title: Fluorescence-based sensing of glucose using engineered glucose/galactose-binding protein: a comparison of fluorescence resonance energy transfer and environmentally sensitive dye labelling strategies.
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.
authors.affiliation: Metabolic Unit, King's College London School of Medicine, Guy's Hospital, London SE1 9RT, UK.
authors.forename: Faaizah
authors.initials: F
authors.lastname: Khan
authors.forename: Luigi
authors.initials: L
authors.lastname: Gnudi
authors.forename: John C
authors.initials: JC
authors.lastname: Pickup
mesh_terms: D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D008954:Models, Biological; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics; D033902:Periplasmic Binding Proteins / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering
chemical_list: D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C023550:galactose-binding protein; D005947:Glucose