A Red-Excitable, Fluorescence Intensity-Based Glucose Sensing Assay for a Proposed Fully Injectable Biosensor

Abstract

Advancements 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. This work focuses on optimizing our fluorescence intensity-based, competitive binding assay by transitioning it to be excitable by red light instead of the previously reported blue light to improve signal within an injectable format. Upon evaluation and validation of an enhanced emission intensity of the biosensor in an injectable format due to a red-excitable design, a new competing ligand was created. The novel approach of designing this competing ligand centers on the use of a linker molecule to bind a single small mannose molecule to a single fluorophore. By including a linking mechanism, the sugar or fluorophore of choice can be easily exchanged in future iterations. Fluorescence anisotropy was used to confirm both the successful binding of the novel competing ligand to ConA and its competitive binding response within a physiological glucose concentration. Computational modeling was utilized to predict optimal concentrations of protein to competing ligand for a sensitive response to glucose, which was directly followed by experimental validation. With the ideal transduction mechanism of the assay being fluorescence emission intensity, the fluorophore labeled mannose molecule was paired with PEGylated ConA and a sensitive glucose response was detected related to the polarity of the environment. This polarity-based response held true at higher concentrations and when the assay was placed beneath rat skin tissues of varying thicknesses and pigmentations. A dual fluorophore assay was then investigated to include the phenomenon of Förster resonance energy transfer (FRET) to potentially increase glucose sensitivity. The assay was modified by binding a FRET pair dye to PEGylated ConA and a glucose response was successfully detected. This work demonstrates the advantages of a red excitable design for a fully injectable fluorescence intensity-based biosensor for continuous glucose monitoring.