Protein:colloid conjugates for surface enhanced Raman scattering:: Stability and control of protein orientation

Conjugates between cytochrome c (Cc) and colloidal Au particles have been prepared. These conjugates, purified by centrifugation/resuspension, provide a reproducible means of positioning a biomolecule analyte between two metal surfaces: the metal nanoparticle to which the biomolecule is bound and an aggregated Ag sol used as a substrate for surface enhanced Raman scattering (SERS). SERS spectra for Ag:Cc:Au sandwiches made by addition of Cc:Au conjugates to aggregated Ag show that the protein retains its native conformation. Conjugation of Cc to colloidal metal nanoparticles prior to exposure to the SERS substrate can be used as a means of controlling protein orientation with respect to the substrate. In addition, Cc molecules bound to 12-nm diameter Au nanoparticles prior to adsorption to aggregated Ag are more stable to changes in the orientation of the heme with respect to the surface and to surface-induced heme spin state conversion than adsorbed, free Cc. These experiments show that protein:Au conjugates offer benefits as reagents for protein SERS.