Journal
ANALYTICA CHIMICA ACTA
Volume 840, Issue -, Pages 28-32Publisher
ELSEVIER
DOI: 10.1016/j.aca.2014.06.020
Keywords
DNA demethylation; DNA demethylase activity; Electrochemical biosensor; Alkaline phosphatase
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Funding
- National Natural Science Foundation of China [21105056, 21375079]
- Project of Development of Science and Technology of Shandong Province, China [2013GZX20109]
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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. (C) 2014 Elsevier B.V. All rights reserved.
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