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Reactive Oxygen Species (ROS) Detection Guide
01 What is Reactive Oxygen Species (ROS)

Reactive oxygen species (ROS), by-products of normal cellular metabolism, partially reduced metabolites of oxygen, a collective term for substances derived from O2 and more reactive than O2 itself[1][2]. Composition: ROS include not only the superoxide radical anion (O2.-) and a number of other oxygen radicals, but also non-radical derivatives of O2, such as hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and peroxynitrite/peroxynitrite (ONOO-/ONOOH).

Source: ROS are mainly produced in mitochondria. In addition to cellular metabolism, ROS are produced by specific plasma membrane oxidative enzymes in response to growth factors and cytokines, and can also be increased by external factors (e.g., environmental stress, radiation, drugs, etc.).

Figure 1. Potential sources of reactive oxygen species[3].

Many enzymes, including those in the mitochondrial electron transport chain, xanthine oxidase, cyclooxygenase, lipoxygenase, myeloperoxidase, cytochrome P450 monooxygenase, uncoupled NOS, heme oxygenase, peroxidase, and NAD(P)H oxidase, are capable of generating ROS.Depending on their location in the cell, these ROS can be generated intracellularly, extracellularly, or in specific intracellular compartments. Depending on their location in the cell, these ROS can be produced inside the cell, outside the cell or in specific intracellular compartments.

Why measure Reactive Oxygen Species (ROS)?

ROS are highly oxidative and perform complex signalling functions at low concentrations, but are harmful to cells at high concentrations. Cells have a defence system to maintain ROS at physiologically normal levels, i.e. enzyme-mediated antioxidants that are responsible for converting free radicals into stable, less harmful molecules. However, when the cell produces ROS in excess of its antioxidant capacity, damage to cellular macromolecules (e.g., lipids, proteins, and DNA) may occur, leading to a state of oxidative stress. ROS perform complex signalling functions when they are present, but are harmful to cells at high concentrations. This damage is thought to be associated with the development of many diseases and the aging process, including pulmonary hypertension, cardiomyopathy, diabetes, Parkinson's disease, and cancer. Oxidative stress plays an important role in cancer characteristics (e.g. angiogenesis, invasiveness, stemness and metastatic capacity): cancer cells are metabolically active and hypoxic, and due to massive growth and insufficient vascular perfusion tend to produce more ROS, which diffuse through the mitochondrial membrane to damage DNA, and also act as signaling messengers involved in cell survival, therapeutic drug resistance, and so on.

Figure 2. Overview of ROS signalling in normal and cancer cells[4].

There are many antioxidants in biology. The main antioxidants in the body are enzyme systems, such as superoxide dismutase (SOD), which converts O2- to H2O2, catalase, which acts on H2O2 to produce H2O and O2, and glutathione peroxidase, which breaks down H2O2 and LOOH. In addition, there are low molecular weight compounds such as N-acetylcysteine (NAC) that are commonly used as 'antioxidants'.

02 ROS Detection

Currently, fluorescent staining, electron paramagnetic (spin) resonance (EPR/ESR), chemiluminescence, chromatography, spectrophotometry, electrochemical biosensors, and fluorescent proteins are some of the main detection methods for ROS.

Table 1. Methods for detection/quantification of reactive oxygen species (different environments) [5].

Specific ROS detection methods

ROS: O2•-

Detection reagents: HKSOX-1,HKSOX-1r,HKSOX-1m

Superoxide anion radical (O2•-) has long been recognised as an important cellular signalling molecule involved in a variety of physiological and pathological processes, including innate immunity and metabolic homeostasis.Abnormal production of O2•- can lead to oxidative damage to biomolecules such as iron-sulphur (Fe-S) proteins and cysteine thiols, or directly induce bacterial and mammalian cell death. Cell death is induced directly in bacterial and mammalian cells. Secondary products, such as hydrogen peroxide, hydroxyl radicals (OH), peroxynitrite (ONOO-), and hypochlorite (HOCI), are also involved in signalling and a variety of pathological conditions.

Figure 3. Confocal imaging of endogenous O2•- imaging of zebrafish embryos 72 h after fertilisation using HKSOX-1[6].

ROS: H2O2

Detection reagents: HKPerox-1, HKPerox-2

H2O2 is produced primarily by NADPH oxidase in conjunction with superoxide dismutase, the mitochondrial electron transport chain, and many other enzymes, and is a strong two-electron oxidant, but its high activation energy limits its reactivity with a few biological targets. H2O2 is relatively stable. It reacts very slowly with glutathione, cysteine and methionine, but depending on the particular protein structure and environment, its reactivity towards cysteines in specific proteins can be greatly increased up to 10 M-1S-1 (about 106 times the average cysteine in proteins), providing the basis for the selectivity and specificity of H2O2 in redox signalling. They play a role in cell signalling, particularly in the immune system, and are involved in cell death through iron sagging.

Figure 4. Molecular imaging of endogenous H2O2 in living cells using the HKPerox probe[6]. RAW264.7 representative confocal images of macrophages co-cultured with HKPerox-1 (10 mm) and CCl3CN.

ROS: •OH

Detection reagents: HKOH-1,HKOH-2

Of all the reactive oxygen ROS, •OH is considered the most reactive and harmful. It has a short life span of about 10-9 seconds, can react with many biomolecules such as DNA bases, lipids and proteins at diffusion-controlled rates, and its overproduction can lead to cellular damage and has been linked to a variety of diseases. On the other hand, there is growing evidence that the production of '•OH' and other ROS can be applied in cancer therapy. Therefore, monitoring intracellular -OH is essential to understand its biological impact and to further investigate its therapeutic use.

Figure 5. Detection of endogenous •OH in RAW264.7 mouse macrophages by HKOH-1r in confocal imaging[8].

Highly sensitive and selective hydroxyl radical detection probes have been developed. The fluorescent probe HKOH-1 has been applied to screen the antioxidant capacity of several hydroxyl radical scavengers. By confocal imaging and flow cytometry, HKOH-1r has detected endogenous hydroxyl radical production in several cell types.

ROS: HCLO

Detection reagents: HKOCl-3, HKOCl-4, HKOCl-4m

Hypochlorous acid and hypochlorite is one of the important intracellular ROS which is catalysed by H2O2 and Cl- via myeloperoxidase, HOCl can act as an immune defence system destroying invasive bacteria while on the other hand it can cause many diseases including cancer due to pathogenic oxidative stress damage.

Figure 6. Confocal fluorescence imaging of endogenous HOCl and HKOCl-4r in RAW264.7 mouse macrophages[9].

ROS: ONOO-

Detection reagents: HKYellow-AM (6/12-mixture)、 HKGreen-4I

Oxidative free radicals including reactive oxygen species (ROS) and reactive nitrogen species (RNS) play a crucial role in the pathological process of brain I/R injury. During the cerebral ischaemia-reperfusion phase, concomitantly generated nitric oxide (NO) and superoxide anion (O2•-) rapidly form peroxynitrite (ONOO-) at a diffusive rate, which can penetrate biological membranes with even higher diffusion capacity than O2•-. ONOO- has a role in mediating apoptotic cell death, inflammation, infarction, and cellular death through the triggering of a series of molecular cascades, which can lead to the formation of a number of oxidative free radicals. ONOO- plays a key role in mediating apoptotic cell death, inflammation, infarct expansion and blood-brain barrier disruption.

Figure 7. Confocal fluorescence imaging of endogenous ONOO- and HKYellow-AM in SH-SY5Y cells[10].

Product Recommendation

HKSOX-1

The HKSOX-1 is an O2•− Probe with Green fluorescence, excited at 509 nm and emitting at 534 nm.

HKPerox-1

The HKPerox-1 serves as an H2O2 probe, emitting yellow fluorescence with excitation at 520 nm and emission at 543 nm.

HKOH-1

The HKOH-1 is an •OH Probe with Green fluorescence, excited at 500 nm and emitting at 520 nm.

HKOCl-3

The HKSOX-3 is an HOCl Probe with Green fluorescence, excited at 490 nm and emitting at 527 nm.

HKSOX-4

The HKSOX-4 is an HOCl Probe with Yellow fluorescence, excited at 530 nm and emitting at 557 nm.
References
[1] Thannickal VJ, et al. Reactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol. 2000 Dec;279(6):L1005-2.
[2] Arfin S,et al. Oxidative Stress in Cancer Cell Metabolism. Antioxidants (Basel). 2021 Apr 22;10(5):642.
[3] Griendling KK, et al. Oxidative stress and cardiovascular injury: Part I: basic mechanisms and in vivo monitoring of ROS. Circulation. 2003 Oct 21;108(16):1912-6.
[4] Khan AQ, et al. Reactive oxygen species (ROS) in cancer pathogenesis and therapy: An update on the role of ROS in anticancer action of benzophenanthridine alkaloids. Biomed Pharmacother. 2021 Nov;143:112142.
[5] Murphy MP, et al. Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo. Nat Metab. 2022 Jun;4(6):651-662.
[6] Hu JJ, et al. Fluorescent Probe HKSOX-1 for Imaging and Detection of Endogenous Superoxide in Live Cells and In Vivo. J Am Chem Soc. 2015 Jun 3;137(21):6837-43.
[7] Ye S,et al. Tandem Payne/Dakin Reaction: A New Strategy for Hydrogen Peroxide Detection and Molecular Imaging. Angew Chem Int Ed Engl. 2018 Aug 6;57(32):10173-10177.
[8] Bai X, et al. HKOH-1: A Highly Sensitive and Selective Fluorescent Probe for Detecting Endogenous Hydroxyl Radicals in Living Cells. Angew Chem Int Ed Engl. 2017 Oct 9;56(42):12873-12877.
[9] Xiaoyu Bai, et al. HKOCl-4: a rhodol-based yellow fluorescent probe for the detection of hypochlorous acid in living cells and tissues. Organic Chemistry_Frontiers. Issue 8, 2020.
[10] Feng J,et al. Naringin Attenuates Cerebral Ischemia-Reperfusion Injury Through Inhibiting Peroxynitrite-Mediated Mitophagy Activation. Mol Neurobiol. 2018 Dec;55(12):9029-9042.