Asphalt carbon-based oxidase-like nano material as well as preparation method and application thereof

Abstract

The invention provides an asphalt carbon-based oxidase-like nano material and a preparation method and application thereof.In the preparation method of the asphalt carbon-based oxidase-like nano material, starting from modification of a coal liquefaction asphalt base material, the asphalt carbon-based oxidase-like nano material is prepared through a series of links of coal liquefaction asphalt activation, metal ion loading, nitrogen doping, heat treatment and the like. And the asphalt carbon-based oxidase-like nano material with high specific surface area, rich porous structure, stable metal active sites and optimized electron distribution is gradually constructed. Through the elaborately designed steps, the material can efficiently catalyze oxidation of a chromogenic substrate without participation of hydrogen peroxide, visual detection of the antioxidant is achieved, and meanwhile it is ensured that the detection method has the remarkable advantages of being low in cost, easy and convenient to operate, high in sensitivity and wide in applicability; and powerful support is provided for technical innovation in the fields of food safety and health monitoring.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biosensors, in particular to an asphalt carbon-based oxidase-like nanomaterial, a preparation method and application thereof. BACKGROUND

[0002] With the rapid development of social economy and the significant improvement of people's living standards, food safety has become the focus of global attention, and its importance cannot be underestimated. During food processing and storage, antioxidants play a crucial role in effectively removing harmful free radicals and slowing down the oxidation rate of food, thereby extending the shelf life of food and maintaining the nutritional quality and sensory characteristics of food. The rational use of antioxidants is crucial to public health, however, once it exceeds the safe intake range or there is illegal addition, it may cause a series of health risks, including allergic reactions, metabolic disorders and even carcinogenic effects. Therefore, it is particularly urgent to establish a system that can quickly and accurately detect the content of antioxidants in food to maintain food safety and public health.

[0003] Traditional antioxidant detection techniques mainly rely on instrument analysis methods such as high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and electrochemical methods. Although these methods can provide accurate analysis results, they face many challenges in practical applications - complex operation procedures, high equipment costs, long analysis periods, and high standards for professional technical personnel. The above problems to some extent hinder their popularization and application in large-scale screening and on-site rapid detection.

[0004] In recent years, nanoenzymes have shown great potential in biological detection, environmental monitoring, and medical diagnosis due to their high catalytic efficiency similar to natural enzymes, higher thermal stability, chemical stability, and ease of preparation. This has attracted widespread attention from the scientific community. In particular, asphalt carbon-based oxidase-like nanomaterials can produce a color signal by catalyzing a specific color substrate and oxygen without the need for additional hydrogen peroxide (H2O2), enabling visual detection of target substances, greatly simplifying the detection process and improving detection efficiency. Currently, the construction methods of such materials mainly include the use of noble metal nanoparticles, transition metal oxides, metal-organic framework materials (MOFs), and nitrogen-containing carbon materials. Although the above materials perform well in improving catalytic activity, they generally have large resource consumption, complex process, high cost, and serious environmental negative impact during preparation, which greatly restricts their application and promotion in large-scale detection scenarios.

[0005] To overcome the limitations of the prior art, researchers have begun to explore more green and sustainable preparation approaches. Biomass carbon materials are considered as ideal carrier materials for the preparation of nanoenzymes due to their extensive natural sources, porous structure, large specific surface area, and rich surface functional groups. Coal liquefaction pitch, as a derivative product in the process of deep processing of coal, not only has abundant resources and low cost, but also is rich in aromatic ring carbon structures and various polar functional groups, which makes it have a significant innate advantage in the preparation of carbon-based catalytic materials. Although existing technologies attempt to modify it to improve performance, such as the preparation method of modified pitch special for high-performance porous graphite material mentioned in patent CN118421101A, its process is complex and tedious, which limits its potential in more extensive applications.

[0006] In summary, how to develop a detection method and material that can not only achieve rapid and accurate detection of antioxidants, but also reasonably utilize resources, be green and low-carbon, and have controllable cost, to overcome the problems of complex operation process, high equipment investment, long detection period, and high professional skill requirements in traditional detection methods, has become a problem to be solved. SUMMARY

[0007] The main purpose of the present application is to provide a pitch carbon-based oxidase-like nanomaterial and its preparation method and application, to solve the problems of complex operation process, high equipment investment, long detection period, and high professional skill requirements in the detection process of antioxidants in the prior art, and to develop a detection method and material that can not only achieve rapid and accurate detection of antioxidants, but also reasonably utilize resources, be green and low-carbon, and have controllable cost.

[0008] The present application provides a preparation method of a pitch carbon-based oxidase-like nanomaterial, which comprises the following steps: mixing coal liquefaction pitch with an activator to obtain a first mixture; the first mixture is subjected to first calcination, acid washing, and first drying to obtain activated coal liquefaction pitch; the activated coal liquefaction pitch is mixed with a soluble salt solution containing transition metal ions to obtain a mixed slurry; the mixed slurry is separated and then subjected to second drying to obtain a pitch carbon-based oxidase-like nanomaterial precursor; the pitch carbon-based oxidase-like nanomaterial precursor is mixed with an organic nitrogen-containing compound to obtain a second mixture; and the second mixture is subjected to second calcination to obtain a pitch carbon-based oxidase-like nanomaterial.

[0009] Further, the soluble salt solution containing transition metal ions contains at least two kinds of transition metal ions; preferably, the soluble salt solution containing transition metal ions contains at least two kinds of transition metal ions selected from iron ions, ferrous ions, copper ions, cuprous ions, zinc ions, cobalt ions, manganese ions and nickel ions; more preferably, the soluble salt solution containing transition metal ions contains at least three kinds of transition metal ions selected from iron ions, ferrous ions, copper ions, cuprous ions, zinc ions, cobalt ions, manganese ions and nickel ions; further preferably, the soluble salt solution containing transition metal ions contains a combination of iron ions, copper ions and zinc ions; or, the soluble salt solution containing transition metal ions contains a combination of iron ions, manganese ions and zinc ions; preferably, when the soluble salt solution containing transition metal ions contains a combination of iron ions, copper ions and zinc ions, the molar ratio of the three is 1:(0.1-10):(0.1-10); or, when the soluble salt solution containing transition metal ions contains a combination of iron ions, manganese ions and zinc ions, the molar ratio of the three is 1:(0.1-10):(0.1-10).

[0010] Further, the weight ratio of the activated coal liquefied pitch to the soluble salt containing transition metal ions is 1:(0.1-5); and / or, the weight ratio of the pitch carbon-based oxidase-like enzyme nanomaterial precursor to the organic nitrogen-containing compound is 1:(0.1-20); preferably, the weight ratio of the activated coal liquefied pitch to the soluble salt containing transition metal ions is 1:(0.2-2); and / or, the weight ratio of the pitch carbon-based oxidase-like enzyme nanomaterial precursor to the organic nitrogen-containing compound is 1:(5-15).

[0011] Further, the weight ratio of the coal liquefied pitch to the activating agent is 1:(1-10); preferably, the activating agent is a bio-source activating agent; preferably, the bio-source activating agent is selected from at least one of the powders of eggshells, oyster shells, salmon bones, cow horns or shrimp shells; more preferably, the bio-source activating agent is selected from at least one of the powders of eggshells, oyster shells or shrimp shells; preferably, the organic nitrogen-containing compound is selected from one or more of urea, dicyandiamide or melamine.

[0012] Further, the first calcination is performed at a temperature of 200-1000℃ for 1-8h; preferably, the first calcination is performed at a temperature of 400-900℃ for 2-6h; preferably, the second calcination comprises a first-stage calcination and a second-stage calcination performed in sequence, the first-stage calcination is performed at a temperature of 400-600℃ for 0.5-2h; the second-stage calcination is performed at a temperature of 700-1000℃ for 3-6h; preferably, the first calcination and the second calcination are performed in an inert gas atmosphere; preferably, the second mixing is performed for 8-20h; preferably, the first drying is performed at a temperature of 60-100℃ for 6-12h; and / or, the second drying is performed at a temperature of 40-80℃ for 4-12h.

[0013] Further, the acid washing solvent is hydrochloric acid aqueous solution; preferably, the concentration of the acid washing solvent is 0.8-1.2mol / L; preferably, the particle size of the coal liquefaction pitch is 40-100 mesh; and / or, the particle size of the activator is 60-100 mesh.

[0014] According to a second aspect of the present application, there is further provided a pitch carbon-based class oxidase nanomaterial prepared by the above preparation method.

[0015] According to a third aspect of the present application, there is further provided an application of the above pitch carbon-based class oxidase nanomaterial in a sensing array detection, the pitch carbon-based class oxidase nanomaterial is used for a three-channel sensing array detection of antioxidant species; wherein the antioxidant is two or more of tannic acid, glutathione, gallic acid, curcumin, quercetin, epigallocatechin gallate, ascorbic acid, protocatechuic acid or ferulic acid; a chromogenic substrate used for establishing the three-channel sensing array is selected from 3,3',5,5'-tetramethylbenzidine, 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt and o-phenylenediamine.

[0016] According to a fourth aspect of the present application, there is further provided an application of the above pitch carbon-based class oxidase nanomaterial in antioxidant concentration detection, the pitch carbon-based class oxidase nanomaterial is used for catalytic oxidation of a chromogenic substrate to detect the concentration of the antioxidant; preferably, the chromogenic substrate is at least one selected from 3,3',5,5'-tetramethylbenzidine, 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt or o-phenylenediamine; more preferably, the chromogenic substrate is selected from 3,3',5,5'-tetramethylbenzidine; preferably, the antioxidant is at least one selected from tannic acid, glutathione, gallic acid, curcumin, quercetin, epigallocatechin gallate, ascorbic acid, protocatechuic acid or ferulic acid.

[0017] According to the fifth aspect of the present application, a method for detecting the concentration of an antioxidant is also provided, which comprises the following steps: placing a chromogenic substrate and the asphalt carbon-based oxidase nanomaterial described above into a solution containing an antioxidant to perform an oxidation reaction, so as to obtain a to-be-tested solution; determining the absorbance of the to-be-tested solution by using a spectrophotometric method, and calculating the concentration of the antioxidant in the to-be-tested solution; wherein the chromogenic substrate is selected from at least one of 3,3',5,5'-tetramethylbenzidine, 2,2-diamino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt or o-phenylenediamine.

[0018] Further, the chromogenic substrate is selected from 3,3',5,5'-tetramethylbenzidine; preferably, the concentration of the asphalt carbon-based oxidase nanomaterial is 0.05-1 g / L; and / or, in the to-be-tested solution, the concentration of the chromogenic substrate is 0.1-2 mmol / L; and / or, the concentration of the antioxidant is 0.01-300 µmol / L; preferably, the temperature of the oxidation reaction is 30-50 °C; preferably, the pH of the to-be-tested solution is 2-8; more preferably, the pH of the to-be-tested solution is 3-5; preferably, the antioxidant is ascorbic acid.

[0019] The preparation method of the asphalt carbon-based oxidase nanomaterial provided in the present application starts from the modification of a coal liquefaction asphalt base material, and through a series of steps such as activation of the coal liquefaction asphalt, loading of metal ions, nitrogen doping and heat treatment, an asphalt carbon-based oxidase nanomaterial with high specific surface area, rich porous structure, stable metal active sites and optimized electronic distribution is gradually constructed. These carefully designed steps enable the material to realize efficient catalysis of the oxidation of the chromogenic substrate without the participation of hydrogen peroxide, realize the visual detection of the antioxidant, and at the same time ensure that the detection method has the remarkable advantages of low cost, simple operation, high sensitivity and wide applicability, thereby providing strong support for the technological innovation in the field of food safety and health monitoring. BRIEF DESCRIPTION OF DRAWINGS

[0020] The accompanying drawings, which form a part of the present application, are used to provide further understanding of the present application, and the illustrative embodiments of the present application and their descriptions are used to explain the present application, and do not constitute improper limitations on the present application. In the drawings:

[0021] Figure 1 A TEM image of the asphalt carbon-based oxidase nanomaterial prepared according to Example 1 of the present application is shown;

[0022] Figure 2 A HAADF-STEM image of the asphalt carbon-based oxidase nanomaterial prepared according to Example 1 of the present application is shown;

[0023] Figure 3 A SAED image of the asphalt carbon-based oxidase nanomaterial prepared according to Example 1 of the present application is shown;

[0024] Figure 4 An EDS graph of the asphalt carbon-based oxidase-like enzyme nanomaterial prepared according to Embodiment 1 of the present application is shown;

[0025] Figure 5 An XRD graph of the asphalt carbon-based oxidase-like enzyme nanomaterial prepared according to Embodiment 1 of the present application is shown;

[0026] Figure 6 A Raman graph of the asphalt carbon-based oxidase-like enzyme nanomaterial prepared according to Embodiment 1 of the present application is shown;

[0027] Figure 7 An FTIR graph of the asphalt carbon-based oxidase-like enzyme nanomaterial prepared according to Embodiment 1 of the present application is shown;

[0028] Figure 8 A BET graph of the asphalt carbon-based oxidase-like enzyme nanomaterial prepared according to Embodiment 1 of the present application is shown;

[0029] Figure 9 An XPS graph of the asphalt carbon-based oxidase-like enzyme nanomaterial prepared according to Embodiment 1 of the present application is shown;

[0030] Figure 10 An EDS element analysis graph of the asphalt carbon-based oxidase-like enzyme nanomaterial prepared according to Embodiment 1 of the present application is shown;

[0031] Figure 11 A standard curve graph of ascorbic acid standard concentration and absorbance is shown;

[0032] Figure 12 A standard curve graph of ascorbic acid standard concentration and three primary colors is shown;

[0033] Figure 13 A schematic diagram of the preparation process of the asphalt carbon-based oxidase-like enzyme nanomaterial and the process of testing antioxidants according to Embodiment 1 of the present application is shown;

[0034] Figure 14 A component analysis graph of the establishment of a three-channel sensing array to distinguish antioxidant types by the asphalt carbon-based oxidase-like enzyme nanomaterial according to Embodiment 1 of the present application is shown;

[0035] Figure 15 A response bubble graph of the establishment of a three-channel sensing array to distinguish antioxidant types by the asphalt carbon-based oxidase-like enzyme nanomaterial according to Embodiment 1 of the present application is shown. DETAILED DESCRIPTION

[0036] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other in the case of no conflict. The present application will be described in detail below with reference to the drawings and in combination with the embodiments.

[0037] As described in the background section, there are problems such as complex operation process, high equipment investment, long detection period and high professional skill requirement in the detection of antioxidants in the prior art. Nanoenzymes have high efficient catalytic characteristics similar to natural enzymes, and have the advantages of higher thermal stability, chemical stability and easy preparation. In many fields such as biological detection, environmental monitoring and medical diagnosis, nanoenzymes have shown great potential. The construction methods of such materials mainly include the use of noble metal nanoparticles, transition metal oxides, metal-organic framework materials (MOFs) and nitrogen-containing carbon materials. Although the above materials perform well in improving catalytic activity, there are problems such as large resource consumption, complex process, high cost and serious environmental negative impact in the preparation process, which greatly restricts the application and promotion of nanoenzymes in large-scale detection scenarios.

[0038] In order to solve the above problems, the present application provides a preparation method of asphalt carbon-based oxidase-like nanomaterial, which comprises the following steps: mixing coal liquefied asphalt with an activator to obtain a first mixed material; the first mixed material is subjected to first calcination, acid washing and first drying to obtain activated coal liquefied asphalt; the activated coal liquefied asphalt is mixed with a soluble salt solution containing transition metal ions to obtain a mixed slurry; the mixed slurry is separated and then subjected to second drying to obtain an asphalt carbon-based oxidase-like nanomaterial precursor; the asphalt carbon-based oxidase-like nanomaterial precursor is mixed with an organic nitrogen-containing compound to obtain a second mixed material; and the second mixed material is subjected to second calcination to obtain an asphalt carbon-based oxidase-like nanomaterial.

[0039] Specifically, in the above preparation method, first, the coal liquefaction pitch is mixed with the activator to enable the coal liquefaction pitch and the activator to be in sufficient contact, and then the obtained mixture is calcined and pickled to obtain the activated coal liquefaction pitch. The activator can improve the microstructure of the coal liquefaction pitch, and can effectively improve the surface energy of the coal liquefaction pitch while making the specific surface area and pore size distribution of the obtained activated coal liquefaction pitch better, thereby facilitating better adsorption and dispersion of transition metal ions and nitrogen elements, and making the structure and performance of the prepared pitch carbon-based oxidase nanomaterial more uniform and better. Then, the activated coal liquefaction pitch is mixed with a soluble salt solution containing transition metal ions to enable the transition metal ions to be better adsorbed or doped on the surface and in the pore of the activated coal liquefaction pitch. After the obtained mixed slurry is separated and dried, a pitch carbon-based oxidase nanomaterial precursor is obtained. Finally, the obtained pitch carbon-based oxidase nanomaterial precursor is mixed with an organic nitrogen-containing compound, and after being calcined again, a pitch carbon-based oxidase nanomaterial is obtained. The material is an artificial biomimetic catalytic material with catalytic function similar to natural oxidase, and can catalyze the oxidation of a color developing substrate (such as 3,3',5,5'-tetramethylbenzidine (TMB), 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS), o-phenylenediamine (OPD), etc.) to generate a colored product or a fluorescent product under the condition of no additional hydrogen peroxide (H2O2). The colored product or the fluorescent product is then reduced by an antioxidant (such as ascorbic acid), and the concentration of the antioxidant is detected according to the above color developing characteristics or fluorescence characteristics.

[0040] In summary, the preparation method of the pitch carbon-based oxidase nanomaterial provided in the present application uses coal liquefaction pitch as a raw material, which is widely available and inexpensive, and has the advantages of efficient resource utilization and cost-effectiveness. The method is simple and easy to operate, and is suitable for large-scale production of pitch carbon-based oxidase nanomaterials. The pitch carbon-based oxidase nanomaterial prepared by the above method is used for detecting antioxidants, and exhibits excellent enzyme kinetics, high anti-interference ability and stable activity, and can realize rapid and accurate detection of antioxidants. The reasons for the above effects may include the following aspects:

[0041] Firstly, the asphalt carbon-based oxidase-like nanomaterial is prepared by taking coal liquefied asphalt as the base material of the catalyst, and activating the coal liquefied asphalt by using an activating agent. The activating agent is decomposed at high temperature to generate a large number of pores, so that the microstructure of the activated coal liquefied asphalt material is changed, and a porous carbon matrix with a high specific surface area is formed, which provides sufficient active sites for subsequent loading of metal ions and doping of nitrogen atoms, and is conducive to better improving the activity and anti-interference ability of the asphalt carbon-based oxidase-like nanomaterial. Moreover, the by-product coal liquefied asphalt is used as a raw material for preparing the catalyst, which solves the problem of its treatment and utilization, realizes high-value utilization of resources, and especially, compared with the traditional nanomaterials for constructing nanoenzymes, which use noble metal nanoparticles and metal-organic framework materials as raw materials, the method provided by the present application has the advantages of lower cost and easier scale production, so that the asphalt carbon-based oxidase-like nanomaterial is more competitive in the rapid detection of antioxidants.

[0042] Secondly, in the preparation of the asphalt carbon-based oxidase-like nanomaterial, the activated coal liquefied asphalt is mixed with a soluble salt solution containing transition metal ions to obtain a precursor of the asphalt carbon-based oxidase-like nanomaterial, and then the precursor is mixed with an organic nitrogen-containing compound and calcined to obtain the asphalt carbon-based oxidase-like nanomaterial. In the above process, the transition metal ions are fixed on the porous surface of the activated asphalt by physical adsorption and chemical bonding. The introduction of the transition metal ions can generate oxidase-like active centers on the surface of the material, which can effectively activate oxygen molecules to convert them into active oxygen species (ROS) with strong oxidizing ability, such as singlet oxygen ( 1 O2), superoxide radical ( ) and hydroxyl radical ( OH), which significantly enhances the enzymatic kinetic activity of the asphalt carbon-based oxidase-like nanomaterial, and is conducive to further enhancing the catalytic efficiency and oxygen activation ability. Moreover, the presence of the transition metal active center in the catalyst can also adjust the electronic structure of the asphalt carbon-based oxidase-like nanomaterial, enhance the activation efficiency of oxygen, and thus improve the activation efficiency and stability of the material. In addition, the addition of nitrogen atoms can promote the flow of electrons on the surface of the material, improve the activity of the transition metal through the property of electron donor, and optimize the electronic structure of the carbon material, which is conducive to further enhancing the stability of the asphalt carbon-based oxidase-like nanomaterial, better promoting the generation of ROS, and thus further improving the rapid and accurate detection ability of the asphalt carbon-based oxidase-like nanomaterial for antioxidants.

[0043] In summary, the preparation method of the asphalt carbon-based oxidase nanomaterial provided in the application starts from modifying the coal liquefaction asphalt base material, and through a series of links such as activation of the coal liquefaction asphalt, loading of metal ions, nitrogen doping, and heat treatment, gradually constructs the asphalt carbon-based oxidase nanomaterial with high specific surface area, rich porous structure, stable metal active sites, and optimized electronic distribution. These carefully designed steps enable the material to realize efficient catalytic oxidation of the chromogenic substrate without the participation of hydrogen peroxide, realize the visual detection of the antioxidant, and at the same time ensure that the detection method has the remarkable advantages of low cost, simple operation, high sensitivity, and wide applicability, thereby providing strong support for the technological innovation in the field of food safety and health monitoring.

[0044] It needs to be further explained here that in the process of preparing the asphalt carbon-based oxidase nanomaterial in the application, coal liquefaction asphalt with a carbon content of >80% and an ash content of less than 0.2% can be used to further improve the asphalt carbon-based oxidase nanomaterial.

[0045] In a preferred embodiment, the soluble salt solution containing transition metal ions contains at least two kinds of transition metal ions; at least two of the transition metal ions are used to prepare the asphalt carbon-based oxidase nanomaterial, which can better adjust the electronic properties and catalytic activity of the prepared asphalt carbon-based oxidase nanomaterial, thereby better activating oxygen to generate active oxygen species (ROS) such as superoxide radicals ( ), which is conducive to further improving the oxidation efficiency of the chromogenic substrate (such as TMB, ABTS, OBD) and achieving better color development effect, and is conducive to further improving the sensitivity and accuracy of the asphalt carbon-based oxidase nanomaterial in the antioxidant detection process.

[0046] Preferably, the soluble salt solution containing transition metal ions contains at least two kinds of transition metal ions selected from iron ions (Fe 3+ ), ferrous ions (Fe 2+ ), copper ions (Cu 2+ ), cuprous ions (Cu + ), zinc ions (Zn 2+ ), cobalt ions (Co 2+ ), manganese ions (Mn 2+ ), and nickel ions (Ni 2+ ). Using at least two of the above specific transition metal ions to prepare the oxidase nanomaterial can achieve better kinetic performance of the obtained oxidase nanomaterial and better improvement effect on the sensitivity and accuracy in the antioxidant detection process.

[0047] More preferably, the transition metal ions in the soluble salt solution containing transition metal ions are selected from at least three of iron ions, ferrous ions, copper ions, cuprous ions, zinc ions, cobalt ions, manganese ions and nickel ions. The use of at least three transition metal ions in combination can synergistically regulate the electronic structure of the asphalt carbon-based oxidase-like nanomaterial, form a multi-metal electronic circulation effect in the asphalt carbon-based oxidase-like nanomaterial, and further improve the active sites on the surface of the material, thereby improving the detection effect of the asphalt carbon-based oxidase-like nanomaterial on antioxidants.

[0048] It should be further pointed out here that when the soluble salt solution containing transition metal ions contains at least two or more transition metal ions, the molar ratio of the metal ions is not limited.

[0049] Further preferably, the transition metal ions in the soluble salt solution containing transition metal ions are iron ions, copper ions and zinc ions in combination; or, the transition metal ions in the soluble salt solution containing transition metal ions are iron ions, manganese ions and zinc ions in combination; preferably, when the transition metal ions in the soluble salt solution containing transition metal ions are iron ions, copper ions and zinc ions in combination, the molar ratio of the three is 1:(0.1-10):(0.1-10); or, when the transition metal ions in the soluble salt solution containing transition metal ions are iron ions, manganese ions and zinc ions in combination, the molar ratio of the three is 1:(0.1-10):(0.1-10). The inventors have found through a large number of experiments that the combination of the above transition metal ions can improve the detection performance of the asphalt carbon-based oxidase-like nanomaterial on antioxidants. In particular, when the ratio of each component is controlled within the above range, the above effect is better. More preferably, when the transition metal ions in the soluble salt solution containing transition metal ions are iron ions, copper ions and zinc ions in combination, the molar ratio of the three is 1:(0.1-5):(5-10); or, when the transition metal ions in the soluble salt solution containing transition metal ions are iron ions, manganese ions and zinc ions in combination, the molar ratio of the three is 1:(0.1-5):(5-10), and the performance of the asphalt carbon-based oxidase-like nanomaterial is better within the above ratio range.

[0050] In a preferred embodiment, the weight ratio of the activated coal liquefied pitch to the soluble salt containing transition metal ions is 1:(0.1-5); and / or, the weight ratio of the pitch carbon-based oxidase-like enzyme nanomaterial precursor to the organic nitrogen-containing compound is 1:(0.1-20). As described above, the introduction of transition metal ions can generate oxidase-like enzyme active centers on the surface of the material, which can effectively activate oxygen molecules into active oxygen species with strong oxidizing ability, enhancing the catalytic efficiency of the pitch carbon-based oxidase-like enzyme nanomaterial and the oxygen activation ability; the addition of nitrogen atoms can promote the flow of electrons on the surface of the material, optimize the electronic structure of the carbon material, improve the activity of the transition metal through the nature of the electron donor, and at the same time enhance the stability of the pitch carbon-based oxidase-like enzyme nanomaterial, form a more complex and effective active center, and better promote the generation of active oxygen species. Controlling the weight ratio of the activated coal liquefied pitch to the soluble salt containing transition metal ions and the weight ratio of the pitch carbon-based oxidase-like enzyme nanomaterial precursor to the organic nitrogen-containing compound in the preparation process of the pitch carbon-based oxidase-like enzyme nanomaterial within the above range can better play the above-mentioned roles, and the performance of the pitch carbon-based oxidase-like enzyme nanomaterial is better. Preferably, the weight ratio of the activated coal liquefied pitch to the soluble salt containing transition metal ions is 1:(0.2-2); and / or, the weight ratio of the pitch carbon-based oxidase-like enzyme nanomaterial precursor to the organic nitrogen-containing compound is 1:(5-15). Controlling the parameters in the preparation process within the above preferred range corresponds to better performance.

[0051] In a preferred embodiment, the weight ratio of the coal liquefied pitch to the activator is 1:(1-10). Controlling the weight ratio of the coal liquefied pitch to the activator within the above range can make the activation effect of the coal liquefied pitch better, thereby facilitating further improvement of the performance of the pitch carbon-based oxidase-like enzyme nanomaterial. More preferably, the weight ratio of the coal liquefied pitch to the activator is 1:(3-10), and controlling the weight ratio of the coal liquefied pitch to the activator within the above range can make the performance of the obtained activated coal liquefied pitch better. Preferably, the activator is a biological source activator; preferably, the biological source activator is selected from the powder of at least one of eggshells, oyster shells, salmon bones, cow horns, or shrimp shells; more preferably, the biological source activator is selected from the powder of at least one of eggshells, oyster shells, or shrimp shells. The above biological source activator contains a large amount of calcium carbonate, and the carbon dioxide generated by the decomposition of calcium carbonate at high temperature can make the pitch form a better porous structure, and compared with traditional activators, the above biological source activator has better environmental protection characteristics while having a better activation effect.

[0052] By way of example but not limitation, the iron-containing metal salt is selected from the group consisting of ferric chloride, ferric nitrate, ferric sulfate; the Cu-containing metal salt is selected from the group consisting of copper nitrate, copper chloride, copper sulfate; the Zn-containing metal salt is selected from the group consisting of zinc nitrate, zinc acetate, zinc chloride. Preferably, the soluble nitrogen-containing substance containing nitrogen element is selected from one or more of urea, dicyandiamide or melamine.

[0053] In a preferred embodiment, the first calcination is performed at a temperature of 200-1000°C for 1-8h; preferably, the first calcination is performed at a temperature of 400-900°C for 2-6h; preferably, the second calcination comprises a first-stage calcination and a second-stage calcination performed sequentially, the first-stage calcination is performed at a temperature of 400-600°C for 0.5-2h; the second-stage calcination is performed at a temperature of 700-1000°C for 3-6h; preferably, the first calcination and the second calcination are performed in an inert gas atmosphere; preferably, the second mixing is performed for 8-20h; preferably, the first drying is performed at a temperature of 60-100°C for 6-12h; and / or, the second drying is performed at a temperature of 40-80°C for 4-12h.

[0054] The first calcination can carbonize the organic matter in the raw material, form a porous structure, increase the specific surface area and surface energy, and provide abundant active sites for the subsequent loading of metal ions and doping of nitrogen-containing elements. The second calcination comprises a first-stage calcination and a second-stage calcination performed sequentially, wherein the first-stage calcination can promote the combination of nitrogen elements and the pitch carbon substrate, form a stable composite structure, and achieve uniform distribution and stable anchoring of the metal or nitrogen-containing elements in the catalyst; the second-stage calcination can carbonize the pitch carbon substrate, so that the nitrogen elements, metal elements and carbon elements in the prepared pitch carbon-based oxidase nanomaterial can be uniformly and orderly dispersed. Controlling the temperature and time of the first calcination and the second calcination within the above ranges can better achieve the above effects, which is conducive to further improving the catalytic performance of the prepared catalyst. In particular, performing the first calcination and the second calcination in an inert gas atmosphere can prevent the catalyst from reacting with oxygen or other components in the air at high temperatures, protect the catalyst from oxidative damage, ensure the activity of the metal elements and the stability of the nitrogen-containing element doping. In addition, the inert atmosphere can also avoid uncontrollable changes in the structure of the catalyst, maintain its designed pore structure and specific surface area, and thus ensure the high performance of the catalyst.

[0055] In a preferred embodiment, the acid pickling solvent is an aqueous hydrochloric acid solution; preferably, the concentration of the acid pickling solvent is 0.8-1.2 mol / L; preferably, the particle size of the coal liquefaction pitch is 40-100 mesh; and / or, the particle size of the activating agent is 60-100 mesh. Controlling the parameters in the process of preparing the pitch carbon-based oxidase nanomaterial within the above ranges can result in a pitch carbon-based oxidase nanomaterial with better performance.

[0056] According to a second aspect of the present application, there is further provided a pitch carbon-based class oxidase nanomaterial, which is prepared by the above method.

[0057] According to a third aspect of the present application, there is further provided an application of the above pitch carbon-based class oxidase nanomaterial in a sensing array detection, wherein the pitch carbon-based class oxidase nanomaterial is used for detecting the types of antioxidants in a three-channel sensing array; the antioxidants are two or more of tannic acid (TA), glutathione (GSH), gallic acid (GA), curcumin (CUR), quercetin (QUE), epigallocatechin gallate (EGCG), ascorbic acid (AA), protocatechuic acid (PA) or ferulic acid (FA); and the chromogenic substrate used for establishing the three-channel sensing array is selected from 3,3',5,5'-tetramethylbenzidine, 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt and o-phenylenediamine. The pitch carbon-based class oxidase nanomaterial provided in the present application has a good response effect on the three chromogenic substrates, i.e. 3,3',5,5'-tetramethylbenzidine, 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt and o-phenylenediamine, and the types of the above nine different antioxidants can be distinguished according to the response of the chromogenic substrate to the antioxidants.

[0058] According to a fourth aspect of the present application, there is further provided an application of the above pitch carbon-based class oxidase nanomaterial in antioxidant concentration detection, wherein the pitch carbon-based class oxidase nanomaterial is used for catalyzing oxidation of a chromogenic substrate to detect the concentration of an antioxidant; the pitch carbon-based class oxidase nanomaterial provided in the present application, when used for catalyzing oxidation of a chromogenic substrate to detect an antioxidant, can not only realize efficient detection of the antioxidant, but also exhibit excellent enzyme kinetics, high anti-interference ability and stable activity. Preferably, the chromogenic substrate is at least one selected from 3,3',5,5'-tetramethylbenzidine, 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt and o-phenylenediamine; more preferably, the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine; and preferably, the antioxidant is at least one selected from tannic acid, glutathione, gallic acid, curcumin, quercetin, epigallocatechin gallate, ascorbic acid, protocatechuic acid and ferulic acid. The pitch carbon-based class oxidase nanomaterial provided in the present application has excellent chromogenic effect on the above types of chromogenic substrates and the above types of antioxidants, and thus can accurately detect the concentration of the above types of antioxidants.

[0059] According to a fifth aspect of the present invention, a method for detecting antioxidant concentration is also provided, the method comprising the following steps: placing a chromogenic substrate and the above-mentioned pitch carbon-based oxidase nanomaterial in a solution containing an antioxidant to carry out an oxidation reaction to obtain a test solution; measuring the absorbance of the test solution by spectrophotometry and calculating the concentration of antioxidant in the test solution; wherein the chromogenic substrate is selected from at least one of 3,3',5,5'-tetramethylbenzidine, 2,2-azido-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt or o-phenylenediamine.

[0060] In a preferred embodiment, the chromogenic substrate in the test solution is selected from 3,3',5,5'-tetramethylbenzidine; preferably, the concentration of the pitch-carbon-based oxidase nanomaterial is 0.05~1 g / L; and / or, the concentration of the chromogenic substrate in the test solution is 0.1~2 mmol / L; and / or, the concentration of the antioxidant is 0.01~300 µmol / L; preferably, the oxidation reaction temperature is 30~50℃; preferably, the pH of the test solution is 2~8; more preferably, the pH of the test solution is 3~5; preferably, the antioxidant is ascorbic acid. Controlling the parameters in the antioxidant concentration detection process within the above ranges results in faster, more accurate, and more efficient detection results.

[0061] The present application will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed in the present application.

[0062] It should be further noted that the coal liquefaction pitch raw material used in this embodiment was provided by China Shenhua Coal-to-Oil Chemical Co., Ltd. The ash content of this coal liquefaction pitch raw material is less than 0.1%, and the weight content parameters of N, C, S, and H are shown below:

[0063]

[0064] Example 1

[0065] (1) Preparation of pitch carbon-based oxidase nanomaterials

[0066] 10g of coal liquefaction pitch raw material was ground into powder and passed through an 80-mesh sieve. Eggshells were ground into powder and passed through an 80-mesh sieve. The pitch powder and eggshell powder were mixed in a 1:3 ratio and calcined at 900℃ under argon for 5 hours at a heating rate of 5℃ / min and an argon flow rate of 70mL / min. The calcined product was washed with 1mol / L hydrochloric acid for 12 hours, then filtered and dried at 80℃ for 12 hours to obtain activated pitch.

[0067] The obtained activated pitch 0.4 g was put into 50 mL of a solution containing soluble salt of transition metal ions (wherein the concentration of ferric nitrate was 10 mmol / L, the concentration of copper nitrate was 10 mmol / L, and the concentration of zinc nitrate was 50 mmol / L), mixed and stirred for 12 h, dried at 80 ℃ for 6 h after filtration, mixed with dicyandiamide 1:10, and then calcined at 550 ℃ for 1 h and then at 900 ℃ for 4 h at an argon atmosphere, a temperature rising rate of 5 ℃ / min, and an argon flow rate of 70 mL / min to obtain pitch carbon-based oxidase-like nanomaterials.

[0068] The obtained pitch carbon-based oxidase-like nanomaterials were tested for relevant performance, and the results were as follows:

[0069] Figure 1 TEM images of the pitch carbon-based oxidase-like nanomaterials are shown, and it can be observed from the images that Fe nanoparticles are formed on the carbon layer;

[0070] Figure 2 HAADF-STEM images of the pitch carbon-based oxidase-like nanomaterials are shown, and it can be further observed from the images that Fe nanoparticles are formed on the carbon layer;

[0071] Figure 3 SAED images of the pitch carbon-based oxidase-like nanomaterials are shown, and it can be seen from the images that there are iron nanoparticles in the material, which corresponds to the results of Figure 1 , 2;

[0072] Figure 4 EDS images of the pitch carbon-based oxidase-like nanomaterials are shown, and it can be seen from the images that C, Fe, N, and Cu elements coexist in the prepared catalyst;

[0073] Figure 5 XRD images of the oxidase-like nanomaterials are shown, and it can be seen from the images that diffraction peaks of copper crystals and iron crystals are observed, indicating the formation of nanoparticles;

[0074] Figure 6 Raman images of the oxidase-like nanomaterials are shown, and it can be seen from the images that the spectrum shows I D / I G (defective carbon / graphitic carbon) = 1.07, indicating that the catalyst has good defect degree;

[0075] Figure 7 FTIR images of the oxidase-like nanomaterials are shown, and it can be seen from the images that the catalyst has abundant functional groups;

[0076] Figure 8 BET images of the oxidase-like nanomaterials are shown, and the images show that the specific surface area of the catalyst is 65.63 m 2 / g;

[0077] Figure 9 XPS diagram of oxidase nanomaterials is shown, which indicates that the catalyst contains C element, N element, O element, Fe element and Cu element;

[0078] Figure 10 EDS element content diagram of oxidase nanomaterials is shown; the diagram can reflect the content of C element, N element, Fe element and Cu element in the catalyst.

[0079] (2) Oxidase-like activity evaluation

[0080] The reaction kinetics of the asphalt carbon-based oxidase-like nanomaterials is analyzed, and the Michaelis constant (K m ) and the maximum reaction rate (V max ) are calculated based on the Lineweaver-Burk double-reciprocal equation. K m can reflect the affinity of the asphalt carbon-based oxidase-like nanomaterials for the color developing substrate TMB; V max can reflect the maximum reaction speed of the asphalt carbon-based oxidase-like nanomaterials; K m and V max can reflect the response activity of the asphalt carbon-based oxidase-like nanomaterials to the substrate TMB. The specific operation includes: adding 0.2 mL of the above asphalt carbon-based oxidase-like nanomaterials with a concentration of 1.5 mg / mL in 1.6 mL of a buffer solution composed of acetic acid / sodium acetate (pH=4), and then adding 0.2 mL of TMB solution with concentrations of 0.5 mmol / L, 1 mmol / L, 1.5 mmol / L, 2 mmol / L, 2.5 mmol / L, 3 mmol / L, 4 mmol / L and 5 mmol / L, respectively, to the above prepared solution, and each reaction is carried out. After the reaction is completed, the absorbance of the obtained solution after the reaction and the Lineweaver-Burk equation are used to calculate K m and V max . The expression of the Lineweaver-Burk double-reciprocal equation is:

[0081] ;

[0082] In the above equation, v: reaction rate; [S]: substrate concentration; V max : maximum reaction rate, reaction speed when the enzyme is completely saturated with the substrate; K m : value of substrate concentration when the reaction speed reaches (V max ) / 2.

[0083] Example 2

[0084] (1) Preparation of asphalt carbon-based oxidase-like nanomaterials

[0085] 10 g of coal liquefaction pitch raw material was ground into powder and passed through an 80 mesh sieve. Eggshells were ground into powder and passed through an 80 mesh sieve. The pitch powder and eggshell powder were mixed at a ratio of 1:3, calcined at 900°C for 5 h under argon, at a heating rate of 5°C / min, with an argon flow rate of 70 mL / min, and the calcined product was washed with 1 mol / L hydrochloric acid for 12 h, then filtered and dried at 80°C for 12 h to obtain activated pitch.

[0086] The obtained activated pitch 0.4 g was placed in 50 mL of a solution of soluble salts containing transition metal ions (wherein the concentration of ferric nitrate was 20 mmol / L, the concentration of copper nitrate was 10 mmol / L, and the concentration of zinc nitrate was 50 mmol / L), mixed and stirred for 12 h, then filtered and dried at 80°C for 6 h. The obtained powder was mixed with dicyandiamide at a ratio of 1:10, then calcined at 550°C for 1 h and at 900°C for 4 h under argon, at a heating rate of 5°C / min, with an argon flow rate of 70 mL / min, to obtain pitch carbon-based oxidase-like enzyme nanomaterials.

[0087] (2) Evaluation of oxidase-like enzyme activity

[0088] The detection method was the same as in Example 1.

[0089] Example 3

[0090] (1) Preparation of pitch carbon-based oxidase-like enzyme nanomaterials

[0091] 10 g of coal liquefaction pitch raw material was ground into powder and passed through an 80 mesh sieve. Eggshells were ground into powder and passed through an 80 mesh sieve. The pitch powder and eggshell powder were mixed at a ratio of 1:5, calcined at 900°C for 5 h under argon, at a heating rate of 5°C / min, with an argon flow rate of 70 mL / min, and the calcined product was washed with 1 mol / L hydrochloric acid for 12 h, then filtered and dried at 80°C for 12 h to obtain activated pitch.

[0092] The obtained activated pitch 0.4 g was placed in 50 mL of a solution of soluble salts containing transition metal ions (wherein the concentration of ferric nitrate was 10 mmol / L, the concentration of copper nitrate was 10 mmol / L, and the concentration of zinc nitrate was 50 mmol / L), mixed and stirred for 12 h, then filtered and dried at 80°C for 6 h. The obtained powder was mixed with dicyandiamide at a ratio of 1:10, then calcined at 550°C for 1 h and at 900°C for 4 h under argon, at a heating rate of 5°C / min, with an argon flow rate of 70 mL / min, to obtain pitch carbon-based oxidase-like enzyme nanomaterials.

[0093] (2) Evaluation of oxidase-like enzyme activity

[0094] The detection method was the same as in Example 1.

[0095] Example 4

[0096] (1) Preparation of asphalt carbon-based oxidase-like enzyme nanomaterials

[0097] 10 g of coal liquefaction asphalt raw material was ground into powder and passed through an 80-mesh sieve. Eggshells were ground into powder and passed through an 80-mesh sieve. The asphalt powder and eggshell powder were mixed at a ratio of 1:2, calcined at 900°C under argon for 5 h at a heating rate of 5°C / min, and the argon flow rate was 70 mL / min. The calcined product was washed with 1 mol / L hydrochloric acid for 12 h, then filtered, and dried at 80°C for 12 h to obtain activated asphalt.

[0098] The obtained activated asphalt 0.4 g was placed in 50 mL of a soluble salt solution containing transition metal ions (wherein the concentration of ferric nitrate was 10 mmol / L, the concentration of copper nitrate was 10 mmol / L, and the concentration of zinc nitrate was 50 mmol / L), mixed and stirred for 12 h, then filtered and dried at 80°C for 6 h. The obtained powder was mixed with dicyandiamide at a ratio of 1:10, then calcined at 550°C for 1 h and at 900°C for 4 h under argon at a heating rate of 5°C / min and an argon flow rate of 70 mL / min to obtain asphalt carbon-based oxidase-like enzyme nanomaterials.

[0099] (2) Evaluation of oxidase-like enzyme activity

[0100] The detection method was the same as in Example 1.

[0101] Example 5

[0102] (1) Preparation of asphalt carbon-based oxidase-like enzyme nanomaterials

[0103] 10 g of coal liquefaction asphalt raw material was ground into powder and passed through an 80-mesh sieve. Eggshells were ground into powder and passed through an 80-mesh sieve. The asphalt powder and eggshell powder were mixed at a ratio of 1:3, calcined at 900°C under argon for 5 h at a heating rate of 5°C / min, and the argon flow rate was 70 mL / min. The calcined product was washed with 1 mol / L hydrochloric acid for 12 h, then filtered, and dried at 80°C for 12 h to obtain activated asphalt.

[0104] The obtained activated asphalt 0.4 g was placed in 50 mL of a soluble salt solution containing transition metal ions (wherein the concentration of ferric nitrate was 10 mmol / L, the concentration of copper nitrate was 50 mmol / L, and the concentration of zinc nitrate was 10 mmol / L), mixed and stirred for 12 h, then filtered and dried at 80°C for 6 h. The obtained powder was mixed with dicyandiamide at a ratio of 1:10, then calcined at 550°C for 1 h and at 900°C for 4 h under argon at a heating rate of 5°C / min and an argon flow rate of 70 mL / min to obtain asphalt carbon-based oxidase-like enzyme nanomaterials.

[0105] (2) Evaluation of oxidase-like enzyme activity

[0106] The detection method is the same as that of Example 1.

[0107] Example 6

[0108] The difference between Example 6 and Example 1 is that the parameters for preparing the asphalt carbon-based oxidase-like nanomaterial are different.

[0109] 10 g of coal liquefaction asphalt raw material was ground into powder and passed through a 40-mesh sieve. Eggshells were ground into powder and passed through a 100-mesh sieve. The asphalt powder and eggshell powder were mixed at a ratio of 1:3, calcined at 200°C under argon for 8 h at a heating rate of 5°C / min, and the argon flow rate was 70 mL / min. The calcined product was washed with 1 mol / L hydrochloric acid for 8 h, then filtered, and dried at 60°C for 12 h to obtain activated asphalt.

[0110] The obtained activated asphalt 0.4 g was placed in 50 mL of a soluble salt solution containing transition metal ions (wherein the concentration of ferric nitrate was 10 mmol / L, the concentration of copper nitrate was 10 mmol / L, and the concentration of zinc nitrate was 50 mmol / L), mixed and stirred for 12 h, and then filtered and dried at 40°C for 12 h. The obtained powder was mixed with dicyandiamide at a ratio of 1:10, and then calcined at 400°C for 2 h and then at 700°C for 6 h under argon atmosphere at a heating rate of 5°C / min and an argon flow rate of 70 mL / min to obtain the asphalt carbon-based oxidase-like nanomaterial.

[0111] Example 7

[0112] The difference between Example 7 and Example 1 is that the parameters for preparing the asphalt carbon-based oxidase-like nanomaterial are different.

[0113] 10 g of coal liquefaction asphalt raw material was ground into powder and passed through a 100-mesh sieve. Eggshells were ground into powder and passed through a 60-mesh sieve. The asphalt powder and eggshell powder were mixed at a ratio of 1:3, calcined at 1000°C under argon for 1 h at a heating rate of 5°C / min, and the argon flow rate was 70 mL / min. The calcined product was washed with 1 mol / L hydrochloric acid for 20 h, then filtered, and dried at 100°C for 6 h to obtain activated asphalt.

[0114] The obtained activated asphalt 0.4 g was placed in 50 mL of a soluble salt solution containing transition metal ions (wherein the concentration of ferric nitrate was 10 mmol / L, the concentration of copper nitrate was 10 mmol / L, and the concentration of zinc nitrate was 50 mmol / L), mixed and stirred for 12 h, and then filtered and dried at 80°C for 4 h. The obtained powder was mixed with dicyandiamide at a ratio of 1:10, and then calcined at 600°C for 0.5 h and then at 1000°C for 3 h under argon atmosphere at a heating rate of 5°C / min and an argon flow rate of 70 mL / min to obtain the asphalt carbon-based oxidase-like nanomaterial.

[0115] Example 8

[0116] Example 8 differs from Example 1 in that the parameters for preparing the asphalt carbon-based oxidase-like nanomaterial are different.

[0117] The 10 g of coal liquefaction asphalt raw material was ground into powder and passed through an 80-mesh sieve. The oyster shell was ground into powder and passed through an 80-mesh sieve. The asphalt powder and eggshell powder were mixed at a ratio of 1:3, calcined at 400°C under argon for 6 h at a heating rate of 5°C / min and an argon flow rate of 70 mL / min, the calcined product was washed with 1 mol / L hydrochloric acid for 12 h, then filtered and dried at 80°C for 12 h to obtain activated asphalt.

[0118] The obtained activated asphalt 0.4 g was placed in 50 mL of a solution of soluble salt containing transition metal ions (wherein the concentration of ferric nitrate was 10 mmol / L, the concentration of copper nitrate was 10 mmol / L, and the concentration of zinc nitrate was 50 mmol / L), mixed and stirred for 12 h, dried at 80°C for 6 h after filtration, and then mixed with urea at a ratio of 1:10, after which it was calcined at 550°C for 1 h and then at 900°C for 4 h under an argon atmosphere at a heating rate of 5°C / min and an argon flow rate of 70 mL / min to obtain an asphalt carbon-based oxidase-like nanomaterial.

[0119] Example 9

[0120] Example 9 differs from Example 1 in that the parameters for preparing the asphalt carbon-based oxidase-like nanomaterial are different.

[0121] The 10 g of coal liquefaction asphalt raw material was ground into powder and passed through an 80-mesh sieve. The eggshell was ground into powder and passed through an 80-mesh sieve. The asphalt powder and eggshell powder were mixed at a ratio of 1:3, calcined at 900°C under argon for 2 h at a heating rate of 5°C / min and an argon flow rate of 70 mL / min, the calcined product was washed with 1 mol / L hydrochloric acid for 12 h, then filtered and dried at 80°C for 12 h to obtain activated asphalt.

[0122] The obtained activated asphalt 0.4 g was placed in 50 mL of a solution of soluble salt containing transition metal ions (wherein the concentration of ferric nitrate was 10 mmol / L, the concentration of copper nitrate was 10 mmol / L, and the concentration of zinc nitrate was 50 mmol / L), mixed and stirred for 12 h, dried at 80°C for 6 h after filtration, and then mixed with melamine at a ratio of 1:10, after which it was calcined at 550°C for 1 h and then at 900°C for 4 h under an argon atmosphere at a heating rate of 5°C / min and an argon flow rate of 70 mL / min to obtain an asphalt carbon-based oxidase-like nanomaterial.

[0123] Example 10

[0124] Example 10 differs from Example 1 in that the weight ratio of asphalt powder to eggshell powder is 1:1 when preparing the asphalt carbon-based oxidase-like nanomaterial.

[0125] Example 11

[0126] Example 11 differs from Example 1 in that the weight ratio of the bitumen powder to the eggshell powder is 1:10 when preparing the bitumen carbon-based oxidase-like nanomaterial.

[0127] Example 12

[0128] Example 12 differs from Example 1 in that the weight ratio of the activated coal liquefaction bitumen to the soluble salt containing transition metal ions is 1:0.1 when preparing the bitumen carbon-based oxidase-like nanomaterial.

[0129] Example 13

[0130] Example 13 differs from Example 1 in that the weight ratio of the activated coal liquefaction bitumen to the soluble salt containing transition metal ions is 1:5 when preparing the bitumen carbon-based oxidase-like nanomaterial.

[0131] Example 14

[0132] Example 14 differs from Example 1 in that the weight ratio of the activated coal liquefaction bitumen to the soluble salt containing transition metal ions is 1:0.2 when preparing the bitumen carbon-based oxidase-like nanomaterial.

[0133] Example 15

[0134] Example 15 differs from Example 1 in that the weight ratio of the activated coal liquefaction bitumen to the soluble salt containing transition metal ions is 1:2 when preparing the bitumen carbon-based oxidase-like nanomaterial.

[0135] Example 16

[0136] Example 16 differs from Example 1 in that the weight ratio of the bitumen carbon-based oxidase-like nanomaterial precursor to the organic nitrogen-containing compound is 1:0.1.

[0137] Example 17

[0138] Example 17 differs from Example 1 in that the weight ratio of the bitumen carbon-based oxidase-like nanomaterial precursor to the organic nitrogen-containing compound is 1:20.

[0139] Example 18

[0140] Example 18 differs from Example 1 in that the weight ratio of the bitumen carbon-based oxidase-like nanomaterial precursor to the organic nitrogen-containing compound is 1:5.

[0141] Example 19

[0142] The difference between Example 19 and Example 1 is that the weight ratio of the asphalt carbon-based oxidaselike nanomaterial precursor to the organic nitrogen-containing compound is 1:15.

[0143] Example 20

[0144] The difference between Example 20 and Example 1 is that the type and content of metal ions in the soluble salt solution containing transition metal ions are different, specifically: in the soluble salt solution containing transition metal ions, the concentration of ferric nitrate is 10 mmol / L, the concentration of copper nitrate is 1 mmol / L, and the concentration of zinc nitrate is 1 mmol / L, and the molar ratio of iron ions, copper ions, and zinc ions is 1:0.1:0.1.

[0145] Example 21

[0146] The difference between Example 21 and Example 1 is that the type and content of metal ions in the soluble salt solution containing transition metal ions are different, specifically: in the soluble salt solution containing transition metal ions, the concentration of ferric nitrate is 1 mmol / L, the concentration of copper nitrate is 10 mmol / L, and the concentration of zinc nitrate is 10 mmol / L, and the molar ratio of iron ions, copper ions, and zinc ions is 1:10:10.

[0147] Example 22

[0148] The difference between Example 22 and Example 1 is that the type and content of metal ions in the soluble salt solution containing transition metal ions are different, specifically: in the soluble salt solution containing transition metal ions, the concentration of ferric nitrate is 10 mmol / L, the concentration of manganese nitrate is 1 mmol / L, and the concentration of zinc nitrate is 1 mmol / L, and the molar ratio of iron ions, manganese ions, and zinc ions is 1:0.1:0.1.

[0149] Example 23

[0150] The difference between Example 23 and Example 1 is that the type and content of metal ions in the soluble salt solution containing transition metal ions are different, specifically: in the soluble salt solution containing transition metal ions, the concentration of ferric nitrate is 1 mmol / L, the concentration of manganese nitrate is 10 mmol / L, and the concentration of zinc nitrate is 10 mmol / L, and the molar ratio of iron ions, manganese ions, and zinc ions is 1:10:10.

[0151] Example 24

[0152] The difference between Example 24 and Example 1 is that the type and content of metal ions in the soluble salt solution containing transition metal ions are different, specifically: in the soluble salt solution containing transition metal ions, the concentration of ferric nitrate is 10 mmol / L, the concentration of nickel nitrate is 10 mmol / L, and the concentration of zinc nitrate is 50 mmol / L, and the molar ratio of iron ions, nickel ions and zinc ions is 1:1:5.

[0153] Example 25

[0154] The difference between Example 25 and Example 1 is that the type and content of metal ions in the soluble salt solution containing transition metal ions are different, specifically: in the soluble salt solution containing transition metal ions, the concentration of ferric nitrate is 10 mmol / L, the concentration of cobalt nitrate is 10 mmol / L, and the concentration of zinc nitrate is 50 mmol / L, and the molar ratio of iron ions, cobalt ions and zinc ions is 1:1:5.

[0155] Example 26

[0156] The difference between Example 26 and Example 1 is that the type and content of metal ions in the soluble salt solution containing transition metal ions are different, specifically: in the soluble salt solution containing transition metal ions, the concentration of ferric nitrate is 20 mmol / L, and the concentration of zinc nitrate is 50 mmol / L, and the molar ratio of iron ions and zinc ions is 2:5.

[0157] Comparative Example 1

[0158] (1) Preparation of asphalt carbon-based oxidase-like enzyme nanomaterials

[0159] 10 g of coal liquefied asphalt raw material was ground into powder and passed through an 80 mesh sieve. Eggshells were ground into powder and passed through an 80 mesh sieve. The asphalt powder and eggshell powder were mixed at a ratio of 1:3, calcined at 900°C for 5h under argon gas, with a heating rate of 5°C / min and an argon gas flow rate of 70 mL / min. The calcined product was washed with hydrochloric acid for 12h, then filtered and dried at 80°C for 12h to obtain activated asphalt.

[0160] The obtained activated asphalt 0.4 g was mixed with dicyandiamide at a ratio of 1:10, then calcined at 550°C for 1h and at 900°C for 4h under argon gas, with a heating rate of 5°C / min and an argon gas flow rate of 70 mL / min, to obtain oxidase-like enzyme N@C for constructing a sensing device.

[0161] (2) Evaluation of oxidase-like enzyme activity

[0162] The detection method is the same as that of Example 1.

[0163] Comparative Example 2

[0164] The difference between Comparative Example 2 and Example 26 is that activated carbon is used instead of bituminous carbon-based oxidase nanomaterials in the preparation of bituminous carbon-based oxidase nanomaterials.

[0165] The Vmaxvalues of the bituminous carbon-based oxidase nanomaterials obtained by testing in the above examples and comparative examples are shown in Table 1. max and K m The results are summarized in Table 1. Among them, the Michaelis constant (K m ) and the maximum reaction rate (V max ), K m can reflect the affinity of the bituminous carbon-based oxidase nanomaterials for the chromogenic substrate TMB, and the lower the parameter, the better the affinity for TMB; V max can reflect the activity of the bituminous carbon-based oxidase nanomaterials, and the higher the parameter, the higher the activity of the bituminous carbon-based oxidase nanomaterials prepared in the present application.

[0166] Table 1

[0167]

[0168] The bituminous carbon-based oxidase nanomaterials in Example 1 above were selected to test the actual sample antioxidant (ascorbic acid), and the preparation process of the bituminous carbon-based oxidase nanomaterials and the following test process are shown in Figure 13 . The specific test steps include:

[0169] 1. Establish a standard curve for sensing detection: In 1.4 mL of a buffer composed of acetic acid / sodium acetate (pH = 4), add 0.2 mL of the above bituminous carbon-based oxidase nanomaterials with a concentration of 1.5 mg / mL, then add 0.2 mL of a TMB solution with a concentration of 2 mmol / L, and then add 0.2 mL of a gradient concentration (0 µmol / L, 20 µmol / L, 40 µmol / L, 60 µmol / L, 80 µmol / L, 100 µmol / L, 120 µmol / L, 140 µmol / L, 160 µmol / L, 180 µmol / L, 200 µmol / L) of ascorbic acid solution. After 5 min of reaction at 35°C, the test solution is obtained by filtration.

[0170] 2. Establish a UV colorimetric detection curve: The absorbance of the test solution obtained in step 1 is detected at 652 nm using a UV spectrophotometer, and the linear relationship between absorbance and concentration is established with ascorbic acid concentration as the abscissa and absorbance as the ordinate. The linear relationship results are shown in Figure 11 .

[0171] 3. Mobile phone colorimetric detection: using the "color recognition" WeChat applet to scan the color of the sample, and analyzing the RGB three primary colors of the sample. Establishing the linear relationship between the ascorbic acid concentration and B / (R+G+B) as the ordinate, and the ascorbic acid concentration as the abscissa. The linear relationship obtained is shown in Figure 12

[0172] 4. Sample detection: replacing the standard concentration of ascorbic acid with real samples for reaction, and then using the linear relationship obtained in steps 2 and 3, the rapid detection of ascorbic acid concentration in actual samples can be realized. The test results are shown in Table 2.

[0173] Table 2

[0174]

[0175] Further, the asphalt carbon-based oxidase nanomaterial in the above embodiment 1 is selected, and three-channel sensing arrays are established by using 3,3',5,5'-tetramethylbenzidine (TMB), 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and o-phenylenediamine (OPD) three color substrates, to distinguish nine antioxidants, namely tannic acid (TA), glutathione (GSH), gallic acid (GA), curcumin (CUR), quercetin (QUE), epigallocatechin gallate (EGCG), ascorbic acid (AA), protocatechuic acid (PA) and ferulic acid (FA). The specific steps are as follows:

[0176] Add 200 μL of asphalt carbon-based oxidase nanomaterial suspension (concentration of 1.5 mg / mL), 1.6 mL of HAc-NaAc buffer solution (concentration of 0.1 mol / L), 200 μL of TMB (2 mmol / L) and 10 mmol / L of antioxidants (antioxidants are tannic acid (TA), glutathione (GSH), gallic acid (GA), curcumin (CUR), quercetin (QUE), epigallocatechin gallate (EGCG), ascorbic acid (AA), protocatechuic acid (PA) and ferulic acid (FA)) into 5 mL centrifuge tubes respectively, and then incubate at room temperature for 10 minutes to construct the TMB detection channel. Using a UV-visible spectrophotometer, record the absorbance spectrum at 652 nm, and process the data to generate the corresponding curve. According to the same method, ABTS detection channel and OPD detection channel are established respectively, and the absorbance is recorded at 415 nm and 450 nm respectively. The composition analysis diagram of the three-channel sensing array obtained according to the response area is shown in Figure 14 The bubble diagram of the three-channel sensing array obtained according to the color intensity is shown in Figure 15

[0177] According to Figure 14 ​​It can be seen that the points corresponding to different antioxidants are located in different regions without intersection, indicating that the asphalt carbon-based oxidase-like nanomaterial provided by the application can clearly distinguish different antioxidants, further indicating that the sensing array has good distinguishing ability. Figure 15 It can be seen that the asphalt carbon-based oxidase-like nanomaterial provided by the application can directly display the response intensity difference of different antioxidants on three substrates; and the unique response mode revealed by the bubble chart further verifies the reliability of the array sensor, which is consistent with the analysis results in Figure 14 .

[0178] From the above description, it can be seen that the above-mentioned embodiments of the application achieve the following technical effects:

[0179] In Example 1 to Example 26, the asphalt carbon-based oxidase-like nanomaterial was prepared by using the preparation method of the asphalt carbon-based oxidase-like nanomaterial provided by the application. According to the data in Table 1, it can be seen that the asphalt carbon-based oxidase-like nanomaterial prepared in the above-mentioned examples has excellent affinity with TMB and also has excellent activity; it indicates that the asphalt carbon-based oxidase-like nanomaterial prepared by the application can have good response with antioxidant TMB. Especially, by controlling the parameters in the preparation process of the asphalt carbon-based oxidase-like nanomaterial within the preferred range, the affinity and activity of the corresponding asphalt carbon-based oxidase-like nanomaterial with TMB are better. In Comparative Example 1, no metal ions were loaded in the process of preparing the asphalt carbon-based oxidase-like nanomaterial; in Comparative Example 2, activated carbon was used as the carbon-based material in the process of preparing the asphalt carbon-based oxidase-like nanomaterial. The related performance of the corresponding asphalt carbon-based oxidase-like nanomaterial has a large gap compared with the examples of the application.

[0180] Further, the asphalt carbon-based oxidase-like nanomaterial prepared in Example 1 was used to test the actual sample, and the test results are shown in Table 2. According to the results in Table 2, the asphalt carbon-based oxidase-like nanomaterial prepared by using the preparation method described in the application has good accuracy in detecting the concentration of ascorbic acid. Especially, the recovery rate is between 95% and 105%, and the accuracy of the test is much better than the conventional standard (85% to 115%).

[0181] In addition, the asphalt carbon-based oxidase-like nanomaterial prepared in Example 1 was used to establish a three-channel sensing array to distinguish nine antioxidants, namely tannic acid (TA), glutathione (GSH), gallic acid (GA), curcumin (CUR), quercetin (QUE), epigallocatechin gallate (EGCG), ascorbic acid (AA), protocatechuic acid (PA) or ferulic acid (FA). According to the results, the asphalt carbon-based oxidase-like nanomaterial in the application has excellent distinguishing effect on the above nine antioxidants.

[0182] In summary, the preparation method of the asphalt carbon-based oxidase nanomaterial provided by the application starts from modifying coal liquefaction asphalt base material, and through a series of links such as activation of coal liquefaction asphalt, metal ion loading, nitrogen doping and heat treatment, an asphalt carbon-based oxidase nanomaterial with high specific surface area, rich porous structure, stable metal active site and optimized electronic distribution is gradually constructed. These carefully designed steps enable the material to realize efficient catalytic oxidation of chromogenic substrate without the participation of hydrogen peroxide, realize the visual detection of antioxidants, and also ensure that the detection method has the remarkable advantages of low cost, simple operation, high sensitivity and wide applicability, providing strong support for the technological innovation in the field of food safety and health monitoring.

[0183] The above merely describes preferred embodiments of the present application but should not be used to limit the present application. Various modifications and changes can be made by those skilled in the art based on the spirit and principles of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a bitumen carbon-based oxidaselike nanomaterial, characterized in that, The preparation method comprises the following steps: The coal liquefaction pitch and the activating agent are mixed to obtain a first mixture; the first mixture is calcined, pickled and dried to obtain activated coal liquefaction pitch; The activated coal liquefaction pitch is mixed with a solution of soluble salt containing transition metal ions to obtain a mixed slurry; the mixed slurry is separated and then dried to obtain a pitch carbon-based oxidase-like nanomaterial precursor; The pitch carbon-based oxidase-like nanomaterial precursor is mixed with an organic nitrogen-containing compound to obtain a second mixture; the second mixture is calcined to obtain the pitch carbon-based oxidase-like nanomaterial.

2. The method for preparing pitch-carbon-based oxidase nanomaterials according to claim 1, characterized in that, The solution of soluble salt containing transition metal ions contains at least two kinds of transition metal ions; Preferably, the transition metal ions in the solution of soluble salt containing transition metal ions are selected from at least two of iron ions, ferrous ions, copper ions, cuprous ions, zinc ions, cobalt ions, manganese ions and nickel ions; More preferably, the transition metal ions in the solution of soluble salt containing transition metal ions are selected from at least three of the iron ions, the ferrous ions, the copper ions, the cuprous ions, the zinc ions, the cobalt ions, the manganese ions and the nickel ions; Further preferably, the transition metal ions in the solution of soluble salt containing transition metal ions are compounded from the iron ions, the copper ions and the zinc ions; or the transition metal ions in the solution of soluble salt containing transition metal ions are compounded from the iron ions, the manganese ions and the zinc ions; Preferably, when the transition metal ions in the solution of soluble salt containing transition metal ions are compounded from the iron ions, the copper ions and the zinc ions, the molar ratio of the three is 1:(0.1-10):(0.1-10); or when the transition metal ions in the solution of soluble salt containing transition metal ions are compounded from the iron ions, the manganese ions and the zinc ions, the molar ratio of the three is 1:(0.1-10):(0.1-10).

3. The method for preparing pitch-carbon-based oxidase nanomaterials according to claim 1, characterized in that, The weight ratio of the activated coal liquefaction pitch to the soluble salt containing transition metal ions is 1:(0.1-5); and / or the weight ratio of the pitch carbon-based oxidase-like nanomaterial precursor to the organic nitrogen-containing compound is 1:(0.1-20); Preferably, the weight ratio of the activated coal liquefaction pitch to the soluble salt containing transition metal ions is 1:(0.2-2); and / or the weight ratio of the pitch carbon-based oxidase-like nanomaterial precursor to the organic nitrogen-containing compound is 1:(5-15).

4. The method of claim 1 to 3, wherein, The weight ratio of the coal liquefaction pitch to the activating agent is 1:(1-10); Preferably, the activating agent is a biological source activating agent; Preferably, the biological source activating agent is powder of at least one of eggshells, oyster shells, salmon bones, cow horns or shrimp shells; more preferably, the biological source activating agent is powder of at least one of the eggshells, the oyster shells or the shrimp shells. Preferably, the organic nitrogen-containing compound is selected from one or more of urea, dicyandiamide or melamine.

5. The method of claim 1 to 3, wherein, The first calcination is performed at a temperature of 200-1000℃ for 1-8h; Preferably, the first calcination is performed at a temperature of 400-900℃ for 2-6h; Preferably, the second calcination comprises a first-stage calcination and a second-stage calcination performed sequentially, the first-stage calcination is performed at a temperature of 400-600℃ for 0.5-2h; and the second-stage calcination is performed at a temperature of 700-1000℃ for 3-6h; Preferably, the first calcination and the second calcination are performed in an inert gas atmosphere; Preferably, the second mixing is performed for 8-20h; Preferably, the first drying is performed at a temperature of 60-100℃ for 6-12h; and / or, the second drying is performed at a temperature of 40-80℃ for 4-12h; Preferably, the pickling solvent is an aqueous hydrochloric acid solution; Preferably, the concentration of the pickling solvent is 0.8-1.2mol / L; Preferably, the coal liquefaction pitch has a particle size of 40-100 mesh; and / or, the activating agent has a particle size of 60-100 mesh.

6. An asphalt carbon-based oxidoenzyme nanomaterial, characterized in that, The pitch carbon-based oxidase-like enzyme nanomaterial is prepared by the preparation method of any one of claims 1-5.

7. Use of the bitumen carbon-based oxidaselike nanomaterial according to claim 6 for the detection of an array of sensors, characterized by, The pitch carbon-based oxidase-like enzyme nanomaterial is used for three-channel sensing array detection of antioxidant species; The antioxidant is two or more of tannic acid, glutathione, gallic acid, curcumin, quercetin, epigallocatechin gallate, ascorbic acid, protocatechuic acid or ferulic acid; The chromogenic substrate used for the three-channel sensing array is selected from 3,3',5,5'-tetramethylbenzidine, 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt and o-phenylenediamine.

8. Use of the bitumen carbon-based oxidaselike nanomaterial according to claim 6 for the detection of antioxidant concentration, characterized in that, The pitch carbon-based oxidase-like enzyme nanomaterial is used for catalytic oxidation of chromogenic substrate to detect the concentration of antioxidant; Preferably, the chromogenic substrate is at least one of 3,3',5,5'-tetramethylbenzidine, 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt or o-phenylenediamine; More preferably, the chromogenic substrate is the 3,3',5,5'-tetramethylbenzidine; Preferably, the antioxidant is at least one of tannic acid, glutathione, gallic acid, curcumin, quercetin, epigallocatechin gallate, ascorbic acid, protocatechuic acid or ferulic acid.

9. A method of detecting an antioxidant concentration, characterized by, The detection method comprises the following steps: The chromogenic substrate and the pitch carbon-based oxidase-like enzyme nanomaterial of claim 6 are placed in a solution containing an antioxidant to perform an oxidation reaction to obtain a solution to be tested; The absorbance of the solution to be tested is determined by spectrophotometry to calculate the concentration of the antioxidant in the solution to be tested; Preferably, the chromogenic substrate is at least one of 3,3',5,5'-tetramethylbenzidine, 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt or o-phenylenediamine.

10. The method of claim 9, wherein the antioxidant concentration is determined by the method comprising: The chromogenic substrate in the solution to be tested is selected from the 3,3',5,5'-tetramethylbenzidine; ​ Preferably, the concentration of the asphalt carbon-based oxidase nanomaterial is 0.05-1 g / L; and / or, the concentration of the chromogenic substrate in the solution to be tested is 0.1-2 mmol / L; and / or, the concentration of the antioxidant is 0.01-300 µmol / L; Preferably, the temperature of the oxidation reaction is 30-50 °C; Preferably, the pH of the solution to be tested is 2-8; more preferably, the pH of the solution to be tested is 3-5; Preferably, the antioxidant is ascorbic acid.

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