Preparation of gold surfaces with biospecific affinity for NAD(H)-dependent lactate dehydrogenase

Gold surfaces with biospecific affinity for NAD(H)-dependent lactate dehydrogenase have been prepared by covalent attachment of several triazine dyes to chemisorbed functionalized alkanethiol self-assembled monolayers. The dyes which work as coenzyme analoga build a site-specific affinity complex with the enzyme by binding through its NAD(+)-binding pocket. This immobilization method implies a biospecific recognition of the enzyme which is expected to yield lactate dehydrogenase self-assembled monolayers in which the enzyme is oriented facing its NAD(+)-binding pocket (and therefore its active site) towards the metal surface. Self-assembled monolayers with Cibacron Blue F3G-A as anchored ligand showed always the highest affinity for lactate dehydrogenase, However, the biospecific affinity of the ligand-anchored monolayer was strongly dependent on the nature of the alkanethiol underlayer used to bind the triazine dye. Higher enzymatic surface coverages were obtained with mixed self-assembled monolayers providing a low amount of ligand (less than a 10% of a densely packed alkanethiol monolayer) bound to the metal surface through a long and flexible spacer. Lactate dehydrogenase-modified gold electrode surfaces catalyzed the electro-oxidation of lactate only when the biological cofactor (NAD(+)) was present in the reaction mixture, thus suggesting that the NAD(+)-binding pocket used to anchor the enzyme to the monolayer is not involved in the enzymatic reaction. (C) 1997 Elsevier Science S.A.