An electrochemical biosensor for detection of T4 polynucleotide kinase activity based on host-guest recognition between phosphate pillar[5]arene and methylene blue.

T4 polynucleotide kinase (T4 PNK) is an important DNA repair-related enzyme that plays a crucial role in DNA recombination, replication and damage repair. Herein, an electrochemical biosensor was developed for detection of T4 PNK activity and inhibitor screening based on supramolecular host-guest recognition between phosphate pillar (Dumitrache and McKinnon, 2017) [5] arene (PP5) and methylene blue (MB). The water-soluble PP5 employed as the host for complexation of MB guest molecules. The substrate DNA with 5'-hydroxyl group was first self-assembled on the gold electrode surface through the chemical adsorption of the thiol group, which was phosphorylated in the presence of T4 PNK and adenosine triphosphate (ATP). TiO served as a bridge to link phosphorylated DNA and PP5 via the robust phosphate-Ti-phosphate chemistry. The immobilized PP5 captured the MB on electrode surface via the supramolecular host-guest recognition interaction, resulting in an enhanced electrochemical response signal. The electrochemical signal is proportional to the T4 PNK concentration in the range of 2 × 10 to 5 U mL with a detection limit of 1 × 10 U mL. It was also successfully used for PNK inhibitor screening and PNK activity assay in HeLa cell lysates sample. The proposed strategy provides a novel sensing platform for kinase activity assay and inhibitor screening, holding a great potential in clinical diagnostics, inhibitor screening, and nucleotide kinase-target drug discovery.