A versatile assay for alkaline phosphatase detection based on thymine-Hg-II-thymine structure generation mediated by TdT
A versatile assay for alkaline phosphatase detection based on thymine-Hg-II-thymine structure generation mediated by TdT
复制标题
基于 TdT 介导的胸腺嘧啶-Hg-II-胸腺嘧啶结构生成的多功能碱性磷酸酶检测方法
DOI：
10.1016/j.talanta.2018.11.061
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发表时间：
2019
期刊：
影响因子：
6.1
通讯作者：
Xing Xiwen
中科院分区：
文献类型：
--
作者：
Wang Yuanyuan;Yang Li;Li Nan;Sun Chuankai;Xia Yiping;Yuan Libo;Lu Jie;Liu Fenyong;Xing Xiwen
碱性磷酸酶（Alkaline phosphatase，ALP）是磷酸盐代谢中的重要水解酶，催化蛋白质、核酸和其他小分子中磷酸酯基团的水解。同时，ALP的异常表达与多种疾病的发生、发展有关。末端脱氧核苷酸转移酶（TdT）是一种广泛应用的工具酶，它以无模板的方式将脱氧核苷三磷酸（dNTPs）随机添加到单链DNA的3′-OH末端。本研究基于ALP、TdT和胸腺嘧啶-汞Ⅱ-胸腺嘧啶（T-Hg ~（2+）-T）结构的特点，设计了一种通用、方便、无标记、高灵敏度的ALP活性荧光增强检测方法。在碱性磷酸酶（ALP）的作用下，ssDNA-p的3′-磷酰基端水解为羟基，并在其3′-端羟基上添加一个聚T尾。然后，带有poly-T尾的DNA可以与Hg ~（2+）相互作用，形成稳定的T-Hg ~（2+）-T介导的金属DNA双链体，使SG的荧光强度增强。在最佳条件下，该系统用于定量监测ALP活性，动态范围为0-2500 mU mL−1，实际检测限可低至0.025 mU mL−1。该传感器对人血清和MCF-7细胞裂解液中ALP活性的测定显示出良好的传感性能，在生化研究和临床诊断中具有潜在的应用价值。同时，该系统也可用于汞离子和TdT的检测。
Alkaline phosphatase (ALP) is a vital hydrolysis enzyme in phosphate metabolism, which catalyzes the hydrolysis of phosphate ester groups in proteins, nucleic acids, and other small molecules. Meanwhile, abnormal ALP expression is associated with occurrence and development of many diseases. Terminal deoxynucleotidyl transferase (TdT) is a widely used tool enzyme in many fields, which randomly adds deoxyribonucleoside triphosphates (dNTPs) at the 3′-OH termini of ssDNA in a template-free manner. In this work, we designed a versatile, convenient, label-free, and highly sensitive fluorescence enhancing assay for ALP activity detection based on the characteristics of ALP, TdT, and thymine-HgII-thymine (T-Hg2+-T) structure. In the presence of ALP, the 3′-phosphoryl end of the ssDNA-p was hydrolyzed to hydroxyl group, followed by addition of a poly-T tail on its 3′ terminal hydroxyl in the mixing solution containing both TdT and dTTPs. Then, the DNA with poly-T tail could interact with Hg2+to form the stable T-Hg2+-T mediated metallo DNA duplex, which enhanced the fluorescence intensity of the SG. Under optimal conditions, the proposed system was employed for quantitatively monitoring ALP activity with a dynamic range of 0–2500 mU mL−1, and the actual detection limit could be down to 0.025 mU mL−1. And the determination of ALP activity in human serum samples and MCF-7 cells lysates exhibited a good sensing performance, demonstrating its potential applications in biochemical research and clinical diagnosis. Meanwhile, this system could also be applied to both TdT and Hg2+detection.