Advances in Fluorescence Techniques for the Detection of Hydroxyl Radicals near DNA and Within Organelles and Membranes

Abstract

Hydroxyl radicals (^•OH), the most potent oxidants among reactive oxygen species (ROS), are a major contributor to oxidative damage of biomacromolecules, including DNA, lipids, and proteins. The overproduction of ^•OH is implicated in the pathogenesis of numerous diseases such as cancer, neurodegenerative disorders, and some cardiovascular pathologies. Given the localized nature of ^•OH-induced damage, detecting ^•OH, specifically near DNA and within organelles, is crucial for understanding their pathological roles. The major challenge of ^•OH detection results from their short half-life, high reactivity, and low concentrations within biological systems. As a result, there is a growing need for the development of highly sensitive and selective probes that can detect ^•OH in specific cellular regions. This review focuses on the advances in fluorescence probes designed to detect ^•OH near DNA and within cellular organelles and membranes. The key designs of the probes are highlighted, with emphasis on their strengths, applications, and limitations. Recommendations for future research directions are given to further enhance probe development and characterization.

Keywords: DNA-targeting; coumarin-based probes; fluorescence detection; hydroxyl radicals; organelle-targeting; oxidative stress; reactive oxygen species.

Figure 1

The Fenton/Haber–Weiss reaction. Superoxide radicals produced from the mitochondria can react with ferritin complexes, releasing ferric ions. These unbound ferric ions can be reduced in the Haber–Weiss reaction to produce ferrous ions. Ferrous ions catalyze the Fenton reaction to generate ^•OH from H₂O₂ produced by mitochondria and NOX oxidases. The ferric ions produced from the Fenton reaction can then be recycled by reduction through superoxide radicals, resulting in continuous ^•OH production.

Figure 2

The hydroxylation of coumarin by ^•OH to produce the fluorescent 7-hydroxycoumarin, along with other hydroxylated products. The coumarin framework is numbered according to the IUPAC numbering system.

Figure 3

Chemical structures of coumarin and its derivatives, which are commonly used for hydroxyl radical detection.

Figure 4

Chemical structures of coumarin–polyamine conjugates 5–8.

Figure 5

Chemical structures of coumarin-based fluorescent probes: compounds 1, 7, and 9.

Figure 6

Schematic illustration of the primary components of the CCA@TPP@CDs nanosensor and its application to ^•OH detection within the mitochondria.

Figure 7

The chemical structure of the dual-emission fluorescent probe NIR-HR and its application to detect ^•OH localized within the mitochondria.

Figure 8

The chemical structure of the Turn-on fluorescent probe RThy and its application to detect ^•OH localized within the mitochondria.

Figure 9

The chemical structure of 1-Red (“off” state) and its application to detect ^•OH localized within the lysosomes of live cells.

Figure 10

The chemical structure of the fluorescent probe HCy-Lyso and its application to target and detect ^•OH localized within the lysosomes of live cells.

Figure 11

The chemical structure of the fluorescent probe HR-DL and its application to target and detect ^•OH localized within the mitochondria and lysosomes of live cells.

Figure 12

The chemical structure of the fluorescent probe PY and its application to simultaneously detect ^•OH localized within the mitochondria and nucleoli of live cells.

Figure 13

The chemical structure of the fluorescent probe DPPEC and its application to detect ^•OH localized within lipid membranes of live cells.