Synthesis of silver nanoparticles using Bombyx mori silk fibroin and antibacterial activity

In the present work, the stable silver nanoparticles (AgNPs) were produced in situ in the presence of white light, using aqueous silk fibroin (SF) acquired from Bombyx mori silk. The UV-Visible spectral study explained the production of AgNPs by displaying a distinctive surface plasmon resonance band (SPR) at the wavelength 424 nm. The crystalline nature of the produced AgNPs have been identified using XRD study. Nanocrystalline phase of silver (Ag) with face centered cubic (FCC) structure was observed by XRD. The shape evolution and size of the formed nanoparticles was studied using transmission electron microscope. The captured images shown the formed particle were spherical in shape in morphology, and diameter in the range 35 to 40 nm. In the application part, an attempt was made to evaluate the potential antibacterial activity of the biogenic silver nanoparticles against pathogenic bacteria’s such as Escherichia coli and Staphylococcus aureus.


Introduction
Nanotechnology is one of the most popular areas of current research and development in all disciplines of science. It has revolutionized in the field of materials science by creating the materials of unusual properties and range of applications. Nanotechnology mainly focuses on the synthesis, characterization and modification of particles size less than 100 nm. In the last two decades the synthesis of noble metal nanoparticles received greater importance because of the wide utility in many fields of science. For the synthesis of metal nanoparticles many physical and chemical methods are available in literature [12]. But due to environmental issues and biological applications several authors searching alternative procedures for the synthesis of metal nanoparticles. Recently, environmentally friendly or biobased methods leading to the evolution of nanoparticles getting more importance [34]. Bio based synthesis methods generally depends on the selection of solvent medium, unhazardous, reducing and stabilizing agents [5]. It is very important to mention here that synthesis of metal nanoparticles using biological/natural materials make nanoparticles more biocompatible, and environmentally friendly. In the current work, we have presented a simple, effective and ICONAMMA2018 IOP Conf. Series: Materials Science and Engineering 577 (2019) 012008 IOP Publishing doi:10.1088/1757-899X/577/1/012008 2 environmentally friendly method for in situ synthesis of silver nanoparticles using SF as a reducing and stabilizing agent at room temperature in the presence of white light condition. SF is a useful biomaterial due to its biocompatible, biodegradability, renewability and unhazardous properties. The produced AgNPs were characterized by using UVVisible, XRD and TEM techniques. The antibacterial activity of the biogenic nanoparticles were carried out with selected bacteria.

Materials
Chemicals like sodium carbonate (Na 2 CO 3 ), silver nitrate (AgNO 3 ) (>99%) and lithium bromide (LiBr) were procured from sigma Aldrich, used without any additional purification. Bombyx mori silk cocoons (Central Sericulture Research 4) were collected from the Department of Sericulture, University of Mysore and the aqueous solutions were prepared using double distilled water.

Preparation of aqueous silk fibroin solution
Detailed preparation method of silk fibroin solution is described in our recent work [6]. In a brief way, Bombyx mori silk cocoons were cut into tiny pieces and treated two times with boiling aqueous solution of 0.02 M sodium carbonate (Na 2 CO 3 , MW: 105.99 g/mole) for 30 min to eliminate the sticky materials. Then the fibroin mass rinsed using deionised water, and it was dried in air at normal temperature and pressure. The degummed SF fiber mass was melted in 9.3 M lithium bromide (LiBr, MW: 86.84 g/mole) salt solution at 70  C for 3 ~ 4 h. The obtained SF and LiBr mixture solution was dialyzed using dialysis cassette (MWCO: 3,500) against double distilled water for 72 h to remove ions present in the solution. Lastly, the attained optically transparent clear solution was centrifuged at 4000 rpm for nearly 15 min to get rid of any silk aggregation during the process. The transparent SF solution was kept at 4 °C for additional use. The initial concentration of SF was about 5 wt% and was diluted to 1 wt% by adding distilled water and used for the preparation of the nanoparticles.

Preparation of colloidal silk fibroinsilver nanoparticles solution
For the preparation of colloidal solution, ten to hundred milligrams of AgNO 3 salt was added into 10 mL of 1 wt% SF solution. The final concentration of AgNO 3 in the solution was 1 and 5 mg/mL. The prepared SFAgNO 3 mixture solution was irradiated using the white light (60 W, from Philips) at normal temperature for 24 h to produce AgNPs in SF. The obtained SFAgNPs colloids were characterized using different analytical techniques.

UVVisible spectroscopy
The UVVisible absorption spectra of the SF and SFAgNPs samples were recorded by using UV visible spectrophotometer (UV1800Shimadzu, Japan), in the wavelength 200 to 800 nm at room temperature.

XRD measurement study
The nanostructural aspects of the silk fibroinsilver nanoparticles were characterized by using Xray diffractometer (Riguku MiniflexII) equipped with Ni filter, CuKα radiation. The sample was scanned in the 2 range of 1080° with scanning rate of 5°/ min.

Transmission electron microscope images
The size and shape of the silver nanoparticles were obtained by using transmission electron microscopy (JEOLJEM 2100 LaB6) operated at 200 keV.

Antimicrobial activity study
Antibacterial properties of the AgNPs was evaluated using disc diffusion method against two bacteria namely, S. aureus and E. coli. The tested bacteria samples were acquired from Microbial Type Culture Collection (MTCC), Chandigarh160036, India.

UVVis study
The production of AgNPs in the SF solution by the reduction of AgNO 3 was evident from the colour change of the reaction mixture after exposed to white light. Originally, the reaction mixture of SF AgNO3 solution was colourless, but subsequently exposed to whitelight, the reaction mixture colour turns to yellow then dark brown within few minutes (figure 1).  The UVVis Absorption spectra of pure SF and SFAgNPs are presented in figure 3. From the spectra it is clear that, pure SF showed a band centered at =275 nm which coincides with the Tyr absorption band due to the excitation of * transition in the SF. On the other hand, SFAgNPs, exhibited a ICONAMMA2018 IOP Conf. Series: Materials Science and Engineering 577 (2019) 012008 IOP Publishing doi:10.1088/1757-899X/577/1/012008 4 single absorption peak in the wavelength range = 424426 nm advocated that formed nanoparticles are in spherical shape in accordance with the Mie theory [11]. This is also confirmed by the TEM study. We have also evaluated the effect of different concentrations of AgNO 3 on the synthesis of AgNPs. As the concentration of AgNO 3 was increased the corresponding peak intensity was also increased. The increasing trend in the intensity of the UVabs spectra denote the increase in the number of AgNPs formed in the SF solution.

XRD study
The crystalline features of the AgNPs formed in this work was recorded and the XRD scans are illustrated in figure 4. From the figure 4, the diffraction peaks observed at 2 = 19.76° and 29.34° are θ corresponding to crystalline region of the pure SF [12]. The intense diffraction peaks riding over a SF domain with an diffraction angles (2 ) about 38.02 θ , 44.35, 64.53°, and 76.34 corresponding to reflections (111), (200), (220), and (311) indicating crystal planes of the metallic silver. These planes confirm the synthesized AgNPs represent a face centred cubic (FCC) structure compared to the pure silver is Joint Committee on Powder Diffraction Standards, silver file No.000040783 [13]. The average crystallite size of the nanoparticles was calculated by using DebyeScherer's equation [14]. The average crystallite size of the AgNPs was found to be 35.23 nm.

TEM analysis
The high resolution TEM images ( figure 5 a, b) illustrate the nature of the silver nanoparticles formed in the silk fibroin solution. Almost all the nanoparticles are formed with the concentration of 5 mg/mL of AgNO 3 are exhibits exactly the spherical in size and shape with smooth faces. The diameter of the nanoparticles was around 35 to 40 nm.

Antimicrobial activity
The antibacterial activity of the formed AgNPs was evaluated using Grampositive and Gramnegative pathogens. The disc diffusion method was used in this study.

Disc diffusion method
Antibacterial study of the biogenic synthesized AgNPs was screened against Gramnegative Escherichia coli (E. coli) and Grampositive Staphylococcus aureus (S. aureus) bacteria. The zone of inhibition (mm) of AgNPs was measured and is tabulated in Table 1. The results indicate that pure SF is not showed activity against any of the tested bacterial strains. But, the prepared SFAgNPs colloidal solutions 1 and 5 mg/mL exhibits growth inhibitory effect on bacterial stains used in the study. However, the prepared samples exhibits bacterial inhibition, and is not uniform for all the tested bacteria.  [15]. In brief, the AgNPs interrupt the cell membrane integrity of the bacterial cells and upon entering into cytosol induces oxidative stress leading to the inhibition of cell advancement, which in turn, leads to cell death. The decomposition of the cell layer and the consequent decomposition of the cell membrane of the bacteria exposed to the AgNPs in the colloidal solution leads to the outflow of cytosolic contents, causing cell collapse [16].

Conclusion
Finally our study, spherical silver nanoparticles (AgNPs) were strongly synthesized by using SF as biotemplate under UVB light at normal pressure and temperature condition. In this study, spherical silver nanoparticles were produced by using SF as biotemplate at room temperature condition. UV Visible study established the production of silver nanoparticles by showing surface plasomn resonance band in the wavelength range 424426 nm. The synthesized AgNPs were highly crystalline nature with average crystallite size was about 35.23 nm. The synthesized silver nanoparticle showed a broad spectrum of antibacterial activity against selected human pathogens.