title: Vinyl functionalized silica hybrid monolith-based trypsin microreactor for on line digestion and separation via thiol-ene "click" strategy.
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.
authors.affiliation: State Key Laboratory of Chemo/Biosensing & Chemometrics, College of Chemistry & Chemical Engineering, Hunan University, Changsha 410082, China.
authors.forename: Yingzhuang
authors.initials: Y
authors.lastname: Chen
authors.forename: Minghuo
authors.initials: M
authors.lastname: Wu
authors.forename: Keyi
authors.initials: K
authors.lastname: Wang
authors.forename: Bo
authors.initials: B
authors.lastname: Chen
authors.forename: Shouzhuo
authors.initials: S
authors.lastname: Yao
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
authors.forename: Lihua
authors.initials: L
authors.lastname: Nie
mesh_terms: D001120:Arginine / Q000737:chemistry; D019149:Bioreactors; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods; D057930:Click Chemistry / Q000379:methods*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D008689:Methacrylates / Q000737:chemistry*; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D010446:Peptide Fragments / Q000378:metabolism; D012821:Silanes / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014106:Tosylarginine Methyl Ester / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism*; D014753:Vinyl Compounds / Q000737:chemistry
chemical_list: D004800:Enzymes, Immobilized; D008689:Methacrylates; D010446:Peptide Fragments; D012821:Silanes; D013438:Sulfhydryl Compounds; D014753:Vinyl Compounds; C017492:methacryloxypropyltrimethoxysilane; C061205:tetramethoxysilane; D014106:Tosylarginine Methyl Ester; D001120:Arginine; D014357:Trypsin