title: Steric crowding effects on target detection in an affinity biosensor.
abstract: This work quantifies the impact of steric crowding on whole antibody (Ab) receptor immobilization and target Ab detection and also demonstrates how the versatile biotin/streptavidin receptor immobilization system must be tuned to optimize target detection in designing biosensors. Results are demonstrated on a label-free optical biosensor fabricated from n-type macroporous porous silicon (PSi) with approximately 88-107 nm diameter pores. We employ a sandwich assay scheme comprising a linking chemistry (biotin/streptavidin) to attach biotinylated anti-rabbit IgG (receptor) to detect rabbit IgG (target). A "bottom-up" approach was taken to investigate each layer of the sandwich assay to optimize target binding. Steric crowding was observed to hinder subsequent layer binding for each layer in the sandwich (biotin, streptavidin, and receptor). Our results give definitive evidence that the onset of steric crowding within the biotin layer occurs at a surface coverage of 57%, which is much higher compared to that from published work on well-ordered self-assembled biotin monolayers on planar gold surfaces. This difference is attributed to the topographical heterogeneity of the PSi substrate. Streptavidin (SA) binding to surface-linked biotin was altered by preblocking the streptavidin binding sites with biotin. Through consistent trends in data, preblocking SA was shown to reduce steric crowding within the SA layer, which translated into increased receptor immobilization. The final detection range of rabbit IgG was 0.07-3 mg mL(-1) (0.4-17 ng mm(-2)), and binding specificity was demonstrated by employing an anti-chicken IgG control receptor. This study underlines the importance of considering binding avidity and surface topography in optimizing chip-based biosensors.
authors.affiliation: Department of Biomedical Engineering, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York 14642, USA.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Bonanno
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Delouise
mesh_terms: D000818:Animals; D000937:Antigen-Antibody Reactions; D001665:Binding Sites; D015374:Biosensing Techniques; D001710:Biotin / Q000737:chemistry*; D006041:Goats; D007074:Immunoglobulin G / Q000737:chemistry*; D007700:Kinetics; D011817:Rabbits; D012680:Sensitivity and Specificity; D012825:Silicon / Q000737:chemistry*; D019809:Streptavidin / Q000737:chemistry*
chemical_list: D007074:Immunoglobulin G; D001710:Biotin; D019809:Streptavidin; D012825:Silicon