Biofabrication of Anisotropic Gold Nanotriangles Using Extract of Endophytic Aspergillus clavatus as a Dual Functional Reductant and Stabilizer
© Verma et al. 2010
Received: 7 July 2010
Accepted: 5 August 2010
Published: 28 August 2010
The Erratum to this article has been published in Nanoscale Research Letters 2011 6:261
Biosynthesis of metal and semiconductor nanoparticles using microorganisms has emerged as a more eco-friendly, simpler and reproducible alternative to the chemical synthesis, allowing the generation of rare forms such as nanotriangles and prisms. Here, we report the endophytic fungus Aspergillus clavatus, isolated from surface sterilized stem tissues of Azadirachta indica A. Juss., when incubated with an aqueous solution of chloroaurate ions produces a diverse mixture of intracellular gold nanoparticles (AuNPs), especially nanotriangles (GNT) in the size range from 20 to 35 nm. These structures (GNT) are of special interest since they possess distinct plasmonic features in the visible and IR regions, which equipped them with unique physical and optical properties exploitable in vital applications such as optics, electronics, catalysis and biomedicine. The reaction process was simple and convenient to handle and was monitored using ultraviolet–visible spectroscopy (UV–vis). The morphology and crystalline nature of the GNTs were determined from transmission electron microscopy (TEM), atomic force spectroscopy (AFM) and X-ray diffraction (XRD) spectroscopy. This proposed mechanistic principal might serve as a set of design rule for the synthesis of anisotropic nanostructures with desired architecture and can be amenable for the large scale commercial production and technical applications.
At present, there is a greater need to develop safe, reliable, clean and eco-friendly methods for the preparation of nanoparticles and other high structured nanomaterials. With the rapid development of new chemical/physical methods, concern for environmental contaminations is regularly heightened as the chemical procedures involved in the synthesis of nanomaterials generates a large amount of hazardous by-products. Thus, there is an urgent need for 'green chemistry' that includes clean, nontoxic and environment-friendly methods of nanoparticle synthesis with precise control over the shape and size. In the recent years, 'green synthesis' of the nanoparticles has paid much more attention in the rapidly growing area of nanoscience and nanotechnology [1–5]. Utilization of cheap nontoxic chemicals, eco-friendly solvents and renewable materials are some of the pivotal issues that merit important concern in a green synthesis strategy for nanomaterials. In this context, biological synthesis of nanoparticles as an emerging highlight of the intersection of nanotechnology and biotechnology has received increasing attention to come up the need of environmentally benign technologies in nanomaterial synthesis, not only because it reduce the use and generation of hazardous substances to human health and environment but also in providing the facile and convenient entry to produce multiple inorganic nanoparticles . Thus, synthesis of nanomaterials using microorganisms is compatible with the green chemistry principles, resulted in a surge of interest in scientists towards biological systems for inspiration [7–9]. Many microbes are known to produce highly structured metallic nanoparticles with very similar properties to that of chemically synthesized materials, while having precise control over size, shape and monodispersity. The magnetosome or the magnetotactic bacteria synthesize the magnetic nanoparticles in nature since long back, which is a very good biosystem to learn the basic principles of biofabrication . Many prokaryotes like Pseudomonas stutzeri  and Schizosacchromyces pombe  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, monocrystalline 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. Earlier, there are many other species of Aspergillus have been reported of their potential to synthesize silver and gold nanoparticles such as Aspergillus niger , A. flavus , A. Fumigatus , A. oryzae var. Viridis . Although this endophytic microbe was earlier investigated by our group for the biofabrication of silver nanoparticles , 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 . 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.
Isolation of Endophytic Aspergillus clavatus
The host plant Azadirachta indica A. Juss. was surveyed, and samples were randomly collected from within the campus premises of Banaras Hindu University, 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 morphotaxonomic features under microscope (Nikon Eclipse E-600), one of the strains is identified as Aspergillus clavatus.
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%), glucose (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 reaction mixture was then put onto a shaker at room temperature 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 mixture and measuring the UV–vis spectra on a Hitachi dual-beam spectrophotometer (Hitachi, UV-2910) operated 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 aqueous 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 similar 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 biomass 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-Kα 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 monochromator in the diffraction beam and a high temperature attachment. For TEM analysis, the samples were prepared by placing 5 μl of gold nanoparticle suspension on a 300-mesh carbon-coated copper grid, and the solution was allowed to stand for 5 min, then excess solution 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 software on a Tecnai G-20 transmission electron microscope, a 200-kV TEM with a W-source and an ultra high-resolution pole piece with a point–point resolution of 1.9 A° (TEM, Tecnai [FEI]-12v.G-20). Surface topology was measured by atomic force microscopy (AFM) in the contact mode on a VEECO Digital Instruments multimode scanning probe microscope equipped with a Nanoscope IV controller.
Results and Discussion
In summary, we have demonstrated the shape controlled biosynthesis of gold nanotriangles using endophytic fungi Aspergillus clavatus, isolated from surface sterilized stem tissues of Azadirachta indica A. Juss. Results showed that triangular gold nanoparticles are formed along with some spherical as well as hexagonal morphology. It was also observed that the synthesis of gold nanotriangles are extracellular and showing a high aspect ratio. The study reported herein serve as a unique single-step green protocol for the generation and stabilization of nontoxic gold nanotriangles (GNT), exploitable in a myriad of diagnostic and therapeutic applications. A. clavatus induced synthesis of GNT will provide unprecedented opportunities towards the design and development of engineered 'green' gold nanotriangles that can be widely utilized in biomedical applications.
This work is a part of the PhD thesis of VCV, and was financially supported from Council of Scientific and Industrial Research (CSIR-09/013(205)/2008/EMR-I, dt.28-09-2008), New Delhi India. Authors are thankful to the Professor-in-charge, Centre of Experimental Medicine and Surgery (CEMS). Authors also extend their thanks to Dr. R. N. Kharwar, Mycopathology and Microbial Technology Laboratory, CAS in Botany, Banaras Hindu University India for his support and help to conduct some part of this work in his laboratory. Authors also extend their thanks to Prof. Dhananjai Pandey, School of Material Science and Technology, Institute of Technology, Banaras Hindu University for assistance with the XRD and AFM studies and to Dr. Madhu Yashpal scientist-in-charge, Electron Microscopy Facility, Department of Anatomy, Institute of Medical Sciences, Banaras Hindu University, India for the TEM analysis of the gold nanoparticles.
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