Nitroreductase Detection and Hypoxic Tumor Cell Imaging by a Designed Sensitive and Selective Fluorescent Probe, 7‑[(5-Nitrofuran-2-yl)methoxy]‑3H‑phenoxazin-3-one

• Published in: Analytical chemistry (Washington) Vol. 85; no. 8; pp. 3926 - 3932
• Main Authors: Li, Zhao, Li, Xiaohua, Gao, Xinghui, Zhang, Yangyang, Shi, Wen, Ma, Huimin
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 16.04.2013
• Subjects: Analytical chemistry; Calibration; Cell Hypoxia; Cell Line, Tumor; Cells; Electrons; Fluorescence; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - metabolism; Humans; Hydrolysis; Hypoxia; Limit of Detection; NAD - chemistry; NAD - metabolism; Neoplasm Proteins - analysis; Neoplasm Proteins - metabolism; Nitrofurans - chemical synthesis; Nitrofurans - metabolism; Nitroreductases - analysis; Nitroreductases - metabolism; Optical Imaging - methods; Optical Imaging - standards; Oxazines - analysis; Oxazines - chemical synthesis; Oxazines - metabolism; Oxidation-Reduction; Tumors
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac400750r

A highly selective and sensitive fluorescence probe, 7-[(5-nitrofuran-2-yl)methoxy]-3H-phenoxazin-3-one (1), is developed for imaging the hypoxic status of tumor cells via the indirect detection of nitroreductase. The detection mechanism is based on the fact that nitroreductase can selectively catalyze the reduction of the nitro group in 1 to a hydroxylamine or amino group in the presence of reduced nicotinamide adenine dinucleotide as an electron donor that is indispensable, followed by the 1,6-rearrangement–elimination and the release of resorufin. As a result, the reaction produces a distinct color and fluorescence change from almost colorless and nonfluorescent to pink and strong red fluorescence. The fluorescence increase of probe 1 at λ550/585 nm is directly proportional to the concentration of nitroreductase in the range of 15–300 ng/mL, with a detection limit of 0.27 ng/mL. The ready reduction of the nitro group in 1 under hypoxic conditions leads to the establishment of a sensitive and selective fluorescence method for imaging the hypoxic status of tumor cells, and with this method Hela and A549 cells under normoxic and hypoxic conditions (even for different extents of hypoxia) can be differentiated successfully. This method is simple and may be useful for the imaging of disease-relevant hypoxia.