Confined-range CoNi alloy-carbon composite nano-enzyme as well as preparation method and application of confined-range CoNi alloy-carbon composite nano-enzyme

Abstract

The invention provides a confinement type CoNi alloy-carbon composite nano enzyme as well as a preparation method and application thereof. The preparation method comprises the following steps: mixing P123, melamine and water to form a solution A; ni (CH3COO) 2.4 H2O and Co (NO3) 2.6 H2O are mixed with water to form a solution B; mixing graphene oxide with water to form a solution C; respectively dripping the solution B and the solution C into the solution A, stirring and mixing at room temperature to obtain a light purple solution, and freeze-drying to obtain a light purple powdery precursor; and heating the yellowish violet powdery precursor in N2 to 450-550 DEG C at a heating rate of 2-5 DEG C / min for carbonization, and then heating to 650-750 DEG C at a heating rate of 2-5 DEG C / min for calcination to obtain black powder with metallic luster, namely the confinement type CoNi alloy-carbon composite nano enzyme.

Description

Technical Field

[0001] The present invention belongs to the technical field of food detection, and specifically relates to a confined CoNi alloy-carbon composite nanozyme, a preparation method thereof, and applications thereof. Background Art

[0002] With increasing attention to sustainable development, the evaluation of food quality and safety, the living environment, and human health has become a hot topic of research. One of the most representative approaches is the assessment of total antioxidant capacity (TAC). It is well known that foods rich in antioxidants play an important role in reducing the risk of cancer, neurological diseases, and cardiovascular diseases. Therefore, TAC is a key indicator for evaluating the antioxidant quality of foods and oxidative stress in healthcare. Antioxidant capacity is an essential characteristic that is related not only to the content of individual antioxidants but also to the structure and potential interactions between different antioxidant components. In real samples, multiple antioxidants are present simultaneously. Therefore, the concept of total antioxidant capacity (TAC), rather than individual antioxidants, has been proposed to evaluate the antioxidant behavior of foods and drugs. To date, various techniques have been used to measure TAC, such as electrochemical, spectroscopic, and chromatographic methods. However, these methods typically require expensive instrumentation, complex sample preparation, and highly trained technicians. The emergence of commercial kits has freed them from the limitations of large instruments, enabling the acquisition of TAC results under simple experimental conditions and providing detailed procedures and instructions. These kits are therefore accessible to even non-experts, demonstrating a certain degree of universality. However, the biological enzymes used in some kits have poor stability, and the reaction solutions require complex pretreatment, which may reduce the accuracy of the results due to operational problems. In addition, there are many types of kits, and the results obtained from each are different. Therefore, there is an urgent need to develop a simpler and faster method for total antioxidant assessment to achieve rapid and accurate assessment of TAC in food or medicine.

[0003] To address the above technical issues, synthesizing efficient nanomaterials with enzyme-like activity to replace natural enzymes and using smartphones as detection devices instead of UV-visible spectrophotometers to construct more convenient and efficient TAC detection methods will have great application potential in the fields of food and drug testing. Summary of the Invention

[0004] The purpose of the present invention is to provide a confined CoNi alloy-carbon composite nanozyme and its application in detecting total antioxidant capacity, providing a highly sensitive, selective, simple and fast method for analyzing the total antioxidant capacity of food or medicine.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a method for preparing a confined CoNi alloy-carbon composite nanozyme, the preparation method comprising:

[0007] P123, melamine and water were mixed to form solution A;

[0008] Ni(CH3COO)2·4H2O and Co(NO3)2·6H2O were mixed with water to form solution B;

[0009] mixing graphene oxide with water to form solution C;

[0010] Solution B and solution C were added dropwise to solution A, respectively, and stirred at room temperature to obtain a lavender solution, which was then freeze-dried to obtain a lavender powder precursor;

[0011] The pale purple powder precursor is carbonized in N2 at a heating rate of 2 to 5°C / min to 450 to 550°C, and then calcined at a heating rate of 2 to 5°C / min to 650 to 750°C. The resulting black powder with a metallic luster is the confined CoNi alloy-carbon composite nanozyme.

[0012] Preferably, in the solution A, the mass ratio of the P123 to the melamine is 1:(8-12).

[0013] Preferably, the concentration of P123 in the solution A is 0.01-0.02 g / mL, and the concentration of melamine is 0.1-0.2 g / mL.

[0014] Preferably, in the solution B, the mass ratio of Ni(CH3COO)2·4H2O to Co(NO3)2·6H2O is 1:(0.8-1.2).

[0015] Preferably, the concentrations of Ni(CH3COO)2·4H2O and Co(NO3)2·6H2O in the solution B are independently 0.02 to 0.05 g / mL.

[0016] Preferably, the concentration of graphene oxide in the solution C is 0.8-1.2 mg / mL.

[0017] Preferably, the volume ratio of solution A, solution B and solution C is (3-5):(0.8-1.2):1.

[0018] Preferably, the feeding mass ratio of P123, melamine, Ni(CH3COO)2·4H2O, 0.4g Co(NO3)2·6H2O and graphene oxide is (0.3-0.6):(3-6):(0.3-0.6):(0.3-0.6):0.01.

[0019] Preferably, the P123, melamine and water are mixed at 70-90° C. and 500-700 r / min.

[0020] Preferably, solution B and solution C are respectively added dropwise to solution A, and then stirred at 200-400 r / min for 8-12 hours at room temperature to obtain the lavender solution.

[0021] Preferably, the carbonization and calcination times are 0.8 to 1.5 hours, respectively.

[0022] Preferably, the preparation method further includes post-treatment, which includes pickling the black powder with metallic luster with 0.5-2 mol / L H2SO4 for 20-30 hours, then washing it alternately with water and ethanol until the solution is neutral, and finally drying it at 50-70°C.

[0023] The second aspect of the present invention provides a confined CoNi alloy-carbon composite nanozyme prepared by the above-mentioned preparation method, wherein the confined CoNi alloy-carbon composite nanozyme contains carbon nanotubes densely covered on a carbon layer, cobalt-nickel alloy particles wrapped in the carbon nanotubes, and the cobalt-nickel alloy particles are located inside the carbon nanotubes. The diameter of the alloy particles is between 10 and 15 nm, and the carbon nanotubes present a multi-node morphology similar to bamboo, with an average diameter of about 35 to 45 nm, and Co, Ni, N, and C are evenly distributed.

[0024] Furthermore, the specific surface area of the confined CoNi alloy-carbon composite nanozyme is 440-460 m 2 / g, and the average pore diameter is 7.5-9.5nm.

[0025] The third aspect of the present invention provides a confined CoNi alloy-carbon composite nanozyme prepared by the above preparation method or the use of the above confined CoNi alloy-carbon composite nanozyme in the evaluation of total antioxidant capacity.

[0026] The fourth aspect of the present invention provides a method for detecting total antioxidant capacity, which detects total antioxidant capacity based on colorimetry. The confined CoNi alloy-carbon composite nanozyme prepared by the above preparation method or the above-mentioned confined CoNi alloy-carbon composite nanozyme is incubated with a chromogenic substrate in a HAc-NaAc buffer at 35-45°C, and then the confined CoNi alloy-carbon composite nanozyme in the incubation solution is separated by a magnet to obtain a chromogenic solution. The chromogenic solution is then mixed with the sample solution to be tested and reacted at room temperature to obtain a reaction solution. Ascorbic acid, reduced glutathione or cysteine are used as antioxidant model compounds, and the total antioxidant capacity is quantified by the expression amount of mM equivalent of the antioxidant model compound per liter of reaction solution. The chromogenic substrate is any one of TMB, ABTS, DAB and OPD, and the sample to be tested includes food and medicine.

[0027] According to some embodiments of the present invention, the total antioxidant capacity is determined by visually observing the color change of the reaction solution before and after the reaction, based on the degree of color change.

[0028] According to other embodiments of the present invention, the absorbance value of the reaction solution at a specific wavelength is measured using an ultraviolet spectrophotometer. When the chromogenic substrate is TMB, the specific wavelength is any one of 649 to 659 nm; when the chromogenic substrate is ABTS, the specific wavelength is any one of 415 to 425 nm; when the chromogenic substrate is DAB, the specific wavelength is any one of 463 to 473 nm; when the chromogenic substrate is OPD, the specific wavelength is any one of 445 to 455 nm. The measured absorbance value is substituted into a standard curve drawn by the absorbance values of antioxidant model compound standards of different concentrations to calculate the total antioxidant capacity.

[0029] According to some further embodiments of the present invention, the reaction solution is photographed and the grayscale value is calculated, and the total antioxidant capacity is obtained using digital imaging colorimetric software in combination with a standard curve drawn by the grayscale values of antioxidant model compound standards at different concentrations.

[0030] The fifth aspect of the present invention provides a kit for detecting total antioxidant capacity, which includes a confined CoNi alloy-carbon composite nanozyme (CoNi@CNTs-N / GO) prepared by the above preparation method or the above confined CoNi alloy-carbon composite nanozyme, an antioxidant model compound standard, an acetic acid-sodium acetate buffer and a chromogenic substrate, wherein the chromogenic substrate is one or more of TMB, ABTS, DAB and OPD.

[0031] Preferably, in the kit, the confined CoNi alloy-carbon composite nanozyme is prepared at a concentration of 1 to 3 mg mL -1The chromogenic substrate is stored in the form of a dispersion (the solvent is water), and the chromogenic substrate is stored in the form of a solution with a concentration of 5 to 8 mM (the solvent is DMSO).

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] The confined CoNi alloy-carbon composite nanozyme prepared by the present invention is different from the traditional peroxide mimic enzyme reaction system which requires the addition of H2O2. It realizes the self-supply of H2O2 and further decomposes it to produce ·OH for catalytic reaction. It can be prepared on a large scale and is easy to store.

[0034] The confined CoNi alloy-carbon composite nanozyme of the present invention is a peroxidase-like enzyme with high enzyme catalytic activity, good affinity for TMB, and high stability. It is universal in catalyzing the color development of chromogenic substrates (TMB, ABTS, DAB, OPD).

[0035] The detection method of the present invention has a wide linear range, a low detection limit, is simple to operate, and has low detection cost. The mobile phone visual colorimetric detection method has good sensitivity and anti-interference properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Analysis of the morphological and structural characteristics of CoNi@CNTs-N / GO: (AB) SEM images of CoNi@CNTs-N / GO, (CE) TEM images of CoNi@CNTs-N / GO and its size distribution (inset) and lattice image (inset), (FH) EDS mapping, (I) XRD pattern, (J) Raman pattern, (K) adsorption-desorption curve, (L) pore size distribution;

[0037] Figure 2 Results of the CoNi@CNTs-N / GO activity study: (A) Enzyme activity diagram of CoNi@CNTs-N / GO nanozyme, (B) Colorimetric agent screening diagram, (C) under different atmosphere conditions (O2, Air, N2), (D) Free radical capture experiment, (EF) Iodine titration method for capturing H2O2, (GH) OH and O2 in the range of 5-30min - EPR test spectrum;

[0038] Figure 3 Steady-state kinetics of CoNi@CNTs-N / GO: (A) Michaelis-Menten curves at different TMB concentrations, (B) double reciprocal Lineweaver-Burk plots;

[0039] Figure 4Detection of TAC levels for CoNi@CNTs-N / GO: (AC) standard curves of different concentrations of AA, GSH, and Cys, (DF) UV absorption spectra of different concentrations of AA, GSH, and Cys;

[0040] Figure 5 Schematic diagram of mobile phone visual detection of TAC levels: (A) CoNi@CNTs-N / GO nanozyme colorimetric detection of TAC; (B) Schematic diagram of the mobile phone visual detection process; (CE) Results of colorimetric, mobile phone visual detection, and commercially available ABTS kit detection methods for detecting TAC levels in actual samples;

[0041] Figure 6 Schematic diagram of the TAC detection principle of CoNi@CNTs-N / GO nanozyme. DETAILED DESCRIPTION

[0042] The present invention will be further described below with reference to the following examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific applications. The implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other. The implementation conditions used in the examples can be further adjusted according to specific requirements. The implementation conditions not specified are generally the conditions used in routine experiments.

[0043] In the following examples, all chemicals were of analytical or chromatographic grade. Ultrapure water (>18.2 MΩ) was produced using a Milli-Q water purifier (Bedford, MA, USA). Scanning electron microscopy (SEM, Quanta 250; FEI, USA); transmission electron microscopy (Talos F200X G2; FEI, USA); X-ray diffractometer (D8-Advance, Bruker, Germany); X-ray photoelectron spectroscopy (K-Alpha+; Thermo Scientific, USA); and Raman spectroscopy (DXR microscope, Thermo Scientific, USA) were performed. Enzyme kinetic data and UV-visible spectra were obtained using a UV-8000 spectrophotometer (Shanghai, China).

[0044] Example 1

[0045] Preparation of confined CoNi alloy-carbon composite nanozyme - CoNi@CNT-N / GO:

[0046] First, weigh 0.5 g of polyether polyol (P123, C5H 10O2) and 5g of melamine were dissolved in 40mL of ultrapure water and magnetically stirred at 600 r / min at 80°C for 30 minutes, designated Solution A. 0.4g of Ni(CH3COO)2·4H2O and 0.4g of Co(NO3)2·6H2O were then dissolved in 10mL of ultrapure water, designated Solution B. 10mg of GO (graphene oxide) was then added to 10mL of ultrapure water and ultrasonicated to obtain a uniform dark brown solution, designated Solution C. Finally, Solutions B and C were slowly added dropwise to Solution A. The mixture was then stirred at 300 r / min at room temperature for 10 hours to obtain a pale purple solution, which was then freeze-dried to obtain a pale purple powdery precursor.

[0047] The pale purple precursor was placed in a tube furnace and heated to 500°C at a rate of 2.5°C / min under a nitrogen atmosphere. The mixture was then carbonized for one hour and then calcined at the same rate to 700°C for one hour. After cooling completely, a black powder with a metallic luster was obtained. The resulting black powder was then acid-washed with 1 mol / L H2SO4 for 24 hours and then washed alternately with ultrapure water and ethanol until the solution was neutral. Finally, the powder was dried in a 60°C oven to obtain the final product, the CoNi@CNTs-N / GO nanozyme.

[0048] Morphological and structural characteristics of CoNi@CNTs-N / GO nanozyme:

[0049] like Figure 1 As shown in the figure, CoNi@CNTs-N / GO nanozymes have a large number of carbon nanotubes with high aspect ratios densely covered on the carbon layer, and cobalt-nickel alloy particles are wrapped in the carbon nanotubes and located inside the carbon nanotubes. The diameter of the alloy particles is between 10-15nm, and the carbon nanotubes have a multi-node morphology similar to bamboo, with an average diameter of about 40nm. Co, Ni, N, and C are evenly distributed. The diffraction peak at around 26° in the XRD test is caused by the diffraction of the (002) crystal plane of graphite carbon. The three obvious diffraction peaks at 44.3°, 51.7°, and 76° are located between the Co metal nanoparticles and the Ni metal nanoparticles. These diffraction peaks correspond to the (111), (200), and (220) crystal planes of Co and Ni, respectively. From the Raman spectrum, it can be seen that I D / l G The value of is 0.91, indicating that there are more defect sites in the CoNi@CNTs-N / GO nanozyme. The specific surface area of the nanozyme is 457.42m 2 / g, with an average pore size of 8.34 nm. The larger specific surface area brings more active sites.

[0050] Example 2

[0051] Study on the activity of CoNi@CNTs-N / GO nanozymes:

[0052] Experimental process: 50 μL of CoNi@CNTs-N / GO nanozyme dispersion (solvent: water, concentration: 1 mg mL -1 ) and 100 μL TMB (solvent is DMSO, concentration is 6 mM) were added to 1850 μL HAc-NaAc buffer (0.1 M, pH 3.2). Incubate at 40 ° C for 30 minutes, and measure the absorbance at 652 nm using a UV-visible spectrophotometer. The CoNi@CNTs-N / GO-TMB system was placed under different atmospheres to verify the main role of O2 in the system. The H2O2 produced during the reaction was captured by iodine titration, and the self-cascade catalytic mechanism of the CoNi@CNTs-N / GO nanozyme was successfully verified, achieving self-supply of H2O2, and further decomposing to produce OH for catalytic reaction ( Figure 2 ).

[0053] Example 3

[0054] Steady-state kinetic parameters of CoNi@CNTs-N / GO nanozyme:

[0055] CoNi@CNTs-N / GO nanozymes have excellent peroxidase-like activity. This is due to the large specific surface area of the nanozymes and the synergistic effect of cobalt and nickel bimetallics. A large amount of dissolved O2 is adsorbed on the surface of CoNi@CNTs-N / GO nanozymes and catalyzed by the nanozymes to generate oxidative O2. - , used to self-produce H2O2, and finally decomposed into OH and then oxidized TMB to generate oxTMB. For TMB, K m 0.33mM, V max 1.89*10 -8 M / s( Figure 3 ).

[0056] Example 4

[0057] CoNi@CNTs-N / GO nanozyme detection of TAC levels in food (colorimetric method):

[0058] (1) Use water as solvent and prepare a concentration of 1 mg mL -1 CoNi@CNTs-N / GO nanozyme dispersion; 6 mM TMB solution was prepared using dimethyl sulfoxide (DMSO) as solvent; HAc-NaAc buffer (0.1 M, pH 3.2);

[0059] (2) Using water as solvent, three antioxidant solutions of ascorbic acid (AA), reduced glutathione (GSH), and cysteine (Cys) with different concentrations (0.01-1.5 mM) were prepared;

[0060] (3) 50 μL of CoNi@CNTs-N / GO nanozyme dispersion and 100 μL of TMB solution were added to 1750 μL of HAc-NaAc (pH = 3.2) and incubated in a 40 °C water bath for 30 min. The resulting color solution was separated from the CoNi@CNTs-N / GO nanozyme using a magnet. Then, 100 μL of AA solution, GSH solution, and Cys solution of different concentrations were added. After reacting for 6 min at room temperature, the color of the solution was measured by UV-visible spectrophotometer. 652 The experiment was repeated three times. 652 The relationship between the values and the concentrations of AA, GSH and Cys was used to establish the standard curves for the detection of the three antioxidants by UV-visible spectrophotometry, and the detection limits and linear ranges were calculated.

[0061] (4) Using the AA antioxidant model compound, three fruits (kiwi, orange, lemon), three effervescent tablets (passion fruit effervescent tablets, orange effervescent tablets, white peach effervescent tablets), and three beverages (Pulpy Orange, Water-soluble C, Suntory Oolong Tea) were selected for total antioxidant capacity (TAC) value testing. The three fruits were peeled and juiced using a juicer. The pulp was removed by centrifugation, and the resulting solution was filtered through a 0.45 μm mixed cellulose filter for later use. One piece of each of the three effervescent tablets was dissolved in 200 mL of ultrapure water for later use. The three beverages were directly filtered through a 0.45 μm mixed cellulose filter for later use. The resulting solution was diluted to the linear range and used as the actual sample solution for testing.

[0062] (5) Referring to step (3), the CoNi@CNTs-N / GO nanozyme was separated from the obtained color solution, and 100 μL of the actual sample solution was added. After reacting for 6 min at room temperature, the A was measured by UV-visible spectrophotometer. 652 The obtained value was substituted into the AA standard curve to obtain the TAC value corresponding to the actual sample, and the TAC content was calculated based on the expression amount of mM equivalent of AA per liter.

[0063] Example 5

[0064] CoNi@CNTs-N / GO nanozyme detects antioxidants in food based on mobile phone visualization (mobile phone visualization detection method):

[0065] First, step (3) of Example 4 was repeated, and the color-developing solution after the reaction with different concentrations of AA was placed in a 96-well plate, placed in a camera darkroom, and a mobile phone was used to take a photo to obtain a color-developing result image; the color-developing result image was imported into the "ThingIdentify" software, and the relationship between the grayscale value of the color-developing result and the AA concentration was constructed. A standard curve for mobile phone visual detection of TAC was established, and the detection limit and linear range were calculated.

[0066] Then, the color-developed solution after the reaction with the actual sample solution (from step (4)) is put into the 96-well plate again and placed in a camera darkroom. The grayscale value of the solution is identified by the "Thing Identify" software and brought into the obtained mobile phone visual detection standard curve to obtain the actual sample TAC value.

[0067] The principle behind the CoNi@CNTs-N / GO nanozyme's TAC detection in Examples 4 and 5 is that the CoNi@CNTs-N / GO nanozyme exhibits a self-cascading catalytic mechanism. Unlike traditional peroxidase mimics, the CoNi@CNTs-N / GO nanozyme self-supplies H₂O₂, which further decomposes to produce ·OH, catalyzing the oxidation of colorless TMB to blue oxTMB. TAC levels were assessed by measuring the effect of reducing AA in the actual sample on the colorimetric solution.

[0068] The process of the colorimetric method of Example 4 and the mobile phone visualization detection method of Example 5 and the results of the colorimetric method, mobile phone visualization detection method and commercially available ABTS kit detection method for detecting TAC levels in actual samples are shown in Figure 5 . Figure 5 The results of Examples 4 and 5 for detecting TAC levels in different real samples showed no significant difference from those of the commercially available ABTS kit. The mobile phone visual colorimetric detection method has improved sensitivity and interference resistance, making it faster, more efficient, and less expensive than other detection methods.

[0069] The above detailed description of the present invention is intended to enable persons familiar with the art to understand the contents of the present invention and implement them. It does not limit the scope of protection of the present invention. Any equivalent changes or modifications made based on the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a confined CoNi alloy-carbon composite nanozyme, characterized in that: The preparation method comprises: P123, melamine and water were mixed to form solution A; Ni(CH3COO)2·4H2O and Co(NO3)2·6H2O were mixed with water to form solution B; mixing graphene oxide with water to form solution C; Solution B and solution C were added dropwise to solution A, respectively, and stirred at room temperature to obtain a lavender solution, which was then freeze-dried to obtain a lavender powder precursor; The pale purple powder precursor is carbonized in N2 at a heating rate of 2 to 5°C / min to 450 to 550°C, and then calcined at a heating rate of 2 to 5°C / min to 650 to 750°C. The resulting black powder with a metallic luster is the confined CoNi alloy-carbon composite nanozyme.

2. The preparation method according to claim 1, characterized in that In the solution A, the mass ratio of the P123 to the melamine is 1:(8-12); and / or, the concentration of P123 in the solution A is 0.01 to 0.02 g / mL, and the concentration of melamine is 0.1 to 0.2 g / mL; and / or, in the solution B, the mass ratio of Ni(CH3COO)2·4H2O to Co(NO3)2·6H2O is 1:(0.8-1.2); and / or, the concentrations of Ni(CH3COO)2·4H2O and Co(NO3)2·6H2O in the solution B are independently 0.02 to 0.05 g / mL; and / or, the concentration of graphene oxide in the solution C is 0.8 to 1.2 mg / mL; and / or, the volume ratio of solution A, solution B and solution C is (3-5):(0.8-1.2):1; And / or, the feeding mass ratio of P123, melamine, Ni(CH3COO)2·4H2O, 0.4g Co(NO3)2·6H2O and graphene oxide is (0.3-0.6):(3-6):(0.3-0.6):(0.3-0.6):0.

01.

3. The preparation method according to claim 1, characterized in that The P123, melamine and water are mixed at 70-90° C. and 500-700 r / min; and / or, solution B and solution C are added dropwise to solution A respectively, and stirred at room temperature at 200-400 r / min for 8-12 hours to obtain the lavender solution; And / or, the carbonization and calcination times are 0.8 to 1.5 hours respectively.

4. The preparation method according to claim 1, characterized in that The preparation method further includes post-treatment, which includes acid washing the black powder with metallic luster with 0.5-2 mol / L H2SO4 for 20-30 hours, then washing with water and ethanol alternately until the solution is neutral, and finally drying at 50-70°C.

5. A confined CoNi alloy-carbon composite nanozyme prepared by the preparation method according to any one of claims 1 to 4, characterized in that: The confined CoNi alloy-carbon composite nanozyme contains carbon nanotubes densely covered on a carbon layer, cobalt-nickel alloy particles wrapped in the carbon nanotubes, and the cobalt-nickel alloy particles are located inside the carbon nanotubes. The diameter of the alloy particles is between 10 and 15 nm, and the carbon nanotubes present a multi-node morphology similar to bamboo, with an average diameter of about 35 to 45 nm, and Co, Ni, N, and C are evenly distributed.

6. The confined CoNi alloy-carbon composite nanozyme according to claim 5, characterized in that The specific surface area of the confined CoNi alloy-carbon composite nanozyme is 440-460 m 2 / g, and the average pore diameter is 7.5-9.5nm.

7. Use of a confined CoNi alloy-carbon composite nanozyme prepared by the preparation method according to any one of claims 1 to 4 or a confined CoNi alloy-carbon composite nanozyme according to claim 5 or 6 in the evaluation of total antioxidant capacity.

8. A method for detecting total antioxidant capacity, characterized in that: The total antioxidant capacity is detected based on the colorimetric method. The confined CoNi alloy-carbon composite nanozyme prepared by the preparation method of any one of claims 1 to 4 or the confined CoNi alloy-carbon composite nanozyme according to claim 5 or 6 is incubated with a chromogenic substrate in a HAc-NaAc buffer at 35-45°C, and then the confined CoNi alloy-carbon composite nanozyme in the incubation solution is separated by a magnet to obtain a chromogenic solution. The chromogenic solution is then mixed with the sample solution to be tested and reacted at room temperature to obtain a reaction solution. Ascorbic acid, reduced glutathione or cysteine are used as antioxidant model compounds, and the total antioxidant capacity is quantified by the expression amount of mM equivalents of the antioxidant model compound per liter of reaction solution. The chromogenic substrate is any one of TMB, ABTS, DAB and OPD, and the sample to be tested includes food and medicine.

9. The detection method according to claim 8, characterized in that The total antioxidant capacity was determined by visually observing the color change of the reaction solution before and after the reaction. Alternatively, the absorbance value of the reaction solution at a specific wavelength is measured using an ultraviolet spectrophotometer. When the chromogenic substrate is TMB, the specific wavelength is any wavelength between 649 and 659 nm; when the chromogenic substrate is ABTS, the specific wavelength is any wavelength between 415 and 425 nm; when the chromogenic substrate is DAB, the specific wavelength is any wavelength between 463 and 473 nm; when the chromogenic substrate is OPD, the specific wavelength is any wavelength between 445 and 455 nm. The measured absorbance value is substituted into a standard curve drawn from the absorbance values of antioxidant model compound standards at different concentrations to calculate the total antioxidant capacity. Alternatively, the reaction solution is photographed and the grayscale value is calculated, and the total antioxidant capacity is obtained by processing the image using digital imaging colorimetric software in combination with a standard curve drawn using the grayscale values of antioxidant model compound standards at different concentrations.

10. A kit for detecting total antioxidant capacity, characterized in that: The kit includes a confined CoNi alloy-carbon composite nanozyme prepared by the preparation method of any one of claims 1 to 4 or a confined CoNi alloy-carbon composite nanozyme according to claim 5 or 6, an antioxidant model compound standard, an acetic acid-sodium acetate buffer and a chromogenic substrate, wherein the chromogenic substrate is one or more of TMB, ABTS, DAB and OPD.

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