Chiral nano-film of anti-staphylococcus aureus biological membrane as well as preparation method and application of chiral nano-film

Abstract

The invention discloses a preparation method of a chiral nano-film of an anti-staphylococcus aureus biological membrane, and belongs to the technical field of nano-materials. According to the method, nanogold is taken as a core, chiral phenylalanine is modified on the surface of the nanogold through water bath heating, and the chiral phenylalanine nanogold anti-biofilm is prepared through a liquid-liquid interface self-assembly method. According to the method disclosed by the invention, the dispersibility, the stability and the antibacterial property of the nanogold and the pure phenylalanine can be remarkably improved, and a remarkable inhibition effect on the formation of a staphylococcus aureus biological membrane is shown.

Description

Technical Field

[0001] The invention belongs to the technical field of nano materials, and in particular relates to a method for preparing a chiral nano film that resists Staphylococcus aureus biofilm. Background Art

[0002] Gold nanoparticles have attracted extensive attention due to their low cytotoxicity, good biocompatibility, and easy surface modification. They have been used in many fields such as biosensors, photothermal therapy, antimicrobial agents, and drug delivery. However, they have low antibacterial activity, are expensive, and are prone to aggregation, making it difficult to meet the needs of practical applications. Chiral phenylalanine amino acids have obvious antibacterial properties, can regulate cell group behavior, and effectively hinder the formation of bacterial biofilms. However, they can only inhibit or degrade biofilms at high concentrations, and have low solubility and are easily oxidized. Summary of the invention

[0003] The purpose of the present invention is to provide a method for preparing a chiral nanofilm against Staphylococcus aureus biofilm, and to obtain a uniform single-layer chiral phenylalanine nanogold film through a liquid-liquid interface self-assembly method, which combines the advantages of nanogold and chiral phenylalanine, reduces the amount of chiral phenylalanine used, and significantly improves the antibacterial activity through their synergistic effect and complementary advantages.

[0004] The purpose of the present invention is achieved through the following technical solutions:

[0005] A method for preparing a chiral nanofilm against Staphylococcus aureus biofilm: The material is prepared by the following method:

[0006] (1) mixing water and chloroauric acid solution, and then stirring at a temperature of 50 to 70° C. for 20 to 40 minutes to obtain a first mixed solution;

[0007] Mix water, tannic acid solution, trisodium citrate solution and potassium carbonate solution, and then stir at a temperature of 50 to 70° C. for 20 to 40 minutes to obtain a second mixed solution;

[0008] The second mixed solution is added to the first mixed solution and stirred for 20 to 40 minutes at a temperature of 50 to 70° C., and then the temperature is raised until the solution boils to obtain a gold seed solution;

[0009] (2) mixing water and chloroauric acid solution, stirring and heating to boiling, then adding trisodium citrate solution and gold seed solution, heating and stirring until the color of the solution changes from blue to stable wine red, stirring at room temperature, adding sodium hydroxide solution, centrifuging to remove the supernatant, and resuspending in water to obtain Au nanoparticle solution;

[0010] (3) Add phenylalanine solution to the Au nanoparticle solution, heat and stir, centrifuge to remove the supernatant, add water to resuspend, and obtain a Phe-Au nanoparticle solution; add n-hexane solution to the Phe-Au nanoparticle solution, then titrate with anhydrous ethanol, and after a film is formed, transfer it to the PDMS film to obtain a Phe-Au nanofilm.

[0011] In the technical solution of the present invention: in the first mixed solution of step (1), the volume ratio of water to chloroauric acid solution is 25-35:1, and the molar concentration of chloroauric acid solution is 10-15 mmol / L;

[0012] In the second mixed solution, the volume ratio of water, tannic acid solution, trisodium citrate solution and potassium carbonate solution is 5-10:0.1-1:1-5:0.05-1;

[0013] The mass fraction of tannic acid is 0.5-5%; the mass fraction of trisodium citrate is 0.5-5%; the molar concentration of potassium carbonate solution is 20-30 mmol / L;

[0014] The volume ratio of the first mixed liquid to the second mixed liquid is 30-50:10.

[0015] In the technical solution of the present invention: the particle size of the gold seed nanoparticles in the gold seed solution in step (1) is 3.5 to 10 nm.

[0016] In the technical scheme of the present invention: the molar concentration of the chloroauric acid solution in step (2) is 8-12 mmol / L; the mass fraction of the trisodium citrate solution is 0.1-3%; the molar concentration of the sodium hydroxide solution is 0.01-1 mol / L;

[0017] The volume ratio of chloroauric acid solution: gold seed solution: trisodium citrate solution: sodium hydroxide solution is 1-10:1-5:0.5-2:0.1-5.

[0018] In the technical solution of the present invention: in step (2), the particle size of the Au nanoparticles in the Au nanoparticle solution is 40 to 50 nm, and the concentration of the Au nanoparticle solution is 0.2 to 0.4 mmol / L.

[0019] In the technical solution of the present invention: the molar concentration of the phenylalanine solution in step (3) is 180-220 mmol / L; the volume ratio of the Au nanoparticle solution: the phenylalanine solution is 1-5:1-5.

[0020] In the technical solution of the present invention: the phenylalanine in step (3) is D-phenylalanine or L-phenylalanine, and the molar concentration of phenylalanine in the Phe-Au nano solution is 30-60 mmol / L.

[0021] A chiral nano film for resisting Staphylococcus aureus biofilm is prepared by the method.

[0022] In the technical solution of the present invention, the chiral nanofilm against Staphylococcus aureus biofilm prepared by the above method can be applied to food packaging materials, food machinery and medical device surfaces, effectively preventing the formation of Staphylococcus aureus biofilm and reducing the probability of disease infection caused by biofilm formation.

[0023] Beneficial effects of the present invention:

[0024] (1) High antibacterial activity: Modifying the surface of gold nanoparticles with phenylalanine effectively improves the antibacterial activity of both. Only a small concentration of chiral phenylalanine is needed to inhibit the adhesion of Staphylococcus aureus and biofilm formation.

[0025] (2) Stable chemical properties: Improve the stability of chiral gold nanofilms, effectively prevent the aggregation of gold nanoparticles, and obtain chiral gold nanofilms with larger areas and higher yields;

[0026] (3) The synthesis method is simple: the reaction conditions are mild, the reagents are green and environmentally friendly, the operation is simple and easy to control. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the TEM image of gold seed nanoparticles with a particle size of 3.5 to 10 nm.

[0028] Figure 2 TEM image of Au nanoparticles with a particle size of 40 to 50 nm.

[0029] Figure 3 SEM images of gold nanofilms with different chirality. Note: (A) D-Phe-Au nanofilm; (B) L-Phe-Au nanofilm.

[0030] Figure 4 Figure 2 shows the number of Staphylococcus aureus adhered to different film surfaces.

[0031] Figure 5 The graphs show the biofilm adhesion rate and colony growth curves of Staphylococcus aureus on different film surfaces. Note: (a) Biofilm adhesion rate on film surface; (b) Colony growth curve on film surface. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto:

[0033] Example 1

[0034] (1) Preparation of gold seed solution: Take a clean conical flask A and add 38.75mL of ultrapure water and 1.25mL of 11.8mmol / L chloroauric acid solution in sequence; take another clean conical flask B and add 7.5mL of ultrapure water, 0.40mL of 1wt% tannic acid solution, 2mL of 1wt% trisodium citrate solution, and 0.10mL of 25mmol / L potassium carbonate solution in sequence. Place solutions A and B on a magnetic stirrer and heat and stir at 60°C for 30 minutes, then quickly add the liquid in bottle B to bottle A, and continue heating and stirring at 60°C for 30 minutes. Then heat the mixed solution to boil until the solution becomes clear and transparent bright red, stop heating, and cool to room temperature to obtain a gold seed solution. Figure 1 The nanoparticles in the gold seed solution have a particle size of 3.5 to 10 nm.

[0035] (2) Preparation of Au nanoparticle solution: Take a clean conical flask and add 195 mL of ultrapure water and 5 mL of 11.8 mmol / L chloroauric acid solution in sequence, then place it on a magnetic stirrer and heat and stir until the solution boils. When boiling occurs, immediately add 0.8 mL of 1% trisodium citrate solution and 2 mL of the gold seed solution obtained in step (1), heat and stir until the color of the solution changes from blue to a stable wine red, cool to room temperature, then add 0.6 mL of 0.1 mol / L sodium hydroxide solution to every 30 mL of the solution, centrifuge at 6000 rpm / min and 6800 rpm / min for 10 min respectively, remove the supernatant, add 3 mL of ultrapure water and resuspend to obtain a 0.294 mmol / L Au nanoparticle solution. Figure 2 , Figure 2 The particle size of the Au nanoparticles in the Au nanoparticle solution is 40 to 50 nm.

[0036] (3) Preparation of L-Phe-Au nanofilm: Add 1 mL of 200 mmol / L L-Phe solution to 3 mL of Au nanoparticle solution obtained in step (2), place in a 60°C water bath for 2 h, centrifuge at 6000 rpm / min and 6800 rpm / min for 10 min respectively, remove the supernatant, add 3 mL of ultrapure water and resuspend to obtain a 37.5 mmol / L (phenylalanine molar concentration) L-Phe-Au solution. Add 10 mL of n-hexane solution to the L-Phe-Au solution, then titrate with anhydrous ethanol. After forming a large-area film, transfer the film to the PDMS film using a glass slide soaked in piranha etching solution to obtain an L-Phe-Au film.

[0037] Example 2

[0038] (1) Preparation of gold seed solution: Take a clean conical flask A and add 38.75mL ultrapure water and 1.25mL 11.8mmol / L chloroauric acid solution in sequence; take another clean conical flask B and add 7.5mL ultrapure water, 0.40mL 1% tannic acid solution, 2mL 1% trisodium citrate solution, and 0.10mL 25mmol / L potassium carbonate solution in sequence. Place solutions A and B on a magnetic stirrer and heat and stir at 60℃ for 30min, then quickly add the liquid in bottle B to bottle A, and continue heating and stirring at 60℃ for 30min. Then heat the mixed solution to boil until the solution turns into a clear and transparent bright red state, stop heating, and cool to room temperature to obtain a gold seed solution.

[0039] (2) Preparation of Au nanoparticle solution: Take a clean conical flask and add 195 mL of ultrapure water and 5 mL of 11.8 mmol / L chloroauric acid solution in sequence, then place it on a magnetic stirrer and heat and stir until the solution boils. When boiling occurs, immediately add 0.8 mL of 1% trisodium citrate solution and 2 mL of the gold seed solution obtained in step (1), heat and stir until the color of the solution changes from blue to a stable wine red, cool to room temperature, then add 0.6 mL of 0.1 mol / L sodium hydroxide solution to every 30 mL of the solution, centrifuge at 6000 rpm / min and 6800 rpm / min for 10 min respectively, remove the supernatant, add 3 mL of ultrapure water and resuspend to obtain a 0.294 mmol / L Au nanoparticle solution.

[0040] (3) Preparation of D-Phe-Au nanofilm: Add 1 mL of 200 mmol / L D-Phe solution to 3 mL of Au nanoparticle solution obtained in step (2), place in a 60°C water bath for 2 h, centrifuge at 6000 rpm / min and 6800 rpm / min for 10 min respectively, remove the supernatant, add 3 mL of ultrapure water and resuspend to obtain a 37.5 mmol / L (phenylalanine molar concentration) D-Phe-Au solution. Add 10 mL of n-hexane solution to the D-Phe-Au solution, then titrate with anhydrous ethanol. After forming a large-area film, transfer the glass slide soaked with piranha etching solution to the PDMS film to obtain a D-Phe-Au film.

[0041] Comparative Example 1

[0042] Taking the pure Au nanofilm as comparative example 1, the particle size of the Au nanoparticles is 45±5 nm.

[0043] The synthesis steps of the Au nanoparticle solution are the same as step (1) and step (2) of Example 1.

[0044] Preparation of Au nanofilm: 10 mL of n-hexane solution was added to the Au nanoparticle solution, followed by titration with anhydrous ethanol. After a large-area film was formed, a glass sheet soaked in piranha etching solution was transferred to the PDMS film to obtain an Au nanofilm.

[0045] Blank group:

[0046] The PDMS film was used as the blank group.

[0047] The synthesis steps are as follows: PDMS matrix and cross-linking agent are mixed at a mass ratio of 10:1, stirred for 20 minutes, ultrasonicated at room temperature (100KHz, 224W) for 15 minutes, and after the bubbles completely disappear, placed in a 60°C oven for curing for 4 hours. 20μL APTES is added to each 20mL of anhydrous ethanol to prepare a 3-aminopropyltriethoxysilane (APTES) solution, and the PDMS film is soaked in the APTES solution for 1 hour, and then soaked in anhydrous ethanol for 30 seconds to obtain a PDMS film, which is placed in a 60°C oven and dried for 1 hour before being taken out for use.

[0048] Performance testing:

[0049] 1) Scanning electron microscope (SEM) test

[0050] The SEM images of the chiral phenylalanine nanogold films prepared in Examples 1 and 2 are as follows: Figure 3 As shown. Figure 3 It can be seen that the D / L-Phe modified chiral gold nanoparticles (Examples 1 to 2) are tightly arranged without stacking. The prepared chiral gold nanofilm exhibits good flatness and compactness, showing the structural characteristics of a single-layer film.

[0051] 2) Anti-Staphylococcus aureus adhesion performance

[0052] Films of the same size were taken from Examples 1 to 2, Comparative Example 1 and the blank group, and mixed with Staphylococcus aureus solution for 4 hours. The anti-adhesion performance of the chiral phenylalanine nanogold film was evaluated by comparing the number of Staphylococcus aureus adhered to the surfaces of different films.

[0053] Examples 1 and 2, Comparative Example 1, the number of Staphylococcus aureus adhered to the film surface of the blank group is as follows: Figure 4 As shown. Figure 4It can be seen that the number of Staphylococcus aureus adhered to the surface of PDMS (blank group) and Au nanofilm (comparative example 1) is significantly greater than that of L-Phe-Au and D-Phe-Au nanofilms (examples 1-2). The number of Staphylococcus aureus adhered to the surface of L-Phe Au and D-Phe Au nanofilms (examples 1-2) is reduced to 36% and 30% respectively compared with PDMS (blank group). Therefore, D / L-Phe Au nanofilms (examples 1-2) have a strong ability to resist Staphylococcus aureus adhesion.

[0054] 3) Anti-Staphylococcus aureus biofilm performance

[0055] Films of the same size were taken from Examples 1 and 2, Comparative Example 1, and the blank group, and mixed and cultured with Staphylococcus aureus solution for 72 hours. The experiment was conducted every 12 hours, and the anti-biofilm performance of the chiral phenylalanine nanogold film was evaluated by comparing the biofilm adhesion rate and colony growth on the surfaces of different films.

[0056] Examples 1 and 2, Comparative Example 1, the adhesion rate of Staphylococcus aureus biofilm on the film surface of the blank group and the growth curve of the colony are as follows Figure 5 As shown. Figure 5 It can be seen that the number of live bacteria adhering to the PDMS (blank group) surface is the largest, and the number of live bacteria adhering to the Au nanofilm (comparative example 1) surface is somewhat lower than that of the PDMS (blank group) surface, but biofilms are formed on both surfaces. 00 The value of the film was the smallest, and the number of Staphylococcus aureus adhered was the least, followed by L-Phe-Au nanofilm (Example 1), and the number of Staphylococcus aureus adhered to the surfaces of the two films never exceeded 10 8 CFU / cm 2 , no Staphylococcus aureus biofilm was formed, which may be the result of the synergistic effect of nano-gold particles and chiral phenylalanine. Nano-gold particles, as a stable carrier, can effectively prevent the oxidation of phenylalanine, and phenylalanine, as a functional factor, can effectively enhance the antibacterial ability of nano-gold particles. Therefore, D / L-Phe Au nanofilms (Examples 1-2) have strong anti-Staphylococcus aureus biofilm performance.

Claims

1. A method for preparing a chiral nanofilm against Staphylococcus aureus biofilm, characterized in that: The material is prepared by the following method: (1) mixing water and chloroauric acid solution, and then stirring at a temperature of 50 to 70° C. for 20 to 40 minutes to obtain a first mixed solution; Mix water, tannic acid solution, trisodium citrate solution and potassium carbonate solution, and then stir at a temperature of 50 to 70° C. for 20 to 40 minutes to obtain a second mixed solution; The second mixed solution is added to the first mixed solution and stirred for 20 to 40 minutes at a temperature of 50 to 70° C., and then the temperature is raised until the solution boils to obtain a gold seed solution; (2) mixing water and chloroauric acid solution, stirring and heating to boiling, then adding trisodium citrate solution and gold seed solution, heating and stirring until the color of the solution changes from blue to stable wine red, stirring at room temperature, adding sodium hydroxide solution, centrifuging to remove the supernatant, and resuspending in water to obtain Au nanoparticle solution; (3) adding phenylalanine solution to the Au nanoparticle solution, heating and stirring, centrifuging to remove the supernatant, and resuspending in water to obtain a Phe-Au nanoparticle solution; A n-hexane solution was added to the Phe-Au nanoparticle solution, followed by titration with anhydrous ethanol. After forming a film, the film was transferred to the PDMS film to obtain a Phe-Au nanofilm.

2. The chiral nanofilm against Staphylococcus aureus biofilm according to claim 1, characterized in that: In the first mixed solution of step (1), the volume ratio of water to chloroauric acid solution is 25-35:1, and the molar concentration of chloroauric acid solution is 10-15 mmol / L; In the second mixed solution, the volume ratio of water, tannic acid solution, trisodium citrate solution and potassium carbonate solution is 5 to 10: 0.1～1：1～5：0.05～1； The mass fraction of tannic acid is 0.5-5%; the mass fraction of trisodium citrate is 0.5-5%; the molar concentration of potassium carbonate solution is 20-30 mmol / L; The volume ratio of the first mixed liquid to the second mixed liquid is 30-50:

10.

3. The chiral nanofilm against Staphylococcus aureus biofilm according to claim 1, characterized in that: The particle size of the gold seed nanoparticles in the gold seed solution in step (1) is 3.5 to 10 nm.

4. The chiral nanofilm against Staphylococcus aureus biofilm according to claim 1, characterized in that: In step (2), the molar concentration of the chloroauric acid solution is 8-12 mmol / L; the mass fraction of the trisodium citrate solution is 0.1-3%; and the molar concentration of the sodium hydroxide solution is 0.01-1 mol / L; The volume ratio of chloroauric acid solution: gold seed solution: trisodium citrate solution: sodium hydroxide solution is 1-10:1-5:0.5-2:0.1-5.

5. The chiral nanofilm against Staphylococcus aureus biofilm according to claim 1, characterized in that: In step (2), the particle size of the Au nanoparticles in the Au nanoparticle solution is 40 to 50 nm, and the concentration of the Au nanoparticle solution is 0.2 to 0.4 mmol / L.

6. The chiral nanofilm against Staphylococcus aureus biofilm according to claim 1, characterized in that: The molar concentration of the phenylalanine solution in step (3) is 180-220 mmol / L; the volume ratio of the Au nanoparticle solution: the phenylalanine solution is 1-5:1-5.

7. The chiral nanofilm against Staphylococcus aureus biofilm according to claim 1, characterized in that: The phenylalanine in step (3) is D-phenylalanine or L-phenylalanine, and the molar concentration of phenylalanine in the Phe-Au nano solution is 30-60 mmol / L.

8. A chiral nanofilm against Staphylococcus aureus biofilm, characterized in that: The film is prepared by the method described in any one of claims 1 to 7.

9. The chiral nanofilm against Staphylococcus aureus biofilm prepared by the method of claim 1 can be applied to food packaging materials, food machinery and medical device surfaces, effectively preventing the formation of Staphylococcus aureus biofilm and reducing the probability of disease infection caused by biofilm formation.

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