High-Efficiency Enzyme Assay and Screening of Enzyme-Inhibiting Nanomaterials Using Capillary Electrophoresis with Hierarchically Porous Metal–Organic Framework-Based Immobilized Enzyme Microreactor

Enzyme-inhibiting nanomaterials have significant potential for regulating enzyme activity. However, a universal and efficient method for systematically screening and evaluating the inhibitory effects of various nanomaterials on drug target enzymes has not been established. While the integrated technique of immobilized enzyme microreactor (IMER) with capillary electrophoresis (CE) serves as an effective tool for enzyme analysis, it still faces challenges such as low enzyme loadability, unsatisfactory stability, and limited applicability. Herein, hierarchical porous metal-organic frameworks (HP-MOFs) were explored as high-performance enzyme immobilization carriers and stationary phases to develop a novel HP-MOFs-based IMER-CE microanalysis system for efficient online enzyme assay and systematic screening of enzyme-inhibiting nanomaterials. As a proof-of-concept demonstration, the model enzyme xanthine oxidase (XOD) was immobilized on a HP-UiO-66-NH₂ coated capillary, serving as an efficient and durable IMER for screening potential XOD-inhibiting nanomaterials. The hierarchically micro- and mesoporous structure and superior enzyme loadability of as-prepared HP-UiO-66-NH₂-IMER was intensively characterized, followed by systematic evaluation of the separation performance of HP-UiO-66-NH₂ coated column and the enzyme kinetics of the immobilized XOD. Compared to the microporous UiO-66-NH₂-IMER, the HP-UiO-66-NH₂-IMER-CE system showed significant improvements in enzyme loading, maximum reaction rate, repeatability, and long-term stability. Furthermore, the established method was effectively employed to screen the XOD inhibitory activity of various nanomaterials, revealing that graphene oxide, single wall carbon nanotube and three other nanomaterials exhibited inhibitory potentials. The HP-MOFs-based microanalysis system can be easily expanded by modifying the types of immobilized enzymes and holds the potential to accelerate the identification and rational design of effective enzyme-inhibiting nanomaterials.