Elsevier

Analytical Biochemistry

Volume 345, Issue 1, 1 October 2005, Pages 133-139
Analytical Biochemistry

Fatty acid sensor for low-cost lifetime-assisted ratiometric sensing using a fluorescent fatty acid binding protein

https://doi.org/10.1016/j.ab.2005.07.030Get rights and content

Abstract

Elevated free fatty acid (FA) levels lead to insulin resistance, hypertension, and microangiopathy, all of which are associated with type 2 diabetes. On the other hand, deficiencies of FA are indicative of certain neurodegenerative diseases, including autism. Thus, free FA levels are a diagnostic indicator for a variety of disorders. Here we describe the use of a commercially available FA binding protein labeled with acrylodan (ADIFAB), which we modified with a ruthenium metal–ligand complex with the intention of creating a low-cost FA sensor. The dual-labeled FA binding protein was used in lifetime-assisted ratiometric sensing (LARS) of oleic acid. For both steady-state and time-resolved luminescence decay experiments, the protein is responsive to oleic acid in the range of 0.02–4.7 μM. The emission at 432 nm, which is associated with the acrylodan occupying the FA binding site, decreases in intensity and red shifts to 505 nm on the addition of oleic acid. The intensities of the 505-nm peak due to the acrylodan displaced from the binding site by FA and of the 610-nm emission peak of ruthenium remained nearly unchanged. Fitting of the fluorescence decay data using the method of least squares revealed three emitting components with lifetimes of approximately 0.60, 4.00, and 370 ns. Fractional intensities of the emitting species indicate that changes in modulation between 2 and 10 MHz on binding of the protein with oleic acid are due mainly to the 4.00-ns component. The 0.60- and 370-ns components are assigned to acrylodan (505 nm) and ruthenium, respectively. Note that because ruthenium has a lifetime that is two orders of magnitude longer than that of acrylodan, the FA measurements were carried out at excitation frequencies lower than what can be done with acrylodan alone. Thus, low-cost instrumentation can be designed for a practical FA sensor without sacrificing the quality of measurements.

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.

References (25)

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