Dual-biomarker-triggered fluorescence probes for differentiating cancer cells and revealing synergistic antioxidant effects under oxidative stress

Hydrogen sulfide (H(2)S) and human NAD(P)H:quinine oxidoreductase 1 (hNQO1) are potential cancer biomarkers and also vital participants in cellular redox homeostasis. Simultaneous detection of these two biomarkers would benefit the diagnostic precision of related cancers and could also help to investigate their crosstalk in response to oxidative stress. Despite this importance, fluorescent probes that can be activated by the dual action of H(2)S detection and hNQO1 activity have not been investigated. To this end, dual-biomarker-triggered fluorescent probes 1 and 2 were rationally constructed by installing two chemoselective triggering groups into one fluorophore. Probe 1 provides a small turn-on fluorescence response toward H(2)S but a much larger response to both H(2)S and hNQO1 in tandem. By contrast, fluorescence probe 2 is activated only in the presence of both H(2)S and hNQO1. Probe 2 exhibits a large fluorescence turn-on (>400 fold), high sensitivity, excellent selectivity as well as good biocompatibility, enabling the detection of both endogenous H(2)S and hNQO1 activity in living cells. Bioimaging results indicated that probe 2 could differentiate HT29 and HepG2 cancer cells from HCT116, FHC and HeLa cells owing to the existence of relatively high endogenous levels of both biomarkers. Expanded investigations using 2 revealed that cells could generate more endogenous H(2)S and hNQO1 upon exposure to exogenous hydrogen peroxide (H(2)O(2)), implying the synergistic antioxidant effects under conditions of cellular oxidative stress.