A ratiometric lysosomal pH probe based on the coumarin–rhodamine FRET system

Abstract

In this study, we developed a coumarin–rhodamine based fluorescence resonance energy transfer (FRET) system RC1 as a ratiometric pH probe. The probe with a pK_a of 4.98 was constructed by integrating a coumarin moiety as an FRET donor with a rhodamine moiety as an FRET accepter. Upon addition of H⁺, the coumarin emission at 477 nm decreased and the rhodamine emission at 582 nm increased simultaneously. The fluorescence intensity ratio (I₄₇₇/I₅₈₂) displayed excellent pH-dependent performance and responded linearly to minor pH changes in the range of 4.20–6.00. The probe exhibited brilliant selectivity among different amino acids, metal cations and the ATP. Moreover, it has been successfully applied in fluorescence imaging in HeLa cells and the results indicated that the probe could selectively stain lysosomes with low cytotoxicity and excellent photostability. We also applied RC1 to monitor intracellular pH variations induced by dexamethasone. Therefore, RC1 could act as a practical tool for the detection of pH in weakly acidic conditions and provide essential information in medicinal analysis and real biological systems.