State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Hunan University, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, Changsha 410082, PR China.
Biosens Bioelectron. 2012 Feb 15;32(1):213-8. doi: 10.1016/j.bios.2011.12.012. Epub 2011 Dec 14.
A novel electrochemical strategy for monitoring the activity and inhibition of T4 polynucleotide kinase (PNK) is developed by use of titanium ion (Ti(4+)) mediated signal transition coupled with signal amplification of single wall carbon nanotubes (SWCNTs). In this method, a DNA containing 5'-hydroxyl group is self-assembled onto the gold electrode and used as substrate for PNK. The biofunctionalized SWCNTs with anchor DNA and ferrocene are chosen as the signal indicator by virtue of the intrinsic 5'-phosphate end of anchor DNA and the high loading of ferrocene for electrochemical signal generation and amplification. The 5'-hydroxyl group of the substrate DNA on the electrode is phosphorylated by T4 PNK in the presence of ATP, and the resulting 5'-phosphoryl end product can be linked with the signal indicator by Ti(4+). The redox ferrocene group on the SWCNTs is grafted to the electrode and generates the electrochemical signal, the intensity of which is proportional to the activity of T4 PNK. This assay can measure activity of T4 PNK down to 0.01 UmL(-1). The developed method is a potentially useful tool in researching the interactions between proteins and nucleic acids and provides a diversified platform for a kinase activity assay.
一种新型电化学策略用于监测 T4 多核苷酸激酶（PNK）的活性和抑制作用，该策略通过使用钛离子（Ti(4+)）介导的信号转换，结合单壁碳纳米管（SWCNTs）的信号放大来实现。在该方法中，含有 5'-羟基的 DNA 自组装到金电极上，并用作 PNK 的底物。具有锚定 DNA 和二茂铁的生物功能化 SWCNTs 被选为信号指示剂，这是由于锚定 DNA 的内在 5'-磷酸末端和二茂铁的高负载量，用于电化学信号的产生和放大。在 ATP 存在的情况下，T4 PNK 将电极上的底物 DNA 的 5'-羟基磷酸化，所得的 5'-磷酸化末端产物可以通过 Ti(4+)与信号指示剂连接。SWCNTs 上的氧化还原二茂铁基团被接枝到电极上，并产生电化学信号，其强度与 T4 PNK 的活性成正比。该测定法可测量低至 0.01 UmL(-1) 的 T4 PNK 活性。该方法为研究蛋白质和核酸之间的相互作用提供了一种潜在有用的工具，并为激酶活性测定提供了多样化的平台。
