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International Journal of Nanotechnology and Application (IJNA) ISSN 2277-4777 Vol. 3, Issue 4, Oct 2013, 1-10 漏 TJPRC Pvt. Ltd.

GREEN SYNTHESIS AND CHARACTERIZATION OF SILVER NANOPARTICLES USING LEAVES OF TECOMA STANS (L.) KUNTH C. ARUNKUMAR, P. NIMA, A. ASTALAKSHMI & V. GANESAN Centre for Research and Postgraduate Studies in Botany, Ayya Nadar Janaki Ammal College, Sivakasi, Tamil Nadu, India

ABSTRACT Plant mediated synthesis of silver nanoparticles have greater applications in the field of Biomedical, Food Packaging and Wound Healing. The present study deals with the synthesis of silver nanoparticles using the leaf broth of Tecoma stans (Family: Bignoniaceae). The observation of colour change in reaction medium from pale yellow to dark brown in the reaction medium which indicates the formation of silver nanoparticles and the synthesized nanoparticles were characterized. UV- Visible spectroscopy showed the 位max at 430 nm. Emission and Excitation peaks obtained in the spectra of photoluminescence study were found at 424 and 430 nm which correlate with UV- Vis absorption spectral patterns. The identification of biomolecules in the leaf broth and reaction medium was analyzed using Fourier Transform Infrared spectroscopy (FT- IR). Energy Dispersive X-Ray (EDX) analysis and Scanning Electron Microscopy (SEM) confirmed the presence of silver nanoparticles. X-ray Diffraction (XRD) and Transmission Electron Microscopy (TEM) analyses shows average particle size of 15nm. This type of plant mediated synthesis appears to be cost effective, eco-friendly and easy alternative green synthesis to conventional, physical and chemical methods to the synthesis of silver nanoparticles

KEYWORDS: Green Synthesis, Tecoma stans, Silver Nanoparticles, Photoluminescence INTRODUCTION There have been tremendous developments in the field of Nanotechnology in the recent past with numerous technologies formulated to synthesize nanoparticles with specific characteristics on Morphology (ie., Shape and Size) and Distribution [1]. Although, there are several methods for the synthesis of pure, well defined nanoparticles, they are very expensive and the use of toxic and hazardous chemicals which cause danger to Environment, Human and Biological means [2]. To overcome this, the eco-friendly synthesis of nanoparticles using environmentally benign materials like Plants [3], Fungi [4], Seaweed [5], Bacteria [6] and Enzymes [7] were employed. It is a single step and offers several advantages such as time reducing, cost effective and Non-toxic. Nanocrystalline silver is a known Noble metal and they have tremendous applications in the field of Detection, Diagnostics, Therapeutics and Antimicrobial activity [8]. Recently, the plant mediated biological synthesis of silver nanoparticles has been reported they are Emblica officinalis [9], Parthenium [10], Aloe vera [11], Pisonia grandis [12], Jatropha curcas [13], Justicia genderussa [14], etc., The present study is therefore aimed to design a protocol for Eco-friendly synthesis of silver nanoparticles using leaf extract of Tecoma stans and their characterization using UV- Visible spectroscopy, Photoluminescence studies, FT-IR (Fourier Transform Infrared) spectroscopy, XRD (X- ray Diffraction), SEM (Scanning Electron Microscopy), EDX (Energy Dispersive X-ray spectroscopy), TEM (Transmission Electron Microscopy) and AAS (Atomic Absorption Spectrophotometer) analyses.

MATERIALS AND METHODS Synthesis of Silver Nanoparticles All the reagents used in the present study were obtained from Himedia Laboratories Pvt Ltd (Mumbai, India). Tecoma stans (L.) Kunth. (Figure 1) belongs to Bignoniaceae. The leaves of Tecoma stans were collected from the


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Botanical garden of Ayya Nadar Janaki Ammal College, Sivakasi, Tamilnadu, India. The collected leaf samples were thoroughly washed with tap water followed by distilled water to remove the surface contaminants and dried for 48 h under the shade. The dried leaves were taken and ground to make fine powder using a mixer and the sieved using 20 mesh sieve to get uniform size range. 10g of sieved leaf powder was added to 100ml of distilled water and boiled at 70 °C for 10 minutes to prepare the leaf broth [15]. 10 ml of freshly prepared leaf broth was resuspended in 190ml of an aqueous solution of silver nitrate and this mixture is used as reaction medium [16]. This medium was kept in an Incubator cum shaker (Orbitek) with 250 rpm at 27°C for 24 h. For this reaction medium a small aliquot of the sample was used to characterize the presence of silver nanoparticles synthesized during the above reaction. The characterization was performed through the following analyses: The Surface Plasmon Resonance (SPR) was analyzed on a Labomed (Model UV- D3200) UV-Visible spectrophotometer with a resolution of 2.0 nm between 300-600 nm possessing a scanning speed of 300 nm per minute. Photoluminescence spectra were recorded in Spectrofluorimeter (ELICO) using 90° illumination to find out the emission and excitation of the nanoparticles. The nature of chemical bonds was characterized using FT-IR spectrophotometer (Shimadzu) using KBr pellet method from the range of 4000- 400 Cm-1. The crystalline nature of the nanoparticles was analyzed using X-ray Diffraction (XRD) (Shimadzu XRD 6000). The surface morphology was characterized using Hitachi S-4500 Scanning Electron Microscope (SEM) and Energy Dispersive X-ray Analyses (EDX). The size distribution of nanoparticles was estimated using Philips-Techno 10 Transmission electron microscope (TEM), in which a drop of leaf broth was placed on carbon coated copper grid and operated at an acceleration voltage of 200KV with resolution of 0.3nm. The quantity of Silver nanoparticles synthesized by leaf tissue was estimated using Atomic Absorption Spectrophotometer (Shimadzu) in accordance with the following equation [17]. q= [(C0 –C) / X] Where: q (mg of metal nanoparticles synthesized by one gram of leaf tissue) is the metal specific uptake, C 0 is the initial metal concentration (mg l-1), C is the residual metal concentration (mg l-1) and X is the biomass concentration of the leaf tissue (g).

RESULTS AND DISCUSSIONS UV- Visible Spectrum of Silver Nanoparticles The leaf broth reduced aqueous silver nitrate into silver ions and formed into silver nanoparticles. The silver nanoparticles were characterized using UV- Visible spectroscopy. The leaf broth had pale yellow colour before the addition of silver nitrate solution which was colorless (Figure 2). After the addition of aqueous silver nitrate, the leaf broth (reaction medium) has gradually changed into dark brown color within 24 h of reaction (Figure 2) and indicates that the silver nitrate is rapidly reduced into silver ions. UV- Visible spectra of the reaction media taken at different time intervals explicit that the Surface Plasmon Resonance (SPR) vibrations are found between 390 nm and 450nm with the λ max at 430 nm which is blue shifted (Figure 3). The Blue shift is related to a decrease in the particle size while the red shift to the increased size of silver nanoparticles [18]. The λmax in SPR bands of silver nanoparticles varies with the substrate or an organism by which they are synthesized. For example the λ max in SPR band is at 400 nm by E.coli [19]; 420 nm by Aspergillus niger [20]; 420 nm by sea weed Kappaphycus alvarezii [5]; 420nm by leaves of Pisonia grandis [12]; 440 nm by leaves of Merrimia tridendata and Citrullus colocynthis [3; 21]; 412nm by leaves of Allium cepa [22] respectively. In the present study, the absorbance of the reaction medium increases up to 0.5 a.u. at 430nm in 24 h of reaction (Figure 3). The capping agents like secondary metabolites or enzymes present in the leaf broth reduce the aqueous silver nitrate rapidly within 24 h and then the gradual decrease in the absorbance was noticed. This may be due to the saturation


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of capping agents in the reaction medium after 24 h which started to decrease to reduce silver nitrate into silver ions. The time taken for the change in colour of the reaction medium varies from plant to plant. Interestingly, the leaf extract of Dioscorea batatas; Euphorbia hirta and Nerium indicum made the colour change within two hours [23; 24]. The leaf extract of Trianthema decandra made the color change within four hours [25] and the leaf extract of Syzygum cumini reduced the silver nitrate and made this colour change within 24 h [26]. Photoluminescence Analysis The optical property of the silver nanoparticles was evaluated using Photoluminescence spectra in which the excitation peak was found at 430 nm, while emission peak was observed at 424 nm. The excitation peak correlates with the absorption spectrum recorded with UV- Vis spectrophotometer (430 nm). The quantum yield (Q= Emission/ Excitation) of the silver nanoparticles was calculated as 0.99 (Figure 4). It indicates that the photons absorbed will be equal to photons emitted. Similarly Kumar et al., [27] and Vigneshwaran et al., [28] have detected the quantum yield around 0.71 and 0.83 respectively. FT-IR Spectroscopic Analysis The biomolecules in the leaf broth of Tecoma stans were responsible for reducing silver nitrate and the synthesized silver nanoparticles. The nature of chemical bonds present in the reaction medium was characterized using FTIR Spectroscopy. The FTIR spectrum of leaf broth before reaction, showed several absorption peaks at 601, 651, 1040, 1193, 1204, 1338, 1747, 2927, 2962, 3126, 3218, 3253, 3315, 3363, 3392, 3421, 3440, 3525 and 3593 cm -1 (Figure 5a). The FT-IR spectrum of purified and dried reaction medium with silver nanoparticles (Figure 5b) shows the absorbance peak at 603, 752, 808, 1112, 1195, 1334, 1398, 1454, 1668, 2106, 2266, 2516, 2883, 2974, 3195 and 3313 cm -1. The total disappearance of the bands at 651, 1050, 1193, 1204, 1338, 1747, 2962 and 3126 cm-1after bio-reduction may be ascribed to the reduction of silver ions into silver nanoparticles, leading to stretching vibrations of unsaturated carbonyl groups with a broad peak at 1668 cm-1. The strong absorbance band at 1386 cm-1 was associated with the stretch of functional groups such as –C-O-C-, -C=O-, -C=C, -C=O- in case of Dioscorea batatas [23]. The absorbance bands are known to be associated with the stretching vibrations for -C-C-O, -C-C-, -C=C-, C=O (esters, ethers) and C-O (polyols) respectively in case of Jatropa curcas [29]. XRD Analysis Figure 6 shows the X-ray diffraction peaks obtained for the synthesized silver nanoparticles using Tecoma stans leaf extract. The Full-Width Half Maximum (FWHM) values measured for the plane of reflection were measured using Debye-Scherrer’s equation, t=0.9λ/βCosθ [30]. The mean size of the nanoparticles was estimated as 15nm using the observed XRD pattern at 2θ= 39.0° marked within (111), 48.0° marked within (200), 67.0° marked within (220) and 78.0° marked within (311). The XRD pattern revealed that the silver nanoparticles formed are face centered cubic (fcc) structures (JCPDS No. 04- 0783). The XRD pattern thus showed that the silver nanoparticles formed are crystalline in nature [31]. Hence, the XRD pattern of the present study clearly elucidates that silver nanoparticles synthesized using Tecoma stans leaf broth are crystalline. SEM and EDAX Analysis The surface morphology (ie. shape and size) of the silver nanoparticles was characterized using Scanning Electron Microscopy (Figure 7). The uniform spherical shape nanoparticles were obtained with the sized ranging from 05-30nm (~). Similarly, the spherical shaped silver nanoparticles with a diameter ranging from 30-40nm were synthesized using


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Boswellia ovalifoliolata [32]; 30-50nm using Merremia tridendata [3]; Carcia papaya [33] and Emblica officinalis [34]. The strong silver peak in the EDX- spectrum (Figure 8) confirms the presence of elemental silver. In the present study the EDX peak of Ag along with Cl and O as the mixed components present in the reaction medium. The strong elemental signal along with weak oxygen that may be originated from the biomolecules bound to the surface of the nanoparticles [23]. TEM Analysis In the present study, size distribution of silver nanoparticles synthesized from the leaf extract of Tecoma stans that are well dispersed with sizes ranging from 5 to 30 nm (~) with the mean 15 Âą 6.99 nm at high resolution is shown in Figure 9a and 9b. Asmathunisha et al., synthesized silver nanoparticles using Sesuvium portulacastrum which ranged from 5 to 20 nm [35]. Chandran et al., shows the size of the silver nanoparticles synthesized using Zingiber officinale ranged from 10 to 30nm were reported [36]. Similarly, the size of the silver nanoparticles synthesized using Elaeagnus latifolia [37]; Emblica officinalis [34] and Carica papaya [33] ranged from 30 to 40nm. The mean size of the silver nanoparticles synthesized using Moringa oleifera was 57 nm and they were spherical in morphology [38]. Atomic Absorption Spectroscopy The amount of silver nanoparticles synthesized was analyzed with Atomic Absorption Spectroscopy after termination of the reduction of silver nitrate in order to find out residual concentration of silver. Interestingly, in this present study one gram dry weight of Tecoma stans leaves could synthesize 1.40 mg of silver nanoparticles within 24 h. The synthesis of silver nanoparticles using plants is an alternate and simple method. Moreover, this is an Eco- friendly method without using toxic chemicals and it is cost effective on production of nanoparticles

CONCLUSIONS Thus the present study, the synthesis of silver nanoparticles using leaf broth of Tecoma stans has been achieved with the rapid reduction of silver nitrate into silver nanoparticles. Interestingly, the reaction medium changed its colour from pale yellow to dark brown within 24 h of reaction. The UV-Visible spectrum of the reaction medium with silver nanoparticles synthesized using Tecoma stans has ď Źmax at 430 nm and absorbance was raised upto 0.5 a.u. The Emission and Excitation spectra obtained from photoluminescence study were found at 424 and 430 nm which correlates with UVVis absorption spectrum. The FT-IR spectrum showed the total disappearance of the bands at 651, 1050, 1193, 1204, 1338, 1747, 2962 and 3126 cm-1 after bio-reduction may be ascribed to the reduction of silver nitrate into silver nanoparticles, leading to unsaturated carbonyl groups with broad peak at 1668 cm-1. The SEM images obtained in the present study elucidate the existence of very small and uniformly spherical nanoparticles with size ranging from 5-30 nm. The XRD and TEM analyses determine the average mean size of the nanoparticles is of 15 nm. The strong silver peak obtained from the EDX spectrum confirms the significant presence of elemental silver. Finally the Atomic Absorption Spectroscopy analysis of the silver nanoparticles synthesized by the leaf tissue of Tecoma stans brings out the ability of one gram of dry weight of leaves to synthesize 1.40 mg of silver nanoparticles. Thus the silver nanoparticles are synthesized using a green resource, Tecoma stans which is an alternate method to physical and chemical syntheses due to its cost effective and eco-friendly nature and they have tremendous applications in the field of Biomedical, Food Packaging and Wound Healing.

ACKNOWLEDGEMENTS Authors acknowledge the financial support by Science and Engineering Research Board, Department of Science


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and Technology, Government of India, New Delhi to carry out this work.

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10. Vyom, P.; Rashmi, P.; Bechan, S. and Avinash, P. Parthenium leaf extract mediated synthesis of silver nanoparticles a novel approach towards weed utilization. Digest Journal of nanomaterials and nanostructures, 2009, 4 : 45-53. 11. Gardea, T.J.L.; Gomez, E.; Peralta, V.J.; Parsons, J.G.; Troiani, H.E. and Jose, Y. Synthesis of Gold nanotriangles and silver nanoparticles using Aloe vera plant extract. Langmuir, 2003, 13, 1357. 12. Jannathul, F.M.; Lalitha, P. and Shubashini, K.S. Novel synthesis of silver nanoparticles using leaf ethanol extract of Pisonia grandis (R.Br). Der Pharma Chemica,2012, 4(6): 2320-2326. 13. Harekrishna, B.; Dipak, K.R.; Priyanka, S.; Sankar, P. and Ajay, M. Green synthesis of Silver nanoparticles using latex of Jatropa curcas. Colloids and Surfaces A: Physicochemical Engineering Aspects, 2009, 339:134-141. 14. Chinna, M. and Hema, P. Green synthesis of highly stable silver nanoparticles using Justicia gendenussa. International Journal of Nanotechnology and Applications. 2012, 39-57.


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15. Sathishkumar, M.; Sneha, K.; Won, S.W.; Cho, C.W.; Kim, S. and Yun, Y.S. Cinnamon zeylanicum bark extract and powder mediated green synthesis of nano-crystalline silver particles and its bactericidal activity. Colloids Surface B. 2009, 73:332–338. 16. Mubayi, A.; Chatterji, S.; Raj, P.K. and Watal, G. Evidence based green synthesis of nanoparticles. VBRI Press, 2012, doi: 10.5185/amlett. Incnano.353. 17. Dias, M.A.; Lacerda, I.C.A.; Pimentel, P.F.; Castro, H.F. and Rosa, C.A. Removal of heavy metals by Aspergillus terrus strain immobilized in a polyurethane matrix. Letters in Applied Microbiology, 2002, 34: 46-50. 18. Mulvaney, P. Surface Plasmon Spectroscopy of nanosized metal particles, Langmuir. 1996, 12: 788-800. 19. Natarajan, K.; Selvaraj, S. and Murty, R.V. Microbial production of Silver nanoparticles. Digest Journal of Nanomaterials and Biostructures. 2010, 1: 135-140. 20. Jaidev, L.R. and Narasimha, G. Fungal mediated biosynthesis of silver nanoparticles, Characterization and Antimicrobial activity. Colloids and surface B: Biointerfaces. 2010, 81: 430- 433. 21. Satyavani, K.; Ramanathan, T. and Gurudeeban, S. Plant mediated synthesis of Biomedical silver nanoparticles by using leaf extract of Citrullus colocynthis. Research Journal of Nanoscience and Nanotechnology. 2011, 1-7. 22. Antariksh, S.; Tripathi, R.M. and Singh, R.P. Biological synthesis of silver nanoparticles using onion (Allium cepa) extract and their Antibacterial activity. Digest Journal of Nanomaterials and Biostructures., 2010,5(2): 427432. 23. Nagajothi, P.C and Lee, K.D. Synthesis of plant mediated silver nanoparticles using Dioscorea batatas Rhizome extract and evaluation of their antimicrobial activities. Journal of nanomaterials. 2011, 1-7. 24. Manopriya, M.; Karunaiselvi, B. and John paul, J.A. Green synthesis of Silver nanoparticles from leaf extracts of Euphorbia hirta and Nerium indicum. Digest Journal of Nanomaterials and Biostructructures, 2011, 6 (2): 869877. 25. Geethalakshmi, R. and Sarada, D.V.L. Synthesis of plant-mediated silver nanoparticles using Trianthema decandra extract and evaluation of their antimicrobial activities. International Journal of Engineering Science and Technology, 2010, 2(5): 970-975. 26. Joyita, B. and Narendhirakannan, R.T. Biosynthesis of silver nanoparticles from Syzygium cimini (L.) seed extract and evaluation of their Invitro Antioxidant activities. Digest Journal of Nanomaterials and Biostructructures. 2011, 6 (3): 961-968. 27. Kumar, S.P.; Darshit, P.; Ankita, P.; Palak, D.; Ram, P.; Pradip, P. and Kaliaperumal, S. Biogenic synthesis of silver nanoparticles using Nicotiana tobaccum leaf extract and study of their antibacterial effect. African Journal of Biotechnology. 2011, 10 (41): 8122- 8130. 28. Vigneshwaran, N.; Kathe, A.; Varadrajan, P.V.; Nachane, R.P. and Balasubramanya, R.H. Biomimetics of silver nanoparticles by white rot fungi Phaenerochaete chrysosporium. Colloids Surfaces B. 53: 55- 59. 29. Bar, H.; Bhui, D.K.; Sahoo, G.P.; Sarkar, P.; Pyne, S. and Misra, A. (2009): Green synthesis of silver nanoparticles using seed extract of Jatropha curcas. Surface Analytical Physicochemical Engineering Aspects. 2009, 348: 212-216.


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APPENDICES

Figure 1: Tecoma stans Leaves


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Figure 2: Colour Change of the Reaction Medium (Leaf Broth of Tecoma stans and 1mM Aqueous Silver Nitrate during the Biological Synthesis of Silver Nanoparticles). A- Control (Aqueous Silver Nitrate), B- Leaf Broth of Tecoma stans C, D, E, F, G and H are the Reaction Media at Different Time Intervals of Reaction Such as 10 Minutes, 30 Minutes, One Hour, Three Hours, 6 Hours and 24 Hours Respectively

Figure 3: UV-Visible Spectra of Silver Nanoparticles Synthesized by Leaf Aqueous Extract of Tecoma stans as a Function of Time

Figure 4: Photoluminescence Spectra of Silver Nanoparticles Using Leaf Extracts of Tecoma stans

Figure 5a: FT-IR Spectrum of Leaf Broth of Tecoma stans


Green Synthesis and Characterization of Silver Nanoparticles Using Leaves of Tecoma stans (L.) Kunth

Figure 5b: FT-IR Spectrum of Silver Nanoparticles Using Leaf Extracts of Tecoma stans Counts 150

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Position [째2Theta] (Copper (Cu))

Figure 6: XRD Pattern of Silver Nanoparticles Formed after Reaction of Leaf Broth of Tecoma stans

Figure 7: SEM Images of Silver Nanoparticles Synthesized Using Tecoma stans Leaf Broth (at 20000X Magnification)

Figure 8: EDX Image of Silver Nanoparticles Synthesized Using Tecoma stans Leaf Broth

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Figure 9a: TEM Image of Silver Nanoparticles Synthesized from the Tecoma stans Leaf Mean 15 SD 6.99

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Figure 9b: Histogram of Silver Nanoparticles Synthesized from Tecoma stans Leaf

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Plant mediated synthesis of silver nanoparticles have greater applications in the field of Biomedical, Food Packaging and Wound Healing. The...