The effective regeneration of nicotinamide adenine dinucleotide, possibly phosphorylated, (NAD(P)H/NAD(P)+) is of great interest, because NAD(P)H/NAD(P)+ acts as an essential cofactor in ubiquitous redox enzymes that are widely applicable in biosensors, bioenergy and bioconversion. However, NAD(P)H oxidation occurs at very high overpotentials, leading to significant energy inefficiencies and increasing the possibility of side reactions. To address these challenges, in this work, we have designed and engineered nanostructured interfaces for high-performance electrochemical cofactor regeneration applicable to bioelectrocatalysis. A high-rate NADH oxidizing electrode was fabricated by incorporating poly(azine) electrocatalysts into a high surface area layer of carboxylated carbon nanotubes (CNTs). Electrodeposition of poly(methylene green) (PMG) and poly(toluidine blue) (PTBO) on the carboxylated CNT-modified electrodes was achieved by cyclic voltammetry. The PMG-CNT interface demonstrates 5.0 mA cm-2 current density for NADH oxidation at 50 mV vs. Ag|AgCl in 20 mM NADH solution.The bioactivity of cofactor electrogenerated NAD+ was verified spectroscopically. A mathematical model calibrated by measurements of NADH oxidation at PMG-CNT-modified glassy carbon electrodes was applied to predict transient NADH consumption. The model showed good agreement with the experimental data, and 80% conversion of NADH was observed after 1 hour of electrochemical oxidation. Using a spectroscopic enzyme cycling assay, the yield of enzymatically active NAD+ was verified at 93% and 87% for applied potentials of 500 mV and 150 mV vs. Ag|AgCl, respectively. To further facilitate NADH electrocatalysis, electrochemical activation of carbon material was found to increase electrode reactivity by introducing carbon-oxygen functionalities. Electrochemically activated carbon electrodes demonstrate enhanced activity toward NADH oxidation, and more importantly, dramatically improved adsorption of bioelectrochemically active azine dyes. Adsorption of methylene green (MG) on an electroactivated carbon electrode yielded a catalyst layer that is 1.8-fold more active toward NADH oxidation than an electrode prepared using electropolymerized MG.A quantitative model was developed to explain and predict experimental phenomena and design bioelectronic interfaces. The kinetics of bioelectrocatalysis can be simplified into two steps: the electrocatalytic regeneration of cofactor NADH/NAD+ and the enzymatic reaction of substrate using dehydrogenase. Planar and porous interface structures are modeled to evaluate the kinetics and diffusion for the bioelectronic interface. Model-based analysis indicates that, bioreactor for dihydroxyacetone production from glycerol is controlled by glycerol dehydrogenase kinetics for enzyme loadings lower than 1 mM and shifted to mass transport and incipient NADH oxidation kinetics control at higher enzyme loadings.This dissertation presents various fabrication, characterization and modeling methods for cofactor regeneration. The findings and designs in this work are highly applicable to dehydrogenase-based economical bioelectrocatalysis.
Read
