Correct cellular function that requires sub-cellular pH, particularly within organelles, is maintained within a tight range.1 Early disease detection is facilitated by the introduction of intensity based fluorescent probes for pH sensing and imaging within organelles.2 The development of fluorescence-based ratiometric sensors utilizing Förster resonance energy transfer (FRET) and photoinduced electron transfer (PET) mechanisms improves accuracy, in turn facilitating the accurate mapping of pH.3 In the past, few researchers have employed the naphthalimide-coumarin FRET pair for precise pH detection4-7, but rational design to optimize the sensing system has not been performed. Here, our work investigates how tuning the FRET-PET mechanism affects the pH sensing ability of naphthalimide-coumarin FRET pairs.
We first investigated how naphthalimide fluorescence is affected by PET quenching. We showed that the greatest PET quenching could be observed by incorporating the PET quencher at the aniline position, with a short linker. We also investigated how the linker between coumarin and naphthalimide affected the FRET efficiency between these two fluorophores. We demonstrated that greater linker rigidity improves energy transfer efficiency.
Combining these two concepts, we have now designed a series of novel FRET-PET based pH sensors. Amongst our probes, NC5_PET showed the clearest ratiometric changes with pH variation, suitable excitation and emission wavelengths for imaging, sufficient quantum yield, lifetime changes with pH change, and biological compatibility. We have therefore applied NC5_PET to confocal microscopy and FLIM studies of cultured cells and shown that we can observe both the ratiometric and lifetime changes with altered pH within cells. These studies illustrate the significance of structural design in the efficacy of FRET-PET based pH sensors and provide a framework for diverse applications in sensing, imaging, and molecular detection.