Copper is an essential redox-active metal that plays integral roles in biology ranging from enzymatic catalysis to mitochondrial respiration. However, if not adequately regulated, this redox activity has the potential to cause oxidative stress through the production of reactive oxygen species. Indeed, the dysregulation of copper has been associated with a variety of disease states including diabetes, neurodegenerative disorders, and multiple cancers. While increasing tools are being developed for illuminating labile intracellular copper pools and the trafficking pathways in which they are involved, significantly less attention has been given to the analogous extracellular labile pool. To address this gap, we have developed a bioluminescence-based imaging probe, picolinic ester caged-diphenylterazine (pic-DTZ) for monitoring labile, extracellular copper using a coelenterazine-like imidazopyrazinone and the genetically-engineered, marine-based luciferase, nanoluciferase. Unlike the more commonly-used firefly luciferase, nanoluciferase does not require ATP, allowing its application to the extracellular milieu. pic-DTZ demonstrates high metal and oxidation state selectivity for Cu(ii) in aqueous buffer as well as selectivity for labile pools over coordinatively inaccessible protein-bound Cu(ii). We demonstrate the potential of pic-DTZ as a diagnostic tool in human serum and plasma for copper-associated diseases. Additionally, we apply pic-DTZ to lend insight into the extracellular copper dynamic in anticancer treatments.
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