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 Ni2 +-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.