Ultrasensitive Wash‐Free Immunoassay Via Constructing Ultrabright AIE Beads Based on Dual‐FRET Process and Cycling Activated Mechanism

ABSTRACT The luminescent oxygen channeling immunoassay (LOCI) is a homogeneous, washing‐free bioanalytical platform renowned for its high convenience and multiple detection capabilities in biomarker detection, but the widespread adoption of LOCI has been constrained by the limited sensitivity originated from the poor luminescence intensity of classical acceptor beads. Herein, we report a reference strategy of constructing ultrabright acceptor beads based on dual‐fluorescence resonance energy transfer (FRET) process and cycling activated mechanism via inserting aggregation‐induced emission (AIE) photosensitive dyes. This design enables the conversion of otherwise wasted energy in conventional Europium‐based microparticles (EuSNPs) luminescence system into luminescence via reactive oxygen species (ROS) activation. By introducing the AIE‐active bridge fluorophore (AG) to acceptor microparticles (TASNPs), the PLQY was remarkably enhanced to 56% compared to AG‐free systems. Based on the established LOCI platform with inserting photosensitive microparticles, an ultrasensitive detection limit of 0.36 pg·mL − 1 toward Interleukin‐6 (IL‐6) is achieved, representing 64‐fold improvement over EuSNPs. The assay could be completed within 5 min and exhibite excellent repeatability (CV < 10%). This work introduces a versatile and efficient strategy‐through synergistic 1 O 2 cycling and dual‐FRET enhancement for advancing the performance of wash‐free luminescent immunoassays, offering a promising pathway toward next‐generation clinical diagnostics.