Verma et al. Nanoscale Res Lett 2011, 6:16 http://www.nanoscalereslett.com/content/6/1/16 pombe [12] are reported to produce silver and cadmium nanocrystals within their periplasmic spaces. Besides these, there are several other eukaryotic microbes such as fungi Verticillium and Fusarium that synthesize the gold nanoparticles with variable shape and size [13,14]. These all examples rectify the importance of bio-systems to get inspiration in fabricating nanomaterials. In this report, we present the single step ‘green synthesis’ protocol for biofabricating highly anisotropic, monocrys- talline gold nanotriangles utilizing extracts of endophytic (endophytes are microbe that resides within the internal living tissues of higher plants as endosymbionts) fungi Aspergillus clavatus, which was isolated from the surface- sterilized stem tissues of Azadirachta indica A. Juss. Ear- lier, there are many other species of Aspergillus have been reported of their potential to synthesize silver and gold nanoparticles such as Aspergillus niger [15], A. flavus [16], A. Fumigatus [17], A. oryzae var. Viridis [9]. Although this endophytic microbe was earlier investigated by our group for the biofabrication of silver nanoparticles [4], but no reports are available about their potential in biofabrication of gold nanoaparticles. This strain is largest among the Aspergillus spp. and conidiophores can be seen from unaided eye. This is first ever report of an endophytic A. Clavatus, in bio-fabricating gold nanoparticles, although some other endophytic fungi like Colletotrichum sp. from Pelargonium graveolens leaves are reported for gold bio-fabrication [18]. Most of the earlier works are emphasizing with the size of nanoparticles in contrast to this report which shows a precise control not only over size but also its shape specially nanotriangle. Experimental Details Isolation of Endophytic Aspergillus clavatus The host plant Azadirachta indica A. Juss. was sur- veyed, and samples were randomly collected from within the campus premises of Banaras Hindu Univer- sity, Varanasi, India. The stem tissues were collected with cut ends sealed with parafilms™ and collected in paper bags. The samples were than washed properly in running tap water for 5–8 min followed by rinse in sterile distilled water to remove the adhered debris and spores. After successive surface sterilization in 75% ethanol (5 min), the stem tissues were rinsed three times in sterilized distilled water and aseptically cut into small pads (0.5 × 0.5 cm2). The small pads were carefully placed onto PDA plates and incubated at 25°C for 20 days until the mycelia of endophytic fungi appeared. Each isolate was then grown and examined to ascertain that it originated from a single spore. Based on literature and other morphotaxo- nomic features under microscope (Nikon Eclipse E-600), one of the strains is identified as Aspergillus clavatus. Page 2 of 7 Biological Synthesis of Gold Nanotriangles The endophytic Aspergillus clavatus strain was grown in 500-ml Erlenmeyer flasks containing 200 ml MGYP medium which is composed of malt extract (0.3%), glu- cose (1%), yeast extract (0.3%) and peptone (0.5%), and after adjusting the pH of the medium to 7.0, the culture was grown with continuous shaking on a rotary shaker (200 rpm) at 27°C for 8 days. After the fermentation of the culture, biomass was harvested by centrifugation (5,000 rpm) at 20°C for 20 min, and then the mycelia were washed thrice with sterile distilled water under aseptic conditions. In the present study, we have used both the biomass (wet mycelia) and the culture-free spent medium (culture extract) as reducing agent. The thoroughly washed and harvested mycelial biomass (10 g wet weight) was suspended in 100 ml of aqueous 1 mM HAuCl4 in 500-ml Erlenmeyer’s flasks. This reac- tion mixture was then put onto a shaker at room tem- perature and 200 rpm. The reaction mixture was routinely monitored by visual colour change as well as periodic sampling of aliquots (2 ml) of the reaction mix- ture and measuring the UV–vis spectra on a Hitachi dual-beam spectrophotometer (Hitachi, UV-2910) oper- ated at a resolution of 1 nm. Similarly, the broth extract of the endophytic strain is also utilized for bioreduction of aqueous gold ion solution. In a flask, 90 ml of aqu- eous 1 mM HAuCl4 solution was taken and 10 ml of fungal extract solution is added, thereafter the reaction mixture is placed on rotary shaker as in conditions simi- lar to the biomass-based reduction. Characterization of Gold Nanotriangles Once the reactions in the flasks have been completed, the nanoparticles formed were accordingly characterized with TEM, XRD and AFM. For XRD studies, the bio- mass of fungal mycelia after the reaction has been taken and dried in sterile condition in hot air oven and ground into fine powder. The characterization of gold nanoparticles was carried out by XRD (Cu-Ka radiation source) using a 12-kW rotoflux rotating Cu anode (Rigaku Tokyo, Japan) powder diffractometer (RINT 2000/PC series) operating in Bragg–Brentano geometry and fitted with a curved crystal graphite monochroma- tor in the diffraction beam and a high temperature attachment. For TEM analysis, the samples were pre- pared by placing 5 μl of gold nanoparticle suspension on a 300-mesh carbon-coated copper grid, and the solu- tion was allowed to stand for 5 min, then excess solu- tion was removed carefully, and the grid was allowed to dry for an additional 5 min; the average size and size distributions of gold nanoparticles were determined by processing the TEM images with image processing soft- ware on a Tecnai G-20 transmission electron micro- scope, a 200-kV TEM with a W-source and an ultra