Photoelectrochemical detection of epigenetic 5-hydroxymethylcytosine based on Cu₂O@CuO@Ag and self-triggered...

Epigenetic DNA hydroxymethylation, particularly 5-hydroxymethylcytosine (5hmC), has emerged as a promising biomarker for early and specific tumor diagnosis. This modification serves as a critical regulator of gene expression, and its aberrant levels correlate directly with tumor initiation, metastasis, and therapeutic resistance. Quantitative analysis of sequence-specific 5hmC modifications offers valuable insights into dynamic epigenetic patterns associated with disease progression. Consequently, developing highly sensitive methods for quantitative detection of 5hmC is crucial for advancing epigenetic detection and diagnosis.

In this study, we engineered a novel photoelectrochemical (PEC) biosensor for ultrasensitive detection of the epigenetic biomarker 5hmC. The sensor utilized a ternary heterojunction Cu₂O@CuO@Ag as the photoactive material, whose unique band structure facilitated efficient charge separation and generated a stable initial photocurrent. The detection mechanism initiated with a click chemistry-mediated azide reaction targeting 5hmC, forming Y-shaped DNA scaffolds. These scaffolds activated a self-triggered isothermal amplification cascade, enzymatically recycling trace targets into abundant trigger P sequences. Subsequently, alkaline phosphatase (ALP)-conjugated probes catalyzed the hydrolysis of ascorbic acid phosphate (AAP), producing ascorbic acid (AA) as an electron donor. The amplified AA significantly enhanced electron transfer to the photoanode, enabling quantification of 5hmC DNA in buffer down to 0.56 fM with high specificity. This integrated signal amplification strategy provides a powerful platform for epigenetic analysis in clinical diagnostics.

In conclusion, this strategy offers a novel approach for ultrasensitive quantification of natural DNA base modifications (e.g., 5hmC). By integrating ternary Cu₂O@CuO@Ag heterojunction photoanodes with self-triggered isothermal amplification, it achieved 0.56 fM detection limit, opening avenues for clinical epigenetics, early cancer screening, and liquid biopsy applications in complex samples.