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

A simple and sensitive electrochemical biosensor was fabricated for hydroxymethylated DNA detection and T4 beta-glucosyltransferase activity assay based on the glycosylation of hydroxymethylated DNA and Hpall 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 beta-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 Hpall, the fragment with 4-MPBA can be released from Fe304 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 beta-glucosyltransferase. The fabricated biosensor also presented high selectivity and acceptable reproducibility. In addition, the inhibition activity of 4-phenylimidazole towards T4 beta-glucosyltransferase was also investigated. This work provides a novel platform for hydroxymethylated DNA detection, T4 beta-glucosyltransferase activity assay and its inhibition screening. (C) 2016 Elsevier B.V. All rights reserved.