Assembly of Dandelion-Like Nanoprobe for Sensitive Detection of N6-Methyladenosine Demethylase by Single-Molecule Counting.

N6-methyladenosine (m6A) demethylase is essential for enzymatically removing methyl groups from m6A modifications and is significantly implicated in the pathogenesis and advancement of various cancers, which makes it a promising biomarker for cancer detection and research. As a proof of concept, we select the fat mass and obesity-associated protein (FTO) as the target m6A demethylase and develop a dandelion-like nanoprobe-based sensing platform by employing biobar-code amplification (BCA) for signal amplification. We construct two meticulously designed three-dimensional structures: reporter-loaded gold nanoparticles (Reporter@Au NPs) and substrate-loaded magnetic microparticles (Substrate@MMPs), which can self-assemble to form dandelion-like nanoprobes via complementary base pairing. In the presence of FTO, the m6A-containing substrates are demethylated, triggering the MazF-assisted cleavage reaction and thereby releasing the Reporter@Au NPs. Furthermore, upon digestion by exonucleases, the Reporter@Au NPs may liberate a significant quantity of Cy3 signals. Remarkably, the combined effects of Au NPs' superior enrichment capacity, MMPs' exceptional magnetic separation efficiency, and the precision of the single-molecule detection platform endow the FTO sensor with exceptional sensitivity and specificity with a detection limit of 7.46 × 10 M. Additionally, this method offers a versatile platform for the detection of m6A demethylase and the screening of corresponding inhibitors, thereby advancing clinical diagnosis and drug development.