Development of new fluorogenic probes for hypochlorous acid activity measurement

Hypochlorous acid (HOCl) and hypochlorite ion (OCl⁻) are among the most important non-radical reactive oxygen species (ROS), produced in vivo from hydrogen peroxide and chloride ions via myeloperoxidase (MPO) catalysis. HOCl acts as a potent oxidant with effective antibacterial activity during microbial invasion. However, excessive HOCl production can lead to tissue damage, contributing to the development of various diseases such as arthritis, kidney diseases, lung injury, cardiovascular diseases, asthma, and cancer. Despite its significance, the precise roles of HOCl and OCl⁻ in these pathological conditions remain unclear due to limitations in current detection technologies. Therefore, developing novel methods for their detection and recognition has gained considerable attention in recent years. Common methods for HOCl and OCl⁻ detection include electroanalysis, potentiometry, and chemiluminescence. Among these, fluorescence techniques offer superior advantages in terms of selectivity, sensitivity, and spectral resolution. Consequently, numerous fluorescent probes for HOCl and OCl⁻ imaging have been rapidly developed. However, designing fluorescent probes suitable for in vivo imaging remains challenging. Due to the very low levels of HOCl formation (for example, 0.47 nmol/min per 106 cells), highly sensitive fluorescent probes are required for cellular pathological studies. Additionally, near-infrared (NIR) fluorescent probes are preferred in clinical applications as they penetrate deeper into tissues while minimizing damage to biological samples. In this study, a novel, rapid-response, highly selective, and sensitive fluorogenic probe was synthesized for the detection of HOCl, and a new spectrofluorometric assay method was developed. A new generation hemicyanine-based NIR fluorogenic probe (NIR-QBH, emission at 660 nm) was designed and synthesized. The chemical characterization of the probe was carried out using ¹³C-NMR, ¹H-NMR, and HRMS molecular spectroscopic techniques. The NIR-QBH probe exhibited strong NIR emission at 660 nm with excitation at 618 nm. The detection mechanism is based on the oxidation of the C=C double bond in the NIR-QBH structure by HOCl, resulting in the formation of non-fluorescent products. The probe demonstrated a detection limit of 0.23 µM and a rapid response time of four minutes. Glutathione (GSH), an important biothiol, was selected as a reference HOCl-scavenging compound, and the relative intensity change of NIR-QBH's initial emission was recorded after a four-minute incubation with GSH. The fluorescence response was proportional to GSH's HOCl-scavenging antioxidant activity, with an IC₅₀ value of 8.97 μM. Additionally, the developed NIR-based fluorescence assay was successfully applied for HOCl detection in fetal bovine serum (FBS) and aqueous solutions. This study contributes to the development of new-generation fluorescent probes for HOCl detection in biological systems.