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Inactivation of MS2 Coliphage by Ferrous Ion and Zero-Valent Iron Nanoparticles Jee Yeon Kim,† Changha Lee,‡ David C. Love,§ David L. Sedlak,§ Jeyong Yoon,*,† and Kara L. Nelson*,§ †
WCU Program of Chemical Convergence for Energy & Environment (C2E2), School of Chemical and Biological Engineering, Seoul National University, 599 Gwanak-ro, Seoul, Republic of Korea ‡ School of Urban and Environmental Engineering, Ulsan National Institute of Science and Technology (UNIST), 100 Banyeon-ri, Eonyang-eup, Ilju-gun, Ulsan 698-805, Republic of Korea § Department of Civil and Environmental Engineering, University of California, Berkeley, California 94720-1710, United States
bS Supporting Information ABSTRACT: This study demonstrates the inactivation of MS2 coliphage (MS2) by nano particulate zerovalent iron (nZVI) and ferrous ion (Fe[II]) in aqueous solution. For nZVI, the inactivation efficiency of MS2 under air-saturated conditions was greater than that observed under deaerated conditions, indicating that reactions associated with the oxidation of nZVI were mainly responsible for the MS2 inactivation. Under air-saturated conditions, the inactivation efficiency increased with decreasing pH for both nZVI and Fe(II), associated with the pHdependent stability of Fe(II). Although the Fe(II) released from nZVI appeared to contribute significantly to the virucidal activity of nZVI, several findings suggest that the nZVI surfaces interacted directly with the MS2 phages, leading to their inactivation. First, the addition of 1,10phenanthroline (a strong Fe(II)-chelating agent) failed to completely block the inactivation of MS2 by nZVI. Second, under deaerated conditions, a linear doselog inactivation curve was still observed for nZVI. Finally, ELISA analysis indicated that nZVI caused more capsid damage than Fe(II).
’ INTRODUCTION Accompanying the rapid advance of nanotechnology, several metal and metal oxide nanoparticles have shown promise as strong antimicrobial agents against a broad spectrum of microorganisms.14 These nanoparticles have potential applications in medical devices, fibers, and water disinfectants. The main advantages of nanoparticles are their high reactivity, and the possibility of rapid diffusion due to their size being similar to biological molecules.5 Recently, several studies have reported on the antimicrobial activity of iron-based nanoparticles against Escherichia coli (E. coli) 68 and bacteriophage.9,10 nZVI rapidly inactivated E. coli in the absence of oxygen, causing serious damage to the integrity of the cell membrane and to respiratory activity.7,8 nZVI also exhibited greater antibacterial activity than iron oxide nanoparticles (e.g., maghemite and magnetite).6 The antibacterial effect of nZVI appears to involve the generation of intracellular oxidants, •OH and Fe(IV), produced by the reaction with hydrogen peroxide or other species, as well as a direct interaction of nZVI with cell membrane components.68 In spite of increasing understanding about the antibacterial activity of nZVI, little is known about the virucidal activity of nZVI or Fe(II). Previous investigators reported that the antimicrobial efficiency and mechanism of novel disinfectants are different for viruses and bacteria.1,11 The objectives of this study were to investigate the virucidal efficiency of nZVI and Fe(II), r 2011 American Chemical Society
and to provide mechanistic interpretations of the virus inactivation. For these purposes, MS2 coliphage (MS2), a common model for pathogenic human enteric RNA viruses, was chosen as a target virus and its inactivation by nZVI and Fe(II) was examined under various conditions. Damage to the protein capsid and ribonucleic acid (RNA) of MS2 was tested using enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR) analyses, respectively.
’ MATERIALS AND METHODS Chemicals and Synthesis of Nano-Fe0. All chemicals were of reagent grade and used without further purification. All chemicals were obtained from Fisher Scientific Inc. except for ferrous sulfate (Sigma-Aldrich Co., USA), and all solutions were prepared using 18 MΩ Milli-Q water from a Millipore system. nZVI was synthesized daily by aqueous-phase reduction of ferrous sulfate with sodium borohydride as described previously.7,8 The diameter of nanoparticles, as was determined with a JEM-2000XII (JEOL Ltd., Japan) transmission electron microscope (TEM), ranged from approximately 10 to 80 nm (average ≈35 nm). The average Received: April 19, 2011 Accepted: July 4, 2011 Revised: June 28, 2011 Published: July 04, 2011 6978
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Environmental Science & Technology nZVI diameter was analyzed by the measurement of over 130 particles in TEM images directly. Most particles (>95%) had diameters less than 60 nm with 70% less than 40 nm. The concentration of nanoparticles was expressed in terms of mM concentration of total iron by measuring Fe(II) concentration after acidification and the reduction of Fe(III) with hydroxylamine hydrochloride. Analytical Methods. The measurement of Fe(II) concentration was carried out using the 1,10-phenanthroline colorimetric method of Tamura et al.12 with absorbance at 510 nm measured with an UVvis spectrophotometer (Perkin-Elmer Lambda 14, USA). MS2 coliphages (ATCC 15597 - B1) were propagated and quantified by the plaque assay method (top and bottom doublesoft agar layer) using an antibiotic (15 mg/L ampicillin and streptomycin) resistant strain of E. coli Famp (ATCC 700891) as the host.13 Inactivation Experiments. All inactivation experiments were performed with 50-mL MS2 suspensions prepared in 3 mM carbonate buffer solution (pH 8.0) at room temperature with the exception of the experiments to assess the effect of pH on inactivation of MS2, in which 2-(N-morpholino) ethanesulfonic acid (MES; pH 5.5 and 6.0) and piperazine-N,N0 -bis (ethanesulfonic acid) (PIPES; pH 7.0) were used as buffers.14 The initial population of MS2 was adjusted to on the order of 106 PFU/mL except for an experiment to compare results of a plaque assay, sandwich ELISA and qRT-PCR analysis in which the initial MS2 population was on the order of 108 PFU/mL. The stock was further purified to remove trace broth components as described in Supporting Information, SI1. The experiments under air saturation conditions were conducted in containers open to the atmosphere, while the reactor was sealed with a rubber septum and ultrapure N2 gas was bubbled with a needle-type diffuser for 15 min before experiments under deaerated conditions. No dissolved oxygen was detected in the reaction solution under deaerated conditions using a DO meter (AP74, Fisher Scientific Inc., USA). The MS2 suspension was vigorously mixed using a magnetic stirrer after adding an aliquot of freshly prepared Fe(II) solution or nano-Fe0 stock suspension (45 mM total Fe). Samples of 1 mL were withdrawn at predetermined timed intervals, and immediately diluted with phosphate buffered saline (PBS, pH 7.2) depending on the expected number of viable MS2 (from 1/10 to 1/106). Triplicate plates were used for counting the plaques of MS2 from the diluted and undiluted 0.1 mL suspensions. A set of triplicate experiments was carried out for each condition, and the average values and standard deviations are presented. It is known that iron species, especially ferric ion (Fe(III)), may cause virus aggregation, and that aggregated viruses form a single plaque in the plaque assay.15,16 To ascertain the effect of aggregation on MS2 inactivation, the concentration of MS2 in samples treated by Fe(II) and nZVI was measured before and after passage through a 0.22-μm filter. No changes in the concentration of MS2 were observed before and after filtration, indicating that aggregation was unlikely to be the main cause for observed decreases in PFUs (Table S2). It was also confirmed that addition of Fe(III) up to a concentration of 1 mM (pH 8.0) did not cause a significant decrease in MS2 under the conditions studied (data not shown), consistent with prior work.17 Antigenicity Measured by Sandwich ELISA. To detect the antigenicity of MS2 treated with Fe(II) or nZVI, an ELISA assay was performed (MS2 virus BioThreat Alert ELISA kit, Tetracore Inc., USA). Rabbit anti-MS2 immunoglobulin G (IgG) (polyclonal
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Figure 1. Inactivation of MS2 coliphage by (a) ferrous (Fe(II)) ion and (b) nZVI as a function of time (pH0 = 8.0 (3 mM carbonate buffer)). No PFUs were detected in solutions inactivated by 0.3 mM Fe(II) under air saturated conditions, thus the minimum inactivation is shown (∼5.3 log).
antibody; 100 μL), diluted to a concentration of 2.5 μg/mL in PBS, was added to each well of 96-well flat-bottom ELISA plates and incubated overnight at 4 °C. The plate was washed 4 times with PBST (PBS with 0.1% Tween 80) and blocked with 150 μL of ELISA dilution/blocking buffer (5 g dry skim milk/100 mL PBST) at 37 °C for 1 h. After washing with PBST (4 times), 100 μL of MS2 sample aliquot and dilutions were inoculated into the well and incubated at 37 °C for 1 h. Then, 2.5 μg/mL of mouse monoclonal anti-MS2 virus was put into the well as a detector antibody after being washed as above. One more washing was followed by addition of 100 μL of rabbit antimouse IgG-HRP for conjugate antibody diluted to 1/5000. Finally, the same volume of ABTS 2-part peroxidase substrate was added to the well and the plate was incubated at 37 °C for 30 min in the dark. Absorbance was detected at a wavelength of 405 nm with a Thermo max microplate reader (Molecular Devices Corp., USA). Quantitative Real-Time PCR Analysis. Viral RNA was extracted from the sample suspensions18,19 and qRT-PCR for MS2 was performed. Details of the extraction procedure of viral RNA are described in SI3. Primers and probe for this study (Table S4) were designed by Gordon Williams,19 and synthesized by Integrated DNA Technologies (Iowa, USA). The standard curve for qRT-PCR was obtained by extracting RNA from a stock solution of MS2, which was serially diluted to give standard 6979
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Figure 2. Inactivation of MS2 coliphage by (a) ferrous (Fe(II)) ion and (b) nZVI as a function of concentration (pH0 = 8.0 (3 mM carbonate buffer), N0 = 12 106 PFU/mL, Contact time = 60 min).
concentration from 102 to 107 copies per 10 μL of reaction solution. The reaction mixture (final volume, 15 μL) consisted of 7.5 μL of 2.0 one step RT-PCR master mix (Applied Biosystems Inc., USA), 0.375 μL of reverse transcriptase, 0.9 μM of forward and reverse primers, 0.25 μM of probe, 1.21 μL of water, and 5 μL of RNA template. Experimental samples and RNA standards were reverse transcribed and amplified using a StepOne Plus real-time PCR detection system (Applied Biosystems, USA) with thermocycling conditions of 30 min at 48 °C, 10 min at 95 °C, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. The amplification efficiency of the qRT-PCR assay was 99%.
’ RESULTS MS2 Phage Inactivation by Fe(II) Ion and nZVI under Air Saturation and Deaeration. The effect of oxygen on the
inactivation of MS2 by two different concentrations of Fe(II) and nZVI is shown in Figure 1. A control test confirmed that no inactivation of MS2 occurred in 3 mM carbonate buffer solution (pH = 8.0) under both air saturation and deaeration conditions. The inactivation of MS2 in the sodium borohydride solution was also negligible after 1 h (pH 6.5). Negligible inactivation of MS2 was observed due to MeOH by itself. The effect of the Fe(II)-complexing ligand 1,10-phenanthroline on the inactivation of MS2 by Fe(II) and nZVI, is shown in Figure 5. MS2 inactivation by Fe(II) was inhibited completely in the presence of excess 1,10-phenanthroline reagent. However, inactivation by nZVI was not completely eliminated with excess 1,10-phenanthroline. Comparison of MS2 Phage Infectivity, Antigenicity, and RNA Reduction. ELISA and qRT-PCR analyses were used to investigate the inactivation mechanism of MS2 caused by Fe(II) and nZVI. The results were compared with the inactivation observed as determined by the standard plaque assay, as depicted in Figure 6. I. Decrease in Virus Culturability. As shown in Figure 6a, the inactivation efficiency increased with the higher doses of Fe(II) or nZVI. The extent of inactivation by 0.2 mM and 0.3 mM of Fe(II) was similar to that observed for 0.45 mM and 0.9 mM of nZVI, respectively. II. Decrease in Virus Antigenicity. The reduction in antigenicity of MS2 inactivated with Fe(II) and nZVI as determined by ELISA is presented in Figure 6b. A Fe(II) concentration of 6981
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between the nZVI surface and the cell membranes were mainly responsible for inactivation. However, in the case of MS2, the higher inactivation observed in the presence of oxygen is consistent with damage by reactive species formed via oxidation of nZVI or Fe(II). In the nZVI/O2 system, nZVI reacts with oxygen to produce Fe(II) and hydrogen peroxide (H2O2) through a two-electron transfer (reaction 1). Hydrogen peroxide then either reacts with nZVI (reaction 2) or is converted to hydroxyl radical (•OH) or ferryl ion (Fe(IV) (e.g., FeO2+)) by the Fenton reaction with Fe(II) (reaction 3a and b).14,20,21 Hydrogen peroxide is also produced by the reaction of Fe(II) with superoxide radical (•O2, reaction 5), which is generated by the oxidation of Fe(II) by oxygen (reaction 4). Fe0ðsÞ þ O2 þ 2Hþ f FeðIIÞ þ H2 O2
ð1Þ
Fe0ðsÞ þ H2 O2 þ 2Hþ f FeðIIÞ þ 2H2 O
ð2Þ
FeðIIÞ þ H2 O2 f FeðIIIÞ þ • OH þ OH-
ð3aÞ
FeðIIÞ þ H2 O2 f FeðIVÞ þ H2 O
ð3bÞ
FeðIIÞ þ O2 f FeðIIIÞ þ• O2
ð4Þ
FeðIIÞ þ• O2 þ 2Hþ f FeðIIIÞ þ H2 O2
ð5Þ
•
Figure 6. Comparison of (A) plaque assay analysis, (B) ELISA analysis, and (C) qRT-PCR analysis of inactivated MS2 coliphage by (a) ferrous (Fe(II)) ion and (b) nZVI (pH0 = 8.0 (3 mM carbonate buffer), N0 = 12 108 PFU/mL, air saturation, contact time = 60 min).
0.1 mM did not significantly change MS2 antigenicity, whereas concentrations of 0.2 and 0.3 mM reduced the antigenicity by about 20 and 50%, respectively, after 60 min exposure. Similarly, the lowest dose of nZVI did not significantly change the antigenicity. However, the higher concentrations of nZVI had larger effects than Fe(II), with 0.45 mM nZVI reducing the antigenicity about 70% and with 0.9 mM of nZVI almost no ELISA signal was observed. (III). Decrease of Virus PCR Target. Figure 6c shows the results from qRT-PCR analysis of MS2 inactivated by Fe(II) and nZVI. A significant reduction in the number of RNA targets was only observed by treatment with 0.9 mM nZVI.
’ DISCUSSION Oxidant Production from nZVI and Fe(II). The inactivation of MS2 by Fe(II) and nZVI was much greater under air-saturated conditions than under deaerated conditions (Figures 1 and 2). This finding is contrary to the results of E. coli,7,8 indicating that viruses are inactivated through a mechanism different from that of bacteria. The greater inactivation of E. coli under deaerated conditions implied that the direct physical and chemical interactions
Previous researchers have concluded that OH and Fe(IV) (e.g., FeO2+) are mainly responsible for the oxidation of organic compounds and inactivation of pathogenic microorganisms.14,17,2025 Under neutral pH conditions, it has been suggested that Fe(IV) rather than •OH is the predominant product of the Fenton reaction.14,20,21,26,27 MS2 Inactivation by •OH and Fe(IV). The inactivation of MS2 increased as pH decreased (Figure 3), which can be explained either by the higher reactivity of •OH than Fe(IV) or by the longer contact time between Fe(II) and MS2 as suggested previously.17 At lower pH values, Fe(II) is more stable (Figure 3), providing more time for Fe(II) to associate with MS2. The effect of MeOH on MS2 inactivation by Fe(II) and nZVI (air-saturation) was also pH dependent, which is likely due to the different radical species that dominated (Figure 4). At pH 5.5 and 6.0, •OH is expected to be the dominant radical, and the near complete inhibition of MS2 inactivation by MeOH suggests a direct role of •OH in inactivation. At pH 6.5, 7.0, and 8.0, however, Fe(IV) is expected to be the dominant radical, and MeOH had no significant effect on MS2 inactivation. We suspect that Fe(IV) was involved in MS2 inactivation at these pH values, but that MeOH did not impact the rates due to its low reactivity with Fe(IV) (rate constants (k, L 3 mol1 3 s1) of MeOH = 9.7 108 with •OH and 5.7 102 with Fe(IV)).2830 Although it has been shown that MeOH can be oxidized to formaldehyde by the reaction with Fe(IV),14,21 30 mM MeOH may have been insufficient to compete with MS2 for Fe(IV). Possible interactions between Fe(II) (or nZVI) and the amino acids in the protein capsid of MS2 may also enhance the selectivity of Fe(IV) toward MS2.17,24 Nonetheless, because we have no direct measure of Fe(IV), it is possible that other reactive species, or direct reduction by Fe(II) contributed to inactivation at pH 6.58.0. MS2 Inactivation under Deaerated Conditions. Up to 1.5 log inactivation of MS2 was observed in deaerated solutions of Fe(II). One possible explanation is that Fenton-type reactions 6982
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Environmental Science & Technology leading to inactivation occurred during the dilution and plating step, after the sample was withdrawn from the deaerated reaction solution, and exposure to oxygen occurred. Below 0.1 mM Fe(II), reduced iron appeared to limit the inactivation, whereas above 0.1 mM Fe(II), the inactivation appears to have been limited either by the oxidant or the reaction time, such that the observed inactivation by Fe(II) was about constant (Figure 2a). Indeed, when 0.1 mM of Fe(II) was added to the reaction suspension after 30 min of reaction time, additional inactivation (approximately 1.5 log) was observed under air-saturation, while no additional inactivation occurred in the deaerated conditions (Figure S1). Another possible explanation is that Fenton-type reactions occurred in the deaerated solutions, due to the presence of residual oxygen or other oxidants (e.g., H2O2) in the MS2 solution that reacted with iron. Thus, we believe the observed inactivation was an experimental artifact. For nZVI, conversely, additional lethal effects originating from the direct interaction between nZVI and MS2 appear to exist (see following section). Direct Interaction between nZVI and MS2. The addition of 1,10-phenanthroline completely blocks oxidant formation from the Fenton reaction (reaction 3). Therefore, the inactivation that occurred in the presence of 1,10-phenanthroline (Figure 5b) must have resulted from the direct interaction between nZVI and MS2, such as physical disruption caused by the nanoparticles. The linear doseresponse inactivation curve under deaerated conditions by nZVI (Figure 2b), which is unlike the behavior of Fe(II) under deaerated conditions (Figure 2a), also supports the hypothesis that inactivation by nZVI involves mechanisms different from or additional to those observed for Fe(II). MS2 consists of an external protein capsid and single-stranded RNA genome of 3569 nucleotides. The capsid morphology is icosahedral, with a diameter of 27 nm, and possessing 32 pores (approximately 1.8 nm in diameter) per capsid.31,32 To investigate further the mechanism of MS2 inactivation by Fe(II) and nZVI, ELISA and qRT-PCR analyses were compared with results of the plaque assay. ELISA has been used to examine the disinfection mechanism of Hepatitis A, since the reactivity of antibodies with virus is likely to depend upon how an intact protein capsid is altered by disinfectants.33 Though a similar degree of inactivation was observed by plaque assay method with Fe(II) (0.1, 0.2, 0.3 mM) and nZVI (0.09, 0.45, 0.9 mM), ELISA analysis indicated a difference between treatment with Fe(II) versus nZVI. As described above (Figure 6B), nZVI reduced the antigenicity of MS2 more than that of Fe(II), which indicates that nZVI caused more damage to the external capsid epitopes than Fe(II). On the other hand, qRT-PCR analysis has been employed in studies investigating the inactivation mechanism by disinfectants such as chlorine, ozone, and UV.3336 With this technique it is possible to determine whether PCR targets (small regions of the RNA or DNA genome) are damaged by exposure to disinfectants. A reduction in the qRT-PCR signal was only observed for the highest nZVI dose, indicating that the RNA target region that was amplified was not damaged by the other treatments (Figure 6C). One explanation is that the higher level of capsid damage at the highest nZVI concentration increased the exposure of the RNA. It is important to note that regions of the RNA genome not targeted by our assay may also have been damaged by Fe(II) and nZVI. Together, the results indicate that inactivation by Fe(II) was due to the Fenton reaction, whereas inactivation by nZVI was due to both Fenton reaction with released Fe(II) and a direct effect of nZVI itself (e.g., physical disruption) on the protein capsid. More research is needed to
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better understand the mechanisms of inactivation, and to quantify damage to the MS2 capsid and RNA. Environmental Implications. nZVI exhibits strong virucidal and bactericidal properties indicating potential for nZVI to be applied as an antimicrobial material; however, more research is needed to characterize the impact of nZVI on other bacterial species, viruses and protozoan cysts, and in complex matrices. The applications of nZVI to inactivate viruses could be broader than for bacteria because nZVI maintains virucidal activity in both the presence and absence of oxygen, whereas aerobic conditions may limit the bactericidal activity of nZVI. One advantage of nZVI over Fe(II) is that it can be immobilized in materials. However, Fe(II) may be a more cost-effective method than nZVI for general purposes of water disinfection, considering the complications involved in preparing nZVI. The impact of engineered nanoparticles on human health and ecosystems is a major concern related to the widespread use of nanomaterials.5,37 It is often unclear whether the cytotoxic effects of metal nanoparticles are due to the toxicity of the metal ion released from the nanoparticles or the size-dependent properties of the nanoparticles.38,39 The results from this study indicate that for nZVI both metal ions released from the particles and a direct interaction of the nanoparticles and MS2 contribute to the toxicity of the nanoparticles.
’ ASSOCIATED CONTENT
bS
Supporting Information. Detailed procedure for propagation and purification of MS2 coliphage stock solution (SI1), table describing the comparison of MS2 coliphage population inactivated by Fe(II) and nZVI before and after filtration (SI2), procedure for extraction of viral RNA from MS2 coliphage (SI3), table about the primers and probe for qRT-PCR assay (SI4), and graph about the effect of one more injection on the inactivation of MS2 coliphage by Fe(II) (SI5). This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: +82-2-880-8927; fax: +82-2-876-8911; e-mail: jeyong@ snu.ac.kr (J.Y.). Phone: +1-510-643-5023; fax: +1-510-643-5264; e-mail:
[email protected] (K.L.N.).
’ ACKNOWLEDGMENT We are grateful to two anonymous reviewers of an earlier version of the manuscript, whose comments were helpful in improving the manuscript. This research was supported by the Korea Research Foundation grant funded by the Korean government (MOEHRD) (KRF-2007-612-D00100). This research was also supported by WCU (World Class University) program through the Korea Science and Engineering Foundation, and Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Education, Science and Technology (400-2008-0230 and 2010-0004946). This support is greatly appreciated. ’ REFERENCES (1) Cho, M.; Chung, H.; Choi, W.; Yoon, J. Different inactivation behaviors of MS-2 phage and Escherichia coli TiO2 photocatalytic disinfection. Appl. Environ. Microbiol. 2005, 71, 270–275. 6983
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