Microchimica Acta

, 186:3 | Cite as

Determination of the activity of T4 polynucleotide kinase phosphatase by exploiting the sequence-dependent fluorescence of DNA-templated copper nanoclusters

  • Xingxing Zhang
  • Qiang Liu
  • Yan Jin
  • Baoxin LiEmail author
Original Paper


A fluorometric method is described for the determination of the activity of the enzyme T4 polynucleotide kinase phosphatase (T4 PNKP). A short 3′-terminus phosphorylated DNA strand is hybridized with a long DNA strand to produce a partially double-stranded DNA (dsDNA) substrate. On addition of T4 PNKP, the substrate is dephosphorylated to generate the long dsDNA, and then the long dsDNA acted as a template for synthesizing copper nanoclusters (CuNCs). The dsDNA-templated CuNCs display fluorescence with excitation/emission peak wavelengths of 340/570 nm. The fluorescence is DNA sequence-dependent. A DNA substrate was designed to enhance fluorescence and to reduce the background in order to improve the sensitivity of the assay. The assay has an analytical range that extends from 0.07 U mL−1 to 15 U mL−1 and a detection limit of 0.06 U mL−1.

Graphical abstract

The sequence-dependent fluorescence of DNA-templated copper nanoclusters, which are in-situ synthesized through the reduction of CuSO4 by ascorbate (Vc) in the presence of dsDNA template, is utilized to obtain the method for sensitive detection of T4 PNKP activity with near-zero background.


T4 polynucleotide kinase phosphatase activity Fluorescent biosensing Label-free Copper nanoclusters Near-zero background 



This work was supported by the National Natural Science Foundation of China (Grant no. 21775099 and 21475083).

Compliance with ethical standards

The author(s) declare that they have no competing interests.

Supplementary material

604_2018_3102_MOESM1_ESM.docx (662 kb)
ESM 1 (DOCX 662 kb)


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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Key laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical EngineeringShaanxi Normal UniversityXi’anChina

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