Thin Solid Films 549 (2013) 93–97

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Thin Solid Films

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Anti-fish bacterial pathogen effect of visible light responsive Fe3O4@TiO2

nanoparticles immobilized on glass using TiO2 sol–gel
N. Yeh a, Y.C. Lee b, C.Y. Chang c, T.C. Cheng d,⁎
a
Center of General Education, MingDao University, Taiwan
b
Graduate Institute of Materials Engineering, National Pingtung University of Science and Technology, Taiwan
c
Center of General Education, National Taitung Junior College, Taiwan
d
Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, Taiwan

a r t i c l e i n f o a b s t r a c t

Available online 12 October 2013 This paper demonstrates a fish pathogen reduction procedure that uses TiO2 sol–gel coating Fe3O4@TiO2 powder
on glass substrate. Such procedure can effectively relieve two constraints that haunt TiO2 sterilization applications:
Keywords: 1) the need for UV for overcoming the wide band gap of pure TiO2 and 2) the difficulty of its recovering from water
TiO2 for reuse. In the process, visible light responsive Fe3O4/TiO2 nanoparticles are synthesized and immobilized on glass
Fe3O4
using TiO2 sol–gel as the binder for fish bacterial pathogen disinfection test. After 3 h of visible light irradiation, the
Visible light
immobilized Fe3O4@TiO2's inhibition efficiencies for fish bacterial pathogen are, respectively, 50% for Edwardsiella
Pathogen
tarda (BCRC 10670) and 23% for Aeromonas hydrophila (BCRC 13018).
© 2013 Elsevier B.V. All rights reserved.

1. Introduction
Bacterial infection has been a major threat to ornamental fish in
aquarium. Reducing the bacterial pathogen loading in water is an en-
vironmental friendly alternative for fish disease control [1]. While TiO2
photocatalysis activity was first discovered in 1921 [2], the photo-
catalysis property of TiO2 for bacteria disinfection has not been iden-
tified until 1985 when Matsunaga et al. demonstrated bactericidal
effects of TiO2 [3]. A 2011 literature review by Foster et al. shows that
a wide range of Gram positive and Gram negative bacterial pathogens
of human, animal, and plant can be photocatalytic disinfected by light
irradiated TiO2 [4].
For TiO2 bactericidal studies that use suspended TiO2 powder in
solution, separation of TiO2 from solution is both difficult and costly
[5]. Therefore, immobilizing TiO2 on proper support becomes necessary.
The most often used strategies to immobilize TiO2 include sputter
coating [6], chemical vapor deposition (CVD) [7], sol–gel coating [8],
binder applying [9,10] and modified so–gel coating [11]. Qualified
substrates for TiO2 deposition include glass [12], metal [13], activated
carbon [14], and polymers [15]. These materials adhere to TiO2 well
and can resist TiO2 photocatalytic activity [16].
The anatase TiO2 has a band gap of 3.23 eV and can generate electron–
hole (e−–h+) pairs when irradiated with UV-A (λ = 315 to 400 nm)
[17]. However, the cost of UV-A is high and the use of UV can be harmful
to human eyes as well as none target organisms. As such, efforts have
been made to reduce the activation energy of TiO2 to the level of visible
light (VL). Among these methods, mixing TiO2 with sensitizer or doping
⁎ Corresponding author.
E-mail address: cheng.tachih@gmail.com (T.C. Cheng).
TiO2 with transition metal or non-metals [18] are often used. To develop
TiO2 photocatalytic aquarium glass to disinfect fish bacteria pathogens,
the research team synthesizes visible responsive Fe3O4/TiO2 powder,
uses glass as supports and TiO2 sol–gel as binder to immobilize Fe3O4/
TiO2 powder on glass, and then evaluated its antimicrobial efficiency.
2. Experimental details
2.1. Synthesis and immobilization of TiO2/Fe3O4 particles
The subject Fe3O4 is synthesized in advance via the following
modified protocol [19]. A 200-ml flask containing 40 ml of distilled
water, 0.008 mol FeCl3 and 0.004 mol FeCl2·H2O is incubated at 80 °C
and stirred with a stir bar at 300 rpm. Then, 5 ml ammonium hydroxide
is added to the solution drop by drop in 30 min. The Fe3O4 precipitation
is collected using magnet and then washed three times with 10 ml of
solution containing acetone and ethanol (volume ratio 1:2). Finally,
the particles are dried at 60 °C after being washed again with 20 ml
distilled water. TEM is used to characterize the particles.
TiO2/Fe3O4 at ratios of 34 mol%, 24 mol%, 0.13 mol% and 0.01 mol%
are synthesized separately using sol–gel method. The solutions con-
taining tetraisopropyl orthotitanate, acetic acid, 2-propanol, and Fe3O4
at the molar ratio of 1:8:2:0.34, 0.24, 0.0013 and 0.0001 are prepared
and sonicated for 8 h at 37 °C. The solutions are baked in the oven for
an hour each at 105 °C and 150 °C. The solid is calcinated in an atmo-
spheric oven heated to 200 °C at the temperature increasing rate
of 5 °C/min, held for 1 h and reheated to 500 °C at the same rate of
5 °C/min, and then held for another 3 h. The resulted solid is grounded
into powder, washed with distilled water, and then dried at 60 °C. These

0040-6090/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.tsf.2013.09.092
94 N. Yeh et al. / Thin Solid Films 549 (2013) 93–97

Fig. 1. TEM images of Fe3O4 (A) and Fe3O4 coated with TiO2 (B).

4 different ratios of TiO2 to Fe3O4 powders are ground using amber λ N 400 nm, 1040 lx) irradiation. The solution's visible light absorption
mortar, then sieved through 250 mesh screen for TEM characterization. at 610 nm is measured.
The TiO2 sol–gel for immobilization is prepared from acetic acid, The degradation efficiency (Ed) of each occasion can be written as:
n-butanol, and Titanium (IV) n-butoxide (TNBT) at 85 °C for 4 h at
150 rpm and aging for 24 h at 25 °C. Each synthesized powder of Ed ¼ f1−ð½Absc−½AbstÞ=½Abscg  100%
different TiO2 to Fe3O4 ratio is individually mixed with the 0.4 g/ml
TiO2 sol–gel and spin coated (1500 rpm) on glass slides (75 mm × where the [Abs]c indicates the absorbance of control groups and [Abs]t
25 mm × 1 mm) followed by calcination at 500 °C for 3 h. The coated the absorbance of experiment groups. The Fe3O4 to TiO2 ratio with the
slides are sonicated in distil water for 20 min to remove unbound
TiO2/Fe3O4 powders. XRD and UV–Vis spectrometer are used to char-
acterize the film. E / eV
4.0 3.5 3.0 2.5 2.0
50
100
2.2. Photocatalytic activity evaluation a
40
80
Reflectivity (%)

Modified indigo carmine dye method [20] is used to identify photo-

catalytic ability of Fe3O4/TiO2 film irradiated with fluorescence light 30
60
(λ N 400 nm) for various time intervals. Briefly, indigo carmine dye b
solution is prepared at 2.2 mg/l using distilled water. The blank glass 20
40
a. TiO2
slides (25 mm by 25 mm) as control and the glass slides with im- c
b. 0.01mol%
mobilized Fe3O4/TiO2 powder are placed in one well of a 6-well plate 10 c. 0.13mol%
d 20
(containing 3 ml of indigo carmine dye solution in each well). 100 μl d. 24mol%
e e. 34mol%
of dye solution is removed from wells at variable time interval dur-
0 0
ing fluorescent light (FL40S · N-EDL · NU, Mitsubishi/Osram, Japan, 300 400 500 600 700 800
Wavelength (nm)

Fig. 3. UV–vis diffuse reflectance spectra of immobilized Fe3O4@TiO2 powders at various

Fe3O4 to TiO2 mol% ratio.

a. TiO2
c b. 0.01mol%
c. 0.13mol%
d
e d. 24mol%
e. 34mol%
(α hv)2

4.0 3.5 3.0 2.5 2.0 1.5

E (eV)

Fig. 2. X-ray diffraction spectrum of immobilized Fe3O4@TiO2 powders at various Fe3O4 to Fig. 4. (αhν)2 vs. (hν) for band gap determination of Fe3O4@TiO2 powder synthesized at
TiO2 mol% ratio. various Fe3O4 to TiO2 mol% ratio and immobilized on glass.
 N. Yeh et al. / Thin Solid Films 549 (2013) 93–97 95

100 0.01 mol% (ASTM D903), and SEM characterization. The best is then used to for
0.13 mol%
Discoloration efficiency (%)
antimicrobial effect evaluation.
80
2.3. Antimicrobial effect evaluation
60 21 mol%
Modified antibacterial drop test [21] is used to evaluate bacterial
inhibition efficiency of Fe3O4/TiO2 film slides. A 100 μl of the phosphate
40
buffered saline (PBS) containing bacteria (105 cells/ml PBS) are depos-
ited on coated as well as uncoated slides as experiment groups, which
34 mol%
20 are then irradiated with fluorescence light (λ N 400 nm) for various
time intervals. Bacteria deposited on non-irradiated slides (coated and
0 uncoated) serve as control. All control and experiment groups have 3
0 6 12 18 24 replicates for each time interval. The bacteria are then washed off
Irradiation time (hr) using 900 μl PBS containing 0.05% Tween-20 at each sampling time
interval. Then, 20 μl of the such solution is added to wells of a 96-well
Fig. 5. Indigo carmine degradation efficiency of immobilized Fe3O4@TiO2 powders at
plates containing 100 μl Luria–Bertani (LB) medium and 2 μl of 0.05%
various Fe3O4 to TiO2 mol%.
2, 3, 5-triphenyl tetrazolium chloride (TTC) and incubated at 28 °C for
16 h. After adding 50 μl of 75% ethanol to each well to terminate reac-
100 0.4 g/ml tion and dissolve violet crystals, the 540 nm wavelength absorbance
Discoloration efficiency (%)

0.6 g/ml
at each well is measured using spectrophotometer.
80 0.2 g/ml The fish pathogen inhibition efficiency (Epi) can be denoted as:

60
Epi ¼ f1−ð½Absi−½AbstÞ=½Absig  100%
0.1 g/ml
where [Abs]i is the absorbance of control group and [Abs]t is the
40
absorbance of experimental group during the same time interval.

20
3. Results and discussion

0 3.1. Synthesis and immobilization of TiO2/Fe3O4 particles

0 6 12
Irradiation time (hr)
Fig. 6. Indigo carmine degradation efficiency of various ratios of Fe3O4@TiO2 powder
(Fe3O4 to TiO2 ratio is 0.01 mol%) to TiO2 sol–gel for immobilization.
highest photocatalytic activity is chosen for further Fe3O4/TiO2 powder
to TiO2 sol–gel ratio optimization. The Fe3O4/TiO2 powder to TiO2 sol–
gel ratio at 0.1, 0.2, 0.4, and 0.6 g/ml are prepared, immobilized on
glasses via spin coating followed by photocatalytic activity mea-
surement. The two with the highest photocatalytic activity are selected
for luminous transmittance measurement (ASTM D1003), peel test
18 24
The Fe3O4 particles are synthesized in advance using co-precipitation
method. Most particles are rough spheres with an average diameter of
16 nm (Fig. 1A). Their average diameter increases to 24 nm (Fig. 1B)
after TiO2 coating, indicating a 4 nm coating layer of TiO2. All coated
Fe3O4 particles are over 16 nm. The thickness of TiO2 coating on the
Fe3O4 may affect the photocatalytic activity of Fe3O4@TiO2 particles.
The photocatalytic activities fluctuate as the thickness of TiO2 increase
from 5 nm to 200 nm. The optimal TiO2 thickness is 10 nm [22]. The
results suggest that the photocatalytic activity of Fe3O4@TiO2 particles
can be improved via TiO2 coating thickness adjustment.

Fig. 7. SEM images of two different concentrations of immobilized Fe3O4@TiO2 powders on glass using TiO2 sol–gel as binder.
96 N. Yeh et al. / Thin Solid Films 549 (2013) 93–97
Fig. 8. Bactericidal effect of immobilized Fe3O4@TiO2 powders using TiO2 sol–gel after
irradiated with visible light.
XRD patterns of Fe3O4 and γ-Fe2O3 signals are detected in the sam-
ple of 34 mol% of Fe3O4 to TiO2 sample immobilized on glass (Fig. 2).
Generation of γ-Fe2O3 from Fe3O4 is possible during Fe3O4 synthesis
via oxidation of Fe3O4 [23]. The XRD patterns of Fe3O4 and γ-Fe2O3 are
similar for their identical crystal structures [24], which make them
difficult to distinguish. However, with X-ray photoelectron spectroscopy
(XPS), a thin layer of α-FeOOH can be identified on the surface of Fe3O4
synthesized using co-precipitation method. The α-FeOOH is converted
to γ-Fe2O3 at 400 °C and led to the formation of Fe3O4@γ-Fe2O3 core/
shell structure [25]. As this study also uses co-precipitation method to
synthesize Fe3O4 for TiO2 coating followed by the 500 °C calcination
described in Section 2.1, the co-existence of Fe3O4 and γ-Fe2O3 is
possible. Other immobilized Fe3O4@TiO2 particles containing lower
Fe3O4 to TiO2 ratio have shown no signals of Fe3O4 and γ-Fe2O3 as the
signals might fall below the XRD detection limit. In addition, only
Fig. 9. SEM images of the intact and the damaged bacterial cells.
anatase TiO2 patterns are found in all samples of immobilized Fe3O4@
TiO2 nanoparticles that contain various Fe3O4 to TiO2 mol%. This result
indicates that Fe3O4 or γ-Fe2O3 does not affect anatase TiO2 formation,
although Eshaghi et al. have found that the adding of TiO2 powder for
immobilization leads to the increase of rutile TiO2 in the composite
nanoparticle film [26].
Fig. 3 shows the red shift of immobilized Fe3O4@TiO2 nanoparticles
comparing with TiO2. The higher Fe3O4 mol% of Fe3O4@TiO2 nano-
particles has more absorption in visible region. The band gap, calculated
for each Fe3O4 mol% of immobilized Fe3O4@TiO2 nanoparticles (Fig. 4),
decreases as the Fe3O4 mol% increases. The band gaps for 0.01 mol%,
0.13 mol%, 24 mol%, 34 mol% of Fe3O4, are 3.4 eV, 2.9 eV, 2.8 ev, 2.3 eV,
and 1.8 eV, respectively. However, the lowest band gap of 34 mol%
Fe3O4, 1.8 eV, might mainly be due to the high Fe3O4 content.
3.2. Photocatalytic activity evaluation
The immobilized Fe3O4@TiO2 nanoparticles that contain lower Fe3O4
mol% led to higher photocatalytic activity (Fig. 5). High concentration
of Fe3+, which is dissociated from Fe3O4, can function as recombina-
tion center for electrons and holes [27,28] and reduces the photocata-
lytic activity. Meanwhile, the increase of Fe3O4 mol% during TiO2
coating may cause the wrapping of multiple Fe3O4 particles within
TiO2. In such case, the surface area reduction and the multiple Fe3O4
cores within TiO2 tend to hinder the light penetration and lead to the
drop of photocatalytic activity. Therefore, the Fe3O4@TiO2 nanoparticles
containing 0.01 mol% Fe3O4 is selected to optimize the amount of
Fe3O4@TiO2 for immobilization.
3.3. Antimicrobial effect evaluation
The optimal Fe3O4@TiO2 to TiO2 sol gel ratio for particle immobili-
zation is 0.4 g/ml (Fig. 6). Increasing that ratio to 0.6 g/ml marks no
significant difference as the Fe3O4@TiO2 immobilized slide is treated
 N. Yeh et al. / Thin Solid Films 549 (2013) 93–97
Table 1
Characterization and comparison of immobilized TiO2/Fe3O4 on glass.
TiO2/Fe3O4 coating
Test
Photo
Luminous transmittance (%)
Peel strength (gf/mm)
Discoloration efficiency (%) (12 h)
Inhibition efficiency (%) (3 h) E. tarda
A. hydrophila
with super-sonication before dye degradation test and the adhesion
between Fe3O4@TiO2 and glass is not strong enough to resist super-
sonication treatment. Chen et al. [29] have found that increasing the
calcination temperature helps to improve adhesion but at eh same
time causes photocatalytic activity reduction. Therefore, 0.2 g/ml and
0.4 g/ml are selected for further characterization.
Fig. 7-A and D demonstrates the SEM illustration of cracked and
rough surfaces of immobilized Fe3O4@TiO2 film. Fig. 7-B and E shows
that the increased Fe3O4@TiO2 loading reduces the size of grain. The
average diameter is 80 nm for the loading of 0.2 g/ml and 30 nm for
0.4 g/ml. Chen et al., [30] have found that the grain size of TiO2 decreases
as the loading of TiO2 increases when using the modified sol–gel meth-
od to immobilize TiO2 on stainless steel. Eshaghi et al. [26] have found
similar trend. The 0.2 g/ml loading demonstrates higher luminous
transmittance (43.2%) than that of 0.4 g/ml loading (19.3%). Higher lu-
minous transmittance causes lower photocatalytic activity. For aquarium
glass, however, high luminous transmittance of coated Fe3O4@TiO2
layer is preferable as long as photocatalytic activity level remains
acceptable. In addition, the adhesion among Fe3O4@TiO2 particles as
well as between Fe3O4@TiO2 and glass is also important. In this study,
for example, the peel strengths for 0.2 g/ml and 0.4 g/ml loading are
30.5 gf/mm and 27.3 gf/mm, respectively. Such strengths are sufficient
to resist the water current in aquarium.
As shown in Fig. 8, both Edwardsiella tarda and Aeromonas
hydrophila are susceptible to photocatalytic disinfection. Significantly
more damage as well as deformed bacteria cells are found on the visible
light irradiated film of immobilized Fe3O4@TiO2 than on the non-
irradiated one (Fig. 9B, D).
In general, Gram-positive is more resilient to the bactericidal effect of
TiO2 due to the cell wall structure and component difference [31–33]. In
this study, both E. tarda and A. hydrophila are Gram-negative. Compared
to A. hydrophila (inhibition efficiency, 23.3%), E. tarda (inhibition effi-
ciency, 50.6%) is less resilient to visible light irradiated Fe3O4@TiO2
film (Fig. 8). This result indicates that bactericidal effect is species
dependent even within the same category of Gram-negative.
4. Conclusion
This study has implemented a procedure that can reduce the limi-
tation accompanied the sterilization applications using TiO2. Such limi-
tations include the need of UV light for its activation and the difficulty of
its recovery from water for reuse. The use of TiO2 sol–gel to immobilize
Fe3O4@TiO2 nanoparticles on glass is optimized and the characteristics
of film listed in Table 1. The results of this study demonstrate that
using TiO2 sol–gel to coat Fe3O4@TiO2 powder on glass to reduce fish
pathogen concentration does help to cut the requirement of UV light
97
Only glass 0.2 g/ml sol–gel 0.4 g/ml sol–gel
100 43.2 19.3
– 30.5 27.3
0 53.5 90.7
0 – 50.6
0 – 23.3
and enhance the rate of recollection. As high intensity LEDs have
evolved to become a prominent light source for scientific research
[34,35], the study conducted in this paper can be followed up using
LEDs in lieu of fluorescence lights for an informative comparison.
Acknowledgment
This study is supported by a grant from the National Science Council
of Taiwan under the contract of NSC: 101-2313-B-020-016.
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