Single-electrode, voltage-switching biosensing of glucose and ethanol enabled by a dual-functional single-atom nanozyme.

Real-time and full concentration monitoring of substrates and products in microbial fermentation remains a fundamental challenge due to complex matrix interference and signal crosstalk. Herein, we report Fe-N-C single-atom site catalyst nanowires (Fe-SASC/NWs) with atomically dispersed Fe-Nx sites exhibiting dual electrocatalytic specificity for H2O2 reduction at +0.05 V and NADH oxidation at +0.2 V with detection limits of 0.2 μM and 0.1 μM by voltage-switching electrocatalysis, respectively. By co-immobilizing glucose oxidase and alcohol dehydrogenase on Fe-SASC/NWs, a single-electrode biosensor achieves interference-free simultaneous detection of glucose (5-30 mM) and ethanol (5-60 mM) via voltage-switching chronoamperometry with corresponding detection limits of 2 mM and 0.2 μM, respectively. Additionally, the developed biosensor successfully monitored glucose and ethanol during a fed-batch ethanol fermentation process, demonstrating excellent agreement (>90%) with gas chromatography (GC) results. This work pioneers a versatile single-electrode multiplexed sensing strategy based on rationally designed multifunctional nanozymes, offering significant potential for real-time monitoring in complex biomanufacturing processes.