A Caged-Fluorophore for Hypochlorous Acid Sensing through Human Serum Albumin Binding

Abstract

Hypochlorous acid （HOCl） is a biologically important reactive oxygen species involved in immune responses and inflammatory processes. However, fluorescence-based detection of HOCl in protein-rich biological fluids, such as plasma and serum, remains challenging. In these environments, fluorescent probes can bind nonspecifically to the hydrophobic pockets of serum albumin, the most abundant protein in plasma, which can increase background fluorescence and distort the sensing signal. To address this challenge, we designed a caged-fluorophore probe, DSH, that uses albumin binding as an integral part of signal generation rather than as a source of background interference. In this system, dansyl-sarcosine （DS）, an albumin-binding fluorophore, was masked with a 2,4- dinitrophenyl group through a HOCl-cleavable hydrazine-based linker to afford DSH. This design suppresses the fluorescence from the intact probe and prevents direct albumin-induced activation before reaction with HOCl. Upon exposure to HOCl, oxidative cleavage of the hydrazine-based linker releases DS. The released fluorophore then binds to albumin, resulting in a clear fluorescence turn-on response in albumin-containing media. This system enables selective detection of HOCl with detection limits of 0.78 µM in PBS/HSA and 0.54 µM in 3% human serum. Collectively, this work demonstrates that albumin binding, which is often considered a source of signal interference in fluorescence sensing, can be incorporated into a controlled signal-generation strategy for HOCl detection in albumin-containing media.