Extracellular bio-synthesis of silver nanoparticles

2.1 Synthesis of silver nanoparticles

To the preparation of aqueous solution of chinnar leaves extract, 10 g fresh leaves collected from the Srinagar, Jammu and Kashmir, India, perfectly washed, chopped into small pieces and added to a Borosil conical flask containing 250 ml double distilled deionized water. The reaction mixture was kept on a boiling water bath for 30 min, cooled and filtered through Whatman filter paper No. 40. The prefect transparent clear filtrate contains only soluble organic moieties of the chinnar leaves and the solid residue is discarded. This resulting aqueous solution used for the reduction of Ag⁺ ions to the metallic Ag. A fresh stock solution of AgNO₃ (molecular weight: 169.87 g/mol, assay: 99%: Merck, India) was prepared by dissolving requisite solid AgNO₃ in double-distilled water. Exactly 5.0 cm³ of the extract was added to 50 cm₃ reaction mixture (0.01 mol dm⁻³; 8.0 cm³ AgNO₃ + 37 cm³ water) in a 250 ml Borosil flask and kept it at room temperature for the reduction process. As the reaction time increases, appearance of pale-yellow observed which indicates the formation of AgNPs (Al-Thabaiti et al., 2008; Khan and Talib, 2010; Khan and Al-Nowaiser, 2011; Rafey et al., 2011; Hussain et al., 2011; Khan et al., 2012, 2013).

2.2 Characterization of silver nanoparticles

The appearance of silver sol (reduction of Ag⁺ ions by leaves extract) was monitored by UV–vis spectroscopy (Shimadzu UV–vis spectrophotometer; model UV-1800, Japan) as a function of time by using a quartz cuvette with water as reference. The morphology of AgNPs was determined by using transmission electron microscope (Hitachi 7600 with an accelerating voltage of 120 kV). The selected area electron diffraction (SADE) data were also recorded. Sample for TEM analysis was prepared by coating of aqueous AgNPs drops on carbon-coated copper grids, and kept for 5 min; the extra solution was removed using blotting paper at room temperature.

3.1 Visual observations and UV–vis spectra

A preliminary visual observation shows that the initial color of the reaction mixture after the addition of leaves extract to the aqueous AgNO₃ solution was nearly colorless. Interestingly, the color of the reaction mixture changed from pale-yellow, pink-yellow, light brown, wine-red to dark brown exponentially with reaction-time as aggregation proceeds. Colourlessness of reaction mixture at the initial stage and the final deep reddish-brown color after the completion of the reaction are shown in (Fig. 1), which indicates the AgNPs shape, size and the size distribution altering with the reaction time at different stages (Khan et al., 2012). The color change might be due to the excitation of SPR in the production of AgNPs (Mulvaney, 1996). The spectra of the biosynthesis production of AgNPs are noted at different time intervals and are shown in Fig. 2 for the 5.0 cm³ of leaves extract. The SRP peak observed at 450 nm confirms the influence of aqueous chinnar leaf extract in reducing Ag⁺ ions to AgNPs from aqueous AgNO₃ solution. Absorbance intensity of broad SRP band increases steadily as a function of reaction time. A weak absorbance peak was observed at 450 nm with 1.0 cm³ of extract after 5 min. Interestingly, the position of the maximum absorption was shifted to a shorter wavelength (from 450 to 425 nm; blue shift). Sharpness of the peak increases with time indicating that much smaller AgNPs were generated with time (Fig. 3). In addition to a sharp peak at 425 nm, a weak shoulder also developed at 475 which might be due to the anisotropic growth of AgNPs. The broadness of the peak indicates the wide size distribution of the nanoparticles in the biomolecules constituents of leaves extract (Song and Kim, 2009).

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Figure 1 Optical images of chinnar leaves extract and its reaction product with AgNO₃ at different time intervals. Reaction conditions: [Ag⁺] = 16.0 × 10⁻⁴ mol dm⁻³, [extract] = 5.0 cm³, temperature = 30 °C.

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Figure 2 UV–vis bioreduction kinetics of the reaction of Oriental plane leaves extract with aqueous AgNO₃ with different time intervals. Reaction conditions: [Ag⁺] = 16.0 × 10⁻⁴, [extract] = 5.0 cm³; (■; only leaves extract), time = 5 (●), 10 (▴), 20 (▾) and 30 min (♦).

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Figure 3 UV–vis bioreduction kinetics of the reaction of Oriental plane leaves extract with aqueous AgNO₃ with different time intervals. Reaction conditions: [Ag⁺] = 16.0 × 10⁻⁴ mol dm⁻³, [extract] = 1.0 cm³; (■; only leaves extract), time = 5 (●), 10 (▴), 20 (▾) and 30 min (♦).

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3.2 Kinetics and mechanism

The reaction-time curves show that the Ag⁺ ions reduction into Ag⁰ occurs very rapidly and more than 90% of the reduction of Ag⁺ ions will be completed in 10 min. From 10 to 30 min, the reaction stops as the intensity of the reaction shows almost a parallel line with x-axis with respect to time (Fig. 4). No appreciable change in absorbance was noticed after 10 min for all the [Ag⁺] used in the entire study, confirming the complete reduction of Ag⁺ ions to AgNPs. The sigmoidal shape of the reaction-time curves clearly suggests that autocatalysis was involved in the path of AgNPs formation (Huang et al., 1993; Mehta et al., 2010). Surprisingly, formation of transparent pale yellow color silver sol was not observed at lower [Ag⁺] (⩽2.0 × 10⁻⁴ mol dm⁻³) whereas, yellowish-white precipitate was also formed at higher [Ag⁺]. Therefore, it can now be stated confidently that formation of AgNPs is not directly proportional to the [Ag⁺]; small [Ag⁺] being enough to initiate the formation of metal nucleation center which acts as a catalyst for the reduction of other Ag⁺ present in the reaction mixture.

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Figure 4 Reaction-time curves showing the effect of [Ag⁺] on the nucleation and growth of Ag-nanoparticles at room temperature. Reaction conditions: [extract] = 5.0 cm³, [Ag⁺] = 4.0 (■), 8.0 (●), 12.0 (▴) and 16.0 × 10⁻⁴ mol dm⁻³ (▾) .

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Before attempting to propose a mechanism for the Ag-nanoparticles (reduction of Ag⁺ ions into Ag⁰; nucleation), it is necessary to discuss the species of silver metal particles existing in aqueous solution. Various species of colloidal silver such as Ag₂⁺, Ag₃²⁺, Ag₄²⁺, Ag₈Ag+ or Ag₉⁺ and Ag₆⁴⁺ exist in aqueous solution and in zeolites, as suggested by various investigators (Khan et al., 2012b; Mulvaney, 1996; Huang et al., 1993; Henglein, 1993). Out of these species, only Ag₄²⁺ can be stabilized for a long time in the presence of a polyanion even under air and growth stops at the stage of this species. On the basis of above results and discussion, Scheme 1 mechanism to the reduction of Ag⁺ ions by constituents of chinnar leaves extract is proposed.

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Scheme 1 Mechanism to the formation of AgNPs.

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Hydroxy groups of reducing sugars and/or platanolic acid reduce Ag⁺ into Ag⁰ (Eq. (1); rate-determining step (RDS)). The neutral atom Ag⁰ reacts with Ag⁺ to form the relatively stabilized Ag₂⁺ clusters as shown in Eq. (3). Ag₂⁺ clusters dimerize to yield Ag₄²⁺ (yellow-color silver sol; stable species) (Eq. (4)) (Henglein, 1993). The intervention of free radical was detected by adding acrylonitrile as a scavenger, and the formation of white precipitate (Signorella et al., 1996) appeared slowly as the reaction proceeded indicating in situ generation of free radicals. Control experiments with Ag⁺ ions and leaves extract did not show formation of a precipitate.

3.3 Shape-directing effect of CTAB

Surfactants are the amphiphilic molecules, which create highly anisotropic interfacial regions lining the boundary formed by the highly polar aqueous and nonpolar hydrocarbon regions. Micelles, dynamic aggregates can alter morphology and other surface optical properties to different extent depending upon their nature of head group, type of counter-ions and length of hydrophobic tail (Yu and Yam, 2004; Pal et al., 1997). Bakshi (2010) and Bakshi (2009) used different surfactants, anionic, cationic and Gemini to the synthesis of multibranched advanced Ag and Au NPs and reported these stabilizers acted as shape-directing agents. In order to see insight into the sub- and post-micellar role of shape-directing CTAB, a series of experiments were performed at two different [CTAB] i.e., 4.0 × 10⁻⁴ and 8.0 × 10⁻⁴ mol dm⁻³ at fixed concentrations of other reagents. The observed results are summarized in Fig. 5 as absorbance–wavelength profiles. The intensity of the SRP band was increased rapidly with time. However, the position of the maximum absorption was shifted to a shorter wavelength with the variation of reaction-time, indicating that much smaller AgNPs were generated in the presence of CTAB. The peak at short wavelength may be attributed to the out-of plane quadrupole (Jin et al., 2001; Kelly et al., 2003). In the present study, we did not observe any significant effect of sub- and post-micellar [CTAB].

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Figure 5 Absorption spectra of extract (■) and its reaction product with [Ag⁺] (=16.0 × 10⁻⁴ mol dm⁻³) at different time intervals. Reaction conditions: [CTAB] = 4.0 (A) and 8.0 × 10⁻⁴ mol dm⁻³ (B); [extract] = 5.0 cm³, time = 5 (●), 10 (▴), 20 (▾) and 30 min (♦).

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3.4 TEM images and SADE patterns

Fig. 6 shows typical TEM images and SADE ring patterns of AgNPs prepared in the absence and presence of CTAB using chinnar leaves extract. These results suggest that the obtained AgNPs were mostly irregular and agglomerated in a unsymmetric manner on to the surface of Ag⁰, reduction species of Ag⁺, with a larger size of 30 nm (Fig. 6A). The reason for the aggregation is due to the presence of excess amounts of reducing moieties. The TEM analyses corroborate well with the results drawn from the corresponding reaction-time curves (Fig. 3). In the presence of CTAB, the interactions and solubilization of reducing molecules occur with the positive head group of CTAB. The AgNPs are also adsorbed and/or bound with the CTAB, which in turn, altered the morphology drastically and the number of spherical shaped AgNPs was also increased (Fig. 6C). TEM micrographs clearly indicate the presence of coatings surrounding the AgNPs which might be due to the terpenoids and/or reducing sugars present in the leaves extract acted as reducing- and capping-agents (Shankar et al., 2004; Rai et al., 2006). Fig 6B indicates that the AgNPs aggregated and/or deposited in a symmetric manner, resulted in the formation of beautiful silver (tiles-like structures with dimensions between 8 and 40 nm). The typical selected-area diffraction pattern is shown in Fig. 6C. The ring patterns are consistent with the plane families {1 1 0}, {1 1 1}, {2 0 0}, {2 2 0}, {3 1 1}, {3 3 1} and {4 2 2}, of pure face-centered cubic silver structure (Huang and Yang, 2008; Guzman et al., 2008; Serra et al., 2009).

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Figure 6 TEM images and SAED patterns of AgNPs synthesized using chinnar leaves extract (5.0 cm³) in the absence (A and B) and presence of CTAB (C and D). Reaction conditions: [Ag⁺] = 16.0 × 10⁻⁴ mol dm⁻³; [CTAB] = 8.0 × 10⁻⁴ mol dm⁻³; [extract] = 5.0 cm³.

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