Combinatorial SiO ₂ ‐Encapsulated Quantum Dot Nanoparticles and their Use in Spectral Unmixing Analysis

Linear unmixing spectral analysis is a technique where signals from tens of fluorophores can be deconvoluted to increase multiplexing by 4–5‐fold. For the mathematical algorithm‐driven analysis to be applied to analytical assays, there is a need to develop spectrally engineered nanoparticle probes. Herein, silica‐encapsulated quantum dot (QD‐SiO 2 ) nanoparticles with tunable spectral emissions are presented. The mechanism and factors for incorporating hydrophobic quantum dots (QDs) in silica in the reverse microemulsion synthesis are investigated, including 1 H NMR study on the interaction of ligands on QDs with the surfactant. The optimized synthesis reduces Förster resonance energy transfer between QDs in silica particles. In combination with linear unmixing analysis, nanoparticles that encapsulate varying ratios of different color QDs enable multiplexing capability up to 8. Their size of ca. 30 nm can enable in vitro imaging in addition to the use in existing immunoassays and analytical platforms.