J Nanopart Res
DOI 10.1007/s11051-011-0428-6
RESEARCH PAPER
Silver nanoparticles with gelatin nanoshells: photochemical
facile green synthesis and their antimicrobial activity
Ali Pourjavadi • Rouhollah Soleyman
Received: 27 October 2010 / Accepted: 16 May 2011
Ó Springer Science+Business Media B.V. 2011
Abstract In the current study, a facile green synthe-
sis of silver-gelatin core–shell nanostructures (spher-
ical, spherical/cubic hybrid, and cubic, DLS diameter:
4.1–6.9 nm) is reported via the wet chemical synthesis
procedure. Sunlight-UV as an available reducing agent
cause mild reduction of silver ions into the silver
nanoparticles (Ag-NPs). Gelatin protein, as an effec-
tive capping/shaping agent, was used in the reaction to
self-assemble silver nanostructures. The formation of
silver nanostructures and their self-assembly pattern
was confirmed by SEM, AFM, and TEM techniques.
Further investigations were carried out using zeta-
potential, UV–Vis, FTIR, GPC, and TGA/DTG/DTA
data. The prepared Ag-NPs showed proper and
acceptable antimicrobial activity against three classes
of microorganisms (Escherichia coli Gram-negative
bacteria, Staphylococcus aureus Gram-positive bacte-
ria, and Candida albicans fungus). The antibacterial
and antifungal Ag-NPs exhibit good stability in
solution and can be considered as promising candidates
for a wide range of biomedical applications.
Keywords Silver nanoparticles Nanoshell Green
synthesis Antimicrobial activity
A. Pourjavadi (&)
R. Soleyman
Polymer Research Laboratory, Department of Chemistry,
Sharif University of Technology, P.O. Box 11365-9516,
Tehran, Iran
e-mail: Purjavad@sharif.edu
Introduction
Nowadays, nanotechnology has become a part of our
daily life in various forms such as cosmetics, electron-
ics, biosensors, pharmaceuticals, and computer sci-
ences. Among the incredible growth of this field of
science, bionanotechnology as a leading science plays
an important role in the development of biosynthetic
and eco-friendly approaches for synthesis of nano-
structures. Silver nanoparticles (Ag-NPs) are promis-
ing agents in bionanotechnology because of their
unique activity against unfavorable processes in bio-
science such as undesirable microbial growth, angio-
genesis in tumors, etc. Ag-NPs have been widely used
in wound dressing; wound healing, water treatment
filters, inks, sensors, catalysts, cosmetics, orthopae-
dics, surgical instruments, and vascular prosthesis.
Ever-increasing applications of Ag-NPs in biomedical
field are due to the antimicrobial (Rai et al. 2009; Kim
et al. 2009; Lara et al. 2010), anti-platelet (Shrivastava
et al. 2009), anti-proliferative (AshaRani et al. 2009),
anti-inflammatory (apoptosis detection) (Nadworny
et al. 2008), and anti-angiogenesis properties (cancer
therapy) (Gurunathan et al. 2009).
Green synthesis of Ag-NPs possesses three main
steps, which were evaluated by green chemistry
perspectives, including the selection of solvent
medium, environmentally benign reducing agent,
and non-toxic capping agent (Sharma et al. 2009).
For the biological green synthesis of Ag-NPs, many
bacteria, fungi, and plants are found as arsenals of
123

Ag-NP synthesizer (Vaidyanathan et al. 2009). In wet
chemical synthesis, various biocompatible materials
such as starch (Kassaee et al. 2008), dextran (Zhang
et al. 2004), sodium alginate (Liu et al. 2009),
chitosan (Wei et al. 2009), gum acacia (Rao et al.
2010), gum arabic (Medina-Ramirez et al. 2009),
natural rubber (AbuBakar et al. 2007), poly (vinyl
alcohol) (PVA) (Filippo et al. 2009), poly (vinyl
pyrrolidone) (PVP) (Liu et al. 2008), carboxymethyl
cellulose (CMC) (Chen et al. 2008), carboxymethyl
curdlan and carboxymethyl fucoidan (Leung et al.
2010), glucose in the form of gluconic acid (Janar-
dhanan et al. 2009), and honey (Philip 2010) were
used as capping agents for the green synthesis of
Ag-NPs. In the category of proteins and their deriv-
atives, wool keratin (Lu and Cui 2010), gelatin
nanofibers (Xu and Zhou 2008), gelatin-g-poly
(methyl methacrylate) white emulsion (Liu et al.
2010), bovine serum albumin (BSA) and poly-L-lysine
(PLL) (Bale et al. 2007), and oligonucleotide based on
DNA (Lee et al. 2007) were utilized for the stabiliza-
tion of Ag-NPs in solution or synthesis of hybrid
nanomaterial Ag-NP conjugates.
In the current research, we synthesized Ag-NPs in
aqueous solution at room temperature based on the
main principles of the green synthesis, using sunlight-
UV as facile non-toxic reducing agent. Gelatin, a
fully biocompatible capping/shaping agent, brought
about stabilization of the Ag-NPs in the solution and
caused self-assembly of the silver nanostructures
(cubic, spherical, and cubic/spherical hybrid). The
self-assembly patterns of the Ag-NPs were proved by
several visual techniques. Also, antimicrobial activity
of Ag-NPs including bactericidal (against Esche-
richia coli and Staphylococcus aureus) and fungicidal
assays (against Candida albicans) showed suitable
and acceptable results.
Materials and methods
Chemicals
Gelatin (GPC data: Mw = 115000 g/mol, Mn = 2800
g/mol, wide distribution) and silver nitrate (AgNO3)
were purchased from Merck (Germany) and all the
solutions were prepared using double distilled–deion-
ized water.
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J Nanopart Res
Preparation of Ag-NPs
In the current study, four samples of Ag-NPs were
synthesized via a facile reagent-free green method.
For the preparation of each sample, 100 mg gelatin
protein was poured into 10 mL tepid water at flask on
a magnetic stirrer and was permitted to dissolve and
homogenize for 30 min. Then, silver nitrate solution
(5, 10, 20 and 40 mg AgNO3 in 1 mL H2O) was
added to gelatin solution at room temperature under
sunlight-UV. After 120 min, the orange-brownish
solution of Ag-NPs was obtained. In the next step,
samples were dialyzed (cut-off: 3000) against double
distilled–deionized water for removing any unre-
duced silver ions for 24 h.
Characterization
UV–visible spectrophotometer (Shimadzu-1650 PC,
Japan) was used for recording absorption spectra in
the solution using a cell of 1.0-cm-path length. FTIR
spectra of the samples in the form of KBr pellets were
recorded on an ABB Bomem MB-100 FTIR spec-
trophotometer. Thermogravimetric analysis (TGA)
was performed on a PL-STA 1500 thermal analyzer
set up at a heating rate of 10 °C/min under nitrogen
atmosphere. The molecular weight distributions of
Ag-NP-gelatin core-shell system were determined by
gel permeation chromatography (GPC, Agilent 1100
series) apparatus using a PL aquagel-OH mixed
column connected to a differential refractometer,
with an RI detector and HPLC grade water as the
mobile phase at 32 °C. Dextran standard samples
were used for calibration. The particle size and zeta
potential of samples were determined using dynamic
light scattering (DLS, zetasizer ZS, Malvern) instru-
ment using a 4-mW He–Ne laser (633 nm wave-
length) with a fixed detector angle of 173°. The
morphology of the Ag-NPs was examined using a
scanning electron microscope (SEM, Philips, XL30)
operated at 20 kV after coating the samples with gold
film. High-resolution surface imaging studies were
performed using atomic force microscopy (AFM) to
estimate the surface morphology and the particle size
distribution. The samples were imaged with the aid of
Dual-scope/Raster-scope C26, DME, using DS
95-50-E scanner with vertical z-axis resolution of
0.1 nm. The TEM images were obtained using a
J Nanopart Res
JEOL JSM-2100 transmission electron microscope
with accelerating voltage of 200 kV. The samples for
TEM analysis were prepared by spotting 10 lL of the
sample solution onto a Holey carbon TEM grid
followed by drying before putting them into the TEM
sample chamber.
Antimicrobial activity
The activity of Ag-NPs against three classes of
microorganisms (Escherichia coli Gram-negative
bacteria, Staphylococcus aureus Gram-positive bac-
teria, and Candida albicans fungi) was assessed.
Antibacterial assay
Escherichia coli (ATCC 51813) and Staphylococcus
aureus (S. aureus, ATCC 27661) were cultivated in
Luria-Bertani (LB) broth medium (containing 10 g/L
peptone, 5 g/L yeast extract, and 10 g/L sodium
chloride). The visual turbidity of the tubes was noted
both before and after incubation. The medium was
solidified by 20 g/L agar. The pH was adjusted to pH
7.0–7.2 with 1 M NaOH before autoclaving. The
autoclaved LB medium (with 1% agar) that had been
allowed to cool to approximately 40 °C was added to
the bacteria-exposed slides. After the agar had
solidified, the slides were incubated at 37 °C for
18 h, and the colonies were quantified.
The Ag-NP samples were diluted and added to
5 mL of LB medium with tested bacterial concen-
trations of 105–106 CFU/mL. Positive control tubes
contained 5 mL of LB medium with tested bacterial
concentrations of 105–106 CFU/mL. Negative control
tubes contained only inoculated broth. The tubes
were incubated at 37 °C with shaking at 250 rpm for
18 h in a constant-temperature incubator. The min-
imum inhibition concentration (MIC) is defined as the
lowest concentration (lg/mL) at which there is no
visible growth, and the minimum bactericidal con-
centration (MBC) is defined as the lowest concentra-
tion at which no colony is observed (more than 99.9%
lethality). The MICs were read by the visual turbidity
of the tubes noted both before and after incubation.
After being properly diluted, aliquots from tubes
(100 lL) that appeared to have little or no cell growth
were plated on LB agar plates to distinguish between
the bacteriostatic or the bactericidal effects. The Ag-
NP sample was used as prepared and tested at final
concentrations (2.0, 5.0, 8.0, 10.0, 12.0, 15.0, and
20.0 lg/mL).
Antifungal assay
Candida albicans (ATCC 90028) was obtained from
the American Type Culture Collection (ATCC).
Fungal cells were cultured in a Mueller-Hinton broth
(MHB, Difco, France) with aeration at 28 °C.
The fungistatic activity of Ag-NPs was performed
by the modified micro-dilution method (Panacek
et al. 2009) which enabled to determine the MICs of
the Ag-NPs inhibiting the growth of the tested
C. albicans. The Ag-NPs were diluted in microtiter
plates by the MHB medium. The obtained concen-
trations of Ag-NPs in the dispersion were in the range
of 2.0, 5.0, 8.0, 10.0, 12.0, 15.0 and 20.0 lg/mL.
After the Ag-NP samples were diluted, a standard
amount of the tested yeast was inoculated onto
microtiter plates so that the inoculum density in the
wells was equal to 105–106 CFU/mL. After 36 h
incubation at 37 °C, the MIC was recorded as the
lowest concentration of the agent inhibiting the
visible growth of microorganisms.
The minimum fungicidal concentration (MFC)
was determined by inoculating the contents of all
wells of the testing plate onto a new microtiter plate
with MHB medium without Ag-NPs. Following 72 h
incubation, the MFC was recorded as the lowest
concentration of the tested agent inhibiting the visible
growth of microorganisms.
Results and discussion
Reaction mechanism
It is well known that ultrasonic waves, prolonged
reflux, UV-irradiation, c-rays, and reducing agents
can reduce the silver cations (Ag?) into Ag-NPs in
the presence of a capping agent. In this study, silver-
gelatin core-shell nanoparticles have been prepared
by sunlight UV-irradiation. Sunlight-UV as a gratis
source of reducing agent was applied and caused
facile and cheap preparation of the Ag-NPs without
any surplus material. Gelatin, as a multi-purpose
(biocompatible, biodegradable, and non-toxic effec-
tive capping/shaping agent) material, was utilized to
inhibit the agglomeration of the Ag-NPs in solution.
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In this approach, the coordinated silver cations were
reduced into the Ag-NPs by photochemical reactions
(Xu and Zhou 2008). The schematic representation of
this conversion was indicated in Scheme 1. A deci-
sive reason which confirms the formation of the
gelatin-capped Ag-NPs was attained from the dialysis
results. The utilized water for dialysis was analyzed
by UV–Vis spectroscopy and interestingly, did not
show any peak due to the exit of Ag-NPs from the
dialysis cassette. Indeed, all produced Ag-NPs had
been surrounded by the gelatins (capping agents) and
any isolated bare Ag-NP did not exist in the solution.
DLS and zeta-potential analysis
The size of four prepared samples of Ag-NPs was
measured by dynamic light scattering (DLS) which are
illustrated in Table 1. Figure 1 shows the DLS and
zeta-potential data for the pure gelatin and the Ag-NPs
prepared by 20 mg AgNO3. Figure 1a obviously
represents that the size of the Ag-NPs (6.9 nm) is
smaller than the size of gelatin molecules (9.9 nm).
Also, the mono-modality and narrow distribution of the
synthesized Ag-NPs is distinctive from the related
curves. Figure 1b indicates the curves of the same
samples in terms of the intensity of scattered light. It is
well known that the proteins can self-assemble and
produce multi-molecular structures with larger sizes.
This phenomena is observed in the aforesaid curve, and
this is a remarkable point that the self-assembly of the
gelatin has caused Ag-NP self-assembly because of the
Scheme 1 Schematic
representation of reaction
mechanism
Table 1 The structural
Ag-NPs sample Morphology by (SEM,
information (shape and
prepared by AFM and TEM)
size) of synthesized Ag-NPs
5 mg AgNO3 Spherical
10 mg AgNO3 Spherical/cubic hybrid
20 mg AgNO3 Cubic/spherical hybrid
40 mg AgNO3 Cubic
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J Nanopart Res
synergy effect between the metal and protein for the
formation of multi-molecular structures. It is well
known that the intensity of scattered light relate with
sixth exponent of particle radius (r6), and it means the
extent of this self-assembly in fresh solutions and under
normal conditions is negligible. The zeta-potential
curves of the same samples (Fig. 1c) give an attractive
information about the silver-gelatin complex and the
mechanism of chelation. The zeta-potential values of
the gelatin and the Ag-NPs prepared by 20 mg AgNO3
are 4.8 and 3.7 mV, respectively. The zeta-potential
value of the gelatin indicates that the surface of the
gelatin molecule has net positive charge because the
number of positive charge fragments (such as [–NH–
CNH2–NH2]? and –NH3?) on the surface of the gelatin
is larger than negative charge fragments (such as
–COO-), and it means that the utilized gelatin is type A
and has isoelectric point greater than 7. The smaller
zeta-potential value of the Ag-NPs indicate that in
addition to non-ionic chelating groups of gelatin (such
as –SH, –OH, ester and amide) and –COO- anionic
groups, the cationic groups of –NH3? in the form of
–NH2 can operate as chelating agent.
SEM/AFM/TEM analysis
The SEM pictures of four prepared samples of Ag-NPs
indicate that the shapes of our nano-objects (Fig. 2a–d)
alter gradually from spherical to spherical/cubic
hybrid, cubic/spherical hybrid, and finally to cubic by
Average core-shell Average core-shell
diameter by DLS (nm) diameter by TEM (nm)
4.1 15
5.6 18
6.9 22
5.2 10
J Nanopart Res
Fig. 1 The DLS curves of the gelatin & the Ag-NPs prepared
by 20 mg AgNO3. a in terms of number (%) and b in terms of
intensity of scattered light and c the zeta-potential curve of the
gelatin & the Ag-NPs prepared by 20 mg AgNO3
Fig. 2 The SEM images of Ag-NPs samples prepared by a 5 mg, b 10 mg, c 20 mg, and d 40 mg of AgNO3
increasing of AgNO3 amount (Table 1). It is obser-
vable that the size of Ag nano-objects is very larger
than which was seen in the DLS data and as talked
about it, this can be attributed to the self-assembly of
silver-gelatin core-shell systems via the synergic effect
created from the electrostatic interactions between the
metal and the protein. With a precise look into the SEM
images, the smaller Ag nano-objects are absolutely
observable and in the Fig. 2d a tetrameric-assembly of
Ag-nanocubes is distinctive. It has been shown that the
secondary interactions between the proteins can play a
significant role in tuning the oligomerization/aggrega-
tion behavior of a protein and finally lead to the self-
assembly of metal-protein system (Salgado et al.
2008). It has been shown that ion metal-bridging and
H-bonding interactions can dictate the geometric
alignment of protein partners, leading to the population
of discrete supramolecular structures over other con-
formations of similar energy (Salgado et al. 2010).
Also, similar self-assembly of metal and protein has
been seen in the directed self-assembly of gold binding
polypeptide-protein (Ko et al. 2009; Grzelczak et al.
2010). For high resolution surface morphology study,
the AFM images of samples were prepared and
compared with SEMs. Figure 3a, b belong to the
SEM images of the Ag-NPs prepared by 5 mg and
40 mg AgNO3, respectively, and Fig. 3c, d belong to
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Fig. 3 Comparison between the SEM images of the Ag-NPs prepared by a 5 mg & b 40 mg of AgNO3 and their AFM pictures;
c 5 mg, and d 40 mg of AgNO3
the AFM images of the same samples. As one can see,
the AFM pictures have the same shape with smaller
size and confirm the self-assembly of the smaller Ag
nano-objects and production of the larger nanoparticles
in the SEM images. To obtain other confirming
evidences about self-assembly, the TEM images were
provided. The TEM pictures (Fig. 4) obviously prove
the self-assembly pattern (Scheme 2) of smaller Ag
nano-objects to larger nano-objects, also. For shell
detection of Ag-NPs, the AFM and TEM techniques
were utilized to measure the thickness of the shells.
Figure 5 represents the AFM and TEM images of the
Ag-NPs prepared by 5 mg AgNO3. In this picture, the
shell thickness for the Ag-NPs was estimated as 3.7 and
5.0 nm from TEM and AFM, respectively. The
attained difference between the two methods can be
attributed to the higher aggregation of gelatin in the
AFM image.
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GPC analysis
The gel permeation chromatography (GPC) curves of
the pure gelatin and the Ag-NPs were provided in
Fig. 6. The blue curve represents the molecular weight
distribution of the gelatin protein as a basis for
comparison. The red curve indicates the GPC curve
of the Ag-NPs prepared by 5 mg AgNO3. Interest-
ingly, it is seen that the apparent molecular weight of
the gelatin has been increased because of the formation
of a multi-molecular layer around Ag-NPs. On the
other hand, the greater hydrodynamic volume of the
silver-gelatin system is due to the self-assembly and
multi-molecular structure of silver-gelatin complex.
The green curve also shows the related curve of the
Ag-NPs prepared by 10 mg AgNO3. As one can see,
the molecular weight of the gelatin has been increased
again dramatically. The increase of the molecular
J Nanopart Res
Fig. 4 The TEM images of the Ag-NPs prepared by a 5 mg and b 40 mg AgNO3 and their self-assembly pattern
Scheme 2 Proposed self-
assembly mechanism for
the formation of larger
silver nano-objects from
smaller ones
weight can be attributed to the inter-molecular silver
chelation which causes hydrodynamic volume incre-
ment and apparent molecular weight growth. Won-
derfully, the violet curve (Ag-NPs prepared by 40 mg
AgNO3) exhibits lower molecular weight for gelatin,
and the reason might be related to the intra-molecular
silver-gelatin chelation due to the higher content of
silver. This intra-molecular silver-gelatin chelation
results in the hydrodynamic volume decrease of silver-
gelatin system and apparent reduction of gelatin
molecular weight.
FTIR and UV–Vis spectroscopy analysis
FTIR measurements were carried out to identify the
interactions between the Ag-NP core and the gelatin
nanoshell (Fig. 7). For the pure gelatin, the strong
broad peak at 3000–3600 cm-1 is characteristic of
the N–H stretching vibration. Two bands at 1651
and 1543 cm-1 are assigned to the amide I and II
bands of proteins (Burt et al. 2004), respectively,
and the band observed at 1438 cm-1 can be
assigned to the C–N stretching vibrations of the
amines (Sanghi and Verma 2009). With a subtle
glance to the spectra, this is obvious that compared
to pure gelatin, all the peaks of gelatin capped
Ag-NPs are sharper and narrower. The sharpness of
the peaks can be explained with this fact that most
of H-bonds between the amide groups in the
proteins will break because of higher tendency of
the amide groups for the formation of stronger
bonds with transition metal atoms (Burt et al. 2004).
In addition to narrowness of the peaks, red or blue-
shifts may occur in their positions, attentive to the
type of bond existing in the chelating groups
(Lu and Cui 2010). Finally, it can be concluded
that the gelatin protein has formed a coating layer
over the Ag-NPs and played the capping agent role.
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Fig. 5 Shell detection of Ag-NPs: the TEM images of the Ag-NPs prepared by 5 mg AgNO3with the magnification of a 250K9 and
b 400K9 and their AFM images (c and d)
Fig. 6 The GPC curves for the gelatin and the Ag-NPs
In Fig. 8a, the curves indicate the characteristic
surface plasmon resonance (SPR) of Ag-NPs between
437 and 449 nm according to their orange-brownish
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Fig. 7 The FTIR spectra of the gelatin and the Ag-NPs
color. Among the metal nanoparticles, Ag-NPs are of
particular interest owing to their third-order optical
nonlinearity and pronounced SPR absorption, which
have promising roles in many applications, such as
optical waveguides and optical switches (Veerapan-
dian et al. 2010). It is well known that the UV–Vis
J Nanopart Res
Fig. 8 a The UV–Vis spectra of the Ag-NPs and b the
changes in UV–Vis spectra of the Ag-NPs prepared by 5 mg
AgNO3 during 15 days
spectrum of metal nanoparticles which is dominated
by the SPR, leads to the distinguished red or blue
shift depending on the dielectric properties of the
surrounding host matrix, or the environmental atmo-
sphere, in addition to the particle size and shape of
nanoparticles. The stability of Ag-NPs preserved in
the dark at room temperature were examined and
showed good stability results. Figure 8b represents
the UV–Vis spectrum of the Ag-NPs prepared by
5 mg AgNO3 for 15 days. The absorbance spectra
show an increase in the absorbance of Ag-NPs
solution and a red shift of kmax with time. It is found
that the solutions stored for 15 days at room temper-
ature have similar absorbance values to those stored
for 1 year at 4.0 °C (Pinto et al. 2010). In compar-
ison to the prior synthesized Ag-NPs, the Ag-NPs
Fig. 9 a The TGA curves of gelatin & the Ag-NPs prepared
by 5 mg AgNO3 and b their DTG/DTA spectra
prepared in this study have stability similar to citrate
stabilized Ag-NPs (Pinto et al. 2010) and have more
stability than the other synthesized by laser ablation
method in organic solvents (Tilaki et al. 2006).
Thermo-Gravimetric Analysis (TGA)/Differential
Thermal Gravimetry (DTG)/Differential Thermal
Analysis (DTA)
TGA traces of gelatin and the Ag-NPs prepared by
5 mg AgNO3 are presented in Fig. 9a. According to
this TGA curves, values related to the pure gela-
tin such as T10 (210.1 °C), T30 (313.4 °C), T50
(356.5 °C), and char yield at 700 °C (1.2%) are
lower compared with gelatin-capped Ag-NPs (T10 =
221.1 °C, T30 = 322.3 °C, T50 = 383.5 °C, Y =
5.8%). As one can see, the gelatin-capped Ag-NPs
were found to be the most thermally stable samples
studied. It is well known that nanocomposite structure
(gelatin-capped Ag-NPs) and the formation of coor-
dination bonds in the composition may act as heat
123

barriers and as a consequence enhance the overall
thermal stability of composition. For the better study
of thermal behavior of samples, their DTG/DTA
curves were provided in Fig. 9b. The DTG curve of
the pure gelatin shows that its maximum decomposi-
tion rate occurs in the two sharp peaks at 315 and
657 °C. Its DTA curve indicates that two decompo-
sition endothermic reactions represented by two broad
peaks occurred at 275–428 and 508–580 °C. In the
Fig. 9b, the DTG/DTA curves of gelatin-capped
Ag-NPs were also represented. The DTG curve
indicates that the maximum decomposition rate of
the gelatin-capped Ag-NPs occurs in the two sharp
peaks at 386 and 667 °C, and according to the DTA, at
this temperature an endothermic reaction causes its
decomposition. Totally, the comparison of TGA/
DTG/DTA curves confirms the higher thermal stabil-
ity of the gelatin-capped Ag-NPs.
Antimicrobial activity investigation
The antibacterial activity of Ag-NPs was tested using
the MIC and MBC tests. Different concentrations of
the tested samples were incubated with E. coli
(as Gram-negative bacteria) and S. aureus (as Gram-
positive bacteria) in LB broth. Bacterial growth was
studied by visual observation as indicated by turbid-
ity. Lack of turbidity may correspond to either the
very low bacterial growth (a bacteriostatic effect) or
the complete killing of bacteria (a bactericidal effect).
If the tested material did not kill but only inhibited the
growth of bacteria (bacteriostatic), bacteria will grow,
and the colonies will be observed upon plating. If the
tested material is bactericidal, no bacterial colony
would be observed. Table 2 shows the bacteriostatic
test results of Ag-NPs toward E. coli. It is obvious that
the MICs against S. aureus are higher than E. coli
Table 2 The anti-bacterial and anti-fungal activity (MIC
values (lg/mL)) of Ag-NPs
Ag-NPs sample E. coli S. aureus C. albicans
prepared by bacteria bacteria fungus
5 mg AgNO3 15 15 12
10 mg AgNO3 10 15 10
20 mg AgNO3 8 12 5
40 mg AgNO3 5 8 2
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J Nanopart Res
relatively, and this can be attributed to the type and
thickness of the S. aureus membrane and its cell wall.
The bactericidal test of the Ag-NPs toward E. coli was
carried out, and the MBC is read by the presence of
live bacteria on the LB agar plate. The first concen-
tration with no indication of live bacteria (the MBC)
for the Ag-NPs prepared by 5 mg AgNO3 toward
E. coli was 15 lg/mL. The MIC and MBC results of
all samples of Ag-NPs against E. coli & S. aureus are
summarized in Table 2 and because the MIC and
MBC values of Ag-NPs toward bacteria were unal-
tered, the MBC results were not shown. According to
the table, it is seen when the shape of Ag-NPs change
from spherical to cubic (from the Ag-NPs prepared by
5 mg AgNO3 toward the one prepared by 40 mg
AgNO3), their antibacterial activities (both in the case
of E. coli and in the case of S. aureus) enhance and
because the size of Ag-NPs is approximately identical
(according to the DLS data), these results can become
a confirmation on the theory of Ag-NP shape can
relate with antibacterial activity (Pal et al. 2007). In
the case of E. coli, it will predict that the Ag-NPs
could have long-lasting activity because it is found
that cationic biopolymers could capture negatively
charged bacteria (bacteria-adsorbing effect), and since
the biopolymer kills/captures the bacteria, the cell
membrane remnants/dead bacteria presumably remain
adsorbed on the polymer surface, preventing further
antibacterial activity. In contrast, the silver nanopar-
ticle-gelatin core-shell system may continue to kill the
bacteria even after the surface is completely covered
by dead bacteria, thereby showing long-lasting activ-
ity (Sambhy et al. 2006). This effect has been proved
for silver-incorporated chitosan film, earlier (Wei
et al. 2009).
The fungistatic activity of the Ag-NPs against
C. albicans as a model for fungi was determined by
the standard micro-dilution method, and the MICs
were provided at Table 2. The Ag-NPs prepared by
20 and 40 mg AgNO3 exhibited a MIC value of 2 and
5 lg/mL, respectively, and the MIC values of this
compound were approximately the same level as
those of amphotericin B, showing MIC values of
2.5–5 lg/mL toward the fungal strains tested. Along
with the study of the fungistatic activity of Ag-NPs,
their fungicidal activity against the tested yeast
was simultaneously assessed. The obtained MFCs
(200 lg/mL) are considerably higher in comparison
to MICs and of course, this is normal behavior and
J Nanopart Res
have been formerly seen (Panacek et al. 2009).
Similar to the antibacterial activity of the Ag-NPs,
these results can communicate to the shape of
Ag-NPs with antifungal activity. The comparison of
the antifungal activity of the Ag-NPs with their
antibacterial activity clearly showed that the Ag-NPs
inhibit yeast growth at lower concentrations than in
the case of the bacterial growth inhibition. This
phenomenon has been previously seen by another
group (Panacek et al. 2009) also.
Although the mechanism by which the nanoparti-
cles are able to penetrate the microorganisms is not
totally understood, yet in the case of Ag-NPs, several
studies have been carried out. These reports illustrate
that Ag-NPs can change the membrane morphology
of microorganisms and may produce a significant
increase in its permeability and affect proper trans-
port through the plasma membrane (Sondi and
Salopek-Sondi 2004). The observation of Ag-NPs
attached to the cell membrane and inside the bacteria
is fundamental and acceptable evidence for the
understanding of the biocidal mechanism of Ag-
NPs (Morones et al. 2005; Li et al. 2010). As
established by the theory of hard and soft acids and
bases (HSAB theory), silver will tend to have a
higher affinity to react with phosphorus and sulfur
compounds. The membrane of the bacteria is well
known to contain many sulfur-containing proteins
which might be preferential sites for the Ag-NPs. On
the other hand, the inside nanoparticles will also tend
to react with other sulfur-containing proteins in the
interior of the cell, as well as with phosphorus-
containing compounds such as DNA (Feng et al.
2000). To conclude, the changes in the morphology
presented in the membrane of the bacteria, as well as
the possible damage caused by the nanoparticles
reacting with the DNA, will affect the bacteria in
processes such as the respiratory chain and cell
division, finally causing the death of the microorgan-
isms. Recently, it has been found that b-cyclodextrin
as capping agent biomaterial can enhance the
antibacterial activity (up to 3.5-fold) of Ag-NPs
produced by reduction of silver salts using NaBH4 via
Trojan horse mechanism (Jaiswal et al. 2010). Based
on this report, in the current study, it will be predicted
that the nano-metric shell of gelatin (3.7 nm) around
Ag-NPs has increased the antibacterial activity of the
Ag-NPs and has positive effect on their antibacterial
activity via Trojan horse mechanism.
Conclusions
In summary, we have developed a highly facile and
inexpensive approach to prepare gelatin-capped silver
nanostructures, which are stable in the aqueous
solutions. It is predicted that this green synthesis
procedure can be easily extended to other similar
systems, which is valuable for the utilization of other
proteinaceous bioresources. Ag-NPs exhibited a
strong anti-microbial activity against E. coli and
S. aureus bacteria and potent antifungal activity toward
C. albicans which suggested that the Ag-NPs could
effectively suppress bacterial/fungal proliferation and
protect wounds from bacterial/fungal invasion.
Acknowledgments The authors thank Dr. G. R. Bardajee,
Dr. M. Adeli, Mr. H. Rezanejad, Mr. H. Abroshan, Mr.
M. Akhlaghi, and Mr. M. Fakourpour for their valuable help in
preparation of this article, making the samples, and data taking.
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