The development of micro–sized enzyme sensor based on direct electron transfer type open circuit potential sensing principle

Abstract

One long term goal for biosensor development is towards implantable, long–term sensing for diagnostics, and monitoring of chronic health. The most common electrochemical sensors employ amperometric principles, although there is a fundamental challenge when trying to minimize the sensor size since the measurement current is directly proportional to the size of the working electrode. An alternative principle to amperometry for enzyme sensor is potentiometry, measuring open circuit potential (OCP). In this study, we demonstrate the size independency of an OCP based enzyme sensor focusing on direct electron transfer (DET) type– and quasi–DET–type oxidoreductases for, glucose and L–lactate, respectively. For glucose detection, we employed DET type flavin adenine dinucleotide glucose dehydrogenase, and the engineered L–lactate oxidase derived from Aerococcus viridans (AvLOx) conjugated with amine–reactive phenazine ethosulfate (arPES) was used as the quasi–DET type enzyme for the L–lactate detection. First, both enzymes were immobilized on 3 mm diameter gold disc electrode (GDE), and glucose and L–lactate were monitored based on OCP and transient potentiometric measurement, dOCP/dt. Then, L–lactate and glucose sensors were constructed using 10 µm GDEs, showing L–lactate and glucose concentration dependent OCP and dOCP/dt changes. The sensitivity was nearly equivalent for ΔOCP and dOCP/dt between the sensor using 3 mm and 10 µm GDE even though amperometric signal was entirely lost when measuring the arPES modified AvLOx on the 10 µm electrode. These results clearly demonstrate the size independency of an OCP based enzyme sensor which will be applied for the implantable and continuous monitoring system for metabolites in vivo.