Single-Molecule Fluorescence Reveals Catalytic Heterogeneity and Active Species in Copper Nanoparticle-Catalyzed Click Reactions

The copper-catalyzed azide–alkyne cycloaddition (CuAAC) is a cornerstone of click chemistry and has been widely employed in many applications. However, its mechanism with heterogeneous catalysts remains incompletely understood. Catalytic reactions mediated by metal nanoparticles may occur on the nanoparticle surface via a heterogeneous catalysis pathway and/or through metal ion leaching that leads to a homogeneous reaction pathway. In this work, we developed a novel fluorogenic CuAAC reaction based on a naphthalimide probe that enables single-molecule visualization using total internal reflection fluorescence (TIRF) microscopy. This approach allows direct observation of catalytic turnover events on individual copper nanoparticles (Cu NPs) with high spatial and temporal resolution. Analysis of single-molecule trajectories revealed catalytic heterogeneity and provided kinetic insights, both inaccessible in ensemble measurements. The reaction kinetics followed a Langmuir–Hinshelwood model with noncompetitive adsorption, indicating that the azide and alkyne substrates occupy distinct active sites on the nanoparticle surface. Comparison between single-molecule and ensemble rates suggested that both heterogeneous and homogeneous reaction pathways contribute to the Cu NP-catalyzed reactions. These findings elucidate structure–activity relationships and mechanistic pathways in Cu NP-catalyzed CuAAC, offering guidance for the rational design of improved heterogeneous catalysts for click reactions or other catalytic transformations.