Drug development studies and other applications in diagnostics/therapeutics require pH measurement down to the micro or nanoscale to provide relevant subcellular information, but the most widely used optical pH sensors (fluorescent dyes, dye-labeled quantum dots) suffer from drawbacks of photobleaching, blinking, and limited tissue penetration. Autofluorescence is also an issue during tissue imaging in the UV-VIS excitation range of these fluorescers. The ability of upconversion nanoparticles (UCNPs) to overcome these challenges through their unique optical properties (NIR excitation, long lifetimes) motivated this work to further the understanding of interactions between particle and pH dye before applying this knowledge to the creation of a novel pH nanosensor for cellular imaging.

High-temperature coprecipitation was used to synthesize b-phase UCNPs with shell layers of different thicknesses ($\leq$1 nm and 3 nm) to decrease surface quenching. The two types of particles were modified with different ligands, polyethylenimine (PEI) and poly(isobutylene-alt-maleic anhydride) (PMA), respectively, to enable dispersion in aqueous solutions. The effect of two anthraquinone dyes, Calcium Red and Alizarin Red S, on UCNPs was measured by spectroscopic techniques at various pHs. When the thick-shell (3 nm), PMA-coated UCNPs and dyes were mixed directly, the green emission band of the UCNPs was quenched by a pH-dependent inner filter effect while the red emission band remained unchanged and acted as the reference signal for ratiometric pH measurements. When the anthraquinones were attached onto the thin-shell (0.4 nm), PEI-coated UCNPs through electrostatic attraction, inner filter effect remained the dominant quenching mechanism compared to resonance energy transfer (RET).

Based on the above findings, UCNPs with an intermediate shell thickness of 1 nm were synthesized to simultaneously enable RET and ensure particle brightness. The PEI-coated UCNPs were attached to a dye called pHAb for pH sensing in live cells. RET was much more significant for UCNP-pHAb compared to either of the UCNP-anthraquinone combinations. Ratiometric sensing was performed with the sensitized pHAb emission and reference UCNP red emission in buffer solutions, then the nanosensors were calibrated in SH-SY5Y cells between pH 4 and 6. The UCNP-pHAb conjugates successfully measured the pH of acidic compartments (lysosomes) after clathrin-mediated endocytosis.