Optimizing FOX Assay Detection Range

To study the effect of perchloric acid concentration on the linear detection range of the FOX assay, xylenol orange reagent was freshly prepared using different concentrations of perchloric acid. The xylenol orange reagent contained 2.0 mM ferrous sulfate, 0.29 mM xylenol orange tetrasodium salt, and perchloric acid (110, 220, 440, or 660 mM) diluted in methanol/water (9:1). The assay was carried out by mixing 30 μL of the sample and 150 μL xylenol orange reagent in a 96-well microplate. The mixture was then incubated for 15 min at room temperature. The absorbance of the samples was read at 570 nm using the Spectramax ID3 multi detection microplate reader. The concentration of perchloric acid which offers a broader linear detection range while maintaining sufficient sensitivity for the measurement (440 mM) was then selected for further experiments.