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 ; dihydrofluorescein diacetates are colorless and nonfluorescent until both of the acetate groups are hydrolyzed and the products are subsequently oxidized to fluorescein derivatives. |