Precise analysis of T4 polynucleotide kinase and inhibition by coupling personal glucose meter with split DNAzyme and ligation-triggered DNA walker

•A precise DNA walker coupling PGM and triple enzyme assisted for T4 PNK analysis is developed.•Split DNAzyme and ligation triggered DNA walker.•A detection limit of 10 mU mL−1 for T4 PNK can be achieved. Monitoring the information of T4 polynucleotide kinase (T4 PNK) activity and its potential inhibitors is of great significance for the therapeutic protocols development, nucleic acid metabolism process investigating, and kinase-targeted drug discovery. In this study, we establish a split DNAzyme and ligation-triggered DNA walker for precise analysis of T4 PNK and inhibition by personal glucose meters (PGM). The DNAzyme is divided into two independent pieces, which can reduce the background signal. After being phosphorylated at 5′-hydroxyl termini by T4 PNK and hybridization with template DNA, a complete DNAzyme unit can be generated and released with the addition of DNA ligase and invasive DNA. It shows high catalytic cleavage toward the substrate strand of the sandwich structure. The releasing invertase of detection probe can catalyze the hydrolysis of sucrose into glucose, which is measured using a pocket PGM. Using this triple enzyme (ligase, DNAzyme, and invertase) assistance catalysis strategy, the T4 PNK detection limit is determined to be 10 mU mL−1. Additionally, the inhibition effects also have been satisfactorily investigated. Furthermore, the practicality of our method is tested toward target spiked in diluted human serum, which indicating that it is promising for biological process investigation and clinic diagnostics.