Considering the important roles of biothiols in lysosomes of live organisms, and unique photophysical/photochemical properties of ruthenium(II) complexes, a novel ruthenium(II) complex, Ru-2, has been developed as a molecular probe for phosphorescence and time-gated luminescence assay of biothiols in human sera, live cells, and in vivo. Ru-2 is weakly luminescent due to the effective photoinduced electron transfer (PET) from Ru(II) luminophore to electron acceptor, 2,4-dinitrobenzene-sulfonyl (DNBS). In the presence of biothiols, such as glutathione (GSH), cysteine (Cys), and homocysteine (Hcy), the emission of Ru-2 solution was switched ON, as a result of the cleavage of quencher to form the product, Ru-1. Ru-2 showed high selectivity and sensitivity for the detection of biothiols under physiological conditions, with detection limits of 62 nM, 146 nM, and 115 nM for GSH, Cys, and Hcy, respectively. The emission lifetimes of Ru-1 and Ru-2 were measured to be 405 and 474 ns, respectively, which enabled them to be used for the background-free time-gated luminescence detection even in the presence of strongly fluorescent dye, rhodamine B. On the basis of this mode, the quantification of biothiols in human serum samples was achieved without interference of background autofluorescence. A morpholine moiety was introduced into the complex to ensure Ru-2 molecules to be driven into lysosomes of live cells. Ru-2 showed low cytotoxicity and excellent membrane permeability toward live cells. Using Ru-2 as an imaging agent, visualizations of biothiols in lysosomes of live cells and in Daphnia magna were successfully demonstrated. The results suggested the potential of Ru-2 for the biomedical diagnosis of biothiol-related human diseases.
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