Synthesis, characterizations of superparamagnetic Fe3O4-Ag hybrid nanoparticles and their application for highly effective bacteria inactivation

Article abstract of DOI:10.1166/jnn.2016.11029

In recent years, outbreaks of infectious diseases caused by pathogenic micro-organisms pose a serious threat to public health. In this work, Fe₃O₄-Ag hybrid nanoparticles were synthesized by simple chemistry method and these prepared nanoparticles were used to investigate their antibacterial properties and mechanism against methicilline-resistant Staphylococcus aureus (MRSA) pathogen. The formation of dimer-like nanostructure of Fe₃O₄-Ag hybrid NPs was confirmed by X-ray diffraction and High-resolution Transmission Electron Microscopy. Our biological analysis revealed that the Fe₃O₄-Ag hybrid NPs showed more noticeable bactericidal activity than that of plain Fe₃O₄ NPs and Ag-NPs. We suggest that the enhancement in bactericidal activity of Fe₃O₄-Ag hybrid NPs might be likely from main factors such as: (i) enhanced surface area property of hybrid nanoparticles; (ii) the high catalytic activity of Ag-NPs with good dispersion and aggregation stability due to the iron oxide magnetic carrier, and (iii) large direct physical contacts between the bacterial cell membrane and the hybrid nanoparticles. The superparamagnetic hybrid nanoparticles of iron oxide magnetic nanoparticles decorated with silver nanoparticles can be a potential candidate to effectively treat infectious MRSA pathogen with recyclable capability, targeted bactericidal delivery and minimum release into environment.

Full text of DOI:10.1166/jnn.2016.11029

Article
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Nanoscience and Nanotechnology
Vol. 16, 5902–5912, 2016
Copyright © 2016 American Scientific Publishers
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Synthesis, Characterizations of Superparamagnetic
Fe3O4–Ag Hybrid Nanoparticles and Their Application for
Highly Effective Bacteria Inactivation
Le Minh Tung1ꢀ∗, Nguyen Xuan Cong2, Le Thanh Huy2ꢀ3, Nguyen Thi Lan2, Vu Ngoc Phan2,
Nguyen Quang Hoa4, Le Khanh Vinh5, Nguyen Viet Thinh1, Le Thanh Tai1, Duc-The Ngo6,
Kristian Mølhave6, Tran Quang Huy7, and Anh-Tuan Le2ꢀ∗
1Department of Physics, Tien Giang University, My Tho City, Tien Giang Province 86000, Vietnam
2Department of Nanoscience and Nanotechnology, Advanced Institute for Science and Technology (AIST),
Hanoi University of Science and Technology (HUST), Hai Ba Trung District, Hanoi 10000, Vietnam
3Faculty of Chemistry and Environment Technology, Hung Yen University of Technology and Education, Hung Yen 16000, Vietnam
4Department of Physics, Hanoi University of Science, 334 Nguyen Trai, Thanh Xuan, Ha Noi 10000, Vietnam
5Institute of Physics at Ho Chi Minh City, Vietnam Academy of Science and Technology (VAST), Ho Chi Minh 70000, Vietnam
6Department of Micro-and Nanotechnology, Technical University of Denmark Building 345Ø,
Ørsted Plads, Kgs Lyngby 2800, Denmark
7National Institute of Hygiene and Epidemiology (NIHE), 1-Yersin Street, Hai Ba Trung District, Hanoi 10000, Vietnam
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In recent years, outbreaks of infectious diseases caused by pathogenic micro-organisms pose a
Copyright: American Scientific Publishers
serious threat to public health. In this work, Fe3O4-Ag hybrid nanoparticles were synthesized by sim-
ple chemistry method and these prepared nanoparticles were used to investigate their antibacterial
properties and mechanism against methicilline-resistant Staphylococcus aureus (MRSA) pathogen.
The formation of dimer-like nanostructure of Fe3O4–Ag hybrid NPs was confirmed by X-ray diffrac-
tion and High-resolution Transmission Electron Microscopy. Our biological analysis revealed that the
Fe3O4–Ag hybrid NPs showed more noticeable bactericidal activity than that of plain Fe3O4 NPs
and Ag-NPs. We suggest that the enhancement in bactericidal activity of Fe3O4–Ag hybrid NPs
might be likely from main factors such as: (i) enhanced surface area property of hybrid nanopar-
ticles; (ii) the high catalytic activity of Ag-NPs with good dispersion and aggregation stability due
to the iron oxide magnetic carrier, and (iii) large direct physical contacts between the bacterial cell
membrane and the hybrid nanoparticles. The superparamagnetic hybrid nanoparticles of iron oxide
magnetic nanoparticles decorated with silver nanoparticles can be a potential candidate to effec-
tively treat infectious MRSA pathogen with recyclable capability, targeted bactericidal delivery and
minimum release into environment.
Keywords: Composite Materials, Magnetic Materials, Chemical Synthesis, Electron Microscopy.
1. INTRODUCTION
drugs in humans as well as in veterinary and agricultural
medicine have led to a rapid increase in new strains of
drug-resistant micro-organisms. Especially in developing
countries, the combination of patients with poor immune
system and cross-infection in several hospitals and health
care systems has given rise to nosocomial infections with
multi-drug-resistant pathogens.2 The global challenge is
facing, the outbreak infections mediated by these highly
resistant pathogens can cause uncontrolled epidemics of
bacterial diseases that can no longer be treated and hence
seriously risk expenses for the society community.1ꢀ2
In recent years, several outbreaks of infectious diseases
were reported, leading to a significant threat on global
economies and public health. The outbreak of infectious
diseases has not only occurred in developing countries
with low levels of hygiene and sanitation, but has also been
recognized in developed countries.1 In addition, the inap-
propriate and unmethodical uses of bactericidal antibiotic
∗Authors to whom correspondence should be addressed.
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doi:10.1166/jnn.2016.11029

Tung et al.
Synthesis, Characterizations of Superparamagnetic Fe3O4–Ag Hybrid Nanoparticles
To eliminate outbreak of infectious pathogens, disinfec-
In this work, we report a facile synthesis of Ag-NPs,
Fe3O4-NPs and their Ag–Fe3O4 hybrid nanoparticles by
modified coprecipitation and photochemical method. To
the best our knowledge, there is no report on photo-
chemical growth of Ag nanocrystals on Fe3O4 seed with
oleic acid as a stabilizer and glucose as reducing agent.
The photochemical method is a promising alternative for
decoration of Ag nanocrystals on the nanosized carri-
ers for various practical applications. This method shows
many advantages over reported previous methods such
as green synthesis, reaction occurring at room temper-
ature, long-term stable dispersions and small particles
sizes. A comparative study of antibacterial activity of
prepared nanoparticles was conducted against Staphylo-
coccus aureus bacteria, which is a the most frequently
drug-resistant pathogen in Vietnam. We provide more
insights in mechanism of bactericidal activity of these
nanoparticles against drug-resistant bacteria in the light of
ultrastructural studies.
tion methods should be done properly to eliminate these
pathogens from infected environmental areas, and effective
treatments should also be carried for patients in hospitals
and in the community.1ꢀ2 Interactions between nanomate-
rials and bio-systems have been intensively researched.3–6
Among them, nano-antimicrobials have been studied for
treatment of these pathogens.7ꢀ8 Among existing nano-
antimicrobials, silver nanoparticles (Ag-NPs) have been
recognized as a promising candidate to fight against resis-
tant pathogens.9ꢀ10 However, the Ag-NPs showed two
major shortcomings for application in practice: first, Ag-
NPs exhibit a highly toxic potential to human cells
and ecology when the use of high concentrations; and
second, due to the protection offered by the biofilm
mode of growth, the Ag-NPs cannot eradicate bacterial
biofilms.11–13
In order to overcome these challenges, hybrid
nanoparticles of silver nanoparticles and magnetic oxide
nanoparticles (i.e., Fe3O4, Fe2O3) were studied for enhanc-
ing antibacterial activity and high compatibility with
human cells.13ꢀ14 For example, Prucek et al.13 produced
two types of magnetic composite nanoparticles including
Ag@Fe3O4 and Fe2O3@Ag. Both synthesized nanocom-
posites exhibited significant antibacterial and antifun-
gal activities against ten tested bacterial strains and
2. EXPERIMENTAL PROCEDURES
2.1. Chemicals
The analytical-grade silver nitrate (AgNO3, 99.9%),
sodium hydroxide (NaOH), ammonium hydroxide (NH3,
25%), ferrous chloride tetrahydrate (FeCl2 ·4H2O, ≥99%),
ferric chloride hexahydrate hydrogen (FeCl3 · 6H2O
four candida species. At the observed minimum inhi-
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≥99%), oleic acid ≥99% and glucose that were used
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bition concentration, these nanocomposites did not dis-
in this study were purchased from Shanghai Chemical
Copyright: American Scientific Publishers
play acute cytotoxicity against mice embryonal fibroblast.
Reagent Co. Ltd.
Chen et al.14 reported one-pot synthesis of Ag@Fe2O3
core–shell and Ag–Fe2O3 heteromer nanoparticles. They
also indicated that the Ag@Fe2O3 core–shell nanoparti-
cles exhibited superior antibacterial property compared to
heteromer Ag–Fe2O3 nanoparticles and plain Ag nanopar-
ticles. The authors proposed that the slow diffusion of
silver ion out of the Fe2O3 shell resulting in enhanced
antibacterial activity. Although some previous results15–18
have been achieved for improving the antibacterial activity
of hybrid nanoparticles by combining the silver nanopar-
ticles and magnetic oxide nanoparticles, but the possible
mechanism for enhanced bactericidal activity of hybrid
nanoparticles has not been fulfilled. The other results19–34
also have proved that the magnetic oxide nanoparticles are
a promising carrier for enhancing biological activity of
hybrid nanoparticles through tuning its surface and inter-
face. However, there is a challenge when synthesizing the
hybrid nanoparticles and applying them in practice is the
aggregation instability of hybrid nanoparticles caused by
magnetic and electrostatic interactions.13ꢀ28ꢀ29
2.2. Synthesis of Fe3O4 Nanoparticles by
Co-Precipitation Method
The superparamagnetic Fe3O4 nanoparticles were synthe-
sized a coprecipitation method as described previously.35
Briefly, 5.41 g (0.02 mol) FeCl3 · 6H2O and 1.99 g
(0.01 mol) FeCl2 · 4H2O were dissolved in the water and
stirred under air in 10 min. In order to obtain inverse
spinel phase, 20 mL of 0.5 M NaOH solution was added
slowly to the solution of iron salts. The color of solu-
tion changed immediately from orange to dark brown after
addition of NaOH indicating the formation of superpara-
magnetic Fe3O4 nanoparticles. The precipitation reaction
was then stirred at temperature about 30 ꢀC for 30 min.
The product of Fe3O4 nanoparticles was separated from
solution by external magnetic field and washed several
times by deionized water and acetone.
2.3. Synthesis of Ag Nanoparticles by Tollens Process
The silver nanoparticles were prepared by using a modi-
fied Tollens process as reported elsewhere.36 Briefly, 1.7 g
(10 mmol) of AgNO3 was dissolved in 100 mL of deion-
ized water. The AgNO3 solution was then precipitated with
0.62 g (15.5 mmol) of sodium hydroxide (Aldrich, >99%).
The obtained precipitate, which is composed of Ag2O,
To prevent their aggregation, several chemical routes
have been made such as the use of polymer matrix as an
effective linker, immobilization of Ag-NPs on the surface
of magnetic silica composite Fe3O4–SiO2–Ag15 or of mag-
netic carbon composite Fe3O4@C@Ag,16 or synthesizing
Ag–Fe3O4 core–shell nanoparticles at high temperature.17
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Synthesis, Characterizations of Superparamagnetic Fe3O4–Ag Hybrid Nanoparticles
Tung et al.
was filtered and dissolved in 100 mL of aqueous ammonia
(0.4% w/w, 23 mmol) until a transparent solution of silver
ammonium complex [Ag(NH3ꢁ2]+(aq) formed. Up to 2.5 g
(8.9 mmol) of oleic acid was then added dropwise into the
complex, and the resulting solution was gently stirred for
2 h at room temperature until the complete homogeneity
of the reaction mixture was achieved. The reduction pro-
cess of the silver complex solution by the addition of 2 g
(11.1 mmol) of glucose was initiated with UV irradiation.
A UV lamp (ꢂ = 365 nm, 35 W) was used as a light source
to stimulate the reduction process.
The background was subtracted using linear interpolation
method. The UV-vis absorbance spectra were recorded
using a HP 8453 spectrophotometer, and the absorp-
tion spectrum of all suspension samples in 10 mm path
length quartz cuvettes was 300 nm to 900 nm. Magnetiza-
tion curves of Fe3O4 nanoparticles and Fe3O4–Ag hybrid
nanoparticles were measured by vibrating system magne-
tometers (VSM, MicroSense).
2.6. Evaluation of Antibacterial Activity
•
Bacterial strain and culture medium
Bacterial strain chosen for this study was Gram-positive
Staphylococcus aureus (ATCC 43300). This strain was
provided from the Department of Virology at the National
Institute of Hygiene and Epidemiology (NIHE) in Hanoi.
The growth of cell cultures was executed in a Luria-
Bertani (LB) medium (1% tryptone, 0.5% yeast extract
and 1% NaCl, pH 7). Next, the culture medium containing
bacteriaꢀwas kept in an incubator for 24 h at the tempera-
ture 37 C; then the content of bacterial culture in it was
108 CFU/ml, where the CFU is the colony forming unit.
2.4. Synthesis of Fe3O4–Ag Hybrid Nanoparticles by
Modified Photochemical Method
A schematic process for synthesis of Fe3O4–Ag hybrid
nanoparticles was shown in Figure 1. First, 30 mg of the
Fe3O4 nanoparticles were added to 100 mL of deionized
water together with 3 mL of acid oleic. In order to well
disperse the Fe3O4 nanoparticles, the solution was contin-
ꢀ
uously stirred at 80 C in 20 hour. Subsequently, 10 mL
of silver ammonium complex [Ag(NH3ꢁ2]+ꢃaqꢁ was added to
the complex of oleic acid-coated Fe3O4 nanoparticles and
the resulting solution was gently stirred for 4 h at room
temperature until the complete homogeneity of the reac-
tion mixture was achieved. After 12 h of UV irradiation,
the colloidal solution of Fe3O4–Ag hybrid nanoparticles
was obtained.
•
Paper-disc diffusion method
The paper-disc diffusion method was used to evaluate
the antibacterial activity of the studied samples against
tested bacteria. Using the spread plate method, nutrient
agar plates were inoculated with 100 ꢇl of bacterial sus-
pension containing 105 CFU. Sterile Whatman No. 1 filter
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paper discs with a diameter of 5 mm each, loaded with
10 ꢇL of samples, were placed on the inoculated plates.
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Copyright: American Scientific Publishers
2.5. Characterization Techniques
Control plates were maintained with discs containing dis-
tilled water. These plates were incubated at 37 ꢀC for 24 h
and the zone of inhibition (ZOI) was measured by sub-
tracting the disc diameter from the total inhibition zone
diameter.
Transmission electron microscopy (TEM, JEOL-JEM
1010) was conducted to investigate the morphology and
size distribution of as-prepared Fe3O4 and Ag nanoparti-
cles. Also, high resolution TEM (Tecnai, G20, 200 kV,
FEI) was used to determine the structure of Fe3O4–Ag
hybrid nanoparticles. The samples for TEM/HRTEM char-
acterization were prepared by placing a drop of col-
loidal solution on a formvar-coated copper grid that
was dried at room temperature. The composition of
the Fe3O4–Ag hybrid nanoparticles was characterized by
energy-dispersive X-ray (5410 LV JEOL). The crystalline
structure of the prepared Fe3O4, Ag nanoparticles and
Fe3O4–Ag hybrid nanoparticles was analyzed by X-ray
diffraction (XRD, Bruker D5005) using CuKꢄ radiation
(ꢂ = 0ꢅ154 nm) at a step of 0.02ꢀ (2ꢆ) at room temperature.
•
Standard microdilution method
The standard microdilution method was applied to
quantitatively compare the bactericidal activity of stud-
ied samples.36 First, the nutrient agar medium was heated
to 50 ꢀC to get a uniform distribution. Next, 10 ml of
each Fe3O4, Ag and Fe3O4–Ag nanoparticles solution was
added into Petri plates containing 25 ml of nutrient agar
medium. Total volume in each Petri plate was kept 35 ml
and the mixing solution was solidified with agar after
15 min. After that 100 ꢇl of a suspension of S. aureus bac-
teria was pipetted and spread on the surface of agar medium
containing Fe3O4, Ag and Fe3O4–Ag nanoparticles. The
ꢀ
Petri plates were incubated at 37 C for 24 h in a shak-
ing incubator (150 rpm) to encourage bacterial cell growth.
The intensity of bacterial growth on agar plates with Fe3O4,
Ag and Fe3O4–Ag nanoparticles was monitored by naked
eye and stereo microscope (ZMS800, Nikon). All the tests
were compared with the S. aureus growth intensity on the
agar plate in the absence of studied nanoparticles.
In order to determine the bactericidal activity of Fe3O4,
Ag and Fe3O4–Ag nanoparticles we used colony count-
ing method for determining the number of CFU which has
Figure 1. Two-step protocol for formation of Fe3O4–Ag hybrid
nanoparticles.
5904
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Tung et al.
Synthesis, Characterizations of Superparamagnetic Fe3O4–Ag Hybrid Nanoparticles
been grown after addition of S. aureus suspension of less
concentration (103 CFU) in agar medium mixed with all
studied nanoparticles. Control solutions were treated sim-
ilarly but without exposure to the nanoparticles for com-
parative purpose. Colonies were counted and compared to
those in control plates to calculate the changes in the bac-
terial growth inhibition. All experiments were performed
under sterile conditions and in triplicate.
It was shown that, in Tollens reaction, the mean diameter,
particle size distribution, and aggregate stability of silver
nanocrystals were strongly dependent on the conditions of
synthesis such as the temperature, ammonia concentration,
and the change in the pH of the medium during the reduc-
tion process. Furthermore, the addition of surfactants into
the reaction medium could control the particle size and
uniform aqueous dispersion of NPs.36–38
In order to control the stable dispersions and sizes of
nanoparticles, we also employed the UV irradiation of the
reagent mixture at the stage of reducing the silver ammo-
nium complex with glucose.39 It was shown that activating
the nanoparticles formation under the UV treatment with
a wavelength of 365 nm made it possible to carry out
the reduction process at room temperature while obtaining
the stable dispersion of NPs.39ꢀ40 Our previous works36–38
revealed that the use of oleic acid as a surfactant, glu-
cose as a reducing agent with simultaneous UV-irradiation
can effectively produce long-term stable dispersion of the
silver NPs in aqueous medium (>1 year) with narrow
average size distribution. In addition, this synthesis route
is environmentally friendly because of use of non-toxic
chemicals.
The percentage reduction ratio of the bacteria has been
expressed as:
A−B
R =
×100%
(1)
A
where R is the percentage reduction ratio, A–the number
of bacterial colonies in the Petri plates without nanopar-
tiles and B–the number of colonies in the Petri plates con-
taining Fe3O4, Ag and Fe3O4–Ag nanoparticles.
2.7. Ultrathin Sectioning Sample Preparation of
MRSA Bacteria Cells
In order to obtain further understanding of the bacte-
ricidal and interaction mechanism of the nanoparticles
samples, ultrathin sectioning technique was carried out
to observe the ultrastructural changes of bacterial cells
destroyed by action of the Ag-NPs and Fe3O4–Ag hybrid
nanoparticles.36 After the S. aureus bacteria were exposed
to the Ag-NPs and Fe3O4–Ag hybrid nanoparticles, the
Figures 2(a), (b) shows a TEM image of the Ag-NPs
and corresponding size distribution of Ag-NPs calculated
from TEM image. It can be seen that the Ag-NPs were
well-formed and well-dispersed. Almost no aggregates
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samples were collected and fixed by 2.5% glutaralde-
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hyde in cacodylate buffer (0.1 M) for 30 min at room
of Ag-NPs were observed through TEM investigation.
Figure 2(b) shows the histogram of the size distribution
of silver NPs obtained from the TEM image. The average
size of Ag-NPs was about 4.5 nm with a relatively nar-
row distribution. The crystallinity of the Ag-NPs was con-
firmed by powder X-ray diffraction (see Fig. 3). It showed
three broad peaks assigned to 111, 200, and 220 planes of
a face centered cubic lattice of bulk silver (JCPDS card,
No. 004-0783). This fact confirmed that the synthesized
NPs consisted of pure silver with high crystallinity. Our
calculation from the XRD patterns according to Scherrer
expression revealed that, the oleic acid-stabilized Ag-NPs
had average-sized particles (∼6–7 nm). This result is fully
consistent with the calculation from TEM image. Further-
more, the broadening of the full width at half maximum
(FWHM) of the XRD pattern revealed the nanoscale sizes
of Ag-NPs.37ꢀ38
Copyright: American Scientific Publishers
temperature. Washing the fixed samples by cacodylate
buffer three times for 10 min each, then transferred to 1%
OsO4/cacodylate buffer for 1 h. The samples were then
dehydrated by using a series of alcohol with 50, 70, 80,
90 and 100% (two times × 5 min), and then propylene
oxide (three times × 5 min). The samples were infiltrated
ꢀ
and finally embedded in Epon 812 at 60 C for 24 h. The
polymerized samples were sectioned into ultrathin slices
60–90 nm in thickness, and placed on the collodion-coated
copper grids (300 meshes). The analyses of ultrastruc-
tural changes of interior of the bacteria cells were con-
ducted by transmission electron microscopy (TEM, JEM
1010, JEOL).
3. RESULTS AND DISCUSSION
3.1. Microstructure Analysis
3.1.2. Synthesis of Fe3O4 Nanoparticles by
Co-Precipitation Method
Magnetic iron oxide NPs (Fe3O4-NPs) were synthesized
using co-precipitation reaction. A fundamental reaction for
formation of iron oxide NPs is as follows:
3.1.1. Synthesis of Ag Nanoparticles by Tollens Process
In our work, silver nanocrystals were synthesized using
Tollens reaction by reduction of silver ions by glucose with
the presence of oleic acid as a stabilizer and UV irradia-
tion. A fundamental reduction reaction involving Tollens
process under UV irradiation is follows:37ꢀ38
Fe2+ +2Fe3+ +8NaOH → Fe3O4 +4H2O
It revealed that in co-precipitation reaction the particles
size and shape of iron oxide nanocrsytals were strongly
dependent on synthesis conditions such as the mol ratio of
hꢊ
0
+
+
˙
ꢈAgꢃNH3ꢁ2ꢉ +RCHOH −→ Ag +2NH3 +H +RCOH
nAg0 → ꢃAgnꢁ0
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Synthesis, Characterizations of Superparamagnetic Fe3O4–Ag Hybrid Nanoparticles
Tung et al.
Figure 2. TEM images and size distribution of (a), (c) Ag NPs and (b), (d) Fe3O4 NPs.
Fe2+/Fe3+, concentration of sodium hydroxide and pH of
solution.19
average-sized particles (∼17–18 nm). This result is con-
sistent with the calculation from TEM image.
Figures 2(c), (d) shows a TEM image of as-prepared
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Fe O -NPs and a histogram of size distribution of Fe O -
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3.1.3. Synthesis of Fe3O4–Ag Hybrid Nanoparticles by
3
4
3
4
Copyright: American Scientific Publishers
NPs calculated from TEM image. It can be seen that the
average size of Fe3O4-NPs obtained about 19.2 nm. The
XRD analysis was conduct to examine the crystallinity of
Fe3O4-NPs as shown in Figure 3. It revealed that there
are eight broad peaks assigned to 111, 220, 311, 222,
400, 422, 511 and 440 planes of a spinel structural lat-
tice of magnetite (JCPDS card, No 11-0614). Our cal-
culation from the XRD patterns according to Scherrer
expression indicated that the as-prepared Fe3O4-NPs had
Modified Photochemical Method
In order to obtain Fe3O4–Ag hybrid nanoparticles, the iron
oxide nanoparticles were also used as seed for the growth
of silver nanoparticles on their surface through the pho-
tochemical reaction of Ag in the Fe3O4 nanoparticle seed
solution. As it can be seen from Figure 3 that seven peaks
are observed in the XRD pattern of Fe3O4–Ag hybrid
nanoparticles. Four of them can be indexed well to the fcc
inverse spinel structure of Fe3O4 phase. They are marked
with crystal planes of (311), (220), (400) and (200) of
magnetite (JCPDS card, No. 11-0614), respectively. The
reflections positioned at 2theta = 38.1ꢀ, 44.3ꢀ and 64.4ꢀ
correspond to (111), (220) and (311) fcc crystal planes of
bulk silver (JCPDS card, No. 004-0783), respectively.
High-resolution transmission electron microscopy
(HRTEM) along with EDS and FFT analyses was
employed to confirm the heterodimer nature of Fe3O4–Ag
nanostructure. Figure 4 shows the bright field TEM image
along with its EDS spectrum and HRTEM images of
nanostructure. Figure 4(a) indicates that formed Fe3O4–Ag
nanostructures have quasi-spherical particles and nearly
uniform distribution. The selected area electron diffraction
(SAED) shows polycrystalline structure (see the inset of
Fig. 4(a)). The EDS spectrum (Fig. 4(b)) of bright field
TEM image reveals that the nanostructure consisted of
main composition of Fe, O and Ag elements indicating
the high purity of sample. However, it is not possible
Figure 3. XRD patterns of Ag NPs, Fe3O4 NPs and Fe3O4–Ag hybrid
nanoparticles.
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Tung et al.
Synthesis, Characterizations of Superparamagnetic Fe3O4–Ag Hybrid Nanoparticles
Figure 4. TEM images and EDS elemental analysis of Fe3O4–Ag hybrid nanoparticles.
to determine the elemental ratio of Fe to Ag from EDS
spectrum because they are recorded at independent loca-
FFT analysis in Gatan Digital Micrographs software we
confirmed the formation of both Ag(111) and Fe3O4(311)
nanoparticles with high crystallinity. Furthermore, the het-
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tions. The HRTEM images (Figs. 4(c) and (d)) confirmed
the exact nature of Fe O and Ag nanoparticles. By using
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erodimer nature of Fe O –Ag nanostructure is evidenced
Copyright: American Scientific Publishers3
4
3
4
Figure 5. High-resolution TEM images of Fe3O4–Ag dimer nanoparticles.
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Synthesis, Characterizations of Superparamagnetic Fe3O4–Ag Hybrid Nanoparticles
Tung et al.
indicates the formation of larger Ag nanoparticles with
different shapes and sizes.36ꢀ37 This is consistent with
results from TEM analysis, the size of Ag-NPs on Fe3O4
seeds (dTEM∼10 nm) is slightly larger than that of Ag-
NPs (dTEM∼5 nm) as calculation of size distribution from
TEM images (see Figs. 2 and 5). The surface plasmon
band shifts are strongly dependent on particle size, shape,
chemical surrounding, and adsorbed species on the surface
and dielectric medium, whereas the plasmon peak and full
width at half maximum depends on the extent of colloid
aggregation.38ꢀ39
3.3. Magnetization Analysis
The magnetic properties of Fe3O4 NPs and Fe3O4–Ag
hybrid NPs were checked by magnetization measurements.
Figure 7 depicts the magnetization curves measured at
room temperature of (a) Fe3O4 NPs and (b) Fe3O4–Ag
hybrid NPs samples. It can be seen that the Fe3O4 NPs
sample were superparamagnetic and saturation magnetiza-
tion value (Ms) was about ∼55.35 emu/g. The interaction
between the nanoscale Ag and Fe3O4 leads to a change on
magnetization behavior of Fe3O4–Ag NPs. Obviously, the
Fe3O4–Ag hybrid NPs also exhibited superparamagnetic
behavior and the Ms value was ∼52,98 emu/g (calcula-
tion was corrected for only amount of Fe3O4 phase). This
result indicates that the formation of silver nanoscrytals on
Fe O seeds does not affect superparamagnetic property
Figure 6. UV-vis spectra of Fe3O4 NPs, Ag NPs and Fe3O4–Ag hybrid
nanoparticles.
from HRTEM analysis as shown in Figure 5. The Ag
nanocrystals with particle sizes about ∼10 nm are stably
formed on the Fe3O4 seeds.
3.2. UV-Vis Analysis
Figure 6 shows (a) the UV-vis spectra of Fe3O4, Ag-NPs,
and Fe3O4–Ag hybrid nanoparticles. Obviously, the Ag-
NPs and Fe3O4–Ag hybrid nanoparticles samples display
strong absorption peaks at 428 and 437 nm, respectively,
because of the surface plasmon resonance (SPR) effect
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4
of sample. In addition, the existence of silver nanoscrytals
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of Ag-NPs. In addition, there is no SPR band observa-
Copyright: American Scientific Publishers
can prevent agglomeration of the Fe3O4 NPs. Noticeably,
tion for Fe3O4 sample. The appearance of characteristic
surface plasmon band at 437 nm indicates the forma-
tion of Ag-NPs on Fe3O4 seeds. The SPR phenomenon
occurs when the incident light interacts with valence elec-
trons at the outer band of Ag-NPs, leading to oscillation
of electrons along with the frequency of the electromag-
netic source.42 However, the absorption band of Fe3O4–
Ag sample (∼437 nm) is shifted to longer wavelength
as compared to Ag-NPs sample alone (∼428 nm). The
shifting of the absorption peak toward longer wavelength
due to high value of magnetization the Fe3O4–Ag hybrid
NPs can be easily removed from solutions and recycled by
exerting an external magnetic field. Thus, the Fe3O4–Ag
hybrid NPs showed high potential to be developed into a
new class of recyclable antibacterial agents.
3.4. Antibacterial Activity Analysis
The antibacterial activities of Fe3O4, Ag-NPs, and Fe3O4–
Ag hybrid nanoparticles were initially assessed with the
Figure 7. Magnetization curves of (a) Fe3O4 NPs and (b) Fe3O4–Ag hybrid nanoparticles.
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Tung et al.
Synthesis, Characterizations of Superparamagnetic Fe3O4–Ag Hybrid Nanoparticles
paper-disc diffusion method, which is widely used for
quick antibiotic susceptibility determinations. Figure 8 dis-
plays the photographs of result of antibacterial test for
all studied samples using paper-disc diffusion method.
As shown in Figure 8(a), the Ag-NPs exhibit significant
inhibitory effects on S. aureus bacteria. Obviously, the
zone of inhibition (ZOI) increases with increasing concen-
tration of Ag-NPs from Ag1 sample (∼10 ꢇg/mL) to Ag4
sample (∼50 ꢇg/mL). A similar tendency was found for
Fe3O4–Ag sample as shown in Figure 8(b). The Fe3O4–Ag
hybrid NPs show noticeable inhibitory effects on S. aureus
bacteria, however, there is no inhibitory effect for bare
Fe3O4 sample (see Fig. 8(b)). This finding suggests that the
antibacterial activity of the hybrid nanoparticles samples
might be resulting from the Ag nanocrystals decorated on
Fe3O4 seeds. The evaluation of ZOI for all studied samples
was summarized in Figure 8(c).
In order to obtain more precise and comprehensive data
for the antibacterial activities of three nanoparticles sam-
ples against S. aureus, we used standard microdilution
method for determining the number of growth bacterial
colonies in these samples. Figure 9 shows the agar plates
treated (a) without nanoparticles and with the (b) Fe3O4,
(c) Ag-NPs, and (d) Fe3O4–Ag hybrid nanoparticles sam-
ples. It can be seen that in the absence of nanoparticles
(Fig. 9(a)), a drastic growth of bacteria was observed. With
the presence of Ag-NPs and Fe3O4–Ag NPs (Figs. 9(c),
(d)), the bacterial growth was completely inhibited, how-
ever for the case of Fe3O4 NPs sample (Fig. 9(b)), no
significant inhibitory effect was also found. The MIC val-
ues were also determined from microdilution method. It is
noted that the MIC values for hybrid nanoparticles sam-
ples are corrected for the effective concentration of Ag
in the heterodimer Fe3O4–Ag. Our obtained results reveal
that the MIC value of Ag-NPs sample (∼8.2 ꢇg/mL) is
slightly lower than that of Fe3O4–Ag hybrid NPs sample
(∼9.7 ꢇg/mL). Moreoever, the percentage reduction ratio
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of the bacteria for both Ag-NPs and Fe O –Ag NPs sam-
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3
4
Copyright: American SpclieesnotibfitcaiPneudbnliesahrelyrs100% as calculation using Eq. (1),
whereas percentage reduction ratio of bacteria for Fe3O4
samples was about 3.4%. This result suggests that Fe3O4
nanoparticles could play a minor role in bactericidal activ-
ity of hybrid nanoparticles.
(c)
Figure 8. Antibacterial activity of Fe3O4 NPs, Ag NPs and Fe3O4–Ag
Figure 9. Agar plates treated (a) without nanoparticles (control),
hybrid nanoparticles.
(b) with Fe3O4 NPs, (c) Ag NPs, and (d) Fe3O4–Ag hybrid nanoparticles.
J. Nanosci. Nanotechnol. 16, 5902–5912, 2016
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Tung et al.
3.5. Proposed Bactericidal Mechanism of Ag-NPs and
Fe3O4–Ag Hybrid NPs
membrane and disruption of membrane integrity, changes
in membrance permeability, interaction with proteins and
disruption of their regular function, interference with DNA
replication and causing DNA damage), and finally result-
ing in cell bacterial death.39ꢀ43
As shown in Figure 10 (left part), at different magnifica-
tions and sections, TEM results showed that some Ag-NPs
bindings around both the S. aureus cell membranes as well
as inside the cells were found. Obviously, the Ag-NPs first
attached to the surface of the cell membrane, and then
penetrated further inside the bacteria. It should be noted
that only Ag-NPs with sufficiently small diameters pene-
trated into the cells (usually < 10 nm). After 30 min, the
cytoplasm was completely destroyed as the silver NPs pen-
etrated the cell [see the left part of Fig. 10]. This analysis
probes how S. aureus bacterial cells can be destroyed by
the Ag-NPs.
To provide more rudimentary insights into the interac-
tion and bactericidal mechanism between the Ag-NPs and
Fe3O4–Ag hybrid nanoparticles with tested S. aureus bac-
teria, the ultrastructural and morphological analyses by
using electron microscopic technique were conducted. The
colloidal solutions of studied samples were dropped onto
the surface of S. aureus bacteria grown on agar plates.
After 0 min, 15 min, and 30 min, S. aureus cells were
taken out and underwent the sectioning method for TEM
observation.
Figure 10 displays the cross-sectional TEM images
showing different stages of interaction of (a) Ag-NPs sam-
ple and (b) Fe3O4–Ag hybrid nanoparticles with S. aureus
bacteria after 0 min, 15 min, and 30 min. It was estab-
lished for the case of bare Ag-NPs that the antibacterial
activity of the Ag-NPs is a complex process, in which the
silver NPs and their ions caused by the release of silver
ions Ag+ can produce free radicals, resulting in induction
of oxidative stress (i.e., reactive oxygen species; ROS).43
The produced ROS can irreversible damage bacteria by
multiple mechanisms (such as direct attachment to cell
In presence of Fe3O4–Ag hybrid nanoparticles [see the
right part of Fig. 10], the surface of S. aureus bacte-
rial cells was significantly modified. In first stage, lots
of hybrid nanoparticles were attached to the surface of
cells membrane. The strong release of Ag+ and Fe2+ ions
made the hybrid nanoparticles positively charged. Because
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Figure 10. The cross-sectional TEM images showing different stages of interaction of (a) Ag-NPs sample and (b) Fe3O4–Ag hybrid nanoparticles
with S. aureus bacteria after 0 min, 15 min, and 30 min.
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Tung et al.
Synthesis, Characterizations of Superparamagnetic Fe3O4–Ag Hybrid Nanoparticles
bacteria cell walls are negatively charged hence they attract
each other by electrostatic interaction, leading to large
density of hybrid nanoparticles attached on the surface of
cells membrane. The enhanced surface area of Fe3O4–Ag
nanoparticles produces large contact area between the bac-
terial cell membrane and nanoparticles.44 The Fe3O4–Ag
hybrid nanoparticles dissolve the outer envelope of bacte-
rial cell wall and thereby leakage of cellular constituents,
resulting in cell death. In our present case, due to large
sizes of Fe3O4–Ag hybrid nanoparticles (25–30 nm), they
can not penetrate into the cells membrane as the case of
Ag-NPs (small sizes ∼ 5 nm).
The authors would like to acknowledge the Center for
Electron Nanoscopy, Technical University of Denmark
(DTU CEN) for HRTEM works. Also, the technical sup-
ports for biological measurements at National Institute
of Hygiene and Epidemiology (NIHE) in Vietnam are
acknowledged.
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ment (NAFOSTED) under grant number 103.99-2012.10.
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Received: 1 February 2015. Accepted: 2 March 2015.
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Copyright: American Scientific Publishers
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J. Nanosci. Nanotechnol. 16, 5902–5912, 2016