Table 18.4 Fluorescence response of 3′-(p-aminophenyl) fluorescein (APF), 3′-(p-hydroxyphenyl) fluorescein (HPF) and dichlorodihydrofluorescein diacetate (H2DCFDA) to various reactive oxygen species (ROS).

Reactive Oxygen Species (ROS) ROS Generation Method APF * HPF * H2DCFDA *
Hydrogen peroxide (H2O2) 100 µM of H2O2 <1 2 190
Hydroxyl radical (HO•) 100 µM of ferrous perchlorate (II) and 1 mM of H2O2 1200 730 7400
Hypochlorite anion (OCl) 3 µM (final) of OCl 3600 6 86
Nitric oxide (NO) 100 µM of 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-methyl-1-triazene (NOC-7) <1 6 150
Peroxyl radical (ROO•) 100 µM of 2,2'-azobis(2-amidinopropane), dihydrochloride (AAPH) 2 17 710
Peroxynitrite anion (ONOO) 3 µM (final) of ONOO 560 120 6600
Singlet oxygen (1O2) 100 µM of 3-(1,4-dihydro-1,4-epidioxy-1-naphthyl)propionic acid 9 5 26
Superoxide anion (•O2) 100 µM KO2 6 8 67
Autooxidation 2.5 hours exposure to fluorescent light source <1 <1 2000
* 10 µm of APF, HPF or DCF (2',7'-dichlorofluorescein) were added to sodium phosphate buffer (0.1 M, pH 7.4); ROS were generated as indicated; and fluorescence was measured using excitation/emission wavelengths of 490/515 nm (for APF and HPF) or 500/520 nm (for DCF). DCF was obtained by hydrolysis of H2DCFDA with base as described in ref; dihydrofluorescein diacetates are colorless and nonfluorescent until both of the acetate groups are hydrolyzed and the products are subsequently oxidized to fluorescein derivatives.