Open Access3D-Printed High-Pressure-Resistant Immobilized Enzyme Microreactor (μIMER) for Protein Analysis
Author(s) -
Tobias Rainer,
Anna-Sophia Egger,
Ricarda Zeindl,
Martin Tollinger,
Marcel Kwiatkowski,
Thomas Müller
Publication year - 2022
Publication title -
analytical chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.117
H-Index - 332
eISSN - 1520-6882
pISSN - 0003-2700
DOI - 10.1021/acs.analchem.1c05232
Subject(s) - chemistry , chromatography , immobilized enzyme , polystyrene , microreactor , polymer , nanotechnology , organic chemistry , enzyme , materials science , catalysis
Additive manufacturing (3D printing) has greatly revolutionized the way researchers approach certain technical challenges. Despite its outstanding print quality and resolution, stereolithography (SLA) printing is cost-effective and relatively accessible. However, applications involving mass spectrometry (MS) are few due to residual oligomers and additives leaching from SLA-printed devices that interfere with MS analyses. We identified the crosslinking agent urethane dimethacrylate as the main contaminant derived from SLA prints. A stringent washing and post-curing protocol mitigated sample contamination and rendered SLA prints suitable for MS hyphenation. Thereafter, SLA printing was used to produce 360 μm I.D. microcolumn chips with excellent structural properties. By packing the column with polystyrene microspheres and covalently immobilizing pepsin, an exceptionally effective microscale immobilized enzyme reactor (μIMER) was created. Implemented in an online liquid chromatography-MS/MS setup, the protease microcolumn enabled reproducible protein digestion and peptide mapping with 100% sequence coverage obtained for three different recombinant proteins. Additionally, when assessing the μIMER digestion efficiency for complex proteome samples, it delivered a 144-fold faster and significantly more efficient protein digestion compared to 24 h for bulk digestion. The 3D-printed μIMER withstands remarkably high pressures above 130 bar and retains its activity for several weeks. This versatile platform will enable researchers to produce tailored polymer-based enzyme reactors for various applications in analytical chemistry and beyond.