Abstract

We report a novel bioelectrode architecture for glucose detection, integrating multi-walled carbon nanotube buckypaper (BP), glucose dehydrogenase (GDH), and covalently immobilized NAD+. The BP was first functionalized with nordihydroguaiaretic acid (NDGA), a naturally occurring quinone, to introduce redox-active anchoring sites. Subsequently, an electrochemical Michael addition of 3-aminophenylboronic acid (3-APBA), pre-complexed with NAD+, enabled the covalent attachment of NAD+ to the surface. This molecular design yielded a robust enzyme electrode operating without exogenous NAD+, thereby eliminating the need for cofactor supplementation during measurements. The resulting sensor demonstrated efficient glucose detection in the 0.1-10 mM range, with a limit of detection (LOD) of 22 μM, and exhibited reliable performance in human serum. This strategy offers a versatile and scalable route for the development of reagentless NAD+-dependent biosensing platforms for glucose determination.