Carbon-based platinum monatomic mimic enzyme as well as preparation method and application thereof

Abstract

The invention relates to a carbon-based platinum monatomic mimic enzyme as well as a preparation method and application thereof. The preparation method of the carbon-based platinum monatomic mimic enzyme comprises the following steps: mixing chloroplatinic acid, carbon quantum dots and ascorbic acid at 25-26 DEG C for reaction, and dialyzing and purifying to obtain the carbon-based platinum monatomic mimic enzyme. Platinum is completely loaded on carbon quantum dots in a monatomic form by adopting an ascorbic acid reduction method, and compared with platinum existing in a monatomic, nanocluster or nanoparticle form in a contrast ratio 1 and a contrast ratio 2, the SOD enzyme activity of the carbon-based platinum monatomic mimic enzyme existing in the monatomic form in the embodiment 1 is higher, and the SOD enzyme activity of the carbon-based platinum monatomic mimic enzyme in the embodiment 2 is higher than that of the platinum existing in the contrast ratio 1 and the contrast ratio 2. The superoxide anions can be better removed. It can be seen from the figure 13 that the SOD enzyme activity of the carbon quantum dots not loaded with the platinum single atoms is 1695 U / mg, the SOD enzyme activity of the carbon quantum dots loaded with the platinum single atoms is 6649 U / mg, and after the carbon quantum dots and the platinum single atoms are compounded, the SOD enzyme activity is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanozyme preparation, and particularly relates to a carbon-based platinum single-atom mimetic enzyme, a preparation method thereof, and an application thereof. Background Art

[0002] Enzymes, as exquisitely ingenious biocatalysts in nature, are renowned for their excellent catalytic selectivity and remarkable ability to accelerate chemical reactions. However, natural enzymes face numerous challenges in practical applications: high preparation and purification costs, limited stability, short storage life, and high sensitivity to environmental conditions (such as temperature, pH value, etc.). These inherent defects severely restrict their widespread application and further development. To break through these bottlenecks, the scientific research community has made great efforts to explore and develop a new type of catalyst - enzyme mimics, which are required to have not only high stability, cost-effectiveness, and simplicity of synthesis, but also controllability of catalytic activity. Against this background, nanozymes have emerged as a bright star in the field of enzyme mimics. Nanozymes, an innovative product integrating the excellent properties of nanomaterials and the catalytic activity of natural enzymes, exhibit unique dual charm. They not only inherit the characteristics of nanomaterials such as small size effect, high specific surface area, good biocompatibility, and easy modification, but also endow stability and environmental adaptability that are difficult to achieve by traditional enzymes. These advantages have enabled nanozymes to shine in multiple fields, including but not limited to biomedicine, chemical industry, food safety assurance, modern agriculture, and environmental pollution control, becoming an important force to promote technological innovation and industrial upgrading in these fields.

[0003] Carbon quantum dots (CDs), a cutting-edge nanomaterial, exhibit extraordinary potential in the fields of biomedicine and catalysis due to their excellent biocompatibility and extremely low toxicity characteristics. The abundant dangling bonds (such as hydroxyl, carbonyl, amino groups, etc.) densely distributed on the surface of carbon quantum dots not only endow carbon quantum dots with rich chemical active sites, but also greatly enhance their surface energy, providing an ideal platform for the firm riveting and efficient exposure of metal active sites. It is precisely these unique surface characteristics that enable carbon quantum dots to exhibit extraordinary catalytic activity when catalyzing various biochemical reactions. The close combination of metal active sites with the surface of carbon quantum dots not only promotes the effective adsorption and activation of reactant molecules, but also accelerates the reaction path and improves the reaction efficiency. Therefore, carbon quantum dots, as a catalyst carrier or itself as a catalyst, exhibit broad application prospects in promoting complex chemical reactions in vivo and in vitro, optimizing biological processes, and environmental governance.

[0004] Among them, due to its unique structural characteristics, single-atom catalysts often exhibit high selectivity for certain chemical reactions. The invention patent with the application publication number CN115957323A discloses a carbon-based platinum single-atom nanozyme and its preparation method and application. The carbon-based platinum single-atom nanozyme uses citric acid and ethylenediamine as carbon sources, prepares carbon-based nanomaterials through a one-step hydrothermal method, dialyzes to remove unreacted precursor substances and small molecules, and freeze-dries to obtain a brownish-yellow solid powder; H 2 PtCl 6 Pt single atoms / nanoclusters are loaded on the surface of the carbon-based nanomaterials by sodium borohydride reduction method to produce Pt SAs / NCs, dialyze to remove unreacted precursor substances and small molecules, and freeze-dry to obtain a black solid powder, which is the carbon-based platinum single-atom nanozyme. The platinum loaded on the carbon quantum dots of the carbon-based platinum single-atom nanozyme exists in the form of single atoms and nanoclusters, and cannot completely exist in the form of platinum single atoms. Summary of the Invention

[0005] The first object of the present invention is to provide a preparation method of a carbon-based platinum single-atom mimic enzyme to solve the technical problem that the platinum loaded on the carbon quantum dots of the existing carbon-based platinum single-atom nanozyme does not completely exist in the form of single atoms.

[0006] The second object of the present invention is to provide a carbon-based platinum single-atom mimic enzyme.

[0007] The third object of the present invention is to provide an application of the carbon-based platinum single-atom mimic enzyme.

[0008] In order to achieve the above objects, the technical solutions adopted by the present invention are as follows:

[0009] A preparation method of a carbon-based platinum single-atom mimic enzyme, comprising the following steps: at 25-26 °C, chloroplatinic acid, carbon quantum dots, and ascorbic acid are mixed and reacted, and then dialyzed and purified to obtain.

[0010] Further, the mass ratio of the chloroplatinic acid to the carbon quantum dots is 5:1-10:1; the molar ratio of the chloroplatinic acid to the ascorbic acid is 1:1-2:1.

[0011] Further, the preparation method of the carbon quantum dots is: cysteine and glucose are dissolved in water to obtain a reaction solution, the pH of the reaction solution is adjusted to alkaline, and then microwave reaction is carried out, cooled, and dialyzed and purified to obtain.

[0012] Further, the power of the microwave reaction is 300-400 W; the temperature of the microwave reaction is 140-160 °C, and the time of the microwave reaction is 10-30 min.

[0013] Further, the pH of the reaction solution is 10-12.

[0014] Furthermore, the molar ratio of cysteine to glucose is 1:1 to 2:1; for every 0.2908 g of cysteine, 10 - 15 mL of water is added.

[0015] The carbon-based platinum single-atom mimetic enzyme is prepared by using the above-mentioned preparation method of the carbon-based platinum single-atom mimetic enzyme.

[0016] Application of the carbon-based platinum single-atom mimetic enzyme in scavenging superoxide anions.

[0017] Advantages of the present invention:

[0018] The present invention uses the ascorbic acid reduction method to load platinum completely in the form of single atoms on carbon quantum dots. Compared with the platinum existing in the form of single atoms, nanoclusters or nanoparticles in Comparative Example 1 and Comparative Example 2, Figure 11 it can be seen that the SOD enzyme activity of the carbon-based platinum single-atom mimetic enzyme existing in the form of single atoms in Example 1 is higher, and it can better remove superoxide anions. From Figure 13 it can be seen that the SOD enzyme activity of the carbon quantum dots without loaded platinum single atoms is 1695 U / mg, while the SOD enzyme activity of the carbon quantum dots loaded with platinum single atoms is 6649 U / mg. After the carbon quantum dots and platinum single atoms are compounded, the SOD enzyme activity is significantly improved.

[0019] The introduction of platinum atoms not only enriches the catalytic active sites of carbon quantum dots, but also promotes charge transfer and efficient adsorption and activation of reactant molecules through the synergistic effect with the carbon quantum dot matrix, thereby significantly improving the catalytic efficiency. Description of the drawings

[0020] Figure 1 It is the particle size diagram of the carbon-based platinum single-atom mimetic enzyme in Example 1, where a is the electron micrograph and b is the particle size distribution diagram;

[0021] Figure 2 It is the particle size diagram of the carbon-based platinum single-atom / nanocluster mimetic enzyme in Comparative Example 1, where a is the electron micrograph and b is the particle size distribution diagram;

[0022] Figure 3 It is the particle size diagram of the carbon-based platinum nanoparticle mimetic enzyme in Comparative Example 2, where a is the electron micrograph and b is the particle size distribution diagram;

[0023] Figure 4 It is the element mapping diagram of the carbon-based platinum single-atom mimetic enzyme in Example 1;

[0024] Figure 5 It is the aberration-corrected electron micrograph of the carbon-based platinum single-atom mimetic enzyme in Example 1;

[0025] Figure 6 It is the element mapping diagram of the carbon-based platinum single-atom / nanocluster mimetic enzyme in Comparative Example 1;

[0026] Figure 7 It is the aberration-corrected electron microscopy image of the carbon-based platinum single atom / nanocluster mimetic enzyme in Comparative Example 1;

[0027] Figure 8 It is the elemental mapping image of the carbon-based platinum nanoparticle mimetic enzyme in Comparative Example 2;

[0028] Figure 9 It is the aberration-corrected electron microscopy image of the carbon-based platinum nanoparticle mimetic enzyme in Comparative Example 2;

[0029] Figure 10 It is the spectrogram of the mimetic enzymes in Example 1 and Comparative Examples 1-2, where a is the ultraviolet-visible spectrogram, b is the emission spectrogram under the excitation wavelength of 360 nm, c is the XRD pattern, and d is the FTIR spectrogram;

[0030] Figure 11 It is the XPS pattern of the mimetic enzymes in Example 1 and Comparative Examples 1-2, where a is the XPS survey spectrum, b is the XPS pattern of C1s, c is the XPS pattern of N1s, d is the XPS pattern of O1s, e is the XPS pattern of S2p, and f is the high-resolution pattern of Pt 4f;

[0031] Figure 12 It is the ESR spectrogram of the mimetic enzymes in Example 1 and Comparative Examples 1-2 for scavenging superoxide anion;

[0032] Figure 13 It is the SOD enzyme activity image of the carbon-based platinum single atom mimetic enzyme in Example 1. Detailed implementation manners

[0033] The present invention will be further described below in conjunction with the embodiments of the present invention and the accompanying drawings.

[0034] Example 1

[0035] The preparation method of the carbon-based platinum single atom mimetic enzyme in Example 1 includes the following steps:

[0036] S1: Preparation of carbon quantum dots: Dissolve 0.2908 g of cysteine and 0.4324 g of glucose in 10 mL of deionized water to obtain a reaction solution, and then add 4 mol / L sodium hydroxide solution to adjust the pH of the reaction solution to 11. Transfer the reaction solution with adjusted pH to the inner lining of a reaction kettle, and heat it in a microwave digestion instrument at 400 W and 140 °C for 10 min. After the reaction is completed and cooled to 60 °C, take it out to obtain a brownish-red CDs solution; after natural cooling to room temperature, dialyze the brownish-red CDs solution in a dialysis bag with a molecular weight cut-off of 500 Da for 24 h, and freeze-dry the solution in the dialysis bag to obtain a yellowish-brown powder.

[0037] S2: Preparation of carbon-based platinum single-atom mimetic enzyme: Under magnetic stirring at 25 °C, 0.95 mL of 38.6 mM aqueous chloroplatinic acid solution, 3 mL of 1 mg / mL aqueous carbon quantum dot solution, and 1.525 mL of 24 mM ascorbic acid (AA) aqueous solution were stirred together and then purified by dialysis to obtain the carbon-based platinum single-atom mimetic enzyme. The carbon-based platinum single-atom mimetic enzyme of Example 1 was named Pt-CDs1. The aqueous carbon quantum dot solution was obtained by dissolving the carbon quantum dots obtained in S1 in water.

[0038] Example 2

[0039] The preparation method of the carbon-based platinum single-atom mimetic enzyme of Example 2 includes the following steps:

[0040] S1: Preparation of carbon quantum dots: 0.2908 g of cysteine and 0.2162 g of glucose were dissolved in 15 mL of deionized water to obtain a reaction solution, and then 4 mol / L sodium hydroxide solution was added to adjust the pH of the reaction solution to 10. The reaction solution with adjusted pH was transferred to the inner liner of the reaction kettle and heated in a microwave digestion instrument at 300 W and 150 °C for 30 min. After the reaction ended and cooled to 60 °C, it was taken out to obtain a brownish-red CDs solution; after natural cooling to room temperature, the brownish-red CDs solution was dialyzed in a dialysis bag with a molecular weight cut-off of 500 Da for 24 h, and the solution in the dialysis bag was freeze-dried to obtain a yellowish-brown powder.

[0041] S2: Preparation of carbon-based platinum single-atom mimetic enzyme: Under magnetic stirring at 25 °C, 1.9 mL of 38.6 mM aqueous chloroplatinic acid solution, 3 mL of 1 mg / mL aqueous carbon quantum dot solution, and 1.525 mL of 24 mM AA solution were stirred together and then purified by dialysis to obtain the carbon-based platinum single-atom mimetic enzyme. The aqueous carbon quantum dot solution was obtained by dissolving the carbon quantum dots obtained in S1 in water.

[0042] Example 3

[0043] The preparation method of the carbon-based platinum single-atom mimetic enzyme of Example 3 includes the following steps:

[0044] S1: Preparation of carbon quantum dots: 0.2908 g of cysteine and 0.3243 g of glucose were dissolved in 10 mL of deionized water to obtain a reaction solution, and then 4 mol / L sodium hydroxide solution was added to adjust the pH of the reaction solution to 12. The reaction solution with adjusted pH was transferred to the inner liner of the reaction kettle and heated in a microwave digestion instrument at 350 W and 160 °C for 10 min. After the reaction ended and cooled to 60 °C, it was taken out to obtain a brownish-red CDs solution; after natural cooling to room temperature, the brownish-red CDs solution was dialyzed in a dialysis bag with a molecular weight cut-off of 500 Da for 24 h, and the solution in the dialysis bag was freeze-dried to obtain a yellowish-brown powder.

[0045] S2: Preparation of carbon-based platinum single-atom mimetic enzyme: Under magnetic stirring at 25 °C, 1.43 mL of an aqueous solution of chloroplatinic acid with a concentration of 38.6 mM, 3 mL of an aqueous solution of carbon quantum dots with a concentration of 1 mg / mL, and 1.345 mL of a 24 mM AA solution were stirred together and then dialyzed and purified to obtain the carbon-based platinum single-atom mimetic enzyme. The aqueous solution of carbon quantum dots was obtained by dissolving the carbon quantum dots obtained in S1 in water.

[0046] Comparative Example 1

[0047] The preparation method of the carbon-based platinum single-atom / nanocluster mimetic enzyme in Comparative Example 1 was roughly the same as that in Example 1. The difference between the preparation method of the carbon-based platinum single-atom mimetic enzyme in Comparative Example 1 and that in Example 1 was that under magnetic stirring at room temperature, 0.95 mL of an aqueous solution of chloroplatinic acid with a concentration of 38.6 mM, 3 mL of an aqueous solution of carbon quantum dots with a concentration of 1 mg / mL, and 33 μL of an aqueous solution of sodium borohydride (NaBH 4 ) with a concentration of 10 mg / mL were stirred together and then dialyzed and purified to obtain the carbon-based platinum single-atom / nanocluster mimetic enzyme. The carbon-based platinum single-atom / nanocluster mimetic enzyme in Comparative Example 1 was named Pt-CDs2.

[0048] Comparative Example 2

[0049] The preparation method of the carbon-based platinum nanoparticle mimetic enzyme in Comparative Example 2 was roughly the same as that in Example 1. The difference between the preparation method of the carbon-based platinum single-atom mimetic enzyme in Comparative Example 2 and that in Example 1 was that under magnetic stirring at room temperature, 0.95 mL of an aqueous solution of chloroplatinic acid with a concentration of 38.6 mM, 3 mL of an aqueous solution of carbon quantum dots with a concentration of 1 mg / mL, 33 μL of an aqueous solution of NaBH 4 with a concentration of 10 mg / mL, and 1.525 mL of a 24 mM AA aqueous solution were stirred together and then dialyzed and purified to obtain the carbon-based platinum nanoparticle mimetic enzyme. The carbon-based platinum nanoparticle mimetic enzyme in Comparative Example 2 was named Pt-CDs3.

[0050] From Figure 1 , Figure 2 , Figure 3 it can be seen that the average sizes of Pt-CDs1 and Pt-CDs2 are 2 - 3 nm, and the average size of Pt-CDs3 is 3 - 5 nm, with good dispersibility. From Figures 4 - 9 it can be seen that C, N, O, S, and Pt elements are evenly distributed in Pt-CDs1, Pt-CDs2, and Pt-CDs3. In the carbon-based platinum single-atom mimetic enzyme using AA as a reducing agent in Example 1, Pt is loaded on the carbon quantum dots in the form of single atoms ( Figure 5 the yellow circles in 4In the mimic enzyme as a reducing agent, Pt exists in the forms of single atoms (A: yellow circles), nanoclusters (B; planar clusters; C: highly disordered clusters). In Comparative Example 2, AA and NaBH 4 In the mimic enzyme with AA and NaBH as reducing agents together, Pt nanoparticles exist in the forms of nanoclusters (A) and particles (B).

[0051] Figure 10 In a, the absorption peak of L-CDs at 284 nm corresponds to the π→π* transition of the C═C bond. In the UV-visible absorption spectrum of Pt-CDs, it is possible that platinum is loaded on the surface of CDs, resulting in the disappearance of the absorption peak at 284 nm. Pt-CDs2 and Pt-CDs3 have a broad absorption peak in the range of 600 - 800 nm. When the excitation wavelength is 360 nm, the maximum emission peak of the CDs solution is located at 443 nm. After loading platinum on CDs, it is found that the emission wavelength decreases. The reason for the decrease in the emission wavelength after loading platinum on CDs may be the combined effect of changes in surface states and defects, regulation of electronic structures, enhancement of quantum size effects, influence of surface functional groups, and changes in charge transfer and energy transfer. Figure 10 In c, the crystal orientation structure of Pt-CDs was determined by XRD. The diffraction peaks in the range of 2θ values from 22° to 25° are composed of the (002) crystal plane of graphite, while the peaks at 2θ values of 39.9, 46.1, and 68.1 correspond to the (111), (200), and (220) crystal planes of platinum, respectively. From Figure 10 In d, it can be seen that the stretching vibrations of N-H / O-H, S-H, and C═O in Pt-CDs1 decrease, possibly due to the coordination of platinum single atoms with the surface functional groups of carbon quantum dots. For Pt-CDs2, the characteristic absorption peaks of N-H / O-H and C═O remain unchanged; the stretching vibration of S-H increases; for Pt-CDs3, the stretching vibrations of N-H / O-H (3450 cm -1 )、S-H and C═O increase significantly. Figure 10 、 12 Both L-CDs in

[0052] and the CDs in the present invention are the carbon quantum dots in Example 1. Figure 12 In b, it can be seen that there are three peaks in the C 1s spectrum, corresponding to C═O, C-S / C-N / C-O, and C═O, respectively; Figure 12 In the N1s spectrum in c, there are two peaks, corresponding to pyrrolic nitrogen and pyridinic nitrogen, respectively; Figure 12 In d, the O1s is split into two peaks, corresponding to C-O and C═O / N═O, respectively; Figure 12 In the S2p spectrum in e, it is split into two peaks, corresponding to C-S and SO 4 2- 。 Figure 12Further proved that the elemental composition of the carbon-based platinum single-atom mimetic enzyme is C, N, S, O, and Pt.

[0053] O 2 - · is generated by the enzymatic xanthine / xanthine oxidase (XOD) system. Mix 10 μL of 5 mM xanthine with 10 μL of 0.25 mM diethylenetriaminepentaacetic acid (DTPA), 5 μL of 250 mM 2-(tert-butoxycarbonyl)-2-methyl-3,4-dihydro-2H-pyrrole 1-oxide (BMPO), and 20 μL of 0.25 mg / mL Pt-CDs in sequence, and then add 5 μL of 0.4 U / mL XOD to initiate the generation of O 2 - ·, and record its ESR spectrum after reacting for 8 minutes. The control group uses 20 μL of H 2 O to replace 20 μL of Pt-CDs. It can be seen from Figure 11 that Pt-CDs1 has a higher ability to scavenge superoxide radicals compared to Pt-CDs3 and Pt-CDs2. Use a commercial SOD assay kit (WST-1) to quantitatively test the SOD enzyme activity of Pt-CDs1. It can be seen from Figure 13 that the SOD enzyme activity of Pt-CDs1 is 6649 U / mg, and the SOD enzyme activity of CDs is 1695 U / mg, indicating that the SOD enzyme activity of Pt-CDs1 comes from CDs rather than platinum single atoms. CDs themselves have a certain SOD enzyme activity, and when compounded with platinum atoms, the SOD enzyme activity is significantly improved.

Claims

1. A method for preparing a carbon-based platinum single-atom enzyme mimic, characterized in that: The method comprises the following steps: at 25-26 DEG C, chloroplatinic acid, carbon quantum dots and ascorbic acid are mixed for reaction, and then dialyzed and purified to obtain the product.

2. The method for preparing the carbon-based platinum single-atom mimetic enzyme according to claim 1, characterized in that: The mass ratio of the chloroplatinic acid to the carbon quantum dots is 5:1 to 10:1; the molar ratio of the chloroplatinic acid to ascorbic acid is 1:1 to 2:

1.

3. The method for preparing the carbon-based platinum single-atom mimetic enzyme according to claim 1, characterized in that: The preparation method of the carbon quantum dots comprises the following steps: dissolving cysteine ​​and glucose in water to obtain a reaction solution, adjusting the pH of the reaction solution to alkaline, then performing microwave reaction, cooling, and dialysis purification to obtain the carbon quantum dots.

4. The method for preparing the carbon-based platinum single-atom mimetic enzyme according to claim 3, characterized in that: The power of the microwave reaction is 300-400W; the temperature of the microwave reaction is 140-160°C; and the time of the microwave reaction is 10-30min.

5. The method for preparing the carbon-based platinum single-atom mimetic enzyme according to claim 3, characterized in that: The pH of the reaction solution is 10-12.

6. The method for preparing the carbon-based platinum single-atom mimetic enzyme according to claim 3, characterized in that: The molar ratio of cysteine ​​to glucose is 1:1 to 2:1; 10 to 15 mL of water is added for every 0.2908 g of cysteine.

7. A carbon-based platinum single-atom enzyme mimic, characterized in that: The enzyme is prepared by the method for preparing the carbon-based platinum single-atom mimetic enzyme described in any one of claims 1 to 6.

8. Use of the carbon-based platinum single-atom mimetic enzyme as claimed in claim 7 in removing superoxide anions.

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