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 beta-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.