Functionalized Glass Micropipettes for Methylated DNA Detection Based on Enzyme-Responsive Signal Switching Effects.

Abstract

DNA methylation is a crucial epigenetic modification whose abnormal alterations are closely associated with various tumors and are considered potential biomarkers for cancer diagnosis. However, achieving highly sensitive and selective detection of low-abundance methylated DNA in complex biological samples remains a significant challenge. This study developed a functionalized glass micropipette electrochemical sensing strategy based on the enzyme-responsive signal switching effect for highly sensitive detection of methylated DNA. This system utilizes glass micropipettes modified with polydopamine nanotubes (PDA-NTs) to construct an ion-transport interface, where the capture probe (cpDNA) is immobilized to achieve specific recognition of target sequences. The detection mechanism relies on sequence differences introduced by sodium bisulfite conversion and the selective cleavage action of the nucleic acid exonuclease Exo III: upon forming stable double-stranded structures with the cpDNA, methylated DNA triggers Exo III-mediated structural changes at the interface. This modulates ion transport behavior within the channels, generating a current response that switches from "Off" to "On", enabling effective differentiation between methylated and unmethylated DNA. This sensing system exhibits excellent linear response to methylated DNA within the 1 aM-100 fM range, with a detection limit as low as 8.5 aM, and enables reliable detection in 10% human serum samples. This strategy achieves sensitive detection through an enzyme-responsive signal switch without requiring PCR amplification or complex labeling procedures, providing a simple and effective method for rapid analysis of DNA epigenetic modifications.