▎ 摘　　要

As a significant potential biomarker for early disease diagnostics, the detection of 5-hydroxymethylcytosine (5hmC) has garnered widespread attention. In this work, a simple and sensitive electrochemiluminescence (ECL) was developed for 5hmC detection. After substrate electrode preparation, anti-5hmC antibody was captured on electrode surface by the formation of amide bond. Then, through immunoreaction, 5hmC was specifically recognized and captured. Utilizing the crosslink effect of Zr4+ with phosphoric acid recognition, aminomethyl phosphate was modified on electrode to further capture PAMAM-silver nanoclusters/Nitrogen-doped graphene nanocomposite (PAMAM-Ag-NG), where this nanocomposi te was employed as ECI.signal probe. The designed immunosensor showed a linear concentration range from 10 pM to 30 nM with a low detection limit of 2.47 pM (S/N = 3). This method presented good selectivity and reproducibility. To prove the applicability, this method was further used to study the influence of phytohormone and avian leukemia virus on 5hmC content in maize seedling leaves and chicken fibroblast cells, respectively.