A self-directed and reconstructible immobilization strategy: DNA directed immobilization of alkaline phosphatase for enzyme inhibition assays

This study focused on the development of a common method for the reversible and self-directed immobilization of enzymes based on a DNA-directed immobilization (DDI) technique. The successful anchoring of alkaline phosphatases to the surfaces of magnetic nanoparticles was confirmed using confocal las...

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Bibliographic Details
Published in:RSC advances 2016-01, Vol.6 (43), p.36849-36856
Main Authors: Yang, Ye, Su, Ping, Zheng, Kangle, Wang, Ting, Song, Jiayi, Yang, Yi
Format: Article
Language:English
Subjects:
Alkaline phosphatase
Assaying
Deoxyribonucleic acid
Enzymes
Immobilization
Inhibition
Nanoparticles
Surface chemistry
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Summary:This study focused on the development of a common method for the reversible and self-directed immobilization of enzymes based on a DNA-directed immobilization (DDI) technique. The successful anchoring of alkaline phosphatases to the surfaces of magnetic nanoparticles was confirmed using confocal laser scanning microscopy, thermogravimetric analysis and a vibrating sample magnetometer. The length of the DNA linker was optimized, with a base number of 24 providing maximum efficiency for the enzymolysis. Notably, the surface of alkaline phosphatase-functionalized magnetic nanoparticles could be regenerated using a mild dehybridization process of DNA. Furthermore, the resulting single-stranded probe DNA-modified magnetic nanoparticles could be reused to immobilize the alkaline phosphatase, which suggested that the DNA-functionalized surface of magnetic nanoparticles exhibited good reversibility. The biological activity of anchored alkaline phosphatases is evaluated in an enzyme inhibition assay. The results revealed that theophylline exhibited greater inhibitory activity than l -phenylalanine. The proposed protocol demonstrates a simple, mild and economic pathway for fabricating enzyme modified nanomaterial and can be applied in the high-throughput screening of inhibitors. A DNA-directed immobilization technique is used to develop a common method for the reversible and self-directed immobilization of enzymes.
ISSN:2046-2069
2046-2069
DOI:10.1039/c6ra01621a