Upconversion nanoparticle-mOrange protein FRET nanoprobes for self-ratiometric/ratiometric determination of intracellular pH, and single cell pH imaging

Fluorescence based intracellular pH nanoprobes have been developed that overcomes the limitations imposed by shallow penetration depth of ultraviolet excitation, photostability, phototoxicity, and interference from background autofluorescence. In this study, we have constructed a Forster Resonance Energy Transfer (FRET) based pH nanoprobe using upconversion nanoparticle (UCNP) as a donor (excitation/emission @ 980/540 nm, green channel), and mOrange fluorescent protein (excitation/emission @ 548/566 nm, red channel) as acceptor. The UCNP-mOrange nanopmbe could be fluorescently imaged with 980 nm excitation, having deep penetration depth, by a fluorescence microscope on a coverslip, or uptaken in a single HeLa cell. The cellular upatake of these nanoparticles were confirmed by transmission electron microscope study. The FRET probes, with a FRET efficiency of similar to 20% at physiological pH of 7.0, have simultaneous self-ratiometric and ratiometric features varying linearly with local pH. The probe exhibits high accuracy, sensitivity, reversibility, and stability over a wide range of pH (3.0-8.0). The fluorescence intensity ratio from individual green, and red channels in fluorescence microscopic images could be used to estimate the pH of the intracellular compartments of HeLa cell from the pH dependent ratiometric calibration. Nigericin mediated intracellular pH (3.0, 5.0, and 7.0) could be accurately estimated from the CLSM derived FRET ratio. The pH probes demonstrate high stability and reversibility when switched between pH 3, and 8 for at least 5 cycles.