A reversible “off-on-off” triphenylamine-based fluorescent bio-probe for specific detection of Cu2+ and H₂S, and its application in paper strip assays, actual food and live animal imaging

The release of heavy metal copper ions (Cu 2+ ) and hydrogen sulfide (H₂S) as industrial byproducts into environmental and biological systems poses serious risks to human health and food safety. In this study, a novel triphenylamine-based fluorescent “on-off-on” bio-probe, ( Z )-N′-((5-(4-(diphenylamino) phenyl) thiophen-2-yl) methylene)-2-hydroxybenzohydrazide ( TMH) was developed for successive determination of Cu 2+ and H 2 S. The probe TMH exhibited highly selective Cu 2+ recognition through the Chelation Caused Emission Quenching ( CHEQ ) mechanism, followed by fluorescence restoration upon subsequent H₂S exposure. This system demonstrated remarkable sensitivity, excellent selectivity among competing ions, and strong anti-interference capability. The reversibility of TMH was rigorously validated through five reversible detection cycles of Cu 2+ and H 2 S. In addition, TMH proved highly effective in practical applications, as a test strip for rapid, on-site detection of Cu 2+ and H₂S, in real water sample analysis, as well as visual monitoring of gaseous H₂S released during food spoilage (pork and shrimp). Meanwhile, TMH exhibited exceptional bioimaging performance, enabling the specific identification of Cu 2+ and H₂S in MCF-7 cells, rice tissue, and live zebrafish and mice. These findings offer novel perspectives for the development of cost-effective, robust and environment-friendly sensing bio-probes. • A novel triphenylamine-based fluorescent bio-probe tracking Cu 2+ and H 2 S. • TMH ensembles exhibited “on-off-on” fluorescence responses for Cu 2+ and H 2 S. • TMH exhibited sensitivity, high anti-interference ability and numerous reversible. • The positive detection in real sample and visual detection of H₂S in food spoilage. • TMH has been applied in living cells, rice roots, zebrafish and mice imaging.