The localization of optical fields is a powerful method of reducing spectroscopic background signals, enabling studies of single fluorescent molecules. Zero-mode waveguides (ZMWs) strongly confine optical fields to zeptoliter (zL, 10−21 L) volumes and can be coupled with fluorescence microscopy to study the dynamics of single enzyme molecules due to their excellent optical confinement, precise positioning, and massive parallelism. The experiments described here exploit arrays of gold-based (Au-based) nanopores derivatized with single copies of the redox enzyme monomeric sarcosine oxidase (MSOX). MSOX contains a covalently bound flavin adenine dinucleotide (FAD) cofactor, which is highly fluorescent in the oxidized state and dark in the reduced state, thus producing a characteristic on-off fluorescence signal synchronous with transitions between oxidation states. Although aluminum (Al) is the common choice for the metallic overlayer in ZMW construction, Au is used here to access its unique surface-binding chemistry. In particular, the signal-to-noise ratio is improved for Au-based ZMWs by selective Au passivation. For MSOX reactions involving both the nominal substrate (sarcosine) and an analogous substrate (proline), statistical analysis of single-molecule temporal trajectories reveals the static heterogeneity of single-enzyme reaction rates, but no dynamic disorder. In addition, the single-molecule data confirm the independence of reduction and oxidation reactions. These structures open the way for systematic studies of the effect of molecular crowding on enzyme dynamics.

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