DNAzyme-driven DNA walker biosensor for amplified electrochemical detection of T4 polynucleotide kinase activity and inhibition

T4 polynucleotide kinase (T4 PNK), a bifunctional repair enzyme, catalyzes the transfer of γ-phosphate residues from adenosine triphosphate (ATP) to 5′- hydroxyl termini in nucleic acids，which plays an essential role in regulating DNA phosphorylation modes during the repair of damaged DNA. Herein, a sensitive DNAzyme-driven DNA walker biosensor was developed for amplified electrochemical detection of T4 PNK activity and inhibition based on T4 PNK-triggered phosphorylation reaction and λ exonuclease (λ exo) cleavage. A novel functional hairpin-shaped swing arm DNA (SA) containing a DNA sequence of the Pb 2+ -dependent DNAzyme is designed to specifically bind to and hydrolyze the substrate strand (rSS) sequence that can drive DNA walker. Upon the reaction of SA， T4 PNK and ATP, λ exo removes the mononucleotides from the stem of the phosphorylated SA, unfolding the hairpin structure and initiating DNAzyme-driven DNA walker. Subsequently, the DNA walker can repeatedly bind and cleave rSS with the aid of Pb 2+ , releasing the methylene blue (MB) - labeled fragment of rSS. The T4 PNK activity can be facilely monitored by recording the electrochemical signal change of MB. The proposed biosensor exhibited a broad linear range from 0.01 U mL −1 to 15 U mL −1 with a low detection limit of 0.001 U mL −1 . The proposed biosensor has also been utilized to screen the T4 PNK inhibitors and exhibited good performance in real sample analysis, making such a strategy hold potential promise in biological fields and clinical diagnosis.