Facile and Sensitive Fluorescence Assay of DNA Polymerase Activity Using Cu2+ and Ascorbate as Signal Developers
Facile and Sensitive Fluorescence Assay of DNA Polymerase Activity Using Cu2+ and Ascorbate as Signal Developers
复制标题
使用 Cu2 和抗坏血酸作为信号显色剂对 DNA 聚合酶活性进行简便、灵敏的荧光测定
DOI：
10.1002/slct.201803850
复制
发表时间：
2019
期刊：
影响因子：
2.1
通讯作者：
Li Baoxin
中科院分区：
文献类型：
--
作者：
Zhang Xingxing;Liu Qiang;Jin Yan;Li Baoxin
我们在这里报告了一种检测DNA聚合酶活性的无标记和开启的荧光方法。在DNA聚合酶存在的情况下，DNA引物可以延长以产生一条长的双链DNA(DsDNA)。然后，以合成的双链DNA为模板，通过Cu2+-抗坏血酸反应(室温下10 m in)原位合成荧光铜纳米团簇(CuNC)，并用荧光强度来定量测定CuNC的聚合酶活性。该方法以廉价的CuSO4和抗坏血酸作为荧光显色剂，成本低，易于实施。有趣的是，基于DNA模板CuNC的序列依赖性荧光，我们合理地设计了DNA底物的序列，以减少背景，增加信号。因此，该方法对DNA聚合酶具有较高的灵敏度，对Klenow片段(KF)聚合酶的检测下限为3.7×10-7U/μL。该策略提出了一种“混合检测”的均一化验形式，不需要沉淀、分离和洗涤。整个过程(包括酶反应和CuNC的合成)可以在一根试管中完成。该方法成本低、操作简便，在一些生物医学研究和临床诊断中具有潜在的应用前景。
We report herein a label‐free and turn‐on fluorescence approach for detecting DNA polymerase activity. In the presence of DNA polymerase, the DNA primer could be elongated to produce one long double‐stranded DNA (dsDNA). Then, the produced dsDNA acted as the template for in‐situ synthesizing fluorescent copper nanoclusters (CuNCs) via Cu2+–ascorbate reaction (10 min at room temperature), and the fluorescent intensity of the CuNCs was used to quantify the activity of DNA polymerase. In this strategy, cheap CuSO4and ascorbate acted as fluorescence developers, so this method is low‐cost and easy to carry out. Interestingly, based on the sequence‐dependent fluorescence of DNA‐templated CuNCs, we rationally designed the sequence of DNA substrate to decrease the background and increase the signal of this assay. Therefore, this proposed method exhibited a high sensitivity toward DNA polymerase, and the limit of detection was 3.7×10–7U/μL for Klenow Fragment (KF) polymerase. The strategy suggests a “mix‐and‐detect” homogenous assay format without precipitation, separation and washing. The whole process (including enzyme reaction and synthesizing CuNCs) can be completed in a single tube. This fluorescence method is low‐cost and simple, and shows potential application in some biomedical studies and clinical diagnostics.