Surface modification with highly-homogeneous porous silica layer for enzyme immobilization in capillary enzyme microreactors

Immobilized enzyme micro-reactors (IMERs) are of vital importance in developing miniaturized bioanalytical systems and have promising applications in various biomanufacturing. An inherent limitation in designing IMERs is the one-dimensional cylindrical geometry of micro-channels that offers limited exposed surface area for molecular reorganization and enzyme immobilization. In this study, we report a robust capillary-IMER based on a three dimensional porous layer open tubular (3D-PLOT) column which is prepared by an easy-to-control surface modification strategy via single-step in situ biphasic reaction. The 3D-PLOT column with highly uniform porous geometry and narrow distribution of porosity can greatly enhance the surface-area-to-volume ratio of the micro-channels, showing the beneficial effects for enzyme immobilization to enhance reaction efficiency and shorten analysis time. Taking trypsin as a model enzyme, enzymatic activities of immobilized enzyme are analyzed. We compare enzyme assays using the proposed 3D-PLOT-IMER with those using normal capillary-IEMR without surface modification as well as free trypsin. The 3D-PLOT-IMER exhibits excellent stability and inter/intra-day reproducibility, and these characteristics imply the reliability of the proposed IMERs for accurate enzyme assay. The feasibility of the proposed method for potential application in biological analysis is demonstrated by coupling the 3D-PLOT-IMER with a nano-LC-MS/MS system for online digestion of standard proteins, cell extraction and living Hela cells. Our study show that the surface modification with the proposed 3D-porous layer is a simple and efficient approach for enzyme immobilization, and could be widely suitable for different kinds of IMERs.