Detection Of ONOO Research Articles

Direct assessment of nitrative stress in lipid environments: Applications of a designer lipid-based biosensor for peroxynitrite

Lipid membranes and lipid-rich organelles are targets of peroxynitrite (ONOO-), a highly reactive species generated under nitrative stress. We report a membrane-localized phospholipid (DPPC-TC-ONOO-) that allows the detection of ONOO- in diverse lipid environments: biomimetic vesicles, mammalian cell compartments, and within the lung lining. DPPC-TC-ONOO- and POPC self-assemble to membrane vesicles that fluorogenically and selectively respond to ONOO-. DPPC-TC-ONOO-, delivered through lipid nanoparticles, allowed for ONOO- detection in the endoplasmic reticulum upon cytokine-induced nitrative stress in live mammalian cells. It also responded to ONOO- within lung tissue murine models upon acute lung injury. We observed nitrative stress around bronchioles in precision cut lung slices exposed to nitrogen mustard and in pulmonary macrophages following intratracheal bleomycin challenge. Results showed that DPPC-TC-ONOO- functions specifically toward iNOS, a key enzyme modulating nitrative stress, and offers significant advantages over its hydrophilic analog in terms of localization and signal generation.

Recent advances in fluorescent probes of peroxynitrite: Structural, strategies and biological applications

Peroxynitrite (ONOO-), owing to its high oxidative and nitrating stress, is associated with several physiological processes in addition to various pathological processes, including those related to neurodegenerative diseases and cancer. Detection of ONOO- at the cellular level is of great significance to understand its pathogenesis. To this end, a variety of fluorescent probes based on small molecules and nanoparticles (NPs) have been engineered and applied as excellent tools for imaging of ONOO- in cells as well as in their diverse biological applications. In this review, we highlight representative cases of fluorescent probes based on recognition mechanism and emphasize their response type (ratiometric, two-photon, long-wavelength/near-infrared, and targeting) in ONOO- detection in the last five years. We further discuss their design strategy, sensing mechanism, and application in bio-imaging and describe NP-based probes according to diverse nanoplatforms.

A mitochondria-targeted ratiometric fluorescent nanoprobe for imaging of peroxynitrite in living cells

Peroxynitrite (ONOO−) is a series of basic biological oxidants involved in physiological and pathological processes. The detection of ONOO− in biological systems has been challenging due to its extremely short half-life and low steady-state concentration. In this work, a ratiometric fluorescent nanoprobe for ONOO− was constructed by coupling covalently of graphene quantum dots (GQDs) with cyanine 5.5 (Cy5.5). This nanoprobe (GQD-Cy5.5) could selectively accumulate in mitochondrial, appears two strong fluorescence emission peaks at 520 and 694 nm. In the presence of ONOO−, the intensity of fluorescence emission peak at 520 nm increased and the intensity of fluorescence emission peak at 694 nm decreased. The ratio (F520 nm/F694 nm) of fluorescence intensity at two emission peaks had a good linear relationship with the concentration of ONOO− in the range of 0–6.0 μM, and the detection limit was 0.03 μM. The excellent properties of the nanoprobe enable its applications in the ratiometric fluorescence imaging of endogenous ONOO− in cell mitochondria.

Elucidating the Structure-Reactivity Correlations of Phenothiazine-Based Fluorescent Probes toward ClO.

In this work, with the aim of developing effective molecular probes and investigating the structure-reactivity correlation, a short series of phenothiazine-based fluorescent probes are designed for the detection of ClO- with differing electron push-pull groups. Sensing experiment results and single-crystal X-ray analysis with the aid of time-dependent DFT (TD-DFT) calculations reveal that substituting groups with increasing electron-withdrawing ability can increase the dihedral angle of the phenothiazine moiety and reduce the gap energy of the probes, leading to enhanced reactivity toward ClO- . Both PT1 and PT2 show two-color switching upon detection of ClO- . PT1, with the strong electron-donating group thiophene, shows a fluorescence color switch from salmon to blue. PT2, with a medium electron-donating/accepting group benzothiazole, shows a fluorescence color switch from red to green. However, both PT1 and PT2 show almost no response to ONOO- . Through the introduction of strong electron-withdrawing ketone combined with a cyano group, PT3 shows a cyan emission upon detection of ClO- and weak red emission upon detection of ONOO- . HRMS and 1 H NMR results confirm that PT1 and PT2 have the same sensing mode, in which the divalent sulfur of phenothiazine can be oxidized to sulfoxide by ClO- . Upon reaction with ClO- , PT3 experiences two-step reactions. It is first oxidized into the sulfone structure by ClO- , and then transformed into sulfoxide phenothiazine aldehyde. Upon encountering ONOO- , PT3 changes into an aldehyde structure and some nonfluorescent byproducts. Owing to their special selectivity and high sensitivity, PT1 and PT2 are applied to image the endogenous ClO- in macrophage cells and zebrafish larvae. This study is expected to provide useful guidelines for probe design for various applications.

Aromatization of 9,10-Dihydroacridine Derivatives: Discovering a Highly Selective and Rapid-Responding Fluorescent Probe for Peroxynitrite.

As part of an effort to develop generally applicable strategies for creating probes suitable for detecting important molecular and ionic species, the oxidative aromatization of nonfluorescent 9,10-dihydroacridine derivatives triggered by peroxynitrite (ONOO-) led to the identification of compound 2H, 9-phenyl-9,10-dihydroacridine-4-carboxylic acid, as a rapid-responding fluorescent probe capable of detecting ONOO- with an extraordinary selectivity. Adding a little more than 1 equiv of ONOO- to a solution of 2H resulted in over 100-fold fluorescence enhancement. In sharp contrast, treating 2H with excessive amounts of other oxidants that often interfere with the detection of ONOO- failed to lead to noticeable fluorescence increase. The reaction of ONOO- with 2H shows a similar efficiency in the pH range of 2-8. Low cytotoxicity was observed for 2H and its aromatized product. Bioimaging experiments revealed the promising potential of 2H as a new fluorescent probe for the selective detection of intracellular ONOO-.

Array of gold ultramicroelectrodes for the simultaneous electrochemical detection of nitric oxide and peroxynitrite

We report here on the design of assembled arrays of ultramicroelectrodes (UMEs) for the simultaneous and dynamic sensing of nitric oxide (NO) and peroxynitrite (ONOO–) released by cultured cells.The arrayed microelectrodes were designed in such a way to solve the sensitivity problem posed by the low concentrations of the target analytes: over 200 UMEs were short-circuited using photolithographic techniques and divided into two sections. Our results show that the short-circuited electrodes behave as typical UMEs over our experimental timescale whilst showing enhanced sensitivity. One section of arrayed UMEs was oriented toward the chemical modification of the electrodes surfaces to obtain selective NO-sensor with the desired selectivity against interfering analytes. To do so, we developed new combination of electropolymerisable membranes for the selective detection of NO, adapted to the arrayed configuration. The second section of arrayed UMEs was devoted to the selective detection of ONOO–. These disposable microelectrode platforms are then integrated within cell culture wells and plates, resulting in an easy-to-handle tool for cellular biology.

Electrochemical approach to detect the presence of peroxynitrite in aerobic neutral solution

This work describes the conditions of use of bare gold electrode to detect electrochemically the presence of peroxynitrite ONOO − in phosphate buffer solution at pH 7.1. As ONOO − is extremely unstable in neutral solution, current–potential curve was reconstructed between −0.5 and 0.7 V vs SCE by amperometry experiments at rotating disk electrode at different potentials. Comparison of this reconstructed curve with voltammograms of the common interfering species (dopamine, hydrogen peroxide, nitrite, ascorbic acid and glutamate) shows that the presence of ONOO − can be selectively determined at −0.1 V vs SCE. This detection occurs through the electrochemical reduction of peroxynitrous acid ONOOH, the conjugated acid of ONOO −. Detection of ONOO − produced in situ by the reaction of nitric oxide with superoxide was also achieved.

Real‐Time Amperometric Analysis of Reactive Oxygen and Nitrogen Species Released by Single Immunostimulated Macrophages

Macrophages are key cells of the immune system. Immunologically activated macrophages are known to release a cocktail of reactive oxygen and nitrogen species. In this work, RAW 264.7 macrophages were activated by interferon-gamma and lipopolysaccharide, and the reactive mixture released by single cells was analyzed, in real time, by amperometry at platinized carbon microelectrodes. In comparison with untreated macrophages, significant increases in amperometric responses were observed for activated macrophages. Nitric oxide (NO*), nitrite (NO2*-), and peroxynitrite (ONOO-) were the main reactive species detected. The amounts of these reactive species were quantified, and their average fluxes released by a single, activated macrophage were evaluated. The detection of ONOO- is of particular interest, as its role and implications in various physiological conditions have been widely debated. Herein, direct evidence for the formation of ONOO- in stimulated macrophages is presented. Finally, the presence of 1400W, a selective inducible nitric oxide synthase (iNOS) inhibitor, led to an almost complete attenuation of the amperometric response of activated RAW 264.7 cells. The majority of the reactive species released by a macrophage are thus likely to be derived from NO* and superoxide (O2*-) co-produced by iNOS.

Endogenous Peroxynitrite Generation Causes a Subsequent Suppression of Coronary Arterial Contraction to Serotonin

High levels of exogenous peroxynitrite (ONOO−) have been reported to cause coronary vascular relaxation by a mechanism that appears to involve the subsequent generation of nitric oxide (NO). In this study, we examined if endogenous vasoactive levels of ONOO−are formed from endogenous superoxide anion (O2⨪) upon exposure of isolated endothelium-removed bovine coronary arteries (BCA) to biological levels of NO. During exposure of BCA to ∼50 nmNO for 2 min, the level of endogenous O2⨪detected by lucigenin-dependent chemiluminescence (CL) was markedly decreased and an increase in luminol-dependent CL was observed, consistent with the detection of ONOO−generation. NO treatment caused a decrease in contraction of BCA to 0.1–3 μmserotonin (5-HT). This suppression of contraction to 5-HT was completely prevented by preincubation prior to NO exposure with agents that prevent endogenous O2⨪production (10 μmdiphenyliodonium) or trap intracellular O2⨪(10 mmTiron) or ONOO−(0.1 mmurate), and by post-NO treatment with an agent that traps NO (1 μmoxyhemoglobin) or prevents the stimulation of cGMP production by NO (10 μmmethylene blue). The NO treatment caused a subsequent release of NO (measured in the head space after a 5-min equilibration with 95% N2–5% CO2), and this subsequent release of NO was reduced by the presence of urate during NO exposure and by depletion of endogenous tissue glutathione (by pretreatment with 7 mmdiethyl maleate). Thus, exposure of BCA to elevated physiological levels of NO causes a prolonged suppression of contraction to 5-HT which appears to result from endogenous ONOO−formation and a thiol-dependent process that traps and subsequently releases vascular relaxant levels of NO.