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Preparation and antibacterial activity of Fe3O4@Ag nanoparticles

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2007 Nanotechnology 18 285604
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IOP PUBLISHING

NANOTECHNOLOGY

Nanotechnology 18 (2007) 285604 (7pp)

doi:10.1088/0957-4484/18/28/285604

Preparation and antibacterial activity of

Fe3O4@Ag nanoparticles
Ping Gong1 , Huimin Li2 , Xiaoxiao He1 , Kemin Wang1,3 ,
Jianbing Hu1 , Weihong Tan1 , Shouchun Zhang1 and Xiaohai Yang1
1

State Key Laboratory of Chemo/Biosensing and Chemometrics, Biomedical Engineering

Centre, College of Chemistry and Chemical Engineering, Hunan University, Research Centre
for Bio-Nanotechnology Engineering of Hunan Province, Changsha 410082,
Peoples Republic of China
2
Biomedical Engineering Center, College of Material Science and Technology,
Hunan University, Changsha, 410082, Peoples Republic of China
E-mail: kmwang@hnu.cn

Received 9 February 2007, in final form 17 May 2007

Published 15 June 2007
Online at stacks.iop.org/Nano/18/285604
Abstract
Bifunctional Fe3 O4 @Ag nanoparticles with both superparamagnetic and
antibacterial properties were prepared by reducing silver nitrate on the
surface of Fe3 O4 nanoparticles using the water-in-oil microemulsion method.
Formation of well-dispersed nanoparticles with sizes of 60 20 nm was
confirmed by transmission electron microscopy and dynamic light scattering.
X-ray diffraction patterns and UVvisible spectroscopy indicated that both
Fe3 O4 and silver are present in the same particle. The superparamagnetism of
Fe3 O4 @Ag nanoparticles was confirmed with a vibrating sample
magnetometer. Their antibacterial activity was evaluated by means of
minimum inhibitory concentration value, flow cytometry, and antibacterial
rate assays. The results showed that Fe3 O4 @Ag nanoparticles presented
good antibacterial performance against Escherichia coli (gram-negative
bacteria), Staphylococcus epidermidis (gram-positive bacteria) and Bacillus
subtilis (spore bacteria). Furthermore, Fe3 O4 @Ag nanoparticles can be
easily removed from water by using a magnetic field to avoid contamination
of surroundings. Reclaimed Fe3 O4 @Ag nanoparticles can still have
antibacterial capability and can be reused.

antimicrobial agent [12] since silver and its compounds have

powerful antimicrobial capability [13] and broad inhibitory
biocidal spectra for microbes including bacteria, viruses and
eukaryotic microorganisms [1416]. Enhanced antibacterial
properties of nanocrystalline silver have been demonstrated
both in vitro and in vivo [1720]. Now various medical
products and industrial devices such as urinary catheters,
suture rings, wood flooring and packaging material have
nanocrystalline silver as an antiseptic component [2124].
However, nanocrystalline silver and silver-containing
products used as drugs or disinfectants of drinking water
and other liquid might have some potential risks, considering
the adverse effects of chronic exposure to silver. Silver is
a recognized cause of argyrosis and argyria [25, 26], and
relevant clinical toxicity of silver to mammiferous cells has
also been reported [27]. So maximum contaminant levels

1. Introduction
With the outbreaks of infectious diseases caused by pathogenic
bacteria and the rise of antibiotic resistance of bacteria [1],
much attention in pharmaceutical and medical fields has been
focused on creating new antibacterial agents [2]. In recent
years, nanoscaled antibacterial materials as novel antimicrobial
species have been seen as promising candidates for application
owing to their high surface-to-volume ratio and their novel
physical and chemical properties on the nanoscale level [3, 4].
Many kinds of nanometre-sized antibacterial materials such
as TiO2 , ZnO, MgO, chitosan, calamine, copper and silver
have been reported on in this area [511]. Among them,
nanocrystalline silver has been proved to be the most effective
3 Author to whom any correspondence should be addressed.

0957-4484/07/285604+07$30.00

2007 IOP Publishing Ltd Printed in the UK

Nanotechnology 18 (2007) 285604

P Gong et al

(MCL) for silver in drinking water (100 ppb) [28] and the
occupational exposure limit to the various forms of silver
(0.01 mg m3 ) [29] have been established in order to avoid
the accumulation of silver in human body. But it is very
difficult to eliminate colloidal silver (<10 nm) from solution
on the basis of the physical or chemical properties of silver
since the particles are too small to be collected by filtering or
centrifugation. One of the key issues for further application
of the nanocrystalline silver as antibacterial agents in liquid
is developing effective carriers, using which nanocrystalline
silver could be removed easily from liquid after bacteria have
been killed. Magnetic carrier use is a very promising choice
for the isolation and removal of environmental contaminants
from water [30]. It has been reported that Ag nanoparticle
loaded hydrogen-bonded multilayers assembled on magnetic
microspheres could be delivered and localized to a specific
region without contaminating the surroundings by using
magnetic fields [31].
In the present study, we employ the micelles technique [32] to fabricate bifunctional Fe3 O4 @Ag nanoparticles with both superparamagnetic and antibacterial properties.
Fe3 O4 @Ag nanoparticles were proved to have excellent antibacterial ability against Escherichia coli (E. coli), Staphylococcus epidermidis (S. epidermis) and Bacillus subtilis (B.
subtilis). Meanwhile, they could be easily separated from liquid on the basis of their superparamagnetic properties. Furthermore, the Fe3 O4 @Ag nanoparticles could be recycled and
reused. These results indicate that Fe3 O4 @Ag nanoparticles
may have potential application as disinfectants for water.

2.2. Characterization of Fe3 O4 @Ag nanoparticles

The morphologies of Fe3 O4 and Fe3 O4 @Ag nanoparticles
were characterized by JEOL-1200EXII transmission electron
microscopy (TEM, Joel Co., Tokyo, Japan). Size analysis of
particles in TEM images was carried out using the ImageJ
software. Additionally, the particle size distribution of
the Fe3 O4 @Ag nanoparticles was determined by dynamic
light scattering (Zetasizer 3000HSA, Malvern, UK). Xray diffraction (XRD) measurement was performed on a
Rigaku D/max 2550 VB+ x-ray diffractometer (Rigaku
Co., Japan) at room temperature. UVvisible absorption
spectra were recorded using a Beckman DU 800 UVvisible
spectrophotometer (Beckman Coulter Inc., Fullerton, USA)
with quartz cuvettes (1 cm optical path) as the containers.
A 7307 vibrating sample magnetometer (VSM, Lake Shore
Cryotronics Inc., Ohio, USA) was used to measure the
magnetic properties of Fe3 O4 @Ag nanoparticles.
2.3. Measurements of antibacterial properties of Fe3 O4 @Ag
nanoparticles
The minimum inhibitory concentration (MIC) values for
E. coli, S. epidermis and B. subtilis were determined. The
inocula of these kinds of bacteria were prepared by growing
strains in LuriaBertani (LB) medium at 37 C until a level
of approximately 108 109 CFU ml1 of bacteria was reached.
Then 75 l 108 CFU ml1 bacterial suspension and 15 ml
LB medium with 0, 10, 20, 30, 40, 50, 60, 70, 80, 90,
100 g ml1 Fe3 O4 @Ag nanoparticles was added to a series
of flasks. The flasks were incubated at 37 C on a rotary shaker
(200 rpm) for 24 h. The MICs of Fe3 O4 @Ag nanoparticles
for three kinds of bacteria were calculated as the lowest
concentrations at which bacterial growth was inhibited. All
assays were carried out in three duplicates.
Flow cytometry was also performed for analysing the
antibacterial properties of Fe3 O4 @Ag nanoparticles. Four
tubes with 1 ml of 5.0 106 cells ml1 live E. coli
suspension was prepared, and 1 ml Fe3 O4 @Ag nanoparticles
in phosphate-buffered saline solution (PBS, pH 7.4) with final
concentrations of 25 and 50 g ml1 were added to two tubes.
As controls, 1 ml phosphate-buffered saline solution without
Fe3 O4 @Ag nanoparticles was added into another two tubes.
The tubes with samples were incubated at 37 C for 1 h to allow
the interaction of Fe3 O4 @Ag nanoparticles with bacteria and
the same procedure was carried out on one of the controls. The
other control tube was boiled for 10 min to kill the bacteria.
To distinguish dead bacteria from live ones, the bacteria were
stained with propidium iodide (PI, Molecular Probes Inc.,
Sydney, Australia). 2 l 1.0 mg ml1 PI dimethyl sulfoxide
solution was added into each tube. The mixed solution
was incubated in the dark at 37 C for 510 min. Stained
samples were analysed using a FACSCalibur flow cytometer
(FCM, Becton Dickinson, San Jose, CA, USA). Fluorescence
emission from PI was collected by a fluorescence detector,
FL3 (>605 nm). All parameters were collected as logarithmic
signals. Data were analysed using CellQuest software
(Becton Dickinson).

2. Experimental details
2.1. Synthesis of Fe3 O4 @Ag nanoparticles
Firstly, Fe3 O4 nanoparticles were synthesized according to
a procedure reported by Kim [33]. 2.7 g (10 mmol) of
FeCl3 6H2 O and 1.0 g (5 mmol) of FeCl2 H2 O were dissolved
in 15 ml of 0.3 M HCl solution (all aqueous solutions and the
water were prepared exclusively with 18 M deionized water
(Barnstead Thermolyne Nanopure)). While stirring under N2 ,
the mixture was titrated to pH 1011 by the dropwise addition
of 6.0 M NH4 OH. Black precipitation formed immediately
and the reaction continued for 30 min. The precipitation
was isolated via magnetic decantation and washed with water.
The large aggregates were then removed by filtration. The
Fe3 O4 nanoparticles were dissolved in 100 ml of 0.01 M
tetramethylammonium hydroxide pentahydrate. The final,
black, 40 mM Fe3 O4 nanoparticle solution was stored in air
under benchtop conditions for further use.
Fe3 O4 @Ag nanoparticles were synthesized by the reverse
micelle method. 100 l of 40 mM Fe3 O4 nanoparticles were
mixed with a W/O microemulsion containing 1.4 ml of Triton
X-100, 1.4 ml of n -hexanol, and 7.5 ml of cyclohexane, with
vigorous stirring. Then, 200 l of 0.10 M AgNO3 (Shanghai
Chemical Reagents Co., Tianjin, China) was added. After
30 min, 200 l of 0.20 M NaBH4 was added to the solution.
The mixture was stirred at room temperature for 4 h. The
black Fe3 O4 @Ag nanoparticles were precipitated by adding
excess acetone, and then centrifuged and repeatedly washed
with ethanol and water to remove surfactant and unreacted
materials. The nanoparticles obtained were suspended in water
for future use.
2

Nanotechnology 18 (2007) 285604

P Gong et al

Figure 1. Schematic illustration of the antibacterial rate for recycled

Fe3 O4 @Ag nanoparticles for different recycling times.

Figure 2. The TEM images of Fe3 O4 nanoparticles (A) and

Fe3 O4 @Ag nanoparticles (B).

2.4. Removal of Fe3 O4 @Ag nanoparticles from water by

magnetically directed separation

(Ncontrol Nsample )/Ncontrol 100%. The experiments were

repeated completely three times.

Five tubes filled with 8 ml 50 g ml1 Fe3 O4 @Ag

nanoparticle suspensions and 5.0 106 cells of ml1 E. coli
were placed into the rack of the magnetic particle concentrator
(manufactured by Dynal Biotech ASA, Oslo, Norway). The
nanoparticles in suspensions were separated from the water
and adsorbed onto the wall of tubes by the magnetic force
of the magnetic particle concentrator. 1 ml water was taken
out from the tubes when the tubes were placed in magnetic
fields for 0, 5, 10, 20, 30 min, respectively. Then the
concentrations of silver in the water were measured with
an IRIS advantage 1000 inductively coupled plasma atomic
emission spectrometer (ICP-AES, Thermo Electron Co.,
Franklin, USA). The experiments were repeated completely,
three times and data were analysed using Microcal Origin 7.0.

3. Results and discussion

3.1. Characteristics of Fe3 O4 @Ag nanoparticles
Fe3 O4 @Ag nanoparticles were prepared by the reverse
micelles technique. Here, Triton X-100 was used as the
surfactant, cyclohexane as the oil phase, and n -hexanol as
the co-surfactant. It is well known that these micelles
could perform as nanoscaled reactors and the reaction
would occur in a controlled manner in the micelles,
resulting in the formation of nanoparticles with controlled
characteristics.
Representative TEM images of Fe3 O4
nanoparticles (magnification: 150 000) and Fe3 O4 @Ag
nanoparticles (magnification: 350 000) are shown in figure 2.
The size of the Fe3 O4 nanoparticles was about 10 4 nm
and particles were non-global and aggregative (figure 2(A)),
while most of the Fe3 O4 @Ag nanoparticles were spherical
and non-aggregative, with an average diameter of about
60 20 nm (figure 2(B)). The morphological analysis by
TEM indicated that Ag+ initially reduced onto the surface of
Fe3 O4 nanoparticles, resulting in larger and more spherical
Fe3 O4 @Ag nanoparticles.
Dynamic light scattering (DLS) measurements for
Fe3 O4 @Ag nanoparticles in water were made to assess the
stability of the colloidal suspension. The size distribution
of Fe3 O4 @Ag nanoparticles is shown in figure 3. The
mean hydrodynamic diameter determined by number was
72.1 nm with a narrow size distribution, which was consistent
with the result from TEM. The polydispersity index was
0.227. These results indicated that the particles were stable
during measurement. Sedimentation could occur when the
nanoparticles with high concentration in water were left for two
days. However, the process of sedimentation for nanoparticles
was slowed in the LB medium and increased in the PBS buffer,
which may imply that high salt concentrations in PBS buffer
damaged the electrostatic stability of the nanoparticles, while
the ingredients of biomacromolecules or polyampholyte in LB
medium cause steric and electrosteric stabilization effects.
To confirm the composition of the particles, the
XRD patterns for Fe3 O4 and Fe3 O4 @Ag nanoparticles
were measured.
The experimentally obtained patterns

2.5. Measurements of the antibacterial rate of recycled

Fe3 O4 @Ag nanoparticles
A schematic diagram of the experimental procedure is
shown in figure 1. 5 ml 1.0 106 CFU ml1 E. coli
and 50 g ml1 Fe3 O4 @Ag nanoparticle suspensions were
incubated at 37 C for 1 h in the tube. Then the tube was placed
into a magnetic field for 30 min and the nanoparticles were
adsorbed onto the wall of the tube. The supernatant containing
bacteria was taken out from the tube and 0.1 ml of it was
spread onto a LB agar plate to perform a bactericidal test by
counting the exact number of colonies of surviving E. coli.
This was the original antibacterial rate of nanoparticles. Then
the nanoparticles adsorbed onto the wall of tube were washed
off with sterile water and recycled. 5 ml of approximately
1.0 106 CFU ml1 E. coli suspension was added into the
tube to incubate with the recycled nanoparticles at 37 C for
1 h. The above steps were repeated to get the antibacterial
rate of recycled Fe3 O4 @Ag nanoparticles for the first cycle.
With the same procedure, the antibacterial rate of the recycled
Fe3 O4 @Ag nanoparticles from the second to the fifth cycle
was obtained. Additionally, 0.1 ml 1.0 106 CFU ml1 of
live E. coli without treatment with Fe3 O4 @Ag nanoparticles
was spread onto the LB agar plate and the number of
colonies was counted as a control. The antibacterial rate
was calculated using the equation antibacterial rate(%) =
3

Nanotechnology 18 (2007) 285604

P Gong et al

Figure 5. UVvisible absorption spectra of aqueous solutions of

Fe3 O4 nanoparticles and Fe3 O4 @Ag nanoparticles.

Figure 3. Size distribution analysis of Fe3 O4 @Ag nanoparticles by

means of dynamic light scattering (DLS).

Figure 6. Hysteresis loops of Fe3 O4 @Ag nanoparticles measured at

300 K.

peaks (at 2 = 38.0 , 44.2 , 64.4 , 77.3 , 81.5 ) revealed

indices corresponding to (111), (220), (311), (400), (422),
(511) for pure silver. The peak at 2 = 35.3 is the only
peak corresponding to the indices (311) for Fe3 O4 . This is
the most intense peak of Fe3 O4 . The occurrence of the most
intense peak of Fe3 O4 indicates the presence of Fe3 O4 as the
core. This means that x-rays might penetrate through the core;
thus the peak at 2 = 35.3 did not completely vanish, but its
intensity decreased. These results are similar to those reported
by Mandal et al [34].
The UVvisible absorption spectra of Fe3 O4 nanoparticles
and Fe3 O4 @Ag nanoparticles solutions are shown in figure 5.
The absorbance of Fe3 O4 nanoparticle increases from 200 to
800 nm with no obvious peak. However, a typical surface
plasmon resonance band of silver nanoparticles at 400 nm was
observed for the Fe3 O4 @Ag nanoparticles. This result further
confirms the deposition of silver on Fe3 O4 nanoparticles.
The magnetic property of Fe3 O4 @Ag nanoparticles was
examined using VSM magnetometry.
Figure 6 shows
the magnetization of Fe3 O4 @Ag nanoparticles versus the
magnetic field at 300 K obtained by cycling the field between
10 and 10 kG. It can be seen that Fe3 O4 @Ag nanoparticles
showed the typical hysteresis loop for superparamagnetic
materials.
This result indicates that the Fe3 O4 @Ag
nanoparticles showed superparamagnetism at 300 K and could
be separated from water by applying a magnetic field.

Figure 4. X-ray diffraction patterns for Fe3 O4 (A) and Fe3 O4 @Ag
nanoparticles (B).

were identified through comparison with standard Fe3 O4

and Ag patterns. XRD patterns for Fe3 O4 nanoparticles
=
in figure 4(A) show characteristic peaks (at 2
18.2 , 30.0 , 35.3 , 43.0 , 53.3 , 56.9 , 62.5 , 73.9 ), marked
with their indices (111), (220), (311), (400), (422), (511), (440)
and (533). These indicated that the nanoparticles were pure
Fe3 O4 with an inverse cubic spinal structure. In figure 4(B),
4

Nanotechnology 18 (2007) 285604

P Gong et al

Figure 7. Flow cytometry analysis for bacteria treated with Fe3 O4 @Ag nanoparticles. The inserted dot plots show forward light scattering
(FSC) on the X -axis versus side light scattering (SSC) on the Y -axis. Region R1 is defined as the gate region for collecting bacteria for
fluorescence measurement. Fluorescence histograms derived from gating on region R1 show red fluorescence (FL3) on the X -axis versus cell
number on the Y -axis. The marker line (M) defines dead bacteria that could be stained by PI. (A) Heat-killed E. coli (boiled for 10 min).
(B) Only Fe3 O4 @Ag nanoparticles in phosphate-buffered saline. E. coli incubated with 25 g ml1 of Fe3 O4 @Ag nanoparticles (C) and
50 g ml1 of Fe3 O4 @Ag nanoparticles (D) at 37 C for 1 h.

3.2. Antibacterial properties of Fe3 O4 @Ag nanoparticles

Table 1. MIC values of three kinds of bacteria.

Silver and silver ions have shown noteworthy antimicrobial

activities, although the mode of action and mechanism
of their antimicrobial activities have not been clarified.
Here, E. coli (gram-negative bacteria), S. epidermidis (grampositive bacteria) and B. subtilis (spore bacteria) were chosen
as representative bacteria for investigating the antibacterial
properties of Fe3 O4 @Ag nanoparticles. As shown in table 1,
the MIC values of Fe3 O4 @Ag nanoparticles for these three
kinds of bacteria were 60 g ml1 for S. epidermidis
and 70 g ml1 for E. coli and B. subtilis. The mass
percentage of Ag in Fe3 O4 @Ag nanoparticles was about 82%,
so the MIC values of Ag in Fe3 O4 @Ag nanoparticles were
about 50 and 60 g ml1 , respectively. These results
indicate that Fe3 O4 @Ag nanoparticles have broad antibacterial
capability for different types of bacteria. As a comparison,
the MIC is approximately 800 g ml1 for commercially
available antibacterial particles [35] and 100 g ml1 for Agloaded multilayer coated magnetic microspheres [31]. Bare
Fe3 O4 nanoparticles did not show any biocidal activity up to
1000 g ml1 .
Antibacterial properties of Fe3 O4 @Ag nanoparticles were
also analysed using the FCM. Generally, live cells with intact
plasma membranes cannot take up PI dye, while dead cells can

Strain
E. coli
S. epidermidis
B. subtilis

MIC value (g ml1 )

70
60
70

be stained with PI and display orange/red fluorescence which

can be collected using the red fluorescence (FL3) channel
of the FCM. Figure 7 shows the results obtained with the
FCM. We can see that there is an overlapping region between
the population of E. coli and that of pure nanoparticles from
scatter dot plots in figures 7(A) and (B), so region R1 was
set to divide bacteria from nanoparticles. As shown in the
fluorescence histogram of figure 7(A), heat-killed cells were
permeabilized and thus showed strong fluorescence, which
is marked as a positive control. When E. coli was treated
with 25 and 50 g ml1 Fe3 O4 @Ag nanoparticles at 37 C
for 1 h, about 36% and 85% bacteria were in the positive
region, respectively (figures 7(C) and (D)). These results show
that these Fe3 O4 @Ag nanoparticles could kill bacteria. They
might destroy the permeability of membranes of bacteria and
lead to cell death, which in part explains the mechanism of
silver antibacterial action [36, 37]. The phenomenon that
5

Nanotechnology 18 (2007) 285604

P Gong et al

Figure 9. Antibacterial rate of recycled Fe3 O4 @Ag nanoparticles for

different recycling times for E. coli.

Figure 8. Concentration of silver in water. Water with

50 g ml1 Fe3 O4 @Ag nanoparticles was placed in magnetic fields
for various times and then the concentration of silver in the water
was measured using ICP-AES.

Fe3 O4 @Ag nanoparticles for different recycling times for E.

coli. It can be seen that even after five cycles, Fe3 O4 @Ag
nanoparticles still had 50% of the antibacterial capability of
the original ones.

the peak in figure 7(C) was not at the same position as for
the dead cells, as seen in figure 7(A), might be the result
of incomplete inactivation of cells in lower concentrations of
Fe3 O4 @Ag nanoparticles. As the concentration of Fe3 O4 @Ag
nanoparticles increased, two peaks occurred in the positive
region in figure 7(D). The occurrence of two peaks might
be due to the aggregation of Fe3 O4 @Ag nanoparticles in
the R1 region (scatter dot plot in figure 7(B)), which were
collected as the cell signal. Additionally, the difference in
death mechanism between heat-killed cells and silver treated
cells or the interference of Fe3 O4 @Ag nanoparticles with cells
for uptake of PI dye might also help explain the occurrence of
two peaks.

4. Conclusions
Fe3 O4 @Ag nanoparticles were prepared using the reverse
micelle method. Their diameters were about 60 20 nm. Both
XRD patterns and UVvisible absorption spectra confirmed
the presence of Fe3 O4 and silver. The bactericidal effect
determined by MIC, FCM analysis and antibacterial rate
proves that the Fe3 O4 @Ag nanoparticles have biocidal activity
as regards S. epidermidis, B. subtilis and E. coli. Due to the
superparamagnetism of these nanoparticles, they were easily
removed from water after the bactericidal effect had occurred.
They could be reused several times. These results indicate that
the Fe3 O4 @Ag nanoparticles may have application as water
disinfectants.

3.3. Magnetically directed antibacterial ability of Fe3 O4 @Ag

nanoparticles
Although silver is known to have outstanding antibacterial
properties, it has been reported that high concentration of silver
is toxic to mammalian cells [27]. So it is necessary to remove
silver from specific targets or surroundings as much as possible
after bacteria have been killed.
In order to verify that silver in Fe3 O4 @Ag nanoparticles
could be removed from water through a magnetic field, water
with 50 g ml1 Fe3 O4 @Ag nanoparticles was placed in a
magnetic field for various times and then the concentration of
silver in the water was measured using ICP-AES. As shown
in figure 8, the amount of silver in water decreased with
increased time of treatment in the magnetic field. Only about
0.045 g ml1 of silver remained in the water after the water
was placed in the magnetic field for 30 min, which was lower
than the MCL for drinking water established by WHO [28].
These results indicate that high dosages of antibacterial agents
could be eliminated through use of a magnetic field easily after
it had exercised its effect.
Fe3 O4 @Ag nanoparticles can also be recycled easily
owing to their superparamagnetism.
Thus, they have
the potential to be reused on the condition that recycled
Fe3 O4 @Ag nanoparticles still have inhibitory bactericidal
effects. The antibacterial properties of recycled Fe3 O4 @Ag
nanoparticles were evaluated by measuring their antibacterial
rate. Figure 9 shows the antibacterial rates of recycled

Acknowledgments
This work was partially supported by the National Key
Basic Research Programme (2002CB513110), Key Project
Foundation of China Education Ministry (107084), Key
Project of International Technologies Collaboration Programme of China (2003DF000039), Programme for New Century Excellent Talents in University (NCET06-0697), National Science Foundation of the Peoples Republic of China
(90606003, 20405005), Outstanding Youth Foundation of Hunan Province (06JJ10004), Key Technologies Research and
Development Programme (2003BA310A16), and Key Project
of Hunan Province Technology Plan of China (02JZY2004,
0399Y1006).

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