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Figure 1 Characterization of SWNTs. (a) Raman spectroscopy (785 nm) of SWNTs before and after purification. (b) TEM image of purified SWNTs.
Figure 2 Cell viability measurements after exposure to SWNTs (5 
g/mL) in an isotonic solution (0.9% NaCl, pH 5.6 ± 0.1). (a) Microscopic image of SWNT aggregates. (b) Fluorescence microscope image of total cells (cells stained with propidium iodide and DAPI). (c) Fluorescence microscope image of dead cells (stained with propidium iodide only). (d) Fluorescence-based assay showing the antimicrobial activity of SWNTs as the percent of cells stained with PI or the percent of loss of viability (with error bars representing 1 SD). Data are shown for suspended SWNT aggregates and for SWNTs deposited layer.
The above studies with human cells may suggest that SWNTs can also interact with microbes and exhibit antimicrobial properties. Surprisingly, however, no published studies exist on the direct interaction of SWNTs with microbes. In this work, we investigate the interaction of well-characterized, low metal content, narrowly distributed, pristine SWNTs with model bacteria Escherichia coli K12. Our experiments provide the first direct evidence that highly purified SWNTs exhibit strong antimicrobial activity and indicate that severe cell membrane damage by direct contact with SWNTs is the likely mechanism responsible for the toxicity to the model bacteria. These observations point to the potential use of SWNTs as building blocks for antimicrobial materials.
To assess the biotoxicity of SWNTs on E. coli K12, we first prepared SWNTs, which had more than 90% selectivity to raw SWNTs, by CO disproportionation over highly dispersed cobalt-substituted MCM-41 and an amorphous silica catalyst.17 Consequently, purification was very mild. Multiwavelength excitation Raman spectra demonstrated that the whole purification procedure did not produce many defects in the SWNTs (the G/D band ratio is essentially unchanged) and that the SWNT structure is well preserved (as evidenced by the Raman breathing mode) (Figure 1a). TEM analysis of the SWNTs shows that the tube diameters ranged from 0.75 to 1.2 nm, with a mean diameter of around 0.9 nm. Using mild acid cleaning, we achieved nearly complete removal of cobalt (less than 0.8 wt % based on elemental analysis) and amorphous carbon (by thermogravimetric analysis, data not shown) for the SWNTs used. SWNTs prepared in this manner are not highly bundled as shown in Figure 1b.
We then tested the viability of E. coli K12 following their interaction with SWNTs in aqueous solution. Large SWNT aggregates were formed during the incubation period (Figure 2a). The favorable attachment and almost complete coverage of E. coli on the exposed surface of the SWNT aggregates (Figure 2b) rendered cell counting techniques, such as the use of a cell-counting chamber and flow cytometry, impractical. Hence, we applied an area-based determination of microbial viability (details in Supporting Information) for these experiments involving the interaction of E. coli cells with suspended SWNTs in the saline solution.
Because SWNTs are capable of interacting with widely used tetrazolium salts (e.g., MTT) and luminescing intrinsically near 520 nm, we selected propidium iodide (PI) and 4'-6-diamidino-2-phenylindole (DAPI) to assess the cytotoxicity of SWNTs. These fluorescent dyes did not significantly interact or overlap with the SWNT emission spectra. Our fluorescence-based assays showed that cells incubated with pristine SWNTs for 60 min exhibited a substantial loss in viability (Figure 2c). The percentage of inactivated or compromised E. coli cells (i.e., PI-stained) on SWNT aggregates averaged 79.9 ± 9.8%, which was significantly higher than the average of the control (7.6 ± 2.1% without SWNT) under similar conditions (Figure 2d). Notably, the free-swimming cells in the sample with SWNTs exhibited no significant difference in viability (7.2 ± 2.4%) compared to that of the control.
Additional experiments with doses of SWNT ranging from 1 to 50 g/mL confirmed that the viability of the free-swimming cells was independent of the SWNT dose whereas cells on SWNT aggregates always exhibited a substantial loss of viability. The above results imply that direct contact between cells and SWNT aggregates was essential for the inactivation of E. coli cells. We also found that the biotoxicity was dependent on the bacterial incubation time with SWNTs. The average percentages of loss of viability (i.e., PI-stained) for cells on SWNT aggregates were 73.1 ± 5.4%, 79.9 ± 9.8%, and 87.6 ± 4.7% for 30, 60, and 120 min incubation times, respectively. No significant change was observed for the control cells under the same conditions.
