RESUMEN
In the present work the preparation of capillary-based microreactors with immobilized trypsin was investigated. The monolithic support was synthesized from 2-hydroxyethyl methacrylate (HEMA) as a functional monomer and N,N'-methylenebis(acrylamide) (MBA) as a hydrophilic crosslinker. Two monomers contents in the polymerization mixture (27% and 35%) at the ratio of HEMA:MBA=3:2 were tested. The results indicated that the heated mixture of the above monomers and the porogen consisting of 40% 1-decanol, 40% 2-propanol and 20% water was the most appropriate polymerization mixture for preparation of HEMA-MBA copolymer. The elaborated novel procedure of HEMA-MBA hydrophilic monolith preparation allowed for the introduction of higher monomers content compared to earlier literature data. The obtained monolith exhibited lower adsorption of BSA in comparison with the commonly used glycidyl methacrylate-co-ethylene dimethacrylate (GMA-EDMA) material. In the second step, the covalent enzyme attachment on the hydroxyl groups via 1,1'-carbonyldiimidazole activation was performed. Two immobilization techniques were tested. The first approach involved direct trypsin attachment to the hydroxyl groups of two-carbons HEMA chain (microreactor T1). In the other strategy, 5-amino-1-pentanol was employed to form a twelve-atom spacer arm (microreactor TS1). The prepared microreactor s activities and specificities were evaluated using low molecular weight trypsin substrate (Nα-benzoyl-l-arginine ethyl ester (BAEE)) and bovine serum albumin (BSA). The chromatographic analysis of the eluates from T1 and TS1 microreactors indicated that the microreactor T1 showed higher activity toward the BAEE than the microreactor TS1. However, the BSA digestion and subsequent MALDI-TOF MS (Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) analysis of the collected eluates resulted in a sequence coverage of 43.9% and 35.7% in the case of TS1 and T1 microreactors, respectively.
Asunto(s)
Reactores Biológicos , Enzimas Inmovilizadas/química , Proteómica/métodos , Tripsina/química , Acrilamidas/química , Animales , Bovinos , Enzimas Inmovilizadas/metabolismo , Espectrometría de Masas , Metacrilatos/química , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Albúmina Sérica Bovina/análisis , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Tripsina/metabolismoRESUMEN
The preparation of capillary microfluidic reactor with co-immobilized trypsin and chymotrypsin with the use of a low-cost commercially available enzymatic reagent (containing these proteases) as well as the evaluation of its usefulness in proteomic research were presented. The monolithic copolymer synthesized from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) was used as a support. Firstly, the polymerization conditions were optimized and the monolithic bed was synthesized in the fused silica capillary modified with 3-(trimethoxysilyl)propyl methacrylate (γ-MAPS). The polymer containing epoxy groups was then modified with 1,6-diaminohexane, followed by the attachment of glutaraldehyde and immobilization of enzymes. The efficiency of the prepared monolithic Immobilized Enzyme Microreactor (µ-IMER) with regard to trypsin activity was evaluated using the low-molecular mass compound (Nα-benzoyl-l-arginine ethyl ester, BAEE). The activities of both enzymes were investigated using a macromolecular protein (human transferrin, Tf) as a substrate. In the case of BAEE, the reaction product was separated from the substrate using the capillary liquid chromatography and the efficiency of the reaction was determined by the peak area of the substrate. The hydrolysis products of transferrin were analyzed with MALDI-TOF which allows for the verification of the prepared enzymatic system applicability in the field of proteomic research.