Electrochemical immunoassays for the detection the activity of DNA methyltransferase by using the rolling circle amplification technique
Electrochemical immunoassays for the detection the activity of DNA methyltransferase by using the rolling circle amplification technique
复制标题
DOI：
10.1007/s00604-013-1141-1
复制
发表时间：
2014-02
影响因子：
5.7
通讯作者：
Zhenning Xu;Huanshun Yin;Z. Tian;Yunlei Zhou;S. Ai
中科院分区：
文献类型：
--
作者：
Zhenning Xu;Huanshun Yin;Z. Tian;Yunlei Zhou;S. Ai
本文报道了一种测定甲基转移酶（MTase）活性的电化学方法。甲基结合结构域-1蛋白被应用于识别ds-DNA的CpG（-C-磷酸-G-）岛中的对称甲基化胞嘧啶，然后特异性地结合抗His标签抗体。超支化滚环扩增（RCA）用于提高灵敏度。当双链DNA被M.Sss I甲基转移酶处理时，5′-CCGG-3′序列被甲基化，并被甲基结合蛋白识别。接着，抗His标签、生物素化IgG、链霉亲和素和生物素化寡核苷酸依次捕获在电极表面上。随后，启动RCA反应，并将链霉亲和素标记的碱性磷酸酶固定在电极表面上。在pH 9.8的条件下，ALP能够催化磷酸1-萘酯水解生成1-萘酚。用1-萘酚的氧化峰电流监测甲基化过程。差示脉冲伏安法得到的响应与M.SssI MTase的浓度在0.1 ~ 40单位mL−1的范围内呈线性关系，检测限为0.03单位mL−1（SNR = 3）。紫杉醇对M.Sss I MTase活性的抑制作用也进行了研究。图描述了一种用于检测DNA甲基化的电化学免疫传感方法，结合滚环扩增技术测定DNA甲基转移酶活性。
We report on an electrochemical method for the determination of the activity of the enzyme methyltransferase (MTase). The methyl-binding domain-1 protein was applied to recognize symmetrically methylated cytosine in CpG (-C-phosphate-G-) islands of ds-DNA which then specifically bind to anti-His tag antibody. Hyperbranched rolling circle amplification (RCA) was used to improve sensitivity. When the dsDNA was treated with M.Sss I methyltransferase, the sequence 5′-CCGG-3′ was methylated and recognized by the methyl binding protein. In turn, the anti-His tag, biotinylated IgG, streptavidin and biotinylated oligonucleotide were captured successively on the surface of an electrode. Subsequently, the RCA reaction was initiated and streptavidin-labeled alkaline phosphatase immobilized on the surface of the electrode. ALP was able to catalyze the hydrolysis of 1-naphthyl phosphate to form 1-naphthol at pH 9.8. The oxidation peak current of 1-naphthol was used to monitor the methylation process. The response obtained by differential pulse voltammetry was linearly related to the concentration of M.SssI MTase in the range from 0.1 to 40 unit mL−1, and the detection limit was 0.03 unit mL−1(at an SNR of 3). The inhibitory action of paclitaxel on the activity of M.Sss I MTase also was investigated.FigureAn electrochemical immunosensing method is described for the detection of DNA methylation, assaying DNA methyltransferase activity by combining the rolling circle amplification technique.