Nanolayered Supramolecular Protein Clusters on Electrodes: A Switchable Cascadic Reaction Scheme for Dual-Analyte Detection

Biochemical signal chains and pathways are characterized by an effective coupling of individual reaction steps resulting in a rather high specificity in signal transduction. Additionally such highly complex systems allow a switching between different reaction cascades, depending on the presence of a certain molecule. Apparently, these efficient biological principles have been adopted for the construction of artificial signal chains.^[1,2] In such a way protein functionalities can be coupled to electrochemical detection schemes, ensuring signal generation in the presence of individual substances.^[3-5]

Here we report on a novel system, which allows an activity-switch between two enzymes co-immobilized in a supramolecular network. This supramolecular network is formed by embedding two different biocatalysts - the multi-copper enzyme laccase (Lac), the multi-domain enzyme cellobiose dehydrogenase (CDH) - and the redox protein cytochrome c (cyt c) in an artificial matrix composed of carboxy-modified silica nanoparticles[6] allowing the immobilization of all the components in a layer-by-layer fashion. The formation of the entity with cyt c, Lac, CDH, and SiNPs was confirmed by quartz crystal microbalance experiments, and investigated by cyclic voltammetry.^[7]

Within this layered architecture two enzymes have been connected to the electrode via cytochrome c. Since the activity of the enzymes is controlled by the delivery or withdrawal of electrons the redox state of cyt c has been used for switching the activity of the biocatalysts on and off. Given that the electron transfer throughout the 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 a non-separated matrix without disturbing the reaction of the other catalyst, allowing lactose and oxygen sensing, respectively.^[7]

The approach is expected to open the way for the development of multiplex biosensors, and also represents a significant advance in mimicking of biological electron transfer cascades.

In addition we also like to show that cyt c has been crystallized on electrodes and that the cyt c molecules in the crystals are in electrical contact with the electrode. Thereby we show for the very first time, that electron transfer within a cyt c-crystal and between the crystal and the electrode is feasible.^[8]

[1] I. Willner, E. Katz, Angew. Chem. Int. Ed. 2000, 39, 1180.

[2] T. Ikeda, K. Kano, J. Biosci. Bioeng. 2001, 92, 9.

[3] E. E. Ferapontova, T. Ruzgas, L. Stoica, A. Christenson, J. Tkac, A. I. Yaropolov, L. Gorton, Perspectives in Bioanalysis, Vol. 1, Elsevier, Dordrecht 2005, p. 517.

[4] J. F. Rusling, Acc. Chem. Res. 1998, 31, 363.

[5] S. C. Feifel, A. Kapp, R. Ludwig, L. Gorton, F. Lisdat, RCS Advances 2013, 3, 3428.

[6] S. C. Feifel, F. Lisdat, J. Nanobiotechnol. 2011, 9:59.

[7] S. C. Feifel, A. Kapp, R. Ludwig, F. Lisdat, Angew. Chem. Int. Ed. 2014, 53, 5676.

[8] Roise McGovern, S. C. Feifel, F. Lisdat, P. Crowley, Angew. Chem. Int. Ed. 2015, DOI: 10.1002/anie.201500191.