Rapid and Label-Free Detection of 5-Hydroxymethylcytosine in Genomic DNA Using an Au/ZnO Nanorods Hybrid Nanostructure-Based Electrochemical Sensor

Ten-eleven-translocation (TET) proteins modify DNA methylation by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Loss of 5hmC, a widely accepted epigenetic hallmark of cancers, is proposed as a biomarker for early cancer diagnosis and prognosis. Thus, precise quantification of 5hmC holds great potential for diverse clinical applications. DNAs containing 5mC or 5hmC display different adsorption affinity toward the gold surface, thus producing different electrochemical responses. Here a novel, label-free electrochemical sensor based on gold nanoparticles (Au NPs)/zinc oxide nanorods (ZnO NRs) nanostructure for the facile and real-time detection of 5hmC-enriched DNAs is reported. The hybrid structure is fabricated by the vertical hydrothermal growth of ZnO NRs onto indium tin oxide glass substrate, followed by the decoration of ZnO NRs with Au NPs via sputtering. Successful fabrication is confirmed by analyzing the morphology and chemical composition of the sensor. By coupling the fabricated sensor with cyclic voltammetry, its functionality in distinguishing genomic DNAs containing different levels of 5hmC is validated. Notably, the sensor device successfully and consistently detects 5hmC loss in primary hepatocellular carcinoma, compared to the normal tissues. Thus, the novel sensing strategy to assess DNA hydroxymethylation will likely find broad applications in early cancer diagnosis and prognosis evaluation.