Integrating Enzymes into Cytochrome c –Based Multilayer Architectures – General Considerations and Example with Fructose Dehydrogenase

Abstract

The creation of artificial electron transfer (ET) chains based on the defined arrangement of enzymes and redox proteins on electrode surfaces represents an interesting field in bioelectronics1. One approach exploits the self exchange properties of cytochrome c (cyt c) to build up new sensing electrodes2. In this study we have investigated the ET reaction of the flavin-dependent enzyme fructose dehydrogenase (FDH) with the redox protein cyt c. Two different pH optima are detected for the reaction of both proteins in solution - at acidic and neutral pH. When one reaction partner - cyt c - is immobilized on a thiol-modified gold electrode and FDH is in solution ET proceeds efficiently at neutral pH which can be concluded from well-shaped catalytic currents obtained in cyclic voltammetry. In addition, a defined dependence on the substrate concentration has been found. In acidic media the reaction can also be verified but appears to be less efficient. Furthermore, it can be demonstrated that both partners can be assembled in a stable multilayer architecture, using the biopolymer DNA as a negatively charged polyelectrolyte building block. The defined layered deposition of DNA and a mixture of FDH and cyt c is confirmed by surface plasmon resonance (SPR) measurements. Prepared on electrodes, substantial catalytic currents are recorded upon addition of fructose. The response can be enhanced by the number of layers deposited on the surface. This shows that an artificial signal chain can be constructed through multiple protein layers. FDH is not the only enzyme which can be immobilized and connected to the electrode by means of the redox protein cyt c. Cellobiose dehydrogenase, sulfite oxidase or billirubin oxidase are other examples3,4. This may indicate that cyt c based multilayers can have some general applicability when some preconditions are fullfilled. 1. Feifel et al , in Biosensors Based on Aptamers and Enzymes, Springer 2014, 140, 253-298 2. Lisdat et al., Chemical Communications 3 2009, 274-283. 3. Feifel et al., Angew. Chemie 126(22) 2014 5782-86 4 Dronov et al. JACS 2008 130(4) 1122.