iLOV3.0 Fluorescent Probes for Monitoring ROS Changes in Mammalian Cells

Monitoring global ROS levels in mammalian cells is essential for capturing redox dynamics that influence both normal physiology and disease progression. Here, we mutated the fluorescent protein iLOV to become more sensitive to ROS in mammalian cells. This mutation enabled the long-term dynamic monitoring of ROS changes in mammalian cells. We confirmed that the mutated iLOV3.0 protein is more sensitive to redox reactions than the iLOV1.0 protein. The iLOV3.0 probe is broadly applicable to mammalian cells, capable of targeting organelles, and exhibits time- and dose-dependent responses to oxidants and reductants while also monitoring ROS changes caused by cellular nutrient stress and alterations in redox-related enzymes. Importantly, the iLOV3.0 probe can track ROS fluctuations during iPSC induction and viral invasion. In summary, this study developed a reactive oxygen species monitoring tool based on the iLOV protein that can be used for mammalian cell imaging. This probe directly utilizes the photophysical properties of iLOV to respond to ROS and undergo fluorescence signal changes without requiring exogenous cofactors. This method is expected to make up for the limitations of existing ROS monitoring tools and provide a new technical means for the dynamic study of the redox states in living cells.