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Microchimica Acta

, 186:229 | Cite as

Electrochemical determination of the activity of DNA methyltransferase based on the methyl binding domain protein and a customized modular detector

  • Lingsong LuEmail author
  • Bei Liu
  • Jianhang Leng
  • Xiao Ma
Original Paper
  • 42 Downloads

Abstract

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 at pH 7.4 in the presence of 50 μM thionine and 0.5 mM 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 μA·μM−1·cm−2.

Graphical abstract

Schematic presentation of the electrochemical immunosensor for the determination of the activity of M. SssI CpG methyltransferase (M. SssI MTase). It is based on an electromagnetic modular detector and the use of glutathione S-transferase tagged methyl CpG binding protein 2 (GST-MeCP2).

Keywords

Graphene oxide Screen printed electrode Glutathione S-transferase Thionine restriction endonuclease 

Notes

Acknowledgements

This work was financially supported by the Public Welfare from Science and Technology Department of Zhejiang Province (No. 2017C33201), National Natural Science Foundation of China (No. 81301514), Medical Health Science and Technology Project of Zhejiang Provincial Health Commission (No. 2017PY024) and Medical Health Science and Technology Project of Zhejiang Provincial Health Commission (No. 2018PY022).

Compliance with ethical standards

The author(s) declare that they have no competing interests.

Supplementary material

604_2019_3309_MOESM1_ESM.doc (338 kb)
ESM 1 (DOC 338 kb)

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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Central Laboratory, Affiliated Hangzhou First People’s HospitalZhejiang University School of MedicineZhejiang ProvincePeople’s Republic of China
  2. 2.Department of Reproductive Genetics, Women’s HospitalZhejiang University School of MedicineZhejiang ProvincePeople’s Republic of China

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