CAPACITIVE MONITORING OF PROTEIN IMMOBILIZATION AND ANTIGEN-ANTIBODY REACTIONS ON MONOMOLECULAR ALKYLTHIOL FILMS ON GOLD ELECTRODES

Self-assembled monolayers of omega-mercaptohexadecanoic acid and omega -mercaptohexadecylamine on gold electrodes are stable at neutral pH an d display pure capacitive behavior at frequencies around 20 Hz. Differ ent methods of covalent immobilization of proteins on these monolayers are compared. Various reagents including succinimides, thionylchlorid e, p-nitrophenol and carbodiimides were used to activate the carboxy g roups of the adsorbed monolayer of omega-mercaptohexadecanoic acid. Gl utaraldehyde, cyanuric chloride and phenylene diisocyanate were used t o activate the amino groups of the monolayer of omega-mercaptohexadecy lamine. The immobilization of albumin on the activated surface was stu died by capacitive measurements. The N-hydroxysuccinimide and carbodii mide methods were identified as most suitable for protein immobilizati on in that they did not compromise the insulating properties of the al kylthiol layer and led to maximal increase of its dielectric thickness ; These approaches were used for a layer-by-layer preparation of a cap acitive immunosensor. Specifically, antibodies to human serum albumin were immobilized on the alkylthiol monolayer. Binding of the antigen l ed to a decrease of the electrode capacitance. The detection limit of the immunosensor is as low as 15 nM (1mg/l). (C) 1997 Elsevier Science Limited.