A heteroatom-containing ligand MOF nanozyme and a preparation method and application thereof

Abstract

The application provides a kind of heteroatom-containing ligand MOF nanozyme and its preparation method and application.The heteroatom-containing ligand MOF nanozyme is prepared as follows: N,N-dimethylformamide is added to a pressure bottle, nitrogen is blown after, ferrous chloride tetrahydrate and 1H-1,2,3-triazole ligand salt, two kinds of mixed ligands of sulfur atom ligand salt are added, and S-MET(Fe) nanozyme doped with sulfur element is synthesized by solvothermal method.The S-MET(Fe) nanozyme doped with sulfur atom can be simply and efficiently prepared by solvothermal method within 130 DEG C, and the synthesis steps are simple and easy to control, the reaction process is stable and controllable, and it can be produced in large scale, solving the problems of complex preparation process, high energy consumption and high cost of existing heteroatom-doped nanozyme.The nanozyme of the application has high affinity with substrate, greatly improved catalytic activity, good antibacterial activity, and has very broad application prospect in the fields of catalysis and biological medicine.

Description

Technical Field

[0001] This invention relates to a nanozyme, its preparation method and application, specifically to a MOF nanozyme containing heteroatom ligands, its preparation method and application. Background Technology

[0002] Nanozymes are a class of nanomaterials capable of catalyzing substrates and converting them into products under both mild and extreme conditions, exhibiting advantages such as high stability, simple preparation, and high catalytic activity. As alternatives to natural enzymes, they hold immense application potential in the biomedical field. Metal-organic framework (MOF)-based nanozymes, as an important class, have attracted widespread attention due to their structural characteristics and composition. However, to date, the catalytic activity of most nanozymes has not been satisfactory in practical applications. Therefore, it is necessary to deeply understand optimization methods for further improving the catalytic activity of nanozymes through various regulatory strategies, including controlling size, morphology, doping, vacancies, surface modification, and composition. The rational development of MOFs with enzyme-like catalytic properties and the optimization of nanozyme activity are of great significance for broadening the application range of nanozymes and promoting their practical application.

[0003] With the deepening research on strategies for regulating the catalytic activity of MOF nanozymes, doping has proven to be an effective strategy for adjusting the structure and catalytic activity of nanozymes. Metal atom doping can affect the surface electronic structure and band structure, thereby regulating the catalytic performance of nanozymes. In addition to metal atoms, heteroatoms such as B, S, and P are introduced as "regulators" to lower the energy barrier in the H2O2 reduction process, thereby improving the activity of nanozymes. By changing the coordination environment, they induce a low energy barrier in the active intermediates, exhibiting excellent POD-like activity.

[0004] Reported sulfur-doped nanozymes, such as metal sulfides, nitrogen-sulfur co-doped carbon materials, and sulfur-doped lignin-derived carbon nanosheets, all require calcination synthesis with the calcination temperature strictly controlled between 400 and 1000°C. This results in high manufacturing costs and unstable reaction processes, making them unsuitable for industrial production. Furthermore, the activity of the prepared nanozymes needs further improvement. Summary of the Invention

[0005] The purpose of this invention is to provide a MOF nanozyme containing heteroatom ligands, its preparation method and application, in order to solve the problems of complex synthesis steps, high energy consumption, high cost and the need to improve enzyme activity of existing heteroatom-doped nanozymes.

[0006] The objective of this invention is achieved as follows: A method for preparing MOF nanozymes containing heteroatom ligands includes the following steps: (a) N,N-dimethylformamide was added to the reaction vessel, nitrogen gas was purged, and then ferrous chloride tetrahydrate was added and ultrasonically dispersed. (b) Add a mixed ligand salt and disperse it by ultrasonication, wherein the mixed ligand salt is a 1H-1,2,3-triazole ligand salt and a sulfur atom ligand salt; (c) Seal the reaction vessel and stir continuously at 110℃~130℃. After the reaction is completed, cool to room temperature, centrifuge to obtain the precipitate, wash the precipitate and vacuum dry it to obtain MOF nanozyme containing heteroatom ligands.

[0007] In step (a), nitrogen is blown for 15–25 minutes.

[0008] The sulfur atom ligand salt mentioned in step (b) is 5-mercapto-1,2,3-triazole monosodium salt, and the molar ratio of 1H-1,2,3-triazole ligand salt and 5-mercapto-1,2,3-triazole monosodium salt is 1:(0.25~4), preferably 1:(0.5~4), and more preferably 1:(1~4).

[0009] The molar ratio of ferrous chloride tetrahydrate to the mixed ligand salt is 1:3.

[0010] The reaction time in step (c) is 12–24 h, and the washing method is to repeatedly wash with DMF and methanol.

[0011] The preparation method of this invention is simple, and sulfur-doped S-MET(Fe) nanozymes can be prepared easily and efficiently at temperatures below 130℃ via a solvothermal method. Furthermore, the synthesis steps are simple and easy to control, the reaction process is stable and controllable, and it can be mass-produced. This solves the problems of complex preparation processes, high energy consumption, and high costs associated with existing heteroatom-doped nanozyme preparation methods.

[0012] The S-MET(Fe) nanozyme prepared by this invention can effectively generate more defects and active sites, and has a high affinity for the substrate, which to some extent solves the problem of insufficient catalytic active sites leading to low catalytic activity in the prior art.

[0013] The S-MET(Fe) nanozyme prepared in this invention exhibits significant antibacterial activity by catalyzing the decomposition of low concentrations of hydrogen peroxide to generate hydroxyl radicals and superoxide anions.

[0014] This invention employs a mixed ligand scheme to prepare S-MET(Fe) nanozymes with high POD-like activity, which have very broad application prospects in catalysis and biomedicine. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the synthesis process of S-MET(Fe) nanozyme.

[0016] Figure 2(a) is a transmission electron microscope image of the MET(Fe) nanozyme in Example 1; (b) is a transmission electron microscope image of the S-MET(Fe) nanozyme prepared by a molar ratio of 1H-1,2,3-triazole ligand salt and 5-mercapto-1,2,3-triazole monosodium salt of 1:2.

[0017] Figure 3 (a) is the PXRD pattern of nanozymes with different proportions of mixed ligands in Example 1; (b) is the TGA pattern of S-MET(Fe) nanozymes prepared by mixing MET(Fe) nanozymes with 1H-1,2,3-triazole ligand salt and 5-mercapto-1,2,3-triazole monosodium salt in a molar ratio of 1:2.

[0018] Figure 4 This refers to the POD-like activity of the nanozyme in Example 2 at room temperature for 10 minutes.

[0019] Figure 5 (a) shows the effect of solution pH on the activity of MET(Fe) nanozyme POD in Example 3; (b) shows the effect of temperature on the activity of MET(Fe) nanozyme POD; (c) shows the effect of solution pH on the activity of S-MET(Fe) nanozyme POD; and (d) shows the effect of solution temperature on the activity of S-MET(Fe) nanozyme POD.

[0020] Figure 6 (a) is the Michaelis-Menten curve of MET(Fe) nanozyme on substrate TMB in Example 3; (c) is the Michaelis-Menten curve of MET(Fe) nanozyme on substrate H2O2; (b) is the Lineweaver-Burk curve of MET(Fe) nanozyme on substrate TMB; (d) is the Lineweaver-Burk curve of MET(Fe) nanozyme on substrate H2O2.

[0021] Figure 7 (a) is the Michaelis-Menten curve of S-MET(Fe) nanozyme on substrate TMB in Example 3; (c) is the Michaelis-Menten curve of S-MET(Fe) nanozyme on substrate H2O2; (b) is the Lineweaver-Burk curve of S-MET(Fe) nanozyme on substrate TMB; (d) is the Lineweaver-Burk curve of S-MET(Fe) nanozyme on substrate H2O2.

[0022] Figure 8(a) shows the effect of different concentrations of active oxygen scavengers on the A / A0 ratio of the MET(Fe)-TMB-H2O2 system in Example 4; (b) shows the effect of different concentrations of active oxygen scavengers on the A / A0 ratio of the S-MET(Fe)-TMB-H2O2 system; (c) shows the ESR spectrum of ·OH detection in the DMPO-H2O2 system; (d) shows the detection of O2 in the DMPO-H2O2 system. •- The ESR spectrum.

[0023] Figure 9 (a) shows the inhibition mechanism of AA on the catalytic activity of S-MET(Fe) nanozyme in Example 5; (b) shows the UV-Vis spectra of the S-MET(Fe) nanozyme catalyzing the TMB-H2O2 oxidation reaction with different concentrations of AA; (c) shows the corresponding linear fitting spectra.

[0024] Figure 10 (a) is the result of the recycling experiment of S-MET(Fe) nanozyme in Example 6; (b) is the result of the stability experiment of S-MET(Fe) nanozyme.

[0025] Figure 11 (a) is in Example 7 E. coli (a) Photographs of plate culture after various treatments; (b) is from Example 8. S. aureus Photos of the plate culture after various treatments. Detailed Implementation

[0026] The present invention will be further illustrated below with reference to comparative examples and embodiments. The following embodiments are for illustrative purposes only and do not limit the scope of protection of the present invention in any way. In the embodiments, all chemicals are analytical or chromatographic grade, and all aqueous solutions are prepared with ultrapure Milli-Q water (ρ>18.0 MΩ·cm). -1 It is prepared by ).

[0027] Example 1: Synthesis and characterization of S-MET(Fe) in different proportions The preparation methods of S-MET(Fe) in different proportions include the following steps: 14 mL of DMF was added to a pressure-resistant bottle, and the mixture was purged with nitrogen for 20 min. Then, 160.1 mg of ferrous chloride tetrahydrate was dissolved in DMF and ultrasonically dispersed until homogeneous. Different proportions of 1H-1,2,3-triazole and 5-mercapto-1,2,3-triazole monosodium salt were added to the solution, and the mixture was ultrasonically dispersed again. The mixture was stirred at 120 °C for 24 h. After cooling, the mixture was centrifuged, washed multiple times with DMF and methanol, and then vacuum dried to obtain S-MET(Fe) in different proportions.

[0028] Here, different ratios refer to different ratios of the two ligands, 1H-1,2,3-triazole and 5-mercapto-1,2,3-triazole monosodium salt, with a total molar amount of 2.4 mmol. The ratio settings are shown in Table 1.

[0029] Table 1. Ratio of 5-mercapto-1,2,3-triazole monosodium salt The overall morphology of the nanozyme was characterized using TEM. MET(Fe) consists of five Fe metal centers connected by bridging 1H-1,2,3-triazole to form octahedral units with a size of approximately 100-130 nm. Figure 2 As shown in (a), compared to MET(Fe), the size of S-MET(Fe) is smaller, approximately 50-60 nm, as... Figure 2 As shown in (b).

[0030] The differences in the crystal structures of the synthesized MET(Fe) and S-MET(Fe) nanozymes were confirmed and compared using PXRD. The PXRD patterns are shown below. Figure 3 As shown in (a), after doping with a sulfur-containing ligand (5-mercapto-1,2,3-triazole monosodium salt), the S-MET(Fe) diffraction peak positions did not shift, retaining the crystal structure of MET(Fe). However, as the proportion of sulfur-containing ligands in the total ligand content gradually increased, the intensity of the diffraction peaks gradually decreased. The TGA pattern is shown below. Figure 3 As shown in (b), the S-MET(Fe) framework collapses at 240℃ after being heated, while MET(Fe) begins to collapse at around 300℃, indicating that the thermal stability of the S-MET(Fe) framework is reduced. This may be attributed to some defects brought about by sulfur-containing ligand doping. Example 2: Enzyme Activity Study The POD-like activity of MET(Fe) and S-MET(Fe) nanozymes with different ligand ratios prepared in Example 1 was studied using TMB and H2O2 as substrates. The activity was quantified by the absorbance of oxTMB at 652 nm. Specifically, an acetate buffer (0.2 mol / L, pH = 4.0) containing 5 μg / mL nanozyme, 0.2 mmol / L TMB, and 0.2 mmol / L H2O2 was incubated at room temperature for 10 min. The absorbance of the reaction solution at 652 nm was then measured using UV-vis. The results are shown below. Figure 4 As shown in the figure. The results indicate that S-MET(Fe) nanozymes, especially those with 5-mercapto-1,2,3-triazole monosodium salt ratios of 1 / 5, 1 / 3, 1 / 2, and 2 / 3, exhibit higher catalytic activity than MET(Fe) nanozymes.

[0031] Compared with other reported nanozymes (see Table 2), S-MET(Fe) has a lower Kc. m The value indicates that S-MET(Fe) has a higher affinity for the substrate and higher catalytic efficiency, suggesting that S doping in S-MET(Fe) nanozymes can enhance the affinity for the substrate, thereby improving POD-like activity.

[0032] Table 2 Comparison of Km values ​​between S-MET(Fe) nanozymes and other reported nanozymes Furthermore, previously reported sulfur-doped nanozymes, such as metal sulfides, nitrogen-sulfur co-doped carbon materials, and sulfur-doped lignin-derived carbon nanosheets, all require calcination synthesis with strictly controlled calcination temperatures between 400 and 1000°C. This results in high manufacturing costs and unstable reaction processes, making them unsuitable for industrial production. The S-MET(Fe) nanozyme prepared in this invention can complete the reaction at temperatures below 130°C, and its synthesis steps are simple, easy to control, and the reaction process is stable, thus solving the problems of manufacturing difficulties and high costs to a certain extent.

[0033] In Examples 3-8, S-MET(Fe) nanozymes all refer to S-MET(Fe) nanozymes when the proportion of sulfur-containing ligands in the mixed ligands is 1 / 3.

[0034] Example 3 Conditional optimization and steady-state dynamics To optimize the catalytic conditions for MET(Fe) and S-MET(Fe), the pH of the acetate buffer solution and the incubation temperature were optimized.

[0035] pH optimization: In a system containing 5 μg / mL MET(Fe) or S-MET(Fe), 0.1 mmol / L TMB, and 0.2 mmol / L H2O2, the pH of the acetate buffer was varied (3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0). After incubation in a 25°C water bath for 20 min, the absorbance of the reaction solution at 652 nm was measured using UV-vis. Incubation temperature optimization: In an acetate buffer solution at pH = 4.0 containing 5 μg / mL MET(Fe) or S-MET(Fe), 0.1 mmol / L TMB, and 0.2 mmol / L H2O2, the incubation temperature was varied (25, 30, 37, 40, 50, 60, and 70°C). After incubation for 15 min, the absorbance of the reaction solution at 652 nm was measured using UV-vis.

[0036] Comprehensive experimental results ( Figure 5As can be seen, S-MET(Fe) and MET(Fe) are similar to natural HRP, with optimal pH and temperature of pH=4 and 40℃, respectively.

[0037] Steady-state kinetics analysis was performed on MET(Fe) and S-MET(Fe): In an acetate buffer solution containing 5 μg / mL MET(Fe) or S-MET(Fe) at pH = 4.0, when TMB was used as the substrate, the concentration of H₂O₂ was kept constant at 1.5 mM while varying the concentration of TMB; when H₂O₂ was used as the substrate, the concentration of TMB was kept constant at 2 mmol / L while varying the concentration of H₂O₂. (Summary) Figure 6 , 7 It can be seen that S doping in S-MET(Fe) nanozymes can not only enhance the affinity for substrates, but also improve POD-like activity.

[0038] Example 4 Free radical capture experiment During the experiment, thiourea (TH) was selected as the ·OH scavenger, and benzoquinone (1,4-Benzoquinone) was used as the scavenger. p -BQ) is O2 •- The cleaning agent. The results were as follows: Figure 8 As shown in (a) and (b), TH is introduced and the pair is... p After -BQ, the absorbance of oxTMB in the MET(Fe)-TMB-H2O2 and S-MET(Fe)-TMB-H2O2 systems decreased, and with TH, p -BQ concentration increases, leading to a decrease. Under N2 conditions, the change in oxTMB in the MET(Fe)-TMB-H2O2 system is more pronounced than that in the S-MET(Fe)-TMB-H2O2 system. The results indicate that the catalytic activity of MET(Fe) and S-MET(Fe) is mainly due to the interaction of ·OH and O2. •- Its function.

[0039] 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) is used as O2. •- In addition, ·OH radical scavengers were used to detect potential ROS in the MET(Fe)-TMB-H2O2 and S-MET(Fe)-TMB-H2O2 systems. Combined with ESR technology to detect the two types of free radicals generated during the reaction, the presence of ·OH and O2 radicals was verified. •- The generation of free radicals, and the fact that S-MET(Fe) has a higher signal value than MET(Fe), indicates that S-MET(Fe) has stronger catalytic ability and generates more ROS. The results are as follows... Figure 8 As shown in (c) and (d).

[0040] Example 5 quenching kinetics experiment To investigate the effect of ascorbic acid (AA) on the catalytic activity of S-MET(Fe)-based PODs, quenching kinetics experiments were conducted. The results are as follows: Figure 9 As shown in (a), the red line represents the kinetic curve of S-MET(Fe) without the addition of AA. As shown by the blue line, after two minutes of reaction, the addition of AA to the TMB-H2O2 catalytic system resulted in a sudden decrease in absorption at 652 nm, corresponding to the blue solution of the catalytic system turning into a colorless solution, indicating that AA can reduce oxTMB to TMB. The black line in the figure indicates that if AA is added at the beginning of the reaction, the oxidation of TMB gradually begins after 6 minutes, indicating that AA consumes ROS in the catalytic system to inhibit the oxidation of TMB.

[0041] Example 6 Construction of an ascorbic acid colorimetric sensing platform based on the S-MET(Fe)-TMB-H2O2 system A highly sensitive AA sensing platform was constructed. Different concentrations of AA solution were added to an acetate buffer containing 10 μg / mL S-MET(Fe), 0.2 mmol / L TMB, and 0.2 mmol / L H₂O₂. After incubating the mixture in a 25°C water bath for 10 min, the absorbance at 652 nm was measured using UV-Vis. The absorbance change ΔA = A₀ - A, where A₀ and A represent the absorbance values ​​without and with AA, respectively. The LOD calculation formula is... The result is as follows Figure 9 As shown in (b) and (c), the results indicate that this detection method exhibits good linearity when the concentration of AA solution is in the range of 2.5–80 μmol / L, with a detection limit of 1.072 μmol / L.

[0042] Cyclic and storage stability of S-MET(Fe) nanozymes The cyclability of S-MET(Fe) nanozymes was evaluated. In an acetate buffer (0.2 mol / L, pH = 4.0) containing 1 mg / mL S-MET(Fe), 0.4 mmol / L TMB and 0.4 mmol / L H₂O₂ were added to a final concentration. After incubation at room temperature for 3 minutes, the mixture was immediately centrifuged. The upper blue liquid was filtered through a membrane, and the filtrate was diluted 4-fold before absorbance measurement at 652 nm. The lower nanomaterial layer was washed twice with anhydrous ethanol by centrifugation, vacuum dried, and then reused in the next catalytic reaction. This cycle was repeated for a total of 5 cycles. Figure 10As shown in (a), after 5 cycles of POD-like catalysis, the enzyme maintained high activity, with the relative activity remaining above 90%, indicating that S-MET(Fe) has good recyclability.

[0043] In addition, the storage stability of S-MET(Fe) as a POD-like nanozyme was investigated. The solid powder was stored at room temperature for 15 days, and its POD-like activity was tested at intervals. An acetate buffer solution (0.2 mol / L, pH = 4.0) containing 5 μg / mL nanozyme, 0.2 mmol / L TMB, and 0.2 mmol / L H2O2 was incubated at room temperature for 10 min. The absorbance of the reaction solution at 652 nm was then measured using UV-vis. Figure 10 As shown in (b), after S-MET(Fe) was stored at room temperature for 15 days, the decrease in its POD-like activity was small, and the relative activity remained above 90%, indicating that the material has good storage stability.

[0044] Example 7 Application of S-MET(Fe) in the fight against Gram-negative bacteria Based on the above studies on the ROS-generating abilities of MET(Fe) and S-MET(Fe), their effects on Gram-negative bacteria (such as...) were further investigated. E. coli) The antibacterial properties. Culture: Thaw the bacterial culture stored at -80℃ at room temperature, then use an inoculation loop to take a small amount of the bacterial solution and streak it overnight. Pick plates and incubate overnight. E. coli Single colonies were reconstituted into Luria-Bertani (LB) liquid medium and incubated overnight at 37°C with continuous shaking. Bacteria were collected and re-inoculated into sterile LB liquid medium, then incubated at 37°C and 180 rpm until the logarithmic growth phase. The diluted bacterial solutions were treated with a control group (acetic acid buffer), H2O2, MET(Fe), S-MET(Fe), MET(Fe), H2O2, S-MET(Fe), and H2O2, respectively, for two hours. After further dilution, 100 μL of the bacterial solution was evenly spread on the surface of a solid medium and incubated overnight at 37°C. Colony counts were then observed and determined.

[0045] Antibacterial results such as Figure 11 As shown in (a), a weakly acidic environment (pH=4.0) does not interfere with the normal growth of Escherichia coli, and similarly, H2O2 alone does not have a significant antibacterial effect. However, under the same experimental conditions, S-MET(Fe) exhibits significant antibacterial activity in the presence of low concentrations of H2O2.

[0046] Example 8 Application of S-MET(Fe) in combating Gram-positive bacteria In addition, tests were also conducted on Gram-positive bacteria (such as...) S. aureus To assess the antibacterial properties of the strain, the following methods were employed: Thaw the strain stored at -80°C at room temperature, then use an inoculation loop to take a small amount of the bacterial solution and streak it overnight. Plates were then picked and incubated overnight. E. coli Single colonies were reconstituted into Luria-Bertani (LB) liquid medium and incubated overnight at 37°C with continuous shaking. Bacteria were collected and re-inoculated into sterile LB liquid medium, then incubated at 37°C and 180 rpm until the logarithmic growth phase. The diluted bacterial solutions were treated with a control group (acetic acid buffer), H2O2, MET(Fe), S-MET(Fe), MET(Fe), H2O2, S-MET(Fe), and H2O2, respectively, for two hours. After further dilution, 100 μL of the bacterial solution was evenly spread on the surface of a solid medium and incubated overnight at 37°C. Colony counts were then observed and determined.

[0047] Antibacterial results such as Figure 11 As shown in (b), S-MET(Fe) in the presence of low concentration H2O2... S. aureus It also exhibits significant antibacterial effects.

[0048] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a heteroatom-containing ligand MOF nanozyme, characterized in that, Includes the following steps: (a) N,N-dimethylformamide was added to the reaction vessel, nitrogen gas was purged, and then ferrous chloride tetrahydrate was added and ultrasonically dispersed. (b) Add a mixed ligand salt and disperse it ultrasonically. The mixed ligand salt is a 1H-1,2,3-triazole ligand salt and a sulfur atom ligand salt. The sulfur atom ligand salt is a 5-mercapto-1,2,3-triazole monosodium salt. The molar ratio of the 1H-1,2,3-triazole ligand salt and the 5-mercapto-1,2,3-triazole monosodium salt is 1:(0.25~4). (c) Seal the reaction vessel and stir continuously at 110℃~130℃. After the reaction is completed, cool to room temperature, centrifuge to obtain the precipitate, wash the precipitate and vacuum dry it to obtain MOF nanozyme containing heteroatom ligands.

2. The production method according to claim 1, characterized by, In step (a), nitrogen is blown for 15–25 minutes.

3. The production method according to claim 1, characterized by, The molar ratio of ferrous chloride tetrahydrate to the mixed ligand salt is 1:

3.

4. The method of claim 1, wherein, The reaction time in step (c) is 12–24 h.

5. The preparation method according to claim 1, characterized in that, The washing method in step (c) is: washing with DMF and methanol.

6. The MOF nanozyme containing heteroatom ligands prepared by any one of claims 1 to 5.

7. The application of the heteroatom-containing MOF nanozyme as described in claim 6 in the colorimetric detection of ascorbic acid.

8. The application of the MOF nanozyme containing heteroatom ligands as described in claim 7 in antibacterial agents.

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