A highly selective and sensitive in vivo fluorosensor for zinc(II) without cytotoxicity

A highly selective and sensitive fluorescent Zn(2+) sensor, 2,6-bis(2-hydroxy-benzoic acid hydrazide)-4-methylphenol (1), was designed and synthesized. In aqueous THF (4 : 6 v/v) ligand 1 induces a 2 : 1 complex formation with respect to Zn(2+) at physiological pH. This probe features visible light excitation(390 nm) and emission (490 nm) profiles, excellent selectivity responses for Zn(2+)over other competing biological metal ions with K(d) < 1 pM(2), LOD < 1 ng L(-1) and about 680 fold enhancement in fluorescent intensity upon Zn(2+) binding. It also exhibits cell permeability and intracellular Zn(2+) sensing in A375 human melanoma cancer cell.     

An efficient sensor for Zn2+ and Cu2+ based on different binding modes

An efficient sensor for Zn(2+) and Cu(2+) was designed based on different binding modes. The sensor displays ratiometric signals for Zn(2+), due to the Zn(2+)-triggered amide tautomerization; while dual-mode selective behaviors for Cu(2+) result from the deprotonation of the amide tautomer.     

An 2-(2'-aminophenyl)benzoxazole-based OFF-ON fluorescent chemosensor for Zn2+ in aqueous solution

A water-soluble fluorescent sensor, 1, based on the "receptor-spacer-fluorophore" [2-(2'-aminophenyl)benzoxazole-amide-2-picolylamine] sensor platform, demonstrates the high sensitivity for Zn(2+) with a 25-fold fluorescence enhancement upon chelation to Zn(2+) and also exhibits high selectivity to Zn(2+) over other metal ions. X-ray crystal structure of Zn(2+) complex reveals that the amide oxygen (O2) cooperates with 2-picolylamine unit (N3, N4) as a receptor bind Zn(2+).     

High-throughput examination of fluorescence resonance energy transfer-detected metal-ion response in mammalian cells

Fluorescence resonance energy transfer (FRET)-based genetically encoded metal-ion sensors are important tools for studying metal-ion dynamics in live cells. We present a time-resolved microfluidic flow cytometer capable of characterizing the FRET-based dynamic response of metal-ion sensors in mammalian cells at a throughput of 15 cells/s with a time window encompassing a few milliseconds to a few seconds after mixing of cells with exogenous ligands. We have used the instrument to examine the cellular heterogeneity of Zn(2+) and Ca(2+) sensor FRET response amplitudes and demonstrated that the cluster maps of the Zn(2+) sensor FRET changes resolve multiple subpopulations. We have also measured the in vivo sensor response kinetics induced by changes in Zn(2+) and Ca(2+) concentrations. We observed an ～30 fold difference between the extracellular and intracellular sensors.     

Reactions of the fluorescent sensor, Zinquin, with the zinc-proteome: adduct formation and ligand substitution

Zinquin (ZQ) is a commonly used sensor for cellular Zn(2+) status. It has been assumed that it measures accessible Zn(2+) concentrations in the nanomolar range. Instead, this report shows a consistent pattern across seven mammalian cell and tissue types that ZQ reacts with micromolar concentrations of Zn(2+) bound as Zn-proteins. The predominant class of products were ZQ-Zn-protein adducts that were characterized in vivo and in vitro by a fluorescence emission spectrum centered at about 470 nm, by their migration over Sephadex G-75 as protein not low molecular weight species, by the exclusion of reaction with lipid vesicles, and by their large aggregate concentration. In addition, variable, minor formation of Zn(ZQ)(2) with a fluorescence band at about 490 nm was observed in vivo in each case. Because incubation of isolated Zn-proteome with ZQ also generated similar amounts of Zn(ZQ)(2), it was concluded that this species had formed through direct ligand substitution in which ZQ had successfully competed for protein-bound Zn(2+). Parallel studies with the model Zn-proteins, alcohol dehydrogenase (ADH), and alkaline phosphatase (AP) revealed a similar picture of reactivity: ZQ(ACID) (Zinquin acid, (2-methyl-8-p-toluenesulfonamido-6-quinolyloxy)acetate)) able to bind to one Zn(2+) and extract the other in Zn(2)-ADH, whereas it removed one Zn(2+) from Zn(2)-AP and did not bind to the other. Zinquin ethyl ester (ethyl(2-methyl-8-p-toluenesulfonamido-6-quinolyloxy)acetate); ZQ(EE)) bound to both proteins without sequestering Zn(2+) from either one. In contrast to a closely related sensor, 6-methoxy-8-p-toluenesulfonamido-quinoline (TSQ), neither ZQ(ACID) nor ZQ(EE) associated with Zn-carbonic anhydrase. A survey of reactivity of these sensors with partially fractionated Zn-proteome confirmed that ZQ and TSQ bind to distinct, overlapping subsets of the Zn-proteome.     

A differentially selective sensor with fluorescence turn-on response to Zn2+ and dual-mode ratiometric response to Al3+ in aqueous media

A novel quinoline-coumarin (QC) fluoroionophore conjugated by means of a triazolyl-pyrrolidinyl linker exhibits differential dual selectivity for Zn(2+) and Al(3+) in mixed media. QC acts as a turn on fluorescence sensor for Zn(2+) while exhibiting overall ratiometric selectivity for Al(3+) in aqueous media. Moreover, QC exhibited preferential second mode of selectivity for Al(3+) as it ratiometrically displaces Zn(2+) from the [QC + Zn(2+)] complex.     

A highly selective fluorescent sensor for distinguishing cadmium from zinc ions based on a quinoline platform

A fluorescent sensor, N-(quinolin-8-yl)-2-(quinolin-8-yloxy)acetamide (HL), based on 8-aminoquinoline and 8-hydroxyquinoline platforms has been synthesized. This sensor displays high selectivity and sensitive fluorescence enhancement to Cd(2+) in ethanol. Moreover, sensor HL can distinguish Cd(2+) from Zn(2+) via two different sensing mechanisms (photoinduced electron transfer for Cd(2+); internal charge transfer for Zn(2+)). The composition of the complex Cd(2+)/HL or Zn(2+)/L(-) has been found to be 1:1, based on the fluorescence/absorption titration and further confirmed by X-ray crystallography.     

Selective zinc sensor molecules with various affinities for Zn2+, revealing dynamics and regional distribution of synaptically released Zn2+ in hippocampal slices

We have developed a series of fluorescent Zn(2+) sensor molecules with distinct affinities for Zn(2+), because biological Zn(2+) concentrations vary over a wide range from sub-nanomolar to millimolar. The new sensors have K(d) values in the range of 10(-8)-10(-4) M, compared with 2.7 nM for ZnAF-2. They do not fluoresce in the presence of other biologically important metal ions such as calcium or magnesium, and they can detect Zn(2+) within 100 ms. In cultured cells, the fluorescence intensity of ZnAF-2 was saturated at low Zn(2+) concentration, while that of ZnAF-3 (K(d) = 0.79 muM) was not saturated even at relatively high Zn(2+) concentrations. In hippocampal slices, we measured synaptic release of Zn(2+) in response to high-potassium-induced depolarization. ZnAF-2 showed similar levels of fluorescence increase in dentate gyrus (DG), CA3 and CA1, which were indistinguishable. However, ZnAF-3 showed a fluorescence increase only in DG. Thus, by using a combination of sensor molecules, it was demonstrated for the first time that a higher Zn(2+) concentration is released in DG than in CA3 or CA1 and that we can easily visualize Zn(2+) concentration over a wide range. We believe that the use of various combinations of ZnAF family members will offer unprecedented versatility for fluorescence-microscopic imaging of Zn(2+) in biological applications.     

A Zn2+-specific turn-on fluorescent probe for ratiometric sensing of pyrophosphate in both water and blood serum

A novel fluorescent sensor composed of a naphthalene functionalized tetraazamacrocycle ligand 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3-methyl naphthalene (1) and Zn(2+) has been designed and prepared, which can be utilized for selective and ratiometric sensing of pyrophosphate (PPi) over other phosphate-containing anions in aqueous solution at physiological pH. Notably, the water soluble 1 itself also exhibits a selective enhanced fluorescent response to Zn(2+), and the complex 1-Zn(2+) thus formed eventually fulfils the synergic Zn(2+) coordination-altered strategy with PPi. Furthermore, the ratiometric sensing of 1-Zn(2+) towards PPi performed well even in blood serum milieu. Finally, the sensor 1-Zn(2+) was successfully employed to monitor a real-time assay of inorganic pyrophosphatase (PPase) by means of ratiometric fluorescent measurements for the first time.     

A minimal core based fluorophore for selective detection of Zn(II) ions in aqueous solution and living cells

A minimal core based fluorophore was introduced as a selectively fluorescent "turn on" sensor for Zn(2+) ions in aqueous solution. Addition of Zn(2+) ions to the fluorophore generates a significant emission through a 1:1 ligand-to-metal complex. The fluorescence titration experiment of the minimal core based fluorophore with various metal ions shows that the pyromellitic diimide derivative also has the advantage of a high selectivity to Zn(2+) ions over other metals such as Ni(2+), or Co(2+), Cu(2+), Fe(3+), Fe(2+). More than 8 fold increase in the intensity of fluorescence was observed for the Zn(2+)-bound fluorophore compared to Zn-free fluorophore. Due to its small molecular size, the fluorophore was cell-permeable and successfully applied to the detection of Zn(2+) in living cells. With its relatively high sensitivity to Zn(2+) in living cells, the synthesized new fluorophore will be very useful in the studies on various biological functions of Zn(2+).     

Zn2+-triggered excited-state intramolecular proton transfer: a sensitive probe with near-infrared emission from bis(benzoxazole) derivative

Near-infrared (NIR) emission can offer distinct advantages for biological applications. A fluorescent sensor, Zinhbo-1, based on bis(benzoxazole) ligand with 2,2'-dipicolylamine (DPA) as receptor, was synthesized. In aqueous solution, Zinhbo-1 demonstrates high sensitivity and selectivity for sensing Zn(2+) with about 10-fold enhancement and nanomolar sensitivity (K(d) = 0.29 nM). Moreover, sensor Zinhbo-1 can detect Zn(2+) in near-infrared region (over 700 nm) with large Stokes shift (ca. 230 nm) attributing to the Zn(2+)-induced excited state intramolecular proton transfer (ESIPT).     

An excitation ratiometric Zn2+ sensor with mitochondria-targetability for monitoring of mitochondrial Zn2+ release upon different stimulations

A mitochondria-targeted fluorescent sensor (Mito-ST), constructed by integrating a sulfamoylbenzoxadiazole fluorophore with a phosphonium group, displays the specific Zn(2+)-induced hypsochromic shifts of both excitation (69 nm) and emission (35 nm) maxima. Its ratiometric Zn(2+) imaging ability via dual excitation mode has been applied in MCF-7 cells to clarify the different behaviours of mitochondrial Zn(2+) release stimulated by H(2)O(2) and SNOC.     

Fluorescent sensors for Zn(2+) based on a fluorescein platform: synthesis, properties and intracellular distribution

Two new fluorescent sensors for Zn(2+) that utilize fluorescein as a reporting group, Zinpyr-1 and Zinpyr-2, have been synthesized and characterized. Zinpyr-1 is prepared in one step via a Mannich reaction, and Zinpyr-2 is obtained in a multistep synthesis that utilizes 4',5'-fluorescein dicarboxaldehyde as a key intermediate. Both Zinpyr sensors have excitation and emission wavelengths in the visible range ( approximately 500 nm), dissociation constants (K(d1)) for Zn(2+) of <1 nM, quantum yields approaching unity (Phi = approximately 0.9), and cell permeability, making them well-suited for intracellular applications. A 3- to 5-fold fluorescent enhancement is observed under simulated physiological conditions corresponding to the binding of the Zn(2+) cation to the sensor, which inhibits a photoinduced electron transfer (PET) quenching pathway. The X-ray crystal structure of a 2:1 Zn(2+):Zinpyr-1 complex has also been solved, and is the first structurally characterized example of a complex of fluorescein substituted with metal binding ligands.     

Coumarin-derived transformable fluorescent sensor for Zn2+

We report a coumarin-derived fluorescent sensor for Zn(2+) termed CTS. CTS shows excellent binding selectivity for Zn(2+) over competing metal ions due to the transformable ability of CTS, that is the displacement of other metal ions by Zn(2+), which induces transformation of chelation from an amide to an imidic acid tautomeric form.     

ZP4, an improved neuronal Zn2+ sensor of the Zinpyr family

A second-generation fluorescent sensor for Zn(2+) from the Zinpyr family, ZP4, has been synthesized and characterized. ZP4 (Zinpyr-4, 9-(o-carboxyphenyl)-2-chloro-5-[2-(bis(2-pyridylmethyl)aminomethyl)-N-methylaniline]-6-hydroxy-3-xanthanone) is prepared via a convergent synthetic strategy developed from previous studies with these compounds. ZP4, like its predecessors, has excitation and emission wavelengths in the visible range ( approximately 500 nm), a dissociation constant (K(d)) for Zn(2+) of less than 1 nM and a high quantum yields (Phi = approximately 0.4), making it well suited for biological applications. A 5-fold fluorescent enhancement is observed under simulated physiological conditions corresponding to the binding of the Zn(2+) cation to the sensor, which inhibits a photoinduced electron transfer (PET) quenching pathway. The metal-binding stereochemistry of ZP4 was evaluated through the synthesis and X-ray structural characterization of [M(BPAMP)(H(2)O)(n)](+) complexes, where BPAMP is [2-(bis(2-pyridylmethyl)aminomethyl)-N-methylaniline]-phenol and M = Mn(2+), Zn(2+) (n = 1) or Cu(2+) (n = 0).     

Aqueous fluorescence turn-on sensor for Zn2+ with a tetraphenylethylene compound

A new sensitive and selective fluorescence turn-on sensor for Zn(2+) (1) was developed by taking advantage of the aggregation-induced emission of the tetraphenylethylene framework. In addition, the corresponding ester precursor of 1 was successfully used for intracellular Zn(2+) imaging.     

Development of a zinc ion-selective luminescent lanthanide chemosensor for biological applications

Detection of chelatable zinc (Zn(2+)) in biological studies has attracted much attention recently, because chelatable Zn(2+) plays important roles in many biological systems. Lanthanide complexes (Eu(3+), Tb(3+), etc.) have excellent spectroscopic properties for biological applications, such as long luminescence lifetimes of the order of milliseconds, a large Stoke's shift of >200 nm, and high water solubility. Herein, we present the design and synthesis of a novel lanthanide sensor molecule, [Eu-7], for detecting Zn(2+). This europium (Eu(3+)) complex employs a quinolyl ligand as both a chromophore and an acceptor for Zn(2+). Upon addition of Zn(2+) to a solution of [Eu-7], the luminescence of Eu(3+) is strongly enhanced, with high selectivity for Zn(2+) over other biologically relevant metal cations. One of the important advantages of [Eu-7] is that this complex can be excited with longer excitation wavelengths (around 340 nm) as compared with previously reported Zn(2+)-sensitive luminescent lamthanide sensors, whose excitation wavelength is at too high an energy level for biological applications. The usefulness of [Eu-7] for monitoring Zn(2+) changes in living HeLa cells was confirmed. This novel Zn(2+)-selective luminescent lanthanide chemosensor [Eu-7]should be an excellent lead compound for the development of a range of novel luminescent lanthanide chemosensors for biological applications.     

Non-covalently functionalized graphene for the potentiometric sensing of zinc ions

The possibility of the application of non-covalently functionalized graphene as a sensing membrane for the potentiometric determination of zinc ions was examined. A graphene carboxylic derivative was functionalized with 1-(2-pyridylazo)-2-naphthol, the Zn(2+) ions complexing ligand, simply by adsorption of ligand molecules due to π-π interactions. This approach has resulted in a potentiometric sensor characterized with significant selectivity for Zn(2+) ions present in solution.     

1,3-Di-amidoquinoline conjugate of calix[4]arene (L) as a ratiometric and colorimetric sensor for Zn2+: spectroscopy, microscopy and computational studies

Carboxamidoquinoline appended calix[4]arene-1,3-di-conjugate (L) has been synthesized and characterized and its single crystal XRD structure has been established. L has been shown to act as selective ratiometric turn-on fluorescence sensor for Zn(2+) up to a lowest concentration of 183 ± 18 ppb (2.82 μM) with a nine-fold enhancement by exhibiting blue-green emission. The coordination features of the species of recognition have been computationally evaluated by DFT methods and found to have distorted tetrahedral Zn(2+) center in an N(4) core. The spherical nano-structural features observed for L in TEM are being transformed into the Koosh nano-flower like structure when complexed with Zn(2+) and hence these two can be easily differentiated. Even the features observed in AFM can distinctly differentiate L from its Zn(2+) complex.     

ZP8, a neuronal zinc sensor with improved dynamic range; imaging zinc in hippocampal slices with two-photon microscopy

The synthesis of a difluorofluorescein monocarboxaldehyde platform and its use for preparing ZP8, a new member of the Zinpyr family of neuronal Zn(2+) sensors, are described. By combining an aniline photoinduced electron transfer (PET) switch and an electron-withdrawing fluorescein scaffold, ZP8 displays reduced background fluorescence and improved dynamic range compared to previous ZP probes. The bright sensor undergoes an 11-fold increase in fluorescence intensity upon Zn(2+) complexation (Phi = 0.03-0.35) with high selectivity over cellular concentrations of Ca(2+) and Mg(2+). In addition, sensors in the ZP family have been utilized for optical imaging in biological samples using two-photon microscopy (TPM). The cell-permeable ZP3 probe is capable of identifying natural pools of labile Zn(2+) within the mossy fiber synapses of live hippocampal slices using TPM, establishing the application of this technique for monitoring endogenous Zn(2+) stores.