A defective magnetic FeNi porous carbon nanozyme with multiple enzyme activities

Abstract

The invention discloses a defective magnetic FeNi porous carbon nanozyme with multiple enzyme activities at the same time. The waste shrimp shells of aquatic waste are used as raw materials, soaked in an ethanol aqueous solution of iron salt and nickel salt, and the waste shrimp shells are modified by iron ions and nickel ions through impregnation and heat-induced assembly to form a metal-like organic framework complex. At the same time, the amino acids, proteins, calcium carbonate, etc. inherent in the waste shrimp shells are used as self-doped nitrogen atoms, self-templates and pore-forming agents, and the chloride ions released by the iron salts form NaCl nanocrystal templates with the sodium ions in the waste shrimp shells. The defective magnetic FeNi porous carbon nanozyme with high specific surface area, large pore capacity and multi-level pore structure can be prepared by high-temperature carbonization with inert gas. The raw materials for preparing the nanozyme of the invention are cheap and easy to obtain, and it has peroxidase-like activity, hydrogen peroxide mimetic enzyme activity, and oxidase-like activity at the same time, and can be used for highly selective colorimetric / photothermal / smartphone three-mode detection of total antioxidant levels and total polyphenol content in beverages and fruits.

Description

Technical Field

[0001] The present invention belongs to the technical field of carbon-based nanozymes, and specifically relates to a defective nitrogen-doped porous carbon-coated magnetic NiFe nanozyme having multiple enzyme activities at the same time. 2 O 4 Nanozymes. Background Art

[0002] Natural enzymes are a type of macromolecular biocatalysts that play a very important role in organisms and have received extensive attention in the fields of disease diagnosis and treatment, food analysis, and environmental science. However, the chemical nature of natural enzymes is protein or RNA, which has bottleneck problems such as high preparation cost, poor stability, harsh storage conditions, and difficulty in large-scale preparation and application. These drawbacks have greatly limited their practical applications in the fields of biomedicine, food safety, and environmental protection.

[0003] In recent years, researchers have discovered that certain nanomaterials have the inherent ability to simulate the catalytic activity of one or more biological enzymes, which scientists call nanozymes. Compared with natural enzymes or traditional enzyme mimics, nanozymes have the advantages of simple preparation, tunable catalytic activity, and not easy to inactivate. Currently developed nanozymes include peroxidase mimics, oxidase mimics, catalase mimics, superoxide dismutase mimics, hydrolase mimics, etc. These nanozymes have attracted widespread attention in many fields such as biomedicine, agriculture, food safety, and environmental governance.

[0004] At present, scientists at home and abroad have successively carried out in-depth explorations in the design and synthesis of nanozymes, the types of enzyme-like catalytic reactions of nanozymes, the regulation of enzyme-like catalytic activity, the revelation of catalytic mechanisms and the expansion of applications. In order to obtain nanozymes with excellent performance, precious metal-based nanozymes, carbon-based nanozymes, metal-organic framework nanozymes and composite nanozymes have been explored one after another. Precious metals and their alloys have excellent catalytic properties, but their high cost greatly limits their large-scale applications. Metal-organic framework nanozymes are expensive and their catalytic activity decreases significantly with large-scale preparation. In contrast, carbon-based nanozymes have unique advantages such as high specific surface area, controllable morphology and pore size, and adjustable catalytic activity. They have shown great application prospects in the fields of food and drug analysis, advanced oxidation of organic poisons, degradation of environmental pollutants, and disease diagnosis and treatment.

[0005] However, the catalytic activity of metal-free carbon nanozymes is often low, the preparation cost is high, and it is difficult to recycle, which makes actual large-scale application quite difficult. In order to break through these bottleneck problems, researchers have found that monometallic or bimetallic nanoparticles doped with carbon skeletons and the introduction of defect engineering can effectively improve the catalytic activity of carbon-based nanozymes. However, the carbon sources for the preparation of carbon-based nanozymes currently mostly use expensive and toxic chemical reagents as raw materials, and large-scale preparation is more difficult, especially under large-scale synthesis conditions. The specific surface area and pore capacity of porous carbon materials prepared will drop sharply. Therefore, this method has certain limitations in the large-scale industrial production of enzymes.

[0006] At present, carbon-based nanozymes mainly include peroxidase mimics, oxidase mimics, catalase mimics, superoxide dismutase mimics, laccase, etc. in terms of simulating the catalytic activity of natural enzymes. Most research focuses on the development and application of peroxidase mimics, but only a few research reports reveal that Fe, Co and N doping can improve the oxidase mimics activity of carbon materials. In fact, oxidase mimics can achieve colorimetric sensing of the target object by using dissolved oxygen in an environment without the introduction of hydrogen peroxide, which is more advantageous than peroxidase mimics (avoiding H 2 O 2 In addition, the easy separation of magnetic carbon-based nanozymes under the action of an external magnetic field can minimize the interference of the nanozyme's own high background signal.

[0007] In recent years, it has become a trend to use biomass waste to replace chemical reagents to synthesize carbon-based nanozymes, because of its cheap and easy availability, low toxicity and the characteristics of containing a variety of biopolymers. Aquatic product waste is rich in a variety of biologically active ingredients and accounts for a considerable proportion of aquatic products. The output of aquatic product waste in my country has shown an increasing trend year by year, but there is currently a lack of advanced conversion technology and high value-added product development. If aquatic product waste is processed and comprehensively utilized, it can not only turn waste into treasure and avoid environmental pollution, but also be a good way for the people to get rich. Summary of the invention

[0008] In view of the bottleneck problems of high preparation cost, low oxidase-like catalytic activity and difficulty in large-scale preparation of the above-mentioned chemical reagent-derived carbon nanozymes, the present invention proposes a defective nitrogen-doped porous carbon-coated magnetic NiFe2O3 derived from waste shrimp shells with high specific surface area, large pore capacity and multi-level pore structure, and simultaneously having peroxidase-like activity, hydrogen peroxide mimetic enzyme activity and oxidase-like activity. 2 O 4 Nanozyme (short for defective magnetic FeNi porous carbon nanozyme).

[0009] In order to achieve the above object, the defective magnetic FeNi porous carbon nanozyme having multiple enzyme activities provided by the present invention is prepared by the following steps:

[0010] Step 1: crushing waste shrimp shells to obtain waste shrimp shell powder;

[0011] Step 2: dissolving iron salt and nickel salt in an ethanol aqueous solution, adding the waste shrimp shell powder prepared in step 1, stirring at room temperature for 2 to 6 hours, and heat-inducing the assembly reaction at 60 to 80° C. for 16 to 24 hours;

[0012] Step 3: After calcining the product after thermal induced assembly in step 2 at 500-700°C for 2-4 hours under inert gas protection, soak it in dilute hydrochloric acid, wash it with distilled water and anhydrous ethanol in turn, and obtain defective magnetic FeNi porous carbon nanozyme after drying.

[0013] In the above step 2, the iron salt and the nickel salt are dissolved in an ethanol aqueous solution, and the waste shrimp shell powder prepared in step 1 is added, preferably stirred at room temperature for 4 hours, and then heat-induced assembly reaction is carried out at 80° C. for 24 hours.

[0014] In the above step 2, the molar ratio of Fe(III) to Ni(II) in the iron salt and the nickel salt is preferably 1.5-2.5:1.

[0015] In the above step 2, the iron salt is any one of anhydrous ferric chloride and ferric nitrate nonahydrate, the nickel salt is any one of nickel nitrate hexahydrate and nickel acetate, and the mass fraction of ethanol in the ethanol aqueous solution is 25% to 40%.

[0016] In the above step 3, the product after the thermal induction assembly in step 2 is preferably calcined at 600° C. for 2.5 hours under the protection of an inert gas.

[0017] In the above step 3, the inert gas is nitrogen or argon.

[0018] In the above step 3, the concentration of the dilute hydrochloric acid is 0.1 mol / L, and the soaking time is 60 to 120 minutes.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The present invention uses waste shrimp shells from aquatic waste as raw materials, soaks the waste shrimp shell powder in an ethanol aqueous solution of iron salt and nickel salt, and promotes iron ions and nickel ions to coordinate and modify the waste shrimp shells to form a metal-like organic framework complex material through impregnation and heat-induced assembly. At the same time, the inherent amino acids, proteins and other bioactive molecules of the waste shrimp shells are used as self-doped nitrogen atoms, and the calcium carbonate rich in itself is used as a self-template and pore-forming agent. In addition, the chloride ions released by the iron salt and the sodium ions in the waste shrimp shells can form a NaCl nanocrystal template, and then a defective magnetic FeNi porous carbon nanozyme with high specific surface area, large pore capacity and multi-level pore structure is prepared by an inert gas high-temperature carbonization method. The defective magnetic FeNi porous carbon nanozyme of the present invention not only has peroxidase-like activity and hydrogen peroxide mimetic enzyme activity, but also has good oxidase-like activity.

[0021] 2. The present invention replaces chemical reagents with low-cost aquatic waste - waste shrimp shells, which can reduce the cost of carbon sources for carbon-based nanozymes, turning waste into treasure and being green and environmentally friendly. The preparation method is simple and the conditions are mild. The obtained defective magnetic FeNi porous carbon nanozyme has stable performance. As a novel oxidase-like mimetic enzyme, it can be used for colorimetric / photothermal / smartphone three-mode selective detection of total antioxidant levels (with polyhydroxy compound ascorbic acid as equivalent) and total polyphenol content (with polyphenol compound tannic acid as equivalent) in beverages and fruits, and has important application value in the field of food quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a transmission electron micrograph of the defective magnetic FeNi porous carbon nanozyme prepared in Example 1.

[0023] Figure 2 3. It is the XRD pattern of the defective magnetic FeNi porous carbon nanozyme and shrimp shell-derived porous carbon prepared in Example 1.

[0024] Figure 3 This is the hysteresis curve of the defective magnetic FeNi porous carbon nanozyme prepared in Example 1.

[0025] Figure 4 This is the infrared spectra of the defective magnetic FeNi porous carbon nanozyme and shrimp shell-derived porous carbon prepared in Example 1.

[0026] Figure 5 This is the nitrogen adsorption-desorption diagram of the defective magnetic FeNi porous carbon nanozyme prepared in Example 1 and the shrimp shell-derived porous carbon.

[0027] Figure 6 It is the Raman spectra of the un-acid-washed magnetic FeNi porous carbon, the defective magnetic FeNi porous carbon nanozyme and the shrimp shell-derived porous carbon in Example 1.

[0028] Figure 7 This is the X-ray photoelectron energy spectrum of the defective magnetic FeNi porous carbon nanozyme and shrimp shell-derived porous carbon prepared in Example 1.

[0029] Figure 8 It is the UV-visible absorption spectrum of the peroxidase-like activity of the defective magnetic FeNi porous carbon nanozyme prepared in Example 1 and its control material.

[0030] Fig. 9 This is the UV-visible absorption spectrum of the oxidase-like activity of the defective magnetic FeNi porous carbon nanozyme prepared in Example 1 and its control material.

[0031] Fig.10 It is the UV-visible absorption spectra of the oxidase-like activity of defective magnetic FeNi porous carbon nanozymes at different calcination temperatures.

[0032] Fig.11 This is a graph of the hydrogen peroxide-mimicking enzyme activity of the defective magnetic FeNi porous carbon nanozyme prepared in Example 1 at different concentrations for decomposing hydrogen peroxide.

[0033] Fig.12 This is the steady-state kinetic curve of the defective magnetic FeNi porous carbon nanozyme prepared in Example 1 towards tetramethylbenzidine.

[0034] Fig.13 This is a concentration-absorbance change curve of the defective magnetic FeNi porous carbon nanozyme prepared in Example 1 for colorimetric analysis of ascorbic acid.

[0035] Fig.14 This is a linear curve diagram of the colorimetric analysis of ascorbic acid by the defective magnetic FeNi porous carbon nanozyme prepared in Example 1, wherein the inset is a physical diagram corresponding to the ascorbic acid concentration.

[0036] Fig.15 This is a linear curve diagram of the photothermal analysis of ascorbic acid by the defective magnetic FeNi porous carbon nanozyme prepared in Example 1.

[0037] Fig.16 This is a linear curve of the concentration-color parameter changes of ascorbic acid analyzed by a smartphone using a defective magnetic FeNi porous carbon nanozyme prepared in Example 1.

[0038] Fig.17 This is a concentration-absorbance change curve of the defective magnetic FeNi porous carbon nanozyme prepared in Example 1 for colorimetric analysis of tannic acid.

[0039] Fig.18 This is a linear curve diagram of the colorimetric analysis of tannic acid by the defective magnetic FeNi porous carbon nanozyme prepared in Example 1, wherein the inset is a physical diagram of the corresponding tannic acid concentration.

[0040] Fig.19 This is a linear curve diagram of the photothermal analysis of tannic acid using the defective magnetic FeNi porous carbon nanozyme prepared in Example 1.

[0041] Fig. 20 This is a linear graph of the concentration-color parameter changes of ascorbic acid analyzed by a smartphone using a defective magnetic FeNi porous carbon nanozyme prepared in Example 1.

[0042] Fig.21 This is a graph showing the selectivity results of the defective magnetic FeNi porous carbon nanozyme prepared in Example 1 for colorimetric analysis of ascorbic acid and tannic acid. DETAILED DESCRIPTION

[0043] The present invention is described below in conjunction with the accompanying drawings and embodiments. The embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0044] Example 1

[0045] Step 1: Dry and crush the collected waste shrimp shells to obtain waste shrimp shell powder.

[0046] Step 2: 9.7 g (0.06 mol) of anhydrous ferric chloride and 8.7 (0.03 mol) of nickel nitrate hexahydrate are completely dissolved in 150 mL of 30% ethanol aqueous solution, 100 g of waste shrimp shell powder is added, and after stirring at room temperature for 4 hours, the mixture is transferred into a constant temperature drying oven and heated at 80° C. At this temperature, the mixture undergoes a heat-induced assembly reaction and gradually evaporates water until a dry brown powder is obtained.

[0047] Step 3: The brown powder obtained after the thermally induced assembly reaction in step 2 was placed in a nitrogen atmosphere and calcined at 600°C for 2.5 hours (recorded as unacid-washed magnetic FeNi porous carbon). Then, 5 g of unacid-washed magnetic FeNi porous carbon was immersed in 100 mL of 0.1 mol / L dilute hydrochloric acid for 90 minutes, and the product was washed with distilled water and anhydrous ethanol. Finally, it was dried at 70°C to obtain a defective magnetic FeNi porous carbon nanozyme.

[0048] The physical adsorption instrument ASAP 2020 and elemental analysis were used to qualitatively and quantitatively analyze the pore structure and chemical composition of the defective magnetic FeNi porous carbon nanozyme prepared above. At the same time, the Michaelis-Menten equation was used to conduct experiments and calculate the Michaelis constant (K) of the prepared defective magnetic FeNi porous carbon nanozyme as a peroxidase-like mimetic enzyme (POD) in the tetramethylbenzidine (TMB) and hydrogen peroxide reaction system. m ) and the maximum reaction rate constant (V max ), and its K as an oxidase-like enzyme (OXD) in TMB m 、V max, the results are shown in Table 1.

[0049] Table 1 Texture characteristics, chemical composition and enzyme catalytic kinetics of defective magnetic FeNi porous carbon nanozymes

[0050]

[0051] Note: In the table [a] is the BET surface area; [b] is the total pore capacity; [c] is the average mesopore size (BJH method).

[0052] As shown in Table 1, the specific surface area of ​​the defective magnetic FeNi porous carbon nanozyme prepared in Example 1 is 241.5 m 2 / g, total pore capacity is 0.24cm 3 / g, an average mesopore size of 6.8nm and a nitrogen content of 8.6wt.%.

[0053] In order to compare the performance of defective magnetic FeNi porous carbon nanozymes, the following control group materials were prepared: (1) Shrimp shell-derived porous carbon: 20 g of crushed shrimp shell powder was placed in a nitrogen atmosphere and calcined at 600 °C for 2.5 hours. After cooling to room temperature, the obtained black solid powder was washed with distilled water and anhydrous ethanol in turn, and finally dried at 70 °C to obtain shrimp shell-derived porous carbon. (2) NiFe 2 O 4 Nanoparticles: 0.727 g nickel nitrate hexahydrate and 0.81 g anhydrous ferric chloride were dissolved in 40 mL ethylene glycol. After complete dissolution, 3.6 g sodium acetate and 2 mL polyethylene glycol 200 were added. The resulting mixture was then hydrothermally reacted at 200 °C for 18 hours. After cooling to room temperature, the resulting precipitate was washed with distilled water and anhydrous ethanol in sequence, and finally dried in a drying oven at 60 °C for 8 hours to obtain NiFe 2 O 4 Nanoparticles.

[0054] Depend on Figures 1 to 5 It can be seen that magnetic NiFe 2 O 4 Nanoparticles are evenly distributed in the porous carbon framework, and magnetic FeNi porous carbon is successfully prepared. Figure 6 It can be seen that the I of the defective magnetic FeNi porous carbon nanozyme obtained after soaking and washing with dilute hydrochloric acid D / I G The value is significantly higher than that of the un-acid-washed magnetic FeNi porous carbon and shrimp shell-derived porous carbon, which indicates that FeNi bimetallic doping and dilute hydrochloric acid immersion washing can improve the defect degree of shrimp shell-derived porous carbon skeleton, especially the combined treatment of the two can significantly enhance the defect degree of carbon materials. Figure 7It can be seen that the shrimp shell-derived porous carbon material mainly contains C, N, O, and Ca elements, while the defective magnetic FeNi porous carbon nanozyme contains C, N, O, Fe, and Ni elements, indicating that N / O co-doped porous carbon confines NiFe 2 O 4 Nanocomposites were successfully prepared.

[0055] In order to demonstrate the beneficial effects of the present invention, the catalytic performance of the defective magnetic FeNi porous carbon nanozyme prepared in the above Example 1 was investigated, and the specific experiment is as follows:

[0056] (1) Evaluation of peroxidase-like enzyme catalytic activity: 100 μL TMB (10 mM), 100 μL H 2 O 2 (6mM) and 100μL of the aqueous dispersion of defective magnetic FeNi porous carbon nanozyme (1mg / mL) were reacted at 40℃ for 15min. The carbon nanozyme was separated by an external magnetic field and the absorbance of the sample solution was measured. 2 O 4 Nanoparticles and un-acid-washed magnetic FeNi porous carbon were used as control experiments. Figure 8 The results showed that under the same experimental conditions, the defective magnetic FeNi porous carbon nanozyme had the highest peroxidase-like mimetic activity, and its catalytic activity was much higher than that of shrimp shell-derived porous carbon and NiFe 2 O 4 Nanoparticles, illustrating shrimp shell-derived porous carbon and NiFe 2 O 4 After hybridization, nanoparticles can exhibit excellent synergistic catalytic effects.

[0057] In addition, the defective magnetic FeNi porous carbon nanozyme prepared in Example 1 (0.055 and 0.549 mM, respectively) showed no significant difference in the activity of TMB and H 2 O 2 The Michaelis constant of the defective magnetic FeNi porous carbon nanozyme is significantly lower than that of natural horseradish peroxidase (0.434 vs. 3.7 mM), indicating that the defective magnetic FeNi porous carbon nanozyme has a strong affinity for TMB and H 2 O 2 Defective magnetic FeNi porous carbon nanozymes have better affinity for TMB and H 2 O 2 The maximum reaction rate constant of the defective magnetic FeNi porous carbon nanozyme is significantly higher than that of natural horseradish peroxidase, indicating that the defective magnetic FeNi porous carbon nanozyme has a strong affinity for TMB and H 2 O 2 Has excellent catalytic activity.

[0058] (2) Evaluation of the catalytic activity of oxide-like enzymes: 100 μL TMB (10 mM) and 400 μL aqueous dispersion of defective magnetic FeNi porous carbon nanozymes (4.5 mg / mL) were added to 2.5 mL NaAc buffer (0.2 M, pH 4.6), reacted at 40 °C for 15 min, and the absorbance of the sample solution was measured after separation by external magnetic field. 2 O 4 Nanoparticles and un-acid-washed magnetic FeNi porous carbon were used as control experiments. Fig. 9 The results showed that under the same experimental conditions, the defective magnetic FeNi porous carbon nanozyme had the highest oxidase-like mimicking activity, and its catalytic activity was much higher than that of shrimp shell-derived porous carbon, NiFe 2 O 4 Nanoparticles and un-acid-washed magnetic FeNi porous carbon materials.

[0059] In addition, the Michaelis constant of the defective magnetic FeNi porous carbon simulated oxidase (0.159 mM) prepared in Example 1 for TMB is significantly lower than that of most of the nano-oxidation simulated enzymes reported so far, such as CeO 2 The Michaelis constants of nanoparticles, Se nanoparticles, Pt nanoclusters and Fe-doped hollow carbon spheres were 0.8, 8.3, 0.63 and 0.21 mM, respectively, indicating that the defective magnetic FeNi porous carbon nanozyme has good affinity for TMB.

[0060] In addition, we also explored the relationship between the calcination temperature and the catalytic activity of the oxidase-like mimic enzyme in the preparation process of Example 1. The results showed that as the calcination temperature increased, the catalytic activity of the oxidase-like enzyme gradually increased. Considering the goal of energy saving and economically friendly preparation of highly catalytically active carbon materials, this study determined 600°C as the optimal calcination temperature (see Fig.10 ).

[0061] (3) Evaluation of the catalytic activity of hydrogen peroxide mimetic enzymes: At room temperature, different doses of defective magnetic FeNi porous carbon nanozymes were mixed with 50 mL of 100 mM PBS buffer (pH 7.4), and then the changes in oxygen content in the solution were monitored using a dissolved oxygen meter. The results showed (see Fig.11 ), as the dosage of defective magnetic FeNi porous carbon nanozyme continued to increase from 50 to 110 μg / mL, the change value of oxygen content under the same test conditions gradually increased, indicating that the hydrogen peroxide-mimicking enzyme activity of defective magnetic FeNi porous carbon nanozyme showed a good dose-dependent relationship.

[0062] In addition, the steady-state kinetic behavior of the defective magnetic FeNi porous carbon nanozyme prepared in Example 1 was studied, and the results showed (see Fig.12), the prepared defective magnetic FeNi porous carbon nanozyme showed a good Michaelis constant curve with the matrix TMB. At the same time, the thermal stability of the prepared defective magnetic FeNi porous carbon nanozyme was tested, and the results showed that it had good thermal stability in the range of 25 to 70°C. However, a large number of studies have shown that the enzymatic activity of natural enzymes decreases significantly with increasing temperature. This shows that the defective magnetic FeNi porous carbon nanozyme of the present invention has good thermal stability.

[0063] In order to further demonstrate the beneficial effects of the present invention, the analytical performance of the prepared defective magnetic FeNi porous carbon simulated oxidase on ascorbic acid and tannic acid was tested by UV-visible spectrophotometry, photothermal analysis and smartphone analysis. The specific experiments are as follows:

[0064] (1) Detection of ascorbic acid: 100 μL TMB (16 mM), 400 μL aqueous dispersion of defective magnetic FeNi porous carbon nanozyme (4.5 mg / mL) and 100 μL ascorbic acid of different concentrations (0.01-250 μmol / L) were added to 2.4 mL NaAc (0.2 M, pH 3.5) buffer and reacted at 25 °C for 20 min. After magnetic separation of carbon nanozyme, the absorbance value was recorded by spectrophotometer (i.e. colorimetric analysis). At the same time, a Huawei smartphone was used to take a photo of the obtained blue sample solution, which was processed by mobile phone software to obtain R, G, and B values, and a related linear curve was drawn (i.e. smartphone mode). In addition, after the obtained blue reaction mixture was separated by an external magnetic field, the filtrate was irradiated with an 808 nm laser (2.42 W) for 5 min, and then the solution temperature was measured with a household thermometer (i.e. photothermal sensing mode).

[0065] like Figures 13-14 As shown in the figure, the experimental results show that the absorbance at 652 nm gradually decreases with the increase of ascorbic acid concentration (1-250 μM) and shows a good linear relationship (R 2 =0.9847), and the minimum detection limit was 1.79 μM. Its determination sensitivity is comparable to that of most metals, precious metals and carbon-based nanozymes. The temperature of the blue solution after magnetic separation gradually decreases with the increase of ascorbic acid concentration (10-120 μM) and shows a good linear relationship (R 2 =0.9920), the minimum detection limit is 8.4 μM (see Fig.15 The results of the smartphone sensing mode test showed that the color parameter B / (R+G+B) gradually decreased with the increase of ascorbic acid concentration (0.01-250 μM), and showed a good linear relationship (R 2 =0.9902), the minimum detection limit is 12.61μM (see Fig.16). These results indicate that the defective magnetic FeNi porous carbon nanozyme of the present invention can realize the analytical requirements of low-cost, high-sensitivity, on-site detection of ascorbic acid.

[0066] (2) Detection of tannic acid: 100 μL TMB (16 mM), 400 μL aqueous dispersion of defective magnetic FeNi porous carbon nanozyme (4.5 mg / mL) and 100 μL tannic acid of different concentrations (0.01-25 μmol / L) were added to 2.4 mL NaAc-HAc (0.2 M, pH 3.5) buffer and reacted at 25 °C for 20 min. Finally, the carbon nanozyme was magnetically separated and the absorbance value was recorded by a spectrophotometer (i.e., colorimetric analysis). At the same time, a Huawei smartphone was used to take a photo of the obtained blue sample solution, and the R, G, and B values ​​were obtained by the mobile phone software and the related linear curve was drawn (i.e., smartphone mode). In addition, the blue mixed solution after magnetic field separation was irradiated with an 808 nm laser (2.42 W) for 5 min, and then the solution temperature was measured with a household thermometer (i.e., photothermal sensing mode).

[0067] like Figures 17-18 As shown in the figure, the experimental results show that the absorbance at 652 nm gradually decreases with the increase of tannic acid concentration (0.01-21 μM) and shows a good linear relationship (R 2 =0.9876), and the minimum detection limit was 0.108 μM. The temperature of the blue solution after magnetic separation gradually decreased with the increase of tannic acid concentration (2-25 μM) and showed a good linear relationship (R 2 =0.9877), the minimum detection limit is 1.2μM (see Fig.19 The results of the smartphone sensing mode test showed that the color parameter B / (R+G+B) gradually decreased with the increase of tannic acid concentration (1-21 μM), and showed a good linear relationship (R 2 =0.9779), the minimum detection limit is 3.1 μM (see Fig. 20 ). These results indicate that the defective magnetic FeNi porous carbon nanozyme of the present invention is expected to be used for monitoring tannic acid in food and medicine.

[0068] In order to clarify the selectivity of the defective magnetic FeNi porous carbon nanozyme prepared in Example 1 for analyzing ascorbic acid and tannic acid, this experiment selected sodium chloride, potassium chloride, copper chloride, calcium chloride, glycine, glucose, and bovine serum albumin as representative interfering substances for testing, and the concentration of the interfering substance was ten times the concentration of ascorbic acid and tannic acid. The reaction conditions were: 2.4mL NaAc buffer (0.2mol / L, pH 3.5) + 100μL TMB (16mM) + 400μL aqueous dispersion of defective magnetic FeNi porous carbon nanozyme (4.5mg / mL) + 100μL interfering reagent (3mM), react at 25℃ for 20min, and record the absorbance and temperature change values ​​respectively.

[0069] Depend on Fig.21 It can be seen that common interfering substances such as sodium chloride, potassium chloride, copper chloride, calcium chloride, glycine (Gly), glucose (Glu), and bovine serum albumin (BSA) have almost no obvious interference with the detection method, indicating that the defective magnetic FeNi porous carbon nanozyme has good selectivity and application potential in the analysis of ascorbic acid and tannic acid.

[0070] In order to explore the application potential of the defective magnetic FeNi porous carbon simulated oxidase prepared in Example 1 in the field of detecting the antioxidant level and total polyphenol content of actual samples, the present invention further established a colorimetric analysis method for the antioxidant level and total polyphenol content in beverages and fruits.

[0071] Experimental method: Two brands of fruit juice drinks, green tea drinks and mandarin oranges were selected as actual samples. The three drinks were diluted 5 times with buffer for later use. Take a piece of mandarin orange and mash it to extract the juice. Pour out the juice and centrifuge it at 6000rmp for 6min and 10000rmp for 5min. After treatment, take the supernatant and dilute it 10 times with NaAc buffer for later use. Take three sample solutions and add them to a mixed solution of 2.4mL NaAc buffer (0.2mol / L, pH 3.5), 100μL TMB (16mM) and 400μL of an aqueous dispersion of defective magnetic FeNi porous carbon simulated oxidase (4.5mg / mL), and react at 25℃ for 20min. Finally, the total antioxidant capacity (in terms of ascorbic acid) and total polyphenol content (in terms of tannic acid) were detected according to the above three-mode sensing method. The results are shown in Table 2.

[0072] Table 2 Performance comparison of defective magnetic FeNi porous carbon nanozymes in sensing total antioxidant capacity and total polyphenol content

[0073]

[0074] As can be seen from Table 2, the antioxidant content in different samples measured with ascorbic acid and tannic acid as indicators is quite different. It should be pointed out that the results of the three-mode analysis method in the actual sample test are relatively consistent, indicating that the colorimetric analysis method currently established can be used to evaluate the antioxidant level and total polyphenol content in beverages and fruits. The sensing platform presents different colors and different temperature changes according to the difference in ascorbic acid and tannic acid content, which is very conducive to the portable monitoring of the colorimetric / photothermal / smartphone APP analysis system. In short, the current sensing platform can achieve the goal of selectively analyzing the total antioxidant capacity in fruits and beverages, indicating that the defective magnetic FeNi porous carbon nanozyme of the present invention has a good oxide-like mimic enzyme activity, and is expected to replace expensive natural enzymes to achieve selective, low-cost, colorimetric, photothermal, and smartphone analysis of the total antioxidant level and total polyphenol content in food.

Claims

1. A defective magnetic FeNi porous carbon nanozyme with multiple enzyme activities simultaneously. Features The nanozyme is prepared by the following steps: Step 1: crushing waste shrimp shells to obtain waste shrimp shell powder; Step 2: dissolving iron salt and nickel salt in an ethanol aqueous solution with a mass fraction of 25% to 40%, adding the waste shrimp shell powder prepared in step 1, stirring at room temperature for 2 to 6 hours, and heat-inducing the assembly reaction at 60 to 80° C. for 16 to 24 hours; the iron salt is anhydrous ferric chloride; Step 3: After the product of step 2 is thermally induced and assembled, it is calcined at 500-700°C for 2-4 hours under inert gas protection, and then soaked in 0.1 mol / L dilute hydrochloric acid for 60-120 minutes, and washed with distilled water and anhydrous ethanol in turn, and dried to obtain a defective magnetic FeNi porous carbon nanozyme, which is a defective nitrogen-doped porous carbon-coated magnetic NiFe 2 O 4 Nanozymes.

2. The defective magnetic FeNi porous carbon nanozyme having multiple enzyme activities according to claim 1, Features: In step 2, iron salt and nickel salt are dissolved in an ethanol aqueous solution, and the waste shrimp shell powder prepared in step 1 is added, and after stirring at room temperature for 4 hours, a heat-induced assembly reaction is performed at 80° C. for 24 hours.

3. The defective magnetic FeNi porous carbon nanozyme having multiple enzyme activities according to claim 1 or 2, Features: In step 2, the nickel salt is any one of nickel nitrate hexahydrate and nickel acetate.

4. The defective magnetic FeNi porous carbon nanozyme having multiple enzyme activities according to claim 1, Features: In step 3, the product after thermal induction assembly in step 2 is calcined at 600° C. for 2.5 hours under the protection of inert gas.

5. The defective magnetic FeNi porous carbon nanozyme having multiple enzyme activities according to claim 1 or 4, Features: In step 3, the inert gas is nitrogen or argon.

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