Rational Engineering of Precision-Tuned Fluoro-Photoacoustic Probe for Physiological-Level Glutathione Dynamics Monitoring

Glutathione (GSH), one of the most abundant antioxidants in eukaryotic cells, maintains redox homeostasis and acts as a signaling molecule at physiological levels. However, most existing GSH-activated probes are highly sensitive with a limit of detection as low as micromoles, making them unable to discriminate different mammalian cell lines based on GSH levels, as GSH concentrations in most cells are approximately 1-2 mM. In this study, we found a novel GSH trigger, 2-furan formate, with moderate reactivity, and developed a precision-tuned fluoro-photoacoustic (PA) probe (HDS-4) by combining with a near-infrared fluorescence (NIRF)/PA dual scaffold (HDS) for accurately monitoring of GSH dynamics. In vitro experiments indicated that HDS-4 could not only enable highly selective detection of GSH but also accurately monitor dynamic changes in GSH concentrations at the millimolar level. In addition, unlike the control probe HDS-1, which employs the conventional GSH-responsive unit 2,4-dinitrobenzenesulfonate, HDS-4 could distinguish not only between normal cells and tumor cells, but also among different tumor cell lines based on their differential GSH expression levels. To evaluate HDS-4's performance in vivo, we successfully employed the cross-sectional PA and NIRF imaging techniques to differentiate different tumors in BALB/c nude mice. Therefore, both in vitro and in vivo experiments have consistently demonstrated that HDS-4 exhibits high selectivity and accuracy for monitoring GSH dynamics at physiological concentrations. These results establish HDS-4 as a promising tool for investigating GSH-associated physiological and pathological processes in living systems.