Layered Protein Assemblies on Electrodes Exploiting Multistep Electron Transfer Chains for Dual Analyte Detection
Authors: Feifel Sven , Kapp Andreas , Ludwig Roland , Lisdat Fred 
Published: February 2020

In recent years there is considerable progress in constructing artificial signal chains which follow natural examples. This can result in sensitive systems which are turned on by the presence of a certain analyte. One approach is based on the construction of protein arrangements using the redox protein cytochrome c (cyt c) [1]. Here electron transfer through several layers of the protein on electrodes has been achieved. The defined arrangement of the biomolecule is an important precondition for a functional system. The layer-by-layer adsorption technique has been shown to be advantageous with this respect. Different building blocks have been applied including polyanilline derivatives, DNA and nanoparticles [2,3]. Furthermore, first examples for the construction of bi-protein assemblies with catalytic properties have been demonstrated, e.g. with sulfite oxidase [4].
In this study it is first demonstrated that bi-protein assemblies using cyt c and the enzyme cellobiose dehydrogenase (CDH) can be constructed in a layered design using modified silica nanoparticles as building block [5]. It is found, that glycosylation of the enzyme significantly influences the layered assembly and the electron transfer chain from the substrate to the electrode. Conditions have been found, so that several catalytically active protein layers can be formed on the electrode surface, thus allowing the adjustment of the electrode response to lactose by the numbers of deposited layers.
Based on these results a tri-protein architecture has been created on a thiol-modified gold electrode. Again the layer-by-layer deposition method is used, but additionally the multi-copper enzyme Laccase has been immobilised within this structure. The formation of the entity with cyt c, Lac, CDH, and SiNPs is first confirmed by quartz crystal microbalance (QCM) experiments, and then investigated electrochemically by cyclic voltammetry (CV).
Within this architecture the two enzymes are connected to the electrode via cyt c. Since the activity of the enzymes is controlled by the delivery or withdrawal of electrons, the redox state of cyt c can be used for switching the activity of the biocatalysts. Since electron transfer throughout the whole layered entity is feasible, Lac and CDH in different distances to the electrode can be addressed. The switchable reaction cascades for Lac and CDH are functioning in an non-separated matrix without disturbing the reaction of the other biocatalyst. Whereas the CDH allows lactose detection, the activity of laccase is influenced by the oxygen content of the solution.
The approach represents an advance in mimicking of biological electron transfer cascades by fixing protein-protein reactions to electrodes in the immobilized state without the need of any mediating shuttle molecule.
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