Highly selective and real-time detection of 5-hydroxymethylcytosine in genomic DNA using a carbon nitride-modified gold transducer-based electrochemical sensor

Cancer biomarkers are crucial indicators of cancer status and progression that aid in early detection and more effective treatment of the disease. The loss of 5-hydroxymethylcytosine (5hmC), an oxidation product of 5-methylcytosine (5mC), is a recurrent epigenetic biomarker across various types of cancers. Therefore, accurately quantifying 5hmC holds great potential for various clinical applications. However, distinguishing 5hmC from 5mC using conventional methods is challenging. In this study, we developed a rapid and highly selective electrochemical sensor for label-free detection of 5hmC-enriched DNAs using a graphitic carbon nitride (g-C3N4)-modified gold transducer. Two-dimensional g-C3N4 sheets were synthesized via direct pyrolysis of urea under ambient or nitrogen atmospheres and drop-cast onto the gold electrode. Subsequently, 5hmC-containing DNAs were immobilized onto g-C3N4 via hydrogen bonding between the –OH of 5hmC and the -NH2 of g-C3N4. The developed sensor demonstrated high sensitivity, selectivity, remarkable reproducibility, and stability, with a low oxidation potential (0.23 V) and an extremely low limit of detection (0.316 pM) for 5hmC. The sensor was also tested for its applicability to real samples using primary liver samples from mouse models, in which 5hmC levels were diminished due to either Tet gene knockout or hepatocellular carcinogenesis. The sensor effectively detected reduced genomic 5hmCs in TET-deficient livers and hepatocellular carcinomas compared to controls. Thus, this novel sensing strategy has the potential to develop clinically applicable sensors for early cancer diagnosis and prognosis evaluation by rapidly quantifying genomic 5hmC. [Display omitted] •A new electrochemical sensor based on a g-C3N4-modified gold transducer was made.•The sensor enabled selective, label-free, and real-time detection of 5hmC in DNA.•5hmC detection with high specificity and stability and a very low LOD was achieved.•The sensing device is highly suitable for assays involving real samples.•It rapidly detects 5hmC depletion in primary liver tissues from TET knockout mice and mice with hepatocellular carcinoma.