Surface-Enhanced Raman Imaging of Intracellular Bioreduction of Chromate in Shewanella oneidensis

This proposed research aims to use novel nanoparticle sensors and spectroscopic tools constituting surface-enhanced Raman spectroscopy (SERS) and Fluorescence Lifetime imaging (FLIM) to study intracellular chemical activities within single bioremediating microorganism. The grand challenge is to develop a mechanistic understanding of chromate reduction and localization by the remediating bacterium Shewanella oneidensis MR-1 by chemical and lifetime imaging. MR-1 has attracted wide interest from the research community because of its potential in reducing multiple chemical and metallic electron acceptors. While several biomolecular approaches to decode microbial reduction mechanisms exist, there is a considerable gap in the availability of sensor platforms to advance research from population-based studies to the single cell level. This study is one of the first attempts to incorporate SERS imaging to address this gap. First, we demonstrate that chromate-decorated nanoparticles can be taken up by cells using TEM and Fluorescence Lifetime imaging to confirm the internalization of gold nanoprobes. Second, we demonstrate the utility of a Raman chemical imaging platform to monitor chromate reduction and localization within single cells. Distinctive differences in Raman signatures of Cr(VI) and Cr(III) enabled their spatial identification within single cells from the Raman images. A comprehensive evaluation of toxicity and cellular interference experiments conducted revealed the inert nature of these probes and that they are non-toxic. Our results strongly suggest the existence of internal reductive machinery and that reduction occurs at specific sites within cells instead of at disperse reductive sites throughout the cell as previously reported. While chromate-decorated gold nanosensors used in this study provide an improved means for the tracking of specific chromate interactions within the cell and on the cell surface, we expect our single cell imaging tools to be extended to monitor the interaction of other toxic metal species

SERS driven cross-platform based multiplex pathogen detection

We demonstrate a cross-platform approach to simultaneously detect three different pathogens using Raman and UV-vis absorption spectroscopy. Gold (Au), silver (Ag), and Ag-Au core-shell nanoparticles were functionalized with anti-Salmonella typhimurium aptamers, anti-Staphylococcus aureus and anti-Escherichia coli O157:H7 antibodies respectively and labeled with unique Raman reporter molecules. A microfiltration step was used to consolidate a highly selective and specific detection platform, with total detection time under 45 min for both species (E. coli O157:H7 vs. S. typhimurium) and strain (E. coli O157:H7 vs. E. coli K12) level sensing at a limit of a detection ranging between 102 and 103 CFU/ml. This simple yet robust multiplex detection platform has the potential to be developed into a rapid and portable pathogen sensor for ultrasensitive detection in liquid samples

Inductively Coupled Mass-Spectrometry.

<p>A. ICP-MS Calibration curve for Cr quantification. B. Intracellularly trapped Cr(VI) and Cr(III) at time t = 0 and t = 12 h after Cr-AuNp treatment.</p

Confocal Fluorescence Lifetime Imaging.

<p><i>S. oneidensis</i> MR-1 incubated with 3.5 nm (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016634#pone-0016634-g007" target="_blank">Fig. 7A & 7C</a>) and 13 nm (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016634#pone-0016634-g007" target="_blank">Fig. 7B & 7D</a>) Cr-AuNp probes show scattered low-lifetime (blue) distribution indicating the presence of gold nanoparticles (both internalized and externally bound) compared to the control incubated with plain gold nanoparticles (inset - 7A).</p

Schematic Illustration.

<p>Representation of passive uptake of Cr-AuNPs by <i>S. oneidensis</i> MR-1 and subsequent Raman Chemical imaging of cells to reveal the intracellular localization of reduced Cr(III) and unreacted Cr(VI).</p

Confocal Raman Mapping.

<p>Raman Intensity Maps averaged over a wide wavenumber region (162–1953 cm⁻¹) covering most of bio-molecular components in cells to obtain a Raman chemical image of the cell (A), Phonon Plasmon peak (207–297 cm⁻¹) originating from gold depicting the presence of Cr-AuNps (B), Cr(VI) - hexavalent chromium (C, 837–873 cm⁻¹), reduced non-toxic trivalent Cr(III) (D, 531–567 cm⁻¹). Raman images in grid format, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016634#pone-0016634-g006" target="_blank">Fig. 6E and 6F</a> are representations of 6B and 6C respectively. 6-E* and F* represent magnified pixel plots to demonstrate the overlap in signal of Au and Cr(VI) peaks within cells.</p

Effect of Cr-AuNPs on the growth of <i>Shewanella oneidensis</i> MR-1.

<p>Either 3.5 nm (closed symbols) or 13 nm (open symbols) Cr-AuNPs were added to wells at volumes of 0, 5, 10, and 50 µl. Error bars represent the standard error from three independent cultures.</p

Effect of Cr-AuNPs on chromate reduction by <i>S. oneidensis</i> MR-1.

<p>Abiotic controls were included to rule out the reduction of chromate by media components and the Cr-AuNPs (open symbols). There was no adverse effect on chromate reduction ability induced by the nanoparticles (closed symbols). In addition, the nanoparticles did not directly reduce the chromate in the medium in the absence of cells (open symbols). Error bars represent the standard error from three independent cultures.</p

Thin-section TEM Images of <i>S. oneidensis</i> MR-1.

<p>A. without particles, B. plain 13 nm gold Nanoparticles, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016634#pone-0016634-g004" target="_blank">Fig. 4C–4D</a>. Chromate coated gold nanoparticles, Cr-AuNp:13 nm, (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016634#pone-0016634-g004" target="_blank">Fig. 4E–4F</a>) 3.5 nm Cr-AuNp. Red arrows indicate extracellularly bound Cr-AuNp and green arrows/circle indicate internalized particles. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016634#pone-0016634-g004" target="_blank">Fig. 4G–4H</a>) show 3.5 nm and 13 nm probes used in Cr-AuNp preparation.</p