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 . 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 . Moreover, TiO2 photo-activity is strongly inﬂuenced by the presence of noble metals . A variety ∗ Corresponding author. Tel.: +86 20 83870387; fax: +86 20 83822807. E-mail addresses: [email protected] (L. Mai), [email protected], [email protected] (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 . 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 . 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 . 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.  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.  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. , 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 . 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 , and then TiO2 have had positive antibacterial ability under visible light after the addition of Ag. 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