Sensitive electrochemical assay for T4 polynucleotide kinase activity based on dual-signaling amplification coupled with exonuclease reaction

Abstract

A novel electrochemical strategy for simple and accurate monitoring of the activity and the inhibition of T4 polynucleotide kinase (PNK) is developed by coupling the λ exonuclease (λ exo) cleavage reaction with a dual-signaling amplification method. In the presence of PNK and adenosine triphosphate, the 5′-hydroxyl group of the substrate DNA is phosphorylated, initiating the hydrolysis of the 5′-phosphoryl termini catalyzed by λ exo, and by combining the resulting G–quadruplex–hemin signal-on probe and the ferrocene signal-off probe, a dual-signaling amplification strategy is realized. This approach exhibits a low detection limit of 0.02U/mL for PNK activity analysis, which is lower than or comparable to that of the previously reported PNK assays. Additionally, the inhibition effects of two known PNK inhibitors, namely ammonium sulfate and sodium hydrogen phosphate, have also been successfully detected. This new dual-signaling electrochemical biosensor provides a facile platform for the detection of the activity and inhibition of nucleotide kinases. With further exploration, it is possible to extend the as-proposed electrochemical biosensor to the detection of other analytes.