Construction and characterization of a multi-layer enzyme electrode: Covalent binding of quinoprotein glucose dehydrogenase onto gold electrodes

Multilayers of quinoprotein glucose dehydrogenase were assembled onto modified gold electrodes. As a primary modifier the bifunctional 3,3′-dithiodipropionic acid bis(N-hydroxysuccinimide ester) was chemisorbed. Glucose dehydrogenase was covalently bound to this activated electrode in a stepwise procedure. In the presence of glucose, the electrode functions as a sensor for electron acceptors. Catalytic current, as observed for p-aminophenol, was used to characterize electrode performance. The dependence of the electrode response on the number of enzyme layers showed that the transition from a kinetic to a diffusion-limited sensor is reached at 6–7 enzyme layers. The response of multilayer electrode is stable over a broad range of pH and ionic strength of the bulk solution. It also shows good stability: after 2 months, 75% of its original activity remained.