期刊
MICROCHIMICA ACTA
卷 186, 期 4, 页码 -出版社
SPRINGER WIEN
DOI: 10.1007/s00604-019-3309-9
关键词
Graphene oxide; Screen printed electrode; Glutathione S-transferase; Thionine restriction endonuclease
资金
- Public Welfare from Science and Technology Department of Zhejiang Province [2017C33201]
- National Natural Science Foundation of China [81301514]
- Medical Health Science and Technology Project of Zhejiang Provincial Health Commission [2018PY022]
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. SssI CpG methyltransferase (M. SssI 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 HpaII restriction endonuclease. After magnetic separation and washing, HRP-labeled GST tag monoclonal antibody and H2O2 were used as a tracer label and enzyme substrate, respectively. Electrochemical measurement was carried out atpH7.4in the presence of 50M thionine and 0.5mM H2O2. 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. SssI MTase can be determined in the activity range of 0.5-125 unit mL (-1) with a detection limit of 0.2 unit mL (-1) (at an S/N ratio of 3). The sensitivity of the immunoassay is 0.489 mu A.mu M-1.cm(.)(-2)
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