Dual-Enhancement Lateral Flow Immunoassay via Gold-Decorated Ferritin Nanoprobes and SpyTag/SpyCatcher Site-Directed Conjugation

Abstract

Abstract: Conventional lateral flow immunoassays (LFIAs) suffer from limited sensitivity due to weak optical signals from colloidal gold, random antibody orientation, and inconsistencies in nanoprobe synthesis. To address these challenges, we introduce a dual-enhancement strategy integrating gold-decorated ferritin nanocages (HFn@Au) with SpyTag/SpyCatcher-mediated oriented antibody immobilization. Engineered ferritin displaying SpyTag peptides on its surface served as a precise template for the controlled nucleation and growth of gold nanoclusters, resulting in highly uniform core-shell nanostructures (PDI = 0.242; size variation coefficient = 12.56%) with significantly amplified optical intensity. Importantly, this deposition process preserved the functionality of the SpyTag sites. Subsequent incubation with SpyCatcher-fused antibodies enabled spontaneous isopeptide bond formation, ensuring site-specific and covalent antibody conjugation. This oriented immobilization enhanced the effective antigen-binding capacity by approximately 8-fold compared to random adsorption. Using human chorionic gonadotropin (hCG) as a model analyte, the optimized LFIA achieved a limit of detection (LOD) of 0.5 mIU/mL, representing a 50-fold sensitivity improvement over commercial gold nanoparticle-based strips (LOD ≈ 25 mIU/mL). The assay demonstrated acceptable linearity from 2.5 to 20 mIU/mL (R² = 0.956) and high reliability in urine matrices, with spike-recovery rates between 91.70% and 108.30%. This synergistic strategy provides a versatile and powerful method for dramatically enhancing the sensitivity of point-of-care diagnostics, representing an encouraging step toward practical real-sample application.