Development, Validation and Applications of a Biological Antioxidant Capacity Assay Using sodA::gfp as a Living Sensor

Two biological antioxidant capacity assays, based on a reactive oxygen species inducible E. coli strain, were described. The strain harbored fusion genes sodA::gfp, and these genes measure oxidative stress by quantitatively responding to superoxide anions. The system measures ascorbate and Trolox antioxidant capacity in response to free radical generation by paraquat. Antioxidant capacity was measured by the degree of inhibition of induced fluorescence. At a fixed oxidant concentration, specific fluorescence decreased as the concentration of the antioxidant increased. Both assays were validated for their reproducibility of the calibration curves, their precision and their accuracy. In both assays, the limit of detection and the limit of quantitation were 2.50mM and 5.00mM, respectively. The hydrophilic antioxidant capacity assay was also correlated with the currently used standard assays, ORAC (r = 0.981) and DPPH assays (r = 0.841). By utilizing the autofluorescent green fluorescent protein (GFP) as the reporter gene and a multi-well plate reader as the screening tool, the method was noninvasive and cost effective. Compared to other available antioxidant capacity assays, most of which need further dilution of the test samples, this assay is one of the few assays that is able to assess samples with high antioxidant concentrations. Furthermore, since living cells are employed, actual bio-availability is assessed. The hydrophilic antioxidant capacity assay was also utilized to compare antioxidant capacity in common teas, wines and juices. Different processing methods of foods, coffees with different roasting levels and different nutritional supplements were investigated for their effects on antioxidant capacity as well