A selenium mono-atom / atom cluster nanosenzyme with enhanced double enzyme activity regulated by pyridine nitrogen and a preparation method and application thereof

Abstract

The application discloses pyridine nitrogen regulation and enhancement of double-enzyme activity selenium single atom / atomic cluster nanometer enzyme and a preparation method and application thereof. The preparation method comprises the following steps: (1) dissolving chitosan in an acetic acid solution, then adding selenium dioxide and g-C3N4, and fully dispersing under the condition of heating and stirring; (2) adding a crosslinking agent into the system obtained in the step (1) to crosslink and form a gel; (3) freeze-drying the obtained gel to obtain a precursor; (4) performing two-step pyrolysis on the precursor under the protection of nitrogen; and (5) obtaining the pyridine nitrogen regulation and enhancement of double-enzyme activity selenium single atom / atomic cluster nanometer enzyme through acid washing, washing and drying of the pyrolysis product. The pyridine nitrogen regulation and enhancement of double-enzyme activity selenium single atom / atomic cluster nanometer enzyme prepared by the application integrates the advantages of structural stability, colorimetric detection and antibacterial activity, and has good comprehensive application value.

Description

Technical Field

[0001] This invention relates to the field of nanoenzyme material preparation technology, specifically to a selenium single-atom / cluster nanoenzyme with pyridine nitrogen regulation to enhance the activity of dual enzymes, its preparation method, and its application. Background Technology

[0002] Carbendazim (CBZ) is a benzimidazole fungicide widely used in agricultural production due to its broad-spectrum fungicidal activity, low cost, and good chemical stability. However, carbendazim degrades slowly in the environment, easily accumulating in soil, water, and agricultural products, and subsequently entering the human body through the food chain, posing a potential threat to the ecological environment and human health. Therefore, establishing a rapid, simple, and reliable detection method for carbendazim is of significant practical value.

[0003] Nanozymes possess significant potential for application in colorimetric detection due to their combination of natural enzyme-like catalytic activity, good stability, and ease of large-scale preparation. Among them, single-atom catalysts, with their atomic-level dispersion and well-defined active centers, can significantly improve catalytic efficiency; while the synergistic structure of single atoms and atomic clusters can further balance high active site density and electron transport capability. Selenium (Se), as an element with unique redox properties and good biocompatibility, has potential applications in constructing efficient catalytic and antibacterial systems; however, Se is prone to volatilization or aggregation during heat treatment, limiting its performance. Furthermore, for Se-based nanozymes, in addition to the Se species itself, the local coordination environment of the support also has a significant impact on its dispersion state, electronic structure, and catalytic behavior, especially the pyridine nitrogen sites in nitrogen-doped carbon materials. Existing studies have shown that pyridine nitrogen can serve as a preferential anchoring site for active centers, enhancing the interaction between the active component and the carbon support, improving dispersion and stability, and promoting the adsorption and activation of small molecules, thereby contributing to improved catalytic activity.

[0004] Existing methods for detecting carbendazim mainly include high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and surface-enhanced Raman spectroscopy (SMR). While these methods offer high sensitivity, they typically rely on expensive instruments, specialized operation, and complex sample pretreatment, making them unsuitable for rapid on-site screening and immediate detection. In contrast, colorimetric sensing methods offer advantages such as simple operation, low cost, intuitive results, and suitability for visual detection, thus attracting widespread attention. Current carbendazim colorimetric detection systems often employ precious metal nanoparticles such as gold and silver, or introduce recognition units such as aptamers and surface functional molecules to achieve signal output. While these methods offer high sensitivity, their effective detection range is narrow, and they often rely on precious metal materials or biorecognition molecules. The system construction process is relatively complex, and most studies focus only on the "detection" itself, with less consideration given to the catalytic stability of the materials, the controllable construction of active sites, and extended functions such as antibacterial activity. However, if the dispersion and electronic structure of the Se active center can be controlled through the pyridine nitrogen site, it is hoped that a wide-range carbendazim colorimetric detection system can be constructed without the need for complex external recognition elements, relying solely on the oxidase-like activity of non-metallic nanozymes to achieve colorimetric amplification.

[0005] On the other hand, nanozymes also show significant application potential in the field of antibacterial research. Existing studies indicate that one of the main mechanisms of antibacterial nanozymes is the catalytic generation of reactive oxygen species such as superoxide anion radicals, hydroxyl radicals, and singlet oxygen through peroxidase-like or oxidase-like activity. These reactive oxygen species then disrupt bacterial cell walls, cell membranes, and intracellular proteins, nucleic acids, and lipids, thereby inhibiting or killing bacteria. However, overall, existing antibacterial nanozymes are mostly based on metals such as Fe, Cu, and Ag, or their composite systems. Some systems suffer from complex compositions, strong dependence on external energy or multi-component synergy, and insufficient stability of the active site. Furthermore, research on using the same nanozyme material for both pesticide colorimetric detection and antibacterial applications remains relatively limited. In contrast, if a Se single-atom / cluster nonmetallic nanozyme regulated by pyridine nitrogen can be constructed, it can not only effectively reduce the amount of metal used, but also have dual-function application performance. On the one hand, it can use its oxidase-like activity to catalyze the oxidation and color development of 3,3',5,5'-tetramethylbenzidine (hereinafter referred to as: TMB) under the condition of no added H2O2, for the colorimetric detection of carbendazim. On the other hand, it can use its peroxidase-like activity to efficiently generate active ·OH in the presence of H2O2 to achieve the inhibition of bacteria, thus having the dual function of "detection-antibacterial".

[0006] Therefore, how to achieve stable anchoring and highly dispersed construction of Se active centers through pyridine nitrogen site regulation, thereby obtaining Se-based non-metallic nanozymes with both oxidase-like and peroxidase-like activities, and applying them to carbendazim colorimetric detection and antibacterial applications, remains a pressing technical problem to be solved in this field. Compared with existing technologies, if the constructed material can possess a pyridine-rich nitrogen coordination environment, Se single-atom / cluster synergistic active centers, and a stable carbon framework support, it is expected to exhibit comprehensive advantages in terms of detection sensitivity, convenient color development, antibacterial efficiency, and material stability. Summary of the Invention

[0007] To overcome at least one technical problem existing in the prior art, the present invention provides a selenium single-atom / cluster nanozyme with pyridine nitrogen regulation to enhance the activity of dual enzymes, its preparation method and application.

[0008] The technical solution of the present invention is as follows:

[0009] This invention first provides a method for preparing selenium single-atom / cluster nanozymes with pyridine nitrogen regulation to enhance the activity of dual enzymes, which includes the following steps:

[0010] (1) Dissolve chitosan in acetic acid solution, then add selenium dioxide and the g-C3N4, and disperse them fully under heating and stirring conditions;

[0011] (2) Add a crosslinking agent to the system obtained in step (1) to cause crosslinking and form a gel;

[0012] (3) The obtained gel was freeze-dried to obtain the precursor;

[0013] (4) The precursor is subjected to two-step pyrolysis under nitrogen protection;

[0014] (5) The pyrolysis product is acid-washed, washed and dried to obtain the selenium single atom / cluster nanozyme with pyridine nitrogen regulation to enhance the activity of the two enzymes.

[0015] Preferably, the g-C3N4 described in step (1) is prepared by the following method:

[0016] A cyanuric acid solution was added dropwise to a melamine solution, and the mixture was stirred to form a white supramolecular complex precipitate. The white supramolecular complex precipitate was then pyrolyzed to obtain the g-C3N4.

[0017] Preferably, the g-C3N4 is prepared by the following method: melamine and cyanuric acid are dispersed separately in dimethyl sulfoxide, and after ultrasonic treatment, a cyanuric acid solution is added dropwise to a melamine solution at 25 °C, and stirred to generate a white supramolecular complex precipitate; the white supramolecular complex precipitate is precipitated, dried, and then subjected to nitrogen atmosphere at 5 °C·min. -1 The temperature was raised to 550 ℃ and held for 4 h. After cooling, the mixture was ground to obtain g-C3N4.

[0018] Preferably, in step (1), the mass ratio of chitosan, selenium dioxide and g-C3N4 is 400-500:10-12:0.1-0.3.

[0019] Most preferably, the mass ratio of chitosan, selenium dioxide and g-C3N4 in step (1) is 450:11.1:0.2.

[0020] Preferably, in step (1), the ratio of chitosan to acetic acid solution is 400-500 mg: 40-60 mL.

[0021] Most preferably, the ratio of chitosan to acetic acid solution in step (1) is 450 mg: 50 mL.

[0022] Preferably, the acetic acid solution is a 1-3% (v / v) acetic acid solution.

[0023] Most preferably, the acetic acid solution is a 2% (v / v) acetic acid solution.

[0024] Preferably, the crosslinking agent in step (2) is a glutaraldehyde solution.

[0025] Preferably, the volume ratio of glutaraldehyde solution to acetic acid solution is 1:40-60.

[0026] Most preferably, the volume ratio of glutaraldehyde solution to acetic acid solution is 1:50.

[0027] Preferably, the glutaraldehyde solution is a 50% (v / v) glutaraldehyde solution.

[0028] Preferably, the specific method for the two-step pyrolysis in step (4) is as follows: under nitrogen protection, at 4-6 °C·min -1 The temperature is increased to 500-600 ℃ at a rate of [temperature value] and held for 1.5-2.5 h, then increased at a rate of 4-6 ℃·min. -1 The temperature is increased to 800-900 ℃ at a rate and held for 1-2 h.

[0029] Most preferably, the specific method for the two-step pyrolysis described in step (4) is as follows: under nitrogen protection, at 5 °C·min -1The temperature was increased to 550 °C and held for 2 h at a rate of 5 °C·min. -1 The temperature was increased to 900 °C at a rate of [missing information] and held for 1.5 h.

[0030] Preferably, the acid washing in step (5) uses 2.0-4.0 mol·L⁻¹ -1 Hydrochloric acid solution, pickling time is 6-10 h.

[0031] Most preferably, the acid washing in step (5) uses 3.0 mol·L⁻¹ -1 Hydrochloric acid solution, pickling time is 8 h.

[0032] The inventors of this invention have discovered that in the nanozymes prepared by the above method, g-C3N4 not only participates in the construction of nitrogen-doped carbon skeleton as a nitrogen source during pyrolysis, but also regulates the formation of pyridine nitrogen sites in the support, thereby promoting the formation of Se-C bonds and improving the anchoring stability of selenium species in the carbon skeleton. This effectively inhibits the volatilization and aggregation of selenium under high temperature conditions, and achieves stable dispersion of selenium active centers in a single-atom / atom cluster synergistic form.

[0033] This invention provides a selenium single-atom / cluster nanozyme with enhanced dual-enzyme activity regulated by pyridine nitrogen prepared by the above-described method.

[0034] The present invention also provides an application of the above-mentioned pyridine nitrogen-regulated and enhanced dual-enzyme selenium single-atom / cluster nanozyme in the colorimetric detection of carbendazim.

[0035] Preferably, the specific method for colorimetric detection of carbendazim is as follows:

[0036] The selenium single-atom / cluster nanozyme with pyridine nitrogen regulation to enhance the activity of dual enzymes was added to an acetate-sodium acetate buffer solution at pH 2.5-3.5 and pre-reacted at 30-40 °C for 10-20 min to allow TMB to oxidize and develop color. Subsequently, the test sample containing carbendazim was added, and the reaction was continued at room temperature for 10-20 min. The absorbance change at 600-700 nm was measured, and the qualitative or quantitative detection of carbendazim was achieved based on the absorbance change.

[0037] Most preferably, the specific method for colorimetric detection of carbendazim is as follows: the selenium single-atom / cluster nanozyme with pyridine nitrogen regulation to enhance the activity of the two enzymes and TMB are added to an acetate-sodium acetate buffer solution at pH 3.0 and pre-reacted at 35 °C for 15 min to allow TMB to oxidize and develop color; then the test sample containing carbendazim is added, and the reaction is continued at room temperature for 15 min, and the absorbance change value at 652 nm is measured. The qualitative or quantitative detection of carbendazim is achieved based on the absorbance change value.

[0038] The present invention also provides an application of the above-mentioned pyridine nitrogen-regulated and enhanced dual-enzyme selenium single-atom / cluster nanozyme in the preparation of antibacterial agents.

[0039] Preferably, the antibacterial agent is an antibacterial agent against Escherichia coli.

[0040] Beneficial effects:

[0041] (1) This invention introduces g-C3N4 rich in pyridine nitrogen sites as a structure regulating component and nitrogen source, which, together with the chitosan-derived carbon framework, constructs a nitrogen-rich carbon support. This effectively regulates the local electronic structure of the support and promotes the formation of Se-C bonds, thereby achieving stable anchoring of the selenium active center in the carbon framework. Through the "ion imprinting-pyrolysis" strategy, this invention effectively suppresses the volatilization and aggregation of Se during high-temperature heat treatment, enabling Se to be highly dispersed in the nitrogen-doped carbon framework in the form of single atoms and atomic clusters coexisting, thus solving the problem of the difficulty in the stable construction of Se-based active centers.

[0042] (2) The pyridine nitrogen-regulated selenium single-atom / cluster nanozyme prepared in this invention has a clear and stable structural feature. No obvious crystalline Se or SeO2 characteristic diffraction peaks were observed in the XRD results, indicating that Se did not undergo large-scale aggregation. AC-HAADF-STEM results showed that isolated and dispersed Se single atoms and local Se clusters coexisted in the material. XPS results further indicated that the introduction of g-C3N4 increased the pyridine nitrogen ratio and the Se-C species ratio in the material, demonstrating that this invention can stabilize the selenium active center through a pyridine nitrogen-rich coordination environment. This structural feature provides an important foundation for subsequent enhancement of catalytic activity.

[0043] (3) The nanozyme prepared in this invention exhibits excellent oxidase-like activity and can directly catalyze the oxidation and color development of TMB without the addition of external H2O2. Compared with the control sample without g-C3N4 or with unsuitable selenium loading, the nanozyme of this invention shows higher TMB catalytic oxidation ability, indicating that the pyridine nitrogen regulation and the Se single-atom / cluster synergistic structure can significantly enhance the adsorption and activation ability of dissolved oxygen and improve the efficiency of the color development reaction. This nanozyme has a strong substrate affinity for TMB, which can provide a guarantee for the subsequent construction of a stable and sensitive colorimetric detection system.

[0044] (4) Based on the oxidase-like activity of the nanozyme described in this invention, a novel colorimetric detection method for carbendazim that is simple to operate and provides intuitive results can be established. This method utilizes the nanozyme described in this invention to catalyze the formation of blue oxidized TMB from TMB, and then utilizes the reducing and decolorizing effect of carbendazim on the oxidized TMB. Qualitative or quantitative analysis of carbendazim is achieved through changes in absorbance at 652 nm. The constructed colorimetric detection system does not rely on large-scale precision instruments, facilitating rapid screening and visual readout; simultaneously, at 200.0 μmol·L⁻¹…-1 –1500.0 μmol·L -1 It exhibits good linearity over a wide concentration range and has a low limit of detection (LOD = 1.16 μmol·L⁻¹). -1 This can meet the needs of rapid detection of carbendazim.

[0045] (5) The nanozyme prepared in this invention also exhibits good peroxidase-like activity, capable of efficiently catalyzing the generation of ·OH in the presence of H2O2. Fluorescent probe experiments show that the nanozyme described in this invention can significantly enhance the ·OH generation capacity and has a high substrate affinity for H2O2. This indicates that the nanozyme described in this invention can not only be used for colorimetric detection but also exert a strong catalytic effect in systems involving H2O2, thereby expanding its application in fields such as antibacterial agents.

[0046] (6) The nanozyme prepared by this invention exhibits excellent antibacterial properties. In the presence of H2O2, the nanozyme can convert H2O2 into highly oxidizing ·OH via a peroxidase-like pathway, thereby destroying the bacterial cell wall, cell membrane, and intracellular biomolecules, effectively inhibiting bacterial growth. Taking *Escherichia coli* as an example, the nanozyme of this invention shows a significant antibacterial effect, with an inhibition rate of up to 87.2%, indicating that it possesses both detection and antibacterial functions.

[0047] (7) The nanozyme described in this invention integrates "structural stability, colorimetric detection, and antibacterial activity," possessing excellent comprehensive application value. On the one hand, it can be used for rapid and visual detection of pesticide residues, especially carbendazim; on the other hand, it can exert antibacterial effects by generating reactive oxygen species, thereby reducing potential interference from microbial growth in complex real-world samples. Therefore, this invention has promising application prospects in fields such as agricultural product safety testing, environmental monitoring, and antibacterial materials. Attached Figure Description

[0048] Figure 1 The powder diffraction (XRD) patterns and Raman spectra of the nanozymes prepared in this invention and the control samples are shown.

[0049] Figure 2The images show TEM, SAED, and AC-HAADF-STEM characterization spectra of the selenium single-atom / cluster nanozyme (SeSAs / ACs@CNC) and a comparative sample described in this invention; wherein, (A) is a high-resolution transmission electron microscope (HRTEM) image of the selenium single-atom / cluster nanozyme (SeSAs / ACs@CNC) described in this invention, (B) is a selected area electron diffraction (SAED) image of the selenium single-atom / cluster nanozyme (SeSAs / ACs@CNC) described in this invention, and (C) is a selenium single-atom / cluster nanozyme described in this invention. (D) is an aberration-corrected high-angle annular dark-field scanning transmission electron microscope (AC-HAADF-STEM) image of (SeSAs / ACs@CNC), (E) is an AC-HAADF-STEM image of the control sample (SeSAs@CNC), and (F) is a transmission electron microscope (TEM) image of the selenium single-atom / cluster nanozyme (SeSAs / ACs@CNC) described in this invention, (G) is an elemental mapping (EDS) image, and (H) is an energy dispersive spectroscopy (EDS) spectrum.

[0050] Figure 3 The images show XPS comparison analysis of the selenium single-atom / cluster nanozyme (SeSAs / ACs@CNC) and the control sample (Se@NC) described in this invention; wherein, (A) is the full XPS spectrum and corresponding high-resolution photoelectron spectroscopy (XPS) spectrum of the selenium single-atom / cluster nanozyme (SeSAs / ACs@CNC) and the control sample (Se@NC) described in this invention; (B) is the C 1s spectrum, (C) is the N 1s spectrum and (D) is the Se 3d spectrum; (E) is the pyridine nitrogen ratio bar chart of the selenium single-atom / cluster nanozyme (SeSAs / ACs@CNC) and the control sample (Se@NC) described in this invention; and (F) is the Se-C species ratio bar chart of the selenium single-atom / cluster nanozyme (SeSAs / ACs@CNC) and the control sample (Se@NC) described in this invention.

[0051] Figure 4 The UV-vis absorption spectra of different nanozymes are shown; in the inset, there are photographs of the corresponding systems.

[0052] Figure 5Figure 1 shows the catalytic mechanism analysis and steady-state kinetics experimental results of the selenium single-atom / cluster nanozyme (SeSAs / ACs@CNC) described in this invention; where (A) is the effect of different scavengers on the UV-vis absorption intensity of the SeSAs / ACs@CNC-TMB system at 652 nm; (B) is the UV-vis absorption spectrum of the selenium single-atom / cluster nanozyme (SeSAs / ACs@CNC) described in this invention; (C) is the Michaelis-Menten curve; and (D) is the corresponding Lineweaver-Burk plot.

[0053] Figure 6 The image shows the colorimetric detection results of carbendazim on the activity of selenium single-atom / cluster nanozymes (SeSAs / ACs@CNC) based on the present invention; where (A) is the UV-vis absorption spectrum of the SeSAs / ACs@CNC-TMB system as the concentration of carbendazim increases; (B) is the logarithm of the decrease in absorbance (ΔA) versus the concentration of carbendazim (CBZ) log(C... CBZ Linear graph of ).

[0054] Figure 7 The figure shows the peroxidase-like activity results of the selenium single-atom / cluster nanozyme (SeSAs / ACs@CNC) described in this invention; where A, B and C are the fluorescence spectra of dihydroxyterephthalic acid generated by the reaction of terephthalic acid (TA) with the ·OH generated by SeSAs / ACs@CNC in the presence of H2O2; (D) is the UV-vis absorption spectrum of SeSAs / ACs@CNC and (E) is the Michaelis-Menten curve, and (F) is the corresponding Lineweaver-Burk plot.

[0055] Figure 8 The figures show the experimental results of the antibacterial performance of the selenium single-atom / cluster nanozymes (SeSAs / ACs@CNC) described in this invention; wherein, (A) is a plate colony image of E. coli after treatment with different nanozyme concentrations (the number of colonies decreased significantly with increasing SeSAs / ACs@CNC concentration, and was compared with the Se@NC control); (B) is a statistical bar chart of the corresponding bacterial survival rate (or inhibition rate) and the results of one-way ANOVA and Tukey post-hoc test (ns, , , (These represent p>0.05, p<0.05, p<0.01, and p<0.001, respectively). Detailed Implementation

[0056] The following specific embodiments are provided to further explain the present invention; however, the embodiments do not limit the scope of protection of the present invention.

[0057] The specific preparation steps of g-C3N4 in this invention and the following embodiments are as follows:

[0058] First, 3.0 g of melamine and 3.06 g of cyanuric acid were weighed and placed in 120 mL and 60 mL of dimethyl sulfoxide (DMSO) solvent, respectively, and then completely dispersed and dissolved using an ultrasonic cleaner. At 25 °C, the cyanuric acid solution was slowly added dropwise to the melamine solution under magnetic stirring for approximately 10 min. The mixture was continuously stirred to form a white supramolecular complex precipitate. After the reaction was complete, the product was collected by centrifugation and repeatedly washed with anhydrous ethanol to remove residual solvent, followed by drying in an 80 °C oven. Finally, the obtained white precursor was placed in a tube furnace and heated at 5 °C / min under a N2 atmosphere. -1 The temperature was increased to 550 °C and calcined at a constant temperature for 4 h. After cooling to room temperature, it was ground to obtain a light yellow powder, which is g-C3N4.

[0059] Example 1: Preparation of selenium single-atom / cluster nanozymes (SeSAs / ACs@CNC) with pyridine nitrogen regulation to enhance dual enzyme activity

[0060] The preparation of SeSAs / ACs@CNC using the "ion imprinting-pyrolysis" strategy described in this embodiment specifically includes the following steps:

[0061] (1) Dissolve 450.0 mg chitosan in 50.0 mL of 2% (v / v) acetic acid solution and stir vigorously until completely dissolved to form a transparent viscous solution;

[0062] (2) Subsequently, 11.1 mg of selenium dioxide (SeO2) powder and 0.2 g g-C3N4 powder were slowly added to the solution, and the mixture was stirred continuously at 50 °C for 1 h to allow the selenium source and nitrogen source to be fully dispersed and adsorbed between the chitosan chains.

[0063] (3) Next, add 1.0 mL of glutaraldehyde solution (50%, v / v) as a crosslinking agent, and continue stirring at 50 °C until the solution undergoes a crosslinking reaction to form a hydrogel;

[0064] (4) The obtained gel was freeze-dried under vacuum to remove moisture and obtain an aerogel precursor;

[0065] (5) Transfer the dried solid to a porcelain boat and place it in a tube furnace; perform programmed pyrolysis under nitrogen protection: first at 5 °C·min -1The temperature was increased to 550 °C at a certain rate and held for 2 h; then the temperature was increased to 900 °C at the same rate and held for 1.5 h.

[0066] (6) After pyrolysis, collect the black powder product and soak it in 3.0 mol·L⁻¹ solution at room temperature. -1 The sample was immersed in hydrochloric acid solution for 8 hours to remove any unstable impurities or agglomerated particles that might form. Finally, it was repeatedly washed with ultrapure water until neutral and dried to obtain the target product SeSAs / ACs@CNC.

[0067] Comparative Example 1: Preparation of SeACs@CNC

[0068] The difference between Comparative Example 1 and Example 1 is that, while keeping other synthesis conditions unchanged, the amount of SeO2 added was adjusted to 111.0 mg. All other steps were the same as in Example 1, and the final sample SeACs@CNC was obtained.

[0069] Comparative Example 2: Preparation of SeSAs@CNC

[0070] The difference between Comparative Example 2 and Example 1 is that, while keeping other synthesis conditions unchanged, the amount of SeO2 added was adjusted to 1.1 mg. All other steps were the same as in Example 1, and the final sample SeSAs@CNC was obtained.

[0071] Comparative Example 3: Preparation of CNC

[0072] The difference between Comparative Example 3 and Example 1 is that SeO2 was not added during the preparation process. All other steps were the same as in Example 1, and the final sample obtained was a CNC sample.

[0073] Comparative Example 4: Preparation of Se@NC

[0074] The difference between Comparative Example 4 and Example 1 is that g-C3N4 was not added during the preparation process; only chitosan and 11.1 mg SeO2 were used for subsequent crosslinking, freeze-drying, pyrolysis, and acid washing. All other steps were the same as in Example 1, ultimately yielding sample Se@NC.

[0075] Example 2: Structural Characterization of SeSAs / ACs@CNC

[0076] To evaluate the crystal structure, microstructure, and surface chemical state of the SeSAs / ACs@CNC of the present invention, the SeSAs / ACs@CNC prepared in Example 1 was characterized by XRD, Raman, TEM, SAED, AC-HAADF-STEM, EDS, and XPS.

[0077] (1) XRD and Raman characterization

[0078] First, the crystal structure and phase composition of the prepared samples were analyzed using XRD technology. For example... Figure 1 As shown in Figure A, the XRD patterns of SeSAs / ACs@CNC and the comparative nanozymes exhibit similar characteristics, namely, two broad and weak diffraction peaks observed at approximately 2θ of 26° and 44°, corresponding to the (002) and (100) crystal planes of the amorphous graphitic carbon material, respectively. This result indicates that the chitosan precursor forms a carbon framework through pyrolysis, while g-C3N4 participates in nitrogen doping and structural regulation during the pyrolysis process, ultimately yielding a nitrogen-rich carbon support with a certain degree of graphitization. Most importantly, no characteristic diffraction peaks of Se or SeO2 were observed in the SeSAs / ACs@CNC spectrum, indicating that the selenium species in the sample did not undergo large-scale aggregation to form crystal particles, but rather existed in a highly dispersed amorphous or atomic-level form within the carbon substrate.

[0079] Subsequently, Raman spectroscopy was used to further investigate the microstructural order and surface defects of the carbon substrate. Figure 1 As shown in B, all samples were at 1350 cm. -1 and 1590 cm -1 The surrounding area exhibits two typical characteristic peaks, which are respectively attributed to the D band representing lattice defects and disordered structure, and the sp band representing sp. 2 The G-band represents the in-plane stretching vibrations of carbon atoms in hybrid graphite. It is generally determined by the intensity ratio of the D-band to the G-band (I0). D / I G This is used to quantify the defect density of carbon materials. The calculation results show that I... D / I G The order of magnitude of the values ​​is: CNC > SeSAs@CNC > SeSAs / ACs@CNC > SeACs@CNC > Se@NC.

[0080] The above results indicate that g-C3N4 is a key factor in introducing defect sites, and the SeSAs / ACs@CNC described in this invention maintains a relatively high Ig. D / I G The value indicates its defect-rich structural features, which provides a structural basis for subsequent efficient catalysis and enzyme-like reactions.

[0081] (2) Characterization by TEM, SAED, AC-HAADF-STEM and EDS

[0082] To clarify the dispersion morphology of Se species, the SeSAs / ACs@CNC samples described in this invention and comparative samples were characterized by TEM, SAED, and AC-HAADF-STEM. Figure 2As shown in Figure A, no clear lattice fringes of Se or SeO2 were observed in the HRTEM image of SeSAs / ACs@CNC; as Figure 2 As shown in Figure B, its SAED image exhibits a diffuse halo, indicating the absence of detectable crystalline structures in the sample. Figure 2 As shown in Figure C, in the AC-HAADF-STEM image of SeSAs / ACs@CNC, numerous isolated bright spots are visible against the carbon support background, with localized areas of even stronger brightness aggregation, indicating the synergistic coexistence of Se single atoms and clusters. For comparison, as shown in Figure C... Figure 2 As shown in D, SeSAs@CNC mainly appears as scattered, isolated bright spots, indicating that it is dominated by single atoms; as Figure 2 As shown in E, the brightness aggregation is more pronounced in SeACs@CNC, indicating a higher proportion of atomic clusters and a further aggregation trend. Figure 2 As shown in F, SeSAs / ACs@CNC exhibits a wrinkled porous carbon framework; as Figure 2 As shown in G, Se, C, and N are uniformly distributed in its EDS element mapping, with no large-scale Se enrichment observed; as Figure 2 As shown in H, the EDS energy spectrum further confirmed the presence of the Se element.

[0083] The above results indicate that in the SeSAs / ACs@CNC described in this invention, Se is highly dispersed in the nitrogen-rich carbon framework in the form of single atoms and atomic clusters coexisting.

[0084] (3) XPS characterization

[0085] To further reveal the regulatory effect of g-C3N4 introduction on the surface elemental composition and chemical valence state of nanozymes, XPS comparative analysis was performed on SeSAs / ACs@CNC and Se@NC. Figure 3 As shown in Figure A, C, N, O, and Se elemental signals were observed in both samples. Further analysis was performed on the C 1s high-resolution spectrum. Figure 3 As shown in B, the spectra of both samples can be deconvolved into five characteristic peaks, which are assigned to CC / C=C (284.8 eV), CN (286.0 eV), CO (287.5 eV), C=O (288.8 eV), and C-Se (290.6 eV), respectively. Figure 3 As shown in Figure C, the N 1s spectrum can be deconvolved into four peaks, attributed to pyridine nitrogen (398.1 eV), pyrrole nitrogen (399.4 eV), graphitic nitrogen (401.0 eV), and nitrogen oxides (402.4 eV), respectively. Quantitative analysis results indicate that the pyridine nitrogen content in SeSAs / ACs@CNC is 30.2%, significantly higher than the 24.1% in Se@NC. Figure 3As shown in Figure D, the Se 3d spectrum reveals two main deconvolution peaks: Se-C at a low binding energy (56.0 eV) and Se-O at a high binding energy (59.4 eV). Further analysis indicates that the proportion of Se-C species in the SeSAs / ACs@CNC described in this invention is 37.83%, significantly higher than the 24.46% in Se@NC. The presence of the Se-C peak and its high relative abundance suggest that Se atoms are not simply physically adsorbed or exist on the surface in oxide form, but are firmly embedded in the carbon-nitrogen framework through Se-C bonds.

[0086] In summary, XPS results show that the introduction of g-C3N4 successfully constructed a carbon skeleton rich in pyridine nitrogen, and then achieved stable anchoring of Se atomic-level species (single atoms / clusters) by regulating the local electronic structure of the carbon skeleton and promoting the formation of Se-C bonds.

[0087] Example 3 Oxidase-like activity of SeSAs / ACs@CNC

[0088] To evaluate the oxidase-like activity of the SeSAs / ACs@CNC described in this invention, TMB was selected as the chromogenic substrate, and the test was performed in HAc-NaAc buffer solution at pH 3.0.

[0089] The specific method is as follows: 5 μL of SeSAs / ACs@CNC dispersion (10 mg·mL) -1 ) and 100 μL TMB solution (10 mmol·L -1 Add 1895 μL of HAc-NaAc buffer system with pH=3.0, mix well, and react at 35 ℃ for 15 min. The catalytic ability is investigated by recording the change of absorbance of the reaction system at 652 nm.

[0090] The results showed that upon addition of the SeSAs / ACs@CNC dispersion, the reaction system rapidly changed from colorless to deep blue, exhibiting a distinct characteristic absorption peak at 652 nm. This colorimetric phenomenon indicates that SeSAs / ACs@CNC can promote the oxidation process involving dissolved oxygen without added H2O2, demonstrating good oxidase-like activity. Under the same conditions, the catalytic activity of SeSAs / ACs@CNC was compared with that of the samples prepared in Comparative Examples 1, 2, 3, and 4. Figure 4As shown, the absorbance of each sample at 652 nm was 1.261 for SeSAs / ACs@CNC, 0.430 for SeACs@CNC, 0.357 for SeSAs@CNC, 0.555 for CNC, and 0.017 for Se@NC. It can be seen that the SeSAs / ACs@CNC described in this invention has the highest absorbance, indicating its strongest catalytic oxidation ability for TMB, significantly better than low-loaded SeSAs@CNC and high-loaded SeACs@CNC, and also significantly better than unloaded CNC and Se@NC without g-C3N4. These results indicate that the pyridine-rich nitrogen-deficient carbon microenvironment constructed after g-C3N4 participation in pyrolysis is beneficial for the stable dispersion of Se species and enhances the adsorption and activation of dissolved oxygen, thereby improving the oxidation and color development efficiency of TMB.

[0091] Example 4: Catalytic Mechanism Analysis and Steady-State Kinetic Parameters

[0092] (1) Catalytic mechanism analysis

[0093] To reveal the intrinsic mechanism by which the SeSAs / ACs@CNC described in this invention exhibit oxidase-like activity, p-benzoquinone (PBQ), L-histidine (L-His), isopropanol (IPA), and disodium ethylenediaminetetraacetate (EDTA) were added as scavengers of different active intermediates to the TMB colorimetric reaction system. Figure 5 As shown in Figure A, the TMB oxidation and color development process was significantly suppressed upon the addition of PBQ, with a substantial decrease in absorbance at 652 nm, indicating that the oxidation of TMB by O2 was significantly inhibited. − It is the main reactive oxygen species generated during the oxidation of TMB catalyzed by SeSAs / ACs@CNC.

[0094] (2) Steady-state dynamics analysis

[0095] Under optimal reaction conditions, the initial reaction rate was recorded as a function of substrate concentration by fixing the nanozyme concentration and changing the substrate TMB concentration. Experimental results show that the catalytic behavior of the SeSAs / ACs@CNC described in this invention conforms to a typical Michaelis-Menten enzymatic kinetic model. Calculations were performed using Lineweaver-Burk double reciprocal plotting. Figure 5 BD), SeSAs / ACs@CNC for substrate TMB K m It is 0.4717 mmol·L -1 V max 43.68 nM·s -1 The K m Value and K of natural horseradish peroxidase on TMB m (0.43 mmol·L) -1The fact that the values ​​are relatively close indicates that the nanozyme prepared in this invention has a strong substrate affinity for TMB.

[0096] Example 5: Colorimetric detection of carbendazim activity based on SeSAs / ACs@CNC oxidases

[0097] Based on the excellent oxidase-like activity of the SeSAs / ACs@CNC nanozyme described in Example 4, a colorimetric detection method for carbendazim (CBZ) was constructed.

[0098] The specific steps are as follows:

[0099] (1) First, according to the activity test system ratio in Example 6, SeSAs / ACs@CNC and TMB were added to HAc-NaAc buffer at pH=3.0 and pre-reacted at 35 °C for 15 min to generate a blue oxidized TMB system;

[0100] (2) Subsequently, different concentrations of carbendazim (CBZ) standard solutions were added to the system, and the reaction was continued at room temperature for 15 min;

[0101] (3) Immediately after the reaction is complete, record the absorbance of the mixed solution at 652 nm;

[0102] (4) Calculate the absorbance difference ΔA=A0-A, where A0 and A are the absorbance when carbendazim (CBZ) is absent and present, respectively, and construct the relationship curve between ΔA and carbendazim (CBZ) concentration.

[0103] like Figure 6 As shown, with the increase of carbendazim (CBZ) concentration in the system, the intensity of the characteristic absorption peak at 652 nm of the solution shows a regular decreasing trend. This indicates that carbendazim (CBZ) has a certain reducing property and can reduce and decolorize the already generated oxidized TMB, thus leading to a decrease in absorbance at 652 nm. Further fitting analysis of the experimental data revealed that ΔA is related to the logarithm of the carbendazim (CBZ) concentration (log C). CBZ There is a good linear relationship between them. (200.0–1500.0 μmol·L⁻¹) -1 Within the concentration range, its linear regression equation is: ΔA = 0.0774 log C CBZ -0.1739, R 2 = 0.9317. Based on the formula LOD = 3σ / S, the detection limit for carbendazim (CBZ) using this colorimetric method is 1.16 μmol·L⁻¹. -1 .

[0104] The above results demonstrate that the colorimetric detection method constructed in this invention can achieve rapid, intuitive, and quantitative analysis of carbendazim (CBZ).

[0105] Example 6: Peroxidase-like activity of SeSAs / ACs@CNC

[0106] In addition to exhibiting excellent oxidase-like activity, the SeSAs / ACs@CNC nanozymes prepared in this invention also possess significant peroxidase-like activity, namely, the ability to catalyze the decomposition of H2O2 to generate ·OH, a highly oxidizing agent. To verify this activity, terephthalic acid (TA) was used as a fluorescent probe for capturing ·OH. Since TA itself is non-luminescent, its reaction with ·OH generates 2-hydroxyterephthalic acid (TAOH), which exhibits significant fluorescence.

[0107] The specific procedure was as follows: TA, H2O2, and SeSAs / ACs@CNC were mixed and reacted, and the changes in their fluorescence spectra were recorded. The experimental results are as follows: Figure 7 As shown in AC, a significantly enhanced fluorescence peak appeared at 435 nm in the TA+H2O2+SeSAs / ACs@CNC system, and its intensity increased with increasing reaction time. In contrast, the fluorescence signals of control samples without nanozymes or with only CNC, Se@NC, SeACs@CNC, or SeSAs@CNC were significantly weaker.

[0108] The above results show that the SeSAs / ACs@CNC described in this invention can efficiently catalyze the bond breaking of H2O2 to generate ·OH, and its catalytic efficiency is significantly better than that of Se@NC without g-C3N4 and other control samples.

[0109] Furthermore, the steady-state kinetics of the peroxidase-like activity of SeSAs / ACs@CNC were studied. The results showed that ( Figure 7 DF), which has a K+ ionization effect on H2O2. m It is 0.2076 mmol·L -1 This indicates that the nanozyme described in this invention has a high substrate affinity for H2O2 and can rapidly initiate the catalytic reaction at a low H2O2 concentration.

[0110] Example 7: Antibacterial properties of SeSAs / ACs@CNC

[0111] Based on the excellent peroxidase-like activity and efficient ·OH generation ability of SeSAs / ACs@CNC described in Example 6, its antibacterial performance against Escherichia coli was further evaluated.

[0112] In a sterile centrifuge tube, 50 μL of diluted E. coli bacterial culture was taken, and different concentrations of SeSAs / ACs@CNC dispersion were added. The final concentration gradients were set to 0, 100, 200, and 400 μg·mL. -1 At the same time, add 50 μL H2O2 (5 mmol·L⁻¹) -1 After thorough mixing, the reaction mixture was incubated overnight at 37 ℃ and 200 rpm in a shaker. After incubation, 50 μL of the mixture was evenly spread onto LB solid agar plates. The plates were then inverted and incubated at 37 ℃ for 18–24 h, and the colony growth was observed and recorded.

[0113] Data statistics and antibacterial rate calculation: By visually observing and counting the colony-forming units (CFU) on the plate, the antibacterial efficiency is calculated using the following formula: Antibacterial rate (%) = (N... control - N sample ) / N control × 100%. Where N is... control N represents the average number of colonies on the control group plates. sample This represents the average number of colonies on the experimental plates. All experiments were repeated in triplicate, and the results were averaged and the standard deviation was calculated.

[0114] like Figure 8 As shown in A and B, as the concentration of SeSAs / ACs@CNC increased from 0 μg·mL... -1 Increase to 400 μg·mL -1 The number of colonies on the agar plates decreased significantly. When the SeSAs / ACs@CNC concentration reached 400 μg·mL... -1 At that time, the survival rate of Escherichia coli was only 12.8%, meaning the inhibition rate was as high as 87.2%. Furthermore, using Se@NC as a control sample, antibacterial experiments were conducted under the same conditions. The results showed that the bacterial survival rate in the Se@NC treatment group was significantly higher than that in the SeSAs / ACs@CNC treatment group, indicating that the SeSAs / ACs@CNC prepared in this invention has stronger antibacterial ability.

[0115] Based on the above results, it can be concluded that the SeSAs / ACs@CNC prepared in this invention can convert H2O2 into highly active ·OH through a peroxidase-like pathway. These free radicals can attack the bacterial cell wall and cell membrane, leading to lipid peroxidation, changes in membrane permeability, and further damage to intracellular proteins and DNA and other biomolecules, ultimately resulting in bacterial death.

Claims

1. A method for preparing a selenium single-atom / cluster nanozyme with pyridine nitrogen-regulated enhanced dual-enzyme activity, characterized in that, Includes the following steps: (1) Dissolve chitosan in acetic acid solution, then add selenium dioxide and the g-C3N4, and disperse them fully under heating and stirring conditions; (2) Add a crosslinking agent to the system obtained in step (1) to cause crosslinking and form a gel; (3) The obtained gel was freeze-dried to obtain the precursor; (4) The precursor is subjected to two-step pyrolysis under nitrogen protection; (5) The pyrolysis product is acid-washed, washed and dried to obtain the selenium single atom / cluster nanozyme with pyridine nitrogen regulation to enhance the activity of the two enzymes.

2. The preparation method according to claim 1, characterized in that, The g-C3N4 mentioned in step (1) is prepared by the following method: A cyanuric acid solution was added dropwise to a melamine solution, and the mixture was stirred to form a white supramolecular complex precipitate. The white supramolecular complex precipitate was then pyrolyzed to obtain the g-C3N4.

3. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of chitosan, selenium dioxide and g-C3N4 is 400-500:10-12:0.1-0.

3.

4. The preparation method according to claim 1, characterized in that, The crosslinking agent mentioned in step (2) is a glutaraldehyde solution.

5. The preparation method according to claim 1, characterized in that, The volume ratio of glutaraldehyde solution to acetic acid solution is 1:40-60.

6. The preparation method according to claim 1, characterized in that, The specific method for the two-step pyrolysis described in step (4) is as follows: under nitrogen protection, at 4-6℃·min -1 The temperature is increased to 500-600℃ at a rate of [temperature value] and held for 1.5-2.5 h, then increased at a rate of 4-6℃ / min. -1 The temperature is increased to 800-900 ℃ at a rate and held for 1-2 h.

7. The selenium single-atom / cluster nanozyme with enhanced dual-enzyme activity regulated by pyridine nitrogen as described in any one of claims 1-6.

8. The application of the selenium single-atom / cluster nanozyme with pyridine nitrogen regulation to enhance the activity of dual enzymes as described in claim 7 in the colorimetric detection of carbendazim.

9. The application according to claim 8, characterized in that, The specific method for colorimetric detection of carbendazim is as follows: The selenium single-atom / cluster nanozyme with pyridine nitrogen-regulated enhanced dual-enzyme activity was added to 3,3',5,5'-tetramethylbenzidine in an acetate-sodium acetate buffer solution at pH 2.5-3.5 and pre-reacted at 30-40℃ for 10-20 min to induce TMB oxidation and color development. Subsequently, the test sample containing carbendazim was added, and the reaction was continued at room temperature for 10-20 min. The absorbance change at 600-700 nm was measured, and the qualitative or quantitative detection of carbendazim was achieved based on the absorbance change.

10. The application of the selenium single-atom / cluster nanozyme with pyridine nitrogen regulation to enhance the activity of dual enzymes as described in claim 7 in the preparation of antibacterial agents.

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