(E-181) Continuous ketone monitoring system based on β-hydroxybutyrate binding protein

Introduction:: Ketone bodies such as β-hydroxybutyrate (BHB) and acetoacetate are important biomarkers for diabetic ketoacidosis (DKA), a life-threatening complication which is characteristic in individuals with insulin deficiency. Although disposable and single-use sensor strips, which can provide information about blood ketone levels, are commercially available, they are limited to providing a snapshot of ketonemia at a specific point in time. Precise dynamic information of blood ketone concentration and ketone variability will be expected to manage and prevent DKA, therefore, the development of a continuous ketone monitoring (CKM) system is required. Current electrochemical sensors for BHB measurement are based on an oxidoreductase which catalyzes the oxidation of BHB; (R)-3-hydroxybutyrate dehydrogenase (BHBDh, E.C. 1.1.1.30). BHBDh is a NAD⁺/NADH dependent oxidoreductase and is the only enzyme specifically recognizing BHB among the enzymes discovered in nature. The present sensors based on BHBDh quantify NADH production, which makes them difficult to configure for continuous monitoring. Therefore, the biggest challenge for CKM is the discovery and utilization of new molecular recognition elements for BHB, that can operate free of soluble cofactors. Recently, we have discovered several solute binding proteins (SBPs) that specifically recognize BHB (BHBBPs). SBPs recognize and bind their target molecules reversibly and specifically without cofactors or catalytic reaction and have been used effectively for construction of continuous biosensing systems. In this study, we aimed to develop an electrochemical sensor for CKM by acknowledging the advantageous characteristics of BHBBPs.


Materials and Methods:: BHBBP was recombinantly produced and immobilized on the gold disk electrode using self-assembled monolayer. Then, BHBBP was modified by the redox probe by covalently bound. Thus redox probe modified BHBBP immobilized gold electrode was then subjected for electrochemical monitoring, such as pulse amperometry and several voltammetry, by applying an alternating potential and monitored the current response in the presence of BHB.


Results, Conclusions, and Discussions::
The redox probe modified BHBBP showed both oxidation and reduction currents, which were altered in the presence of BHB in the solution. The BHB dependent current increase was observed not only in the buffer solution, but also in the artificial interstitial. Since SBPs are known to change their conformation upon the binding the ligand, the access of the redox probe modified on the surface of BHBBP to the electrode might be modulated by the conformational change in BHBBP and can be monitored electrochemically. These results suggest the feasibility of electrochemical sensor employing redox probe modified BHBBP for CKM system.




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