Abstract
Simple SummaryGenomic profiling of cancer-derived materials in circulation has become an alternative approach for tumour genotyping. The detection of tumour origin markers such as DNA methylation in bodily fluids enables cancer screening, early-stage diagnosis and evaluation of therapy response. The development of broad platform technologies that underpin many in vitro clinical diagnostic tests has brought about a paradigm shift in cancer management and diagnosis. This study developed a multifaceted technology platform based on bioengineered polymer nanobeads for efficient capture and electrochemical detection of DNA methylation in ovarian cancer patient samples. This could be a versatile diagnostic platform for detecting numerous disease biomarkers, thus allowing several disease diagnoses.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 quantifying 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 patients’ 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 peroxidase (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 peroxide/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 Coulombic 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 bioengineered polymer nanobeads with high surface modification versatility could be a useful alternative platform for the electrochemical detection of varying molecular biomarkers.
Highlights
Epigenetic regulation of gene expression is expedited by mechanisms such as DNA methylation, histone modification and nucleosome positioning along the DNA [1]
The peroxidase activity of horseradish peroxidase (HRP) via the H2 O2 /HRP/HQ redox cycling system was used to achieve an electrochemical quantification of methylated DNA sequences present in the cell line and plasma solutions
The increased current response for Whole-genome amplification (WGA) could be due to the HRP saturation level on the electrode surface, which allows a huge amount of available HQ to participate in the redox cycling processes
Summary
Epigenetic regulation of gene expression is expedited by mechanisms such as DNA methylation, histone modification and nucleosome positioning along the DNA [1]. Regulating DNA clusters ensures proper maintenance of precise chromosome replication, gene expression, and stable gene silencing [3]. DNA methylation is one of the most commonly occurring epigenetic events in the mammalian genome that plays a critical role in normal cell physiology [4]. DNA methylation is essential for several cellular regulatory pathways such as X-chromosome inactivation, genomic imprinting, long term gene silencing and regulation of chromatin structure [5]. While appropriate DNA methylation is essential for normal biological processes, distinct and aberrant methylation patterns can result in many diseases, such as autoimmune diseases, diabetes, and cancer [2,6]. Aberrant DNA methylation patterns are a promising biomarker for early detection and assessment of future cancer risk owing to their early manifestation in carcinogenesis [7]
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