Benchtop microplate reader

The principle of the antioxidant assay is based on the formation of a ferryl myoglobin radical from metmyoglobin and hydrogen peroxide, which oxidizes the 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) to produce a radical cation, ABTS⁺, a soluble chromogen that is green in color and can be measured in a spectrophotometer at 405 nm. Antioxidants suppress the production of the radical cation in a concentration dependent manner and the color intensity decreases proportionally. Trolox, a water-soluble vitamin E analog, was used as a control antioxidant for analyzing the antioxidant activity of HNK, MCP, and MCP : HNK (9 : 1). In a 96-well plate, the assay was set up with 10 µl of increasing concentrations of compounds (0–500 µg/ml) and 20 µl of myoglobin working solution per the protocol described in Antioxidant Assay Kit (Sigma-Aldrich, St. Louis, MO). Afterward, 150 μl of ABTS working solution containing 0.0075% H₂O₂ was added and incubated at room temperature for 5 min. The reaction was stopped by adding 100 µl stop solution and absorbance was measured at 405 nm in a Bio-Rad Benchtop microplate reader. The decrease in absorbance indicated the antioxidant activity of HNK, MCP, and MCP : HNK (9 : 1) equivalent to the Trolox standard, which was plotted against concentrations of compounds [42 (link)].