NIR Long-lived Luminescent Upconversion Nanoparticle-Based Time-Gated LRET for Ultrasensitive Detection of MicroRNAs

Upconversion nanoparticle (UCNP)-based luminescence resonance energy transfer (LRET) biosensing offers advantages, including high sensitivity, simplicity, wash-free detection, and precise biomolecule quantification. However, conventional LRET assays using sensitizer (Yb³⁺)/activator (Tm³⁺) co-doped UCNPs under continuous wave excitation of 980 nm have limitation in detection sensitivity due to the energy harvesting and recharging properties of dopant ions. To address this, we proposed a time-gated LRET approach utilizing NIR long-lived luminescent UCNP donors (L-donors) under 980 nm pulsed excitation. This approach, referred to as L-TG-LRET, leverages the long luminescence lifetime and nanostructure of the L-donor (NaYbF₄@NaYF₄:10%Yb,1%Tm@NaYF₄) to prevent the immediate recharging of Tm³⁺ ions deactivated by LRET. This significantly improves energy transfer efficiency from Tm³⁺ (800 nm emission) to IRDye800 acceptors (650–850 nm absorption). As a proof of concept, we applied the L-TG-LRET assay for detecting microRNA (miRNA), demonstrating sub-attomolar sensitivity for three cancer-related miRNAs within 10 minutes with a broad dynamic range (10 aM to 100 fM). This assay successfully quantified miRNA expression levels in cancer cells, patient plasma, and exosomes, distinguishing lung and breast cancer patients from healthy donors. This approach also showed higher sensitivity than PCR for detecting low levels of exosomal miRNAs even in 1000-fold diluted samples. These results highlight the potential of the L-TG-LRET system as a powerful tool for sensitive biomolecular detection in clinical diagnostics.