Limits...
Surface-enhanced Raman imaging of intracellular bioreduction of chromate in Shewanella oneidensis.

Ravindranath SP, Henne KL, Thompson DK, Irudayaraj J - PLoS ONE (2011)

Bottom Line: Second, we demonstrate the utility of a Raman chemical imaging platform to monitor chromate reduction and localization within single cells.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.

View Article: PubMed Central - PubMed

Affiliation: Bindley Bioscience Center, Birck Nanotechnology Center, Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana, United States of America. josephi@purdue.edu

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.

Show MeSH

Related in: MedlinePlus

Schematic Illustration.Representation of passive uptake of Cr-AuNPs by S. oneidensis MR-1 and subsequent Raman Chemical imaging of cells to reveal the intracellular localization of reduced Cr(III) and unreacted Cr(VI).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3045368&req=5

pone-0016634-g001: Schematic Illustration.Representation of passive uptake of Cr-AuNPs by S. oneidensis MR-1 and subsequent Raman Chemical imaging of cells to reveal the intracellular localization of reduced Cr(III) and unreacted Cr(VI).

Mentions: As illustrated in Figure 1, in order to track the cellular sites of reductive reactions, chromate-coated gold nanoparticles (Cr-AuNps) 3.5 and 13 nm in diameter [38] were fabricated. The negatively charged gold nanospheres were first stabilized with multifunctional PEG (Polyethylene glycol) molecules (SH-PEG-NH2), which have dual functionality with gold-binding thiol groups as well as surface accessible amine groups.These probes were first incubated in MES (Morpholineethanesulfonic acid) buffer to activate the amine groups, followed by prolonged interaction with chromate molecules to form a uniform layer of chromate on the nanoparticle surfaces. The electrostatic interaction between chromate (oxyanion) and amine groups on the particles form a strong linkage to hold chromate onto the surface of the particles. Cr-AuNps were found to be highly stable based on their zetapotential values (∼32 mV), indicative of the strength of the colloidal suspension. Ultimately, the Cr-AuNp probes were purified using multiple centrifugation steps to remove free chromate in the suspension before the probes could be used for cellular studies.


Surface-enhanced Raman imaging of intracellular bioreduction of chromate in Shewanella oneidensis.

Ravindranath SP, Henne KL, Thompson DK, Irudayaraj J - PLoS ONE (2011)

Schematic Illustration.Representation of passive uptake of Cr-AuNPs by S. oneidensis MR-1 and subsequent Raman Chemical imaging of cells to reveal the intracellular localization of reduced Cr(III) and unreacted Cr(VI).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045368&req=5

pone-0016634-g001: Schematic Illustration.Representation of passive uptake of Cr-AuNPs by S. oneidensis MR-1 and subsequent Raman Chemical imaging of cells to reveal the intracellular localization of reduced Cr(III) and unreacted Cr(VI).
Mentions: As illustrated in Figure 1, in order to track the cellular sites of reductive reactions, chromate-coated gold nanoparticles (Cr-AuNps) 3.5 and 13 nm in diameter [38] were fabricated. The negatively charged gold nanospheres were first stabilized with multifunctional PEG (Polyethylene glycol) molecules (SH-PEG-NH2), which have dual functionality with gold-binding thiol groups as well as surface accessible amine groups.These probes were first incubated in MES (Morpholineethanesulfonic acid) buffer to activate the amine groups, followed by prolonged interaction with chromate molecules to form a uniform layer of chromate on the nanoparticle surfaces. The electrostatic interaction between chromate (oxyanion) and amine groups on the particles form a strong linkage to hold chromate onto the surface of the particles. Cr-AuNps were found to be highly stable based on their zetapotential values (∼32 mV), indicative of the strength of the colloidal suspension. Ultimately, the Cr-AuNp probes were purified using multiple centrifugation steps to remove free chromate in the suspension before the probes could be used for cellular studies.

Bottom Line: Second, we demonstrate the utility of a Raman chemical imaging platform to monitor chromate reduction and localization within single cells.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.

View Article: PubMed Central - PubMed

Affiliation: Bindley Bioscience Center, Birck Nanotechnology Center, Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana, United States of America. josephi@purdue.edu

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.

Show MeSH
Related in: MedlinePlus