Nitroxidative chemistry interferes with fluorescent probe chemistry: Implications for nitric oxide detection using 2,3-diaminonaphthalene

Simultaneous production of nitric oxide (NO) and superoxide generates peroxynitrite and causes nitroxidative stress. The fluorometric method for NO detection is based on the formation of a fluorescent product from the reaction of a nonfluorescent probe molecule with NO-derived nitrosating species. Here, we present an example of how nitroxidative chemistry could interact with fluorescent probe chemistry. 2,3-Naphthotriazole (NAT) is the NO-derived fluorescent product of 2,3-diaminonaphthalene (DAN), a commonly used NO-detecting molecule. We show that NO/superoxide cogeneration, and particularly peroxynitrite, mediates the chemical decomposition of NAT. Moreover, the extent of NAT decomposition depends on the relative fluxes of NO and superoxide; the maximum effect being reached at almost equivalent generation rates for both radicals. The rate constant for the reaction of NAT with peroxynitrite was determined to be 2.2 × 10³ M⁻¹ s⁻¹. Further, various peroxynitrite scavengers were shown to effectively inhibit NO/superoxide- and peroxynitrite-mediated decomposition of NAT. Taken together, the present study suggests that the interference of a fluorometric NO assay can be originated from the interaction between the final fluorescent product and the formed reactive nitrogen and oxygen species.     

Synthesis of a novel fluorescent probe useful for DNA detection

In this paper, a novel fluorescent probe 2-methylbenzo[b][1,10] phenanthrolin-7(12H)-one (m-BPO) is synthesized, and its molecular structure has been characterized by IR, UV, MS, (1)H-NMR and elements analysis. The fluorescent characteristics of m-BPO were investigated in detail. It was found that DNA had the ability to quench the fluorescence of m-BPO at 411 nm (lambda(ex)=286 nm), and the quenched intensity of fluorescence was proportional to the concentration of DNA. Based on this fact, m-BPO has been used as the fluorescent probe for detection of calf thymus DNA (ctDNA) and fish semen DNA (fsDNA). Under the optimal conditions, the calibration curves are linear up to 15.0 microg/ml for both ctDNA and fsDNA. The corresponding detection limits are 3.6 ng/ml for ctDNA and 5.5 ng/ml for fsDNA, respectively. The interaction mechanism for the binding of m-BPO to ctDNA was studied in detail, and the results suggested that the interaction mode between m-BPO and ctDNA was groove binding.     

A novel highly specific colorimetric fluorescent probe for the detection of nitrite in aqueous solution

Developing an effective method for the detection of nitrite (NO₂⁻) ions in the natural environment especially in environmental waters and soils is very necessary, since they will cause serious damage to human health once excess NO₂⁻ ions enters the human body. Therefore, a new colorimetric fluorescent probe NB-NO₂⁻ for determining NO₂⁻ ions was designed, which possesses good water-solubility and satisfactory selectivity over other common ions for NO₂⁻ ions. The addition of NO₂⁻ ions changed the color of solution from blue to colorless seen by the naked-eye. Furthermore, through test and calculation, the detection limit of the probe NB-NO₂⁻ is 129 nM. Based on the earlier excellent characteristics, the probe NB-NO₂⁻ was successfully used for monitoring NO₂⁻ ions in environmental waters and soils.     

Visualization of nitric oxide in living cells by a copper-based fluorescent probe

Nitric oxide (NO) serves as a messenger for cellular signaling. To visualize NO in living cells, we synthesized a turn-on fluorescent probe for use in combination with microscopy. Unlike existing fluorescent sensors, the construct--a Cu(II) complex of a fluorescein modified with an appended metal-chelating ligand (FL)--directly and immediately images NO rather than a derivative reactive nitrogen species. Using spectroscopic and mass spectrometric methods, we established that the mechanism of the reaction responsible for the NO-induced fluorescence involves reduction of the complex to Cu(I) with release of the nitrosated ligand, which occurs irreversibly. We detected NO produced by both constitutive and inducible NO synthases (cNOS and iNOS, respectively) in live neurons and macrophages in a concentration- and time-dependent manner by using the Cu(II)-based imaging agent. Both the sensitivity to nanomolar concentrations of NO and the spatiotemporal information provided by this complex demonstrate its value for numerous biological applications.     

A homogeneous fluorescent cell-based assay for detection of heterologously expressed nitric oxide synthase activity

Arhodamine-derived, membrane-permeable fluorophore (DAR-4M AM) sensitive to nitric oxide production has been developed recently. The authors evaluated this reagent in both 96 and 384-well formats using heterologously expressed neuronal nitric oxide synthase (nNOS). nNOS transfected into HEK-293T cells was stimulated by the addition of ionomycin. The calcium mobilization resulting from ionomycin treatment of nNOS-expressing 293T cells induced a robust increase in emission intensity, as measured using a standard rhodamine filter set. The effect was time dependent, and a 3 to 4-fold stimulation could be achieved in a 2-h time period. Ionomycin-dependent nitric oxide (NO) production was completely inhibited by several arginine analogs at micromolar concentrations (e.g., L-NAME IC 50=3.0 micro M). Several arginine analog inhibitors of nNOS were revealed to be differentially reversible over increasing substrate concentrations. The assay is a facile method for characterizing inhibitors of nNOS in a relatively unperturbed cell environment.     

A novel adaptive fluorescent probe for cell labelling

In this study we describe the synthesis and characterisation of a new hydrazone-based fluorescent compound that is able to selectively label the endoplasmic reticulum (ER) in yeast and mammalian living cells. The fluorescence properties of the compound depended on the DMSO/water ratio and on the pH. NMR experiments allowed determination of the conformation adopted in various environments. Apart from the convenient synthetic procedure, our compound displays low cell toxicity and blue emission compatible with filters routinely used in fluorescence microscopy.     

Preparation of a copper-based fluorescent probe for nitric oxide and its use in mammalian cultured cells

A procedure for the preparation of a copper(II) complex (CuFL) as a fluorescent nitric oxide (NO) detector is described. The fluorescein-based ligand FL can be synthesized in seven reaction steps (overall yield approximately 20%), typically requiring a total time of 9 days. The CuFL probe allows for the detection of NO produced in mammalian cultured cells. The detailed protocol for the use of CuFL for imaging NO in human neuroblastoma SK-N-SH cells takes a total time of approximately 26 h. This includes plating cells on six-well tissue culture plates or imaging dishes, treatment with CuFL, stimulation of NO synthases and imaging by fluorescence microscopy.     

Reliable in vitro measurement of nitric oxide released from endothelial cells using low concentrations of the fluorescent probe 4,5-diaminofluorescein

4,5-Diaminofluorescein (DAF-2) and its membrane-permeable derivate DAF-2 diacetate are fluorescent probes that have been developed to perform real-time biological detection of nitric oxide (NO). Their use for intracellular imaging, however, has recently been seriously questioned and data using DAF-2 for extracellular NO detection at low levels, as for example released from endothelial cells, are rare. Here we show that a reliable detection of low levels of NO in biological systems by DAF-2 is possible (a) by using low DAF-2 concentrations (0.1 microM) and (b) by subtracting the DAF-2 auto-fluorescence from the measured total fluorescence. The described method allows easy real-time detection of endothelial NO formation.     

In vivo detection of nitric oxide distribution in mice

This paper discusses in vivo detection of nitric oxide (NO) distribution in endotoxin-treated mice using L-band (1.1 GHz) electron paramagnetic resonance spectroscopy (EPR) in combination with the hydrophilic NO trapping complex: N-methyl-D-glucamine dithiocarbamate and iron (MGD-Fe). MGD-Fe-NO complex is found in the upper abdomen (liver region), lower abdomen (kidney and urinary bladder) and head region of ICR mice. Experiments with nitric oxide synthase (NOS) inhibition and ¹⁵N-labeled L-arginine as NOS substrate verify the origin of trapped NO from L-arginine. However, contribution from a 'nonenzymatic' NO generation pathway can not be ruled out. This paper further examines potential artifacts, which may arise in experiments using dithiocarbamate-iron complexes as NO trapping agents.     

In vivo detection of nitric oxide distribution in mice

This paper discusses in vivo detection of nitric oxide (NO) distribution in endotoxin-treated mice using L-band (1.1 GHz) electron paramagnetic resonance spectroscopy (EPR) in combination with the hydrophilic NO trapping complex: N-methyl-D-glucamine dithiocarbamate and iron (MGD-Fe). MGD-Fe-NO complex is found in the upper abdomen (liver region), lower abdomen (kidney and urinary bladder) and head region of ICR mice. Experiments with nitric oxide synthase (NOS) inhibition and 15N-labeled L-arginine as NOS substrate verify the origin of trapped NO from L-arginine. However, contribution from a 'nonenzymatic' NO generation pathway can not be ruled out. This paper further examines potential artifacts, which may arise in experiments using dithiocarbamate-iron complexes as NO trapping agents.     

A novel aggregation-induced emission fluorescent probe for nucleic acid detection and its applications in cell imaging

A new kind of aggregation-induced emission compound was synthesized and used as the probe of nucleic acid. The characterization of this compound was studied. Both the RNA and DNA were detected by using this probe. And the detection scope of DNA and RNA was different. We researched the selectivity of our probe in double and single strand DNA sequences. The visualization of gel electrophoresis and the cell nucleus imaging were researched as well. Compared with the traditional nucleus dye Hoechst 33258, our probe also has the potential to be nucleus dye. And the cell toxicity was well performed by MTT assays.     

A dual-function fluorescent probe for the detection of pH values and formaldehyde

A dual-function fluorescent probe (Probe 1) was developed in this work for the separate detection of pH value and formaldehyde (HCHO). Probe 1 could recognize HCHO and the pH value from the amino group. The colour of the probe solution was changed from grey blue to light blue with the increase in the pH value, and luminous intensity became larger with the increase in formaldehyde concentration. The curve function relationship between fluorescence intensity and the pH value was also determined. A smartphone containing a colour detector for imaging was used to record the values of the three primary colours (R value, G value, and B value) for the probe solution in formaldehyde. Importantly, there was a linear functional relationship between the B*R/G value with HCHO concentration. Therefore, the probe could be used as a rapid tool for the detection of formaldehyde. More importantly, Probe 1 was successfully used to detect formaldehyde in an actual distilled liquor sample.     

A colorimetric fluorescent probe for rapid and specific detection of nitrite

The method of fluorescent probes has been an important technique for detection of nitrite (NO₂^?). As an important inorganic salt, excessive nitrite would threaten humans and the environment. In this paper, a colorimetric fluorescent probe P‐N (1,2‐diaminoanthraquinone) with rapid response and high selectivity, which could detect NO₂^? by visual colour changes and fluorescence spectroscopy is presented. The probe P‐N solution (pH 1) changed from pink to colourless with the addition of NO₂^? and fluorescence intensity at 639 nm clearly decreased. Good linear exists between fluorescence intensities and NO₂^? concentrations for the range 0–16 μM, and the detection limit was 54 nM (based on a 3σ/slope). Moreover, probe P‐N could also detect NO₂^? in real water samples, and results were all satisfactory. Probe P‐N shows great practical application value for detecting NO₂^? in the environment.     

A ratiometric fluorescent probe for rapid and specific detection of hypochlorite

Hypochlorite (ClO⁻), as a kind of essential reactive oxygen species, plays a crucial role in vitro and in vivo. Here, a ratiometric fluorescent probe ( TPAM ) was designed and constructed for sensing ClO⁻ based on substituted triphenylamine and malononitrile, which exhibited obvious colour transfer from orange to colourless under daylight accompanied by noticeable fluorescence change from red to green in response to ClO⁻. TPAM could effectively monitor ClO⁻ with the merits of fast response, excellent selectivity, high sensitivity and a low detection limit of 0.1014 μM. ¹H NMR, mass spectra and theoretical calculations proved that ClO⁻ caused the oxidation of the carbon–carbon double bond in TPAM , resulting in compound 1 and marked changes in colour and fluorescence. In addition, TPAM was utilized for imaging ClO⁻ in living cells successfully with good photostability and biocompatibility.     

A coumarin‐based fluorescent turn‐on probe for detection of biothiols in vitro

A novel fluorescent probe (CA‐N) was designed and synthesized for detection of biothiols. CA‐N displayed a strong fluorescence in the presence of biothiols with high sensitivity, and the mechanism for detection biothiols was based on the Michael addition reaction of a thiol group to α,β‐unsaturated ketones. CA‐N showed low detection limit for cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), which were calculated as 3.16, 0.19 and 5.15 μM, respectively. At the same time, CA‐N exhibited high selectivity toward biothiols compared with other biological amino acids. In vitro cell experiments proved that CA‐N had no cytotoxicity, high cell permeability and could be employed in living cell imaging for biothiols. Copyright © 2015 John Wiley & Sons, Ltd.     

A novel fluorescent probe with large Stokes shift for the detection of viscosity changes and its imaging in living cells

As an important cellular microenvironmental parameter, viscosity could reflect the status of living cells. Small molecular fluorescent probes are a vital tool to measure the change of viscosity in living cells. A novel fluorescence probe ZL-1 with a large Stokes shift (in methanol it reached to 153 nm and in glycerol it reached to 125 nm) and excellent sensitivity toward viscosity was developed. The sharp enhancement of the emission intensity for the probe ZL-1 from low viscous methanol to high viscous glycerol indicated that the probe ZL-1 could respond to the viscosity variations. Moreover, the probe ZL-1 has been successfully utilized to detect of the viscosity variations in living cells.     

A novel viscosity-sensitive fluorescent probe for monitoring the changes of mitochondrial viscosity

As a fundamental physical parameter, viscosity influences the diffusion in biological processes. The changes in intracellular viscosity led to the occurrence of relevant diseases. Monitoring changes in cellular viscosity is important for distinguishing abnormal cells in cell biology and oncologic pathology. Here, we devised and synthesized a viscosity-sensitive fluorescent probe LBX-1 . LBX-1 showed high sensitivity, providing a large Stokes shift as well as an enhancement in fluorescent intensity (16.1-fold) from methanol solution to glycerol solution. Furthermore, the probe LBX-1 could localize in mitochondria because of the ability of the probe to penetrate the cell membrane and accumulate in mitochondria. These results suggested that the probe could be utilized in monitoring the changes in mitochondrial viscosity in complex biological systems.     

A highly selective fluorescent probe for pyrophosphate detection in aqueous solutions

A novel and simple fluorescence enhancement method is introduced for selective pyrophosphate (PPi) sensing in an aqueous solution. The method is based on a 1:1 metal complex formation between tris(8‐hydroxyquinoline‐5‐sulphonate) thulium(III) [Tm(QS)₃] and PPi ion. The linear response covers a concentration range of 1.6 × 10^?7–1.0 × 10^?5 mol/L PPi and the detection limit is 2.3 × 10^?8 mol/L. The association constant of Tm(QS)₃–PPi complex was calculated as 2.6 × 10⁵ mol/L. Tm(QS)₃ shows a selective and sensitive fluorescence enhancement toward PPi ion in comparion with I₃^?, NO₃^?, CN^?, CO₃^2?, Br^?, Cl^?, F^?, H₂PO₄^? and SO₄^2?, which is attributed to higher stability of the inorganic complex between pyrophosphate ion and Tm(QS)₃. Copyright © 2011 John Wiley & Sons, Ltd.     

Development of a novel fluorescent probe for fluorescence correlation spectroscopic detection of kinase inhibitors

We have developed a fluorescently labeled probe for high-throughput screening of kinase inhibitors using fluorescence correlation spectroscopy. With this probe, we have successfully evaluated the inhibitory activities of known inhibitors of a model kinase, ASK1. Because the probe contains a general kinase inhibitor, staurosporine, we believe that this homogeneous, high-throughput, and simple method can be applied to the inhibitor screening of other kinases as well.     

In vivo detection of inducible nitric oxide synthase in rodent gliomas

Increased iNOS expression is often found in brain tumors, such as gliomas. The goal of this study was to develop and assess a novel molecular MRI (mMRI) probe for in vivo detection of iNOS in rodent models for gliomas (intracerebral implantation of rat C6 or RG2 cells or ethyl nitrosourea-induced glioma). The probe we used incorporated a Gd-DTPA (gadolinium(III) complex of diethylenetriamine-N,N,N′,N″,N″-pentaacetate) backbone with albumin and biotin moieties and covalent binding of an anti-iNOS antibody (Ab) to albumin (anti-iNOS probe). We used mMRI with the anti-iNOS probe to detect in vivo iNOS levels in gliomas. Nonimmune normal rat IgG coupled to albumin–Gd-DTPA–biotin was used as a control nonspecific contrast agent. By targeting the biotin component of the anti-iNOS probe with streptavidin Cy3, fluorescence imaging confirmed the specificity of the probe for iNOS in glioma tissue. iNOS levels in glioma tumors were also confirmed via Western blots and immunohistochemistry. The presence of plasma membrane-associated iNOS in glioma cells was established by transmission electron microscopy and gold-labeled anti-iNOS Ab. The more aggressive RG2 glioma was not found to have higher levels of iNOS compared to C6. Differences in glioma vascularization and blood–brain barrier permeability between the C6 and the RG2 gliomas are discussed. In vivo assessment of iNOS levels associated with tumor development is quite feasible in heterogeneous tissues with mMRI.