Electrochemical biosensor for hydroxymethylated DNA detection and ß-glucosyltransferase activity assay based on enzymatic catalysis triggering signal amplification

A simple and sensitive electrochemical biosensor was fabricated for hydroxymethylated DNA detection and T4 β-glucosyltransferase activity assay based on the glycosylation of hydroxymethylated DNA and HpaII restriction endonuclease system triggering signal amplification. After immobilizing double-str...

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Bibliographic Details
Published in:Sensors and actuators. B, Chemical Chemical, 2017-05, Vol.B243, p.602
Main Authors: Yin, Huanshun, Yang, Zhiqing, Wang, Haiyan, Zhou, Yunlei, Ai, Shiyun
Format: Article
Language:English
Subjects:
Amplification
Assaying
Biosensors
Catalysis
Deoxyribonucleic acid
DNA
DNA methylation
Glucose
Gold
Iron compounds
Iron oxides
Magnetic separation
Nanoparticles
Reproducibility
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Summary:A simple and sensitive electrochemical biosensor was fabricated for hydroxymethylated DNA detection and T4 β-glucosyltransferase activity assay based on the glycosylation of hydroxymethylated DNA and HpaII restriction endonuclease system triggering signal amplification. After immobilizing double-stranded DNA (dsDNA) on the surface of carboxyl functionalized Fe3O4 nanoparticle, the glycosylation reaction of hydroxymethylated cytosine can be performed with the catalytic effect of T4 β-glucosyltransferase using uridine diphosphoglucose as glucose donor. Then, 4-mercaptophenylboronic acid (4-MPBA) can be further reacted with glucose and captured on Fe3O4 nanoparticle surface. After the dsDNA was digested by HpaII, the fragment with 4-MPBA can be released from Fe3O4 nanoparticle surface. After magnetic separation, this fragment can be further modified on gold electrode surface through the interaction between Au and −SH, which can result in a decreased electrochemical signal of [Fe(CN)6]3−. According to this detection strategy, the electrochemical biosensor showed high sensitivity with the low detection limits of 0.14 nM and 0.27 unit/mL for hydroxymethylated DNA and T4 β-glucosyltransferase. The fabricated biosensor also presented high selectivity and acceptable reproducibility. In addition, the inhibition activity of 4-phenylimidazole towards T4 β-glucosyltransferase was also investigated. This work provides a novel platform for hydroxymethylated DNA detection, T4 β-glucosyltransferase activity assay and its inhibition screening.
ISSN:0925-4005
1873-3077