Synthesis and bactericidal ability of Ag/TiO2 composite films

Applied Surface Science 257 (2010) 974–978
Contents lists available at ScienceDirect
Applied Surface Science
journal homepage: www.elsevier.com/locate/apsusc
Synthesis and bactericidal ability of Ag/TiO2 composite films deposited on
titanium plate
Lixiang Mai a , Dawei Wang a , Sheng Zhang b , Yongjian Xie a , Chunming Huang c , Zhiguang Zhang a,∗
a
b
c
Hospital of Stomatology, Guanghua College of Stomatology, Institute of Stomatological Research, Sun Yat-sen University, 56 Lingyuan West Road, Guangzhou 510055, PR China
Guangdong Provincial Stamotological Hospital, Guangzhou 510280, PR China
Guangzhou Institute of Energy Conversion, Chinese Academy of Science, Guangzhou 510640, PR China
a r t i c l e
i n f o
Article history:
Received 5 November 2009
Received in revised form 31 July 2010
Accepted 1 August 2010
Available online 7 August 2010
Keywords:
Thin films
Sol–gel preparation
Titanium dioxide
Bactericidal ability
Ag nanoparticles
a b s t r a c t
In this study, we develop a bactericidal coating material for micro-implant, TiO2 films with Ag deposited
on were prepared on titanium plates by sol–gel process. Their anti-microbial properties were analyzed
as a function of the annealed temperature using Escherichia coli as a benchmark microorganism. Ag
nanoparticles deposited on TiO2 film were of metallic nature and could grow to larger ones when the
annealed temperature increased. The results indicated that the smaller size of Ag nanoparticles, the better
bactericidal ability. On the other hand, the positive antibacterial effect of TiO2 enhanced the bactericidal
effect of Ag.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Micro-implants have become very popular in the orthodontic community in recent years as skeletal anchorage devices and
have shown encouraging results [1–3]. Titanium and its alloys are
commonly used as micro-implant biomaterials for their combination of mechanical stability and fine biocompatibility [4]. However,
peri-implantitis often arises clinically from infection, which is usually caused by adherence and colonization of bacteria on implants.
Morphological and chemical nature of Ti micro-implant surface
are important in relation to their effects on bacteria. Therefore,
to prevent peri-implantitis, some kinds of biomaterials should
be developed to modify the implant surface to achieve excellent
antibacterial activity and biocompatibility.
Application of photocatalysis as a remedy to the implant infection has increased tremendously in the recent past [5,6]. TiO2 is the
most widely employed photocatalyst, considering its high stability,
low cost and widespread availability. However, with the band gap
ranging from 3.0 to 3.2 eV, the efficiency of photocatalytic reaction
is limited by recombination processes and by absorption capability in visible light region [7]. Moreover, TiO2 photo-activity is
strongly influenced by the presence of noble metals [8]. A variety
∗ Corresponding author. Tel.: +86 20 83870387; fax: +86 20 83822807.
E-mail addresses: mailixiang@163.com (L. Mai), drzhangzg@163.com,
65071863@qq.com (Z. Zhang).
0169-4332/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.apsusc.2010.08.003
of attempts have been made to introduce various metal species
into the TiO2 matrix with an effort to enhance the photocatalytic
activity or broaden the absorption of the solar spectrum by the
doped TiO2 . Ag nanoparticles, one of the most important noble
metal nanoparticles, due to their outstanding physical and chemical properties, have attracted considerable interest in many fields,
especially in electrocatalytic activity and antibacterial effects [9].
Researches approved that Ag played an important role in improving
photocatalytic activity of TiO2 [10,11] since it can serve as electron
trap aiding electron–hole separation, and also facilitate electron
excitation by creating a local electric field [12,13].
It is well known that the TiO2 in anatase form is capable of
oxidizing and decomposing various organisms including virus, bacteria, fungi, algae, and cancer cell [14,15]. A treatment to reduce
bacterial infection is the synthesis of a thin TiO2 coating on biomaterial [16]. To achieve bactericidal properties more effectively,
silver doped titanium oxide coatings were developed. The promotion of the disinfection capability of TiO2 -anatase by addition of
silver has been confirmed [17–19]. The researchers also provided
the photo-killing activity by the preparation method, silver content
or heated at different temperatures and heated for different time.
In this study, Ag/TiO2 composite films were prepared by a sol–gel
spin-coating technique. Their anti-microbial properties were analyzed as a function of the annealed temperature using Escherichia
coli as a benchmark microorganism. Our aim is to seek for a better understating of the interaction of silver with anatase and the
potential effects in the anti-microbial properties of the system.
L. Mai et al. / Applied Surface Science 257 (2010) 974–978
975
Fig. 1. SEM images of the Ag/TiO2 samples annealed at different temperature (a) Sample A; (b) Sample B; and (c) Sample C.
2. Experimental procedure
2.1. Preparation of TiO2 films
The TiO2 thin films were synthesized by the sol–gel spin-coating
method [20]. First, TiO2 sol was prepared from the hydrolysis of
tetrabutyl titanate [Ti(OC4 H9 )4 , Aldrich]. 0.15 mol of ethanol was
added to 0.01 mol of Ti(OC4 H9 )4 which was cooled with ice and
stirred. 0.1 mol of ethanol was mixed with 0.02 mol of deionized
water and 0.01 mol of acetylacetone (acac). The ethanol/H2 O/acac
solution was added to the Ti(OC4 H9 )4 /ethanol solution under
stirring and cooling with ice. The Ti(OC4 H9 )4 /ethanol/H2 O/acac
solution was stirred for 2 h. The TiO2 sol was aged for about
48 h before coating. In this case, molar ratio of sol is 1:25:2:1 of
Ti(OC4 H9 )4 :ethanol:H2 O:acac. Deionized water and acac are used
for hydrolysis polycondensation reaction, which acac acts as the
chelating agent to decrease the reactivity of Ti(OC4 H9 )4 . TiO2 thin
films were coated on titanium plate by the sol–gel spin-coating
method with a rotating speed of 2000 rpm/min. The resulting films
were subjected to heat treatment at 100 ◦ C for 15 min. By repeating
this process, TiO2 thin films of different thickness were obtained.
Finally the films were annealed at 500 ◦ C for 2 h in air for crystallization.
2.2. Preparation of Ag/TiO2 composite films
Ag/TiO2 composite films were prepared by the reaction between
TiO2 thin films and 1 M AgNO3 solution for 5 min. The resulting
composite films were subjected to heat treatment at 120 ◦ C for 48 h.
Then, these films were annealed at 200 ◦ C and 300 ◦ C for 6 h. The
samples annealed at 120 ◦ C, 200 ◦ C, 300 ◦ C, were denoted as Sample
A, Sample B, Sample C, respectively. Also, the bare TiO2 film was
denoted as Control.
2.3. Morphology and bactericidal ability characterization
The morphology of the samples was investigated using Quanta
400F field-emission scanning electron microscopy (FESEM). Crystallinity and phase analyses of the films were carried out by XRD
˚ as an X-ray
(Philips X Pert) using Cu K␣ radiation ( = 1.54056 A)
source. The X-ray photoelectron spectroscopy (XPS, ESCALab 250)
method was applied to determine the valence state of Ti and Ag.
The transmittance and absorption spectra of films were measured
by a UV-VIS spectrophotometer (UV-260, Shimadzu).
The in vitro antibacterial tests were considered as preliminary
Chinese Industrial Standard (qbt2591-2003) for the evaluation of
bactericidal ability of photocatalytic titania. E. coli (BL21, TAKARA)
was employed as standard strain. All glasswares and materials were
autoclaved at 120 ◦ C for 30 min to ensure the sterility for testing.
E. coli was inoculated and grew aerobically in 50 ml liquid nutrient
broth at 37 ◦ C on a rotary shaker (120 rpm/min) for 18 h. The bac-
teria was subcultured from 50 ml to 500 ml flask with 250 ml broth
and incubated aerobically. At exponential growth phase, bacterial
cells were collected by centrifugation at 4000 rpm (10 min, 4 ◦ C),
then the bacterial pellet was washed three times with phosphatebuffer solution (0.2 mol/L, pH = 7.0). Finally the resulting pellet was
resuspended in sterile PBS and serial dilution of the cells were performed to obtain initial concentration of 106 colonies forming units
per milliliter (CFU/ml) for photocatalytic bactericidal experiment.
The experiments were carried out under two irradiation conditions: visible light and the dark. The visible light source for
photocatalysis was a 350-W Xe arc lamp (Shanghai Photoelectron
Device Ltd.). Light passed through a water filter and a UV cutoff filter
(JB420, > 420 nm, Shanghai Kawa Optical Equipment Co., Ltd.). All
plates were sterilized by autoclaving under 120 ◦ C for 30 min. The
experimental procedures were as follows. A total of 10 ␮l E. coli suspension (106 CFU/ml) was dropped onto the Ag/TiO2 coated plates
and control plates, and then being covered quickly by the prepared
cover films. After visible light irradiation or in the dark for 5, 10,
15, 20, 25, 30 min, respectively, the samples were rinsed by 10 ml
sterilized PBS to collect the survival E. coli. After being stirred, 10 ␮l
of the diluted suspension was seeded onto nutrient agar medium.
The inoculum was spread and incubated aerobically under 37 ◦ C for
24 h. At last the survival number of E. coli was obtained by counting colony. All of the above procedures were repeated three times.
The number of killed E. coli were averaged over three respective
experiments for both two conditions.
3. Results and discussion
The FESEM images of the Ag/TiO2 samples are shown in Fig. 1. In
Sample A, outer diameters of these Ag nanoparticles are between
20 nm and 30 nm. The size of Ag nanoparticles in Sample B is
between 60 nm and 80 nm. As for Sample C, the size is more than
100 nm, which is slightly larger than that of Sample B. The result
revealed that the nanoparticles could grow to the larger ones with
the annealed temperature heightened.
XRD can provide an effective method of determining the phase
transformation of TiO2 . Fig. 2 shows the XRD patterns of TiO2 and
Ag/TiO2 thin films annealed at different temperatures. In Fig. 2, XRD
patterns of TiO2 thin films annealed at 500 ◦ C exhibited anatase
phase. Ag/TiO2 thin films annealed at different temperatures all
showed anatase. Also, with increasing the annealing temperature,
the intensities of the Ag peaks are increased, implying an improvement in crystallinity and growth of Ag nanoparticles. This result is
same with FESEM.
The chemical states of elements in the Sample A and Control
were analyzed by XPS. The positions of the XPS peaks were corrected using the C 1 s core level taken at 284.8 eV as the binding
energy reference. As shown in Fig. 3a, the XPS result of Control
sample shows the core levels of Ti2p1/2 and Ti2p3/2 to be approximately at 464.6 eV and 458.9 eV, respectively, which was assigned
976
L. Mai et al. / Applied Surface Science 257 (2010) 974–978
Fig. 4. Transmittance spectra of Ag/TiO2 thin films annealed at different temperatures.
Fig. 2. XRD patterns of TiO2 and Ag/TiO2 thin films annealed at different temperatures.
to the Ti4+ in anatase TiO2 [21,22]. The line separation between
Ti2p1/2 and Ti2p3/2 was 5.7 eV, which is consistent with 5.7 eV as
the standard binding energy. After the addition of Ag nanoparticles, the binding energies of Ti2p peaks shift to higher energies.
This observation may be induced by the electron transfer between
TiO2 and Ag in metal-semiconductor contact.
Fig. 3b shows the high-resolution original XPS curve of Ag
3d region. Two peaks centered at 368.1 eV and 374.1 eV were
Fig. 3. XPS spectra of the Sample A and Control (a) Ti2p and (b) Ag 3d.
observed, corresponding to Ag 3d5/2 and Ag 3d3/2, respectively.
The splitting of the 3d doublet was 6.0 eV. No peak corresponding to Ag2 O (367.8 eV) or AgO (367.4 eV) was observed, indicating
that Ag nanoparticles deposited on TiO2 film was of metallic nature
[23–25]. Also, with use of XPS, the atomic concentration ratio of
Ti/Ag obtained for this resulting thin film is 17:7.
Fig. 4 shows the UV–vis transmittance spectra of Ag/TiO2 thin
films annealed at different temperatures. The transmittance of
Ag/TiO2 thin film increases with the annealing temperature heightened. In other words, Ag/TiO2 thin films can respond to visible light.
Moreover, the smaller size of Ag nanoparticles, the more strength of
light absorption. It is founded that the addition of Ag nanoparticles
have been shown to make TiO2 have a visible light photoresponse.
The bactericidal ability results of a (in the dark) and b (under
visible light irradiation for 20 min) are exemplified in Fig. 5. Spread
plate method denotes the survival number of E. coli. Bacteria were
almost completely killed within 20 min in Sample A in the dark,
while sterilization rate was more than 86% in Sample B and about
60% in Sample C. Under visible light irradiation, more than 98%
of E. coli were killed after 20 min on three kinds of Ag/TiO2 thin
films. Neither under visible light irradiation nor in the dark did the
control plat showed any bactericidal effects. So we concluded that
the Ag/TiO2 thin films can significantly reduce the risk of bacterial
infection. The E. coli survival curves as a function of time for the
different samples are presented in Fig. 6. The viability of cells was
determined by colonies counting. The plots provide evidence that
Sample A with larger killed-values have better bactericidal ability
than Sample B and Sample C in the dark.
Using E. coli as a model microorganism, Pratap et al. [19] showed
that under UV irradiation and with titania-loaded, the time taken
for complete inactivation of bacteria was found to be only 16 min,
20 min and 2 min for AT1, Ag–HAP and Ag–TiO2 /HAP, respectively.
But they did not study the bactericidal ability in the dark or under
visible light irradiation. Kubacka et al. [17] showed that a silver content around 1 wt.% maximized the photo-killing activity
of Ag–TiO2 , irrespective of the preparation method. However, the
distribution and size of Ag nanoparticles could be controlled by
altering the annealed temperature. Recall the FESEM result of our
study revealed that the nanoparticles could grow to the larger ones
with the annealed temperature heighten. According to the previous report of Oya et al. [26], antibacterial activity of silver/activated
carbon fiber would be enhanced by decreasing the size of silver particle. Our study is in agreement with previous results, showing that
the smaller size of Ag nanoparticles, the better bactericidal ability.
L. Mai et al. / Applied Surface Science 257 (2010) 974–978
977
Fig. 5. Photographs of the antibacterial test of the samples on E. coli after 20 min (a) in the dark and (b) at visible light irradiation.
Furthermore, when the three kinds of samples were exposed
under visible light, all of them showed perfect bactericidal effects.
As already discussed above, the UV–vis showed Ag/TiO2 thin films
had a strong visible light photoresponse. The UV–vis result could
help to explain why these films work very well under visible light.
These results indicated that TiO2 have had positive antibacterial
ability which could enhance the bactericidal effect of Ag.
Generally speaking, illuminated TiO2 photocatalysts decompose
organic compounds by oxidation, with holes (h+ ) generated in the
valence band and with conduction hydroxyl radical (OH·) produced
by the oxidation water. It has been reported that Ag-doped TiO2
films show enhanced photocatalytic efficiency [27]. In this paper,
our results indicated that Ag/TiO2 thin films can respond to visible light and the positive antibacterial effect of TiO2 enhanced
the bactericidal effect of Ag. This effect is due to the electron–hole
separation which occurs with the presence of Ag particle on TiO2
under visible light. As we know, the electrons transfer from TiO2 to
the metallic Ag particles coated on TiO2 results in a space charge
layer at the boundaries between Ag and TiO2 . Thus Ag can help
the electron–hole separation by attracting the photoelectrons [28],
and then TiO2 have had positive antibacterial ability under visible
light after the addition of Ag. In summary, the bactericidal effect of
Ag/TiO2 under visible light originates from the synergistic effect of
Ag and TiO2 .
4. Conclusion
The Ag/TiO2 composite films were prepared by sol–gel method.
It had been confirmed that Ag nanoparticles of smaller size had better bactericidal ability. Moreover, TiO2 had a positive antibacterial
ability, which could enhance the bactericidal effect of Ag. Hence,
we concluded that the Ag/TiO2 composite film would be a potential
coating material for micro-implant anchorage.
Acknowledgement
The authors gratefully acknowledge the financial support of this
work by the Sun Yat-Sen University Clinical Research 5010 Program
(2007050).
References
Fig. 6. Survival curves of E. coli for samples (a) in the dark and (b) at visible light
irradiation.
[1] R. Kanomi, J. Clin. Orthod. 31 (1997) 763.
[2] U. Fritz, A. Ehmer, P. Diedrich, J. Orofac. Orthop. 65 (2004) 410.
[3] L. Huang, J.L. Shotwell, H. Wang, Am. J. Orthod. Dentofac. Orthop. 127 (2005)
713.
[4] L. Carlsson, T. Röstlund, B. Albrektsson, T. Albrektsson, P.I. Brånemark, Acta
Orthop. Scand. 57 (1986) 285.
[5] L. Montanaro, D. Campoccia, C.R. Arciola, Biomaterials 28 (2007) 5155.
[6] A. Mo, J. Liao, W. Xu, S. Xian, Y. Li, S. Bai, Appl. Surf. Sci. 255 (2008) 435.
[7] B. Xin, L. Jing, Z. Ren, B. Wang, H. Fu, J. Phys. Chem. B 109 (2005) 2805.
[8] S.K. Lee, A. Mill, Platinum Met. Rev. 47 (2003) 61.
978
L. Mai et al. / Applied Surface Science 257 (2010) 974–978
[9] A. Isse, S. Gottardello, C. Maccato, A. Gennaro, Electrochem. Commun. 8 (2006)
1707.
[10] B. Ohtani, K. Iwai, S.I. Nishimoto, S. Sato, J. Phys. Chem. B 101 (1997) 3349.
[11] C. He, Y. Xiong, X. Zhu, Thin Solid Films 422 (2002) 235.
[12] G. Zhao, H. Kozuka, T. Yoko, Thin Solid Films 277 (1996) 147.
[13] J.M. Herrmann, H. Tahiri, Y. Ait-Ichou, G. Lassaletta, A.R. Gonzalez-Elipe, A.
Fernandez, Appl. Catal. B 13 (1997) 219.
[14] I. Sopyan, S. Murasawa, K. Hashimoto, A. Fujishima, Chem. Lett. 23 (1994) 723.
[15] O. Carp, C.L. Huisman, A. Reller, Prog. Solid State Chem. 32 (2004) 33.
[16] D.M. Alrousan, P.S. Dunlop, T.A. McMurray, J.A. Byrne, Water Res. 43 (2009) 47.
[17] A. Kubacka, M. Ferrer, A. Martinez-Arias, M. Fernández-García, Appl. Catal. B 84
(2008) 87.
[18] B. Peng, J. Wang, L. Chai, A. Mao, Y. Wang, Trans. Nonferr. Met. Soc 18 (2008)
986.
[19] M. Pratap Reddy, A. Venugopal, M. Subrahmanyam, Water Res. 41 (2007) 379.
[20] L. Mai, C. Huang, D. Wang, Z. Zhang, Y. Wang, Appl. Surf. Sci. 255 (2009) 9285.
[21] A. Manivannan, M.S. Seehra, S.B. Majumder, R.S. Katiyar, Appl. Phys. Lett. 83
(2003) 111.
[22] B.S. Jeong, Y.W. Heo, D.P. Norton, J.G. Kelly, R. Rairigh, A.F. Hebard, J.D. Budai,
Y.D. Park, Appl. Phys. Lett. 84 (2004) 2608.
[23] K.V. Kumar, J. Electron Spectrosc. Relat. Phemon. 56 (1991) 273.
[24] Y. Lai, Y. Chen, H. Zhuang, C. Lin, Mater. Lett 62 (2008) 3688.
[25] F. Zhang, G.K. Wolf, X. Wang, X. Liu, Surf. Coat. Tech 148 (2001) 65.
[26] A. Oya, M. Kimura, I. Sugo, A. Katakai, Y. Abe, T. Lizuka, N. Makyama, J. Mater.
Sci 28 (1993) 4731.
[27] C. Chang, C. Lin, C. Chan, C. Hsu, C. Chen, Thin Solid Films 494 (2006) 274.
[28] I.M. Arabatzis, T. Stergiopoulos, M.C. Bernard, D. Labou, S.G. Neophytides, P.
Falaras, Appl. Catal. B: Environ. 42 (2003) 187.