Label-Free DNA Biosensor Using Modified Reduced Graphene Oxide Platform as a DNA Methylation Assay
Eliska Sedlackova,
Zuzana Bytesnikova,
Eliska Birgusova,
Pavel Svec,
Amir M. Ashrafi,
Pedro Estrela,
Lukas Richtera
Affiliations
Eliska Sedlackova
Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1665/1, 613 00 Brno, Czech Republic
Zuzana Bytesnikova
Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1665/1, 613 00 Brno, Czech Republic
Eliska Birgusova
Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1665/1, 613 00 Brno, Czech Republic
Pavel Svec
Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1665/1, 613 00 Brno, Czech Republic
Amir M. Ashrafi
Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1665/1, 613 00 Brno, Czech Republic
Pedro Estrela
Centre for Biosensors, Bioelectronics and Biodevices (C3Bio) and Department of Electronic and Electrical Engineering, University of Bath, Bath BA2 7AY, UK
Lukas Richtera
Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1665/1, 613 00 Brno, Czech Republic
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.