Continuous glucose monitoring is desired to avoid the myriad of diabetes complications such as blindness, amputation, heart and kidney diseases. Despite the availability of glucose monitoring kits in the market today, the search for continuous minimally invasive glucose monitoring devices continues. We present here our initial study to develop a continuous quantitative fluorescence detection of glucose using immobilized dicysteine bearing periplasmic glucose binding protein (GBP). Two amino acid mutations (L255C/E149C and L255C/A71C) were achieved by site-directed mutagenesis. The L255C mutation is located in a more obscure position near the hinge of the GBP while the other mutation (E149C or A71C) is positioned opposite L255C is more exposed to the solvent. Covalent immobilization was done by conjugation of the more exposed thiol groups (E149C or A71C) to dextran modified glass capillary surface. The immobilized GBP was subsequently labeled with acrylodan at position L255C. The resulting system binds glucose with a corresponding decrease in acrylodan fluorescence. Results suggest that immobilization did not hinder substrate binding to the conjugated protein.

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