Turn-On Fluorescence Probe for Nitric Oxide Detection and Bioimaging in Live Cells and Zebrafish

An effective bioanalytical method for rapid, sensitive, specific, and in situ sensing of nitric oxide (NO) is the key for further unveiling the biological functions of this gasotransmitter molecule in vitro and in vivo. In this contribution, a new fluorescence probe for sensing and imaging of NO in live systems was developed. The probe, FP-NO, was designed by exploring a novel sensing mechanism, i.e., the rotation of the N-N single bond of a coumarin derivative. FP-NO was prepared by incorporating a recognition unit, thiosemicarbazide moiety into a coumarin fluorophore. The weakly fluorescent FP-NO quickly and selectively reacts with NO to form a highly fluorescent product, FP-P. Such an enhancement of fluorescence emission allows NO detection with high sensitivity. The detection limit was 47.6 nM. The reaction mechanism was validated by HRMS titration analysis and the "OFF-ON" fluorescence response mechanism was rationalized by theoretical computation. FP-NO is biocompatible and live cell membrane permeable. The feasibility of FP-NO as the fluorescence probe for imaging and flow cytometry analysis of exogenous NO in MCF-7 cells and exogenous NO production in inflamed J774A.1 macrophage cells was then evaluated. Visualization of exogenous and endogenous NO production in live zebrafish was then achieved, implying the potential application of FP-NO in the studies of the NO roles in live organisms.