Immobilization of proteolytic enzymes on replica-molded thiol-ene micropillar reactors via thiol-gold interaction
| Autor: | Jouko Peltonen, Jawad Sarfraz, Sari Tähkä, Tiina Sikanen, Riccardo Provenzani, Susanne K. Wiedmer, Lauri Urvas, Ville Jokinen |
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| Přispěvatelé: | University of Helsinki, Åbo Akademi University, Department of Chemistry and Materials Science, Aalto-yliopisto, Aalto University, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, Divisions of Faculty of Pharmacy, Department of Chemistry, Susanne Wiedmer / Principal Investigator, Tiina Sikanen / Chemical Microsystems Lab |
| Rok vydání: | 2019 |
| Předmět: |
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Molecular Immobilized enzyme PROTEINS Microfluidics FABRICATION Metal Nanoparticles 02 engineering and technology TRYPSIN Bradykinin 01 natural sciences Biochemistry Soft lithography Analytical Chemistry Hydrolysis CHEMISTRY Lab-On-A-Chip Devices Animals Chymotrypsin Enzyme immobilization Gold nanoparticles Sulfhydryl Compounds Thiol-enes chemistry.chemical_classification Mass spectrometry Chemistry SOFT-LITHOGRAPHY 010401 analytical chemistry Proteolytic enzymes PERFORMANCE Enzymes Immobilized Microreactors 021001 nanoscience & nanotechnology 0104 chemical sciences INTERFACE Chemical engineering 317 Pharmacy Colloidal gold Thiol Cattle MICROCHIP ELECTROPHORESIS Gold Microreactor 0210 nano-technology |
| Zdroj: | Analytical and Bioanalytical Chemistry |
| ISSN: | 1618-2650 1618-2642 |
| Popis: | openaire: EC/H2020/| 311705/EU//CUMTAS We introduce rapid replica molding of ordered, high-aspect-ratio, thiol-ene micropillar arrays for implementation of microfluidic immobilized enzyme reactors (IMERs). By exploiting the abundance of free surface thiols of off-stoichiometric thiol-ene compositions, we were able to functionalize the native thiol-ene micropillars with gold nanoparticles (GNPs) and these with proteolytic α-chymotrypsin (CHT) via thiol-gold interaction. The micropillar arrays were replicated via PDMS soft lithography, which facilitated thiol-ene curing without the photoinitiators, and thus straightforward bonding and good control over the surface chemistry (number of free surface thiols). The specificity of thiol-gold interaction was demonstrated over allyl-rich thiol-ene surfaces and the robustness of the CHT-IMERs at different flow rates and reaction temperatures using bradykinin hydrolysis as the model reaction. The product conversion rate was shown to increase as a function of decreasing flow rate (increasing residence time) and upon heating of the IMER to physiological temperature. Owing to the effective enzyme immobilization onto the micropillar array by GNPs, no further purification of the reaction solution was required prior to mass spectrometric detection of the bradykinin hydrolysis products and no clogging problems, commonly associated with conventional capillary packings, were observed. The activity of the IMER remained stable for at least 1.5 h (continuous use), suggesting that the developed protocol may provide a robust, new approach to implementation of IMER technology for proteomics research. [Figure not available: see fulltext.]. |
| Databáze: | OpenAIRE |
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