A glucose affinity sensor based on a homogeneous fluorescence resonance energy transfer (FRET) assay system was developed to monitor the competitive binding between concanavalin A (con A), a sugar-binding protein labeled with acceptor fluorophore, Alexa Fluor 647 (AF647) and polysaccharides conjugated with donor fluorophore, Alexa Fluor 568 (AF568). Confounding factors such as: (i) the impact of scattering due to tissue optical properties; (ii) the reabsorption of propagated donor fluorescence by the acceptor fluorophore; (iii) photobleaching; and (iv) fluorophore loading must be accounted for before quantitative glucose measurements can be made from fluorescence intensity measurements. Fluorescence lifetime spectroscopy made in the frequency domain circumvents most of these artifacts by measuring phase-shift and modulation ratio related with the fluorophore lifetime change. Experiments were performed to assess the FRET effects of this affinity sensing system, using dextran (MW 2000K) labeled with donor molecule, AF568 (donor-dextran), and con A labeled with acceptor molecule, AF647 (acceptor- con A). Herein we demonstrate that the FRET decay kinetics can indicate changes in the competitive binding of 0.09 μM dextran as con A concentration (from 0 to 10.67 μM) and glucose concentration (from 0 to 224 mg/dL) are changed. Preliminary work also presented here shows that quantitative frequency-domain lifetime measurement of FRET changes could be achieved in tissue-like scattering media using the photon diffusion equation.
Frequency-domain photon migration measurements across the surface of a tissue-mimicking, semi-infinite phantom are acquired via an intensified charge-coupled device (ICCD) detection system and used in conjunction with the diffusion approximation to determine the optical properties. The absorption and reduced scattering coefficients are determined least accurately when relative measurements of average light intensity I are employed either alone or in a combination with relative modulation amplitude data I and/or relative phase shift data rel. The absorption and reduced scattering coefficients are found accurate to within 15 and 11%, respectively, of the values obtained from standard single-pixel measurements when rel measurements are employed alone or in combination with I data.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.