Emerging strategies for the formulation of antibody–nanoparticle conjugation in lateral flow immunoassays

Lateral flow immunoassays (LFIAs) are indispensable point-of-care diagnostic tools due to their simplicity, fast readout and low cost. Traditionally based on gold nanoparticles (AuNPs) for visual detection, recent advances in nanotechnology have expanded the LFIA toolkit to include a wide range of nanoparticles (NPs), such as carbon NPs, quantum dots, upconversion NPs, nanozymes, aggregation-induced emission nanoparticles, and bimetallic or hybrid structures. These novel nanomaterials improve assay sensitivity and allow for multimodal detection (using fluorescence, surface-enhanced Raman scattering or photothermal detection). Central to the performance of NP-based LFIAs is the protein corona, typically formed by antibodies, which mediates antigen recognition. Immobilization strategies range from simple physisorption to more controlled chemisorption techniques (e.g., 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide carbodiimide coupling, thiol- or glycan-directed attachment) and bioaffinity methods (e.g., biotin–streptavidin). Although chemisorption is often assumed superior, a critical comparison of recent studies reveals that optimized physisorption can achieve similar or better detection limits in many cases. Conjugation strategy, NP morphology, and surface chemistry collectively influence probe stability, orientation, and binding efficiency. This review critically evaluates recent innovations in nanoparticle selection, conjugation methods, and signal generation strategies.