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Int J Biol Macromol. 2018 Jul 1;113:38-44. doi: 10.1016/j.ijbiomac.2018.02.057. Epub 2018 Feb 11.

DNA-directed trypsin immobilization on a polyamidoamine dendrimer-modified capillary to form a renewable immobilized enzyme microreactor.

Author information

1
College of Science, Beijing University of Chemical Technology, Beijing 100029, China.
2
The MOH Key Laboratory of Geriatrics, Beijing Hospital, National Center of Gerontology, Beijing 100730, China.
3
College of Science, Beijing University of Chemical Technology, Beijing 100029, China. Electronic address: suping@mail.buct.edu.cn.
4
College of Science, Beijing University of Chemical Technology, Beijing 100029, China. Electronic address: yangyi@mail.buct.edu.cn.

Abstract

A novel type of trypsin capillary microreactor was developed based on a DNA-directed immobilization (DDI) technique applied to a fused-silica capillary modified with polyamidoamine (PAMAM) dendrimers. Trypsin binding to the inner wall of the capillary was confirmed by confocal laser scanning microscopy. The properties of the trypsin-DNA conjugated, PAMAM-modified capillary microreactor were investigated by monitoring hydrolysis of Nα-benzoyl-L-arginine ethyl ester. Through the hybridization and dehybridization of the DNA, the inner wall of the capillary functionalized with trypsin can be regenerated, thus indicating the renewability of this enzyme microreactor. In addition, these results demonstrated that introduction of PAMAM enabled higher amounts of trypsin to be immobilized, markedly improving the enzymolysis efficiency, compared with traditional modified capillaries. The digestion performance of the trypsin capillary microreactor was further evaluated by digesting cytochrome C, and a peptide numbers of 8, and a sequence coverage of 59% were obtained. This renewable and efficient immobilized trypsin capillary microreactor combines advantages of both DDI technology and PAMAM, and is potentially adaptable to high-throughput enzyme assays in biochemical and clinical research.

KEYWORDS:

Capillary enzyme microreactor; DNA-directed immobilization; Trypsin

PMID:
29444474
DOI:
10.1016/j.ijbiomac.2018.02.057
[Indexed for MEDLINE]

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