期刊
BIOSENSORS & BIOELECTRONICS
卷 150, 期 -, 页码 -出版社
ELSEVIER ADVANCED TECHNOLOGY
DOI: 10.1016/j.bios.2019.111936
关键词
T4 PNK activity; Dark field microscopy; Gold nanoparticles; Magnetic bead
类别
资金
- Shenzhen Municipal government [JCYJ20160301153959476, JCYJ20160324163734374]
- Shenzhen Reform Commission (Disciplinary Development Program for Chemical Biology)
T4 polynucleotide kinase (T4 PNK) plays an essential role in DNA phosphorylation during the DNA repair process. Therefore, the sensitive, selective and convenient detection of T4 PNK activity is of great significance. In this work, we proposed a sensitive non-amplification strategy for the sensing of T4 PNK activity via dark field microscope (DFM) based on magnetic bead (MB)-gold nanoparticle (AuNP) hybrids probe, MB-dsDNA-AuNP (MDA). In the presence of T4 PNK, the 5'-OH termini of DNA are phosphorylated and cleaved into oligonucleotides by lambda exonuclease (lambda exo), resulting in the destruction of the MDA probe and the separation of AuNP from the MB. Through automatic counting of AuNPs from DFM images, T4 PNK activity can be quantitatively measured. This strategy revealed a limit of detection (LOD) as low as 0.0058 U/mL and exhibited a dynamic range from 0.01 to 1 U/mL. The strategy presents an excellent ability to discriminate T4 PNK from the other proteins and enzymes. Notably, this strategy was applied to screen the T4 PNK inhibitors and test the activity in cell lysates, showing great potential for the discovery of new anticancer drugs and other related research field.
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