Activators Confined Upconversion Nanoprobe with Near-Unity Förster Resonance Energy Transfer Efficiency for Ultrasensitive Detection

Lanthanide-doped upconversion nanoparticles (UCNPs) as energy donors for Förster resonance energy transfer (FRET) are promising in biosensing, bioimaging, and therapeutic applications. However, traditional FRET-based UC nanoprobes show low efficiency and poor sensitivity because only partial activators in UCNPs possessing suitable distance with energy acceptors (<10 nm) can activate the FRET process. Herein, a novel excited-state energy distribution-modulated upconversion nanostructure is explored for highly efficient FRET. Integration of the optimal 4% Er³⁺doped shell and 100% Yb³⁺core achieves ∼4.5-fold UC enhancement compared with commonly used NaYF₄:20%Yb³⁺,2%Er³⁺nanoparticles, enabling maximum donation of excitation energy to an acceptor. The spatial confinement strategy shortens significantly the energy-transfer distance (∼4.5 nm) and thus demonstrates experimentally a 91.9% FRET efficiency inside the neutral red (NR)-conjugated NaYbF₄@NaYF₄:20%Yb³⁺,4%Er³⁺nanoprobe, which greatly outperforms the NaYbF₄@NaYF₄:20%Yb³⁺,4%Er³⁺@SiO₂@NR nanoprobe (27.7% efficiency). Theoretical FRET efficiency calculation and in situ single-nanoparticle FRET measurement further confirm the excellent energy-transfer behavior. The well-designed nanoprobe shows a much lower detection limit of 0.6 ng/mL and higher sensitivity and is superior to the reported NO₂^-probes. Our work provides a feasible strategy to exploit highly efficient FRET-based luminescence nanoprobes for ultrasensitive detection of analytes.