title: Enzyme-based electrochemical biosensor for sensitive detection of DNA demethylation and the activity of DNA demethylase.
abstract: A novel electrochemical method is developed for detection of DNA demethylation and assay of DNA demethylase activity. This method is constructed by hybridizing the probe with biotin tagged hemi-methylated complementary DNA and further capturing streptavidin tagged alkaline phosphatase (SA-ALP) to catalyze the hydrolysis reaction of p-nitrophenyl phosphate. The hydrolysate of p-nitrophenol (PNP) is then used as electrochemical probe for detecting DNA demethylation and assaying the activity of DNA demethylase. Demethylation of target DNA initiates a degradation reaction of the double-stranded DNA (dsDNA) by restriction endonuclease of BstUI. It makes the failed immobilization of ALP, resulting in a decreased electrochemical oxidation signal of PNP. Through the change of this electrochemical signal, the DNA demethylation is identified and the activity of DNA demethylase is analyzed with low detection limit of 1.3 ng mL(-1). This method shows the advantages of simple operation, cheap and miniaturized instrument, high selectivity. Thus, it provides a useful platform for detecting DNA demethylation, analyzing demethylase activity and screening inhibited drug.
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China.
authors.forename: Yunlei
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China.
authors.forename: Bingchen
authors.initials: B
authors.lastname: Li
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China.
authors.forename: Mo
authors.initials: M
authors.lastname: Wang
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China.
authors.forename: Zhiqing
authors.initials: Z
authors.lastname: Yang
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China. Electronic address: yinhs@sdau.edu.cn.
authors.forename: Huanshun
authors.initials: H
authors.lastname: Yin
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China. Electronic address: ashy@sdau.edu.cn.
authors.forename: Shiyun
authors.initials: S
authors.lastname: Ai
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D019175:DNA Methylation / Q000502:physiology*; D015254:DNA Modification Methylases / Q000032:analysis* / Q000378:metabolism*; D055664:Electrochemical Techniques / Q000379:methods*; D004789:Enzyme Activation / Q000502:physiology
chemical_list: D015254:DNA Modification Methylases
keywords: Alkaline phosphatase; DNA demethylase activity; DNA demethylation; Electrochemical biosensor