Immobilization of Enzyme Cascades with DNA Scaffolds

Biofuel cells (BFCs) have been gaining popularity as a means of harvesting energy from renewable fuel sources. Even though a wide variety of BFC types have been developed, enzymatic biofuel cells (EBFCs) that employ enzymes as biocatalysts have become appealing technologies due to their ability to convert chemical energy stored in organic substrates into electrical energy with high turnover rates and easy control over the system. However, the commercial feasibility of EBFCs has been hampered by the poor energy density caused by the partial oxidation of fuels. The utilization of multi-enzyme cascades to perform sequential oxidation of fuels is an attractive approach to enhance the energy density of EBFCs. Nevertheless, the mass transport of intermediates between enzymes is a limiting factor of these enzyme systems. DNA scaffolds offer a suitable approach to partly overcome this obstacle as they allow for the precise control of enzyme arrangements, which facilitates the timely interaction of reaction intermediates and enzymes in close proximity. In this chapter, we describe protocols for the assembly of an invertase (Inv)/glucose oxidase (GOx) enzyme cascade on a DNA scaffold and the preparation of a bioanode using the assembled enzyme-DNA complex for improved bioelectrocatalysis.