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BioNanoSci. (2013) 3:67 72 DOI 10.1007/s12668-012-0070-5 Silver Nanoparticles and Their Antimicrobial Activity on a Few Bacteria Ratan Das & Mitu Saha & Syed Arshad Hussain & Siddhartha S. Nath Published online: 4 January 2013 # Springer Science+Business Media New York 2012 Abstract The antimicrobial effects of silver (Ag) ion or salts are well known, but the effects of silver nanoparticles on microorganisms have not been revealed clearly. Here, the antimicrobial properties of silver nanoparticles have been investigated using Bacillus subtilis and methicillin-resistant Staphylococcus aureus. Linoleic acid-protected fine and uniform silver nanoparticles have been prepared through the chemical reduction of silver ions by ethanol. X-ray diffraction study confirms the formation of silver nanoparticles. Transmission electron microscopic image shows a good size distribution of the particles in the range of 4 15 nm. Further, the antimicrobial activity of silver nanoparticles shows that these nanoparticles can be used as effective growth inhibitors. Keywords Antimicrobial. Silver nanoparticles. Linoleic acid PACS 61.05.cp. 68.37.Lp. 61.82.Rx 1 Introduction Nanotechnology deals with the preparation of materials at atomic level to attain unique properties, which can be suitably manipulated for the desired applications [1 23]. Recently, nanotechnology is expected to open some new aspects to prevent diseases using nanomaterials. The dimension of nanomaterials is comparable to that of most biological molecules and structures, and therefore, nanomaterials R. Das (*) : M. Saha : S. A. Hussain Department of Physics, Tripura University, Tripura, India e-mail: dasratanpj@gmail.com S. S. Nath CIL, Assam University, Silchar, Assam, India can be useful for biomedical research and applications. The integration of nanomaterials with biology has led to the development of diagnostic devices, many analytical tools, therapy applications, and drug delivery vehicles. For antimicrobial properties [22, 23], metallic nanoparticles show good activity. The increase in chemical activity of nanoparticles is due to their large surface to volume ratio. The importance of antimicrobial study is because of the increase in new resistant strains of bacteria against most antibiotics. Thus, we have studied the antibacterial activity of linoleic acid-capped silver nanoparticles against a few bacteria. Noble metal nanoparticles such as silver, gold and copper show unique and considerably changed physical, chemical and biological properties compared to their bulk counterparts [2 7]. Recently, inorganic nanoparticles capped by organic ligands have attracted great interest due to their manifold technological applications [8 10]. Here, silver nanoparticles have been synthesized through the chemical reduction of silver ions by ethanol [11] to study the antimicrobial effect of this fatty acid (linoleic acid)-capped silver nanoparticles. The prepared silver nanoparticles have been examined using X-ray diffraction (XRD), transmission electron microscope (TEM) and Fourier transform infrared spectroscopy (FTIR). Finally, antibacterial effects of these linoleic acid-capped silver nanoparticles have been tested against Bacillus subtilis and methicillin-resistant Staphylococcus aureus, which show that linoleic acid-protected silver nanoparticles are good inhibitors against all these bacteria. 2 Sample Preparation (Silver Nanoparticles) For the preparation of silver nanoparticles, linoleic acid is used as a stabilising agent, silver nitrate solution as a precursors and ethanol as a reducing agent. Silver nanoparticles can be obtained through the reduction of silver ions by

68 BioNanoSci. (2013) 3:67 72 ethanol in between the temperatures 90 and 100 C under hydrothermal conditions in presence of sodium linoleate [11]. In this preparation method, 10 ml of aqueous solution containing silver nitrate (AgNO 3 ) having 2.75 10 5 M concentration, 1.2 g sodium linoleate (C 18 H 32 ONa), 7 ml ethanol (C 2 H 5 OH) and 1.5 ml linoleic acid (C 18 H 32 O 2 )areaddedinacappedtube under stirring condition at constant rate. The system is kept between 90 and 100 C for 1.5 h. In the pure solution of silver nitrate, sodium linoleate and separate mixture of linoleic acid and ethanol are added one after the other. Ethanol reduced the silver ions in the solution into silver nanoparticles. A solid phase of sodium linoleate, a liquid phase of ethanol and linoleic acid, and water ethanol solution phase containing silver ions, formed in the solution. A phase transfer process of silver ions takes place continuously due to ion exchange. This process gives a major way for the formation of silver linoleate and entering of sodium ions into the aqueous phase. Then, ethanol in the liquid and solution phases reduced silver ions into silver nanoparticles of uniform size. The linoleic acid caps the silver nanoparticles along with the reduction process through steric stabilization. FTIR spectroscopic analysis confirms the capping of linoleic acid. The chemical structure of linoleic acid is shown in Fig. 1. Cooling to normal temperature, silver nanoparticles can be dispersed in non-polar solvents like chloroform, toluene and cyclohexane to form a homogenous colloidal solution. Here, silver nanoparticles are dispersed in chloroform, which show reddish yellow colour. This colour clearly indicates the formation of silver nanoparticles. The role of linoleic acid is to protect the silver nanoparticles from agglomeration by making a layer over it with its alkyl chains on the outside. This layer of linoleic acid provides a hydrophilic environment to the nanoparticles and the produced nanoparticles gain a hydrophobic surface [11]. After the synthesis, samples of silver nanoparticles are characterized by the following experimental processes. 3 Different Characterization 3.1 X-Ray Diffraction Study in the Joint Committee on Powder Diffraction Standard correspond to the planes of (111), (200) and (220), respectively, and confirming the formation of silver nanoparticles. Measuring the line broadening at 39.9, 45.2 and 67.9, average particle size D is estimated by using Debye Scherrer formula [12 15] D ¼ 0:9l ð1þ W cos θ Where 1 is the wavelength of the X-ray used (0.1541 nm), W is the full width at half maximum and θ is the diffraction angle or glancing angle in radians. Considering the instrumental broadening at 39.9, 45.2 and 67.9 as 0.00451, 0.00629 and 0.00793 respectively, the average particle size is calculated to be around 13±1 nm with 6 % error in the measurement. 3.2 TEM Image Analysis Morphology of the silver nanoparticles has been obtained from transmission electron microscopic image, which was performed on a JEM 1000C X II model instrument. Samples for TEM studies are prepared by placing drops of the silver nanoparticles solutions on carbon-coated TEM grids. TEM micrograph of the prepared colloidal solution of silver nanoparticles shows that nanoparticles are spherical in shape as shown in Fig. 2a. Further, it indicates that the size distribution of silver nanoparticles is narrow as shown in Fig. 2b, ranging from 4 15 nm. This TEM image suggests that no clustering of nanoparticles takes place as they are well separated from each other. Capping of linoleic acid restrict the nanoparticles from agglomeration. Histograms of size distribution are calculated from the TEM images by measuring the diameters of at least 35 nanoparticles. 3.3 FTIR Spectral Analysis Capping of linoleic acid on silver nanoparticle has been examined by FTIR spectroscopy. The FTIR absorption spectra of the samples are shown in Fig. 3 with resolution of 4 cm 1, which was performed in Spectrum BX series. The peak at 3443 cm 1 of the FTIR spectra contains OH stretching modes [16]. The peak around 3027 cm 1 is due to C=C Formation and the structure of prepared samples have been investigated by Bruker AXS model X-ray diffractometer. The XRD patterns of the sample prepared by the present reduction method are shown in the Fig. 2. The XRD pattern clearly indicates crystallinity of the particles as the peaks at 39.9, 45.2 and 67.9 match with those of a face-centred cubic Ag as Fig. 1 Chemical structure of linoleic acid Fig. 2 XRD pattern of silver nanoparticles

BioNanoSci. (2013) 3:67 72 69 Fig. 3 a TEM micrograph of silver nanoparticles. b Histograms of particle size distribution stretching mode. The lack of broad peak due to OH stretching of the free ligand in the range 3000 to 3100 cm 1 is due to the chemisorptions of linoleic acid on the copper nanoparticles, which is an indicator for the conformational ordering of the metal-linked alkyl chains of linoleic acid (Fig. 4). 3.4 UV/Vis Study of Silver Nanoparticles Noble metal nanoparticles are well known for its strong absorption band in the visible range. This absorption is due to surface plasmon resonance (SPR), which is the collective oscillation of conduction band electron in resonance with the frequency of the light wave [17 20]. When the dimensions of nanoparticles become smaller than the wavelength of the exciting light, energy can be confined in small spatial regions through the local excitation of SPR. When the frequency of the light wave become resonant with the electron motion, a strong absorption occurs gives the reason for the origin of the observed colour of colloids [17 20]. Here silver nanoparticles dispersed in chloroform show a reddish yellow colour because of this absorption. The UV/Vis absorption spectra of the silver nanoparticles dispersed in chloroform are shown in Fig. 5. The absorption (SPR) peak is obtained in the visible range at 427 nm. A good symmetry of the absorption spectra around the peak position indicates the nearly uniform distribution of the particles produced. 4 Assay for Antimicrobial Activity of Ag Nanoparticles Against Microorganisms The antimicrobial activity of silver nanoparticles was evaluated against the bacterial strains B. subtilis and methicillin-resistant S. Fig. 4 FTIR spectra of linoleic acid-protected silver nanoparticles dispersed in chloroform Fig. 5 UV/Vis spectra of silver nanoparticles dispersed in chloroform

70 BioNanoSci. (2013) 3:67 72 aureus by the Kirby Bauer diffusion method [22, 23]. The agar used was Meuller Hinton agar that was rigorously tested for composition and ph value. Approximately 10 7 colony-forming units (CFU) of each microorganism were inoculated on Muller Hinton agar (MHA) plates, and Ag nanoparticles-laden discs of concentration 60 μg/ml were then placed on the different plates. Kanamycin and penicillin were used as positive controls.the plates with the discs were incubated at 35 C for 24 h, after which the average diameters of the inhibition zones surrounding the disk were measured with a standard Himedia ruler. 5 Antimicrobial Activity of Silver Nanoparticles With the increase of microorganisms resistant to different antibiotics, new effective antimicrobial reagents free of resistance have been tried to develop. Recently, the emergence of nanotechnology explored the bactericidal effect of metal nanoparticles. The bactericidal effect of metal nanoparticles has been attributed to their small size and high surface to volume ratio, which allows them to interact closely with microbial membranes [24]. In this work, antimicrobial activity of these linoleic acid-capped spherical silver nanoparticles has been investigated against B. subtilis and methicillin-resistant S. aureus. The bacterial suspension was applied uniformly on the surface of a MHA plate at a concentration of 10 7 CFU/mL. Soon after, kanamycin- and penicillin-impregnated discs and well-prepared silver nanoparticles-laden disk (5 mm diameter) also have been placed on the same plate. The plates with the discs were incubated at 35 C for 24 h, after which the average diameter of the inhibition zone surrounding the disk was measured with a ruler. The plates in which B. subtilis and methicillin-resistant S. aureus bacterial suspension were applied with nanoparticles-laden disk and antibiotic impregnated discs are shown in Fig. 6. The diameter of inhibition zones around the disk containing silver nanoparticles in bacterial suspension are shown in Table 1, which is based on a single experiment, the inhibition zone of which is shown in Fig. 6. Finally, we have determined the minimum inhibition concentration (MIC) using the standard dilution micromethod. The MIC, defined as the lowest concentration of material that inhibits the growth of an organism, has been determined using varying concentration of silver nanoparticles in suspension. The MIC has also been determined after 24 h of incubation at 35 C, which is summarized in Table 2. For this MIC test, the experiment has been repeated seven times. 6 Discussions A very well-known fact is that Ag ions and different Agbased compounds have strong antimicrobial effects [25]. Fig. 6 Silver nanoparticles-laden disk (a) and antibiotic kanamycin (b) and Penicillin (c) impregnated disk placed on the surface of Bacillus subtilis (1), methicillin-resistant S. aureus (2) bacterial suspension on Muller Hinton agar (MHA) plate after incubation at 35 C for 24 h But nowadays researchers are using inorganic nanoparticles such as silver and copper nanoparticles as an antibacterial agent because these nanoparticles have great advantage over the general chemical antibacterial agents. In general, antibacterial activity of chemical agents depends on the special binding with the surface of the microorganism and hence subsequently different microorganisms have become drug resistant with time. Therefore, to overcome the drug resistant Table 1 Diameter of inhibition zones Bacteria Diameter of inhibition zones of the discs containing Silver nanoparticles (mm) Kanamycin (mm) Bacillus subtilis 9 11 5 Methicillin-resistant 8 18 5 S. aureus Penicillin (mm)

BioNanoSci. (2013) 3:67 72 71 Table 2 Minimum inhibitory concentration (MIC) of silver nanoparticles Bacteria Bacillus subtilis 40 Methicillin-resistant S. aureus 55 Minimum inhibition concentration (μg/ml) of various microorganisms, we need different types of antibacterial agents such as silver nanoparticles. Here, the costeffective preparation method of linoleic acid-capped silver nanoparticles shows antibacterial activity against various microorganisms. It is found that silver nanoparticles are not equally effective against B. subtilis and methicillin-resistant S. aureus as inhibition zones are different for different microorganisms. This test indicates that antimicrobial activity of silver nanoparticles must be associated with specific characteristics of specific bacteria. It further indicates that silver nanoparticles affects differently for Gram-positive and - negative bacteria. As Gram-positive and -negative bacteria have different membrane structures, hence they show different responses against silver nanoparticles. One specific feature of bacterial species is that it has a special peptidoglycan layer in the membrane structure [26], which is not present in mammalian cells. It may be further tested whether the antibacterial activity of silver nanoparticles is associated with this peptidoglycan layer. If it happens, then silver nanoparticles can specifically be used as an antibacterial agent. Though silver nanoparticles are well known for its antibacterial effects, the mechanism of the inhibitory effects is not very well known. It was reported that cellular proteins of the bacteria become inactive on treating with silver nanoparticles [27]. It was also reported that antimicrobial activity is due to the electrostatic attraction between negatively charged cell membrane of microorganism and positively charged nanoparticles [28]. On the other hand, Sondi and Salopek-Sondi [29] reported that the antimicrobial activity of silver nanoparticles on Gramnegative bacteria was dependent on the concentration of Ag nanoparticle, and was due to the formation of pits in the cell wall of bacteria. Thereby, silver nanoparticles accumulated in the bacterial membrane caused the permeability, triggering cell death [26]. Therefore, it is clear that both types of bacteria can get affected by silver nanoparticles. Our experimental result shows that linoleic acid-capped silver nanoparticles can be used as effective growth inhibitors against B. subtilis and methicillin-resistant S. aureus, making them applicable to different antimicrobial control systems. Further, Yoon et al. [30] reported the antibacterial activity of silver nanoparticles of 40 nm size against Escherichia coli and B. subtilis; butin this study, silver nanoparticles of 4 15 nm have been prepared with linoleic acid as a capping agent, which shows antibacterial activity against B. subtilis as well as methicillin-resistant S. aureus, as antibacterial activity of silver nanoparticles is common against E. coli. Lok et al. [31] reportedelaborately the antibacterial activity against E. coli only, where they have prepared the silver nanoparticles by citrate reduction. But in this work, a simple process of reduction method by ethanol has been adopted with linoleic acid as a capping agent. Gogoi et al. [32] reported the investigation of the bactericidal effect of Ag nanoparticles on GFP (green fluorescent protein) expressing recombinant E. coli where they have prepared silver nanoparticles by reduction method using sodium borohydride (NaBH 4 ). Kim et al. [26] again reported the antibacterial activity of silver nanoparticles against E. coli and Staphylococcus aureus, where they again prepared the silver nanoparticles by reduction method using sodium borohydride (NaBH 4 ). Among all these preparation methods, preparation by ethanol reduction is very simple, less toxic and more stable. Compared to the antibacterial activity of silver nanoparticles in these reports [26, 30 32], the work has been extended to the methicillin-resistant S. aureus and B. subtilis instead of the much-studied E. coli. Though silver nanoparticles are much known for its broad spectrum of antimicrobial activity, on the other hand, a few research groups investigated the toxic effect of silver nanoparticles [33 35]. In spite of that, little is known about the mechanism of toxicity of silver nanoparticles. AshaRanietal.[33] reported the cytotoxicity (low metabolic activity), genotoxicity (DNA damage and chromosomal aberrations) of silver nanoparticles. They have also reported an active repair pathway, i.e. the cells which successfully repair the damage will re-enter the cell cycle and those with massive damage will not be able to repair the DNA effectively [33]. Hence, toxicity of silver nanoparticles is dose dependent. Though silver nanoparticles are equally toxic to normal cells, still deeper understanding is required to achieve the toxic nature of silver nanoparticles. Even though it is well established from many reports that silver nanoparticles are effective as an antibacterial agent and in this work we re-established this fact of antibacterial activity of silver nanoparticles, extending the work to other bacteria such as to methicillin-resistant S. aureus and B. subtilis. 7 Conclusion Silver nanoparticles have been prepared through the reduction of silver ions by ethanol. XRD study and TEM micrograph indicates that the prepared linoleic acid-capped silver nanoparticles are spherical in shape having nearly uniform size distribution with size range 4-15 nm. Further, FTIR spectra confirm the capping of linoleic acid on the nanoparticles surfaces. These linoleic acid-capped silver nanoparticles are tested for antimicrobial activity against B. subtilis and methicillin-resistant S. aureus and the result shows that these linoleic acid-capped silver nanoparticles can be used as effective growth inhibitors against various microorganisms.

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