A bioreference electrode; a reference electrode which shifts its redox potential symmetrically with the working...

Potentiometric bioelectrochemical sensors offer advantages over amperometric ones due to their size-independent Nernstian response. However, their reliance on Ag/AgCl reference electrodes presents instabilities with environmental changes and prolonged use. Ag/AgCl instabilities are due to their reliance on a stable equilibrium between Ag⁺ and Cl^- which may shift with fluctuations in chloride. To address this issue, we propose a reference electrode that incorporates an "inactivated" biological recognition element (inBRE) harboring identical redox species to the active BRE on the working electrode but lacking catalytic activity, thereby expecting a shift in its redox potential symmetrically with the working electrode, termed as a bioreference electrode. As proof of concept, we developed a potentiometric glucose bioelectrochemical sensor using a genetically and structurally inactivated direct electron transfer (DET)-type Burkholderia cepacia glucose dehydrogenase (inBcGDH) as the inBRE. Mutations (αHis476Ala/Asn519Ala) rendered BcGDH catalytically inactive while preserving its redox cofactors, creating a stable redox potential against which to measure. The inBcGDH was covalently immobilized on a 2 mm diameter gold electrode to form a bioreference electrode and integrated into an open-circuit potential (OCP)-based glucose sensor, which was evaluated for stability over 10 days and tested in various environmental factors (ionic strength, pH, and temperature) against traditional Ag/AgCl electrodes. Compared to Ag/AgCl, which experienced 67% signal loss over 10 days, the bioreference electrode exhibited only 23% loss. Additionally, after glucose addition (1 mM) in NaCl concentrations ranging from 50 to 500 mM, the bioreference electrode had only 6.2% signal bias, while Ag/AgCl showed up to 100% signal bias. Further, the bioreference electrode was able to be miniaturized using a 100 μm electrode, maintaining a linear signal response with glucose addition. These results demonstrate the bioreference electrode's ability to enhance potentiometric sensor stability and accuracy under varying conditions.