Multicolor Upconversion Förster Resonant Energy Transfer Using Optimized Yb@YbTm Core@Shell Nanoparticles

Upconverting nanoparticles (UCNPs) have emerged as promising alternative donors for resonance energy transfer (FRET)-based biosensing. However, employing UCNPs in FRET assays remains challenging because they display relatively small absorption cross sections and are relatively large as compared to the Förster distance. Thousands of individual donor ions in each UCNP are located within various distances from surface-bound acceptors, complicating the data analysis. While previous studies have explored how the composition and architecture of UCNPs influence FRET, many reports remain qualitative, and multicolor UC-FRET systems involving a single donor and multiple acceptors are less commonly studied than single-donor-single-acceptor systems. To address these challenges, we synthesized UCNPs with an absorbing core (Yb³⁺-doped)/active shell (Yb³⁺, Tm³⁺-doped) nanoparticles systematically varying Tm³⁺ concentrations to optimize the FRET efficiency to surface-bound organic acceptors. A shell composition containing 4% Tm³⁺ yielded the highest FRET efficiency. Moreover, four distinct ATTO dyes showing spectral overlap with the blue emission of Tm³⁺ were used as acceptor dyes on the surface of UCNPs to evaluate FRET efficiencies in spectral and time domains. The differentiation of the four ATTO dyes on one type of upconverting donor nanoparticles using a simple ratiometric approach lays the foundation for the design of multiplexed bioassays. Our results offer a strategy for improving UC-FRET sensitivity through smart core-shell UCNPs designs, donor concentration tuning, and provide important insights into the rational design of more efficient, multicolor, and wash-free UC biosensing platforms.