A fast-responsive fluorescent turn-on probe for nitroreductase imaging in living cells

Nitroreductase (NTR) may be more active under the environment of hypoxic conditions, which are the distinctive features of the multiphase solid tumor. It is of great significance to effectively detect and monitor NTR in the living cells for the diagnosis of hypoxia in a tumor. Here, we synthesized a novel turn-on fluorescent probe NTR-NO₂ based on a fused four-ring quinoxaline skeleton for NTR detection. The highly efficient probe can be easily synthesized. The probe NTR-NO₂ showed satisfactory sensitivity and selectivity to NTR. Upon incubation with NTR, NTR-NO₂ could successively undergo a nitro reduction reaction and then generate NTR-NH₂ along with significant fluorescence enhancement (30 folds). Moreover, the fluorescent dye NTR-NH₂ exhibits a large Stokes shift (Δλ = 111 nm) due to the intramolecular charge transfer (ICT) process. As a result, NTR-NO₂ displayed a wide linear range (0–4.5 μg mL⁻¹) and low detection limit (LOD = 58 ng mL⁻¹) after responding to NTR. In addition, this probe was adopted for the detection of endogenous NTR within hypoxic HeLa cells.

Probe NTR-NO₂ was effectively reduced in the presence of NTR generating a highly fluorescent product.