Engineering an asymmetric rhodamine dye suitable for developing ratiometric fluorescent probe

Abstract Fluorescent probes based on rhodamine skeleton are extensively used in biological imaging. However, the construction of ratiometric fluorescent probes based on the rhodamine skeleton without introducing additional fluorophores is still challenging. Herein, we propose an effective method to construct a rhodamine‐based ratiometric fluorescent probe through the regulation of electron cloud density. A ratiometric fluorescent probe RDQF‐RB‐NTR was successfully constructed for the detection of nitroreductase (NTR). RDQF‐RB‐NTR exhibits good sensitivity, high selectivity, and ratiometric response to NTR. Cell imaging experiments showed that RDQF‐RB‐NTR can rapidly and accurately detect the fluctuation of NTR in cells and difference of NTR levels between normal cells and cancer cells. In addition, RDQF‐RB‐NTR was successfully applied to the imaging of NTR in liver tissue slices, and we found that the level of NTR was upregulated in liver cirrhosis.