Novel fluorescence probe for ClO- in living cells: Based on FRET mechanism.

Hypochlorous acid (HClO) as a kind of reactive oxygen species (ROS) plays a vital role in many biological processes. Organic fluorescence probes have attracted great interests for the detection of HClO, due to their relatively high selectivity and sensitivity, satisfactory spatiotemporal resolution and good biocompatibility. Constructing fluorescence probes to detect HClO with advantages of large Stokes shift, wide emission gap, near infrared emission and good water solubility is still challenging. In this work, a new ratiometric fluorescence probe (named HCY) for HClO was developed. FRET-based HCY was constructed by bonding a coumarin and a flavone fluorophore. In absence of HClO, HCY exists FRET process, however, FRET is inhibited in the presence of HClO because the conjugated double bond broke. Due to the good match of the emission spectrum of the donor and the absorption spectrum of the acceptor, the FRET system appears favorable energy transfer efficiency. HCY showed high sensitivity and rapid response time. The linearity between the ratios of fluorescence intensity and concentration of HClO was established with a low limit of detection. What's more, HCY was also applied for fluorescence images of HClO in RAW264.7 cells.

A near-infrared fluorescent probe based FRET for ratiometric sensing of H2O2 and viscosity in live cells.

Hydrogen peroxide (H2O2) and viscosity play vital roles in the cellular environment as signaling molecule and microenvironment parameter, respectively, and are associated with many physiological and pathological processes in biological systems. We developed a near-infrared fluorescent probe, CQ, which performed colorimetric and ratiometric detection of H2O2 and viscosity based on the FRET mechanism, and was capable of monitoring changes in viscosity and H2O2 levels simultaneously through two different channels. Based on the specific reaction of H2O2 with borate ester, CQ exhibited a significant ratiometric response to H2O2 with a large Stokes shift of 221 nm, a detection limit of 0.87 µM, a near-infrared emission wavelength of 671 nm, a response time of 1 h, a wide detection ranges of 0.87-800 µM and a high energy transfer efficiency of 99.9 %. CQ could also recognize viscosity by the TICT mechanism, and efficiently detect viscosity changes caused by food thickeners. More importantly, CQ could successfully detect endogenous/exogenous H2O2 and viscosity in live HeLa cells, which was expected to be a practical tool for detecting H2O2 and viscosity in live cells.

Dual-response and lysosome-targeted fluorescent probe for viscosity and sulfur dioxide derivatives.

Viscosity and sulfur dioxide levels are important factors to evaluate the changes of cell micro-environment because a series of diseases usually occur when they are abnormal. At present, dual-response probes that can detect both viscosity and sulfur dioxide are rare. Therefore, we developed a novel fluorescent probe CBN for simultaneous detection of sulfur dioxide and viscosity. Besides, probe CBN could target lysosome of which normal function will be disrupted by the abnormality of viscosity. Therefore, probe CBN has the potential to be served as an effective biological tool to monitor the intracellular micro-environment.

FRET-based fluorescent probe with favorable water solubility for simultaneous detection of SO2 derivatives and viscosity.

The intracellular viscosity is an important parameter of the microenvironment and SO2 is a vital gas signal molecule. At present, some dual-response fluorescence probes for simultaneous measurements of viscosity and SO2 derivatives (HSO3-/SO32-) possessed poor water solubility. In this work, we developed a water-soluble fluorescence probe CIJ (0.0864 g/100 mL of water at 20 °C) for simultaneous measurements of viscosity and SO2 derivatives. CIJ exhibited a sensitive fluorescence enhancement to environmental viscosity from 0.97 to 28.04 cP based on a twisted intramolecular charge transfer mechanism and was applied to effective measurement of viscosity in vitro and in vivo. CIJ could also respond to SO2 derivatives with a low detection limit (44 nM) and a fast response time (5 min) based on the nucleophilic addition reaction. Furthermore, CIJ was applied to monitor SO2 derivatives in ratiometric response manner in living cells.

A quinoline-coumarin near-infrared ratiometric fluorescent probe for detection of sulfur dioxide derivatives.

Sulfur dioxide derivatives (HSO3- and SO32-) play an important role in food preservative, antibacterial, antioxidant and other aspects, so it is urgent for us to develop more efficient detection methods to broaden their application in biochemical research and related disease diagnosis. Fluorescent probes are of particular interest because of their simplicity and high temporal and spatial resolution. Herein, we constructed a new near-infrared (NIR) fluorescence probe, CQC, composed of coumarin fluorophore and quinoline fluorophore, for detecting SO2 derivatives. The near-infrared emission probe CQC with a large Stokes shift (260 nm) not only kept the distance between the two emission peaks large enough (165 nm), but also had a particularly high energy transfer efficiency (99.5%), and was particularly sensitive to the detection of HSO3-/SO32- (LOD: 0.1 µM). The powerful probe CQC succeeded in real-time visualizing endogenous HSO3-/SO32- in living cells.

A ratiometric fluorescent probe for selective detection of thiophenol derivatives.

Though a number of on-off or off-on fluorescent probes have been developed for the detection of thiophenol by using its unique recognition groups, such as 2, 4-dinitrophenyl ether, 2, 4-dinitrophenyl sulfonamide, and 2, 4-dinitrophenyl sulfonate, up to now, there are few probes that can detect thiophenol by the proportional fluorescence signal. We developed a ratiometric fluorescent probe with coumarin pyridine derivative as fluorophore and 2, 4-dinitrophenyl ether moiety as the sensing unit which could be used to detect thiophenol derivatives by the aromatic nucleophilic substitution reaction. This probe (CPBPN) displayed significant change in fluorescence ratio (256 fold) to result in a more reliable analysis by self-calibration and a relatively low detection limit of 24 nM toward 4-methylthiophenol (MTP) within 30 min to achieve more sensitivity. Besides, the probe was also applied to detect the presence of thiophenol derivatives in actual water samples and fluorescence imaging in living cells. The present work is of great importance for monitoring environmental pollutants and studying their biological function.

Two simple but effective turn-on benzothiazole-based fluorescent probes for detecting hydrogen sulfide in real water samples and HeLa cells.

Two simple turn-on fluorescent probes, containing a benzothiazole and the 2,4-dinitrobenzenesulfonyl group, were designed for detecting H2S. Two probes exhibited good selectivity and high sensitivity, which were applied to detect the H2S in real water samples. Probe P2 with a positive charge had better solubility than probe P1 in water; therefore, probe P2 was successfully applied to detect both the endogenous and exogenous H2S in lysosomes of living HeLa cells.

A synergetic FRET/ICT platform-based fluorescence probe for ratiometric imaging of bisulfite in lipid droplets.

A deep-red emission and lipid droplets-targeted fluorescence probe (named ZFPy) for effective bioimaging of bisulfite was developed from flavone moiety and benzoindole derivative based on intramolecular charge transfer (ICT) and Förster resonance energy transfer (FRET) platform. ZFPy displayed promising fluorescence parameters including bright deep red fluorescence (615 nm), large Stokes shift (205 nm), extended emission window gap (140 nm), high absolute fluorescence quantum yield (4.1%) and stable emission signal output. In addition, ZFPy realized ratiometric fluorescence monitoring for SO2 derivatives with low detection limit (30 nM), preferable linearity, high sensitivity and selectivity. Interestingly, dual fluorophores (i.e. the donor moiety and 1,1,2,3-tetra-substituent-1H-benzo[e]indol-3-ium iodide moiety) released the same emission band about 475 nm to enhance the emission signal when ZFPy reacted with SO2 derivatives, to the best of our knowledge, this is the first synergetic FRET/ICT platform for fluorescence probe, which might effectively offer ZFPy a high sensitivity and low detection limit in the detection of SO2 derivatives. More importantly, ZFPy could image exogenous and endogenous SO2 derivatives in living HeLa, HepG2 and L-O2 cells with good biocompatibility and photostability. ZFPy also preferred to load on lipid droplets with high Pearson's coefficient (0.95).

A novel mitochondria-targeted ratiometric fluorescent probe for endogenous sulfur dioxide derivatives as a cancer-detecting tool.

A new mitochondria-targeted fluorescent probe RBC, constructed using a coumarin moiety which was selected as the donor and a benzothiazole derivative as the acceptor, for SO2 derivatives (HSO3-/SO32-) was presented. The probe designed on a new FRET platform showed high selectivity and a low detection limit. Importantly, the probe could respond to HSO3-/SO32- within 35 s. Furthermore, the probe could target mitochondria and was successfully used for fluorescence imaging of endogenous bisulfite in HepG2 with low cytotoxicity, which significantly assisted in cancer diagnosis.

A mitochondria-targeted fluorescent probe for the detection of endogenous SO2 derivatives in living cells.

A unique fluorescent probe (ZACA) for the monitoring of SO2 derivatives was developed from coumarin and benzoindoles based on FRET and ICT. ZACA exhibited an active emission signal, large Stokes shift, wide emission window distance, and high photostability. It also possessed many advantages in the ratiometric detection of HSO3-/SO32- including low detection limit and high selectivity and sensitivity. Importantly, ZACA was successfully applied in the ratiometric detection of endogenous HSO3-/SO32- in living cells with excellent cellular imaging capability (1 µM) and mitochondria-targeting ability (co-localization coefficient: 0.91).

A near-infrared and mitochondria-targeted fluorescence probe for ratiometric monitoring of sulfur dioxide derivatives in living cells.

A promising near-infrared emissive and mitochondria-targeted fluorescence probe (SNB) for the ratiometric detection of sulfur dioxide derivatives with a novel reaction mechanism was developed on the basis of FRET and the ICT platform. Probe SNB showed favorable fluorescence properties, including an active NIR emission signal (671 nm), ultra-large Stokes shift (251 nm) and an ultra-broad emission band gap (193 nm). Furthermore, SNB was applied to detect sulfur dioxide derivatives with a low detection limit (17 nM), excellent sensitivity and exceptional selectivity. Of greater significance, SNB also successfully implemented the real-time and ratiometric surveillance for exogenous or endogenous sulfur dioxide derivatives in living HeLa, L-O2 and HepG2 cells. Interestingly, SNB possessed a subcellular organelle targeting ability with a preferred co-localization coefficient of 0.91 for mitochondria, which revealed its promising potential in the detection of subcellular sulfur dioxide derivatives.

Enhancing autophagy protects platelets in immune thrombocytopenia patients.

BACKGROUND: Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder and involves increased apoptosis of platelets. Autophagy is an essential process for platelets to maintain their life and physiological functions. However, the role of autophagy in ITP platelets was previously unclear. METHODS: In the present study, the expression of autophagy-related protein and autophagy flux were detected in platelets from ITP patients and healthy controls by immunofluorescence staining and immunoblotting, and the influence of autophagy on the viability and apoptosis of ITP platelets was further explored. RESULTS: We found that platelet autophagy was diminished in ITP patients. Platelet autophagy in ITP was regulated by the PI3K/AKT/mTOR pathway, with mTOR (mammalian target of rapamycin) as a negative regulator and class III PtdIns3K playing a crucial role in the process. Importantly, the small-molecule compound ABO (6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine) enhanced autophagy in ITP platelets. Enhancing platelet autophagy alleviated platelet destruction by inhibiting apoptosis and improving platelet viability. CONCLUSIONS: These results suggest a role for autophagy regulation in the pathogenesis of ITP, and offer a novel treatment for these patients.

A new FRET-based ratiometric fluorescence probe for hypochlorous acid and its imaging in living cells.

A novel ratiometric fluorescence probe for hypochlorous acid was constructed by coumarin and pyridinium fluorophore based on the Forster resonance energy transfer (FRET) and intramolecular charge transfer (ICT) platform. In this ICT/FRET system, the energy transfer efficiency is high to 94.3%. Moreover, the probe could respond to hypochlorous acid with high selectivity and sensitivity, and exhibited a large Stokes shift. It was interesting to find that the probe could recognize hypochlorous acid via a new mechanism, in which the α-position of carbonyl group was oxidized to form a diketone derivative. More importantly, the probe was successfully applied to the ratiometric imaging of both exogenous and endogenous hypochlorous acid in living RAW 264.7 cells, with low toxicity and high photo-stability.

A ratiometric fluorescence probe based on a novel recognition mechanism for monitoring endogenous hypochlorite in living cells.

A ratiometric fluorescence probe (named ZOC) for the fast detection of HClO/ClO- was constructed by coumarin (donor) and pyridinium (acceptor) based on Forster resonance energy transfer (FRET) and intramolecular charge transfer (ICT) platform. ZOC possessed red emission signal (610 nm), large Stocks shift (190 nm), high energy transfer efficiency (95.3%), high selectivity and sensitivity, low detection limit (25 nM), wider detection range (from 25 nM to 30 µM), rapid response (within 13 S), and good biocompatibility. It was very interesting that the recognition mechanism involved a new organic reaction in which olefin double bond reacted first with HClO/ClO- regioselectively, followed by cyclization. ZOC was successfully used to the real time detection of endogenous HClO/ClO- in RAW 264.7 cells.

A novel lipid droplets-targeting ratiometric fluorescence probe for hypochlorous acid in living cells.

Lipid droplets were found to be involved in many organism activities. Here, a lipid droplets-targeted near-infrared fluorescence probe (named XHZ) for ratiometric detection of endogenous hypochlorous acid/hypochlorite (HClO/ClO-) in living cells was developed, which was constructed by a coumarin moiety and a malononitrile derivative. XHZ could detect HClO/ClO- with high selectivity and sensitivity in a ratiometric manner based on FRET (Förster Resonance Energy Transfer) mechanism. The two well-resolved emission (470/672 nm) bands could ensure accurate detection of HClO/ClO- in vitro as well as in vivo. XHZ was successfully used for ratiometric fluorescence imaging of exogenous and endogenous HClO/ClO- in RAW264.7 cells. A good linear relationship between the fluorescence intensity ratios of the two emissions and HClO/ClO- concentrations from 0 to 40 µM was obtained. Importantly, XHZ could localize mainly in lipid droplets of RAW264.7 cells. To the best of our knowledge, XHZ is the first lipid droplets-targeted ratiometric fluorescence probe for HClO/ClO-.

Discovery of novel HSP90 inhibitors that induced apoptosis and impaired autophagic flux in A549 lung cancer cells.

Heat shock protein 90 (HSP90) inhibition has aroused increasing enthusiasm in antitumor strategies in recent years. According to our previous studies, we synthesized a series of coumarin pyrazoline compounds HCP1-HCP6 that might be HSP90 inhibitors. Interactions between HCP1-HCP6 and HSP90 were examined and antitumor activities of them were investigated in A549 lung cancer cells. Results showed that all the six derivatives could interact with HSP90, in which HCP1 exhibited the best binding ability and inhibited the activity of HSP90. Meanwhile, HCP1-HCP6 reduced the cell viability of A549 cells and HCP1 possessed the lowest IC50 value. Above all HCP1 exerted better HSP90 inhibitory and anticancer effects than our initially identified HSP90 inhibitor DPB. As to the underlying mechanism, HCP1-HCP6 not only induced apoptosis as DPB but also blocked autophagic flux in A549 cells. Therefore, we discovered a novel HSP90 inhibitor HCP1 that had better biological activity and provided us a useful tool to explore the underlying mechanism of lung cancer therapy.

A new fluorescent probe for colorimetric and ratiometric detection of sulfur dioxide derivatives in liver cancer cells.

A new ratiometric fluorescent probe was constructed with hemicyanine and 7-nitrobenzofurazan for detection of sulfur dioxide derivatives (HSO3-/SO32-). The ratiometric response mode could be attributed to the efficient FRET (Förster resonance energy transfer) platform. The probe exbihited some desirable properties including fast response (within 2 minutes), good selectivity and high sensitivity. Moreover, the probe could detect endogenous HSO3- in liver cancer cells rather than normal liver cells, implying the diagnosal potential of the probe.

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.

Fluorescence detection of endogenous bisulfite in liver cancer cells using an effective ESIPT enhanced FRET platform.

Probe L-HF1, which featured large (pseudo) Stokes shifts and high FRET efficiency, was designed on a new ESIPT enhanced FRET platform for the detection of HSO3-/SO32-. L-HF1 could detect endogenous bisulfite in HepG2 cells but not in L-02 cells, implying the different bisulfite levels in normal and cancer cells of liver.

A mitochondria-targeted fluorescence probe for ratiometric detection of endogenous hypochlorite in the living cells.

A mitochondria-targeted fluorescence probe (CPBT) for ratiometric detection of endogenous hypochlorite in the living cells was developed. CPBT could detect hypochlorite with high selectivity and sensitivity in a ratiometric manner based on FRET mechanism. In absence of hypochlorite, when CPBT was excited with absorption maximum wavelength of the donor moiety, it showed the emission of acceptor moiety because of FRET process. However, in the presence of hypochlorite, the reaction of CC double bond with hypochlorite interrupted the conjugation system resulting in the inhibition of FRET process and the emission of the donor moiety. The two well-resolved emission bands can ensure accurate detection of hypochlorite. A good linear relationship between the fluorescence intensity ratios of the two emissions and the ClO- concentrations in the range from 41.8 nM (detection limit) to 12.5 µM was established. Importantly, CPBT could localize mainly in the mitochondria of RAW264.7 cells. CPBT was successfully used to fluorescence ratiometric imaging of endogenous hypochlorite in RAW264.7 cells.