Direct electrochemical detection of 5-hydroxymethylcytosine on electrochemically reduced graphene oxide modified pyrolytic graphite electrodes in biological samples

The 5-hydroxymethylcytosine is a significant epigenetic DNA modification in mammalian genomes, and the dysregulation of the 5-hydroxymethylcytosine levels is closely related to various human diseases. The determination of the 5-hydroxymethylcytosine in human urine can serve as a non-invasive diagnostic of diseases. In this study, for the first time, a rapid and sensitive voltammetric method was developed to quantify the 5-hydroxymethylcytosine in human urine based on a reduced graphene oxide modified electrode, through which the modification was obtained by reduction process through the cyclic voltammetry on the pyrolytic graphite electrode in the presence of graphene oxide. The effects of pH and scan rate on the voltammetric signal of the 5-hydroxymethylcytosine were tested on the fabricated modified sensor. The electrochemical oxidation process of the 5hydroxymethylcytosine was realized under diffusion-adsorption mixed control. The stripping conditions and square wave voltammetric parameters were optimized in a pH 7.0 phosphate buffer. The calibration curve demonstrated a linear range between 0.6 and 4.0 ppm for the 5-hydroxymethylcytosine in human urine, with a satisfactory recovery value of 101.21 %. Furthermore, the proposed method was successfully applied to the analysis of a calf thymus DNA sample.