World Journal of Nano Science and Engineering, 2012, 2, 196-200
http://dx.doi.org/10.4236/wjnse.2012.24026 Published Online December 2012 (http://www.SciRP.org/journal/wjnse)
Characterization and Evaluation of Antibacterial Activities
of Chemically Synthesized Iron Oxide Nanoparticles
Sudhanshu Shekhar Behera1, Jayanta Kumar Patra1, Krishna Pramanik2, Niladri Panda2,
1Department of Biotechnology, College of Engineering and Technology, Biju Patnaik University of Technology, Bhubaneswar, India
2Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, India
Received September 11, 2012; revised October 8, 2012; accepted October 30, 2012
The iron oxide nanoparticles have been synthesized in co-precipitation method using aqueous solution of ferric and fer-
rous ions with sodium salt. The synthesis of iron-oxide nanoparticles were validated by UV-Visible spectroscopy which
showed higher peak at 370 nm as valid standard reference. An average size of iron oxide nanoparticle found by diffrac-
tion light scattering (DLS) particle size analyser, ranges approximately between 10 nm to 120 nm with mean particle
size of 66 nm. The X-ray power diffraction (XRD) analysis revealed the crystallographic structure of magnetic particles.
Characterization of the mean particle size and morphology of iron oxide nanoparticles confirmed that the iron oxide
nanoparticles are nearly spherical and crystalline in shape. Further the antibacterial effect of iron oxide nanoparticles
was evaluated against ten pathogenic bacteria which showed that the nanoparticles have moderate antibacterial activity
against both Gram positive and Gram negative pathogenic bacterial strains and retains potential application in pharma-
ceutical and biomedical industries.
Keywords: Iron Oxide; Co-Precipitation; Nanoparticles; Antibacterial Activity
Nanometer-size metallic nanoparticles have been the
subject to research in recent years because these materi-
als represent an intermediate dimension between bulk
materials and atoms/molecules . Among these metallic
nanoparticles, iron oxide (IO) have received special at-
tention because of their variety of scientific and techno-
logical applications such as biosensor , antimicrobial
activity , food preservation , magnetic storage me-
dia, ferrofluids, magnetic refrigeration, magnetic reso-
nance imaging, hyperthermic cancer treatments, cell sort-
ing and targeted drug delivery [5-7]. Besides, it has also
been widely used in biomedical research because of its
biocompatibility and magnetic properties . The syn-
thesis of these IO nanoparticles are carried out by differ-
erent chemical approaches such as coprecipitation, Sol-
gel and forced hydrolysis, hydrothermal, surfactant me-
diated/template synthesis, microimulsion, electrochemi-
cal and laser pyrolysis. Among these, the co-precipitation
technique is probably the simplest and most efficient
chemical pathway through which a larger amount of
nanoparticles can be synthesized .
The development of new resistant strains of bacteria to
current antibiotics has become a serious problem in pub-
lic health; therefore there is a strong incentive to develop
new bacteriocides from various sources . Recent ad-
vancement in the field of nanotechnology has provided
attractive method for synthesizing alternative antimicro-
bial agents and reducing biofilm formation . Al-
though nanoparticles have long been known to exhibit a
strong toxicity to a wide range of micro-organisms [10,
12], very little is known about the toxicity of iron oxide
nanoparticles towards these microorganisms.
In the present study, an attempt has been made to syn-
thesize iron-oxide nanoparticles in co-precipitation method
and characterize it by absorption spectrophotometer (UV-
VIS), particle size analyzer (PD), X-ray diffraction (XRD),
and scanning electron microscope (SEM) along with the
evaluation of their antibacterial activity against ten human
pathogenic Gram positive and Gram negative bacteria
with a view to explore their pharmaceutical applications.
2. Materials and Methods
All the chemicals used in this work were analytical re-
agent grade from commercial market. Distilled water was
used for preparation of the solutions after deoxygenation
opyright © 2012 SciRes. WJNSE
S. S. BEHERA ET AL. 197
with dry N2 for 10 min. The divalent (FeCl2·4H2O), tri-
valent (FeCl3·6H2O) iron salts, 2 M HCl solution and
aqueous NaOH (25% - 28%, w/w) were also deoxygen-
ated with dry nitrogen before use.
2.2. Synthesis of Iron-Oxide Nanoparticles
Iron-oxide (IO) nanoparticles were synthesized by co-
precipitation method as reported by Predoi . The
synthesis was carried out by coprecipitation of ferrous
and ferric ion salts in aqueous solution by adding base at
room temperature with flowing N2 gas. Briefly, 4.0 ml of
1 M FeCl3 and 1.0 ml of 2 M FeCl2 solution were dis-
solved in deionised deoxygenated (DD) water followed
by adding 200 ml of 0.02 M HCl solution under vigorous
stirring at 8000 rpm for about 30 min. The resulting
brown precipitate was added with 200 ml of 1.5 M
NaOH solution, the color of the mixture then turned from
brown to black.
2FeFe8OHFeO 4 HO
The Fe3O4 (IO) nanoparticles were finally collected as
power after oven dried at 50˚C (Figure 1).
2.3. Characterization Techniques
2.3.1. UV-VIS Spectra Analysis (UV-VIS)
The reduction of pure Fe3+ ions was monitored by meas-
uring the UV-VIS spectrum of the reaction medium after
diluting a small aliquot of the sample into distilled water
at wave length 330 - 450 nm. UV-VIS spectral analysis was
done by using UV-VIS spectrophotometer (Systronis-117).
2.3.2. Particle S i ze Analysis (PD)
In order to determine the average particle size distribu-
tion, the milled powder of iron oxide nanoparticles was
measured by ZETA Sizer Nanoseries (Malvern instru-
ments Nano ZS). Initially, the liquid dispersant contain-
ing 500 ml of deionized water and 25 ml of sodium hexa-
metaphosphate was kept in the sample holder and then
iron oxide (IO) nanoparticles were dispersed in deionised
water followed by ultrasonication.
2.3.2. X-Ra y Diffr ac ti on (XRD )
In order to obtain the structural information of the pro-
Figure 1. Synthesized ironoxide nanopart ic le s.
duct, the crystallographic structure of magnetic particles
was analyzed by X-ray power diffraction (XRD). The
crystallographic analysis of samples in diffraction pa-
tterns were recorded from 10˚ to 70˚ with a panalytical
system diffractometer (Model: DY-1656) using Cu Kα (λ
= 1.542 Ao) with an accelerating voltage of 40 KV. Data
were collected with a counting rate of 1˚/min. The Kα
doublets were well resolved.
2.3.3. S c anning Electron Microsc o p e ( SEM)
To characterize mean particle size and morphology of
Iron oxide nanoparticles, SEM (scanning electron micro-
scope) was performed using Jeol JSM-6480 LV SEM
machine of 20 KV of accelerating voltage.
2.3.4. Screening of Antimicrobial Act ivit y
Ten pathogenic bacteria viz. Staphylococcus aureus
(MTCC 1144), Shigella flexneri (Lab isolate), Bacillus
licheniformis (MTCC 7425), Bacillus brevis (MTCC
7404), Vibrio cholerae (MTCC 3904), Pseudomonas
aeruginosa (MTCC 1034), Streptococcus aureus (Lab
isolate), Staphylococcus epidermidis (MTCC 3615), Baci-
llus subtilis (MTCC 7164) and E. coli (MTCC 1089) used
in the study were obtained from Institute of Microbial
Technology, Chandigarh or lab isolates. The organisms
were maintained on nutrient agar (Hi Media, India)
slopes at 4˚C and subcultured before use.
Agar cup plate method of Khalid et al.  was
carried out to establish the antibacterial activity of the
iron oxide (IO) nanoparticles against the test pathogens.
Wells of 6 mm diameter were punched over the agar
plates using sterile gel puncher (cork borer) 100 μl (50
mg/ml) of nanoparticle powder in sterile distilled water
were poured into the wells. The plates were incubated at
37˚C for 24 h. The zone of the clearance around each
well after the incubation period, confirms the antimicro-
bial activity of the IO nanoparticle extract. Neomycin (30
µg/disc) was taken as standard.
3. Results and Discussion
The iron oxide nanoparticles (Fe3O4) synthesized by co-
precipitation of ferric and ferrous chloride was validated
by UV-Visible spectroscopic analysis and their scanning
absorbance vs wave length (λ) has been established
(Figure 2). The characteristics peaks of IO nanoparticles
were observed at 370 nm, which is due to charge transfer
spectra. The particle size distribution of the iron oxide
nanoparticles determined by laser diffraction method
with a multiple scattering technique revealed that the
particle size distribution of iron oxide nanoparticles
ranges approximately from 10 nm to 120 nm with mean
particle size of 66 nm and the distribution of oxide
nanoparticle is more uniform with a narrow distribution
range (Figure 3).
Copyright © 2012 SciRes. WJNSE
S. S. BEHERA ET AL.
The XRD analysis of IO nanoparticles shown in Fig-
ure 4, were made to detect the diffraction angles at 31.5˚,
35˚, 37˚, 45.2˚ and 53˚ which implies the diffraction sur-
faces of the nanoparticle crystal. The diffraction angles
of different peaks are corresponds to Fe3O4 nanopar-
This data is very close to the American Society for
Testing and Materials (ASTM) data of iron oxide [(Fe3O4)]
nanoparticles, which could be a good evidence to prove
that the prepared nanoparticles, was made of iron oxide.
The X-ray power diffraction (XRD) results of nano-
particles confirmed that the synthesized product was a
magnetite (Fe3O4) .
Further analysis of the SEM image of synthesized iron
oxide nanoparticles, showed a clear image of highly
dense IO nanoparticles which are almost spherical in size
(Figure 5). The size of most of the nanoparticles ranges
from 30 nm to 110 nm. However the percentage of
nanoparticles beyond 100 nm is very less. The average
percentage of nanoparticles present in our synthesized
Figure 2. The UV-VIS spectrum of Fe3O4 naoparticles.
Figure 3. DLS particle size analysis curve of iron oxide
Figure 4. XRD of Fe3O4 nanoparticles.
sample is 66 nm. From the image it is confirmed that the
sample contains various sizes of nanoparticles which are
indeed agreement with the result obtained from DLS
particle analyser. Similar results on SEM analysis of IO
nanoparticles has also been reported by other workers .
The antibacterial activities of the iron oxide nanopar-
ticle evaluated against ten pathogenic bacteria (six Gram
positive and four Gram negative) are presented in (Table
1 and Figure 6). The result of antibacterial activity of IO
nanoparticle showed moderate antimicrobial activity
against eight pathogenic strains (six gram positive and
two gram negative) with zone of inhibition ranging from 9
mm to 22 mm (Table 1).
Figure 5. SEM image of synthesized iron oxide nanoparti-
Figure 6. Study of antibacterial activity (zone of inhibition)
of Fe3O4 nanoparticles.
Table 1. Antibacterial activity of iron oxide nanoparticle
and standard antibiotics.
Strains Iron oxide
nanoparticles (50 mg/ml)
neomycin (30 µg/disc)
Staphylococcus aureus12 ± 0.35 17 ± 0.70
Shigella flexneri 0 ± 0.0 18 ± 0.35
Bacillus licheniformis22 ± 0.70 21 ± 1.4
Bacillus brevis 9 ± 0.15 27 ± 0.35
Vibrio cholerae 9 ± 0.0 18 ± 0.70
aeruginosa 0 ± 0.0 18 ± 0.35
Streptococcus aureus12 ± 0.35 16 ± 0.35
epidermidis 14 ± 0.44 15 ± 0.07
Bacillus subtilis 20 ± 1.11 16 ± 1.4
Escherichia coli 11 ± 0.44 14 ± 0.07
Copyright © 2012 SciRes. WJNSE
S. S. BEHERA ET AL. 199
The present results are comparable with that of the
standard antibiotic Neomycin (30 µg/disc). The IO nano-
particles do not show any activity against two Gram
negative bacteria viz. Shigella flexneri and Pseudomonas
aeruginosa (Table 1). There are many factors respon-
sible for the antibacterial activity of iron oxide nano-
The main mechanism by which these particles showed
antibacterial activity might be via oxidative stress
generated by ROS [10,12]. ROS, including superoxide
radicals (O2–), hydroxyl radicals (–OH), hydrogen pero-
xide (H2O2), and singlet oxygen (1O2), can cause damage
to proteins and DNA in bacteria. In the present study,
metal oxide (FeO) could be the source that created ROS
leading to the inhibition of most of the pathogenic
bacteria including Staphylococcus aureus. A similar pro-
cess was also described by Kim et al. (2007) in which
Fe2+ reacted with oxygen to create hydrogen peroxide
(H2O2). This H2O2 consequently reacted with ferrous
irons via the Fenton reaction and produced hydroxyl
radicals which are known to damage biological macro-
Some authors have demonstrated that the small size of
nanoparticles can also contribute to bactericidal effects.
For example, Lee et al.  reported that the inactivation
of Escherichia coli by zero-valent iron nanoparticles 
could be because of the penetration of the small particles
(sizes ranging from 10 - 80 nm) into E. coli membranes.
Nano scale zero valent iron (NZVI) could then react with
intracellular oxygen, leading to oxidative stress and
eventually causing disruption of the cell membrane.
Studies on ZnO and MgO nanoparticles have also shown
that antibacterial activity increased with decreasing
particle size [18,19]. In the present study, the concen-
tration of nanoparticles was a major factor for anti-
bacterial activity of the nanoparticle. A similar con-
centration-dependent behavior was observed by Kim et al.
 when they investigated the antimicrobial effects of
Ag and ZnO nanoparticles on S. aureus and E. coli
[18,19]. Similarly, in a study of bactericidal effects of
iron noxide nanoparticles on S. epidermidis, Taylor and
Webster , also reported concentration dependent bac-
terial inhibition. It is also important to note that IO nano-
particles do not negatively influence all cells and thus it
can be said that with an appropriate external magnetic
field, FeO nanoparticles may be directed to kill bacteria
as needed throughout the body.
Application of Iron Oxide nanoparticle shows zone of
inhibition comparable to that of other nanoparticle (Ag)
of topical use. Furthermore it shows better bactericidal
activity in Gram-positive bacteria as compared to Gram-
negative bacteria. The present study highlights the poten-
tial application of IO nanoparticles as antibacterial agents
which can be explored for its topical application in
pharmaceutical and biomedical industries and opens the
path for further research regarding the toxicity and carci-
nogenicity properties for its use in human being.
Authors are grateful to the authorities of NIT, Rourkela
and College of Engineering and Technology, Bhubanes-
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