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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

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Abstract

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-Zr4+-phosphate chemistry allowed Zr4+ 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.

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Funding

This work was supported by the National Natural Science Foundation of China (Nos. 21665027, 21565031), Applied Basic Research Project of Yunnan Provincial Science and Technology Department (No. 202001AT070012), Graduate Scientific Research Foundation of Yunnan Minzu University (Nos. 2023SKY028, 2023SKY027), and YMU-DEAKIN International Associated Laboratory on Functional Materials.

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  1. Key Laboratory of Environmental Functional Materials of Yunnan Province Education Department, Yunnan Minzu University, Kunming, 650504, People’s Republic of China

    Yongju Wu, Jinfei Yi, Aiwen Su, Yanli Zhang, Hongbin Wang, Lijuan Yang & Pengfei Pang

  2. School of Life and Environmental Sciences, Deakin University, Geelong, VIC, 3217, Australia

    Wenrong Yang

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  1. Yongju Wu
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Wu, Y., Yi, J., Su, A. et al. 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. Microchim Acta 190, 394 (2023). https://doi.org/10.1007/s00604-023-05983-w

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