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 β-glucosyltransferase activity assay based on the glycosylation of hydroxymethylated DNA and Hpa II restriction endonuclease system triggering signal amplification. After immobilizing double-stranded DNA (dsDNA) on the surface of carboxyl functionalized Fe 3 O 4 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 Fe 3 O 4 nanoparticle surface. After the dsDNA was digested by Hpa II, the fragment with 4-MPBA can be released from Fe 3 O 4 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.