Accurate Quantitative Sensing of Intracellular pH based on Self-ratiometric Upconversion Luminescent Nanoprobe

Accurate quantitation of intracellular pH (pH_i) is of great importance in revealing the cellular activities and early warning of diseases. A series of fluorescence-based nano-bioprobes composed of different nanoparticles or/and dye pairs have already been developed for pH_i sensing. Till now, biological auto-fluorescence background upon UV-Vis excitation and severe photo-bleaching of dyes are the two main factors impeding the accurate quantitative detection of pH_i. Herein, we have developed a self-ratiometric luminescence nanoprobe based on förster resonant energy transfer (FRET) for probing pH_i, in which pH-sensitive fluorescein isothiocyanate (FITC) and upconversion nanoparticles (UCNPs) were served as energy acceptor and donor, respectively. Under 980 nm excitation, upconversion emission bands at 475 nm and 645 nm of NaYF₄:Yb³⁺, Tm³⁺ UCNPs were used as pH_i response and self-ratiometric reference signal, respectively. This direct quantitative sensing approach has circumvented the traditional software-based subsequent processing of images which may lead to relatively large uncertainty of the results. Due to efficient FRET and fluorescence background free, a highly-sensitive and accurate sensing has been achieved, featured by 3.56 per unit change in pH_i value 3.0-7.0 with deviation less than 0.43. This approach shall facilitate the researches in pH_i related areas and development of the intracellular drug delivery systems.