3.3. Luminol Chemiluminescence Assay

Luminol was obtained from Sigma-Aldrich (Burlington, MA, USA), 3% solution of H₂O₂ from Tula Pharmaceutical Factory (Tula, Russia), and potassium hydroxide from Khimreactiv (Nizhny Novgorod, Russia).

Stock luminol solution (10⁻² M) was prepared as follows: luminol powder was dissolved in 5 mL in KOH (Khimreactiv, Nizhny Novgorod, Russia) and then 5 mL of distilled water was added. The 5.4·10⁻⁵ M alkaline luminol solution was applied to measure the chemiluminescence signal.

The chemiluminescence luminol reaction was initiated by 1.8·10⁻⁴ M K₃[Fe(CN)₆]; the maximum chemiluminescence intensity was determined. All chemiluminescence measurements were conducted in 10–15 replicates using the biochemiluminometer Luminoskan Ascent (Thermo Electron Corporation, Solon, OH, USA) with an injector system. SD values did not exceed 0.1, GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA).

Initially, the dependence of chemiluminescence intensity on the concentration of H₂O₂ was determined; it was used as a calibration dependence in the following experiments to evaluate concentrations of peroxide compounds in the solutions of F10-12. Peroxides were considered as the constituents of the ROS group.

The ROS content was studied in the bioluminescence assay system in the presence of F10-12 and/or 1,4-benzoquinone. Registration of chemiluminescence signal was provided after bioluminescence signal in the bioassay system.

All experiments with solutions of fullerenol F10-12 excluded the effect of “optic filter”, and this effect did not skew the results of ROS measurements. Optical density of solutions analyzed did not exceed 0.1 in the wavelength region of the chemilunescence light emittitance.

The ROS content was plotted vs. concentration of F10-12.