Detection and Imaging of Nitric Oxide with Novel Fluorescent Indicators: Diaminofluoresceins

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• 作者：Hirotatsu Kojima ; Naoki Nakatsubo ; Kazuya Kikuchi ; Shigenori Kawahara ; Yutaka Kirino ; Hiroshi Nagoshi ; Yasunobu Hirata ; and Tetsuo Nagano
• 刊名：Analytical Chemistry
• 出版年：1998
• 出版时间：July 1, 1998
• 年：1998
• 卷：70
• 期：13
• 页码：2446 - 2453
• 全文大小：225K
• 年卷期：v.70,no.13(July 1, 1998)
• ISSN：1520-6882

文摘

Nitric oxide is a gaseous, free radical which plays a roleas an intracellular second messenger and a diffusableintercellular messenger. To obtain direct evidence forNOfunctions in vivo, we have designed and synthesizeddiaminofluoresceins (DAFs) as novel fluorescent indicators for NO. The fluorescent chemical transformationofDAFs is based on the reactivity of the aromatic vicinaldiamines with NO in the presence of dioxygen. TheN-nitrosation of DAFs, yielding the highly green-fluorescent triazole form, offers the advantages of specificity,sensitivity, and a simple protocol for the directdetectionof NO (detection limit 5 nM). The fluorescencequantumefficiencies are increased more than 100 times after thetransformation of DAFs by NO. Fluorescence detectionwith visible light excitation and high sensitivity enabledthe practical assay of NO production in living cells.Membrane-permeable DAF-2 diacetate (DAF-2 DA) canbe used for real-time bioimaging of NO with fine temporaland spatial resolution. The dye was loaded intoactivatedrat aortic smooth muscle cells, where the ester bonds arehydrolyzed by intracellular esterase, generating DAF-2.The fluorescence in the cells increased in a NOconcentration-dependent manner.