Detection Of Superoxide Radical Anion Research Articles

A Nile red-based near-infrared fluorescent probe for the detection of superoxide radical anion in living cells

Reactive oxygen species (ROS) are important products of aerobic metabolism and play important roles in many physiological and pathological processes. Among the ROS, superoxide radical anion (O 2 •‒ ) is a primary ROS and precursor of a few other kinds of ROS. O 2 •‒ is widely involved in regulating cell functions and is a critical biomarker for monitoring cellular stress responses. Thus, a real-time quantitative detection of O 2 •‒ is of great significance in ascertaining the pathological roles of O 2 •‒ in related diseases. Herein we report a near-infrared (NIR) fluorescent probe NR1 for the sensitive detection and imaging of O 2 •‒ , which is rationally designed and synthesized through an esterification reaction of a Nile red dye and the diphenyl phosphinyl group. The NR1 probe exploits the strong nucleophilic mechanism of O 2 •‒ as a novel detection strategy, which leads to the cleavage of NR1 to yield Nile red as the fluorescence reporter and diphenyl phosphinyl functional group as the responsive unit. The developed NR1 probe exhibits highly sensitive, selective and rapid fluorescence turn-on response on addition of O 2 •‒ in the presence of potential ROS interfering species. Furthermore, the probe NR1 is successfully applied in imaging of various cancer cell lines (4T1 and Hela) and excellent imaging ability of O 2 •‒ in vitro is achieved. Biocompability of NR1 is also evaluated through MTS assay. The present study provides a novel strategy for molecular design of NIR fluorescent probe and the resulting probe shows great potential for practical application in complex bio-systems to study the development and progression of related diseases.

A sensitive and selective fluorescence probe for the detection of superoxide radical anion in vivo based on a protection-deprotection process

Superoxide radical anion (O2·−) plays vital roles in numerous physiological and pathological processes. Thus, it is in great demand to develop an efficient fluorescence probe for the measurement of O2·−. In the investigation, a new fluorescence probe NAP-SCM was developed for detection of O2·− on the basis of a protection-deprotection process. This probe, which utilizes coumarin as the fluorescent platform and 1,8-naphthalimidesulfonyl as the masking moiety, could monitor O2·− with good specificity, rapid response and low detection limit. More importantly, probe NAP-SCM displayed negligible cytotoxicity, good bio-compatibility and excellent capability of tracking the endogenous superoxide radical anion in live cells and live animals. The favorable observations illustrated that this probe has the promising potential to provide a valuable support for exploring the role(s) played by O2·− in the biologically functional processes.

Comment on "Water-Soluble Fluorescent Probe with Dual Mitochondria/Lysosome Targetability for Selective Superoxide Detection in Live Cells and in Zebrafish Embryos".

Recently, a new water-soluble, fluorescein-based probe for the detection of superoxide radical anion in aqueous media was developed by Lu et al. (ACS Sens. 2018, 3, 59-64). The probe was proven to be selective for superoxide and was used successfully also in cells and zebrafish embryos. To characterize the response of the probe to superoxide, Lu et al. used KO2 dissolved in deionized water as a surrogate. In testing this probe in different applications, we repeated some of these experiments and came to realize that the fluorescence signal observed by the Authors in their experiments with KO2 was incorrectly attributed to the reaction of the probe with superoxide and is due instead to its reactions with HO- and HO2-. We show that indeed under the conditions used in these assays KO2 undergoes very fast reaction with water to form HO- and HO2-. On the other hand, by using a proper surrogate, namely, KO2 dissolved in DMSO, and spin trapping experiments, we confirmed the ability of the probe to detect superoxide.

HPLC-Based Monitoring of Oxidation of Hydroethidine for the Detection of NADPH Oxidase-Derived Superoxide Radical Anion.

Hydroethidine is a fluorogenic probe that in the presence of the superoxide radical anion is oxidized to a red fluorescent product, 2-hydroxyethidium. In cells, hydroethidine is also oxidized to other products, including red fluorescent ethidium. Thus, selective monitoring of 2-hydroxyethidium is required for specific detection of the superoxide radical anion. Here, we provide protocols for HPLC- and LC-MS-based quantitation of 2-hydroxyethidium, among other oxidation products. Also, a protocol for continuous sampling for real-time monitoring of superoxide production using rapid HPLC measurements of 2-hydroxyethidium is described.

P-370 - The pulse radiolytic study of one-electron oxidation of hydropropidine and hydroethidine – implications for the detection of superoxide radical anion in biological systems

Hydroethidine (HE) is a fluorogenic probe widely used for the detection of superoxide radical anion in cellular studies. Recently, we have synthesized cell-impermeant analogue of HE called hydropropidine (HPr +). HPr+ is a water-soluble molecule, possessing highly localized positive charge on the nitrogen atom of the alkyl group, that prevents its cellular uptake. Analogously to its precursor, HPr+ reacts with superoxide forming a specific fluorescent product 2-hydroxypropidine (2-OH-Pr++). According to the proposed mechanism of superoxide-mediated oxidation of HE and HPr+, the probe undergoes one-electron oxidation to the radical cation, and next, in the presence of superoxide, is converted to the appropriate hydroxylation product. Here, we show the spectroscopic characetrization of radical cation of HPr+ and HE by pulse radiolysis technique. The reactivity of both probes toward biologically relevant radicals, e.g. nitrogen dioxide (•NO2) and carbonate (CO3•−) radicals (products of peroxynitrite decomposition), as well as glutathionyl radicals (GS•), will be presented. These results will be discussed in relation to the mechanism of oxidation of hydroethidine based probes in biological milieu.

A fast-responsive two-photon fluorescent probe for in vivo imaging superoxide radical anion with a large stokes shift

A novel and simple two-photon fluorescent probe NS-O for the detection of superoxide radical anion (O2˙−) with a large turn-on fluorescence signal is constructed to monitor endogenous superoxide radical anions in living cells, tissues and zebrafish.

77 - N,N,N,N-tetramethylhydroethidine (TMHE) - in search for Novel Probes for the Detection of Superoxide Radical Anion

77 - N,N,N,N-tetramethylhydroethidine (TMHE) - in search for Novel Probes for the Detection of Superoxide Radical Anion

P 061 - N,N.N′,N′-tetramethylhydroethidine (TMHE) - in search for better probes for the detection of superoxide radical anion

The superoxide radical anion is an important reactive species produced by one-electron reduction of molecular oxygen. The main sources of superoxide radical anion in vivo are the enzymes NADPH oxidases (NOX) and mitochondria (mitochondrial electron transport chain). Superoxide radical anion reacts rapidly with nitric oxide with the formation of peroxynitrite (ONOO−). It can also undergo spontaneous or SOD-catalyzed dismutation with the formation of hydrogen peroxide (H2O2) and thus it is a precursor of other biologically relevant oxidants playing an important role in various pathologies. Due to its reactivity and short lifetime in vivo, its detection and quantitation is difficult and demands special and sensitive techniques. One of the approaches is the use of fluorogenic probes, the compounds which themselves are not fluorescent but in the reaction with superoxide are oxidized to the fluorescent products that can be directly detected. Among various profluorescent probes available hydroethidine (HE) seems to be a gold standard for detection of superoxide in biological systems. In the presence of superoxide radical anion, HE undergoes oxidative transformation into 2-hydroxyethidium (2-OH-E+), a specific marker of superoxide radical anion production. In the reaction with other radical, one-electron oxidants, HE is oxidatively transformed into ethidine and dimeric products. Here we present a spectroscopic and chemical characterization of new analogue of hydroethidine - N,N,N,N-tetramethylhydroethidine (TMHE).

Recent developments in detection of superoxide radical anion and hydrogen peroxide: Opportunities, challenges, and implications in redox signaling

In this review, some of the recent developments in probes and assay techniques specific for superoxide (O2–) and hydrogen peroxide (H2O2) are discussed. Over the last decade, significant progress has been made in O2– and H2O2 detection due to syntheses of new redox probes, better understanding of their chemistry, and development of specific and sensitive assays. For superoxide detection, hydroethidine (HE) is the most suitable probe, as the product, 2-hydroxyethidium, is specific for O2–. In addition, HE-derived dimeric products are specific for one-electron oxidants. As red-fluorescent ethidium is always formed from HE intracellularly, chromatographic techniques are required for detecting 2-hydroxyethidium. HE analogs, Mito-SOX and hydropropidine, exhibit the same reaction chemistry with O2– and one-electron oxidants. Thus, mitochondrial superoxide can be unequivocally detected using HPLC-based methods and not by fluorescence microscopy. Aromatic boronate-based probes react quantitatively with H2O2, forming a phenolic product. However, peroxynitrite and hypochlorite react more rapidly with boronates, forming the same product. Using ROS-specific probes and HPLC assays, it is possible to screen chemical libraries to discover specific inhibitors of NADPH oxidases. We hope that rigorous detection of O2– and H2O2 in different cellular compartments will improve our understanding of their role in redox signaling.

22 - Spin Trapping with Fluorescence Detection: Novel Method for Specific and Sensitive Fluorimetric Detection of Superoxide Radical Anion

22 - Spin Trapping with Fluorescence Detection: Novel Method for Specific and Sensitive Fluorimetric Detection of Superoxide Radical Anion

Superoxide Radical Anion Adduct of 5,5-Dimethyl-1-pyrroline N-Oxide. 4. Conformational Effects on the EPR Hyperfine Splitting Constants

Spin trapping has been commonly employed in the detection of superoxide radical anion in chemical and biological systems; hence, accurate interpretation of the hyperfine splitting constants (hfsc's) arising from the O(2)(*-) adducts (also referred to as hydroperoxyl (HO(2)(*)) radical adducts) of various nitrones is important. In this work, the nature of the relevant hfsc's was investigated by examining the effect of conformational changes in the hydroperoxyl moiety of the O(2)(*-) adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide (EMPO), 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), 5-carbamoyl-5-methyl-1-pyrroline N-oxide (AMPO), and 7-oxa-1-azaspiro[4.4]non-1-en-6-one N-oxide, (CPCOMPO) on the magnitude of a(N), a(beta-H), and a(gamma-H). Conformational change around the substituents and their effect on the hfsc's were also explored. Results indicate that a(beta-H) is most sensitive to conformational changes of the hydroperoxyl and substituent groups relative to hfsc's of other nuclei. The orbital overlap between the C-H sigma-orbital and the SOMO of the nitroxyl nitrogen plays a crucial factor in determining the magnitude of the a(beta-H). The hfsc values for the O(2)(*-) adducts were predicted with high accuracy by using a low-cost computational method at the PCM(water)/BHandHLYP/EPR-III//B3LYP/6-31G* level of theory without taking into account the explicit water interaction.

Superoxide Radical Anion Adduct of 5,5-Dimethyl-1-pyrroline N-Oxide (DMPO). 3. Effect of Mildly Acidic pH on the Thermodynamics and Kinetics of Adduct Formation

The nitrone, 5,5-dimethylpyrroline N-oxide (DMPO), is a commonly used spin trap for the detection of superoxide radical anion (O2*-) using electron paramagnetic resonance spectroscopy. This work investigates the reactivity of DMPO to O2*- in mildly acidic pH (5.0-7.0). Mild acidity is characteristic of acidosis and has been observed in hypoxic systems, e.g., ischemic organs and cancer cells. Although the established pKa for O2*- is 4.8, the pKa for DMPO is unknown. The pKa of the conjugate acid of DMPO was determined to be 6.0 using potentiometric, spectrophotometric, 1H and 13C NMR, and computational methods. 1H and 13C NMR were employed to investigate the site of protonation. An alternative mechanism for the spin trapping of O2*- in mildly acidic pH was proposed, which involves protonation of the oxygen to form the N-hydroxy imino cation and subsequent addition of O2*-. The exoergicity of O2*- addition to protonated DMPO was rationalized using density functional theory (DFT) at the PCM/B3LYP/6-31+G**//B3LYP/6-31G* level of theory.