Electrochemical detection of global dna methylation using biologically assembled polymer beads

Narshone Soda, Zennia Jean Gonzaga, Amandeep Singh Pannu, Navid Kashaninejad, Richard Kline, Carlos Salomon, Nam Trung Nguyen, Prashant Sonar, Bernd H.A. Rehm, Muhammad J.A. Shiddiky

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DNA methylation is a cell‐type‐specific epigenetic marker that is essential for transcriptional regulation, silencing of repetitive DNA and genomic imprinting. It is also responsible for the pathogenesis of many diseases, including cancers. Herein, we present a simple approach for quan-tifying global DNA methylation in ovarian cancer patient plasma samples based on a new class of biopolymer nanobeads. Our approach utilises the immune capture of target DNA and electrochemical quantification of global DNA methylation level within the targets in a three‐step strategy that involves (i) initial preparation of target single‐stranded DNA (ss‐DNA) from the plasma of the pa-tients’ samples, (ii) direct adsorption of polymer nanobeads on the surface of a bare screen‐printed gold electrode (SPE‐Au) followed by the immobilisation of 5‐methylcytosine (5mC)‐horseradish pe-roxidase (HRP) antibody, and (iii) immune capture of target ss‐DNA onto the electrode‐bound PHB/5mC‐HRP antibody conjugates and their subsequent qualification using the hydrogen perox-ide/horseradish peroxidase/hydroquinone (H2O2/HRP/HQ) redox cycling system. In the presence of methylated DNA, the enzymatically produced (in situ) metabolites, i.e., benzoquinone (BQ), binds irreversibly to cellular DNA resulting in the unstable formation of DNA adducts and induced oxidative DNA strand breakage. These events reduce the available BQ in the system to support the redox cycling process and sequel DNA saturation on the platform, subsequently causing high Cou-lombic repulsion between BQ and negatively charged nucleotide strands. Thus, the increase in methylation levels on the electrode surface is inversely proportional to the current response. The method could successfully detect as low as 5% methylation level. In addition, the assay showed good reproducibility (% RSD ≤ 5%) and specificity by analysing various levels of methylation in cell lines and plasma DNA samples from patients with ovarian cancer. We envision that our bioengi-neered polymer nanobeads with high surface modification versatility could be a useful alternative platform for the electrochemical detection of varying molecular biomarkers.

Original languageEnglish
Article number3787
Number of pages15
Issue number15
Publication statusPublished - 01 Aug 2021


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