Coupling of pyrroloquinoline quinone dependent glucose dehydrogenase to (cytochrome c/DNA)-multilayer systems on electrodes

The redox protein cytochrome c (cyt c) assembles into electro-active multilayers on gold electrodes by the help of deoxyribonucleic acid (DNA) as a negatively-charged building block. The feasibility of this electro-active system as a novel interface for the immobilization of enzymes on electrodes is investigated in this study. Therefore the known reaction of cyt c and PQQ-GDH is confined to the immobilized state of both molecules. We find that electron-transfer from the substrate via PQQ-GDH and cyt c molecules, towards the electrode occurs; thus the system can be considered as an artificial signal chain.First, a monolayer of cyt c is prepared on a thiol-modified gold electrode and investigated with PQQ-GDH in solution. Cyclic voltammetric measurements prove that a small catalytic current occurs in the presence of the substrate. Next, both proteins are immobilized. We use the layer-by-layer deposition technique to assemble cyt c with DNA in multiple layers and a terminal layer of PQQ-GDH: (cyt c/DNA)n/PQQ-GDH. It is found that a catalytic current flows when glucose is present, proving that this system relies on inter-protein electron-transfer. The current intensity can be increased from 0.1 nA, at the monolayer system, up to 3.7 nA, at the (cyt c/DNA)4/PQQ-GDH electrode. This bi-protein multilayer system can follow different glucose concentrations in a linear dynamic range between 25 nM and 0.5 μM at its pH optimum, i.e. pH 6. Therefore this system is of limited importance for sensing but it represents a new biomimetic signal chain by arranging proteins in multiple layers on electrodes, making direct electron exchange feasible.

► Novel electro-active interface for the immobilization of enzymes is created.
► Direct electron exchange between PQQ-GDH and cyt c is achieved in immobilized state.
► Biomimetic signal chain from glucose via PQQ-GDH and cyt c is established.
► Sensitivity of the system is tunable to a certain extent by the number of layers.