Oriented immobilization of antibodies on suspended nanoparticle and substrate surfaces via one-step catch-and-link approach for development of biosensors

• Boronic acid-tosylate linker facilitates robust antibody orientation on suspended gold nanoparticles and solid substrates. • The method avoids the aggregation effect of gold nanoparticles caused by positive charges. • The amount of antibody required for conjugation was reduced by 100-fold. • Enhances the biosensor performance as compared to the traditional EDC/NHS coupling approach. • Antibodies remain securely attached in procalcitonin-spiked serum samples during sensing. In immunoassay, the orientation of the antibody is usually random, and the restricted orientation of the antibody may affect the antibody activity, which may lead to the decrease of analytical sensitivity. In this study, a boronic acid-tosylate linker (abbreviated as BA-Ts linker) was used to conjugate the antibody to the substrate. The BA-Ts linker structure contains a boronic acid to form a reversible bond with the glycan at the Fc-terminal end of an antibody and an azide group for copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction to anchor the linker to an alkynylated surface. The feasibility and advantages of using this linker for antibody conjugation are illustrated by using the Fiber Optic Particle Plasmon Resonance (FOPPR) biosensor in both the FOPPR direct assay and fiber optic nanogold-linked immunosorbent assay (FONLISA) methods. The results of this study successfully demonstrated that the use of the BA-Ts linker improved the analytical sensitivity of the FOPPR biosensor for procalcitonin (PCT) detection and reduced the amount of antibody required for conjugation on optical fibers (from 10 −4 g/mL to 10 −6 g/mL). In FOPPR direct assay, the limit of detection (LOD) improved from 110 nM to 42.5 nM. In FONLISA, the LOD improved from 13.4 fM to 1.7 fM. Furthermore, good recoveries between 94.1 % and 101.8 % were obtained in PCT spiked serum samples, indicating great potential for future clinical applications.