Fluorogenic Linkage Integration for Nonfluorescent Transformations (FLINT).

Since its creation, single-molecule optical imaging has revolutionized the study of catalytic processes, yet its application largely relies on probing fluorogenic reactions. To overcome this limitation, we propose the Fluorogenic Linkage Integration for Nonfluorescent Transformation (FLINT) approach, an imaging method to resolve nonfluorogenic reactions at the single-molecule level. Using glucose oxidation as a model reaction, we coupled this nonfluorogenic reaction with a fluorogenic Amplex Red (AR) → resorufin (RF) transformation to create a cascading reaction. This integration allowed us to monitor single-turnover events and extract key kinetic parameters for glucose oxidation despite their being invisible under the optical microscope. Our ensemble measurements combining cyclic voltammetry and fluorescence spectroscopy confirmed the cascade reaction mechanism and revealed first-order kinetics for both elementary reaction steps. At the single-molecule level, turnover time analysis provided detailed information on the reaction kinetics, distinguishing the relatively fast glucose oxidation from slower AR oxidation. We further confirmed the validity of the FLINT approach by comparing the catalytic performances of 5 nm gold nanoparticles (AuNPs) against that of 18 × 52 nm gold nanorods (AuNRs) and AuNP@DNA coronazymes. Furthermore, FLINT was used to evaluate the chiral selectivity of d- and l-glucose on coronazymes, suggesting the potential application of FLINT in enantioselective reactions. The FLINT approach is a significant advancement in single-molecule imaging as it enables the study of nonfluorogenic reactions with high spatiotemporal resolution.