Synthesis Method of a Copper Single-Atom Nanozyme and Its Catalytic Antibacterial Application

Abstract

The present invention belongs to the technical field of sheet-like single-atom nanomaterials, and relates to a synthesis method of copper single-atom nanozyme and its catalytic antibacterial application. First, the present invention synthesizes copper single-atom nanozyme by a salt template method using copper salt, potassium chloride and degummed silk fibroin. The obtained copper single-atom nanozyme simultaneously has biomimetic nitrite reductase activity and near-infrared photothermal conversion performance, and is used as a catalytic antibacterial agent and near-infrared photothermal to enhance the bactericidal effect. The ultrathin sheet-like copper single-atom nanozyme generates nitric oxide in the reaction system of catalyzing nitrite and ascorbic acid for sterilization. Under near-infrared light irradiation, the copper single-atom nanozyme can generate local overheating, and the rapid sterilization effect can be achieved through the increase in temperature. In the present invention, bacteria with a concentration of 5*10<supgt;7< / supgt> cfu / mL are mixed with the ultrathin sheet-like copper single-atom nanozyme in the reaction system of catalyzing nitrite and ascorbic acid, and after being irradiated under near-infrared light conditions for a period of time, the bactericidal effect is good.

Description

Technical Field

[0001] The present invention belongs to the technical field of sheet-shaped single-atom nanomaterials, and relates to a synthesis method of a copper single-atom nanozyme and its catalytic antibacterial application. Specifically, it relates to a preparation method of a copper single-atom nanozyme with bionic nitrite reductase activity and near-infrared photothermal conversion performance, and an application of near-infrared photothermal enhanced sterilization. Background Art

[0002] Due to the problem of bacterial drug resistance caused by the abuse of antibiotics, antibacterial materials mainly develop in the nano-size direction. Copper-based nanozymes are a kind of artificially synthesized antibacterial material at the nano level with catalytic activity and antibacterial functions similar to natural enzymes. Benefiting from the advantages of nanomaterials, their cost, production, storage, and stability in harsh environments are superior to natural enzymes.

[0003] However, nanozymes also have certain defect problems. Compared with natural enzymes, the lack of high-activity centers and low active-site density on the surface of nanomaterials leads to relatively low enzyme activity of nanozymes. And currently, the antibacterial methods of antibacterial nanozymes are relatively single, mostly based on peroxidase-like enzymes, and sterilization is achieved by catalyzing the cleavage of hydrogen peroxide to generate reactive oxygen free radicals. Since the lifespan of reactive oxygen free radicals is only dozens of nanoseconds. It has been found that nitric oxide (NO) is an endogenously produced diatomic free radical with a lifespan of 3 - 5 seconds, which plays a key role in the immune response of mammals to pathogens. Therefore, we enhance the catalytic NO process of the enzyme by increasing the number of enzyme centers of the nanozyme, reducing the size, and moderately increasing external stimuli such as near-infrared (NIR) irradiation. Single atoms are a new generation of nanozymes formed by combining advanced single-atom technology with enzyme-like active sites. Their advantage lies in having 100% atomic utilization rate and fully exposed metal atom active sites, showing significant catalytic activity and stability. We load metal copper atoms onto silk fibroin with good biocompatibility to form two-dimensional sheet-shaped copper single-atom nanozymes.

[0004] In the antibacterial process of the ultra-thin sheet-shaped copper single-atom nanozyme of the present invention, the copper single-atom nanozyme sterilizes by catalyzing the nitrite-ascorbic acid reaction system to generate nitric oxide, and then using near-infrared light irradiation, the solution temperature of the nitrite-ascorbic acid reaction system will rise to achieve the purpose of efficient sterilization. Thus, an ultra-thin sheet-shaped copper single-atom nanozyme with good biocompatibility and high sterilization efficiency is prepared. There is no relevant report yet. Summary of the Invention

[0005] The present invention synthesizes a method for an ultra-thin sheet-shaped copper single-atom nanozyme with bionic nitrite reductase activity and near-infrared photothermal conversion performance, and can be used to synergistically enhance the sterilization effect by photothermal. This sheet-shaped copper single-atom nanozyme has good biocompatibility, strong interaction with bacteria, and low cytotoxicity, and can quickly kill bacteria in a short time.

[0006] In the above technical solution, the copper single-atom nanozyme with an ultrathin flake shape is synthesized by a salt template method using a copper salt, potassium chloride, and degummed silk fibroin.

[0007] The present invention provides a method for synthesizing a copper single-atom nanozyme, and the steps are as follows:

[0008] (1) First, prepare a methanol solution containing copper ions as solution A. After adding potassium chloride to solution A and stirring vigorously until homogeneous, perform vacuum drying to obtain a solid salt loaded with potassium chloride and copper ions.

[0009] (2) Prepare a methanol solution of degummed silk fibroin as solution B. Then add the solid salt loaded with potassium chloride and copper ions prepared in step (1) to solution B and stir vigorously until homogeneous, followed by vacuum drying to obtain a potassium chloride salt loaded with copper-silk fibroin.

[0010] (3) Place the potassium chloride salt loaded with copper-silk fibroin obtained in step (2) in a tube furnace, heat it to the reaction temperature, and react under an argon gas flow to obtain black powder doped in the white potassium chloride salt. After removing the potassium chloride salt by washing with deionized water, a liquid containing the black powder-like material is obtained. Perform vacuum drying, then carry out a reflux reaction in a dilute sulfuric acid solution, and subsequently wash with deionized water and perform vacuum drying to obtain the copper single-atom nanozyme.

[0011] In step (1), the mass ratio of the amount of potassium chloride used to the mass of copper ions is 1000 / 1.

[0012] In step (1), the volume ratio of solution A to solution B in step (2) is 1:1; in solution A, the concentration of copper ions is 8 - 10 mg / mL; in solution B, the concentration of the methanol solution of degummed silk fibroin is 25 - 30 mg / mL.

[0013] In step (2), the degummed silk fibroin is synthesized by a method of boiling with sodium carbonate salt. The specific preparation method is as follows: Place the silkworm cocoons in a sodium carbonate aqueous solution with a concentration of 0.1 - 0.3 mol / L and heat at 100 °C for 2 - 4 h to obtain a silk fibroin suspension. Centrifuge and purify it at 14000 revolutions per minute for more than 2 times, 10 - 15 minutes each time, to obtain a degummed silk fibroin aqueous solution. Finally, perform vacuum drying at a temperature below 85 °C to obtain a solid of degummed silk fibroin.

[0014] In step (3), the reaction temperature under the argon gas flow is above 700 °C, and the reaction time is 1 - 3 h.

[0015] In step (3), the volume percentage concentration of the dilute sulfuric acid solution is 10% - 20%; the temperature of the reflux reaction is 100 - 150 °C, and the time is 10 - 15 h.

[0016] In steps (1)-(3), the temperature of vacuum drying is greater than 60 °C.

[0017] The copper single-atom nanozyme prepared by the present invention has an ultrathin flake structure with a thickness of 1-4 nm. Copper is dispersed in the nitrogen-doped carbon carrier in the form of single atoms, and the copper content is 0.5-1.5%. It has a significant absorption of near-infrared light with a wavelength of 800-1200 nm.

[0018] The application of the copper single-atom nanozyme prepared by the present invention in photothermal sterilization is carried out according to the following steps:

[0019] First, add ascorbic acid and the aqueous solution of copper single-atom nanozyme, then add the bacterial suspension, and finally add the nitrite solution. Mix them evenly. Under the near-infrared light with a wavelength of 1086 nm and a power of 1 W / cm 2 Irradiate for 10-15 min. After the solution temperature rises to 50 °C, dilute it 10,000 times with phosphate buffer. Take 100 μL of the diluted suspension and place it on the Luria Bertani solid medium. The medium is cultured in an incubator at 37 °C for 18-24 h, and the number of colonies is calculated.

[0020] The volume ratio of the ascorbic acid, the aqueous solution of copper single-atom nanozyme, the bacterial suspension, and the nitrite solution is 60 μL:60 μL:20 μL:60 μL. Among them, the concentration of ascorbic acid is 15 mmol / L, the concentration of the aqueous solution of copper single-atom nanozyme is greater than 60 μg / mL, the concentration of the bacterial suspension is 4-7×10 7 cfu / mL, and the concentration of the nitrite solution is 15 mmol / L.

[0021] The above-mentioned bacteria are one of Escherichia coli and Staphylococcus aureus.

[0022] The beneficial effects of the present invention are as follows:

[0023] (1) The copper single-atom nanozyme in the present invention has bionic nitrite reductase activity and near-infrared photothermal conversion performance, and has a dual orthogonal bactericidal effect by catalyzing the release of NO and near-infrared photothermal ablation, and has a good bactericidal effect.

[0024] (2) When the molar ratio of ascorbic acid to nitrite is 1:1, the copper single-atom nanozyme can catalyze the reduction of nitrite to produce NO, and the nitrite conversion rate is as high as more than 70% within 15 min. Description of the Drawings

[0025] Figure 1 It is the transmission diagram (a) and the H-TEM dark field diagram (b) of the ultrathin flake copper single-atom nanozyme;

[0026] Figure 2TEM images of the interaction between ultrathin flake copper single-atom nanozyme and Escherichia coli (a) and Staphylococcus aureus (b);

[0027] Figure 3 Bar chart of the synergistic photothermal sterilization of ultrathin flake copper single-atom nanozyme at different concentrations;

[0028] Figure 4 Bar chart of the synergistic photothermal sterilization of ultrathin flake copper single-atom nanozyme at different photothermal powers;

[0029] Figure 5 Bar chart of the synergistic photothermal sterilization of ultrathin flake copper single-atom nanozyme at the same photothermal power with different illumination times;

[0030] Figure 6 Bar chart of the catalytic efficiency of copper single-atom nanozyme and CuO, Cu 2 O in catalyzing NO in the reaction system of nitrite and ascorbic acid. Specific implementation mode

[0031] The technical solution of the present invention will be further described below with reference to the drawings and embodiments.

[0032] Example 1

[0033] Preparation of antibacterial ultrathin flake copper single-atom nanozyme:

[0034] Weigh 6 grams of silkworm cocoons and heat them in 100 mL of 0.2 mol / L Na 2 CO 3 aqueous solution at 100 °C for 3 hours. Obtain a silk fibroin suspension, thoroughly rinse the silk fibroin suspension with deionized water, and purify it by centrifugation at 14,000 revolutions per minute twice, 10 minutes each time, to remove the colloidal silk fibroin protein and obtain degummed silk fibroin. Then put the degummed silk fibroin into a vacuum oven at 80 °C to dry and obtain dry degummed silk fibroin.

[0035] Dissolve 0.7275 g of Cu(NO 3 ) 2 in 30 mL of anhydrous methanol as solution A. Take 500 g of KCl as a template and stir it vigorously with solution A. Dry it at 80 °C to fix Cu 2+ onto the surface of potassium chloride.

[0036] Take 0.821 g of degummed silk fibroin added in 30 mL of methanol as solution B, add solution B to the potassium chloride loaded with copper ions, and stir vigorously. Then vacuum-dry the potassium chloride salt at 80 °C to obtain Cu(SF) / KCl.

[0037] The obtained Cu(SF) / KCl was annealed for 2 hours under an Ar gas flow at 750 °C with a heating rate of 5 °C / min. Then it was refluxed in a 10% (v / v) dilute sulfuric acid solution at 120 °C for 12 h. Finally, the bulk sample was washed with deionized water multiple times and dried to obtain the copper single-atom nanozyme.

[0038] From Figure 1 As can be seen from the transmission image (a) and the H-TEM dark field image (b) of the copper single-atom nanozyme, the nanozyme presents an ultrathin sheet-like shape, and copper single atoms can be observed.

[0039] Example 2

[0040] Photothermal sterilization of copper single-atom nanozyme:

[0041] First, 60 μL of aqueous solutions of copper single-atom nanozyme with concentrations of 15 μg / mL, 30 μg / mL, 60 μg / mL, 120 μg / mL, and 240 μg / mL were separately prepared for standby.

[0042] Using a 1.5 mL centrifuge tube as the reaction vessel, in sequence, first add 60 μL of ascorbic acid with a concentration of 15 mmol / L, then 60 μL of the aqueous solution of copper single-atom nanozyme, and then add 20 μL of a bacterial suspension of Staphylococcus aureus or Escherichia coli with a concentration of 5×10 7 cfu / mL. Finally, add 60 μL of a nitrite solution with a concentration of 15 mmol / L. Mix the four evenly in the centrifuge tube. Thus, there is a final 200 μL of mixed solution in the 1.5 mL centrifuge tube. Under near-infrared light with a wavelength of 1086 nm and a power of 1 W / cm 2 Irradiate for 10 min. After the solution temperature rises to 50 °C, dilute it 10,000 times with phosphate buffer. Take 100 μL of the diluted suspension and place it on Luria Bertani solid medium. Incubate the medium in a 37 °C incubator for 18 - 24 h and calculate the number of colonies.

[0043] The obtained bacterial survival rates are shown in Table 1. The results show that, as shown Figure 3 below: Compared with the first group without the catalyst copper single-atom nanozyme, as the concentration of the copper single-atom nanozyme increases, the sterilization efficiency of the solution increases.

[0044] Table 1 Influence of different copper single-atom nanozyme concentrations on the catalytic NO sterilization

[0045] Copper single-atom nanozyme (μg / mL) Bacterial survival rate (%) 0 85.04 15 36.14 30 29.54 60 2.49 120 1.63 240 0

[0046] Example 3

[0047] Same as Example 2, only changing the near-infrared light radiation power to 0, 0.5, 1.0, 1.5 W / cm 2, the concentration of the immobilized copper single-atom nanozyme was fixed at 60 μg / mL;

[0048] The obtained bacterial survival rates are shown in Table 3. As can be seen from the appendix Figure 4 As can be seen, as the power of the near-infrared light radiation increases, the photothermal sterilization efficiency gradually increases, which can be attributed to the fact that a higher radiation power is conducive to the catalyst generating higher heat, thereby enhancing the sterilization efficiency of the copper single-atom nanozyme.

[0049] Table 3 Effects of different near-infrared light radiation powers on photothermal sterilization

[0050] <![CDATA[Radiation power (W / cm 2 )]]> Bacterial survival rate (%) 0 41.8 0.5 27.36 1.0 2.49 1.5 0

[0051] Example 4

[0052] Same as Example 2, the final concentration of the copper single-atom nanozyme was fixed at 60 μg / mL, and only the near-infrared light irradiation time in the photothermal sterilization step of the copper single-atom nanozyme was changed, which were 0, 2, 4, 6, 8, and 10 min respectively, and the infrared light radiation power was 1 W / cm 2 The obtained bacterial survival rates are shown in Table 4. As can be seen from the appendix Figure 5 As can be seen, as the irradiation time increases, the bacterial survival rate decreases, but after 8 min, the bacterial survival rate remains basically unchanged. Therefore, appropriately extending the near-infrared light radiation time is beneficial to increasing the sterilization efficiency of the copper single-atom nanozyme.

[0053] Table 4 Effects of different near-infrared light radiation times on photothermal sterilization

[0054] Irradiation time (min) Bacterial survival rate (%) 10 2.49 8 2.89 6 6.15 4 19.73 2 37.04 0 41.8

[0055] Example 5

[0056] Same as Example 2, only the bacteria in the photothermal sterilization step were changed to Escherichia coli and Staphylococcus aureus respectively, and the obtained bacterial survival rates are shown in Table 5. It can be seen that under the same conditions, under the irradiation of near-infrared light with a wavelength of 1086 nm and a power of 1 W / cm 2 The 60 μg / mL antibacterial copper single-atom nanozyme can kill a variety of bacteria. The interaction between the material and the two bacteria is as Figure 2 shown, the darker color is the bacteria, and the lighter gray is the material.

[0057] Table 5 Effects of photothermal sterilization on different bacteria

[0058]

[0059]

[0060] Example 6

[0061] Same as Example 2, only change the catalyst copper single-atom nanozyme in the photothermal sterilization step of the antibacterial single-atom nanozyme loaded with copper, and replace it with CuO and Cu with the same copper content (10 μg / mL, the copper loading of the copper single-atom nanozyme is approximately in the range of 0.5 - 1.5%, calculated as 1% here). 2 O, the NO generation rate in the catalytic nitrite and ascorbic acid system is shown in Table 6 and Figure 6 (the copper single-atom nanozyme in the figure is represented by Cu-N-C). It can be seen that the catalytic efficiency of the ultrathin flake copper single-atom nanozyme is much higher than that of CuO and Cu 2 O.

[0062] Table 6 Effects of different coppers on the generation of NO in the reaction system of sodium nitrite and ascorbic acid

[0063] Catalyst type NO production rate (%) CuO 44.48 <![CDATA[Cu 2 O]]> 53.14 Copper single-atom nanozyme (Cu-N-C) 71.69

Claims

1. Application of copper single-atom nanozyme in photothermal sterilization, Characterized in that, The specific steps are as follows: First, add ascorbic acid and the aqueous solution of copper single-atom nanozyme, then add the bacterial suspension, and finally add the nitrite solution. Mix them evenly. Under the irradiation of near-infrared light with a wavelength of 1086 nm and a power of 1 W / cm 2 for 10 - 15 min. After the solution temperature rises to 50 °C, dilute it 10,000 times with phosphate buffer. Take 100 μL of the diluted suspension and place it on Luria Bertani solid medium. Incubate the medium in an incubator at 37 °C for 18 - 24 h, and calculate the number of colonies; The synthesis of the copper single-atom nanozyme includes the following steps: (1) First, prepare a methanol solution containing copper ions as solution A. After adding potassium chloride to solution A and stirring vigorously until evenly mixed, vacuum dry to obtain a solid salt with potassium chloride loaded with copper ions; (2) Prepare a methanol solution of degummed silk fibroin as solution B. Then add the solid salt with potassium chloride loaded with copper ions prepared in step (1) to solution B and stir vigorously until evenly mixed, and vacuum dry to obtain a potassium chloride salt loaded with copper-silk fibroin; (3) Place the potassium chloride salt loaded with copper-silk fibroin obtained in step (2) in a tube furnace, heat up to the reaction temperature, and react under an argon gas stream to obtain black powder doped in the white potassium chloride salt. After removing the potassium chloride salt by washing with deionized water, obtain a liquid containing the black powder-like material, vacuum dry, then carry out a reflux reaction in a dilute sulfuric acid solution, and then wash with deionized water and vacuum dry to obtain the copper single-atom nanozyme.

2. The application according to claim 1, Characterized in that, The volume ratio of the ascorbic acid, the aqueous solution of copper single-atom nanozyme, the bacterial suspension, and the nitrite solution is 60 μL: 60 μL: 20 μL: 60 μL. Among them, the concentration of ascorbic acid is 15 mmol / L, the concentration of the aqueous solution of copper single-atom nanozyme is greater than 60 μg / mL, the concentration of the bacterial suspension is 5×10 7 -7×10 7 cfu / mL, and the concentration of the nitrite solution is 15 mmol / L; The bacteria are one of Escherichia coli and Staphylococcus aureus.

3. The application according to claim 1, Characterized in that, In step (1), the mass ratio of the amount of potassium chloride used to the mass of copper ions is 1000:

1.

4. The application according to claim 1, Characterized in that, In step (1), the volume ratio of solution A to solution B in step (2) is 1:1; in solution A, the concentration of copper ions is 8-10 mg / mL; in solution B, the concentration of the methanol solution of degummed silk fibroin is 25-30 mg / mL.

5. The application according to claim 1, Characterized in that, In step (2), the degummed silk fibroin is synthesized by the method of boiling with sodium carbonate salt. The specific preparation method is: place the cocoon in a sodium carbonate aqueous solution with a concentration of 0.1-0.3 mol / L and heat at 100 °C for 2-4 h to obtain a silk fibroin suspension. Centrifuge and purify it at 14000 revolutions per minute for more than 2 times, 10-15 minutes each time, to obtain a degummed silk fibroin aqueous solution. Finally, vacuum dry at a temperature below 85 °C to obtain a solid of degummed silk fibroin.

6. The application according to claim 1, Characterized in that, In step (3), The reaction temperature under the argon gas stream is above 700 °C, and the reaction time is 1-3 h; The volume percentage concentration of the dilute sulfuric acid solution is 10%-20%; the temperature of the reflux reaction is 100-150 °C, and the time is 10-15 h.

7. The application according to claim 1, Characterized in that, In steps (1)-(3), the temperature of vacuum drying is greater than 60 °C.

Images