Biogenic Synthesis of Au Nanoparticles Using Vascular Plants

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Ind. Eng. Chem. Res. 2010, 49, 12762–12772

Biogenic Synthesis of Au Nanoparticles Using Vascular Plants Roza Bali and Andrew T. Harris* Laboratory for Sustainable Technology, School of Chemical and Biomolecular Engineering, UniVersity of Sydney, NSW, 2006, Australia

The known metallophytes Brassica juncea (B. juncea) and Medicago satiVa (M. satiVa) were investigated for their ability to accumulate and sequester gold (Au) from aqueous solutions of KAuCl4. Once sequestered, some of the metal was stored as nanoparticles, throughout the epidermis, cortex, and vascular tissue for both species, but predominantly located in the xylem parenchyma cells. Nanoparticle size distribution within the plant tissues was determined. In general, particle sizes ranged between 2 nm to 2 µm in B. juncea and 2 nm to 1 µm in M. satiVa and was location dependent; root located nanoparticles had similar size distributions in both species, whereas the distribution within above ground tissues differed between M. satiVa and B. juncea, with B. juncea showing a much broader range of particle sizes. Au(0) nanoparticles were also formed ex vivo following contact between root exudates and an aqueous solution of KAuCl4 resulting in the reduction of Au(III) to Au(0). The largest proportion of particles was in the range 5-10 nm (B. juncea) and 10-20 nm (M. satiVa). The mechanism of growth of Au(0) nanoparticles using live plants, both in vivo and ex vivo is consistent with Turkevich [Gold Bull. 1985, 18 (3), 86-91], who suggested that the process of Au(0) particle formation involved the interplay of crystal nucleation, growth, and coagulation. 1. Introduction The exceptional properties of gold (Au) at the nanoscale are of fundamental interest to researchers. Consequently, Au(0) nanoparticles are used in a wide range of applications including opto-electronic devices,1,2 catalysis,3 biomedical including DNA labeling and drug delivery,4-6 cell imaging,7 immunostaining, and biosensors.8 These new uses of Au require techniques to synthesize nanoparticles that are both cost-effective and environmentally benign. Conventional preparative methods for nanoparticle synthesis include physical and chemical techniques; physical methods include evaporation and laser ablation, whereas chemical methods involve the reduction of a salt with reducing agents, e.g., NaBH4.9,10 Varying the size, shape, and structural organization within the material can modulate nanoparticle properties. To date, a wide range of processes for metallic nanoparticle synthesis have been reported, however, the synthesis of nanoparticles with precise control over size distribution, shape selectivity, and stability remains a challenge. The growing demand for environmentally benign synthesis techniques has led to the use of biological entities for nanoparticle synthesis. Uni- and multicellular organisms (e.g., Escherichia coli, Shewanella algae, Rhodopseudomonas capsulata, Actinomycetes (Thermomonospora sp.), Algae (Sargassum wightii GreVille), and Fungi (Verticillium sp, Fusarium oxysporum) have been used to synthesize inorganic materials, both intra- and extra-cellularly.11-15 However, the use of plants and their extracts for nanoparticle synthesis is a comparatively new and under-researched technique. The majority of research on the use of plants to synthesize nanoparticles has investigated ex vivo synthesis, i.e., contacting a broth of plant leaves with metal salts.16-21 Only Gardea-Torresday et al.;22,23 Sharma et al.;24 Marshall et al.;25 Manceau et al.;26 Haverkamp and Marshall,27 and Harris and Bali28 have investigated the synthesis of metallic nanoparticles inside live plants. Recently, Sharma et al.24 confirmed the catalytic function of the biomatrix containing in vivo generated Au nanoparticles by reducing the * To whom correspondence should be addressed. Tel.+61 2 93512926. Fax +61 2 93512854. E-mail: [email protected].

toxic pollutant aqueous 4-nitrophenol (4-NP) to 4-aminophenol (4-AP). This work demonstrates there are potential uses of in vivo generated nanoparticles without the need for their removal from the plant biomatrix. Previous research on M. satiVa and B. juncea has shown their remarkable ability to accumulate heavy elements, e.g., Pb, Zn, Cu, Ag, Pt, and Au, in their tissues.28-33 Both these metallophytes (metal loving plants) have previously been used for in vivo silver (Ag) and platinum (Pt) nanoparticle synthesis.28,34 In addition, our previous studies demonstrated their potential to hyperaccumulate Au over a range of concentrations and exposure times.33 Therefore, both species make a good choice to investigate the biogenic synthesis of Au nanoparticles. In the present study, we extend these earlier studies by investigating structural, mechanistic, and compositional information for the nanoparticles that are formed. We determine the size distributions of the nanoparticles formed both in vivo and ex vivo and develop a new understanding of how altering the experimental conditions can influence these particle size distributions. We further propose a mechanism for in vivo Au(0) nanoparticle growth in live plants. 2. Experimental Section Plant samples were prepared according to Bali et al. as follows: B. juncea and M. satiVa seeds were surface sterilized in hydrogen peroxide to avoid fungal contamination, washed with deionized water, and germinated for two days at 25 °C.33 Seedlings were then transplanted into vessels containing 250 mL of full strength Hoagland’s medium (Hoaglands Basal No 2, Sigma Aldrich). All experiments were performed in the controlled environment of a plant growth chamber (Contherm Scientific Ltd.) with a 12/12 light/dark cycle (25 °C/18 °C). Seedlings were harvested between two and three weeks following germination and transferred to Petri plates containing aqueous solutions of KAuCl4 (Sigma Aldrich, 99.99%). The effects of exposure time (24, 48, and 72 h) at 1000 ppm (5.07 mM) on Au nanoparticle formation in both B. juncea and M. satiVa was investigated.

10.1021/ie101600m  2010 American Chemical Society Published on Web 11/02/2010

Ind. Eng. Chem. Res., Vol. 49, No. 24, 2010

In order to examine the effect of concentration, structural alterations, and the morphology of the nanoparticles formed, scanning electron microscopy (SEM) analysis of roots of B. juncea and M. satiVa seedlings was performed. For SEM analysis, root sections of M. satiVa and B. juncea were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer for 1 h. The samples were then rinsed with the same buffer three times for 5 min, followed by dehydration through a graded series of ethanol substitutions (30%, 50%, 70% twice for 5 min at each step and then immersed in 90% and finally 100% ethanol four times for 10 min). The tissues were then transferred to a critical point dryer (CPD, BAL-TEC 030) where liquefied carbon dioxide was used as a transitional fluid. The dehydrated samples were mounted on aluminum stubs and carbon coated using a sputter coater (Emitech K950X Carbon Coater) prior to visualization under SEM (Philips XL 30 CP and Zeiss Ultra Plus.). Root sections from control plants and plants exposed to 40 ppm, 100 ppm, and 1000 ppm Au concentration for 24 h were prepared for this analysis. For Transmission Electron Microscopy (TEM, Philips CM12), samples were fixed in 2.5% glutaraldehyde followed by dehydration with ethanol, embedded in synthetic resin, and cured at 60 °C for 24 h. Thin sections, approximately 50-80 nm thick, were cut from the resin block using a microtome, prior to loading onto carbon coated copper grids for analysis. Dark- and bright-field images of some samples were recorded using a JEOL 3000F equipped with X-ray energy dispersive spectroscopy (EDS). High-resolution images were obtained using Scanning Transmission Electron Microscopy (STEM) with a probe diameter ranging between 0.22-1.0 nm. Selected area electron diffraction (SAED) patterns were measured to determine the crystallinity of the nanoparticles. The effect of Au(III) concentration on nanoparticle formation in the solution was also investigated. Whole plants (with their roots inside the solution) of 14-day-old B. juncea and M. satiVa seedlings were exposed to 40 mL of 5 ppm (0.025 mM), 20 ppm (0.1 mM) and 40 ppm (0.2 mM) KAuCl4 solution for a period of 24, 48, and 72 h. This resulted in a reaction between the exudates (most likely released from the roots) and the Au(III) salt forming nanoparticles ex vivo. The resulting solution was subjected to ultrasonication for 10 min to minimize primary particle agglomeration and then TEM samples were prepared by drop coating the solution directly onto carbon coated copper grids. Heavy metal speciation was evaluated using X-ray photoelectron spectroscopy (XPS, VG ESCALAB 220iXL). Au nanoparticles synthesized ex vivo after 24 h exposure of B. juncea to a 40 ppm Au stock solution were studied by depositing four droplets of the nanoparticle solution onto clean glass slides and then drying overnight. Elemental and chemical information was extracted from the surface layer (to a depth of ∼5 - 10 nm). The XPS instrument used an X-ray monochromator (Al Ka, photon energy ) 1486.6 eV) as electron source. Region scan spectra were recorded at 20 eV pass energy. The binding energy scale was referenced to that of C 1s (285.0 eV). X-ray diffraction (XRD) patterns of purified Au(0) nanoparticle solutions formed from the reaction of B. juncea and M. satiVa plant exudates (40 ppm after 24, 48, and 72 h) were measured by depositing samples onto clean silicon wafers. The purification process entailed 3 cycles of centrifugation and redispersion of Au particles in deionized water. The XRD pattern was recorded on a Siemens diffractometer D5000 using Cu-KR (λ ) 1.5418 Å) radiation operated at 40 kV and 30 mA in the diffraction angle range of 2θ ) 30-80°.

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Figure 1. SEM image of root cross section of (a) B. juncea and (b) M. satiVa showing the basic structure of two week old seedlings.

3. Results and Discussion Both B. juncea and M. satiVa demonstrated increased uptake of Au with increased Au(III) concentration in the media.33 The high influx of Au in both B. juncea and M. satiVa after 24 h is likely due to nutrient starvation of the cells. Once the cells are saturated with metal, uptake is then dependent upon the external concentration (i.e., the concentration driving force) as well as the availability of free ion exchange sites at the root surface. The initial rapid uptake of Au may be because of diffusion, metal homeostasis or an ion exchange mechanism.35 As a first step to analyze the Au present in the plants, SEM/EDS analysis was performed to investigate specific locations with accumulated Au. In B. juncea and M. satiVa across all concentrations and exposure times, a change in root color from white to purple was observed, indicative of the formation of Au(0) nanoparticles within the root structure. SEM micrographs of root sections of M. satiVa and B. juncea showed the characteristic feature of roots; the exarch condition, e.g., xylem and phloem strands on alternate radii (Figure 1). However, with an increase in concentration the roots showed a decrease in the intercellular spaces as compared to the control plant. In addition, cell breakdown was observed and this effect was remarkably significant in plants exposed to 1000 ppm. The vascular cylinder was severely affected at 1000 ppm, where no cellular differentiation could be observed (Figure 2a). Interestingly, the cellular structure of root samples exposed to 40 ppm and 100 ppm was intact (Figure 2b). As shown in Figure 2, Au(0) particles were present throughout the cross section ranging from

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Figure 2. (a) SEM image of B. juncea root exposed to 1000 ppm Au solution for 24 h, (b) SEM image of root cross section of M. satiVa exposed to 40 ppm of Au solution, (c) higher magnification of parts of Figure 2b, (d) single cortical cell filled with Au particles, (e) Au nanoparticles of varying morphologies, and (f) EDS image confirming nanoparticles contained Au.

vascular cylinder to cortex and outer epidermal layer (Figure 2a-e). Higher magnification micrographs showed the presence of nanoparticulate Au in epidermal and layers of cortical cells near the epidermis (Figure 2c). Particles of varying sizes and shapes were observed in M. satiVa root section at 40 ppm (Figure 2d,e). In general, across different concentrations particles ranging from 20 nm to 2 µm were observed. This result was confirmed by EDS analysis of the particles, which clearly demonstrated the presence of Au in the sections (Figure 2f). TEM images of cross sections of the roots and shoots of both plants revealed the presence of nanoparticles of varying sizes and shapes in large quantities (Figures 3, 4, and 5). A significant number of particles were observed in the root cell walls of both B. juncea and M. satiVa, consistent with previous research

suggesting heavy metals are immobilized in the root cell walls.22-36 However, nanoparticles were observed both extracellulary and intracellularly; in particular within the epidermal cell walls, xylem cell walls, cell membranes, and vacuoles. Larger aggregates of particles were observed in the vacuoles as expected (vacuoles are the site for metal sequestration). The mean particle size was 44 nm in M. satiVa roots, 38 nm in M. satiVa shoots, 43 nm in B. juncea roots, 61 nm in B. juncea shoots, within a range of 18 to 252 nm in all cases measured using SIMAGIS image analysis software from a minimum of six TEM images from the same sample. Individual particles as small as 5 nm and as large as 2 µm were observed. TEM analysis of B. juncea and M. satiVa root cells indicated the presence of large quantities of nanoparticles (Figures 3 and


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Figure 3. (a) TEM analysis of B. juncea root cells indicating the presence of large quantities of nanoparticles, the majority spherical in shape, (b) Higher magnification TEM image showing agglomeration of particles forming hexagonal shape and high resolution image of hexagonal particle in inset, and (c) Higher magnification TEM image showing agglomeration of particles forming spherical shapes in B. juncea root cells and high resolution spherical nanoparticle shown in inset.

Figure 4. (a) Gold nanoparticles formed in epidermal B. juncea root cells with extensions after 24 h exposure to 1000 ppm, (b) High magnification TEM image of cell wall extensions, showing gold nanoparticles, and (c) Higher magnification TEM image of cell structures B. juncea root cell walls showing presence of gold nanoparticles.

4). High-resolution transmission electron microscopy (HRTEM) analysis showed that the majority of them were spherical in shape as shown in inset of Figure 3c. The mechanistic aspects of Au(0) nanoparticle formation inside live plants have not yet been fully elucidated, although Haverkamp and Marshall27 have shown that in the case of Ag, the maximum quantity of Ag(0) present in B. juncea up to 8 h after exposure, was ∼0.355 wt-% (dry basis) with the remainder present as a complexed Ag salt. In the case of Au, previous research has demonstrated that Au is not absorbed by plant roots in its metallic form, but rather as a complex.37-39 Shacklette et al.40 further suggested that the root cell membranes allow a maximum of 10 nm sized particles to enter into the plant. Thus the presence of larger sized nanoparticles suggests these are synthesized in vivo, due to coalescence of smaller nanoparticles. The presence of such a large quantity of Au(0) particles in the root and stem cross sections suggests that reduction occurs within the cell structures due to action by secondary metabolites. In support of this we note the work of Turkevich41 who described the process of ex situ Au(0) nanoparticle growth. The three images in Figure 3,

at increasing resolution, illustrate the step-by-step process of Au(0) nanoparticle formation in B. juncea roots. We examined hundreds of images under TEM and observed this mechanism of nanoparticle formation in B. juncea only. The first stage is nucleation, a redox reaction, where a reducing agent reacts with Au ions forming thermodynamically stable nuclei of a critical size. In this case the formation of nuclei depends upon the reducing capacity of the plants. A variety of reductants are generated inside the plants when exposed to heavy metals, including amino acids, e.g. histidines and peptides, e.g., phytochelatins and glutathione.42,43 The different ionic forms of Au possess strong affinity to N-containing and S-containing ligands; during ligand exchange reactions at appropriate binding sites these ionic forms are reduced.44,45 Once reduced, both temperature and the presence of ketones/aldehydes might play an important role in directing nanoparticle shape toward nanoprisms. Figure 3b,c shows the second step of the growth stage, where these small particle seeds aggregate together to form larger sized spherical and hexagonal nanoplates. These in turn exhibit well-defined edges; the smaller nanoplates form trian-

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Figure 5. (a) STEM analysis showing the presence of spherical, hexagonal, triangular and irregularly shaped nanoparticles in the B. juncea root cell walls formed after 24 h exposure at 1000 ppm, (b) HRTEM image of truncated triangular particle formed inside B. juncea root cell walls, (c) HRTEM image of triangular particle formed inside root cell walls, (d) selected area diffraction pattern of the triangular nanoparticle which is shown in inset, (e) Bright field STEM image showing varying shapes of Au nanoparticles formed inside M. satiVa root cells at 1000 ppm after 24 h exposure, (f) High resolution TEM image of triangular Au nanoparticle formed inside M. satiVa root, (g) High resolution TEM image of showing lattice of irregular Au nanoparticle which is shown in inset, and (h) selected area diffraction pattern of hexagonal nanoparticle shown in inset.

gular structures while the larger nanoplates form triangular, truncated triangular, hexagonal and truncated hexagonal shapes. The nanotriangles and hexagons show plate surfaces bound by stable (111) facets. In this case, for shape controlled Au(0) nanoparticle synthesis, the degree of coagulation and aggregation needs to be controlled. Figure 4 shows TEM images of Au(0) particles formed in B. juncea root structures after 24 h exposure to a stock solution containing 1000 ppm Au. Images of the epidermal cell wall indicate large quantities of spherical nanoparticles. Figure 4a also shows the cell wall extensions which are formed as a result of heavy metal stress, while the images in Figure 4b,c indicate the large number of nanoparticles which are present across these extensions. Examples of the different Au(0) nanoparticle sizes and shapes in B. juncea and M. satiVa roots are shown in Figure 5. STEM analysis shows the presence of spherical, hexagonal, triangular and irregularly shaped nanoparticles in the root cell walls (Figure 5a); Gardea-Torresday et al.22 have previously observed similar morphologies in M. satiVa when exposed to 320 ppm Au solutions. Figure 5b,c shows HRTEM micrographs

of truncated triangular nanoplates and triangular nanoplates, respectively, both showing the dark contrast bands and stripes attributable to strain in the crystal lattice.19 It was observed that the larger nanoparticles formed inside the roots and stem sections were polycrystalline with multiple grain boundaries observed by selected area electron diffraction (SAED). Gardea-Torresday et al.46 made similar observations of the nanoparticles produced after contacting M. satiVa biomass with Au(III) salts. HRTEM analysis of M. satiVa roots showed the presence of a large number of spherical nanoparticles with some triangular and hexagonal particles also observed (Figure 5e-h). The Au(0) nanoparticles in M. satiVa roots were either monocrystalline or polycrystalline. Most of the irregular particles were polycrystalline, while the larger nanoparticles exhibited regular morphologies, e.g., the triangular or hexagonal particles were monocrystalline (Figure 5g,h). The elemental composition of these particles was assessed using EDS. Figure 6 confirms that the particles are crystalline Au(0); the only other signal obtained being from the Cu TEM support grids. We acknowledge that the EDS technique used here is less sensitive than synchrotron


Figure 6. Representative EDS of a discrete particle identified under TEM showing it to be pure Au. The peaks for Cu are due to the sample support grid.

based speciation methods;22,24,25 however, there was no indication of the presence of anything other than Au(0) and Cu in our measurements, collectively, across all of samples analyzed, suggesting that; (i) any ligands present were removed during the TEM sample preparation process, or (ii) these were present at values below the detection limit of the instrument (Figure 6). Figure 7 shows the size distribution of Au(0) nanoparticles formed in M. satiVa and B. juncea roots and shoots after 24 h exposure to a 1000 ppm Au stock solution. In the case of M. satiVa the root and shoot distributions are quite similar, with the most common size being 20-30 nm in diameter. This suggests there is little change in the particle size distribution during metal translocation. However for B. juncea, the distributions were location dependent; the particle size distribution within above ground tissues for B. juncea showed a much broader range of particle sizes suggesting that nanoparticle growth continued during metal translocation. Ex vivo synthesis of Au(0) nanoparticles was observed by a change in the color of the broth solution ranging from deep red to dark purple, indicative of the formation of colloidal Au(0). TEM analysis showed the presence of different nanoparticle

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shapes and morphologies in solution, consistent with those observed in vivo. Similar morphologies have been produced by wheat biomass.47 Spherical, triangular, and irregular nanoparticles were the most abundant. At lower Au concentrations (5, 20, and 40 ppm) in the broth, TEM analysis showed the formation of Au(0) nanoparticles by both B. juncea and M. satiVa. Root exudates seem to play an important role in metal uptake,48 e.g., these functionally enriched compounds are likely to be responsible for reduction of Au(III) to Au(0), although a systematic study of root exudates and their activity toward metal reduction has not yet been undertaken. There is, however, a confirmed correlation between root exudates and metal tolerance. Au forms complexes with organic ligands; a characteristic of Group IB metals is their ability to strongly bind to organic matter.49,50 The interaction of Au and organic matter involves mostly electron donor elements, such as N, O, or S, rather than C.51,52 Vlassopoulos et al.53 showed that Au binds preferentially to organic S under reducing conditions, whereas under oxidizing conditions it binds mostly to organic N. Au(III) reduction is linked to S- and N-containing ligands and hence amino acids are likely to be important for the reduction of Au(III). Previously S-containing amino acids cysteine and methionine demonstrated their capability to reduce Au(III) to Au(I) and ultimately to Au(0).54 However, recent studies conducted by Bhargava et al.55 showed the ability of tyrosine, arginine and mixture of tyrosineglycine to effectively reduce Au(III) to Au(0). Moreover, surface complexation of Au(0) particles with amino acid lysine can render them stability and water dispersibility.56,57 In the case of B. juncea, the majority of the particles formed were spherical in the range 5-10 nm (Figure 8), with a small proportion of irregular triangular, hexagonal or decahedral nanoparticles also observed. At 5 ppm, the majority of the nanoparticles were 5-10 nm, with a maximum size of 37 nm (Figure 8a). At 20 ppm, large numbers of particles were in the range 5-15 nm, with the largest observed being 44 nm (Figure 8c). Figure 8e shows the highest percentage of particles formed at 40 ppm, in the range of 5-10 nm, however particles of different sizes were observed.

Figure 7. Particle size distributions of Au nanoparticles after 24 h of exposure to 1000 ppm Au salt solution formed in: (a) B. juncea roots; (b) B. juncea shoots; (c) M. satiVa roots; and (d) M. satiVa shoots.

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Figure 8. Particle size distributions and TEM images of Au nanoparticles formed as a function of concentration after 24 h of exposure time by B. juncea: (a,b) 5 ppm, (c,d) 20 ppm, and (e,f) 40 ppm.

In general across all concentrations, Au(III) solutions exposed to M. satiVa roots showed large numbers of spherical and irregular nanoparticles (Figure 9). At 5 ppm, the majority of the nanoparticles were triangular or spherical, with a size distribution around 10-20 nm, although particles as large as 200 nm were observed (Figure 9a). At 20 ppm, lesser numbers of triangular, hexagonal, and decahedral particles were observed; the majority of the particles were either spherical or irregular, in 10 - 20 nm range (Figure 9c). Only spherical and irregular particles were observed at 40 ppm solution concentration, with a size distribution centered on the 10-20 nm range (Figure 9e). Figure 9 also shows the percentage of nanoparticles in 10-20 nm range increased with an increase in concentration, suggesting increasing monodispersity with increasing Au(III) concentration. Although with an increase in concentration, particle size remained nearly the same in B. juncea but monodispersity was achieved at 5 ppm. In M. satiVa, monodispersity increased with increased solution Au concentration. In general, the geometry of the particles depends on the quality of the precursor, whereas the particle size depends on the precursor/reducing agent ratio. The rate of reduction of a metal salt is greatly influenced by (i) initial concentration of the metal salt, (ii) concentration and type of reducing agent, (iii) temperature, and (iv) pH.58,59 Previous studies suggest that the formation of smaller particles depends on the type and amount of the reducing agent. In general, the

rate of reduction increases with stronger/higher concentrations of the reducing agent and ultimately yields monodispersed smaller particles.60,61 We hypothesize that increased Au concentration in the solution triggers a dramatic increase in exudation of an array of biologically active compounds (enriched with reducing agents) from the roots. Similar observations of increased release of citrate to aluminum stress in Cassia tora was reported by Ishikawa et al.62 This in turn resulted in a near constant reducing agent to Au molar ratio (even though the solution Au concentration was increased) and ultimately increased monodispersity in M. satiVa. The different behavior depicted by B. juncea may be attributed to the different type of reducing agent released by the B. juncea roots in response to Au concentration. XPS spectra of Au(0) nanoparticles resulting from the reaction of B. juncea plant exudates after 24 h exposure to 40 ppm Au solution is shown in Figure 10. The high resolution spectrum of Au4f shows two chemically distinct peaks at 84.8 and 87.4 eV assigned to the spin orbit splitting component of Au (4f 7/2) level in metallic Au.63 Similar observations of Au4f peaks at 84.8 eV were reported by Lengke et al.64The typical binding energy (BE) for metallic Au is 84 eV, however the positive shift (0.8 eV) of the BE obtained in our results may be attributed to the small particle size,65 which correlates well to the observed nanoparticle range (5-10 nm) from the TEM analysis. The XPS


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Figure 9. Particle size distributions and TEM images of Au (0) nanoparticles formed as a function of concentration after 24 h of exposure time by M. satiVa: (a,b) 5 ppm, (c,d) 20 ppm, and (e,f) 40 ppm.

spectra also clearly depicts the presence of four different BEs of C1s with the major peak at 284.98 eV BE assigned to alkyl carbon (C-C) or hydrocarbon (C-H),66 the peak at 286.58 BE, attributed to carbons in hydroxyl groups (C-OH), 287.98 eV to amino groups (C-N), and 289.14 eV to carbonyl groups (CdO). These groups are the backbone of many biomolecules including, monosaccharides, amino acids (serine, threonine), proteins and aldehydes/ketones, respectively.67-69 N1s exhibited peaks centered at bindings energies of 400.18 and 401.83 eV. The peak observed at 400.18 is characteristic of C-N chemical bonds in an organic matrix, while the 401.83 eV BE can be ascribed to organic N from compounds in the exudates containing amine functional groups, e.g., amino acids and proteins.70 These results collectively suggest the capping of nanoparticles by these biomolecules ultimately providing stability to the nanoparticles. The crystalline nature of the ex vivo synthesized Au nanoparticles after 24 h exposure to 40 ppm stock solution by B.

juncea was characterized by the corresponding XRD pattern. Peaks appear at 38.3°, 44.4°, 64.7°, 77.8°, and 81.8°, and are assigned to (111), (200), (220), (311), and (222) lattice planes of the face centered (FCC) Au crystals,71,72 respectively, as shown in Figure 11. The (111) diffraction peak exhibited the greatest intensity, which implies that the as-synthesized Au nanoparticles were primarily dominated by Au(111) facets.71 4. Conclusions B. juncea and M. satiVa can accumulate and sequester Au(0) in a process which is both time and concentration dependent. Both plants demonstrated the formation of colloidal Au(0) particles inside live specimens. TEM analysis confirmed the presence of Au(0) nanoparticles in different orientations and sizes in root and stem sections of B. juncea and M. satiVa. In general, in vivo synthesized Au particle size ranged between 2 nm to 2 µm in B. juncea and 2 nm to 1 µm in M. satiVa. B.

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Figure 10. (a) XPS survey scan, (b) Au 4f7/2, (c) C1s, and (d) N1s spectra from B. juncea experiment after 24 h exposure to a 40 ppm Au stock solution.

juncea, and M. satiVa roots exudates reacted differently to Au(III) solutions, the former producing uniform sized particles at low concentration (5 ppm) and the latter at higher concentrations (40 ppm). Acknowledgment R.B. is grateful for the support of the Richard Claude Mankin Scholarship fund at the University of Sydney. The authors are grateful for the assistance of S. Bulcock from Electron Microscopy Unit with STEM analysis and B. Gong from UNSW Analytical Centre with XPS analysis. Literature Cited

Figure 11. XRD pattern of Au(0) from B. juncea experiment after 24 h exposure time to a 40 ppm Au stock solution.

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ReceiVed for reView July 27, 2010 ReVised manuscript receiVed October 11, 2010 Accepted October 17, 2010 IE101600M

Biogenic Synthesis of Au Nanoparticles Using Vascular Plants

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