Alkaline phosphatase (ALP) is widely expressed in human tissues. ALP plays an important role in the dephosphorylation of proteins and nucleic acids. Therefore, quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods. Terminal deoxynucleotidyl transferase (TdT) catalyzes continuous polymerization of deoxynucleotide triphosphates at the 3′-OH end of single-stranded DNA in the absence of a template. In this study, we developed a highly sensitive and selective method based on TdT and endonuclease IV (Endo IV) to quantify ALP activity. After ALP hydrolyzes the 3′-PO₄ end of the substrate and generates 3′-OH, TdT can effectively elongate the 3′-OH end with deoxynucleotide adenine triphosphate (dATP) and produce a poly A tail, which can be detected by the poly T probes. Endo IV digests the AP site in poly T probes to generate a fluorescent signal and a new 3′-OH end, leading to the generation of exponential fluorescence signal amplification. The substrate for TdT elongation was optimized, and a limit of detection of 4.3 × 10⁻³ U/L was achieved for ALP by the optimized substrate structure. This method can also detect ALP in the cell lysate of a single cell. This work has potential applications in disease diagnosis and biomedical detection.

碱性磷酸酶 (ALP) 在人体组织中广泛表达。ALP 在蛋白质和核酸的去磷酸化中起重要作用。因此，ALP的定量分析在疾病诊断和生物检测方法的发展中起着至关重要的作用。在没有模板的情况下，末端脱氧核苷酸转移酶 (TdT) 在单链 DNA 的 3'-OH 末端催化脱氧核苷酸三磷酸的连续聚合。在这项研究中，我们开发了一种基于 TdT 和核酸内切酶 IV (Endo IV) 的高灵敏度和选择性方法来量化 ALP 活性。ALP 水解 3'-PO _4后在底物末端产生 3'-OH，TdT 可以有效地用脱氧核苷酸三磷酸腺嘌呤 (dATP) 拉长 3'-OH 末端并产生 poly A 尾，可被 poly T 探针检测到。Endo IV 消化 poly T 探针中的 AP 位点以产生荧光信号和新的 3'-OH 末端，从而产生指数荧光信号放大。^{优化了TdT延伸的底物，通过优化的底物结构实现了ALP的4.3×10 -3} U/L的检测限。该方法还可以检测单个细胞的细胞裂解液中的ALP。这项工作在疾病诊断和生物医学检测方面具有潜在应用。