Development of an endoplasmic reticulum-lipid droplet network-targetable ratiometric fluorescent probe for evaluating hypochlorous acid fluctuations during oxidative stress.

Synchronous and highly sensitive detection of hypochlorous acid (HOCl) dynamics in living cells is of fundamental significance for understanding intracellular oxidative stress processes. In this work, we present a phenothiazine-based ratiometric fluorescent probe, ER-LDs, designed for the rapid and selective sensing of HOCl with subcellular targeting abilities. Spectroscopic studies demonstrated that ER-LDs exhibits an ultra-sensitive ratiometric fluorescence response to HOCl within 60 s, accompanied by a distinct 110 nm hypsochromic emission shift from 560 nm (yellow) to 450 nm (blue). The limit of detection (LOD) was calculated to be as low as 10.40 nM. High-resolution mass spectrometry (HRMS) and Density Functional Theory (DFT) calculations revealed that the sensing mechanism relies on the stepwise oxidation of the sulfur atom within the phenothiazine core, triggering a transition from an intramolecular charge transfer (ICT) state to a locally excited (LE) state. Furthermore, systematically evaluating its photophysical behavior confirmed that local polarity modulates baseline emission intensity without interfering with the HOCl-induced 110 nm emission shift, ensuring self-calibrated ratiometric readouts (I450/I560). Biological experiments confirmed low cytotoxicity, stable performance across physiological pH ranges (5.0-9.0), and effective dual-localization capability toward the endoplasmic reticulum (PCC = 0.80) and lipid droplets (PCC = 0.92). ER-LDs was successfully applied to visualize endogenous and exogenous HOCl fluctuations under variable cellular redox states in HepG2 cells, providing a reliable chemical tool for evaluating subcellular oxidative microenvironments.