Triggering the redox reaction of cytochrome c on a biomimetic layer and elimination of interferences for NADH detection

Biomimetic layers triggering the redox process of cytochrome c (cyt c) by β-nicotinamide adenine dinucleotide (NADH) were fabricated and applied for the detection of NADH. A probe was constructed based on a conducting polymer (poly-5,2′:5′,2″-terthiophene-3′-carboxylic acid, poly-TTCA) formed on the Au nanoparticles, which were deposited on a screen-printed carbon electrode; the probe was modified with biomaterials including cyt c, lipids (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cardiolipin), and ubiquinone, which are involved in electron transfer sequence in the cell membrane. To eliminate affection of foreign biological species, we assembled a lipid bilayer using the Langmuir–Blodgett technique by controlling the density of the outward lipid layer. The characteristics of the biomimetic layers were investigated by cyclic voltammetry, impedance spectrometry, quartz crystal microbalance, and X-ray photoelectron spectroscopy. The electron transfer process triggered by the presence of NADH was used to determine the NADH concentration. l-ascorbic acid, uric acid, and NADPH did not show any affection during the detection of NADH at the density controlled-lipid bilayer. Real sample analysis was successfully performed to evaluate the reliability of the proposed biomimetic probe.