Measurements of FRET in a glucose-sensitive affinity system with frequency-domain lifetime spectroscopy.
We report measurements of fluorescence resonance energy transfer (FRET) for glucose sensing in an established concanavalin A-dextran affinity system using frequency-domain lifetime spectroscopy. A dextran (MW 2,000,000) labeled with a small fluorescent donor molecule, Alexa Fluor 568, was used to competitively bind to a sugar-binding protein, concanavalin A, labeled with acceptor molecule, Alexa Fluor 647, in the presence of glucose. The FRET-quenching kinetics of the donor were analyzed from frequency-domain measurements as a function of both glucose and acceptor-protein concentrations using a Förster-type decay kinetics model. The results show that the frequency-domain measurements and donor decay kinetics can quantitatively indicate changes in the competitive binding of 0.09 microM dextran to labeled concanavalin A at a solution concentration of 10.67 microM in the presence of glucose at concentrations ranging from 0 to 224 mg/dL.
Liang
Feng
F
The Photon Migration Laboratory, Department of Chemistry, Texas A&M University, College Station, USA.
Pan
Tianshu
T
Sevick-Muraca
Eva M
EM
D001667:Binding, Competitive; D002846:Chromatography, Affinity / Q000379:methods*; D003208:Concanavalin A / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D005947:Glucose / Q000032:analysis*; D007700:Kinetics; D013057:Spectrum Analysis / Q000379:methods*
D005456:Fluorescent Dyes; D003208:Concanavalin A; D005947:Glucose