Borinic Acid-Based Fluorogenic Probes as an Alternative to the Amplex Red Assay for Real-Time H₂O₂ Monitoring in Live Cells

Hydrogen peroxide (H 2 O 2 ) is a crucial reactive oxygen species (ROS) involved in regulating both physiological and pathological processes. Excessive H 2 O 2 production can lead to oxidative stress, contributing to aging, cancer, and neurodegenerative diseases. In contrast to other ROS exhibiting short lifespans, H 2 O 2 is relatively stable, and its spatial and temporal dynamics are central to understanding its pathophysiological role. Therefore, the development of fluorescent probes that are highly selective, sensitive, and capable of a rapid response is still required. To date, numerous fluorescent probes have been developed. Among them, boronic acid triggers have attracted considerable attention but often suffer from limited reactivity, preventing real-time H 2 O 2 monitoring. To overcome this lack of reactivity, we report the design and synthesis of new borinic acid-based fluorogenic probes for H 2 O 2 detection in cellular environments. These probes are based on a hemicyanine scaffold functionalized with the borinic acid trigger, which demonstrated superior kinetics compared to its boronic counterpart. These probes enable efficient real-time monitoring of H 2 O 2 in cellular models, both extracellularly and intracellularly. The kinetics of these enzyme-free chemical probes matched that of the gold standard Amplex UltraRed/horseradish peroxidase (HRP) assay, representing a significant advancement in the field and offering a versatile and sensitive tool for studying H 2 O 2 -mediated cell signaling.