点击化学连接启动的无亚硫酸盐测定法，通过纳米珠实现对位点特异性5 mC的高灵敏度检测。

Abstract

Gentle TAPβ conversion and PNA-chemical ligation dual recognition enables highly specific identification of 5 mC sites. • The synergy of physical enrichment and template-free amplification achieves an ultrahigh sensitivity down to 1 fM. • Quantification via flow cytometric MFI on nanobeads demonstrates high practical applicability. • This method enables robust 5 mC detection in complex biological samples. 5-Methylcytosine (5 mC) represents the most prevalent and functionally significant DNA modification in mammalian genomes. Aberrations in its status are intricately associated with the pathogenesis of various diseases, including cancer and neurodegenerative disorders. The ultrasensitive detection of low-abundance 5 mC in trace biological fluids, such as plasma, holds great potential for early disease diagnosis and may drive the evolution of clinical diagnostic methodologies. Therefore, we have developed a β-glucosyltransferase and Ten-eleven translocation (TET) dioxygenases-assisted pyridine borane conversion and click chemistry ligation-initiated flow cytometry assay (TAPβ-CCLFC) for site-specific 5 mC analysis. In this method, TAPβ conversion gently converts DNA epigenetic modifications into distinguishable base variations. Furthermore, a peptide nucleic acid (PNA) clamp acts synergistically with click chemistry to enable single-base discrimination and further improve assay specificity. Ligation products are efficiently enriched on magnetic nanobeads, followed by template-independent, primer-free isothermal amplification via terminal deoxynucleotidyl transferase (TdT) to achieve high sensitivity. Consequently, the TAPβ-CCLFC enables precise discrimination of 5 mC at a frequency as low as 1.4%, and achieves a detection limit as low as 1 fM. Its practical utility has been further validated using human plasma samples. Collectively, the TAPβ-CCLFC strategy provides a robust and convenient platform with a simplified workflow for the sensitive, specific, and quantitat