Fatty acid sensor for low-cost lifetime-assisted ratiometric sensing using a fluorescent fatty acid binding protein
Section snippets
Materials and methods
ADIFAB was purchased from Molecular Probes [15]. RuMLC was prepared as described previously [17]. Fluorescence measurements were carried out using 0.02 μM ADIFAB in buffer containing 50 mM Tris, 1 mM ethyleneglycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), and 0.05% sodium azide adjusted to pH 8 using a Varian Cary Eclipse spectrofluorometer (Varian Instruments, Walnut Creek, CA, USA). Dual-labeled ADIFAB was prepared by adding RuMLC (0.53 μM final concentration) to the ADIFAB
Results and discussion
ADIFAB is a rat intestinal FA binding protein covalently labeled with acrylodan. ADIFAB is used in dual-wavelength (432 and 505 nm) measurements of free FAs C-12 to C-24 [15]. Acrylodan, a polarity-sensitive fluorophore, changes its intensity at 432 nm in inverse proportion to free FA concentrations. This 432-nm emission was attributed to acrylodan occupying the FA binding site in the absence of FA. As FA is added, the acrylodan is displaced from the binding site, resulting in a decrease in
Acknowledgments
The study was supported by grants from the U.S. Army (W81XWH-04-1-0781) and the National Institutes of Health (DK062990) to Leah Tolosa. Cameron Bardliving was supported by the MARC U*STAR Program at the University of Maryland Baltimore County. Yordan Kostov contributed valuable technical support and insightful discussions.
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