Electrochemical Detection of Global DNA Methylation Using Biologically Assembled Polymer Beads

Abstract

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.

Recent advances in liquid biopsy technologies for cancer biomarker detection

“…Recently, we designed a versatile technology platform based on bioengineered polymer nanobeads for efficient capture and electrochemical detection of DNA methylation in ovarian cancer patient samples. 227 Engineered polyhydroxybutyrate (PHB) nanobeads were utilized as electrode surface modifiers that facilitated the adsorption of 5 mC antibody (specific antibody for methylated DNA). This method could detect methylation changes as low as 5% in ovarian cancer cell lines and plasma DNA samples.…”

Toll like receptor-based electrochemical sensors via N-heterocyclic carbene-modified surfaces: towards improved sensing of DNA molecules

“…27,28 Although effective, the use of an NHC with a maleimide end group for immobilization of the TLR protein moiety through Michael addition reaction required the use of thiol nucleophiles that provided a point of potential instability due to reversible reactions. 27,29 Despite the current precedents, such as DNA hybridization, [30][31][32] nanobeads, 33 or a reverse method of using DNA platform to detect proteins, 34 there has not yet been a reported example of electrochemical biosensors using organic ligand SAM technology along with TLRs to detect DNA molecules.…”

Correction: Soda et al. Electrochemical Detection of Global DNA Methylation Using Biologically Assembled Polymer Beads. Cancers 2021, 13, 3787

“…In the original publication [1], reference [55] was not cited. The citation has now been inserted in 3.…”