Label-Free DNA Biosensor using Modified Reduced Graphene Oxide Platform as a DNA Methylation Assay

This work reports using modified reduced graphene oxide (rGO) as a platform for label-free DNA-based electrochemical biosensor as a possible diagnostic tool for DNA methylation assay. The biosensor sensitivity wasenhanced by variously modified rGO. The rGO decorated with three nanoparticles (NPs)—gold (AuNPs), silver (AgNPs), and copper (CuNPs) were implemented to increase the electrode surface area. Subsequently, the thiolated DNA probe (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 HpaII endonuclease specific site sequence of CpG islands 5’-CCGG-3’. For the monitoring of the MTase activity was used differential pulse voltammetry (DPV), whereas the best results were obtained by rGO-AuNPs. This assay is rapid, cost-effective, sensitive, selective, highly specific, and displays a low detection limit (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.