Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 114 (2013) 144147

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Spectrochimica Acta Part A: Molecular and

Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa

Biological synthesis of silver nanoparticles using the fungus Humicola sp.

and evaluation of their cytoxicity using normal and cancer cell lines
Asad Syed a, Supriya Saraswati b, Gopal C Kundu b, Absar Ahmad a,
a
Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India
b
National Centre for Cell Science, Pune 411 007, India

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 First report on biosynthesis of silver The thermophilic fungus Humicola sp. was successfully employed rst time in the laboratory for the bio-
nanoparticles using Humicola sp. synthesis of extracellular protein capped, water dispersible and well dispersed silver nanoparticles.
 Non toxic, highly stable, protein
capped, cheap synthesis of silver
nanoparticles.
 Nanoparticles are non toxic to normal
and cancer cells up to 50 lg/ml
concentrations.

a r t i c l e i n f o a b s t r a c t

Article history: Nanoscience is a new born science of the modern era and taps into the potential of particles at nanoscale.
Received 11 October 2012 Bulk materials reduced to nanoscale dimensions thus obtain unique properties such as electronic, optical,
Received in revised form 16 February 2013 magnetic and chemical. As far as synthesis of nanoparticles is concerned, biological synthesis has recently
Accepted 13 May 2013
sparked a great interest as compared to other available chemical and physical methods on account of its
Available online 25 May 2013
eco-friendliness and cost-effectiveness. Here we report, for the rst time, the biosynthesis of silver nano-
particles by the thermophilic fungus Humicola sp. The fungus when reacted with Ag+ ions reduces the
Keywords:
precursor solution and leads to the formation of extracellular nanoparticles as monitored by ultra violet
Nanoparticles
Silver
visible spectroscopy (UVVis). The morphology of nanoparticles is found to be spherical with good dis-
Fungus persity as revealed by transmission electron microscopy (TEM). Cell viability assays were carried out
Humicola sp. to assess the cytotoxicity of silver nanoparticles on NIH3T3 mouse embryonic broblast cell line and
Cell viability MDA-MB-231 human breast carcinoma cell line.
2013 Elsevier B.V. All rights reserved.

Introduction
Nanoscale materials exhibit novel properties different from
their bulk counterparts. The change in chemical, physical, optical,
electronic and magnetic properties signicantly depends upon
Corresponding author. Tel.: +91 020 25902226; fax: +91 020 25902648.
E-mail address: a.ahmad@ncl.res.in (A. Ahmad).
the particle size and shape. The creative use of microorganisms
for the biosynthesis of nanomaterials over other available methods
has sparked great interest and impact upon their potential to be
explored as an inexhaustible source of fundamental nanoparticles
which can be used for various drug delivery applications. Some
well known examples of microbes which synthesize nanoscale
materials are the magnetotactic bacteria (for magnetite nanoparti-
cles) [1], diatoms (siliceous materials) [2], S-layer bacteria

1386-1425/$ - see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.05.030
 A. Syed et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 114 (2013) 144147
(gypsum and calcium carbonate layers) [3], etc. To replicate these
nanostructures in laboratory, bacteria [4] actinomycetes [5], fungi
[6] and even higher plants [7] have been employed for the produc-
tion of nanomaterials.
Among bacteria, Pseudomonas stutzeri AG256 from silver mine
[8] and Lactobacillus found in buttermilk [9] have been found to
produce silver nanoparticles. Besides bacteria-mediated biosyn-
thesis, eukaryotic organisms (fungi) based approach is also a good
choice for the production of nanomaterials as they secrete more
enzymes, are easier to handle and grow on simple media. Recently,
a novel bio-inspired process has been reported for the intra and ex-
tra-cellular synthesis of nanoparticles using the fungus Verticillium
sp. [6] and Fusarium oxysporum [10] respectively. Apart from
microbial systems, biosynthesis of nanoparticles is also possible
using plants such as Alfalfa [11], Geranium (Pelargonium graveo-
lens) [12], lemongrass plant (Cymbopogon exuosus) [13] extracts,
Azadirachta indica [14], Emblica ofcinalis [15], Aloe vera [16] and
Cinnamomum camphora plant extracts [17]. In addition to the
above mentioned routes for nanomaterials synthesis, enzymes
puried from fungus were also successfully employed for the
in vitro synthesis of silver nanoparticles [18].
Many microbes, plants and their extracts are being regularly
investigated for the biosynthesis of nanomaterials. Bio-based ap-
proaches have been successful in synthesizing nanomaterials of
different chemical composition, size and shape alongwith a signif-
icant improvement in their dispersity. In the current manuscript,
we report for the rst time, the extracellular biosynthesis of pro-
tein capped, highly stable, water soluble, non toxic and highly dis-
persed silver nanoparticles using the thermophilic fungus
Humicola sp. alongwith the evaluation of its cytotoxicity. More-
over, the nanoparticles synthesized by our method are non-toxic
to normal and cancer cells up to 50 lg/ml concentrations unlike
the nanoparticles which were synthesized by various other routes
in the past and caused toxicity to the cells [1921].
Experimental details
Materials
Silver nitrate (AgNO3) was obtained from Sigma Aldrich. Malt
extract, yeast extract, glucose and peptone were obtained from
HiMedia and used as-received.
Fungal growth and maintenance
The fungus was maintained on MGYP [malt extract (0.3%), glu-
cose (1%), yeast extract (0.3%), and peptone (0.5%)] agar slants.
Stock cultures were maintained by sub-culturing at monthly inter-
vals. After growing the fungus at pH 9 and 50 C for 4 days, the
slants were preserved at 15 C. From an actively growing stock cul-
ture, subcultures were made on fresh slants and after 4 days of
incubation at pH 9 and 50 C, the same were used as the starting
material for biosynthesis of silver nanoparticles.
Extracellular biosynthesis of silver nanoparticles by Humicola sp.
For the biosynthesis of silver nanoparticles, the fungus Humicola
sp. was grown in 250 ml Erlenmeyer asks containing 100 ml of
MGYP [malt extract (0.3%), glucose (1%), yeast extract (0.3%), and
peptone (0.5%)] medium. Sterile 10% sodium carbonate was used
to adjust the pH of the medium to 9. After the pH of the medium
was adjusted, the culture was grown with continuous shaking on
a rotary shaker (200 rpm) at 50 C for 96 h. After 96 h of fermenta-
tion, mycelia were separated from the culture broth by centrifuga-
tion (5000 rpm) at 20 C for 20 min and then the mycelia were
145
washed thrice with sterile distilled water under sterile conditions.
The harvested mycelial mass (20 g of wet mycelia) was then resus-
pended in 100 ml (1 mM AgNO3 from Sigma Aldrich) solutions in
250 ml Erlenmeyer asks and the same was put onto a shaker at
50 C (200 rpm). The reaction was carried out for a period of 96 h
and fungal biomass was separated by lter-paper to collect bio-
mass and ltrate in sterile conditions. Periodically, aliquots of the
reaction solution were removed and subjected to UVVis spectros-
copy to check the formation of nanoparticles extracellularly.
Characterization of biosynthesized silver nanoparticles
The UVVis spectroscopy measurements were performed on a
Shimadzu dual-beam spectrophotometer (model UV-1601 PC)
operating at a resolution of 1 nm. The biosynthesized silver nano-
particles solution was drop cast on a carbon coated copper grid for
Transmission Electron Microscopy (TEM) and analyzed using JEOL
1200EX instrument operated at a voltage of 80 kV. The Selected
Area Electron Diffraction (SAED) analysis was carried out on the
same grid. HR-TEM images were scanned on FEI Technai G2 system
operating at an accelerating voltage of 300 kV at room tempera-
ture. The X-ray Diffraction (XRD) patterns were obtained on a Phi-
lips PW 1830 instrument operating at 40 kV and at a current of
30 mA with Cu Ka radiation (k = 1.5404 ). X-ray photoelectron
spectra were recorded on VG MicroTech ESCA 3000 instrument.
FTIR spectra of metal nanoparticle solution was recorded on Perkin
Elmer spectrum one B in diffuse reectance (DRS) mode at a reso-
lution of 2 cm 1. Energy Dispersive Analysis of X-ray (EDAX) anal-
ysis was carried out on a Leica Stereoscan-440 SEM equipped with
a Phoenix EDAX attachment. EDAX spectra were recorded in the
spot-prole mode by focusing the electron beam onto a region
on the surface coated with nanoparticles.
Cell viability assay
The cytotoxicity of silver nanoparticles was assessed by cell via-
bility assay [MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide] carried on NIH3T3 mouse embryonic
broblast cell line and MDA-MB-231 human breast carcinoma cell
line. In brief, the cells (2  104 for NIH3T3 and 1  105 for MDA-
MB-231) grown in 96-well plates were treated with varying con-
centrations of nanoparticles (501000 lg/ml) for 24 h at 37 C.
The cells were further incubated with MTT (0.5 mg/ml) at 37 C
for 3 h followed by addition of 200 ll of isopropanol. The color
intensity was measured at 570 nm using an enzyme linked immu-
nosorbent assay (ELISA) reader. The experiments were performed
in triplicates. The cell viability was plotted as percent of control
[22].
Results and discussion
Our group has already synthesized a range of inorganic nanom-
aterials of different sizes, shapes and chemical compositions using
microbes and plant extracts [5,6,1214,16]. However, the unique-
ness about the present work using Humicola sp. is that we have
achieved superior control over the size of these nanoparticles,
focusing upon them to be in the size range of 525 nm, so that
these inorganic particles when employed in biomedical applica-
tions may not block the glomerulus of the kidneys and may easily
pass through urine within a short period of time. This is the rst
report of its kind where silver nanoparticles have been synthesized
using a biological route employing the thermophilic fungus Humi-
cola sp.
Fig. 1a shows two conical asks with the fungal biomass before
(1) and after (2) exposure to 1 mM AgNO3 solution at temperature
146 A. Syed et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 114 (2013) 144147

Fig. 1. (a) Conical asks with fungus (Humicola sp.) before exposure (1) and after (2) exposure to AgNO3 ions. (b) UVVis spectrum for the silver nanoparticles. Inset shows
ltrate of the control ask (1) and ltrate of treated ask (2).

Fig. 2. (a) Representative TEM micrograph of silver nanoparticles (inset shows particle size distribution histogram). (b) HR-TEM micrograph (inset shows Selected Area
Electron Diffraction pattern), (c) XRD pattern and (d) XPS spectrum of the silver nanoparticles.

50 C and pH 9 for 96 h under shaking condition. Formation of sil-
ver nanoparticles is clearly demonstrated by the change in color
from yellow to brown. This is because of the surface plasmon
vibrations [23]. After ltration, it was observed that the aqueous
solution contained the silver nanoparticles, characterized by an in-
tense brown color. This demonstrates that the reduction of the Ag+
ions takes place extracellularly (inset in Fig. 1b).
Bio-reduction of the aqueous Ag+ ions after exposure to the fun-
gal biomass was monitored by UVVis spectroscopy (Fig. 1b). The
surface plasmon band was found to be centered at 415 nm which
corresponds to the silver nanoparticles [10]. These silver nanopar-
tices are stabilized by the secreted protein in the reaction mixture
which appears at around 270 nm in the UV spectrum which is
characteristic for proteins.
A representative TEM micrograph (Fig. 2a) shows very well dis-
persed nanoparticles. The morphology is predominantly spherical
in size ranging from 525 nm as seen in particle size histogram (in-
set in Fig. 2a). HR-TEM micrograph (Fig. 2b)) shows the inteplanar
distance which matches with (1 1 1) plane of the silver nanoparti-
cles (inset shows the SAED pattern). The Scherer ring pattern char-
acteristic of face centered cubic (fcc) silver is clearly observed,
showing that the structures seen in TEM images are nanocrystal-
line in nature. In XRD pattern (Fig. 2c), the presence of Braggs
reections arises due to (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes
and agrees well with those reported for fcc silver. The XRD pattern
clearly shows the crystalline nature of the silver nanoparticles
formed by the fungus Humicola sp. [7]. X-ray photoelectron spec-
trum represents the decomposed Ag3d into Ag 3d5/2 and 3d3/2 with
binding energies (BE) at 367.8 eV and 374.2 eV respectively
(Fig. 2d) and are assigned to the metallic Ag. It clearly indicates
that all the silver ions are reduced by the fungus [18].
The fungus secretes proteins in the reaction mixture which sta-
bililze these nanoparticles and prevent their agglomeration. FTIR
analysis was carried out to further conrm the presence of proteins
and shows two bands at around 1644 and 1523 cm 1 (Fig. 3a);
these bands are assigned to amide I and amide II of the proteins
 A. Syed et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 114 (2013) 144147 147

Fig. 3. (a) FTIR spectrum, (b) EDAX spectrum and (c) Cell viability assay of silver nanoparticles against NIH3T3 mouse embryonic broblast cell line and MDA-MB-231 human
breast carcinoma cell line. The data is represented in the form of a bar graph and plotted using means + S.E. of triplicate determinations. The values were analyzed by
Students t-test (p < 0.005). Statistical analysis, P values for signicantly different means, P < 0.05 and P > 0.05 vs control.

and are due to AC@O and ANAH stretch vibrations present in the Department of Biotechnology, Govt. of India (New Delhi) for the
amide linkages of the proteins respectively. EDAX spectrum Tata Innovation Fellowship award and nancial support through
(Fig. 3b) represents the signal from Ag, together with C, O and Si. NWP0035 CSIR, New Delhi. The authors thank the Centre for Mate-
Signals appear from C and O due to the X-ray emission from the rials Characterization (CMC) N.C.L. Pune for assistance regarding
biomolecules (likely to be of proteins) and Si signal due to the glass TEM measurements.
substrate used in analysis.
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A.S. thanks the Council of Scientic and Industrial Research

(CSIR), New Delhi for Senior Research Fellowship. A.A thanks the