NAD is a cofactor that maintains cellular redox homeostasis and has immense industrial and biological significance. It acts as an enzymatic mediator in several biocatalytic electrochemical reactions and undergoes oxidation/reduction to form NAD + or NADH, respectively. The NAD redox couple (NAD + /NADH) mostly exists in enzyme-assisted metabolic reactions as a coenzyme during which electrons and protons are transferred. NADH shuttles these charges between the enzyme and the substrate. In order to understand such complex metabolic reactions, it is vital to study the bio-electrochemistry of NADH. In addition, the regeneration of NADH in industries has attracted significant attention due to its vast usage and high cost. To make biocatalysis economically viable, primary methods of NADH regeneration including enzymatic, chemical, photochemical and electrochemical methods are widely used. This review is mainly focused on the electrochemical reduction of NAD + to NADH with specific details on the mechanism and kinetics of the reaction. It provides emphasis on the different routes (direct and mediated) to electrochemically regenerate NADH from NAD + highlighting the NAD dimer formation. Also, it describes the electrocatalysts developed until now and the scope for development in this area of research.
In this study, the authors report a simple fabrication of thermoplastic polyurethane (TPU) nanofibres-based kit for cholesterol detection. TPU is a polymer that is highly elastic, resistant to microorganisms, abrasion and compatible with blood; thus, making it a natural selection as an immobilisation matrix for cholesterol oxidase (ChOx) enzyme. The nanofibre was fabricated by electrospinning process and was characterised using scanning electron microscopy and Fourier transform-infrared spectroscopy. ChOx was covalently immobilised on TPU nanofibre and cholesterol level/concentration was visually found using 4-aminoantipyrine, a dye that reacts with HO produced from the oxidation of cholesterol by ChOx and changes colour from yellow to red. The efficacy of the nanofibre to act as a detecting substrate was compared with cellulose acetate (CA) membrane, a well-documented enzyme immobilisation matrix. The optimisation of enzyme concentration and dye quantity were performed using standard ChOx spectrophotometric assay and the same was used in CA membrane and TPU nanofibre. The ChOx immobilised nanofibre showed good linear range from 2 to 10 mM with a lower detection limit of 2 mM and was highly stable compared to that of CA membrane. The enzyme immobilised nanofibre was further validated in serum samples.
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