Year of Publication
Doctor of Philosophy (PhD)
Arts and Sciences
Proteins possess a broad range of structural and functional properties and, therefore, can be employed in a variety of biomedical applications. While a good number of protein-based biosensing systems and biosensors for target analytes have been developed, the search for versatile, highly sensitive and selective sensors with long term stability able to provide fast detection of target analytes continues to be a challenge. To that end, we now report the design and development of modified proteins with tailored characteristics and their further utilization in the development of biosensing systems.
We take advantage of binding proteins that undergo a change in conformation upon binding to their respective target ligand analytes for the development of highly selective biosensing systems. The first class of binding proteins that was explored for this purpose was antibodies. A non-canonical site in the variable region of a monoclonal antibody was tagged with a fluorescent probe to sense the binding of analyte to its corresponding antigen-binding site. The strategy employed for designing antibodysensing molecules is universal as it can be employed for sensing any biomolecule of interest provided that there is an available antibody against the target ligand analyte.
In a second strategy, we utilized designer glucose recognition proteins (GRPs) that were prepared by incorporation of unnatural amino acids in the glucose/galactose binding protein (GBP) of Escherichia coli and its truncated fragments. By taking advantage of the global incorporation method, we were able to fine-tune the binding affinity and thermal stability of the proteins, thus, allowing for the development of a reagentless fluorescence based fiber optic glucose biosensor capable of monitoring glucose in the hypoglycemic, normal, and hyperglycemic range, as well as in the hypothermic and hyperthermic temperature range.
Joel, Smita, "ENGINEERING PROTEINS WITH UNIQUE CHARACTERISTICS FOR DIAGNOSTICS AND BIOSENSORS" (2011). University of Kentucky Doctoral Dissertations. 180.