Environ. Sci. Technol. 2009, 43, 2905–2910
Potent Antibacterial Activities of Ag/TiO2 Nanocomposite Powders Synthesized by a One-Pot Sol-Gel Method HUANJUN ZHANG AND GUOHUA CHEN* Department of Chemical and Biomolecular Engineering, the Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, People’s Republic of China
Received December 4, 2008. Revised manuscript received February 23, 2009. Accepted February 26, 2009.
The antimicrobial properties of Ag-based materials have been actively investigated recently. In such materials, control of the size of the Ag particles is critical to their bactericidal performance. A novel one-pot sol-gel scheme is described here. It incorporates room-temperature ionic liquids (RTILs) to synthesize Ag/TiO2 nanocomposite powders. The presence of RTILs is indispensable to the control of the size of the Ag particles. Highly dispersed, metallic Ag nanoclusters are formed on the TiO2 nanoparticle surface after calcination of the gel. The average cluster size of Ag can be controlled to be below 5 nm with high Ag loading (7.4wt%). Antibacterial tests using 7.4wt% Ag/TiO2 on 105 CFU/mL Escherichia coli (E. coli) strains incubated on Luria-Bertani (LB)/agar plates show that bacterial growth was inhibited by 98.8% at an Ag concentration of 1.2 µg/mL. Complete inhibition was achieved at 2.4 µg(Ag)/mL. At this concentration, a 3.9wt% Ag/TiO2 sample, with a smaller Ag cluster size (2.4 µg/
SCHEME 1. Postulated Evolution of Ag/TiO2 Formation in the Presence of [bmim]PF6
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FIGURE 3. Results of the antibacterial tests (105 CFU/mL) using Ag(7.4wt%)/TiO2. Samples: (A) blank, (B) pure TiO2, and (C-F) Ag(7.4wt%)/TiO2 at various Ag concentrations (1.2, 2.4, 3.0, and 3.6 µg/mL for C, D, E, and F, respectively). mL) lead to complete suppression of E. coli growth. These data show that the Ag(7.4wt%)/TiO2 nanocomposite powders possess highly potent antibacterial properties against E. coli. The antibacterial activity of the Ag/TiO2 powders in this work appears to be superior to certain pure Ag nanoparticles (4, 36, 37). Under similar testing conditions (e.g., E. coli CFU), Sondi et al. (4) reported that pure Ag nanoparticles (∼12 nm in size) inhibited bacterial growth by 70% at a concentration of 10 µg/mL (∼8 times that of sample C in Figure 3). Complete inhibition was observed only when the Ag concentration reached 50 µg/mL. The smaller Ag particle size should account for the higher antibacterial activity of our Ag/TiO2. This size effect was also reported by other researchers (5, 38). The dependence on particle size of antibacterial activity can be understood from two arguments: (1) smaller particles have more surface atoms that are expected to be active upon contact with bacterial cells; (2) smaller particles also have a larger fraction of atoms on low-coordination and high-energy sites (corners, edges, steps, kinks and adatoms etc.), which makes them more active than larger particles (39). With the Ag particle size controlled below 5 nm, both arguments are expected to contribute to the potent antibacterial activity of Ag/TiO2. The unique structure of TiO2 nanoparticles supporting highly dispersed Ag clusters should also lead to superior bactericidal properties. In such a configuration, the Ag clusters are largely fixated on TiO2 particle surfaces, which minimizes their aggregation in aqueous suspensions. Unsupported Ag particles, however, tend to aggregate to reduce their effective surface area. Modifications of Ag nanoparticles have been addressed by introducing surfactants or polymer stabilizers against aggregation (40). In our study, the TiO2 particles served as solid antiaggregation supports to stabilize Ag dispersion. More importantly, the Ag clusters covered a large portion of the TiO2 surfaces and were exposed to the surroundings, as revealed by XPS and TEM studies. This configuration should promote Ag/bacteria contact and bactericidal efficiency in attacking and destructing bacterial cell membranes (4, 5). These actions have been reported to increase membrane permeability, leading to uncontrolled mass transport through the membranes, Ag penetration into the cells, Ag interactions with P- and S-containing compounds, and finally cell death (6, 36, 37). Thiel et al. (10) reported the antibacterial performance of Ag/TiO2 composite powders without such structural features. Their most effective sample (Ag0.96wt%/TiO2) inhibited bacterial growth by ∼90% (total colony number ∼400) at an Ag concentration of 3.06 × 10-2 µg/cm2. Comparatively, the Ag(7.4wt%)/TiO2 sample in our study had inhibition of ∼99%, with the Ag concentration 1 order of magnitude lower (2.11 × 10-3 µg/cm2) and the total colony number 25 times larger. Impact of Ag Particle Size on Ag/TiO2 Performance. To study the effect of Ag particle size on bactericidal activity, two sets of E. coli samples were treated with Ag(3.3wt%)/ TiO2 and Ag(3.9wt%)/TiO2, respectively, and bacterial colonies on the LB/agar plates were counted after 24 h incubation 2908
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(Supporting Information Figure S5). The control experiments estimated ∼3 × 106 CFU per mL of E. coli in the blank sample. After the treatment using Ag3.3wt%/TiO2 with 2.4 µg/mL Ag, the colonies were still too crowded to be accurately counted. With 1.6 µg/mL Ag, the number of colonies counted indicates a killing ratio of ∼99.9% after treatment using Ag3.9wt%/ TiO2. When the Ag concentration was increased to 2.4 µg/ mL, practically all the bacteria were killed. The significantly different bactericidal activity between the two Ag/TiO2 samples was largely attributed to the difference in the Ag particle size. This argument is supported by the following observations: assuming that the Ag particles are spherical and that the estimated average sizes in the two samples are 3 and 15 nm, respectively, the number of Ag particles in Ag(3.9wt%)/TiO2 would be ∼125 times that in Ag(3.3wt%)/ TiO2 at equal Ag concentrations. Their exposed surface areas differed significantly as well. The Ag-bacteria interaction for the [bmim]PF6-derived sample would be much more efficient than that for the other. The mechanism of the antibacterial functions of silver is not yet fully understood. In Ag nanoparticles, it is also unclear if the function is specifically related to nanoparticle properties or due to the effects of released Ag+ ions (41). At least three mechanisms have been proposed to interpret the antibacterial activities of silver. First, Ag nanoparticles can attach to the bacterial cell membrane, causing structural changes or functional damages. The membrane includes many sulfurcontaining proteins, which could be the preferential sites for Ag particle attachment due to sulfur-Ag affinity (42). Morphological studies (4, 5) on the bacteria after Ag treatment reveal that small (
