Zinc Probe Research Articles

An experimental and computational study on naphthylideneimine based pH sensitive fluorescence probe for zinc.

Rational design chelating fluorescent sensors probing metal ions in biological system are continuously hot essays nowadays, especially for zinc detection. Herein, a naphthylideneimine based zinc fluorescence probe (3) was prepared and characterized in this work. Structural features and optical properties of 3 and its metallic complexes were characterized. Fluorescent experiment indicates 3 is extremely sensitive and selective for Zn2+ with a strong fluorescence enhancement (∼34 folds) in aqueous buffer solution with a limit of detection (LOD) of 3.78 × 10-7 mol L-1. Formation constant (logKa) of the chelating complex of 3 and Zn2+ ion was determined to be 4.45. Theoretical studies were carried out to get deep insight into the response mechanism in the sensing process. Density functional theory (DFT) methods calculated formation Gibbs free energy (ΔrGmө) of the deprotonated complexes model (32- ⊃ Zn) is-2.9 kcal/mol, which is in good agreement with the experimental result. The calculation results show that the low excitation states can be ascribe to S0 → T2 and S0 → S1 at 390-430 nm and 310-330 nm, respectively, due to the π → π∗ transition. Finally, yeast cell imaging experiments indicate that 3 can monitor intracellular Zn2+ as well. These findings would enable this fluorescent probe to be used as a Zinc sensor.

A benzothiazole-based fluorescent probe for efficient detection and discrimination of Zn2+ and Cd2+, using cysteine as an auxiliary reagent

This paper describes the design and synthesis of a benzothiazole-based fluorescent probe (H1) for zinc and cadmium ion detection and discrimination, using cysteine as an auxiliary reagent. Among the various metal ions tested, zinc and cadmium ions induced blue-shifted emissions of probe H1 from 573 nm to 520 nm and 540 nm, respectively. The addition of cysteine caused the emission of H1-Cd2+ to red-shift back to 573 nm. In contrast, the emission of H1-Zn2+ with the addition of cysteine only decreased without any red-shift of wavelength. Furthermore, confocal laser scanning micrographs of HeLa cells demonstrated satisfactory cell permeability of H1 and selectivity toward zinc and cadmium ions in living cells. Test strip experiments suggest that probe H1 can recognise zinc and cadmium ions together by a remarkable fluorescence change, thus providing a convenient method for tracking zinc and cadmium ions in biological systems.

Characterization of Caco-2 cells stably expressing the protein-based zinc probe eCalwy-5 as a model system for investigating intestinal zinc transport

Intestinal zinc resorption, in particular its regulation and mechanisms, are not yet fully understood. Suitable intestinal cell models are needed to investigate zinc uptake kinetics and the role of labile zinc in enterocytes in vitro. Therefore, a Caco-2 cell clone was produced, stably expressing the genetically encoded zinc biosensor eCalwy-5. The aim of the present study was to reassure the presence of characteristic enterocyte-specific properties in the Caco-2-eCalwy clone. Comparison of Caco-2-WT and Caco-2-eCalwy cells revealed only slight differences regarding subcellular localization of the tight junction protein occludin and alkaline phosphatase activity, which did not affect basic integrity of the intestinal barrier or the characteristic brush border membrane morphology. Furthermore, introduction of the additional zinc-binding protein in Caco-2 cells did not alter mRNA expression of the major intestinal zinc transporters (zip4, zip5, znt-1 and znt-5), but increased metallothionein 1a-expression and cellular resistance to higher zinc concentrations. Moreover, this study examines the effect of sensor expression level on its saturation with zinc. Fluorescence cell imaging indicated considerable intercellular heterogeneity in biosensor-expression. However, FRET-measurements confirmed that these differences in expression levels have no effect on fractional zinc-saturation of the probe.

Quinoline Fluorescent Probes for Zinc - from Diagnostic to Therapeutic Molecules in Treating Neurodegenerative Diseases.

Fluorescent compounds had gained strong attention due to their wide and appealing applications. Microscopic techniques and visualization are good examples among others. Introduction of fluorescent dyes into microbiology opens the possibility to observe tissues, organisms or organelle with exceptional sensitivity and resolution. Probes for detection of biologically relevant metals as zinc, iron or copper seems to be particularly important for drug design and pharmaceutical sciences. Quinoline derivatives are well known for their good metal affinity and wide spectrum of biological activity. In this regard, molecular sensors built on this scaffold may be useful not only as analytical but also as therapeutic agents. In the present review, application of quinoline moiety in designing of novel fluorescent probes for zinc is presented and discussed. Zinc cations are relevant for vast majority of processes and recently attract a great deal of attention for their role in neurodegenerative diseases. Compounds interacting with Zn2+ may be used for early diagnosis of such disorders, for example the Alzheimer disease. Quinoline-based zinc probes may exert some beneficial role in organism acting as theranostic agents. First preliminary drugs for Alzheimer therapy that are based on quinoline moiety are good example of this trend.

Modulation of Zinc Homeostasis in Acanthamoeba castellanii as a Possible Antifungal Strategy against Cryptococcus gattii.

Cryptococcus gattii is a basidiomycetous yeast that can be found in the environment and is one of the agents of cryptococcosis, a life-threatening disease. During its life cycle, cryptococcal cells take hold inside environmental predators such as amoebae. Despite their evolutionary distance, macrophages and amoebae share conserved similar steps of phagocytosis and microbial killing. To evaluate whether amoebae also share other antifungal strategies developed by macrophages, we investigated nutritional immunity against cryptococcal cells. We focused on zinc homeostasis modulation in Acanthamoeba castellanii infected with C. gattii. The intracellular proliferation rate (IPR) in amoebae was determined using C. gattii R265 and mutants for the ZIP1 gene, which displays defects of growth in zinc-limiting conditions. We detected a reduced IPR in cells lacking the ZIP1 gene compared to wild-type strains, suggesting that amoebae produce a low zinc environment to engulfed cells. Furthermore, flow cytometry analysis employing the zinc probe Zinpyr-1 confirmed the reduced concentration of zinc in cryptococcal-infected amoebae. qRT-PCR analysis of zinc transporter-coding genes suggests that zinc export by members of the ZnT family would be involved in the reduced intracellular zinc concentration. These results indicate that amoebae may use nutritional immunity to reduce fungal cell proliferation by reducing zinc availability for the pathogen.

Zinc as an Imaging Biomarker of Prostate Cancer.

Zinc has long been the focus of many biological investigations because of its essential role in biology including a catalytic role in many enzymes, a structural role in the many zinc finger proteins, and a physiological role in many secretory cell processes. Divalent zinc is known to be highly abundant in healthy prostate tissues and lower in prostate cancer (PCa). Given the need for newer diagnostic methods for detection of prostate cancer, zinc-responsive probes of various types have been considered as imaging tools for detecting tissue levels of zinc. Among them, recent zinc-responsive MRI probes show great promise for non-invasive detection of zinc ion secretion from the prostate and other tissues in vivo. In this review, we summarize the need for new diagnostic tools and demonstrate how responsive zinc probes and MRI could satisfy this unmet need.

Selective and sensitive fluorescence “turn-on” Zn2+ probes based on combination of anthracene, diphenylamine and dipyrrin

Two fluorescence “turn-on” Zn2+ probes were developed by introducing an anthracenyl fluorophore through the linkage of a diphenylamino moiety at the 5-position of a dipyrrin moiety. Thus, two compounds with weak fluorescence were designed, synthesized, and employed as CHEF (chelation enhanced fluorescence) type fluorescence “turn-on” Zn2+ probes, which exhibit dramatic fluorescence enhancement upon addition of Zn2+, showing high sensitivities and impressive detection limits of 13 and 12 nM, respectively, better than their analogues containing simple aryl substituents at the 5 positions of a di- or tripyrrin moiety. In addition, both of the probes exhibit good selectivity, short response time of less than 10 s and wide applicable pH ranges. Furthermore, the weak fluorescence nature of the probes was rationalized based on viscosity dependence measurements and theoretical calculations. These results provide further insight into the development of selective and sensitive zinc probes.

Low-Affinity Zinc Sensor Showing Fluorescence Responses with Minimal Artifacts.

The study of the zinc biology requires molecular probes with proper zinc affinity. We developed a low-affinity zinc probe (HBO-ACR) based on an azacrown ether (ACR) and an 2-(2-hydroxyphenyl)benzoxazole (HBO) fluorophore. This probe design imposed positive charge in the vicinity of a zinc coordination center, which enabled fluorescence turn-on responses to high levels of zinc without being affected by the pH and the presence of other transition-metal ions. Steady-state and transient photophysical investigations suggested that such a high tolerance benefits from orchestrated actions of proton-induced nonradiative and zinc-induced radiative control. The zinc bioimaging utility of HBO-ACR has been fully demonstrated with the use of human pancreas epidermoid carcinoma, PANC-1 cells, and rodent hippocampal neurons from cultures and acute brain slices. The results obtained through our studies established the validity of incorporating positively charged ionophores for the creation of low-affinity probes for the visualization of biometals.

Intracellular zinc trafficking and metallothionein gene activation in HUVEC treated with Crotalus atrox venom

Hemorrhagic snake venom, such as Crotalus atrox, destroys local tissue and disrupts hemostasis. In this study, we examined zinc trafficking in human umbilical vein endothelial cells (HUVEC) stimulated with C. atrox snake venom. We utilized MTS cytotoxicity assays to monitor the cytotoxic range of C. atrox venom. HUVEC monolayers stimulated with 10 μg/ml C. atrox venom for 3 hours displayed cellular retraction, which coincided with 53.0±6.5 percent viability. In contrast, venom concentrations of 100 μg/ml produced a complete disruption of cellular adherence and viability decreased to 36.6±1.0. Venom induce cellular retraction involves cytoskeletal rearrangements. Therefore, we used the zinc probe FluorZin‐3AM to compare intracellular free zinc in HUVEC stimulated with 10 μg/ml C. atrox venom or 1 μg/ml cytochalasin D for 1 hour. Fluorescent intensity analysis returned values of 287.1±57.7 for C. atrox and 261.7±33.3 for cytochalasin D, demonstrating an increase in labile zinc compared to non‐stimulated controls. Labile intracellular zinc or other metals can induce the gene activation of intracellular metal binding proteins known as metallothioneins. Thus, we measured the gene and protein expression of metallothionein 1X (MT1X) and metallothionein 2A (MT2A) in HUVEC stimulated with C. atrox venom. MT2A gene expression increase by 4.4±0.41 fold compared to 96.1±3.0 for MT1X. This data suggest cell death from C. atrox venom involves intracellular zinc trafficking, and metallothionein gene activation.Support or Funding InformationCETL, OVPR Grant

Combination of pyrrole and pyridine for constructing selective and sensitive Zn2+ probes

The selective and sensitive detection of zinc ions has been an important research topic because of the vital role played by zinc ions in numerous physiological activities, and fluorescent probes have emerged as effective and powerful tools for zinc detection because of their simplicity andcereal-time monitoring nature. In this work, with the purpose to improve the binding affinities and sensitivities of dipyrrin based fluorescent Zn2+ probes, strongly coordinating pyridyl moieties were introduced into dipyrrins. Aroylation of 5-pentafluorophenyl and 5-unsubstituted dipyrromethanes with picolinoyl chloride afforded two corresponding dipicolinoyl dipyrromethanes, which were further oxidized with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) to afford the expected 5-pentafluorophenyl-substituted dipicolinoyl dipyrrin and a unique unexpected dipicolinoyl dipyrrolyl ketone, respectively. Both of these compounds exhibited noticeable fluorescence “turn-on” responses specifically to Zn2+ over other metal cations, with fluorescence enhancements of 87 fold and 119 fold, respectively. However, because of the different substituents at the 5 positions and the different conjugation frameworks, distinct Zn2+ sensing behavior was observed. For the 5-pentafluorophenyl-substituted dipicolinoyl dipyrrin, the sensing of Zn2+ induced a color change from light orange to pink accompanied with enhanced red fluorescence. Whereas, the sensing of Zn2+ by the dipicolinoyl dipyrrolyl ketone induced the color change from nearly colorless to bright yellow and enhanced green fluorescence. Based on high binding affinities and the dramatic spectral responses, both of the probes exhibit high sensitivities towards Zn2+ with detection limits of 9.8 nM and 6.3 nM, respectively. In addition, fast responses within 10 s and wide applicable pH ranges are indicative of their potential applications as promising zinc probes. Finally, the 5-pentafluorophenyl dipicolinoyl dipyrrin was successfully applied to cell imaging in Hela cells, demonstrating its practical applicability. It is noteworthy that the single crystal structure of a Zn2+ complex of the dipicolinoyl dipyrrolyl ketone was successfully analyzed by X-ray diffraction, which revealed an interesting tetranuclear Zn2+ coordination structure. These results provide further insights into the design of highly sensitive Zn2+ probes by introducing pyridyl moieties into dipyrrin platforms.

Zinc supplementation augments TGF‐β1‐dependent regulatory T cell induction

Regulatory T cells (Treg) play a pivotal role in immune regulation. For proper immune function, also trace elements such as zinc, and anti-inflammatory cytokines, including transforming growth factor beta 1 (TGF-β1) and interleukin (IL)-10 are indispensable. Hence, in this study the influence of TGF-β1, IL-10, and zinc supplementation on Treg cells differentiation was investigated. A synergistic effect of a combined zinc and TGF-β1 treatment on Foxp3 expression in peripheral blood mononuclear cells and mixed lymphocyte cultures (MLC) was found by performing Western blot analysis. Additionally, combined treatment causes elevated Smad 2/3 phosphorylation, which plays an important role in Foxp3 expression. This is due to a TGF-β1-mediated increase of intracellular-free zinc measured by zinc probes Fluozin3-AM and ZinPyr-1. Moreover, zinc as well as TGF-β1 treatment caused significantly reduced interferon (IFN)-γ secretion in MLC. Combined zinc and TGF-β1 treatment provoked an increased Treg cell induction due to a triggered intracellular zinc signal, which in association with an increased Smad 2/3 activation leads to a boosted Foxp3 expression and resulting in an ameliorated allogeneic reaction in MLC. Thus, zinc can be used as a favorable additive to elevate the induction of Treg cells in adverse immune reactions.

Development of an Optical Zn2+ Probe Based on a Single Fluorescent Protein

Various fluorescent probes have been developed to reveal the biological functions of intracellular labile Zn2+. Here, we present Green Zinc Probe (GZnP), a novel genetically encoded Zn2+ sensor design based on a single fluorescent protein (single-FP). The GZnP sensor is generated by attaching two zinc fingers (ZF) of the transcription factor Zap1 (ZF1 and ZF2) to the two ends of a circularly permuted green fluorescent protein (cpGFP). Formation of ZF folds induces interaction between the two ZFs, which induces a change in the cpGFP conformation, leading to an increase in fluorescence. A small sensor library is created to include mutations in the ZFs, cpGFP and linkers between ZF and cpGFP to improve signal stability, sensor brightness and dynamic range based on rational protein engineering, and computational design by Rosetta. Using a cell-based library screen, we identify sensor GZnP1, which demonstrates a stable maximum signal, decent brightness (QY = 0.42 at apo state), as well as specific and sensitive response to Zn2+ in HeLa cells (Fmax/Fmin = 2.6, Kd = 58 pM, pH 7.4). The subcellular localizing sensors mito-GZnP1 (in mitochondria matrix) and Lck-GZnP1 (on plasma membrane) display sensitivity to Zn2+ (Fmax/Fmin = 2.2). This sensor design provides freedom to be used in combination with other optical indicators and optogenetic tools for simultaneous imaging and advancing our understanding of cellular Zn2+ function.

Effects of Zinc Chelators on Aflatoxin Production in Aspergillus parasiticus.

Zinc concentrations strongly influence aflatoxin accumulation in laboratory media and in food and feed crops. The presence of zinc stimulates aflatoxin production, and the absence of zinc impedes toxin production. Initial studies that suggested a link between zinc and aflatoxin biosynthesis were presented in the 1970s. In the present study, we utilized two zinc chelators, N,N,N′,N′-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN) and 2,3-dimercapto-1-propanesulfonic acid (DMPS) to explore the effect of zinc limitation on aflatoxin synthesis in Aspergillus parasiticus. TPEN but not DMPS decreased aflatoxin biosynthesis up to six-fold depending on whether A. parasiticus was grown on rich or minimal medium. Although we observed significant inhibition of aflatoxin production by TPEN, no detectable changes were observed in expression levels of the aflatoxin pathway gene ver-1 and the zinc binuclear cluster transcription factor, AflR. Treatment of growing A. parasiticus solid culture with a fluorescent zinc probe demonstrated an increase in intracellular zinc levels assessed by increases in fluorescent intensity of cultures treated with TPEN compared to controls. These data suggest that TPEN binds to cytoplasmic zinc therefore limiting fungal access to zinc. To investigate the efficacy of TPEN on food and feed crops, we found that TPEN effectively decreases aflatoxin accumulation on peanut medium but not in a sunflower seeds-derived medium. From an application perspective, these data provide the basis for biological differences that exist in the efficacy of different zinc chelators in various food and feed crops frequently contaminated by aflatoxin.

Zinc and diabetes

Zn2+ ions are essential for the normal processing and storage of insulin and altered pancreatic insulin content is associated with all forms of diabetes mellitus. Work of the past decade has identified variants in the human SLC30A8 gene, encoding the zinc transporter ZnT8 which is expressed highly selectively on the secretory granule of pancreatic islet β and α cells, as affecting the risk of Type 2 Diabetes. Here, we review the regulation and roles of Zn2+ ions in islet cells, the mechanisms through which SLC30A8 variants might affect glucose homeostasis and diabetes risk, and the novel technologies including recombinant targeted zinc probes and knockout mice which have been developed to explore these questions.

Revised stability constant, spectroscopic properties and binding mode of Zn(II) to FluoZin-3, the most common zinc probe in life sciences

2-[2-[2-[2-[bis(carboxylatomethyl)amino]-5-methoxyphenoxy]ethoxy]-4-(2,7-difluoro-3-oxido-6-oxo-4a,9a-dihydroxanthen-9-yl)anilino]acetate (FluoZin-3) is used very broadly in life sciences as intra- and extracellular Zn(II) sensor selective for Zn(II) over Co(II), Ca(II) and Mg(II) ions at their physiological concentrations. It has been used for determination of relative and absolute levels of exchangeable Zn(II) in cells and extracellular fluids. Despite its popularity, the knowledge of its acid/base and Zn(II) coordination abilities and of its spectroscopic properties remained very limited. Also the published conditional dissociation constant (CKd) values at pH7.4 are slightly discrepant, (15nM or 8.9nM). In this work we determined the CKd for Zn(II) complexation by FluoZin-3 at pH7.4 with nitrilotriacetic acid (NTA) as competitor using two independent methods: fluorimetry and UV–Vis spectroscopy. For the first time, we investigated FluoZin-3 alone and complexed with Zn(II) in the wide range of pH, determining the total of eight pKa values from fluorescence spectra and from various regions of UV–Vis spectra. The validated values of CKd (9.1±0.4nM; −log CKd=8.04) and of the absolute (pH-independent) stability constant log βZnL (8.16±0.05) were provided by fluorescence spectroscopy experiments performed at 1μM concentrations. Our experiments demonstrated that both of aminocarboxylate moieties of FluoZin-3 bind the Zn(II) ion synergistically.

Reaction-Based Probes for Imaging Mobile Zinc in Live Cells and Tissues.

Chelatable, or mobile, forms of zinc play critical signaling roles in numerous biological processes. Elucidating the action of mobile Zn(II) in complex biological environments requires sensitive tools for visualizing, tracking, and manipulating Zn(II) ions. A large toolbox of synthetic photoinduced electron transfer (PET)-based fluorescent Zn(II) sensors are available, but the applicability of many of these probes is limited by poor zinc sensitivity and low dynamic ranges owing to proton interference. We present here a general approach for acetylating PET-based probes containing a variety of fluorophores and zinc-binding units. The new sensors provide substantially improved zinc sensitivity and allow for incubation of live cells and tissue slices with nM probe concentrations, a significant improvement compared to the μM concentrations that are typically required for a measurable fluorescence signal. Acetylation effectively reduces or completely quenches background fluorescence in the metal-free sensor. Binding of Zn(II) selectively and quickly mediates hydrolytic cleavage of the acetyl groups, providing a large fluorescence response. An acetylated blue coumarin-based sensor was used to carry out detailed analyses of metal binding and metal-promoted acetyl hydrolysis. Acetylated benzoresorufin-based red-emitting probes with different zinc-binding sites are effective for sensing Zn(II) ions in live cells when applied at low concentrations (∼50–100 nM). We used green diacetylated Zinpyr1 (DA-ZP1) to image endogenous mobile Zn(II) in the molecular layer of mouse dorsal cochlear nucleus (DCN), confirming that acetylation is a suitable approach for preparing sensors that are highly specific and sensitive to mobile zinc in biological systems.

Phosphorescent Zinc Probe for Reversible Turn-On Detection with Bathochromically Shifted Emission.

Phosphorescent molecules are attractive complements to fluorescent compounds for bioimaging. Time-gated acquisition of the long-lived phosphorescence signals provides an effective means to eliminate unwanted background noises due to short-lived autofluorescence. We have previously investigated the molecular principles governing modulation of photoinduced electron transfer in phosphorescence zinc probes that were based on biscyclometalated Ir(III) complexes (Woo, H. et al. J. Am. Chem. Soc. 2013, 135, 4771-4787). The studies established that phosphorescence turn-on responses would be attainable for Ir(III) complexes with high triplet-state energies. This sets an upper limit to an emission wavelength, restricting the development of red- or near-IR-phosphorescence turn-on probes. To address this challenge, we designed and synthesized a new phosphorescent probe having an electron-deficient 2-(2-pyridyl)pyrazine diimine ligand tethering a di(2-picolyl)amine (DPA) zinc receptor. This ligand control led to red phosphorescence emission (λ(ems) = 596 nm), with an excited-state reduction potential (E*(red)) retained as high as 1.44 V versus standard calomel electrode (SCE). The E*(red) value was more positive than the ground-state oxidation potential of DPA (1.05 V vs SCE), permitting an occurrence of photoinduced electron transfer at a rate of 2 × 10(7) s(-1). Zinc binding at DPA abolished the electron transfer to produce phosphorescence turn-on signaling. The probe was capable of detecting zinc ions selectively over other competing biological metal ions in aqueous buffer solutions (pH 7.4, 20 mM piperazine-N,N'-bis(2-ethanesulfonic aid)) with the zinc dissociation constant of 109 pM. Finally, bioimaging utility of the probe has been successfully demonstrated by visualizing exogenously supplied zinc ions in live HeLa cells. The research described in this paper demonstrates that judicious ligand control enables retention of turn-on responses in the low-energy phosphorescence region.

Fluorescent carbon dots for sensitive determination and intracellular imaging of zinc(II) ion

We describe the preparation of carbon dots (CDs) from glucose that possess high stability, a quantum yield of 0.32, and low toxicity (according to an MTT assay). They were used, in combination with the fluorogenic zinc(II) probe quercetin to establish a fluorescence resonance energy transfer (FRET) system for the determination of Zn(II). The CDs are acting as the donor, and the quercetin-Zn(II) complex as the acceptor. This is possible because of the strong overlap between the fluorescence spectrum of CDs and the absorption spectrum of the complex. The method enables Zn(II) to be determined in the 2 to 100 μM concentration range, with a 2 μM detection limit. The method was applied to image the distribution of Zn(II) ions in HeLa cells.

Responsive mechanism of 2-(2′-hydroxyphenyl)benzoxazole-based two-photon fluorescent probes for zinc and hydroxide ions**Project supported by the National Basic Research Program of China (Grant No. 2011CB808105) and the National Natural Science Foundation of China (Grant No. 21303096).

Response theory is used to investigate one- and two-photon absorption (TPA) as well as the emission properties of a series of potential zinc ion and pH sensitive materials containing 2-(2′-hydroxyphenyl)benzoxazole (HPBO) end groups. Special emphasis is placed on the evolution of their optical properties upon combining with zinc ions or deprotonation. Our calculated results indicate that upon combining with zinc ions or deprotonation, these HPBO derivatives show drastic changes in their one-photon absorption (OPA), emission, and TPA properties. Moreover, the mechanisms of the probes are analyzed and found to be an intramolecular charge transfer. These compounds are thus proved to be excellent candidates for two-photon fluorescent zinc and pH probes.

An efficient vanillinyl Schiff base as a turn on fluorescent probe for zinc(ii) and cell imaging

Vanillinyl Schiff base (H2L), a nontoxic probe and a fluorescent sensor to Zn2+, LOD 0.018 μM is used for living cell imaging.