Wash-free electrochemical biosensor for CpG methylation using a quasi-direct electron transfer type enzyme as the...

We developed an electrochemical detection system for CpG methylation using an enzyme with the quasi-direct electron transfer (quasi-DET) ability for labeling. We directly fused the methyl-CpG binding domain (MBD), which binds to the oligonucleotide bearing methylated CpG, and glucose dehydrogenase (GDH), which was used for labeling, to construct MBD-GDH. MBD-GDH was directly modified with amine-reactive phenazine ethosulfate (arPES) as a mediator to add its quasi-direct electron transfer (quasi-DET) ability. When MBD bound to the methylated CpG oligonucleotide, which was immobilized on the electrode, MBD-GDH was brought into close proximity to the electrode. Upon adding glucose to the electrode, electrons from GDH and arPES were transferred to the electrode surface. The arPES-modified MBD-GDH showed the quasi-DET ability when the target DNA was immobilized on the electrode and it showed a linear response to the increase in methylation level in the range of 0-100 %. We succeeded in developing a wash-free electrochemical detection system using labeling enzymes with the quasi-DET ability.