An electrochemical 3-hydroxybutyrate biosensor using phenazine-based electron mediators

Accurate quantification of 3-hydroxybutyrate (3-HB) is essential for the diagnosis and management of diabetic ketoacidosis and related metabolic disorders. As the predominant ketone body during ketosis, blood 3-HB provides a more reliable clinical indicator than conventional ketone assays, motivating the development of rapid point-of-care detection platforms. Herein, we report an electrochemical biosensor that integrates phenazine-based redox mediators with NAD-dependent 3-hydroxybutyrate dehydrogenase (HBDH) for the sensitive and selective detection of 3-HB. Three phenazine derivatives were systematically evaluated using molecular docking, solubility assessment and electrochemical characterization. Among them, compound 3 exhibited superior aqueous solubility, high electrochemical stability, and a strong linear response (R² = 0.9993) across a 3-HB concentration range of 0.05-14 mM within 20 s. When incorporated into a screen-printed test strip, the biosensor demonstrated consistent performance after five days of incubation at 60 °C with minimal signal drift. Interference testing confirmed less than ±15 % deviation in the presence of clinically relevant concentrations of glucose, uric acid, ascorbic acid, lactic acid, and creatinine. These results establish compound 3 as a highly effective electron mediator, supporting its potential application in reliable point-of-care and clinical 3-HB monitoring.