As a ubiquitous signal molecule in biosystems, nitric oxide (NO) plays an important role in many physiological and pathological processes. Therefore, it is of great significance to detect NO in organisms for the study of related diseases. Currently, a variety of NO fluorescent probes have been developed based on several types of reaction mechanisms. However, due to the inherent disadvantages of these reactions, like potential interference by biologically related species, there is a great need to develop NO probes based on the new reactions. Herein, we report our discovery of the unprecedented reaction between a widely used fluorophore of 4-(dicyanomethylene)-2-methyl-6-(p-(dimethylamino)styryl)-4H-pyran (DCM) and NO under mild conditions with fluorescence changes. By the analysis of the structure of the product, we proved that DCM undergoes a particular nitration process and proposed a mechanism for fluorescence changes due to the interruption of the intramolecular charge transfer (ICT) process of DCM by the nitrated product of DCM-NO₂. Based on the understanding of this specific reaction, we then easily constructed our lysosomal-localized NO fluorescent probe LysoNO-DCM by linking DCM and a morpholine group, a lysosomal-targeting functional group. LysoNO-DCM exhibits excellent selectivity, sensitivity, pH stability, and outstanding lysosome localization ability with Pearson's colocalization coefficient of up to 0.92 and is successfully applied to the imaging of exogenous and endogenous NO in cells and zebrafish. Our studies expand design methods for NO fluorescence probes based on the novel reaction mechanism and will benefit the studies of this signaling molecule.

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