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3D-Printed High-Pressure-Resistant Immobilized Enzyme Microreactor (μIMER) for Protein Analysis
Tobias Rainer1, Anna-Sophia Egger2, Ricarda Zeindl1
1Institute of Organic Chemistry and Center for Molecular Biosciences (CMBI), Leopold-Franzens University Innsbruck, 6020 Innsbruck, Austria.
Analytical Chemistry
|June 9, 2022
Summary
Stereolithography 3D printing can now be used for mass spectrometry (MS) applications. A novel washing protocol removes contaminants, enabling the creation of 3D-printed microscale immobilized enzyme reactors for faster protein digestion.
Area of Science:
- Analytical Chemistry
- Materials Science
- Biotechnology
Background:
- Additive manufacturing, specifically stereolithography (SLA) printing, offers high resolution and cost-effectiveness.
- SLA prints often contain contaminants like urethane dimethacrylate, hindering applications in sensitive techniques such as mass spectrometry (MS).
- Developing robust and contamination-free materials is crucial for advancing analytical instrumentation.
Purpose of the Study:
- To develop a method for preparing SLA-printed devices suitable for MS analysis.
- To create a microscale immobilized enzyme reactor (μIMER) using SLA printing for enhanced protein digestion.
- To evaluate the performance and durability of the 3D-printed μIMER in a liquid chromatography-MS/MS system.
Main Methods:
- A stringent washing and post-curing protocol was implemented to eliminate contaminants from SLA prints.
- SLA printing was utilized to fabricate 360 μm I.D. microcolumn chips.
- Columns were packed with polystyrene microspheres and pepsin was covalently immobilized to create the μIMER.
- The μIMER was integrated into an online liquid chromatography-MS/MS setup for protein digestion and peptide mapping.
Main Results:
- The developed protocol successfully mitigated contamination, rendering SLA prints compatible with MS.
- The 3D-printed μIMER demonstrated excellent structural integrity, withstanding pressures over 130 bar.
- The μIMER enabled reproducible protein digestion and peptide mapping with 100% sequence coverage for recombinant proteins.
- Compared to traditional methods, the μIMER achieved 144-fold faster protein digestion for complex proteome samples.
- The μIMER maintained activity for several weeks, showcasing its stability and reusability.
Conclusions:
- A robust protocol enables the use of SLA-printed materials in MS-based analyses.
- 3D-printed μIMERs offer a highly efficient and rapid solution for protein digestion.
- This versatile platform facilitates the creation of customized enzyme reactors for diverse analytical applications.
