Mycosynthesis of selenium nanoparticles J. Sarkar, P. Dey, S. Saha, K. Acharya Molecular and Applied Mycology and Plant Pathology Laboratory, Department of Botany, University of Calcutta, Kolkata 700019, India E-mail:
[email protected] Published in Micro & Nano Letters; Received on 28th April 2011; Revised on 10th June 2011
The culture filtrate of the fungus, Alternaria alternata, was used for the bio-reduction of sodium selenate to produce selenium nanoparticles. Change in colour to dark red of the reaction mixture signifies the development of nano-a-selenium. Dynamic light scattering experiments, atomic force microscopy, scanning and transmission electron microscopic images explained the formation of monodisperse spherical a-selenium nanoparticles in the range of 30 –150 nm. X-ray diffraction spectrum of the nano-Se exhibited a broad peak at the 2u angles of 15 –358, signifying its amorphous nature. Energy-dispersive X-ray study revealed the presence of selenium in the nanoparticles. Fourier transform infrared spectroscopy confirmed the presence of a protein shell outside the nanoparticles, which in turn support their stabilisation. A novel method has been presented here to synthesise nano-selenium in which the fungal culture filtrate was used and up-to-date literature survey showed that this is the first report on mycosynthesis of selenium nanoparticles.
1. Introduction: Nanotechnology is considered to be the next industrial revolution and is believed to have an enormous impact on society, economy and life in general, at present as well as in future. This area has had tremendous impact on varied fields such as energy, chemical, medicine, information technology, electronics, environment application, security, space, drug and gene delivery, biotechnology etc. The synthesis of nanoparticles of different chemical compositions, sizes and controlled monodispersity is an important area of research in nanotechnology. Nanoparticles production has traditionally been through chemical and physical methods. However these routes for synthesis of particles/crystallites require tedious and environmentally challenging techniques. These methods invariably involve toxic chemicals and radiations [1]. Selenium nanoparticle is a novel selenium species with unique biological activities and low toxicity [2]. It has been confirmed that a-Se can improve the activity of the seleno-enzyme, glutathione peroxidase, and prevent free radicals [3]. It has also been found to have important applications in rectifiers, solar cells, photographic exposure meters [4], xerography [5] and glass industry [6]. In the past few years, there have been several reports on the synthesis of selenium nanostructures by different ways. The selenium nanoparticles occur in its allotropes: the principal one is a trigonal phase consisting of helical chains and the less stable one is a monoclinic phase, which consists of Se8 rings [7]. Monoclinic selenium (m-Se) comes in three forms: a, b and g, which differ only in the way the rings are packed [8]. Amorphous selenium is composed of a mixture of disordered chains. Trigonal selenium is black in colour whereas amorphous and monoclinic selenium are orange red or brick red in colour [9]. Previous attempts have been made to produce chemically synthesised selenium nanoparticles either in trigonal [10] or in amorphous [11] condition. Selenium nanoparticles could be electrochemically synthesised by electrolysis of an aqueous solution of selenium dioxide [12]. Biogenic synthesis is an interesting and exciting way to prepare various inorganic like selenium nanoparticles. The method is a clean, non-toxic and environment-friendly (‘green chemistry’) procedure for the synthesis and assembly of nanoparticles, and most significantly, the synthesised materials are biologically compatible [13–15]. Biologically selenium nanoparticles were synthesised by micro-organisms like bacteria either in their cytoplasm [16] or in their periplasmic space [17]. Recent reports also suggested that some plants [18] are also capable to produce selenium nanoparticles.
Micro & Nano Letters, 2011, Vol. 6, Iss. 8, pp. 599 –602 doi: 10.1049/mnl.2011.0227
Working towards the goal to enlarge the scope of bio-organisms in the biosynthesis of nanomaterials, we explore the potential of a phyto-pathogenic fungus, Alternaria alternata, culture filtrate for the first time to reduce sodium selenate to selenium nanoparticles. 2. Materials and methods 2.1. Isolation of the pathogen and culture maintenance: The pathogen, A. alternata isolated previously by Maiti et al. [19], was grown aerobically in 50 ml of fully autoclaved potato-dextrose broth (HiMedia Laboratories Pvt. Ltd., Mumbai, India) in Erlenmeyer flasks having the capacity of 250 ml and kept for 12 days at 308C temperature. After the incubation period, the media was filtered with Whatman filter paper no. 1. A volume of 100 ml of that fungal culture filtrate (FCF) was taken in a sterilised Erlenmeyer flask, which was further used for the reduction of the sodium selenate aqueous solution. 2.2. Synthesis of selenium nanoparticles: The chemical sodium selenate (Na2SeO4) was purchased from Sigma (St Louis, MO, USA). A volume of 100 ml of FCF was taken in each different Erlenmeyer flask and mixed with sodium selenate solution (1 mM final concentration). The FCF containing flask was agitated for 24 h at room temperature. Simultaneously, two positive controls of the FCF and uninoculated culture media, respectively, and a negative control of only sodium selenate solution were maintained under same conditions. The selenium nanoparticles were separated out by centrifugation (at 12 000 g for 10 min), and the settled nanoparticles were washed in deionised water (three times). The purified selenium nanoparticles were resuspended in deionised water and ultrasonicated by Piezo-u-sonic ultrasonic cleaner (Pus-60w). 2.3. Size measurement by dynamic light scattering experiment: Particle size was measured by laser diffractometry using a Nano Size Particle Analyser (Zen 1600 Malvern, USA) in the range between 0.6 nm and 6.0 mm, under the following conditions: particle refractive index 1.590, particle absorption coefficient 0.01, water refractive index 1.33 and temperature 258C. 2.4. X-ray diffraction analysis: After bio-reduction, the liquid reaction mixture was dried at 458C in a vacuum drying oven. The vacuum-dried mixture was then collected and used for powder X-ray diffraction (XRD) analysis. The spectra were recorded in a PW 3040/60 PANalytical X-ray diffractometer (Cu Ka radiation,
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l 1.54443) running at 45 kV and 30 mA. The diffracted intensities were recorded from 2 to 908 2u angles. 2.5. Fourier transform infrared spectroscopic analysis: For Fourier transform infrared spectroscopic (FTIR) analysis, the vacuumdried selenium nanoparticles were mixed with potassium bromide at a ratio of 1:100 and the spectra were recorded with a Shimadzu 8400S FTIR using a diffuse reflectance accessory under normal pH conditions (pH 7.2). The scanning data were obtained from the average of 50 scans in the range between 4000 and 400 cm21. 2.6. Atomic force microscopic observation of selenium nanoparticles: The size and surface topography of the drop-coated film of the selenium nanoparticles was investigated with atomic force microscope (AFM) (Nanoscope (R) 111a Veeco multimode, USA) and high-resolution surface images were produced. In AFM characterisation, the tapping mode with RTESP tip was used. The resonance frequency was 287.64 and the scanning range was 1.25 mm.
Figure 1 Histogram of particle size distribution as obtained from light scattering of the selenium nanoparticles produced by A. alternata. Inset represents the appearance of red colour due to the addition of A. alternata FCF to 1024 M aqueous Na2SeO4 solution after 24 h incubation period
2.7. Scanning electron microscopy–energy-dispersive X-ray observation of selenium nanoparticles: Scanning electron microscopy (SEM) images were taken using a Hitachi S 3400N instrument (Japan). Samples were filtered and dried before measurements. The material was gold coated using Ion Sputter Coater Hitachi E1010. Energy-dispersive X-ray (EDX) analysis was carried out by the same instrument and employed to know the elemental compositions of the particles. 2.8. Zeta potential measurement: Charge distribution (zeta potential) was analysed using Beckman Coulter DelsaTM Nano Particle Analyser (USA) by illuminating the solution of selenium particles with He–Ne laser (658 nm) in a sample cell. 2.9. Transmission electron microscopic observation of selenium nanoparticles: Transmission electron microscopic (TEM) samples of the aqueous suspension of selenium nanoparticles were prepared by placing a drop of the suspension on carbon-coated copper grids and allowing the water to evaporate. The micrographs were obtained by Tecnai G2 spirit Biotwin (FP 5018/40) TEM, operated at 80 kV accelerating voltage. 3. Results and discussion 3.1. Characterisation and identification: The reaction of SeO−2 4 ions with FCF occurred rapidly and stably in the solution at room temperature and the reaction solution displayed a time-dependent colour change. At the beginning of the reaction, the solution was light yellow and became reddish yellow after 1 h of incubation at room temperature; the reddish yellow colour changed to dark red colour (Fig. 1 inset) gradually at 24 h and then the red colour did not change with increasing incubation time. The appearance of the red colour indicated the occurrence of the reaction and the formation of a-Se. In case of positive control (only FCF or uninoculated culture media) and negative control (sodium selenate solution alone), no change in colour was observed. 3.2. Size measurement by DLS: Particle size was determined by dynamic light scattering (DLS) measurement. Laser diffraction studies revealed that particle size obtained from highly dispersed mixture was in the range of 30 –150 nm (Fig. 1). 3.3. EDX and XRD characterisation: An elemental composition analysis employing SEM –EDX showed the presence of a strong signal from Se atoms (81.54%) (Fig. 2a). This analysis indicated that the nanostructures were composed solely of selenium. Other EDX peaks such as Si, P, Cl and Ca peaks were also found, 600 & The Institution of Engineering and Technology 2011
Figure 2 Elemental composition analysis employing SEM–EDX a Energy-dispersive X-ray spectrum b Typical X-ray diffraction pattern of selenium nanoparticles
suggesting that they were mixed precipitates of the FCF and selenium salt. XRD further confirmed the generation of Se0. XRD measurements can describe an accurate analysis for the formation of new compounds and its phase. In this analysis, there was no sharp Bragg’s reflection except for a broad peak at the 2u angles of 15 –358, indicating that the reaction product was not a crystalline phase (Fig. 2b). This analysis revealed that the red nano-selenium was amorphous in nature [18]. 3.4. FTIR characterisation of significant functional groups: FTIR measurements were carried out to identify the possible interactions between selenium salt with culture filtrate under normal pH conditions (pH 7.2). Typical FTIR absorption spectra of fungal biomass before (Fig. 3a) and after (Fig. 3b) bioreduction of sodium selenate were shown in the given figure. Both of them showed the presence of bands at around 675, 1050, 1250, 1396, 1460, 1550, 1650, 2890, 2950 and 3340 cm21. The bands at around 1650, 1550 and 1250 indicated the presence of amide I, II and III of proteins, respectively [20]. The strong broad peak at 3000 –3500 cm21 was characteristic of the N– H stretching Micro & Nano Letters, 2011, Vol. 6, Iss. 8, pp. 599 –602 doi: 10.1049/mnl.2011.0227
Figure 5 Tapping mode AFM height images of 2D graphics showing the clean symmetrical and spherical shaped selenium nanoparticles
come together [23]. The slightly negative charge on Se0 particles is probably resulting in the high stability of the selenium nanoparticles without forming aggregates and these particles do not transform to black amorphous form when kept for prolonged period of time of more than a month.
Figure 3 FTIR absorption spectra of fungal biomass a Before bio-reduction of sodium selenate b After bio-reduction of sodium selenate
vibration [21]. Infrared active modes attributed to side-chain vibrations include symmetric and anti-symmetric modes of C –H stretching at around 2950 and 2890 cm21 corresponding to aliphatic and aromatic modes, respectively [20]. The bands at 1454 cm21 confirmed the symmetrical stretching vibrations of –COO [22]. The two bands at 1396 and 1050 cm21 showed a resemblance to the C –N stretching vibrations of aromatic and aliphatic amines, respectively [20]. The band at around 675 cm21 might be the plane bending vibration of N – H groups of protein. With the overall observations, it can be concluded that the proteins might have formed a coating over the nano-selenium, which in turn supports their stabilisation. Owing to this, the nano-Se persisted for several months in liquid suspension.
3.6. AFM images of selenium nanoparticles: Results obtained from the AFM study represent a clear concept regarding the shape as shown in Fig. 5. Majority of the particles were symmetrical, spherical in shape and well distributed without aggregation. The size of the selenium nanoparticles was found to be similar with the data collected from the DLS study and the variation in size of nanoparticles was commonly found during biological synthesis. 3.7. SEM and TEM images of selenium nanoparticles: SEM images (Fig. 6a) and TEM images (Fig. 6b) recorded different sizes of selenium nanoparticles that arose from the bio-reduction of sodium selenate by FCF at room temperature for 24 h. These observations revealed that homogeneous and dispersed spherical structures of the a-selenium nanoparticles were formed in the reaction solution. The diameters of these selenium nanoparticles were measured and the size was in the range of 30–150 nm. The average diameter of these selenium nanoparticles was of 90 + 10 nm. The formation of Se0 by different bacteria has been known for sometimes. The biogenesis of abundant Se0 nanospheres on the exterior of the cell envelope of a gram-positive rod, Bacillus
3.5. Zeta potential value of selenium nanoparticles: The zeta potential measurements indicate negative charge (213.48 mV) on the selenium nanoparticles (Fig. 4). If all the particles in suspension have a negative or positive zeta potential, then they will tend to repel each other and there is little tendency for the particles to
Figure 4 Zeta potential measurement of the selenium nanoparticles
Micro & Nano Letters, 2011, Vol. 6, Iss. 8, pp. 599 –602 doi: 10.1049/mnl.2011.0227
Figure 6 SEM and TEM images of selenium nanoparticles a Scanning electron micrographs of selenium nanoparticles b Transmission electron micrographs of selenium nanoparticles
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selenitireducens was demonstrated by Oremland et al. [16]. They have also reported similar external and internal accumulation of selenium nanospheres in selenite-grown cells of the gram-negative species Selenihalanaerobacter shriftii and Sulfurospirillum barnesii [16]. Dungan et al. [24] reported the formation of Se0 after 28 h during studies with Stenotrophomonas maltophilia in the presence of selenite. Reports of such formation are noted from Enterobacter cloacae [25] and Pseudomonas aeruginosa [26]. However, the use of fungus for the production of Se nanoparticles has not been examined previously. 4. Conclusion: Biomimetic synthesis of nanoparticles has opened its doors to a world of nanoparticles with easy preparation protocols, less toxicity and a wide range of applications according to their size and shape. To date, extracellular synthesis of stable and uniform selenium nanoparticles by a fungus, A. alternata has not been reported and thus is reported here for the first time. AFM, SEM and TEM analysis confirmed the uniform distribution of nanoparticles. Characterisation by other techniques confirmed the presence of a protein matrix as a stabilising agent. This methodology could be used for synthesising a number of other metallic nanoparticles with good size and shape morphology. 5. Acknowledgments: The authors thank Dr Aparna Laskar for carrying out TEM images and measurements using TEM facility of the Indian Institute of Chemical Biology, Kolkata, India. 6
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