A novel ratiometric pH probe for extreme acidity based on FRET and PET

In this study, a novel ratiometric pH probe RC1 was successfully developed. RC1 was constructed by integrating a coumarin fluorophore as a fluorescence resonance energy transfer (FRET) donor into a rhodamine B fluorophore as a FRET acceptor, which is associated with rhodamine B dyes possessing spirocyclic (non-fluorescent) and ring-opening (fluorescent) forms with response to pH. At weak basic pH, the photo-induced electron transfer (PET) process of the N atom of aromatic imino in the rhodamine moiety partly quenches the coumarin emission. At acidic pH, the PET process is gradually inhibited upon acidification, enhancing the fluorescence intensity of coumarin remarkably; at the same time, the spirolactam form of rhodamine changes to a ring-opening form followed by the FRET process between coumarin and rhodamine. Hence, the emission intensities of coumarin and the rhodamine moiety simultaneously increase along with the pH decrease. The sensing mechanism is an integration of the PET and FRET processes. Based on the ratios of fluorescence intensity at 583 nm and 470 nm (I-583/I-470), RC1 with a pK(a) of 3.21 could be used in the ratiometric detection of pH in the range 2.20-4.20 with high selectivity. Furthermore, it can be applied to visualize extreme acidity in bacteria. The results demonstrate that RC1 can serve as an ideal probe for extremely acidic pH levels with excellent biological significance.