Toward more efficient bioelectrocatalytic oxidation of ethanol for amperometric sensing and biofuel cell technology

The integrated, structured, and multifunctional bioelectrocatalytic system for effective oxidation of ethanol is developed here. The concept is based on the layer-by-layer (LbL) assembly through electrostatic attraction of positively charged, multiwalled carbon nanotubes and the controlled combination of dehydrogenase enzymes. More specifically, the LbL technique was employed for sequential immobilization of two dehydrogenase enzymes and poly(diallyldimethylammonium chloride)-covered multiwalled carbon nanotubes onto a glassy carbon electrode substrate. Both monoenzymatic [utilizing a single enzyme, alcohol dehydrogenase (ADH)] and bienzymatic (anchoring sequentially both ADH and aldehyde dehydrogenase) systems were tested. Multilayers were characterized using scanning electron microscopy, infrared spectroscopy, and cyclic voltammetry. The results are consistent with the view that our approach enables good control of distribution and efficient utilization of both enzymes within the biocomposite film and leads to sizable enhancement of the oxidation of ethanol through significant (more than 2-fold) increase of bioelectrocatalytic currents and by shifting the ethanol oxidation potential to 0.1 V (vs Ag/AgCl) or decreasing the overvoltage by ca. 200 mV in comparison with the monoenzymatic electrode system. This simple biocomposite (enzyme-cascade) system permits fabrication of highly sensitive ethanol biosensors based on nicotinamide adenine dinucleotide coenzyme-dependent dehydrogenases. Our ethanol biosensor exhibited a good linearity ranging from 50 to 300 μM, and it was characterized by a high sensitivity of 118.8 μA mM(-1) cm(-2) as well as a low detection limit of 24 μM.