Electrochemical biosensors for polynucleotide kinase activity assay and inhibition screening based on phosphorylation reaction triggered λ exonuclease and exonuclease Ⅰ cleavage

摘要

A simple and sensitive electrochemical strategy was developed for detecting T4 polynucleotide kinase (PNK) activity and screening inhibitors based on phosphorylation reaction triggered λ exonuclease and exonuclease Ⅰ cleavage. Firstly, a double strand DNA was designed with 5′-SH modification of probe DNA and 5′-OH modification of target DNA. After assembly on the electrode surface, the double strand DNA can block the diffusion of the electrochemical redox probe of Fe(CN)_6~(3-), and lead to a weak electrochemical signal. However, the assembled target DNA can be phosphorylated at its 5′-OH terminal, which was catalyzed by PNK in the presence of ATP as phosphate group donor. Then, the phosphorylated target DNA can be digested by λ exonuclease and the probe DNA was remained on the electrode surface, which can be further digested by exonuclease Ⅰ. After the above two kinds of digestion reactions, the interface transfer resistance of the electrode was decreased greatly and the electrochemical signal was increased significantly. The electrochemical signal intensity was proportional to the activity of PNK. The proposed method showed wide linear range of 0.005-5 unit/mL and low detection limit of 0.0018 unit/mL (S/N = 3). The inhibition activity of (NH_4)_2SO_4, ADP, Na_2HPO_4 and EDTA on PNK was also verified using this method.