In Situ Localization of Enzyme Activity in Live Cells by a Molecular Probe Releasing a Precipitating Fluorochrome

Current enzyme-responsive, fluorogenic probes fail to provide in situ information, because the released fluorophores tend to diffuse away from the reaction sites. The problem of diffusive signal dilution can be addressed by designing a probe, which upon enzyme conversion releases a fluorophore that precipitates. In this work, we developed a novel ESIPT-based solid-state fluorophore HTPQ, which is strictly insoluble in water and emits intense fluorescence in the solid state, with λex/em = 410 / 550 nm, thus making it far better suited to the use with a commercial confocal microscope. We further utilized HTPQ in the design of an enzyme-responsive, fluorogenic probe (HTPQA), targeting alkaline phosphatase (ALP) as a model enzyme. HTPQA allowed for diffusion-resistant in situ detection of endogenous ALP in live cells. It was also employed in the visualizing of different levels of ALP in osteosarcoma cells and tissue, thus demonstrating its interest for the diagnosis of this type of cancer.