Label-Free and Homogeneous Sensing Platform with Template-Independent Cascade Amplification Strategy for Genome-Wide 5-Hydroxymethylcytosine Analysis.

5-Hydroxymethylcytosine (5hmC) is an important epigenetic mark in DNA, and its dysregulation is closely associated with cancer and neurodegenerative diseases. However, due to the structural similarity with 5-methylcytosine (5mC), existing detection techniques for distinguishing 5hmC from 5mC often involve either cumbersome separation steps or DNA-damaging treatments. In this work, we developed a template-independent cascade amplification-based sensing platform for homogeneous, label-free, and genome-wide detection of 5hmC. The platform integrated terminal transferase (TdT)-mediated template-independent nucleic acid amplification with APE1-driven cyclic cleavage for cascaded signal enhancement. The assay required only a signal probe containing an apurinic/apyrimidinic (AP) site, with no need for separation steps. This method achieved a detection limit as low as 6.34 × 10-17 M and could specifically identify as low as 0.099% 5hmC in the presence of 5mC and cytosine (C). At the cellular level, the assay exhibited a sensitivity of 2.33 × 10-5 ng/μL for U-118MG cells and successfully distinguished different tumor cell lines and cancerous/normal tissues. Our results were consistent with those obtained by commercial ELISA kits, but the cost was reduced by 3.6-fold. Moreover, this method can accurately distinguish cancer patients from healthy individuals (P < 0.001), with an area under the receiver operating characteristic curve (AUC) of 0.993, holding potential applications in tumor liquid biopsy and epigenetic marker screening.