A fluorescent nanoprobe for single bacterium tracking: functionalization of silver nanoparticles with tryptophan to probe the nanoparticle accumulation with single cell resolution

Abstract

The investigation of the interaction of silver nanoparticles and live bacteria cells is of particular importance for understanding and controlling their bactericidal properties. In this study, the process of internalization of silver nanoparticles in Escherichia coli cells was followed by means of synchrotron excitation deep ultraviolet (DUV) fluorescence imaging. Antimicrobial nanostructures that can absorb and emit light in the UV region were prepared by functionalization of silver nanoparticles with tryptophan amino acid and used as environmentally sensitive fluorescent probes. The nanostructures were characterized by morphological (TEM) and spectroscopic methods (UV-vis, FTIR, XPS, and photoluminescence). The TEM images and the analyses of the UV-vis spectra suggested that the addition of tryptophan led to the formation of hybrid nanostructures with pronounced eccentricity and larger sizes with respect to that of the initial silver nanoparticles. The DUV imaging showed that it was possible to distinguish the fluorescent signal pertaining to silver-tryptophan nanostructures from the autofluorescence of the bacteria. The spatial resolution of the fluorescence images was 154 nm which was sufficient to perform analyses of the accumulation of the nanostructures within a single bacterium. The DUV imaging results imply that the tryptophan-functionalized silver nanoparticles interact with cell membranes via insertion of the amino acid into the phospholipid bilayer and enter the cells.

Toxicity of Food-Grade TiO2 to Commensal Intestinal and Transient Food-Borne Bacteria: New Insights Using Nano-SIMS and Synchrotron UV Fluorescence Imaging

“…Under these conditions, the photoluminescence of TiO 2 particles enables tracking their accumulation in bacteria with a higher spatial resolution compared to conventional optical methods ( Jamme et al, 2010 ). In previous works, this technique has been successfully employed to probe the distribution of antimicrobials in Enterobacter aerogenes ( Kašèáková et al, 2012 ) as well as the accumulation of tryptophan-functionalized gold and silver nanoparticles in E. coli ( Pajović et al, 2015 ; Dojčilović et al, 2016 ). Here, DUV fluorescence imaging with single-cell resolution revealed TiO 2 /cell co-localization by enabling to distinguish between the fluorescent signal pertaining to TiO 2 particles and the autofluorescence of treated E. coli or L. lactis bacterial cells.…”

“…Deep ultraviolet fluorescence imaging of E. coli K12 MG1655 and L. lactis IBB477 cells after TiO 2 exposure was carried out on a Zeiss Axio Observer Z-1 microscope at the DISCO beamline ( Giuliani et al, 2009 ) of the SOLEIL synchrotron facility, as previously described for tryptophan-functionalized gold ( Pajović et al, 2015 ) and silver nanoparticles ( Dojčilović et al, 2016 ). Bacterial cells were cultured in appropriate medium containing TiO 2 at a concentration of 320 μg/mL (aggregated vs. dispersed forms, E171 vs. P25), collected at the stationary phase of growth and washed twice in phosphate buffered saline (5000 g, 5 min, room temperature).…”

Tryptophan Silver Nanoparticles Synthesized by Photoreduction Method: Characterization and Determination of Bactericidal and Anti-Biofilm Activities on Resistant and Susceptible Bacteria

“…The effect of TrpAgNPs synthetized by a femtosecond laser system proved high bactericidal efficiency against E. coli . 24 Dojčilović et al 25 showed an increase in the intensity of fluorescence toward the interior of the cells, indicating that E. coli internalized the tryptophan functionalized nanoparticles.…”

Enhanced resonance energy transfer in gold nanoparticles bifunctionalized by tryptophan and riboflavin and its application in fluorescence bioimaging

“…So far, structural techniques such as transmission electron microscopy [19], as well as noninvasive optical methods that include surface-enhanced Raman scattering, Rayleigh light scattering and confocal Raman microscopy [1], were used to investigate the nanoparticle-cell interactions and the migration of nanoparticles in cells. Our recent results demonstrated that synchrotron radiation deep-ultraviolet (DUV) fluorescence microscopy is a convenient method for determination of the accumulation of antibiotics [20,21,22,23,24], and nanoparticles [25,26,27,28,29] within an individual cell with a spatial resolution of ∼150 nm. For example, DUV fluorescent imaging studies of bacteria incubated with tryptophan functionalized silver [27] and gold [29] nanoparticles and showed that it was possible to distinguish the fluorescent signals of the nanostructures from the autofluorescence of the cells, due to a relatively simple morphology and small size (∼3 µm) of these prokaryotic cells.When it comes to more complex cells, such as human tissue cells, the signal differentiation is far from a trivial since the biomaterial is highly heterogeneous.…”