Fluorescence Off-on Response Research Articles

A highly specific red-emitting fluorescent probe for monitoring of Cu2+ in living cells and zebrafish.

A novel seminaphthorhodafluor-based fluorescent probe was designed and prepared. It exhibited an outstanding off-on fluorescence response (about 45-fold enhancement) to Cu2+ because the structure changed from a spirocyclic form to a ring-open state. It showed satisfactory detection performances for Cu2+ with fast response speed, high selectivity and anti-interference, and a low detection limit (22.9nM) due to its long emission wavelength and off-on fluorescence response mode. This probe was also satisfactorily applied for the monitoring of Cu2+ in living cells and zebrafish.

Ratiometric fluorescent biosensor for detection and real-time imaging of nitric oxide in mitochondria of living cells

Nitric oxide (NO), a ubiquitous gaseous messenger, plays critical roles in various pathological and physiological progresses. The abnormal levels of NO in organisms are closely related to a large number of maladies. Mitochondria are the main area that produce NO in mammalian cells. Thus, detecting and real-time imaging of NO in mitochondria is of great significance for exploring the biological functions of NO. Herein, a ratiometric fluorescent biosensor (Mito-GNP-pNO520) is developed for sensitive and selective detection and real-time imaging of NO in mitochondria of living cells. The detection is achieved through the fluorescence off-on response of Mito-GNP-pNO520 toward NO. This biosensor shows excellent characteristics, such as high sensitivity toward NO with a low detection limit of 0.25 nM, exclusive selectivity to NO without interference from other substances, good biological stability and low cytotoxicity. More importantly, the biosensor is specifically located in mitochondria, enabling the detection and real-time imaging of endogenous and exogenous NO in mitochondria of living cells. Therefore, our biosensor offers a new approach for dynamic detecting and real-time imaging of NO in subcellular organelles, providing an opportunity to explore new biological effects of NO.

Rational design of a lysosome-targeted fluorescent probe for monitoring the generation of hydroxyl radicals in ferroptosis pathways.

Ferroptosis is a newly discovered iron-dependent form of regulated cell death associated with high levels of hydroxyl radical (˙OH) production. Meanwhile, lysosome dysfunction has been shown to be one of the causes of ferroptosis. Although a variety of ˙OH-responsive fluorescent probes have been developed for detecting intracellular ˙OH in living cells, there are still only few lysosome-targeted probes to monitor the variation in lysosomal ˙OH levels during ferroptosis. Herein, we report a novel ˙OH-specific fluorescent probe HCy-Lyso, which is composed of the hydrocyanine and morpholine moiety. Upon treatment with ˙OH, its hydrocyanine unit was converted to the corresponding cyanine group, thus leading to a large π-conjugation extension of HCy-Lyso, accompanied by a significant fluorescence off-on response. Moreover, after reacting with ˙OH in an acidic environment, the protonation product of HCy-Lyso exhibits a higher fluorescence enhancement, which is suitable for detecting lysosomal ˙OH variation. HCy-Lyso has been utilized for imaging endogenous ˙OH in living cells under phorbol myristate acetate (PMA) stimuli and monitoring the changes in lysosomal ˙OH levels during ferroptosis. Thus, our study proposes a new strategy to design lysosome-targeted and ˙OH-responsive fluorescent probes to investigate the relationship between lysosomes and ferroptosis.

Simple dihydropyridine-based colorimetric chemosensors for heavy metal ion detection, biological evaluation, molecular docking, and ADMET profiling

In this study, two novel chemosensors containing dihydropyridine fragment namely; (2E, 2Eʹ)-1,1ʹ-(2,6-dimethyl-1,4-dihydropyridine-3,5-diyl)bis(3-(4-(dimethylamino)phenyl)prop-2-en-1-one) (1), (2E,2E',4E,4E')-1,1ʹ -(2,6-dimethyl-1,4-dihydropyridine-3,5-diyl)bis(5-(4-(dimethylamino)phenyl)penta-2,4-dien-1-one) (2) have been synthesized and characterized. The solvatochromic behavior was explored in different solvents of various polarities. The visual detection, as well as UV–Vis and fluorescence measurements were carried out to explore the colorimetric and optical sensing properties of the investigated chemosensors towards various metal ions such as Al3+, Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Mg2+, Hg2+ and Zn2+. The chemosensors 1 and 2 have strong detecting abilities, with excellent sensitivity and selectivity for Cu2+ and Fe3+, respectively, over the other metal ions. The chemosensors were totally reversible upon addition of EDTA to the formed complexes and displayed a turn on–off-on fluorescence response based on an effect of chelation-quenching fluorescence. The antioxidant activities of the investigated chemosensors were assessed. They were examined in-silico for their capacity to block the Akt signaling pathway, which is involved in cancer proliferation with interpreting their pharmacokinetics aspects. Furthermore, in-vitro antitumor evaluation against a panel of cancer cell lines for the investigated chemosensors has been examined. Conclusively, chemosensor 1 was more effective at scavenging free radicals and as an anticancer agent and could be exploited as a therapeutic candidate for cancer therapy than chemosensor 2 due to its potential inhibition of Akt protein.

Ligand-Directed H2 S Probe and Scavenger for Specific Tumor Imaging.

An expanding body of evidence suggests that specifically targeting hydrogen sulfide (H2 S) might potentially benefit both tumor diagnosis and treatment, but there is still a lack of cancer-targeted molecular tools for in vivo applications. Here, we report the first ligand-directed H2 S-specific near-infrared fluorescent sensor PSMA-Cy7-NBD and scavenger PSMA-Py-NBD that target the prostate-specific membrane antigen (PSMA). PSMA-Cy7-NBD displays a 53-fold off-on fluorescence response to H2 S at 803 nm with high specificity. PSMA-Py-NBD can scavenge H2 S fast (k2 =30.8 M-1 s-1 at 25 °C) without interference from biothiols. Both tools are highly water-soluble and can be transported selectively into PSMA-expressing prostate cancer cells. Endogenous H2 S levels in murine 22Rv1 tumor models can be imaged and downregulated by intravenous injection of PSMA-Cy7-NBD and PSMA-Py-NBD, respectively. These tools could potentially help to investigate H2 S cancer biology and with related therapies.

Mitochondrial Membrane Potential Independent Near-Infrared Mitochondrial Viscosity Probes for Real-Time Tracking Mitophagy.

Mitophagy is a vital cellular process playing vital roles in regulating cellular metabolism and mitochondrial quality control. Mitochondrial viscosity is a key microenvironmental index, closely associated with mitochondrial status. To monitor mitophagy and mitochondrial viscosity, three molecular rotors (Mito-1, Mito-2, and Mito-3) were developed. All probes contain a cationic quinolinium unit and a C12 chain so that they can tightly bind mitochondria and are not affected by the mitochondrial membrane potential. Optical studies showed that all probes are sensitive to viscosity changes with an off-on fluorescence response, and Mito-3 shows the best fluorescence enhancement. Bioimaging studies showed that all these probes can not only tightly locate and visualize mitochondria with near-infrared fluorescence but also effectively monitor the mitochondrial viscosity changes in cells. Moreover, Mito-3 was successfully applied to visualize the mitophagy process induced by starvation, and mitochondrial viscosity was found to show an increase during mitophagy. We expect Mito-3 to become a useful imaging tool for studying mitochondrial viscosity and mitophagy.

Rational design of metal-free fluorescent probe for tracking CO-releasing molecule-3 in vitro and in vivo

Carbon monoxide (CO), a potential therapeutic gaseous molecule for the treatment of inflammation-related diseases, is difficult to deliver directly into the body. CO-releasing molecule (CORM)− 3 is an alternative molecule for in vivo delivery of CO. To track the distribution of CORM-3 in vitro and in vivo, we reasonably designed and synthesized a series of coumarin-pyridine dyads (1−3) by DFT calculation. Applying the 4-nitrobenzyl group as the recognition site, two new metal-free fluorescent probes (MFP-1/2) were used for CORM-3 detection. As expected, probe MFP-1/2 showed an obvious off-on fluorescent response after the reaction with CORM-3. Meanwhile, probe MFP-2 exhibited good selectivity and sensitivity to CORM-3 in PBS. With the aid of its large stock shift and red emitting wavelength, MFP-2 was successfully applied to track the distribution of CORM-3 in living cells and in mice.

A fluorescein-2-(Pyridin-2-ylmethoxy) benzaldehyde conjugate for fluorogenic turn-ON recognition of Hg2+ in water and living cells with logic gate and memory device applications

An effective Hg2+ specific probe, fluorescein-2-(Pyridin-2-ylmethoxy)-benzaldehyde conjugate (FO511), was designed, synthesized and characterized by various instrumental techniques. The sensing behavior of FO511 was investigated by fluorescence technique which clearly established the high selectivity towards Hg2+ through OFF–ON fluorescence response in the presence of other metal ions like Zn2+, Mg2+, Co2+, Ni2+, Cu2+, Pb2+, Al3+, Cr3+, Cd2+, Pd2+, Fe2+ and Fe3+ in aqueous medium (pH 7.2, 10 mM HEPES buffer). The sensing mechanism could be attributed to the Hg2+ triggered spirolactam ring opening of the fluorescein moiety and simultaneous formation of a 1:1 FO511–Hg2+ complex. The interaction and formation of FO511–Hg2+ species was supported by the observations gained from fluorescence titrations, Job’s plot, 1H NMR and HRMS, and other spectroscopic studies. For the Hg2+ interaction towards FO511 the binding constant was evaluated to be (3.21 ± 0.05) × 104 M−1 with detection limit 92.7 nM. On addition of S2− to the FO511–Hg2+complex, the fluorescence intensity was totally quenched due to removal of Hg2+ from the complex by S2− ion arising out of stronger affinity of Hg2+ towards S2− resulting concomitant formation of ring closed non-fluorescent form, FO511. The tentative coordination environment in the FO511–Hg2+complex was established by DFT studies. FO511 exhibits low cytotoxicity and cell permeability, which makes it capable for bioimaging applications in living HepG2 cells.

A new fast-responding fluorimetric “turn-on” sensor based on benzothiazole-phenanthridine for the sensitive, selective, and reversible detection of Cu2+ in real water samples and its use in bio-imaging

This article describes a new chemosensor, 2-(benzo[d]thiazol-2-yl)-6-(7,8,13,14-tetrahydrodibenzo [a, i] phenanthridin-5-yl) phenol (BTPP), which is based on the combination of phenanthridine and benzothiazole groups for the sensitive and selective detection of Cu2+. Following the successful synthesis of BTPP in a single step, the molecular structure of BTPP was verified by 1H NMR, 13C NMR, mass spectrometry, and X-ray single-crystal analysis. These sensors exhibit high optical selectivity for the Cu2+ ion across a broad spectrum of other common metal ions in ACN/H2O (1:1) solution, resulting in an extraordinary fluorescence off-on response with a very low Limit of Detection (LOD) value. When Cu2+ was added to the compound BTPP, it displayed outstanding properties such as bright yellow emission, rapid reaction time (10 s), detection over a wide pH range (pH = 6–12), and reversibility with glutathione(GSH). FT-IR analysis, 1H NMR titration experiments, HRMS, and Job's plot analysis were used to determine the mechanism of BTPP's binding to Cu2+. Inspired by these remarkable sensing characteristics, we successfully used BTPP to detect Cu2+ in paper strip-based detection, ambient water samples, and fluorescent bio-imaging in E. coli bacteria with satisfying results.

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.

New fluorescent probe with recognition moiety of bipiperidinyl reveals the rise of hepatocellular carboxylesterase activity during heat shock

Heat shock is a heat-related pathology characterized by a high body temperature and an obvious change of many enzymatic activities. Carboxylesterase (CE), as the major hydrolase in liver, is responsible for the hydrolysis of many drugs or the detoxification of various toxins from all organs. However, the correlation between heat shock and the CE activity in cells remains unknown, mainly due to the lack of a suitable research approach. Herein, a new water-soluble fluorescence probe, MYO-CE, with a specific bipiperidinyl recognition moiety has been developed for detecting the CE activity. MYO-CE reacted selectively with CE instead of other esterase, causing a large fluorescence off-on response at 560 nm with a detection limit of 0.39 U/mL. The applicability of MYO-CE for cell imaging was demonstrated by monitoring the alteration of the hepatocellular CE activity under inflammation. More importantly, we investigated the change of the CE activity during heat shock, uncovering a significant increase for the first time. This finding was further validated by a commercial colorimetric kit assay. The proposed probe shows a promising prospect for the CE study in cells under different pathological conditions.

A rhodamine derivative-based fluorescent probe for visual monitoring of pH changes in the Golgi apparatus

The pH homeostasis of Golgi plays a vital role in maintaining normal cell functions. An effective near-infrared fluorescent probe that can sense pH changes in Golgi apparatus is of great significance. Here, a Golgi-targeting fluorescent probe (Golgi-hNR) based on a rhodamine derivative is presented to show a highly sensitive response to pH variations. Golgi-hNR displays a conspicuous pH-dependent behavior at 664 nm from pH 7.4–3.0 with a remarkable fluorescence off-on response. The pKa value of Golgi-hNR is measured to be 3.62. Confocal fluorescence imaging reveals that Golgi-hNR diffuses into Golgi with high selectivity. Using Golgi-hNR, we have successfully visualized real-time tracking of Golgi pH variations for medical therapy in living cells.

Heavy Atom-Free, Mitochondria-Targeted, and Activatable Photosensitizers for Photodynamic Therapy with Real-Time In-Situ Therapeutic Monitoring.

Based on spin-orbit charge transfer intersystem crossing mechanism, two heavy-atom-free photosensitizers (PSs) BDP1/BDP2 with absorption maxima at 506 nm/660 nm were constructed for photodynamic therapy (PDT). The long triplet state lifetimes and large singlet oxygen quantum yields, coupled with the mitochondria-targeted feature, made them highly phototoxic toward cancer cells. Moreover, the PDT-promoted cell apoptosis could be monitored by an obvious fluorescence off-on response of the two PSs due to the concomitant activation of extensive mitophagy, thus facilitating timely therapeutic feedback to avoid under- or over-treatment. Importantly, such design allows the activatable PSs Glu-BDP1/Glu-BDP2 to be fabricated by attaching γ-glutamyl, a substrate of γ-glutamyltranspeptidase, to the alkoxyaniline unit of BDP1/BDP2, and their ability in either selectively killing cancer cells over normal cells or in ablating implanted tumour without damage to healthy tissue was demonstrated.

A novel near-infrared fluorescence off-on probe for imaging hypoxia and nitroreductase in cells and in vivo

Nitroreductase (NTR) is a highly expressed endogenous enzyme in hypoxic tumor cells, which is associated with the hypoxic degree in tumors. Therefore, it is particularly important to develop low-toxicity and high-sensitivity fluorescent probes for NTR. Herein, we report an NTR-activated near-infrared probe with good selectivity and sensitivity. More importantly, the background fluorescence signal is effectively reduced due to the quenching effect of nitro group as well as the spironolactone (ring-closed) form of rhodamine moiety. Upon reaction with NTR, the probe exhibits a remarkable fluorescence off-on response at 740 nm (about 32-fold intensity enhancement), with an extremely low detection limit of 1.09 ng/mL. Moreover, the probe is able to target mitochondria. With this probe, we have achieved the visualization of NTR overexpression in hypoxic cancer cells, as well as in hypoxic tumors in vivo as revealed in the tumor-bearing mouse model. By using this probe, some essential information may be revealed about hypoxia-relevant diseases.

On-off-on fluorescence detection for biomolecules by a fluorescent cage through host-guest complexation in water

Detection of nucleoside derivatives has paramount importance because they are the essential biomolecular units for all life. Herein, we report a host-guest approach by using a fluorescent tetraphenylethene-based octacationic cage as host and 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) as guest and fluorescent indicator to form non-fluorescent 1:1:1 host-(endo-exo)guest complex in water. This new host-(endo-exo)guest complex can be successfully used for detecting nucleosides (e.g., ATP and GTP), DNA (e.g., sm-DNA), and antibiotics (e.g., Penicillin G) with off-on fluorescence response via a competitive host-guest exchange with HPTS as exo-guest in water. Furthermore, this on-off-on fluorescent host-guest complex is also used for cell imaging based on ATP concentration in HeLa cells. Therefore, this study not only provides insight into the construction of a supramolecular probe with on-off-on fluorescence via host-guest complexation and exchange in solution, but also realizes a universal method for detecting and monitoring biomolecules.

Real-time imaging mitochondrial viscosity dynamic during mitophagy mediated by photodynamic therapy

Photodynamic therapy has been generally developed and approved as a promising theranostic technique in recent years, which requires photosensitizers to bear high efficiency of reactive oxygen species production, precisely targeting ability and excellent biocompatibility. The real-time monitoring the microenvironments such as viscosity dynamic involved in mitophagy mediated by photodynamic therapy is significantly important to understand therapeutic process but barely reported. In this work, a pyridinium-functionalized triphenylamine derivative, (E)-4-(2-(4'-(diphenylamino)-[1,1′-biphenyl]-4-yl)vinyl)-1-methylpyridin-1-ium iodide (Mito-I), was exploited as photosensitizer for mitochondria-targeted photodynamic therapy and as fluorescent probe for imaging the mitochondrial viscosity dynamic during mitophagy simultaneously. The results indicated that the additional phenyl ring in Mito-I was beneficial to promote its efficiency of singlet oxygen production. The excellent capability of targeting mitochondria and singlet oxygen generation allowed Mito-I for the specifically mitochondria-targeted photodynamic therapy. Moreover, Mito-I displayed off-on fluorescence response to viscosity with high selectivity and sensitivity. The observed enhancement in fluorescence intensity of Mito-I revealed the increasingly mitochondrial viscosity during mitophagy mediated by the photodynamic therapy of Mito-I. As a result, this work presents a rare example to realize the mitochondria-targeting photodynamic therapy as well as the real-time monitoring viscosity dynamic during mitophagy, which is of great importance for the basic medical research involved in photodynamic therapy.

Viscosity sensitive fluorescent dyes with excellent photostability based on hemicyanine dyes for targeting cell membrane

Abstract In this paper, three viscosity sensitive cell membrane fluorescent dyes (1a-c) were obtained. Dyes 1a-c were linked by benzaldehyde (5) and benzoindole (2a), benzoxazole (2b) and benzothiazole (2c), with double bond, respectively. The optical properties showed that dyes 1a-c had larger molar extinction coefficients (3.34 × 104-2.64 × 105 M−1 cm−1) and excellent photostability (89–93 %). In particular, dyes 1a-c had obvious specific response to viscosity. More specifically, they indicated significant OFF-ON fluorescent response at 563 nm, 599 nm and 601 nm to the increase of viscosity (0.89–865 cp), respectively, and displayed an excellent linear relationship with viscosity. Further, the excited-state lifetime of dye 1b was evaluated as long as 828 ps in high viscosity environment by transient absorption spectra. The low cytotoxicity of dyes 1a-c, provided the possibility to be used in living cells. Accordingly, laser confocal experiments clearly exhibited that dyes 1a-c could be well located in the cell membrane. The results can assist in our understanding of the functions of cell membrane as well as application in biosensor and diagnostics.

Near-Infrared Fluorescent Probe Activated by Nitroreductase for In Vitro and In Vivo Hypoxic Tumor Detection.

Tumor hypoxia is correlated with increased resistance to chemotherapy and poor overall prognoses across a number of cancer types. We present here a cancer cell-selective and hypoxia-responsive probe (fol-BODIPY) designed on the basis of density functional theory (DFT)-optimized quantum chemical calculations. The fol-BODIPY probe was found to provide a rapid fluorescence "off-on" response to hypoxia relative to controls, which lack the folate or nitro-benzyl moieties. In vitro confocal microscopy and flow cytometry analyses, as well as in vivo near-infrared optical imaging of CT26 solid tumor-bearing mice, provided support for the contention that fol-BODIPY is more readily accepted by folate receptor-positive CT26 cancer cells and provides a superior fluorescence "off-on" signal under hypoxic conditions than the controls. Based on the findings of this study, we propose that fol-BODIPY may serve as a tumor-targeting, hypoxia-activatable probe that allows for direct cancer monitoring both in vitro and in vivo.

Real-Time Monitoring Mitochondrial Viscosity during Mitophagy Using a Mitochondria-Immobilized Near-Infrared Aggregation-Induced Emission Probe.

Mitophagy plays a crucial role in maintaining intracellular homeostasis through the removal of dysfunctional mitochondria and recycling their constituents in a lysosome-degradative pathway, which leads to microenvironmental changes within mitochondria, such as the pH, viscosity, and polarity. However, most of the mitochondrial fluorescence viscosity probes only rely on electrostatic attraction and readily leak out from the mitochondria during mitophagy with a decreased membrane potential, thus easily leading to an inaccurate detection of viscosity changes. In this work, we report a mitochondria-immobilized NIR-emissive aggregation-induced emission (AIE) probe CS-Py-BC, which allows for an off-on fluorescence response to viscosity, thus enabling the real-time monitoring viscosity variation during mitophagy. This system consists of a cyanostilbene skeleton as the AIE active core and viscosity-sensitive unit, a pyridinium cation for the mitochondria-targeting group, and a benzyl chloride subunit that induces mitochondrial immobilization. As the viscosity increased from 0.903 cP (0% glycerol) to 965 cP (99% glycerol), CS-Py-BC exhibited an about 92-fold increase in fluorescence intensity at 650 nm, which might be attributed to the restriction of rotation and inhibition of twisted intramolecular charge transfer in a high viscosity system. We also revealed that CS-Py-BC could be well immobilized onto mitochondria, regardless of the mitochondrial membrane potential fluctuation. Most importantly, using CS-Py-BC, we have successfully visualized the increased mitochondrial viscosity during starvation or rapamycin-induced mitophagy in real time. All these features render CS-Py-BC a promising candidate to investigate mitophagy-associated dynamic physiological and pathological processes.

Boronate based sensitive fluorescent probe for the detection of endogenous peroxynitrite in living cells.

In this work, a novel boronate-based fluorescent probe (FAM) for the endogenous detection of peroxynitrite (ONOO-) has been developed by using anthracycline as the fluorophore, arlyboronate as the recognition moiety, lipophilic cation as the mitochondrial targeting moiety. Upon reaction of the probe with ONOO-, the oxidation and subsequent hydrolysis of ONOO- to arlyboronate triggers quite rapid fluorescence off-on response, providing a sensitive and highly selective method for the detection of ONOO-. In addition, probe holds high sensitivity with the detection limit of 3.2nM and excellent specificity including a series of biologically relevant reactive oxygen species. Importantly, FAM with good water solubility displays excellent performances for imaging endogenous peroxynitrite produced by RAW264.7 cells.