Zhou Hong, Han Tongqian, Wei Qin, Zhang Shusheng
Shandong Provincial Key Laboratory of Detection Technology for Tumor Markers, School of Chemistry and Chemical Engineering, Linyi University , Linyi 276005, People's Republic of China.
Key Laboratory of Chemical Sensing and Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan , Jinan 250022, People's Republic of China.
Anal Chem. 2016 Mar 1;88(5):2976-83. doi: 10.1021/acs.analchem.6b00450. Epub 2016 Feb 17.
Herein, an original electrochemiluminescence (ECL) method for the detection of DNA methyltransferase (MTase) activity is presented based on the efficient enhanced ECL of CdS quantum dots (QDs) through catalytic generation of coreactant and energy transfer by glucose oxidase mimicking gold nanoparticles (Au NPs). Briefly, a double-stranded DNA (ds-DNA) containing the symmetric sequence of 5'-CCGG-3' was bonded to the CdS QDs modified glassy carbon electrode (GCE). After that, the electrode was incubated with M.SssI CpG MTase which catalyzed the methylation of the specific CpG dinucleotides. Subsequently, the electrode was treated with a restriction endonuclease HpaII which could recognize and cut off the 5'-CCGG-3' sequence. Once the CpG site in the 5'-CCGG-3' was methylated, the recognition function of HpaII was blocked, and it could not cut off the ds-DNA. Later, Au NPs were combined with the end of the ds-DNA section which was not cut off and has -SH groups. Therefore, the higher M.SssI MTase activity could lead to more Au NPs immobilized on ds-DNA. Au NPs could not only catalyze the oxidation of glucose with cosubstrate oxygen, producing gluconate and hydrogen peroxide (H2O2) which served as the ECL coreactant of CdS QDs, but also enhanced CdS QDs ECL via energy transfer (ET). Thus, the methylation event corresponding to the MTase activity could be monitored and amplified by this method. Finally, a logarithmic linear correlation between the ECL intensity of CdS QDs and the activity of M.SssI MTase that ranged from 1.0 to 120 U mL(-1) with the detection limit of 0.05 U mL(-1) was obtained.
在此，基于通过模拟葡萄糖氧化酶的金纳米颗粒（Au NPs）催化生成共反应剂和能量转移实现的硫化镉量子点（QDs）高效增强电化学发光（ECL），提出了一种用于检测DNA甲基转移酶（MTase）活性的原创ECL方法。简而言之，将含有5'-CCGG-3'对称序列的双链DNA（ds-DNA）连接到修饰有CdS QDs的玻碳电极（GCE）上。之后，将电极与M.SssI CpG MTase孵育，该酶催化特定CpG二核苷酸的甲基化。随后，用限制性内切酶HpaII处理电极，该酶可识别并切断5'-CCGG-3'序列。一旦5'-CCGG-3'中的CpG位点被甲基化，HpaII的识别功能就会被阻断，无法切断ds-DNA。之后，Au NPs与未切断且带有-SH基团的ds-DNA片段末端结合。因此，更高的M.SssI MTase活性会导致更多的Au NPs固定在ds-DNA上。Au NPs不仅可以与共底物氧气一起催化葡萄糖氧化，生成葡萄糖酸和过氧化氢（H2O2），作为CdS QDs的ECL共反应剂，还可以通过能量转移（ET）增强CdS QDs的ECL。因此，通过该方法可以监测和放大与MTase活性对应的甲基化事件。最后，获得了CdS QDs的ECL强度与M.SssI MTase活性之间的对数线性相关性，其范围为1.0至120 U mL(-1)，检测限为0.05 U mL(-1)。
