One-pot synthesis of trypsin-based magnetic metal-organic frameworks for highly efficient proteolysis

Immobilization of enzymes onto metal-organic frameworks (MOFs) through a biomimetic mineralization approach can preserve biological functionality in harsh environments. Despite the success of this approach, the alkaline environment of the reaction system, which is caused by the organic monomers of MOFs, makes it unsuitable for some pH-sensitive enzymes, especially for trypsin. Herein, we reported a facile approach for the one-pot synthesis of trypsin-immobilized magnetic zeolite imidazolate framework-8 (iron oxide@ZIF-8@trypsin), where the growth of ZIF-8 around the citric acid-modified iron oxide and immobilization of trypsin occurred simultaneously when the pH of the reaction system was changed to some extent. With a large specific surface area and a high enzyme loading capacity, the resultant iron oxide@ZIF-8@trypsin exhibited 2.6 times higher enzymatic activity than free trypsin. Moreover, it showed a favourable magnetic response (43 emu g⁻¹) which made the operation and recycling easy and convenient. In addition, iron oxide@ZIF-8@trypsin could be applied as an immobilized enzyme microreactor (IMER) to rapidly and efficiently digest proteins and complex human serum samples with satisfactory results, showing great promise for application in proteomic analysis.