Rosindol: A fluorogen for the quantitative measurement of reactive oxygen species in living cells

Reactive oxygen species (ROS) are key mediators of disease, yet accurate characterization in living systems remains challenging because current probes lack oxidation specificity and produce nonlinear, pH-dependent signals. Here we introduce Rosindol, a novel thioacetal-based fluorogenic probe that overcomes these limitations. Rosindol undergoes an umpolung oxidation in the presence of ROS to generate fluorescence, displaying dose-linear responses to H ₂ O ₂ , O ₂ •⁻, OH•, and HOCl with minimal background signal. Unlike conventional probes, Rosindol is pH-independent, photostable, water soluble, and agnostic to glucose concentration, esterase expression, and ambient oxygen. Validation in human cells—including PMA-stimulated neutrophils and SOD knockout models—confirms accurate detection of cytosolic and mitochondrial ROS. In pancreatic cancer cells, Rosindol reveals a fourfold increase in mitochondrial O ₂ •⁻ generation capacity via Complex I of the electron transport chain. Glucose stimulation induces twofold higher ROS generation in malignant cells, highlighting a connection between Warburg metabolism and the etiology of oxidative stress in pancreatic cancer. These studies illustrate the utility of Rosindol to provide valuable insight to oxidative stress processes in complex biological environments.