An ascorbate oxidase mimetic enzyme and preparation thereof

Abstract

The application belongs to the technical field of nanomaterials, and provides ascorbic acid oxidation mimetic enzyme and preparation thereof. The application adopts a methanol solution of zinc nitrate, copper acetylacetonate and 2-methyl imidazole to perform a hydrothermal reaction, and obtains the ascorbic acid oxidation mimetic enzyme by oxygen isolation calcination of a hydrothermal product. The copper-nitrogen-carbon nanomaterial obtained by the application has a rhombohedron structure, can specifically oxidize ascorbic acid, and can be applied to ascorbic acid content detection, oxidized ascorbic acid preparation and dehydroascorbic acid preparation as ascorbic acid oxidation enzyme mimetic enzyme. The nanomaterial can maintain ascorbic acid oxidation mimetic enzyme activity under acid, alkaline and high temperature conditions; the steady-state kinetic parameter (Michaelis constant is 0.012 mM) is better than that of natural ascorbic acid oxidation enzyme, and the ascorbic acid oxidation mimetic enzyme activity of the material has a broad application prospect in the fields of nanozyme, nanotechnology and biological analysis detection.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials technology, specifically relating to a copper-nitrogen-carbon structured nanomaterial and its application as an ascorbic acid oxidase. Background Technology

[0002] Natural enzymes possess advantages such as high efficiency and specificity; however, due to their protein-based chemical nature, they are easily inactivated in complex environments such as acidity, alkalinity, and high temperature. Nanozymes are enzyme mimics that combine the properties of nanomaterials with catalytic activity. Compared to natural enzymes, nanozymes exhibit better catalytic performance, stability in acidic, alkaline, and high-temperature environments, and advantages such as low cost and recyclability. Based on these advantages, the application research of nanozymes has expanded to multiple fields such as bioanalysis, agriculture, medicine, and environmental remediation, gradually forming a new field of nanozyme research.

[0003] Ascorbate oxidase is a copper-containing enzyme that catalyzes the oxidation of ascorbic acid to dehydroascorbic acid in the presence of oxygen. It can also couple with other redox reactions, acting as a terminal oxidase and possessing anti-aging properties, playing a crucial role in plant metabolism. However, similar to many natural enzymes, ascorbate oxidase is easily inactivated by various factors, and the experimental conditions for its application are quite demanding, greatly limiting its applicability.

[0004] Since the first reports of peroxide nanozymes, an increasing number of nanozymes have been studied and reported. However, nanomaterials with ascorbic acid oxidase mimicry activity still require further research and development. Given the advantages of nanozymes, exploring nanomaterials with ascorbic acid oxidase mimicry activity is of great significance, as they can be used to construct different detection methods or applied to various future fields. Summary of the Invention

[0005] To provide nanoenzymes with better performance, this invention provides a copper-nitrogen-carbon nanomaterial that can function as ascorbic acid oxidase and is superior to natural ascorbic acid oxidase in terms of steady-state kinetic parameters and stability.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned ascorbic acid oxidase mimicry.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] A method for preparing copper-nitrogen-carbon nanomaterials includes the following steps: a hydrothermal reaction is carried out with a methanol solution of zinc nitrate, copper acetylacetonate, and 2-methylimidazole, and the hydrothermal product is calcined in an oxygen-free environment.

[0009] The molar ratio of zinc nitrate, copper acetylacetonate, and 2-methylimidazole is 4:(0-0.4):16, and the proportion of copper acetylacetonate is not 0.

[0010] The hydrothermal reaction temperature is 110℃-150℃, and the reaction time is 3-5 hours. As the reaction temperature or reaction time increases, the particle size of the product tends to increase. To obtain a better particle size, the preferred reaction temperature is 120℃-130℃. The calcination temperature is 900℃.

[0011] A copper-nitrogen-carbon nanomaterial was obtained by the above preparation method. The morphology of the nanomaterial is a rhombic dodecahedral particle with a particle size of about 300-1500 nm.

[0012] The aforementioned copper-nitrogen-carbon structure has a specific oxidizing effect on ascorbic acid and can be used as an ascorbic acid oxidase mimic enzyme for ascorbic acid content detection, oxidation of ascorbic acid, and preparation of dehydroascorbic acid.

[0013] The aforementioned copper-nitrogen-carbon structure can be reacted with peroxides, such as hydrogen peroxide, and can be used as a peroxidase mimic enzyme for peroxide content detection and peroxide removal.

[0014] The present invention has the following advantages:

[0015] This invention discovers a copper-nitrogen-carbon material with ascorbic acid oxidase mimicry activity. Under oxygen-containing conditions, it can efficiently catalyze the oxidation of ascorbic acid to dehydroascorbic acid, maintaining its ascorbic acid oxidase mimicry activity under acidic, alkaline, and high-temperature conditions. The steady-state kinetic parameters (Michaelis constant of 0.012 mM) of the copper-nitrogen-carbon ascorbic acid oxidase are better than those of the natural ascorbic acid oxidase, and the stability of the copper-nitrogen-carbon ascorbic acid oxidase is significantly stronger than that of the natural ascorbic acid oxidase. The ascorbic acid oxidase mimicry activity of this material shows broad application prospects in the fields of nanozymes, nanotechnology, and bioanalytical detection. Attached Figure Description

[0016] Figure 1 Scanning electron microscope (SEM) image (a), transmission electron microscope (TEM) image (b), and aberration-corrected TEM image (c) of copper-nitrogen-carbon nanomaterials.

[0017] Figure 2 The activities of copper-nitrogen-carbon ascorbic acid oxidase (a) and peroxidase-like activities (b) synthesized with different ratios;

[0018] Figure 3 The graph shows the change in ultraviolet absorbance at 265 nm in the copper-nitrogen-carbon catalytic oxidation of ascorbic acid.

[0019] Figure 4For the determination of the activities of copper-nitrogen-carbon peroxidase and oxide-mimicking enzymes;

[0020] Figure 5 The catalytic oxidation of ascorbic acid is specific to copper-nitrogen-carbon.

[0021] Figure 6 Figure showing the effects of oxygen and nitrogen on the copper-nitrogen-carbon catalytic oxidation of ascorbic acid.

[0022] Figure 7 The graph shows the effect of temperature on copper-nitrogen-carbon as an ascorbic acid oxidase mimicry.

[0023] Figure 8 The effect of pH on copper-nitrogen-carbon as an ascorbic acid oxidase mimicry is shown in the figure.

[0024] Figure 9 Figure 1 shows the effect of reaction time on the effect of nano-copper oxide as an ascorbic acid oxidase mimicry.

[0025] Figure 10 The figure shows the effect of material concentration on the role of nano-copper oxide as an ascorbic acid oxidase mimicry.

[0026] Figure 11 Steady-state kinetics curves for copper-nitrogen-carbon as an ascorbic acid oxidation mimic enzyme;

[0027] Figure 12 The stability of copper-nitrogen-carbon at different times;

[0028] Figure 13 The oxidation of ascorbic acid to dehydroascorbic acid, catalyzed by copper-nitrogen-carbon, and the subsequent reaction with o-phenylenediamine to produce a fluorescent product (excitation wavelength 350 nm). Detailed Implementation

[0029] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0030] Example 1: Preparation of copper-nitrogen-carbon nanomaterials

[0031] (1) 1.019 g zinc nitrate hexahydrate and 0.2 mM copper acetylacetonate were dissolved in 30 mL of methanol; 1.314 g 2-methylimidazole was dissolved in 15 mL of methanol; the two solutions were mixed evenly to obtain the raw material solution;

[0032] (2) Pour the raw material liquid into a Teflon-lined reactor and heat it at 120°C for 5 hours. Immediately centrifuge the solution obtained after heating, wash it three times with methanol and DMF respectively, and dry it for 24 hours to obtain the precursor.

[0033] (3) The precursor was placed in a tube furnace and heated at 900°C for 3 hours in an argon atmosphere to obtain copper-nitrogen-carbon nanomaterials, denoted as 0.2 copper-nitrogen-carbon.

[0034] Scanning electron microscope (SEM) images, transmission electron microscope (TEM) images, and aberration-corrected TEM images of the above materials are respectively... Figure 1 As shown in image ac. The image shows that the obtained material is granular, with each particle having a rhombic dodecahedral structure and a particle size distribution of approximately 500-1000 nm. From Figure 1 In image c, uniform and dispersed bright spots can be seen, marked with circles, proving that copper exists in the form of single atoms.

[0035] Example 2 Preparation of copper-nitrogen-carbon nanomaterials

[0036] (1) 1.019 g zinc nitrate hexahydrate and 0.1 mM copper acetylacetonate were dissolved in 30 mL of methanol; 1.314 g 2-methylimidazole was dissolved in 15 mL of methanol; the two solutions were mixed evenly to obtain the raw material solution;

[0037] (2) Pour the raw material liquid into a Teflon-lined reactor and heat it at 130°C for 4 hours. Immediately centrifuge the solution obtained after heating, wash it three times with methanol and DMF respectively, and dry it for 24 hours to obtain the precursor.

[0038] (3) The precursor was placed in a tube furnace and heated at 900°C for 3 hours in an argon atmosphere to obtain copper-nitrogen-carbon nanomaterials, denoted as 0.1 copper-nitrogen-carbon. Scanning electron microscopy and transmission electron microscopy images showed that the above material was granular, each particle had a rhombic dodecahedral structure, and the particle size was distributed at about 500-1000 nm. Through aberration-corrected transmission electron microscopy, copper in the material existed in the form of single atoms.

[0039] Example 3 Preparation of copper-nitrogen-carbon nanomaterials

[0040] (1) 1.019 g zinc nitrate hexahydrate and 0.4 mM copper acetylacetonate were dissolved in 30 mL of methanol; 1.314 g 2-methylimidazole was dissolved in 15 mL of methanol; the two solutions were mixed evenly to obtain the raw material solution;

[0041] (2) Pour the raw material liquid into a Teflon-lined reactor and heat it at 150°C for 3 hours. Immediately centrifuge the solution obtained after heating, wash it three times with methanol and DMF respectively, and dry it for 24 hours to obtain the precursor.

[0042] (3) The precursor was placed in a tube furnace and heated at 900°C for 3 hours in an argon atmosphere to obtain copper-nitrogen-carbon nanomaterials, denoted as 0.1 copper-nitrogen-carbon. Scanning electron microscopy and transmission electron microscopy images showed that the above material was granular, each particle had a rhombic dodecahedral structure, and the particle size was distributed at about 600-1500 nm. Through aberration-corrected transmission electron microscopy, copper in the material existed in the form of single atoms.

[0043] Comparative Example 1: Preparation of Nitrogen-Carbon Nanomaterials

[0044] (1) Dissolve 1.019 g of zinc nitrate hexahydrate in 30 mL of methanol; dissolve 1.314 g of 2-methylimidazole in 15 mL of methanol; mix the two solutions thoroughly to obtain the raw material solution;

[0045] (2) Pour the raw material liquid into a Teflon-lined reactor and heat it at 120°C for 5 hours. Immediately centrifuge the solution obtained after heating, wash it three times with methanol and DMF respectively, and dry it for 24 hours to obtain the precursor.

[0046] (3) The precursor is placed in a tube furnace and heated at 900°C for 3 hours in an argon atmosphere to obtain nitrogen-carbon nanomaterials, denoted as nitrogen-carbon.

[0047] Example 4: Copper-nitrogen-carbon ascorbic acid oxidase and peroxidase-like activities synthesized with different ratios

[0048] In the presence of oxygen, the activity of the copper-nitrogen-carbon ascorbic acid oxidase-like enzyme prepared in Examples 1-3 was examined: at a water bath temperature of 35°C, 15 µL of copper-nitrogen-carbon material with different material ratios at a concentration of 1 mg / mL and 50 μL of ascorbic acid at a concentration of 2 mM were added to 935 μL of phosphate buffer (pH = 7.0, 0.2 M), and the absorbance at 265 nm was immediately measured using a UV spectrophotometer for 180 s.

[0049] The peroxidase-like activity of copper-nitrogen-carbon was investigated in the presence of oxygen: 15 µL of copper-nitrogen-carbon material with different material ratios at a concentration of 1 mg / mL, 50 μL of hydrogen peroxide at a concentration of 0.1 M, and 50 μL of TMB at a concentration of 5 mM were added to 885 μL of acetate buffer (pH = 3.6, 0.2 M). The absorbance at 652 nm was immediately measured using a UV spectrophotometer for 180 s.

[0050] The results are as follows Figure 2As shown, after adding different material ratios in the copper-nitrogen-carbon reaction, the absorbance of the system at a wavelength of 265 nm showed different trends of decrease as the reaction time increased, while the absorbance at 652 nm showed different trends of increase. The two enzymes showed better activity when the molar ratio of zinc nitrate: copper acetylacetonate: 2-methylimidazole was 4:0.2:16.

[0051] Example 5: Detection of copper-nitrogen-carbon as ascorbic acid oxidase

[0052] In the presence of oxygen, the activity of the copper-nitrogen-carbon ascorbic acid oxidase-like material prepared in Example 1 was examined: at a water bath temperature of 35°C, 15 µL of copper-nitrogen-carbon material with a concentration of 1 mg / mL and 50 μL of ascorbic acid with a concentration of 2 mM were added to 935 μL of phosphate buffer (pH = 7.0, 0.2 M), and the absorbance at 265 nm was immediately measured using a UV spectrophotometer for 180 s.

[0053] The results are as follows Figure 3 As shown, after the addition of copper-nitrogen-carbon reaction, the absorbance of the system at a wavelength of 265 nm decreased significantly with the extension of reaction time, indicating that ascorbic acid in the system was significantly consumed, proving that copper-nitrogen-carbon has the function of ascorbic acid oxidase.

[0054] Example 6: Peroxide-mimicking enzyme and oxide-mimicking enzyme activity of copper-nitrogen-carbon materials

[0055] In the presence of oxygen, the peroxidase-like activity of the copper-nitrogen-carbon prepared in Example 1 was examined: 15 μL of the copper-nitrogen-carbon aqueous solution (1 mg / mL) prepared in Example 1, 50 μL of TMB (5 mM), and 50 μL of H2O2 (0.1 M) were added to 885 μL of acetate buffer (pH=3.6 0.2 M), and the absorbance at 652 nm was measured using a UV spectrophotometer for 120 s.

[0056] In the presence of oxygen, the oxidase-like activity of the copper-nitrogen-carbon prepared in Example 1 was examined: 15 μL of the copper-nitrogen-carbon aqueous solution (1 mg / mL) prepared in Example 1 and 50 μL of 5 mM TMB were added to 935 μL of acetate buffer (pH=3.6 0.2 M), and the absorbance at 652 nm was measured using a UV spectrophotometer for 120 s.

[0057] The results are as follows Figure 4As shown, in the presence of H2O2, the absorbance of the system at 652 nm increases significantly with the extension of reaction time, indicating that copper-nitrogen-carbon has peroxidase-mimicking activity, which can catalyze the decomposition of hydrogen peroxide to generate free radicals that cause TMB to turn blue, and has virtually no peroxidase activity.

[0058] Example 7: Specificity of copper-nitrogen-carbon materials in oxidizing ascorbic acid

[0059] Add 185 μL of phosphate buffer (pH 7.0, 0.2 M), 15 µL of the copper-nitrogen-carbon material prepared in Example 1 with a concentration of 1 mg / mL, and 50 μL of ascorbic acid with a concentration of 2 mM to 2 mL test tubes respectively. Replace these with cysteine, homocysteine, glutathione, fructose, maltose, uric acid, citric acid, glucose, and lactose respectively. Figure 5 After reacting for 10 minutes (as in step 2-10), 700 μL of phosphate buffer (pH = 6.0, 0.2 M) and 50 μL of 8 mM o-phenylenediamine were added. After reacting at 35°C for 15 minutes, the fluorescence intensity was measured at 425 nm excitation wavelength in the range of 365-680 nm. The fluorescence intensity at 425 nm excitation wavelength was measured at 350 nm.

[0060] The results are as follows Figure 5 As shown, when the substrate is ascorbic acid, the system produces strong fluorescence. When the substrate is replaced with other solutions, the system hardly emits fluorescence, demonstrating that the fluorescence method is selective for detecting ascorbic acid oxidative mimicry catalyzed by copper-nitrogen-carbon as an ascorbic acid oxidative mimicry.

[0061] Example 8: The effect of oxygen presence or absence on the oxidation of ascorbic acid by copper-nitrogen-carbon

[0062] The activity of the copper-nitrogen-carbon ascorbic acid oxidase-like enzyme prepared in Example 1 was measured under nitrogen and oxygen atmospheres, respectively: 935 μL of phosphate buffer (pH 7.0, 0.2 M), 15 µL of copper-nitrogen-carbon material with a concentration of 1 mg / mL and 50 μL of ascorbic acid with a concentration of 2 mM were added to test tubes, respectively. After reacting in a 35°C water bath with nitrogen, oxygen, and sealed for 10 minutes, the absorbance at 265 nm was measured using a UV spectrophotometer.

[0063] The results are as follows Figure 6 As shown, the absorbance at 265 nm only decreases significantly when oxygen and copper-nitrogen-carbon are present simultaneously, indicating that copper-nitrogen-carbon catalyzes the oxidation of ascorbic acid.

[0064] Example 9 Effect of temperature on copper-nitrogen-carbon oxidation of ascorbic acid

[0065] The activity of the copper-nitrogen-carbon ascorbic acid oxidase mimic prepared in Example 1 was examined in the presence of oxygen:

[0066] Add 150 µL of the copper-nitrogen-carbon (1 mg / mL) solution prepared in Example 1 and 500 μL of ascorbic acid (2 mM) to 9.35 mL of phosphate buffer (pH = 7.0, 0.2 M). Incubate the reaction solution in a water bath at 25–55 °C for 10 minutes. Measure the absorbance at 265 nm using a UV spectrophotometer, with 2 mM ascorbic acid as a control. Calculate ΔA according to the following formula:

[0067]

[0068] Let ΔA represent the catalytic activity of copper-nitrogen-carbon as an ascorbic acid oxidation mimic enzyme in the oxidation of ascorbic acid.

[0069] The results are as follows Figure 7 As shown, the temperature reaches its maximum plateau at 45℃ and then tends to stabilize. The main reason why temperature affects enzyme activity is that, as with general chemical reactions, increased temperature leads to faster molecular motion and thus a faster reaction rate; however, excessively high temperatures can cause ascorbic acid decomposition, resulting in a decrease in the measured activity.

[0070] Example 10 Effect of pH on copper-nitrogen-carbon oxidation of ascorbic acid

[0071] The activity of the copper-nitrogen-carbon ascorbic acid oxidase mimic prepared in Example 1 was investigated in the presence of oxygen: 15 µL of copper-nitrogen-carbon (1 mg / mL) and 50 μL of ascorbic acid (2 mM) were added to 935 μL of phosphate buffer (pH = 7.0, 0.2 M) in a water bath at 35°C. Reaction solutions were prepared using buffer solutions at pH 4.5, 5.0, 5.5, 6.0, 6.5, 7.5, 8.0, and 8.5, respectively. After reacting for 10 minutes, the absorbance at 265 nm was measured using a UV spectrophotometer, with 2 mM ascorbic acid as a control. ΔA was calculated according to the formula in Example 4.

[0072] The results are as follows Figure 8 As shown, the catalytic activity of copper-nitrogen-carbon continuously increases in the pH range of 4.5-6.0, while the effect on oxidation activity is relatively small in the pH range of 6-8.5, remaining at a high level. However, acidic conditions affect the dissociation state of the substrate, thereby affecting the binding of the copper-nitrogen-carbon material to the substrate and thus the reaction rate.

[0073] Example 11: Changes in ascorbic acid oxidized by copper-nitrogen-carbon over time

[0074] In the presence of oxygen, the activity of the copper-nitrogen-carbon ascorbic acid oxidase mimic prepared in Example 1 was investigated: At a water bath temperature of 35°C, 15 µL of the copper-nitrogen-carbon prepared in Example 1 (1 mg / mL) and 50 μL of ascorbic acid (2 mM) were added to 935 μL of phosphate buffer (pH = 7.0, 0.2 M). Timing was started after the addition of ascorbic acid. After thorough mixing, the absorbance at 265 nm was measured using a UV spectrophotometer at 2 min, 5 min, 8 min, 10 min, 15 min, 20 min, and 25 min of reaction time, with 2 mM ascorbic acid as a control. ΔA was calculated according to the formula in Example 4.

[0075] like Figure 9 As shown, the reaction proceeds rapidly from 2 to 10 minutes, gradually slows down after 10 minutes, and reaches a plateau at 15 minutes.

[0076] Example 12 Effect of copper-nitrogen-carbon concentration on oxidized ascorbic acid

[0077] In the presence of oxygen, the activity of the copper-nitrogen-carbon ascorbic acid oxidase mimic prepared in Example 1 was investigated: At a water bath temperature of 35°C, different volumes of copper-nitrogen-carbon aqueous solution (concentration 1 mg / mL) and 50 μL of ascorbic acid (2 mM) were added to phosphate buffer (pH = 7.0, 0.2 M) to achieve copper-nitrogen-carbon concentrations of 2.5, 5, 7.5, 10, 12.5, 15, and 17.5 mg / L, respectively. After reacting for 10 minutes, the absorbance at 265 nm was measured using a UV spectrophotometer, with 2 mM ascorbic acid as a control. ΔA was calculated according to the formula in Example 4.

[0078] The results are as follows Figure 10 As shown, the reaction concentration gradually reached a plateau after the concentration of nanomaterials reached 15 mg / L.

[0079] Example 13 Kinetics of copper-nitrogen-carbon oxidation of ascorbic acid

[0080] In the presence of oxygen, the steady-state kinetic parameters of the copper-nitrogen-carbon ascorbic acid oxidase mimicry prepared in Example 1 were investigated: 15 µL of copper-nitrogen-carbon (1 mg / mL) and 50 μL of ascorbic acid (2 mM) were added to 935 μL of phosphate buffer (pH = 7.0, 0.2 M) in a 35°C water bath. Immediately afterward, the absorbance at 265 nm was measured using a UV spectrophotometer, and the degradation rate was calculated. The Michaelis-Menten equation was then used to determine the degradation rate. V = V max ×[S] / ( K m+[S]), V max [S] represents the reaction rate when the enzyme is saturated with substrate, and [S] represents the substrate concentration. Steady-state kinetic parameters of copper-nitrogen-carbon anteroascorbic acid were obtained by fitting.

[0081] The results are as follows Figure 11 As shown: Compared to natural ascorbic acid oxidase... V max =0.4 μM / s, K m =0.003 mM, Example 1 yielded the maximum kinetic reaction rate for the copper-nitrogen-carbon oxidation of ascorbic acid. V max = 2.754 μM / s, K m = 0.012mM; has a large V max and smaller K m Due to natural ascorbic acid oxidase.

[0082] Example 14 Stability of copper-nitrogen-carbon

[0083] The copper-nitrogen-carbon material prepared in Example 1 was prepared into a 1 mg / L solution and stored for different number of days. Then, the catalytic ascorbic acid oxidation was measured. In the presence of oxygen, at a water bath temperature of 35°C, 15 µL of the copper-nitrogen-carbon material prepared in Example 1 (1 mg / mL) and 50 μL of ascorbic acid (2 mM) were added to 935 μL of phosphate buffer (pH = 7.0, 0.2 M). After reacting for 10 minutes, the ultraviolet-visible absorption spectrum and absorbance at a wavelength of 265 nm were measured using an ultraviolet spectrophotometer.

[0084] The results are as follows Figure 12 As shown, the stability of copper-nitrogen-carbon can be maintained for at least 30 days.

[0085] Example 15: Copper-nitrogen-carbon as an ascorbic acid oxidase mimicking enzyme for ascorbic acid oxidation

[0086] Ascorbic acid oxidase can oxidize ascorbic acid to dehydroascorbic acid, while o-phenylenediamine can react with dehydroascorbic acid to form a fluorescent product. A fluorescence method is used to detect whether copper-nitrogen-carbon oxidizes ascorbic acid to dehydroascorbic acid.

[0087] 185 μL of phosphate buffer (pH 7.0, 0.2 M), 15 μL of the copper-nitrogen-carbon material prepared in Example 1 with a concentration of 1 mg / mL, and 50 μL of ascorbic acid with a concentration of 2 mM were added to 2 mL test tubes. After reacting for 10 minutes, 700 μL of phosphate buffer (pH = 6.0, 0.2 M) and 50 μL of o-phenylenediamine with a concentration of 8 mM were added. After reacting at 35°C for 15 minutes, fluorescence scanning was performed at an excitation wavelength of 425 nm in the range of 365-680 nm.

[0088] The results are as follows Figure 13 As shown, the product of ascorbic acid oxidative mimicry catalyzed by copper-nitrogen-carbon can react with o-phenylenediamine to form a distinct blue fluorescent product, proving that nano-copper oxide, as an ascorbic acid oxidative mimicry, can catalyze the oxidation of ascorbic acid to dehydroascorbic acid.

Claims

1. The application of a copper-nitrogen-carbon nanomaterial as an ascorbic acid oxidase mimic or peroxidase mimic, characterized in that, A method for preparing copper-nitrogen-carbon nanomaterials includes the following steps: A methanol solution of zinc nitrate, copper acetylacetonate, and 2-methylimidazole was subjected to a hydrothermal reaction, and the hydrothermal product was calcined in the absence of oxygen. The molar ratio of zinc nitrate, copper acetylacetonate, and 2-methylimidazole is 4:(0-0.4):16, and the proportion of copper acetylacetonate is not 0. The hydrothermal reaction temperature is 110℃-150℃; The calcination temperature is 900℃.

2. The application according to claim 1, characterized in that, The hydrothermal reaction temperature is 120℃-130℃; the hydrothermal reaction time is 3-5 hours.

3. The application according to claim 1, characterized in that, The morphology consists of rhombic dodecahedral particles with a particle size of 300-1500 nm.

4. The application according to claim 1, characterized in that, The applications include ascorbic acid content detection, oxidative ascorbic acid, preparation of dehydroascorbic acid, peroxide content detection, or peroxide removal.

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