Electrochemical Determination of the Activity of DNA Methyltransferase Based on the Methyl Binding Domain Protein and a Customized Modular Detector

Overview

Journal Mikrochim Acta

Specialties Biotechnology
Chemistry

Date 2019 Mar 9

PMID 30848391

Citations 3

Authors

Lingsong Lu

Bei Liu

Jianhang Leng

Xiao Ma

Affiliations

Soon will be listed here.

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 HO 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 HO. 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 with a detection limit of 0.2 unit·mL (at an S/N ratio of 3). The sensitivity of the immunoassay is 0.489 μA·μM·cm 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).

Citing Articles

HpaII-assisted and linear amplification-enhanced isothermal exponential amplification fluorescent strategy for rapid and sensitive detection of DNA methyltransferase activity.

An Y, Yu Z, Liu D, Han L, Zhang X, Xin X Anal Bioanal Chem. 2023; 415(12):2271-2280.

PMID: 36961574 DOI: 10.1007/s00216-023-04647-1.

Overview on the Design of Magnetically Assisted Electrochemical Biosensors.

Chang Y, Wang Y, Zhang J, Xing Y, Li G, Deng D Biosensors (Basel). 2022; 12(11).

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Fluorescence determination of the activity of O-methylguanine-DNA methyltransferase based on the activation of restriction endonuclease and the use of graphene oxide.

Le D, Jiang J Mikrochim Acta. 2020; 187(5):300.

PMID: 32347374 DOI: 10.1007/s00604-020-04280-0.

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