Evaluating Metabolite-Related DNA Oxidation and Adduct Damage from Aryl Amines Using a Microfluidic ECL Array

Abstract

Damage to DNA from the metabolites of drugs and pollutants constitutes a major human toxicity pathway known as genotoxicity. Metabolites can react with metal ions and NADPH to oxidize DNA or participate in SN2 reactions to form covalently linked adducts with DNA bases. Guanines are the main DNA oxidation sites, and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) is the initial product. Here we describe a novel electrochemiluminescent (ECL) microwell array that produces metabolites from test compounds and measures relative rates of DNA oxidation and DNA adduct damage. In this new array, films of DNA, metabolic enzymes, and an ECL metallopolymer or complex assembled in microwells on a pyrolytic graphite wafer are housed in dual microfluidic chambers. As reactant solution passes over the wells, metabolites form and can react with DNA in the films to form DNA adducts. These adducts are detected by ECL from a RuPVP polymer that uses DNA as a coreactant. Aryl amines also combine with Cu2+ and NADPH to form reactive oxygen species (ROS) that oxidize DNA. The resulting 8-oxodG was detected selectively by ECL-generating bis(2,2′-bipyridine)-(4-(1,10-phenanthrolin-6-yl)-benzoic acid)Os(II). DNA/enzyme films on magnetic beads were oxidized similarly, and 8-oxodG determined by LC/MS/MS enabled array standardization. The array limit of detection for oxidation was 720 8-oxodG per 106 nucleobases. For a series of aryl amines, metabolite-generated DNA oxidation and adduct formation turnover rates from the array correlated very well with rodent 1/TD50 and Comet assay results.

Organ-Specific Screening for Protein Damage Using Magnetic Bead Bioreactors and LC–MS/MS

“…In our previous work using a similar methodology to form metabolites from test chemicals using CYPs and microsomes, we found that rates of DNA adduction and oxidation correlated very well with Comet DNA damage assays and rodent TD 50 genotoxicity assays, as did the reactivities of CYPs and microsomes. − In the present work, we are only substituting proteins for DNA, so we expect the reactivities of CYPs and microsomes to also correlate with natural systems. We previously showed that CYPs and supersomes provide the same or better activity and the same selectivity when on magnetic beads compared to dispersed in solutions. − …”

Confined Electrochemiluminescence Generation at Ultra-High-Density Gold Microwell Electrodes

“…Electrochemiluminescence (ECL) is light emission from excited state luminophores generated by electrochemical reactions, which possesses better spatiotemporal control in comparison with chemiluminescence ( Richter, 2004 ; Hu and Xu, 2010 ; Hesari and Ding, 2017 ; Liu et al, 2019 ). Because no external light illumination is required to generate ECL emission, ECL has unique advantages, such as low background and high sensitivity, et al It has manifested itself to be a powerful transduction signal for the determination of small biomolecules, DNA and protein biomarkers in biosensing and clinical diagnostics ( Doeven et al, 2014 ; Wu et al, 2014 ; Lim et al, 2016 ; Bist et al, 2017 ; Ji et al, 2017 ; Yang et al, 2018 ; Zhou et al, 2018 ; Zhang et al, 2019a ; Jones et al, 2020 ). Moreover, ECL has recently emerged as a useful surface analysis technology because of its intrinsic surface-confined and surface-sensitive nature ( Guo et al, 2017 ; Zhang et al, 2019b ; Zanut et al, 2019 ; Ma et al, 2020 ).…”

Dye‐Doped Silica Nanoparticles for Enhanced ECL‐Based Immunoassay Analytical Performance

“…Indeed, ECL has been used for immunoassay and ultrasensitive detection of a wide range of analytes in different fields like medical diagnostics, environmental analysis, and (bio)sensors fabrication [4–8] . The subsequent implementation of ECL principles in microscopy allowed new frontiers and new applications in particular for multiplexing analysis, [9, 10, 11] making the investigation of the ECL mechanisms possible at nanoscale level, especially for sensor application and for biological characterizations. For these reasons, ECL microscopy is a very promising technique for the surface‐confined mapping and quantification of several extremely diluted analytes [12–19] …”