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Vinyl Functionalized Silica Hybrid Monolith-Based Trypsin Microreactor for on Line Digestion and Separation via Thiol-Ene "Click" Strategy

Vinyl Functionalized Silica Hybrid Monolith-Based Trypsin Microreactor for on Line Digestion and Separation via Thiol-Ene "Click" Strategy

Yingzhuang Chen, Minghuo Wu, Keyi Wang, Bo Chen, Shouzhuo Yao, Hanfa Zou, Lihua Nie

J Chromatogr A. 2011 Nov 4;1218(44):7982-8.

PMID: 21937052

Abstract:

A novel thiol-ene "click" strategy for the preparation of monolithic trypsin microreactor was proposed. The hybrid organic-inorganic monolithic capillary column with ene-functionality was fabricated by sol-gel process using tetramethoxysilane (TMOS) and γ-methacryloxypropyltrimethoxysilane (γ-MAPS) as precursors. The disulfide bonds of trypsin were reduced to form free thiol groups. Then the trypsin containing free thiol groups was attached on the γ-MAPS hybrid monolithic column with ene-functionality via thiol-ene click chemistry to form a trypsin microreactor. The activity of the trypsin microreactor was characterized by detecting the substrate (Nα-p-tosyl-L-arginine methyl ester hydrochloride, TAME) and the product (Nα-p-tosyl-L-arginine, TA) with on-line capillary zone electrophoresis. After investigating various synthesizing conditions, it was found that the microreactor with poly(N,N'-methylenebisacrylamide) as spacer can deliver the highest activity, yielding a rapid reaction rate. After repeatedly sampling and analyzing for 100 times, the monolithic trypsin microreactor still remained 87.5% of its initial activity. It was demonstrated that thiol-ene "click" strategy for the construction of enzyme microreactor is a promising method for the highly selective immobilization of proteins under mild conditions, especially enzymes with free thiol radicals.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP1784038	Nα-p-Tosyl-L-arginine methyl ester hydrochloride		1784-03-8	Price

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