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Sensors 2010, 10(7), 6290-6306; doi:10.3390/s100706290

A Bacterial Biosensor for Oxidative Stress Using the Constitutively Expressed Redox-Sensitive Protein roGFP2

1
Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan
2
Environmental Risk Management Authority, PO BOX 131, Wellington 6140, New Zealand
3
Faculty of Allied Health Sciences/UKM Medical Molecular Biology Institute, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, Kuala Lumpur 50300, Malaysia
4
Department of Materials Science and Chemical Engineering, Shizuoka University, Hamamatsu 432-8561, Japan
*
Author to whom correspondence should be addressed.
Received: 17 May 2010 / Revised: 8 June 2010 / Accepted: 21 June 2010 / Published: 24 June 2010
(This article belongs to the Section Biosensors)
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Abstract

A highly specific, high throughput-amenable bacterial biosensor for chemically induced cellular oxidation was developed using constitutively expressed redox-sensitive green fluorescent protein roGFP2 in E. coli (E. coli-roGFP2). Disulfide formation between two key cysteine residues of roGFP2 was assessed using a double-wavelength ratiometric approach. This study demonstrates that only a few minutes were required to detect oxidation using E. coli-roGFP2, in contrast to conventional bacterial oxidative stress sensors. Cellular oxidation induced by hydrogen peroxide, menadione, sodium selenite, zinc pyrithione, triphenyltin and naphthalene became detectable after 10 seconds and reached the maxima between 80 to 210 seconds, contrary to Cd2+, Cu2+, Pb2+, Zn2+ and sodium arsenite, which induced the oxidation maximum immediately. The lowest observable effect concentrations (in ppm) were determined as 1.0 x 10−7 (arsenite), 1.0 x 10−4 (naphthalene), 1.0 x 10−4 (Cu2+), 3.8 x 10−4 (H2O2), 1.0 x 10−3 (Cd2+), 1.0 x 10−3 (Zn2+), 1.0 x 10−2 (menadione), 1.0 (triphenyltin), 1.56 (zinc pyrithione), 3.1 (selenite) and 6.3 (Pb2+), respectively. Heavy metal-induced oxidation showed unclear response patterns, whereas concentration-dependent sigmoid curves were observed for other compounds. In vivo GSH content and in vitro roGFP2 oxidation assays together with E. coli-roGFP2 results suggest that roGFP2 is sensitive to redox potential change and thiol modification induced by environmental stressors. Based on redox-sensitive technology, E. coli-roGFP2 provides a fast comprehensive detection system for toxicants that induce cellular oxidation. View Full-Text
Keywords: oxidative biosensor; redox-sensitive GFP; ratiometric measurement; ROS; environmental stressors oxidative biosensor; redox-sensitive GFP; ratiometric measurement; ROS; environmental stressors
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MDPI and ACS Style

Arias-Barreiro, C.R.; Okazaki, K.; Koutsaftis, A.; Inayat-Hussain, S.H.; Tani, A.; Katsuhara, M.; Kimbara, K.; Mori, I.C. A Bacterial Biosensor for Oxidative Stress Using the Constitutively Expressed Redox-Sensitive Protein roGFP2. Sensors 2010, 10, 6290-6306.

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