Functionalization of gold surfaces for specific and reversible attachment of a fused beta-galactosidase and choline-receptor protein

A method that allows the specific immobilization of proteins onto a gold electrode has been developed. Mixed self-assembled monolayers of thiol chains functionalized with choline and hydroxyl groups have been synthesized step-by-step over a template of thiocarboxylic acid adsorbed onto gold. Choline-functionalized monolayers displayed affinity for a chimera protein made by the fusion of the beta-galactosidase (beta-Gal) from Escherichia coli and the choline-binding domain of the (acetylmuramoyl)-L-alanine amidase (C-LYTA) from Streptococcus pneumoniae. This chimera maintains both the hydrolase activity and the affinity for choline, respectively, of its parent proteins. The binding of the protein to the tared interface was specific and could be inhibited either by soluble choline or by saturating the monolayer choline groups with the C-LYTA fragment. Using an S-35-labeled chimera, saturation coverage was found under optimized binding conditions. The activity of the immobilized chimera was determined with (p-aminophenyl)-beta-D-galactopyranoside, a synthetic substrate of beta-galactosidase. The product of the enzymatic reaction, p-aminophenol, was detected electrochemically using the functionalized gold surface with bound chimera protein as a working electrode in a conventional electrochemical cell. Gold electrodes covered with chimera protein were very stable and gave fast and reproducible electrochemical response to the addition of beta-Gal substrate in a conventional flow injection analysis system.