CN113575612A

CN113575612A - Antibacterial film with gold nanosheet loaded on bacterial cellulose and preparation method and application thereof

Info

Publication number: CN113575612A
Application number: CN202110814479.2A
Authority: CN
Inventors: 周满; 孟珂卉; 李忠玉; 梁倩; 徐松; 梁玄; 薛苏婷
Original assignee: Changzhou University
Current assignee: Changzhou University
Priority date: 2021-07-19
Filing date: 2021-07-19
Publication date: 2021-11-02

Abstract

The invention belongs to the field of microbial technology and inorganic materials, and relates to a bacterial cellulose gold nanosheet-loaded antibacterial film, and a preparation method and application thereof, wherein the preparation method comprises the following steps: immersing the bacterial cellulose membrane into 0.1M NaOH solution with the temperature of 90-95 ℃, then washing the bacterial cellulose membrane to be neutral by using distilled water, and drying the bacterial cellulose membrane in vacuum to obtain a pure bacterial cellulose sample; and then, immersing the obtained pure bacterial cellulose sample into a chloroauric acid solution, irradiating by using ultraviolet light, washing by using deionized water after the ultraviolet light irradiation, and then drying in vacuum to obtain the antibacterial film with the bacterial cellulose loaded with the gold nanosheets. The preparation method of the gold nanoplate reduced by the ultraviolet light-induced bacterial cellulose is simple, environment-friendly and reusable, and the gold nanoplate loaded by the bacterial cellulose has better antibacterial ability and self-degradation ability because the bacterial cellulose has good biodegradability.

Description

Antibacterial film with gold nanosheet loaded on bacterial cellulose and preparation method and application thereof

Technical Field

The invention relates to an antibacterial film with gold nanosheets loaded on bacterial cellulose, and a preparation method and application thereof, and belongs to the fields of microbial technology and inorganic materials.

Background

The antibacterial material has important significance for solving the problems of infectious diseases and bacteria which harm human life health, so that the development of the reusable and environment-friendly antibacterial film becomes a research hotspot in many other related fields such as biochemistry and environmental chemistry. The Bacterial Cellulose (BC) is a natural nano biological material which is rich in reserves in the nature, and has biodegradability and good biocompatibility. However, the bacterial cellulose has the disadvantages that the bacterial cellulose does not have antibacterial capability per se, the bacterial cellulose cannot inhibit or kill bacteria, and the bacterial cellulose is easy to be corroded by microorganisms and difficult to deal with wounds caused by bacterial infection. Therefore, the endowment of the bacterial cellulose with unique antibacterial property has important significance in life science. At present, the antibacterial material is more a novel functional material which has the capability of inhibiting or killing bacteria on the surface of the material by adding a certain antibacterial substance (antibacterial agent), such as antibacterial plastic, antibacterial fiber, antibacterial ceramic and the like. In recent years, the use of bacterial cellulose for photocatalysts and the like has been reported by our group. However, as far as we know, the progress of using bacterial cellulose for antibacterial and disease prevention is very slight.

Gold is a transition metal that, when dissolved, can form trivalent or monovalent positive ions (+1/+ 3/zero valence). The gold has a reflectivity of approximately 100% for infrared light. Partial gold salts have the property of preventing inflammation and are used for treating arthritis, and researches show that the gold nanoparticles have wide antibacterial spectrum, various antibacterial mechanisms and good biocompatibility.

Gold nanoparticles (AuNPs) are typically prepared by reduction of gold salts (e.g., tetrachloroauric acid (HAuCl)₄) Or synthesized in situ using a seed growth method, in which the particles are gradually enlarged. Depending on their concentration and particle size and surrounding surface functional groups (ligands), gold nanoparticles can be biocompatible and chemically inert, and can be used for medical applications such as cellular imaging and photothermal therapy.

Over the past 10 years, studies have shown that AuNPs are also very effective against bacteria, including multi-drug resistant bacteria, without binding to commercial antibiotics. In a word, AuNPs have wide antibacterial spectrum, various antibacterial mechanisms and good biocompatibility. Hydroxyl on the surface of bacterial cellulose has reducibility, chloroauric acid is reduced into gold nanoplatelets with controllable appearances, the gold nanoplatelets are embedded in a three-dimensional framework of the bacterial cellulose, and the gold nanoplatelets are applied to inhibiting the growth of common escherichia coli (E.coli) and staphylococcus aureus (S.aureus).

Disclosure of Invention

The invention aims to provide an antibacterial film with gold nano-sheets loaded on bacterial cellulose, and a preparation method and application thereof. Under the ultraviolet illumination, time regulation and control are carried out, the induced bacterial cellulose can self-reduce chloroauric acid to generate gold nanoplates with controllable shapes, and the gold nanoplates can repeatedly generate better inhibiting effect on escherichia coli and staphylococcus aureus after simple treatment.

A preparation method of a bacterial cellulose loaded gold nano-sheet antibacterial film comprises the following steps:

a. purifying bacterial cellulose: soaking the bacterial cellulose membrane into 0.1M NaOH solution with the temperature of 90-95 ℃ (the soaking time is preferably 2 hours), washing the bacterial cellulose membrane to be neutral by using distilled water, and drying the bacterial cellulose membrane in vacuum to obtain a pure sample of the bacterial cellulose.

b. Preparing a bacterial cellulose gold nanosheet antibacterial film (AuNSs @ BC for short): and (b) immersing the pure bacterial cellulose sample obtained in the step (a) into a chloroauric acid solution (preferably, the concentration of the chloroauric acid solution is 30-100 mM), irradiating by using ultraviolet light, washing by using deionized water after the ultraviolet irradiation, and drying in vacuum to obtain the AuNSs @ BC.

Preferably, the bacterial cellulose is immersed into 100ml of the chloroauric acid solution in the step b, and the mass of the bacterial cellulose is 6-8 g.

Preferably, the ultraviolet irradiation time in the step b is 12 hours, wherein the wavelength of the ultraviolet light is 254 nm-365 nm.

A bacterial cellulose loaded gold nano-sheet antibacterial film is prepared based on the method.

The application of the bacterial cellulose loaded gold nanosheet antibacterial film is used for inhibiting escherichia coli and staphylococcus aureus.

The invention has the beneficial effects that: the preparation method of the antibacterial film of the gold nanoplatelets loaded by the bacterial cellulose is simple and environment-friendly, and can be repeatedly used, and the gold nanoplatelets loaded by the bacterial cellulose have better antibacterial capability and self-degradation capability because the bacterial cellulose has good biodegradability. Furthermore, the AuNSs @ BC had better tensile strength and elongation than the pure BC film. The gold nanoplate prepared by the method has high dispersibility, no obvious agglomeration phenomenon, and the side length range of the gold nanoplate is 4-12 nm.

Drawings

FIG. 1 is a scanning electron microscope image of AuNSs-S @ BC obtained by reducing gold nanoplates with ultraviolet light induced bacterial cellulose in the first embodiment.

FIG. 2 shows the X-ray diffraction (XRD) spectra of AuNPs @ BC and AuNSs-S, AuNSs-L @ BC obtained by immersing the bacterial cellulose in 30mM, 60mM and 100mM chloroauric acid solution in example two, (AuNSs @ BC obtained after bacteriostasis test in example 2).

FIG. 3 is a TEM image of UV-induced reduction of gold nanoplates AuNSs-S @ BC by bacterial cellulose in example II.

FIG. 4 is a histogram of AuNSs side length size distribution from optical microscope image statistics in example II.

FIG. 5 shows the inhibition bands of AuNPs @ BC, AuNSs-S @ BC and AuNSs-L @ BC on Staphylococcus aureus and Escherichia coli in example II.

FIG. 6 shows the inhibition bands of AuNSs-S @ BC against Staphylococcus aureus and Escherichia coli in example III, which were repeated three times.

FIG. 7 is a graph showing the growth of AuNPs @ BC, AuNSs-S @ BC, AuNSs-L @ BC on Staphylococcus aureus and Escherichia coli in example II. Detailed Description

The invention will now be further illustrated by reference to specific examples, which are intended to be illustrative of the invention and are not intended to be a further limitation of the invention.

The bacterial cellulose in the following examples was purchased from Guilin Qi scientific and technology, Inc.

The antibacterial effect evaluation of the antibacterial film with gold nanosheets loaded on bacterial cellulose prepared in the following examples adopts a disc diffusion method, wherein the preparation method of the solid medium is as follows:

preparing a solid culture medium: weighing nutrient broth and agar powder by using an electronic balance, dissolving in deionized water, and placing into a high-pressure steam sterilization pot for sterilization for 30min to form an original sterile culture medium. Pouring the warm original sterile culture medium into a culture dish sterilized by ultraviolet for 30min on an ultra-clean workbench, and forming a solid culture medium after the culture dish is cooled and solidified. Wherein the weight of the nutrient broth is 3.6g, the weight of the agar powder is 3.0g, and the volume of the deionized water is 200 mL.

Preparing materials of escherichia coli and staphylococcus aureus: the culture is carried out by using a streak plate method, and staphylococcus aureus and escherichia coli are respectively taken and placed in nutrient pure broth (without agar) to be cultured in a constant temperature bacterial incubator at 37 ℃ in a shaking way. Wherein the mass of the nutrient broth is 9g, the volume of the deionized water is 500mL, and the incubation time of the bacteria is 12 h.

The antibacterial effect of the prepared material on escherichia coli and staphylococcus aureus is as follows: and respectively adding 1mL of staphylococcus aureus and escherichia coli diluent into the AuNPs @ BC, AuNSs-S @ BC and AuNSs-L @ BC cut into the round pieces, adding the mixture into a solid culture medium, and carrying out shake culture in a constant-temperature bacterial incubator at 37 ℃. Wherein AuNPs @ BC, AuNSs-S @ BC and AuNSs-L @ BC are cut into disks with the size of 6mm, and the incubation time of the bacteria is 24 h. The width H of the bacteriostatic band is (D-D)/2, wherein H is the width (mm) of the bacteriostatic band; d-average value (mm) of the outer diameter of the zone; d-sample diameter (mm);

TABLE 1 evaluation of antibacterial Effect

Description of the drawings: bacteria can grow rapidly in the culture dish, but because the dressing sample has antibacterial capacity, the bacteria can generate a remarkable bacteria inhibition zone around the sample, namely a bacteriostasis zone.

The first embodiment is as follows:

the preparation method of the antibacterial film with the bacterial cellulose loaded with the gold nanosheets comprises the following steps:

(1) weighing a sample with the mass of 0.6-0.8 g of bacterial cellulose, placing the sample in a beaker, immediately adding 100mL0.1mol of NaoH solution, boiling at the constant temperature of 95 ℃ for 2h, cooling to room temperature to obtain an opaque suspension, and vacuum-drying for 24h to obtain a pure Bacterial Cellulose (BC) sample.

(2) Transferring 1mL, 2mL and 3.4mL of chloroauric acid solution with the mass content of 10% by using a liquid transfer gun, respectively transferring the chloroauric acid solution into a 10mL volumetric flask, preparing 30mM, 60mM and 100mM chloroauric acid solution, soaking the pure bacterial cellulose sample in the step (1) in the 30mM chloroauric acid solution for 12h of ultraviolet lamp illumination to be AuNPs @ BC, in the 60mM chloroauric acid solution for 12h of ultraviolet lamp illumination to be AuNSs-S @ BC, and in the 100mM chloroauric acid solution for 12h of ultraviolet lamp illumination to be AuNSs-L @ BC, wherein the wavelength of the ultraviolet lamp is 254nm, after the ultraviolet lamp illumination is carried out at the power of 8W, taking out the pure bacterial cellulose sample, washing the pure bacterial cellulose sample by using distilled water, and carrying out vacuum drying to obtain a sample of the bacterial cellulose-loaded nano-sheet antibacterial gold film (AuNPs @ BC, AuNSs-S @ BC and AuNSs-L @ BC).

FIG. 1 is a field emission scanning electron microscope image of a bacterial cellulose loaded gold nanosheet antibacterial film obtained by dipping a bacterial cellulose pure sample in a 60mM chloroauric acid solution through ultraviolet lamp illumination for 12 hours, and is recorded as AuNSs-S @ BC and AuNSs-S @ BC, and the shape of the gold nanosheet reduced by ultraviolet light induced bacterial cellulose is shown in FIG. 1 to include regular polygons, quadrangles and triangles.

Example two:

the AuNPs @ BC, AuNSs-S @ BC and AuNSs-L @ BC prepared in the first embodiment are placed in a solid culture medium containing bacterial liquid, and incubated in a constant-temperature incubator for 24 hours at 37 ℃ and the oscillation frequency of the incubator of 78r/min, and the result shows that the prepared AuNSs-S @ BC has an obvious bacteriostatic effect. Referring to fig. 5, the bacterial cellulose-loaded gold nanosheet antibacterial film (marked as the antibacterial effect of AuNSs-S @ BC) is obtained by soaking a pure bacterial cellulose sample in a 60mM chloroauric acid solution and then performing ultraviolet illumination for 12 hours, and as can be seen from the drawing of fig. 5, the AuNSs-S @ BC antibacterial film has a good antibacterial effect and an obvious antibacterial zone.

Example three:

and (3) taking the AuNSs @ BC obtained in the second embodiment after incubation for 24h in a solid culture medium containing a bacterial liquid, washing with deionized water and ethanol, and drying at 60 ℃ to obtain the reusable AuNSs @ BC. Repeated experiments are carried out under the same conditions of the second embodiment, and the results prove that the reusable AuNSs-S @ BC product still has a good bacteriostatic effect after being repeatedly used, as shown in figure 6, the AuNSs-S @ BC product is the bacteriostatic effect of the reusable AuNSs-S @ BC product after being washed, and as can be seen from the figure 6, the AuNSs-S @ BC product still has an obvious bacteriostatic effect.

Example four:

the AuNSs @ BC and the BC prepared in the first embodiment are respectively placed in a solid culture medium containing bacterial liquid and incubated in a constant-temperature incubator for 24 hours to obtain an antibacterial effect, the temperature is 37 ℃, the oscillation frequency of the incubator is 78r/min, and a test result shows that the AuNSs @ BC has a more obvious antibacterial ring compared with the BC, the antibacterial effect comparison of the AuNSs-S @ BC and the BC is specifically shown in figure 6, and as can be seen from the figure 6, the AuNSs-S @ BC has a good antibacterial effect and an obvious antibacterial ring compared with the BC.

FIG. 7 shows the growth inhibition zone of Escherichia coli and Staphylococcus aureus, in which AuNSs-S @ BC is obtained by soaking pure bacterial cellulose sample in 60mM chloroauric acid solution and irradiating with ultraviolet light for 12 h.

In addition, the composite membrane prepared by the preparation and catalytic performance of the composite membrane of bacterial cellulose-loaded gold nanoparticles published by synthetic fibers of chenyan, chen shi yan and the like is proved to be unsuitable for the application, and AuNPs ultrasonically treated on the composite membrane have a spherical structure, uniform particle size, regular shape, small particle size, high dispersibility and no obvious agglomeration phenomenon. The average grain size of AuNPs is 5.3nm, the standard deviation is 1.23nm, and the interplanar spacing is 0.23 nm; however, the AuNSs structure under the ultraviolet irradiation method is basically flaky, the particle size distribution is wide, the shape is controllable, the average particle size of the AuNSs is 7.4nm (see figure 7), and the interplanar spacing is 0.21 nm. The method disclosed by the document has the advantages that the method shows higher catalytic efficiency in a catalytic reduction 4-NP test; however, the use effect of the method of the present application is against Escherichia coli and Staphylococcus aureus.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Claims

1. A preparation method of an antibacterial film with gold nanosheets loaded on bacterial cellulose is characterized by comprising the following steps: the method comprises the following steps:

a. purifying bacterial cellulose: purifying bacterial cellulose, namely immersing the bacterial cellulose membrane into 0.1M NaOH solution at 90-95 ℃, washing the bacterial cellulose membrane to be neutral by using distilled water, and drying the bacterial cellulose membrane in vacuum to obtain a pure bacterial cellulose sample;

b. preparing an antibacterial film with gold nanosheets loaded on bacterial cellulose: and (b) immersing the pure bacterial cellulose sample obtained in the step (a) into a chloroauric acid solution, irradiating by using ultraviolet light, washing by using deionized water after the ultraviolet light irradiation, and then drying in vacuum to obtain the antibacterial film with the bacterial cellulose loaded with the gold nanosheets.

2. The method for preparing an antibacterial film with gold nanosheets loaded on bacterial cellulose according to claim 1, wherein: and (b) soaking in the NaOH solution for 2h in the step a.

3. The method for preparing an antibacterial film with gold nanosheets loaded on bacterial cellulose according to claim 1, wherein: the concentration of the chloroauric acid solution in the step b is 30-100 mM.

4. The method for preparing an antibacterial film with gold nanosheets loaded on bacterial cellulose according to claim 1, wherein: in the step b, the wavelength of ultraviolet light is 254 nm-365 nm, and the ultraviolet illumination time is 12 h.

5. The method for preparing an antibacterial film with gold nanosheets loaded on bacterial cellulose according to claim 1, wherein: in the step b, the mass of bacterial cellulose dipped into each 100ml of chloroauric acid solution is 6-8 g.

6. A bacterial cellulose loaded gold nanosheet antibacterial film is characterized in that: the antibacterial film is prepared by the method for preparing the antibacterial film with gold nano-sheets loaded on the bacterial cellulose of any one of the claims 1 to 5.

7. The use of a bacterial cellulose-loaded gold nanoplate antimicrobial film according to claim 6, wherein: can be used for inhibiting Escherichia coli and Staphylococcus aureus.