Browsing by Author "Ngwadom, Chiagoziem"

Now showing 1 - 1 of 1
  • Thumbnail Image
    ItemEmbargo
    Engineering Glucose Enzymes Through Domain Insertion for Adaptive Bioelectronic Sensors
    (2024-12-03) Ngwadom, Chiagoziem; Ajo-Franklin, Caroline; Warmflash, Aryeh
    Biosensors are essential in diagnostics, monitoring, and therapeutics. A major example is the glucometer, which effectively utilizes glucose-oxidizing enzymes to generate accurate electrical signals that report blood sugar levels. This bioelectrochemical sensor’s affordability, manufacturability, and suitability for patient-side use make glucose enzymes highly appealing for broader sensing applications. Although existing studies have explored mutagenizing glucose redox enzymes to enhance their stability and activity, significant obstacles remain in repurposing these enzymes to detect other biomarkers. These challenges stem from an incomplete understanding of glucose enzyme design and the limited effectiveness of current protein engineering approaches. This thesis addresses these challenges by using pyrroloquinoline quinone glucose dehydrogenase (PQQ-GDH) as a robust platform for glucose-dependent oxidoreductase applications. Through comprehensive methods developed to identify structural elements crucial to its function, this work demonstrates the repurposing of PQQ-GDH to produce electrochemical output for non-glucose analytes. Additionally, a high-throughput screening system is introduced to accelerate the development of a broad range of bioelectronic sensors. In Chapter 2, we integrate small peptide sequences into PQQ-GDH to investigate the structure-sequence-function relationships at various structural levels. In Chapter 3, we engineer PQQ-GDH conformational switches to create electronic sensors capable of detecting cancer therapeutics in blood samples, pushing the boundaries of traditional glucose sensing. In Chapter 4, we establish a high-throughput selection system for glucose enzyme variants by manipulating glucose metabolism and NADPH regeneration in E. coli through targeted knockouts. Our research explores multiple strategies for functionalizing PQQ-GDH to enhance bioelectronic diagnostics. These findings provide critical insights into how the structure and sequence of PQQ-GDH influence its function—particularly at the active site and dimerization interface, which are essential for enzyme activity and stability. When integrated onto electrode interfaces, our functionalized PQQ-GDH variants demonstrate a significant electrical response to the cancer therapeutic 4-hydroxytamoxifen in blood. This advancement lays a solid foundation for real-time, point-of-care diagnostics in therapeutic monitoring. Additionally, our innovative growth complementation assay enriches enzyme variants in direct proportion to their activity levels, establishing a novel selection method for variants that exhibit superior performance. These contributions advance biosensing technologies and significantly expand the application scope of bioelectrochemical systems. We are paving the way for reliable point-of-care diagnostic devices and therapeutic monitoring platforms that promise to transform future healthcare solutions