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Rapid, multiplexed, sensitive and specific identification and quantitative detection of nitric oxide (NO) are in great demand in biomedical science. Herein, a novel multifunctional probe based on the intramolecular LRET (luminescence resonance energy transfer) strategy, TRP-NO , was designed for the highly sensitive and selective ratiometric and luminescence lifetime detection of lysosomal NO. Before reaction with NO, the emission of the rhodamine moiety in TRP-NO is switched off, which prevents the LRET process, so that the probe emits only the long-lived Tb 3+ luminescence. However, upon reaction with NO, accompanied by the turn-on of rhodamine emission, the LRET from the Tb 3+ -complex moiety to rhodamine moiety occurs, which results in a remarkable increase of the rhodamine emission and decrease of the Tb 3+ emission. After the reaction, the intensity ratio of the rhodamine emission to the Tb 3+ emission, I  565 / I  540 , was found to be 28.8-fold increased, and the dose-dependent enhancement of the I  565 / I  540 value showed a good linearity upon the increase of NO concentration. In addition, a dose-dependent luminescence lifetime decrease was distinctly observed between the average luminescence lifetime of the probe and NO concentration, which provides a ∼10-fold contrast window for the detection of NO. These unique properties allowed TRP-NO to be conveniently used as a time-gated luminescence probe for the quantitative detection of NO using both luminescence intensity ratio and luminescence lifetime as signals. The applicability of TRP-NO for ratiometric time-gated luminescence imaging of NO in living cells was investigated. Meanwhile, dye co-localization studies confirmed a quite precise distribution of TRP-NO in lysosomes by confocal microscopy imaging. Furthermore, the practical applicability of TRP-NO was demonstrated by the visualization of NO in Daphnia magna . All of the results demonstrated that TRP-NO could serve as a useful tool for exploiting and elucidating the function of NO at sub-cellular levels with high specificity, accuracy and contrast.

Development of a novel lysosome-targetable time-gated luminescence probe for ratiometric and luminescence lifetime detection of nitric oxide in vivo