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 0.1nA, at the monolayer system, up to 3.7nA, 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 25nM and 0.5μM 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.
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