Head-to-Head In Vitro Characterization of Novel HOCl-Targeted and Literature-Derived Fluorescent Probes Using Standardized Assay Conditions

Abstract Hypochlorous acid (HOCl), the primary oxidant generated by myeloperoxidase (MPO) in activated neutrophils, plays a central role in the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Excessive HOCl production contributes to oxidative tissue damage, yet no clinically validated fluorescent probe currently exists to monitor MPO derived HOCl in vivo. In this study, we performed a rigorous, head-to-head comparison of 11 HOCl-responsive fluorescent probes─including three newly synthesized rhodamine-based structures─under a unified experimental framework designed to minimize inter assay variability. We systematically assessed probe stability, activation kinetics, fluorescence contrast, solvent effects, pH-dependence, and detection limits and tested the activation specificity. Our results reveal substantial discrepancies between previously reported probe performance and behavior under standardized conditions, highlighting the importance of harmonized benchmarking. Among the tested candidates, several probes demonstrated rapid activation, high quenching efficiency (>98%), and sub micromolar detection limits suitable for detecting biologically relevant HOCl concentrations. This work establishes a robust methodological framework for comparative probe evaluation and identifies promising candidates for future translational development toward real-time optical imaging of neutrophil-derived HOCl in ALI/ARDS.