Environ. Sci. Technol. 2010, 44, 5649–5654
Antibacterial Activity of Nanosilver Ions and Particles GEORGIOS A. SOTIRIOU AND SOTIRIS E. PRATSINIS* Particle Technology Laboratory, Institute of Process Engineering, Department of Mechanical and Process Engineering, ETH Zurich, Sonneggstrasse 3, CH-8092 Zurich, Switzerland
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Received April 5, 2010. Revised manuscript received June 7, 2010. Accepted June 14, 2010.
The antibacterial activity of nanosilver against Gram negative Escherichia coli bacteriaisinvestigatedbyimmobilizingnanosilver on nanostructured silica particles and closely controlling Ag content and size. These Ag/SiO2 nanoparticles were characterized by S/TEM, EDX spectroscopy, X-ray diffraction the exposed Ag surface area was measured qualitatively by O2 chemisorption. Furthermore, the fraction of dissolved nanosilver was determined by measuring the released (leached) Ag+ ion concentration in aqueous suspensions of such Ag/SiO2 particles. The antibacterial effect of Ag+ ions was distinguished from that of nanosilver particles by monitoring the growth of E. coli populations in the presence and absence of Ag/SiO2 particles. The antibacterial activity of nanosilver was dominated by Ag+ ions when fine Ag nanoparticles (less than about 10 nm in averagediameter)wereemployedthatreleasehighconcentrations of Ag+ ions. In contrast, when relatively larger Ag nanoparticles were used, the concentration of the released Ag+ ions was lower. Then the antibacterial activity of the released Ag+ ions and nanosilver particles was comparable.
Introduction The unique physicochemical properties of silver nanoparticles (nanosilver) have made them one of the most commercialized nanomaterials in health care (1). Apart from nanosilver’s “traditional” applications in heterogeneous catalysis (2), its antibacterial properties make it attractive in new applications as an additive in textiles (3) and food packaging (4). This antibacterial activity, however, is undesirable when nanosilver is disposed and ends up dissolving and leaching ions (3, 5-7) acting against aquatic microorganisms (8). So regulatory agencies worldwide monitor nanosilver (9) prompting research for a detailed understanding of its toxicity (10, 11). That way, correct risk assessments can be made and its safe use can be established for minimal, if any, environmental impact. There is an ongoing debate regarding the role of released Ag+ ions from nanosilver and its toxicity against microorganisms (12). More specifically, Navarro et al. (13) concluded that nanosilver alone has minimal toxicity and it serves mostly as a source of Ag+ ions. Miao et al. (14) showed also that dissolved Ag+ ions dictate nanosilver’s toxicity. In contrast, Fabrega et al. (5) concluded that the effect of released Ag+ ions is not significant and thus, the dominating factor for this toxicity is bacterial contact with the nanosilver particles themselves. Furthermore, Kawata et al. (15) also * Corresponding author e-mail:
[email protected]. 10.1021/es101072s
2010 American Chemical Society
Published on Web 06/28/2010
stated that the toxicity induced by nanosilver cannot be attributed solely to the released Ag+ ions but rather to nanosilver particles, in agreement with Laban et al. (7). All these studies, however, employ commercially available nanosilver (5, 7, 13-15) having limited, if any, control over Ag size, morphology and degree of agglomeration. This makes difficult to draw universally accepted conclusions regarding the toxicity mechanism of nanosilver. For example, it is not uncommon to have nanosilver flocculation in bacterial suspensions unless its surface is modified with surfactants (16) that may alter again the antibacterial activity of nanosilver. Here this potential drawback is overcome by immobilizing nanosilver on an inert, nanostructured support (SiO2) upon its synthesis by flame aerosol technology (17) that allows close control of product particle size and morphology (18). The role of this silica support with its corrugated texture is to prevent nanosilver particle growth by sintering or coalescence during its characterization and to hinder nanosilver agglomeration (flocculation) in bacterial suspensions. It should be noted, however, that such composite nanoparticles can be made also by wet chemistry methods (19). The nanoparticle properties are measured systematically focusing, first on the nanosilver size and exposed surface area qualitatively (20) and second, on the Ag+ ion release as determined by ion analysis with an Ag+ ion selective electrode (13) as a function of average nanosilver particle size (4-15 nm). So, the antibacterial activity of nanosilver against Escherichia coli is correlated to particle properties and its mechanism is elucidated by quantitatively distinguishing, perhaps for the first time, between the role of Ag+ ions from that of nanosilver particles (21), the “holy grail” in this field so-to-speak.
Materials and Methods Particle Synthesis. Nanosilver on nanostructured silica was made in one step by flame spray pyrolysis (FSP) of appropriate precursor solutions as described elsewhere (17). Silver acetate (Aldrich, purity >99%) and hexamethyldisiloxane (HMDSO, Aldrich, purity >97%) were used as silver and silicon precursors, respectively. Appropriate amounts of silver acetate were dissolved in a 1:1 mixture of 2-ethylhexanoic acid (Aldrich, purity >98%) and acetonitrile (Aldrich, purity >98%). The corresponding amount of HMDSO for a given Ag-content product was added and stirred for a few minutes just before that solution was fed into the FSP reactor. The total precursor (HMDSO and Ag-acetate) concentration was 0.5 M. The precursor solution was fed through the FSP capillary nozzle at 5 mL/min, dispersed to a fine spray by 5 L/min oxygen (Pan Gas, purity >99%) and combusted to produce Ag/SiO2 nanoparticles that were collected on a filter downstream. The Ag-content of product Ag/SiO2 particles ranged from x ) 0 to 98 wt % and their composition corresponds to the nominal Ag- and Si-content of the FSP precursor solution used here (4). The notation for such particles is xAg/SiO2. Particle Characterization. High resolution transmission electron microscopy (HRTEM) was performed on a CM30ST microscope (FEI; LaB6 cathode, at 300 kV, point resolution ∼2 Å) and scanning transmission electron microscopy (STEM) on a Tecnai F30 (FEI; 300 kV) with a high-angle annular dark field (HAADF) detector with bright Z contrast and energy dispersive X-ray spectroscopy (EDXS; detector (EDAX)). Product particles were dispersed in ethanol and deposited onto a perforated carbon foil supported on a copper grid. VOL. 44, NO. 14, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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The crystallite size of silver was determined by XRD (17) using the TOPAS 3 software and fitting its (111) diffraction peak (2θ ) 36-40°) with the Inorganic Crystal Database [ICSD Coll. Code.: 064995]. The exposed surface area of Ag (AgSSAE) per unit mass of Ag was measured with O2 pulse chemisorption (Micromeritics Autochem II 2920). The samples were reduced at 350 °C for 3 h under flowing H2 (20 mL/min) followed by 30 min flushing by He. Then the O2-pulse chemisorption at 150 °C was performed, with 50 mL/min He (22) and 10 pulses of 0.5 mL/min (5% O2 in He), assuming (20) a stoichiometric ratio (O:Ag) of 0.5. The Ag+ ion concentration was measured with an ion selective electrode and an ion meter (Metrohm 781) (13). The measurements were calibrated using silver containing aqueous solutions (silver standard, Aldrich). Centrifugation (Rotina 35, Hettich, 10 000 rpm, 100 min) of aqueous solutions containing Ag/ SiO2 nanoparticles along with UV/vis spectroscopy (Cary Varian 500) were employed to ensure the removal of the Ag/SiO2 nanoparticles. Error bars correspond to the standard deviation of, at least, three measurements. For the UV/vis diffuse reflectance measurements, samples were diluted with barium sulfate (if needed) and placed in a dry powder sample holder (Praying Mantis). Antibacterial Activity. The antibacterial activity of the Ag/SiO2 nanoparticles was obtained by a growth inhibition assay. E. coli JM101 bacteria synthesizing a green fluorescent protein (GFP) from a plasmid-encoded gene were grown in Luria-Bertani (LB) broth at 37 °C overnight (23). The culture was subsequently diluted with LB to an optical density (OD) of 0.05 at 600 nm, which corresponds to about 107 colony forming units (CFU)/mL. The Ag/SiO2 nanoparticles were homogeneously dispersed in deionized water by ultrasonication (Sonics vibra-cell) for 20 s at 75% amplitude with a pulse configuration on/off of 0.5/0.5 s. For the assay, 50 µL of these nanoparticle-containing solutions were added to 50 µL of the diluted cells. The bacterial growth was monitored by the fluorescent signal of the GFP (Perkin-Elmer 1420). The data were corrected for background fluorescence and normalized for the control measurement. The error bars for each data point were the standard deviation of four measurements.
Results and Discussion Nanosilver Morphology and Size. Figure 1a shows an STEM image and EDX spectra of two areas of a sample with 2 wt % Ag (2Ag/SiO2). The nanosilver particles (bright contrast) are dispersed on the SiO2 matrix, as verified also by EDX. In the spectra of area 1 from Figure 1a which includes a bright spot, there is a clear peak of Ag confirming its presence there along with Si and O which correspond to SiO2 (2, 4). In contrast, in the spectra of area 2 that contains no bright spots, only peaks of Si and O are present. The Cu peaks come from the carbon coated copper foil that was used to obtain the electron microscopy images. Figure 1b shows diffuse reflectance UV/vis spectra of the xAg/SiO2 particles for x ) 0, 1, 2, and 10. Even for the lowest Ag-content particles (1Ag/SiO2), the plasmon absorption band at 410 nm (24) indicates metallic Ag, without excluding, however, that its surface can be oxidized (2). The inset of Figure 1b shows a representative TEM image of FSP-made 6Ag/SiO2 sample where dark Ag nanoparticles are dispersed on the gray nanostructured SiO2 particles exhibiting a surface with corrugated texture. The XRD patterns revealed the characteristic peaks attributed to silver metal, for x g 10 wt % (25). For lower Ag-contents the crystal concentration was below the XRD detection limit. No silver oxides were detected by XRD at all Ag-contents, x. The obtained nanosilver particles had a unimodal size distribution (4) (Supporting Information (SI) Figure S1). Figure 2 shows the average nanosilver particle 5650
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FIGURE 1. (a) STEM image of the 2Ag/SiO2 and the EDX spectra of silver-containing (area 1) and pure SiO2 (area 2) locations (the Cu peaks arise from the carbon coated copper grid). (b) Diffuse reflectance UV/vis spectra of the xAg/SiO2 for x ) 0, 1, 2, and 10 show the plasmon absorption band of Ag metal at 410 nm in all Ag-containing samples with a TEM image (inset) of 6Ag/SiO2 showing Ag nanoparticles (dark dots) dispersed on the nanostructured silica (gray) support. diameter as determined by XRD (circles, dXRD) and electron microscopy (triangles, dS/TEM) while SI Table S1 shows detailed particle counts and geometric standard deviations for each x. The two average diameters are in good agreement indicating monocrystalline nanosilver. For an increasing Agcontent in the composite xAg/SiO2 particles the average nanosilver size increases monotonically so it can be closely controlled from 4 to about 16 nm (25). Figure 2 (left ordinate) shows also the exposed nanosilver surface area (AgSSAE) per unit mass of Ag as a function of Ag-content, x. Lower Ag-content particles have higher AgSSAE. By increasing x, the AgSSAE decreases, as larger nanosilver particles are formed exposing less surface area per unit mass of Ag. Therefore, by varying x it is possible to precisely control the exposed surface area of Ag. Additionally, a TEM image is presented as inset in Figure 2 showing a single nanosilver particle attached on the amorphous SiO2, having a fraction of its surface exposed. XRD spectra taken before and after O2 chemisorption were identical indicating no bulk Ag oxidation by O2 chemisorption that took place solely on the surface of nanosilver. Ag+ Ion Release. Figure 3 shows the Ag+ ion concentration (filled symbols) in aqueous suspensions containing xAg/SiO2 (x ) 1-98 wt %) particles at constant C ) 20 mg/L of Ag in solution as a function of x. All Ag+ ion concentrations were rapidly attained (within few minutes upon dispersing) and
FIGURE 2. The exposed specific surface area (squares) of nanosilver per unit mass of Ag (as determined by O2 chemisorption) AgSSAE, and average nanosilver particle diameter by electron microscopy (triangles, dS/TEM) and X-ray diffraction (circles, dXRD) of the xAg/SiO2 composite nanoparticles as a function of their Ag-content, x. The close agreement between microscopy and XRD indicates monocrystalline particles. In the inset, a TEM image of a single nanosilver particle attached on amorphous SiO2. were stable for at least 24 h (SI Figure S2). Similarly, the stability of the dispersed Ag/SiO2 nanoparticles was monitored by dynamic light scattering (SI Figure S3), verifying that the suspensions were stable over 24 h (long enough time for the antibacterial experiments). The inset of Figure 3 shows pictures of the corresponding suspensions containing xAg/SiO2 particles. At low Ag-contents (x ) 1 or 2 wt %), the suspensions are nearly colorless indicating very few and small Ag metal nanoparticles. As x increases, the suspension color darkens (Figure 3, inset) consistent with the plasmonic behavior of nanosilver (24) that indicates here the presence of larger silver particles. In the right ordinate of Figure 3, the corresponding percentage of released Ag+ ions over the total nanosilver
mass is shown. For small Ag-contents (x < 10 wt %) and particles, most of nanosilver is in the form of ions in solution, for example, for the 6Ag/SiO2, ∼50% of nanosilver is as ions in solution. This is consistent with Gunawan et al. (ref 6 Figure 3) who reported 38% as Ag+ ions from their 5 at % Ag/TiO2 that would correspond to 6.64Ag/SiO2 here. Such high Ag leaching and ion concentrations have also been observed when employing rather fine Ag nanoparticles (3). High Ag-content (x > 95 wt %) composite Ag/SiO2 particles contain rather large nanosilver particles (Figure 2, circles and triangles) that hardly contribute to the exposed Ag surface area (Figure 2, squares) and rather little on the released Ag+ ions in solution. This is also consistent with reported low Ag+ ion concentrations, when bigger particles (>50 nm) were dispersed in water by ultrasonication (5, 7), as in here. Figure 3 also shows the Ag+ ion concentration after centrifugation of the suspension and removal of the xAg/ SiO2 particles (Ag+ ions only, open symbols). The Ag+ ion concentration remains practically the same, indicating that by centrifugation, only Ag/SiO2 particles are removed but not Ag+ ions. Furthermore, after centrifugation all suspensions become colorless and transparent indicating particle removal. To verify that all xAg/SiO2 particles were removed from the suspensions, their plasmon absorption band was monitored before and after centrifugation. Figure 4 shows exemplarily the UV/vis spectra of aqueous solutions containing 2 (thin lines) and 20 mg/L of Ag (bold lines) of 25Ag/ SiO2 particles, before (solid line) and after centrifugation (broken line). In the inset of Figure 4, images before and after removal of particles are presented for C ) 2 mg/L of Ag. The peak intensity for C ) 20 mg/L of Ag is too high and has been cut in Figure 4 to distinguish also the spectrum with C ) 2 mg/L. Before centrifugation (solid lines), the characteristic plasmon peak at ∼410 nm attributed to Ag nanoparticles (24) exists for both concentrations. After centrifugation (broken lines) this peak has been drastically reduced for both concentrations, indicating that there are very few, if any, Ag nanoparticles in solution. Also, both yellow-colored suspensions become colorless after centrifugation proving further the removal of Ag nanoparticles. The fraction of released Ag+ ions (Figure 3) follows closely the AgSSAE (Figure 2). In fact, it appears a linear relation (R2 ) 0.96) between the Ag+ ion fraction and AgSSAE (Figure 5),
FIGURE 3. The Ag+ ion concentration (left ordinate) of aqueous suspensions containing 20 mg/L of Ag with xA/SiO2 particles as a function of their Ag-content, x, along with the corresponding released (leached) nanosilver mass fraction (right ordinate), before (filled symbols) and after the nanosilver removal by centrifugation (open symbols). As inset, images of the corresponding aqueous suspensions are presented before the removal of the Ag/SiO2 particles. VOL. 44, NO. 14, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. The UV/vis spectra of aqueous suspensions containing 2 (thin lines) and 20 mg/L of Ag (bold lines) with 25Ag/SiO2 nanoparticles before (solid lines) and after centrifugation (broken lines). In the inset, images of the suspensions of C ) 2 mg/L is shown before and after the removal of the 25Ag/SiO2 nanoparticles. Removing these nanoparticles converted the yellowish suspension to a transparent one.
FIGURE 5. The released Ag+ ion percentage as a function of the AgSSAE. For increasing exposed nanosilver surface area, higher fraction of Ag is present as ions. at constant Ag concentration in solution, C. There is, however, significant scatter and quite likely a deviation from this at smaller nanosilver sizes as discussed later on. Antibacterial Activity of Nanosilver: Ag+ Ions and Ag Nanoparticles. The E. coli growth was investigated by monitoring the fluorescence intensity of suspensions of E. coli cells at 37 °C which encode the green fluorescent protein (GFP). Thus, the fluorescence intensity directly correlates with the E. coli population (26) and the initial fluorescence corresponds to approximately 107 CFU/mL. Figure 6 shows the E. coli growth as a function of time up to 330 min in the absence (control, stars) and presence of xAg/SiO2 nanoparticles for x ) 1 (inverse triangles), 6 (squares), 10 (diamonds), 25 (circles), and 50 (triangles) wt % at constant Ag mass concentration, C ) 1 mg/L. The E. coli growth in the absence 5652
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FIGURE 6. The evolution of E. coli growth (fluorescence) for 330 min at 37 °C in the presence of the xAg/SiO2 nanoparticles for x ) 0-50 wt % for an Ag mass concentration of C ) 1 mg/L. of Ag (stars) exhibited the characteristic exponential bacterial growth (26). The E. coli growth in the presence of pure SiO2 (hexagons, normalized to the highest concentration of all Ag-containing nanoparticles, specific surface area of 295 m2/ g) is identical with the control one (stars) indicating that SiO2 is indeed inert and does not influence the process (27). In the presence, however, of the smaller Ag-content and size particles (Figure 2), a much stronger antibacterial activity is observed than that of larger ones. Such larger Ag-content Ag/SiO2 particles have lower exposed nanosilver specific surface area and release less Ag+ ions (Figure 3) exhibiting lower antibacterial activity than smaller nanosilver particles. This result is in line with studies exhibiting also a stronger antibacterial activity for smaller nanosilver particles (28). Therefore, by controlling the Ag-content in the Ag/SiO2 particles, the antibacterial activity of nanosilver can be controlled also: From no or little E. coli growth for x ) 1-10 wt % Ag (Figure 6, inverse triangles, squares, diamonds), nearly maximum E. coli growth is reached for x ) 50 (triangles) at C ) 1 mg/L of Ag (Figure 6). The toxicity of nanosilver is investigated against E. coli suspensions containing equal Ag+ ion concentration in the presence and absence of nanosilver particles. Figure 7 shows the final E. coli population after 330 min before (Ag+ ions and particles, filled squares) and after removal of xAg/SiO2 (Ag+ ions only, open squares) by centrifugation. At C ) 1 mg/L of Ag, the E. coli population is identical in the presence and absence of nanosilver particles but always in the presence of Ag+ ions that are not removed by centrifugation (Figure 3). For the low Ag-content xAg/SiO2, very small silver particles (4 nm) are present that largely dissociate into Ag+ ions dominating the toxicity of nanosilver, so particles alone do not have a chance to play a significant role on toxicity against E. coli. This indicates that the antibacterial activity is dictated by Ag+ ions alone. This result is consistent with studies (13, 14) reporting that Ag+ ions released from the nanosilver surface play the most important role. Since the higher Ag-content xAg/SiO2 particles (e.g., x > 40 wt %) do not exhibit practically any E. coli growth inhibition for C ) 1 mg/L of Ag and 330 min, their Ag mass concentration C in solution needs to be increased substantially. So, at C ) 20 mg/L of Ag (filled triangles) all the low Ag-content (x e 75 wt %) Ag/SiO2 particle suspensions exhibit strong antibacterial activity that totally prevents E. coli growth for 330 min (Figure 7). At x ) 95 and 98 wt % Ag, however, the E.
FIGURE 7. The final E. coli population after 330 min in the presence of 1 mg/L (squares), 20 mg/L (triangles) and 30 mg/L (circles) of nanosilver before (Ag+ ions and particles, filled symbols) and after removal of the nanosilver particles (Ag+ ions only, open symbols) by centrifugation. The E. coli growth of the control samples (no silver) is also displayed (star, broken line). The graph is separated in two areas, area I (the antibacterial activity is dictated by the released Ag+ ions from the nanosilver surface), and area II (the antibacterial activity by Ag+ ions and nanosilver particles is comparable). coli growth is only partially suppressed. For C ) 30 mg/L (filled circles), again all samples exhibit strong antibacterial activity and practically zero E. coli growth. When, however, the Ag/SiO2 particles are removed by centrifugation (open symbols) and solutions become transparent, then significant E. coli growth takes place for both Ag mass concentrations of 20 and 30 mg/L at x > 90 wt %. For example, for x ) 95 wt % and C ) 20 mg/L when both Ag+ ions and particles are present (filled triangles), the E. coli growth is inhibited by ∼40%. When only Ag+ ions are present (open triangles), this inhibition reaches half of it, ∼20%. At these Ag concentrations and particle sizes, the antibacterial activity of Ag+ ions alone is comparable to that in the presence of particles, indicating that the latter also play a strong antibacterial role in agreement with recent studies (5, 7, 15), employing relatively large (dp ∼50 nm) nanosilver particles that had released also a minimal fraction (∼1%) of Ag+ ions (5). This emphasizes the importance of the released Ag+ ions and particles in the mechanism of the antibacterial activity of such nanosilver particles. All the above indicate that the mechanism of the antibacterial activity of nanosilver particles seems to depend quite clearly on their size. When nanosilver particles are small and release many Ag+ ions, the antibacterial activity is dominated by these ions rather the nanosilver particles (Figure 7, yellow area I). This high release rate of such small nanosilver particles could be related to their enhanced curvature that facilitates mass transfer from their surface (Kelvin effect) and/or the presence of an oxide layer on their surface (2). However, when relatively large (average size larger than about 10 nm) nanosilver particles are employed with a low of Ag+ ion release, the particles themselves also influence the antibacterial activity of nanosilver (Figure 7, blue area II). This result may explain seemingly contradicting studies that support either the Ag+ ions (13, 14) or the Ag particles (5, 7, 15) as the dominant source of nanosilver toxicity. In fact, it appears that a large part of the dispute in the literature may be traced to the lack of closely sizecontrolled nanosilver or its actual state of agglomeration or flocculation. Finally, it should be noted that these studies use as model biological systems other strains of bacteria (5),
algae (13, 14), fish embryos (7), or human cells (15), corroborating the validity of the present nanosilver toxicity mechanism to such systems beyond the E. coli tested here.
Acknowledgments We thank Dr. Andreas Meyer and Prof. Dr. Sven Panke (Bioprocess Laboratory, ETH Zurich) for their help with the toxicity measurements and Dr. Frank Krumeich for the electron microscopy analysis. Financial support by the Swiss National Science Foundation (No. 200020-126694) is gratefully acknowledged.
Supporting Information Available An STEM image and the nanosilver particle size distributions of the 10Ag/SiO2 and the summary of the XRD and S/TEM analysis of all xAg/SiO2 composite particles for all x used here. Additionally, the time evolution of the Ag+ ion concentration in aqueous suspensions containing the Ag/ SiO2 particles and the stability of the 50Ag/SiO2 aqueous suspension by dynamic light scattering (DLS) is presented exemplarily. Finally, the composite Ag/SiO2 size distributions determined by DLS are also presented. This material is available free of charge via the Internet at http://pubs.acs.org.
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ES101072S
