This paper describes the determination of limits of detection (LODs) of interactions between an antigen, human chorionic gonadotrophin (hCG), and antibodies, anti-alpha-hCG and anti-beta-hCG, using a sandwich assay by surface plasmon field-enhanced fluorescence spectroscopy (SPFS). Randomly biotinylated antibodies were adsorbed onto a structured self-assembled monolayer (SAM)-streptavidin matrix, tethered to gold via a SAM consisting of biotinylated thiol molecules interspersed with hydroxyalkanethiol molecules. The influence of the concentration of biotinylated thiol on the binding of biotinylated antibody and its functionality, in terms of its ability to bind to the hCG antigen, was studied. This allowed determination of the optimum biotin-thiol mole fraction in the mixed thiol solution and consequently in the SAM, to maximize binding of hCG of the streptavidin-bound antibody. SPFS studies of the binding of a secondary fluorescently labeled antibody to hCG immobilized on the optimized SAM-streptavidin-antibody layer showed that a LOD of hCG of 2 mIU mL(-1) (4 x 10(-12) mol L(-1)) could be realized. The system was further optimized by using a more oriented and organized surface by adsorbing monobiotinylated Fab-hCG in place of the whole antibody. A LOD of 0.3 mIU mL(-1) (6 x 10(-13) mol L(-1)) was achieved for this system. This work illustrates the importance of antibody orientation, both on the planar surface and in terms of position of binding site, in maximizing sensor sensitivity.