Ferrocene-functionalized SWCNT for electrochemical detection of T4 polynucleotide kinase activity.
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.
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.
Yonghong
Y
Wang
Xiaoxiao
X
He
Kemin
K
Wang
Xiaoqi
X
Ni
Jing
J
Su
Zhifeng
Z
Chen
D017122:Bacteriophage T4 / Q000201:enzymology*; D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D055664:Electrochemical Techniques / Q000379:methods*; D005296:Ferrous Compounds / Q000737:chemistry*; D000075163:Metallocenes; D037742:Nanotubes, Carbon / Q000737:chemistry*; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012680:Sensitivity and Specificity; D014025:Titanium / Q000737:chemistry
D005296:Ferrous Compounds; D000075163:Metallocenes; D037742:Nanotubes, Carbon; D004247:DNA; D014025:Titanium; D011116:Polynucleotide 5'-Hydroxyl-Kinase; C004998:ferrocene