Micro-immobilized enzyme reactors for mass spectrometry proteomics.

Micro-immobilized enzyme reactors (μ-IMERs) have proven to be superior to traditional enzymatic methods by offering improved enzyme stability, less reagent consumption, and higher reaction efficiency. This review aims to provide an assessment of recent advances in μ-IMER design, fabrication, and proteomic applications. Common immobilization methods including adsorption, covalent binding, affinity binding, and entrapment are discussed, and their respective advantages, challenges and possible future development directions are summarized. The review also covers different μ-IMER designs, including open-tube, packed, monolithic reactors, and membrane-immobilized enzymes, and provides a detailed analysis of their structural architectures and operational performance characteristics. In addition, μ-IMER applications in proteomics are presented, demonstrating their potential for improving sample preparation and analytical workflows. Finally, we summarize the current state of development and the challenges faced, offering new insights into potential future directions.