Letter pubs.acs.org/journal/estlcu
Cationic Fullerene Aggregates with Unprecedented Virus Photoinactivation Efficiencies in Water Samuel D. Snow,† KyoungEun Park,† and Jae-Hong Kim*,†,‡ †
Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, United States Department of Chemical and Environmental Engineering, Yale University, New Haven, Connecticut 06520, United States
‡
S Supporting Information *
ABSTRACT: Of the many fullerene derivatives that have been examined, cationic functionalization has proven to be most promising for aqueous or biological applications. Until recently, however, no cationic colloidal fullerene aggregates in the nanosize regime have been characterized in the aqueous phase. The results presented here represent the most rapid and efficient, to the best of our knowledge, viral inactivation reported for any colloidal fullerene aggregates. Tris-adducted fulleropyrrolidinium aggregates are prepared and analyzed for concentration-dependent singlet oxygen (1O2) production and MS2 bacteriophage inactivation. Experiments are performed under visible, UVA, and sunlight irradiation with the addition of natural organic matter (NOM) to simulate environmental conditions. Viral inactivation was observed at sensitizer concentrations in the nanomolar range. A 5-log inactivation of MS2 was observed after 4 or 1 min of sunlight exposure with 250 nM fullerenes with or without NOM, respectively. The environmental implications of these results are discussed in the context of previously reported 1O2-mediated MS2 inactivation.
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INTRODUCTION Given their unique photochemical properties,1 fullerenes have received much attention as prospective materials for a variety of light-activated applications.2 However, their use in aqueous or biological media is limited by drastic decreases in these properties upon spontaneous aggregation in water.3 Hydrophilic functionalization has been primarily pursued to minimize aggregation and restore the photoactivity of the fullerene cage for environmental and biomedical applications. Some functionalizations can also enhance C60’s ability to absorb visible wavelengths,4−6 another important consideration, especially for solar applications. While improved photochemical activity may be advantageous for some applications, a concurrent concern over environmental implications arises, as the functionalized fullerenes are tuned to have greater dispersivity and reactivity in environmental media. C60’s propensity to efficiently sensitize ground-state molecular oxygen to yield singlet oxygen (1O2) under UVA and visible light irradiation is the characteristic of greatest interest for applications and, at the same time, concern for environmental impact.7 Cationic functionalization of the C60 cage has been the most promising strategy for adapting the hydrophobic fullerene cage to aqueous systems, particularly for pharmaceutical and disinfection applications.8−15 Utilizing quaternary ammonium moieties within functional groups attached to the cage, several researchers have shown cationically functionalized fullerenes to exhibit a remarkable photobiological activity, suitable for various applications.8,10−14 These studies, however, are limited © 2014 American Chemical Society
in their relevance to direct environmental dispersion scenarios because of the use of unreasonably high concentrations (e.g., 15 μM or 28 mg/L),8 a surfactant for micelle formation,16 or an organic solvent such as dimethyl sulfoxide for stabilization.10,14 Few researchers have studied cationic fullerenes as aqueous aggregates in the nanosize regime prior to our recent report of the photophysical properties of a variety of functionalized fullerenes (Figure S1 of the Supporting Information).6 In the study presented here, we highlight one derivative’s extremely potent viral photoinactivation under environmentally relevant conditions at concentrations as low as 10 nM. These findings are significant not only for the unprecedented photoinactivation kinetics useful for environmental and biological applications but also for the consequent ecological risks associated with unintended release into the environment.
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EXPERIMENTAL SECTION C60 tris-functionalized with methylpyrrolidinium groups, called B3 to be consistent with the nomenclature of our previous work (Figure S1 of the Supporting Information),6 is the molecule highlighted herein, while B2 (bis-functionalized methylpyrrolidinium) and other fullerenes are also briefly mentioned. Details of the preparation and characterization of Received: Revised: Accepted: Published: 290
December 11, 2013 May 28, 2014 May 28, 2014 May 28, 2014 dx.doi.org/10.1021/ez5001269 | Environ. Sci. Technol. Lett. 2014, 1, 290−294
Environmental Science & Technology Letters
Letter
min with a concentration of 100 nM. No significant inactivation was observed for 10 and 50 nM over 2 h. When lowconcentration suspensions were exposed to the UVA irradiation instead of visible light, efficient inactivation was observed, with an exceptional 4-log kill in 4 min at a concentration of 50 nM. The inactivation for 10 nM under UVA was also increased but plateaued at 2 log after ∼20 min. It is remarkable that B3 achieved a 2-log within tens of minutes at 10 nM, or ∼9 μg/L. As a reference, a recent study reported that wastewater NOM, a known 1O2 sensitizer, achieves a 2-log MS2 inactivation in the range of milligrams per liter under simulated sunlight after 12 h.19 Extremely efficient viral inactivation necessitates the questions of efficiency under environmentally relevant conditions. Figure 2 displays the results from experiments
the aggregates can be found in the Supporting Information. Viral inactivation experiments were performed using MS2 bacteriophage, grown with Escherichia coli as the virus host. MS2 and fullerenes were added to a reactor containing 10 mM phosphate-buffered saline (PBS) at pH 7.2 and placed under various light conditions to induce 1O2 sensitization and subsequent MS2 inactivation. E. coli inactivation was performed in an identical manner under UVA irradiation. Additional details of the MS2 and E. coli strains and culturing techniques can be found in the Supporting Information. Standard natural organic matter (NOM) from the Suwannee River (International Humic Substance Society, RO isolation) was used at a concentration of 5 mg/L. The photoinactivation system used for experiments consisted of three 6 W fluorescent lamps (FLs) with a UV cutoff filter (400 nm) or three 4 W Black Light Blue (BLB) lamps oriented directly above a magnetically stirred reaction vessel. Emission spectra are provided in Figure S2 of the Supporting Information, along with the absorption spectra of B3 aggregates. Incident intensities measured at 365 nm using a UVX radiometer (UVP, LLC) were 38.0 μW/cm2 and 1.17 mW/cm2 for the FL (without the UV cutoff filter) and BLB lamps, respectively. A blue light sensor (PMA 2121, Solar Light Co.) was also used for the FLs, with an average value of 482 μW/cm2. Sunlight experiments were performed on the campus of the Georgia Institute of Technology in Atlanta, GA (33°46′25″N 84°23′38″W) using a reaction vessel on a magnetic stirrer in open sunlight. Production of 1O2 was measured using furfuryl alcohol (FFA) as a probe [k(FFA + 1 O2) = 1.2 × 108 M−1 s−1].17,18
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RESULTS AND DISCUSSION Light and Concentration Dependence of MS2 Inactivation. Photoinactivation of MS2 by B3 under visible irradiation exhibited extremely fast kinetics (Figure 1), e.g., 5-
Figure 2. MS2 inactivation by 250 nM (chosen to achieve an ideal experimental time frame to minimize evaporation) B3 under sunlight, with and without 5 mg/L NOM (10 mM PBS, pH 7.2, 36 °C) on September 4 and 12, 2013. On both days, experiments were conducted between 1:00 and 1:30 pm EST, the ambient temperature was measured at 36 °C, the blue light intensity averaged ∼2.8 mW/cm2, and the UVA intensity averaged ∼1.0 mW/cm2. The maximal UVA intensity reached high marks of 1.20 and 1.01 mW/cm2 on September 4 and 12, respectively.
conducted under sunlight with 250 nM B3 with and without 5 mg/L NOM. Sunlight had comparable visible and UV light intensities, with UVA intensities slightly lower than that of the laboratory BLB reactor. Remarkably, a 2-log inactivation was almost immediately observed, even though the first sample was withdrawn in
