Investigation of antibacterial properties silver nanoparticles prepared via green method
© Shameli et al.; licensee Chemistry Central Ltd. 2012
Received: 20 May 2012
Accepted: 10 July 2012
Published: 27 July 2012
This study aims to investigate the influence of different stirring times on antibacterial activity of silver nanoparticles in polyethylene glycol (PEG) suspension. The silver nanoparticles (Ag-NPs) were prepared by green synthesis method using green agents, polyethylene glycol (PEG) under moderate temperature at different stirring times. Silver nitrate (AgNO3) was taken as the metal precursor while PEG was used as the solid support and polymeric stabilizer. The antibacterial activity of different sizes of nanosilver was investigated against Gram–positive [Staphylococcus aureus] and Gram–negative bacteria [Salmonella typhimurium SL1344] by the disk diffusion method using Müeller–Hinton Agar.
Formation of Ag-NPs was determined by UV–vis spectroscopy where surface plasmon absorption maxima can be observed at 412–437 nm from the UV–vis spectrum. The synthesized nanoparticles were also characterized by X-ray diffraction (XRD). The peaks in the XRD pattern confirmed that the Ag-NPs possessed a face-centered cubic and peaks of contaminated crystalline phases were unable to be located. Transmission electron microscopy (TEM) revealed that Ag-NPs synthesized were in spherical shape. The optimum stirring time to synthesize smallest particle size was 6 hours with mean diameter of 11.23 nm. Zeta potential results indicate that the stability of the Ag-NPs is increases at the 6 h stirring time of reaction. The Fourier transform infrared (FT-IR) spectrum suggested the complexation present between PEG and Ag-NPs. The Ag-NPs in PEG were effective against all bacteria tested. Higher antibacterial activity was observed for Ag-NPs with smaller size. These suggest that Ag-NPs can be employed as an effective bacteria inhibitor and can be applied in medical field.
Ag-NPs were successfully synthesized in PEG suspension under moderate temperature at different stirring times. The study clearly showed that the Ag-NPs with different stirring times exhibit inhibition towards the tested gram-positive and gram-negative bacteria.
Silver nanoparticles (Ag-NPs) have been known for its inhibitory and bactericidal effects in the past decades . Antibacterial activity of silver containing materials can be applied in medicine for reduction of infections on the burn treatment [2, 3], prevention of bacteria colonization on catheters [4, 5] and elimination of microorganisms on textile fabrics [6, 7] as well as disinfection in water treatment . Besides that, Ag-NPs were also being reported in the literature to exhibit a strong cytoprotective activity towards human immunodeficiency virus (HIV) infections . Polyethylene glycol (PEG) is frequently used in the polymer blends production to improve the biocompatibility of its film due to its wide range of molecular weights, excellent solubility in aqueous medium, low toxicity, chain flexibility and biocompatibility properties. Although PEG has non biodegradability properties, it is readily excreted from the body and forms non-toxic metabolites . Besides that, PEG was able to act both as reducing agent and stabilizer . In several research studies [12, 13], researchers proposed that longer polymer chain of PEG exhibits higher reducing activity and provides higher stability in forming Ag-NPs. These can effectively prevent agglomeration of Ag-NPs.
There are numerous techniques to perform antibacterial and antimicrobial susceptibility tests. The techniques include agar disk diffusion, broth dilution (macrodilution and microdilution), agar dilution and E test method (modification of the disk diffusion and the agar dilution method) . Agar disk diffusion is a traditional and routine method for antimicrobial susceptibility tests . It has advancement to be used in this project because of its reliability, low cost and simplicity [16, 17]. Mueller Hinton agar is chosen among the culture media because it gives satisfactory growth for most nonfastidious organisms like Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli and it shows good bacteria culture reproducibility .
There are many synthetic routes that have been developed to synthesize Ag-NPs due to the applications found tremendously in wide range of fields. Among the synthetic routes includes chemical reduction [19, 20], thermal decomposition , electrochemical , sonochemical , photochemical , microwave , radiation assisted process [26, 27] and currently by green chemistry synthesis [28–31].
Chemical reduction method is widely used to synthesize Ag-NPs because of its readiness to generate Ag-NPs under gentle conditions and its ability to synthesize Ag-NPs on a large scale . However, these chemical synthesis methods employ toxic chemicals in the synthesis route which may have adverse effect in the medical applications and hazard to environment. Therefore, preparation of Ag-NPs by green synthesis approach has advantages over physical and chemical approaches as it is environmental friendly, cost effective and the most significant advantage is that conditions of high temperature, pressure, energy and toxic chemicals are not required in the synthesis protocol .
In this work, we reported “green” synthesis of Ag-NPs using sugar and PEG. This method was performed by reducing the silver nitrate (AgNO3) in different stirring times of reaction at moderate temperature with sugar and PEG used as green reducing agent and polymeric stabilizer. The antibacterial activity of silver/polyethylene glycol [Ag(PEG)] were tested with Mueller-Hinton agar disc diffusion method against Staphylococcus aureus (S. aureus), and Salmonella typhimurium SL1344 (S. typhimurium SL1344).
Results and discussion
After dispersion of silver ions in the PEG aqueous solution matrix (Equation 1), PEG reacted with the Ag to form a PEG complex [Ag(PEG]+, which reacted with sugar to form [Ag(PEG)] due to the reduction of silver ions through the oxidation of sugar to gluconic acid (Equation 2).
Thus, there is a normal case in this situation for the SPR absorption band for the particles, which agreed with the TEM results, whereby red–shifts were observed as size increased in the during the reaction after 1, 3, 6, 24 and 48 h respectively. This can be explained by the multilayer Mie theory model, which theorizes that the chemical interaction caused the lowered electron conductivity in the outermost atomic layer and consequently caused the red–shifts . As seen from the Figure 2C, it can be observed that 24 h had large absorbance compared to 48 h because the particle size of Ag-NPs after 48 h were larger than those at 24 h. Also, absorption spectra of larger metal colloidal dispersions can exhibit broad peaks or additional bands with the lower absorbance in the UV-visible range due to the excitation of plasma resonances or higher multipole plasmon excitation . This phenomenon could be due to the fact that, after reaching a certain particle size, the stabilizer was not able to withhold the nanoparticle’s size effectively, which resulted in its very large size.
Powder X–ray diffraction
Where K is the Scherrer constant with value from 0.9 to 1 (shape factor), where λ is the X-ray wavelength (1.5418 Å), β1/2 is the width of the XRD peak at half height and θ is the Bragg angle. From the Scherrer equation the average crystallite size of Ag-NPs for 6, 24 and 48 h times of reaction are found to be around 10–25 nm, which are also in line with the observation of the TEM results discussed later.
Zeta potential measurement
FT-IR chemical analysis
Thus, as shown hydroxyl group of PEG as capping agent can make a cover in the surface of Ag-NPs. This is possible because the surface of Ag-NPs is positively charged. Certainly, we suppose that colloidal stabilization for [Ag (PEG)] occur due to the presence of van der waals forces between the oxygen negatively charged groups present in the molecular structure of the PEG, and the positively charged that surround the surface of the inert Ag-NPs [45, 47]. Therefore, the FT-IR spectra showed the existence molecular interactions between the Ag-NPs with the chain of polymeric media . As shown in the Figure 6, schematic illustrated the interaction between the charged of Ag-NPs that capped with PEG [28, 48].
Average inhibition zone and standard deviation for PEG, [Ag (PEG)] + (A0) and [Ag (PEG)] suspension (A2–A5) at different stirring times (3, 6, 24 and 48 h), respectively
11.65 ± 0.56
12.78 ± 0.12
13.64 ± 0.29
9.71 ± 0.14
9.67 ± 0.33
9.44 ± 0.36
11.51 ± 0.43
10.64 ± 0.39
10.62 ± 0.36
In summary, we have described a simple and green method of colloidal Ag-NPs synthesis by using green reducing agents which requires no special physical conditions. Ag-NPs were successfully synthesized under moderate temperature (45°C) at different stirring times of reaction. The formation of Ag-NPs was confirmed in the UV-visible absorption spectra, which showed the SPR band characteristics of Ag-NPs in the range of 412–437 nm. The XRD results confirmed that the Ag-NPs possessed a face-centered cubic crystal structure (fcc). In addition, this also revealed that Ag-NPs were the main composition present in the nanocomposites without any contamination peaks. The TEM images showed that the Ag-NPs were in spherical shape and the average diameters of the particles were 10.60, 11.23, 12.95 and 25.31 nm for the stirring times of 3, 6, 24 and 48 h, respectively. FTIR spectrum suggested the complexation present between PEG and Ag-NPs to form metallopolymer [Ag (PEG)] and the stability of the Ag-NPs was confirmed with the zeta potential measurements. The antibacterial activities of [Ag (PEG)] at the different particle size of Ag-NPs were showed antibacterial activity against the Gram-positive and Gram-negative bacteria. These results show that the antibacterial activities of Ag-NPs in PEG can be modified with the size of Ag-NPs and it decreases with the increase in the particle size. Needless to say, further studies are required to investigate the biological effects of [Ag (PEG)] suspension on the types of bacteria for potential widening of this subject area.
All reagents in this effort were analytical grade and were used as received without further purification. AgNO3 (99.98%) was used as a silver precursor, and was provided by Merck, Germany. PEG (Mw 3,350) used as a stabilizer for the preparation of Ag-NPs which was purchased from Sigma–Aldrich (USA). Meanwhile, the sugar was used as a green reducing of silver ions to Ag atoms and was obtained from BDH Chemical Ltd., Poole, UK. All solutions were freshly prepared using double distilled water and kept in the dark to avoid any photochemical reactions. All glassware used in experimental procedures were cleaned in a fresh solution of HNO3/HCl (3:1, v/v), washed thoroughly with double distilled water, and dried before use.
Synthesis of Ag-NPs by using green method
The preparation of Ag-NPs in the PEG matrix is quite directly forward. In a typical synthesis, a 10 mL of a 1.0 M solution of AgNO3 was added to 200 mL of a 0.1 wt.% aqueous solution of soluble PEG to obtain the clear solution . After complete dissolution of these components, 20 mL of a 1.0 M aqueous solution of sugar was then added and further stirred. The solution obtained was distributed into 5 cuvettes, which were stirred and maintained at different periods of time: 1 (a), 3 (b), 6 (c), 24 (d), and 48 h (e), respectively. Throughout the reduction process, all solutions were kept at a constant temperature of 60°C in the dark to avoid any photochemical reactions. All solution components were purged with nitrogen gas prior to use. Subsequently, reduction proceeded in the presence of nitrogen to eliminate oxygen. The obtained colloidal suspensions of [Ag (PEG)] were then centrifuged at 20000 rpm for 15 min and washed four times to remove silver ion residue. The precipitate nanoparticles were then dried overnight at 40°C under vacuum overnight to obtain the Ag-NPs.
Evolution of antibacterial activity
The in vitro antibacterial activity of the samples was evaluated by utilizing the disc diffusion method using Müeller–Hinton Agar (MHA) with determination of inhibition zones in millimeter (mm), which conform with recommended standards of the National Committee for Clinical Laboratory Standards (NCCLS; now renamed as Clinical and Laboratory Standards Institute, CLSI, 2000). Salmonella typhimurium (S. typhimurium SL1344) and Staphylococcus aureus (S. aureus) (ATCC 25923) were used for the antibacterial effect assay. Briefly, the sterile paper discs (6 mm) impregnated with 20 μl of PEG, and [Ag (PEG)] (3, 6, 24 and 48 h) with different treatment times were suspended in sterile distilled water and were left to dry at 37°C for 24 h in a sterile condition. The bacterial suspension was prepared by making a saline suspension of isolated colonies selected from tryptic soy agar plate, the agar plates were grown for 18 to 24 h. The suspension was adjusted to match the tube of 0.5 McFarland turbidity standard using the spectrophotometer of 600 nm, which equals to 1.5 × 108 colony–forming units (CFU)/ml. The surface of MHA was completely inoculated using a sterile swab, which steeped in the prepared suspension of bacterium. Finally, the impregnated discs were placed on the inoculated agar and incubated at 37°C for 24 h. After incubation, the diameter of the growth inhibition zones was measured. Chloramphenicol (30 μg) and Cefotaxime (30 μg) were used as the positive standards in order to control the sensitivity of the bacteria. All tests were done in triplicate.
Characterization methods and instruments
The prepared Ag-NPs were characterized by using the X–ray diffraction (XRD), transmission electron microscopy (TEM), ultraviolet–visible spectroscopy, Fourier transform infrared (FT–IR) spectroscopy and zeta potential measurements. The XRD patterns were recorded at a scan speed of 2° min–1. Meanwhile, the structures of the produced Ag-NPs were examined using Shimadzu PXRD–6000, powder X–ray diffraction. Moreover, TEM observations were carried out using the Hitachi H–7100 electron microscopy, whereas the particle size distributions were determined using the UTHSCSA Image Tool software (Version 3.00). To make sure the formation of Ag-NPs, the colloids solutions were tested for their optical absorption property using a Shimadzu H.UV, 1650 PC UV–visible spectrophotometer over the range of 300 to 700 nm. The FT–IR spectra were however recorded over the range of 200–4000 cm−1 utilizing the Series 100 Perkin Elmer FT–IR 1650 spectrophotometer. The zeta potential measurements were also performed using a Zetasizer Nano–ZS (Malvern Instruments).
The authors thank from University Putra Malaysia (UPM) for its financial support (RUGS, Project No. 9199840). The authors are also grateful to the staff of the Department of Chemistry UPM for their help in this research, Institute of Bioscience (IBS/UPM) for technical assistance.
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