True FRET-based sensing of pH via Separation of FRET and Photon Reabsorption

Förster Resonance Energy Transfer (FRET)-based devices have been extensively researched as potential biosensors due to their highly localized responsivity. In particular, dye-conjugated upconverting nanoparticles (UCNPs) are among the most promising FRET-based sensor candidates. UCNPs have a multi-modal emission profile that allows for ratiometric sensing, and by conjugating a biosensitive dye to their surface, this profile can be used to measure localized variations in biological parameters. However, the complex nature of the UCNP energy profile as well as reabsorption of emitted photons must be taken into account in order to properly sense the target parameters. To our knowledge, no proposed UCNP-based sensor has accurately taken care of these intricacies. In this article, we account for these complexities by creating a FRET-based sensor that measures pH. This sensor utilizes Thulium ( $$ )-doped UCNPs and the fluorescent dye Fluorescein Isothiocyanate (FITC). We first demonstrate that photon reabsorption is a serious issue for the 475 nm $$ emission, thereby limiting its use in FRET-based sensing. We then show that by taking the ratio of the 646 and 800 nm emissions rather than the more popular 475 nm one, we are able to measure pH exclusively through FRET.