Methods for reactive oxygen species (ROS) detection in aqueous environments

Endogenously produced pro-oxidant reactive species are essential to life, being involved in several biological functions. However, when overproduced (e.g. due to exogenous stimulation), or when the levels of antioxidants become severely depleted, these reactive species become highly harmful, causing oxidative stress through the oxidation of biomolecules, leading to cellular damage that may become irreversible and cause cell death. The scientific research in the field of reactive oxygen species (ROS) associated biological functions and/or deleterious effects is continuously requiring new sensitive and specific tools in order to enable a deeper insight on its action mechanisms. However, reactive species present some characteristics that make them difficult to detect, namely their very short lifetime and the variety of antioxidants existing in vivo, capable of capturing these reactive species. It is, therefore, essential to develop methodologies capable of overcoming this type of obstacles. Fluorescent probes are excellent sensors of ROS due to their high sensitivity, simplicity in data collection, and high spatial resolution in microscopic imaging techniques. Hence, the main goal of the present paper is to review the fluorescence methodologies that have been used for detecting ROS in biological and non-biological media.

The enzymatic determination of hydrogen peroxide can be accomplished with high sensitivity and specificity using N-acetyl-3, 7-dihydroxyphenoxazine (Amplex Red), a highly sensitive and chemically stable fluorogenic probe for the enzymatic determination of H2O2. Enzyme-catalyzed oxidation of Amplex Red, which is a colorless and nonfluorescent derivative of dihydroresorufin, produces highly fluorescent resorufin, which has an excitation maximum at 563 nm and emission maximum at 587 nm. The reaction stoichiometry of Amplex Red and H2O2 was determined to be 1:1. This probe allows detection of 5 pmol H2O2 in a 96-well fluorescence microplate assay. When applied to the measurement of NADPH oxidase activation, the Amplex Red assay can detect H2O2 release from as few as 2000 phorbol myristate acetate-stimulated neutrophils with a sensitivity 5- to 20-fold greater than that attained in the scopoletin assay under the same experimental conditions. Furthermore, the oxidase-catalyzed assay using Amplex Red results in an increase in fluorescence on oxidation rather than a decrease in fluorescence as in the scopoletin assay. In comparison with other fluorometric and spectrophotometric assays for the detection of monoamine oxidase and glucose oxidase, this probe is also found to be more sensitive. Given its high sensitivity and specificity, Amplex Red should have a broad application for the measurement of H2O2 in a variety of oxidase-mediated reactions and very low levels of H2O2 in food, environmental waters, and consumer products. Copyright 1997 Academic Press.