N6-甲基腺苷 (m6A) 已被证明是 mRNA 代谢中关键的转录后调节因子，其失调与多种人类疾病相关。本文中，我们构建了一个单分子荧光生物传感器，用于无抗体检测癌细胞和组织中的特定位点 m6A。设计了一个 5'-生物素化捕获探针和一个 3'-羟基化助剂探针，用于识别特定的 m6A-mRNA。敏感于 m6A 的内切核糖核酸酶 MazF 可以鉴别和切割未甲基化的 mRNA，而保留的完整的 m6A-mRNA 可以与辅助探针和捕获探针杂交形成夹心杂交体。夹心杂交体被固定在磁珠上，以启动末端脱氧核苷酸转移酶 (TdT) 辅助聚合反应，促进 Cy5-dATP 的连续合成形成长的 Cy5-polyA 尾部，产生珠上放大的荧光信号。经过磁分离和外切核酸酶消化后，释放出大量的 Cy5 荧光团，随后进行单分子检测。特别地，这种生物传感器实现简单且等温，不涉及放射性标记或 m6A 特异性抗体的参与。此外，这种生物传感器具有超高的灵敏度，检测限为 2.24 × 10-17 M，能够区分存在于大量共存对应物中的 0.01% m6A 水平。此外，这种生物传感器可用于监测细胞 m6A-mRNA 的表达并区分乳腺癌患者组织中的 m6A 水平与健康人体组织中的不同，为临床诊断和表观转录组研究提供了新的途径。

N6-Methyladenosine (m6A) has emerged as a key post-transcriptional regulator in mRNA metabolism, and its dysregulation is associated with multiple human diseases. Herein, we construct a single-molecule fluorescent biosensor for antibody-free detection of locus-specific m6A in cancer cells and tissues. A 5'-biotinylated capture probe and a 3'-hydroxylated assistant probe are designed for the recognition of specific m6A-mRNA. The m6A-sensitive endoribonuclease MazF can identify and cleave the unmethylated mRNA, and the retained intact m6A-mRNA can hybridize with assistant probes and capture probes to achieve sandwich hybrids. The sandwich hybrids are immobilized on magnetic beads (MBs) to initiate the terminal deoxynucleotidyl transferase (TdT)-assisted polymerization, facilitating the continuous incorporation of Cy5-dATP to form long Cy5-polyA tails for the production of an on-bead amplified fluorescence signal. After magnetic separation and exonuclease digestion, numerous Cy5 fluorophores are released and subsequently measured by single-molecule detection. Especially, this biosensor is implemented simply and isothermally without the involvement of either radiolabeling or m6A-specific antibody. Moreover, this biosensor shows ultrahigh sensitivity with a detection limit of 2.24 × 10-17 M, and it can discriminate a 0.01% m6A level from a large pool of coexisting counterparts. Furthermore, this biosensor can be used for monitoring cellular m6A-mRNA expression and differentiating the m6A level in the breast cancer patient tissues from that in the healthy person tissues, providing a new avenue for clinical diagnosis and epitranscriptomic research.