Application of an open-tube immobilized enzyme microreactor constructed by magnetic macroporous silica beads in...

Fast and efficient protein digestion is important in proteomics to improve the performance of protein analysis. In this work, a series of magnetic macroporous silica carriers with pore sizes ranging from 32 nm to 184 nm was synthesized, among which the silica bead with the most suitable pore size and pore structure (SiO₂@Fe₃O₄-650 °C/650 °C, 184 nm) exhibits high enzyme immobilization capacity (76.96 µg mg^-1) and great digestion activity. Trypsin was adsorbed on the silica beads through the formation of a protein corona to prepare an IMER (SiO₂@Fe₃O₄-nIMER). The non-covalent immobilization strategy could maximally preserve the enzymatic activity. SiO₂@Fe₃O-nIMER retained 97% of its enzymatic hydrolysis efficiency even after five cycles, demonstrating excellent reusability. SiO₂@Fe₃O₄-nIMER was further immobilized in the capillary (75 µm i.d.) with the assistance of permanent magnets, constructing an open-tube immobilized enzyme microreactor (SiO₂@Fe₃O₄-cIMER) for dynamic protein digestion. Under the optimal enzymatic digestion conditions (effective length of the enzyme reactor: 5.4 cm and flow rate of the protein sample: 10 µL min^-1), the enzymatic digestion performance of SiO₂@Fe₃O₄-cIMER was evaluated using bovine serum albumin (BSA) and cytochrome C (Cyt C) as model proteins, and the sequence coverages were 97% for BSA (168 peptides matched) and 92% for Cyt C (26 peptides matched). Using a dynamic enzymatic digestion mode, SiO₂@Fe₃O₄-cIMER identified 509 proteins from human serum samples, which was significantly higher than that identified by SiO₂@Fe₃O₄-nIMER (444 proteins) and free-solution enzymatic digestion (359 proteins). The novel enzyme reactor has the advantages of a simple preparation process, good reproducibility, stability and high digestion efficiency, making it suitable for large-scale proteomics research.