An electrochemical biosensor for T4 polynucleotide kinase activity identification according to host–guest recognition among phosphate pillar[5]arene@palladium nanoparticles@reduced graphene oxide nanocomposite and toluidine blue

T4 polynucleotide kinase (T4 PNK) helps with DNA recombination and repair. In this work, a phosphate pillar[5]arene@palladium nanoparticles@reduced graphene oxide nanocomposite (PP5@PdNPs@rGO)–based electrochemical biosensor was created to identify T4 PNK activities. The PP5 used to complex toluidine blue (TB) guest molecules is water-soluble. With T4 PNK and ATP, the substrate DNA, which included a 5ʹ-hydroxyl group, initially self-assembled over the gold electrode surface by chemical adsorption of the thiol units. Strong phosphate-Zr 4+ -phosphate chemistry allowed Zr 4+ to act as a bridge between phosphorylated DNA and PP5@PdNPs@rGO. Through a supramolecular host–guest recognition connection, TB molecules were able to penetrate the PP5 cavity, where they produced a stronger electrochemical response. With a 5 × 10 −7 U mL −1 detection limit, the electrochemical signal is linear in the 10 −6 to 1 U mL −1 T4 PNK concentration range. It was also effective in measuring HeLa cell lysate–related PNK activities and screening PNK inhibitors. Nucleotide kinase–target drug development, clinical diagnostics, and screening for inhibitors all stand to benefit greatly from the suggested technology, which offers a unique sensing mechanism for kinase activity measurement. Graphical Abstract