Insight into the spatial interaction of D-π-A bridge derived cyanines and nitroreductase for fluorescent cancer hypoxia detection

•Two new trimethincyanine (Cy3) probes were developed for fluorescent detection of hypoxia in tumor tissue.•The para-nitro substituent Cy3 exhibited higher affinity than meta-nitro substituent Cy3 toward nitroreductase (NTR).•The hydrophobic match-mismatch between Cy3 and NTR was modulated by the position of nitro substituent.