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 5'-hydroxyl group, initially self-assembled over the gold electrode surface by chemical adsorption of the thiol units. Strong phosphate-Zr-phosphate chemistry allowed Zr 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 U mL detection limit, the electrochemical signal is linear in the 10 to 1 U mL 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.