Observation of Catalytic Variability of Single Enzyme Zeolitic Imidazole Framework‐8 Nanoparticles

Abstract Metal–organic frameworks (MOFs), such as zeolitic imidazolate framework‐8 (ZIF‐8), are an emerging class of advanced materials for enzyme encapsulation due to their protective properties, stability, and biocompatibility. As a range of enzyme@ZIF‐8 nanoparticles (NPs) with specific functions for practical applications has been demonstrated, scientists aim to strategically design enzyme@ZIF‐8 NPs with optimized catalytic performance. Beyond demonstrating enzyme and ZIF‐8 combinations, a mechanistic understanding of catalytic performance is necessary for the strategic design of novel enzyme@ZIF‐8 NPs. Conventional methods for monitoring the catalytic performance of enzyme@ZIF‐8 NPs rely on ensemble‐averaged measurements, where variation in catalytic activity between individual particles remains unresolved and limits the ability to study catalytic performance. Here, the catalytic variability of single enzyme@ZIF‐8 NPs is investigated using a microwell‐based fluorescence method. Bovine serum albumin (BSA) is utilized to load horseradish peroxidase (HRP) as our model enzyme into ZIF‐8 to synthesize colloidal HRP+BSA@cZIF‐8 NPs. Single‐particle analysis shows distributions of activity in single HRP+BSA@cZIF‐8 NPs, and experiments provide insights into the subpopulations of NPs that produce catalytic variability. This work highlights the need for single‐particle methodologies to understand the mechanisms of catalytic activity of enzyme@MOF NPs, enabling the design of optimized, tailorable enzyme@MOF NPs and enhancing their potential in practical applications.