Fluorescence-based sensing of glucose using engineered glucose/galactose-binding protein: a comparison of fluorescence resonance energy transfer and environmentally sensitive dye labelling strategies |
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Authors: | Khan Faaizah Gnudi Luigi Pickup John C |
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Affiliation: | Metabolic Unit, King’s College London School of Medicine, Guy’s Hospital, London SE1 9RT, UK |
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Abstract: | Fluorescence-based glucose sensors using glucose-binding protein (GBP) as the receptor have employed fluorescence resonance energy transfer (FRET) and environmentally sensitive dyes, but with widely varying sensitivity. We therefore compared signal changes in (a) a FRET system constructed by transglutaminase-mediated N-terminal attachment of Alexa Fluor 488/555 as donor and QSY 7 as acceptor at Cys 152 or 182 mutations with (b) GBP labelled with the environmentally sensitive dye badan at C152 or 182. Both FRET systems had a small maximal fluorescence change at saturating glucose (7% and 16%), badan attached at C152 was associated with a 300% maximal fluorescence increase with glucose, though with badan at C182 there was no change. We conclude that glucose sensing based on GBP and FRET does not produce a larger enough signal change for clinical use; both the nature of the environmentally sensitive dye and its site of conjugation seem important for maximum signal change; badan-GBP152C has a large glucose-induced fluorescence change, suitable for development as a glucose sensor. |
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Keywords: | Glucose sensing Glucose/galactose-binding protein Fluorescence resonance energy transfer Fluorescence Dye Badan |
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