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- Introduction

sis of silver-gelatin core–shell nanostructures (spher-
ical, spherical/cubic hybrid, and cubic, DLS diameter: Nowadays, nanotechnology has become a part of our
4.1–6.9 nm) is reported via the wet chemical synthesis daily life in various forms such as cosmetics, electron-
procedure. Sunlight-UV as an available reducing agent ics, biosensors, pharmaceuticals, and computer sci-
cause mild reduction of silver ions into the silver ences. Among the incredible growth of this field of
nanoparticles (Ag-NPs). Gelatin protein, as an effec- science, bionanotechnology as a leading science plays
tive capping/shaping agent, was used in the reaction to an important role in the development of biosynthetic
self-assemble silver nanostructures. The formation of and eco-friendly approaches for synthesis of nano-
silver nanostructures and their self-assembly pattern structures. Silver nanoparticles (Ag-NPs) are promis-
was confirmed by SEM, AFM, and TEM techniques. ing agents in bionanotechnology because of their
Further investigations were carried out using zeta- unique activity against unfavorable processes in bio-
potential, UV–Vis, FTIR, GPC, and TGA/DTG/DTA science such as undesirable microbial growth, angio-
data. The prepared Ag-NPs showed proper and genesis in tumors, etc. Ag-NPs have been widely used
acceptable antimicrobial activity against three classes in wound dressing; wound healing, water treatment
of microorganisms (Escherichia coli Gram-negative filters, inks, sensors, catalysts, cosmetics, orthopae-
bacteria, Staphylococcus aureus Gram-positive bacte- dics, surgical instruments, and vascular prosthesis.
ria, and Candida albicans fungus). The antibacterial Ever-increasing applications of Ag-NPs in biomedical
and antifungal Ag-NPs exhibit good stability in field are due to the antimicrobial (Rai et al. 2009; Kim
solution and can be considered as promising candidates et al. 2009; Lara et al. 2010), anti-platelet (Shrivastava
for a wide range of biomedical applications. et al. 2009), anti-proliferative (AshaRani et al. 2009),
anti-inflammatory (apoptosis detection) (Nadworny
Keywords Silver nanoparticles Nanoshell Green et al. 2008), and anti-angiogenesis properties (cancer
synthesis Antimicrobial activity 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
A. Pourjavadi (&)
R. Soleyman medium, environmentally benign reducing agent,
Polymer Research Laboratory, Department of Chemistry,
and non-toxic capping agent (Sharma et al. 2009).
Sharif University of Technology, P.O. Box 11365-9516,
Tehran, Iran For the biological green synthesis of Ag-NPs, many
e-mail: [email protected] bacteria, fungi, and plants are found as arsenals of

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Ag-NP synthesizer (Vaidyanathan et al. 2009). In wet Preparation of Ag-NPs

chemical synthesis, various biocompatible materials
such as starch (Kassaee et al. 2008), dextran (Zhang In the current study, four samples of Ag-NPs were
et al. 2004), sodium alginate (Liu et al. 2009), synthesized via a facile reagent-free green method.
chitosan (Wei et al. 2009), gum acacia (Rao et al. For the preparation of each sample, 100 mg gelatin
2010), gum arabic (Medina-Ramirez et al. 2009), protein was poured into 10 mL tepid water at flask on
natural rubber (AbuBakar et al. 2007), poly (vinyl a magnetic stirrer and was permitted to dissolve and
alcohol) (PVA) (Filippo et al. 2009), poly (vinyl homogenize for 30 min. Then, silver nitrate solution
pyrrolidone) (PVP) (Liu et al. 2008), carboxymethyl (5, 10, 20 and 40 mg AgNO3 in 1 mL H2O) was
cellulose (CMC) (Chen et al. 2008), carboxymethyl added to gelatin solution at room temperature under
curdlan and carboxymethyl fucoidan (Leung et al. sunlight-UV. After 120 min, the orange-brownish
2010), glucose in the form of gluconic acid (Janar- solution of Ag-NPs was obtained. In the next step,
dhanan et al. 2009), and honey (Philip 2010) were samples were dialyzed (cut-off: 3000) against double
used as capping agents for the green synthesis of distilled–deionized water for removing any unre-
Ag-NPs. In the category of proteins and their deriv- duced silver ions for 24 h.
atives, wool keratin (Lu and Cui 2010), gelatin
nanofibers (Xu and Zhou 2008), gelatin-g-poly Characterization
(methyl methacrylate) white emulsion (Liu et al.
2010), bovine serum albumin (BSA) and poly-L-lysine UV–visible spectrophotometer (Shimadzu-1650 PC,
(PLL) (Bale et al. 2007), and oligonucleotide based on Japan) was used for recording absorption spectra in
DNA (Lee et al. 2007) were utilized for the stabiliza- the solution using a cell of 1.0-cm-path length. FTIR
tion of Ag-NPs in solution or synthesis of hybrid spectra of the samples in the form of KBr pellets were
nanomaterial Ag-NP conjugates. recorded on an ABB Bomem MB-100 FTIR spec-
In the current research, we synthesized Ag-NPs in trophotometer. Thermogravimetric analysis (TGA)
aqueous solution at room temperature based on the was performed on a PL-STA 1500 thermal analyzer
main principles of the green synthesis, using sunlight- set up at a heating rate of 10 °C/min under nitrogen
UV as facile non-toxic reducing agent. Gelatin, a atmosphere. The molecular weight distributions of
fully biocompatible capping/shaping agent, brought Ag-NP-gelatin core-shell system were determined by
about stabilization of the Ag-NPs in the solution and gel permeation chromatography (GPC, Agilent 1100
caused self-assembly of the silver nanostructures series) apparatus using a PL aquagel-OH mixed
(cubic, spherical, and cubic/spherical hybrid). The column connected to a differential refractometer,
self-assembly patterns of the Ag-NPs were proved by with an RI detector and HPLC grade water as the
several visual techniques. Also, antimicrobial activity mobile phase at 32 °C. Dextran standard samples
of Ag-NPs including bactericidal (against Esche- were used for calibration. The particle size and zeta
richia coli and Staphylococcus aureus) and fungicidal potential of samples were determined using dynamic
assays (against Candida albicans) showed suitable light scattering (DLS, zetasizer ZS, Malvern) instru-
and acceptable results. 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
Materials and methods scanning electron microscope (SEM, Philips, XL30)
operated at 20 kV after coating the samples with gold
Chemicals film. High-resolution surface imaging studies were
performed using atomic force microscopy (AFM) to
Gelatin (GPC data: Mw = 115000 g/mol, Mn = 2800 estimate the surface morphology and the particle size
g/mol, wide distribution) and silver nitrate (AgNO3) distribution. The samples were imaged with the aid of
were purchased from Merck (Germany) and all the Dual-scope/Raster-scope C26, DME, using DS
solutions were prepared using double distilled–deion- 95-50-E scanner with vertical z-axis resolution of
ized water. 0.1 nm. The TEM images were obtained using a

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JEOL JSM-2100 transmission electron microscope concentrations (2.0, 5.0, 8.0, 10.0, 12.0, 15.0, and
with accelerating voltage of 200 kV. The samples for 20.0 lg/mL).
TEM analysis were prepared by spotting 10 lL of the
sample solution onto a Holey carbon TEM grid Antifungal assay
followed by drying before putting them into the TEM
sample chamber. Candida albicans (ATCC 90028) was obtained from
the American Type Culture Collection (ATCC).
Antimicrobial activity Fungal cells were cultured in a Mueller-Hinton broth
(MHB, Difco, France) with aeration at 28 °C.
The activity of Ag-NPs against three classes of The fungistatic activity of Ag-NPs was performed
microorganisms (Escherichia coli Gram-negative by the modified micro-dilution method (Panacek
bacteria, Staphylococcus aureus Gram-positive bac- et al. 2009) which enabled to determine the MICs of
teria, and Candida albicans fungi) was assessed. the Ag-NPs inhibiting the growth of the tested
C. albicans. The Ag-NPs were diluted in microtiter
Antibacterial assay plates by the MHB medium. The obtained concen-
trations of Ag-NPs in the dispersion were in the range
Escherichia coli (ATCC 51813) and Staphylococcus of 2.0, 5.0, 8.0, 10.0, 12.0, 15.0 and 20.0 lg/mL.
aureus (S. aureus, ATCC 27661) were cultivated in After the Ag-NP samples were diluted, a standard
Luria-Bertani (LB) broth medium (containing 10 g/L amount of the tested yeast was inoculated onto
peptone, 5 g/L yeast extract, and 10 g/L sodium microtiter plates so that the inoculum density in the
chloride). The visual turbidity of the tubes was noted wells was equal to 105–106 CFU/mL. After 36 h
both before and after incubation. The medium was incubation at 37 °C, the MIC was recorded as the
solidified by 20 g/L agar. The pH was adjusted to pH lowest concentration of the agent inhibiting the
7.0–7.2 with 1 M NaOH before autoclaving. The visible growth of microorganisms.
autoclaved LB medium (with 1% agar) that had been The minimum fungicidal concentration (MFC)
allowed to cool to approximately 40 °C was added to was determined by inoculating the contents of all
the bacteria-exposed slides. After the agar had wells of the testing plate onto a new microtiter plate
solidified, the slides were incubated at 37 °C for with MHB medium without Ag-NPs. Following 72 h
18 h, and the colonies were quantified. incubation, the MFC was recorded as the lowest
The Ag-NP samples were diluted and added to concentration of the tested agent inhibiting the visible
5 mL of LB medium with tested bacterial concen- growth of microorganisms.
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 Results and discussion
tubes contained only inoculated broth. The tubes
were incubated at 37 °C with shaking at 250 rpm for Reaction mechanism
18 h in a constant-temperature incubator. The min-
imum inhibition concentration (MIC) is defined as the It is well known that ultrasonic waves, prolonged
lowest concentration (lg/mL) at which there is no reflux, UV-irradiation, c-rays, and reducing agents
visible growth, and the minimum bactericidal con- can reduce the silver cations (Ag?) into Ag-NPs in
centration (MBC) is defined as the lowest concentra- the presence of a capping agent. In this study, silver-
tion at which no colony is observed (more than 99.9% gelatin core-shell nanoparticles have been prepared
lethality). The MICs were read by the visual turbidity by sunlight UV-irradiation. Sunlight-UV as a gratis
of the tubes noted both before and after incubation. source of reducing agent was applied and caused
After being properly diluted, aliquots from tubes facile and cheap preparation of the Ag-NPs without
(100 lL) that appeared to have little or no cell growth any surplus material. Gelatin, as a multi-purpose
were plated on LB agar plates to distinguish between (biocompatible, biodegradable, and non-toxic effec-
the bacteriostatic or the bactericidal effects. The Ag- tive capping/shaping agent) material, was utilized to
NP sample was used as prepared and tested at final inhibit the agglomeration of the Ag-NPs in solution.

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In this approach, the coordinated silver cations were synergy effect between the metal and protein for the
reduced into the Ag-NPs by photochemical reactions formation of multi-molecular structures. It is well
(Xu and Zhou 2008). The schematic representation of known that the intensity of scattered light relate with
this conversion was indicated in Scheme 1. A deci- sixth exponent of particle radius (r6), and it means the
sive reason which confirms the formation of the extent of this self-assembly in fresh solutions and under
gelatin-capped Ag-NPs was attained from the dialysis normal conditions is negligible. The zeta-potential
results. The utilized water for dialysis was analyzed curves of the same samples (Fig. 1c) give an attractive
by UV–Vis spectroscopy and interestingly, did not information about the silver-gelatin complex and the
show any peak due to the exit of Ag-NPs from the mechanism of chelation. The zeta-potential values of
dialysis cassette. Indeed, all produced Ag-NPs had the gelatin and the Ag-NPs prepared by 20 mg AgNO3
been surrounded by the gelatins (capping agents) and are 4.8 and 3.7 mV, respectively. The zeta-potential
any isolated bare Ag-NP did not exist in the solution. value of the gelatin indicates that the surface of the
gelatin molecule has net positive charge because the
DLS and zeta-potential analysis number of positive charge fragments (such as [–NH–
CNH2–NH2]? and –NH3?) on the surface of the gelatin
The size of four prepared samples of Ag-NPs was is larger than negative charge fragments (such as
measured by dynamic light scattering (DLS) which are –COO-), and it means that the utilized gelatin is type A
illustrated in Table 1. Figure 1 shows the DLS and and has isoelectric point greater than 7. The smaller
zeta-potential data for the pure gelatin and the Ag-NPs zeta-potential value of the Ag-NPs indicate that in
prepared by 20 mg AgNO3. Figure 1a obviously addition to non-ionic chelating groups of gelatin (such
represents that the size of the Ag-NPs (6.9 nm) is as –SH, –OH, ester and amide) and –COO- anionic
smaller than the size of gelatin molecules (9.9 nm). groups, the cationic groups of –NH3? in the form of
Also, the mono-modality and narrow distribution of the –NH2 can operate as chelating agent.
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 SEM/AFM/TEM analysis
well known that the proteins can self-assemble and
produce multi-molecular structures with larger sizes. The SEM pictures of four prepared samples of Ag-NPs
This phenomena is observed in the aforesaid curve, and indicate that the shapes of our nano-objects (Fig. 2a–d)
this is a remarkable point that the self-assembly of the alter gradually from spherical to spherical/cubic
gelatin has caused Ag-NP self-assembly because of the hybrid, cubic/spherical hybrid, and finally to cubic by

Scheme 1 Schematic
representation of reaction
mechanism

Table 1 The structural

Ag-NPs sample Morphology by (SEM, Average core-shell Average core-shell
information (shape and
prepared by AFM and TEM) diameter by DLS (nm) diameter by TEM (nm)
size) of synthesized Ag-NPs
5 mg AgNO3 Spherical 4.1 15
10 mg AgNO3 Spherical/cubic hybrid 5.6 18
20 mg AgNO3 Cubic/spherical hybrid 6.9 22
40 mg AgNO3 Cubic 5.2 10

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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
Fig. 1 The DLS curves of the gelatin & the Ag-NPs prepared
compared with SEMs. Figure 3a, b belong to the
by 20 mg AgNO3. a in terms of number (%) and b in terms of SEM images of the Ag-NPs prepared by 5 mg and
intensity of scattered light and c the zeta-potential curve of the 40 mg AgNO3, respectively, and Fig. 3c, d belong to
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

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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, GPC analysis
the AFM pictures have the same shape with smaller
size and confirm the self-assembly of the smaller Ag The gel permeation chromatography (GPC) curves of
nano-objects and production of the larger nanoparticles the pure gelatin and the Ag-NPs were provided in
in the SEM images. To obtain other confirming Fig. 6. The blue curve represents the molecular weight
evidences about self-assembly, the TEM images were distribution of the gelatin protein as a basis for
provided. The TEM pictures (Fig. 4) obviously prove comparison. The red curve indicates the GPC curve
the self-assembly pattern (Scheme 2) of smaller Ag of the Ag-NPs prepared by 5 mg AgNO3. Interest-
nano-objects to larger nano-objects, also. For shell ingly, it is seen that the apparent molecular weight of
detection of Ag-NPs, the AFM and TEM techniques the gelatin has been increased because of the formation
were utilized to measure the thickness of the shells. of a multi-molecular layer around Ag-NPs. On the
Figure 5 represents the AFM and TEM images of the other hand, the greater hydrodynamic volume of the
Ag-NPs prepared by 5 mg AgNO3. In this picture, the silver-gelatin system is due to the self-assembly and
shell thickness for the Ag-NPs was estimated as 3.7 and multi-molecular structure of silver-gelatin complex.
5.0 nm from TEM and AFM, respectively. The The green curve also shows the related curve of the
attained difference between the two methods can be Ag-NPs prepared by 10 mg AgNO3. As one can see,
attributed to the higher aggregation of gelatin in the the molecular weight of the gelatin has been increased
AFM image. again dramatically. The increase of the molecular

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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 and 1543 cm-1 are assigned to the amide I and II
chelation which causes hydrodynamic volume incre- bands of proteins (Burt et al. 2004), respectively,
ment and apparent molecular weight growth. Won- and the band observed at 1438 cm-1 can be
derfully, the violet curve (Ag-NPs prepared by 40 mg assigned to the C–N stretching vibrations of the
AgNO3) exhibits lower molecular weight for gelatin, amines (Sanghi and Verma 2009). With a subtle
and the reason might be related to the intra-molecular glance to the spectra, this is obvious that compared
silver-gelatin chelation due to the higher content of to pure gelatin, all the peaks of gelatin capped
silver. This intra-molecular silver-gelatin chelation Ag-NPs are sharper and narrower. The sharpness of
results in the hydrodynamic volume decrease of silver- the peaks can be explained with this fact that most
gelatin system and apparent reduction of gelatin of H-bonds between the amide groups in the
molecular weight. proteins will break because of higher tendency of
the amide groups for the formation of stronger
FTIR and UV–Vis spectroscopy analysis bonds with transition metal atoms (Burt et al. 2004).
In addition to narrowness of the peaks, red or blue-
FTIR measurements were carried out to identify the shifts may occur in their positions, attentive to the
interactions between the Ag-NP core and the gelatin type of bond existing in the chelating groups
nanoshell (Fig. 7). For the pure gelatin, the strong (Lu and Cui 2010). Finally, it can be concluded
broad peak at 3000–3600 cm-1 is characteristic of that the gelatin protein has formed a coating layer
the N–H stretching vibration. Two bands at 1651 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. 7 The FTIR spectra of the gelatin and the Ag-NPs

color. Among the metal nanoparticles, Ag-NPs are of

Fig. 6 The GPC curves for the gelatin and the Ag-NPs
particular interest owing to their third-order optical
nonlinearity and pronounced SPR absorption, which
In Fig. 8a, the curves indicate the characteristic have promising roles in many applications, such as
surface plasmon resonance (SPR) of Ag-NPs between optical waveguides and optical switches (Veerapan-
437 and 449 nm according to their orange-brownish dian et al. 2010). It is well known that the UV–Vis

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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

Fig. 8 a The UV–Vis spectra of the Ag-NPs and b the stabilized Ag-NPs (Pinto et al. 2010) and have more
changes in UV–Vis spectra of the Ag-NPs prepared by 5 mg
stability than the other synthesized by laser ablation
AgNO3 during 15 days
method in organic solvents (Tilaki et al. 2006).

spectrum of metal nanoparticles which is dominated Thermo-Gravimetric Analysis (TGA)/Differential

by the SPR, leads to the distinguished red or blue Thermal Gravimetry (DTG)/Differential Thermal
shift depending on the dielectric properties of the Analysis (DTA)
surrounding host matrix, or the environmental atmo-
sphere, in addition to the particle size and shape of TGA traces of gelatin and the Ag-NPs prepared by
nanoparticles. The stability of Ag-NPs preserved in 5 mg AgNO3 are presented in Fig. 9a. According to
the dark at room temperature were examined and this TGA curves, values related to the pure gela-
showed good stability results. Figure 8b represents tin such as T10 (210.1 °C), T30 (313.4 °C), T50
the UV–Vis spectrum of the Ag-NPs prepared by (356.5 °C), and char yield at 700 °C (1.2%) are
5 mg AgNO3 for 15 days. The absorbance spectra lower compared with gelatin-capped Ag-NPs (T10 =
show an increase in the absorbance of Ag-NPs 221.1 °C, T30 = 322.3 °C, T50 = 383.5 °C, Y =
solution and a red shift of kmax with time. It is found 5.8%). As one can see, the gelatin-capped Ag-NPs
that the solutions stored for 15 days at room temper- were found to be the most thermally stable samples
ature have similar absorbance values to those stored studied. It is well known that nanocomposite structure
for 1 year at 4.0 °C (Pinto et al. 2010). In compar- (gelatin-capped Ag-NPs) and the formation of coor-
ison to the prior synthesized Ag-NPs, the Ag-NPs dination bonds in the composition may act as heat

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barriers and as a consequence enhance the overall relatively, and this can be attributed to the type and
thermal stability of composition. For the better study thickness of the S. aureus membrane and its cell wall.
of thermal behavior of samples, their DTG/DTA The bactericidal test of the Ag-NPs toward E. coli was
curves were provided in Fig. 9b. The DTG curve of carried out, and the MBC is read by the presence of
the pure gelatin shows that its maximum decomposi- live bacteria on the LB agar plate. The first concen-
tion rate occurs in the two sharp peaks at 315 and tration with no indication of live bacteria (the MBC)
657 °C. Its DTA curve indicates that two decompo- for the Ag-NPs prepared by 5 mg AgNO3 toward
sition endothermic reactions represented by two broad E. coli was 15 lg/mL. The MIC and MBC results of
peaks occurred at 275–428 and 508–580 °C. In the all samples of Ag-NPs against E. coli & S. aureus are
Fig. 9b, the DTG/DTA curves of gelatin-capped summarized in Table 2 and because the MIC and
Ag-NPs were also represented. The DTG curve MBC values of Ag-NPs toward bacteria were unal-
indicates that the maximum decomposition rate of tered, the MBC results were not shown. According to
the gelatin-capped Ag-NPs occurs in the two sharp the table, it is seen when the shape of Ag-NPs change
peaks at 386 and 667 °C, and according to the DTA, at from spherical to cubic (from the Ag-NPs prepared by
this temperature an endothermic reaction causes its 5 mg AgNO3 toward the one prepared by 40 mg
decomposition. Totally, the comparison of TGA/ AgNO3), their antibacterial activities (both in the case
DTG/DTA curves confirms the higher thermal stabil- of E. coli and in the case of S. aureus) enhance and
ity of the gelatin-capped Ag-NPs. 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
Antimicrobial activity investigation
relate with antibacterial activity (Pal et al. 2007). In
the case of E. coli, it will predict that the Ag-NPs
The antibacterial activity of Ag-NPs was tested using
could have long-lasting activity because it is found
the MIC and MBC tests. Different concentrations of
that cationic biopolymers could capture negatively
the tested samples were incubated with E. coli
charged bacteria (bacteria-adsorbing effect), and since
(as Gram-negative bacteria) and S. aureus (as Gram-
the biopolymer kills/captures the bacteria, the cell
positive bacteria) in LB broth. Bacterial growth was
membrane remnants/dead bacteria presumably remain
studied by visual observation as indicated by turbid-
adsorbed on the polymer surface, preventing further
ity. Lack of turbidity may correspond to either the
antibacterial activity. In contrast, the silver nanopar-
very low bacterial growth (a bacteriostatic effect) or
ticle-gelatin core-shell system may continue to kill the
the complete killing of bacteria (a bactericidal effect).
bacteria even after the surface is completely covered
If the tested material did not kill but only inhibited the
by dead bacteria, thereby showing long-lasting activ-
growth of bacteria (bacteriostatic), bacteria will grow,
ity (Sambhy et al. 2006). This effect has been proved
and the colonies will be observed upon plating. If the
for silver-incorporated chitosan film, earlier (Wei
tested material is bactericidal, no bacterial colony
et al. 2009).
would be observed. Table 2 shows the bacteriostatic
The fungistatic activity of the Ag-NPs against
test results of Ag-NPs toward E. coli. It is obvious that
C. albicans as a model for fungi was determined by
the MICs against S. aureus are higher than E. coli
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
Table 2 The anti-bacterial and anti-fungal activity (MIC 5 lg/mL, respectively, and the MIC values of this
values (lg/mL)) of Ag-NPs compound were approximately the same level as
Ag-NPs sample E. coli S. aureus C. albicans those of amphotericin B, showing MIC values of
prepared by bacteria bacteria fungus 2.5–5 lg/mL toward the fungal strains tested. Along
5 mg AgNO3 15 15 12
with the study of the fungistatic activity of Ag-NPs,
10 mg AgNO3 10 15 10
their fungicidal activity against the tested yeast
was simultaneously assessed. The obtained MFCs
20 mg AgNO3 8 12 5
(200 lg/mL) are considerably higher in comparison
40 mg AgNO3 5 8 2
to MICs and of course, this is normal behavior and

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have been formerly seen (Panacek et al. 2009). Conclusions

Similar to the antibacterial activity of the Ag-NPs,
these results can communicate to the shape of In summary, we have developed a highly facile and
Ag-NPs with antifungal activity. The comparison of inexpensive approach to prepare gelatin-capped silver
the antifungal activity of the Ag-NPs with their nanostructures, which are stable in the aqueous
antibacterial activity clearly showed that the Ag-NPs solutions. It is predicted that this green synthesis
inhibit yeast growth at lower concentrations than in procedure can be easily extended to other similar
the case of the bacterial growth inhibition. This systems, which is valuable for the utilization of other
phenomenon has been previously seen by another proteinaceous bioresources. Ag-NPs exhibited a
group (Panacek et al. 2009) also. strong anti-microbial activity against E. coli and
Although the mechanism by which the nanoparti- S. aureus bacteria and potent antifungal activity toward
cles are able to penetrate the microorganisms is not C. albicans which suggested that the Ag-NPs could
totally understood, yet in the case of Ag-NPs, several effectively suppress bacterial/fungal proliferation and
studies have been carried out. These reports illustrate protect wounds from bacterial/fungal invasion.
that Ag-NPs can change the membrane morphology
of microorganisms and may produce a significant Acknowledgments The authors thank Dr. G. R. Bardajee,
Dr. M. Adeli, Mr. H. Rezanejad, Mr. H. Abroshan, Mr.
increase in its permeability and affect proper trans- M. Akhlaghi, and Mr. M. Fakourpour for their valuable help in
port through the plasma membrane (Sondi and preparation of this article, making the samples, and data taking.
Salopek-Sondi 2004). The observation of Ag-NPs
attached to the cell membrane and inside the bacteria
is fundamental and acceptable evidence for the
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