In developing novel designs for semiconductor-based biosensors and for biofunctionalization of semiconductors in general, it is extremely important to be able to block the reaction sites present on a surface following the biomodification from further chemical transformations. This procedure is required both to protect the surface from oxidation and to allow for molecular-level control of the biomolecular interactions at the topmost layer. In this work, the biosensor model system is designed based on a single-strand biotin-modified thiol-DNA attached to the silicon substrate. The binding of this thiol-DNA to the surface is performed through the cross-linker sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SSMCC) attached to the 11-amino-1-undecene monolayer on Si(111) surface. Streptavidin-coated gold nanoparticles are used to test the reactivity of the surface and to examine the role of passivation in the entire scheme. The passivation of the remaining surface reactive sites is achieved via a reaction with 1-octadecanethiol (ODT). This approach tests both the stability of the silicon/organic layer interface and the passivation of the biofunctionalized surface on top of the organic layer. Microscopy and spectroscopy studies are combined to interrogate this model system before and after surface passivation.