Mechanistic insights into iontronic glucose biosensing in enzyme-functionalized single nanochannels

Iontronic biosensing strategies based on enzyme-functionalized nanochannels rely on the transduction of local pH variations generated by enzymatic reactions at confined interfaces. Despite glucose oxidase (GOx) being one of the most extensively studied enzymes in biosensing, its implementation in iontronic single-nanochannel sensors remains comparatively limited and the factors governing signal generation are still poorly understood. Here, we investigate the mechanisms underlying glucose sensing in GOx-functionalized solid-state single nanochannels. GOx was immobilized within bullet-shaped nanochannels using a cationic polyelectrolyte that promotes enzyme adsorption while amplifying local pH-induced variations in surface charge density. Systematic studies were performed to evaluate the influence of the amount of enzyme, dissolved oxygen availability, and substrate concentration on the sensing response. The results are consistent with a mechanism in which the iontronic response is governed by a steady-state proton concentration established near the nanochannel interface, resulting from the balance between enzymatic proton generation and mass transport. Consequently, both the amount of immobilized enzyme and the local oxygen availability strongly influence signal generation. Based on these findings, a mechanistic framework is proposed that rationalizes the observed dependencies and provides quantitative guidelines for sensor optimization. Based on this understanding, a glucose-dependent iontronic response was demonstrated over the 50-5000 μM range without external oxygen supplementation. More broadly, the results establish design principles for iontronic biosensors based on oxidoreductases and pH-coupled signal transduction.