Voltammetric Microwell Array for Oxidized Guanosine in Intact ds-DNA

Abstract

Oxidative stress in humans causes damage to biomolecules by generating reactive oxygen species (ROS). DNA can be oxidatively damaged by ROS, which may lead to carcinogenesis. Here we report a microfluidic electrochemical array designed to rapidly detect oxidation in intact DNA in replicate measurements. Sensor arrays were fabricated by wet-chemistry patterning of gold compact discs. The 8-sensor array is incorporated into a 60 µL microfluidic channel connected to a pump and sample valve. The array features 7 nm thick osmium bipyridyl poly(vinylpyridine) chloride [Os(bpy)2(PVP)10Cl]+ films assembled layer-by-layer with polyions onto the gold sensors. 7,8-dihydro-8-oxoguanine (8-oxodG) in ds-DNA is selectively oxidized by [Os(bpy)2(PVP)10Cl]+ in intact ds-DNA to provide catalytic square wave voltammograms (SWV). The device is easy-to-use, fast, inexpensive, reusable and can detect one 8-oxodG per 6600 nucleobases. The mass detection limit is 150-fold lower than a previously reported dip-and-read voltammetric sensor for oxidized DNA. Fast assays (<1 min) and moderate sample consumption (15 pmol DNA) suggest potential for research and clinical applications. Practical use is illustrated by detecting DNA oxidation from cigarette smoke and ash extracts in dispersions with NADPH and Cu2+.

Direct LC-MS/MS Detection of Guanine Oxidations in Exon 7 of the p53 Tumor Suppressor Gene

“…The active oxidant in Fenton’s reagent is thought to be • OH, 60,61 and oxidation products of 8-oxodG have also been identified. 9,62,63 We found none of these overoxidation products under our conditions.…”

Electrochemiluminescent Array to Detect Oxidative Damage in ds-DNA Using [Os(bpy)2(phen-benz-COOH)]2+/Nafion/Graphene Films

“…DNA used is significantly less than that in our microfluidic voltammetric array that required 2.5 µg. 22 …”

“…Hydrolysis of oxidized DNA was done with DNase I (400 unit mg −1 of DNA) for 3 h, followed by addition of phosphodiesterase I (0.2 unit mg −1 of DNA) and alkaline phosphatase (1.2 unit mg −1 of DNA). 22 DNA samples were then incubated overnight in a rotor at 37 °C, then vacuum filtered using a 96-well filtration plate (3 kDa MW cutoff), and analyzed via UPLC-MS/MS. A Hypersil Gold C18 (100 mm × 0.3 mm i.d., 3 µm) reversed phase column was used.…”

Microfluidic array for simultaneous detection of DNA oxidation and DNA-adduct damage

“…18,19 Herein we report a new array that employs thin films of metabolic enzymes, DNA and catalytic Ru and Os metallopolymers to measure metabolite-related DNA damage from both adduct formation and oxidation simultaneously.…”