CN114452386B - Preparation methods and applications of gold-copper bimetallic nanoenzyme composite materials - Google Patents

Abstract

The invention belongs to the technical field of nanomaterials and discloses a preparation method and application of gold-copper bimetallic nanoenzyme composite materials. In the present invention, a mixed solution of chloroauric acid and copper chloride is added to the lysozyme fiber solution, and after letting it stand for full mixing, a freshly prepared sodium borohydride solution is added as a reducing agent to obtain a gold-copper bimetallic nanoenzyme composite material. In the gold-copper bimetallic nanoenzyme composite material, the lysozyme fiber is uniform in size, and the small-sized gold-copper nanoparticles are uniformly distributed at high density on the surface of the lysozyme fiber, which can significantly enhance peroxide-like properties under near-infrared light radiation. Enzyme catalytic activity to achieve efficient sterilization.

Description

Preparation methods and applications of gold-copper bimetallic nanoenzyme composite materials

Technical field

The invention belongs to the technical field of nanomaterials, and relates to a gold-copper bimetallic nanozyme composite material and its catalytic/photothermal antibacterial application, specifically using gold-copper bimetallic composite nanoparticles (Au@Cu) as a catalyst, in the near infrared Under light (NIR) irradiation, the efficiency of catalyzing hydrogen peroxide is improved by enhancing peroxidase activity, thereby achieving an efficient antibacterial method.

Background technique

Nanozymes are nanomaterials with similar enzyme activity. As a substitute for natural enzymes, they have attracted great interest due to their obvious advantages over natural enzymes, such as simple synthesis, adjustable catalytic activity, and stability in harsh environments. Good to wait. The functional diversity of nanomaterials gives nanozymes a variety of functions, making them widely studied in the biomedical field. They are mainly used in biomolecule detection, biosensors, antibacterial, immune analysis, cancer diagnosis and treatment, and environmental monitoring. Generally, peroxidase mimics can specifically catalyze the conversion of hydrogen peroxide into highly toxic reactive oxygen species (ROS) such as hydroxyl radicals (·OH), which attack the cell membrane of microorganisms at weakly acidic infection sites. The nanobiocatalytic system with nanozymes as the core has a variety of oxidoreductase activities and can adjust the level of ROS according to pH and other conditions. Therefore, based on this principle, it can quickly kill a variety of super-resistant bacteria and clear biofilms.

Similar to natural enzymes, the activity of nanozymes can be regulated by a variety of factors, such as pH, temperature, surrounding environment, and metal ions. In addition, the activity of nanozymes can also be adjusted by changing their physical and chemical properties and structure. Typical nanoscale factors, such as size, morphology, surface modification and valence state, and the composition and configuration of the active center, significantly affect the performance of nanozymes. active. Therefore, it is of great significance to research and develop a new nanozyme material that has both good bacterial binding ability and enhanced catalytic activity.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings of low catalytic efficiency and weak interaction with bacteria of gold-based nanozymes in the prior art, provide a gold-copper nanoparticle composite material using lysozyme as a template, and use it to catalyze antibacterial activity; The gold-copper nanoparticle composite material synthesized by the invention has a small dosage, high photothermal enhanced catalytic efficiency, strong interaction with bacteria, and can kill bacteria efficiently. The present invention uses lysozyme fiber as a template to regulate the size and distribution of gold and copper nanoparticles, effectively improves the catalytic efficiency, has good interaction with bacteria, and can achieve the purpose of efficient sterilization.

The technical solution of the present invention is as follows:

The present invention first provides a biological macromolecule lysozyme fiber (LNFs) as a template. At the same time, it can regulate the high-density distribution of metal particles on the fiber to obtain smaller-sized metal nanoparticles. The bimetallic nanoparticles can be uniformly modified on the lysozyme. fiber surface. The specific preparation method is as follows:

Take the synthesized lysozyme fiber solution, add a certain proportion of chloroauric acid and copper chloride mixed solution, let it stand for more than 30 minutes to fully mix, then add freshly prepared sodium borohydride solution as a reducing agent to obtain a gold-copper double solution. Metal nanoenzyme composite material, namely LNFs@Au/Cu composite material.

Wherein, the volume ratio of lysozyme fiber solution, chloroauric acid and copper chloride mixed solution and sodium borohydride solution is 1:1:1, wherein the concentration of the lysozyme fiber solution is 5mg/mL, chloroauric acid and chlorine The total concentration of metal ions in the copper chloride mixed solution is 0.15mM, and the concentration of the sodium borohydride solution is 0.01mM.

Further, the preparation method of lysozyme fiber described in the test is as follows:

Prepare 10 mL of hydrochloric acid solution with a concentration of 1M, add 0.015g glycine, and prepare solution A; prepare 1 mL of glacial acetic acid solution with a concentration of 1mM, add 0.1396g of choline chloride, and prepare solution B; take 0.01g of lysozyme, and add 4750 μL Dissolve the solution and 250 μL of B solution, stir and react in an oil bath at 70°C for 5 hours. After the reaction is completed, centrifuge at 12,000 rpm and wash twice with ultrapure water, 20 min each time.

In the gold-copper bimetallic nanozyme composite material prepared by the present invention, the lysozyme fiber is uniform in size, and the small-sized gold-copper nanoparticles are uniformly distributed at high density on the surface of the lysozyme fiber. Under near-infrared light radiation, the process can be significantly enhanced. Oxidase catalytic activity achieves the purpose of efficient sterilization.

The invention also provides a gold-copper bimetallic nanoenzyme composite (LNFs@Au/Cu) catalytic/photothermal sterilization method, which is carried out according to the following steps:

(1) The solvent of LNFs@Au/Cu composite material is replaced with pH=5.5, 0.1M sodium acetate-acetic acid buffer solution;

(2) Place the bacterial suspension into the LNFs@Au/Cu composite material treated in step (1), add a certain concentration of hydrogen peroxide and let it stand for a while, then react for a period of time under near-infrared light irradiation, and then use phosphoric acid to Dilute with salt buffer, place the diluted suspension into Luria Bertani solid medium, incubate at 37°C for 12 hours, and count the number of colonies.

Wherein, the volume ratio of the bacterial suspension and LNFs@Au/Cu composite material is 1:9;

Further, the concentration of LNFs@Au/Cu composite material was 70-80 μg/ml, and the concentration of bacterial suspension was 10 ⁸ /mL.

After adding hydrogen peroxide, the final concentration of hydrogen peroxide is 200 μM.

The conditions for the near-infrared light irradiation are: power is 2W, irradiation is 10 minutes, and the wavelength of the near-infrared light is 808nm;

The dilution is 10,000 times.

The bacterium is one of Pseudomonas aeruginosa, Salmonella, Escherichia coli or Staphylococcus aureus.

Compared with the existing technology, the beneficial effects of the present invention are as follows:

Currently, the use of antibiotics is a commonly used sterilization method, but it can easily lead to bacterial resistance. In the gold-copper bimetallic nanozyme composite material prepared by the present invention, the gold-copper nanoparticles are small in size and uniformly modified on the surface of the lysozyme fiber (as shown in Figure 1), which increases the distribution density of metal active centers, thereby enhancing the catalytic effect. . At the same time, lysozyme fiber not only serves as a biological template, but also can effectively adhere to bacteria, has strong interaction with bacteria, and can be combined on the surface of bacteria (as shown in Figure 2). The presence of LNFs@Au/Cu nanozyme, It promotes the Fenton-like reaction of hydrogen peroxide, improves the generation efficiency of hydroxyl radicals, and can effectively sterilize. Under near-infrared light irradiation, not only can the photothermal effect be utilized, but the photothermal temperature can also improve the catalytic activity, and a lower concentration of hydrogen peroxide can be used to achieve a better sterilization effect (as shown in Figure 3) without causing bacterial resistance. Medicine is a new, highly efficient and green antibacterial method.

Description of the drawings

Figure 1 shows the TEM image of LNFs@Au/Cu composite material.

Figure 2 is a TEM image of the interaction between LNFs@Au/Cu composite and Staphylococcus aureus.

Figure 3 shows the sterilization effects of LNFs@Au/Cu composites using different sterilization methods.

Detailed ways

The technology of the present invention will be further described below in conjunction with the accompanying drawings and examples.

Example 1:

Preparation of lysozyme fiber:

Prepare 10 mL of hydrochloric acid solution with a concentration of 1 M, and add 0.015 g of glycine to prepare solution A. Prepare 1 mL of glacial acetic acid solution with a concentration of 1 mM, and add 0.1396 g of choline chloride to prepare solution B. Take 0.01g of lysozyme, add 4750μL of LA solution and 250μL of B solution to dissolve. Stir the reaction in an oil bath at 70°C for 5 hours. After the reaction is completed, centrifuge at 12,000 rpm and wash with ultrapure water twice, 20 min each time. 8 ml of the final lysozyme fiber aqueous solution was obtained.

Preparation of LNFs@Au/Cu nanozyme composite:

Take 800 μL of lysozyme fiber solution, add a total of 800 μL of chloroauric acid and copper chloride solution, react for 30 minutes, add 800 μL of freshly prepared sodium borohydride solution, and quickly reduce to obtain a metal nanoparticle composite material.

Wherein, the concentration of the lysozyme fiber is 5mg/mL, the total concentration of the metal salt solution is 0.15mM, and the concentration of the sodium borohydride solution is 0.01mM.

LNFs@Au/Cu nanoenzyme composite catalytic/photothermal synergistic sterilization:

20 μL of Staphylococcus aureus suspension was placed in 180 μL of LNFs@Au/Cu with a concentration of 80 μg mL ^–1 . There was 200 μM hydrogen peroxide in the system. It was left to stand for 10 min and irradiated with near-infrared light with a wavelength of 808 nm and a power of 2 W. , irradiate for 10 minutes, continue to act for 10 minutes, and finally dilute 10,000 times with phosphate buffer, take 100 μL of the diluted suspension and place it on Luria Bertani solid medium, culture it at 37°C for 12 hours, and count the number of colonies. The obtained bacterial survival rates are shown in Table 1.

Example 2:

Same as Example 1, except that the proportion of the gold-copper salt solution in the preparation step of the gold-copper nanozyme composite material was changed, and LNFs@Au/Cu photothermal sterilization was performed. The obtained bacterial survival rate is shown in Table 1. The results show that compared with unmodified gold nanoparticle materials, as the amount of copper added increases, the bactericidal efficiency of LNFs@Au/Cu increases, but further increasing the copper content, its bactericidal efficiency decreases, which can be attributed to the higher copper amount. The addition of LNFs@Au/Cu metal nanoparticles increases the size, reduces near-infrared absorption, and reduces the photothermal conversion efficiency.

Table 1 Effect of different copper addition amounts on LNFs@Au/Cu photothermal sterilization

Copper addition amount (molar ratio of Au to Cu) Bacterial survival rate (%) 1:0 65.13 4:1 28.24 3:1 27.5 2:1 56.21 1:1 76.3 0:1 100

Example 3:

Same as Example 1, except that the ratio of the gold-copper salt solution in the preparation step of the gold-copper nanozyme composite material was changed, and LNFs@Au/Cu catalyzed hydrogen peroxide sterilization was performed. The obtained bacterial survival rate is shown in Table 2. It can be seen that with the increase in the amount of copper added, the catalytic bactericidal efficiency of LNFs@Au/Cu increases, but further increasing the copper content, the bactericidal efficiency decreases, which can be attributed to the addition of higher copper amounts that promote LNFs@Au/Cu metal As the size of the nanoparticles increases, the catalytic activity decreases.

Table 2 Effects of different copper addition amounts on LNFs@Au/Cu catalytic sterilization

Copper addition amount (molar ratio of Au to Cu) Bacterial survival rate (%) 1:0 87.26 4:1 68.13 3:1 61.34 2:1 73.46 1:1 91.74 0:1 100

Example 4:

Same as Example 1, except that the near-infrared light radiation power in the photothermal sterilization step of the LNFs@Au/Cu nanozyme composite material was changed. The obtained bacterial survival rate is shown in Table 3. It can be seen that as the near-infrared light radiation power increases, the photothermal sterilization efficiency gradually increases, which can be attributed to the fact that higher radiation power is conducive to the photothermal agent generating higher heat.

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

Example 5:

Same as Example 1, except that the concentration of hydrogen peroxide in the sterilization step catalyzed by the LNFs@Au/Cu nanozyme composite material was changed. The obtained bacterial survival rate is shown in Table 4. It can be seen that higher hydrogen peroxide concentration can improve sterilization efficiency.

Table 4 Effects of different hydrogen peroxide concentrations on catalytic sterilization

H ₂ O ₂ concentration (μM) Bacterial survival rate (%) 50 89.12 100 64.05 200 51.75 300 38.48 400 22.41

Example 6:

Same as Example 1, under the final reaction conditions, the concentration of LNFs@Au/Cu was 80 μg/ml and the hydrogen peroxide concentration was 200 μM. The sterilization was carried out through catalysis and photothermal synergistic sterilization method. The obtained bacterial survival rate is shown in Table 5. It can be seen that photothermal has an enhanced effect on catalytic sterilization.

Table 5 Bactericidal effects of different sterilization types

Sterilization type Bacterial survival rate (%) Photothermal sterilization 27.8 Catalytic sterilization 60.36 Catalytic/photothermal synergistic sterilization 0

Example 7:

Same as Example 1, except that the bacteria in the collaborative sterilization step of the LNFs@Au/Cu nanozyme composite material were changed to Pseudomonas aeruginosa, Escherichia coli, and Salmonella. The obtained bacterial survival rates are shown in Table 6. It can be seen that under the same conditions, LNFs@CuS can kill a variety of bacteria.

Bacteria type Bacterial survival rate (%) Staphylococcus aureus 0 Pseudomonas aeruginosa 0 E. coli 0 salmonella 0

Comparing the results of Examples 1, 2 and 3, changing the preparation parameters of gold and copper nanoparticles has an important impact on the photothermal and catalytic sterilization efficiency.

Figure 1 is a TEM image of LNFs@Au/Cu nanozyme, which shows that the Au/Cu nanoparticles are relatively uniform in size and are relatively uniformly loaded on the lysozyme fiber.

Figure 2 is a TEM image of the interaction between LNFs@Au/Cu nanozyme and Staphylococcus aureus, indicating that LNFs@Au/Cu has good interaction with bacteria.

Figure 3 shows the sterilization effect of LNFs@CuS nanozyme using different mechanisms of photothermal and catalysis. It shows that photothermal has a certain enhancement effect on catalysis, and the two synergistically can kill bacteria efficiently.

Claims (8)

1. A method for preparing a gold-copper bimetallic nanoenzyme composite material, which is characterized in that the steps are: Take the lysozyme fiber solution, add a mixed solution of chloroauric acid and copper chloride, let it stand and mix it thoroughly, then add a freshly prepared sodium borohydride solution as a reducing agent to obtain a gold-copper bimetallic nanoenzyme composite material, or LNFs. @Au/Cu composite material; The volume ratio of the lysozyme fiber solution, the mixed solution of chloroauric acid and copper chloride and the sodium borohydride solution is 1:1:1, Wherein, the concentration of the lysozyme fiber solution is 5mg/mL, The total concentration of metal ions in the mixed solution of chloroauric acid and copper chloride is 0.15mM, and the concentration of the sodium borohydride solution is 0.01mM. 2. The preparation method according to claim 1, characterized in that the standing time is more than 30 minutes. 3. The preparation method as claimed in claim 1, characterized in that the preparation method of lysozyme fiber is: Prepare 10 mL of hydrochloric acid solution with a concentration of 1M, add 0.015g glycine to prepare solution A; prepare 1mL of glacial acetic acid solution with a concentration of 1mM, add 0.1396g of choline chloride, and prepare solution B; take 0.01g of lysozyme and add 4750 μL Dissolve solution A and 250 μL solution B, stir and react in an oil bath at 70°C for 5 hours. After the reaction is completed, centrifuge at 12,000 rpm and wash twice with ultrapure water, 20 minutes each time. 4. The gold-copper bimetallic nanozyme composite material prepared by the preparation method according to any one of claims 1 to 3, characterized in that, in the gold-copper bimetallic nanozyme composite material, the lysozyme fiber is uniform in size, and the gold-copper bimetallic nanozyme composite material is uniform in size. Copper nanoparticles were evenly distributed on the surface of lysozyme fibers. 5. The use of the gold-copper bimetallic nanozyme composite material according to claim 4 for preparing a catalytic/photothermal bactericide, characterized in that the specific steps are: (1) The solvent of LNFs@Au/Cu composite material is replaced with pH=5.5, 0.1M sodium acetate-acetic acid buffer solution; (2) Place the bacterial suspension into the LNFs@Au/Cu composite material treated in step (1), add hydrogen peroxide and let it stand for a while, then react for a period of time under near-infrared light irradiation, and use phosphate buffer The solution was diluted, and the diluted suspension was placed on Luria Bertani solid medium, cultured at 37°C for 12 hours, and the number of colonies was counted. 6. The use according to claim 5, wherein in step (2), the volume ratio of the bacterial suspension to the LNFs@Au/Cu composite material is 1:9; wherein, LNFs@Au/Cu The concentration of the composite material was 70-80 μg/ml, and the concentration of the bacterial suspension was 10 ⁸ cells/mL. 7. The use according to claim 5, characterized in that, in step (2), after adding hydrogen peroxide, the final concentration of hydrogen peroxide is 200 μM. 8. The use according to claim 5, characterized in that in step (2), The conditions for near-infrared light irradiation are: power is 2W, irradiation is 10 minutes, and the wavelength of near-infrared light is 808 nm; The dilution is 10,000 times; The bacterium is one of Pseudomonas aeruginosa, Salmonella, Escherichia coli or Staphylococcus aureus.