Use of Prussian Blue pseudocapacitive properties to amplify the pulsed amperometric readout of biosensors

Enzymatic electrochemical biosensors are a cornerstone technology in enabling further advancements in the field of Continuous Glucose Monitoring (CGM). Pulsed amperometric methods improve the sensitivity and accuracy of electrochemical biosensors. The literature shows that pulsed amperometry increases the sensitivity of enzymatic glucose biosensors based on Prussian Blue (PB). However, the underlying mechanism responsible for this improvement is poorly understood, which impedes further development of this promising measurement method. The present work elucidates the role of the spontaneous reaction between hydrogen peroxide (H₂O₂) and Prussian White (PW) in the sensitivity improvement observed with pulsed amperometry. A charged working electrode (WE) containing PW can catalyze the H₂O₂ reduction in the open-circuit regime (OCP). The consumption of H₂O₂ over a 30-min contact at OCP was 65 % at a PW WE, compared to 13 % at a PB WE. This spontaneous process is associated with a partial discharge of the WE (PW → PB) between the amperometric pulses. The subsequent re-charging (PB → PW) yields the current amplification observed with pulsed amperometry. Based on this consideration, we developed and validated a model for glucose quantification using pulsed amperometry that considers the spontaneous reaction of H₂O₂ with PW. The model achieves 0.998 determination coefficient between glucose concentration and four analytical signals. The insights presented in this work support the optimization and development of the pulsed amperometric detection method in enzymatic glucose biosensors. Additionally, this work advances the understanding of H₂O₂ detection at PB-based sensors, and contributes to the development of precise and accurate enzymatic glucose biosensors.