ATPase inhibitor based luciferase assay for prolonged and enhanced ATP pool measurement as an efficient fish freshness indicator

• Published in: Analytical and bioanalytical chemistry 2014-07, Vol.406 (18), p.4541-4549
• Main Authors: Ranjan, Rajeev, Priyanka, B. S., Thakur, M. S.
• Format: Article
• Language: English
• Subjects: Acids; Adenosine Triphosphatases - antagonists & inhibitors; Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - analysis; Analysis; Analytical Chemistry; Animals; Apyrase - metabolism; Assaying; ATP; Biochemistry; Biological assay; Bisphenol A; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Degradation; Degradation products; Edetic Acid - pharmacology; Enzyme Inhibitors - pharmacology; Enzymes; Fish; Fish as food; Fish harvest; Fish Products - analysis; Fishes - metabolism; Food Analysis - methods; Food Quality; Food Science; Food Storage; Freshness; Indicators; Inhibitors; Laboratory Medicine; Limit of Detection; Low temperature; Luciferase; Luciferases - metabolism; Luminescent Measurements - methods; Mass spectrometry; Measurement; Monitoring/Environmental Analysis; Nitrogen; Quality control; Research Paper; Sample preparation; Sensitivity and Specificity; Sodium; Vanadates - pharmacology
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-014-7840-6

The nucleotide degradation pathway in somatic cells leads to the accumulation of products such as hypoxanthine and inosine, which are commonly used as fish and meat freshness indicators. Assays based on these molecules cannot differentiate the postmortem time over a short period of time (5–10 h). Further, quantification of these degradation products is cumbersome, costly and time-consuming. For the proposed assay, optimal concentrations of 30 and 2 mM, respectively, for the ATPase inhibitors sodium orthovanadate and EDTA were found. Further, it was observed that a firefly luciferase based assay could enhance the sensitivity levels up to 165-fold at 30 °C. In addition, it was observed that the sensitivity for ATP assay was enhanced up to 60-fold even after 12 h. The limit of detection for the ATP assay was 1 pM, unlike other conventional methods, which are sensitive only up to micromolar levels. Moreover, as little as 0.044 g fish fillet was required for the assay, and no time-consuming sample preparation was necessary. Luminescence of prolonged duration was observed in harvested fish kept at -20 °C in comparison with fish kept at 4 and 30 °C, which reflects the shelf life of fish preserved at lower temperatures. Figure ATPase inhibitor based firefly luciferase enzyme assay for prolonged and enhanced ATP pool measurement as an efficient fish freshness indicator