Iron-manganese bimetal monatomic nano-enzyme as well as preparation method and application thereof

Abstract

The invention discloses an iron-manganese double-metal monatomic nano-enzyme as well as a preparation method and an application of the iron-manganese double-metal monatomic nano-enzyme. The preparation method of the iron-manganese bimetallic monatomic nano-enzyme comprises the following steps: (1) dissolving a NaCl template, metal ions, a functional monomer and a sulfur-nitrogen doping agent in an acetic acid solution together to obtain an iron-manganese metal ion-chitosan ion imprinting pre-coordination compound; then, a cross-linking agent is added into the iron and manganese metal ion-chitosan ion imprinting pre-coordination compound, and after freeze drying, a carbonized precursor with NaCl as a template is obtained; (2) putting the carbonized precursor taking NaCl as a template into a tubular furnace for heat treatment to obtain black powder; and dispersing the black powder in an acid solution to remove impurities, finally separating the powder, and washing and drying the powder to obtain the iron-manganese bimetallic monatomic nano-enzyme. The nano enzyme shows excellent multi-enzyme activity, can simulate cytochrome P450 enzyme, and shows excellent O2 activation capability in O2-driven oxidative dehydrogenation reaction.

Description

Technical Field

[0001] This invention relates to the field of biomimetic catalysis and catalytic material preparation technology, specifically to an iron-manganese bimetallic single-atom nanozyme, its preparation method, and its application. Background Technology

[0002] 2,6-Dimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid diethyl ester (1,4-DHP), a potent calcium channel blocker, is widely used in the treatment of cardiovascular diseases. Cytochrome P450 enzymes (CYPs) are a class of heme proteins widely distributed in the body. Their active centers are composed of iron porphyrin complexes, which can efficiently activate molecular oxygen (O2) and catalyze various oxidation reactions, such as 1,4-DHP metabolism. However, the direct application of bioenzymes as catalysts is often limited by high cost, instability, and poor recyclability, which greatly restricts their widespread application in biomimetic catalysis (such as 1,4-DHP metabolic performance). Therefore, developing nanozymes that can mimic CYP functions is crucial for promoting the research and application of biomimetic catalysis and drug metabolism. However, achieving this goal remains a challenge because most nanozymes do not have molecular structures similar to natural enzymes. Fe-NC materials not only possess enzyme-like properties but also exhibit Fe-N x The active site possesses a similar structure to CYP heme factors, which offers the possibility of mimicking advanced functions of CYPs. However, high-temperature pyrolysis easily leads to the aggregation of metal particles in the precursor into strongly magnetic clusters, reducing catalytic performance; further improvements are needed. Summary of the Invention

[0003] In order to overcome at least one technical problem existing in the prior art, the present invention provides an iron-manganese bimetallic single-atom nanozyme, its preparation method and application.

[0004] The technical solution of the present invention is as follows: This invention first provides a method for preparing iron-manganese bimetallic single-atom nanozymes, which includes the following steps: (1) Dissolve the NaCl template, metal ions, functional monomers and sulfur and nitrogen dopants together in acetic acid solution to obtain iron-manganese metal ion-chitosan ion-imprinted pre-coordinated complex; then, add crosslinking agent to iron-manganese metal ion-chitosan ion-imprinted pre-coordinated complex, and freeze-dry to obtain a carbonized precursor with NaCl as template. (2) The carbonized precursor with NaCl as template is placed in a tube furnace for heat treatment to obtain black powder; the black powder is dispersed in an acid solution to remove impurities, and finally the powder is separated, washed and dried to obtain the iron-manganese bimetallic single-atom nanozyme.

[0005] This invention provides a novel iron-manganese bimetallic single-atom nanozyme, which exhibits high catalytic activity for diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid (1,4-DHP), and can completely metabolize 1,4-DHP into DDPD within 40 min. Its catalytic performance is significantly better than that of iron or manganese single-atom nanozymes.

[0006] Furthermore, the aforementioned iron-manganese bimetallic single-atom nanozyme also possesses oxidase-like and catalase-like activities. Its catalase-like activity can efficiently decompose H₂O₂ into O₂, introducing an oxygen source for the reaction and thus significantly accelerating the 1,4-DHP metabolism rate, shortening the metabolism time to 10 minutes. Therefore, based on the aforementioned iron-manganese bimetallic single-atom nanozyme, a novel colorimetric sensing method for dynamically monitoring the 1,4-DHP metabolic process can be developed, enabling real-time monitoring of the 1,4-DHP metabolic process without relying on large instruments.

[0007] Preferably, in step (1), the ratio of NaCl template, metal ions, functional monomers, sulfur and nitrogen dopants and acetic acid solution is: 5-10g: 0.02-0.2mmol: 0.3-0.6g: 0.3-0.6g: 30-60mL.

[0008] Most preferably, the ratio of NaCl template, metal ions, functional monomers, sulfur and nitrogen dopant and acetic acid solution in step (1) is 8g:0.1mmol:0.5g:0.5g:50mL.

[0009] Preferably, the metal ion mentioned in step (1) is Fe. 3+ and Mn 2+ .

[0010] Preferably, the functional monomer mentioned in step (1) is chitosan.

[0011] Preferably, the sulfur-nitrogen dopant mentioned in step (1) is thiourea.

[0012] Preferably, the concentration of the acetic acid solution in step (1) is 1-3% (w / v).

[0013] Most preferably, the concentration of the acetic acid solution in step (1) is 2% (w / v).

[0014] Preferably, the volume ratio of the crosslinking agent to the acetic acid solution in step (1) is 0.5-2:1.

[0015] Most preferably, the volume ratio of the crosslinking agent to the acetic acid solution in step (1) is 1:1.

[0016] Preferably, the crosslinking agent is an aqueous solution of glutaraldehyde with a volume fraction of 40-60%.

[0017] Most preferably, the crosslinking agent is a 50% (v / v) aqueous solution of glutaraldehyde.

[0018] Preferably, the heat treatment mentioned in step (2) specifically refers to heat treatment under N2 conditions at 3-6℃·min. -1 The temperature is increased from 20-35℃ to 700-900℃ and maintained at 700-900℃ for 1-3 hours.

[0019] Most preferably, the heat treatment mentioned in step (2) specifically refers to heat treatment under N2 conditions at 5°C·min. -1 The temperature was increased from 30°C to 800°C and maintained at 800°C for 2 hours.

[0020] In the field of biomimetic catalysis, those skilled in the art know that for the biomimetic catalysis of 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid diethyl ester (1,4-DHP) to diethyl-2,6-dimethyl-3,5-pyridinedicarboxylate (DDPD) using nanozymes, the inventor needs to prepare nanozyme materials with high catalytic activity according to the specific requirements of the biomimetic catalytic reaction. The selectivity and catalytic activity of the prepared nanozyme biomimetic sensing system for the desired catalytic reaction are mainly determined by the preparation method of the nanozyme material. The preparation method of nanozyme materials mainly includes the selection of methods, raw materials, proportions, composition and morphology of the obtained raw materials, and reaction conditions such as pyrolysis temperature. For nanozyme materials used in biomimetic catalytic systems, differences in the preparation method, raw material selection, proportions, composition and morphology of the obtained raw materials, and reaction conditions such as pyrolysis temperature will lead to significant differences in the catalytic performance of the subsequently prepared nanozymes, resulting in significant differences in catalytic selectivity and catalytic activity. Through extensive experiments, the inventors of this invention continuously adjusted the preparation method, raw material composition, ratio, and pyrolysis process parameters during the preparation process, and obtained iron-manganese bimetallic single-atom nanozymes under the above conditions. Compared with nanozymes obtained under other conditions, the biomimetic catalytic system constructed with iron-manganese bimetallic single-atom nanozymes can significantly enhance its catalytic activity.

[0021] The present invention also provides an iron-manganese bimetallic single-atom nanozyme prepared by the above preparation method.

[0022] The present invention also provides an application of the above-mentioned iron-manganese bimetallic single-atom nanozyme in the biomimetic catalysis of the metabolism of diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid (1,4-DHP) to diethyl-2,6-dimethyl-3,5-pyridinedicarboxylate (DDPD).

[0023] The present invention also provides an application of the above-mentioned iron-manganese bimetallic single-atom nanozyme in the dynamic monitoring of the metabolism of 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid diethyl ester (1,4-DHP).

[0024] Beneficial effects: (1) In order to overcome the limitations of high cost, instability and poor recycling of biological enzymes, this invention first provides a novel ion imprinting technology combined with space engineering strategy to synthesize an iron-manganese bimetallic single-atom nanozyme. This nanozyme exhibits excellent multi-enzyme activity and can simulate the function of cytochrome P450 enzyme (CYP). It shows excellent O2 activation ability in O2-driven oxidative dehydrogenation reaction; (2) The iron-manganese bimetallic single-atom nanozyme of this invention has high catalytic activity for the in vitro metabolic reaction of 1,4-DHP. It can completely metabolize 1,4-DHP into DDPD within 40 min, and its catalytic activity is significantly better than that of iron or manganese single-atom nanozymes; (3) The iron-manganese bimetallic single-atom nanozyme of this invention also has catalase-like activity. Its catalase-like activity can efficiently decompose H2O2 into O2, introduce oxygen source for the reaction, and thus significantly accelerate the metabolism of 1,4-DHP. The metabolism time can be shortened to 10 min. min; (4) The iron-manganese bimetallic single-atom nanozyme described in this invention also has an oxidase-like activity. Based on the oxidase-like activity, a new colorimetric method for dynamically monitoring the 1,4-DHP metabolic process has been developed, which can realize real-time monitoring of the 1,4-DHP metabolic process without relying on large instruments. Attached Figure Description

[0025] Figure 1 (A) Fe / Mn (2.0) -CNS (0.5) (A) Transmission electron microscopy (TEM) image at -800°C; (B) High-resolution transmission electron microscopy (HRTEM) image; and (C) Fe / Mn (2.0) -CNS (0.5) Selected area electron diffraction (SAED) pattern at -800°; (D) Fe / Mn (2.0) -CNS (0.5) -800 spherical aberration electron microscopy (AC-STEM) image; (E) Fe / Mn (2.0) -CNS (0.5) -800 AC-TEM energy dispersive spectroscopy (EDS); (FH)Fe / Mn (2.0) -CNS (0.5) Electron energy loss spectrum (EELS) at -800°C; (I) Fe / Mn (2.0) -CNS (0.5) -800 high-angle annular dark field image (HAADF-STEM) and its corresponding (JP)EDS elemental mapping.

[0026] Figure 2 (A, C, E, G) UV-Vis absorption spectra of different nanozymes in 3,3',5,5'-tetramethylbenzidine (TMB) reaction solution (reaction carried out at 40 °C for 15 min, nanozyme concentration was 25 μg·mL). -1 (D) In ​​the presence of 25 μg·mL -1 Fe / Mn (2.0) -CNS (0.5) UV-Vis absorption spectra of TMB reaction solutions at different concentrations at -800°C; (B, D, F, H) comparison of Michaelis constants of different nanozymes; (I) Fe / Mn (2.0) -CNS (0.5) UV-Vis absorption spectra of the -800 system in the presence and absence of isopropanol (IPA), histidine (HIS), p-benzoquinone (BQ) and disodium ethylenediaminetetraacetate (EDTA-2Na); (J) Comparison of UV-Vis absorbance; (K) Changes in fluorescence intensity after terephthalic acid (TA) captures ·OH.

[0027] Figure 3 (A, B, C, D) Reaction time curves of H2O2 decomposition catalyzed by different nanozymes (the inset corresponds to the photographs of O2 bubbles generated by H2O2 degradation catalyzed by all catalysts); (EF) Ultraviolet absorption spectra of H2O2 at different concentrations and the fitted standard curves.

[0028] Figure 4 1H NMR spectra (CDCl3) of the oxidation products (DDPD) of 1,4-DHP in the presence of all catalysts and the corresponding conversion bar charts.

[0029] Figure 5 (A) UV absorption spectra of 1,4-DHP at different concentrations and their fitted standard curves (inset); (B) Fe / Mn were added after all nanozymes reacted with 1,4-DHP for 40 min. (2.0) -CNS (0.5) (C) Comparison of the conversion rates of 1,4-DHP metabolism catalyzed by all nanozymes measured by UV method; (D) Fe / Mn ratio after adding different concentrations of H2O2. (2.0) -CNS (0.5) -800 The time required for complete conversion of 1,4-DHP; (E) All nanozymes with 1,4-DHP after addition of 50 mmol·L -1 After H2O2 reacts for 10 min, Fe / Mn is added. (2.0) -CNS (0.5) The UV absorption spectrum of the -800-TMB system; (F) Measurement by UV method of the addition of 50 mmol·L-1 Comparison of the conversion rates of 1,4-DHP metabolism catalyzed by all nanozymes after H2O2 treatment; (G)Fe / Mn (2.0) -CNS (0.5) -800 types of oxidases' visual response color maps to different concentrations of 1,4-DHP, and the linear relationship between grayscale values ​​obtained in RGB mode and 1,4-DHP concentration; (H) Comparison of the conversion rates of 1,4-DHP metabolism catalyzed by all nanozymes measured colorimetrically; (I) Measurement of the conversion rates of 1,4-DHP metabolism after adding 50 mmol·L⁻¹ of 1,4-DHP by colorimetric method. -1 Comparison of the conversion rates of 1,4-DHP metabolism catalyzed by all nanozymes after H2O2 treatment.

[0030] Figure 6 A comparison of radar graphs for detecting 1,4-DHP conversion using three methods. Detailed Implementation

[0031] The present invention will be further explained below with reference to specific embodiments, but the embodiments do not limit the scope of protection of the present invention.

[0032] Example 1: Preparation of iron-manganese bimetallic single-atom nanozymes The first step involves adding 8 g of NaCl template and 0.1 mmol of metal ions (Fe2+) to the sample. 3+ and Mn 2+ The concentration formed was 2.0 mmol·L⁻¹ -1 0.5 g of functional monomer (chitosan) and 0.5 g of sulfur-nitrogen dopant (thiourea) were dissolved together in 50 mL of 2% (w / v) acetic acid solution to obtain iron-manganese metal ion-chitosan ion-imprinted pre-coordination complex. Then, 0.5 mL of the crosslinking agent glutaraldehyde (50% aqueous solution) was added to the solution; after 24 hours of freeze drying, a carbonized precursor with NaCl as a template was obtained. The second step involves, under N2 conditions, at 5 °C·min -1 The precursor was heated from 30 °C to 800 °C in a tube furnace at a certain rate and held at 800 °C for 2 hours to obtain a black powder; After cooling, the black powder was dispersed in 3 mol·L⁻¹ -1 The powder was incubated overnight in HCl solution to remove impurities; the powder was collected by centrifugation, washed with water, and dried to obtain Fe / Mn. (2.0) -CNS (0.5) -800, which refers to the iron-manganese bimetallic single-atom nanozyme.

[0033] Fe / Mn were studied using transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM). (2.0) -CNS (0.5)The microstructure at -800 was characterized in detail. TEM results show ( Figure 1 A), the nanozymes prepared in this invention possess a unique honeycomb interconnected framework. HRTEM image ( Figure 1 B) Confirmed the absence of any metal nanoparticles or clusters in the sample and observed a typical carbon lattice structure (lattice spacing of 0.34–0.38 nm). Atomic-level characterization of the prepared nanozymes was performed using AC-STEM; high-density bright spots were observed on the carbon matrix, confirming the presence of isolated individual Fe and Mn atoms. Figure 1 D). In addition, the SAED map ( Figure 1 C) shows a typical carbon ring diffraction pattern, but no metal clusters or metal nanoparticle electron diffraction flowers are observed. EDS results ( Figure 1 E) indicates that the percentage of Mn atoms is approximately 0.08% and the percentage of Fe atoms is approximately 0.17%, indicating that both Fe and Mn atoms are present in the nanozyme prepared in this invention, and the Fe content is higher than that of Mn. Furthermore, EELS spectroscopy at atomic resolution (E) shows that... Figure 1 F) further confirms the coexistence of Fe and Mn atoms, with Fe atoms being more abundant than Mn. N and SK edge signals can be detected near the metal atoms. Figure 1 G and H indicate that Fe and Mn atoms are stabilized through coordination with heteroatoms such as N / S. Atomic-resolution AC-STEM images and their corresponding EDS maps reveal the uniform distribution of Fe, Mn, N, S, and C elements in the sample. Figure 1 These results confirm that the Fe and Mn bimetals prepared in this invention are dispersed in a carbon matrix in the form of single atoms.

[0034] Example 2: Preparation of iron-manganese bimetallic single-atom nanozymes The difference between Example 2 and Example 1 is that in the first step, 0.05 mmol of metal ions (Fe) are used. 3+ and Mn 2+ The concentration formed was 1.0 mmol·L⁻¹ -1 Fe / Mn was prepared (1.0) -CNS (0.5) -800; Everything else is the same as in Example 1.

[0035] Example 3: Preparation of iron-manganese bimetallic single-atom nanozymes The difference between Example 3 and Example 1 is that 0.2 mmol of metal ions (Fe) were used in the first step. 3+ and Mn 2+ The concentration formed was 4.0 mmol·L⁻¹ -1 Fe / Mn was prepared(4.0) -CNS (0.5) -800; Everything else is the same as in Example 1.

[0036] Example 4: Preparation of iron-manganese bimetallic single-atom nanozymes The difference between Example 4 and Example 1 is that 0.4 mmol of metal ions (Fe) were used in the first step. 3+ and Mn 2+ The concentration formed was 8.0 mmol·L⁻¹ -1 Fe / Mn was prepared (8.0) -CNS (0.5) -800; Everything else is the same as in Example 1.

[0037] Example 5: Preparation of iron-manganese bimetallic single-atom nanozymes The difference between Example 5 and Example 1 is that the heat treatment conditions in the second step are different, namely, under N2 conditions, at 5 °C·min -1 The precursor was heated from 30 °C to 700 °C in a tube furnace at a certain rate, and held at 700 °C for 2 hours to obtain a black powder; the Fe / Mn was finally prepared. (2.0) -CNS (0.5) -700.

[0038] Everything else is the same as in Example 1.

[0039] Example 6 Preparation of iron-manganese bimetallic single-atom nanozymes The difference between Example 6 and Example 1 is that the heat treatment conditions in the second step are different, namely, under N2 conditions, at 5 °C·min -1 The precursor was heated from 30 °C to 900 °C in a tube furnace at a certain rate, and held at 900 °C for 2 hours to obtain a black powder; the Fe / Mn was finally prepared. (2.0) -CNS (0.5) -900.

[0040] Everything else is the same as in Example 1.

[0041] Comparative Example 1: Preparation of Iron-Manganese Bimetallic Single-Atom Nanozymes The difference between Comparative Example 1 and Example 1 is that no metal ions (Fe) are added in the first step. 3+ and Mn 2+ CNS was prepared. (0.5) -800; Everything else is the same as in Example 1.

[0042] Comparative Example 2: Preparation of Iron-Manganese Bimetallic Single-Atom Nanozymes The difference between Comparative Example 2 and Example 1 is that 0.1 mmol of metal ions (Fe) were used in the first step. 3+ The concentration formed was 2.0 mmol·L⁻¹ -1 Fe was prepared (2.0) -CNS (0.5) -800; Everything else is the same as in Example 1.

[0043] Comparative Example 3: Preparation of Iron-Manganese Bimetallic Single-Atom Nanozymes The difference between Comparative Example 3 and Example 1 is that 0.1 mmol of metal ions (Mn) was used in the first step. 2+ The concentration formed was 2.0 mmol·L⁻¹ -1 Mn was prepared (2.0) -CNS (0.5) -800; Everything else is the same as in Example 1.

[0044] Example 7 Oxidase-like activity of iron-manganese bimetallic single-atom nanozymes Oxidases can use O2 from the air to oxidize colorless TMB to blue oxidized TMB (oxTMB), simultaneously producing a strong UV absorption peak at 652 nm (the underlying solution is an acetate buffer solution at pH 3.0). Figure 2 As shown in Figure A, by observing the color changes of TMB solutions under different nanozyme conditions and measuring the UV absorbance of oxTMB at 652 nm, the Fe / Mn ratio was demonstrated. (2.0) -CNS (0.5) -800, Fe (2.0) -CNS (0.5) -800, Mn (2.0) -CNS (0.5) -800 and CNS (0.5) Comparison of oxidase-like activities at -800 nm. The nanozyme containing both Fe and Mn bimetallic single atoms exhibited the strongest UV absorption signal at 652 nm (absorbance 3.5), superior to that of Fe single atoms alone (absorbance 2.6) or Mn single atoms alone (absorbance 1.7), and the nanozyme without any metal single atoms (absorbance 0.7). This indicates that the Fe / Mn nanozyme described in this invention... (2.0) -CNS (0.5) -800 exhibits the highest oxidase-like activity, attributed to a synergistic effect between Fe and Mn atoms. Furthermore, increasing thiourea doping from 0 g to 0.5 g significantly improved the Fe / Mn ratio. (2.0) -CNS (0.5) -800 material oxidase activity ( Figure 2(C) This enhanced activity may be due to the fact that S / N doping further optimizes the three-dimensional structure of the carbon support, providing more metal single-atom immobilization sites, thereby improving the catalytic activity of the nanozyme. The oxidase-like activity of the nanozyme decreases when the thiourea concentration is 1.0 g. Therefore, while maintaining a thiourea doping amount of 0.5 g, the activity of the nanozyme was explored with different metal contents of Fe and Mn (1, 2, 4, and 8 mmol·L⁻¹). -1 The effect of TMB oxidation. For example... Figure 2 As shown in E, the nanozymes synthesized under different metal concentrations all exhibited certain outstanding oxidase activities, among which the Fe / Mn nanozymes described in this invention... (2.0) -CNS (0.5) The highest activity was observed at -800 °C. Furthermore, the pyrolysis temperature during nanozyme preparation also significantly affected the catalyst activity. Control samples were synthesized at 700 °C and 900 °C. Figure 2 G shows that the activity order of these catalysts is: Fe / Mn (2.0) -CNS (0.5) -800 > Fe / Mn (2.0) -CNS (0.5) -900 > Fe / Mn (2.0) -CNS (0.5) -700; this result indicates that the optimal temperature for synthesizing nanozyme materials is 800 ℃. Subsequently, all nanozymes (25 μg·mL⁻¹) were studied. -1 Steady-state kinetic parameters of catalytic TMB oxidation were determined. The Michaelis constant (Km) for each sample was determined using a double reciprocal Lineweaver-Burk plot. m ) value; K value of nanozymes m The smaller the value, the stronger the catalytic activity. It can be seen that the order of oxidase activity using TMB as a substrate is: Fe / Mn (2.0) -CNS (0.5) -800 (0.32 mmol·L -1 ) > Fe (2.0) -CNS (0.5) -800 (0.62 mmol·L -1 CNS (0.5) -800 (1.18 mmol·L -1 Mn (2.0) -CNS (0.5) -800 (2.31 mmol·L -1 )(See Figure 2 B), Fe / Mn (2.0) -CNS (0.5) -800 (0.32 mmol·L -1 Fe / Mn (2.0)-CN-800 (0.54 mmol·L -1 Fe / Mn (2.0) -CNS (1.0) -800 (0.64 mmol·L -1 Fe / Mn (2.0) -CNS (0.2) -800 (0.95 mmol·L -1 )(See Figure 2 D), Fe / Mn (2.0) -CNS (0.5) -800 (0.32 mmol·L -1 Fe / Mn (4.0) -CNS (0.5) -800 (0.48 mmol·L -1 Fe / Mn (1.0) -CNS (0.5) -800 (0.76 mmol·L -1 Fe / Mn (8.0) -CNS (0.5) -800 (0.85 mmol·L -1 )(See Figure 2 F), Fe / Mn (2.0) -CNS (0.5) -800 (0.32 mmol·L -1 Fe / Mn (2.0) -CNS (0.5) -900 (0.54 mmol·L) -1 Fe / Mn (2.0) -CNS (0.5) -700 (0.56 mmol·L -1 )(See Figure 2 H). Fe / Mn (2.0) -CNS (0.5) -800 K m The value is the lowest, and also lower than the K of natural horseradish peroxidase. m Value (0.43 mmol·L) -1 This indicates that the Fe / Mn of the present invention (2.0) -CNS (0.5) -800 °C exhibits optimal affinity for the catalytic substrate. These comparative results confirm that the present invention achieves optimal performance at 800 °C, with a thiourea content of 0.5 g and Fe and Mn metal concentrations of 2 mmol·L⁻¹. -1 The iron-manganese bimetallic single-atom nanozyme synthesized in this invention exhibits superior catalytic ability for O2 reactions. This is to clarify the Fe / Mn reaction in the presence of O2. (2.0)-CNS (0.5) The types of reactive oxygen species (ROS) generated by -800-TMB were analyzed, and specific quenchers were selected to detect ROS. Specifically, HIS was used to quench the ROS. 1 O2, BQ is used to quench O2. · - IPA is used to quench ·OH, and EDTA-2Na is used to quench h. + .like Figure 2 As shown in I and J, the introduction of BQ and EDTA-2Na reduced the production of oxTMB, indicating that O2 was generated in the system. ·- and h + The most significant decrease in absorbance at 652 nm was observed after the introduction of BQ, indicating that O2... ·- It participates in Fe / Mn (2.0) -CNS (0.5) The main ROS types oxidized by -800-TMB. In addition, Figure 2 K represents the change in fluorescence intensity after capturing ·OH with terephthalic acid (TA), which also indicates the Fe / Mn ratio. (2.0) -CNS (0.5) The oxidation process of -800-TMB does not involve ·OH.

[0045] Example 8: Catalase-like activity of iron-manganese bimetallic single-atom nanozymes Catalase catalyzes the decomposition of H₂O₂ to produce O₂, and increased O₂ content in the substrate can further enhance the biomimetic catalytic activity of the nanozyme. The decomposition of H₂O₂ was monitored by changes in absorbance at 240 nm using ultraviolet spectroscopy, and the catalase-like activity of the iron-manganese bimetallic single-atom nanozyme was determined (the substrate was a Na₂CO₃-NaHCO₃ buffer solution at pH 11.5). Figure 3 As shown in A, with Fe (2.0) -CNS (0.5) -800, Mn (2.0) -CNS (0.5) -800 and CNS (0.5) Compared to the -800 nano-enzyme, the Fe / Mn of this invention (2.0) -CNS (0.5) The decomposition rate of H2O2 is fastest at -800°C (producing the most and fastest O2, see...). Figure 3 Illustration A shows superior catalase-like activity, indicating a synergistic effect between Fe and Mn metal atoms, which significantly reduces the activation energy of H₂O₂ decomposition, thereby improving catalytic efficiency. Furthermore, doping with thiourea from 0.0 g to 0.5 g significantly enhanced the catalase-like activity of the nanozyme. Figure 3(B and its illustration). This is likely because S / N doping introduces more defect sites, optimizes the three-dimensional structure of the carbon support, and increases the dispersion and stability of Fe and Mn single atoms, thereby improving the Fe / Mn ratio. (2.0) -CNS (0.5) -800 catalytic activity. However, adding excessive thiourea (1.0 g) may lead to over-adsorption on the catalyst surface, covering the active sites of Fe and Mn single atoms. Therefore, the thiourea doping amount was fixed at 0.5 g. Different metal content levels (1, 2, 4, and 8 mmol·L⁻¹) were used to determine the catalytic activity. -1 The synthesized nanozymes all exhibited catalase-like activity in relation to the decomposition of H2O2. Among them, the Fe / Mn nanozymes described in this invention... (2.0) -CNS (0.5) -800 type catalase activity is higher than that of nanozymes prepared with other three metal contents ( Figure 3 (C and its illustration). The pyrolysis temperature during catalyst preparation also has a significant impact on catalyst activity. Control samples were synthesized at 700 °C and 900 °C, and their catalytic activity in H₂O₂ decomposition was investigated. Figure 3 D and its illustration show that the activity order of these catalysts is: Fe / Mn (2.0) -CNS (0.5) -800 > Fe / Mn (2.0) -CNS (0.5) -900> Fe / Mn (2.0) -CNS (0.5) The samples pyrolyzed at -700 and 800 °C exhibited the best catalytic activity, indicating that 800 °C is the optimal pyrolysis temperature. Further investigation into the catalase-like kinetics of the nanozyme revealed that the Fe / Mn nanozyme described in this invention... (2.0) -CNS (0.5) -800 exhibits the best catalytic performance, K m Only 4.44 mmol·L -1 The lowest value was achieved compared to other control nanozymes (wherein, Fe... (2.0) -CNS (0.5) -800 catalase K m It is 6.78 mmol·L -1 Mn (2.0) -CNS (0.5) -800 is 8.13 mmol·L -1 CNS (0.5) -800 is 5.17 mmol·L -1 Fe / Mn (2.0) -CN-800 is 5.77 mmol·L. -1 Fe / Mn (2.0)-CNS (0.2) -800 is 434.6 mmol·L -1 Fe / Mn (2.0) -CNS (1.0) -800 is 91.6 mmol·L -1 Fe / Mn (1.0) -CNS (0.5) -800 is 22.0 mmol·L -1 Fe / Mn (4.0) -CNS (0.5) -800 is 25.9 mmol·L -1 Fe / Mn (8.0) -CNS (0.5) -800 is 48.4 mmol·L -1 Fe / Mn (2.0) -CNS (0.5) -700 is 8.58 mmol·L -1 Fe / Mn (2.0) -CNS (0.5) -900 is 203.6 mmol·L -1 ); indicating that the Fe / Mn of the present invention (2.0) -CNS (0.5) -800 has an extremely high affinity for H2O2, and can catalyze the decomposition of H2O2 to produce O2 with the highest efficiency; it can also increase the O2 content in the bottom liquid most effectively.

[0046] Example 9: Biomimetic catalytic activity of iron-manganese bimetallic single-atom nanozymes To investigate the biomimetic catalytic activity of the iron-manganese bimetallic single-atom nanozyme described in this invention, its catalytic ability in the oxidative dehydrogenation reaction of 1,4-DHP metabolism in the presence of O2 was studied. In the absence of any nanozyme, 1,4-DHP remained almost unchanged; however, with the addition of the Fe / Mn nanozyme described in this invention… (2.0) -CNS (0.5) After reacting at -800°C for 40 min, 1,4-DHP was completely metabolized and converted to DDPD; the conversion rate of 1,4-DHP catalyzed by all nanozymes was determined using NMR internal standard method. Figure 4 As shown in A, B, C, and D, within the same reaction time (40 min), only the Fe / Mn described in this invention... (2.0) -CNS (0.5) -800 can completely metabolize 1,4-DHP into DDPD (100%), while the catalytic efficiency of other control nanozymes is significantly lower than that of Fe / Mn. (2.0) -CNS (0.5) -800 (where Fe (2.0) -CNS(0.5) -800 metabolized 1,4-DHP to DDPD with an efficiency of 42%, Mn (2.0) -CNS (0.5) -800 is 72%, CNS (0.5) -800 is 43%, Fe / Mn (2.0) -CNS (1.0) -800 is 59%, Fe / Mn (2.0) -CNS (0.2) -800 is 42%, Fe / Mn (2.0) -CN-800 is 44%, Fe / Mn (8.0) -CNS (0.5) -800 is 50%, Fe / Mn (4.0) -CNS (0.5) -800 is 67%, Fe / Mn (1.0) -CNS (0.5) -800 is 28%, Fe / Mn (2.0) -CNS (0.5) -700 is 25%, Fe / Mn (2.0) -CNS (0.5) The percentage of -900 is 34%, indicating that iron-manganese bimetallic single-atom nanozymes have higher catalytic metabolic capacity and CYP-mimicking enzyme activity than Fe or Mn single-metal single-atom nanozymes synthesized under other conditions. Figure 4 This indicates that the iron-manganese bimetallic single-atom nanozyme prepared in this invention is the most suitable catalyst for this biomimetic catalytic 1,4-DHP metabolic reaction.

[0047] Example 10: Application of Oxidase-like and Catalase-like Activities in Biomimetic Catalytic Reactions (1) Construct a colorimetric sensing real-time monitoring system based on the oxidase-like activity of the iron-manganese bimetallic single-atom nanozyme obtained in Example 1. 1,4-DHP possesses antioxidant properties, capable of reducing blue oxTMB to colorless TMB, thus lowering the intensity of its characteristic absorption peak at 652 nm. DDPD, however, does not exhibit this reducing ability. Based on this phenomenon, a Fe / Mn... (2.0) -CNS (0.5) The linear relationship between the absorbance of oxTMB and the concentration of 1,4-DHP in the -800–TMB system was established, and a standard curve was plotted using the UV method. Figure 5 A and illustrations; linear range: 6–200 mmol·L⁻¹ -1 The linear relationship is A = -0.0128 C. 1,4DHP + 2.7599, R 2 = 0.9915; A represents the ultraviolet absorbance at 652nm, C 1,4DHP(representing 1,4-DHP concentration), a Fe / Mn-based method was developed. (2.0) -CNS (0.5) A novel UV method for detecting the activity of 800 types of oxidases was used to quantitatively determine the conversion rate of 1,4-DHP. The results are as follows: Figure 5 As shown in C. In addition, Fe / Mn was also established. (2.0) -CNS (0.5) The linear relationship between the color grayscale value I and the 1,4-DHP concentration in the -800–TMB system was established, and the corresponding colorimetric standard curve was plotted. Figure 5 G and illustrations; linear range: 8–200 mmol·L⁻¹ -1 The linear relationship is ∆I = 0.2254 C 1,4DHP + 0.4284, R 2 = 0.9673; I represents the grayscale value of the color, calculated using the formula I = 0.3R + 0.59G + 0.11B. RGB values ​​are color values; ∆I is Fe / Mn (2.0) -CNS (0.5) (The gray value of the -800–TMB system is obtained by subtracting the gray value of the system when different concentrations of 1,4-DHP are added). A simpler colorimetric method was developed to quantitatively detect the conversion rate of 1,4-DHP. The detection results are listed below. Figure 5 H. Both of the above detection methods utilize the Fe / Mn ratio. (2.0) -CNS (0.5) The activity of oxidase-like enzymes at -800 nm was measured using ultraviolet light and color changes to determine the remaining content of 1,4-DHP in the reaction system after the reaction, thereby utilizing... Figure 5 A or Figure 5 The conversion rate is calculated using the standard curve in G, avoiding the need for large-scale equipment such as NMR or HPLC. Comparison via radar charts ( Figure 6 The results of UV and colorimetric methods for determining the conversion rate of 1,4-DHP oxidation metabolism catalyzed by different nanozymes under the same reaction conditions ( Figure 5 C represents the result determined by ultraviolet (UV) method. Figure 5 H is the result of colorimetric determination) and NMR internal standard method ( Figure 4 The results are basically consistent, which verifies the reliability of the colorimetric real-time monitoring method proposed in this invention.

[0048] (2) The catalase-like activity of the iron-manganese bimetallic single-atom nanozyme obtained in Example 1 can accelerate the biomimetic catalytic rate. 1,4-DHP can be metabolized into DDPD more quickly in an oxygen-rich environment, while the Fe / Mn of this invention... (2.0) -CNS (0.5)-800 exhibits excellent catalase activity, enabling it to efficiently catalyze the decomposition of H2O2 into O2. Therefore, utilizing Fe / Mn (2.0) -CNS (0.5) This enzymatic property of -800 allows for the effective promotion of the conversion of 1,4-DHP to DDPD by introducing H2O2 as an oxygen source into the reaction system. The effect of the amount of H2O2 added on the reaction process was investigated (e.g., Figure 5 (As shown in D). The results showed that with the increase of H2O2 addition (from 8 to 200 mmol·L), -1 The time required for complete metabolism of 1,4-DHP oxidation gradually decreased (from 40 min to 5 min). Specifically, when the H2O2 concentration was 50 mmol·L⁻¹, the time required for complete metabolism decreased. -1 At that time, Fe / Mn (2.0) -CNS (0.5) The -800 catalyst can completely convert 1,4-DHP to DDPD in just 10 minutes; the H2O2 concentration is 100 mmol·L⁻¹. -1 The metabolic transformation can be completed in just 5 minutes. Considering that a high H2O2 concentration would not be conducive to saving reagents, 50 mmol·L⁻¹ was chosen. -1 The concentration of H2O2 is sufficient. Further measurements were performed on all control sample nanozymes under the same conditions (i.e., 50 mmol·L⁻¹). -1 The conversion rate of 1,4-DHP to DDPD catalyzed by H2O2 (reaction time 10 min) was measured by ultraviolet (UV) method. Figure 5 E, F) and colorimetry ( Figure 5 I) Quantitative analyses were performed separately. The results showed that, in addition to the Fe / Mn ratio described in this invention... (2.0) -CNS (0.5) Apart from -800, the conversion rates of other comparative nanozymes decreased compared to those without H2O2 after 40 min of reaction. This phenomenon may be due to two factors: first, the shortened reaction time makes the conversion process more compact, requiring higher biomimetic catalytic activity from the nanozymes; second, compared to the Fe / Mn nanozyme described in this invention... (2.0) -CNS (0.5) Compared to -800, other comparative nanozymes exhibited weaker catalase activity, making it difficult to decompose H2O2 and generate sufficient O2 in a short time. This confirms the Fe / Mn nanozyme described in this invention. (2.0) -CNS (0.5) -800 Excellent catalase activity and overall superior efficiency in catalyzing 1,4-DHP.

Claims

1. A method for preparing an iron-manganese bimetallic single-atom nanozyme, characterized in that, It includes the following steps: (1) Dissolve the NaCl template, metal ions, functional monomers and sulfur and nitrogen dopants together in acetic acid solution to obtain iron-manganese metal ion-chitosan ion-imprinted pre-coordinated complex; then, add crosslinking agent to iron-manganese metal ion-chitosan ion-imprinted pre-coordinated complex, and freeze-dry to obtain a carbonized precursor with NaCl as template. (2) The carbonized precursor with NaCl as template is placed in a tube furnace for heat treatment to obtain black powder; the black powder is dispersed in an acid solution to remove impurities, and finally the powder is separated, washed and dried to obtain the iron-manganese bimetallic single-atom nanozyme.

2. The method for preparing iron-manganese bimetallic single-atom nanozymes according to claim 1, characterized in that, In step (1), the ratio of NaCl template, metal ions, functional monomers, sulfur and nitrogen dopants and acetic acid solution is: 5-10g: 0.02-0.2mmol: 0.3-0.6g: 0.3-0.6g: 30-60mL.

3. The method for preparing iron-manganese bimetallic single-atom nanozymes according to claim 1, characterized in that, The metal ion mentioned in step (1) is Fe 3+ and Mn 2+ .

4. The method for preparing iron-manganese bimetallic single-atom nanozymes according to claim 1, characterized in that, The functional monomer mentioned in step (1) is chitosan.

5. The method for preparing iron-manganese bimetallic single-atom nanozymes according to claim 1, characterized in that, The sulfur-nitrogen dopant mentioned in step (1) is thiourea.

6. The method for preparing iron-manganese bimetallic single-atom nanozymes according to claim 1, characterized in that, The volume ratio of the crosslinking agent to the acetic acid solution in step (1) is 0.5-2:

1.

7. The method for preparing iron-manganese bimetallic single-atom nanozymes according to claim 1, characterized in that, The heat treatment mentioned in step (2) specifically refers to heat treatment under N2 conditions at 3-6℃·min. -1 The temperature is increased from 20-35℃ to 700-900℃ and maintained at 700-900℃ for 1-3 hours.

8. The iron-manganese bimetallic single-atom nanozyme prepared by the preparation method according to any one of claims 1-7.

9. The application of the iron-manganese bimetallic single-atom nanozyme of claim 8 in the biomimetic catalysis of the metabolism of diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid (1,4-DHP) to diethyl-2,6-dimethyl-3,5-pyridinedicarboxylate (DDPD).

10. The application of the iron-manganese bimetallic single-atom nanozyme of claim 8 in the dynamic monitoring of the metabolism of 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid diethyl ester (1,4-DHP).

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