5-羟甲基尿嘧啶 (5hmU) 是多种生物体基因组中胸腺嘧啶的氧化衍生物，可作为表观遗传标记和癌症生物标记。然而，目前的5hmU检测通常存在操作繁琐、特异性低、灵敏度不理想的缺点。在此，我们展示了点击化学介导的超支化扩增驱动的树突纳米组装，用于乳腺细胞系和人类乳腺组织中 5hmU 的全基因组分析。该策略具有良好的选择性、超低背景和高灵敏度，检测限为83.28 aM。该方法甚至可以准确检测混合物中 0.001% 5hmU 的水平。此外，它还可以在单​​细胞水平上测定5hmU，区分正常人和乳腺癌患者组织中5hmU的表达，在5hmU相关的生物学研究和临床诊断方面具有广阔的前景。

5-Hydroxymethyluracil (5hmU) is an oxidation derivative of thymine in the genomes of various organisms and may serve as both an epigenetic mark and a cancer biomarker. However, the current 5hmU assays usually have drawbacks of laborious procedures, low specificity, and unsatisfactory sensitivity. Herein, we demonstrate the click chemistry-mediated hyperbranched amplification-driven dendritic nanoassembly for genome-wide analysis of 5hmU in breast cell lines and human breast tissues. The proposed strategy possesses good selectivity, ultralow background, and high sensitivity with a detection limit of 83.28 aM. This method can accurately detect even a 0.001% 5hmU level in the mixture. Moreover, it can determine 5hmU at single-cell level and distinguish the expressions of 5hmU in tissues of normal persons and breast cancer patients, holding great promise in 5hmU-related biological research and clinical diagnosis.