Tracking Globally 5-Methylcytosine and Its Oxidized Derivatives in Colorectal Cancer Epigenome Using Bioelectroanalytical Technologies

This work presents the first electroanalytical bioplatforms to track individually or simultaneously at a global level all four methylation marks involved in the DNA methylation-demethylation cycle: 5-methylcytosine (5mC) and their sequential oxidative derivatives (5-hydroxymethyl-(5hmC), 5-formyl-(5fC), and 5-carboxyl-(5caC) cytosines). The bioplatforms employed direct competitive immunoassay formats implemented on the surface of magnetic microparticles (MBs) and involved capture antibodies specific to each epimark as well as synthetic biotinylated DNA oligomers with a single epimark that were enzymatically marked with horseradish peroxidase (HRP) to perform an amperometric readout on disposable platforms for single or multiplexed detection. These new electroanalytical biotechnologies, groundbreaking from analytical and clinical perspectives, achieved attractive operational characteristics, reaching detection limits at pM levels for synthetic single epimark-bearing DNA oligomers. The developed methodology was applied to track globally all four target epimarks in a fast, simple, sensitive, and selective way while their correlation in genomic DNA extracted from paired healthy and tumor tissues of patients with colorectal cancer (CRC) was established for the first time.