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

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.