4.7 Article

Electrochemical detection of T4 polynucleotide kinase activity based on magnetic Fe3O4@TiO2 nanoparticles triggered by a rolling circle amplification strategy

Journal

TALANTA
Volume 241, Issue -, Pages -

Publisher

ELSEVIER
DOI: 10.1016/j.talanta.2022.123272

Keywords

T4 polynucleotide kinase; Fe3O4@TiO2 core-shell nanoparticles; Rolling circle amplification; Magnetic gold electrode; Electrochemical sensors

Funding

  1. National Natural Science Foundation of China [21665027, 21565031]
  2. Applied Basic Research Project of Yunnan Provincial Sci-ence and Technology Department [202001AT070012]
  3. Yunnan Provincial Department of Education Science Research Fund Project [2021Y665]
  4. YMU-DEAKIN International Associated Laboratory on Functional Materials

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An ultrasensitive electrochemical detection method using Fe3O4@TiO2 core-shell nanoparticles was developed for monitoring the activity and inhibition of T4 PNK. The method showed high sensitivity and low detection limit for T4 PNK activity, making it useful for screening T4 PNK inhibitors and potential applications in nucleotide kinase-target drug discovery and early clinical cancer diagnosis.
An ultrasensitive electrochemical detection of the activity and inhibition of T4 polynucleotide kinase (T4 PNK) was developed by using magnetic Fe3O4@TiO2 core-shell nanoparticles, which was triggered by a rolling circle amplification strategy (Fe3O4@TiO2-RCA). We used Fe3O4@TiO2 as a substrate to anchor a DNA primer. DNA S1 with 5'-OH termini was phosphorylated in the presence of T4 PNK and ATP, which was adsorbed on the surface of Fe3O4@TiO2 NPs and served as the primer for subsequent RCA reactions. After adding circular template DNA S2, RCA was initiated in the presence of phi29 DNA polymerase and dNTPs. Then, Fc-labeled DNA S3 (Fc-S3) was hybridized with RCA. The obtained Fe3O4@TiO2-RCA was adsorbed on the surface of a magnetic gold electrode (MGE) by magnetic enrichment, resulting in an enhanced electrochemical signal. The T4 PNK activity can be monitored by measuring the electrochemical signal generated. This electrochemical assay is sensitive to the activity of T4 PNK with a dynamic linear range of 0.00001-20 U/mL and a low detection limit of 3.0 x 10(-6) U/mL. The proposed strategy can be used to screen the T4 PNK inhibitors, so it has great potential in the discovery of nucleotide kinase-target drug and early clinical diagnosis of cancer.

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