3.3. Luminol Chemiluminescence Assay

Luminol was obtained from Sigma-Aldrich, potassium hydroxide from Khimreactiv (Russia), and 3% solution of hydrogen peroxide from Tula Pharmaceutical Factory (Russia). The 10⁻⁴ M aqueous alkaline luminol solution was used.

The chemiluminescence luminol reaction was initiated by a solution of K₃[Fe(CN)₆] and maximal value of chemiluminescence intensity was determined. All measurements were carried out in 25–40 replicates using TriStar LB 941 bioluminometer with injector system. Average and SD values did not exceed 0.05.

The dependence of chemiluminescence intensity on H₂O₂ concentration was initially determined and it was used as a calibration dependent in the following experiments to evaluate concentrations of peroxide compounds in the solutions of fullerenols. Peroxides were considered to be components of the ROS group. The ROS content was plotted vs. concentrations of fullerenols.

To compare the effects of fullerenols on ROS content, their effective concentrations that decreased chemiluminescence intensity by 50%, EC₅₀, were determined.