Colloids and Surfaces B: Biointerfaces 85 (2011) 360–365

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Plant extract mediated synthesis of silver and gold nanoparticles and its
antibacterial activity against clinically isolated pathogens
D. MubarakAli a , N. Thajuddin a,∗ , K. Jeganathan b , M. Gunasekaran c
a
Department of Microbiology, School of Life Sciences, Bharathidasan University, Palkaliperur, Tiruchirappalli 620 024, India
b
Centre for Nanoscience and Nanotechnology, Department of Physics, Bharathidasan University, Tiruchirappalli 620 024, India
c
Department of Biology, Fisk University, Nashville, TN 37208, USA

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

Article history: Biosynthesis of nanoparticles is under exploration is due to wide biomedical applications and research
Received 2 February 2011 interest in nanotechnology. Bioreduction of silver nitrate (AgNO3 ) and chloroauric acid (HAuCl4 ) for the
Received in revised form 9 March 2011 synthesis of silver and gold nanoparticles respectively with the plant extract, Mentha piperita (Lamiaceae).
Accepted 9 March 2011
The plant extract is mixed with AgNO3 and HAuCl2 , incubated and studied synthesis of nanoparticles using
Available online 17 March 2011
UV–Vis spectroscopy. The nanoparticles were characterized by FTIR, SEM equipped with EDS. The silver
nanoparticles synthesized were generally found to be spherical in shape with 90 nm, whereas the synthe-
Keywords:
sized gold nanoparticles were found to be 150 nm. The results showed that the leaf extract of menthol is
Silver and gold nanoparticles
Plant extracts
very good bioreductant for the synthesis of silver and gold nanoparticles and synthesized nanoparticles
FTIR active against clinically isolated human pathogens, Staphylococcus aureus and Escherichia coli.
SEM Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.
Biosynthesis
Menthol
Antibacterial

1. Introduction
Nanotechnology provides the tools and technology platform for
the investigation and transformation of biological systems, and
biology offers inspiration models and bio-assembled components
to nanotechnology. Nanobiotechnology is defined as a filed that
applies the nanoscale principle and techniques to understand and
transform bio systems (living and non-living) and which uses bio-
logical principles and materials to create new devices and systems
integrated from the nanoscale [1].
Key advances have been made in the ability to make measure-
ments at the sub-cellular level and in understanding the cell as
highly organized, self-repairing, self-replicating, information-rich
molecular machines [2,3]. Smalley, classified nanotechnologies into
wet and dry nanotechnology, the first one describes the living
bio systems and the second one deals with man-made objects at
nanoscale structures [4].
A number of living organisms are already well-known to elab-
orate nanostructured composites such as cyanobacteria, bacteria,
fungi, actinomycetes, biomolecules and various plant materi-
als such as Cinnamomum camphora [5], Medicago sativa [6–8],
Pelargonium graveolens [9], Avena sativa [10], Azardirachta indica
∗ Corresponding author. Tel.: +91 431 2407082; fax: +91 431 2407045.
E-mail address: thajuddin@gmail.com (N. Thajuddin).
[11], Tamarindus india [12], Emblica offcinalis [13], Aloe vera [14],
Coriandrum sativum [15], Carica papaya [16]. Parthenium hys-
terophorus [17], Tritium vulgare [18], Acanthella elongata [19],
Sesuvivm potulacastrum [20] and gold nanoparticles also synthe-
sized by biomolecules like honey [21] (Table 1). Gold nanoparticles
of different sizes, ranging from 1 nm to 8 ␮m and shapes includ-
ing spherical, octahedral, sub-octahedral, decahedral multiple
twinned, icosahedral multiple twinned, irregular shape, nano-
triangles and nanoprisms, tetra hedral, hexagonal platelets and
nanorods [22]. Rapid and green synthesis methods using biological
extracts have shown a great potential in nanoparticle synthe-
sis, however understanding the mechanism of involvement of
biomolecules is lacking [19]. Nanostructured materials showed
many aspects of interesting characteristics, i.e., optical, catalytic,
that greatly depends on the size and shape of nanoparticles as an
effect of quantum confinement of electrons. Metal nanoparticles
are extensively used in many electrochemical, electroanalytical and
bioelectrochemical applications owing to their extraordinary elec-
trocatalytic activity. Although metal is a poor catalyst in bulk form,
nanometer-sized particles can exhibit excellent catalytic activity
due to their relative high surface area-to-volume ratio and their
interface-dominated properties, which significantly differ from
those of the bulk material [23].
The gold nanoparticles are used for the development of
biosensors, DNA labeling and vapor sensing [24–26] and silver
nanoparticles also applied to the determination of ct-DNA and

0927-7765/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2011.03.009
 D. MubarakAli et al. / Colloids and Surfaces B: Biointerfaces 85 (2011) 360–365 361

Table 1
Uses of various plant materials in the biosynthesis of nanoparticles and its size range.

S. no. Plant materials used Nanoparticle produced Size References

1 Cinnamomum camphora Ag and Au 55–80 nm [5]

2 Medicago sativa Ag 2–3 nm [6]
3 Medicago sativa Ti/Ni bimetallic 1–4 nm [7]
4 Medicago sativa Ag 86–108 nm [8]
5 Pelargonium graveolens Ag 16–40 nm [9]
6 Avena sativa Au 5–20 nm (at pH 3 and 4), 25–85 nm (at pH 2) [10]
7 Azadirachta indica Ag, Au and Ag–Au bimetallic 50–100 nm [11]
8 Tamarindus indica Au 20–40 nm [12]
9 Emblica officinalis Ag and Au (10–20 nm) and (15–25 nm) [13]
10 Aloe vera Ag and Au 15.2 ± 4.2 nm [14]
11 Coriandrum sativum Au 6.75–57.91 nm [15]
12 Carica papaya Ag 60–80 nm [16]
14 Parthenium hysterophorus Ag 52–80 nm [17]
15 Avena sativa and Tritium vulgare Au <20 nm [18]
16 Acanthella elongate (Dendy, 1905) Au 7–20 nm [19]
17 Sesuvium portulacastrum L Ag 5–20 nm [20]

used as potential biological labels [27,28]. Rearranging matter at
the nanoscale using ‘weak’ molecular interactions, such as Van der
Waal forces, hydrogen bonds, electrostatic dipoles, fluidics and var-
ious surface forces requires low-energy consumption and allows for
reversible or other subsequent changes. This present investigation
deals with Mentha piperita plant extract mediated synthesis and
characterization of silver and gold nanoparticles and their biomed-
ical application.
isolated Gram negative (Escherichia coli) and Gram positive (Staphy-
lococcus aureus) microorganisms. The pathogenic cultures were
bringing into broth culture for antibacterial assay. Approximately
7-mm diameter of well was made on Muller Hinton Agar plate with
the help of gel puncture. The cultures were swabbed on test media
with sterile cotton swab. 25 ␮l of synthesized particles were inoc-
ulated to the well, and then the plates were incubated in incubator
for 37 ◦ C for 24 h, the zones of inhibition was discussed.

2. Methods 3. Results

2.1. Chemical 3.1. Biosynthesis of silver and gold nanoparticles

The silver nitrate (AgNO3 ), chloroauric acid (HAuCl4 ) and other When the extract was subjected to AgNO3 and HAuCl2 , the
components were purchased from HiMedia (Mumbai, India). biosynthesis reaction started within few minutes and the color
reaction was observed in which clear AgNO3 solution changed into
brown color whereas pale yellowish HAuCl4 solution turned to ruby
2.2. Preparation of plant extract
red colored solution which indicates that formation of correspond-
ing nanoparticles (Fig. 1). The UV–Vis spectra of silver and gold
The leaves of M. piperita were washed thoroughly thrice with
nanoparticles synthesized by M. piperita are shown in Fig. 2. The
distilled water and were shade dried for 5 days. The fine powder
distinct peak observed at 450 nm, that is surface plasmon resonance
was obtained from the dried leaves by using kitchen blender. The
of the silver nanoparticles (Fig. 2a). Whereas, after the addition of
leaf powder was sterilized at 121 ◦ C for 15 min. 20 g of powder
M. piperita extract, the color of HAuCl4 changed from light yel-
was taken and mixed with 200 ml of Milli Q water and kept in
low to pink indicated the synthesis of gold nanoparticles in the
boiling water bath at 60 ◦ C for 10 min. The extracts were filtered
aqueous solution. A broad peak was observed at 530 nm in gold
with Whatman filter paper No 1. The filtered extract was stored in
nanoparticles (Fig. 2b).
refrigerator at 4 ◦ C for further studies.

2.3. Biosynthesis of silver and gold nanoparticles

For the biosynthesis silver nanoparticles, 1.5 ml of plant extracts

is mixed with 30 ml of AgNO3 solution (1 mM/ml) and incubated at
28 ◦ C for 24 h. Small aliquot of solution is used for the UV–Vis spec-
troscopy and FTIR is performed to the extract which was exposed
before and after addition to the silver nitrate solution. The reactions
mixture is centrifuged at 6000 rpm for 10 min and the pellet was
resuspended in small amount of sterilized double distilled water
and then small amount of suspension was sprayed on glass slide to
make thin film. The thin film was kept in hot air oven to dry and
then the thin film was used for the SEM analysis equipped with EDS
(Model JEOL, JSM-5610). The same procedure is followed for gold
nanoparticles synthesis.

2.4. Screening of antibacterial property in synthesized

nanoparticles
Fig. 1. Synthesis of silver and gold nanoparticles (a) HAuCl4 solution, (b) synthesized
Antibacterial activity was analyzed with synthesized silver and gold nanoparticle in ruby red color solution, (c) synthesized silver nanoparticles in
gold nanoparticles by well diffusion method against clinically brown color solution and (d) AgNO3 solution.
362 D. MubarakAli et al. / Colloids and Surfaces B: Biointerfaces 85 (2011) 360–365

7.00 a 100.0
6.5 90 1091.61

6.0 1404.90
80
5.5 2079.07
70
5.0
60
4.5

4.0
%T 50
40 677.84
A 3.5
3.0 30
2.5 20
2.0
10 1637.93
1.5
0.0
3394.11 a
4000.0 3000 2000 1500 1000 400.0
1.0
cm-1
0.5
100.0
0.00
200.0 250 300 350 400 450 500 550 600 650 700 750 800 850 900.0
nm 90 2358.58
5.00 80 2076.07
b
4.5 70

60
4.0 666.69

%T 50
3.5
40
3.0
30
1638.32
A 2.5 20

2.0 10
3436.89 b
0.0
1.5 4000.0 3000 2000 1500 1000 400.0
cm-1
1.0

100.0
0.5
90 2358.58
0.00
200.0 250 300 350 400 450 500 550 600 650 700 750 800 850 900.0 80 2076.07
nm
70
Fig. 2. UV–Vis spectrum analysis. (a) Plasmon resonance of silver nanoparticles
reduced by M. piperta at 450 nm and (b) plasmon resonance of gold nanoparticles 60
666.69
reduced by M. piperta at 540 nm.
%T 50
40

3.2. Characterization of silver and gold nanoparticles 30

1638.32
20
FT IR spectroscopic studies were carried out to investigate to
find possible bioreducing agents present in the extract. The spec- 10
tra of extracts were recorded before and after adding silver nitrate 3436.89 c
0.0
(a) and chloroauric acid (b) solution (Fig. 3). Both the interferro- 4000.0 3000 2000 1500 1000 400.0
cm-1
gram exhibit a broad at 3394 cm−1 is assigned to the N–H group
from peptide linkage present in the extract. Formation of C C Fig. 3. FTIR analysis of extracts of plant, Mentha piperta. Extract with silver nitrate
bonds is energetically favored over S C bonds, as the latter will solution (a) and extract with chloroauric acid (b).
impose severe geometrical constraints on the molecule more spe-
cific in thiol group and less in acidic as compared to alcohols
and that makes elimination of hydrogen attached to sulfur group.
There is a decrease in the concentration of the amide linkage gold nanoparticles indicating that they are also spherical in shape in
in the aqueous solution after the formation of silver nanoparti- range of 150 nm. Above results suggested that the silver and gold
cles. nanoparticles are synthesized due to the action of plant extract,
According to the scanning electron micrograph, the morphology M. piperita, which act as good bioreductant for biosynthesis. In the
of the silver and gold nanoparticles was observed and approxi- analysis by energy dispersive spectroscopy (EDS) of the silver and
mately spherical, in which the silver nanoparticles is in aggregated gold nanoparticles the presence of elemental metal signal was con-
form (Fig. 4a). This reveals that the powder particles are slightly firmed. Although silver and gold signal are present, the presence of
agglomerated but its size range of 90 nm and the closed view of silicon signal may be due to the thin film made on the glass slide
spherical nanoparticle has showed and Fig. 5a is a micrograph of the taken for the EDS (Figs. 4b and 5b).
 D. MubarakAli et al. / Colloids and Surfaces B: Biointerfaces 85 (2011) 360–365
Fig. 4. SEM images of silver nanoparticles. (a) The spherical shaped nanoparticles in
range of 90 nm synthesized from M. piperita and also showed closed view of silver
nanoparticle which is spherical in shape. (b) EDS spectrum showed silver signal.
3.3. Antibacterial efficacies of synthesized nanoparticles
The antibacterial activity of silver nanoparticles against E. coli
is higher than that against S. aureus is due to the variation in the
cell wall composition between Gram positive and negative bac-
teria. Whereas in gold nanoparticles showed antibacterial activity
363
Fig. 5. SEM images of gold nanoparticles. (A) The spherical shaped nanoparticles
in range of 150 nm synthesized from M. piperita. (B) EDS spectrum showed higher
percentage of gold signal.
against E. coli not in S. aureus, in plant extract was not found any of
antibacterial patterns (Fig. 6).
4. Discussion
Based on the studies of synthesis of silver and gold nanopar-
ticles and its biomedical property, it was confirmed with various
reports the appearance of weak intensity peak at 439 nm corre-
sponds to the absorption intensity steadily increased as a function
of time reaction without any shift in the peak position. The peak
at 440 nm has due to excitation of longitudinal plasmon vibrations
[29,11]. This shift in the plasmon band for higher wavelength is
associated with the larger particle sizes obtained by the slow diffu-
sion by reducing agent glucose, compared to sodium borohydride
inside the agarose matrix [30]. It has been reported that the callus
and leaf extract from the coastal saltmarsh plant, Sesuvivm portu-
lacastrum L was reduced silver ions and it was revealed that the
420 nm range of peak was observed in broth callus and leaf extract
through spectrophotometer [20].
The band at 240 cm−1 is a direct evidence for the formation of
a chemical bond between silver nanoparticles and the nitrogen of
the amide group present in the amino acids [31]. It has been stated
that either through free amino groups or cysteine residues, the pro-
tein can bind to gold nanoparticles that lead to the stabilization of
gold nanoparticles by surface bound protein [32]. Various phyto-
chemicals like alkaloids, flavones, steroids, polysaccharides, amino
acids, oximes and proteins in general and menthol in particular
364 D. MubarakAli et al. / Colloids and Surfaces B: Biointerfaces 85 (2011) 360–365
Fig. 6. Antibacterial assay of M. piperita extract (Ext) and silver (Ag) and gold (Au)
nanoparticles using well diffusion test against E. coli (a), Staphylococcus aureus (b).
Plant extract is used as control.
are present in the extract may reduce Au+ into Au0 . In C. camphora
leaf, the polyol in lixivium played a critical role in reduction of sil-
ver ions [33]. Quercetin, an antioxidant compound present in the
plant, M. sativa is possess the readily oxidized hydroxyl group in
the C-ring next to the carboxyl group was proposed that the pos-
sible mechanism of silver nanoparticles synthesis by plant extract
[8]. It also been proposed that the M. piperita may reduce the silver
and gold nanoparticle into metallic nanoparticles. It was reported
that the ketone is the major component in Cymbopogon flexuosus
extract that renders the liquid like characteristics of the spherical
gold nanoparticle [34].
It has been reported earlier that the mechanism of colloidal
gold fixation by Bacillus cells is dependent on the cell surface and
involves a number of functional groups are provided by proteins
and carbohydrates [35]. The surfactants such as cetyltrimethylam-
moniumbromide (CTAB) and sodium citrate are commonly used in
the preparation of gold nanoparticles in chemical synthesis. These
chemicals are commonly used as capping agents for the stabiliza-
tion of gold nanoparticles in aqueous solutions and to transfer
nanoparticles from a water phase to a liquid organic phase [36–38].
A biological route of gold nanoparticles to promote the anisotropic
growth of different crystal planes under ambient condition.
Shanker et al. have reported that the synthesis of a high per-
centage of thin, flat, and single-crystalline gold nanotriangles by
using the lemongrass plant [39]. Very recently, Rai et al. have
demonstrated that the presence of halide ions and modulation of
temperature can control the morphology of biologically synthe-
sized gold triangles using lemongrass leaf extract [40]. It has also
been shown that the size of nanoparticles using this technique can
be controlled through pH adjustment, exposure time, and temper-
ature reaction.
The enhanced antibacterial effects of novel silver nanoparticles
is characterized and also stated that once inside the cell, nanopar-
ticles would interfere with the bacterial growth signaling pathway
by modulating tyrosine phosphorylation of putative peptides sub-
strate critical for cell viability and division and the nanoparticles
were not in direct contact even within the aggregates, indicating
stabilization of the nanoparticles by a capping agent [41,42].
Shahverdi et al. reported that the silver nanoparticles have an
antimicrobial effect on S. aureus and E. coli [43]. The oligodynamic
effect of silver has antibacterial activity against microorganisms.
The changes in the local electronic structure on the surface of the
smaller sized particles lead to the enhancement of their chemi-
cal reactivity leading to bactericidal effect [44]. It was suggested
that the pre and post washed silver nanoparticles delayed bacte-
ria growth at the interface of silver nanoparticles as reported by
Morones et al. [45] and direct usage of the silver nanoparticles from
the native reaction solution warranted more effective [46].
5. Conclusion
To summarize, we succeeded in the biological reduction of silver
and gold nanoparticles by M. piperita leaf extract. Silver and gold
nanoparticles were synthesized in ambient conditions and charac-
terization of synthesized nanoparticles was carried out by UV–Vis
spectroscopy, FT IR and SEM equipped with EDS. It is believed that
phytochemicals present in the extract of M. piperita has reduced
the silver and gold ions into metallic nanoparticles. This may be a
first report that leaf had been sterilized before the extraction, we
assumed to decant surface inhabitant microorganisms. The synthe-
sized silver and gold exhibited a strong antibacterial activity against
both E. coli and S. aureus. The process for the synthesis of nanopar-
ticles in large scale using these readily available plant extract may
have commercial viability and to develop studies in the interface
between biology and material science. By using such plant extracts
to develop nanomedicine against various human and veterinary
pathogens.
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