Microplate ORAC-pyranine spectrophotometric assay for high-throughput assessment of antioxidant capacity

Abstract

Antioxidant activity assessment is crucial to establish the protective role of bioactive compounds against free radical-induced oxidative damage, a serious threat to health and food quality. For oxygen radical absorbance capacity (ORAC) assays, the target molecule employed has been indicated as a key factor to characterize free-radical scavenging mechanisms. The combined use of target molecules such as pyranine (PYR) and pyrogallol red (PGR) allows to distinguish between the stoichiometry and the chemical reactivity of free radical scavenging by antioxidants. In this work, a high-throughput microplate-based ORAC-PYR method was developed for the estimation of antioxidant capacity (stoichiometry) in a miniaturized fashion. Kinetic studies of pyranine consumption by AAPH-derived radicals were carried out to achieve high sensitivity, reduced consumption of reagents, and low generation of waste. The use of low amounts of target molecule and radical generator (2.5 ng and 2.5 µg, respectively) was feasible with a maximum time to result of 3 h for 96 simultaneous determinations. The limit of detection was 1 µM for Trolox and RSD values were <5.4% and <6.4% for intra- and inter-assay precision, respectively. A dual-probe approach (ORAC-PGR and ORAC-PYR) was implemented in microplate format and a comparative study of structure-antioxidant activity of catechol-derived compounds was performed. This strategy was also extended to the analysis of antioxidant-rich beverages for assessing the applicability of the proposed method to complex matrices.