Green Chemistry Dynamic Article Links Cite this: Green Chem., 2012, 14, 1322 www.rsc.org/greenchem PAPER Synthesis, characterization and catalytic activity of gold nanoparticles biosynthesized with Rhizopus oryzae protein extract† Sujoy K. Das, a Calum Dickinson, b Fathima Lafir, b Dermot F. Brougham c and Enrico Marsili* a Received 28th December 2011, Accepted 3rd February 2012 DOI: 10.1039/c2gc16676c A simple one-pot green chemical method for the biosynthesis of gold nanoparticles (AuNPs) by reducing chloroauric acid (HAuCl 4 ) with protein extract of Rhizopus oryzae to produce novel gold nano-bio- conjugates (AuNBC) is described. AuNBCs, having sizes ranging from 5 to 65 nm, were synthesized by altering the HAuCl 4 –protein extract ratio. The conjugates were characterized by spectroscopic, electron microscopic, light scattering and electrophoretic mobility measurements. It was found that the aqueous AuNBC suspensions exhibited excellent stability over a wide range of ionic strength, pH and temperature. The effect of pH and ionic strength indicated that stabilization is due to electrostatic repulsion arising from the negative chargeof the conjugate proteins. The AuNBCs were stable at temperatures lower than the denaturation temperature of the fungal proteins. The catalytic activity of the as-synthesized AuNBCs was quantified by analysing the reduction of p-nitrophenol by borohydride. The conjugates exhibited interesting size and shape dependent catalytic activity, which was stronger than that observed for AuNPs prepared by conventional chemical methods. The catalytic activity was found to be sensitive to both the surface-area-to-volume ratio and the thickness of the protein coating on the NP. Introduction In the ever-expanding field of nanomaterial research, gold nano- particles (AuNPs) have attracted great attention because of their stability, oxidation resistance, and biocompatibility. 1,2 AuNPs have applications in electronics and photonics, 3 catalysis, 4 sensing, 5 imaging, 6 and biomedicine. 7 The size, shape, and crystal structure of AuNPs govern their physicochemical proper- ties along with their catalytic activity. 8 Consequently, synthetic protocols for the production of size- and shape-controlled mono- disperse nanoparticles are of paramount importance. In most cases, AuNPs are synthesized by reducing gold ions with reduc- tants such as borohydride, hydrazine, citrate, etc., followed by surface modification with suitable capping ligands to prevent self-aggregation. 9 In recent years, sol–gel methodology has been utilized to control the nucleation and growth process, thus gener- ating AuNPs of desired size and shape. 10 However, the use of organic solvents in these synthetic processes raises environ- mental questions. 11 Besides, the conventional approaches often result in the formation of polydisperse nanoparticle populations and require additional separation steps to obtain monodisperse populations. 12 Biosynthetic routes generate negligible quantities of hazardous waste, and consume far less energy than chemical synthesis routes, thereby reducing the AuNPs production cost. Biosynthesis and/or biomineralization processes of inorganic materials are quite common in viable microorganisms. Typical examples include the formation of magnetite, gypsum, calcium carbonate, siliceous materials, metal sulphides, etc., in numerous uni- and multi-cellular microorganisms such as Magnetospiril- lum magnetotacticum, Bacillus stearothermophilus and Cylin- drotheca fusiformis. 13–15 These biomineralization processes exploit the specific interactions among biomolecular templates (e.g., proteins) and inorganic materials, thus favouring controlled and efficient synthesis. 16 The template molecules provide a syn- thetic microenvironment where the inorganic phase morphology is highly controlled due to physicochemical interactions. Thus, the application of biosynthesis processes in metal nanoparticle preparation can yield novel nanostructured materials, which could then be further tuned by synthetic chemistry, if required. 17 Different types of microorganisms such as bacteria, fungus, yeast, virus and biomolecules have been used to synthesize metal nanoparticles. 18–20 In the landmark work of Klaus et al., 21 Pseudomonas stutzeri AG259 was used to generate silver nano- particles (AgNPs) intracellularly. Enzymatically catalysed for- mation of AuNP has been observed in thermophilic and hyperthermophilic bacteria and archaea such as Thermotoga maritima and Pyrobaculum islandicum. 19 Formation of AuNPs on the cell wall of Gram-negative β-proteobacterium Cupriavi- dus metallidurans has been reported by Reith et al. 22 Saraf and co-workers reported the assembly of Au nanorods on the surface of bacterium to obtain a highly conductive hybrid system. 23 † Electronic supplementary information (ESI) available: Synthesis and characterization details of gold nanoparticles using XPS, UV-vis spec- troscopy and TEM (Tables S1–S2, Fig. S1–S5). See DOI: 10.1039/ c2gc16676c a School of Biotechnology, National Centre for Sensor Research, Dublin City University, Ireland. E-mail: enrico.marsili@dcu.ie b Materials and Surface Science Institute, University of Limerick, Ireland c National Institute for Cellular Biotechnology, School of Chemical Science, Dublin City University, Ireland 1322 | Green Chem., 2012, 14, 1322–1334 This journal is © The Royal Society of Chemistry 2012 Downloaded by Central Leather Research Institute (CLRI) on 07 November 2012 Published on 14 March 2012 on http://pubs.rsc.org | doi:10.1039/C2GC16676C View Online / Journal Homepage / Table of Contents for this issue