A bacterial biosensor for oxidative stress using the constitutively expressed redox-sensitive protein roGFP2

Carlos R. Arias-Barreiro, Keisuke Okazaki, Apostolos Koutsaftis, Salmaan H. Inayat-Hussain, Akio Tani, Maki Katsuhara, Kazuhide Kimbara, Izumi C. Mori

Research output: Contribution to journalArticle

21 Citations (Scopus)

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 × 10-7 (arsenite), 1.0 × 10-4 (naphthalene), 1.0 × 10-4 (Cu2+), 3.8 × 10-4 (H2O2), 1.0 × 10-3 (Cd2+), 1.0 × 10-3 (Zn2+), 1.0 × 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.

Original languageEnglish
Pages (from-to)6290-6306
Number of pages17
JournalSensors
Volume10
Issue number7
DOIs
Publication statusPublished - Jul 2010

Keywords

  • Environmental stressors
  • Oxidative biosensor
  • ROS
  • Ratiometric measurement
  • Redox- sensitive GFP

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Atomic and Molecular Physics, and Optics
  • Instrumentation
  • Electrical and Electronic Engineering

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