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Measurements of FRET in a Glucose-sensitive Affinity System with Frequency-domain Lifetime Spectroscopy

Authors

  • Feng Liang,

    1. The Photon Migration Laboratory, Department of Chemistry, Texas A&M University, College Station, TX
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    • Current address: Division of Molecular Imaging, Department of Radiology, Baylor College of Medicine, Houston, TX, USA.

  • Tianshu Pan,

    1. The Photon Migration Laboratory, Department of Chemistry, Texas A&M University, College Station, TX
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    • Current address: Division of Molecular Imaging, Department of Radiology, Baylor College of Medicine, Houston, TX, USA.

  • Eva M. Sevick-Muraca

    Corresponding author
    1. The Photon Migration Laboratory, Department of Chemistry, Texas A&M University, College Station, TX
      *To whom correspondence should be addressed: One Baylor Plaza, MS BCM 360, Houston, TX 77030-3411. USA. Fax: 713-798-8050; e-mail: evas@bcm.edu
    Search for more papers by this author
    • Current address: Division of Molecular Imaging, Department of Radiology, Baylor College of Medicine, Houston, TX, USA.


*To whom correspondence should be addressed: One Baylor Plaza, MS BCM 360, Houston, TX 77030-3411. USA. Fax: 713-798-8050; e-mail: evas@bcm.edu

ABSTRACT

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 2000000) 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 μM dextran to labeled concanavalin A at a solution concentration of 10.67 μM in the presence of glucose at concentrations ranging from 0 to 224 mg/dL.

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