Near-infrared fluorescence glucose sensing based on glucose/galactose-binding protein coupled to 651-Blue Oxazine

Near-infrared (NIR) fluorescent dyes that are environmentally sensitive or solvatochromic are useful tools for protein labelling in in vivo biosensor applications such as glucose monitoring in diabetes since their spectral properties are mostly independent of tissue autofluorescence and light scattering, and they offer potential for non-invasive analyte sensing. We showed that the fluorophore 651-Blue Oxazine is polarity-sensitive, with a marked reduction in NIR fluorescence on increasing solvent polarity. Mutants of glucose/galactose-binding protein (GBP) used as the glucose receptor were site-specifically and covalently labelled with Blue Oxazine using click chemistry. Mutants H152C/A213R and H152C/A213R/L238S showed fluorescence increases of 15% and 21% on addition of saturating glucose concentrations and binding constants of 6 and 25mM respectively. Fluorescence responses to glucose were preserved when GBP-Blue Oxazine was immobilised to agarose beads, and the beads were excited by NIR light through a mouse skin preparation studied in vitro. We conclude GBP-Blue Oxazine shows proof-of-concept as a non-invasive continuous glucose sensing system.