Bioactive nano-composite material and method for synthesizing same

Abstract

Disclosed are a bioactive nano-composite material and a method for synthesizing same, wherein the material minimizes damage to oral tissue while achieving a long-lasting whitening effect. The bioactive nanocomposite material of the present invention may comprise an inorganic material, a first enzyme bound to the surface of the inorganic material, and a second enzyme bound to the surface of the inorganic material.

Description

Bioactive nanocomposite material and its synthesis method

[0001] The present invention relates to a bioactive nanocomposite material that achieves a long-term whitening effect while minimizing damage to oral tissue and a method for synthesizing the same.

[0002] Conventional whitening agents, such as hydrogen peroxide, carbamide peroxide, and sodium perborate, produce free radicals and reactive oxygen species (ROS) that can oxidize organic pigments in teeth, resulting in a whitening effect. Consequently, they are widely used in various fields, including the environment, medicine, and biotechnology.

[0003] However, these conventional materials generate high concentrations of free radicals and reactive oxygen species (ROS), which produce a whitening effect, and can be detrimental to the body. To address this issue, the development of materials that generate reactive oxygen species (ROS) that produce a whitening effect while exhibiting low cytotoxicity is essential. Therefore, the development of materials that simultaneously achieve these two properties (ROS generation and low cytotoxicity) is urgently needed.

[0004] One object of the present invention is to provide a bioactive nanocomposite material having a high whitening effect and low cytotoxicity by applying a dual enzyme to the surface of an inorganic material.

[0005] Another object of the present invention is to provide a method for synthesizing the bioactive nanocomposite material.

[0006] In one aspect, the present invention provides a bioactive nanocomposite material comprising an inorganic material, a first enzyme bound to a surface of the inorganic material, and a second enzyme bound to the surface of the inorganic material, wherein the first enzyme is selected from glucose oxidase or a glucose oxidase-like nanozyme, and the second enzyme is selected from catalase, peroxidase, catalase-like nanozyme, and peroxidase-like nanozyme.

[0007] In one embodiment, the present invention provides a bioactive nanocomposite material comprising hydroxyapatite nanoparticles, glucose oxidase bound to the surface of the hydroxyapatite nanoparticles, and catalase bound to the surface of the hydroxyapatite nanoparticles.

[0008] In one embodiment, the weight ratio of the first enzyme and the second enzyme may be 9:1 to 5:5. When the first enzyme is glucose oxidase and the second enzyme is catalase, the weight ratio of the glucose oxidase and catalase may be 9:1 to 5:5.

[0009] In one embodiment, the bioactive nanocomposite material can be used for tooth whitening purposes.

[0010] In another aspect, the present invention provides a method for synthesizing a bioactive nanocomposite material, comprising a first step of functionalizing an amine group on the surface of hydroxyapatite nanoparticles, a second step of replacing the surface functional groups of the nanoparticles with carboxyl groups, and a third step of binding glucose oxidase and catalase to the surface of the nanoparticles through an EDC / NHS ((1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) / N-hydroxysuccinimide) reaction.

[0011] In one embodiment, the first step may include adding alendronate hydrate or pamidronate hydrate to a hydroxyapatite nanoparticle dispersion and reacting the same.

[0012] In one embodiment, the concentration of alendronate hydrate or pamidronate hydrate added in the first step is 0.015 to 0.15 mg / ml, and the reaction in the first step can be performed at 30 to 60°C for 6 to 24 hours.

[0013] In one embodiment, the second step may comprise reacting the reactant of the first step with succinic anhydride in a buffer solution.

[0014] In one embodiment, the concentration of the succinic anhydride is 0.046 to 0.115 mg / ml, and the second step reaction can be performed at room temperature for 2 to 6 hours.

[0015] In one embodiment, the third step may include a step of performing a first reaction by adding the reactant of the second step to an EDC solution, a step of performing a second reaction by adding NHS to the first reaction solution, and a step of performing a third reaction by adding glucose oxidase and catalase to the second reaction solution.

[0016] In one embodiment, in the third step, the EDC solution concentration may be 0.088 to 0.22 mg / ml, NHS may be added at a concentration of 0.25 to 0.625 mg / ml, and glucose oxidase and catalase may be added at a concentration of 0.135 to 1.35 mg / ml, respectively.

[0017] In one embodiment, in the third step, the first reaction may be performed for 10 minutes to 2 hours, the second reaction may be performed for 10 minutes to 2 hours, and the third reaction may be performed for 1 to 2 hours.

[0018] In one embodiment, the weight ratio of glucose oxidase and catalase bound to the surface of the nanoparticle may be 9:1 to 5:5.

[0019] The bioactive nanocomposite material according to the present invention uses glucose oxidase (GOD) (or glucose oxidase-like nanozyme) and catalase (CAT) (or peroxidase, catalase-like nanozyme, peroxidase-like nanozyme) as a dual enzyme to functionalize the surface of an inorganic material, and can be utilized as a whitening material.

[0020] The bioactive nanocomposite of the present invention possesses the bioactivity of both GOD and CAT or similar enzymes at the nanoscale interface. Therefore, it generates reactive oxygen species suitable for a whitening effect while having little effect on cell toxicity. In particular, in the case of conventional whitening materials, the concentration of free radicals and reactive oxygen species generated is very high, so the enzyme itself is damaged, and a long-term whitening effect cannot be realized. However, the bioactive nanocomposite of the present invention not only causes minimal damage to the enzyme itself that realizes the whitening effect, but also enables the immobilization of two enzymes together on the surface of the nanoparticles, enabling long-term implementation of a whitening effect. Therefore, the bioactive nanocomposite of the present invention can be applied to the medical and bio fields where the implementation of appropriate reactive oxygen species is very important.

[0021] Figure 1 shows SEM images (a, b), TEM images (c, d) before and after surface functionalization of a bioactive nanocomposite of the present invention, surface charge analysis (e) during the surface functionalization process of a bioactive nanocomposite, and the results of surface functional group analysis through FT-IR analysis (f).

[0022] Figure 2 is an SEM image (right) that confirms the presence of dentinal tubules (left) present in a tooth and the presence of bioactive nanocomposite materials remaining within the dentinal tubules.

[0023] Figure 3 shows the results of functionalizing the dual enzyme at various ratios and comparing the activity of bioactive nanocomposites according to the ratios through TMB-HRP analysis.

[0024] Figures 4 and 5 show the results of evaluating the whitening performance of a bioactive nanocomposite material using a footbath.

[0025] Figure 6 shows the results of evaluating the biocompatibility of the bioactive nanocomposite material of the present invention.

[0026] Figure 7 shows the results of confirming the antibacterial properties of the bioactive nanocomposite material of the present invention.

[0027] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. The present invention is susceptible to various modifications and variations, and thus specific embodiments are illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific disclosed form, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. Throughout the description of each drawing, similar reference numerals have been used to designate similar components.

[0028] The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, it should be understood that the terms "comprise" or "have" indicate the presence of a feature, step, operation, component, part, or combination thereof described in the specification, but do not preclude the possibility of the presence or addition of one or more other features, steps, operations, components, parts, or combinations thereof.

[0029] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and will not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0030] One object of the present invention is to provide a bioactive nanocomposite material having a high whitening effect and low cytotoxicity by applying a dual enzyme to the surface of an inorganic material.

[0031] A bioactive nanocomposite material according to one embodiment of the present invention may include an inorganic material, a first enzyme bound to the surface of the inorganic material, and a second enzyme bound to the surface of the inorganic material.

[0032] The inorganic material used in the present invention may include ceramic materials, metal nanoparticles, silica particles, etc. In one embodiment, the inorganic material may include hydroxyapatite nanoparticles.

[0033] The surface of the above-mentioned inorganic material is conjugated with a dual enzyme having different bioactivities. When the surface of the inorganic material is functionalized with the dual enzyme, the bioactivities of the different enzymes generate a biological synergy effect, effectively controlling the concentration of reactive oxygen species at the nanointerface, thereby simultaneously realizing a long-term whitening effect and low cytotoxicity.

[0034] The first enzyme and the second enzyme of the present invention may include a protein-based enzyme or a nanozyme, which is an enzyme analogue having a function similar to that of the enzyme. In one embodiment, the first enzyme may be selected from glucose oxidase or a glucose oxidase-like nanozyme, and the second enzyme may be selected from catalase, a peroxidase-based enzyme, a catalase-like nanozyme, and a peroxidase-like nanozyme. However, the present invention is not limited thereto, and any enzyme or analogue that can perform a function similar to that of the respective enzymes may be substituted.

[0035] More specifically, a bioactive nanocomposite material according to one embodiment of the present invention may include hydroxyapatite nanoparticles, glucose oxidase bound to the surface of the hydroxyapatite nanoparticles, and catalase bound to the surface of the hydroxyapatite nanoparticles.

[0036] The bioactive nanocomposite material of the present invention comprises dual enzymes, each possessing a different bioactivity, conjugated to the surface of hydroxyapatite nanoparticles. The bioactivities of these different enzymes generate a biological synergy, effectively controlling the concentration of reactive oxygen species at the nanointerface, thereby simultaneously achieving a long-term whitening effect and low cytotoxicity.

[0037] In one embodiment, the weight ratio of the first enzyme and the second enzyme, which are the dual enzymes used in the present invention, may be 9:1 to 5:5. For example, the weight ratio of glucose oxidase and catalase selected as the dual enzymes may be 9:1 to 5:5. Preferably, the weight ratio of glucose oxidase and catalase may be 7:3. The bioactive nanocomposite material of the present invention, in which such dual enzymes are functionalized at various ratios, has a superior effect in generating reactive oxygen species compared to a composite material based on a single enzyme. However, the present invention is not limited thereto, and even when the weight ratio of glucose oxidase and catalase, which are the dual enzymes used in the present invention, is 4:6 to 1:9, the whitening effect may be exhibited although the activity is lower than that of the content of the preferred weight ratio.

[0038] In this way, the bioactive nanocomposite material of the present invention can be used for teeth whitening purposes because it not only causes minimal damage to the enzyme itself but also enables long-term implementation of a whitening effect, and can be applied to medical and bio fields where implementation of appropriate reactive oxygen species is important.

[0039] Meanwhile, a method for synthesizing a bioactive nanocomposite material according to one embodiment of the present invention may include a first step of functionalizing an amine group on the surface of hydroxyapatite nanoparticles, a second step of replacing the surface functional group of the nanoparticles with a carboxyl group, and a third step of binding glucose oxidase and catalase to the surface of the nanoparticles through an EDC / NHS ((1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) / N-hydroxysuccinimide) reaction.

[0040] The first step involves functionalizing the surface of hydroxyapatite nanoparticles with amine groups. Since it is not easy to directly introduce carboxyl groups onto the surface of hydroxyapatite nanoparticles, the amine groups are first functionalized, and then the amine groups are replaced with carboxyl groups in the second step, thereby allowing the dual enzyme to be bound to the surface.

[0041] In one embodiment, the first step includes adding alendronate hydrate or pamidronate hydrate to a hydroxyapatite nanoparticle dispersion and reacting the same. Through the reaction, an amine group may be functionalized on the surface of the nanoparticle. Here, the concentration of the added alendronate hydrate or pamidronate hydrate may be 0.015 to 0.15 mg / ml. In addition, the reaction of the first step may be performed at 30 to 60°C for 6 to 24 hours. Preferably, alendronate hydrate or pamidronate hydrate may be added at 0.015 mg / ml and reacted at 30°C for 12 hours. However, the present invention is not limited thereto, and an amine group-containing substance may be used to substitute the surface of the hydroxyapatite nanoparticle with an amine group.

[0042] The second step involves replacing the surface functional groups of the surface-functionalized nanoparticles after the first step with carboxyl groups.

[0043] In one embodiment, the second step may include reacting the reactant of the first step with succinic anhydride in a buffer solution. Here, the concentration of the succinic anhydride may be 0.046 to 0.115 mg / ml, and the reaction of the second step may be performed at room temperature for 2 to 6 hours. Preferably, succinic anhydride in a concentration of 0.046 mg / ml in the buffer solution may be reacted with the reactant of the first step for 3 hours at room temperature. However, the present invention is not limited thereto, and a surface functional group of the reactant of the first step may be substituted with a carboxyl group using a carboxyl group-containing material.

[0044] The third step involves conjugating glucose oxidase and catalase to the surface of the nanoparticles via an EDC / NHS reaction. The EDC / NHS reaction allows for the conjugation and functionalization of dual enzymes onto the surface of hydroxyapatite nanoparticles.

[0045] Specifically, the third step may include the following steps:

[0046] First, a first reaction step is performed by adding the reactants of the second step to the EDC solution. Here, the concentration of the EDC solution may be 0.088 to 0.22 mg / ml, and the first reaction may be performed for 10 minutes to 2 hours. Preferably, the 0.088 mg / ml EDC solution may be reacted with the reactants of the second step for 20 minutes.

[0047] Next, a second reaction step is performed by adding NHS to the first reaction solution. Here, the NHS can be added at a concentration of 0.25 to 0.625 mg / ml, and the second reaction can be performed for 10 minutes to 2 hours. Preferably, a 0.25 mg / ml NHS solution can be added and reacted for 20 minutes.

[0048] Thereafter, a third reaction step is performed by adding glucose oxidase and catalase to the second reaction solution. Here, the glucose oxidase and catalase may be added at a concentration of 0.135 to 1.35 mg / ml, respectively, and the third reaction may be performed for 1 to 2 hours. Preferably, the dual enzymes (glucose oxidase and catalase) may be added at a concentration of 0.675 mg / ml, respectively, and the reaction may be performed for 2 hours.

[0049] In one embodiment, the concentrations of glucose oxidase and catalase added to the second reaction solution are not particularly limited. By appropriately adjusting the concentrations of the two enzymes, bioactive nanocomposites functionalized at various ratios can be synthesized. Bioactive nanocomposites functionalized at various ratios have superior reactive oxygen species generation ability compared to single-enzyme-based composites. Preferably, the weight ratio of glucose oxidase and catalase bound to the surface of the nanoparticles may be 9:1 to 5:5.

[0050] According to the present invention, a bioactive nanocomposite material can be provided that can simultaneously implement a whitening effect and low cytotoxicity characteristics by functionalizing the surface of an inorganic material with a dual enzyme having different bioactivities and effectively controlling the concentration of reactive oxygen species at the nanointerface.

[0051] The bioactive nanocomposite material of the present invention can prevent the side effects of conventional whitening materials due to excessive generation of reactive oxygen species, a drawback of conventional whitening materials, by simultaneously functionalizing two enzymes to induce bioactivity, and can induce long-term whitening effects. This is because the bioactivities of different enzymes generate a biological synergy effect.

[0052] Hereinafter, embodiments of the present invention will be described in detail. However, the embodiments described below are merely some embodiments of the present invention, and the scope of the present invention is not limited to the embodiments described below.

[0053]

[0054] Example

[0055] Hydroxyapatite nanoparticles were mixed in 3-distilled water at a concentration of 3 mg / ml and dispersed by vortexing and sonication. Then, alendronate hydrate or pamidronate hydrate was added at a concentration of 0.015 to 0.15 mg / ml and reacted at 30 to 60°C for 6 to 24 hours. Next, succinic anhydride was added at a concentration of 0.046 to 0.115 mg / ml in 1 x PBS buffer and reacted with the reactants at room temperature for 2 to 6 hours.

[0056] Afterwards, EDC was prepared as a 0.088 to 0.22 mg / ml solution and reacted with the reactant. After 10 minutes to 2 hours, NHS was added at a concentration of 0.25 to 0.625 mg / ml and reacted with stirring for 10 minutes to 2 hours. Afterwards, an enzyme mixture of glucose oxidase and catalase was added to the reactant at a concentration of 0.135 to 1.35 mg / ml and reacted for 2 hours.

[0057]

[0058] Experimental example

[0059] ① Analysis of the components of bioactive nanocomposite materials

[0060] Figure 1 shows SEM images (a, b), TEM images (c, d) before and after surface functionalization of a bioactive nanocomposite, surface charge analysis (e) during the surface functionalization process of a bioactive nanocomposite, and the results of surface functional group analysis through FT-IR analysis (f).

[0061] The hydroxyapatite particles of the present invention are rod-shaped with a needle shape, and it was confirmed through (a)-(d) of Fig. 1 that there was no significant difference in the particle shape before and after the reaction.

[0062] Meanwhile, Fig. 1(e) shows the analysis of the surface charge during the process of functionalizing the surface of hydroxyapatite particles, showing that hydroxyapatite with a negative charge becomes positively charged as it is functionalized with an amine group, and then the amine group is replaced with a negative charge again as it is functionalized with a carboxyl group.

[0063] Figure 1(f) shows the analysis of functional groups on the particle surface using FT-IR, and peaks of amine and carboxyl groups are observed at each stage, and ultimately, it was confirmed that the dual enzyme was successfully bound to the surface of hydroxyapatite nanoparticles.

[0064] ② Dental application of bioactive nanocomposite materials

[0065] Figure 2 is a SEM image (right) that confirms the presence of dentinal tubules (left) present in a tooth and the presence of bioactive nanocomposite materials remaining within the dentinal tubules. The present invention can cause nanoparticles to remain within the dentinal tubules by brushing teeth using a bioactive nanocomposite material.

[0066] ③ Activity analysis of bioactive nanocomposite materials

[0067] Dual enzymes (glucose oxidase and catalase) were functionalized at various ratios ranging from 10:0 to 5:5, and the activities of the bioactive nanocomposites according to the ratios were compared through TMB-HRP analysis. Specifically, the bioactive nanocomposite was mixed with TMB, HRP, and glucose at an appropriate ratio. The bioactive nanocomposite decomposes glucose to generate hydrogen peroxide, which is then decomposed into radicals by catalase and HRP to generate the color of TMB. The higher the intensity of the graph shown in Fig. 3, the higher the enzyme activity, and the longer the intensity is maintained, the longer the enzyme activity is maintained. Through these results, it was confirmed that the bioactive nanocomposite functionalized with dual enzymes has a superior long-term oxygen radical generation performance than the case where a single enzyme is functionalized.

[0068] ④ Confirmation of the whitening performance of bioactive nanocomposite materials using a dental floss

[0069] Figures 4 and 5 show the results of evaluating the whitening performance of a bioactive nanocomposite material using a footbath.

[0070] Referring to Fig. 4, it can be confirmed that the color of teeth gradually becomes brighter as they are whitened by the bioactive nanocomposite material of the present invention. The Vita shade guide is a guide that dentists use in clinical practice to compare the color of patients' teeth. Referring to Fig. 4, it was confirmed that the bioactive nanocomposite material of the present invention can whiten teeth with a color of 5M 3, which corresponds to the darkest, to 1M 1, which is the brightest.

[0071] Fig. 5 shows the results of comparing the dE*ab, dL, a, b values, and reflectance of teeth. dL indicates a change in brightness, and unlike the control group where there is no change, a continuous change in dL is observed in the whitening group. a and b each indicate a change in tooth color, and it can be confirmed that it changes to the color before staining. Reflectance analyzes the light reflectance of teeth at each wavelength, and it can be confirmed that whitening is progressing at all wavelengths. dE*ab is a value widely used to express color as a single number, and as whitening progresses, the change in number becomes greater, and through the results of Fig. 5, it can be confirmed that whitening is progressing due to the bioactive nanocomposite material of the present invention.

[0072] ⑤ Confirmation of biocompatibility of bioactive nanocomposite materials using cell experiments

[0073] Figure 6 shows the results of confirming the biocompatibility of the bioactive nanocomposite material of the present invention through CCK-08 analysis and FDAEB Live / Dead cell analysis. Specifically, MC3T3-E1 was seeded in a 6-well plate (1 x 10 5 The cells were seeded on a well (25 ?? 400 μg / ml) and cultured with nanoparticles at each concentration (25 ?? 400 μg / ml) for 24 hours, and the cell survival rate and morphology were observed.

[0074] Referring to Fig. 6 showing the results, the cell viability of the particles of the present invention was close to 100% at a glucose concentration similar to that of saliva (approximately 1.23 mg / L) compared to the control group, and the FDAEB analysis results confirmed that there were no problems with the live / dead cell ratio and cell morphology. In this way, it was confirmed that the generation of reactive oxygen species of the bioactive nanocomposite of the present invention did not affect cell death and that it had excellent biocompatibility.

[0075] ⑥ Antibacterial properties of bioactive nanocomposite materials

[0076] Figure 7 shows the results of confirming the antibacterial properties of the bioactive nanocomposite material of the present invention.

[0077] Figure 7 shows the cell activity graph (top) for E. coli and S. aureus strains treated with bioactive nanocomposites, and the visual data (bottom) after 9 hours of measuring CFU by counting the number of bacteria, allowing for visual confirmation of the strain ratio. Compared to the control (leftmost), it was confirmed that the number of bacteria decreased after 9 hours at each concentration. Based on Figure 7, the antibacterial properties were confirmed at various concentrations from 6.25 to 200 μg / ml, and in particular, it was confirmed that strong antibacterial properties were shown for more than 12 hours at 6.25 to 50 μg / ml.

[0078] Although the present invention has been described above with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and changes may be made to the present invention without departing from the spirit and scope of the present invention as set forth in the claims below.

Claims

1. Weapon material; A first enzyme bound to the surface of the above inorganic material; and A second enzyme bound to the surface of the above-mentioned inorganic material; The first enzyme is selected from glucose oxidase or glucose oxidase like nanozyme, The second enzyme is selected from catalase, peroxidase, catalase-like nanozyme and peroxidase-like nanozyme. Bioactive nanocomposites.

2. In paragraph 1, Hydroxyapatite nanoparticles; Glucose oxidase bound to the surface of the above hydroxyapatite nanoparticles; and Catalase bound to the surface of the above hydroxyapatite nanoparticles; Bioactive nanocomposites.

3. In paragraph 1, The weight ratio of the first enzyme and the second enzyme is 9:1 to 5:5, Bioactive nanocomposites.

4. In paragraph 1, The above bioactive nanocomposite material is used for teeth whitening purposes. Bioactive nanocomposites.

5. The first step is to functionalize amine groups on the surface of hydroxyapatite nanoparticles; A second step of replacing the surface functional group of the above nanoparticles with a carboxyl group; and A third step of binding glucose oxidase and catalase to the surface of the nanoparticles through an EDC / NHS ((1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) / N-hydroxysuccinimide) reaction; Method for synthesizing bioactive nanocomposite materials.

6. In paragraph 5, The first step includes adding alendronate hydrate or pamidronate hydrate to a hydroxyapatite nanoparticle dispersion and reacting the same. Method for synthesizing bioactive nanocomposite materials.

7. In paragraph 6, The concentration of alendronate hydrate or pamidronate hydrate added in the first step is 0.015 to 0.15 mg / ml, The above first step reaction is carried out at 30 to 60°C for 6 to 24 hours. Method for synthesizing bioactive nanocomposite materials.

8. In paragraph 5, The second step comprises a step of reacting the reactant of the first step with succinic anhydride in a buffer solution. Method for synthesizing bioactive nanocomposite materials.

9. In paragraph 8, The concentration of the above succinic anhydride is 0.046 to 0.115 mg / ml, The above second step reaction is carried out at room temperature for 2 to 6 hours. Method for synthesizing bioactive nanocomposite materials.

10. In paragraph 5, The third step above is, A step of performing a first reaction by adding the reactants of the second step to the EDC solution; A second reaction step is performed by adding NHS to the first reaction solution; and A third reaction step comprising adding glucose oxidase and catalase to the second reaction solution; Method for synthesizing bioactive nanocomposite materials.

11. In paragraph 10, In the third step above, The EDC solution concentration is 0.088 to 0.22 mg / ml, NHS is added at a concentration of 0.25 to 0.625 mg / ml, Glucose oxidase and catalase are added at concentrations of 0.135 to 1.35 mg / ml, respectively. Method for synthesizing bioactive nanocomposite materials.

12. In paragraph 10, In the third step above, The first reaction is carried out for 10 minutes to 2 hours, The second reaction is carried out for 10 minutes to 2 hours, The third reaction is carried out for 1 to 2 hours, Method for synthesizing bioactive nanocomposite materials.

13. In paragraph 5, The weight ratio of glucose oxidase and catalase bound to the surface of the above nanoparticles is 9:1 to 5:

5. Method for synthesizing bioactive nanocomposite materials.

14. Manufactured according to the method of any one of clauses 5 to 13, Bioactive nanocomposites.

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