Fatty acid sensor for low-cost lifetime-assisted ratiometric sensing using a fluorescent fatty acid binding protein Amelita Bartolome, Cameron Bardliving, Govind Rao, Leah Tolosa * Center for Advanced Sensor Technology, Chemical and Biochemical Engineering, University of Maryland Baltimore County, Baltimore, MD 21250, USA Received 4 May 2005 Available online 9 August 2005 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 lM. 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 inten- sities 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, respec- tively. Note that because ruthenium has a lifetime that is two orders of magnitude longer than that of acrylodan, the FA measure- ments 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. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Fatty acid binding protein; Lifetime-assisted ratiometric modulation-based sensing; Ruthenium; Acrylodan Fatty acids (FAs) 1 yield large amounts of ATP through b-oxidation and the tricarboxylic acid (TCA) cycle during metabolism. Along with glucose, free FAs are major sources of energy for many cell types. In fact, heart and skeletal muscles preferably rely on free FAs for their energy requirement. However, in cases of over- nutrition and sedentary lifestyles, elevated FA levels lead to insulin resistance, hypertension, and microangi- opathy [1–3]. Insulin resistance is a primary event lead- ing to the development of type 2 diabetes. Even in healthy individuals who are genetically predisposed to type 2 diabetes, reduction of plasma free FA increases insulin sensitivity, thereby preventing or prolonging the onset of the disease [4]. Thus, type 2 diabetes can be classified not only as impairment of glucose metabo- lism but also of FA metabolism. Alarmingly, obesity, a risk factor in the aforementioned diseases, is a global 0003-2697/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.07.030 * Corresponding author. Fax: +1 410 455 6500. E-mail address: leah@umbc.edu (L. Tolosa). 1 Abbreviations used: FA, fatty acid; TCA, tricarboxylic acid; FABP, fatty acid binding protein; ADIFAB, acrylodated intestinal fatty acid binding protein; LARS, lifetime-assisted ratiometric sensing; RuMLC, ruthenium bis(2,2 0 -bipyridyl)-1,10-phenathroline-9-isothiocyanate; LED, light-emitting diode; EGTA, ethyleneglycol-bis(b-aminoethyle- ther)- N,N,N 0 ,N 0 -tetraacetic acid; PMT, photomultiplier tube; FRET, fluorescent resonance energy transfer. ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 345 (2005) 133–139 www.elsevier.com/locate/yabio