Environ. Sci. Technol. 2010, 44, 5210–5215
Silver Nanocrystallites: Biofabrication using Shewanella oneidensis, and an Evaluation of Their Comparative Toxicity on Gram-negative and Gram-positive Bacteria A N I L K . S U R E S H , * ,† D A L E A . P E L L E T I E R , † WEI WANG,‡ JI-WON MOON,† BAOHUA GU,‡ NINELL P. MORTENSEN,† D A V I D P . A L L I S O N , § D A V I D C . J O Y , §,| TOMMY J. PHELPS,† AND M I T C H E L J . D O K T Y C Z * ,†,| Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831-6445, Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831-6422, Department of Biochemistry & Cellular & Molecular Biology, University of Tennessee, Knoxville, Tennessee, 37996-0840, and Center for Nanophase Materials Science, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831-6488
Received December 7, 2009. Revised manuscript received May 5, 2010. Accepted May 14, 2010.
Microorganisms have long been known to develop resistance to metal ions either by sequestering metals inside the cell or by effluxing them into the extracellular media. Here we report the biosynthesis of extracellular silver-based single nanocrystallites of well-defined composition and homogeneous morphology utilizing the γ-proteobacterium, Shewanella oneidensis MR-1, upon incubation with aqueous silver nitrate solution. Further characterization of these particles revealed that the crystals consist of small, reasonably monodispersed spheres in the 2-11 nm size range (average of 4 ( 1.5 nm). The bactericidal effect of these nanoparticles (biogenic-Ag) is compared to chemically synthesized silver nanoparticles (colloidalAg and oleate capped silver nanoparticles, oleate-Ag) and assessed using Gram-negative (E. coli and S. oneidensis) and Gram-positive (B. subtilis) bacteria. Relative toxicity was based on the diameter of inhibition zone in disk diffusion tests, minimum inhibitory concentrations, live/dead assays, and atomic force microscopy. From a toxicity perspective, straindependent inhibition depended on the synthesis procedure and the surface coat. Biogenic-Ag was found to be of higher toxicity compared to colloidal-Ag for all three strains tested, whereas E. coli and S. oneidensis were found to be more resistant to either of these nanoparticles than B. subtilis. In contrast, oleate-Ag was not toxic to any of the bacteria.
* Address correspondence to either author. E-mail: doktyczmj@ ornl.gov (M.J.D.);
[email protected] (A.K.S.). † Biosciences Division, Oak Ridge National Laboratory. ‡ Environmental Sciences Division, Oak Ridge National Laboratory. § University of Tennessee. | Center for Nanophase Materials Science, Oak Ridge National Laboratory. 5210
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These findings have implications for the potential uses of Ag nanomaterials and for their fate in biological and environmental systems.
Introduction Nanoparticles are of tremendous interest to pursuits in biology, medicine (1-3), and electronics (4) due to their sizeand shape-dependent physical, chemical, and biological properties. Characteristics such as high surface to volume ratios and quantum confinement result in materials that are qualitatively different from their bulk counterparts (5). These properties make them suitable for various biomedical applications including cell imaging, drug delivery, biomedical targeting, labeling experiments, and biosensors. For example, because of enhanced effectiveness, pharmaceuticals based on nanoparticles of polymers, metal or metal oxide nanoparticles, liposomes, micelles, quantum dots, or dendrimers are being considered for combating diseases such as cancer (6, 7) and fighting bacterial pathogens (8-11). To facilitate nanoparticle applications, investigators are looking for new synthesis strategies. A commonly employed method for producing size- and shape-distributed forms is the chemical reduction of salts (12). Other techniques include UV-irradiation, aerosol techniques, lithography, laser ablation, and photochemical reduction (12). These methods, however, are complex and can lead to variable results. Further, the resulting nanocrystallites are mostly hydrophobic and often not compatible with biological materials. To overcome these problems, an alternative approach exploits biological methods of synthesis and utilizes microorganisms or plant extracts. Though there are several reports on the microbial-based biosynthesis of silver nanoparticles using Fusarium (13), Enterobacteria (14), Pseudomonas (15), Cyanobacteria (16), Bacillus (17), Aspergillus (18) and Geobacter (19); all these methods produce either polydispersed or larger particles (>20 nm). The present investigation details for the first time the facile biosynthesis of small, spherical, nearly monodispersed silver nanocrystallites in the size range from ∼2 to 11 nm (average size of 4 ( 1.5 nm) using the metal-reducing bacterium, Shewanella oneidensis MR-1, seeded with a silver nitrate solution. The biogenic-Ag particles are extracellular, highly stable, hydrophilic, and capped by a protein coat. Additionally, the antibacterial properties of these biogenic silver nanoparticles are compared with chemically synthesized nanoparticles (colloidal-Ag) and (oleate-Ag) on E. coli, S. oneidensis, and B. subtilis. The different chemical/biological coatings on the nanoparticles significantly influence their toxicity and may in turn provide a means for adapting nanoparticles for different applications or for influencing their fate in biological and environmental systems.
Materials and Methods Bacterial Cultures. All bacterial strains used were wild-type strains purchased from the American Type Culture Collection, Manassas (BD Biosciences, Franklin Lakes, NJ)snamely, Shewanella oneidensis MR-1, Escherichia coli (ATCC 25922), and Bacillus subtilis (ATCC 9372). Biosynthesis of Silver Nanoparticles. S. oneidensis was maintained on Luria-Bertani agar (LBA) Petri dishes at 30 °C. A single bacterial colony from an overnight LBA Petri dish served as inoculum for 100 mL of LB broth in a 500 mL Erlenmeyer flask, followed by incubation at 30 °C on a shaker (200 rpm) for 24 h. The bacteria were collected by centrifugation (5000g, 25 °C, 20 min) and washed with distilled water 10.1021/es903684r
2010 American Chemical Society
Published on Web 05/28/2010
under sterile conditions. In a 500 mL Erlenmeyer flask, ∼3-5 g of wet bacterial biomass was suspended in 100 mL of 1 mM AgNO3 solution and incubated at 30 °C under shaking (200 rpm) conditions. Biosynthesis of silver nanoparticles was monitored by UV-vis absorption spectra (200-700 nm), as a function of time. After completion of the reaction process (48 h), the reaction mixture was first centrifuged (5000g, 20 min) to remove the bacteria, filtered using a sterile 0.1 µM syringe filter, and the particles were collected by high-speed centrifugation (100,000g, 45 min) using an ultracentrifuge. After washing twice with Milli Q water the biogenic-Ag nanoparticles were used for further characterization and toxicity experiments. The colloidal-Ag and oleate-Ag nanoparticles used in the toxicity comparison studies were synthesized as described earlier (20, 21) and purified as described above for the biogenic nanoparticles. Bacteriological Toxicity Assessment. Disc Diffusion Tests. Bacterial sensitivity to different Ag nanoparticles was tested by a disk diffusion test as described by Ruparelia et al. (22). First, stocks of equal concentrations (25 µg/mL) were made for the three types of silver nanoparticles. Then, small Whatman filter paper discs of uniform size (6 mm diameter) were placed separately in stock solutions of the nanoparticles for 5 min; the discs were removed carefully using sterile forceps. After plating the bacterial suspension (100 µL of 104-105 CFU mL-1) uniformly on the LBA Petri dishes, a disk containing nanoparticles was placed at the center of each plate and incubated at 37 °C for 18 h. The average diameter of the inhibition zone (DIZ) surrounding the discs was measured to assess toxicity. Determination of Minimum Inhibitory Concentration. The minimum inhibitory concentration (MIC), defined as the lowest concentration of compound that inhibits the growth of an organism (23), was determined using 100-well bioscreen plates containing 200 µL of the logarithmic phase (∼0.096 OD) bacterial culture and varying concentrations of Ag nanoparticles or silver nitrate in suspension. Bacterial growth was monitored every 15 min for 8 h at OD 600 nm using a bioscreen plate reader (Thermo Labsystems, Finland). Each treatment was performed in 8 wells, and every experiment was repeated at least three times to ensure reproducibility. Live/Dead Viability Assay. Bacterial cultures were grown to logarithmic phase in LB medium and subsequently treated with different concentrations of the various silver nanoparticles for 15 h. Following exposure to silver nanoparticles, the impact on bacterial membrane integrity was assessed using a Live/Dead BacLight Bacterial Viability Kit (Invitrogen) following the manufacturer’s protocol. To quantify the relative number of live and dead cells, the relative fluorescence intensities were measured using a fluorescence plate reader (excitation at 485 nm; emission at 525 and 625 nm). Dissolution of Nanoparticles Using Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). To determine the dissolution of Ag+ from the three types of Ag nanoparticles, the total Ag+ concentration was quantified using ICP-MS (ICP-MS, Perkin-Elmer, Shelton) as described previously (24) (see the Supporting Information (SI) for details). Immobilization of Bacteria onto Gelatin-Coated Mica for Atomic Force Microscopy (AFM) Measurements. The bacteria were immobilized onto gelatin-coated mica as described previously (25, 26). Briefly, freshly cleaved mica surfaces were dipped into 0.5% gelatin (Sigma-Aldrich G6114) solution in Milli Q water at 60 °C and dried overnight. Untreated or silver nanoparticle-treated bacterial cultures were washed and suspended in Milli Q water. A 100 µL portion of the suspension was applied to the gelatin-coated mica surface, allowed to stand for 10 min, rinsed in Milli Q water, and used for AFM imaging. Physical Characterization. UV-vis absorbance was recorded on a CARY 100 Bio spectrophotometer (Varian
FIGURE 1. UV-vis, FTIR, XRD, and EDS measurements of the silver nanoparticles formed by the reaction of AgNO3 with bacterial biomass. (a) UV-vis spectrum recorded from the external medium after 48 h; an intense peak at 418 nm indicates the formation of silver nanoparticles. The inset shows test tubes containing AgNO3 solution before (test tube on the left) and after (test tube on the right) reaction with the biomass. (b) Fourier transform spectra of the silver nanoparticles. Significant vibration bands are labeled. (c) XRD analysis of the silver nanoparticle powder. (d) Energy dispersive X-ray spectra of the silver nanoparticles; strong signals from silver can be observed. Smaller peaks for Si and Cl are also observed and are likely due to common contaminants or artifacts. Instruments, CA) operated at a resolution of 1 nm. Fourier transform infrared (FTIR) analysis of the samples deposited on a ZnSe window was carried out on a Nicolet Magna-IR 760 spectrophotometer at 4 cm-1 resolution. Dynamic light scattering (DLS) and zeta potential measurements were performed on a Brookhaven 90 Plus/BI-MAS Instrument (Brookhaven Instruments, NY). X-ray diffraction (XRD) of dried silver nanoparticle powder was performed on a Discover D8 X-ray diffractometer with a Xe/Ar gas-filled Hi-Star area detector and an XYZ platform, operated at 40 kV and at a current of 40 mA. Transmission electron microscopy (TEM) measurements for the samples prepared on carbon-coated copper grids were performed on a Hitachi HD-2000 STEM operated at an accelerating voltage of 200 kV. For AFM, samples were imaged in either contact or intermittent contact mode with a PicoPlus AFM (Aligent Technologies, Tempe, AZ) using a 100 µm scanning head at 128-512 pixels per line scan and a scan speed of 0.5 line/s. The cantilevers used were Veeco silicon nitride probes (MLCT-AUHW, Veeco, Santa Barbara, CA).
Results and Discussion When microorganisms are incubated with silver ions, extracellular silver nanoparticles can be generated as an intrinsic defense mechanism against the metal’s toxicity. This defense mechanism can be exploited as a method of nanoparticle synthesis and has advantages over chemical routes of synthesis. Environmentally harmful surfactants and solvents are avoided, the method is highly reproducible, and the nanoparticles are hydrophilic. We found that when silver nitrate was added to actively growing S. oneidensis in solution, the medium turned from colorless to brown (inset of Figure 1a) over a period of 48 h. UV-vis spectra showed the presence of a surface plasmon resonance (SPR) band at 418 nm, as shown in Figure 1a and suggests the presence of silver VOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. TEM and AFM analysis of the silver nanoparticles formed after the reaction of AgNO3 with bacterial biomass. (a,b) TEM images of particles from different locations on the TEM grid at increasing magnifications. (c) SAED pattern from one of the particles in (a). (d) AFM topographical (left) and deflective (right) images of the biogenic-Ag nanoparticles. (e) Surface height measurements obtained from the topographical AFM image by counting ∼100 particles to obtain surface diameter. The deflection image was used to differentiate between single particles and clumps of particles. nanoparticles (27). The nanoparticle solution was stable, with no evidence of particle flocculation even months after the reaction. The possible synthesis mechanism may involve reduction of silver ions to convert toxic Ag+ to stable Ag0 and subsequent stabilization of particles using capping proteins/ peptides secreted by the bacteria under metal stress (28). Previous studies indicated that such biotransformation of metals involves the secretion of factors such as NADHdependent reductases, quinines, soluble electron-shuttles that reduce iron, silver, and gold, and capping peptides (28-30). A similar strategy was also hypothesized for magnetotactic bacteria in the formation of magnetosomes (31). To further elucidate the presence of a capping protein/ peptide surrounding the biogenic-Ag nanoparticles, FTIR spectra revealed vibration bands at 1075, 1240, 1565, 1650, and 2935 cm-1 along with an intense, broad band at 3320 cm-1 (Figure 1b). The band at 1650 cm-1 arises due to carbonyl stretch and -N-H stretch vibrations in the amide linkages, clearly indicating the presence of protein/peptide on the surface that appears to be acting as a capping/stabilizing agent (28, 29, 32). The bands at 1075 and 1240 cm-1 correspond to the carbonyl group and alcoholic groups respectively. The band at 3320 cm-1 is characteristic of the hydroxyl functional group in alcohols and phenolic compounds. XRD analysis of biogenic-Ag nanoparticle powder showed intense peaks at (111), (200), and (220). These Bragg reflections in the 2θ range of 30°-70° are shown in Figure 1c; these reflections agree with values reported for silver nanocrystals (28). Energy dispersive X-ray spectra (EDS) of the particles showed a strong silver signal for the nanoparticles (Figure 1d). TEM images showed monodispersed spheres (Figure 2a, b). Figure 2 shows single silver nanoparticles as well as a number of aggregates. The selected-area electron diffraction (SAED) pattern showed that the particles were crystalline (Figure 2c). The hexagonal nature of the diffraction spots indicated that the particles were highly oriented (111), with the top normal to the electron beam. The spots could be indexed based on a face-centered-cubic (f.c.c.) structure of silver. A particle size histogram plot from one of the TEM images showed the size distribution of biogenic silver nanoparticles to range from ∼2 to 11 nm with the largest number of particles being 4 ( 1.5 nm (SI Figure 1d). The nanoparticles were not in direct contact, even within the aggregates. This separation could be due to a capping protein/ peptide consistent with the UV-vis and FTIR spectroscopy measurements. AFM of the biogenic-Ag nanoparticles showed well-dispersed, uniformly shaped nanoparticles with a 5212
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particle height ranging from ∼2 to 11 nm (Figure 2d). A particle size histogram of the nanoparticles, obtained from AFM height data by counting ∼100 particles from the AFM images revealed an average size of 4 ( 1 nm (Figure 2e), similar to the size distribution observed in TEM images. A bacterial toxicity study was conducted with the biogenicAg nanoparticles and chemically synthesized (colloidal-Ag and oleate-Ag) nanoparticles to compare the effects of surface chemistries. Regardless of the synthesis methodology employed, all the nanoparticles examined were less than 20 nm and had a narrow size distribution, as can be inferred from the TEM and particle size histogram analysis (SI Figure 1). However, hydrodynamic sizes of the nanoparticles, as determined by DLS, appeared to be larger: biogenic-Ag, ∼82.5 nm; colloidal-Ag, ∼62.6 nm; and oleate-Ag, ∼46.3 nm (SI Table 1) when compared to the TEM measurements (SI Figure 1), this is potentially attributed to particle aggregation in the DLS while TEM allows latitude for eliminating clumps of particles from the analysis. The antibacterial potential of the variously synthesized silver nanoparticles on Gram-negative (E. coli) and Grampositive (B. subtilis) bacteria were compared using the diameter of the inhibition zone (DIZ), a measure of the magnitude of the susceptibility of the bacteria. The DIZ of disks with biogenic-Ag was larger than the DIZ of disks with colloidal-Ag for both E. coli and B. subtilis (SI Figure 2a,b). The DIZ of disks with biogenic-Ag on B. subtilis was almost 20-30% greater than that observed with E. coli. Similarly, the colloidal-Ag was found to be more effective on B. subtilis than on E. coli; however, the difference in the DIZ was merely 10-15%. The oleate-Ag showed almost no DIZ for both E. coli and B. subtilis (SI Figure 2c). The method illustrates the potential enhancement in the bactericidal activity of biogenic-Ag nanoparticles on both E. coli and B. subtilis strains. However, the DIZ is susceptible to complications originating from the diffusion rate and the hydrophobic or hydrophilic nature of the individual nanoparticles. To further elucidate the antibacterial activity of the nanoparticles, the MIC was determined for E. coli, B. subtilis, and S. oneidensis with varying concentrations of the three types of silver nanoparticles as well as silver ions in the form of silver nitrate (Figure 3). A greater lag phase was observed with increasing concentrations of biogenic and colloidal-Ag nanoparticles for all three bacterial strains tested (Figure 3a,b). The MIC for E. coli, B. subtilis, and S. oneidensis with biogenic-Ag was determined to be 2.0, 0.5, and 3.0 µg/mL, respectively (Figure 3a). These are in agreement with the MIC previously reported in the literature for biosynthesized silver nanoparticles (18, 33, 34). Clearly, the biogenic-Ag nanoparticles were toxic to S. oneidensis, the strain used to synthesize them. Whereas, for colloidal-Ag the MIC concentrations were much higher: 6.0, 2.0, and 6.5 µg/mL, respectively (Figure 3b), implying that biogenic-Ag nanoparticles are more toxic toward the three bacterial strains than the colloidal-Ag. Oleate-Ag, despite its small size (∼3-8 nm), showed no toxicity (at the concentrations tested) toward any of the strains (Figure 3c). The MIC for E. coli, B. subtilis, and S. oneidensis using AgNO3 was found to be 7.5, 5, and 8 µg/mL, respectively (Figure 3d). These are similar to the MIC previously reported in the literature for Ag+ toxicity (35). Overall, B. subtilis appears to be more susceptible to AgNO3, biogenic-Ag, and colloidal-Ag than either E. coli or S. oneidensis. The silver nanoparticles may be interacting with the bacterial membrane, causing structural changes, dissipation of the proton motive force, and finally cell death (36). The finding that E. coli and S. oneidensis are more tolerant of silver nanoparticles is consistent with previously published reports that reveal that the lipopolysaccharides of the outer membrane of Gram-negative bacteria provides resistance against nanoparticles (22, 36, 37).
FIGURE 3. Bacterial dynamic growth curve for E. coli, B. subtilis, and S. oneidensis in LB medium at varying concentrations of the three types of silver nanoparticles and silver nitrate (AgNO3): (a) biogenic-Ag nanoparticles, (b) colloidal-Ag nanoparticles, (c) oleate-Ag nanoparticles, and (d) AgNO3 (positive control). Observations from the live/dead viability assay (SI Figure 3) were similar to observations from the MIC assay discussed above. A substantial loss of cell viability was observed with biogenic-Ag treatment of both E. coli (2-fold loss) and B. subtilis (3- and 4-fold loss) and with colloidal-Ag treatment of E. coli (onefold) and B. subtilis (2-fold). Oleate-Ag did not produce a loss of cell viability greater than that of the controls using any of the bacterial strains. To investigate if dissolution of Ag+ from the nanoparticles contribute to the observed toxicity, dissolution experiments for the three types of nanoparticles were carried out using ICP-MS. Dissolution experiments indicate the concentration of Ag+ in LB media containing Ag nanoparticles (100 µg/mL) to be 4.9 × 10-6 M (0.53 µg/mL) for biogenic-Ag, 4.4 × 10-6 M (0.48 µg/mL) for colloidal-Ag, and 4.3 × 10-6 M (0.47 µg/ mL) for oleate-Ag (SI Figure 4). Even at a nanoparticle concentration that is ∼50 fold higher than the MIC, the dissolved silver ion concentration is 10-fold below that needed to induce toxicity (Figure 3d). Also, there was little difference in the measured dissolution of Ag+ from the different nanoparticles indicating that the relative increased toxicity
of the biogenic silver nanoparticles is not due to the dissolution of Ag+ in media. This is not to say that Ag+ does not have a role in nanoparticle toxicity as Navarro et al. have demonstrated that biotic interactions with Ag-nanoparticles can influence toxicity (38). Certainly, other nanoparticle characteristics besides dissolution and size, such as surface area, bioavailability, or structural distortion, can impact their potential toxicity. For example, Xia et al. have demonstrated that material charge can affect interactions between the nanomaterial and the cell surface (39). Biogenic-Ag was found to have a zeta potential of -12 ( 2 mV which is less negative than colloidal and oleate-Ag, which were -42 ( 5 mV and -45 ( 4 mV, respectively (SI Table 1). As the overall bacterial charge is negative, there may be greater repulsion between the bacteria and the colloidal or oleate-Ag when compared to biogenicAg. This in turn may influence the toxicity of the material. Alternatively, the capping agent may influence nanoparticle reactivity to the bacteria. To further understand the interaction between the bacteria and the silver nanoparticles, AFM imaging experiments of the VOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. AFM images showing the interaction of E. coli and B. subtilis with silver nanoparticles. AFM topographical (left) and deflective (right) images are shown for (a) E. coli alone (control), (b) biogenic-Ag treated, and (c) colloidal-Ag treated E. coli. (d) B. subtilis alone (control), (e) biogenic-Ag treated, and (f) colloidal-Ag treated B. subtilis. The arrows indicate lethal effects of nanoparticles on bacteria. Lethal effects of both biogenic-Ag and colloidal-Ag nanoparticles on E. coli is evidenced by loss in the cell height and rod shaped morphology while on B. subtilis the cells developed lumps but basically retain their rod shaped morphology. nanoparticle treated bacteria were carried out. AFM is a suitable tool for investigating changes in cell membrane morphology and surface structure (10, 25, 40-42) and can aid in elucidating early morphological changes induced by bactericidal agents on bacterial cells. AFM images of E. coli and B. subtilis that were either untreated or incubated with lethal concentrations of biogenic-Ag or colloidal-Ag are shown in Figure 4. After 20 min of exposure, most of the E. coli and B. subtilis cells appeared unchanged and had surface morphologies that were similar to the untreated controls. However, some cells (∼10%) in the nanoparticle treated samples did show damage. For E. coli treated with biogenic-Ag, collapsed cells are shown (arrows) along with rod shaped cell that are similar to the untreated controls. B. subtilis damaged from treatment with colloidal-Ag (arrow) show nodules appearing along the length of the damaged cell while other cells appear normal. We also found that biogenic-Ag and colloidal-Ag had similar effects on these two bacterial species. Others have reported membrane rupture and shredded flagella (10, 11). We also found broken flagella but could not rule out sample preparation as the cause. Resolvable holes or tears in the cell surface were not found. Though the exact mechanism of the bacterial-nanoparticle interaction is still unclear, the nanoparticles do appear to cause structural changes to the cell surface that may eventually lead to cell death. In conclusion, the metal reducing bacterium S. oneidensis has been shown to synthesize small (∼4 ( 1.5 nm), nearly monodispersed silver nanoparticles. This bacterially based method of synthesis is economical, simple, reproducible, and requires less energy when compared to chemical synthesis routes. The silver nanoparticles exhibit useful propertiessthey are hydrophilic, stable, and have a large 5214
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surface area. The exact mechanism of the formation of nanocrystals, the precise makeup of the protein cap, and the genetics involved are currently under investigation. Scaleup of this synthesis approach can lead to large-scale production without hazardous chemical wastes. Bacterial toxicity assessments showed that biogenic-Ag nanoparticles had a greater bactericidal activity on E. coli, S. oneidensis, and B. subtilis strains than did chemically synthesized colloidal-Ag nanoparticles. The nanoparticle surface coating appears to play a prominent role in toxicity. Despite its small size oleate-Ag showed no significant toxicity toward any of the bacteria studied. These observations can be exploited when defining nanoparticles for intended applications. Coatings that lead to increased toxicity may find use as effective bactericidal agents, while coatings that render the nanoparticle benign may find use in drug delivery and labeling applications. Further investigation on the biological effects of nanoparticles and their surface coatings at protein and gene expression levels may provide insight into the precise mechanism of toxicity.
Acknowledgments We acknowledge support from the Office of Biological and Environmental Research, U.S. Department of Energy (DOE). Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. DOE under contract DE-AC05-00OR22725. We thank Ms. X. Yin for the ICP-MS measurements. N.P.M. thanks Lundbeck Foundation for financial support.
Supporting Information Available TEM images and size distribution histogram analysis of the three types of silver nanoparticles, diameter of zone of
inhibition, live/dead assay results, ICP-MS measurements, AFM images, and a table showing the size range, MIC, DLS, and zeta potentials. This material is available free of charge via the Internet at http://pubs.acs.org.
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