Adsorption and immobilization of cytochrome c on nanodiamonds

Methods have been developed to immobilize proteins onto the surfaces of nanodiamonds with an average size of 5 +/- 1 nm. The immobilization started with carboxylation/oxidization of diamonds with strong acids, followed by coating the surfaces with poly-L-lysine (PL) for covalent attachment of proteins using heterobifunctional cross-linkers. The feasibility of this approach is proven with fluorescent labeling of the PL-coated diamonds by Alexa Fluor 488 and subsequent detection of the emission using a confocal fluorescence microscope. Immobilization of proteins onto the surfaces is also demonstrated with yeast cytochrome c, which possesses a free SH group for linkage and a characteristic Soret absorption band for observation.