A highly sensitive fluorescent probe for tracking intracellular zinc ions and direct imaging of prostatic tissue in mice

A highly sensitive fluorescent sensor ZnDN was designed, synthesized and used for tracking intracellular zinc ions in various living cells and direct imaging of prostatic tissue in mice. ZnDN was prepared from the heterocyclic-fused naphthalimide fluorophore, and the zinc receptor, N,N-bis(2-pyridylmethyl)ethyl-enediamine (BPEN). Upon addition of Zn²⁺ to the solutions of ZnDN, a remarkable fluorescence enhancement was observed, which could be attributed to the photo-induced electron transfer (PET) mechanism. Since ZnDN exhibited high sensitivity toward Zn²⁺ in phosphate buffer solution, with a limit of detection of 4.0×10^-9 mol/L, it was further applied for the imaging of exogenous and endogenous Zn²⁺ in different living cells. Living cells imaging experiments suggested that ZnDN could image the changes of intracellular free zinc ions, and could be used for two-photon imaging. Moreover, flow cytometry suggested that ZnDN could distinguish cancerous prostate cells from normal cells. Animal experiments indicated that ZnDN had the potential in imaging prostate tissue in vivo.     

A quinoline-based selective ‘turn on’ chemosensor for zinc(II) via quad-core complex,and its application in live cell imaging

An efficient quinoline-based fluorescent chemosensor (QLNPY) was successfully developed for the detection of zinc ions (Zn²⁺). This novel chemosensor displayed higher sensitivity and selectivity toward Zn²⁺ over other competitive metal ions accompanying with obvious fluorescence enhancement. The QLNPY-Zn²⁺ complex can be further used as a new fluorescent “turn-off” sensor for pyrophosphate (PPi) and sulfur ion (S^2?) via a Zn²⁺ displacement approach. The limits of detection were calculated to be 3.8 × 10^?8 M for Zn²⁺, 3.7 × 10^?7 M for PPi and 4.9 × 10^?7 M for S^2?. The binding mechanism of QLNPY and Zn²⁺ was investigated through NMR, HR-MS analysis and further studied by crystallographic analysis. Additionally, further application of QLNPY for sequential bioimaging of Zn²⁺ and PPi was studied in HepG2 cells, suggesting that the quinoline-based chemosensor possesses great potential applications for the detection of intracellular Zn²⁺ and PPi in vivo.     

Gadolinium(III)–fluorescein complex as a dual modal probe for MRI and fluorescence zinc sensing

A bimodal imaging Gd–Zpy probe based on magnetic resonance imaging and fluorescence sensing has been synthesized and characterized. Gd–Zpy features a bright green emission and a turn-on fluorescent response manner with high sensitivity for Zn²⁺ in aqueous solution and is able to luminescent imaging intracellular Zn²⁺ levels within living cells. It exhibits a 130% increase of the longitudinal relaxation time and a 115% increase of transverse relaxive time upon addition of Zn²⁺. The results demonstrated that the incorporating of the fluorescein dye having the efficient chelators within a high-spin Gd³⁺ system was a powerful approach to achieve dual modal probes for MRI and fluorescence sensing.     

Design and Synthesis of a Highly Sensitive Off–On Fluorescent Chemosensor for Zinc Ions Utilizing Internal Charge Transfer

Fluorescence imaging is a powerful tool for the visualization of biological molecules in living cells, tissue slices, and whole bodies, and is important for elucidating biological phenomena. Furthermore, zinc (Zn²⁺) is the second most abundant heavy metal ion in the human body after iron, and detection of chelatable Zn²⁺ in biological studies has attracted much attention. Herein, we present a novel, highly sensitive off–on fluorescent chemosensor for Zn²⁺ by using the internal charge transfer (ICT) mechanism. The rationale of our approach to highly sensitive sensor molecules is as follows. If fluorescence can be completely quenched in the absence of Zn²⁺, chemosensors would offer a better signal‐to‐noise ratio. However, it is difficult to quench the fluorescence completely before Zn²⁺ binding, and most sensor molecules still show very weak fluorescence in the absence of Zn²⁺. But even though the sensor shows a weak fluorescence in the absence of Zn²⁺, this fluorescence can be further suppressed by selecting an excitation wavelength that is barely absorbed by the Zn²⁺‐free sensor molecule. Focusing on careful control of ICT within the 4‐amino‐1,8‐naphthalimide dye platform, we designed and synthesized a new chemosensor ( 1 ) that shows a pronounced fluorescence enhancement with a blueshift in the absorption spectrum upon addition of Zn²⁺. The usefulness of 1 for monitoring Zn²⁺ changes was confirmed in living HeLa cells. There have been several reports on 4‐amino‐1,8‐naphthalimide‐based fluorescent sensor molecules. However, 1 is the first Zn²⁺‐sensitive off–on fluorescent sensor molecule that employs the ICT mechanism; most off–on sensor molecules for Zn²⁺ employ the photoinduced electron transfer (PeT) mechanism.     

A water-soluble,small molecular fluorescence probe based on 2-(2′-hydroxyphenyl) benzoxazole for Zn2+ in plants

A water-soluble, small molecular zinc fluorescence probe (ZFP) based on 2-(2′-hydroxyphenyl) benzoxazole was prepared. It exhibited high selectivity and sensitivity to Zn²⁺ than the other metal ions. The highest fluorescence enhancement was observed in the presence of Zn²⁺ owing to the inhibition of excited-state intramolecular proton transfer (ESIPT). Furthermore, fluorescence imaging experiments confirmed that ZFP can be used to monitor Zn²⁺ in biological systems.     

Two‐Photon Probes for Zn2+ Ions with Various Dissociation Constants. Detection of Zn2+ Ions in Live Cells and Tissues by Two‐Photon Microscopy

A series of Zn²⁺‐selective two‐photon fluorescent probes (AZnM1−AZnN) that had a wide range of dissociation constants (K_d^TP=8 nm‐ 12 μM ) were synthesized. These probes showed appreciable water solubility (>3 μM ), cell permeability, high photostability, pH insensitivity at pH>7, significant two‐photon action cross‐sections (86–110 GM) upon complexation with Zn²⁺, and can detect the Zn²⁺ ions in HeLa cells and in living tissue slices of rat hippocampal at a depth of >80 μm without mistargeting and photobleaching problems. These probes can potentially find application in the detection of various amounts of Zn²⁺ ions in live cells and intact tissues.     

Circular Polarized Light Activated Chiral Satellite Nanoprobes for the Imaging and Analysis of Multiple Metal Ions in Living Cells

Here, we construct a handedness‐dependent circular polarized light (CPL)‐activated chiral satellite assemblies formed from DNAzymes and spiny platinum modified with gold nanorods and upconversion nanoparticles (UCNPs), enabling the simultaneous quantitative analysis of multiple divalent metal ions in living cells. The chiral nanoprobes, in coordination with their corresponding divalent metal ions under 980 nm left circular polarized (LCP) light illumination, served as an in situ confocal bioimaging platform for the quantitation of the given intracellular metal ions. The limit of detection (LOD) of the chiral probes in living cells is 1.1 nmol/10⁶ cells, 1.02 nmol/10⁶ cells and 0.45 nmol/10⁶ cells for Zn²⁺, Mg²⁺, and Cu²⁺, respectively.     

A far-red emitting probe for unambiguous detection of mobile zinc in acidic vesicles and deep tissue

Imaging mobile zinc in acidic environments remains challenging because most small-molecule optical probes display pH-dependent fluorescence. Here we report a reaction-based sensor that detects mobile zinc unambiguously at low pH. The sensor responds reversibly and with a large dynamic range to exogenously applied Zn²⁺ in lysosomes of HeLa cells, endogenous Zn²⁺ in insulin granules of MIN6 cells, and zinc-rich mossy fiber boutons in hippocampal tissue from mice. This long-wavelength probe is compatible with the green-fluorescent protein, enabling multicolor imaging, and facilitates visualization of mossy fiber boutons at depths of >100 μm, as demonstrated by studies in live tissue employing two-photon microscopy.     

A FRET-based fluorescent Zn2+ sensor: 3D ratiometric imaging,flow cytometric tracking and cisplatin-induced Zn2+ fluctuation monitoring

Monitoring labile Zn²⁺ homeostasis is of great importance for the study of physiological functions of Zn²⁺ in biological systems. Here we report a novel ratiometric fluorescent Zn²⁺ sensor, CPBT, which was constructed based on chelation-induced alteration of FRET efficiency. CPBT was readily cell membrane permeable and showed a slight preferential localization in the endoplasmic reticulum. With this sensor, 3D ratiometric Zn²⁺ imaging was first realized in the head of zebra fish larvae via Z-stack mode. CPBT could track labile Zn²⁺ in a large number of cells through ratiometric flow cytometric assay. More interestingly, both ratiometric fluorescence imaging and flow cytometric assay demonstrated that the labile Zn²⁺ level in MCF-7 cells (cisplatin-sensitive) decreased while that in SKOV3 cells (cisplatin-insensitive) increased after cisplatin treatment, indicating that Zn²⁺ may play an important role in cisplatin induced signaling pathways in these cancer cells.

A Zn²⁺ sensor exhibiting 3D ratiometric imaging and flow cytometric ability was constructed based on the FRET mechanism, and cisplatin-induced endogenous labile Zn²⁺ fluctuations were monitored in real time.     

Carbazole-driven ratiometric fluorescence turn on for dual ion recognition of Zn2+ and Hg2+ by thiophene-pyridyl conjugate in HEPES buffer medium: spectroscopy,computational, microscopy and cellular studies

Carbazole-based thiophene-pyridyl conjugate (L) was synthesised and characterised. The complexation between L with Zn²⁺ and Hg²⁺ was studied in HEPES buffer medium by fluorescence, absorption and visual colour change with the detection limit of ~3.7 and ~4.8 μM, respectively. The L detects Zn²⁺ by bringing ratiometric change in the fluorescence signals at 418 and 515 nm, but in the case of Hg²⁺, the signals are observed at 418 and 365 nm, while no new band is observed at 515 nm. The structure of L has been established by single-crystal XRD and that of complexes [ZnL] and [HgL] by density functional theory calculations. TDDFT calculations were performed in order to demonstrate the electronic properties of receptors and their zinc and mercury complexes. The isolated fluorescent complexes [ZnL] and [HgL] were found to be sensitive and selective towards phosphate-bearing ions and sulfide ions, respectively, among the other anions studied. The nanostructural features such as shape and size obtained using atomic force microscopy distinguish L from its complexes formed between L and Zn²⁺ from that formed with Hg²⁺. Moreover, the utility of the conjugate L in showing the zinc recognition in live cells has also been demonstrated using RAW cells as monitored by fluorescence imaging.     

Pre-potassiated hydrated vanadium oxide as cathode for quasi-solid-state zinc-ion battery

Zinc-ion batteries（ZIBs）, in particular quasi-solid-state ZIBs, occupy a crucial position in the field of energy storage devices owing to the superiorities of abundant zinc reserve, low cost, high safety and high theoretical capacity of zinc anode. However, as divalent Zn²⁺ions experience strong electrostatic interactions when intercalating into the cathode materials, which poses challenges to the structural stability and higher demand in Zn²⁺ions diffusion kinetics of the ...     

A new turn-on fluorescence probe for Zn in aqueous solution and imaging application in living cells

We designed and synthesized a new pyrazoline-based turn-on fluorescence probe for Zn²⁺ by the reaction of chalcone and thiosemicarbazide. The structure of the probe was characterized by IR, NMR and HRMS spectroscopy. The probe (L) exhibits high selectivity and sensitivity for detecting Zn²⁺ in buffered EtOH/HEPES solution (EtOH/HEPES = 1/1, pH 7.2) with 80-fold fluorescence enhancement, which is superior to previous reports. Job’s plot analysis revealed 1:1 stoichiometry between probe L and Zn²⁺ ions. The association constant estimated by the Benesi–Hildebrand method and the detection limit were 3.92 × 10³ M⁻¹ and 5.2 × 10⁻⁷ M, respectively. A proposed binding mode was confirmed by ¹H NMR titration experiments and density functional theory (DFT) calculations. The probe is cell-permeable and stable at the physiological pH range in biological systems. Because of its fast response to Zn²⁺, the probe can monitor Zn²⁺ in living cells. Moreover, the selective binding of L and Zn²⁺ was reversible with the addition of EDTA in buffered EtOH/HEPES solution and Zn²⁺ could be imaged in SH-SY5Y neuron cells.     

Subcellular localised small molecule fluorescent probes to image mobile Zn2+

Zn²⁺, as the second most abundant d-block metal in the human body, plays an important role in a wide range of biological processes, and the dysfunction of its homeostasis is related to many diseases, including Type 2 diabetes, Alzheimer''s disease and prostate and breast cancers. Small molecule fluorescent probes, as effective tools for real-time imaging, have been widely used to study Zn²⁺ related processes. However, the failure to control their localisation in cells has limited their utility somewhat, as they are generally incapable of studying individual processes in a specific cellular location. This perspective presents an overview of the recent developments in specific organelle localised small molecule fluorescent Zn²⁺ probes and their application in biological milieu, which could help to extend our understanding of the mechanisms that cells use to respond to dysfunction of zinc homeostasis and its roles in disease initiation and development.

A number of recently developed subcellular localised small molecule fluorescent probes to image mobile Zn²⁺ are reviewed in this perspective.     

A turn-on fluorescent probe for Au3+ based on rodamine derivative and its bioimaging application

A new fluorescent probe RY was synthesized for the detection of Au³⁺ ions based on a rhodamine B derivative. The fluorescent probe showed good selectivity and sensitivity to Au³⁺ ions. Obvious color and fluorescence changes could be observed with the naked eye while the fluorescent probe reacted with the Au³⁺ ions. The detection limit of the probe was determined to be 36 ppb by the fluorescence titration; the excellent linear relationship suggests that the probe is potentially useful for quantitative detection of Au³⁺ in vitro. We also demonstrated its bioimaging application in both living cells and mice; this was the first time that a fluorescent probe was successfully applied to imaging Au³⁺ in living animals.     

Interaction of angiotensin peptides and zinc metal ions probed by electrospray ionization mass spectrometry

Electrospray ionization-tandem mass spectrometry experiments were used to provide evidence regarding the sites of interactions between zinc metal ions and angiotensin peptides. The electrospray ionization mass spectra of histidine-containing human angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) and angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) in the presence of zinc show abundant multiply charged ions for the zinc-attached peptide [M + aZn²⁺ +(c ? 2a)H⁺]^c+, where a = 1, 2 and c is charge. From collisionally activated dissociation experiments, with both low energy (triple quadrupole mass spectrometry) and high energy collisions (linked scan at constant B/E with a double focusing instrument) of the [M + Zn]²⁺ and [M + Zn + H]³⁺ ions for angiotensin II, a [b ₆ + Zn]²⁺ species is produced as the most abundant product ion, suggesting that the zinc interaction site is in the vicinity of the His⁶ residue. Additionally, tandem mass spectra from the zinc-attached ions for angiotensin I show abundant [b ₆ + Zn]²⁺ and [b ₉ + Zn]²⁺ products, providing evidence that both His⁶ and His⁹ are involved in zinc coordination.     

Development of Solid Contact Micropipette Zn-Ion Selective Electrode for Corrosion Studies

Micron-size ion selective micropipettes can be used in scanning electrochemical microscopy (SECM). They can provide excellent spatial resolution. Unfortunately the resistance of these small sensors is high. Their application needs special shielding and slow scanning rates. Usually their lifetime hardly exceeds a few days.

Zinc layer or dispersed zinc particles containing films are often used for providing cathodic protection against corrosion in case of metal surfaces. Therefore, in corrosion studies, measurements of local zinc ion concentration can give important information about the nature of the process. For corrosion studies we needed SECM measuring tips for imaging concentration profiles of Zn²⁺ions involved in surface processes. Based on our earlier experience, solid contact micropipettes for selective measurements of Zn²⁺ion concentration were prepared with a tip size of a few micrometers. The properties of the micropipettes were investigated. They were also used in SECM imaging. In this paper, details of Zn²⁺ion selective microelectrode preparation are described. Data about their properties, lifetime, resistance, and ion activity response are shown. Preliminary findings in SECM imaging of zinc ion concentration profiles are shown. The improvement of the scanning rate achieved by lowering tip resistance is a main advantage in potentiometric SECM.     

基于苯并噻唑Zn2+荧光探针及其细胞造影应用

设计合成基于苯并噻唑Zn²⁺荧光增强型探针BHP,在HEPES缓冲液中测其对Zn²⁺识别性能。实验结果表明,BHP对Zn²⁺有较高的选择性,对其他金属离子如Cd²⁺,Fe²⁺,Ni²⁺,Pb²⁺,Hg²⁺,Al³⁺,Mn²⁺,Ag⁺,Cu²⁺,Co²⁺,Na⁺,K⁺,Mg²⁺和Ca²⁺无明显荧光增强响应。BHP与Zn²⁺按1：1计量比配位,在生理条件下荧光强度不受pH值影响。在HeLa细胞中对Zn²⁺的造影表明BHP可用于生物体Zn²⁺检测。     

Fluorescent sensors based on controllable conformational change for discrimination of Zn2+ over Cd2+

A new coumarin-derived imine IC1 was designed for highly selective sensing of Zn²⁺ over Cd²⁺ ions in aqueous solution based on a controllable CN isomerization mechanism. Methyl as a fine controllable unit was incorporated into IC1 to tune the conformational change of imine and thus significantly improve its selectivity to Zn²⁺ compared with methyl-free imine IC2. Sensor IC1 was also utilized to image intracellular Zn²⁺ ions in HepG2 cells with a good performance.     

Binding of the Zn2+ ion to ferric uptake regulation protein from E. coli and the competition with Fe2+ binding: a molecular modeling study of the effect on DNA binding and conformational changes of Fur

The three dimensional structure of Ferric uptake regulation protein dimer from E. coli, determined by molecular modeling, was docked on a DNA fragment (iron box) and Zn²⁺ ions were added in two steps. The first step involved the binding of one Zn²⁺ ion to what is known as the zinc site which consists of the residues Cys 92, Cys 95, Asp 137, Asp141, Arg139, Glu 140, His 145 and His 143 with an average metal-Nitrogen distance of 2.5 Å and metal-oxygen distance of 3.1–3.2 Å. The second Zn²⁺ ion is bound to the iron activating site formed from the residues Ile 50, His 71, Asn 72, Gly 97, Asp 105 and Ala 109. The binding of the second Zn²⁺ ion strengthened the binding of the first ion as indicated by the shortening of the zinc-residue distances. Fe²⁺, when added to the complex consisting of 2Zn²⁺/Fur dimer/DNA, replaced the Zn²⁺ ion in the zinc site and when a second Fe²⁺ was added, it replaced the second zinc ion in the iron activating site. The binding of both zinc and iron ions induced a similar change in Fur conformations, but shifted residues closer to DNA in a different manner. This is discussed along with a possible role for the Zn²⁺ ion in the Fur dimer binding of DNA in its repressor activity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A Microporous Anionic Metal–Organic Framework for Sensing Luminescence of Lanthanide(III) Ions and Selective Absorption of Dyes by Ionic Exchange

Herein, a novel anionic framework with primitive centered cubic (pcu) topology, [(CH₃)₂NH₂]₄[(Zn₄dttz₆)Zn₃]?15 DMF?4.5 H₂O, ( IFMC‐2 ; H₃dttz=4,5‐di(1H‐tetrazol‐5‐yl)‐2H‐1,2,3‐triazole) was solvothermally isolated. A new example of a tetranuclear zinc cluster {Zn₄dttz₆} served as a secondary building unit in IFMC‐2 . Furthermore, the metal cluster was connected by Zn^II ions to give rise to a 3D open microporous structure. The lanthanide(III)‐loaded metal–organic framework (MOF) materials Ln³⁺@IFMC‐2 , were successfully prepared by using ion‐exchange experiments owing to the anionic framework of IFMC‐2 . Moreover, the emission spectra of the as‐prepared Ln³⁺@IFMC‐2 were investigated, and the results suggested that IFMC‐2 could be utilized as a potential luminescent probe toward different Ln³⁺ ions. Additionally, the absorption ability of IFMC‐2 toward ionic dyes was also performed. Cationic dyes can be absorbed, but not neutral and anionic dyes, thus indicating that IFMC‐2 exhibits selective absorption toward cationic dyes. Furthermore, the cationic dyes can be gradually released in the presence of NaCl.