A bifunctional naphthalimide-based fluorescent probe for imaging lysosomal peroxynitrite and viscosity in living cells and zebrafish.

Peroxynitrite (ONOO-) and viscosity are critical indicators of lysosome functionality, intimately linked to numerous diseases' pathophysiological processes. Hence, creating reliable analytical techniques to observe fluctuations in lysosomal ONOO- and viscosity is highly important. This study presents the development of a novel naphthalimide-based fluorescent probe, Nap-Cy, specifically designed to target lysosomes and simultaneously detect both ONOO- and viscosity. Nap-Cy displayed a near-infrared fluorescence "turn-on" response to viscosity (ranging from 1.0 to 1410 cp) with an approximately 400-fold increase in intensity. At the same time, it functioned as a ratiometric probe with high sensitivity for detecting ONOO-, featuring a quick response time of approximately 10 min, a low detection limit of 42 nM, a broad pH range (5-11), and excellent selectivity for ONOO- over other chemical and biological species. Additionally, Nap-Cy was successfully applied for fluorescence imaging to monitor ONOO- and viscosity variations in SH-SY5Y cells and zebrafish across multiple channels. This research introduces a valuable molecular probe for investigating the biological functions and interactions of ONOO- and viscosity within lysosomes.