An approach is introduced for studying the adsorption and recognition mechanisms of biomolecules, without using any markers. We show for the first time, that the sum frequency generation spectroscopy performed in the total internal reflection (TIR-SFG) geometry, combined with a regular close-packed array of gold nanoparticles allows to probe with a high sensitivity the changes in conformation and orientation induced by the recognition process of avidin by biocytin. This approach represents a new platform with potential use in biosensors, diagnostics and bioactive layers.