Fluorescent and Luminescent Probes for ROS, RNS, Thiols, and Metal Ions

Oxidative stress is brought on by elevated levels of reactive oxygen species (ROS), reactive nitrogen species (RNS), depleted availability of detoxifying thiols, and dysregulation of metal ions (both redox-active iron and copper as well as non-redox active calcium and zinc). Isolating and detecting these culprits is often difficult, which can lead to misreporting or misinterpretation of data. However, proper utilization of a well-designed probe can lead to a wealth of information about oxidative biology. Cayman offers several classes of small molecule fluorescent and chemiluminescent probes designed to detect these various molecules. Use the tables below to navigate to the most appropriate probes for your particular experiment.

Fluorescent and Luminescent Probes for ROS and RNS

ROS, generated during both physiological and pathological processes, reacts with cellular components (amino acids, proteins, lipids, and nucleic acids) to participate in redox signaling and other key events that are advantageous to the cellular system. During stress, however, ROS can participate in reactions that lead to cell damage. Hydroxyl radicals (OH^•) are presumed to be the species that causes direct damage to biomolecules. Unfortunately, most probes available to detect oxidative activity are not specific for any individual ROS and may require conditional activation to produce a signal.

For example, one of the most commonly used probes, 2,7-dichlorodihydrofluorescein diacetate (DCFH) does not react directly with H₂O₂ but requires a catalyst such as cytochrome c or a redox-active metal cation to be oxidized by two electrons to produce a fluorescent product. As a complicating factor, one electron oxidation of DCFH yields a radical intermediate that reacts with O₂ to produce superoxide (O₂^•ˉ), and DCFH itself is susceptible to auto-oxidation, which can turn on a false fluorescence response.

Two compounds that offer more limited reactivity to ROS are APF and HPF. Both probes are resistant to autooxidation and fluoresce upon interaction with OH^•, ONOO^ˉ, or singlet oxygen ¹O₂. In addition, APF can react with hypochlorite anion (OCl^ˉ). With this difference in reactivity, APF used in conjunction with HPF can differentially detect OCl^ˉ.

A group of boronate-containing fluorogenic compounds (e.g., coumarin boronic acid) are emerging as one of the most effective probes for detecting and quantifying peroxynitrite (ONOO^ˉ), OCl^ˉ, and hydrogen peroxide (H₂O₂). Boronates react with ONOO^ˉ nearly one million times faster than with H₂O₂ and have been used to monitor real-time generation of ONOO^ˉ in cells and for imaging in living animals.

Pentafluorobenzenesulfonyl fluorescein was designed for even greater selectivity for H₂O₂. It utilizes fluorescein protected within a pentafluorbenzenesulfonyl ester. The sulfonyl group’s reactivity, which is H₂O₂-responsive, is enhanced by the presence of the pentafluorobenzene ring. With the addition of a protecting group that reacts exclusively with H₂O₂, this probe overcomes many of the limitations of DCFH and other commonly used probes such as dihydroethidium (DHE). Pentafluorobenzenesulfonyl fluorescein can be used as a probe for H₂O₂ in whole cell systems.

Chemiluminescence can also be a sensitive detection method for radical production. For example. Lucigenin is a luminescent indicator of O₂^•ˉ production by both enzymatic and cellular sources. Although extremely sensitive to O₂^•ˉ, H₂O₂ as well as other reducing agents and numerous nucleophiles can also induce its luminescence. The luminophore coelenterazine and MCLA (hydrochloride) can also produce chemiluminescence in response to O₂^•ˉ generation in cells. MCLA (hydrochloride) offers the advantage of requiring an optimum pH for luminescence generation that is closer to the physiological range of cells.

To directly assess the extent of lipid peroxidation, researchers can measure the amount of lipid hydroperoxides using fluorescence quenching of the fatty acid analog cis-parinaric acid, whose extensive saturation is highly susceptible to oxidation. Alternatively, lipid soluble DPPP fluoresces upon oxidation and has been used to detect picomole levels of hydroperoxides by HPLC. C11 BODIPY 581/591 also serves as a highly sensitive probe for imaging lipid peroxidation in live cells. It undergoes a shift from red to green fluorescence emission upon oxidation of the phenylbutadiene segment of the fluorophore. Cayman also carries assay kits to detect the presence of secondary reaction products of lipid peroxidation (e.g., hydroxy-2-nonenal or malonaldehyde).

Nitric oxide radicals (NO^•) are relatively short-lived and occur at low concentrations, making detection particularly challenging. NO^• is readily oxidized to NO⁺, which is moderately stable but highly reactive to nucleophiles and other NOs. However, NO^• can be trapped by aromatic amines (e.g., DAF-2 diacetate, DAF-FM diacetate) to form fluorescent diazonium salts or by aromatic 1,2-diamines to form fluorescent benzotriazoles. These compounds must first be nonspecifically oxidized to an anilinyl radical, which then reacts with NO^• to form the fluorescent product.

Cayman’s fluorescent and luminescent probes for the detection of ROS and RNS represent the wide-ranging diversity in strategies to detect these elusive oxidants. We also offer a ROS Detection Cell-Based Assay Kit (DCFDA), ROS Detection Cell-Based Assay Kit (DHE), and many additional assays that conveniently package detection probes together with all the necessary reagents to detect the generation of ROS. Download our guide to find the right assay for application.

ROS and RNS Probes

Fluorescent Probes for Intracellular Thiols

Intracellular thiols like cysteine or glutathione play an important role in antioxidant defense. Any disruption to their homeostasis can lead to an imbalance of cellular redox and are linked to disease. Cayman’s thiol-reactive fluorogenic probes can be used to quantify a variety of compounds containing reactive sulfur or thiol groups. In addition, our Thiol Detection Assay Kit conveniently packages a proprietary fluorometric detector along with all the reagents needed to assay free thiol content in a wide range of samples.

Fluorescent Probes for Metal Cations

Calcium

Intracellular calcium concentrations are tightly regulated at low levels. Fluxes in concentration are controlled by the introduction of extracellular calcium from various pumps, exchangers, and channels or from intracellular stores. The mechanisms for controlling extracellular calcium import are susceptible to oxidative stress. Significant damage is evident with transient calcium increases.

Most calcium probes are designed by linking a fluorophore to a calcium chelator (e.g., BAPTA) to generate a calcium-dependent response. Cayman’s calcium probes have affinities ranging from high to low to allow for calcium detection in a wide variety of intracellular systems. High-affinity calcium indicators display high selectivity for calcium and are not affected by sodium gradients, membrane potential, or intracellular pH, which is ideal for monitoring low levels of calcium, such as in resting cells. Low-affinity calcium indicators are particularly useful for studying compartments with high concentrations of calcium, such as endoplasmic reticulum, where high-affinity dyes will be insensitive to luminal fluctuations. Cayman’s probes offer sensitive calcium detection with excitation and emission wavelengths that span the UV and visible light spectrum. Although UV-excitable probes must be used with caution in cells, since UV light can be damaging and can excite autofluorescence. Some of these probes exhibit a ratiometric response (a shift in their excitation or emission profile), which is useful for determining calcium concentration and for normalizing any background interference.

Ratiometric Calcium Indicators

Excitation and/or emission wavelengths shift in response to calcium binding.

UV-excitable Calcium Indicators (Single Wavelength)

While providing sensitivity, use with caution in cells since UV light can be damaging and can excite autofluorescence.

Medium-Wavelength Calcium Indicators

Exhibits >100-fold calcium-dependent green fluorescence emission with minimal fluorescence at resting calcium levels.

Long-Wavelength Calcium Indicators

Large increase in fluorescence emission intensity upon binding calcium, useful for multiplexing with green fluorescent dyes or in cells with high autofluorescence.

Zinc

Zinc is an indirect antioxidant whose deficiency is linked to elevated ROS. It is important for the activity of zinc-dependent enzymes such as superoxide dismutase and for the production of metallothionein. Because many calcium sensors (e.g., carboxylate-based probes) can also bind zinc, it is possible to discriminate between these two metal ions using probes that are selective for zinc. By possessing little affinity for calcium, zinc probes can be used to cross-validate a particular response as calcium- or zinc-dependent. Cayman offers fluorescent zinc probes that enable the visualization of zinc in live cells. They act as high-affinity sensors for applications where only trace amounts of zinc are expected.

High-Affinity Zinc Indicators

Copper

Copper is an essential transition metal with a key role as a redox cofactor for various cellular processes. Its uptake, transport, and storage must be tightly regulated, since it can displace other metal ions that act as cofactors in enzyme-catalyzed reactions. Also, copper ions can react with H₂O₂via the Fenton reaction to produce reactive OH^• and OH^ˉ and the corresponding oxidized metal, Cu²⁺ that can damage proteins, DNA, etc. at high concentrations. There are few copper probes available that can select for labile copper (Cu⁺). Cayman offers a highly sensitive Cu²⁺ probe that acts as a chemodosimeter, relying on Cu²⁺-promoted hydrolysis to irreversibly open a rhodamine spirolactam ring to trigger fluorescence.

Copper Chemodosimeter

Iron

Iron is an essential transition metal that is necessary for processes such as oxygen transport, metabolism, electron transfer, and enzymatic reactions within the mitochondrial respiratory chain. The majority of iron is tightly bound to storage proteins or enzymes, but a small percentage remains chelatable and redox-active in a steady-state, labile, ferrous iron (Fe²⁺) pool to serve as a transition point for cellular iron metabolism. Under oxidative stress, this equilibrium is disrupted. Excess Fe²⁺ can lead to oxidative damage, since it will catalyze the toxic production of OH^• and OH^ˉ and ferric iron (Fe³⁺) via the Fenton reaction. Cayman’s iron probes rely on a fluorescence quenching mechanism wherein a known iron chelator is tagged with a fluorophore whose signal is turned off upon iron binding. These probes can be used to detect either ferrous or ferric iron and are available with excitation and emission wavelengths that span the full range of the fluorescent spectrum.

Fe²⁺/Fe³⁺ Sensors

Suggested Reading

Hyman, L.M. and Franz, K.J. Probing oxidative stress: Small molecule fluorescent sensors of metal ions, reactive oxygen species, and thiols. Coord. Chem. Rev.256(19-20), 2333–2356 (2012).

Kaur, K., Saini, R., Kumar, A., et al. Chemodosimeters: An approach for detection and estimation of biologically and medically relevant metal ions, anions and thiols. Coord. Chem. Rev. 256(17-18), 1992-2028 (2012).

Nauseef, W.M. Detection of superoxide anion and hydrogen peroxide production by cellular NADPH oxidases. Biochim. Biophys. Acta.1840(2), 757-767 (2014).

Paredes, R.M., Etzler, J.C., Watts, L.T., et al. Chemical calcium indicators. Methods. 46(3), 143-151 (2008).

Wardman, P. Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: Progress, pitfalls, and prospects. Free Radic. Biol. Med.43(7), 995-1022 (2007).

Zielonka, J., Sikora, A., Hardy, M., et al. Boronate probes as diagnostic tools for real time monitoring of peroxynitrite and hydroperoxides. Chem. Res. Toxicol.25(9), 1793–1799 (2012).