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Shape-specific silver nanoparticles prepared by

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microwave-assisted green synthesis using

Cite this: RSC Adv., 2015, 5, 95433
pomegranate juice for bacterial inactivation and
removal†
Ekta Roy,a Santanu Patra,a Shubham Saha,a Rashmi Madhuri*a
and Prashant K. Sharmab

Received 10th September 2015
Accepted 16th October 2015
DOI: 10.1039/c5ra18575k
www.rsc.org/advances
Herein, we have synthesized four different shapes (spherical, oval, rod and flower shape) of silver
nanoparticles (AgNPs) using a green synthesis approach via microwave-assisted synthesis. The
nanoparticles were synthesized using pomegranate juice as a novel reducing agent. The synthesized
AgNPs were characterized by UV-vis spectroscopy, X-ray diffraction (XRD), scanning electron
microscopy (SEM) and tunnelling electron microscopic (TEM) analysis. The as-prepared AgNPs show
a very rapid, effective, shape-specific and dose-dependent bacteriostatic/bactericidal effect towards four
different bacterial strains (two Gram negative and two Gram positive). The analysis was determined by
different methods such as a disc diffusion study, growth curve analysis, minimum inhibitory
concentration and minimum bactericidal concentration determinations. Amongst the four different
shaped AgNPs, the flower shaped AgNPs demonstrated the best results and mediated the fastest
bactericidal activity against all the tested strains at similar bacterial concentrations. The results were also
confirmed by confocal microscopic analysis. Additionally, the flower shaped AgNPs have the ability to
non-specifically remove various bacterial strains from a real water sample with
98% removal efficiency,
as compared to other synthesized different shaped nanoparticles.

reported that silver nanoparticles (AgNPs) with a size in the

Introduction
A big challenge in the eld of pharmaceuticals and biomedi-
cines is resistant of human beings towards pathogens or bac-
teria's. In this, the anti-biotic resistance has acquired a large
range of fear for emergence and re-emergence of multidrug-
resistant (MDR) pathogens and parasites.1,2 When a person gets
infected with MDR bacteria, their cure becomes very difficult,
resulting in loss of money as well as time in hospitals. The MDR
bacteria infected patient requires multiple treatments of broad-
spectrum antibiotics, which are less effective, more toxic and
more expensive.3,4 Therefore, some modications and develop-
ment in anti-bacterial compounds with improved bacteriostatic
and bactericidal effects are now a high priority in the eld of
research.5,6
From past decades, silver has been used as an antimicrobial
compound to deal with infections, burns and chronic wounds
due to its low toxicity to the human body. Many scientists have
a
Department of Applied Chemistry, Indian School of Mines, Dhanbad, Jharkhand 826
004, India. E-mail: rshmmadhuri@gmail.com; Tel: +91 9471191640
b
Functional Nanomaterials Research Laboratory, Department of Applied Physics,
Indian School of Mines, Dhanbad, Jharkhand 826 004, India
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra18575k
range of 10–100 nm showed strong bactericidal potential
against both Gram-positive and Gram-negative bacteria.7
Though the mechanism of antibacterial killing or prevention is
not very clear to date, some of the authors have discussed the
mechanism based on their own obtained results. Accordingly,
AgNPs may penetrate the bacterial cell wall and modulate
cellular signaling by dephosphorylating peptide substrates on
tyrosine residues.8–10 During the study, it was also found that
the antibacterial property of AgNPs depends up on the size,
morphology, stability and (chemical and physical) properties of
the nanoparticles. The shape, size and other factors are
strongly inuenced by the experimental conditions viz.,
kinetics of the interaction of metal ions with reducing agents,
and adsorption processes of stabilizing agents with metal
nanoparticles.11 Generally, specic control of the shape, size
and distribution of the produced nanoparticles could be ach-
ieved by changing the methods of synthesis, the reducing
agents and stabilizers.12–15
During the last three decades, synthesis of metal nano-
particles using natural resources i.e. green technology and/or
synthesis has been increased a lot. Using natural precursors
or natural compounds as intermediate chemicals in nano-
particle synthesis is not only an environmental friendly step, it

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is a step towards less-harmful and more cost-effective chemical Experimental section

synthesis.16 In the literature, several works have been reported
for the synthesis of AgNPs using a green synthesis approach.
Vinod et al. have reported the synthesis of Ag, Au and Pt
nanoparticles using a natural hydrocolloid gum kondagogu
(Cochlospermum gossypium) as a precursor molecule.17 Loges-
wari et al. reported the synthesis of AgNPs from commercially
available plant powders i.e. Solanum trilobatum, Syzygium
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Reagents and instruments
Silver nitrate (AgNO3) and cetyltrimethylammonium bromide
(CTAB) were purchased from Fluka. All chemicals and reagent
used here were of analytical grade or higher.
The synthesized nanoparticles were characterized by Field
emission scanning electron microscope (FE-SEM, Zeiss, model

cumini, Centella asiatica and Citrus sinensis.18 AgNPs have also Super 55). The powder X-ray diffraction (XRD) study was carried
been synthesised from three medicinal leaf extracts, Musa bal- out on a Bruker D8 Focus X-ray diffractometer instrument using
bisiana (banana), Azadirachta indica (neem) and Ocimum tenui- a Cu target radiation source. Transmission electron microscopy
orum (black tulsi), by Banerjee et al.19 (TEM) studies were performed on a Tecnai 30 G2 S-Twin elec-
Although, several studies have been reported the synthesis of tron microscope operated at 300 kV accelerating voltage. UV-
AgNPs using chemical routes and their applications in anti- visible spectroscopic characterization was done on a Perkin-
bacterial studies, the synthesis of shape-specic nanoparticles Elmer Lambda 35 (Singapore) spectrophotometer.
using a green synthesis approach is rarely reported. The effect of
shape specic nanoparticles on bacterial growth was rst
Preparation of the pomegranate extract
explored by Pal et al.,20 where three different shaped AgNPs
(spherical, rod shaped and truncated) were reported. It was The pomegranate was collected from a local market on the basis
found that the truncated AgNPs had a better response for of cost effectiveness and ease of availability. Fresh and healthy
antibacterial purposes (Sodium borohydride). Similarly, Dong pomegranate were collected and rinsed thoroughly rst with tap
et al. have reported the synthesis of triangular nanoprism and water followed by distilled water to remove all the dust and
spherical AgNPs and it was reported that the triangular-shape unwanted visible particles. Seeds were separated from the fruit,
nanoparticle had a better antibacterial property than the crushed and juice was collected in a beaker (250 mL). To this,
spherical one due to its geometric structure and geometric 100 mL distilled water was added and the mixture was boiled for
plane.21 To the best of our knowledge, a green chemistry about 20 min. Aer that, the juice was ltered and stored at
approach for the synthesis of shape-specic AgNPs as antimi- 4  C, before use.
crobial agents has not been reported yet.
The present work has focused on the development of an Synthesis of the different shaped silver nanoparticles (AgNPs)
easy and one-pot synthesis of AgNPs by an environmentally
For the synthesis of the different shaped AgNPs, AgNO3 was
friendly procedure (microwave assisted method) with pome-
used as the precursor compound with CTAB as the stabilizer
granate fruit juice as the reducing agent. Microwave irradiation
and pomegranate juice as the reducing agent. The amount of
has several advantages in comparison to the traditional heat-
constituents and reaction conditions were varied to synthesize
ing methods. It produced uniform heating of the solution
the different shaped AgNPs. The procedures are given below
(short thermal induction period) and as a result, more homo-
and shown in Scheme 1.
geneous nucleation (less-time) with a short crystallization time
Spherical shaped AgNPs (S-AgNPs). For this, an aqueous
is obtained at a much lower cost.22 In addition, herein,
solution of AgNO3 (0.01% by weight) was prepared in a 250 mL
pomegranate fruit juice was used as a reducing agent which
contains mainly gallic acid, ellagic acid and avonol glycosides
that are able to reduce silver nitrate into AgNPs very effi-
ciently.23 The nanoparticles were prepared as four different
shapes, namely, spherical, oval, rod and ower. The synthe-
sized AgNPs have been characterized by eld emission scan-
ning electronic microscopy (FE-SEM), transmission electron
microscopy (TEM), UV-visible spectroscopy and powder X-ray
diffraction (XRD) techniques. The bactericidal activity of the
as prepared different shaped AgNPs against the pathogenic,
MDR as well as multi-drug susceptible strains of bacteria such
as Pseudomonas aeruginosa (Gram negative), ampicillin-
resistant Escherichia coli (Gram negative), Bacillus subtilis
(Gram positive), and methicillin-resistant Staphylococcus
aureus (Gram positive) were studied. It was found that the
shape of the nanoparticle plays a very important role in the
killing of the bacterial strain. In addition, some real water
samples were also used to explore the water purication
impact of the synthesized nanoparticles. Scheme 1 Graphical representation for synthesis of shape-specific
silver nanoparticle using green synthesis approach.

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volumetric ask. 50 mL was then taken into a conical ask into
which 1.82 g of CTAB was added. To the mixture, 3.0 mL of
freshly prepared pomegranate juice was added and placed
inside a microwave oven for complete bioreduction at 300 W for
5 min. The color of the solution changed to brown, which
indicated the formation of AgNPs. The solution was kept under
stirring at room temperature for a further 12 h. The resulting
nanoparticles were collected, centrifuged and stored in vacuum
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desiccators.
Oval shaped AgNPs (O-AgNPs). Oval shaped silver nano-
particles were synthesized by a seed-mediated growth method.24
Firstly, a seed solution for O-AgNPs was prepared by addition of
1.0 mL cold aqueous solution of pomegranate seed extract into
a mixture of 0.25 mM AgNO3 and 0.25 mM trisodium citrate
(10.0 mL each). The resultant mixture was stirred for 2 hours
and stored as a seed solution. Similarly, a growth solution was
also prepared by mixing 7.0 mL of 0.020 M CTAB, 0.50 mL of
10.0 mM AgNO3 and 0.50 mL of pomegranate juice. To the as
prepared growth solution, 0.50 mL of the seed solution was
injected and incubated at 27  C for 12 h. The resulting nano-
particles (O-AgNPs) were collected by centrifugation, re-
dispersed in deionised water, washed, dried and stored in
vacuum desiccators.
Rod shaped AgNPs (R-AgNPs). For the preparation of the rod
shaped AgNP growth solution, 0.2 M CTAB (10 mL) and 4.0 mM
AgNO3 (1.2 mL) were added to 25 mL distilled water. To the
mixture, 0.5 mL pomegranate juice and 20 mL oval shaped
nanoparticle solution was added and the mixture was kept in
a water bath at 30  C for 24 h. The resulting nanoparticles (R-
AgNPs) were collected by centrifugation, re-dispersed in
deionised water, washed, dried and stored in vacuum
desiccators.
Flower shaped AgNPs (F-AgNPs). For the synthesis of the
ower shaped AgNPs, 0.2 M CTAB (5 mL) and 4.0 mM AgNO3 (3
mL) were added to 33.0 mL distilled water. To the mixture, 8.0
mL pomegranate juice and 10.0 mL of rod shaped nanoparticle
solution was added and the solution was kept at 30  C for 24 h.
The resulting nanoparticles (F-AgNPs) were collected by centri-
fugation, re-dispersed in deionised water, washed, dried and
stored in vacuum desiccators.
Preparation of bacterial samples and antibacterial study
The various shaped AgNPs synthesized using pomegranate juice
were applied to explore their shape-specic properties towards
different bacterial strains i.e. Pseudomonas aeruginosa (Gram
negative), Escherichia coli (Gram negative), Bacillus subtilis
(Gram positive), and Staphylococcus aureus (Gram positive). The
study was performed by different analysis methods: (1) a disc
diffusion method, (2) minimum inhibition concentration
(MIC), (3) minimum bactericidal concentration (MBC) and (4)
growth kinetics.
Disc diffusion study (DDS). The disc diffusion study (DDS)
was carried out with a bacterial strain (105–106 CFU mL1)
cultured separately on Luria–Bertani (LB) agar plates. Sepa-
rately, 6.0 mm lter paper disks were impregnated with 1.0 mL
of the different shaped AgNP sample solutions and gently
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placed on the center of the bacteria growth on the LB agar plates
and incubated overnight at 37  C. The inhibition zone was
monitored and the diameter of the inhibition zone was calcu-
lated by visualizing the blank space around the lter paper to
evaluate the shape-specic antibacterial performance of the
AgNPs.
Growth curve. For the growth kinetic test, the bacterial
strains of all four bacteria [Pseudomonas aeruginosa (P. aerugi-
nosa), Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), and
Staphylococcus aureus (S. aureus)] were prepared in LB medium
and incubated overnight at 37  C. Aer that, the incubated
bacteria were injected into fresh media and grown at 37  C with
shaking at 200 rpm. To the solution, various concentrations of
the different shaped AgNPs were added and optical density
(O.D.) was measured at different time intervals. Bacterial
growth rates were measured by monitoring the optical density
at 600 nm (O.D600) using a UV-visible spectrophotometer.
Determination of minimum inhibitory concentration (MIC).
To examine the minimum inhibitory concentration (MIC) for all
four bacterial strains, silver nanoparticles of various concen-
trations (0.05–25.00 mg L1) were introduced into sterile asks
containing 50.0 mL nutrient broth. The mixture was sonicated
for 15 minutes to avoid any type of aggregation in the solution.
To the ask, 5.0 mL of freshly prepared bacterial suspension (1
 105 to 1  106 CFU mL1) was added and the culture media
was incubated for 24 hours (37  C) in an orbital shaker at high
rotation speed (120 rpm). Aer 24 h incubation, 5.0 mL of fresh
bacterial solution was further added to the ask and incubated
at similar conditions for the next 24 h. The growth or no growth
of the bacterial colony was determined by visual observation.
The lowest concentration that is the highest dilution required to
arrest the growth of bacteria was regarded as the minimum
inhibitory concentration (MIC). For accuracy of result, all of the
concentration values were analyzed three times and the relative
standard deviation was less than 1%. The aqueous solution of
AgNO3 was used as a control or standard.
Estimation of minimum bactericidal concentration (MBC).
The minimum bactericidal concentration (MBC) can be dened
as the lowest concentration of AgNPs that kills 99.9% of the
bacteria and it was determined from the batch culture studies
done for the measurement of MIC values. The sample asks
that appeared to have little or no bacterial growth were selected
and a loop full from each ask was plated on fresh solid media
(2% agar in nutrient broth) and incubated at 37  C for 24 hours.
The nanoparticle concentration causing a bactericidal effect
was selected based on the absence of colonies on the agar plate
and the lowest concentration causing a bactericidal effect was
reported as the MBC. For accuracy, each experiment was done
thrice to assess the MBC values.
Cell death or % killing. To study the cell death or percentage
killing of bacteria, rstly, the bacterial strain was spread on the
agar plates and incubated at 37  C, followed by addition of 100.0
mL of the nanoparticle (20 mg L1). The plate was nally incu-
bated at 37  C for 24 h and the bacterial colony was counted at
regular time intervals of 2 hours. The antibacterial drug
(streptomycin sulphate) was used as a positive control and each
result was presented as an average of three independent
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experiments. The death rate of the bacteria was calculated using
the following eqn (1).7
Death rate (%) ¼ [(counts in the control)
 (counts upon incubation with the nanoparticle)]/
(counts in the control) (1)
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Removal of bacteria by ower shaped AgNPs
Nowadays, the water purication system including ltration
membranes etc. are modied with nanoparticles to remove
bacterial contaminations more efficiently. Herein, we have also
tried to explore the water purication aspect of the ower sha-
ped AgNPs for removal of bacterial contamination from water
samples collected from different areas of our state. For the
analysis, the diluted bacterial solution (1  103 CFU mL1) was
added in a glass vial containing 10.0 mL of F-AgNPs (20.0 mg
L1). The mixed solution was placed in a rotary shaker for 15
min to ensure the effective binding between the bacteria and
nanoparticles. Aer that, the nanoparticles were ltered and
separated from the mixture solution and the remaining super-
natant was used to analyze the bacterial count by a conventional
surface plate count method. The bacteria removal efficiency of
the F-AgNPs was calculated using eqn (2):
Removal efficiency (%) ¼ (CFU0  CFUt)/CFU0  100 (2)
where CFU0 and CFUt are the initial and residual numbers of
bacterial colonies in the samples.25
Result and discussion
Characterization of AgNPs
UV-visible spectroscopy. Fig. S1,† shows the UV-vis spectra of
the silver nanoparticles synthesized with the help of pome-
granate juice as a reducing agent. The UV-vis spectrum of
pomegranate juice solution was taken as a reference and no
absorption spectrum was observed. UV-visible spectroscopy is
one of the basic techniques for structural characterization of
metal nanoparticles due to their surface plasmon resonance
(SPR) behavior.26 As shown in Fig. S1,† all the spectra possess
the characteristic SPR band of AgNPs at 420 nm, which conrms
the successful synthesis of nanoparticles.27 According to the
literature, the absorption spectra of metal nanoparticles, gov-
erned by SPR, show a shi to a longer wavelength with an
increase in the particle size.21 However, according to Mie’s
theory, only a single SPR band is expected in the absorption
spectra of the spherical shaped nanoparticles, whereas other
nanoparticles could give rise to two or more SPR bands
depending on their shape, due to the decrease in the symmetry
of the nanoparticles.28 Herein, the spherical and oval shaped
nanoparticles show one adsorption band at 420 and 430 nm,
respectively, whereas the rod and ower shaped nanoparticles
show two adsorption bands at 437 and 660 nm and 450 and 680
nm, respectively. This conrms the large size and multi-plane
structure of the synthesized nanoparticles. Herein, the various
shapes of the AgNPs were developed due to the agglomeration
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of the spherical shaped nanoparticles, which rst forms a rod
shaped nanoparticle and then these rod shaped nanoparticles
agglomerate and lead to the ower shaped structure. It may be
possible that during this structural transformation, some
amount of spherical as well as rod-shaped nanoparticles may be
present along with nanoowers, which leads to weak UV
adsorption peaks at 660 and 680 nm, respectively. However,
most of the spherical/rod shaped particles are transformed to
ower shaped nanoparticles, which gives an additional peak in
the UV spectra and is later on also supported by SEM images. In
addition, the red-shi in wavelength of the nanoparticle also
supports the mechanism shown for preparation of different
shaped nanoparticles i.e. multi-nanoparticle aggregation
(Scheme 1).
XRD spectra. Crystalline information of the synthesized
nanoparticles was obtained by XRD measurement. Fig. 1 shows
the XRD patterns of the spherical (curve 1), oval (curve 2), rod
(curve 3), and ower shaped nanoparticles (curve 4). All the
synthesized nanoparticles show ve distinct diffraction peaks at
38.5 , 44.4 , 64.6 , 77.4 and 81.7 , which correspond to the
(111), (200), (220), (311) and (222) planes of face centered cubic
silver, respectively. The resultant data was matched with the
JCPDS le number 87-0720. The XRD patterns clearly suggest
that the AgNPs synthesized from the pomegranate seed extract
were crystalline in nature and were well matched with
a previous report.29
SEM analysis. An SEM technique was employed to visualize
the size and shape of the synthesized silver nanoparticles. The
corresponding SEM images of the synthesized AgNPs (various
shapes) are shown in Fig. 2. Fig. 2A shows the SEM image of the
spherical shaped AgNPs. The S-AgNPs are round in shape and
have an average size of
50 nm. As shown in Fig. 2B, the O-
AgNPs have a slightly larger size and an elongated shape in
comparison to the S-AgNPs. However, the rod shaped AgNPs
have a diameter of
100 nm and length of
250 nm (Fig. 2C).
The rod shaped AgNPs get agglomerated to form a ower sha-
ped nanoparticle, which is clearly shown in the FE-SEM image
(Fig. 2D). To get a closer view and a more magnied image of the
Fig. 1 Powder X-ray diffraction patterns of (1) spherical, (2) oval, (3)
rod and (4) flower shaped silver nanoparticles.
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Fig. 2 FE-SEM images of the (A) spherical, (B) oval, (C) rod and (D) flower shaped silver nanoparticles at low and (E) high magnifications.
F-AgNPs, their high magnication image was also recorded and
is shown in Fig. 2E. The SEM images also support the weak UV
peaks obtained at 660 and 680 nm for the R-AgNPs and F-
AgNPs, respectively. Therefore, the SEM study clearly supports
the formation of different shaped silver nanoparticles with their
morphological dimensions, as shown in Scheme 1.
TEM analysis. The TEM images of the different shaped
AgNPs (spherical, oval, rod and ower) are illustrated in Fig. 3,
where it can be easily visualized that well-dened shapes of
AgNPs are formed. As shown in the Fig. 3A and B, the spherical
and oval shape nanoparticles have small size in which approx-
imately 85–90% of nanoparticles possess the spherical and/or
oval shape. A single TEM image of the rod shape is presented in
Fig. 3C, and reveals that the diameter of the rod shape is
100
nm. Fig. 3D, shows a monodispersed large ower shaped AgNP
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with an average size of 550 nm. The ower shaped AgNP has
a very dark contrast image, which indicates the rich three-
dimensional (3D) structures of the F-AgNPs, likely to be
formed by self-assembly of two-dimensional (2D) rod shaped
AgNPs.30 The single-crystalline nature of the synthesized AgNPs
was also conrmed by a selected area electron diffraction
(SAED) pattern shown in Fig. S2.† It also indicates that the pure
face-centered cubic (fcc) silver structure were oriented with
(1 1 1) planes as the basal plane.31 The obtained TEM images are
in good agreement with the earlier synthesized AgNPs by other
research groups like Bakshi,32 Lou et al.,33 Zaheer and
Rauddin,31 and Mirkin et al.34 This suggests that natural
resources, either in the form of a precursor or reducing agent,
have much potential in the eld of nanomaterial synthesis.
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Fig. 3 HR-TEM images of (A) spherical, (B) oval, (C) rod and (D) flower
shape silver nanoparticles.
Antibacterial study
Disc diffusion method. The antibacterial activity of different
shaped AgNPs were studied by both disc diffusion or the inhi-
bition zone method. The nanoparticles were impregnated in
a small disc, placed on the plates occupied by different bacterial
Fig. 4 Disk diffusion test of different shaped silver nanoparticles
against different bacterial strains: (A) E. coli, (B) Pseudomonas aeru-
ginosa, (C) B. subtilis and (D) S. aureus. Different shaped-AgNPs were
used in each plate and they were represented by numbers: (1)
spherical, (2) oval, (3) rod and (4) flower shaped AgNPs.
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strains and the zone of inhibition around the disk (the clear
zone around the disc) was monitored, calculated and portrayed
in Fig. 4A–D. The different nanoparticles are depicted by
a number i.e. (1) represents S-AgNPs, (2) O-AgNPs, (3) R-AgNPs
and (4) F-AgNPs. From the gure, it is clear that in the case of
all four bacteria, the bacterial growth inhibition was lowest in
the case of the spherical shaped AgNPs, for which the zones of
inhibition were 8 mm (E. coli), 10 mm (Pseudomonas aerugi-
nosa), 11 mm (B. subtilis) and 6 mm (S. aureus), and highest with
the ower shaped AgNPs, for which the zones of inhibition were
15 mm (E. coli), 15 mm (Pseudomonas aeruginosa), 17 mm (B.
subtilis) and 13 mm (S. aureus). The maximum antibacterial
activity of nanoparticles can be ordered in the following
sequence:
S-AgNPs < O-AgNPs < R-AgNPs < F-AgNPs
Here, the maximum zone of inhibition (17 mm) was observed
for F-AgNPs and the minimum for S-AgNPs, which may be
attributed to the rounded edges of the spherical nanoparticles,
which had poor interaction with bacterial cells, resulting in low
bactericidal activity.35 Similarly, amongst all the four bacterial
strains, the best performance was observed with B. subtilis.
Growth curve analysis. A bacterial inhibition growth curve
was used to study the growth kinetics of E. coli, P. aeruginosa, B.
subtilis and S. aureus with prepared bacterial samples (Fig. S3†).
The optical density at 600 nm (OD600) was measured to monitor
bacterial growth. As shown in Fig. S3A–D,† the bacterial growth
(for all four strains) was delayed when the different shaped
AgNPs were added at the same concentration as compared to
the control. The growth of all the bacterial strains was
completely inhibited by the F-AgNPs, however, the least effect
was observed with S-AgNPs. The results conrm that the F-
AgNPs possessed a much more enhanced antimicrobial prop-
erty than the S-AgNPs. This may be due to the large surface area
provided by the F-AgNPs, which results in better interaction
with bacterial membranes.
MIC and MBC test. For the evaluation of the minimum
concentration of all the AgNPs required for inhibition of
bacterial growth, a MIC test was performed. For such a study, an
aqueous solution of AgNO3 was used as a standard or reference.
The results are portrayed in Table 1, in terms of positive (+) and
negative () symbols, where “” represents no growth and “+”
represents the growth of bacteria. As shown in the table, the
Gram-negative bacterial strain of E. coli and P. aeruginosa show
a 0.6 mg L1 MIC value for F-AgNPs, 1.0 mg L1 for R-AgNPs, 2.0
mg L1 for O-AgNPs and 3.0 mg L1 for S-AgNPs in comparison to
an 8.0 mg L1 MIC for AgNO3 aer 24 h incubation (Table 1),
which is approximately, 16, 8, 4, and 3 times lower than the
standard solution of silver. It was also found that MIC values at
48 h were higher than those at 24 h because of the addition of
a fresh bacterial strain as well as more time for growth. Even at
48 h, a low MIC value of 1.0 mg L1 was observed for F-AgNPs, to
inhibit the growth of Gram negative bacteria, which suggests
the very good antibacterial properties of F-AgNPs.
Similarly, for the Gram positive bacterial strains (B. subtilis
and S. aureus), the lowest MIC values were found for F-AgNPs,
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Table 1 Minimum inhibitory concentration tests of various shape silver nanoparticles on Gram negative and Gram positive bacteria

Gram negative bacterial strain

E. coli P. aeruginosa

S-AgNPs O-AgNPs R-AgNPs F-AgNPs AgNO3 S-AgNPs O-AgNPs R-AgNPs F-AgNPs AgNO3
Concentration
(mg L1) 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h
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25.0                    
20.0                    
15.0                    
12.0                    
10.0                    
8.0          +          +
6.0         + +         + +
3.0  +       + +  +       + +
2.0 + +  +     + + + +  +     + +
1.0 + + + +  +   + + + + + +  +   + +
0.60 + + + + + +  + + + + + + + + +  + + +
0.50 + + + + + + + + + + + + + + + + + + + +
0.40 + + + + + + + + + + + + + + + + + + + +
0.30 + + + + + + + + + + + + + + + + + + + +
0.10 + + + + + + + + + + + + + + + + + + + +
0.05 + + + + + + + + + + + + + + + + + + + +

Gram positive bacterial strain

Concentration
(mg L1) B. subtilis S. aureus

25.0                    
20.0                    
15.0                    
12.0                    +
10.0                   + +
8.0                   + +
6.0           +        + +
3.0          + + +  +     + +
2.0          + + + + +  +   + +
1.0          + + + + + + +  + + +
0.60  +       + + + + + + + + + + + +
0.50 + +       + + + + + + + + + + + +
0.40 + +  +     + + + + + + + + + + + +
0.30 + + + +  +   + + + + + + + + + + + +
0.10 + + + + + +  + + + + + + + + + + + + +
0.05 + + + + + + + + + + + + + + + + + + + +

0.1 mg L1 and 1.0 mg L1, respectively, and an increase in the
value was obtained with same trend as presented for Gram
negative bacteria i.e. F-AgNPs < R-AgNPs < O-AgNPs < S-AgNPs
(Table 1). Therefore, it is clearly demonstrated that the shape
of nanoparticles plays a very important role on their antibac-
terial activity and herein, the multi-plane structure i.e. F-AgNPs
shows the maximum response towards all the four bacterial
strains.
As discussed in the UV and SEM analysis, during the struc-
tural transformation, the rod-shaped or ower-shaped AgNPs
may contain some amount of oval or spherical nanoparticles.
Their separation is not possible. Therefore, to study their
coordinated effect on the antibacterial activity, we have con-
ducted a MIC study with different mixtures of AgNPs. The cor-
responding data are portrayed in Table S1.† As shown in the
table, a mixture containing the same concentration of S-AgNPs
and F-AgNPs was unable to inhibit the growth of bacteria
(1.0 mg L1 for Gram negative and 0.1 mg L1 for Gram positive),
however, a similar concentration of F-AgNPs could effectively
inhibit the growth (Table S1† and Table 1). The study suggests
that the mixing of nanoparticles was unable to show a strong
antibacterial activity, due to the presence of different surface
areas and shapes. However, the individual nanoparticles, with
large surface areas, have more potential towards inhibiting the
growth of bacterial strains.
Comparative study for antibacterial potential of different
shaped silver nanoparticles. It was already demonstrated by
disc diffusion and a growth kinetics study that bactericidal
efficacy of AgNPs is signicantly enhanced as the shape of
nanoparticles is changes from spherical to ower shaped. To

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RSC Advances
further explore the shape-dependent antibacterial efficacy of the
synthesized AgNPs, a test was performed against four bacterial
strains. For comparison, the MBC value of individual nano-
particles was considered for a particular bacterial strain, which
could be sufficient to not only resist the growth but kill the
bacteria. The initial bacterial concentration was adjusted to 105
to 106 CFU mL1 in 50.0 mL nutrient broth, while the time
period for the antibacterial activity was estimated up to 120
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minutes, as the minimum time necessary to achieve a 99%
bacteriostatic effect and 99.9% bacterial killing (bactericidal
effect) is expected to fall within this duration.7
Fig. 5, represents the size dependent killing kinetics of S-
AgNPs, O-AgNPs, R-AgNPs and F-AgNPs against the E. coli (A),
P. aeruginosa (B), B. subtilis (C) and S. aureus (D) bacterial strains
used in the earlier studies. In the case of Gram negative
bacteria, F-AgNPs and R-AgNPs showed bacteriostatic (99.36%
and 99.00% killing) and bactericidal effects (99.90% and
99.00% killing) in 60 and 90 minutes respectively, while, O-
AgNPs and S-AgNPs displayed almost similar bactericidal effi-
cacy and requires 120 minutes to achieve
99.9% bacterial
reduction. This clearly indicates that the ower shaped AgNPs
mediate the fastest bactericidal action by rst quickly inhibiting
bacterial proliferation within 60 minutes and thereaer
achieving bacterial reduction over the next half an hour.
Similarly, for the Gram positive bacteria, F-AgNPs and R-
AgNPs show antibacterial activity by achieving bacteriostatic
(99.42% and 99.21% killing) and bactericidal effects (99.93%
and 99.30% killing) within 90 and 120 minutes, respectively.
Fig. 5 Bar diagrams representing the killing kinetics of bacterial strains exposed to different silver nanoparticles at their MBC values. (A) E. coli, (B)
P. aeruginosa, (C) B. subtilis and (D) S. aureus.
95440 | RSC Adv., 2015, 5, 95433–95442
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Although, the similar efficacy for all bacterial strains was ach-
ieved in 120 minutes using O-AgNPs and S-AgNPs, out of the
four bacterial strains tested, S. aureus was found to be the least
sensitive against AgNPs. This can be explained on the basis of
the time taken to achieve the bacteriostatic effect, which was
extended from 60 minutes for F-AgNPs and R-AgNPs to 90 and
120 minutes for O-AgNPs and S-AgNPs, respectively.
Mechanism for antibacterial activity of different shaped
nanoparticles. During the various antibacterial studies in this
work, we have found that different shaped nanoparticles show
different results and on that basis we have concluded that the F-
AgNPs have the best activity, in comparison to the other AgNPs.
The observed trend can be explained on the basis of the percent
active facets present in the different shapes AgNPs. According to
Pal et al., the reactivity of silver is favored by high-atom-density
facets such as (111).22 Morones et al. have proved the faceting of
nanoparticles as well as the direct interaction of the (111) facets
with the bacterial surface.8 Herein, the ower shaped AgNPs
shows the maximum intensity of the (111) facet i.e. more facets,
in comparison to other nanoparticles that contain a low inten-
sity for the (111) facet, like spherical or oval shaped AgNPs.
Therefore, the trend of antibacterial activity (obtained by
experimental data): S-AgNPs < O-AgNPs < R-AgNPs < F-AgNPs,
could be attributed to the presence of different effective
surface areas in terms of active facets.
Confocal microscopic analysis. The antibacterial efficiency
of the synthesized AgNPs was further conrmed by confocal
microscopy analysis using back light uorescent staining
This journal is © The Royal Society of Chemistry 2015

Paper
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Fig. 6 Confocal images of live (green) and dead (red) E. coli bacterial
cells: (A) without F-AgNPs, (B) after 5 min, (C) 15 min and (D) 30 min
incubation with F-AgNPs.
(Fig. 6). Green uorescence indicates live bacterial cells (E. coli),
however, the dead ones are depicted by a red colour. As shown
in the gure, initially the all the bacterial cells are alive and
green in colour in the absence of nanoparticles (Fig. 6A). But,
when the cells were treated with F-AgNPs, aer just 5 minutes,
the cells started dying, represented by the presence of a red
colour (Fig. 6B). Aer increasing the incubation time to 15
minutes, more red colour appears and aer 30 minutes of
incubation the samples demonstrated an almost complete
eradication of the bacterial cells (Fig. 6C and D). The analysis
fully supports the high and rapid antibacterial efficacy of the
synthesized ower shaped AgNPs.
Non-specic removal of bacteria from aqueous and real
water samples. According to the standard of water, a limit of
less than 100 CFU E. coli per mL of water was employed for
general use. The water sample, containing an E. coli concen-
tration more than the limit, is taken as polluted or contami-
nated water. We also aware of the fact that E. coli is an
enterohemorrhagic strain responsible for mortality in the whole
world.36 Therefore, in this work, we have also tried to remove the
bacterial contamination from aqueous as well as real water
samples. Herein, various bacteria were removed using different
shaped AgNPs (concentration 1.0 mg mL1). As shown in the
Fig. S3,† the F-AgNPs have the ability to non-selectively remove
all four of the bacterial strains with
98% removal efficiency.
We have also employed a bacterial colony count method to
explore the real world applicability of the synthesized F-AgNPs.
The water samples used in the experiment were collected from
the local pond and river (Dhanbad, Jharkhand). The F-AgNPs
(different concentrations) was incubated with 5.0 mL of the
collected water sample for 15 min, then the nanoparticles were
ltered from the solution and the remaining water sample was
cultured on a LB plate for 24 h at 37  C. The number of colonies
grown on the surface of LB plate was used to monitor the
bactericidal efficiency of the F-AgNPs. As expected, we found
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that the F-AgNPs have a very high bacterial capture ability and
the bacterial cells were removed with 90–95% efficiency.
Conclusions
In summary, we represent an environmental friendly method to
synthesize different shaped AgNPs with the help of pome-
granate juice as a reducing agent and a microwave assisted
method. No toxic reagents were used to synthesize the different
shaped AgNPs (spherical, oval, rod and ower). All the AgNPs
were found to be highly effective as antibacterial agents to the
all studied bacterial strains and their antibacterial efficacy
increased when the shapes of the AgNPs were changed (F-AgNPs
> R-AgNPs > O-AgNPs > S-AgNPs). The bacteria killing efficiency
was also visualized, herein, using confocal microscopic anal-
ysis. Additionally, the synthesized F-AgNPs were also applied to
the capture and rapid removal of pathogenic bacteria from real
water samples.
Acknowledgements
The authors are thankful to the Department of Science and
Technology, Government of India for the sanction of the Fast
Track Research Project for Young Scientists to Dr Rashmi
Madhuri (Ref. No.: SB/FT/CS-155/2012) and Dr Prashant K.
Sharma (Ref. No.: SR/FTP/PS-157/2011). Dr Sharma (FRS/34/
2012-2013/APH) and Dr Madhuri (FRS/43/2013-2014/AC) are
also thankful to the Indian School of Mines, Dhanbad for
a Major Research Project grant under the Faculty Research
Scheme. We are also thankful to the Board of Research in
Nuclear Sciences (BRNS), Department of Atomic Energy,
Government of India, for the major research project (34/14/21/
2014-BRNS).
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