The Continuous 3 Month Operation of Open Circuit Potential Based Glucose Sensor Employing Direct Electron Transfer Type Fad Dependent Glucose Dehydrogenase

Abstract

Introduction To control glycemic levels of patients with type 1 diabetes, continuous monitoring of blood glucose concentrations is inevitable for the subsequent infusion of insulin. Since the amount of insulin being supplied is calculated based on the continuous glucose monitoring (CGM) results, CGM is considered as the core component for the continuous and accurate diabetes management. With the increase of the demands on convenient and user-friendly system, much accurate and stable sensing systems are required. Current commercially available CGM sensors employing amperometric enzyme sensor principles, are operated under continuous potential application to the glucose oxidoreductases and redox mediators, to keep their continuous electrochemical-catalytic reaction. Under such harsh conditions, inactivation of enzymes and mediators is unavoidable during continuous operation. Additionally, potential application to the system may cause oxidation of interfering substances directly on the electrode surface. Consequently, these disadvantage of amperometric enzyme sensor principle are the inherent reason for the limitation of current CGM stability and accuracy.We are currently engaged in the development of open circuit potential (OCP) based enzyme glucose sensing principle, which can solve the current issues in the glucose sensors based on amperometric principle. We previously reported OCP based glucose sensor employing direct electron transfer type FAD-dependent glucose dehydrogenase complex (FADGDH) derived from Burkholderia cepacia 1-5. By combining direct electron transfer type FADGDH with OCP principle, the sensor signal could be obtained without being affected by ascorbic acid and acetaminophen. In this study, we demonstrated a long-term operation, over 3 months, using OCP based CGM sensor employing direct electron transfer type FADGDH. Methods Recombinantly prepared bacterial FADGDH, capable of direct electron transfer, was used in this study, and the enzyme was immobilized onto gold electrode by self-assembled monolayer. OCP of the enzyme electrode was monitored continuously toward the sample containing various concentration of glucose, against Ag/AgCl reference electrode. We calculated the ∆OCP as difference between the OCP value at each glucose concentration and the OCP value in presence of 0.1 mM glucose, and then calibration curve was prepared based on plotting ∆OCP versus glucose concentration. Continuous operation was carried out in 100 mM phosphate buffer (pH 7.0) including 20 mM glucose for 108 days. Results and Discussion ∆OCP increased depending on glucose concentration (0.1 mM~20 mM) without being affected by ascorbic acid and acetaminophen which are representative interference of glucose sensor as we previously observed. This sensor could be operated continuously for 108 days without showing any significant decrease on the slope of calibration curve. Since there were no current flow during open circuit state, the turnover number of enzyme reaction could be significantly minimized. Consequently, stable OCP signal could be obtained for 108 days without significant decrease of OCP signal. These results revealed that OCP principle could prevent enzyme inactivation during continuous operation, thereby lead to improve long-term stability of sensor. This result indicated that the OCP based glucose sensor employing direct electron transfer type FADGDH could measure glucose concentration continuously, accurately and stably over 3 months, and this long-term stability could be achieved by employing OCP measurement principle.