copper oxide nanoparticles

BIOLOGICAL SYNTHESIS OF COPPER/COPPER OXIDE NANOPARTICLES 1

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Rajesh Ramanathan , Suresh K. Bhargava , and Vipul Bansal 1

School of Applied Sciences, RMIT University, GPO Box 2476V, Melbourne, VIC 3001, Australia *E-mail: [email protected] / [email protected] ABSTRACT Metal nanoparticles have gained significant interest in the past decade because of their unique physio-chemical properties which has a potential to be used in a wide range of applications. Recent efforts have gained success in synthesising nanoparticles with excellent shape and size control; however they are largely restricted to chemical methods. There is an increasing focus on eco-friendly ‘green’ routes for the production of metal nanoparticles, e.g. using biological entities. Morganella sp., a known silver resistant bacterium, was recently reported by our group to produce anisotropic silver nanoparticles by controlling the bacterial growth kinetics. Interestingly, due to structural similarity between silver and copper ions, bacteria that are silver resistant might also be able to uptake copper ions which can then be reduced to synthesize copper nanoparticles. Although similarities in the resistance machinery for silver and copper have been established, their role in synthesizing copper nanoparticles is not well understood. In an effort to synthesize copper nanoparticles via a green biological route, we herein demonstrate for the first time that exposing silver resistant bacteria Morganella sp. to Cu+2 ions can enable aqueous phase synthesis of Cu nanoparticles. The results obtained were characterized using a wide range of techniques such as TEM, HR-TEM, UV-vis spectroscopy and XPS. INTRODUCTION Metal nanoparticles have gained significant interest in materials science, as controlling shape and size confers them with unique physio-chemical properties that have direct impacts on their use in molecular diagnostics (Hill and Mirkin 2006; Thaxton, Georganopoulou et al. 2006; Hill, Vega et al. 2007; Seferos, Giljohann et al. 2007), catalysis (Shiraishi and Toshima 1999; Astruc, Lu et al. 2005; Somorjai, Tao et al. 2008), electronics (Schmid and Corain 2003; Somorjai, Tao et al. 2008), photonics (Millstone, Hurst et al. 2009), biological tagging (Thaxton, Georganopoulou et al. 2006) and surface enhanced Raman scattering (Dick, McFarland et al. 2001; Chen, Wang et al. 2007) applications. There have been efforts to synthesize metal nanoparticles with considerable success, but they are largely limited to chemical methods (Parikh, Singh et al. 2008; Millstone, Hurst et al. 2009). Some of the limitations of using chemical synthesis methods include the use of toxic solvents, noxious precursor chemicals and harmful by-products (Thakkar, Mhatre et al. 2010). Hence there is an increasing focus on developing eco-friendly ‘green synthesis’ routes for the production of metal nanoparticles, for example using biological systems.

R. Ramanathan, S.K. Bhargava, V. Bansal

Among all the biological systems used so far for the synthesis of nanoparticles, bacteria have gained considerable importance (Sastry, Ahmad et al. 2005). Ease of culturing, extracellular production of nanoparticles, its requirement of mild experimental conditions such as pH, pressure, temperature, and easy downstream processing as well as short generation time explains the potential for use of bacteria in nanoparticle synthesis (Parikh, Singh et al. 2008). In the past, microorganisms have been shown to produce a large array of metal as well as metal oxide nanoparticles (Klaus, Joerger et al. 1999; Ahmad, Mukherjee et al. 2003; Shankar, Rai et al. 2004; Bansal, Rautaray et al. 2005; Bansal, Ahmad et al. 2006; Parikh, Singh et al. 2008; Bansal, Ramanathan et al. 2011; Ramanathan, O'Mullane et al. 2011). For example silver resistant bacteria synthesizing anisotropic silver nanoparticles (Ramanathan, O'Mullane et al. 2011), sulfate reducing bacteria Desulfovibrio desulfuricans for the production of palladium nanoparticles (Yong, Rowson et al. 2002), and so on. Till date, the research in the field of biosynthesis has been mainly focused on Ag and Au nanoparticles, and there have been very few reports on the synthesis of Cu/CuO nanoparticles (Thakkar, Mhatre et al. 2010). The few papers in the literature on synthesis of Cu nanoparticles have been able to synthesize them in their oxide form (Hasan, Singh et al. 2008; Singh, Patil et al. 2010). This is probably because copper is well known to be susceptible to oxidation and most successful chemical synthesis of metallic copper nanoparticles is either carried out in organic phases (Yang, Zhou et al. 2006) or require elaborate setups for aqueous phase synthesis to avoid potential oxidation of Cu into its oxide forms (Huang, Yan et al. 1997). The synthesis of pure metallic copper nanoparticles in aqueous phase is therefore still an open challenge for bionanotechnologists. There has been an important observation that molecules possessing thiol groups are produced by the bacteria in response to oxidative stress (Hasan, Singh et al. 2008). These molecules have been associated with playing the role of capping agents in bacteria-mediated synthesis of nanoparticles where they prevent the oxidation of metal nanoparticles (Hasan, Singh et al. 2008; Manceau, Nagy et al. 2008). It has been long known that silver and copper ions share structural similarities (Riggle and Kumamoto 2000). Interestingly the molecular basis for resistance to silver cations in bacteria also showed similarity to that of copper resistance machinery (Brown, Barrett et al. 1995; Gupta, Matsui et al. 1999). Especially the silver binding protein (silE), which is believed to be present in the periplasmic space and is specific to Ag+ binding, showed 47 % identity to copper binding protein (PcoE) (Brown, Barrett et al. 1995; Riggle and Kumamoto 2000). In our previous studies we have shown the presence of silE protein in Morganella sp., which is responsible for its resistance to silver ions. Considering its identity with copper binding protein and the ability of bacteria to synthesize technologically important and complex nanoparticles, we envisage that growing Morganella sp., a silver resistant bacterium in the presence of copper ions might provide a potential tool for the synthesis of copper nanoparticles. In an effort to synthesize metallic copper nanoparticles via a green biological route, we herein demonstrate for the first time that by growing two bacterial strains viz. M. psychrotolerans and M. morganii RP42 in the presence of Cu+2 ions, metallic copper nanoparticles can be obtained. This is particularly important considering that there has

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so far been very few reports demonstrating aqueous phase synthesis of copper nanoparticles and more so no report using a green biological approach. MATERIALS AND METHODS Silver resistant bacteria Morganella morganii RP42 and Morganella psychrotolerans were sub-cultured and maintained in LB agar medium. All chemicals were obtained from USB chemicals and were used without further treatment. Silver resistant bacteria Morganella morganii RP4 and Morganella psychrotolerans were grown in LB broth, pH 7 without added NaCl at 37 °C and 20 °C (the optimum growth temperatures for these bacteria) respectively for 24 hours in an incubator shaker set at 200 rpm, following which aqueous CuSO4 solution at a final concentration of 5 mM was added. The reaction was continued for further 24 hours. After the reaction, bacteria were removed by centrifugation, and the coloured supernatant containing the nanoparticles were used for further analysis. In a control experiment, bacterial growth medium in the presence of 5 mM CuSO4 but in the absence of bacteria was maintained under similar reaction conditions. Following the reaction, bacterial culture supernatant was centrifuged at 3000 rpm for 5 min to remove bacterial cells from the suspension. This homogenous coloured colloidal solution of copper nanoparticles was used for further characterization using Perkin Elmer UV-Vis spectroscopy, Jeol 1010 Transmission Electron Microscope, Jeol 2100F high resolution transmission electron microscope, and Thermo K-alpha X-ray photoelectron spectrometer. Samples for transmission electron microscopy (TEM) were prepared by drop casting sonicated samples on lacy carbon TEM grids. TEM and selected area electron diffraction (SAED) were performed using Jeol 1010 TEM instrument operated at an accelerating voltage of 100 kV. HR-TEM imaging was performed using Jeol 2100F at an operating voltage of 200 kV. For X-ray photoemission spectroscopy (XPS), samples were prepared by drop casting the sample on a Si substrate, and measurements were carried out using Thermo K-Alpha XPS instrument at a pressure better than 1 x 10–9 Torr (1 Torr = 1.333 × 102 Pa). The general scan and C 1s, N 1s, O1s, and Cu 2p core level spectra for the samples were recorded with un-monochromatized Mg Kα radiation (photon energy of 1253.6 eV) at a pass energy of 20 eV and electron take off angle of 90°. The overall resolution was 0.1 eV for XPS measurements. The core level spectra were background corrected using Shirley algorithm and chemically distinct species were resolved using a nonlinear least squares fitting procedure. The core level binding energies (BE) were aligned with adventitious carbon binding energy of 285 eV. RESULTS AND DISCUSSION Addition of 5 mM copper sulphate solution to the flask containing Morganella sp. led to appearance of a dark green colour solution indicating the formation of nanoparticles. The UV-Vis spectroscopy recorded from this solution shows the characteristic surface plasmon resonance (SPR) spectra with absorbance at ca. 590 – 630 nm and peak maximum at 610 nm, which can be confidently attributed to the formation of Cu nanoparticles (Banerjee and Chakravorty 2000; Mott, Galkowski et al. 2007; Hasan,

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Singh et al. 2008). The exact position of the SPR band may shift depending on the individual particle properties including size, shape, and capping agents (Mott, Galkowski et al. 2007). Figure 1 shows the optical absorption spectra of the copper nanoparticles.

Fig. 1: UV-visible spectra of copper nanoparticles synthesized from Morganella morganii RP42 and Morganella psychrotolerans in the presence of Cu+2 salt. The shape and size of the copper nanoparticles biosynthesized using Morganella spp. were further analysed by transmission electron microscopy (TEM), as shown in Figure 2a. In both cases, the particles synthesized are polydispersed and vary from 3-10 nm. As the samples were difficult to analyse under TEM due to their small size and presence of organic matter in the samples, we dialyzed the samples to remove the extracellular proteins that were still present in the supernatant. Following the dialysis, the particles increased in size and were found to be in the size range of 15-20 nm. Selected area electron diffraction (SAED) analysis of the as-prepared nanoparticles clearly shows the crystalline nature of the nanoparticles (Figure 2a insert). Due to the presence of proteins surrounding the particles, the SAED showed characteristics similar to that found in amorphous materials. This observation was further confirmed by Fourier Transform Infra-red (FTIR) spectroscopy, which showed the presence of 1650 and 1540 cm-1 amide I and II signatures (data not shown), arising from peptides/proteins bound to Cu nanoparticles, which suggests the possibility of these agents acting as a capping agents (Bansal, Ahmad et al. 2006).

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Fig. 2: (a) TEM image of biogenic Cu nanoparticles biosynthesized by M. morganii RP42. The inset shows the SAED ring pattern obtained from these particles. (b) HRTEM image of biogenic Cu particles showing lattice planes. To gain more structural information, HR-TEM was performed on the dialysed samples. Figure 2b shows the crystal lattice structure of the as synthesized particles. The dspacing obtained from the HR-TEM corresponds to lattice parameters of Cu nanoparticles in the [111] crystal plane (PCPDF number: 85-1326). The HR-TEM also confirms the presence of proteins bound to these particles which is can be seen around the edges of the particle. Although crystallinity of Cu nanoparticles was evident from HR-TEM imaging, we could not observe any diffraction peaks during XRD analysis, which was most possibly due to the extremely small size of these particles and/or presence of an organic matter (protein) coating around as-synthesized Cu nanoparticles. Further, we performed XPS analysis to substantiate the formation of metallic copper nanoparticles. Figure 3 (a, b, c, and d) show the C 1s, N 1s, O 1s and Cu 2p core level XPS spectra respectively from the as synthesized Cu nanoparticles.

Fig. 3: XPS data showing the (a) C 1s, (b) N 1s, (c) O 1s, and (d) Cu 2p core level spectra recorded from biogenic Cu nanoparticles film cast on to a Si substrate.

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The C1s core level spectra could be deconvoluted into three chemically distinct components centered at 285 eV, 286.3 eV, and 288 eV. The component at 285 eV is due to the presence of adventitious carbon in the sample (Bansal, Rautaray et al. 2005; Hasan, Singh et al. 2008). The components at 286.3 eV and 288 eV originate due to αcarbon and carbonyl groups present in the proteins that are bound to these nanoparticles (Bansal, Rautaray et al. 2005; Hasan, Singh et al. 2008). The O 1s core level spectra could be deconvoluted in to two chemically distinct components which correspond to the carbonyl species from the proteins. The Cu 2p core level spectra showed the presence of only one species with spin-orbit pair of 2p3/2 and 2p1/2 (spin-orbit splitting ~ 19.8 eV) with distinct binding energies of 932.6 eV and 952.3 eV respectively, which are in strong agreement with the reported values of metallic copper (Liou, Lo et al. 2007; Park, Jeong et al. 2007; Hasan, Singh et al. 2008). XPS analysis did not reveal any signatures of satellite peaks that are always present in copper oxide species, thereby suggesting that the synthesized particles are metallic copper in nature. In addition to these peaks, we also found peaks from S 2p (data not shown for brevity) suggesting the involvement of thiol rich proteins which might be acting as capping agents and have the ability to prevent oxidation (Hasan, Singh et al. 2008). Although interesting, it is not possible at this stage to elucidate a plausible mechanism correlating the synthesis of metallic copper nanoparticles by this organism. But some of the preliminary electrochemistry experiments suggest that copper ions are taken up by the bacteria and the nanoparticle formation is an intracellular process. Copper is well known to be susceptible to oxidation and most successful chemical synthesis of metallic copper is either carried out in organic phases or requires elaborate setups for aqueous phase synthesis. It is fascinating that a bacterium known to be silver resistant is able to synthesize metallic copper nanoparticles under aqueous conditions at physiological pH and temperature. Further studies are being carried out to find out whether copper resistant machinery is also present along with silver resistant machinery in these bacteria, and also whether there is a possibility that silver machinery has a role to play in the synthesis of metallic copper nanoparticles. CONCLUSION We have demonstrated for the first time biosynthesis of metallic copper nanoparticles by bacteria Morganella sp. under aqueous physiological conditions. The ability of bacteria to synthesize metallic copper nanoparticles in an aqueous environment would open up exciting new avenues for the potential large scale ‘green’ synthesis. This concept can in the future be extended to other difficult to synthesize nanomaterials. Studies on the biomacromolecules involved in preventing these nanoparticles from oxidising and the exact mechanism for the synthesis are currently under investigation, which will advance our knowledge towards the fundamental understanding of copper resistance. ACKNOWLEDGEMENTS Rajesh Ramanathan thanks the Government of Australia for an Australian Postgraduate Award (APA) for his PhD. Vipul Bansal gratefully acknowledge the Australian Research Council (ARC) for an APD Fellowship and financial support through the ARC-Discovery Project (DP0988099).

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