Displacement of molecules near a metal surface as seen by an SPR-SPFS biosensor

Movement of a fluorophore-labeled antibody on the surface of a self-assembled monolayer (SAM) was observed by surface plasmon resonance and surface-plasmon field-enhanced fluorescence spectroscopy (SPFS). At an extremely low surface coverage, the antibody lies close to the biotin-functionalized SAM surface. As additional nonlabeled antibodies were bound, the fluorophore-labeled antibody was displaced away from the SAM surface (and thus the gold surface) due to the constraint imposed by the neighboring nonlabeled antibody. A greater SPFS fluorescence signal was observed due to the weaker fluorescence quenching at large distances from the gold surface. The magnitude of the displacement is proportional to the available biotin binding sites on the sensor surface. The displacement is theoretically explained on the basis of the relationship between the fluorescence intensity and the evanescent field amplitude within the dielectric medium.