Fluorimetric Methods

Fluorimetry is an analytical technique that measures the fluorescence at a specific emission wavelength in compounds after induction with a light pulse of a specific excitation wavelength. Fluorescent probes used in oxidative stress (OS) assessment are based on redox processes between OS biomarker (hydrogen peroxide, NO•, superoxide, S-nitrosothiols, protein carbonyls, malondialdehyde (MDA), advanced glycation end-products (AGEs), glutathione (GSH)) and the reduced probe, which fluoresces upon oxidation. Although fluorescence is among the most sensitive techniques for radical detection, it has multiple limitations. Due to the lack of selectivity of most sensors (e.g., dichlorodihydrofluorescein), these chemicals are more appropriately called “redox probes” to indicate their general reactivity to numerous reactive species (RS). The probe should be compatible with multicolor detection to combine RS detection with other parameters such as pH indicators or cellular differentiation markers. Many probes for the detection of RS are commercially available, but most of them do not meet the ideal criteria. In this chapter, we describe the most important fluorimetric sensors for quantifying OS biomarkers. We discuss their advantages and limitations as well as the reaction involved in the generation of the fluorescence.