The technology comprises a new class of reaction-based fluorescent sensors that selectively detect zinc ions in live cells. The invention can be applied to a variety of fluorophore scaffolds including fluorescein, coumarin, resorufin, and benzoresorufin. These probes could be used for applications such as live cell imaging, flow cytometry, and the detection of zinc in biological fluids.
Zinc is an essential nutrient. Chelatable or “mobile” forms of zinc can act as signaling agents, the actions of which have been directly implicated in numerous physiological and pathological processes, such as neurotransmission, neurogenesis, cancer, diabetes, and acute inflammation. Understanding the role of mobile zinc in biology requires tools that can be used to visualize and track the changes of mobile zinc levels in diverse cellular and subcellular environments. Unfortunately, most commercially available small-molecule probes for mobile zinc have a narrow dynamic range, adventitious localization, and/or a pH-sensitivity, which limits their utility.
A zinc-reactive protecting group is added to the sensor scaffold, thereby creating a non-fluorescent sensor precursor. Upon binding of zinc, the ester is hydrolyzed via a zinc-mediated process, resulting in a restoration of fluorescence. This modification increases the dynamic range, reduces the pH-sensitivity of the metal-free state, and improves the efficiency with which the probes can be targeted to intracellular locales by reducing endo/lysosomal entrapment. These probes also offer improved solubility and can be applied to cells at lower probe concentrations than are typical. Furthermore, unlike most reaction-based probes, the zinc-induced fluorescence can be partially reversed in the presence of a chelator.
- A significantly improved dynamic range and pH profile
- Resistance to water and esterase-mediated hydrolysis both in vitro and in vivo
- The ability to target subcellular organelles such as mitochondria