Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 1175–1180

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular and

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

Intracellular synthesis of silver nanoparticle by actinobacteria and its

antimicrobial activity
S.V. Otari a, R.M. Patil a, S.J. Ghosh a, N.D. Thorat a,b,⇑, S.H. Pawar a
a
Center for Interdisciplinary Research, D.Y. Patil University, Kolhapur 416 006, Maharashtra State, India
b
Samsung Biomedical Research Institute, Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 440-746, South Korea

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

 Green, cost-effective, clean method

for silver nanoparticle synthesis is
presented.
 Intracellular synthesis of
nanoparticles by actinomycetes is
demonstrated.
 Synthesis is occurring in the cell
cytoplasm, not at cell membrane is
proved.
 Bacteriostatic and bactericidal
activity of silver nanoparticle is
showed.

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

Article history: Intracellular synthesis of silver nanoparticles (AgNPs) using Rhodococcus spp. is demonstrated. The syn-
Received 6 July 2014 thesized nanoparticles were characterized by UV–Vis spectroscopy, X-ray diffraction, energy dispersive
Received in revised form 23 August 2014 spectroscopy, Fourier trans-form infrared spectroscopy, and transmission electron microscopy. Transmis-
Accepted 4 October 2014
sion electron microscopy study of microorganisms’ revealed synthesis of nanoparticle was occurring
Available online 4 November 2014
inside the cell, in the cytoplasm. AgNPs ranged from 5 to 50 nm. Formed nanoparticles were stable in
the colloidal solution due to presence of proteins on the surface. AgNPs showed excellent bactericidal
Keywords:
and bacteriostatic activity against pathogenic microorganisms.
Green synthesis
Intracellular synthesis
Ó 2014 Elsevier B.V. All rights reserved.
AgNPs
DLS
Actinobacteria

Introduction
The intense light emission properties of noble metals (gold, sil-
ver, etc.) nanoparticles have caught a lot of attention. Nobel metals
nanostructures have contributed for the great enhancement of the
⇑ Corresponding author at: Center for Interdisciplinary Research, D.Y. Patil
University, Kolhapur 416 006, Maharashtra State, India. Tel.: +91 0231 260122;
fax: +91 231 2601595.
E-mail addresses: thoratnd@yahoo.com (N.D. Thorat), pawar_s_h@yahoo.com
(S.H. Pawar).
nanobiology field, [1] which have proven to be highly versatile and
tunable materials for a range of bioapplications including biophys-
ical studies, [2], biological sensing [3], imaging [4], medical diag-
nostics [5] and cancer therapy [6]. Among various metals, silver
has been known since ancient times as effective antimicrobial
agent for the treatment of diseases, for food preservation and
water purification [7].
The recent advancements in the field of nanotechnology have
made silver nanoparticles to be widely used as a novel therapeutic
agent as antibacterial, antifungal, antiviral, anti-inflammatory and
anti-cancerous agents. Various methods chemical and physical

http://dx.doi.org/10.1016/j.saa.2014.10.003
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
1176 S.V. Otari et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 1175–1180

methods have been employed for synthesis of silver nanoparticles
which are energy and capital intensive, employ toxic chemicals,
and often yield particles in non-polar organic solutions, precluding
their biomedical applications. On the contradictory, biological syn-
theses are not only a good way to fabricate benign nanostructure
materials, but also to reduce the use or generation of hazardous
substances to human health and the environment. Thus the focus
for nanoparticles synthesis is transferring from physical and chem-
ical processes towards ‘green’ chemistry i.e. bioprocesses. In vari-
ety of biological techniques of nanoparticle synthesis, microbial
synthesis would help to circumvent many of the detrimental fea-
tures by enabling synthesis at mild pH, pressure and temperature
and at a substantially lower cost. Active metal transformation pro-
cesses require viable microbes which enzymatically catalyze the
alteration of the metal. The microorganisms probably play a role
in providing a huge number of nucleation centers and establish
conditions for obtaining highly disperse nanoparticle systems.
They entirely prevent or minimize the rate of aggregation by
immobilizing the particles, and providing a viscous medium con-
taining extracellular secreted proteins, enzymes and carbohydrates
[8]. There are several reports on use of microorganisms such as
Bacillus koriensis and Bacillus subtilis, and fungi such as Aspergillus
fumigatus and Fusarium oxysporum for the biosynthesis of AgNPs
[9] and marine alga, Sargassum wightii Greville for gold nanoparti-
cles [10].
In the present study, we show for the first time that the versa-
tile genus Rhodococcus spp. when exposed to AgNO3 ions results in
the reduction of silver ions and formation of monodispersed intra-
cellular AgNPs. Rhodococcus strains may be utilized as industrial
organisms, primarily for biotransformations and biodegradation
of many organic compounds, since they are equipped with a large
number of enzymatic activities, unique cell wall structure and suit-
able biotechnological properties [11]. Rhodococci can thus be
applied in environmental remediation and in the pharmaceutical
and chemical industries [12]. In our previous study we have dem-
onstrated that the synthesis of AgNPs can be achieved extracellu-
larly using phenol degraded broth to make the synthesis method
very cost effective and clean [13]. So synthesis of silver nanoparti-
cles with Rhodococcus spp. can be done using environmental haz-
ardous materials. The intracellular accumulation of gold
nanoparticles with a dimension of 5–15 nm by an alkalotolerant
actinomycete, Rhodococcus sp. has been demonstrated where the
available reductase on the cell wall reduced Au+3 and accumulated
as Au0 on the cell wall and on cytoplasmic membrane [14].
The synthesized nanoparticles were characterized using stan-
dard analytical techniques, viz, UV–Visible spectroscopy, Transmis-
sion Electron Microscopy (TEM), energy dispersive spectroscopy
(EDS), X-ray Diffraction (XRD), and Fourier transform infra red spec-
troscopy (FT-IR). The particle size and the stability of the as-synthe-
sized AgNPs in cell free culture broth were analyzed by using
Dynamic Light Scattering (DLS) and zeta potential respectively.
The nanoparticles were also evaluated for antimicrobial activity
against pathogenic Gram-positive and Gram-negative bacteria.
Experimental methods
Microorganism and chemicals
An actinobacteria Rhodococcus NCIM 2891 was obtained from
NCIM (National Collection of Industrial Microorganisms), National

Fig. 1. (a) Change in the color of the broth from white to brown confirming formation of AgNPs; (b) UV–Vis spectra of AgNPs synthesized by Rhodococcus sp. for 72 h where
the absorption at 405 nm goes on increasing as the time of incubation increases; (c) XRD pattern of AgNPs showing the crystalline nature of nanoparticles; (d) FT-IR of AgNPs.
The FT-IR study of AgNPs showed major peaks around 1550, 1650, 2903 and 3310 cm 1 demonstrating presence amides stretching and banding on the surface. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
 S.V. Otari et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 1175–1180 1177

Fig. 2. (a) EDS spectra of cell free extract. EDS spectrum shows the presence carbon (C), oxygen (O), silicon (Si) and silver (Ag); (b) TEM micrograph of AgNPs s, TEM
micrograph demonstrates the spherical AgNPs in the range 5–50 nm; (c) DLS of AgNPs in colloidal solution, DLS study shows the presence of AgNPs in the range of 10–50 nm
in colloidal solution; (d) Zeta potential of AgNPs in colloidal solution confirming the stability of the AgNPs in colloidal solution due to presence of biomolecules on the surface
of particle.

Chemical Laboratory (NCL), Pune, India. The culture was main-
tained on GYEME (Glucose Yeast extract Malt extract) agar con-
taining 1% glucose, 0.3% yeast extract, 0.5% peptone and 0.3%
malt extract and 1.5% agar-agar at 4 °C. Chemicals were purchased
from Lobachemie Pvt. Ltd. of GR grade and media components
were obtained from Himedia Laboratories Pvt. Ltd.
Synthesis of silver nanoparticles
Rhodococcus was aerobically grown in M9 medium containing
Sodium acetate (0.420%), NaNO3 (0.3%), K2HPO4 (0.3%), Na2HPO4
(0.6%), MgSO4 (0.05%), NaCl (0.05%) in double distilled water for
24 h at 30 °C and agitated at 130 rpm. After 24 h, the grown culture
was used as the inocula for the synthesis media which is slight
modification of the M9 media. In 500 ml Erlenmeyer flask, 95 ml
synthesis media was added with 5 ml inocula at pH 7.0 containing
1 mM AgNO3. The change in the color of the medium from white to
brown in incubation period was observed. Then microorganism
was further incubated for 24 h. For the TEM studies, some micro-
bial culture was collected centrifugally after 10 h incubation and
fixed by 2.5% glutaraldehyde.
Characterization of AgNPs
The formation of the AgNPs was monitored by UV–visible
(UV–Vis) spectroscopy of the cell supernatant by recording spectra
between wavelength 300–600 nm and simultaneously monitoring
the appearance of the characteristic peak at 400–500 nm using a
Schimadzu (Model No. UV 1800) double beam spectrophotometer.
XRD pattern of AgNPs drop coated and air- dried on the glass sub-
strate was recorded to study the structural and phase analysis by
Philips PW-3710 diffractometer using Cu Ka radiation in the 2h
range from 20° to 80°. The X-ray Diffraction (XRD) patterns were
evaluated by X’pert high score software and compared with JCPDS
Card No. 04-0783. Energy-dispersive analysis of X-ray spectroscopy
(EDX, JEOL JSM 6360) of the freeze dried cell free extract. The Fou-
rier transform infrared (FTIR) spectra of were recorded in transmit-
tance mode with Alpha ATR Bruker (Eco ATR 500–400 cm 1)
spectrum of freeze dried AgNPs. For the Transmission Electron
Microscopy (TEM) of actinobacteria, pellets of freshly harvested
Ag-loaded actinobacteria were fixed in 2.5% (w/v) aqueous glutaral-
dehyde, centrifuged, re-suspended in 1.5 ml of 0.1 M phosphate
buffer (pH-7.2) at 4 °C and post fixed in 1% Osmium tetraoxide at
4 °C in 0.1 M phosphate buffer. Samples were dehydrated using a
graded series of acetone. After two 15 min washes in acetone, cells
were embedded in fresh araldite followed by polymerization at
60 °C for 24 h. Ultrathin sections (90 nm) were cut on a Leica Ultra-
cut R microtome, and mounted on pioloform-coated Cu grids. The
sections were stained with 2% aqueous uranyl acetate for 10 min
and triple lead stain for 5 min. Also for the determination of the
morphology and size of the AgNPs in the colloidal solution, the col-
loidal solution of the AgNPs was transferred on to a carbon coated
copper grid and allowed to air dry. The grids were then scanned
using Philips CM200 model transmission electron microscopy,
operating voltage 20–200 kV with resolution 2.4 Å. DLS measure-
ments and zeta potential of AgNPs in colloidal solution were per-
formed using a NICOMPTM 380 ZIS (Santa Barbara, California,
USA) for the determination of hydrodynamic diameter (HDD).
1178 S.V. Otari et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 1175–1180
Fig. 3. TEM micrograph of the Rhodococcus sp. demonstrating that the synthesis is occurring in the cytoplasm of microorganisms and SAED pattern of AgNPs signifying
crystalline nature of nanoparticles.
Antimicrobial activity
The antibacterial activity of AgNPs was studied by both growth
curve method and agar diffusion method with different concentra-
tions of AgNPs (30, 50 and 100 lg ml 1). Pathogenic Gram positive
bacteria (Staphylococcus aureus), and Gram negative bacteria (Kleb-
siella pneumoniae, Proteus vulgaris, Enterococcus faecalis, Pseudomo-
nas aeruginosa, and Escherichia coli) were chosen for the assay. The
above strains (105–107 CFU ml 1) cells were inoculated in nutrient
broth and the growth was monitored at OD 600 nm at every hour
interval for 50 h using a Bio-Rad iMARK micro plate reader.
Results and discussions
Synthesis and characterization of AgNPs
Biological rout is an ideal way for the green chemistry approach
for the synthesis of nanoparticles. Ease in genetic manipulation,
short doubling time, the ease in the downstream processing and
easy manipulation of the environmental conditions are the major
advantages of the bacterial mediated synthesis of nanoparticles
among other biological routs [15]. The formation of AgNPs after
reduction of aqueous AgNO3 by Rhodococcus sp. was indicated by
change in the color from white to brown (Fig. 1a) after 24 h incu-
bation. The colorless media was appeared light yellow color after
incubation of 10 h. After 24 h incubation the change in the color
from colorless to brown was observed demonstrating formation
of nanoparticles (Fig. 1a) which is the typical optical property of
AgNPs [16]. The UV–Vis spectra of the suspension showed typical
surface plasmon resonance (SPR) around 405 nm (Fig. 1b) which
is characteristic surface plasmon resonance of the AgNPs. The
organism required more than 10 h for the synthesis of detectable
amount of AgNPs. The synthesis of the AgNPs continued for 72 h
as the SPR spectra continued to increase for 72 h and no further
increase was observed. The AgNPs show characteristic peaks
between 300 and 500 nm due to surface plasmon resonance
(SPR) effects. The SPR spectra suggest these AgNPs are spherical
in shape [17].
XRD pattern taken using Cu Ka target in the range 30–80° of
AgNPs is shown in Fig. 1d revealing crystalline nature of the nano-
particles. XRD pattern taken using Cu Ka target in the range 30–80°
of AgNPs is shown in Fig. 1d. The peaks match with JCPDF Card No.
89-3722 which exhibits the characteristic peaks of silver crystal-
lites observed at 2h values of 37.8°, 44.1°, 62.9° and 75.9°. The
obtained pattern is for fcc cubic crystal structure. The crystallite
size was calculated from the full-width at half-maximum (FWHM)
of the diffraction peaks using the Debye–Sherrer formula; D = 0.9k/
bcos h, where D is the mean grain size, k is the X-ray wavelength for
Cu target, b is the FWHM of diffraction peak and h is the diffraction
angle. In order to measure the size of nanoparticles accurately each
peak is Gaussian fitted and also the instrumental broadening is
subtracted using Si standard sample broadening. The size of nano-
particles from value measured for (1 1 1) plane of reflection is in the
range of 25 nm.
The FT-IR study of AgNPs showed major peaks around 1550,
1650, 2903 and 3310 cm 1 demonstrating presence amides on
the surface (Fig. 1a). The presence of the biomolecules on AgNPs
was determined using FT-IR measurement of the freeze dried cell
free extract containing AgNPs which may be responsible for the
stability (capping material) of the nanoparticles in the medium.
The biomolecules may be the peptides, proteins, carbohydrates
present in cell free extract. The amide linkages between amino acid
residues in proteins give rise to well known signatures in the infra-
red region of the electromagnetic spectrum. FTIR spectrum reveals
two bands at 1650.5 and 1550 cm 1 that corresponds to the bend-
ing vibrations of the amide I and amide II bands of the proteins
respectively; while their corresponding stretching vibrations were
seen at 3310 and 2903.6 cm 1 respectively (Fig. 2a). The protein-
nanoparticle interactions can occur either through free amine
groups or cysteine residues in proteins and via the electrostatic
attraction of negatively charged carboxylate groups in enzymes
[18]. The two bands observed around 1381 and 1038 cm 1 can
be assigned to the C–N stretching vibrations of the aromatic and
aliphatic amines, respectively [19]. These results indicated that
the carbonyl group of proteins adsorbed strongly to metals, dem-
onstrating that proteins could have also formed a layer along with
other bio-organic molecules, securing nanoparticles from aggrega-
tion and subsequently adds advantage for the stabilization of
AgNPs.
The elemental analysis was done by EDS, showed characteristic
signals of crystalline AgNPs at 3 keV (Fig. 1c) [20]. The strong peaks
of carbon (C) and oxygen (O) with silver (Ag) in EDS of AgNPs
 S.V. Otari et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 1175–1180
Fig. 4. Antimicrobial activity of AgNPs on (a) Escherichia coli, (b) Enterococcus faecalis, (c) Staphylococcus aureus, (d) Klebsiella pneumoniae, (e) Pseudomonas aeruginosa, and (f)
Proteus vulgaris with different concentration as control,
(Fig. 2a) are indication of existences of biological material on the
nanoparticles. The TEM of AgNPs showed that nanoparticles are
spherical in shape and less than 50 nm, ranged from 5 to 50 nm
(Fig. 2b). DLS study was well in agreement with TEM study show-
ing the size of nanoparticles in the rage of 10–50 nm (Fig. 2c). The
zeta potential is an indication of the stability of colloidal aqueous
dispersions, and usually particles with zeta potential
(n > ± 30 mV) are considered to be stabilized due to electrostatic
repulsion [21]. Here, the zeta potential of as-formed AgNPs in col-
loidal solution was -30 mV which shows the excellent stability of
the AgNPs in the colloidal solution (Fig. 2d).
The biosynthesis of AgNPs is possible in two ways viz enzymatic
and non-enzymatic synthesis. One of the disadvantages of the
enzymatic reduction of silver is the slow rate of the reaction. The
time required for reduction ranges has varied between 24 and
120 h. The non-enzymatic reduction is often fast, taking only a
couple of minutes. The non-enzymatic reduction of silver is based
on the chemical reduction where the reducing and stabilizing com-
pounds are produced by microorganisms or plants [22,23]. Within
5 min the supernatant of Klebsiella pneumonieae, E. coli, and Enter-
obacter cloacae turned brown, indicating the formation of nanosil-
ver. Mukherjee et al. first time suggested that enzymes might be
responsible for the reduction of silver ions using Verticillium sp.
[23]. The NADPH-dependant reductase was involved in the reduc-
tion of the AgNO3 with fungus F. oxysporum [24]. The Bacillus spp.
has been demonstrated for the intracellular synthesis of AgNPs at
the cell membrane [25]. Form the current experiments it is evident
that there is a role of enzyme in the reduction of Ag+ ions as it has
taken 10 h for the synthesis of detectable amount of AgNPs. Fig. 3 is
showing TEM micrograph of the microorganisms, showing the
nanoparticles in the cytoplasm of microorganisms and SAED pat-
tern of AgNPs. Ahmad et al. showed that alkalotolerant actinomy-
cete Rhodococcus spp. synthesized gold nanoparticles
intracellularly but the synthesis was occurred at cell membrane
1179
10 lg/ml, 30 lg/ml, 50 lg/ml.
and cell wall [13]. Blasco et al. [26] demonstrated that Rhodococcus
RB1 and Bacillus substilis are carrying NADH-dependant nitrate
reductase inside the cell not at the cell membrane [26]. So herein
intracellular synthesis of AgNPs, it evident that the NADH-depen-
dant nitrate reductase present in the cell cytoplasm of Rhodococcus
spp. plays major role in the reduction of the Ag+ ions to form
AgNPs.
Antimicrobial activity
Silver is known for its antimicrobial properties and has been
used for years in the medical field for antimicrobial applications.
The different mechanisms of the bactericidal effect of AgNPs have
been proposed from the several studies but the exact mechanism
remains to be understood. Many literatures stated that AgNPs
may attach to the surface of the cell membrane disturbing perme-
ability and respiration functions of the cell [27]. It is also possible
that AgNPs not only interact with the surface of membrane, but can
also penetrate inside the bacteria [28]. Smaller AgNPs having the
large surface area available for interaction would give more bacte-
ricidal effect than the larger AgNPs [27]. The shape of AgNPs has
been shown very essential for the effective antibacterial activity
[29]. A triangular nanoplate has predominately desirable activity
than spherical and rod-shaped AgNPs [30]. Here in this study the
bactericidal and bacteriostatic effect of AgNPs were studied by
observing the effect on the different growth phases of these path-
ogenic microorganisms using different concentrations of AgNPs
(10, 30 and 50 lg ml 1 of medium). Fig. 4 demonstrates the anti-
bacterial activity of the AgNPs. The lowest concentration i.e.
10 lg ml 1 was found most effective for K. pneumoniae where
organism showed growth after 48 h of incubation. For other organ-
isms this low concentration of AgNPs affected log phase causing
delay. S. aureus and E. faecalis attained the stationary phase earlier
than control one. So for these two organisms, 10 lg ml 1 AgNPs
1180 S.V. Otari et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 1175–1180
acted as bacteriostatic concentration. E. coli, S. aureus and Pseudo-
monas arugenosa showed some growth after 50 h incubation for
30 lg ml 1 of AgNPs. The higher concentration (50 lg ml 1) of
the AgNPs completely arrested the growth of the all organisms.
So the AgNPs synthesized using green and eco-friendly rout can
be used for the biomedical purpose.
Conclusions
The present study demonstrates the intracellular synthesis of
AgNPs by Rhodococcus spp. TEM micrographs of microorganisms
revealed that the synthesis of AgNPs was occurring in cytoplasm
of microbial cell, not at cell membrane or at cell wall of microbial
cells. The size of AgNPs was found in the range 10–30 nm charac-
terized using TEM. DLS study showed the distribution of AgNPs in
rage 30–50 nm and zeta potential showed very good stability in
colloidal solution. As-synthesized AgNPs showed excellent bacte-
riostatic and bactericidal activity against pathogenic Gram positive
and Gram negative microorganism. The approach of the synthesis
of AgNPs is much beneficial using Rhodococcus sp. than other
microorganisms and conventional chemical and physical methods
as synthesis can be done coast effectively with bioremediation.
Acknowledgments
Authors are very grateful to Board of Research in Nuclear Sci-
ences (BRNS) and Department of Science and Technology (DST)
for providing the funds to carry out the research activities. Also
authors are thankful to SAIF IIT-Bombay for TEM facility. Authors
are very grateful to Department of Microbiology, Department of
Biochemistry and Department of Biotechnology, Shivaji University,
Kolhapur for allowing the use of facilities to carry out experiments.
References
[1] M.A. El-Sayed, Acc. Chem. Res. 34 (2001) 257–264.
[2] B.M. Reinhard, S. Sheikholeslami, A. Mastroianni, A.P. Alivisatos, J. Liphardt,
Proc. Natl. Acad. Sci. USA 104 (2007) 2667–2672.
[3] R. Elghanian, J.J. Storhoff, R.C. Mucic, R.L. Letsinger, C.A. Mirkin, Science 277
(1997) 1078–1081.
[4] K. Sokolov, M. Follen, J. Aaron, I. Pavlova, A. Malpica, R. Lotan, R. Richards-
Kortum, Cancer Res. 63 (2003) 1999–2004.
[5] X. Xu, N.L. Rosi, Y. Wang, F. Huo, C.A. Mirkin, J. Am. Chem. Soc. 128 (2006)
9286–9287.
[6] L.R. Hirsch, R.J. Stafford, J.A. Bankson, S.R. Sershen, B. Rivera, R.E. Price, et al.,
Proc. Natl. Acad. Sci. USA 100 (2003) 13549–13554.
[7] S. Silver, FEMS Microbiol. Rev. 27 (2003) 341–353.
[8] Y. Sun, Y. Xia, Science 298 (2002) 2176–2179.
[9] N.I. Hulkoti, T.C. Taranath, Colloid Surf. B 121 (2014) 474–483.
[10] G. Singaravelu, J.S. Arockiamary, V.G. Kumar, K. Govindaraju, Colloid Surf. B 57
(2007) 97–101.
[11] K.S. Bell, J.C. Philp, D.W.J. Aw, N. Christofi, J. Appl. Microbiol. 85 (1998) 195–
210.
[12] R. Van Der Geize, L. Dijkhuizen, Curr. Opin. Microbiol. 7 (2004) 255–261.
[13] A. Ahmad, S. Senapati, M.I. Khan, R. Kumar, R. Ramani, V. Srinivas, M. Sastry,
Nanotechnology 14 (2003) 824–828.
[14] S.V. Otari, R.M. Patil, N.H. Nadaf, S.J. Ghosh, S.H. Pawar, Environ. Sci. Pollut. Res.
21 (2014) 1503–1513.
[15] M. Sastry, A. Ahmad, M.I. Khan, R. Kumar, Curr. Sci. 85 (2003) 162–170.
[16] P. Mulvaney, Langmuir 12 (1996) 788–800.
[17] K.G. Stamplecoskie, J.C. Scaiano, J. Am. Chem. Soc. 132 (2010) 1825–1827.
[18] A. Gole, C. Dash, V. Ramakrishnan, S.R. Sainkar, A.B. Mandale, M. Rao, M. Sastry,
Langmuir 17 (2001) 1674–1679.
[19] N. Vigneshwaran, A.A. Kathe, P.V. Varadarajan, R.P. Nachane, R.H.
Balasubramanya, Langmuir 23 (2007) 7113–7117.
[20] P. Magudapathy, P. Gangopadhyay, B.K. Panigrahi, K.G.M. Nair, S. Dhara, Phys.
B Condens. Matter. 299 (2001) 142–146.
[21] M. Instruments, Zetasizer Nano Serles Tech. Note. MRK654-01. 2 (2011) 1–6.
[22] V.K. Sharma, R.A. Yngard, Y. Lin, Adv. Colloid Interface Sci. 145 (2009) 83–96.
[23] P. Mukherjee, A. Ahmad, D. Mandal, S. Senapati, S.R. Sainkar, M.I. Khan, et al.,
Nano Lett. 1 (2001) 515–519.
[24] A. Ahmad, P. Mukherjee, S. Senapati, D. Mandal, M.I. Khan, R. Kumar, M. Sastry,
Colloids Surf. B 28 (2003) 313–318.
[25] N. Pugazhenthiran, S. Anandan, G. Kathiravan, N.K. Udaya Prakash, S. Crawford,
M. Ashokkumar, J. Nanoparticle Res. 11 (2009) 1811–1815.
[26] R. Blasco, M. Martínez-Luque, M.P. Madrid, F. Castillo, C. Moreno-Vivián, Arch.
Microbiol. 175 (2001) 435–440.
[27] L. Kvítek, A. Panáček, J. Soukupová, M. Kolář, R. Večeřová, R. Prucek, M.
Holecová, R. Zbořil, J. Phys. Chem. C 112 (2008) 5825–5834.
[28] J.R. Morones, J.L. Elechiguerra, A. Camacho, K. Holt, J.B. Kouri, J.T. Ramírez,
et al., Nanotechnology 16 (2005) 2346–2353.
[29] S. Pal, Y.K. Tak, J.M. Song, Appl. Environ. Microbiol. 73 (2007) 1712–1720.
[30] B. Wiley, Y. Sun, B. Mayers, Y. Xia, Chem. – A Eur. J. 11 (2005) 454–463.