An Optimized Firefly Luciferase Bioluminescent Assay for the Analysis of Free Fatty Acids

• Type: Article in International Scientific Journal
• Date: 2015

Authors

Simone M Marques (Author) Other; Luis M Goncalves (Author) Other; Joaquim C G E Esteves da Silva (Author) FCUP

Journal

Title: Photochemistry and Photobiology

Vol. 91

Pages: 980-984

ISSN: 0031-8655

Publisher: Wiley-Blackwell

Indexing

ISI Web of Knowledge - 3 Citations

Scopus - 3 Citations

Scientific classification

FOS: Natural sciences > Biological sciences

Other information

Authenticus ID: P-00G-CKK

DOI: 10.1111/php.12458

Abstract (EN): 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 (O-2), 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 2L of sample per tube in a final reaction volume of 50L. It is linear in the concentration range from 1 to 20m, with limits of detection (LOD) and quantitation (LOQ) of 1.3 and 4.5m, 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.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 5