Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai, P.R. China.
Bioconjug Chem. 2012 Jan 18;23(1):141-5. doi: 10.1021/bc200523p. Epub 2011 Dec 30.
We herein report a novel electrochemical method in this paper to monitor protein phosphorylation and to assay protein kinase activity based on Zr(4+) mediated signal transition and rolling circle amplification (RCA). First, substrate peptide immobilized on a gold electrode can be phosphorylated by protein kinase A. Then, Zr(4+) links phosphorylated peptide and DNA primer probe by interacting with the phosphate groups. After the introduction of the padlock probe and phi29 DNA polymerase, RCA is achieved on the surface of the electrode. As the RCA product, a very long DNA strand, may absorb a large number of electrochemical speices, Ru(NH(3))(6), via the electrostatic interaction, localizing them onto the electrode surface, initiated by protein kinase A, a sensitive electrochemical method to assay the enzyme activity is proposed. The detection limit of the method is as low as 0.5 unit/mL, which might promise this method as a good candidate for monitoring phosphorylation in the future.
本文报道了一种基于 Zr(4+)介导的信号转换和滚环扩增（RCA）的新型电化学方法，用于监测蛋白质磷酸化和测定蛋白激酶活性。首先，固定在金电极上的底物肽可被蛋白激酶 A 磷酸化。然后，Zr(4+)通过与磷酸基团相互作用将磷酸化肽与 DNA 引物探针连接。引入发夹探针和 phi29 DNA 聚合酶后，在电极表面实现 RCA。由于 RCA 产物是一条非常长的 DNA 链，它可以通过静电相互作用吸附大量电化学物种Ru(NH(3))(6)，将它们定位到电极表面，这是由蛋白激酶 A 引发的，提出了一种用于测定酶活性的灵敏电化学方法。该方法的检测限低至 0.5 单位/mL，有望成为未来监测磷酸化的候选方法。
