Synthesis of a monolithic, micro-immobilised enzyme reactor via click-chemistry

Abstract

An immobilised enzyme reactor (IMER) in the form of capillary monolith was developed for a micro-liquid chromatography system. The plain monolith was obtained by in situ thermal copolymerisation of glycidyl methacrylate and ethylene dimethacrylate in a fused silica capillary (200 × 0.53 mm ID) by using n-propanol/1,4-butanediol as porogen. The enzyme, α-chymotrypsin (CT), was covalently attached onto the monolith via triazole ring formation by click-chemistry. For this purpose, the monolithic support was treated with sodium azide and reacted with the alkyne carrying enzyme derivative. CT was covalently linked to the monolith by triazole-ring formation. The activity behaviour of monolithic IMER was investigated in a micro-liquid chromatography system by using benzoyl-L-tyrosine ethyl ester (BTEE) as synthetic substrate. The effects of mobile-phase flow rate and substrate feed concentration on the final BTEE conversion were investigated under steady-state conditions. In the case of monolithic IMER, the final substrate conversion increased with increasing feed flow rate and increasing substrate feed concentration. Unusual behaviour was explained by the presence of convective diffusion in the macropores of monolith. The results indicated that the monolithic-capillary IMER proposed for micro-liquid chromatography had significant advantages with respect to particle-based conventional high-performance liquid chromatography-IMERs.

A one-step preparation method of monolithic enzyme reactor for highly efficient sample preparation coupled to mass spectrometry-based proteomics studies

“…Compared to other previously published work on IMER columns, the NAS-AAm-Bis column features mild fabrication condition and an open tubular inner structure with higher protein sequence coverage as observed with BSA analysis [28,29]. We further applied IMER digestion to more complex samples followed by LC-ESI-Orbitrap detection to test if IMER digestion can be applied to large-scale proteomics studies.…”

Various Strategies in Post-Polymerization Functionalization of Organic Polymer-Based Monoliths Used in Liquid Phase Separation Techniques

“…The results revealed that the final substrate conversion increased with increasing the feed flow rate and substrate feed concentration due to the increase of the interaction between the substrate and the enzymatic monolith reactor. The preparation of a monolithic-immobilized enzyme reactor by click-chemistry for micro-LC was recently reported [22]. It involved the preparation of a poly(GMA-co-EDMA) monolith via in situ thermal polymerization in capillary format.…”

Is click chemistry attractive for separation sciences?

“…Soybean trypsin inhibitor was clicked on this support after conjugation through the N ‐hydroxysuccinimide ester of 4‐pentynoic acid to produce an affinity sorbent for trypsin. Porous (GMA‐co‐EDMA) monolithic capillary columns were used to click an alkyne‐carrying enzyme derivative (α‐chemotrypsin) to prepare immobilized enzyme reactors . In both cases, biomolecules have to be derivatized with heterobifunctional reagents carrying the alkyne moiety.…”

“…Such reactions afford high yields and can be carried out in various solvents such as aqueous solutions at room temperature. Due to these numerous benefits, "click reactions" have received more and more interest in many areas such as nanotechnology [2,3], bioconjugation [4,5], DNA functionalization [6,7], surface modification [8][9][10], polymers [11][12][13][14], and others [15][16][17]. Over the last 10 years, click chemistry has also spread to separative sciences and more particularly for the preparation of chromatographic materials [18][19][20][21][22][23].…”

“…As described by these authors, it refers to a “set of powerful, virtually 100% reliable, selective reactions for the rapid synthesis of new compounds via heteroatom links (C–X–C).” Such reactions afford high yields and can be carried out in various solvents such as aqueous solutions at room temperature. Due to these numerous benefits, “click reactions” have received more and more interest in many areas such as nanotechnology , bioconjugation , DNA functionalization , surface modification , polymers , and others . Over the last 10 years, click chemistry has also spread to separative sciences and more particularly for the preparation of chromatographic materials .…”