A firefly luciferase (LUC)-based bioluminescent assay for total free fatty acids (FFA) is presented. It is based on LUC's capability of converting FFA into fatty acyl-adenylates with consumption of adenosine 5'-triphosphate (ATP). Since ATP is a cosubstrate in LUC's bioluminescent reaction, together with firefly D-luciferin (D-LH2 ) and atmospheric oxygen (O2 ), any reduction in the assay's ATP content will lead to a decrease in the bioluminescent signal, which is proportional to the amount of FFA. Using FFA mixtures containing myristic (14:0), palmitic (16:0), stearic (18:0), oleic (18:1) and arachidonic acid (20:4) in ethanol, the assay was optimized through statistical experimental design methodology, namely fractional factorial (screening) and central composite (optimization) designs. The optimized method requires 2 μL of sample per tube in a final reaction volume of 50 μL. It is linear in the concentration range from 1 to 20 μm, with limits of detection (LOD) and quantitation (LOQ) of 1.3 and 4.5 μm, respectively. The method proved to be simple to perform, demands low reagent volumes, it is sensitive and robust and may be adapted to high-throughput screening.
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