Journal of Nanoparticle Research

, Volume 11, Issue 5, pp 1075–1085

Phyllanthin-assisted biosynthesis of silver and gold nanoparticles: a novel biological approach

Authors

  • J. Kasthuri
    • Department of Chemistry, Faculty of Engineering and TechnologyS.R.M. University
  • K. Kathiravan
    • Department of BiotechnologyUniversity of Madras
    • Department of Polymer ScienceUniversity of Madras
Research Paper

DOI: 10.1007/s11051-008-9494-9

Cite this article as:
Kasthuri, J., Kathiravan, K. & Rajendiran, N. J Nanopart Res (2009) 11: 1075. doi:10.1007/s11051-008-9494-9

Abstract

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.

Graphical abstract

Anisotropic gold and silver nanoparticles were synthesized by a simple procedure using phyllanthin extract as reducing agent. The rate of bioreduction of AgNO3 is lower than the HAuCl4 at constant concentration of phyllanthin extract. The required size of the nanoparticles can be prepared by varying the concentration of phyllanthin with AgNO3 and HAuCl4.
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Keywords

PhyllanthinGold nanoparticle synthesisBioreduction methodSilver colloidsGreen chemical synthesis

Introduction

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).

Experimental section

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

UV–visible absorption spectroscopy is one of the main techniques to examine the size and shape of the NPs in aqueous suspensions (Wiley et al. 2006). Figure 1 shows the change in the UV–visible–NIR optical response recorded after complete reduction of constant concentration of HAuCl4 (2 × 10−3 M) with increased concentration of phyllanthin extract [(a) 0.0016, (b) 0.0018, (c) 0.002, (d) 0.0022, (e) 0.0024 and (f) 0.0026 M], respectively. The figure inset shows photographs of the test tubes (labelled a–f) containing different concentration of phyllanthin-reduced gold NPs (a–f), respectively. The pink to green colour observed is characteristic of the SPR band of different size of gold NPs in solution. There were two significant observations noted while varying the concentration of plant extract in the reaction medium. The transverse plasmon resonance absorption peak appeared at 540 nm is slightly shifted to shorter wavelength along with increasing the intensity. The relative intensity and the position of the second band appeared in the NIR region of the electromagnetic spectrum along with red shift, known as the longitudinal plasmon resonance band, which occurred due to the formation of triangular- and hexagonal-shaped anisotropic NPs, respectively (Link et al. 1999). This can be achieved with low concentration of phyllanthin extract (curve a–d of Fig. 1). When the concentration of extract increased, the peak appeared in the NIR region was completely suppressed and the visible peak shifted towards shorter wavelength along with increasing intensity. This might be due to the formation of spherical gold NPs which resulted in the colour variation from pink to green.
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Fig. 1

UV–visible–NIR spectra of gold nanoparticles synthesized using different concentration of phyllanthin extract [(a) 0.0016, (b) 0.0018, (c) 0.002, (d) 0.0022, (e) 0.0024 and (f) 0.0026 M] with HAuCl4 (2 × 10−3 M) at 25 °C. The inset photos of the different nanoparticle solutions after 20 min of the reaction whose labels correspond to spectra shown in the main part of the figure

Figure 2a shows the stacked UV–visible–NIR spectra recorded from the HAuCl4 (1 × 10−3 M)–phyllanthin extract (0.0016 M) in aqueous medium as a function of time of reaction. After the addition of phyllanthin extract, the colour of HAuCl4 changed from light yellow to pink. As a result, the gold SPR band occurred initially at 549 nm after 1 min of reaction, increased in intensity with time, accompanied by a broad absorbance band at 1,000 nm after 12 min of commencement of the reaction (not shown in figure). However, by changing the concentration of the extract from 0.0016 to 0.002 M in HAuCl4 (1 × 10−3 M), the intensity of transverse plasmon resonance peaks which centred at 539 nm increased along with very small shift towards shorter wavelength. At the same time, the unidirectional longitudinal plasmon resonance centred at 817 nm shifts considerably towards longer wavelength region (Fig. 2b). The observed shift might resulted from the formation of spherical seeds that act as nuclei growth centres upon which deposition of gold in the form of triangle or hexagonal occurred with shape directing agent present in the solution. Thus, the coexistence of hexagonal, triangular and spherical particles shows two absorption bands in the UV–visible–NIR absorption spectra.
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Fig. 2

The stacked UV–visible–NIR spectra of different concentration of phyllanthin extract [(a) 0.0016 and (b) 0.002 M] with HAuCl4 (1 × 10−3 M) at various time intervals. The inset of the plot shows corresponding absorbance variation with respect to time

TEM analysis results of gold nanoparticles

The shift in the spectral changes was further confirmed by representative TEM images (Fig. 3a–e) of gold NPs resulting from the reduction of HAuCl4 (1 × 10−3 M) by varied concentration of extract. It has been confirmed that the gold NPs mostly exist in the hexagonal shape structure with the lowest concentration of plant extract (0.0016 M). However, gradual increase of the extract in the reaction medium (0.0018, 0.002 and 0.0022 M), the conversion of gold particles from hexagonal to triangular forms increased. These changes are reflected in the increasing intensity along with red shift in the visible–NIR region. Higher concentration of extract (0.0026 M) offers to more number of spherical particles with sizes from 18 to 38 nm with an average particle size of 29 nm as shown in Fig. 3e. The spherical shape of the NPs was confirmed from UV–visible spectra (Fig. 1e, f). This may be because the presence of low concentration of phyllanthin extract, the rate of formation of spherical nuclei should be slow enough to enable deposition of gold in the form of triangles around the spheres. However, a high concentration of phyllanthin leads to strong interaction between biomolecules and surfaces of the shaped NPs, preventing nascent gold nanocrystals from rapid sintering.
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Fig. 3

TEM images of gold nanoparticles synthesized using different concentration of phyllanthin extract [(a) 0.0016, (b) 0.0018, (c) 0.0022, (d) 0.0024 and (e) 0.0026 M] with HAuCl4 (1 × 10−3 M) at 25 °C. Scale bar: (a, b) 50 nm, (c) 100 nm, (d, e) 50 nm

Different magnification of the TEM images of the gold NPs synthesized by treating HAuCl4 (1 × 10−3 M) solution with phyllanthin extract (0.0018 M) for 24 h has been shown in Fig. 4a–d. In this figure, it is possible to identify large population of polydispersed gold NPs consisted of spherical-, triangular-, hexagonal- and rod-shaped with irregular contours. The resultant histograms representing the size distribution of the particles were obtained by digital analysis of images containing at least 253 particles (Fig. 4e). The distribution of the particle diameters showed as main peak located between 32 and 43 nm and the sizes ranging from 10 to 110 nm with an average particles size of 37.5 nm. A percentage of distribution for each different shape of particles is shown in inset of Fig. 4e. From this figure, it can be seen that the irregular and spherical particles are more than that of triangular and hexagonal shapes. The stability of the resultant solution was confirmed by UV–visible and TEM analysis. In the UV–visible spectrum, it shows same absorption intensity even after 3-month time. Moreover, in the TEM analysis the particle size remains same and is agreeable with the reported size.
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Fig. 4

(ae) TEM images and corresponding size distribution of gold particles obtained by reduction of HAuCl4 (1 × 10−3 M) salt with phyllanthin (0.0018 M) at different magnification. The inset of e shows percentage distribution of different size of gold nanoparticles

Figure 5a shows the TEM image of the biogenic gold NPs and the corresponding electron diffraction patterns (inset figure) of the particle obtained using low concentration of phyllanthin extract (0.0018 M). The SAED pattern was obtained by aligning the electron beam perpendicular to the triangular facet of the nanoplate. The hexagonal symmetry of the diffracted spots suggests the single crystalline nature of gold nanotriangle lying flat on the TEM grid (Germain et al. 2003). There are three sets of spots that could be identified from this diffraction pattern. The inner set with weak intensity was caused by reflections from the 1/3{422} planes (triangle); the set with the strong intensity could be indexed to the {200} planes (hexagonal) of fcc gold; and the outer set with weak intensity is reflection from {111} planes (square). These results are close to the reported standard data (JCPDS File No. 893697). Figure 5b shows the HRTEM image of the vertex of triangular gold NP. A d-spacing of ~2.362 Å for adjacent lattice planes corresponds to the {111} planes of single crystalline fcc structure.
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Fig. 5

(a) TEM image of the plate like triangular gold nanoparticles prepared from HAuCl4 (1 × 10−3 M) with phyllanthin extract (0.0018 M). The inset figure shows the electron diffraction pattern of gold nanotriangle. (b) HRTEM image from the vertex of one of the triangular gold nanoparticle

UV–visible and TEM analysis results of silver nanoparticles

Figure 6a and b shows the stacked UV–visible spectra of silver NPs formed from the reaction of aqueous AgNO3 (1 × 10−3 M) with different concentrations of phyllanthin extract (0.002 and 0.004 M) at various time intervals. After the addition of phyllanthin extract (0.002 M) to the aqueous AgNO3, the solution changed from colourless to pale orange which is indicative of the formation of silver NPs. The appearance of weak intensity peak at 439 nm (Fig. 6a) corresponds to the SPR of silver NPs. Thus, the absorption intensity steadily increased as a function of time of reaction without any shift in the peak position, and it attained maximum at 30 min of incubation. No change was observed after this time.
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Fig. 6

Stacked UV–visible spectra of different concentration of phyllanthin extract (a) 0.002 and (b) 0.004 M with AgNO3 (1 × 10−3 M) at different time intervals. The inset of b shows increased absorption variation with respect to time

Figure 6b shows the stacked absorption spectra of silver NP formed at high concentration of phyllanthin extract (0.004 M) with respect to time variation. The absorption peak increases with time, and the position of the absorption maxima at 446 nm gradually shifted towards longer wavelength. The shift towards longer wavelength indicates increasing particle size (aggregation) due to rapid reduction of Ag+ ion in the reaction medium. Thus, the colour of the corresponding solution rapidly changed from colourless to red in about 40 min. Figure 7a and b shows the TEM image of silver NPs obtained from 0.002 and 0.004 M of phyllanthin extract reduced with AgNO3. The resulting NPs for low concentration of phyllanthin (0.002 M) exhibit quasi-spherical, and the average size is about 30 nm. This is may be due to mild reduction of silver ions with 0.002 M of phyllanthin extract which was reflected in the weak intensity absorption maxima (Fig. 6a). However, at high concentration of phyllanthin extract (0.004 M) the Ag+ ions were reduced rapidly that leads to ellipsoidal NPs with low density dispersion as shown in Fig. 7b. It has been supported by UV–visible spectra (Fig. 6b) showing a very broad band towards longer wavelength region.
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Fig. 7

TEM images of silver nanoparticles synthesized using various concentration of phyllanthin extract (a) 0.002 M and (b) 0.004 M with AgNO3 (1 × 10−3 M)

CV analysis of silver and gold nanoparticles formation

The change in the oxidation state of the metal ion was studied by CV technique using platinum electrode with fresh surface at a rate of 25 mV s−1 in the potential range between 0 and 1.0 V (Ma et al. 2004). In Fig. 8a, curve 1 shows the cyclic voltammograms of AgNO3 (1 × 10−3 M) in aqueous medium. The peak observed at 0.54 and 0.25 V is correspond to oxidation and reduction potential of AgNO3. In the phyllanthin-free solution, all the metal ions are reduced to lower oxidation state, since there is no possibility for formation of NPs. Upon addition of phyllanthin extract in the reaction medium, the cathodic peak shifted towards the negative potential direction, implying that the reduced silver NPs are stabilized by phyllanthin extract (curve 2). The extent of decrease in anodic peak current is greater than that of the cathodic peak current due to fact that the rate of reduction of silver ion may be greater than its oxidation. In Fig. 8b, curve 1 shows stable irreversible cyclic voltammogram of HAuCl4 (1 × 10−3 M) in the potential range 0.3–1.0 V. The redox potential of Au3+–Au0 was found to be 0.69 V (Newman and Blanchard 2006). After the addition of phyllanthin in the reaction medium, the redox peak current of HAuCl4 gradually decreased without any potential change which confirms the reduction of HAuCl4 ions to Au0 state (curve 2). This might be because the electron-donating methoxy (–OCH3) groups containing phyllanthin extract can provide a suitable environment for the formation of NPs.
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Fig. 8

Cyclic voltammograms of silver and gold nanoparticles synthesized by reacting 1 × 10−3 M of metal salt (AgNO3 and HAuCl4) in presence of 0.002 M of phyllanthin extract. Curve 1 is 1 × 10−3 M of metal salt (AgNO3/HAuCl4) with supporting electrolyte KNO3 (0.1 mol dm−3) and curve 2 is 1 × 10−3 M of metal salt (AgNO3/HAuCl4) with phyllanthin extract at different time intervals. (a) Silver nanoparticles and (b) gold nanoparticles

FT-IR analysis and TGA results of gold and silver nanoparticles

Further analyses towards the mode of interactions between NPs and phyllanthin extract were studied by using FT-IR spectrophotometer. A previous report confirmed that the hydroxyl group (–OH) from saccharides has a stronger ability to bind Au3+ ion (Lin et al. 2005). Therefore, the affinity of phyllanthin containing methoxy group (–OCH3) to metal ions was studied by comparing phyllanthin-reduced silver and gold NPs with pure phyllanthin as shown in Fig. 9a–c. The band appeared at 1,088 cm−1 (Fig. 9a) corresponds to the –OCH3 group of the phyllanthin extract. After the bioreduction with the AgNO3 and HAuCl4, the shift in the peak at 1,088 cm−1 towards lower frequency is attributed to the binding of –OCH3 group with NPs as shown in Fig. 9b and c. However, the extent of shift towards lower frequency in the gold NPs is slightly higher than the silver NPs which indicate the efficient adsorption of phyllanthin extract on the surface of the gold NPs than the silver NPs. The mechanism has been proposed in Scheme 1.
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Fig. 9

Typical FT-IR spectra of the phyllanthin extract (a), phyllanthin-reduced silver nanoparticles (b) and phyllanthin-reduced gold nanoparticles (c)

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Scheme 1

Schematic diagram of the formation of phyllanthin stabilized gold and silver nanoparticles

Figure 10a and b shows the TGA of the phyllanthin extract loaded with gold and silver NPs when heated from 35 to 800 °C. The initial weight loss observed at 150 °C was attributed to the water molecules present in the phyllanthin extract which reduced gold and silver NPs. Also, there is a steady weight loss until 800 °C. The observed behaviour is most likely as a consequence of the surface desorption of bioorganic compounds present in the NP powder. Thus, the phyllanthin extract-stabilized NPs are expected to be made up of molecules responsible for the reduction of metal ion and stabilizing particles in the solution.
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Fig. 10

Thermogravimetric analysis of phyllanthin-stabilized (a) gold nanoparticles and (b) silver nanoparticles in the form of dried powder at 25 °C

Conclusions

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.

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