Purple‑, Blue‑, and Green-Emitting Multishell Alloyed Quantum Dots: Synthesis, Characterization, and Application for Ratiometric Extracellular pH Sensing

We report the synthesis of a series of Cd_xZn_1–xSe/Cd_yZn_1–yS/ZnS and ZnSe/Cd_yZn_1–yS/ZnS multishell alloyed luminescent semiconductor quantum dots (QDs) with fluorescence maxima ranging from 410 to 530 nm which cover the purple, blue, and green portion of the spectrum. Their subsequent surface modification to prepare water-soluble blue-emitting QDs, characterization, and application for ratiometric pH sensing in aqueous buffers and in an extracellular environment are further described. QDs were synthesized starting from ZnSe cores, and the fluorescence peak positions were tuned by (i) cation exchange with cadmium ions and/or (ii) overcoating with Cd_yZn_1–yS layers. The as-prepared QDs had reasonably high fluorescence quantum yields (∼30–55%), narrow fluorescence bands (fwhm ∼25–35 nm), and monodispersed semispherical shapes. Ligand exchange with hydrophilic compact ligands was successfully carried out to prepare a series of water-soluble blue-emitting QDs. QDs coated with the hydrophilic compact ligands preserved the intrinsic photophysical properties well and showed excellent colloidal stability in aqueous buffers for over a year. The blue-emitting QDs were further conjugated with the pH-sensitive dye, fluorescein isothiocyanate (FITC), to construct a fluorescence resonance energy transfer-based ratiometric pH sensing platform, and pH monitoring with the QD-FITC conjugates was successfully demonstrated at pHs ranging between 3 and 7.5. Further assembly of the QD-FITC conjugates with membrane localization peptides allowed monitoring of the pH in extracellular environments. High quality, water-soluble blue-emitting QDs coated with compact ligands can help expand the practical fluorescence range of QDs for a variety of biological applications.