1. Silver nanoparticles and Type of UV using in This Study

The AgNPs used in this study were prepared as previously reported ^[1]. The resulting AgNPs had the same characteristics as those obtained in our previous experiments (maximum absorption at 390 nm, and particle sizes were ~5 nm ^[2][3][4][5]. To produce UV radiation, we used a universal handy-type UV lamp that was able to generate has a wavelength of 365 nm, putting it in the range of UVA (320 to 400 nm), which is safer than other types of UV radiation (i.e., UVC and UVB). UVB (280 to 320 nm) can cause a variety of damaging effects in various biological cells, whereas UVC (200 to 280 nm) is very hazardous to most organisms ^[6][7].

We first examined whether hydroxyl radicals are generated when a solution containing AgNPs is subjected to UV irradiation. Hydroxyl radicals were detected when a UV-irradiated solution was subjected to an electron paramagnetic resonance (EPR) analysis (Figure 1A). We measured the amounts of hydroxyl radicals in solutions of AgNPs by varying the time of UV irradiation from 0 to 30 min. The presence of hydroxyl radicals was discernible after adding AgNPs to PBS (“0 min required for UV irradiation” in Figure 1B). We observed UV irradiation-mediated enhanced production of hydroxyl radicals from AgNPs. The proportion of hydroxyl radicals increased as UV irradiation continued for up to 1 min and subsequently plateaued (Figure 1B). The amounts of hydroxyl radicals generated by UV irradiation for 1 and 3 min did not differ significantly. The lifespan of hydroxyl radicals generated was very short, at around 30 s (Figure 1C).

There are several possible mechanisms underlying the generation of hydroxyl radicals induced by UV irradiation of AgNPs. An surface plasmon resonance-mediated collective oscillation of conduction electrons may be generated on the surface of UV-irradiated AgNPs, leading to local generation of an enhanced electric field. Homolytic cleavage, a process in which the covalent bonds of water surrounding AgNPs are broken, may occur in response to the collective oscillation of conduction electrons, resulting in the generation of unpaired electrons as well as radicals. Unfortunately, the current results do not support this hypothesis, and further analysis is required to elucidate the mechanism of action.

Some reports claim that AgNPs yield reactive oxygen species, which act as oxidative stressors to kill various types of bacteria ^{[8][9][10][11]}. However, these reports appear to rely on the indirect action of Ag, in which antibacterial activity might be elicited through oxidized peripheral substances or modified materials. Electron paramagnetic resonance spectroscopy is considered one of the standard methods for the quantification of ROS; however, the equipment is very expensive. To our knowledge, there has only been one report indicating that this equipment is useful to detect radicals ^[12]. In this study, we demonstrated the usefulness of an EPR spectroscopy-based direct detection approach. Our present study differs from the experiments of Zhang et al. ^[12], who explored the mechanism by which ROS exerts microbicidal activity. We observed a 1.7-fold enhanced production of hydroxyl radicals when AgNPs were irradiated for 0.5 min, and 4.5-fold enhanced production when AgNPs were UV-irradiated for 1 min (Figure 1B). The increased levels of hydroxyl radicals should reflect enhanced microbicidal activity of AgNPs.

We investigated whether UV-irradiated AgNPs inhibit the survival of the bacterium E. coli. Experiments were carried out using two approaches: In the first set of experiments, solutions containing AgNPs were UV-irradiated, and then mixed with E. coli prior to seeding onto agar plates (Figure 2). In the second approach, agar plates that had their surface coated with AgNPs were irradiated with UV and plated with E. coil (Figure 3).

In the first approach, PBS or PBS-containing AgNPs (PBS/AgNPs) was subjected to UV irradiation for 0, 1, or 3 min, after which, each solution was mixed with E. coli prior to plating onto agar plates. After overnight incubation at 37 °C, the number of E. coli colonies growing on plates coated with the AgNPs, which had been UV-irradiated for 1 min (Figure 2A-e,B), was significantly lower than that of untreated E. coli colonies (“UV irradiation for 1 min” in Figure 2A-b,B). Similar results were observed when UV irradiation was performed for 3 min (“UV irradiation for 3 min” in Figure 2A-f,B). The number of E. coli colonies grown in the “AgNPs” group that had not been irradiated was lower than that of those grown in the “PBS” group (PBS vs. AgNPs in Figure 2A-a,d,B). When E. coli were grown on plates coated with PBS, the number of E. coli colonies was not significantly different, even after UV irradiation for 3 min (Figure 2A-a–c,B). These results indicate that the antibacterial effects of AgNPs were significantly enhanced after exposure to UV for 1 min.