3D streptavidin surfaces for higher capacity and enhanced kinetics in solid-phase immunoassays

Abstract

The streptavidin-biotin system is commonly used in solid-phase immunoassays to immobilize biomolecules. The conventional streptavidin (SA) coatings form two-dimensional surfaces which may limit binding site accessibility due to steric hindrance and mass transport limitations. Polystreptavidin (polySA) coatings can instead create a more three-dimensional architecture that may improve binding site accessibility and increase binding capacity. The aim of this study was to evaluate whether three-dimensional polySA surfaces can increase the solid-phase surface binding capacity and improve the performance of solid-phase immunoassays. SA and polySA surfaces were prepared using direct and indirect coating strategies. Indirect coatings utilized biotinylated bovine serum albumin (bio-BSA) and thyroglobulin (bio-Tg) as anchoring layers. Binding capacity of the surfaces was assessed using europium-labelled biotin (Eu-biotin) and europium-labeled biotinylated IgG antibodies (Eu-bio-IgG). The functional performance and kinetics were evaluated using a troponin I immunoassay with time-resolved fluorescence detection at various incubation times. PolySA had consistently higher binding capacity than conventional SA. Surface measurements showed 6.5- and 4-fold increases with Eu-biotin and Eu-bio-IgG, respectively, while solution measurements showed corresponding increases of 2.9- and 2.3-fold. Improvements in immunoassay performance were more moderate with approximately 1.3–1.5-fold increase in assay response but kinetic analysis showed consistently faster binding for polySA surfaces at all incubation times. Indirect coating strategies produced comparable or slightly reduced performance depending on the anchor protein and its biotinylation degree. Overall, the results indicate that polySA substantially enhances solid-phase binding capacity while maintaining functional immunoassay performance, supporting its use for improving assay kinetics.