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Proceedings Paper

Fluorescent proteins as singlet oxygen photosensitizers: mechanistic studies in photodynamic inactivation of bacteria
Author(s): Rubén Ruiz-González; John H. White; Aitziber L. Cortajarena; Montserrat Agut; Santi Nonell; Cristina Flors
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Paper Abstract

Antimicrobial photodynamic therapy (aPDT) combines a photosensitizer, light and oxygen to produce reactive oxygen species (ROS), mainly singlet oxygen (1O2), to photo-oxidize important biomolecules and induce cell death. aPDT is a promising alternative to standard antimicrobial strategies, but its mechanisms of action are not well understood. One of the reasons for that is the lack of control of the photosensitizing drugs location. Here we report the use of geneticallyencoded fluorescent proteins that are also 1O2 photosensitizers to address the latter issue. First, we have chosen the red fluorescent protein TagRFP as a photosensitizer, which unlike other fluorescent proteins such as KillerRed, is able to produce 1O2 but not other ROS. TagRFP photosensitizes 1O2 with a small, but not negligible, quantum yield. In addition, we have used miniSOG, a more efficient 1O2 photosensitizing fluorescent flavoprotein that has been recently engineered from phototropin 2. We have genetically incorporated these two photosensitizers into the cytosol of E. coli and demonstrated that intracellular 1O2 is sufficient to kill bacteria. Additional assays have provided further insight into the mechanism of cell death. Photodamage seems to occur primarily in the inner membrane, and extends to the outer membrane if the photosensitizer’s efficiency is high enough. These observations are markedly different to those reported for external photosensitizers, suggesting that the site where 1O2 is primarily generated proves crucial for inflicting different types of cell damage.

Paper Details

Date Published: 21 February 2013
PDF: 7 pages
Proc. SPIE 8596, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications V, 859609 (21 February 2013); doi: 10.1117/12.2000695
Show Author Affiliations
Rubén Ruiz-González, Univ. Ramon Llull (Spain)
John H. White, The Univ. of Edinburgh (United Kingdom)
Aitziber L. Cortajarena, Madrid Institute for Advanced Studies in Nanoscience (Spain)
Montserrat Agut, Univ. Ramon Llull (Spain)
Santi Nonell, Univ. Ramon Llull (Spain)
Cristina Flors, Madrid Institute for Advanced Studies in Nanoscience (Spain)

Published in SPIE Proceedings Vol. 8596:
Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications V
Samuel Achilefu; Ramesh Raghavachari, Editor(s)

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