This work reports the use of modified reduced graphene oxide (rGO) as a platform for a label-free DNA-based electrochemical biosensor as a possible diagnostic tool for a DNA methylation assay. The biosensor sensitivity was enhanced by variously modified rGO. The rGO decorated with three nanoparticles (NPs)-gold (AuNPs), silver (AgNPs), and copper (CuNPs)-was implemented to increase the electrode surface area. Subsequently, the thiolated DNA probe (single-stranded DNA, ssDNA-1) was hybridized with the target DNA sequence (ssDNA-2). After the hybridization, the double-stranded DNA (dsDNA) was methylated by M.SssI methyltransferase (MTase) and then digested via a HpaII endonuclease specific site sequence of CpG (5'-CCGG-3') islands. For monitoring the MTase activity, differential pulse voltammetry (DPV) was used, whereas the best results were obtained by rGO-AuNPs. This assay is rapid, cost-effective, sensitive, selective, highly specific, and displays a low limit of detection (LOD) of 0.06 U·mL-1. Lastly, this study was enriched with the real serum sample, where a 0.19 U·mL-1 LOD was achieved. Moreover, the developed biosensor offers excellent potential in future applications in clinical diagnostics, as this approach can be used in the design of other biosensors.
Keywords: DNA methylation; biomedical applications; electrochemical biosensor; nanomaterials; rGO.