Citation: Dailu Jia, Wenjiao Fan, Wei Ren, Chenghui Liu. One-step detection of T4 polynucleotide kinase activity based on single particle-confined enzyme reaction and digital particle counting[J]. Chinese Chemical Letters, ;2023, 34(4): 107673. doi: 10.1016/j.cclet.2022.07.016 shu

One-step detection of T4 polynucleotide kinase activity based on single particle-confined enzyme reaction and digital particle counting

Dailu Jia ^{a, b} ,
Wenjiao Fan ^{a, b} ,
Wei Ren ^{a, b} ,
Chenghui Liu ^{a, b, *,}

a.
Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Xi'an 710119, China
b.
Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710119, China

liuch@snnu.edu.cn

Received Date: 22 April 2022
Revised Date: 22 June 2022
Accepted Date: 8 July 2022
Available Online: 10 July 2022

Figures(4)

T4 polynucleotide kinase (T4 PNK) is a pivotal enzyme for DNA replication, recombination, and DNA damage repair. Herein, a robust single particle counting-based assay has been developed for the high-sensitive determination of T4 PNK activity through only a simple one-step reaction. Taking benefit of the exceptional space-confined enzymatic property of T4 PNK towards DNA substrates on a single nanoparticle, the T4 PNK activity can be precisely determined by counting the fluorescence-positive nanoparticles in a digital manner with a total internal reflection fluorescent microscope (TIRFM). Due to the featured spatial-confined enzymatic property of T4 PNK and the single particle counting-based signal readout, T4 PNK can be effectively differentiated from other interfering enzymes. This facile strategy has been also successfully applied to screen T4 PNK inhibitor and accurately determine T4 PNK activity in complex biological samples, paving a potential avenue for the digital analysis of biomarkers.
- T4 PNK,
- Single particle counting,
- Total internal reflection fluorescent microscope,
- Space-confined enzyme reaction,
- Nanoparticles
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