Label-Free Fluorescence-Based Enzyme Assays Using Supramolecular Host-Guest Approaches

Monitoring enzymatic reactions is crucial in enzymology, drug metabolism, and bioengineering. Supramolecular tandem enzyme assays (STA), based on indicator displacement assays, leverage the differential binding affinities of supramolecular hosts for enzymatic substrates and products. STA enables label-free, real-time, and rapid monitoring of enzymatic reactions, offering advantages such as simplicity, high sensitivity, and strong specificity. STA has been extensively employed to determine enzymatic kinetic parameters, screen inhibitors, diagnose biomarkers, and analyze enantiomeric excess. This review summarizes recent advancements in STA strategies across various enzyme types and provides current challenges and perspectives for future development.