Metallic single-atom nanoscale enzyme composite material, preparation method and application thereof

Abstract

The application provides a metal single-atom nanoscale enzyme composite material, a preparation method and application thereof, raw materials of the metal single-atom nanoscale enzyme composite material include: a metal source, a non-metallic heteroatom source and a carbon-based carrier. The metal source includes one or more of an iron source, a manganese source, a cobalt source, a gold source, a platinum source, a palladium source and a copper source; the non-metallic heteroatom source includes one or more of a nitrogen source, a sulfur source, a phosphorus source and a boron source; and the carbon-based carrier includes one or more of MXene, graphene oxide, reduced graphene oxide, graphite-like phase carbon nitride and carbon nanotubes. The nanoscale enzyme composite material MX@C synthesized through the design of a metal single-atom M active center mediated by a non-metallic heteroatom X has both oxidase-like and peroxidase-like characteristics, can simultaneously activate dissolved oxygen and low-dose H2O2, significantly improves the ROS yield and the pollutant removal efficiency, and overcomes the problems of single function and poor adaptability of traditional nanoscale enzymes.

Description

Technical Field

[0001] This invention relates to the fields of environmental functional material preparation technology and water pollution control technology, and in particular to a metal single-atom nanoenzyme composite material, its preparation method and application. Background Technology

[0002] Water pollution, especially complex pollution caused by a variety of persistent pollutants, has become one of the most severe environmental challenges facing the world today. The complexity of this pollution lies in the wide range of pollutant sources, the diverse types of pollutants, and their immense harm. Among these, harmful algal blooms are a prominent problem. With the long-term accumulation of pollution in watersheds, persistently high nutrient inputs, and the combined effects of climate warming and the trend towards water body stabilization, algal blooms, including cyanobacteria, in lakes, reservoirs, and river networks are exhibiting a trend of increasing frequency, persistence, and spatial expansion.

[0003] To address the simultaneous occurrence of sudden and periodic algal blooms, remediation technologies must meet the engineering requirements of rapid effectiveness and wide applicability, while also ensuring ecological safety and process control, avoiding secondary pollution and rebound risks. On the other hand, emerging organic pollutants (OPCs) have attracted significant attention due to their widespread detection in the environment, potential persistence, bioaccumulation, and ecotoxicity. These pollutants are diverse, including widely used industrial dyes (such as methylene blue MB), large-scale emissions of antibiotics (such as tetracycline TC and ciprofloxacin CIP), pharmaceuticals and personal care products that frequently enter water bodies (such as the analgesic ibuprofen IBU), industrial chemicals with endocrine disrupting effects (such as the plastic additive bisphenol A BPA), and ubiquitous plasticizers (such as phthalates PAEs). They enter the aquatic environment through various pathways such as wastewater discharge, agricultural runoff, and landfill leachate, and even at low concentrations, they can pose long-term, potentially chronic hazards to aquatic organisms and human health. Furthermore, due to their stable chemical structure, conventional treatment methods often fail to completely remove them.

[0004] To address these challenges, existing water pollutant treatment technologies mainly revolve around physical, chemical, and biological methods. However, these technologies generally face numerous bottlenecks: homogeneous Fenton oxidation requires acidic conditions, iron sludge production is large and difficult to recover, and catalyst loss causes secondary pollution; ozone oxidation equipment has high investment and operating costs and may produce toxic byproducts; photocatalysis technology is limited by problems such as rapid recombination of photogenerated electron-hole pairs, low quantum efficiency, low utilization of visible light, and difficulty in catalyst recovery.

[0005] In recent years, nanoenzyme materials have provided a new approach to green catalytic algae removal. However, most materials exhibit a single enzymatic function (such as only having peroxidase-like or peroxidase-like properties), which is not suitable for the multi-factor oxidation process in complex water bodies. At the same time, traditional nano or multinuclear metal active sites have the risk of leaching, and the material synthesis process is lengthy, lacking reproducibility and large-scale manufacturing capabilities, which limits the engineering application. Summary of the Invention

[0006] In view of this, the present invention proposes a metal single-atom nanoenzyme composite material, its preparation method and application, with the aim of providing an environmental functional material with dual enzyme characteristics that combines high activity and high stability, in order to solve the technical bottlenecks in the current treatment of water pollutants, such as insufficient efficiency in generating reactive oxygen species under low-dose hydrogen peroxide conditions, limited resistance to interference in complex water bodies, and difficulty in scale-up.

[0007] This invention utilizes a non-metallic heteroatom X-mediated strategy to achieve stable anchoring and electronic structure regulation of a single metal atom M on the surface of a carbon-based support C. This enables the material to possess dual-enzyme properties, exhibiting both oxidase-like and peroxidase-like characteristics. Under environmental conditions, it can efficiently catalyze the generation of reactive oxygen species from dissolved oxygen and hydrogen peroxide, achieving rapid oxidation and removal of pollutants in water. This invention overcomes the problems of poor adaptability of single enzyme functions, complex material preparation, and insufficient stability in existing technologies, while also considering emergency response efficiency, ecological safety, and process scalability.

[0008] The technical solution of the present invention is implemented as follows: In a first aspect, the present invention provides a metal single-atom nanoenzyme composite material MX@C, the raw materials of which include: a metal source (M), a non-metal heteroatom source (X) and a carbon-based support (C).

[0009] Specifically, the MX@C of this invention anchors metal single-atom sites firmly on a carbon-based support modified with non-metallic heteroatoms (X). It utilizes the coordination environment provided by the non-metallic heteroatoms to regulate the local electronic structure of the metal single atoms, giving it dual-enzyme characteristics: on the one hand, it catalyzes the activation of dissolved oxygen to generate ROS; on the other hand, it efficiently catalyzes the generation of ROS in the presence of low-dose H2O2. The generated ROS damages water pollutants, thereby achieving the oxidative removal of water pollutants. The material employs a one-pot molten salt etching method to simultaneously complete the etching of the carbon-based support, doping of non-metallic heteroatoms, and anchoring of metal single atoms, resulting in high site density and stability. Therefore, it can achieve efficient pollutant removal and reduce ecological risks even with low H2O2 dosage.

[0010] Based on the above technical solutions, preferably, the metal source includes one or more of iron, manganese, cobalt, gold, platinum, palladium, and copper; the non-metallic heteroatom source includes one or more of nitrogen, sulfur, phosphorus, and boron. The carbon-based support includes one or more of MXene, graphene oxide, reduced graphene oxide, graphitic carbon nitride, and carbon nanotubes.

[0011] Based on the above technical solutions, preferably, the mass ratio of the metal source, the non-metal heteroatom source and the carbon-based support is 0.1~50:1:1~30.

[0012] Based on the above technical solutions, preferably, the iron source is selected from one or more of ferric chloride, ferrous chloride, ferrous sulfate, ferric sulfate, ferric nitrate, and ferric citrate; the manganese source is selected from one or more of manganese nitrate, manganese acetate, manganese chloride, and manganese sulfate; the cobalt source is selected from one or more of cobalt nitrate, cobalt acetate, cobalt chloride, and cobalt sulfate; the gold source is selected from one or more of chloroauric acid, sodium chloroaurate, gold nitrate, and gold acetate; the platinum source is selected from one or more of chloroplatinic acid, platinum acetylacetonate, and tetraammineplatinum nitrate; the palladium source is selected from one or more of palladium chloride, palladium nitrate, and palladium acetylacetonate; and the copper source is selected from one or more of copper nitrate, copper acetate, copper chloride, and copper sulfate.

[0013] Based on the above technical solutions, preferably, the nitrogen source is selected from one or more of urea, melamine, ammonia, polyethyleneimine, and amino acids; the sulfur source is selected from one or more of thiourea, thioacetamide, and sodium sulfide; the phosphorus source is selected from one or more of sodium hypophosphite, phosphoric acid, and organophosphonic acid; and the boron source is selected from one or more of boric acid, boron oxide, and sodium tetraborate.

[0014] Secondly, the present invention provides a method for preparing a metal single-atom nanoenzyme composite material, comprising the following steps:

[0015] S1, a composite material is obtained by mixing a metal source, a non-metal heteroatom source and a carbon-based support and grinding them, and then calcining the composite material under an inert atmosphere;

[0016] S2, the calcined composite material is added to hydrochloric acid and stirred to remove metal nanoparticles, and then washed by centrifugation multiple times with ultrapure water.

[0017] S3, freeze-dry the washed material to obtain a nanoenzyme composite material.

[0018] Based on the above technical solutions, preferably, in step S1, the calcination temperature is 400~800℃ and the time is 2~10h.

[0019] Based on the above technical solutions, preferably, in step S1, the inert atmosphere is nitrogen or argon.

[0020] Based on the above technical solutions, preferably, in step S2, the mass-to-volume ratio of the composite material to hydrochloric acid is 1g:50-500mL.

[0021] Thirdly, this invention provides an application of a metal single-atom nanoenzyme composite material in water pollution control.

[0022] Based on the above technical solutions, preferably, the pollutant is at least one of algae and chemical pollutants.

[0023] Based on the above technical solutions, preferably, the algae is at least one of Microcystis aeruginosa, Oscillatoria, Nostoc, Anabaena, Trichophyton, Chlorella, Diatoms and Cladophora; and the chemical pollutant is at least one of algal toxins MC-LR, methylene blue, tetracycline, ciprofloxacin, ibuprofen, bisphenol A and phthalates.

[0024] The metal single-atom nanoenzyme composite material of the present invention, its preparation method, and its application have the following advantages over the prior art:

[0025] (1) The nanoenzyme composite material MX@C synthesized by the non-metallic heteroatom X-mediated metal single atom M active center design of the present invention has both oxidase-like and peroxidase-like properties. It can simultaneously activate dissolved oxygen and low dose H2O2, significantly improve ROS yield and pollutant removal efficiency, and overcome the problem of traditional nanoenzymes having single function and poor adaptability.

[0026] (2) The present invention synthesizes nanoenzyme composite material MX@C by metal single atom M and non-metal heteroatom X with carbon-based carrier C. The synthesis method adopts the one-pot method of molten salt etching, which is simple and scalable. The substrate construction, introduction of non-metal heteroatom and anchoring of metal single atom are completed simultaneously. The process is short, has few steps and is highly efficient. Compared with the traditional route of multi-step synthesis and post-processing, this method has better batch stability, lower cost and is convenient for large-scale preparation and field application.

[0027] (3) The method adopted in this invention achieves higher pollutant removal efficiency with equivalent or lower oxidant dosage. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 FeN@Ti3C2T in Embodiment 1 of the present inventionx Aberration electron micrograph;

[0030] Figure 2 FeN@Ti3C2T in Embodiment 1 of the present invention x UV-Vis and intuitive diagrams in TMB and TMB-H2O2 systems;

[0031] Figure 3 FeN@Ti3C2T in Example 12 of this invention x A visual representation of the oxidase-like properties that inhibit Microcystis aeruginosa;

[0032] Figure 4 The FeN@Ti3C2T in Example 13 of this invention x A visual representation of the synergistic effect of oxidase-like and peroxidase-like properties in inhibiting the algae growth of Microcystis aeruginosa;

[0033] Figure 5 FeN@Ti3C2T in Example 14 of this invention x Visual representation of algae-killing flocculants. Detailed Implementation

[0034] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0035] The MAX phase ceramic Ti3C2T used in this invention x The graphitic carbon nitride (GCN) and reduced graphene oxide (rGO) used to prepare the MXene carrier were purchased from Aladdin Reagent (Shanghai) Co., Ltd.; the remaining carbon-based carriers, graphene oxide (GO) and carbon nanotubes (CNT), were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0036] Example 1

[0037] This embodiment prepares a metal single-atom nanoenzyme composite material FeN@Ti3C2T. x The preparation method includes the following steps:

[0038] (1) Take 25 g of ferric chloride, 2.5 g of urea and 60 g of Ti3AlC2 and place them in an agate mortar. Grind them thoroughly and mix them evenly.

[0039] (2) Transfer the uniformly ground powder to a quartz tube furnace and heat it to 500°C at a heating rate of 5°C / min. Then, keep it heated in a N2 atmosphere for 4 hours.

[0040] (3) Weigh 5 g of the calcined product and place it in 300 mL of 3M HCl solution. Stir and acid wash at room temperature for 6 h. Then centrifuge at 8000 rpm for 10 min and discard the supernatant. Resuspend the precipitate in deionized water (30 mL each time) and repeat centrifugation (8000 rpm, 10 min) three times. After the final wash, collect the solid and freeze-dry to obtain FeN@Ti3C2T. x .

[0041] The FeN@Ti3C2T synthesized in this embodiment x Perform spherical aberration electron microscopy characterization, such as Figure 1 As shown, individual dispersed Fe single atoms can be observed, indicating the successful preparation of Fe single atoms.

[0042] Testing FeN@Ti3C2T x The peroxidase-like activities and peroxidase-like activities were shown in the results. Figure 2 As shown.

[0043] FeN@Ti3C2T x Peroxidase-like catalytic ability: Utilizing the TMB-mediated H2O2 oxidation colorimetric reaction, the catalytic activity of FeN@Ti3C2T x The peroxidase-like catalytic performance was evaluated. In a 96-well plate, 50 μL of FeN@Ti3C2T was first introduced sequentially. x The reaction mixture consisted of a dispersion (0.5 mg / mL), an appropriate amount of acetate-sodium acetate buffer (pH = 3.0, 0.2 M), and 44 μL of freshly prepared 3 mM TMB solution. Different volumes of H2O2 solution were added to achieve final concentration gradients of 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, and 5.0 mM in the final reaction system (200 μL). After initiating the reaction at room temperature, the absorbance of the reaction system was continuously monitored over time at 652 nm using a microplate reader, and the reaction progress curve was recorded (sampling interval 6 s; total monitoring time 10 min). The apparent Michaelis constant was obtained using Lineweaver–Burk double reciprocal plotting. K m With maximum reaction rate V max .

[0044]

[0045] in, V 0 [S] represents the initial reaction rate, and [S] represents the substrate concentration. V max Indicates the maximum reaction rate.K m This represents the Michaelis constant.

[0046] FeN@Ti3C2T x Oxidase-like catalytic ability: Utilizing TMB-mediated oxidation-colorimetric reaction, the catalytic activity of FeN@Ti3C2T x The catalytic performance of FeN@Ti3C2T was evaluated. x 50 μL of dispersion (1 mg / mL) was added to a 96-well plate, along with a certain amount of 6 mM TMB solution and acetate-sodium acetate buffer (pH=3, 0.2 M) to construct a total reaction volume of 200 μL. The final TMB concentrations were 0, 0.1, 0.3, 0.6, 0.9, 1.3, and 1.8 mM, respectively. After the reaction started, the absorbance was continuously monitored at 652 nm (sampling interval 6 s, continuous monitoring for 10 min). The initial rate was calculated from the initial linear segment of the absorbance-time curve. V 0 The apparent Michaelis constant was obtained by using Lineweaver–Burk double reciprocal plotting. K m With maximum reaction rate V max .

[0047]

[0048] in, V 0 [S] represents the initial reaction rate, and [S] represents the substrate concentration. V max Indicates the maximum reaction rate. K m This represents the Michaelis constant.

[0049] Investigating FeN@Ti3C2T using the TMB colorimetric system x enzyme-like activity, Figure 2 As shown, curves a and b are gentle curves, curve c shows an absorption peak at 652 nm, while curve d exhibits a more pronounced absorption at 652 nm. The corresponding solution color changes are as follows: solutions a and b are colorless, solution c is blue, and solution d is a distinct blue. This indicates that FeN@Ti3C2T x It can catalyze the production of ROS from dissolved oxygen in water, and it can also catalyze the production of ROS from H2O2, causing TMB to oxidize and produce blue oxidized TMB (oxTMB) products.

[0050] Example 2

[0051] This embodiment prepares a novel metal single-atom nanoenzyme composite material FeNS@Ti3C2T. x The preparation method includes the following steps:

[0052] (1) Take 3 g of ferric chloride, 1.5 g of thiourea and 10 g of Ti3AlC2 and place them in an agate mortar. Grind them thoroughly and mix them evenly.

[0053] (2) Transfer the uniformly ground powder to a quartz tube furnace and heat it to 600°C at a heating rate of 5°C / min. Then, keep it heated in an Ar atmosphere for 4 hours.

[0054] (3) Weigh 5 g of the calcined product and place it in 250 mL of 4 M HCl solution. Perform a magnetic stirring and acid washing treatment at room temperature for 10 h. Then centrifuge at 8000 rpm for 10 min and discard the supernatant. Resuspend the precipitate in deionized water (30 mL each time) and repeat the centrifugation three times (8000 rpm, 10 min). After the final wash, collect the solid and freeze-dry it to obtain FeNS@Ti3C2T. x .

[0055] Testing FeNS@Ti3C2T x The catalytic activity of oxidase-like and peroxidase-like enzymes was determined using the same method as in Example 1, and the results are shown in Tables 1 and 2.

[0056] Table 1. Oxidase-like enzymes under different catalysts K m , V max Comparison of values

[0057]

[0058] Table 1 shows the results: In the oxidase-like reaction, FeNS@Ti3C2T x The nanozyme exhibited significantly superior catalytic properties compared to the control material and natural horseradish peroxidase (HRP). Specifically, its apparent Michaelis constant... K m Significantly reduced, indicating higher substrate affinity; while the maximum reaction rate V max The significant improvement reflects the simultaneous enhancement of intrinsic site activity and overall catalytic efficiency.

[0059] Table 2. Peroxidase-like enzymes under different catalysts K m , V max Comparison of values

[0060]

[0061] Table 2 shows the results for FeNS@Ti3C2T. x Calculations for H2O2 and TMB K m The values ​​were 0.73 mM and 0.41 mM, respectively. V max The value is 10.01 × 10 -8 Ms -1 and 22.9×10 -8 Ms -1 FeNS@Ti3C2T x It exhibits superior affinity for both the substrate H₂O₂ and TMB compared to similar nanozymes. Specifically, its apparent Michaelis constant is higher. K m Significantly reduced, indicating higher substrate affinity; while the maximum reaction rate V max The significant improvement reflects the simultaneous enhancement of intrinsic site activity and overall catalytic efficiency.

[0062] Example 3

[0063] This embodiment prepares a novel metal single-atom nanoenzyme composite material CuP@GO. The preparation method includes the following steps:

[0064] (1) Take 12 g of copper nitrate, 3 g of sodium phosphate and 50 g of GO and put them into an agate mortar. Grind them thoroughly and mix them evenly.

[0065] (2) Transfer the uniformly ground powder to a quartz tube furnace and heat it to 700°C at a heating rate of 5°C / min. Then, keep it at the temperature for 5 h under an Ar atmosphere.

[0066] (3) Weigh 5g of the calcined product and place it in 1000mL of 4M HCl solution. Stir and acid wash at room temperature for 5h. Then centrifuge at 8000rpm for 10min and discard the supernatant. Resuspend the precipitate in deionized water (30mL each time) and repeat centrifugation (8000rpm, 10min) three times. Collect the solid after the last wash and freeze-dry to obtain CuP@GO.

[0067] Example 4

[0068] This embodiment prepares a novel metal single-atom nanoenzyme composite material AuPtB@GCN. The preparation method includes the following steps:

[0069] (1) Take 5g of gold acetate, 5g of chloroplatinic acid, 2g of boric acid and 50g of GCN and place them in an agate mortar. Grind them thoroughly and mix them evenly.

[0070] (2) Transfer the uniformly ground powder to a quartz tube furnace and heat it to 800°C at a heating rate of 5°C / min. Then, keep it heated in an Ar atmosphere for 9 hours.

[0071] (3) Weigh 2g of the calcined product and place it in 100 mL of 4 M HCl solution. Stir and acid wash at room temperature for 5 h. Then centrifuge at 8000 rpm for 10 min and discard the supernatant. Resuspend the precipitate in deionized water (30 mL each time) and repeat centrifugation (8000 rpm, 10 min) three times. Collect the solid after the last wash and freeze-dry to obtain AuPtB@GCN.

[0072] Example 5

[0073] This embodiment prepares a novel bimetallic single-atom nanoenzyme composite material AuPtB@GCN. The preparation method includes the following steps:

[0074] (1) Take 10g of gold acetate, 5g of chloroplatinic acid, 2g of boric acid and 50g of GCN and place them in an agate mortar. Grind them thoroughly and mix them evenly.

[0075] (2) Transfer the uniformly ground powder to a quartz tube furnace and heat it to 800°C at a heating rate of 5°C / min. Then, keep it heated in an Ar atmosphere for 9 hours.

[0076] (3) Weigh 5g of the calcined product and place it in 1000mL of 4M HCl solution. Stir and acid wash at room temperature for 5h. Then centrifuge at 8000rpm for 10min and discard the supernatant. Resuspend the precipitate in deionized water (30mL each time) and repeat centrifugation (8000rpm, 10min) three times. Collect the solid after the last wash and freeze-dry to obtain AuPtB@GCN.

[0077] Example 6

[0078] This embodiment prepares a novel bimetallic single-atom nanoenzyme composite material MnCoS@rGO. The preparation method includes the following steps:

[0079] (1) Take 50g of manganese nitrate, 50g of cobalt sulfate, 2g of sodium sulfide and 60g of rGO and put them into an agate mortar. Grind them thoroughly and mix them evenly.

[0080] (2) Transfer the uniformly ground powder to a quartz tube furnace and heat it to 700°C at a heating rate of 5°C / min. Then, keep it heated in an Ar atmosphere for 8 hours.

[0081] (3) Weigh 5g of the calcined product and place it in 1000mL of 4M HCl solution. Stir and acid wash at room temperature for 5h. Then centrifuge at 8000rpm for 10min and discard the supernatant. Resuspend the precipitate in deionized water (30mL each time) and repeat centrifugation (8000rpm, 10min) three times. Collect the solid after the last wash and freeze-dry to obtain MnCoS@rGO.

[0082] Example 7

[0083] This embodiment prepares a novel metal single-atom nanoenzyme composite material PdP@CNT. The preparation method includes the following steps:

[0084] (1) Take 50g palladium nitrate, 2.5g sodium hypophosphite and 75g CNT and place them in an agate mortar. Grind them thoroughly and mix them evenly.

[0085] (2) Transfer the uniformly ground powder to a quartz tube furnace and heat it to 600°C at a heating rate of 5°C / min. Then, keep it heated in a N2 atmosphere for 10 h.

[0086] (3) Weigh 8g of the calcined product and place it in 2000 mL of 4 M HCl solution. Stir and acid wash at room temperature for 4 h. Then centrifuge at 8000 rpm for 10 min and discard the supernatant. Resuspend the precipitate in deionized water (30 mL each time) and repeat centrifugation (8000 rpm, 10 min) three times. Collect the solid after the last wash and freeze-dry to obtain PdP@CNT.

[0087] Example 8

[0088] Compared with Example 2, Example 8 has an increased content of metal source, specifically: 22.5 g ferric chloride, 1.5 g thiourea, and 10 g Ti3AlC2, with a ratio of 15:1:6.67. The rest of the contents are the same as in Example 2.

[0089] Example 9

[0090] Compared with Example 2, Example 9 has an increased content of metal source, specifically: 45 g ferric chloride, 1.5 g thiourea, and 10 g Ti3AlC2, with a ratio of 30:1:6.67. The rest of the contents are the same as in Example 2.

[0091] Example 10

[0092] Compared with Example 2, Example 10 has an increased content of metal source, specifically: 75 g ferric chloride, 1.5 g thiourea, and 10 g Ti3AlC2, with a ratio of 50:1:6.67. The rest of the contents are the same as in Example 2.

[0093] Example 11

[0094] This embodiment verifies the FeN@Ti3C2T prepared in Example 1. x It has good thermal stability and resistance to acids and alkalis.

[0095] FeN@Ti3C2T x Its thermal stability and acid and alkali resistance were evaluated by incubating it for 1 h at different temperatures (25-80 ℃) and different pH (3.0-11.0).

[0096] Its oxidase-like and peroxidase-like activities were evaluated at seven temperature points between 25 and 80 °C, with the highest activity set at 100%. The results are shown in Table 3.

[0097] Table 3 FeN@Ti3C2T at different temperatures and pH values x Thermal stability and acid and alkali resistance

[0098]

[0099] Table 3 shows that FeN@Ti3C2T x The material maintains stable and high activity of two types of enzymes in the ambient temperature range of 25–50 °C. When the temperature reaches 60–70 °C, the two activities jump simultaneously and approach or reach their peak values ​​respectively. Even at a high temperature of 80 °C, the oxidase-like enzyme is still at its peak and the peroxidase-like enzyme only drops slightly. Overall, the material can maintain a high level of activity in the low, medium and high temperature ranges.

[0100] FeN@Ti3C2T x The oxidase-like activity reached its maximum relative activity at pH 4.0, while the peroxidase-like activity was highest at pH 3.0. Under moderately alkaline conditions, the material still exhibited stable and significant catalytic activity: the oxidase-like activity ranged from 75.20% to 88.28%, and the peroxidase-like activity ranged from 79.21% to 90.88%. This indicates that the nanocatalytic system is functional across a wide pH range, possessing both high activity in the acidic range and maintaining over 75% relative activity under alkaline conditions, demonstrating good environmental adaptability and engineering application potential. Example

[0101] This embodiment verifies the FeN@Ti3C2T prepared in Example 1. x The algicidal effect of microcystis aeruginosa, taking Microcystis aeruginosa, Oscillatoria, and Nostoc as examples, is demonstrated through the following steps:

[0102] Microcystis aeruginosa, Oscillatoria, and Nostoc are cultured to the logarithmic growth phase, and FeN@Ti3C2T is then applied. x(50, 100, 150 mg / L) were added to the Microcystis aeruginosa solution (algal OD). 680 The value was 0.1. Algal solution without any nanozyme was used as the control group. Samples were taken on days 0, 1, and 2 to test the chlorophyll a content and calculate the inhibition rate. The results are shown in Table 4.

[0103] Table 4 Algal suppression efficiency at different culture times

[0104]

[0105] Based on the data in Table 4, it can be seen that FeN@Ti3C2T x The inhibitory effects on *Microcystis aeruginosa*, *Oscillatoria*, and *Nostoc* showed significant time- and dose-dependent characteristics: at the same dosage, the inhibition rate significantly increased from day 1 to day 2 at a constant dose. At a constant time point, the inhibition rate significantly increased with increasing dose, while the control group showed negative growth (−0.33% to −2.49%), indicating that the inhibitory effect was genuine rather than a natural fluctuation. High concentrations exhibited rapid onset of action (over 65% was achieved on day 1 at 150 mg / L). A visual representation of the corresponding *Microcystis aeruginosa* is shown below. Figure 3 The results indicate that this material has a significant and quantifiable time / dose-dependent inhibitory effect on Microcystis aeruginosa, Oscillatoria, and Nostoc.

[0106] Example 13

[0107] This embodiment verifies the synergistic algicidal effect of the nanoenzyme composite materials prepared in Examples 1-10, exhibiting both oxidase-like and peroxidase-like properties. First, it uses *Microcystis aeruginosa*, *Anabaena*, and *Gnaphalium* as examples, specifically the FeN@Ti3C2T nanoenzyme composite material prepared in Example 1. x For the test object, the specific steps are as follows:

[0108] Using *Microcystis aeruginosa*, *Anabaena*, and *Gnaphalium* in their logarithmic growth phase as models, the initial algal solution was prepared with OD... 680 Under the condition of characterization and standardization to 0.1, blank control (CK), H2O2-only control (uniform concentration 6 mg / L), and synergistic treatment with different nanozyme doses (1, 10, 20 mg / L) and H2O2 were set up. Samples were taken on days 0, 1, and 2 after treatment, and the algae inhibition rate was calculated based on chlorophyll a content. The results are shown in Table 5.

[0109] Table 5 Algal suppression efficiency at different culture times

[0110]

[0111] Table 5 shows that the algae inhibition rate of H2O2 alone was negative on day 2, indicating that low doses of hydrogen peroxide promoted algae growth. However, under the condition of 1 mg / L nanozyme + 6 mg / L H2O2, the algae inhibition rate reached over 50% on day 2, and increased to approximately 60% and 70% respectively at higher nanozyme concentrations (10 and 20 mg / L). This indicates a significant synergistic effect between nanozyme and H2O2, and the inhibitory effect is time-dependent. A visual representation of Microcystis aeruginosa is shown below. Figure 4 .

[0112] The algae inhibition rate of 20 mg / L nanozyme + H2O2 was tested using algae cultured for 2 days. The nanozyme composite materials prepared in Examples 1-10 were used as test materials. Microcystis aeruginosa, Anabaena aeruginosa, Fibrocystis pilosa, green algae and diatoms were used as examples. The results are shown in Table 6.

[0113] Table 6. Inhibitory effect of nanoenzyme composite materials on algae

[0114]

[0115] Table 6 shows that the nanoenzyme composite materials prepared in this invention have good inhibitory effects on *Microcystis aeruginosa*, *Anabaena*, *Hylocereus*, green algae, and diatoms. Among them, the AuPtB@GCN prepared in Example 4 showed the best inhibitory effect on *Microcystis aeruginosa*, *Anabaena*, and *Hylocereus*, reaching over 80%. The increase in metal source and algicidal efficiency are not entirely positively correlated. This is because the anchoring sites on the carrier surface are limited during the synthesis of metal single-atom nanoenzymes. Excessive metal source not only fails to form single atoms but also aggregates into clusters or particles, thus reducing the catalytic activity for generating reactive oxygen species.

[0116] Example 14

[0117] This embodiment verifies the FeN@Ti3C2T prepared in Example 1. x To demonstrate the algae-inhibiting and flocculation effects, taking Microcystis aeruginosa, a common algae in cyanobacterial blooms, as an example, the specific steps are as follows:

[0118] Using FACHB-905 in the logarithmic growth phase as a model, the initial algal solution was prepared with OD... 680 Characterized and standardized to 0.1, a blank control (CK) and a nanozyme group (FeN@Ti3C2T) were set up. x ).like Figure 5 As shown, the control group showed that the algal solution before treatment was a uniform light green suspension with no visible flocs or sedimentation layer; the solid-liquid separation state observed after the treatment group was allowed to stand clearly showed a nearly colorless and clear supernatant and a dense yellowish-brown floc sedimentation zone formed at one end, with a distinct interface between the two, indicating that the nanomaterials prepared in this invention can effectively induce algal cell flocculation and achieve gravity sedimentation.

[0119] Example 15

[0120] This embodiment verifies the degradation effect of the nanoenzyme composite materials prepared in Examples 1-10 on chemical pollutants (algal toxin MC-LR, methylene blue, tetracycline, ciprofloxacin, ibuprofen, bisphenol A, and phthalates). The test results are shown in Table 7, and the test steps are as follows:

[0121] The degradation efficiency of the algal toxin MC-LR was determined by HPLC. The 400 μL reaction system consisted of 50 mM buffer (pH 7.0) containing MC-LR (0.25 mg / mL) and 0.02 mg of nanozyme (prepared in Examples 1-10). After reacting the reaction mixture at 25 °C for 5 min, 4 μL of 20% phosphate aqueous solution was added to terminate the reaction. After centrifugation at 12000 rpm for 60 min at 4 °C, the residual MC-LR in the supernatant was detected by HPLC at 238 nm, and the degradation rate of MC-LR was obtained by peak area.

[0122] The methylene blue was determined by UV-Vis method. 0.02 mg of nanozyme (prepared in Examples 1-10) and methylene blue were added to a 200 μL system to make the final concentration of both 1 mg / L. After reacting at room temperature for 5 min, the absorbance at 664 nm was measured using a UV-Vis spectrophotometer, and the degradation rate of methylene blue was calculated.

[0123] The degradation efficiency of tetracycline was determined by UV-Vis method. 0.02 mg of nanozyme (prepared in Examples 1-10) and tetracycline were added to a 200 μL system to make the final concentration of both 10 mg / L. After reacting at room temperature for 30 min, the absorbance at 357 nm was measured by UV-Vis spectrophotometer, and the degradation rate of tetracycline was calculated.

[0124] The degradation efficiency of ciprofloxacin was determined by UV-Vis method. 0.02 mg of nanozyme (prepared in Examples 1-10) and ciprofloxacin were added to a 200 μL system to make the final concentration of both 20 mg / L. After reacting at room temperature for 1 h, the absorbance at 277 nm was measured by UV-Vis spectrophotometer, and the degradation rate of ciprofloxacin was calculated.

[0125] The ibuprofen degradation efficiency was determined by HPLC. The 400 μL reaction system consisted of 50 mM buffer (pH 3.0) containing ibuprofen (0.5 mg / mL) and 0.02 mg of nanozyme (prepared in Examples 1-10). The reaction mixture was reacted at 25 °C for 5 minutes. After centrifugation at 12000 rpm for 60 min at 4 °C, the residual ibuprofen in the supernatant was detected by HPLC at 220 nm, and the degradation rate of ibuprofen was obtained from the peak area.

[0126] The degradation efficiency of bisphenol A was determined by HPLC. The 400 μL reaction system consisted of 50 mM buffer (pH 3.0) containing bisphenol A (0.5 mg / mL) and 0.02 mg of nanozyme (prepared in Examples 1-10). After reacting the reaction mixture at 25 °C for 5 min, the mixture was centrifuged at 12000 rpm for 60 min at 4 °C. The residual bisphenol A in the supernatant was detected by HPLC at 276 nm, and the degradation rate of bisphenol A was obtained from the peak area.

[0127] The degradation efficiency of phthalic acid esters was determined by HPLC. A 400 μL reaction system consisted of 50 mM buffer (pH 3.0) containing phthalic acid esters (20 mg / mL) and 0.02 mg of nanozyme (prepared in Examples 1-10). The reaction mixture was reacted at 25 °C for 5 min. After centrifugation at 12000 rpm for 60 min at 4 °C, the residual phthalic acid esters in the supernatant were detected by HPLC at 227 nm. The degradation efficiency of phthalic acid esters was obtained from the peak area.

[0128] Table 7 summarizes the degradation performance of the nanozyme composite materials prepared in different embodiments for seven typical environmental chemical pollutants. Experimental results show that the nanozyme composite material exhibits excellent broad-spectrum catalytic degradation capabilities. Specifically, the nanozyme showed the highest removal efficiency for antibiotic pollutants (such as tetracycline and ciprofloxacin). In Examples 3 and 7, the degradation rates of tetracycline and ciprofloxacin reached the highest, reaching 92.1% and 92.5%, respectively. This is attributed to the effective destruction of the molecular structure of antibiotics by the large number of reactive oxygen species (ROS) generated by the material in an acidic microenvironment. Furthermore, the material also maintained good catalytic activity for more structurally stable and recalcitrant algal toxins and endocrine disruptors (such as bisphenol A and dimethyl phthalate). In conclusion, although the performance of different embodiments fluctuated slightly due to differences in preparation conditions, all examples demonstrate the great application potential of this nanozyme composite material in treating complex aquatic pollutants.

[0129] Table 7. Degradation effect of nanozyme composite materials on chemical pollutants

[0130]

[0131] Comparative Example 1

[0132] The difference between this comparative example and Example 1 is that the amount of metal source used exceeds the specified range, specifically: 150g ferric chloride, 2.5g urea, 60g Ti3AlC2, in a ratio of 60:1:24. The rest of the contents are the same as in Example 2.

[0133] Comparative Example 2

[0134] The difference between this comparative example and Example 1 is that the amount of non-metallic heteroatom source used exceeds the specified range, specifically: 25g ferric chloride, 2.5g urea, 125g Ti3AlC2, in a ratio of 10:1:50. The rest of the contents are the same as in Example 2.

[0135] FeN@Ti3C2T prepared in Comparative Examples 1-2 x The peroxidase-like activity of FeN@Ti3C2T was detected using the same steps as in Example 1. The results showed that the FeN@Ti3C2T prepared in Comparative Examples 1-2 exceeded the limits defined by this invention. x It cannot catalyze the oxidation and discoloration of TMB and has no catalytic ability similar to an oxidase.

[0136] For the prepared FeN@Ti3C2T x The peroxidase-like activity of FeN@Ti3C2T was detected using the same steps as in Example 1. The results showed that the FeN@Ti3C2T prepared in Comparative Examples 1-2 exceeded the limits defined by this invention. x It cannot catalyze the oxidation and discoloration of TMB in the presence of H2O2, and has no peroxidase-like catalytic ability.

[0137] Comparative Example 3

[0138] Compared with the visible light-activated carbon nanoparticle nanozyme VCN reported in the literature (which only exhibits single enzyme catalytic properties similar to oxidases [Wang et al., Chem. Eng. J., 2023, 472:145029]), the FeNS@Ti3C2T constructed in Example 2 of this invention... x Under the same testing conditions, it exhibits superior enzyme kinetics and application performance (see Table 8). Its Michaelis constant is 62.1% lower than that of VCN, indicating a significant enhancement in substrate affinity; the maximum reaction rate is 11.6 times that of VCN, confirming a substantial improvement in catalytic efficiency.

[0139] Table 8. Different types of oxidases K m , V max Comparison of values

[0140]

[0141] At the application level, VCN needs to be at 1.5 g / L to generate sufficient reactive oxygen species to effectively destroy algal cells, while the nanozyme in this study can achieve a comparable effect at 50 mg / L (about 1 / 30 of VCN). When synergistically used with a low dose of H2O2 (6 mg / L), the effective working concentration is further reduced to 1 mg / L, demonstrating significant potential in low-dose, resource-friendly application scenarios.

[0142] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a metal single-atom nanoenzyme composite material in water pollution control, characterized in that, The raw materials for this composite material include: a metal source, a non-metallic heteroatom source, and a carbon-based support; The metal source includes one or more of iron, manganese, cobalt, gold, platinum, palladium, and copper; the non-metallic heteroatom source includes one or more of nitrogen, sulfur, phosphorus, and boron; the carbon-based support includes one or more of MXene, graphene oxide, reduced graphene oxide, graphitic carbon nitride, and carbon nanotubes; the MXene includes MAX phase ceramic Ti3C2T. x ; The preparation method of the metal single-atom nanoenzyme composite material includes the following steps: S1, a composite material is obtained by mixing a metal source, a non-metal heteroatom source and a carbon-based support and grinding them, and then calcining the composite material under an inert atmosphere; S2, the calcined composite material is added to hydrochloric acid and stirred to remove metal nanoparticles, and then washed by centrifugation multiple times with ultrapure water. S3, freeze-dry the washed material to obtain nanoenzyme composite material; The pollutant is at least one of algae and chemical pollutants, wherein the chemical pollutant is at least one of algal toxin MC-LR, methylene blue, tetracycline, ciprofloxacin, ibuprofen, bisphenol A, and phthalates.

2. The application as described in claim 1, characterized in that, The mass ratio of the metal source, the non-metal heteroatom source, and the carbon-based support is 0.1~50:1:1~30.

3. The application as described in claim 1, characterized in that, The iron source is selected from one or more of ferric chloride, ferrous chloride, ferrous sulfate, ferric sulfate, ferric nitrate, and ferric citrate; the manganese source is selected from one or more of manganese nitrate, manganese acetate, manganese chloride, and manganese sulfate; the cobalt source is selected from one or more of cobalt nitrate, cobalt acetate, cobalt chloride, and cobalt sulfate; the gold source is selected from one or more of chloroauric acid, sodium chloroaurate, gold nitrate, and gold acetate; the platinum source is selected from one or more of chloroplatinic acid, platinum acetylacetonate, and tetraammineplatinum nitrate; the palladium source is selected from one or more of palladium chloride, palladium nitrate, and palladium acetylacetonate; and the copper source is selected from one or more of copper nitrate, copper acetate, copper chloride, and copper sulfate. The nitrogen source is selected from one or more of urea, melamine, ammonia, polyethyleneimine, and amino acids; the sulfur source is selected from one or more of thiourea, thioacetamide, and sodium sulfide; the phosphorus source is selected from one or more of sodium hypophosphite, phosphoric acid, and organophosphonic acid; and the boron source is selected from one or more of boric acid, boron oxide, and sodium tetraborate.

4. The application as described in claim 1, characterized in that, In step S1, the calcination temperature is 400~800℃ and the time is 2~10h.

5. The application as described in claim 1, characterized in that, In step S1, the inert atmosphere is nitrogen or argon.

6. The application as described in claim 1, characterized in that, In step S2, the mass-to-volume ratio of the composite material to hydrochloric acid is 1g:50-500mL.

7. The application as described in claim 1, characterized in that, The algae mentioned are at least one of Microcystis aeruginosa, Oscillatoria, Nostoc, Anabaena, Trichophyton, Chlorella, Diatoms and Cladophora.

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