Phyllanthin-assisted biosynthesis of silver and gold nanoparticles: a novel biological approach
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- Kasthuri, J., Kathiravan, K. & Rajendiran, N. J Nanopart Res (2009) 11: 1075. doi:10.1007/s11051-008-9494-9
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The anisotropic gold and spherical–quasi-spherical silver nanoparticles (NPs) were synthesized by reducing aqueous chloroauric acid (HAuCl4) and silver nitrate (AgNO3) solution with the extract of phyllanthin at room temperature. The rate of reduction of HAuCl4 is greater than the AgNO3 at constant amount of phyllanthin extract. The size and shape of the NPs can be controlled by varying the concentration of phyllanthin extract and thereby to tune their optical properties in the near-infrared region of the electromagnetic spectrum. The case of low concentration of extract with HAuCl4 offers slow reduction rate along with the aid of electron-donating group containing extract leads to formation of hexagonal- or triangular-shaped gold NPs. Transmission electron microscopy (TEM) analysis revealed that the shape changes on the gold NPs from hexagonal to spherical particles with increasing initial concentration of phyllanthin extract. The Fourier transform infrared spectroscopy and thermogravimetric analyses reveal that the interaction between NPs and phyllanthin extract. The cyclic voltammograms of silver and gold NPs confirms the conversion of higher oxidation state to zero oxidation state.
KeywordsPhyllanthinGold nanoparticle synthesisBioreduction methodSilver colloidsGreen chemical synthesis
The syntheses of metal nanoparticles (NPs) and nanostructured materials are attracting attention in recent research because of their valuable properties which make them useful for catalysis (Narayanan and EI-Sayed 2004), sensor technology (Gomez-Romero 2001), biological labelling (Qiu et al. 2004), optoelectronics recording media and optics (Gracias et al. 2002). The size, shape and surface morphology play pivotal roles in controlling the physical, chemical, optical and electronic properties of these nanoscopic materials (Kamat 2002; EI-Sayed 2001). This is particularly important for noble metals such as Au and Ag which have strong surface plasmon resonance (SPR) oscillations. The shape-selective metal NPs such as rods, tubes, wires, triangles, prisms, hexagons and cubes can be regularly synthesized by chemical, biological and physical methods (Lim et al. 2008; Shiv Shankar et al. 2004a, b). In the last decade, biosynthesis of NPs have been received considerable attention due to the growing need to develop clean, nontoxic chemicals, environmentally benign solvents and renewable materials (Harris and Bali 2008; Gericke and Pinches 2006).
As a result, researchers in the field of NP synthesis and assembly have turned towards the utilization of biological system such as yeast, fungi, bacteria and plant extracts for the synthesis of biocompatible metal and semiconductor NPs through control nucleation and growth of inorganic NPs (Shiv Shankar et al. 2003a, b; Lengke et al. 2006; Shahverdi et al. 2007).
Jose Yacaman and co-workers (2002) established the synthesis of gold NPs within the live alfalfa plants by gold uptake from solid media. This method can be very efficient in decontaminating the soil polluted with heavy metal ions. Klaus et al. (1999) has described the significance of biosorption and bioreduction of silver ions by dried Pseudomonas stutzeri AG259. Liz-Marzan and co-workers (2002) have shown that triangular/hexagonal Au NPs are preferentially absorbed on a thin film polyelectrolyte compared to their spherical counterpart. Later, Mirkin et al. (1996) developed anisotropic NPs of Ag and Au in the form of triangular prisms and nanoframes. On the other hand, magnetostatic bacteria have been used to synthesize magnetic NPs. It has been reported that the fungus Verticillium isolated from the Taxus plant facilitates an intracellular reduction of Ag ions from aqueous solution producing silver NPs.
Importantly, Mukherjee et al. (2001a, b) have reported the synthesis of metal NPs using eukaryotic system and successfully accomplished extracellular synthesis of Ag or Au NPs using the fungal system. More specifically, they studied bioreduction of chloroaurate ions or silver ions by the broths of geranium and neem (Shiv Shankar et al. 2004a, b). In addition, they synthesized gold nanotriangles using Tamarind leaf extract and studied their potential application in vapour sensing (Ankamwar et al. 2005). Very recently, they have demonstrated synthesis of gold nanotriangles and silver NPs using Aloe vera plant extracts (Chandran et al. 2006). Most of the above-mentioned reports on the biological synthesis of metal NPs utilizing plant extract employed broths obtained from boiled fresh plant leaves.
In this present study, we report on the synthesis of gold nanotriangle and silver NPs using purified phyllanthin extract at ambient conditions. The approach is a simple, cost-effective, stable for long time, reproducible and previously unexploited method. This plant is a predominant species in South India especially in Tamil Nadu. It has been used for many medical applications such as antiviral activity of hepatitis B, gastropathy, diarrhoea, dysentery, scabies, ulcers, asthma and wounds (Venkateswaran et al. 1987; Rajakannan et al. 2003). We found that by changing the concentration of phyllanthin extract in the reaction medium, the change of hexagonal-shaped gold NP to spherical particle was greater and the size of the NPs can be modulated. Accordingly, the longitudinal SPR in the near-infrared (NIR) region can be easily turned. The prominent disparities of shape control between gold and silver NPs are discussed. The stabilizations of gold and silver NPs by phyllanthin have been kinetically monitored and characterized using UV–visible, Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), cyclic voltammetry (CV) and thermogravimetric analysis (TGA).
Chloroauric acid trihydrate (HAuCl4 · 3H2O) and silver nitrate (AgNO3) were obtained from Sigma-Aldrich and used as such. UV–visible spectra were recorded on Shimadzu UV-1601 spectrophotometer containing double beam in identical compartments each for reference and test solution fitted with 1-cm path length quartz cuvettes. The FT-IR spectra were recorded using Perkin-Elmer FT-IR spectrophotometer. The HAuCl4- and AgNO3-reduced phyllanthin solutions were centrifuged at 10,000 rpm for 15 min individually. The deposited residue was dried and grinned with KBr to obtain pellet for the purpose of FT-IR analysis. Transmission electron microscopic images were collected with a JEOL 3010 UHR TEM equipped with a Gatan Imaging Filter. The phyllanthin-stabilized silver and gold NPs were prepared for TEM measurement by placing a drop of the NP bearing different sizes on copper-grid precoated formvarfilms, followed by solvent evaporation under vacuum. Deionized water and KNO3 (Sigma-Aldrich) were used in this study. All the experiments were carried out at ambient temperature of 25 ± 1 °C. A conventional three-electrode electrochemical cell was used for the CV measurements, with a high surface area Pt counter electrode (area = 2 mm dia), and Ag/AgCl wire was used as reference electrode, powered by a CHI model 600 voltammetric analyser. Cyclic voltammograms of 0.1 mol dm−3 KNO3 + AgNO3 (1 × 10−3 M) mixed solution with and without phyllanthin extract were obtained through linear potential scan from positive vertex potential to the negative vertex potential and finally back to the initial potential at a scan rate of 25 mV s−1. The powders of biologically synthesized gold and silver NP were subjected to TGA on a Seiko Instrument at heating rate of 10 °C min−1 under nitrogen atmosphere.
Separation of phyllanthin from the Phyllanthus amarus plant
Isolation of phyllanthin and hypophyllanthin was followed according to the procedure described by Rajakannan et al. (2003). The leaf powder was mixed with slaked lime prepared in petroleum ether in the proportion of 10:3, to prevent extraction of chlorophyll. The extract consisted of phyllanthin, hypophyllanthin, carotenoids and wax. Waxes were removed from the extraction by adding methanol, and phyllanthin and hypophyllanthin could thus be secured in fairly pure conditions. The individual separation was effected by fractional crystallization from petroleum ether; hypophyllanthin separates out first; and the subsequent fractions was collected later. However, several crystallization from petroleum ether or methanol was necessary to obtain pure sample of hypophyllanthin and phyllanthin. The purity of the phyllanthin was checked by UV–visible spectroscopy [λmax = 278 nm (0.226); 210 nm (2.360)] and mass spectroscopy.
Synthesis of nanoparticles
Solutions of HAuCl4 were prepared with deionized triple distilled water to obtain all experimental data. A series concentration (0.0016–0.0026 M) of phyllanthin extract (dissolved in 0.1% MeOH) was added into 1 mL of 10−3 M aqueous HAuCl4 solution, and the final volume was adjusted to 5 mL with water at room temperature. The time of addition of extract into the metal ion solution was considered as the start of the reaction. Under continuous stirring conditions, after 1 min, the light yellow colour of HAuCl4 gradually changes to pink colour indicates the formation of gold NPs. The efficiency of the phyllanthin extract was compared with commercial phyllanthin (purchased from Natural Remedies (Pvt), Bangalore, India) by doing reduction of HAuCl4 salt with constant concentration of phyllanthin. The resulting solutions of both commercial and extracted phyllanthin show same absorbance in the UV–visible spectra. The same procedure was employed for synthesis of silver NPs.
Results and discussion
UV–visible spectra of gold nanoparticles
TEM analysis results of gold nanoparticles
UV–visible and TEM analysis results of silver nanoparticles
CV analysis of silver and gold nanoparticles formation
FT-IR analysis and TGA results of gold and silver nanoparticles
A bioreductive approach of anisotropic gold and silver NPs utilizing the phyllanthin extract has been demonstrated which provides a simple and efficient way for the synthesis of nanomaterials with tunable optical properties directed by particle shape. The reduction rate of HAuCl4 by phyllanthin extract was greater than AgNO3 at constant amount which was confirmed by the increasing intensity of the absorption peak. The presence of small amount of phyllanthin extract leads to slow reduction of HAuCl4 ions which facilitated the formation of triangular- or hexagonal-shaped NPs. Whereas greater amount of phyllanthin extract leads to higher population of spherical NPs and was confirmed from the UV–visible and TEM analysis. The electron-donating nature of –OCH3 group of the phyllanthin extract plays a leading role for the formation and stabilization of NPs, respectively. The interaction of –OCH3 group with the metal ion was confirmed from FT-IR spectra. The electrochemical characterizations performed by using CV show significant responses for change in the reduction potential of the metal ion from higher oxidation state to zero oxidation state.