Date Published: February 25, 2011
Publisher: Public Library of Science
Author(s): Sandeep P. Ravindranath, Kristene L. Henne, Dorothea K. Thompson, Joseph Irudayaraj, Wei-Chun Chin. http://doi.org/10.1371/journal.pone.0016634
Abstract: 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.
Partial Text: Chromium (Cr) is an important industrial metal used in the fabrication of a wide range of products and applications including alloys, leather tanning, textile processing, electroplating, printing inks, refractories and several other industries . Due to its widespread use in industry, chromate [hexavalent chromium, Cr(VI)] has become a pervasive contaminant in the environment, making it a serious public health and environmental concern . Chromium (Cr) can exist in valence states ranging from −2 to +6, of which Cr(VI) and Cr(III) are the most stable forms . Chromium (III) is an essential nutrient used by the human body in processing sugar, protein, and fat . Hexavalent chromium [Cr(VI)], on the other hand, is a known “human carcinogen”  whose inhalation has been linked to lung cancer according to the International Agency for Research on Cancer. Cr(VI) is readily soluble in alkaline environments , posing a threat to ground water quality as it can mobilize and spread quickly. For these reasons, the U.S. EPA (Environmental Protection Agency) has designated chromium as a “priority pollutant”  and considerable measures have been taken to effectively remediate and safely detoxify chromium-polluted soil and aquatic environments. Bioremediation is a promising approach for cheap, effective, and rapid in situ remediation of polluted environments . Bioremediation offers multiple advantages over competing technologies by way of in situ decontamination, utilization of natural processes that are specific to the target contaminant , and significant reduction of additional environmental stresses . Although bioremediation has vast potential in dealing with intractable environmental problems, much of this promise has yet to be realized. Specifically, much needs to be learned about what drives remediating microorganisms and their interactions with their surrounding chemical and biological environment .