Electrochemical detection of T4 polynucleotide kinase based on target-assisted ligation reaction coupled with silver nanoparticles.

Phosphorylation of DNA is a critical event for normal cell processes such as DNA replication, DNA repair and DNA recombination. It is important to develop accurate and convenient approach for the detection of kinase and inhibition. In this work, we have described a novel sensing strategy for T4 polynucleotide kinase. A single-stranded DNA probe is firstly immobilized on the surface of the electrode. After phosphorylated by target kinase, it could be ligated with amino group labeled DNA probe by certain hybridization. Denaturation process is then proceeded to release unligated DNA probes, which may help reduce the background signal. The remained amino groups could be used to adsorb silver nanoparticles (AgNPs) as electrochemical species for the quantification of T4 polynucleotide kinase. This biosensor shows high sensitivity and selectivity. It may also be used for the screening kinase inhibitors. Therefore, it shows great potential utility in biological process investigation, drug discovery and clinical diagnosis.