Electrochemical detection of natural DNA damage induced by in situ peroxidase-generated reactive nitrogen species in DNA layer-by-layer films.

The NO2• radical is one kind of reactive nitrogen species and can be generated by horseradish peroxidase (HRP)-catalyzed oxidation of nitrite (NO2−) in the presence of hydrogen peroxide (H2O2) and may cause DNA damage in living system. In the present work, {PDDA/DNA}4 layer-by-layer (LBL) films assembled with poly(diallyldimethylammonium chloride) (PDDA) and natural double-stranded DNA on electrodes were used to detect DNA damage induced by NO2• radicals generated from the HRP+H2O2+NO2− incubation system using cyclic voltammetry with Ru(bpy)32+ as the electroactive catalyst in solution. HRP and DNA were then further assembled into PDDA/DNA/{HRP/DNA}2 LBL films, and the damage of DNA in the films caused by the NO2• radicals produced in situ by incubation of the films with H2O2+NO2− solutions was detected electrochemically. When catalase layers were assembled on the surface of PDDA/DNA/{HRP/DNA}2 films, the DNA damage induced by the H2O2+NO2− system was inhibited since the catalase in the films could effectively decompose H2O2, and the NO2• radicals could not be generated. This work not only provides a foundation for fabricating electrochemical biosensors in detecting NO2•-induced DNA damage, but also offers an in vitro model to simulate the pathway of DNA damage and protection in living process.