ALBERT

Description: 〈h3〉Abstract〈/h3〉 〈p〉An electrochemical method is described for the determination of the activity of the DNA methyltransferase (MTase). The assay was based on the use of a commercially available customized electromagnetic modular detector, which consisted of a magnetic switch, electrical connectors and a screen-printed electrode modified with graphene oxide. The biotinylated single-strand DNA (ss-DNA) S1 was absorbed by streptavidin-modified magnetic beads (MBs) via streptavidin-biotin interaction. The biotinylated ss-DNA S1 was hybridized with the complementary ss-DNA S2. After the symmetrical sequences 5′-CCGG-3′ of the duplex DNA (ds-DNA) were methylated by M. 〈em〉Sss〈/em〉I CpG methyltransferase (M. 〈em〉Sss〈/em〉I MTase), the symmetrical sequences 5′-CCGG-3′ in the ds-DNA were recognized by glutathione S-transferase (GST) tagged methyl CpG binding protein 2 (MeCP2). The unmethylated 5′-CCGG-3′ sequences were specifically cleaved by 〈em〉Hpa〈/em〉II restriction endonuclease. After magnetic separation and washing, HRP-labeled GST tag monoclonal antibody and H〈sub〉2〈/sub〉O〈sub〉2〈/sub〉 were used as a tracer label and enzyme substrate, respectively. Electrochemical measurement was carried out at pH 7.4 in the presence of 50 μM thionine and 0.5 mM H〈sub〉2〈/sub〉O〈sub〉2〈/sub〉. Stepwise changes in the microscopic features of the SPE surface upon the formation of each layer were studied by scanning electron microscopy. Cyclic voltammetry and differential pulse voltammetry were used to characterize the electrochemical behavior of the different modified electrodes. Under the optimal conditions, the activity of M. 〈em〉Sss〈/em〉I MTase can be determined in the activity range of 0.5–125 unit〈strong〉·〈/strong〉mL〈sup〉−1〈/sup〉 with a detection limit of 0.2 unit〈strong〉·〈/strong〉mL〈sup〉−1〈/sup〉 (at an S/N ratio of 3). The sensitivity of the immunoassay is 0.489 μA·μM〈sup〉−1〈/sup〉·cm〈sup〉−2〈/sup〉〈sub〉.〈/sub〉〈/p〉 〈p〉 〈span〉 〈caption〉 〈strong〉Graphical abstract〈/strong〉 〈em〉 〈div〉Schematic presentation of the electrochemical immunosensor for the determination of the activity of M. 〈em〉Sss〈/em〉I CpG methyltransferase (M. 〈em〉Sss〈/em〉I MTase). It is based on an electromagnetic modular detector and the use of glutathione S-transferase tagged methyl CpG binding protein 2 (GST-MeCP2).〈/div〉 〈/em〉 〈/caption〉 〈span〉 〈img alt="" src="https://static-content.springer.com/image/MediaObjects/604_2019_3309_Figa_HTML.png"〉 〈/span〉 〈/span〉 〈/p〉