The catalyst concentration of sulfuric acid in the reaction solution was 9 mmol / L. It was confirmed that carbon nanoparticles were generated when the reaction solution changed from transparent to opaque brown.

[0091] [Comparative Example 2] For 10 mL of dimethylacetamide, microwave irradiation was performed at 180 °C for 1 hour using a microwave synthesizer manufactured by Anton Paar. The reaction solution remained almost transparent and hardly emitted fluorescence even when irradiated with ultraviolet light of 365 nm.

[0092] [Example 7] 5 μL of 98% sulfuric acid was added to 10 mL of dimethylformamide, and microwave irradiation was performed at 180 °C for 1 hour using a microwave synthesizer manufactured by Anton Paar. The catalytic concentration of sulfuric acid in the reaction solution was 9 mmol / L. It was confirmed that carbon nanoparticles were generated by the change of the reaction solution from transparent to opaque brown.

[0093] [Comparative Example 3] For 10 mL of dimethylformamide, microwave irradiation was performed at 180 °C for 1 hour using a microwave synthesizer manufactured by Anton Paar. The reaction solution remained almost transparent and hardly emitted fluorescence even when irradiated with ultraviolet light of 365 nm.

[0094] [Example 8] 5 mg of dimethylaminopyridine was added to 10 mL of acetic anhydride, and microwave irradiation was performed at 180 °C for 1 hour using a microwave synthesizer manufactured by Anton Paar. It was confirmed that carbon nanoparticles were generated by the change of the reaction solution from transparent to opaque brown.

[0095] (Fluorescence measurement) Thereafter, the reaction solution was mixed with 2 times the volume of chloroform to obtain a uniform mixed solution. Further, 2 times the volume of water was added, and the chloroform phase was washed in the order of 5% aqueous NaHCO3 solution, saturated brine, and magnesium sulfate in a separatory funnel to remove acetic anhydride and dimethylaminopyridine in the mixed solution.

[0096] When the chloroform extract was diluted 50 times with chloroform, it became a pale orange transparent solution. When the chloroform solution of the carbon nanoparticles of Example 8 was irradiated with ultraviolet light of 365 nm, it emitted yellow-green light.

[0097] [Example 9] 5 mg of p-toluenesulfonic acid was added to 10 mL of acetic anhydride, and microwave irradiation was carried out at 180 °C for 1 hour using a microwave synthesizer manufactured by Anton Paar. It was confirmed that carbon nanoparticles were generated when the reaction solution changed from transparent to opaque brown.

[0098] (Fluorescence measurement) Subsequently, the reaction solution was mixed with 2 volumes of chloroform to obtain a uniform mixed solution. Further, 2 volumes of water was added, and the chloroform phase was washed with 5% aqueous NaHCO3 solution, saturated brine, and magnesium sulfate in this order using a separatory funnel to remove acetic anhydride and p-toluenesulfonic acid in the mixed solution.

[0099] When the chloroform extract was diluted 50-fold with chloroform, it became a pale orange transparent solution. When the chloroform solution of the carbon nanoparticles of Example 9 was irradiated with ultraviolet light of 365 nm, it emitted yellow-green light.

[0100] [Examples 10 to 17] Except that the temperature reaching temperature of 180 °C in Example 1 was changed as shown in Table 1, microwave irradiation was carried out for 1 hour in the same manner as in Example 1. The catalyst concentration of sulfuric acid in the reaction solution was 9 mmol / L. The reaction solution was mixed with 2 volumes of chloroform to obtain a uniform mixed solution. Further, 2 volumes of water was added, and the chloroform phase was washed with 5% aqueous NaHCO3 solution, saturated brine, and magnesium sulfate in this order using a separatory funnel to remove acetic anhydride and sulfuric acid in the mixed solution.

[0101] In Examples 10 and 11, the chloroform phase became a pale orange transparent solution, and a chloroform solution of the carbon nanoparticles of Examples 10 and 11 could be prepared.

[0102] In Examples 12 and 13, the chloroform phase became dark orange, and a chloroform extract of the carbon nanoparticles of Examples 12 and 13 was prepared. When the chloroform extract was diluted 20-fold with chloroform, it became a pale orange transparent solution. These were used as the chloroform solutions of the carbon nanoparticles in Examples 12 and 13 and subjected to fluorescence measurement.

[0103] In Examples 14 to 17, the chloroform phase became brownish, and chloroform extracts of the carbon nanoparticles in Examples 14 to 17 were prepared. When the chloroform extract was diluted 50-fold with chloroform, it became a pale orange transparent solution. These were used as the chloroform solutions of the carbon nanoparticles in Examples 14 to 17 and subjected to fluorescence measurement.

[0104] (Fluorescence measurement) When these chloroform solutions of the carbon nanoparticles in Examples 10 to 17 were irradiated with ultraviolet light of wavelength 365 nm using an Ultra-Lum UV transilluminator device (model number 900-1211-02), it was visually confirmed that all of them emitted yellow-green fluorescence. The intensities of these yellow-green fluorescences were visually observed, and the fluorescence intensities were evaluated according to the following evaluation criteria. The results are shown in Table 1. ◎: The fluorescence intensity was very strong. ○: The fluorescence intensity was strong. △: The fluorescence intensity was weak.

[0105] [Table 1]

[0106] [Examples 18 to 27] Except that the catalyst concentration of sulfuric acid in Example 2 was changed as shown in Table 2, it was heated under reflux for 1 hour in the same manner as in Example 2. The reaction solution was mixed with 2 times the volume of chloroform to obtain a uniform mixed solution. Further, 2 times the volume of water was added, and the chloroform phase was washed in a separatory funnel with 5% aqueous NaHCO3 solution, saturated brine, and magnesium sulfate in this order to remove acetic anhydride and sulfuric acid in the mixed solution.

[0107] In Example 18, the chloroform phase became a light orange transparent solution, and a chloroform solution of the carbon nanoparticles of Example 18 could be prepared.

[0108] In Example 19, the chloroform phase became a dark orange color, and a chloroform extract of the carbon nanoparticles of Example 19 was prepared. When the chloroform extract was diluted 20-fold with chloroform, it became a light orange transparent solution. This was used as the chloroform solution of the carbon nanoparticles of Example 19 for fluorescence measurement.

[0109] In Examples 20 to 23, the chloroform phase became brown, and chloroform extracts of the carbon nanoparticles of Examples 20 to 23 were prepared. When the chloroform extracts were diluted 50-fold with chloroform, they became light orange transparent solutions. These were used as the chloroform solutions of the carbon nanoparticles of Examples 20 to 23 for fluorescence measurement.

[0110] In Example 24, a small amount of black lumps were observed in the reaction solution. The chloroform phase became brown, and a chloroform extract of the carbon nanoparticles of Example 24 was prepared. When the chloroform extract was diluted 50-fold with chloroform, it became a light orange transparent solution. This was used as the chloroform solution of the carbon nanoparticles of Example 24 for fluorescence measurement.

[0111] In Examples 25 to 27, a large amount of black lumps were observed in the reaction solution. The chloroform extracts became brown, and chloroform extracts of the carbon nanoparticles of Examples 25 to 27 were prepared. When the chloroform extracts were diluted 50-fold with chloroform, they became light orange transparent solutions. These were used as the chloroform solutions of the carbon nanoparticles of Examples 25 to 27 for fluorescence measurement.

[0112] (Fluorescence measurement) When the chloroform solutions of the carbon nanoparticles of Examples 18 to 27 were irradiated with ultraviolet light having a wavelength of 365 nm using a UV transilluminator device manufactured by Ultra-Lum, it was visually confirmed that all of them emitted yellow-green fluorescence. The intensities of this yellow-green fluorescence were visually observed, and the fluorescence intensity was evaluated according to the following evaluation criteria. The results are shown in Table 2. ◎: The fluorescence intensity was very strong. ○: The fluorescence intensity was strong. △: The fluorescence intensity was weak.

[0113]

Table 2

[0114] [Experimental Example 1] (Antibacterial Activity of Carbon Nanoparticles) Using the following Escherichia coli and medium, one colony of Escherichia coli was inoculated into the medium and cultured with shaking at 37°C for 16 hours to prepare an Escherichia coli solution. Escherichia coli: Strain K12, obtained from the National Institute of Technology and Evaluation. Medium: 10 g of sodium chloride (FUJIFILM Wako Pure Chemical Corporation), 10 g of tryptone (Nacalai Tesque, Inc.), and 5 g of yeast extract (Nacalai Tesque, Inc.) were dissolved in 1 L of water and sterilized at 121°C for 20 minutes using an autoclave device manufactured by Yamato Corporation.

[0115] The carbon nanoparticles of Example 1 were dispersed in dimethyl sulfoxide (DMSO) to prepare dispersions with carbon nanoparticle concentrations of 50 μg / mL, 100 μg / mL, 150 μg / mL, and 200 μg / mL. 10 μL of the carbon nanoparticle dispersion or 10 μL of dimethyl sulfoxide (DMSO) (control experiment: Control) was added to 1 mL of the Escherichia coli solution, and the mixture was cultured with shaking at 37°C for 6 hours. The absorbance of the Escherichia coli solution at a wavelength of 620 nm was measured every hour to evaluate the growth of Escherichia coli. The measurement results of the absorbance are shown in Fig. 11. When the concentration of the carbon nanoparticles reached 200 μg / mL, the growth of Escherichia coli was completely suppressed even after 6 hours. The antibacterial activity of the carbon nanoparticles against Escherichia coli was confirmed.

[0116] In addition, using Salmonella (LT2 strain, obtained from the National Institute of Technology and Evaluation) and Staphylococcus aureus (ATCC25923, obtained from the National Institute of Technology and Evaluation), when the same experiments as in the case of E. coli were conducted, similarly, the antibacterial activity of the carbon nanoparticles against Salmonella and Staphylococcus aureus in Example 1 was confirmed for the carbon nanoparticles.

[0117] [Experimental Example 2] (Antiviral Activity of Carbon Nanoparticles) Using the following bacteriophage Qβ and medium, bacteriophage Qβ was inoculated into the medium to prepare a bacteriophage Qβ solution (10 6 pfu / mL). Bacteriophage Qβ: Obtained from the National Institute of Technology and Evaluation. Medium: 1 g of peptone (Nacalai Tesque, Inc.) and 8.5 g of sodium chloride (FUJIFILM Wako Pure Chemical Corporation) were dissolved in 1 L of water and sterilized at 121 °C for 20 minutes using an autoclave device manufactured by Yamato.

[0118] The carbon nanoparticles (GQDs) of Example 1 were dispersed in dimethyl sulfoxide (DMSO) to prepare dispersions with carbon nanoparticle concentrations of 100 μg / mL and 200 μg / mL. 10 μL of these dispersions or 10 μL of dimethyl sulfoxide (DMSO) (control experiment: Control) was added to 1 mL of the bacteriophage Qβ solution, which is a virus that does not infect humans, and mixed, followed by shaking culture at 37 °C for 3 hours. Thereafter, bacteriophage Qβ was mixed with the host Escherichia coli (K12 strain, obtained from the National Institute of Technology and Evaluation) and cultured at 37 °C for 16 hours in a soft agar medium (the above medium with 0.33% by mass of agar powder added). Plaques lysed by the infection of bacteriophage Qβ were formed, and the number of bacteriophage Qβ was quantified by counting the number of plaques. Figure 12 is a graph showing the results of the antiviral activity evaluation of the carbon nanoparticles (GQDs).

[0119] As shown by the results in Fig. 12, for the control experiment, when the concentration of carbon nanoparticles (GQDs) was 100 μg / mL, bacteriophage Qβ decreased to nearly one-tenth, and when the concentration of carbon nanoparticles reached 200 μg / mL, bacteriophage Qβ decreased to nearly one-thousandth. Thus, the antiviral effect of carbon nanoparticles was confirmed.

[0120] 10 μL of the dispersion with a carbon nanoparticle (GQDs) concentration of 200 μg / mL in Example 1, or 10 μL of dimethyl sulfoxide (DMSO) (control experiment: Control) was added to 1 mL of the bacteriophage Qβ solution, and the mixture was cultured with shaking at 37 °C for 30 minutes, 3 hours, and 6 hours. Subsequently, bacteriophage Qβ was mixed with the host Escherichia coli and cultured at 37 °C for 16 hours in a soft agar medium (the medium with 0.33% by mass of agar powder added). Plaques lysed by the infection of bacteriophage Qβ were formed, and the number of bacteriophage Qβ was quantified by counting the number of plaques. The results are shown in Fig. 13. Carbon nanoparticles showed antiviral activity from as short a time as 30 minutes.

[0121] [Experimental Example 3] (Antibacterial Activity of Composite Films of Carbon Nanoparticles and Polymers) Poly(lactic-co-glycolic acid) (PLGA): with a mass average molecular weight of 100,000, obtained from LACTEL. Poly(l-lactic acid) (PLLA): with a mass average molecular weight of 100,000, obtained from LACTEL. Polycaprolactone (PCL): with a mass average molecular weight of 80,000, obtained from LACTEL. Silk fibroin (SF): obtained from Atsumaru Holdings Co., Ltd. Escherichia coli: K12 strain, obtained from the National Institute of Technology and Evaluation. Culture medium: Sodium chloride (FUJIFILM Wako Pure Chemical Corporation), tryptone (Nacalai Tesque, Inc.), and yeast extract (Nacalai Tesque, Inc.) were dissolved in water and sterilized using an autoclave device manufactured by Yamato Corporation at 121 °C for 20 minutes.

[0122] 18 mg of each of the four types of polymers (polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLLA), polycaprolactone (PCL), or silk fibroin (SF)) and 3 mg of the carbon nanoparticles (GQDs) of Example 1 were dispersed in 1 mL of hexafluoro-2-propanol (HFIP) to prepare respective dispersions. 100 μL of these dispersions was applied onto the surface of a glass plate with φ10 mm × t1 mm and dried to produce a composite film of approximately 0.0268 mg / mm 2 The composite film on the glass plate was immersed in 1 mL of Escherichia coli solution and cultured with shaking at 37 °C for 6 hours, and the absorbance of the Escherichia coli solution was measured every 2 hours. Also, as a control experiment (Control), without immersing the film, it was directly cultured with shaking at 37 °C for 6 hours, and the absorbance of the Escherichia coli solution was measured every 2 hours. Figure 14 is a graph showing the results of the antibacterial activity evaluation of each composite film.

[0123] From the results in Figure 14, it was shown that the absorbance of each composite film was suppressed compared to the control experiment (Control), indicating antibacterial activity. In addition, the composite films of polylactic acid (PLLA) + carbon nanoparticles (GQDs) and polycaprolactone (PCL) + carbon nanoparticles (GQDs) showed higher antibacterial activity than the composite films of polylactic acid-glycolic acid copolymer (PLGA) + carbon nanoparticles (GQDs) and silk fibroin (SF) + carbon nanoparticles (GQDs). The reason why the antibacterial activity against Escherichia coli differed depending on the type of polymer in this way is considered to be that the release rate of carbon nanoparticles (GQDs) from the polymer is different.

[0124] Using the composite film of the four types of polymers on the glass plate described above, the adhesion of Escherichia coli was observed with a scanning electron microscope manufactured by Hitachi High-Technologies Corporation. Escherichia coli on the film was fixed overnight with 4%-paraformaldehyde phosphate buffer (Fuji Film Wako Pure Chemical Industries, Ltd.), and dehydrated with 25%, 50%, 75%, 90%, and 100% ethanol (Fuji Film Wako Pure Chemical Industries, Ltd.). Then, it was observed with a scanning electron microscope (Figs. 15 to 18). The composite film mixed with carbon nanoparticles (GQDs) (the right side of Figs. 15 to 18) had significantly fewer Escherichia coli on the film surface than the one without carbon nanoparticles (GQDs) (the left side of Figs. 15 to 18), indicating that carbon nanoparticles (GQDs) inhibited the formation of Escherichia coli biofilm.

[0125] 18 mg of polycaprolactone (PCL) and 500 μg, 1000 μg, 1500 μg, 2000 μg, 2500 μg of the carbon nanoparticles (GQDs) of Example 1 were dispersed in 1 mL of hexafluoro-2-propanol (HFIP) to prepare respective dispersions. 100 μL of these dispersions was applied to a glass plate of φ10 mm × t1 mm and dried, and composite films with a mass (mg) of about 0.0236 mg / mm 2 , about 0.0242 mg / mm 2 , about 0.0248 mg / mm 2 , about 0.0255 mg / mm 2 , about 0.0261 mg / mm 2 , and about 0.0261 mg / mm 2 per unit area (1 mm Fig. 19 is a graph showing the evaluation results of the content dependence of the antibacterial activity of the composite film of carbon nanoparticles (GQDs) and polycaprolactone (PCL). The composite film of polycaprolactone (PCL) mixed with 150 μg or more of carbon nanoparticles (GQDs) showed an antibacterial activity of 50% or more even after 6 hours.

[0126] [Experimental Example 4] The same experiment as in Experimental Example 3 was conducted, except that the Escherichia coli in Experimental Example 3 was changed to Salmonella (LT2 strain, obtained from the National Institute of Technology and Evaluation), or Staphylococcus aureus (ATCC25923, obtained from the National Institute of Technology and Evaluation). Similar results were obtained for Salmonella and Staphylococcus aureus. The absorbance of each composite film was suppressed compared to the control experiment (Control), indicating antibacterial activity against Salmonella and Staphylococcus aureus. In addition, the composite films of polylactic acid (PLLA) + carbon nanoparticles (GQDs) and polycaprolactone (PCL) + carbon nanoparticles (GQDs) showed higher antibacterial activity against Salmonella and Staphylococcus aureus than the composite films of poly (lactic-co-glycolic acid) (PLGA) + carbon nanoparticles (GQDs) and silk fibroin (SF) + carbon nanoparticles (GQDs).

[0127] [Experimental Example 5] (FT-IR Characterization of Composite Films) 3 mg of the carbon nanoparticles (GQDs) of Example 1 and 18 mg of polylactic acid (PLLA) were dispersed in 1 mL of hexafluoro-2-propanol (HFIP) to prepare a dispersion. 100 μL of this dispersion was applied to a glass plate with a diameter of 10 mm and a thickness of 1 mm and dried to prepare a composite film. 18 mg of polylactic acid (PLLA) was dispersed in 1 mL of hexafluoro-2-propanol (HFIP) to prepare a dispersion. 100 μL of this dispersion was applied to a glass plate and dried to obtain a polymer film with a film mass (mg) per unit area (1 mm 2 ) of approximately 0.0229 mg / mm 2 . The structural analysis of these films was carried out by Fourier transform infrared spectroscopy (FT-IR). Figure 20 shows the FT-IR spectra of the composite film of carbon nanoparticles (GQDs) and poly(lactic acid) (PLLA), as well as the poly(lactic acid) (PLLA) film. As a result, when carbon nanoparticles (GQDs) were mixed, peaks of C=C and C=O derived from carbon nanoparticles (GQDs) were detected around 1640 cm -1 and 1570 cm -1 .

[0128] Similarly, a composite film of carbon nanoparticles (GQDs) and polycaprolactone (PCL), as well as a polycaprolactone (PCL) film were prepared and their FT-IR spectra were obtained. Similarly, a composite film of carbon nanoparticles (GQDs) and poly(lactic-co-glycolic acid) (PLGA), as well as a poly(lactic-co-glycolic acid) (PLGA) film were prepared and their FT-IR spectra were obtained. In all cases, when carbon nanoparticles (GQDs) were mixed, peaks of C=C and C=O derived from carbon nanoparticles (GQDs) were detected around 1640 cm -1 and 1570 cm -1 .

[0129] Also, similarly, a composite film of carbon nanoparticles (GQDs) and silk fibroin (SF), as well as a silk fibroin (SF) film were prepared and their FT-IR spectra were obtained. The results are shown in Figure 21. Interestingly, no peak around 1570 cm -1 derived from carbon nanoparticles (GQDs) was observed. Also, the peak around 1640 cm -1 showing the random structure of fibroin shifted to 1620 cm -1It branched to the nearby peak. From this result, it is considered that by mixing carbon nanoparticles (GQDs) into silk fibroin, a part of the fibroin structure may have changed to a β-sheet. Thus, by adding carbon nanoparticles (GQDs) to the polymer, not only can antibacterial activity be imparted, but it is also possible to change the properties of the polymer.

[0130] [Experimental Example 6] (Fluorescent Staining of Silk Using Carbon Nanoparticles) 5 μL of sulfuric acid was added to 10 mL of acetic anhydride to prepare a mixed solution. 30 mg of silk fiber (commercially available thread) was immersed in this mixed solution, and microwave irradiation was performed at 180 °C for 1 hour using a microwave synthesizer manufactured by Anton Paar. Thereafter, the dyed silk fiber was taken out and washed with a large amount of water. When ultraviolet light was irradiated on the fiber, it emitted orange light. Also, the fluorescence could be observed even when observed with a fluorescence microscope (FITC filter, 488 nm). It is considered that the carbon nanoparticles (GQDs) were adsorbed onto the silk fiber immediately after being synthesized.

[0131] [Experimental Example 7] (Preparation of Composite Nanoparticles of Carbon Nanoparticles (GQDs) and Poly(lactic-co-glycolic acid) (PLGA)) 5 mg of the carbon nanoparticles (GQDs) of Example 1 and 10 mg of the poly(lactic-co-glycolic acid) copolymer (PLGA) were dispersed in 1 mL of chloroform to prepare a chloroform solution of the carbon nanoparticles (GQDs) and the poly(lactic-co-glycolic acid) copolymer (PLGA). While irradiating with ultrasonic waves, the chloroform solution of GQDs and PLGA was dropped into 1 mL of a 1 mass% aqueous polyvinyl alcohol solution cooled to 4°C. After stirring for 3 hours, this mixed solution was centrifuged three times to remove the supernatant, and the precipitate was redispersed in water to synthesize composite nanoparticles of carbon nanoparticles (GQDs) and poly(lactic-co-glycolic acid) copolymer (PLGA) that were uniformly dispersed in water. When the dispersion of the composite nanoparticles was diluted five-fold, a yellow solution (five-fold dilution) was obtained. When this solution was irradiated with ultraviolet light of 365 nm, it emitted light yellow. The measurement results of the fluorescence spectrum measured with a spectrofluorometer (FP-6600 manufactured by JASCO Corporation) are shown in Fig. 22. It was found that when excited at 520 nm, the maximum fluorescence was emitted around 595 nm. In the fluorescence spectrum of the carbon nanoparticles (GQDs) in Fig. 8, the maximum fluorescence was emitted around 506 nm when excited at 440 nm, whereas it was shown that the maximum fluorescence wavelength of the composite nanoparticles of carbon nanoparticles (GQDs) and poly(lactic-co-glycolic acid) copolymer (PLGA) shifted from around 506 nm to around 595 nm.

[0132] Generally, since water-soluble carbon nanoparticles (GQDs) are non-selectively taken up by cells and emit fluorescence, it is considered difficult to actually apply them to bioimaging. On the other hand, poly(lactic-co-glycolic acid) (PLGA) is a biodegradable polymer, and nanoparticles of poly(lactic-co-glycolic acid) (PLGA) are selective for cells and are easily taken up. The carbon nanoparticles (GQDs) according to the present invention are hydrophobic and soluble in organic solvents, so they can be encapsulated in composite nanoparticles of carbon nanoparticles (GQDs) and poly(lactic-co-glycolic acid) (PLGA) and are expected to be applied to bioimaging. Also, general fluorescent labeling substances (such as rhodamine and phalloidin) are easily soluble in water, so the fluorescence stability in nanoparticles is low. In contrast, the carbon nanoparticles (GQDs) according to the present invention are hydrophobic and soluble in organic solvents, so they can be stably encapsulated in composite nanoparticles and are considered to have emitted fluorescence.

[0133] The average particle size of the composite nanoparticles of carbon nanoparticles (GQDs) and poly(lactic-co-glycolic acid) (PLGA) measured by the dynamic light scattering method was 400 nm. The polydispersity index measured by the dynamic light scattering method was 0.15, and it was confirmed that the dispersion had a uniform particle size distribution.

[0134] [Experimental Example 8] (Uptake of Composite Nanoparticles of Carbon Nanoparticles (GQDs) and Poly(lactic-co-glycolic acid) (PLGA) into HeLa Cells) 1×10 5 cells / mL of HeLa cells (obtained from JCRB9004, National Institute of Biomedical Innovation, Health and Nutrition, National Institutes of Biomedical Innovation, Health and Nutrition) were dispersed in a medium (D-MEM, obtained from Fujifilm Wako Pure Chemical Corporation) and cultured at 37 °C in a 5% CO 2 environment for 24 hours, adhered to a dish manufactured by AS ONE, and a HeLa cell culture solution was prepared.

[0135] Composite nanoparticles (100 μg / mL) of carbon nanoparticles (GQDs) and poly(lactic-co-glycolic acid) (PLGA) were added to the HeLa cell culture medium and cultured at 37 °C in a 5% CO2 environment for 2 hours. Also, 10 mg of poly(lactic-co-glycolic acid) (PLGA) was dispersed in 1 mL of chloroform to prepare a chloroform solution of poly(lactic-co-glycolic acid) (PLGA). While irradiating with ultrasonic waves, the chloroform solution of PLGA was dropped into a 1 wt% aqueous polyvinyl alcohol solution (1 mL) cooled to 4 °C. After stirring for 3 hours, this mixed solution was centrifuged 3 times to remove the supernatant, and the precipitate was redispersed in water to synthesize nanoparticles of poly(lactic-co-glycolic acid) (PLGA) that were uniformly dispersed in water. Nanoparticles of poly(lactic-co-glycolic acid) (PLGA) (100 μg / mL) were added to the HeLa cell culture medium and cultured in the same manner for 2 hours. Thereafter, uptake into cells was evaluated using an optical microscope and a fluorescence microscope.

[0136] Figure 23(A) is an optical micrograph when nanoparticles of poly(lactic-co-glycolic acid) (PLGA) were added to HeLa cells and cultured for 2 hours. Figure 23(B) is a fluorescence micrograph when nanoparticles of poly(lactic-co-glycolic acid) (PLGA) were added to HeLa cells and cultured for 2 hours. Figure 23(C) is an optical micrograph when composite nanoparticles of carbon nanoparticles (GQDs) and poly(lactic-co-glycolic acid) (PLGA) were added to HeLa cells and cultured for 2 hours. Figure 23(D) is a fluorescence micrograph when composite nanoparticles of carbon nanoparticles (GQDs) and poly(lactic-co-glycolic acid) (PLGA) were added to HeLa cells and cultured for 2 hours. As a result, the composite nanoparticles of carbon nanoparticles (GQDs) and poly(lactic-co-glycolic acid) (PLGA) were taken up into HeLa cells, and fluorescence imaging was confirmed.

Industrial Applicability

[0137] Since the method for producing carbon nanoparticles of the present invention uses an organic solvent as a carbon source, carbon nanoparticles dispersible in the organic solvent can be produced simply and in a short time. The carbon nanoparticles obtained from the method for producing carbon nanoparticles of the present invention are expected to be used as medical materials, fluorescent materials, dyes, insect repellents, and the like. In particular, since the carbon nanoparticles of the present invention emit green fluorescence, they have the potential to be used as a contrast agent for bioimaging. In addition, since the carbon nanoparticles of the present invention have antibacterial activity and antiviral activity, they have the potential to be used as antibacterial agents and antiviral agents.

Claims

1. A method for producing carbon nanoparticles, comprising heating an organic solvent in the presence of a catalyst of a strong acid or a strong base to form carbon nanoparticles, wherein the organic solvent is an organic acid anhydride.

2. The method for producing carbon nanoparticles according to claim 1, wherein the strong acid or strong base is sulfuric acid, p-toluenesulfonic acid, or dimethylaminopyridine.

3. The method for producing carbon nanoparticles according to claim 1 or 2, wherein heating the organic solvent is heating the organic solvent by irradiating the organic solvent with microwaves.

4. The method for producing carbon nanoparticles according to any one of claims 1 to 3, wherein the organic acid anhydride is acetic anhydride.

5. The method for producing carbon nanoparticles according to any one of claims 1 to 4, wherein the carbon nanoparticles are soluble in an organic solvent.

6. The method for producing carbon nanoparticles according to any one of claims 1 to 5, wherein the average particle diameter of the carbon nanoparticles is 5 to 1000 nm.

7. When the carbon nanoparticles are measured by Raman spectroscopy, the intensity I of the G band derived from the graphite structure Ｇ and the intensity I of the D band caused by the defect of the graphite structure Ｄ The relative ratio (I Ｄ / I Ｇ ) is 0.3 to 0.

9. The method for producing carbon nanoparticles according to any one of claims 1 to 6.

8. When the carbon nanoparticles are measured by X-ray photoelectron spectroscopy (XPS) and quantified by the Shirley method from the obtained XPS C1s spectrum, the total amount of the C-O amount and the C=O amount is 60% or less with respect to 100% of the total amount of the C-C amount, the C=C amount, the C-O amount, and the C=O amount. The method for producing carbon nanoparticles according to any one of claims 1 to 7.

9. When the carbon nanoparticles are measured by Fourier transform infrared spectroscopy, a peak of C-H bond appears in the vicinity of 2900 cm －１ and a peak of O-H bond does not appear in the vicinity of 3300 cm －１ The method for producing carbon nanoparticles according to any one of claims 1 to 8.

10. The method for producing carbon nanoparticles according to any one of claims 1 to 9, wherein the organic solvent does not contain N atoms and S atoms.

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