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Biosens Bioelectron. 2019 Feb 15;127:155-160. doi: 10.1016/j.bios.2018.12.022. Epub 2018 Dec 16.

An electrochemical DNA biosensor analytic technique for identifying DNA methylation specific sites and quantify DNA methylation level.

Author information

1
Department of Clinical and military Laboratory Medicine, Army Medical University, 30 Gaotanyan Street, Shapingba District, Chongqing 400038, China; Department of Clinical Biochemistry, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China.
2
Department of Clinical Laboratory, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China; Medical Laboratory, Guizhou Medical University, Guiyang 550525, China.
3
Medical Laboratory, Guizhou Medical University, Guiyang 550525, China.
4
Department of Clinical and military Laboratory Medicine, Army Medical University, 30 Gaotanyan Street, Shapingba District, Chongqing 400038, China.
5
Department of laboratory medicine, the General Hospital of Chinese People's Armed Police Forces, Beijing 100039, China. Electronic address: yangxiaolitwins@163.com.
6
Department of Clinical and military Laboratory Medicine, Army Medical University, 30 Gaotanyan Street, Shapingba District, Chongqing 400038, China. Electronic address: zhengalpha@sina.com.

Abstract

We herein developed a novel electrochemical biosensor to detect DNA methylation level, and to quantitatively analyze multiple methylated sites. Graphene oxide was modified with anti-5-methylcytosine antibody to specifically bind CpG methylation sites, and horseradish peroxidase (HRP)-labeled IgG secondary antibody was bound to the former antibody. In buffer containing H2O2 and hydroquinone, HRP-IgG catalyzed the oxidation of hydroquinone into benzoquinone over H2O2, thereby generating electrochemical reduction signals. The number of 5-methylcytosine was directly proportional to current signal, thereby allowing accurate quantification of methylation level. We also analyzed monomethylated target sequences with different sites. After different methylated sites were captured by the probe, the steric hindrance differences between -CH3 hydrophobic sphere and the electrode surface were induced. The peak current decreased with reducing distance from the electrode surface, so DNA methylation sites were identified by measuring corresponding peak current responses. With a low detection limit (1 fM), this DNA biosensor was suitable for ultrasensitive DNA methylation detection. The linear detection range was 10-15 M to 10-8 M. Meanwhile, this method had high specificity, stability and repeatability, thus being widely applicable to the clinical detection of DNA methylation.

KEYWORDS:

DNA methylation; Electrochemical biosensor; Hydrophobic sphere; Steric hindrance

PMID:
30597434
DOI:
10.1016/j.bios.2018.12.022
[Indexed for MEDLINE]

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