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 p repared. These conjugates, purified by centrifugation/resuspension, provide a reproducible means of positioning a biomolecule analyte between two meta l surfaces: the metal nanoparticle to which the biomolecule is bound and an aggregated Ag sol used as a substrate for surface enhanced Raman scatterin g (SERS). SERS spectra for Ag:Cc:Au sandwiches made by addition of Cc:Au co njugates to aggregated Ag show that the protein retains its native conforma tion. Conjugation of Cc to colloidal metal nanoparticles prior to exposure to the SERS substrate can be used as a means of controlling protein orienta tion with respect to the substrate. In addition, Cc molecules bound to 12-n m diameter Au nanoparticles prior to adsorption to aggregated Ag are more s table 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 reagent s for protein SERS.