A Rationally-designed Fluorescence Competitive Binding Assay for Continuous Glucose Monitoring Applications
Cummins, Brian Michael
MetadataShow full item record
Improved continuous glucose monitoring devices have the potential to increase patient compliance and improve the management of diabetes. One type of sensing chemistry that has been recognized as having significant amount of potential is based on the protein, Concanavalin A. However, to date, this assay has continually shown problems with sensitivity, stability, and reversibility in free solution. This work uses rational design to generate a new version of the competitive binding assay that can allow for its full potential to be shown. The first part of this work uses a glycosylated dendrimer as the competing ligand in the assay. This assay is optimized with regards to its fluorescence response and encapsulated within microporated microspheres to allow for the glucose-dependent aggregative response to occur. The exact mechanism of this assay was explored, and the stability issues were identified to be a direct result of the electrostatic interactions and multivalent presentation of sugar residues. The second part of this work generated a model for the unique recognition and transduction mechanisms of the ConA-based assay, in an attempt to generate maps to identify desirable qualities to generate an optimized assay. This model was validated with experimental data, and used to optimize a ConA-based assay to track glucose concentrations with anisotropy. They were also used to explain the problems with previous ConA-based approaches. The core trimannose of N-linked glycans was recognized as a high-affinity ligand that achieved the required affinities without leading to the aggregation that has caused previous assays to fail. A smartly-designed fluorescent ligand was generated based on this core trimannose to achieve the desired qualities as defined by the previous models, and it was used in a ConA-based assay to track glucose concentrations with anisotropy and F?rster Resonance Energy Transfer (FRET). Finally, in an attempt to generate a cost-effective smartly-designed fluorescent ligand that could be encapsulated with a size exclusion membrane, the glycated fraction of ovalbumin was fluorescently labeled and used in a ConA-based assay. This work proves that the smartly-designed fluorescent ligand concept can overcome the problems of previous ConA-based assays, and such an assay is expected to be capable of translation to practical applications.