Photocaged Fluorescent Chemosensor for Sequential Activation and Precise Monitoring of Mitochondrial ATP

Abstract Adenosine triphosphate (ATP), primarily produced in mitochondria, serves as the main energy source for cellular processes. Conventional mitochondrial ATP chemosensors often exhibit low accuracy and limited specificity, because they can react with cytosolic ATP and become prematurely activated. Herein, a trackable, mitochondria‐targeted ATP chemosensor is reported, equipped with a light‐activatable ortho‐nitrobenzyl cage group that enables spatiotemporal control. In its dormant state, the chemosensor emits green fluorescence from the tracer 1‐methyl‐3‐[2‐(4‐pyridinyl)ethenyl] (λ ex = 450 nm, λ em = 535 nm), allowing real‐time tracking and confirmation of mitochondrial localization. Upon confirmation of mitochondrial uptake, light irradiation removes the photoactivatable cage, exposing the ATP‐reactive moiety. This activation triggers the reaction between Rhodamine and ATP, producing red fluorescence (λ ex = 560 nm, λ em = 581 nm), which ensures exclusive mitochondrial ATP detection while eliminating cytosolic interference. As the first platform enabling precise mitochondrial ATP monitoring, this chemosensor revealed higher mitochondrial ATP levels in cancer cell lines than in normal cell lines, thereby facilitating cancer cell discrimination. The “lock‐activate‐detect” design provides both spatial accuracy and temporal control, effectively preventing off‐target activation. This innovation offers a reliable tool for investigating mitochondrial energy dynamics and holds strong potential for applications in mitochondria‐associated biomedical research.