Distance-Independent Synergistic Fluorescence Quenching Lateral Flow Immunoassay for Sensitive Detection of Creatine Kinase-MB Based on Gold–Iridium Oxide Nanoparticles

Fluorescence quenching lateral flow immunoassay (FQ-LFIA) has emerged as a powerful tool for the rapid detection of disease biomarkers due to its high signal-to-noise ratio and multimodality signal output. However, conventional fluorescent quenchers are constrained by specific distances and narrow absorption peaks, hindering their quenching efficiency and sensitivity for trace analyte detection. In this study, we synthesized bimetallic gold-iridium oxide nanoparticles (Au@IrO₂ NPs) via a seed-mediated growth method as the quenchers and constructed a distance-independent synergistic FQ-LFIA (SFQ-LFIA) platform. The Au@IrO₂ NPs significantly reduced the background fluorescence signal on the test line (T-line) through the inner filter effect (IFE), while their excellent catalytic activity oxidized the substrate 3,3',5,5'-tetramethylbenzidine (TMB) to generate the colored product oxidized TMB (oxTMB), which further synergistically quenched background fluorescence via a secondary IFE pathway. Using creatine kinase-MB (CK-MB) as a model biomarker, the detection limit of the Au@IrO₂ NPs-based SFQ-LFIA in fluorescence mode (0.71 pg/mL) was 13-fold lower than in the precatalysis (8.91 pg/mL) and at least 215-fold lower than in colorimetric mode. The platform demonstrated high accuracy in quantifying CK-MB levels in clinical serum samples, providing a novel strategy for developing advanced in vitro rapid diagnostic systems.