Versatile bioelectronic interfaces based on heterotrifunctional linking molecules

Bioelectronic interfaces that allow dehydrogenase enzymes to communicate with electrodes have potential applications such as biosensors and biocatalytic reactors. A major challenge in creation of such bioelectronic interfaces is to orient the enzyme, its cofactor, and an electron mediator properly with respect to the electrode in order to achieve efficient, multistep electron transfer. This paper describes a versatile, new method that uses cysteine, an inexpensive, branched amino acid having sulfhydryl, amino, and carboxyl functional groups, to achieve such orientation. This approach provides greater flexibility in assembling complex bioelectronic interfaces than previously reported approaches that bind the enzyme, cofactor, and mediator in a linear chain. Cysteine was attached to a gold electrode through the sulfhydryl groups, to the electron mediator toluidine blue O (TBO) through the carboxyl group, and to the cofactor (e.g., NAD(P)+) through the amino group. Cyclic voltammetry, impedance spectroscopy, chronoamperometry and quartz crystal microbalance gravimetry were used to demonstrate the sequential assembly steps and the electrical activity of the resulting bioelectronic interface.