Carbazole-based mitochondria-targeted fluorescent probes for in vivo viscosity and cyanide detection in cells and zebrafish.

Cells of most eukaryotic species contain mitochondria, which play a role in physiological processes such as cellular senescence, metabolism, and autophagy. Viscosity is considered a key marker for many illnesses and is involved in several crucial physiological processes. Cyanide (CN-) can target cytochrome-c oxidase, disrupting the mitochondrial electron transport chain and causing cell death through asphyxiation. In this study, a fluorescent probe named HL-1, which targets mitochondria and measures viscosity and CN- levels, was designed and synthesized. HL-1 is viscosity-sensitive, with a linear correlation coefficient of up to 0.992. In addition, HL-1 was found to change color substantially during a nucleophilic addition reaction with CN-, which has a low detection limit of 47 nM. HL-1 not only detects viscosity and exogenous CN- in SKOV-3 cells and zebrafish but also monitors viscosity changes during mitochondrial autophagy in real time. Furthermore, HL-1 has been used successfully to monitor changes in mitochondrial membrane potential during apoptosis. Endogenous CN- in plant samples was quantified. HL-1 provides new ideas for studying viscosity and CN-.

A novel pyrazoline-based selective fluorescent probe for detecting reduced glutathione and its application in living cells and serum.

A new fluorescent probe, N-(4-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-2,4-dinitrobenzenesulfonamide (probe 3), was designed and synthesized as a highly sensitive and selective fluorescent probe for recognizing and detecting glutathione among biological thiols in aqueous media. Probe 3 is a nonfluorescent compound. On being mixed with biothiols under neutral aqueous conditions, the 2,4-dinitrobenzenesulfoyl moiety can be cleaved off by glutathione, and the blue emission of the pyrazoline at 464 nm is switched on, with a fluorescence enhancement of 488-fold for glutathione. Furthermore, probe 3 was highly selective for glutathione without interference from some biologically relevant analytes. The detection limit of glutathione was 4.11 × 10(-7) M. The emission of the probe is pH independent in the physiological pH range. Moreover, the probe can be used for fluorescent imaging of cellular glutathione and can be used for detecting glutathione in calf serum.

A highly sensitive NIR fluorescence probe for hypoxia imaging in cells and ulcerative colitis.

Ulcerative colitis, a kind of inflammatory bowel disease (IBD), is caused by dysregulated immune response of intestinal bacteria. This chronic disorder can lead to a deficiency of O2 (hypoxia) in the colon microenvironment. Nitroreductase (NTR) is a highly expressed endogenous enzyme under hypoxia, so the detection of NTR can provide diagnostic information about ulcerative colitis. Herein, an ultrasensitive NTR-triggered fluorescence probe (WS-1-NO2) is developed for hypoxia imaging in ulcerative colitis. The probe shows a significant fluorescence enhancement (45-fold) after reacting with NTR, with an extremely low detection limit of 0.096 ng/mL. Furthermore, we apply it for fluorescence imaging of hypoxia in living cells, tumors and dextran sulphate sodium (DSS)-induced ulcerative colitis mouse models. We believe that the probe may be investigated as an effective potential tool for gaining insight into the hypoxia-relevant diseases, such as cancer and ulcerative colitis.

Activatable near-infrared fluorescent probe triggered by nitroreductase for in vivo ulcerative colitis hypoxia imaging.

Ulcerative colitis is a prevalent inflammatory disease caused by the intestinal bacterial infection. And it is related to the hypoxic degrees in the colon microenvironment. Hypoxia, a condition of imbalance in O2 supply and consumption, is accompanied by the overexpressed level of nitroreductase (NTR). Therefore, the NTR detection has been widely applied for the diagnosis of hypoxia-related diseases. In this study, we developed a novel near-infrared fluorescent probe (IW-1) for NTR. Upon reaction with NTR, IW-1 exhibited a significant fluorescence off-on response at 740 nm with a low detection limit of 0.043 µg/mL. Confocal fluorescence imaging verified its ability to detect the overexpression of NTR in cancer cells. More significantly, IW-1 was applied for in vivo hypoxia imaging in tumors and dextran sulphate sodium (DSS)-induced ulcerative colitis mouse model. We expect that the probe may present a new tool for better understanding the biological functions of NTR as well as revealing essential information about hypoxia-related pathological processes, including cancer and ulcerative colitis.

A sensitive NIR mitochondria-targeting fluorescence probe for visualizing viscosity in living cells and mice.

Mitochondria are the powerhouses in cells, providing the energy needed for cellular activities. However, the abnormalities in the mitochondrial microenvironment (e.g., the increased viscosity) can lead to mitochondrial dysfunctions and diseases. Herein, we develop a series of near-infrared (NIR) fluorescence probes for the detection of viscosity. After screening, probe CQ-4 is selected since it shows a great fluorescence enhancement (89-fold) in the NIR window. Its specific response to viscosity is not influenced by pH, polarity and biological species. Under stimulation with monensin or nystatin, CQ-4 can measure the cellular viscosity changes with good biocompatibility. In addition, we can observe an increase of viscosity during starvation. CQ-4 is applied to distinguish cancer cells from normal cells based on the viscosity differences. Furthermore, the probe has been successfully applied to image viscosity in inflamed and tumor-bearing mice in vivo. Therefore, CQ-4 may contribute to the future study about viscosity in the physiological and pathological processes.

Visual monitoring of the mitochondrial pH changes during mitophagy with a NIR fluorescent probe and its application in tumor imaging.

Mitophagy, a mitochondria-selective autophagy process, plays critical roles in maintaining intracellular homeostasis by removing the damaged mitochondria and recycling the nutrients in a lysosome-dependent manner. Mitophagy process could result in the changes of mitochondrial pH. So fluorescent probes for detecting mitochondrial pH during mitophagy are highly needed for exploring the functions of mitochondria. Herein, a series of near-infrared pH probes were designed based on the rhodamine framework. The probes showed high sensitivity for pH with the tunable pKa from 4.74 to 6.54. Particularly, for probe 5 (with the pKa of 6.54), a linear relationship between fluorescence intensity and pH in the range of 5.6-7.2 was observed, which was suitable for mitochondrial pH detection. The probe displayed excellent mitochondria-targeting ability. It was applied to monitor pH changes during mitophagy caused by starvation. Besides, in vivo non-invasive visualization of tumor pH variations was achieved via the fluorescence imaging in the near-infrared region. We anticipate that the probe may be a useful tool for revealing essential information about mitophagy-related research and clinical tumor diagnosis.

A near-infrared fluorescent probe based on the hemicyanine skeleton for the detection of hydrogen peroxide in vivo.

As a member of the reactive oxygen species, hydrogen peroxide (H2O2) plays critical roles in oxidative stress and cell signaling. Intracellular abnormal levels of H2O2 production are closely related to many diseases. Therefore, the real-time monitoring of H2O2 in the cells is important. In this work, we designed a novel fluorescent probe (Mito-H2O2) for the specific detection of H2O2 based on the hemicyanine skeleton, with bright near-infrared fluorescence emission. Mito-H2O2 displayed fast response, excellent water-solubility and great fluorescence intensity enhancement after the addition of H2O2. Furthermore, Mito-H2O2 has been successfully applied to image both of the exogenous and endogenous H2O2 in cells and mice with negligible cytotoxity.

Benzoindoxazine derivatives containing carbazole for detection of CN- and its application in plant seed extracts and cell imaging.

Cyanide (CN-) is a highly toxic compound that exists in many substances and is harmful to the environment and human health. Therefore, it is of great significance to develop excellent CN- ion probes, especially solvent-induced on-off fluorescent probes. Based on the condensation reaction of indolo[2,1-b][1,3]oxazine molecules with aldehydes, probes (E)-13a-(2-(9-ethyl-9H-carbazol-3-yl)vinyl)-14,14-dimethyl-10-nitro-13a,14-dihydro-8H-benzo[e]benzo[5,6][1,3]oxazino[3,2-a]indole (NCO) and (E)-13a-(2-(9-benzyl-9H-carbazol-3-yl)vinyl)-14,14-dimethyl-10-nitro-13a,14-dihydro-8H-benzo[e]benzo[5,6][1,3]oxazino[3,2-a]indole (NBO) were synthesized to detect CN-. Compared with other cyanogen ion probes, NCO and NBO have special carbazole ring structures and large conjugate systems. When CN- is added to the probe-detection solution, color changes that are visible to the naked eye can occur. The UV-vis spectrum test using differential spectroscopy shows that the probe (i) has excellent solvent-induced switching characteristics and stability (CH3OH-H2O) and (ii) high selectivity, anti-interference ability, and sensitivity for the detection of CN-. The fluorescence limit of detections (LODs) are 1.05 µM for NCO and 1.34 µM for NBO. The UV LODs are 0.83 µM for NCO and 0.87 µM for NBO. Fluorescence spectroscopy shows that the probe has remarkable fluorescence properties. Fluorescence titration experiments, liver cancer cell (Hep G2) imaging, and cytotoxicity experiments all show that the probes have high biocompatibility, low toxicity, high cell permeability, and high sensitivity for the detection of CN- in cells. In addition, NCO and NBO have been successfully used for the detection of cyanogenic glycosides in the seeds of ginkgo, crabapple, apple, and cherry. Test strips were fabricated to detect CN-. After adding CN-, the color of the test strip changed significantly-from brown to light yellow; thus, the test strips have a high application value in the fields of drug quality control, drug safety testing, and pharmacological research.

Rapid and selective visualization of mitochondrial hypochlorite by a red region water-soluble fluorescence probe.

Hypochlorite (-OCl) has long been recognized as an effective microbicidal agent in immune system. Herein, we report the design, preparation and spectral characteristics of a -OCl fluorescent probe (FI-Mito). The probe exhibited remarkable fluorescence turn-on signal in the red region upon -OCl titration with the detection limit as low as 0.9 nM. FI-Mito displayed specific response for -OCl in completely aqueous solution. Meanwhile, the introduction of quaternized pyridine realized mitochondria-targeting ability. FI-Mito was further applied to monitor the generation of endogenous -OCl in the mitochondria of macrophage cells and mice. Therefore, it was established that FI-Mito may serve as a useful molecular tool for -OCl detection in vivo.

A near-infrared rhodamine-based lysosomal pH probe and its application in lysosomal pH rise during heat shock.

Heat shock is a potentially fatal condition characterized by high body temperature (>40 °C), which may lead to physical discomfort and dysfunctions of organ systems. Acidic pH environment in lysosomes can activate enzymes, thus facilitating the degradation of proteins in cellular metabolism. Owing to the lack of a practical research tool, it remains difficult to exploit relationship between heat shock and lysosome. Herein, a NIR lysosomal pH chemosensor (NRLH) was developed. One typical lysosome-locating group, morpholine, was incorporated into NRLH. The fluorescence intensity showed pH-dependent characteristics and responded sensitively to pH fluctuations in the pH range of 3.0-5.5. NRLH with a pKa of 4.24 displayed rapid response and high selectivity for H+ among common species. We also demonstrated NRLH was capable of targeting lysosomes. Importantly, NRLH was applied in cellular imaging and the data revealed that lysosomal pH increased but never decreased during the heat shock. Therefore, NRLH may act as an effective molecular tool for exploring the mechanisms of heat-related pathology in bio-systems.

A ratiometric fluorescent probe for lysosomal hypochlorous acid based on through-bond energy transfer strategy.

In this paper, we synthesized a ratiometric fluorescence probe (IRh-Ly) for lysosomal hypochlorous acid (HOCl) by adopting a through-bond energy transfer (TBET) strategy on rhodamine-imidazo[1,5-a]pyridine platform. IRh-Ly showed brilliant selectivity, rapid response for HOCl. The probe also exhibited high sensitivity with the detection limit calculated to be 10.2 nM. Moreover, we demonstrated its successful application of detecting lysosomal HOCl in living RAW264.7 cells. Notably, the morpholine was integrated into the fluorescent probe IRh-Ly and the results revealed that IRh-Ly could target lysosome and detect the lysosomal HOCl. All the unique features made IRh-Ly particularly suitable for ratiometric HOCl detection and bio-imaging applications.

A rhodamine B-based probe for the detection of HOCl in lysosomes.

A rhodamine B-based derivative (RL1) was developed as a specific fluorescent probe for HOCl. Meanwhile, morpholine moiety was introduced into the probe. It was found that the probe could display highly selective, sensitive and naked-eye detection upon the addition of HOCl. And the detection limit (LOD) was calculated to be as low as 2.8 nM. Furthermore, cellular confocal microscopic studies revealed that the introduction of morpholine moiety realized the lysosome-targeting capability. Moreover, RL1 was successfully applied for the imaging of endogenous HOCl with low cytotoxicity. Therefore, all the desirable features made probe RL1 particularly suitable for HOCl detection in aqueous buffer solution samples as well as the bio-imaging applications.

A new ratiometric fluorescent probe for sensing lysosomal HOCl based on fluorescence resonance energy transfer strategy.

In this paper, ratiometric imaging of lysosomal HOCl was realized with a molecular probe (CR-Ly) based on fluorescence resonance energy transfer by using coumarin as the donor and rhodamine as acceptor. CR-Ly showed high sensitivity and fast response to HOCl. Moreover, CR-Ly exhibited excellent selectivity and sensitivity for HOCl over other biologically relevant species. Furthermore, it was successfully utilized to image the endogenous HOCl with low cytotoxity. And CR-Ly was capable of targeting lysosomes and monitoring lysosomal hypochlorous acid changes owing to the presence of the morpholine moiety. We believe that probe CR-Ly would be helpful to further research on the HOCl-associated diseases in lysosomes.

A mitochondria-targeted ratiometric fluorescent probe for hypochlorite and its applications in bioimaging.

A novel ratiometric probe (RCP) for -OCl was developed based on the fluorescence resonance energy transfer (FRET) platform. The probe was constructed by integrating the coumarin moiety (FRET donor) with the rhodamine moiety (FRET acceptor). Upon treatment with -OCl, the coumarin emission at 483 nm decreased and the rhodamine emission at 570 nm increased, enabling the probe to provide accurate detection of -OCl (in the concentration range of 0-50 µM). The probe exhibited brilliant selectivity and sensitivity, rapid response and low cytotoxicity. More importantly, the introduction of the quaternized pyridine moiety can not only manage to increase the solubility, but also achieve mitochondria-targeting. The probe was applied successfully to imaging endogenous -OCl in mitochondria, highlighting its potential applications in bioanalysis.

A rhodamine B-based lysosomal pH probe.

A novel rhodamine B-based fluorescent probe (RML) for lysosomal pH was developed by integrating a 4-(2-aminoethyl)morpholine moiety, which is a lysosome-targetable group, into a rhodamine B fluorophore, which is associated with rhodamine B dyes possessing spirocyclic (non-fluorescent) and ring-opening (fluorescent) forms with response to pH. The probe responded to acidic pH at low concentration in a short amount of time. In addition, RML showed good membrane permeability and brilliant selectivity among various amino acids and metal cations. RML exhibited an 80-fold increase in fluorescence intensity at 583 nm throughout the pH range of 7.40-4.00 with a pKa of 5.16, which indicates that RML is valuable for studying intracellular acidic organelles. Moreover, RML has been successfully applied in HeLa cells, and the results demonstrated that RML could selectively stain lysosomes in living HeLa cells. Note that RML could be used to detect the pH increase in lysosomes induced by bafilomycin A1 within HeLa cells.

A ratiometric lysosomal pH probe based on the naphthalimide-rhodamine system.

In this study, a novel ratiometric pH probe RNL based on fluorescence resonance energy transfer (FRET) was well developed. It was fabricated by integrating the naphthalimide moiety as an FRET donor with the rhodamine moiety as an FRET acceptor. Meanwhile, 4-(2-aminoethyl)morpholine, which was a lysosome-locating group, was introduced. The sensing mechanism was the integration of PET and FRET processes and the comprehensive effect led to the simultaneous intensity enhancement of naphthalimide and rhodamine along with the pH value decrease. With a pKa of 4.82, the fluorescence intensity ratio (I529/I580) of the probe changed significantly within the pH range from 4.50 to 5.50. The probe showed excellent selectivity among various metal cations, amino acids and ATP. Moreover, RNL has been successfully applied in HeLa cells, and the results demonstrated that it could be used to detect lysosomal pH changes. The probe could also selectively stain lysosome in HeLa cells. Besides, the probe exhibited low cytotoxicity and satisfactory photostability in living HeLa cells.

Novel pyrazoline-based fluorescent probe for detecting glutathione and its application in cells.

A novel compound, 2-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl acrylate (probe L), was designed and synthesized as a highly sensitive and selective fluorescent probe for recognizing and detecting glutathione among cysteine, homocysteine and other amino acids. The structures of related compounds were characterized using IR, NMR and HRMS spectroscopy analysis. The probe is a non-fluorescent compound. On being mixed with glutathione in buffered EtOH:PBS=3:7 solution at pH 7.4, the probe exhibited the blue emission of the pyrazoline at 474 nm and a 83-fold enhancement in fluorescence intensity. This probe is very sensitive and displayed a linear fluorescence off-on response to glutathione with fluorometric detection limit of 8.2 × 10(-8)M. The emission of the probe is pH independent in the physiological pH range. Live-cell imaging of HeLa cells confirmed the cell permeability of the probe and its ability to selectively discriminate GSH from Cys and Hcy in cells. The toxicity of the probe was low in cultured HeLa cells.

Fluorescence turn-on chemodosimeter for rapid detection of mercury (II) ions in aqueous solution and blood from mice with toxicosis.

The heavy metal mercury (Hg) is a threat to the health of people and wildlife in many environments. Among various chemical forms, Hg(2+) salts are usually more toxic than their counterparts because of their greater solubility in water; thus, they are more readily absorbed from the gastrointestinal tract into circulation. Therefore, new chemical receptors for detecting Hg(2+) ions in circulation are needed. In this study, we developed a rhodamine-based turn-on fluorescence probe to monitor Hg(2+) in aqueous solution and in blood of mice with toxicosis. The chemodosimeter responds to Hg(2+) ions stoichiometrically, rapidly, and irreversibly at room temperature as a result of a chemical reaction that produces strongly fluorescent oxadiazole. The new fluorescent probe shows good fluorescence response, with high sensitivity and selectivity, toward Hg(2+) ions in aqueous solution and in blood from mice with toxicosis and facilitates the naked-eye detection of Hg(2+) ions.

Novel chiral ferrocenylpyrazolo[1,5-a][1,4]diazepin-4-one derivatives--synthesis, characterization and inhibition against lung cancer cells.

A series of novel 2-ferrocenyl-7-hydroxy-5-phenethyl-5,6,7,8-tetrahydro-4H-pyrazolo[1,5-a][1,4]diazepin-4-one derivatives with optical activity (2) was synthesized in the microwave-assisted condition and characterized by means of IR, (1)H NMR and mass spectroscopy, and furthermore confirmed by X-ray analysis of a representative compound (R)-2a. Preliminary biological evaluation showed that some compounds could suppress the growth of A549, H322 and H1299 lung cancer cells. Among the tested compounds, 2b-d were more effective and might perform their action through cell cycle arrest for A549 cell. Whereas these compounds inhibited growth of H1299 and H322 cells by inducing apoptosis. The anti-tumor activities of these compounds were related to the nature of substituents in benzene moiety. In addition, the results indicated also that compounds 2b-d possessed notable cytotoxicity and selectivity for A549 vs H1299 and H322 lung cancer cells.

Synthesis, X-ray crystal structure and optical properties of novel 1,3,5-triarylpyrazoline derivatives and the fluorescent sensor for Cu2+.

A series of novel 1,3,5-triarylpyrazoline derivatives was synthesized by the reaction of chalcone and 5-aryl-2-hydrazinyl-1,3,4-thiadiazole in 43.3-84.7% yields. The structures of compounds were characterized using IR, (1)H NMR and HRMS spectroscopy and X-ray diffraction analysis. The absorption and fluorescence characteristics of the compounds were investigated in dichloromethane, toluene, acetonitrile, N,N-dimethylformamide and tetrahydrofuran. The results showed that the absorption maxima of the compounds vary from 366 to 370nm depending on the group bound to benzene rings. The maximum emission spectra of the compounds in dichloromethane were dependent on nature of groups in benzene ring. Furthermore, the compound 3b can be used to determine Cu(2+) ion with high selectivity and a low detection limit in the DMF:H2O=1:1 (v/v) solution.