ENGINEERING OF A DIRECT ELECTRON TRANSFER TYPE FAD-DEPENDENT GLUCOSE DEHYDROGENASE FOR ITS APPLICATION IN ELECTROCHEMICAL GLUCOSE SENSORS

The development of continuous glucose monitors (CGMs) are the pinnacle achievement in wearable sensors and the gold standard for diabetes management. The advancement of currently available CGM devices which are absent from hydrogen peroxide or toxic, artificial mediators is the future of CGM technologies. Critically important to this work is the development and improvement of an enzyme to use in a 3rd generation sensor, direct electron transfer (DET)-type enzyme, Burkholderia cepacia derived glucose dehydrogenase (BcGDH). The information contained in this thesis therein details the efforts to make this enzyme suitable for its future application to construct an electrochemical glucose sensor for continuous glucose monitoring, by focusing on its beneficial and inferior features. To complete these tasks, the BcGDH enzyme must undergo improvements—through engineering strategies—to make it suitable for a 3rd generation sensor to be used in CGMs. This BcGDH enzyme was engineered to increase its specific activity towards glucose by minimizing its catalytic activity towards galactose, maltose, and xylose, alike. This enzyme was used to determine the causality of electrochemical inactivation set forth under continuous applied potential using chronoamperometry and mitigate this inactivation by engineering strategies. Enzyme engineering strategies and knowledge of electrochemical inactivation were used to create a novel biological reference electrode, a Bioreference, to enhance the stability of long term, continuous operation of a glucose sensor. This iterative improvement towards a 3rd generation sensor for CGMs showcases the ability to move towards a future sensing modality with extended sensor lifetimes and is devoid of hydrogen peroxide or toxic, artificial mediators.