Poster W11 - Fluorescence Intensity-based Glucose Sensing Assay for a Proposed Implantable Biosensor

Introduction: Research advancements in continuous glucose monitoring (CGM) for diabetes management have focused on developing accurate, sensitive, cost-effective biosensors with fast response times and minimal discomfort to improve patient quality of life. The growing prevalence of diabetes, which affects 1 in 10 adults worldwide, has underscored the need for alternatives to the inconvenient and painful finger-prick method. Advancement in fluorescence intensity-based, fully injectable biosensors for continuous glucose monitoring for diabetes management has shown great promise over the past several years. Our group has extensively reported on methods to improve the stability and sensitivity of glucose biosensing assays which utilize the protein Concanavalin A (ConA). This work focuses on optimizing our red-excitable, fluorescence intensity-based, competitive binding assay to improve the signal within an implantable biosensor.

Materials and

Methods: Four mannose molecules (alpha-mannotetraose, beta-mannotetraose, alpha-mannopentaose and beta-mannopentaose) were chosen for Cyanine5.5 (Cy5.5) labeling. The synthesis, purification and characterization of Cy5.5-mannose protocols are very well established in the group. To confirm the synthesized novel fluorescent competing ligand is able to bind successfully bind to the recognition element of the assay, PEGylated ConA, and unbind in the presence of physiological glucose concentration ranges, the polarization-based method of fluorescence anisotropy (FA) was utilized. All equilibrium binding was conducted using a QuantaMaster spectrofluorometer (PTI). To identify the best ratio of the Cy5.5-mannose to PEG-ConA, the change fluorescence mission intensity was monitored by varying the concentrations of the PEG-ConA with a constant concentration of each Cy5.5-mannose (0.1uM) and adding a physiologically high concentration of glucose to each assay sample (800mg/dL). The optimum concentrations are used to measure the glucose response among the physiological glucose range. The Tecan Infinite 200 Pro plate reader was used for fluorescence emission readings and all samples were excited at 655nm.

Results, Conclusions, and Discussions: The four different Cy5.5-mannoses were synthesized, purified and characterized successfully. The fluorescence anisotropy data allowed us to better understand the interaction of the recognition element with the competing ligands. In general, alpha-mannoses show better results when compared to beta-mannoses. Cy5.5-mannopentaose in particular is a better candidate in terms of cost and glucose sensitivity. Improved stability of the assay has been shown by increasing the number of poly(ethylene glycol) (PEG) chains on ConA. Future work will include testing the optimized assay encapsulated within the proposed biosensor using the in-house optical system for glucose sensitivity, reversibility, and leaching studies.

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