The goal of this project is to develop new approaches to fluorescence- based glucose sensors of possible use in the treatment of type 1 diabetes. The maintenance of euglycemia in type 1 diabetes patients requires the continuous measurement and monitoring of blood glucose levels, which would be facilitated by the development of an appropriate glucose sensor. Fluorescence-based approaches have certain advantages over other types of glucose sensors. However, a functional fluorescence-based glucose sensor for use in diabetes patients has not yet been developed. This project is based on previous observations that the fluorescence of an arylboronic acid may be modified upon cyclic boronate ester formation with a sugar molecule. The change in fluorescence is due to quenching of the fluorescence of a fluorophore by photoinduced electron transfer (PET). While the observed fluorescence change may be used to quantitative glucose in solution, these simple sensors are not specific for glucose as other compounds present in biological systems can elicit the same response. This project will explore two novel approaches for building in high selectivity for glucose in systems chosen such that the extent of glucose binding can be conveniently quantitated by fluorescence measurements. (1) The first approach will make use of an antibody have an arylboronic acid moiety covalently attached. A combinatorial approach to antibody evolution will be used, along with phage display technology. To discover antibody-fluorophore conjugates exhibiting fluorescence-sensitive glucose binding. This will require the synthesis of fluorescent arylboronic acids designed for covalent attachment to an antibody. This will be followed by screening of a combinatorial library of antibody-fluorophore conjugates for binding to a solid support having appended glucose molecules. 2) The second approach will involve a computer-based design method for the discovery of small molecules for specific recognition of glucose with fluorescence-sensitive binding. This will involve the design and synthesis of arylboronic acids having a second functional group appropriately positioned to form favorable interactions with functional groups of the glucose molecule remote from the arylboronic acid binding site. The binding interaction of this second functional group will provide additional affinity and the desired specificity in glucose recognition.
