Ratiometric fluorescent biosensor for detection and real-time imaging of nitric oxide in mitochondria of living cells

Nitric oxide (NO), a ubiquitous gaseous messenger, plays critical roles in various pathological and physiological progresses. The abnormal levels of NO in organisms are closely related to a large number of maladies. Mitochondria are the main area that produce NO in mammalian cells. Thus, detecting and real-time imaging of NO in mitochondria is of great significance for exploring the biological functions of NO. Herein, a ratiometric fluorescent biosensor (Mito-GNP-pNO520) is developed for sensitive and selective detection and real-time imaging of NO in mitochondria of living cells. The detection is achieved through the fluorescence off-on response of Mito-GNP-pNO520 toward NO. This biosensor shows excellent characteristics, such as high sensitivity toward NO with a low detection limit of 0.25 nM, exclusive selectivity to NO without interference from other substances, good biological stability and low cytotoxicity. More importantly, the biosensor is specifically located in mitochondria, enabling the detection and real-time imaging of endogenous and exogenous NO in mitochondria of living cells. Therefore, our biosensor offers a new approach for dynamic detecting and real-time imaging of NO in subcellular organelles, providing an opportunity to explore new biological effects of NO.