Bioelectronic interfaces that facilitate electron transfer between the electrode and a dehydrogenase enzyme have potential applications in biosensors, biocatalytic reactors, and biological fuel cells. Secondary alcohol dehydrogenase (2° ADH) from Thermoanaerobacter ethanolicus is well suited for the development of such bioelectronic interfaces because of its thermostability and facile production and purification. The development of bioelectronic interfaces could lead to significant advances in biosensors, biocatalytic reactors, and biological fuel cells.

Development of a bioelectronic interface is especially challenging for cofactor-dependent dehydrogenase enzymes, whose activity requires the presence of an electron carrying cofactor. Direct electron transfer between the cofactor and the electrode is not kinetically favored, requiring the use of high overpotentials which may lead to cofactor degradation. These problems can be circumvented by using an electron mediator to shuttle electrons between the electrode and cofactor at moderate potentials. We capitalized upon the formation of an amide linkage to form a bioelectronic interface capable of electron transfer at moderate potentials while providing greater flexibility in the assembly of the bioelectronic interfaces. However, enzymes and cofactors have limited useful lifetimes, due to natural degradation processes. The abovementioned interface fabrication method involved the covalent linkage of the enzyme to the surface making no provision for removal and replacement of degraded components. However, for long-term operation, new interface-assembly methods must be developed that allow facile removal and replacement of the cofactor and enzyme.

We present the fabrication of a renewable bioelectronic interface in which poly(ethyleneimine) (PEI) was used to couple the cofactor and enzyme to the electrode. By decreasing the pH the surface-bound carboxylic acid group protonates disrupting the ionic bonds and releasing the enzyme and cofactor. After neutralization, fresh PEI, enzyme, and cofactor can be reassembled, allowing the interface to be reconstituted. Renewable bioelectronic interfaces were also fabricated on a glassy carbon electrode. To increase the performance parameters (saturation current and sensitivity) exfoliated graphite nanoplatelets were incorporated into the bioelectronic interface. However, these interfaces are limited in their reaction capacity, because it contains a single enzyme monolayer. A novel approach, in which multiple, nanostructured bioelectronic cassettes are stacked in series to yield multilayered bioelectronic interfaces having higher reaction capacities is also described. An approximate analytical solution for bioelectronic interfaces containing reversible enzymes and mediators; the general approach developed takes into account reversible enzyme kinetics, reversible mediator kinetics, substrate diffusion, product diffusion, and electron diffusion was also developed.