Nanopore and Self‐Assembled Monolayer Engineering of l ‐Lactate Oxidase Gold Electrodes for Enhanced l ‐Lactate Biosensing

Nanoporous architectures offer unique opportunities to spatially organize biocatalysts while preserving efficient interfacial charge transfer. Here, we introduce a tiered enzyme–electrode interface based on nanoporous gold (np‐Au) that enables high‐density, activity‐retaining immobilization of lactate oxidase for sensitive l ‐lactate detection. The np‐Au scaffold, prepared by controlled dealloying of Ag‐Au, exhibits average pore diameter of ∼170 nm, providing geometric confinement compatible with the tetrameric structure of Aerococcus viridans l ‐lactate oxidase (LOx). A mixed self‐assembled monolayer composed of 1‐thioglycerol and Ni 2 + ‐nitrilotriacetic acid‐terminated 12‐thiododecaneamide establishes a bifunctional interface that combines hydrophilic‐antifouling properties with site‐directed immobilization of N‐terminal His‐tagged LOx. This architecture affords high immobilization yield (64%), elevated surface loading (7.44 mg m − 2 ; ∼9 mg g −1 ), and superior retention of enzymatic activity. Electrochemical impedance spectroscopy reveals controlled modulation of interfacial charge‐transfer resistance upon sequential SAM formation and enzyme coupling, confirming the formation of a structurally defined biointerface. The resulting bioelectrode exhibits stable and sensitive amperometric l ‐lactate detection with a linear dynamic range of 0.12 – 3.0 mM and a sensitivity of 75 µA mM −1 cm − 2 , covering clinically relevant concentrations in saliva and blood. The presented tiered design strategy establishes a modular platform for high‐performance nanoporous metal bioelectrodes with controlled enzyme orientation and enhanced operational stability.