Green Synthesis (Ocimum tenuiflorum)

International Journal of NanoScience and Nanotechnology. ISSN 0974-3081 Volume 2, Number 2 (2011), pp. 103-117 © International Research Publication House http://www.irphouse.com

Green Synthesis (Ocimum tenuiflorum) of Silver Nanoparticles and Toxicity Studies in Zebra Fish (Danio rerio) Model S.C.G. Kiruba Daniel1, R. Kumar2, V. Sathish2, M. Sivakumar1, S. Sunitha2 and T. Anitha Sironmani3* 1

Dept. Nanoscience and Nanotechnology, Anna University of Technology, Trichy, India 2 Dept. of Biotechnology, St. Michael College of Engg. & Tech., Kalayarkoil, Sivagangai, India 3* School of Biotechnology, Madurai Kamaraj University, Madurai 625021, India E-mail: [email protected] / [email protected]

Abstract Silver nanoparticles of different size and shape were synthesized using Ocimum tenuiflorum leaf extract. The nanoparticles were characterized by UV– visible, TEM, XRD and FTIR measurements. The toxicity of the silver nanoparticles was evaluated against zebra fish Danio rerio using direct exposure to silver nanoparticles and indirectly through food chain via feeding silver nanoparticles exposed chironomous larva) There was no toxicity developed against Ocimum tenuiflorum stabilized silver nanoparticles and it could penetrate all tissues including the brain through BBB Life time protection can be given to healthy young ones at a very low concentration (160ug) by simple bathing method.

Introduction Compared with larger particles of the bulk material Nanoparticles exhibit completely new or improved properties based on specific characteristics such as size, distribution and morphology. Nanoparticles present a higher surface-to-volume ratio is relevant for catalytic reactivity and other related properties such as antimicrobial activity in silver nanoparticles. Generally, nanoparticles are prepared by a variety of chemical and physical methods such as chemical reduction [1-4], photochemical reduction [46], electrochemical reduction [7, 8] heat evaporation [9,10] etc., which are not environmental friendly..

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Biological methods of nanoparticles synthesis using microorganisms [11-13,] enzymes [14] and plants or plant extracts [15] have been suggested as possible ecofriendly alternatives to chemical and physical methods. Nanoparticles. synthesis using plants for nanoparticles can be advantageous over other biological processes because it eliminates the elaborate process of maintaining cell cultures and can also be suitably scaled up for large-scale synthesis of nanoparticles [15]. Bioreduction of gold and silver ions to yield metal nanoparticles using living plants [ 16,17] Geranium leaf broth [18], Neem leaf broth [19] lemongrass extract [ 20], Tamarind leaf extract [21] and Aloe vera plant extracts [22] have been reported. Kasthuri et al [23] adopted a bioreductive approach of anisotropic gold and quasispherical silver nanoparticles by using apiin compound, Kasthuri et al.,[24] synthesized the anisotropic gold and spherical– quasi-spherical silver nanoparticles (NPs) using extract of phyllanthin at room temperature. Spent mushroom substrate [25] Gliricidia sepium extract [26,27] and C. zeylanicum bark powder[28] were used to synthesize nanoparticles. Krishna raj et al., [29] studied the rapid synthesis of silver nanoparticles using aqueous leaves extract of A. indica and evaluated its antibacterial activity against water borne pathogens such as Escherichia coli and Vibrio cholerae. Daizy Philip[30] studied mushroom mediated green chemistry approach towards the synthesis of Au, Ag and Au–Ag nanoparticles Synthesis of metallic nanoparticles using green resources like Jatropha ( J. curcas latex_)[31] and Hibiscus [32]. The silver nitrate solution incubated with SMS changed to a yellow color from 24 h onward, indicating the formation of silver nanoparticles. The purified solution yielded the maximum absorbance at 436 nm due to surface plasmon resonance of the silver nanoparticles. X-ray analysis of the freeze-dried powder of silver nanoparticles confirmed the formation of metallic silver. In the present study we used common Ocimum tenuiflorum plant extracts to synthesize silver nanoparticles and could obtain synthesis rates comparable to those of chemical methods. Synthesis of metallic nanoparticles using green resources like Ocimum tenuiflorum is a challenging alternative to chemical synthesis, since this novel green synthesis is pollutant free and eco-friendly synthetic rote for silver nanoparticles.

Experimental work Preparation of leaf broth The broth was prepared by taking 10 gm G. sepium leaves by boiling with 10ml of sterile distilled water. The mixture was cooled and filtered through Whatman filter paper No. 1. The solutions of Leaf broth and 1mM silver nitrate were used for base line correction for spectral studies. Synthesis of silver nanoparticles For synthesis of silver (Ag) nanoparticles, 50mL of a 1 mM solution of silver nitrate was taken along with 500microliters of plant broth. The reduction reaction was carried out at room temperature for 10 min

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Analysis of bioreduced silver nanoparticles UV-Vis- Spectroscopy Studies UV-Vis- Spectroscopy measurements (Shimadzu UV 1700) were carried out at a resolution of 1nm. Transmission Electron Microscopy (TEM) Measurements A drop of nanoparticle colloidal solution was loaded on carbon-coated copper grids and solvent was allowed to evaporate. TEM measurements were performed on Philips model CM 200 instrument operated at an accelerating voltage at 200 kV. Fourier Transform Infrared (FTIR) Spectroscopy Measurements FTIR spectroscopic analyses were carried out using a Jasco Fourier Transform Infrared Spectrometer 410. FTIR spectrophotometer was connected to a photoacoustic cell in the spectral range from 4000 to 400 cm-1. Toxicity of silver nanoparticles in Danio rerio The toxicity of the silver nanoparticles was evaluated against zebra fish Danio rerio. Two methods have been carried out for the toxicity studies. First method was direct method where Danio rerio was directly incubated in silver nanoparticles. One set of Danio rerio were used as control and other five sets were maintained at different time intervals as 5,10,20,30 and 60 respectively. Other method has been carried out through chironomas larva - the food chain of Zebra fish Danio rerion at different time period. The six sets of worms were maintained in a titre well plate along with the silver nanoparticle samples and each set consists of five worms. One set of worms were used as control and other five sets were maintained at different time period such as 5,10,20,30 and 60 respectively. Among those worms, two worms were fed to Danio rerio and kept under observations for sometimes.

Results and discussions Biosynthesis of nano-scale silver particles On challenging, leaf broth of Ocimum tenuiflorum and aqueous AgNO3 (1mM) solution changed from yellowish green to brown, the final color appeared immediately. The entire reaction mixture turned to brown color within 10 min. of reaction. The Ag nanoparticles produced by the Ocimum tenuiflorum leaf was observed to be very stable in the solution, even 3 months after their synthesis, which validates the application of Ocimum tenuiflorum as biomaterials for the synthesis of nano-sized Ag particles. The silver particles were observed to be extremely stable even after 6 weeks. Rapid synthesis of stable silver nanoparticles using Geranium leaf broth (20 g of leaf biomass) and 1mM aqueous AgNO3 have been reported by Sastry et al.[33], where bioreduction was found to be completed within 24 hrs. Similarly, Shivshankar et al.[19] reported rapid synthesis of stable silver, gold and bi-metallic Ag/Au core shell nanoparticles using 20 g of leaf biomass of Azadirachta indica and 1mM aqueous AgNO3, within 4 hrs with 90% reduction of the metal ions. On

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challenging the broth with Ag+ ions, the solution changed from yellowish green to brown the final brownish color appeared gradually with time. The entire reaction mixture turned to brown color after 12 hrs of reaction. The complete reduction was observed with in 10 min. of incubation with the plant extract. Though the reduction was immediate maximum time of 10 min.was given for completion of the reduction process. The water-soluble fractions comprised of complex polyols [34] in the biomass were believed to have played a major role in the bioreduction of Ag ions. UV-Visible Spectrum of silver particles UV–vis spectroscopy is an indirect method to examine the bioreduction of Ag nanoparticles from aqueous AgNO3 solution. The Ag nanoparticles (figure1) exhibited an absorbance peak around 446 nm characteristic of Ag nanoparticle, its surface plasmon absorbance and due to different shapes of lone spherical or roughly spherical Ag nanoparticles. The band at 446 nm can be attributed to Mie scattering which responds only to silver metal. According to Mie's theory, only a single SPR band is expected in the absorption spectra of spherical nanoparticles, whereas anisotropic particles could give rise to two or more SPR bands depending on the shape of the particles.. In present investigation, the reaction mixtures showed a single SPR band revealing spherical shape of silver nanoparticles, which was further confirmed by TEM images.

Figure 1: UV-Visible spectrum of Ocimum tenuiflorum prepared silver nanoparticles.

TEM analysis Figure 2 shows the TEM images of the Ag nanoparticles produced by Ocimum tenuiflorum which illustrate that the formed nanoparticles were quantitatively more concentrated than normal. There was no marked difference in the shape. Most of the nanoparticles were circular in shape with rough edges. Some nanoparticles showed aggregation and had nanostructures with irregular contours. The shape of the nanoparticles produced was further verified at higher magnification, which revealed a


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size of 20nm. These structures were identical with that of the Ag nanoparticles produced from the leaves of Cinnamomum camphora, which was attributed to a similarity in the reductive agents present in plant species [34]. At low magnification very large density of silver nanoparticles can be seen (Figure.2). Thus silver nanoparticles are polydispersed and ranged in size from 10-50 nm with a calculated average size of 27 nm. At higher magnification, the morphology of silver nanoparticles is more clearly seen, the particles are being predominantly spherical These results were in agreement with those reported by Huang et al.[34], Basavaraja et al.[35] and Mukherjee et al. [36]. The TEM images at higher resolution also revealed that nanoparticles are not in uniform shape and the images clearly showed the presence of thick secondary material capping. This capping may be assigned to bioorganic compounds present in the leaf broth [19]

Figure 2: TEM image of the Ocimum tenuiflorum synthesized silver nanoparticles with a view at higher magnification.

FTIR measurements FTIR measurements were carried out to identify the potential biomolecules in Ocimum tenuiflorum leaf responsible for reduction and capping of the bio reduced silver nanoparticles. The FTIR spectra of untreated and treated leaves extract samples containing AgNPs is depicted in Figure.3 and Figure 4. The untreated leaves extract sample show absorption bands at 3395, 2815, 2726, 2098,1623,1350,1230 and 672cm1 . For untreated sample the strongest absorption bands can be assigned carbonyl peak (C O stretching) at 1623cm-1 is indicating carboxylate content in plant based samples, which may be responsible for the reduction of metal ion to metal nanoparticles. Bands originating from hydroxyl group (free water and/or alcohols) at 1395 and 1230cm-1, as well as medium band at 1350 cm-1 indicating presence of amine groups, as would be expected due to plant-origin of the these samples These amide I and II b and s arise

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due to carboxyl stretch and N–H deformation vibrations in the amide linkages of the proteins [37] present in it.. Comparison between spectra of untreated sample to the treated sample AgNPs reveal only minor changes in the positions as well as on the magnitude of the absorption bands; wave numbers varying typically about ±1–10 cm-1. With closer examination in the spectrum of AgNPs, the absorption band at 1230 cm-1 was absent in comparison to the untreated one with stronger band at 1638 and 1350 cm1 , Minor shift on carbonyl peak at 1638 and at the same time weaker absorption on 1350 cm-1. Shift can also be observed on the broad band at 3405 and 2813 cm-1 for AgNPs which have been shifted from 3395 cm-1 and 2726 cm-1of untreated one. The C– O–C and C–OH vibrations [38] of the protein in the leaf appear as a very strong IR band at 1230 cm-1.(Figure 4) The prominent appearance of the amide I and amide II bands with a slight shift from that of the plain leaf indicate the possibility that silver nanoparticles are bound to proteins through free amine groups However, the role of malicacid molecules present in the leaf extract on bioreduction and stabilization cannot be ruled out and needs further study.

Figure 3: FTIR pattern of Ocimum tenuiflorum plant extract and silver nanoparticles synthesized using the Ocimum tenuiflorum plant


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Figure 4: Comparative bar diagram of FTIR peak concentration with wave number for both Ocimum tenuiflorum plant extract and Ocimum tenuiflorum plant extract synthesized silver nanoparticles.

Toxicity studies in Zebra fish Danio rerio model The toxicity of the silver nanoparticles was evaluated against zebra fish Danio rerio. Two methods (direct exposure to silver nanoparticles and indirectly through food chain via feeding silver nanoparticles exposed chironomous larva) were used for the toxicity studies. Interestingly during the period of study, the fishes did not show any significant changes in behavior that might have indicated the neurotoxic effects. The UV-Visible Florescence spectrum of worms and whole body tissue of Danio rerio fed with worms are shown in Figure 5 a and 5b. Increase in incubation time showed a very insignificant increase in binding of Ag nanoparticles ( Figure7). A shift in the absorption spectrum and florescence spectrum were observed in worm fed fishes because of the binding of proteins to silver nanoparticles [39]. Figure 6 shows the absorbance spectrum of the blood and whole body homogenate of fishes exposed directly to silver nanoparticles. Nanoparticles have diverse applications in life sciences such as drug development, protein detection and gene delivery. Drug targeting through nanoparticles may improve therapies yet a thorough understanding of the feature that regulates the effect of carrier nanoparticle is needed to translate this approach into the clinical application.

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In the present, study Silver nanoparticles were distributed in all organs as observed in whole body X-Ray (Figure8) similar to that observed by Daniel et al.[40, 41]; in the case of silver nanoparticles in mice and rat. Experiments on medaka fish [42]; using fluorescent solid latex nanoparticles confirmed a homogeneous distribution of the particles [42, 43].

Figure 5a: The fluorescence spectral pattern of silver nanoparticle fed( at different time period) Chiranamous larvae.

Figure 5b: The fluorescence spectral pattern of silver nanoparticles treated worms (at different time period) fed Zebra fish.


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Figure 6: The fluorescence spectral pattern of silver nanoparticle exposed Zebra fish.

Figure 7: Silver nanoparticle incorporation into Zebra fish by direct and indirect method at different time intervals.

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Figure 8: The whole body X-ray pattern of different time period treated worm fed Zebra fish.

The nanoparticles were detected in the brain indicating that silver nanoparticles have the ability to penetrate blood brain barrier as observed in Danio rerio[43-45], mice and rat model[40,41] It was suggested that the nanoparticles could enter the cells through many routes, some of which include diffusion or endocytosis through the skin of embryos. Both nanocopper and nanosilver exposures increased metal content associated with gill tissue, though silver concentrations were much higher following nanosilver exposures suggesting that intact silver nanoparticles are associated with the gill.[42,43] Comparative toxicity of several metal oxide nanoparticle aqueous suspensions to Danio rerio early developmental stage was reported earlier. The embryo toxicity test revealed that nano-ZnO killed Danio rerio embryos (50 and 100mg/L), retarded the embryo hatching (1–25 mg/L), reduced the body length of larvae, and caused tail. The embryo toxicity of nano-Cu at 0.01 and 0.05 mg/L showed no significant difference from Cu2+ at the corresponding concentrations (0.006 and 0.03 mg/L), but 0.1 mg/L nano-Cu had a greater toxicity than 0.06 mg/L Cu2+.(44) As nanoparticle concentration increased, the number of normally developed Danio rerio decreased, while the number of dead Danio rerio increased. [46-50] But the real time study of transport and biocompatablity in early embryonic development in Zebra fish embryo single silver nanoparticles (5-46nm) showed at 0.19nm concentration showed no toxicity.[50 ] As observed in this and our earlier study in gold fish, the 160ug nanoparticle concentration is the critical concentration of Ag nanoparticles in the adults of Danio rerio. Asharani et al.[45] reported that the Ag-np treated embryos showed a normal cardiac morphology, with atria and ventricle differentiated normally with proper


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orientation with time. Only at higher concentrations of Ag-Nps resulted in significant growth retardation, which could be due to delay or inhibition of cell division In our study the Danio rerio were let to swim in silver nanoparticle solution for one hour and they are still alive and healthy without any immuno pathological symptoms. The above results clearly indicated that there was no toxicity developed against Ocimum tenuiflorum stabilized silver nanoparticles and it could penetrate all tissues including the brain through BBB Life time protection can be given to diseased fishes or healthy young ones at a very low concentration by simple bathing method.(51 paper communicated)

Conclusions Nanoparticles have diverse applications in life sciences such as drug development, protein detection and gene delivery. Drug targeting through nanoparticles may improve therapies yet a thorough understanding of the feature that regulates the effect of carrier nanoparticle is needed to translate this approach into the clinical application. There are many groups world wide who are selecting Zebra fish as model system for various diseases. Hence in this study silver nanoparticles synthesized via green route was characterized and zebra fish was selected for toxicity studies. Silver nanoparticles of different size and shape were synthesized using Ocimum tenuiflorum leaf extract. Water-soluble organics present in the plant materials were mainly responsible for the reduction of Ag ions to nanosized Ag particles. The nanoparticles were characterized by UV– visible, TEM, XRD and FTIR measurements.TEM images and FTIR spectra There was no toxicity developed against Ocimum tenuiflorum stabilized silver nanoparticles and it could penetrate all tissues including the brain through BBB Life time protection can be given to healthy young ones at a very low concentration (160ug) by simple bathing method.

Acknowledgement The authors thank Madurai Kamaraj University UPE facility for all spectroscopic studies. And Tamilnadu Veterinary University for TEM study. No financial support was received from funding agency.

Reference [1] D.G. Yu, “Formation of colloidal silver nanoparticles stabilized by Na+-poly (glutamic acid) silver nitrate complex via chemical reduction process”, Colloids Surf.Vol. B 59, 2007,pp.171–178. [2] Y. Tan, Y. Wang, L. Jiang, et al., “Thiosalicylic acid-functionalized silver nanoparticles, synthesized in one-phase system”, J. Colloid Interface Sci. Vol.249,2002,pp.336–345. [3] C. Petit, P. Lixon and M.P. Pileni, “In situ synthesis of silver nanocluster inAOTreverse micelles”, J. Phys. Chem.Vol.97,1993,pp.12974–12983.

114


[4] S.A. Vorobyova, A.I. Lesnikovich and N.S.Sobal, “Preparation of silver nanoparticles by interphase reduction”, Colloids Surf.Vol. A 152,1999, pp.375–379. [5] K. Mallick, M.J. Witcombb and M.S. Scurrella, “Self-assembly of silver nanoparticles in a polymer solvent: formation of a nanochain through nanoscale soldering”,Mater. Chem. Phys.Vol.90,2005,pp. 221–224. [6] S. Keki, J. Torok, G. Deak, et al., “Silver nanoparticles by PAMAM-assisted photochemical reduction of Ag+”, J. Colloid Interface Sci.Vol.229, 2000,pp.550–553. [7] Y.C. Liu and L.H. Lin, “New pathway for the synthesis of ultrafine silver nanoparticles from bulk silver substrates in aqueous solutions by sonoelectrochemical methods”,Electrochem. Commun. Vol.6, 2004, pp.1163– 1168. [8] G. Sandmann, H. Dietz and W. Plieth, “Preparation of silver nanoparticles on ITO surfaces by a double-pulse method”,J.Electroanal. Chem.Vol.491, 2000,pp. 78–86. [9] C.H. Bae, S.H. Nam and S.M. Park, “Formation of silver nanoparticles by laser ablation of a silver target in NaCl solution”, Appl. Surf. Sci.Vol.197, 2002,pp. 628–634. [10] A.B. Smetana, K.J. Klabunde and C.M. Sorensen, “Synthesis of spherical silver nanoparticles by digestive ripening, stabilization with various agents, and their 3-D and 2-D superlattice formation”, J.Colloid Interface Sci.Vol. 284,2005,pp. 521–526 [11] T.Klaus, R. Joerger, E.Olsson and C.Granqvist, “Silver-based crystalline nanoparticles, microbially fabricated”. Proc. Natl. Acad. Sci. USA,Vol.96, No.24,1999,pp.13611-13614. [12] Y.Konishi, K.Ohno, N.Saitoh, T.Nomura, S.Nagamine, H.Hishida,Y.Takahashi, and T.Uruga,(2007) “Bioreductive deposition of platinum nanoparticles on the bacterium Shewanella algae.” J Biotechnol.Vol.128,2007,pp.648–653. [13] B.Nair and T.Pradeep, “Coalescence of nanoclusters and formation of submicron crystallites assisted by Lactobacillus strains”.Crystal Growth Des.Vol.Vol.2,No.4, 2002,pp. 293-298. [14] I.Willner, R.Baron and B.Willner, “Growing metal nanoparticles by enzymes”,Adv. Mater.Vol.18,2006,pp.1109–1120. [15] S.Shivshankar, A.Rai, A.Ahmad and M.Sastry, “Rapid synthesis of Au, Ag, and bimetallic Au core–Ag shell nanoparticles using Neem (Azadirachta indica)leaf broth”, J. Colloid Interface Sci.,Vol.275,2004,pp. 496-502. [16] J.L.Gardea-Torresdey, E.Gomez, J.R.Peralta-Videa, J.G.Parsons, H.Troiani and M.Jose-Yacaman, “Alfalfa sprouts: A natural source for the synthesis of silver nanoparticles”, Langmuir,Vol.19,2003,pp.1357-1361. [17] J.L.Gardea-Torresdey, E.Rodriguez, G.Parsons-Jason, J.R.Peralta-Videa, E.G.Meitzner and G.Cruz-Jimenez, “Use of ICP and XAS to determine the enhancement of gold phytoextraction by chilopsis linearisusing thiocyanate as a complexing agent”, Anal. Bioanal. Chem.,Vol.382,2005,pp.347-352.


115

[18] S.Shivshankar, A.Ahmad and M.Sastry, “Geranium leaf assisted biosynthesis of silver nanoparticles”, Biotechnol. Prog.Vol.19,2003,pp.1627-1631. [19] S.Shivshankar, A.Rai, A.Ahmad and M.Sastry, “Rapid synthesis of Au, Ag,and bimetallic Au core–Ag shell nanoparticles using Neem (Azadirachta indica)leaf broth”, J.Colloid Interface Sci., Vol.275,2004,pp. 496-502. [20] S.Shivshankar, A.Rai, A.Ahmad and M.Sastry, “Controlling the optical properties of lemongrass extract synthesized gold nanotriangles and potential application in infrared-absorbing optical coatings”,Chem. Mater.,Vol.17, 2005,pp.566-572. [21] B.Ankamwar, M.Chaudhary and M.Sastry, “Gold nanotriangles biologically synthesized using tamarind leaf extract and potential application in vapor sensing”,Synth. React. Inorg. Metal-Org. Nanometal. Chem.,Vol.35, 2005, pp.19-26. [22] S.C.Prathap, M.Chaudhary, R.Pasricha, A.Ahmad and M.Sastry, “Synthesis of gold nanotriangles and silver nanoparticles using Aloe vera plant extract”, Biotechnol. Prog.,Vol.22, 2006, pp.577-583. [23] J.Kasthuri, S.Veerapandian and N. Rajendiran, “Biological synthesis of silver and gold nanoparticles using apiin as reducing agent”, Colloids and Surfaces B: Biointerfaces. Vol.68,2009, pp. 55–60. [24] J. Kasthuri, K.Kathiravan and N.Rajendiran. “Phyllanthin-assisted biosynthesis of silver and gold nanoparticles: a novel biological approach”, J Nanopart Res. Vol.11,2009,pp.1075–1085. [25] Nadanathangam Vigneshwaran, Arati A. Kathe, Perianambi Varadarajan,Rajan P. Nachane, and Rudrapatna H. Balasubramanya, “Silver-Protein (Core-Shell) Nanoparticle Production Using Spent Mushroom Substrate”, Langmuir,Vol. 23,2007, pp.7113-7117. [26] Jae Yong Song and Beom Soo Kim. “Biological synthesis of bimetallic Au/Ag nanoparticles using persimmon (Diopyros kaki) leaf extract”, Korean J. Chem. Eng.,Vol.25,No.4,2008,pp. 808-811. [27] W.Raut Rajesh, R.Lakkakula Jaya, S.Kolekar Niranjan, D.Mendhulkar Vijay and B.Kashid Sahebrao, “Phytosynthesis of Silver Nanoparticle Using Gliricidia sepium (Jacq.)”, Current Nanoscience,Vol.5, 2009,pp. 117-122. [28] M.Sathishkumar, K.Sneha, S.W.Won, C.-W. Cho, S. Kim and Y.-S. Yun, “Cinnamon zeylanicum bark extract and powder mediated green synthesis of nano-crystalline silver particles and its bactericidal activity”, Colloids and Surfaces B: Biointerfaces,Vol.73, 2009,pp.332–338. [29] C.Krishnaraj, E.G.Jagan, S.Rajasekar, P.Selvakumar, P.T.Kalaichelvan and N. Mohan, “Synthesis of silver nanoparticles using Acalypha indica leaf extracts and its antibacterial activity against water borne pathogens”, Colloids and Surfaces B: Biointerfaces,Vol.76, 2010, pp 50–56. [30] Daizy Philip, “Biosynthesis of Au, Ag and Au–Ag nanoparticles using edible mushroom extract”, Spectrochimica Acta Part A.Vol.73,2009, pp.374–381. [31] Harekrishna Bar, Dipak Kr. Bhui, Gobinda P. Sahoo, Priyanka Sarkar, Santanu Pyne and Ajay Misra, “Green synthesis of silver nanoparticles using seed

116

[32] [33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42] [43] [44]

S.C.G. Kiruba Daniel et al extract of Jatropha curcas”, Colloids and Surfaces A: Physicochem. Eng. Aspects, Vol.348,2009,pp. 212–216. Daizy Philip, “Green synthesis of gold and silver nanoparticles using Hibiscus rosasinensis” Physica E Vol.42, 2010,pp. 1417–1424. M.Sastry, A.Ahmad, M.I.Khan and R.Kumar,“Biosynthesis of metal nanoparticles using fungi and actinomycete”, Curr. Sci.Vol.85,2003,pp.162– 170. J.Huang, Q.Li, D.Sun, Y.Lu, Y.Su, X.Yang, H.Wang, Y.Wang, W.Shao, N. He, J.Hong and C.Chen, “Biosynthesis of silver and gold nanoparticles by novel sundried Cinnamomum camphora leaf”, Nanotechnology Vol.18,2007,pp. 105104-105114., doi:10.1088/09574484/18/10/105104 S. Basavaraja, S.D. Balaji, A. Lagashetty, A.H. Rajasab and A. Venkataraman, “Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium semitectum”,Mater. Res. Bull. Vol.43,2008,pp.1164–1170. P. Mukherjee, M. Roy, B.P. Mandal, G.K. Dey, P.K. Mukherjee, J. Ghatak, A.K. Tyagi and S.P. Kale, “Green synthesis of highly stabilized nanocrystalline silver particles by a non-pathogenic and agriculturally important fungus T. asperellum”, Nanotechnology Vol.19,2008,pp. 075103, doi:10.1088/09574484/19/7/075103. F. Caruso, D.N. Furlong, K. Ariga, I. Ichinose and T. Kunitake, “Characterization of polyelectrolyte-protein multilayer films by atomic force microscopy, scanning electron microscopy and Fourier transformed infrared reflection-absorption spectroscopy”, Langmuir Vol.14,1998,pp. 4559–4565. C.Gole, V.Dash, V.Ramakrishnaan, S.R.Sainkar, A.B.Mandal, M.Rao and M. Sastry, “Pepsin–gold colloid conjugates: preparation, characterization and enzymatic activity”, Langmuir Vol.17,2001,pp. 1674–1679. A.Nimrodh Ananth, S.C.G.Kiruba Daniel, T.Anitha Sironmani and Umapathi, “PVA and BSA stabilized silver nanoparticles based Surface – Enhanced Plasmon Resonance probes for protein detection”, Colloids and Surfaces B: Biointerfaces, Colloids and Surfaces B: Biointerfaces,Vol.85,2011,pp.138–144. S.C.G. Kiruba Daniel, V.Tharmaraj, T.Anitha Sironmani and K.Pitchumani, “Toxicity and Immunological activity of Silver Nanoparticles”, Applied Clay Science Vol.48,2010, 547. S.C.G. Kiruba Daniel, T.Anitha Sironmani,Tharmaraj, & K.Pitchumani, Synthesis, characterization and In vivo studies of Fluorophore attached Silver Nanoparticles. Bulletin of Mat.Sci. 2010,(in press ) G.Oberdorster, Z.Sharp, V.Atudorei, A.Elder, R.Gelein, W.Kreyling, et al. Inhal. Toxicol.Vol.16,2004,pp. 437 S.Kashiwada, “Distribution of nanoparticles in the see-through medaka (Oryzias latipes) Environ”, Health Perspect.Vol.114,2006,pp.1697–702. Karl Fent, Christin J. Weisbrod, Amina Wirth-Heller and Uwe Pieles, “Assessment of uptake and toxicity of fluorescent silica nanoparticles in zebrafish (Danio rerio) early life stages”, Aquatic Toxicology of Nanomaterials, Vol.100,No.2,2010,pp.218-28.


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[45] P.V.Asharani, Y.L.Wu, Z.Gong and V.Suresh, “Toxicity of silver nanoparticles in Zebra fish models” Nanotechnology, Vol.19,2008, 255102 doi:10.1088/0957-4484/19/25/255102. [46] Bai Wei, Tian Wenjing, Zhang Zhiyong, He Xiao, Ma Yuhui, Liu Nianqing, and Chai Zhifang. Effects of Copper Nanoparticles on the Development of Zebrafish Embryos. Journal of Nanoscience and Nanotechnology.Vol.10,No.12,2010, pp.8670-8676. [47] X.Zhu, L.Zhu, Z.Duan, R.Qi, Y.Li and Y.Lang, “Comparative toxicity of several metal oxide nanoparticle aqueous suspensions to Zebrafish (Danio rerio) early developmental stage”, J Environ Sci Health A Tox Hazard Subst Environ Eng. Vol.43,No.3,2008,pp.278-284. [48] Wei Bai,Zhiyong Zhang, Wenjing Tian,Xiao He, Yuhui Ma, Yuliang Zhao and Zhifang Chai, “Toxicity of zinc oxide nanoparticles to zebrafish embryo: a physicochemical study of toxicity mechanism”, J Nanopart Res.Vol.12,2010, pp.1645–1654. [49] Cristina Ispas, Daniel Andreescu, Avni Patel, Dan V. Goia, Silvana Andreescu and Kenneth N. Wallace, “Toxicity and Developmental Defects of Different Sizes and Shape Nickel Nanoparticles in Zebrafish”, Journal of Nanoscience and Nanotechnology, Vol.10,No.12,2010,pp. 8670-8676. [50] Kerry J.Lee,Prakash D.Nallathambi,Lauren M.Browning,Christober J.Osgood and Xiao-Hong Nancy Xu, “In Vivo Imaging of transport and biocompatibility of single silver Nanoparticles in early development of Zebrafish Embryos”,ACS Nano.Vol.1,No.2,2007,pp. 133-143. [51] S.C.G.Kiruba Daniel, T.Anitha Sironmani and Dhinagaran (2011), Silver nanoparticles as curative and lifelong protective agent for red spot and white spot diseases of ornamental gold fish Carassius auratus (Under revision)