ABSTRACT
As one of endogenous reactive oxygen species (ROS), peroxynitrite (ONOO-) performs various functions in both pathological and physiological mechanisms. In this work, an optical and near-infrared (NIR) fluorescent probe (NX), which based on 3-dihydro-1H-xanthene and 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) group was designed and prepared to detect ONOO-. This probe revealed an obvious optical and a fluorescent response when ONOO- was present and it exhibited higher selectivity over other ROS. Especially, the dual NIR fluorescence changes at 660 and 800 nm allowed quantitative detection of ONOO- in the range of 15-40 µM, and the detection limit was 82 nM. Finally, the probe was effectively employed to visualize exogenous and endogenous ONOO- in HepG2 cells and zebrafish, respectively. All the results indicated the dual NIR-channel probe could serve as a potent detecting tools in studying ONOO- in vitro and in vivo.
Subject(s)
Fluorescent Dyes , Peroxynitrous Acid , Zebrafish , Fluorescent Dyes/chemistry , Fluorescent Dyes/chemical synthesis , Humans , Hep G2 Cells , Peroxynitrous Acid/analysis , Animals , Spectrometry, Fluorescence , Limit of Detection , Spectroscopy, Near-Infrared/methodsABSTRACT
A near-infrared fluorescent probe (TX-P) for detecting peroxynitrite is constructed. The probe has a near-infrared emission (725 nm), large Stokes shift (125 nm) and excellent sensitivity and selectivity. In addition, TX-P can be used to visualize ONOO- in living cells, image ONOO- in paw edema mice and evaluate anti-inflammatory drugs.
Subject(s)
Edema , Fluorescent Dyes , Peroxynitrous Acid , Animals , Peroxynitrous Acid/metabolism , Peroxynitrous Acid/analysis , Fluorescent Dyes/chemistry , Fluorescent Dyes/chemical synthesis , Mice , Edema/diagnostic imaging , Edema/drug therapy , Edema/chemically induced , Infrared Rays , Humans , Optical Imaging , RAW 264.7 Cells , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/therapeutic useABSTRACT
Nitric oxide (NO) is an inorganic signaling molecule that plays a crucial role in the regulation of numerous physiological functions. An oxidation product of the cytoprotective NO is cytotoxic peroxynitrite (ONOO-). In biological systems, the concentrations of NO and ONOO- are typically transient, ranging from nanomolar to micromolar, and these increases are normally followed by a swift return to their basal levels due to their short life spans. To understand the vital physiological role of NO and ONOO- in vitro and in vivo, sensitive and selective methods are necessary for direct and continuous NO and ONOO- measurements in real time. Because electrochemical methods can be adjusted for selectivity, sensitivity, and biocompatibility in demanding biological environments, they are suitable for real-time monitoring of NO and ONOO- release. Metalloporphyrin nanosensors, described here, have been designed to measure the concentration of NO and ONOO- produced by a single human neural progenitor cell (hNPC) in real time. These nanosensors (200-300 nm in diameter) can be positioned accurately in the proximity of 4-5 ± 1 µm from an hNPC membrane. The response time of the sensors is better than a millisecond, while detection limits for NO and ONOO- are 1 × 10-9 and 3 × 10-9 mol/L, respectively, with a linear concentration response of up to about 1 µM. The application of these metalloporphyrin nanosensors for the efficient measurement of the concentrations of NO and ONOO- in hNPCs is demonstrated, providing an opportunity to observe in real time the molecular changes of the two signaling molecules in situ.
Subject(s)
Metalloporphyrins , Neural Stem Cells , Nitric Oxide , Peroxynitrous Acid , Peroxynitrous Acid/analysis , Humans , Nitric Oxide/analysis , Neural Stem Cells/cytology , Metalloporphyrins/chemistry , Biosensing Techniques/methods , Electrochemical Techniques/methods , Nanotechnology/methodsABSTRACT
Given its pivotal role in modulating various pathological processes, precise measurement of nitric oxide (âNO) levels in physiological solutions is imperative. The key techniques include the ozone-based chemiluminescence (CL) reactions, amperometric âNO sensing, and Griess assay, each with its advantages and drawbacks. In this study, a hemin/H2O2/luminol CL reaction was employed for accurately detecting âNO in diverse solutions. We investigated how the luminescence kinetics was influenced by âNO from two donors, nitrite and peroxynitrite, while also assessing the impact of culture medium components and reactive species quenchers. Furthermore, we experimentally and theoretically explored the mechanism of hemin oxidation responsible for the initiation of light generation. Although both hemin and âNO enhanced the H2O2/luminol-based luminescence reactions with distinct kinetics, hemin's interference with âNO/peroxynitrite- modulated their individual effects. Leveraging the propagated signal due to hemin, the âNO levels in solution were estimated, observing parallel changes to those detected via amperometric detection in response to varying concentrations of the âNO-donor. The examined reactions aid in comprehending the mechanism of âNO/hemin/H2O2/luminol interactions and how these can be used for detecting âNO in solution with minimal sample size demands. Moreover, the selectivity across different solutions can be improved by incorporating certain quenchers for reactive species into the reaction.
Subject(s)
Hemin , Molecular Probes , Nitric Oxide , Hemin/chemistry , Hydrogen Peroxide/chemistry , Hydrogen Peroxide/analysis , Kinetics , Luminescent Measurements , Luminol/chemistry , Molecular Probes/chemistry , Nitric Oxide/analysis , Oxidation-Reduction , Peroxynitrous Acid/analysis , Peroxynitrous Acid/chemistry , SolutionsABSTRACT
Peroxynitrite is one of the important reactive oxygen species in the human body and is closely related to the physiological and pathological processes of many diseases. Therefore, the development of probes to detect peroxynitrite is important for diagnostic and pathologic studies of many diseases. In this work, a ratiometric probe was designed using benzopyran as the recognition site, and the sensitivity and selectivity of the probe were tuned by modification of substituents on benzopyran. Upon reaction with peroxynitrite, the color of the solution changes to the naked eye (from blue to yellow), and the fluorescence changes from red to blue. The probe SJ has the advantages of large Stokes shift (237 nm), fast response (≤10 s), wide linear range, good selectivity, low detection line (21.3 nm), and low cytotoxicity. Probe SJ has been successfully used for bioimaging of endogenous and exogenous peroxynitrite.
Subject(s)
Fluorescent Dyes , Peroxynitrous Acid , Spectrometry, Fluorescence , Peroxynitrous Acid/analysis , Fluorescent Dyes/chemistry , Fluorescent Dyes/chemical synthesis , Humans , Animals , Benzopyrans/chemistry , Mice , Limit of DetectionABSTRACT
Superoxide anion (O2·-) and peroxynitrite (ONOO-), two important oxidants under oxidative stress, coexist in complex cell and organism systems, playing crucial roles in various physiological and pathological processes, particularly in neurodegenerative diseases. Despite the absence of robust molecular tools capable of simultaneously visualizing O2·- and ONOO- in biosystems, the relationship between these two species remains understudied. Herein, we present sequentially activated fluorescent probe, DHX-SP, which exhibits exceptional selectivity and sensitivity toward O2·- and ONOO-. This probe enables precise imaging of these species in living PC12 cells under oxidative stress conditions using distinct fluorescence signal combinations. Furthermore, the probe DHX-SP has the ability to visualize changes in O2·- and ONOO- levels during ferroptosis of PC12 cells and in the Parkinson's disease model. These findings establish a connection between the crosstalk of the phosphorus group of O2·- and ONOO- in PC12 cells under oxidative stress.
Subject(s)
Fluorescent Dyes , Oxidative Stress , Peroxynitrous Acid , Superoxides , PC12 Cells , Peroxynitrous Acid/analysis , Peroxynitrous Acid/metabolism , Animals , Rats , Oxidative Stress/drug effects , Fluorescent Dyes/chemistry , Superoxides/metabolism , Superoxides/analysis , Optical ImagingABSTRACT
Ferroptosis is a type of lipid peroxidation-induced apoptosis brought on by imbalances in iron metabolism and redox. It involves both the thiol-associated anti-ferroptosis pathway and the excessive buildup of reactive oxygen species (ROS), which stimulates the ferroptosis pathway. Determining the precise control mechanism of ferroptosis requires examining the dynamic connection between reactive sulfur species (RSS) and ROS. Cysteine (Cys) and peroxynitrite (ONOO-) are highly active redox species in organisms and play dynamic roles in the ferroptosis process. In this study, a coumarin dye was conjugated with specific response sites for Cys and ONOO-, enabling the simultaneous detection of Cys and ONOO- through the green and red fluorescence channels, respectively (λem = 498 nm for Cys and λem = 565 nm for ONOO-). Using the probe LXB, we monitored the changes in Cys and ONOO- levels in the ferroptosis pathway induced by erastin. The results demonstrate a significant generation of ONOO- and a noticeable decrease in intracellular Cys levels at the beginning upon erastin treatment and finally maintains a relatively low level. This study presents the first probe to investigate the intracellular redox modulation and control between Cys and ONOO- during ferroptosis, providing valuable insights into the potential mutual correlation between Cys and ONOO- in this process.
Subject(s)
Cysteine , Ferroptosis , Fluorescent Dyes , Peroxynitrous Acid , Ferroptosis/drug effects , Fluorescent Dyes/chemistry , Cysteine/metabolism , Cysteine/analysis , Humans , Peroxynitrous Acid/analysis , Peroxynitrous Acid/metabolism , Spectrometry, Fluorescence , Oxidation-Reduction , Piperazines/pharmacology , Piperazines/chemistry , Coumarins/chemistry , Coumarins/pharmacologyABSTRACT
Peroxynitrite (ONOO-) is one of the most significant reactive oxygen species (ROS) in living cells. Zn2+ in living cells plays an essential part in different physiological processes. The abnormal concentration of ONOO- and Zn2+ in living cells are related to many kinds of diseases, such as anemia, epilepsy, diarrhea, Alzheimer's disease, and so on. The relationship of ONOO- and Zn2+ in living cells when the relative disease occurs remains unknown. So we develop the first probe H-1 for detecting ONOO- and Zn2+ at the same time. The probe H-1 shows high selectivity, good anti-interference capability, low detection limit and short response time to ONOO- and Zn2+. When the probe was applied to detect ONOO- and Zn2+ in HeLa cells, we could observe the fluorescence changing in the green and blue channels separately without interference in real time. It has the potential to employ the relation of ONOO- and Zn2+ in some disease mechanism research.
Subject(s)
Fluorescent Dyes , Peroxynitrous Acid , Spectrometry, Fluorescence , Zinc , Humans , Fluorescent Dyes/chemistry , Fluorescent Dyes/chemical synthesis , Zinc/analysis , HeLa Cells , Peroxynitrous Acid/analysis , Limit of DetectionABSTRACT
In the current situation, peroxynitrite (ONOO-) is drawing the increasing attention of researchers for its pivotal role in diverse pathological and physiological processes on grounds of robust oxidation and nitrification. Herein, we have successfully designed and synthesized a phenanthrenequinone benzyl borate-based chemosensor for fast and selective detection of ONOO-. The probe PTDP itself had an orange fluorescence, which was changed to strong blue fluorescence upon the addition of ONOO-, indicating the ratiometric response of the probe. This is so because of the cleavage of the benzyl boronate-protecting group of PTDP upon the addition of ONOO- with simultaneous releasing of pyridinyl-based chemosensor PPI. The PTDP showed outstanding performance in the various photophysical studies such as good selectivity, excellent sensitivity with a very low detection limit of 2.74 nM, and a very fast response time (<15 s). Furthermore, for practical applicability, it was successfully applied in the ratiometric detection of ONOO- in osteoblast precursor cells.
Subject(s)
Fluorescent Dyes , Osteoblasts , Peroxynitrous Acid , Phenanthrenes , Fluorescent Dyes/chemistry , Fluorescent Dyes/chemical synthesis , Peroxynitrous Acid/analysis , Osteoblasts/drug effects , Phenanthrenes/chemistry , Molecular Structure , Optical Imaging , Limit of Detection , Animals , Humans , Spectrometry, FluorescenceABSTRACT
An AIE-based fluorescent probe was designed to evaluate peroxynitrite levels in complex biological samples. The newly synthesized hydrazone-conjugated probe fluoresces strongly in the presence of peroxynitrite. Clinically, the peroxynitrite levels can be measured in human serum and cellular mitochondria with an LOD of 6.5 nM by fluorescence imaging in vitro.
Subject(s)
Fluorescent Dyes , Optical Imaging , Peroxynitrous Acid , Humans , Peroxynitrous Acid/blood , Peroxynitrous Acid/analysis , Fluorescent Dyes/chemistry , Fluorescent Dyes/chemical synthesis , Mitochondria/metabolism , Mitochondria/chemistry , Limit of Detection , Hydrazones/chemistry , Hydrazones/chemical synthesis , HeLa Cells , Molecular StructureABSTRACT
Excessive peroxynitrite (ONOO-) is closely related to the occurrence and progression of inflammation. Therefore, the development of an efficacious ONOO- activatable probe holds great potential for the early diagnosis of pathological inflammation, and the direct evaluation of the therapeutic efficacy of active protectants. In this work, a new ONOO--activated fluorescent probe (SZP) which greatly improved the specificity and sensitivity (LOD = 8.03 nM) with large Stokes shift (150 nm) through introducing two reaction triggers (diphenyl phosphinate moiety, CC unsaturated bond) was rationally designed for rapid detecting ONOO- (within 2 min). The excellent properties of probe SZP enable it to realize the fluorescence-guided diagnosis of inflammation. More importantly, probe SZP has also been utilized to assess the anti-inflammatory efficacy of traditional Chinese medicines (TCMs) active ingredients for the remediation of inflammation by monitoring ONOO- fluctuation for the first time.
Subject(s)
Fluorescent Dyes , Inflammation , Peroxynitrous Acid , Peroxynitrous Acid/analysis , Peroxynitrous Acid/antagonists & inhibitors , Fluorescent Dyes/chemistry , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/pharmacology , Inflammation/drug therapy , Animals , Molecular Structure , Mice , Humans , RAW 264.7 Cells , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/therapeutic use , Optical Imaging , Dose-Response Relationship, Drug , Structure-Activity Relationship , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , MaleABSTRACT
ONOO-, a bioactive molecule, plays a critical role in inflammation-related signaling pathways and pathological mechanisms. Numerous studies have established a direct correlation between elevated ONOO- levels and tumor progression. Therefore, investigating ONOO- levels in inflammation and tumors is of utmost importance. Fluorescence imaging presents a highly sensitive, non-invasive, easily operable, selective, and efficient method for ONOO- detection in situ. In this study, we designed and synthesized a rhodamine-based probe, NRho, which effectively identifies tumors, inflammatory cells, tissues, and organs by detecting ONOO- content. The synthesis process of NRho is simple, yielding a probe with favorable spectral characteristics and rapid response. Our cell imaging analysis has provided novel insights, revealing distinct ONOO- levels among different types of cancer cells, with hepatocellular carcinoma cells exhibiting higher ONOO- content than the others. This observation marks the proposal of such variations in ONOO- levels across cancer cell types. Furthermore, our study has showcased the practicality of our probe in live organ imaging, enabling the identification of tumors from living organs within a brief 5-minute incubation period. Additionally, our findings highlight the rapid detection capability of the probe NRho in various tissue samples, effectively identifying inflammation. This research holds important promise in advancing biomedical research and clinical diagnosis.
Subject(s)
Fluorescent Dyes , Peroxynitrous Acid , Humans , Peroxynitrous Acid/analysis , Rhodamines , HeLa Cells , Inflammation/diagnostic imagingABSTRACT
Abnormal changes occurring in the mitochondrial microenvironment are important markers indicating mitochondrial and cell dysfunction. Herein, we designed and synthesized a multifunctional fluorescent probe DPB that responds to polarity, viscosity, and peroxynitrite (ONOO-). DPB is composed of an electron donor (diethylamine group) and electron acceptor (coumarin, pyridine cations, and phenylboronic acid esters), in which the pyridine group with a positive charge is responsible for targeting to mitochondria. D-π-A structure with strong intramolecular charge transfer (ICT) and twisted intramolecular charge transfer (TICT) properties give rise to respond to polarity and viscosity. The introduction of cyanogroup and phenylboronic acid esters increases the electrophilicity of the probe, which is prone to oxidation triggered by ONOO-. The integrated architecture satisfies the multiple response requirements. As the polarity increases, the fluorescence intensity of probe DPB at 470 nm is quenched by 97%. At 658 nm, the fluorescence intensity of DPB increases with viscosity and decreases with the concentration of ONOO-. Furthermore, the probe is not only successfully used to monitor mitochondrial polarity, viscosity, and endogenous/exogenous ONOO- level fluctuations but also to distinguish cancer cells from normal cells by multiple parameters. Therefore, as-prepared probe provides a reliable tool for better understanding of the mitochondrial microenvironment and also a potential approach for the diagnosis of disease.
Subject(s)
Fluorescent Dyes , Mitochondria , Fluorescent Dyes/chemistry , Viscosity , Mitochondria/chemistry , Microscopy, Fluorescence/methods , Optical Imaging/methods , Pyridines/analysis , Peroxynitrous Acid/analysisABSTRACT
Boronic acid-containing fluorescent molecules have been widely used to sense hydrogen peroxide and peroxynitrite, which are important reactive oxygen and nitrogen species in biological systems. However, it has been challenging to gain specificity. Our previous studies developed genetically encoded, green fluorescent peroxynitrite biosensors by genetically incorporating a boronic acid-containing noncanonical amino acid (ncAA), p-boronophenylalanine (pBoF), into the chromophore of circularly permuted green fluorescent proteins (cpGFPs). In this work, we introduced pBoF to amino acid residues spatially close to the chromophore of an enhanced circularly permuted red fluorescent protein (ecpApple). Our effort has resulted in two responsive ecpApple mutants: one bestows reactivity toward both peroxynitrite and hydrogen peroxide, while the other, namely, pnRFP, is a selective red fluorescent peroxynitrite biosensor. We characterized pnRFP in vitro and in live mammalian cells. We further studied the structure and sensing mechanism of pnRFP using X-ray crystallography, 11B-NMR, and computational methods. The boron atom in pnRFP adopts an sp2-hybridization geometry in a hydrophobic pocket, and the reaction of pnRFP with peroxynitrite generates a product with a twisted chromophore, corroborating the observed "turn-off" fluorescence response. Thus, this study extends the color palette of genetically encoded peroxynitrite biosensors, provides insight into the response mechanism of the new biosensor, and demonstrates the versatility of using protein scaffolds to modulate chemoreactivity.
Subject(s)
Biosensing Techniques , Peroxynitrous Acid , Animals , Peroxynitrous Acid/analysis , Hydrogen Peroxide/metabolism , Green Fluorescent Proteins/metabolism , Fluorescent Dyes/chemistry , Boronic Acids , Phenylalanine/chemistry , Biosensing Techniques/methods , Mammals/metabolismABSTRACT
Viscosity and peroxynitrite (ONOO-) are two significant indicators to affect and evaluate the mitochondrial functional status, which are nearly relational with pathophysiological process in many diseases. Developing suitable analytical methods for monitoring mitochondrial viscosity changes and ONOO- is thus of great importance. In this research, a new mitochondria-targeted sensor DCVP-NO2 for the dual determination of viscosity and ONOO- was exploited based on the coumarin skeleton. DCVP-NO2 displayed a red fluorescence "turn-on" response toward viscosity along with about 30-fold intensity increase. Meanwhile, it could be used as ratiometric probe for detection of ONOO- with excellent sensitivity and extraordinary selectivity for ONOO- over other chemical and biological species. Moreover, thanks to its good photostability, low cytotoxicity and ideal mitochondrion-targeting capability, DCVP-NO2 was successfully utilized for fluorescence imaging of viscosity variations and ONOO- in mitochondria of living cells through different channels. In addition, the results of cell imaging revealed that ONOO- would lead to the increase of viscosity. Taken together, this work provides a potential molecular tool for researching biological functions and interactions of viscosity and ONOO- in mitochondria.
Subject(s)
Fluorescent Dyes , Peroxynitrous Acid , Fluorescent Dyes/chemistry , Peroxynitrous Acid/analysis , Nitrogen Dioxide/analysis , Viscosity , Mitochondria/chemistryABSTRACT
Ferroptosis is an iron-dependent process that regulates cell death and is essential for maintaining normal cell and tissue survival. The explosion of reactive oxygen species characterizes ferroptosis in a significant way. Peroxynitrite (ONOO-) is one of the endogenous reactive oxygen species. Abnormal ONOO- concentrations cause damage to subcellular organelles and further interfere with organelle interactions. However, the proper conduct of organelle interactions is critical for cellular signaling and the maintenance of cellular homeostasis. Therefore, investigating the effect of ONOO- on organelle interactions during ferroptosis is a highly attractive topic. To date, it has been challenging to visualize the full range of ONOO- fluctuations in mitochondria and lysosomes during ferroptosis. In this paper, we constructed a switchable targeting polysiloxane platform. During the selective modification of NH2 groups located in the side chain, the polysiloxane platform successfully constructed fluorescent probes targeting lysosomes and mitochondria (Si-Lyso-ONOO, Si-Mito-ONOO), respectively. Real-time detection of ONOO- in lysosomes and mitochondria during ferroptosis was successfully achieved. Remarkably, the occurrence of autophagy during late ferroptosis and the interaction between mitochondria and lysosomes was observed via the differentiated responsive strategy. We expect that this switchable targeting polysiloxane functional platform will broaden the application of polymeric materials in bioimaging and provide a powerful tool for further deeper understanding of the ferroptosis process.
Subject(s)
Ferroptosis , Siloxanes , Reactive Oxygen Species/metabolism , Mitochondria/metabolism , Lysosomes/chemistry , Fluorescent Dyes/chemistry , Peroxynitrous Acid/analysisABSTRACT
Peroxynitrite (OONO-) is closely related to the occurrence and development of health and inflammatory diseases. The physiological and pathological results of OONO- are related to the local concentration of ONOO-. Therefore, to develop of a simple, rapid and reliable OONO- detection tool is badly needed. In this work, we developed a small-molecule near-infrared (NIR) turn-on fluorescence sensor (NN1), harnessing a well-known response group phenylboronic acid response toward OONO-. It shows high detection sensitivity and yields a ratio (I658/I0) fluorescence enhancement (â¼280-fold). In addition, NN1 can be effectively used to detect endogenous and exogenous ONOO- in living inflammatory cells. Notably, NN1 can be applied to OONO- imaging analysis in drug-induced inflammatory mice model with satisfactory results. Therefore, NN1 is a robust molecular biological tool, which has a good prospect in the study of ONOO- and the occurrence and development of inflammatory diseases.
Subject(s)
Diagnostic Imaging , Fluorescent Dyes , Animals , Mice , Fluorescence , Peroxynitrous Acid/analysis , Optical ImagingABSTRACT
There is an urgent need to develop highly sensitive and selective fluorescence probes for ONOO- in mitochondria. Herein, we reported a ratiometric fluorescent probe COUS with coumarin-cyanine hybrid as fluorophore and C = C bonds as reaction sites of ONOO-. The probe COUS was sensitive and selective to ONOO-, and had a large fluorescence emission shift (239 nm) as well as a low detection limit (41.88 nM). Moreover, COUS showed the mitochondrial targeting ability, and the targeting moiety could dissociate from the probe when reacting with ONOO-, which enabled COUS to accurately detect ONOO- in mitochondria.
Subject(s)
Fluorescent Dyes , Peroxynitrous Acid , Fluorescent Dyes/chemistry , Peroxynitrous Acid/analysis , Mitochondria/chemistry , Coumarins/analysis , FluorescenceABSTRACT
Peroxynitrite (ONOO-), as a strong oxidizing reactive nitrogen substance (RNS), is generated endogenously by cells. Its visualization research is crucial to understand relevant disease processes. Herein, we reported a long-wavelength mitochondria-targeted fluorescence "turn on" probe TL. The probe TL could react with ONOO- by using 4-(Bromomethyl)benzeneboronic as a reactive site, which exhibited outstanding characteristics for detection of ONOO-, thus improving response time (about 50 s), sensitivity (DL, 10.1 nM), and emission wavelength (667 nm). Besides, TL displayed well mitochondria targeting and biological visualizing of exogenous and endogenous ONOO- in biological systems. Finally, TL was used to monitor high concentration of dexamethasone-induced an up-regulation of ONOO-. This indicated that TL has excellent potential to study the fluctuation of ONOO- in the physiological and pathological system.
Subject(s)
Fluorescent Dyes , Peroxynitrous Acid , Fluorescent Dyes/chemistry , Peroxynitrous Acid/analysis , Mitochondria/chemistry , Microscopy, Fluorescence/methods , Optical Imaging , Dexamethasone/analysisABSTRACT
Ratiometric luminescence (fluorescence/phosphorescence) probes have attracted widespread attention of researchers in the field of biological detection and noninvasive imaging of bioactive molecules in living systems. However, most of them suffer from some defects such as small emission shift, different excitation wavelength and spectral overlap, which eventually affect the luminescence ratio, thus leading to limitations in ratiometric bioimaging applications. In this paper, we present a novel "ruthenium(II) complex-fluorescein" scaffold probe (Ru-FL-ONOO) for ratiometric luminescence detection of peroxynitrite (ONOO-), in which a Ru(II) complex was conjugated to fluorescein serving as the dual-emissive moiety and the spirocyclic structure of fluorescein-phenylhydrazine was used as the specifically-reactive moiety for recognizing ONOO-. The probe possesses not only favourable specificity but also high sensitivity for responding to ONOO-, exhibiting a large emission shift (Δλem > 120 nm) at a single excitation wavelength. After being transferred into living cells, the probe localized within lysosomes, allowing ONOO- therein to be imaged at ratiometric mode. The imaging results reveal that the ratiometric probe bearing the Ru(II) complex-fluorescein scaffold could be a useful approach for overcoming the drawback of spectral overlap of dual-emissive moiety under single-wavelength excitation so as to improve the signal-to-noise ratio, thus benefiting the development of ratiometric bioimaging.