Platinum cluster with nano-enzyme and visible light catalysis function as well as preparation method and application of platinum cluster

Abstract

The invention belongs to the technical field of functional metal cluster preparation, and particularly relates to a platinum cluster with nano-enzyme and visible light catalysis functions and a preparation method and application thereof. The platinum cluster takes 6-aza-2-thiothymine as a ligand and platinum salt as a precursor, arginine is added to modify the ligand, and the platinum cluster is synthesized through a hydrothermal method. The platinum cluster is suitable for an environment with a pH value of 3-7.4. The platinum cluster disclosed by the invention has relatively high peroxidase activity and visible light catalytic activity.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional metal cluster preparation, and specifically relates to a platinum cluster with nanozyme and visible light catalytic functions, and a preparation method and application thereof. Background Art

[0002] Ultrasmall metal nanoclusters (1nm-3nm) have recently shown promising applications in metal catalysis due to their abundant active sites, large surface area, and strong quantum confinement. Among them, green and nontoxic platinum nanoclusters (Pt NCs) boast not only excellent biocompatibility but also tunable catalytic activity, and are frequently used in photodynamic therapy, electrocatalysis, and chemical catalysis. The unique core-shell structure of Pt NCs is similar to that of natural enzymes, enabling them to exhibit enzyme-like activity. Currently, Pt NCs have been shown to exhibit catalytic activity similar to that of various enzymes, including superoxide dismutase, oxidase, and peroxidase. However, the catalytic activity of most Pt NCs is relatively low and, like natural enzymes, is significantly affected by environmental factors. For example, the natural enzyme horseradish peroxidase is inactive at pH 3, its activity drops to approximately 40% at pH 6, and is less than 20% at pH 7. Pt NCs exhibit considerable peroxidase-like activity in acidic environments between pH 3 and 4, but their activity is negligible under neutral and near-neutral conditions between pH 5 and 7.4. Although optimizing the cluster structure can improve the catalytic activity of Pt NCs to some extent, such regulation is often fraught with uncertainty. Designing nanoclusters with multimodal catalytic functions is considered an effective strategy to enhance cluster catalytic activity. Therefore, incorporating efficient photocatalysis into Pt NC systems with enzymatic catalytic activity is of great significance for promoting the practical application of metal clusters in catalysis.

[0003] To obtain Pt NCs with multimodal catalytic activity, the synthesis process typically requires strict control of the selection and dosage of reducing agents and ligands to regulate the composition, size, and shape of the clusters. When sodium borohydride is used as the classic reducing agent, its excessively strong reducing power rapidly converts high-valent Pt ions into zero-valent Pt atoms. The strong catalytic activity of the Pt atoms themselves further accelerates the reduction process, leading to their own in situ deposition or catalytic decomposition of the reducing agent, resulting in uncontrollable synthesis of Pt NCs. Furthermore, the high surface energy of ultrasmall Pt NCs leads to a strong tendency to aggregate, which inevitably reduces the specific surface area of the clusters and, consequently, eliminates their catalytic activity. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a platinum cluster with nanozyme and visible light catalytic functions, as well as its preparation method and application. A platinum cluster with high peroxidase catalytic activity and visible light catalytic activity in the pH range of 3-7.4 is synthesized by a simple hydrothermal method, which has high application value in the field of catalysis.

[0005] To achieve the above object, the present invention adopts the following technical solutions: A platinum cluster with nanozyme and visible light catalytic functions, wherein the platinum cluster is synthesized by a hydrothermal method using 6-aza-2-thiothymine as a ligand and a platinum salt as a precursor, and arginine is added to modify the ligand; The platinum cluster is suitable for an environment with a pH value of 3 to 7.4.

[0006] The present invention utilizes 6-aza-2-thiothymine as a ligand and adds arginine to modify the ligand, resulting in platinum clusters that maintain good enzymatic activity in the pH range of 3 to 7. 6-aza-2-thiothymine not only serves as a ligand but also acts as a reducing agent and stabilizer, thus avoiding the uncontrollable synthesis of Pt NCs caused by the strong reducing ability of sodium borohydride as a reducing agent. The Pt-S bond formed between the platinum salt and 6-aza-2-thiothymine exhibits excellent strength and anti-interference properties, while platinum also exhibits good corrosion resistance. This allows the resulting platinum clusters to maintain good catalytic activity across a wide range of pH values, expanding the pH range of platinum cluster applications. Furthermore, the platinum salts and nitrogen and sulfur atoms in the 6-aza-2-thiothymine molecule possess lone pairs of electrons, which can form coordination bonds with platinum atoms. This coordination allows 6-aza-2-thiothymine to be stably adsorbed on the surface of the platinum clusters, preventing aggregation of the cluster particles and thus improving their dispersibility. Arginine forms hydrogen bonds with the 6-aza-2-thiothymine molecules on the Pt surface, increasing the rigidity of the structure and forming a more stable spatial configuration. This further enhances the coordination stabilization of the platinum clusters, allowing them to maintain a well-dispersed state in different environments and impart multimodal catalytic capabilities, namely, dual nanozyme and visible light catalysis, effectively addressing the problem of reduced catalytic activity due to aggregation.

[0007] In another preferred embodiment, the platinum salt is chloroplatinic acid; The mass ratio of the 6-aza-2-thiothymine, the chloroplatinic acid and the arginine is 5.2-6.2:2.8-3.2:7.9-8.9.

[0008] In another preferred embodiment, the particle size of the platinum clusters is 1 nm to 1.4 nm.

[0009] The second aspect of the present invention provides a method for preparing the platinum cluster having nanozyme and visible light catalytic function, characterized in that it comprises the following steps: 6-aza-2-thiothymine and chloroplatinic acid are stirred and reacted in an aqueous environment at 30° C. to 40° C. for 0.75 to 1.25 hours, and then sodium hydroxide is added and reacted for 22 to 26 hours to obtain a primary product solution; the mass ratio of the 6-aza-2-thiothymine to chloroplatinic acid and sodium hydroxide is 5.2 to 6.2:2.8 to 3.2:5.1 to 6.1; the mass volume ratio of the 6-aza-2-thiothymine to the water is 5.2 mg to 5.7 mg:7.3 mL to 7.7 mL; The initial product solution was dialyzed, the dialyzate was centrifuged, and the supernatant was collected. Arginine was added and the mixture was reacted at 30° C. to 40° C. for 22 h to 26 h to obtain the platinum clusters.

[0010] In another preferred embodiment, the dialysis uses a 3500Da dialysis bag.

[0011] The third aspect of the present invention provides the use of the platinum cluster in a photocatalytic reaction.

[0012] In another preferred embodiment, the substrates of the photocatalytic reaction are levodopa and 3,3',5,5'-tetramethylbenzidine.

[0013] The fourth aspect of the present invention provides the use of the platinum cluster in the oxidation reaction of 3,3',5,5'-tetramethylbenzidine.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses a simple hydrothermal method to co-modify 6-aza-2-thiothymine and arginine to synthesize platinum clusters with both nanozyme and visible light dual catalytic functions. The 6-aza-2-thiothymine molecules can form Pt-S coordination bonds with platinum atoms in platinum salts, which not only prevents agglomeration, but also has high strength and anti-interference properties. The Pt-S bond can exhibit good activity in the pH range of 3 to 7. The ligand is further modified by adding arginine to increase the rigidity of the structure, resulting in a more stable spatial structure and further improving the stability of the platinum cluster, resulting in a platinum cluster with both nanozyme and visible light catalytic functions.

[0015] Compared with horseradish peroxidase, the platinum clusters in the present invention not only show a higher substrate affinity Km of 0.098 mM and a faster reaction rate of 27.8×10 -8M / s, and still has a catalytic activity of about 80% at a neutral acidity of pH 6, and the average catalytic activity is above 50% in the neutral to neutral acidity range of pH 6 to 7; in addition, the platinum clusters in the present invention also exhibit considerable photocatalytic activity. Under visible light irradiation, TMB can be directly catalyzed and oxidized to oxTMB without the participation of hydrogen peroxide. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Figures 1 and 2 show the AFM, TEM, and XPS images and particle size statistics of L-Arg / ATT-Pt NCs prepared in the present invention. (a) shows the AFM image of L-Arg / ATT-Pt NCs, (b) shows the cross-sectional height of the line corresponding to (a), (c) shows the TEM image of L-Arg / ATT-Pt NCs, where c1 is an enlarged image of the platinum cluster, (d) shows the particle size statistics of L-Arg / ATT-Pt NCs, (e) shows the full XPS spectrum of L-Arg / ATT-Pt NCs, and (f) shows the fine spectrum of Pt 4f orbitals. L-Arg / ATT-Pt NCs represents platinum clusters.

[0017] Figure 2 The diagram shows the release analysis of the L-Arg / ATT-Pt NCs of the present invention simulating the peroxidase properties; wherein, (a) is the absorption diagram after TMB is treated with different systems, and a1 in the figure represents the photos of the solution color changes corresponding to the different systems, from left to right are the photos of the TMB system, the L-Arg / ATT-Pt NCs system, the L-Arg / ATT-Pt NCs+TMB system, the H2O2+TMB system, and the H2O2+L-Arg / ATT-Pt NCs+TMB system; (b) is the absorption diagram of TMB and H2O2 changing with time in the presence of L-Arg / ATT-PtNCs, and the curves of different colors in the figure represent the changes in absorbance values at different times, and the times from bottom to top are 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, and 10 min, respectively. b1 in the figure is the absorption diagram of the solution after L-Arg / ATT-Pt NCs is added to the TMB and H2O2 mixture. Photos before and after NCs, where the left one is the photo without adding L-Arg / ATT-Pt NCs, and the right one is the photo after adding L-Arg / ATT-Pt NCs.

[0018] Figure 3Graphs showing changes in the enzyme catalytic activity of L-Arg / ATT-Pt NCs of the present invention with pH and storage time; wherein, (a) is a graph showing changes in the enzyme catalytic activity of L-Arg / ATT-Pt NCs with pH, and (b) is a graph showing changes in the enzyme catalytic activity of L-Arg / ATT-Pt NCs with storage time.

[0019] Figure 4 These are steady-state kinetic analysis diagrams of the oxidation of TMB by L-Arg / ATT-Pt NCs of the present invention in the presence of H2O2; wherein, (a) is a steady-state kinetic analysis diagram for different TMB concentrations, (b) is a steady-state kinetic analysis diagram for different H2O2 concentrations, (c) is a double reciprocal graph of (a), and (d) is a double reciprocal graph of (b).

[0020] Figure 5 These are diagrams showing the visible light catalytic effects of L-Arg / ATT-Pt NCs of the present invention on different substrates; wherein, (a) is an absorption diagram of the TMB reaction catalyzed by L-Arg / ATT-Pt NCs under xenon lamp irradiation, and (b) is an absorption diagram of the DOPA reaction catalyzed by L-Arg / ATT-Pt NCs under xenon lamp irradiation changing with time. Curves of different colors represent the corresponding absorption changes under different irradiation times, and the times from bottom to top are 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min, respectively; b1 is a photograph of the color change of the solution before and after xenon lamp irradiation, the left side is a photograph without irradiation, and the right side is a photograph after 30 minutes of irradiation. DETAILED DESCRIPTION

[0021] The technical solutions of the present invention are clearly and completely described in conjunction with the specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0022] Unless otherwise specified, the methods described in the embodiments of the present invention are conventional methods. The materials and reagents used are all commercially available unless otherwise specified.

[0023] The present invention is a ligand by 6-aza-2-thiothymine, and arginine is used to modify the ligand, so that the platinum clusters obtained are still have good enzymatic activity in the pH value of 3~7.4 scope, wherein the intensity and anti-interference performance of the Pt-S bond formed by platinum salt and 6-aza-2-thiothymine are good, and platinum has good corrosion resistance, so that the platinum clusters obtained can still maintain good catalytic activity under different pH values. In addition, the nitrogen, sulfur and other atoms in platinum salt and 6-aza-2-thiothymine molecules have lone pairs of electrons and can form coordination bonds with platinum atoms. This coordination allows 6-aza-2-thiothymine to be stably adsorbed on the platinum cluster surface, prevents the aggregation of platinum cluster particles, thereby improving its dispersibility. Arginine forms hydrogen bonds with the 6-aza-2-thiothymine molecules on the Pt surface, which increases the rigidity of the structure and forms a more stable spatial configuration, further enhancing the coordination stabilization effect on the platinum clusters, allowing the platinum clusters to maintain a good dispersion state in different environments, thereby effectively avoiding the problem of reduced catalytic activity due to agglomeration.

[0024] The Pt-S bond formed by the platinum cluster obtained in the present invention has higher strength and anti-interference ability than other metals, such as the Au-S bond formed by replacing platinum salt with gold salt and 6-aza-2-thiothymine. In addition, platinum has stronger corrosion resistance than gold, thereby being able to achieve excellent catalytic activity in an environment with a pH value of 3 to 7.4.

[0025] 6-Aza-2-thiothymidine was purchased from Shanghai Bidex Pharmaceutical Technology Co., Ltd., tetrachloroplatinic acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., arginine was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., 3,3',5,5'-tetramethylbenzidine was purchased from Sangon Biotechnology Co., Ltd., and L-dopa was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0026] 6-Aza-2-thiothymine was denoted as ATT; 3,3',5,5'-tetramethylbenzidine was denoted as TMB.

[0027] Example 1 A method for preparing platinum clusters having nanozymes and visible light catalytic functions comprises the following steps: S1. Dissolve 5.71 mg of ATT in 7.5 mL of ultrapure water under ultrasonication at 500 W for 3 min. Add 160 μL of 50 mM tetrachloroplatinic acid and stir in a 35°C water bath for 1 h. Then, add 800 μL of 175 mM sodium hydroxide and react for 24 h to obtain the initial product solution, which is recorded as ATT-Pt NCs.

[0028] S2. The initial product solution was placed in a 3500Da dialysis bag and dialyzed for purification for 24 hours, with water changed every 4 hours, repeated 4 times, and the last water change was followed by overnight dialyzation to obtain a dialyzed solution.

[0029] S3. Centrifuge the dialyzed solution, take the supernatant, add 100 μL of 40 mM arginine to 1 mL of the supernatant, and react at 35°C for 24 h to obtain platinum clusters with both nanozyme and visible light dual catalytic functions, which are recorded as L-Arg / ATT-Pt NCs.

[0030] The excellent peroxidase catalytic and visible light catalytic properties of L-Arg / ATT-Pt NCs were characterized by UV-visible absorption spectroscopy, Michaelis equation, and photocatalytic experiments.

[0031] Figure 1 AFM, TEM and XPS images of L-Arg / ATT-Pt NCs prepared in the present invention and the corresponding particle size statistics. From the particle size statistics of TEM and AFM, it can be seen that the diameter of L-Arg / ATT-Pt NCs is 1.2±0.2nm. In the wide scan measurement of XPS, the peak at 73.23eV corresponds to Pt 4f, revealing the presence of Pt element in the final product. By peak fitting the fine XPS spectrum, it can be found that the Pt 4f peak is caused by Pt 0 and Pt 2+ The cluster is composed of characteristic peaks of , which means that the cluster contains zero-valent Pt and divalent Pt. Further quantitative calculation results show that the proportions of the two are 89.8% and 10.2%, respectively.

[0032] Figure 2 Results show the use of L-Arg / ATT-Pt NCs to mimic the properties of peroxidase. Different systems were prepared and their absorbance was measured: TMB, L-Arg / ATT-Pt NCs, L-Arg / ATT-Pt NCs + TMB, H₂O₂ + TMB, and H₂O₂ + L-Arg / ATT-Pt NCs + TMB. The volume of each system was 2.5 mL. The concentration of TMB in each system was 0.5 mM, the concentration of H₂O₂ was 30 mM, and the amount of L-Arg / ATT-Pt NCs used was 20 mL.

[0033] from Figure 2 (a) The changes in the absorption spectra of different systems and the corresponding solution color changes show that L-Arg / ATT-Pt NCs can simulate peroxidase to oxidize TMB to produce a strong characteristic absorption peak at 652 nm, and the H2O2 and TMB mixture changes from colorless to blue, as shown in Figure 2. Figure 2 As shown in (b) in .

[0034] Figure 3 The figure shows the changes of enzyme catalytic activity of L-Arg / ATT-Pt NCs with pH and storage time. Figure 3 As can be seen from (a), the platinum clusters of the present invention exhibit high catalytic activity in an acidic environment. Compared with the natural enzyme horseradish peroxidase, which has no catalytic activity at pH 3, its catalytic activity drops to about 40% at pH 6, and its catalytic activity is less than 20% at pH 7, L-Arg / ATT-Pt NCs have high catalytic activity in a wider pH range. This is mainly because after being modified with dual ligands, the rigidity of L-Arg / ATT-Pt NCs is greatly improved, which enhances its stability and is less affected by the environment. This greatly broadens the scope of application of platinum clusters, especially in weakly acidic and near-neutral environments close to physiological conditions and tumor environments. In addition, Figure 3 (b) shows the activity results of L-Arg / ATT-Pt NCs at 4°C. As can be seen from the figure, at 4°C, the activity of L-Arg / ATT-Pt NCs is not affected over time, and it still has more than 95% catalytic activity after being stored for half a year.

[0035] 1. Nanozyme catalytic activity of L-Arg / ATT-Pt NCs The peroxidase catalytic activity of L-Arg / ATT-Pt NCs was evaluated using a TMB oxidation reaction model in the presence of H₂O₂. The catalytic performance was calculated by UV-visible absorption spectroscopy, recording the change in oxTMB absorbance at 652 nm. Specifically, 20 μL of L-Arg / ATT-Pt NCs was added to 2.3 mL of pH buffer (0.2 M, pH 4.0) at room temperature. The experiment was performed by varying the amounts of H₂O₂ (0–60 mM) and TMB (0.05–1.0 mM). The absorbance at 652 nm was recorded over increasing reaction time intervals (0–10 min). The initial reaction rate was calculated using the Beer-Lambert law: c = A / (εb), where c is the substrate concentration, A is the absorbance, and ε is the molar absorption coefficient of the colorimetric substrate used (39000 M at 652 nm for oxTMB). −1 cm −1 The reaction rate data were fitted using the Michaelis-Menten equation to determine the enzymatic parameters.

[0036] .

[0037] Where V is the initial reaction rate, V max is the maximum reaction rate, [S] is the substrate concentration. K mis the Michaelis-Menten constant, which is directly related to the affinity of the nanozyme for the substrate.

[0038] Finally got Figure 4 and Table 1, from which we can determine the K of L-Arg / ATT-Pt NCs when TMB is used as substrate m 0.098mM, V max 27.8×10 -8 M / s, it has a faster reaction rate and higher substrate affinity than the natural enzyme horseradish peroxidase.

[0039] Table 1 Michaelis-Menten parameters of L-Arg / ATT-Pt NCs Note: HRP stands for horseradish peroxidase.

[0040] 2. Visible light photocatalytic performance of L-Arg / ATT-Pt NCs It is the same as the enzyme catalysis system except that H2O2 is no longer used. Instead, the catalytic reaction is carried out by irradiation with a xenon lamp.

[0041] 200 μL of L-Arg / ATT-Pt NCs were added to 2 mL of levodopa (2 mM, pH = 7.0). After irradiation with a xenon lamp for 1 h, the changes in oxTMB absorption at 652 nm were monitored by UV-visible absorption spectroscopy. Figure 5 The figure shows the visible light catalytic effect of the platinum clusters of the present invention on different substrates. The results show that in the absence of H2O2, through photocatalysis, L-Arg / ATT-Pt NCs can not only oxidize TMB into oxTMB, but also convert L-DOPA into dopachrome, which means that the platinum clusters of the present invention have significant universality and can still have a good catalytic effect under neutral conditions.

[0042] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, it is of course possible to easily make other implementation methods by replacing or changing the technical content disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A platinum cluster with nanozyme and visible light catalytic function, characterized in that: The platinum cluster is synthesized by a hydrothermal method using 6-aza-2-thiothymine as a ligand, platinum salt as a precursor, and arginine to modify the ligand; The platinum cluster is suitable for an environment with a pH value of 3 to 7.

4.

2. The platinum cluster having nanozyme and visible light catalytic function according to claim 1, characterized in that: The platinum salt is chloroplatinic acid; The mass ratio of the 6-aza-2-thiothymine, the chloroplatinic acid and the arginine is 5.2-6.2:2.8-3.2:7.9-8.

9.

3. The platinum cluster having nanozyme and visible light catalytic function according to claim 2, characterized in that: The particle size of the platinum clusters is 1 nm to 1.4 nm.

4. A method for preparing platinum clusters having nanozymes and visible light catalytic functions according to claim 3, characterized in that: The following steps are involved: 6-aza-2-thiothymine and chloroplatinic acid are stirred and reacted in an aqueous environment at 30°C to 40°C for 0.75h to 1.25h, sodium hydroxide is added, and the reaction is carried out for 22h to 26h to obtain a primary product solution; the mass ratio of 6-aza-2-thiothymine, chloroplatinic acid, and sodium hydroxide is 5.2-6.2:2.8-3.2:5.1-6.1; The initial product solution was dialyzed, the dialyzate was centrifuged, and the supernatant was collected. Arginine was added and the mixture was reacted at 30° C. to 40° C. for 22 h to 26 h to obtain the platinum clusters.

5. The method for preparing platinum clusters having nanozymes and visible light catalytic functions according to claim 3, characterized in that: The dialysis was performed using a 3500Da dialysis bag.

6. Use of the platinum cluster according to any one of claims 1 to 3 in a photocatalytic reaction.

7. The use according to claim 6, characterized in that The substrates of the photocatalytic reaction are levodopa and 3,3',5,5'-tetramethylbenzidine.

8. Use of the platinum cluster according to any one of claims 1 to 3 in the oxidation reaction of 3,3',5,5'-tetramethylbenzidine.

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