JOURNAL OF INTERNATIONAL ACADEMIC RESEARCH FOR MULTIDISCIPLINARY Impact Factor 1.393, ISSN: , Volume 2, Issue 3, April 2014

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1 STUDY OF THE EFFECT OF CONCENTRATION OF GREEN STABILIZING/CAPPING AGENTS ON THE SIZE OF BIOSYNTHESIZED SILVER NANOPARTICLES SHWETA RAJAWAT* SONALI SHAH** DR.M.S.QURESHI*** *Dept. of Physics, M.A.N.I.T., Bhopal, M.P, India **Dept. of Physics, M.A.N.I.T., Bhopal, M.P, India ***Dept. of Physics, M.A.N.I.T., Bhopal, M.P India ABSTRACT Stabilizing/capping agents play important role in the synthesis of nanoparticles. Degree of agglomeration and shape and size of the nanoparticles can be tuned by varying the concentration of the stabilizing agents/capping agents. In the present work synthesis of silver nanoparticles is done by electrolytic deposition method using silver nitrate as a precursor and onion extract as capping agent. The work focuses on the study of the effect of concentration of the capping agent on the morphology of as-synthesized silver nanoparticles. Various characterization techniques such as XRD, TEM, AFM, UV-visible and PL-spectroscopy are used for the analysis of as-synthesized silver nanoparticles. It was found that mono dispersed spherical silver nanoparticles of smaller size, for higher concentration of capping agent, and polydispersed spherical nanoparticles of larger size, for lower concentration of capping agent, were formed. Capping agent at higher concentration encapsulates nanoparticles in the nucleation stage itself inhibiting them to enter into growth stage. KEYWORDS: Stabilizing Agents, Concentration, Silver Nanoparticles, INTRODUCTION Size and shape of the nanoparticles play detrimental role in case of extraordinary properties of the nanoparticles. A stabilizer or a capping agent, often a polymer, provides a protective shell around the forming nanoparticle. Nanoparticle in its formation process undergoes two phases: nucleation and growth phase. Capping agents cap the nanoparticles immediately after getting placed in the synthesis process. When the capping agent is effective on the nanoparticles at the nucleation stage, the nanoparticle is not able to enter the growth phase and smaller nanoparticles are synthesized. In case when the nanoparticles are capped after entering into growth phase, large sized nanoparticles are synthesized. Stabilizer, by encapsulating nanoparticles, prevents them from agglomerating. The stabilizer is attached to the particle at one end of the polymer chain, making the polymer look like tentacles on the particle. When the two particles approach each other, such that the distance (d) between the 155

2 particles becomes shorter than twice the thickness (L) of the polymer layer on the particles, an interaction between them occurs. This interaction between the two particles increases the Gibbs free energy and they repel each other. If there is not enough stabilizer present to cover the surface more than for instance 50% the polymers are inclined to interpenetrate in order to reduce the unoccupied space between polymers. If the particle is not covered 100% and two particles move towards each other the result will be a decrease in Gibbs energy showing that the polymers on the surface of nanoparticle promote a further coiling. This in turn results in the two particles bundling together[2].therefore study of effect of the capping agents on the size and shape of nanoparticles at the time of synthesis finds important place in today s scenario. Materials and Methods A set of experiment was designed to observe the effect of the concentration of the capping agent, onion extract, on the size of nanoparticles using silver nitrate precursor solution (0.005N). Onion (Allium cepa) is the most widely cultivated species of the genus Allium. Wideranging claims have been made for the effectiveness of onion against conditions ranging from the common cold to heart disease, diabetes, osteoporosis, and other diseases. It contains chemical compounds having anti-inflammatory, anti cholesterol, anticancer, and antioxidant properties, such as quercetin. Quercetin, aflavonol, is a plant-derived flavonoid. Molecular structure of the Quercetin is shown in Fig 2. Hydroxyl group has appreciable affinity with silver nanoparticles thus rendering it as an effective capping agent to prevent agglomeration. In onions, higher concentrations of quercetin occur in the outermost rings. Preliminary studies have shown increased consumption of onions reduces the risk of head and neck cancers. Onion extract, shown in Fig 1, is prepared by grating the onion and squeezing the grated onion using a filter. The per cent concentration of the capping agent was varied over 1.25%, 2.5%, 6.25%. In the experimental setup, two electrodes: silver wire (99% pure), as anode, and carbon rod wrapped by LDPE (Low Density Poly Ethylene)material, as cathode, were used. LDPE material is used in the synthesis process for easy extraction of as-synthesized silver nanoparticles. The distance between the two electrodes is 1cm. The diameter of the silver wire is1.04mm and the diameter of the carbon rod used is 4mm. The length of the carbon rod as well as silver wire is 4.5 cm. Onion extract is added to the electrolyte as a capping agent. The whole assembly is placed on magnetic stirrer which keeps the solution in the beaker 156

3 stirring continuously. A power supply of rating 15volts and 3A is used. Copper wires are used to connect the components of the circuit. Schema and the setup of the experiment are shown in Fig.3 and Fig.4 respectively. All the parameters were same for the samples synthesized, except the concentration of the capping agent; onion extract. Fig. 1 Onion extracts Fig. 2 Molecular structure of Quercetin Fig. 3 Schematic picture of experimental setup Fig. 4 Experimental setup Results and discussions Corresponding to three different concentrations, solutions with different colours were obtained. The colour of the solution, as shown in Fig.5, for lowest percent of concentration was dark grey. The colour changes to brown and light brown as the concentration is increased. The different colours of the colloidal solutions of silver nanoparticles are due to the difference in size of as-synthesized nanoparticles. To determine the morphology of silver nanoparticles various characterizations (e.g. TEM, UV-vis spectroscopy) were done.. Fig 5 Colour of the solution obtained for 1.25, 2.5 and 6.25 percent concentration of onion extract for 0.005N Silver Nitrate 157

4 XRD characterization Wide angle XRD technique is used for the characterization of the samples. This technique is used for elemental analysis only i.e. to determine the purity of the sample as we cannot determine the size of nanoparticles using it. Fig 6 (a, b, c) give XRD graphs for samples with variation in the concentration of onion extract. A characteristic XRD pattern of the as-synthesized silver nanoparticles had Bragg reflections at 2θ = 38.18, 44.37, and 77.63, which can be indexed to the (111), (200),(220), (311) and (222) orientations respectively for all three concentrations. The diffraction profiles of as-prepared silver are obviously broadened as compared with bulk silver confirming the formation of silver nanoparticles. The XRD results show Face-Centred-cubic (fcc) structure of as-synthesized silver nanoparticles The graphs, on comparing with standard JCPDS file no , show that the particles synthesized are highly pure. To determine the size and shape of assynthesized silver nanoparticles TEM characterization is done. Fig 6 (a) Fig 6 (b) Fig 6 (c) Fig 6 (a, b, c) XRD graphs of as-synthesized silver nanoparticles for 1.25, 2.5 and 6.25 per cent concentration of onion extract TEM and AFM characterization For the morphological studies of as-synthesized silver nanoparticles, TEM and AFM characterization were done. Fig 7 (a, b, c) show the TEM images of silver nanoparticles synthesized using 1.25 per cent concentration of onion extract as capping agent. The size of the nanoparticle lies in the range of 5nm-25nm with an average size of 20nm. The spherical 158

5 shape of the particle is due to the fact that the spherical shapes correspond to a state of minimum potential energy. As onion is very effective capping agent, negligible agglomeration is observed in the TEM images. TEM pictures, in case of onion extract, show nano particles surrounded by a faint thin layer of proteins and metabolites such as terpenoids having functional groups of amines, alcohols, ketones, aldehydes and carboxylic acids. Fig 7(a) Fig 7(b) Fig 7(c) Fig 7 (a, b, c) TEM images of silver nanoparticles of size 5-25nm for 1.25 per cent concentration of onion extract TEM images in fig 8 (a, b, c) show that the particles size obtained for 2.5 per cent concentration of onion extract lies in almost same range as that for the 1.25 per cent concentration of onion extract. Since the system is in low energy state, the silver nanoparticles remain stable over a longer period of time. Fig 8 (d, e) shows the AFM images of the same sample. Nanoparticles observed are very small (<20nm) in size. The 3d image shows the nanoparticles of almost uniform morphology. Fig 8(a) Fig 8(b) 159

6 Fig 8(c) Fig 8(d) Fig 8 (e) Fig 8 (a, b, c, d, e) AFM images of silver nanoparticles synthesized of size 2-20nm using 2.5%concentration of onion extract as capping agent TEM images of the silver nanoparticles for 6.25% concentration are shown in Fig 9 (a, b, c). The spherical nanoparticles are synthesized with size distribution in the range of 5-8nm. Greater concentration of onion extract removes agglomeration completely and caps assynthesized silver nanoparticles immediately as the silver nitrate gets reduced giving no time to the nanoparticle to enter the growth phase after nucleation. 160

7 Fig 9(a) Fig 9(b) Fig 9(c) Fig 9 (a, b, c) TEM and AFM images of silver nanoparticles of size 2-10nm for 6.25% concentration of onion extract as capping agent respectively UV-Visible characterization UV-Visible spectroscopy was done for the as-synthesized silver nanoparticles to confirm the results of XRD and TEM results. Fig 10 shows the UV-Visible graphs for silver nanoparticles. The peak values obtained for three different percent concentrations, 1.25, 2.5 and 6.25 were 388.4nm, 371nm and 365nm. Blue shift in the peaks with respect to the increase in concentration is quite obvious in Fig 10 which confirms the decrease in size of nanoparticle with increase in the concentration of capping agent. Fig. 10 UV-graphs of silver nanoparticles for different concentration of onion extract 161

8 Table 1: Effect of concentration of onion extract on the size of nanoparticles and UV-Visible peaks for set 6, method 1, with parametric values: 1.25% concentration, 0.01N silver nitrate, 200mA current and 30ºC temperature S. Per cent concentration of Size of the Peak value (λ max ) No. onion extract nanoparticle(nm) (nm) Anti-bacterial studies The antibacterial and antiviral actions of silver, silver ion, and silver compounds have been thoroughly investigated [8].The bactericidal properties of the nanoparticles are size dependent, since the only nanoparticles that present a direct interaction with the bacteria preferentially have a diameter of approximately 1-10 nm [209]. The reason for this size dependency is probably a combination of the nanoparticles ability to react with and penetrate the cell. The size of silver nanoparticles ensures that a significantly large surface area of the particles is in contact with the bacterial cells. Such a large contact surface is expected to enhance the extent of bacterial elimination [210]. Silver nanoparticles can easily reach the nuclear content of bacteria and they present the greatest surface area; therefore the contact with bacteria is the greatest [37]. Anti- bacterial study was done for the pure sample and sample in combination with ampicillin and gentamicin, synthesized using 6.25 per cent concentration of onion extract, 0.01N silver nitrate, 200mA current and 30ºC temperature, as the smallest particle size (2-10nm) is obtained. The results of the studies show that the sample in its pure form is highly effective against the E.coli, S. Aureus, S. Typhi and P. Aeruginosa. To verify whether the antibacterial activities of silver nanoparticles are enhanced in combination with standard antibiotics, synergistic effects were studied. Two standard antibiotics were selected. Ampicillin is a beta-lactam antibiotic that has been used extensively to treat bacterial infections since 1961and is relatively non-toxic and gentamicin is active against a wide range of human bacterial infections. The fig 12 (a, b, c, d, e, f, g, h) show the pictures of inhibition zones corresponding to the combination with gentamicin and ampicillin for E.coli, S. typhi, S.aureus and P. Aeruginosa. The table 2, 3, 4, 5, 6 show the results of antibacterial studies corresponding to pure sample and to the combination of as-synthesized nanoparticles with ampicillin and gentamicin. 162

9 Fig 12(a) Fig 12 (b) Fig 12(c) Fig 12(d) Fig 12(e) Fig 12(f) Fig 12 (g) Fig 12 (h) Fig 12(a, b, c, d, e, f, g, h) Inhibition zones for silver nanoparticles in combination with antibiotics. Table 2:1ml Pure Sample ml Culture [kept for 15 minutes] total solution plated on Soya bean casein digest agar incubated at 37 0 C for 48 hrs S. No. Organism Culture/ml Growth on plate Killing % 1. E. Coli 12 x x S. Aureus 8 x 10 6 No growth P. Aeruginosa 16 x10 6 2x S. Typhi 3 x 10 7 No growth 100 As per the study, it is observed from table 1 and 2 that S aureus and S typhi are inhibited totally. 163

10 TEST FOR SAMPLE WITH AMPICILLIN: Table 3:1ml Sample ml Culture + Ampicillin [10 mcg] [kept for 15 minutes] total solution plated on Soya bean casein digest agar incubated at 37 0 C for 48 hrs S. No. Organism Culture/ml Growth on plate Killing % 1. E. Coli 12 x 10 6 No growth S. Aureus 6 x x P. Aeruginosa 2 x10 6 4x S. Typhi 8 x 10 6 No growth 100 TEST FOR SAMPLE WITH GENTAMICIN: Table 4: 1ml Sample ml Culture + Gentamicin [10 mcg] [kept for 15 minutes] total solution plated on Soya bean casein digest agar incubated at 37 0 C for 48 hrs S. No. Organism Culture/ml Growth on Killing % plate 1. E. Coli 12 x 10 6 No growth S. Aureus 6 x 10 6 No growth P. Aeruginosa 2 x10 6 No growth S. Typhi 8 x 10 6 No growth 100 From the tables 3 and 4, it is observed that gentamicin is comparatively more effective than ampicillin. The slightly lower killing percentage observed in case of pure sample can be attributed to poor dispersion of the silver nanoparticles in agar plate. Tables 5 and 6 give the details of the comparative study in terms of zone size. Comparative tabulation Table 5: 1ml sample + ampicillin [10 mcg] [kept for 15 minutes]and zone readings taken S. No. Organism Zone for Sample + Ampicillin R1 R2 Mean 1. E. Coli S. Aureus P. Aeruginosa S. Typhi It is observed from the above table that Pseudomonas is resistant to Ampicillin = RE Table 6: 1ml sample + gentamicin [10 mcg] [kept for 15 minutes]and zone readings taken S. No. Organism Zone for Sample + Gentamicin R1 R2 Mean 1. S. Aureus E. Coli P. Aeruginosa S. Typhi Plate 1=P1 Plate 2=P2 164

11 It is observed that the two antibiotics, in combination with silver nanoparticles, are equally effective except for S.aureus where gentamicin slightly takes an upper edge. Conclusion We conclude from the present work that silver nanoparticles can be synthesized using electrolytic deposition method using onion extract as capping agent. We can tune the size of the nanoparticles by varying the concentration of the capping agent. Decrease in the concentration of the onion extract increases the size of nanoparticles and vice-versa. Assynthesized silver nanoparticles capped with onion extract are environmentally friendly and find more applications in the field of medicine compared to silver nanoparticles capped with hazardous chemicals. Antibacterial studies show killing % in S. Aureus, E.coli, P. Aeruginosa, S. Typhi. The synergistic studies show that combination of silver nanoparticles with ampicillin has slightly an upper edge as compared to the combination with gentamicin. Acknowledgements The author acknowledges Dr Appukuttan K.K., Director, MANIT for the support in best possible ways, IUC-DAE Indore for providing XRD facilities, HSADL, IVRI, Bhopal for TEM facility, Mukul Kulshrestha, Professor, Civil Engineering, MANIT for UV-Visible facility, SIERT-R, Bhopal for Pl studies and Micro-Bio Labs, Mumbai, for Anti-Bacterial testing of samples of silver nanoparticles. References 1. Silver Nanoparticles: No Threat to the Environment, George J Maass, URL: 2. Nikolaj L. Kilde by, Ole Z. Andersen, Rasmus E. Røge, Tom Larsen, Ren e Petersen, Jacob F. Riis, Title: Silver Nanoparticles, , Project Group: N344,Aalborg University Faculty of Physics and Nanotechnology. 3. N. L. Rosi, D. A. Giljohann, C. S. Thaxton, A. K. R. Lytton-Jean, M. S. Han, and C. A. Mirkin, Oligonucleotide-modified gold nanoparticles for infracellular gene regulation, Science, 2006, 312, 5776, pp.no MuthuIrulappanSriram, Selvaraj BarathManiKanth. Antitumor activity of silver nanoparticles in Dalton s lymphomaascitestumor model, International Journal of Nanomedicine,2010: Amruta S. Lanje, Satish J. Sharma and Ramchandra B. Pode. Synthesis of silver nanoparticles: A safer alternative to conventional antimicrobial and antibacterial agents, J.Chem.Pharm.Res.,2010,2(3): XC Jiang, WM Chen, CY Chen, SX Xiong, AB Yu, Role of Temperature in the Growth of Silver Nanoparticles Through a Synergetic Reduction Approach, Nanoscale Res Lett, 2011, 6, M.Raffi, F.Hussain, T.M.Bhatti, J.I.Akhter, A. Hameed and M.M.Hasan, Antibacterial Characterization of Silver Nanoparticles against E: Coli ATCC-5224,J. Mater. Sci. Technol., 2008, 24, 2, pp.no Oka, M., T. Tomioka, K. Tomita, A. Nishino, and S. Ueda, Inactivation of enveloped viruses by a silver-thiosulfate complex, Metal-Based Drugs, 1994, 1, pp.no