Elucidating Protein/Ligand Recognition with Combined Surface Plasmon Resonance and Surface Enhanced Raman Spectroscopy.

The ability to distinguish between specific and nonspecific binding is important for assessing the interactions between protein receptors and ligands. Surface plasmon resonance (SPR) spectroscopy is an advanced tool to measure binding events, yet the ability to distinguish between specific and nonspecific binding remains a limitation. To address this problem, we use SPR spectroscopy correlated with surface enhanced Raman scattering (SERS). The chemical information present in SERS spectra provides insight into the molecular interactions between functionalized nanoparticles and proteins, which are not detectable by SPR alone. Using a custom instrument with the Kretschmann configuration, we successfully demonstrate simultaneous affinity and the chemical characterization of streptavidin-functionalized gold nanoparticles (STV-NPs) binding to biotin immobilized on a gold film in both air and flowing phosphate buffered saline (PBS). The SPR performance is consistent with that of previous reports. The association constant (K _A ) for streptavidin/biotin and STV-NPs/biotin interactions observed (2 ± 1 × 10 ⁷ M ^-1 and 2.4 ± 0.3 × 10 ¹⁰ M ^-1 , respectively) agree with literature values and show a strong avidity effect associated with the STV-NPs. The SERS scattering from STV-NPs is excited by the surface plasmon polariton and collected from an objective lens mounted over the fluidic channel. The SERS spectra are recorded simultaneously with the SPR sensorgram, and the detected Raman bands provide chemical insight into the binding event. Multivariate curve resolution analysis of the spectra can differentiate specific from nonspecific binding. This label-free, real time, and surface sensitive detection method provides chemical information to protein/ligand binding affinity measurements.