ORIENTATIONAL ASPECTS OF ANTIBODY IMMOBILIZATION AND IMMUNOLOGICAL ACTIVITY ON QUARTZ-CRYSTAL MICROBALANCE ELECTRODES

The resonant frequency shift of a quartz crystal microbalance (QCM) ha s been used to monitor the immobilization of immunoglobulin G (IgG) an d anti-IgG antibodies onto unmodified and modified gold QCM electrodes , IgG and anti-IgG antibodies have been immobilized onto, the gold QCM surfaces by four separate methods. First, the antibodies were immobil ized directly onto unmodified gold QCM surfaces by physical adsorption . Second, 12-mercaptododecan-1-ol was reacted with the gold surface, a nd after reaction with bromoacetic acid, the antibodies were coupled t o the thiolated surface using a water soluble carbodiimide hydrochlori de and N-hydroxysuccinimide. Third, the antibodies were modified by th e direct attachment of thiol groups using 2-iminothiolane and subseque ntly immobilized onto the Bold surface. Fourth, the gold QCM surface w as modified by the direct adsorption of Protein A, followed by antibod y adsorption. Physical adsorption of the antibodies onto gold QCM elec trodes resulted in 30% more bound antibody compared with the covalent attachment and Protein A immobilization methods, Atomic force microsco py( AFM) images of adsorbed IgG onto gold-coated mica revealed the ant ibodies to be aggregated and randomly distributed on the surface, Tile immobilized receptor layers, prepared by the above four procedures, w ere allowed to interact with their biospecific partner species, The re sulting QCM frequency shifts were used to evaluate both the orientatio nal aspects of antibody immobilization and immunological activities of the immobilized layers. Receptor layers formed by the immobilization of antibodies onto thiolated QCM electrodes did not increase the amoun t of antibody subsequently bound compared with receptor layers prepare d by direct physical adsorption of antibodies, Receptor layers of thio lated antibodies yielded varied immunological activities, The protein A coupling layer between the antibody receptor layer and gold QCM surf ace facilitated subsequent antibody binding, (C) 1996 Academic Press, Inc.