Cyclic voltammetric detection of chemical DNA damage induced by styrene oxide in natural dsDNA layer-by-layer films using methylene blue as electroactive probe

In this work, an electrochemical sensor for detecting damage of natural double-stranded DNA (dsDNA) was constructed based on PSS/{PDDA/dsDNA}(3) layer-by-layer films, where PSS was poly(styrene sulfonate) and PDDA was poly(diallyldimethyl ammonium). When the PSS/{PDDA/dsDNA}(3) films assembled on pyrolytic graphite (PG) electrodes were immersed into methylene blue (MB) solution and loaded MB into the films, the formed PSS/{PDDA/dsDNA}(3)-MB films in blank buffers at pH 7.0 showed a reversible cyclic voltammetric (CV) peak pair at -0.23 V vs. SCE for MB redox couple. In blank solutions, the MB loaded into the films was released gradually from the films, but the complete reloading of MB into the films could be realized by immersing the films into MB solution again, indicating the good reversibility of MB incorporation. However, after incubation in the solution of known genotoxic agent styrene oxide (SO), the damaged PSS/(PDDA/dsDNA)3-MB films could not return to their original and fully-loaded state with reloading of MB, and showed smaller CV peak currents than those of intact PSS/{PDDA/dsDNA}(3)-MB films. The relative peak current ratio, I-p,I-I/I-p,I-III, where I-pI was the anodic peak current of intact DNA films in blank buffers after fully loading with MB and I-p,I-III was that of SO-damaged DNA films after reloading with MB, increased linearly with the incubation time with SO in the range of 5-40 min with a damage rate of 0.0099 min(-1). While the formation of SO adducts with dsDNA had no substantial effect on the dsDNA conformation, the steric hindrance of SO adducts with guanine or adenine blocked the intercalation of MB into the base pairs of dsDNA, resulting in the decrease of CV peak currents of loaded MB. The specific intercalation of MB into dsDNA base pairs and the sensitive electrochemical response of MB, combined with the unique feature of loading reversibility of MB in the DNA layer-by-layer films, make the difference in CV response between the intact and damaged dsDNA films become pronounced in the "loading/release/reloading" procedure. This may provide a new and general approach for constructing a biosensor for screening genetoxic chemicals in vitro. (c) 2006 Elsevier B.V. All rights reserved.