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.