Interface-Limited Amperometric Cholesterol Biosensing in Ultrathin Pd-NPs-Based-Enzyme Films

High Resolution Image Download MS PowerPoint Slide This manuscript investigates the limitations of enzyme–electrocatalyst coupling in one-step designed amperometric cholesterol biosensors. One-step electrodeposition from Pd-ChOx-Nafion electrolyte produces a bioinorganic hybrid sensing layer, while preserving the enzyme’s native conformation and biocatalytic activity. Despite structural coupling between Pd and ChOx, the amperometric response remains dominated by enzymatic H 2 O 2 production, indicating that direct electronic communication between ChOx and the electrode is not established. More specifically, we show that direct electron transfer (DET) cannot occur even in ultrathin electrodeposited nanostructured films, likely due to oxygen-dominated electrochemistry. Furthermore, the sensitivity of cholesterol determination using Pd-NPs/ChOx/Nafion-modified electrodes was independent of layer thickness and architecture, highlighting the limitations of the Pd-NPs–enzyme interface. These findings provide mechanistic insight into ultrathin enzyme–electrocatalyst interfaces and are expected to impact the development of next-generation cholesterol biosensors.