Antibacterial property of Ag nanoparticle-impregnated N-doped titania films under visible light

Photocatalysts produce free radicals upon receiving light energy; thus, they possess antibacterial properties. Silver (Ag) is an antibacterial material that disrupts bacterial physiology. Our previous study reported that the high antibacterial property of silver nanoparticles on the surfaces of visible light-responsive nitrogen-doped TiO2 photocatalysts [TiO2(N)] could be further enhanced by visible light illumination. However, the major limitation of this Ag-TiO2 composite material is its durability; the antibacterial property decreased markedly after repeated use. To overcome this limitation, we developed TiO2(N)/Ag/TiO2(N) sandwich films in which the silver is embedded between two TiO2(N) layers. Various characteristics, including silver and nitrogen amounts, were examined in the composite materials. Various analyses, including electron microscopy, energy dispersive spectroscopy, X-ray diffraction, and ultraviolet–visible absorption spectrum and methylene blue degradation rate analyses, were performed. The antibacterial properties of the composite materials were investigated. Here we revealed that the antibacterial durability of these thin films is substantially improved in both the dark and visible light, by which bacteria, such as Escherichia coli, Streptococcus pyogenes, Staphylococcus aureus, and Acinetobacter baumannii, could be efficiently eliminated. This study demonstrated a feasible approach to improve the visible-light responsiveness and durability of antibacterial materials that contain silver nanoparticles impregnated in TiO2(N) films.

ious sputtering periods, namely 0, 15, 30, 60, and 120 s, were examined. X-ray diffraction patterns of the sandwich films revealed that the anatase phase of titania was formed after annealing ( Supplementary Fig.  S1). Energy-dispersive X-ray spectroscopy analysis indicated that the amount of Ag deposition increased with time ( Supplementary Fig. S2). Furthermore, a field-emission scanning electron microscopy analysis revealed that the thickness of the sandwiched thin films was nearly 700 ± 50 nm, in which the thickness for the silver layers are 1.9, 3.8, 7.5 and 15 nm for the specimens prepared under various sputtering time periods of 15, 30, 60, 120 s, respectively ( Fig. 1A-J). In addition, various amounts of Ag nanoparticles were observed on the TiO 2 (N) surfaces of some films before and after annealing ( Fig. 1K-T, white spots). An ultraviolet-visible (UV-Vis) absorption spectroscopy analysis revealed no significant difference after annealing; whereas the Ag-containing samples showed a redshift, indicating an increased visible light absorption ( Fig. 2A,B). The visible-light-responsive photocatalytic property was determined using a methylene blue (MB) degradation analysis as described 18 . Data revealed that Ag doping did not markedly increase the visible-light-responsive photocatalysis (Fig. 2C). Despite this, the antibacterial property was determined. Because of the lack of the visible-light-induced photocatalytic activity, no significant difference was observed between the groups with or without visible light illumination (Fig. 2D). Because these thin films contained Ag, the antibacterial property was detectable even in the dark conditions. The NTA120s sample, which contained the highest Ag levels ( Supplementary Fig. S1) exhibited the most antibacterial activity compared with the other tested samples (Fig. 2D).
Effect of nitrogen level on photocatalytic and antibacterial properties of TiO 2 (N)/Ag/TiO 2 (N) sandwich films. To increase the visible-light responsiveness, N doping is critical 6 . The nitrogen content in these films was minimal when oxygen flow was high [5 sccm (standard cubic centimeters per minute)] during film deposition. We hypothesized that this was caused by an excess supply of oxygen, rendering N unable to be efficiently integrated into the lattice structure of TiO 2 . Thus, various low-O 2 -supplying conditions, including the O 2 flow rate of 2, 3, 4, and 5 sccm, were tested (Supplementary Table 1). Consistent with our hypothesis, an X-ray photoelectron spectroscopy analysis revealed that reduced O 2 supply improved N levels in composite thin films (Fig. 3A). However, the Ti-to-O ratio was also changed, with the N(4)TA and N(5)TA samples exhibiting a relatively 1:2 ratio, whereas the N(2)TA and N(3)TA samples exhibited a ratio of approximately 1:1 ( Fig. 3A; the amount of oxygen supplied was indicated in brackets, e.g., N(4)TA was prepared with 4 sccm O 2 ). These results indicated that 2 and 3 sccm O 2 supply were insufficient to oxidize Ti and produce TiO 2 ; instead, TiN or TiON was formed. Consistently, X-ray photoelectron spectroscopy analysis for the 1 s atomic orbital of N indicated a significant increase in the formation of TiN (Fig. 3B). X-ray diffraction analysis further revealed the production of TiN in N(2)TA and N(3)TA samples before and after annealing (Fig. 3C,D). Because TiO 2 was not efficiently Scientific RepoRts | 5:11978 | DOi: 10.1038/srep11978 produced in the N(2)TA and N(3)TA samples, the rutile and anatase TiO 2 signals were observed only in the N(4)TA and N(5)TA thin films after annealing (Fig. 3D). The field-emission scanning electron microscopy analysis indicated that the N(2)TA and N(3)TA samples formed thicker films, with less surface-exposed Ag nanoparticles compared with the N(4)TA and N(5)TA samples ( Fig. 4A-E). A UV-Vis absorption spectroscopy analysis indicated that reduced O 2 supply caused remarkable redshifts in the samples (Fig. 5A,B). The band gaps were calculated using the UV-Vis spectroscopy and Tauc plots 19 . However, the band gaps for the N(2)TA and N(3)TA were not derived, because the two samples produced under 2 and 3 sccm O 2 supply were insufficient to form TiO 2 ; instead, TiN or TiON was formed with high amount of nitrogen of 18.8% and 13.5% and thus, with high absorbance (Fig. 3A,B). These results indicated that the increased N content reduced the band gap of the thin films (Fig. 5C, Supplementary Table S1). Analyses of MB and Hoechst dye degradation rates further revealed that the N(4)TA sample exhibited a superior visible-light-induced photocatalytic ability than did the other samples ( Fig. 5D; Supplementary Fig. S3), suggesting that the balance of the N-doping amount and TiO 2 production was critical. In accordance with the aforementioned analyses, antibacterial experiments demonstrated that the N(4)TA sample exhibited the highest antibacterial activity among the thin film samples under visible light illumination (Fig. 5E, N(4)TA-N500). In addition, visible light illuminating the N(4)TA sample exerted higher bactericidal activity than did the control groups with the N(4)TA sample in the dark (Fig. 5E, N(4)TA-N500; *P < 0.05).
Durability of visible-light-responsive antibacterial property. The visible-light-responsive antibacterial property of these novel sandwich films after repeated usages was compared with that of our previously developed single-layer thin films containing Ag nanoparticles 6 . Although the first use of the single-layer thin films could achieve an approximately 5-log reduction in bacteria 6 , the data indicated that these thin films could not efficiently eliminate the bacteria after frequently use (Fig. 6, secondand third-cycle use of single-layer groups). By contrast, favorable performance was observed after these multilayer films were repeated used, which eliminated E. coli efficiently after the second and third use ( Fig. 6, second-and third-cycle use of multilayer groups). Scanning electron microscopy was employed to investigate the E. coli cell damage caused by the sandwich films. We observed that if the bacterial cells were not treated with Ag present in the N-doped TiO 2 sandwich films with or without visible light illumination, they displayed relatively smooth surfaces (Fig. 7A,B). Because of the antibacterial property of Ag, bacterial cells displayed rough surfaces after being treated with a sandwich film in the dark (Fig. 7C). More vigorous changes, which were showed as unique cracks, such as structures on their surfaces, were observed after the bacterial cells were illuminated with visible light on a sandwich film (Fig. 7D, arrows).

Pathogen analyses.
To investigate the performance of TiO 2 (N)/Ag/TiO 2 (N) sandwich films in eradicating pathogenic bacteria and human pathogens, including S. pyogenes, S. aureus, and A. baumannii, the films were subjected to visible-light-induced catalysis. Among these, S. aureus and A. baumannii are pathogenic bacteria with a high antibiotic resistance rate, causing increased incidence of nosocomial infections 20 . We demonstrated that all the tested pathogens were efficiently eliminated after exposure to the sandwich films illuminated with visible light (Fig. 8, dark vs. light groups). The effectiveness showed a nearly 1-log reduction of the bacterial population. In addition, the TiO 2 (N)/Ag/TiO 2 (N) sandwich films appeared equally potent in eliminating the bacteria when applied to both the Gram-positive (S. aureus and S. pyogenes) and Gram-negative (E. coli and A. baumannii) bacteria (Fig. 8).

Discussion
Silver has been used to prevent infections for thousands of years 21 , with Hippocrates describing the antimicrobial properties of the metal in 400 BC. Antibacterial silver products have been widely used   for the handling and cleaning of burn, trauma, catheter, and dental amalgam 22,23 . Previous studies have demonstrated that both Ag + ions and Ag nanoparticles possess antibacterial properties [22][23][24] . Silver disrupts various bacterial physiologies, including disulfide bond formation, metabolism, and iron homeostasis; these changes increase production of ROS and membrane permeability and disrupt membrane respiratory electron transport chains and DNA replication components 23,25 . Thus, silver is widely used as tableware and a hygiene product. In these cases, the durability of the antibacterial property is not a Figure 5. Influence of nitrogen content in TiO 2 (N)/Ag/TiO 2 (N) on the film property. Analyses of UV-visible absorption spectroscopy before (A) and after (B) annealing, the band gaps (C) the methylene blue degradation (D) and the bacterial survival (CFU) (E) were shown. Control groups on the glass were normalized to 100%. *P < 0.05, **P < 0.01, compared to respectively dark groups. n = 3.
problem because the entire metal product is able to release a sufficient amount of Ag ions and nanoparticles with time. However, this is not the case when only a nanoscaled layer of silver is used.
Previous studies have indicated that Ag particles or their coating on TiO 2 surfaces can enhance the light-driven photoinactivation of bacteria 2,6,26-30 . However, the antibacterial activity of such thin films after repeated use has not been investigated thoroughly. In addition, the antibacterial property of Ag-TiO 2 composite thin films remains to be elucidated. For example, our previous study reported that  TiO 2 containing nanoscale Ag wires exhibited strong visible-light enhanced antibacterial properties 6 . The durability of these thin films is always a problem; because their antibacterial properties rapidly decline after repeated use (Fig. 6, second-and third-cycle use of single-layer groups). Therefore, we described the influence of the various amounts of silver and nitrogen on the antibacterial property of the sandwich films. Analysis results indicated that silver depositions improved the antibacterial activity of the thin films (Fig. 2). In addition, the amount of nitrogen supplied is crucial for the formation of high-performance antibacterial thin films under visible light illumination (Fig. 5). Thus, we developed the TiO 2 (N)/Ag/ TiO 2 (N) sandwich films, which exhibited sustainable antibacterial properties after repeated use.
Nanoscaled thin films have been used to achieve a controlled release of embedded materials [31][32][33] . In the original experimental design, the silver layer was embedded in the middle of TiO 2 (N)/Ag/TiO 2 (N) sandwich films. Notably, in addition to the embedded Ag layer, we observed certain scattered Ag nanoparticles on the surface of the as-deposited sandwich films and a higher density of Ag nanoparticles on annealed films. Ag nanoparticles appeared to emerge on the film surface by atomic diffusion through the grain boundaries of the upper TiO 2 (N) layer. In addition, this could happen when silver atoms gain sufficient energy from the bombardment of atoms and ions during sputtering growth or from thermal annealing. In either case, the Ag nanoparticles are impregnated in the TiO 2 (N) films and not merely placed or attached on the film surface, which could be the main reason for the high durability of the photocatalytic TiO 2 (N)/Ag/TiO 2 (N) sandwich films.
The silver-induced bactericidal effect is a complex response, involving the disruption of bacterial physiologies and abnormal elicitation of ROS in bacteria 23,25,34 . Combined treatment with exogenous ROS and silver revealed a synergistic antibacterial effect 35 . ROS production is the main antibacterial mechanism of photocatalysts 2,36,37 . Thus, it is reasonable that the silver synergizes with the photocatalytic components of the sandwich films to efficiently eradicate pathogenic bacteria under the visible light (Fig. 8). However, nanoscale Ag particles and TiO 2 appear to exert a synergistic impact on the environment 38,39 . The increased production and use of silver nanoparticles in products leads to the inevitable increase in the release of these particles into the environment through the lives of these products, from the raw material stage to disposal 40 . Likewise, the increased disposal of TiO 2 nanoparticles exerts a considerable impact on the ecosystem 41 . Although the mechanism remains to be further elucidated, the coexistence of Ag and TiO 2 nanoparticles has been shown to exert increased environmental impact under sunlight 38,39 . Furthermore, exposure to Ag nanoparticles was shown to influence the immune system 42 . However, previously reported Ag-TiO 2 thin films have typically had a surface-exposed Ag layer 6,[43][44][45][46] . Thus, the increase reusability of the nanoscaled Ag-TiO 2 composite material with a TiO 2 -covered layer will reduce the environmental impact of products and will benefit human health.
In conclusion, we successfully demonstrated the antibacterial properties of TiO 2 (N)/Ag/TiO 2 (N) sandwich films, which could be optimized through Ag and N depositions on titania substrates. The silver and TiO 2 (N) composite materials exhibited synergistic antibacterial activity under visible light illumination. These findings suggest that the concepts used in this study and multilayer TiO 2 (N)/Ag/TiO 2 (N) composite materials have potential applications in the developing alternative disinfectants. Methods Various thin films, including single layers of Ag, TiO 2 , and TiO 2 (N), and TiO 2 (N)/Ag/TiO 2 (N) sandwich layers were prepared in a reactive magnetron sputtering system. As Supplementary   Fig. S4 illustrates, five sputtering targets exist in the deposition system, but only two titanium (Ti) targets and one silver (Ag) target were used, and the base pressure was below 1.3 × 10 −5 Pa. To prepare pure TiO 2 or TiO 2 (N) and Ag, two Ti targets and one Ag target were used, respectively. Before sputtering, we used argon plasma to etch the surface of the substrate for 10 min to remove residual particles on the surface. The substrate holder was rotated at a speed of 5 rpm without applying substrate bias during deposition. Silicon wafer (100) and glass substrates were used. The substrate was nearly at room temperature without external heating. The target powers were set at 250 W each and 20 W in the DC mode for the two Ti targets and one Ag target, respectively. For pure TiO 2 deposition, the gas contained argon and oxygen with the fixed flow rates of Ar at 20 sccm and O 2 at 7 sccm, and the total pressure was approximately 4.7 × 10 −1 Pa (3.5 × 10 −3 Torr). For the TiO 2 (N) films, the gas contained Ar, O 2 , and N 2 at the flow rates of 20, 5-2, and 8 sccm, respectively, whereas for the Ag film, the gas used contained only Ar with a flow rate of 20 sccm without adding other gases.
In addition, for forming a monolithic layer of Ag, TiO 2 , and TiO 2 (N), two series of sandwiched TiO 2 (N)Ag/TiO 2 (N) films were prepared. Furthermore, one series was prepared with various amounts of Ag in the sandwiched films, whereas the other series was prepared with a fixed amount of Ag but with various nitrogen contents in the sandwiched films. In the first series, the amount of Ag was regulated by changing the deposition time from 30 s to 60, 90, and 120 s, and the corresponding sample IDs were denoted as NTAxxxs, where xxx was the Ag deposition time. In the second series, the amount of Ag was fixed with the deposition time of 120 s, and the nitrogen contents in the TiO 2 (N) films were regulated by reducing the oxygen flow rates from 5 sccm to 4, 3, and then 2 sccm, and the corresponding sample IDs were denoted as N (x)TA, where x was the oxygen flow rate. The as-deposited films were further annealed at 500 °C for 1 h in the nitrogen atmosphere by using a conventional vacuum furnace.
The deposition rate of TiO 2 (N) films varied with the oxygen flow rate and was approximately 350 nm/h with the oxygen flow rate of 4 sccm. The deposition rate of silver was approximately 7.5 nm/min. The deposition times of the typical sandwich TiO 2 (N)/Ag/TiO 2 (N) film used in this study were 1 h and 2 min, and 1 h. Thus, an average thickness of the Ag impregnated TiO 2 (N) films was 700 ± 50 nm. The thickness for the silver layer are 1.9, 3.8, 7.5 and 15 nm for the specimens prepared under various sputtering time periods of 15, 30, 60, 120 s, respectively. Characterization analysis. The structure and crystallinity of films were analyzed using X-ray diffraction measurements recorded using the Rigaku X-ray diffractometer D/MAX-2500 V with a Cu Kα radiation (40 kV, 100 mA) source. The surface morphology and the cross-sectional view of the films were observed using the JEOL JEM-6500 F field-emission scanning electron microscopy. The UV-Vis absorption spectra of the films were recorded using a JASCO V-650 spectrophotometer ranging from 300 to 900 nm. The composition of samples was determined using an energy dispersive spectroscopy (Horiba, EMAX-ENERGY) and a K-Alpha ™ X-ray photoelectron spectrometer using an AlK α X-ray radiation source to estimate elements semi-quantitatively.
Antibacterial experiment. Bacterial culturing and plating were performed following the previously described standard methods 5,47-49 . The bacterial concentration was determined using the standard plating method or from optical density readings at 595 nm (OD 595 ). For example, the conversion factor for E. coli BL 21 was calculated to be 1 × 10 9 colony-forming unit (CFU)/mL at OD 595 , and the cultures were diluted with the culture medium to 1 × 10 7 CFU/mL. The 1 × 10 6 CFU culture was allowed to drip on the sample (approximately 6.25 cm 2 ) and was then placed in a dark room or exposed under the visible light and at room temperature. The visible light source was an incandescent lamp (Classictone incandescent lamp, 60 W, Philips Taiwan; Taipei, Taiwan), and the illumination density was recorded using a light meter (model LX-102; Lutron Electronic Enterprises, Taipei, Taiwan). In the photocatalytic reaction, the illumination distance between the sample and lamp was approximately 10 cm, which was exposed for 30 min, and the light intensity on the sample surface was nearly 1.2 × 103 lux (lumen/m 2 ) (30 mW/cm 2 ). After illumination, 100 μ L of the bacterial solution was recovered from the sample. Finally, the bacterial concentration was determined using standard dilution and plating methods, and the percentage of surviving bacteria was calculated 18,19 . S. pyogenes (strain M29588), pandrug-resistant A. baumannii (strain M36788), and S. aureus (strain SA02) were the clinical isolates provided by Buddhist Tzu-Chi General Hospital in Hualien, Taiwan 5 . E. coli and A. baumannii were grown and maintained in the lysogeny broth (LB) medium or LB agar (BD Diagnostics, Sparks, MD, USA) at 37 °C. By contrast, S. pyogenes and S. aureus were grown in the trypticase soy broth with yeast extract (TSBY) medium or TSBY agar (MDBio, Inc., Taipei, Taiwan) at 37 °C. When the visible light was used to elicit the photocatalysis reaction, a UV cut-off filter (400 nm; Edmund Optics, Barrington, NJ, USA) was used to prevent the illumination of small fractions with UV-range wavelength in the photocatalytic experiments.
Photocatalytic properties. Analysis methods were based on recently reported literatures 4,50,51 , which used the MB degradation rate to analyze the photocatalytic performance of impurity-doped TiO 2, in accordance with the generally accepted method and reported format of MB photodecomposition. Photocatalytic efficiency was evaluated by examining the decomposition of 10 ppm MB (Sigma-Aldrich, St. Louis, MO, USA). The MB concentration was determined according to the intensity of the light-absorption peak at 664 nm wavelength, as measured using a UV-Vis spectrometer. A fixed size (1 cm × 1 cm) of the sample sank in 2 mL of the MB aqueous solution. The visible light illumination was conducted using a fluorescent lamp (Philips; P-LF27W/865) with a wavelength distribution of approximately 400-750 nm and a maximum intensity at the range of 543-611 nm, producing an average power density of 4.2 mW/cm 2 at a distance of 8 cm between the visible light source and sample.
Statistical analysis. The means, standard deviations, and statistics of the quantifiable data were calculated using Microsoft Office Excel 2003, SigmaPlot 10, and SPSS 19. The significance of data was determined using a one-way analysis of variance followed by a post hoc Bonferroni corrected test. The probability of type I error α = 0.05 was recognized as statistically significant.