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 0.1nA, 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.
Biosystems Technology, Technical University of Applied Sciences Wildau, Bahnhofstraße 1, 15745 Wildau, Germany. Christoph.Wettstein@th-wildau.de
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D016954:Acinetobacter calcoaceticus / Q000201:enzymology; D000818:Animals; D002417:Cattle; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004563:Electrochemistry; D004566:Electrodes; D004579:Electron Transport; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D005947:Glucose / Q000032:analysis / Q000737:chemistry; D042843:Glucose 1-Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D045542:PQQ Cofactor / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation
D004800:Enzymes, Immobilized; D045542:PQQ Cofactor; D045304:Cytochromes c; D004247:DNA; D042843:Glucose 1-Dehydrogenase; D005947:Glucose