Electrogenerated chemiluminescence biosensing method for the discrimination of DNA hydroxymethylation and assay of the β-glucosyltransferase activity.

An electrogenerated chemiluminescence (ECL) biosensing method for highly sensitive discrimination of DNA hydroxymethylation and assay of the β-glucosyltransferase (β-GT) activity was developed. The ECL biosensing electrode was fabricated by gold nanoparticles (AuNPs)/Nafion film, and then, tris(2, 2'-ripyridine) dichlororuthenium(II) (Ru(bpy)3(2+)) was electrostatically adsorbed into the AuNPs/Nafion film, finally, the hydroxymethylated double-stranded DNA (ds-DNA)-tagged with ferrocene was self-assembled onto the surface of the AuNPs. When β-GT and uridine diphosphoglucose (UDP-Glu) were introduced, the hydroxymethylcytosine residues within 5'-CCGG-3' of ds-DNA on the biosensing electrode were glucosylated. After the glucosylated biosensing electrode was treated by MspI endonuclease, the unglucosylated hydroxymethylcytosine was cleaved, leading to the quencher leaving the electrode, resulting in an increased ECL signal. For the ECL biosensing method, it showed an extremely low detection limit of 0.04 U/mL for β-GT, and offered a good discrimination toward cytosine, 5-methylcytosine, and 5-hydroxymethylcytosine. This work demonstrates that the combination of the enzyme-linkage reactions with the highly sensitive ECL method is a promising strategy for the discrimination of DNA hydroxymethylation, assay of the activity of β-GT, and evaluation of the capability of inhibitors for the β-GT.