Recent advances of capillary electrophoresis for enzyme analysis (2021–2024)

• Comparative review of capillary electrophoresis in homogeneous vs. heterogeneous enzyme analysis methods. • Stepwise dissection of pre-capillary enzyme analysis workflows: from incubation to detection. • Classification framework for emerging in-capillary enzyme analysis methodologies. • Immobilization strategies for enzyme microreactors. Capillary electrophoresis (CE) is a robust and versatile analytical technique that has demonstrated significant potential and emerging trends in enzyme analysis. This review comprehensively summarizes the latest advancements in CE for enzyme analysis from 2021 to 2024, covering both homogeneous and heterogeneous analysis. The advancements in homogeneous CE enzyme analysis, including both pre-capillary enzyme analysis and in-capillary enzyme analysis, are thoroughly examined. Specifically, an in-depth analysis is provided of the general principles, characteristics, and key parameters at each step of the pre-capillary analysis process, such as incubation, termination, separation, and detection. Moreover, this review introduces various novel developments and applications in different mixed modes of in-capillary enzyme analysis. Additionally, it discusses diverse immobilization methods, such as adsorption, cross-linking, covalent bonding, encapsulation, and the recently developed nanogels method, aiming to construct highly efficient and stable immobilized enzyme microreactors (IMERs). This review presents an up-to-date overview of CE applications in enzyme analysis, highlighting its significant potential in this field.