Table 1.

Direct methods for detection of ROS in CVDs.

Methods	ROS detected	Applications/mechanism	Reference
Fluorescent protein-based redox probes	Cytoplasmic and mitochondrial H2O2	Used to detect redox status and ROS by introducing adenoviruses or plasmids inside cells. Afterwards, cells form chimeric proteins efficient to detect alteration in the redox status or ROS.	[25, 26]
Dihydroethidium (DHE) and mitochondrion-targeted probe mitoSOX	Cellular and mitochondrial O2 ∙−	Can detect mitochondrial O2 ∙− by adding a triphenylphosphonium group for promoting its collection in the mitochondria. Similar to DHE, mitoSOX reacts with O2 ∙− to give 2-hydroxy-mito-ethidium (2-OH-Mito-E+) so as to be identified and measured using HPLC.	[27–29]
Cyclic hydroxylamine spin probes	Total cellular and mitochondrial O2 ∙−	Allows measurement of O2 ∙− in tissue, in in vitro cells, and in vivo.	[30–32]
Boronate-based fluorescent probes	H2O2 and ONOO∙−	As probes have a fluorophore which is secured by boronate, when subjected to H2O2, the boronate encounters a nucleophilic attack, followed by its displacement from the fluorophore, thus causing emission of light.	[33, 34]
Immunospin trapping	Free radical adduct formation in the mitochondria, cells, and tissue samples	5,5-Dimethyl-1-pyrroline-N-oxide reacts with protein radicals to form epitopes which can be particularly characterized immunologically.	[35, 36]
In vivo using X- and L-band ESR spectroscopy	Short-lived free radicals in whole living animals	Detection is done in vivo by infusion of cyclic hydroxylamines or nitrone spin traps, followed by ex vivo study of the tissue or blood using X-band (9 GHz) electron spin resonance spectroscopy.	[37, 38]