Near-Infrared Long Lifetime Upconversion Nanoparticles for Ultrasensitive microRNA Detection via Time-Gated Luminescence Resonance Energy Transfer

Abstract Although upconversion nanoparticle (UCNP)-based luminescence resonance energy transfer (LRET) biosensing offers advantages such as wash-free detection and precise biomolecule quantification, there is still room for enhancing its sensitivity by preventing continuous energy transfer in co-doped UCNPs during LRET. In this study, we report a time-gated LRET approach using near-infrared (NIR) long-lived luminescent UCNP donors (L-TG-LRET), achieving an 8-fold increase in luminescence lifetime without compromising emission intensity. The extended and slower energy migration and transfer pathway of the L-TG-LRET approach significantly enhances sensitivity by preventing rapid Tm3+ reactivation of UCNPs during LRET from Tm3+ ions (800 nm emission) to IRDye800 acceptors (650–850 nm absorption). The developed L-TG-LRET system was applied to the detection of microRNAs (miRNAs), providing 17.9-fold higher sensitivity than conventional steady-state methods. Furthermore, this method has been successfully employed to determine the miRNA expression levels in cancer cells, patient plasma, and exosomes, enabling the differentiation of patients with lung and breast cancers from healthy donors. Notably, this approach demonstrated enhanced sensing performance compared to polymerase chain reaction (PCR) for the detection of low levels of exosomal miRNAs. These results highlighted the potential of the L-TG-LRET system as a valuable tool for sensitive biomolecular detection in clinical diagnostics.