Abstract
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.
Highlights
In the last decade, research has confirmed the crucial role of epigenetics in the origin of cancer, its progression, and treatment
The changes in the electrochemical features of the interfacial electrode surface due to the modifications were characterized via electrochemical impedance spectroscopy (EIS)
The methylation assay is based on the differential pulse voltammetry (DPV) response, which is correlated to the amount of the intercalated methylene blue (MB)
Summary
Research has confirmed the crucial role of epigenetics in the origin of cancer, its progression, and treatment. Epigenetics play an important role in genetic expressions, which are not affected by changes in DNA sequences. The most significant epigenetic mechanisms are DNA methylation, histone modification, and gene silencing related to RNA. DNA methylation’s essential functions in cells occur under physiological and pathological conditions [1]. DNA methylation is a heritable post-translational covalent modification of DNA catalyzed by DNA methyltransferase (MTase). The process of methylation includes DNA MTase as the carrier of the methyl (−CH3 ) group from S-adenosyl-methionine (SAM), which is a donor to the carbon in the 5th cytosine position in the dinucleotide field of CpG islands. Afterwards, MTase binds the methyl group to carbon in the cytosine’s
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