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Measurements of FRET in a Glucose‐sensitive Affinity System with Frequency‐domain Lifetime Spectroscopy
Author(s) -
Liang Feng,
Pan Tianshu,
SevickMuraca Eva M.
Publication year - 2005
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1562/2005-02-14-ra-440
Subject(s) - förster resonance energy transfer , alexa fluor , chemistry , acceptor , concanavalin a , kinetics , fluorescence , dextran , spectroscopy , quenching (fluorescence) , fluorescence spectroscopy , biophysics , analytical chemistry (journal) , photochemistry , biochemistry , chromatography , biology , physics , quantum mechanics , in vitro , condensed matter physics
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) 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 μ M dextran to labeled concanavalin A at a solution concentration of 10.67 μ M in the presence of glucose at concentrations ranging from 0 to 224 mg/dL.