Visualizing the hypoxic heterogeneity for distinguishing the cancer tissues with a two-photon nitroreductase-H 2 S logic probe via intramolecular isomerization

The uncontrollable growth of tumor leads to the heterogeneity of hypoxia, which is more complicated than researchers have detected using a single factor. Using two factors to reveal the heterogeneous hypoxic environment would be more accurate for distinguishing the cancerous tissues. Herein, we develop a two-photon fluorescence probe (E)-1-(2-(2,4-dinitrophenoxy)naphthalen-1-yl)-2-(4-nitrophenyl)diazene ( DNHF ) featuring the logic responses toward two factors nitroreductase (NTR) and H 2 S via intramolecular isomerization, for the imaging of the hypoxic heterogeneity. DNHF is transformed respectively by H 2 S or NTR to a hydrazone structure (DND) or a reduced product DAHF. Interestingly, DND and DAHF is almost quenched due to the interinhibitive effect or the hydrazone isomerization. While the product AHF triggered by the coexistent high level of NTR and H 2 S shows strong fluorescence via an intermolecular isomerization to the azo-structure AHF-2. These properties enable DNHF to reveal the severe hypoxia (high NTR and H 2 S) by releasing the strong fluorescence and the mild hypoxia (low NTR or H 2 S) by releasing the weak fluorescence, thereby visualizing the hypoxic heterogeneity by two factors. With the DNHF, the heterogeneity difference between normal and tumor tissues is enhanced. We envision this probe may serve as an effective tool for the accurate analysis of cancer tissues.