Electrochemical determination of the activity of DNA methyltransferase based on the methyl binding domain protein and a customized modular detector
- 42 Downloads
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.
KeywordsGraphene oxide Screen printed electrode Glutathione S-transferase Thionine restriction endonuclease
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.
- 4.Doerfler W (1983) DNA methylation and cene activity. Annu Rev Biochem 52:93–124. https://doi.org/10.1146/annurev.bi.52.070183.000521 CrossRefPubMedGoogle Scholar
- 8.Hou P (2003) Detection of methylation of human p16Ink4a gene 5’-CpG islands by electrochemical method coupled with linker-PCR. Nucleic Acids Res 31: 92e–992. https://doi.org/10.1093/nar/gng092
- 16.Gebhard C, Schwarzfischer L, Pham TH, Schilling E, Klug M, Andreesen R, Rehli M (2006) Genome-wide profiling of CpG methylation identifies novel targets of aberrant hypermethylation in myeloid leukemia. Cancer Res 66:6118–6128. https://doi.org/10.1158/0008-5472.CAN-06-0376 CrossRefPubMedGoogle Scholar
- 19.Yin H, Sun B, Zhou Y, Wang M, Xu Z, Fu Z, Ai S (2014) A new strategy for methylated DNA detection based on photoelectrochemical immunosensor using Bi2S3nanorods, methyl bonding domain protein and anti-his tag antibody. Biosens Bioelectron 51:103–108. https://doi.org/10.1016/j.bios.2013.07.040 CrossRefPubMedGoogle Scholar
- 20.Xu Z, Yin H, Huo L, Zhou Y, Ai S (2014) Electrochemical immunosensor for DNA methyltransferase activity assay based on methyl CpG-binding protein and dual gold nanoparticle conjugate-based signal amplification. Sensors Actuators B Chem 192:143–149. https://doi.org/10.1016/j.snb.2013.10.099 CrossRefGoogle Scholar
- 21.Lu L, Liu B, Leng J, Wang K, Ma X, Wu S (2016) Electrochemical sandwich immunoassay for human epididymis-specific protein 4 using a screen-printed electrode modified with graphene sheets and gold nanoparticles, and applying a modular magnetic detector device produced by 3D laser sintering. Microchim Acta 183:837–843. https://doi.org/10.1007/s00604-015-1727-x CrossRefGoogle Scholar
- 22.Yang X, Wu F, Chen D-Z, Lin H-W (2014) An electrochemical immunosensor for rapid determination of clenbuterol by using magnetic nanocomposites to modify screen printed carbon electrode based on competitive immunoassay mode. Sensors Actuators B Chem 192:529–535. https://doi.org/10.1016/j.snb.2013.11.011 CrossRefGoogle Scholar
- 24.Barallat J, Olivé-Monllau R, Gonzalo-Ruiz J, Ramírez-Satorras R, Muñoz-Pascual FX, Ortega AG, Baldrich E (2013) Chronoamperometric magneto immunosensor for myeloperoxidase detection in human plasma based on a magnetic switch produced by 3d laser sintering. Anal Chem 85:9049–9056. https://doi.org/10.1021/ac401549d CrossRefPubMedGoogle Scholar
- 30.Wu H, Liu S, Jiang J, Shen G, Yu R (2012) A sensitive electrochemical biosensor for detection of DNA methyltransferase activity by combining DNA methylation-sensitive cleavage and terminal transferase-mediated extension. Chem Commun 48:6280–6282. https://doi.org/10.1039/c2cc32397d CrossRefGoogle Scholar
- 32.Yin H, Zhou Y, Xu Z, Wang M, Ai S (2013) Ultrasensitive electrochemical immunoassay for DNA methyltransferase activity and inhibitor screening based on methyl binding domain protein of MeCP2 and enzymatic signal amplification. Biosens Bioelectron 49:39–45. https://doi.org/10.1016/j.bios.2013.04.040 CrossRefPubMedGoogle Scholar
- 34.Yang Z, Xie L, Yin H, Zhou Y, Ai S (2015) Methyltransferase activity assay based on the use of exonuclease III, the hemin/G-quadruplex system and reduced graphene oxide on a gold electrode, and a study on enzyme inhibition. Microchim Acta 182:2607–2613. https://doi.org/10.1007/s00604-015-1645-y CrossRefGoogle Scholar