2.9. Amplex Red Assay for Hydrogen Peroxide

Amplex red hydrogen peroxide/peroxidase assay kit was used to detect the production of H₂0₂ from fullerene + KI mediated PDT. The colorless probe Amplex red (10-acetyl-3, 7-dihydroxy-phenoxazine) reacts with H₂O₂ in the presence of peroxidase and forms resorufin (7-hydroxy-3H-phenoxazin-3-one). The detection process after 10 μM fullerene + 400 mMKI mediated PDT was according to manufacturer’s instructions. The reaction volume contained 10 μM BB4 or BB4-PPBA with added 400 mM KI were illuminated with increasing fluence of 415 nm blue light and aliquots withdrawn and added to 50 μM Amplex Red reagent and O.lU/mL horseradish peroxidase (HRP) in Krebs-Ringer phosphate (145 mM NaCl, 5.7 mM Na₃P0₄, 4.86 mM KC1, 0.54 mM CaCl₂, 1.22 mM MgS0₄, 5.5 mM glucose, pH 7.35). After 30 min incubation, a fluorescence microplate reader (excitation 530 nm and emission 590 nm) was used to measure incremental fluorescence after each fluence of blue light was delivered. Controls were [1] fullerene + light, [2] KI + light, and [3] Amplex red reagent alone. Each experiment was performed at least three times.