Mimicking enzymatic effects of cytochrome P450 by an efficient biosensor for in vitro detection of DNA damage

Abstract

A novel biosensor for detecting DNA damage induced by benzo(a)pyrene (BP) and its metabolite was presented 18 in this work. The nafion-solubilized single wall carbon nanotubes-ionic liquid (SWCNTs-NA-IL) composite film 19 was prepared and then horseradish peroxidase (HRP) and double-stranded DNA were alternately assembled on 20 the composite film by the layer-by-layer method. The biosensor was characterized by cyclic voltammetry (CV), 21 electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV), scanning electron 22 microscopy (SEM) and computational methods. UVvis spectrophotometry was also used to investigate DNA 23 damage induced by BP and its metabolites in solution. The DNA biosensor was treated separately in BP, 24 hydrogen peroxide (H2O2) and in their mixture, respectively. The EIS analysis showed a decrease in the charge 25 transfer resistance at the DNA/HRP/SWCNTs-NA-IL/GCE incubated in a mixture of HRP and H2O2, because 26 HRP in the presence of H2O2 could mimic enzymatic effects of cytochrome P450 (CYP450) to metabolize BP 27 which could cause significant DNA damage and the exposed DNA bases reduced the electrostatic repulsion of the 28 negatively charged redox probe and leads to Faradaic impedance changes. Finally, a novel biosensor for BP 29 determination was developed and this method provided an indirect, and quantitative estimate of DNA damage in 30 vitro.