Spatial organization of glucose oxidase and catalase in layered polyethylenimine coated carbon nanotube electrodes affects coupled reaction kinetics and oxygen recycling in glucose biosensors.

The buildup of H2O2 often diminishes the sensitivity and stability of glucose oxidase (GOx)-based glucose biosensors. This work assessed four carbon nanotubes (CNT) and polyethylenimine (PEI) - based biocatalyst configurations using a layer-by-layer approach (CNT/PEI/GOx; CNT/PEI/GOx-Cat; CNT/PEI/GOx/PEI/Cat; CNT/PEI/Cat/PEI/GOx) to modulate the H2O2 diffusion route and O2 regeneration via catalase (Cat). Voltammetric and amperometric analyses were conducted in 0.1 M phosphate-buffered saline at pH 7.4 (aerobic conditions) at a potential of -0.45 V vs. Ag/AgCl. The optimal configuration was CNT/PEI/GOx/PEI/Cat, exhibiting analytical performance metrics of Jmax of 22.0 μA cm-2, Kmapp of 6.55 mM, sensitivity of 3.34 μA×cm-2×mM-1, and LOD of ≈ 44.84 μM. This structure exhibited an active enzyme coverage (as FADH2) of 16.47 pM·cm-2 and maintained signal retention of around 90% through the fourth cycle, showing that the rate-limiting factor under amperometric circumstances is governed by the generation, diffusion, and conversion of H2O2, as well as the availability of O2, rather than exclusively by direct electron transfer (DET). Positioning catalase as the topmost layer above GOx (isolated from PEI) efficiently inhibits H2O2 buildup next to the electrode while regenerating local O2, hence optimizing catalytic current and stability. This configuration also achieved the most accurate glucose recovery (average error ∼3%) in spiked solutions containing common electroactive interferents, confirming its practical reliability for complex sample analysis. These results provide a structured framework for the development of more sensitive and resilient glucose biosensors.