Endogenous ONOO Research Articles

A borate-based peroxynitrite fluorescent probe and its application in fluorescence imaging of living cells.

As a bioactive species with high oxidation capacity, peroxynitrite (ONOO-) plays a crucial role in the regulation of diverse pathophysiological processes, and the overproduction of ONOO- is closely associated with various physiological diseases such as liver injury, pulmonary fibrosis and so on. Herein, two borate-based fluorescent probes 3a and 3b were synthesized for monitoring ONOO- by a simple substitution reaction. The experimental results showed that 3a and 3b had high selectivity and sensitivity for ONOO-. The detection limits of 3a and 3b were 79.46 nM and 32.12 nM, respectively. Moreover, the recognition was not disturbed by other active oxygen groups and common ions. More importantly, the probes 3a and 3b had low cytotoxicity and were successfully used to detect endogenous and exogenous ONOO-. They would provide an efficient detection method for further exploring the physiological and pathological role of ONOO- in complex biological systems and related diseases.

A FRET assembly of fluorescent gold nanoclusters for ratiometric monitoring peroxynitrite in living cells and zebrafish

Developing reliable probes for accurate monitoring peroxynitrite (ONOO–) in living systems with high sensitivity and selectivity is imperative for both elucidating its functions in many life-threatening diseases and improving therapeutic interventions. Herein, a novel nano-assembly structure (d-AuGP) was developed by electrostatic interaction-induced assembly between dichromatic gold nanoclusters (Au NCs). The assembly further induced fluorescence resonance energy transfer (FRET) and endowed d-AuGP with a well single-excitation, dual-emission fluorescence characteristic. The prepared nanostructure showed a quantitative ratiometric fluorescence response toward ONOO– with a remarkable decrease of emission peak at 685 nm and a stable peak at 470 nm. A good linear relationship between the ratio of emission intensities at 685 and 470 nm (I685/I470) with ONOO– concentration in the range of 0–100 μM was achieved and the limit of detection (LOD) was 0.39 μM. The possible fluorescence response mechanism was ascribed to the combined effects of dissociation-disturbed FRET and surface structure reconstruction. The Au NCs-based probe was demonstrated to possess high stability, low cytotoxicity, good cell and tissue permeability and high biocompatibility. It was effectively employed for biological imaging and monitoring exogenous and endogenous ONOO– levels in living cells and zebrafish with high contrast.

Multifunctional lysosome-targetable fluorescent probe for imaging peroxynitrite in acute liver injury model

Liver injury is usually caused by different sources (such as drugs, food, and alcohol), which is regarded as a serious threat to human health. Since the current indicators are not well defined, it is crucial to develop novel probes to characterize the kinetic process of liver injury. In this paper, a novel probe with fluorescence signals, Lyso-ONOO, was synthesized to detect the fluctuating peroxynitrite (ONOO−) level in various acute injury model mice. With the addition of ONOO−, the fluorescence response of Lyso-ONOO exhibited a large Stokes shift of 105 nm under 450 nm excitation. The reaction had the merits of high sensitivity, high selectivity, rapid reaction, and stability under various pH conditions. In addition, Lyso-ONOO had prominent lysosome-targeting ability. It achieved monitoring the exogenous and endogenous ONOO− in human hepatic stellate cells. Further, Lyso-ONOO could display endogenous ONOO− in models of 4T1-xenograft and inflammation in mice. More importantly, Lyso-ONOO could gauge the changing levels of ONOO− in three types (induced by CCl4, APAP, and alcohol) of acute liver injury models and treatments (silybin and bicyclol) of CCl4-induced liver injury mice. Both in vivo imaging and depth-of-penetration fluorescence imaging demonstrated that Lyso-ONOO could characterize the developmental and therapeutic status during the liver injury process by measuring the dynamic change of ONOO−. The monitoring of Lyso-ONOO appeared to be useful in inferring the order of severity in diverse acute liver injury processes and taking into account the efficacy of treatment regimens, which might provide important information for corresponding research.

An Ultrafast Fluorescent Probe for the Detection of Peroxynitrite in Living Cells

Peroxynitrite (ONOO−), a highly reactive nitrogen species, which plays a crucial role in numerous physiological and pathological processes of cell functionalization. The anomalous concentration of ONOO− may result in a range of diseases, such as arthritis, neurological disorders, and even cancer. Therefore, it is urgent to develop a simple and effective tool to monitor the fluctuation of ONOO− levels in biological systems. Herein, an ultrafast fluorescent probe (HND-ONOO) is proposed to detect ONOO−, which displays brilliant fluorescence in less than 30 s with a large Stokes shift. Furthermore, the probe exhibited the lower detection limit (48 nM) and satisfactory results in differentiating ONOO− from other related species. The probe that possesses good biocompatibility and low toxicity was employed to monitor the level of exogenous and endogenous ONOO− in living cells. Thus, the probe HND-ONOO could be served as a potential imaging tool to visualize intracellular ONOO− and understand the relationship between ONOO− and inflammation.

Dual-Channel Fluorescent Probe for the Detection of Peroxynitrite and Glutathione in Mitochondria: Accurate Discrimination of Inflammatory and Progressing Tumor Cells.

Distinguishing between normal, inflammatory, and progressing tumor cells plays a vital role in early diagnoses and clinical studies. The simultaneous quantification of multiple biomarkers in cells can reveal cellular heterogeneity, which contributes to the discrimination of different types of cells. Herein, a dual-channel fluorescent probe has been developed for monitoring peroxynitrite (ONOO-) and glutathione (GSH) to accurately discriminate normal cells, inflammatory cells, and progressing cancer cells. The probe can monitor exogenous and endogenous mitochondrial GSH and ONOO- in living cells and zebrafish by green (530 nm, G530) and red (630 nm, R630) emission based on its good selectivity and low biotoxicity. GSH and ONOO- are visualized via fluorescence imaging, and the corresponding output signals can be employed to differentiate nontumorigenic, malignant, and metastatic breast cells in cocultured cells. Furthermore, the accurate discrimination among normal, inflammatory, and cancerous cells is achieved through the changes in the dual-channel fluorescence signal, which shows great potential for the diagnosis of inflammation and cancer diseases.

Construction of a ratiometric two-photon ER-targeting fluorescent probe for the imaging of peroxynitrite in living systems

Peroxynitrite (ONOO-) could deplete ER-associated Ca2+ and cause ER stress, and thereby precise and in situ detection of ONOO- at ER is highly important for the study of its biological roles. Herein, we present a ratiometric two-photon ER-targeting fluorescent probe (NAB) for the detection of ONOO- in living cells and brain tissues. NAB utilized 4-bromomethylphenyl borate as a recognition site for ONOO-, and utilized p-toluenesulfonamide as ER-targeting group. After the response of NAB to ONOO-, the 4-bromomethylphenyl borate converted to hydroxyl, resulting in the change of the fluorescence color from blue to yellow. The probe NAB could show highly sensitive response to ONOO- within 6 s, and exhibited excellent selectivity to ONOO-. The probe NAB mainly located at ER and showed excellent ER-targeting property. Ratiometric two-photon imaging demonstrated that the probe NAB could selectively detect exogenous ONOO- at ER, and also could be applied to image metformin-induced ONOO- in living HepG2 cells. Furthermore, NAB can effectively detect the changes of ONOO- level in brain tissues, and revealed that the endogenous ONOO- level at ER showed increase in hippocampus of the depressive mouse.

Dual-targetable fluorescent probe for mapping the fluctuation of peroxynitrite in drug-induced liver injury model

Drug-induced liver injury (DILI) is one of the most common and serious adverse drug reactions which can cause acute liver failure or even death in severe cases. With the incidence rate increasing over the years, DILI has became a frequent clinical liver disease and a global public health problem. As a biomarker of DILI, the detection of peroxynitrite (ONOO−) has became a powerful tool for the early diagnosis of liver injury. Here, we synthesized five mitochondria-targetable probes, 1–5, for detecting endogenous ONOO−. Through dye-screening, probe 5 was stood out by its excellent performance. In the presence of ONOO−, the fluorescence signal of probe 5 reduced 40-fold in 19 s with a low detection limit (9.36 nM). At the same time, the transformation can be observed with the naked eye under sunlight or UV lamp without being affected by the other reactive species. Even better, with low toxicity and high biocompatibility, probe 5 could successfully detect endogenous ONOO− in the mitochondrion of cells. Finally, probe 5 could specifically target the liver, and can be employed for monitoring the therapeutic effect of hepatoprotective medicine after drug-induced hepatotoxicity in vivo. In brief, probe 5 has the practical application capability for diagnosing the severity of the liver injury and researching the therapeutic effect of antidote in complex bio-systems.

Spying on the peroxynitrite fluctuation during hearing loss enabled by rationally designed fluorescent carbon dots

Hearing loss is a common type of sensory deprivation. Peroxynitrite (ONOO−) is a biologically generated reactive oxygen species that can behave as a deleterious species due to its abnormal regulation associated with the pathogenesis of many human diseases, including hearing loss. Despite the importance of ONOO− and its biological functions, accurate detection methods for monitoring ONOO− in real time and exploring the roles of ONOO− in living systems are lacking. Herein, we report a novel carbon dots (CDs)-based fluorescence turn-on ONOO− probe for tracking endogenous and exogenous ONOO− produced by RAW264.7 cells. The probe exhibited considerably enhanced emission intensity, fast response rate, outstanding selectivity, and good sensitivity. Owing to these attractive sensing properties, peroxynitrite fluctuations in a hearing loss model were monitored for the first time. Therefore, CDs are potentially effective tools for assessing fluctuations in ONOO− under different physiological and pathological conditions.

A benzothiazole-based fluorescence probe for imaging of peroxynitrite during ferroptosis and diagnosis of tumor tissues.

Ferroptosis, as a new regulated mode of cell death, is mainly characterized by iron-dependent accumulation of lipid peroxides related to reactive oxygen species (ROS). However, the changes of peroxynitrite (ONOO-) during ferroptosis are not very clear due to the limited reports. On the other hand, ONOO- has become an endogenous toxicant leading to cell apoptosis and necrosis. Thus, it is urgent to develop molecular tools with high selectivity and sensitivity to monitor intracellular ONOO-. Herein, we presented a fluorescent probe, BTMO-PN, by introducing a phenylboronic acid ester as the ONOO- recognition site into benzothiazolyl derivative. BTMO-PN exhibited a rapid and marked fluorescence enhancement signal toward ONOO-, owing to the ONOO--triggered the cleavage of phenylboronic acid ester to release strongly fluorescent BTMO. Moreover, BTMO-PN could image endogenous and exogenous ONOO- changes in live cells. Importantly, using BTMO-PN, we demonstrated the up-generation of ONOO- levels in cancer cells during ferroptosis. Furthermore, BTMO-PN has successfully been applied for distinguishing tumor tissues from normal tissues with excellent contrast, making it great potential for cancer diagnosis by detecting the ONOO- changes.

Development of peroxynitrite-responsive fluorescence probe for recognition of drug-induced liver injury

Peroxynitrite (ONOO−) as an active substance, is produced during normal physiological process, which plays an important role in maintaining cell REDOX balance and cell function. Moreover, the peroxynitrite is involved in many diseases and especially can be used as a biomarker of drug-induced liver injury (DILI). Therefore, in this work, we synthesized a fluorescent probe JQ-3 for detecting ONOO−. The results showed the probe JQ-3 possessed excellent selectivity, fast response time (10 min) and low detection limit (32 nM). The probe JQ-3 is almost unaffected by pH, showing the potential application in biological systems. Moreover, the probe JQ-3 can be successfully used for the detection of exogenous and endogenous ONOO− in living cells and zebrafish. At the same time, the DILI was successfully recognized by visualizing ONOO− with JQ-3 in living cells and zebrafish. Therefore, the probe JQ-3 provides a potential tool for detecting ONOO− to understand physiological and pathology processes of disease.

A Two-Photon Fluorescent Probe for the Visual Detection of Peroxynitrite in Living Cells and Zebrafish.

Peroxynitrite (ONOO−), as an important reactive oxygen species (ROS), holds great potential to react with a variety of biologically active substances, leading to the occurrence of various diseases such as cancer and neurodegenerative diseases. In this work, we developed a novel mitochondria-localized fluorescent probe, HDBT-ONOO−, which was designed as a mitochondria-targeting two-photon fluorescence probe based on 1,8-naphthylimide fluorophore and the reactive group of 4-(bromomethyl)-benzene boronic acid pinacol ester. More importantly, the probe exhibited good biocompatibility, sensitivity, and selectivity, enabling its successful application in imaging the generation of intracellular and extracellular ONOO−. Furthermore, exogenous and endogenous ONOO− products in live zebrafish were visualized. It is greatly expected that the designed probe can serve as a useful imaging tool for clarifying the distribution and pathophysiological functions of ONOO− in cells and zebrafish.

Enhancing probe's sensitivity for peroxynitrite through alkoxy modification of dicyanovinylchromene.

An intramolecular charge transfer (ICT)-based fluorescent probe P-ONOO- was synthesized to detect ONOO-. After responding to peroxynitrite, the dicyano-vinyl group of P-ONOO- generates the aldehyde group, emitting strong green fluorescence accompanied by quenching of the yellow fluorescence. According to the calculated Fukui function, the modification of the alkoxy group can enhance the f+ of P-ONOO-, which can enhance the probe's nucleophilic addition reactivity with ONOO-. It has been experimentally verified that P-ONOO- shows fast response (within 30s), excellent sensitivity (the detection limit = 10.4nM), and good selectivity towards ONOO-. Additionally, the probe P-ONOO- has high membrane permeability and good biocompatibility, which can image endogenous ONOO- and exogenous ONOO- in HeLa cells.

A highly selective probe for ratiometric imaging peroxynitrite in living cells and in vivo

In this study, we constructed and displayed a ratiometric fluorescent probe JQ-2 for detecting ONOO−. The probe JQ-2 showed a ratiometric signal for visualizing ONOO− with a rapid response and high selectivity over a panel of biological analytes. Moreover, the JQ-2 has near-infrared emission (657 nm), which provides an excellent basis for the practical application in biological systems. The probe JQ-2 possessed low cytotoxicity and excellent cell membrane permeability, which can specifically visualize the exogenous and endogenous ONOO− in vitro and vivo by emission in two channels. Meanwhile, JQ-2 can be used for diagnosing drug-induced liver injury by visualizing and monitoring the fluctuations of endogenous ONOO−. Therefore, JQ-2 provided a potential tool for precisely detecting the fluctuation of ONOO− in biological systems to understand physiological and pathological process.

A highly selective and sensitive fluorescence probe based on Rhodol for imaging of endogenous peroxynitrite in living cells

Peroxynitrite (ONOO−), as one of the important reactive oxygen/nitrogen species, is associated with many physiological and pathological activities. Therefore, effective detection methods with high sensitivity and rapid response time are essential to reveal its functions. A novel Rhodol-based fluorescent probe RN is constructed and synthesized by introducing of 1,1-dimethylhydrazone as a recognition group. Due to the oxidative breakage of the CN bond by ONOO−, the probe has significant fluorescence enhancement change with excellent detection properties such as rapid response time, high sensitivity and selectivity. Moreover, probe RN show low cytotoxicity and is successfully capable of imaging endogenous and endogenous ONOO− in living cells. These properties indicate that RN has great application potential for imaging of ONOO− in biological systems.

Rationally designed meso-benzimidazole-pyronin with emission wavelength beyond 700 nm enabling in vivo visualization of acute-liver-injury-induced peroxynitrite

Fluorescent dyes with fluorescence emission above 700 nm are favorable for bio-imaging due to the higher tissue transparency and lower background fluorescence. In this study, we present a meso-benzimidazole-pyronin platform (SiBMs) with fluorescence emission maxima above 700 nm, which possess good cell permeability, photostability, and lysosomal localization. The great photophysical properties of the SiBMs encouraged us to further exploit their application toward bio-imaging. We synthesized the reduced ‘dihydro’ derivative HSiBM3 for sensing ONOO−, with high selectivity and sensitivity and a fast fluorescence “off-on” response (within 2 s). Then, we confirmed the potential of HSiBM3 for visualizing exogenous and endogenous ONOO− in cells and mice. More importantly, HSiBM3 was successfully employed for visualizing acute-liver-injury-induced peroxynitrite.

Imaging pulmonary fibrosis with a practical probe for the detection of peroxynitrite in living cells and mice

Idiopathic pulmonary fibrosis (IPF) is a frequent chronic trouble, during which the fibrogenic process was associated to the chronic inflammation and oxidative stress, especially peroxynitrite. In this work, we intended to image the pulmonary fibrosis with a practical probe, YXP, for the detection of peroxynitrite. The probe showed the emission at 535 nm when being excited at 397 nm. The fluorescence quantum yield was calculated as 0.31 and the limit of detection was determined as 0.81 μM with a linear range of 2–10 μM. The response time was within 300 s, and the fluorescence intensity was steady over 48 h and within the range of 6–10. With high selectivity and non-interference, YXP was applied in the applications in living BEAS cells from human lung and in pulmonary fibrosis model in mice. In living cells, YXP could exhibit dose-dependent response towards ONOO− with steady signal output, and reflect the fluctuation of endogenous ONOO− in the physiological procedures. In the successfully constructed pulmonary fibrosis model, YXP could hint the development of the damage by the detection of peroxynitrite. The findings and applications in this work might provide useful information for the early diagnosis of IPF.

Construction of a Mitochondria-Endoplasmic Reticulum Dual-Targeted Red-Emitting Fluorescent Probe for Imaging Peroxynitrite in Living Cells and Zebrafish.

Peroxynitrite (ONOO- ) is one of the important reactive oxygen species, which plays a vital role in the physiological process of intracellular redox balance. Revealing the biological functions of ONOO- will contribute to further understanding of the oxidative process of organisms. In this work, we designed and synthesized a novel red-emitting fluorescent probe MCSA for the detection of ONOO- , which could rapidly respond to ONOO- within 250 s and exhibited high sensitivity to ONOO- with a low detection limit of 78 nM. Co-localization experiments demonstrated MCSA had the ability to localize into the mitochondria and endoplasmic reticulum. What's more, MCSA enabled monitoring ONOO- level changes during tunicamycin-induced endoplasmic reticulum stress. We have also successfully achieved the visual detection of exogenous and endogenous ONOO- in living cells and zebrafish. This work presented a chemical tool for imaging ONOO- in vitro and in vivo.

A novel “dual-locked” fluorescent probe for ONOO– and viscosity enables serum-based rapid disease screening

Peroxynitrite (ONOO–) plays important roles in the progression of important disease such as inflammation, cancer, and diabetes, which made it an attractable target for biosensor development. However, to detect ONOO– solely is highly dependent on the sensitivity of the detection method and may be disturbed by unwillingly false-positive signal. Cellular viscosity is an important microenvironmental parameter and its abnormal changes are closely related to diseases such as diabetes and cancer. In this case, to construct a “dual-locked” molecular tool for both ONOO– and viscosity sensing and to evaluate the performance of such strategy in disease diagnosis is of great importance. We herein firstly reported the construction of a novel “dual-locked” probe DCI-OV which showed capability for simultaneous measuring ONOO– concentration and system viscosity with high sensitivity (LOD = 4.7 nM) and high specificity. Moreover, both exogenous and low level of endogenous ONOO– in living cells could be detected using DCI-OV due to viscosity amplified signal. Furthermore, cancer cells and insulin-resistant cells could be easily distinguished using DCI-OV. By taking advantage of the “dual-locked” sensing strategy, a total of 85 samples of human serum were screened using DCI-OV based rapid disease screening method and it was capable of differentiated and subdivided patients into specific type of disease, indicating the great potential of application of DCI-OV into clinical related disease diagnosis.

An ESIPT-based fluorescent probe with large Stokes shift for peroxynitrite detection in HeLa cells and zebrafish

The quick coupled reaction of nitric oxide (NO) with superoxide free radicals (·O2−) produces peroxynitrite (ONOO−), it is a powerful biological oxidant. And according to interact with proteins, lipids, nucleic acids and other organisms, the ONOO− eventually leads to all sorts of diseases, for instance, Alzheimer's disease, diabetes, cancer, inflammation. To fully understand the multiple pathological processes of ONOO− in vivo, it is imperative to exploit effective tools to realize the precise, fast and sensitive detection of endogenous ONOO−. We described a novel fluorescent probe named YV in this work, covering the synthesis, characterization and biological application. YV can detect ONOO− by means of fluorescence imaging. Because of the borate group cleavage, YV exhibited a fluorescence intensity change toward ONOO−. It was, the principle of ESIPT that resulted in fluorescence enhancement. The probe YV shows a series of advantages such as high selectivity, fast response, significant Stokes shift and low detection limit. Fluorescence imaging can be used to identify variations in ONOO− in Hela cells and zebrafish. YV will be a robust imaging tool for detecting endogenous ONOO−.

An activatable reporter for fluorescence imaging drug-induced liver injury in diverse cell lines and in vivo

Peroxynitrite (ONOO−) is considered to be an important indicator of the early stage of drug-induced liver injury (DILI). Therefore, the establishment of a reliable ONOO− detection method is of great significance for the early prediction and diagnosis of DILI. In this paper, a red fluorescence probe BDP-PN based on BODIPY dye was constructed studiously for large signal-to-noise ratio, highly selective and sensitive recognition of ONOO−in vitro. Furthermore, the probe has been successfully used to image exogenous and endogenous ONOO− in different cell lines. More importantly, in the APAP-induced liver injury model of diverse cell lines and mice, BDP-PN not only could monitor the up-regulated expression of ONOO−, but also could evaluate the remediation of NAC. In addition, BDP-PN could distinguish cancer cells from normal cells by the different levels of ONOO− expression. All the results demonstrated the wider universality of probe, which may as a promising tool to diagnose drug-induced liver injury by means of monitoring fluctuation of ONOO−.