Ratiometric and Near‐Infrared Molecular Probes for the Detection and Imaging of Zinc Ions

The detection and imaging of Zn²⁺ in biological samples are of paramount interest owing to the role of this cation in physiological functions. This is possible only with molecular probes that specifically bind to Zn²⁺ and result in changes in emission properties. A “turn‐on” emission or shift in the emission color upon binding to Zn²⁺ should be ideal for in vivo imaging. In this context, ratiometric and near‐IR probes are of particular interest. Therefore, in the area of chemosensors or molecular probes, the design of fluorophores that allow ratiometric sensing or imaging in the near‐IR region is attracting the attention of chemists. The purpose of this Focus Review is to highlight recent developments in this area and stress the importance of further research for future applications.     

基于2-(2′-吡啶基)苯并咪唑的Zn2+比例计量型荧光识别

本文以荧光分子2-(2'-吡啶基)苯并咪唑(2-PBI)作为1,1'-联(2-芳杂环)类化合物模型.研究利用其2,2'-N原子螯合Zn2 导芳环共面化和荧光发射红移实现Zn2 比例计量检测的可行性.2-PBI在不同体系中的Zn2 荧光响应行为表明Zn2 结合将导致最大发射波长明显红移(乙腈36 nm;HEPES缓冲液39 nm).具有比例计量型Zn2 荧光探针的基本特点.2-PBI还具有显著的Zn2 荧光响应选择性,可以同时作为构建比例计量型探针的信号团和受体的基本骨架.通过对2-BPI的荧光机制和Zn2 识别行为的分析,提出了以2-PBI为基本骨架构建实用化比例计量型探针的途径.     

表面活性剂增溶比率荧光Zn2+检测方法研究

合成了一种新的Zn2+荧光检测试剂8-(2-(十八氨基)乙酰氨基)喹啉(AQZ-18)。通过自发荧光的非离子表面活性剂OP-10增溶AQZ-18,获得了一个与Zn2+结合后在320 nm和505 nm分别有两个荧光发射峰的溶液体系。短波长荧光峰来自OP-10,荧光峰强度不随Zn2+浓度变化;长波长荧光峰来自AQZ-18,荧光峰强度随Zn2+浓度增加而增强。利用上述两个荧光峰强度随Zn2+浓度变化时的比值变化建立了一种新的比率荧光Zn2+检测方法。研究表明,Zn2+与AQZ-18形成1∶1型基态配合物,其表观结合常数为1.1×106L/mol。常见金属离子对Zn2+荧光检测无干扰,Zn2+浓度在0～1.1×10-5mol/L范围内与荧光强度变化的比值呈良好的线性关系,相关系数(r2)为0.996 2,检出限为55 nmol/L。该方法可用于水样中Zn2+的检测。     

5-氨基-1,10-菲咯啉：可异构化杂环螯合荧光载体作为比例计量型锌离子荧光探针的一种原型

5-amino-l,10-phenanthroline （5-AP）, as a tautomeric heterocyclic aromatic chelating fluorophore （THACF）, can sense Zn^2＋ selectively by shifting emission from 495 to 564 nm upon Zn^2＋ addition in ethanol. The ratiometric fluorescent sensing behavior has been correlated to the tautomerization of 5-AP affected by solvents and metal chelation. The strategy using THACF as ratiometric fluorescent sensor for Zn^2＋ not only simplifies the synthetic procedure but also gives a promising alternative for Zn^2＋ ratiometric fluorescent sensor design.     

Construction of Fluorescent Probes Via Protection/Deprotection of Functional Groups: A Ratiometric Fluorescent Probe for Cu2+

Herein a ratiometric fluorescent Cu²⁺ probe was rationally constructed in a straightforward manner with the concept of aldehyde group protection/deprotection. The probe showed a ratiometric fluorescent response to Cu²⁺ with a large emission wavelength shift (>100 nm) and displayed high selectivity for Cu²⁺ over other metal ions due to distinct deprotection conditions. In addition, a Cu²⁺‐promoted dethioacetalization mechanism was proposed.     

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.     

Water‐Soluble Polymer Functionalized CdTe/ZnS Quantum Dots: A Facile Ratiometric Fluorescent Probe for Sensitive and Selective Detection of Nitroaromatic Explosives

A ratiometric fluorescent probe based on dual luminescence QD/CPL for selective sensing of the nitroaromatic explosive picric acid (PA) was constructed. The observed ratiometric fluorescence intensity change allows the quantitative detection of PA with a detection limit of 9 nM .     

Amino Acid-Derived Sensors for Specific Zn2+ Detection Using Hyperpolarized 13C Magnetic Resonance Spectroscopy

Alterations in Zn²⁺ concentration are seen in normal tissues and in disease states, and for this reason imaging of Zn²⁺ is an area of active investigation. Herein, enriched [1-¹³C]cysteine and [1-¹³C₂]iminodiacetic acid were developed as Zn²⁺-specific imaging probes using hyperpolarized ¹³C magnetic resonance spectroscopy. [1-¹³C]cysteine was used to accurately quantify Zn²⁺ in complex biological mixtures. These sensors can be employed to detect Zn²⁺ via imaging mechanisms including changes in ¹³C chemical shift, resonance linewidth, or T₁.     

High-Affinity Ratiometric Fluorescence Probe Based on 6-Amino-2,2′-Bipyridine Scaffold for Endogenous Zn2+ and Its Application to Living Cells

Zinc is an essential trace element involved in many biological activities; however, its functions are not fully understood. To elucidate the role of endogenous labile Zn²⁺, we developed a novel ratiometric fluorescence probe, 5-(4-methoxyphenyl)-4-(methylsulfanyl)-[2,2′-bipyridin]-6-amine (6 (rBpyZ)) based on the 6-amino-2,2′-bipyridine scaffold, which acts as both the chelating agent for Zn²⁺ and the fluorescent moiety. The methoxy group acted as an electron donor, enabling the intramolecular charge transfer state of 6 (rBpyZ), and a ratiometric fluorescence response consisting of a decrease at the emission wavelength of 438 nm and a corresponding increase at the emission wavelength of 465 nm was observed. The ratiometric probe 6 (rBpyZ) exhibited a nanomolar-level dissociation constant (K_d = 0.77 nM), a large Stokes shift (139 nm), and an excellent detection limit (0.10 nM) under physiological conditions. Moreover, fluorescence imaging using A549 human lung adenocarcinoma cells revealed that 6 (rBpyZ) had good cell membrane permeability and could clearly visualize endogenous labile Zn²⁺. These results suggest that the ratiometric fluorescence probe 6 (rBpyZ) has considerable potential as a valuable tool for understanding the role of Zn²⁺ in living systems.     

Sensing of Phosphates by Using Luminescent EuIII and TbIII Complexes: Application to the Microalgal Cell Chlorella vulgaris

Phenanthroline‐based chiral ligands L¹ and L² as well as the corresponding Eu^III and Tb^III complexes were synthesized and characterized. The coordination compounds show red and green emission, which was explored for the sensing of a series of anions such as F^?, Cl^?, Br^?, I^?, NO₃^?, NO₂^?, HPO₄^2?, HSO₄^?, CH₃COO^?, and HCO₃^?. Among the anions, HPO₄^2? exhibited a strong response in the emission property of both europium(III) and terbium(III) complexes. The complexes showed interactions with the nucleoside phosphates adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP). Owing to this recognition, these complexes have been applied as staining agents in the microalgal cell Chlorella vulgaris. The stained microalgal cells were monitored through fluorescence microscopy and scanning electron microscopy. Initially, the complexes bind to the outer cell wall and then enter the cell wall through holes in which they probably bind to phospholipids. This leads to a quenching of the luminescence properties.     

Synthesis of PAF,an Antifungal Protein from P. chrysogenum,by Native Chemical Ligation: Native Disulfide Pattern and Fold Obtained upon Oxidative Refolding

The folding of disulfide proteins is of considerable interest because knowledge of this may influence our present understanding of protein folding. However, sometimes even the disulfide pattern cannot be unequivocally determined by the available experimental techniques. For example, the structures of a few small antifungal proteins (PAF, AFP) have been disclosed recently using NMR spectroscopy but with some ambiguity in the actual disulfide pattern. For this reason, we carried out the chemical synthesis of PAF. Probing different approaches, the oxidative folding of the synthetic linear PAF yielded a folded protein that has identical structure and antifungal activity as the native PAF. In contrast, unfolded linear PAF was inactive, a result that may have implications concerning its redox state in the mode of action.     

Poly(arginine)‐Selective Coprecipitation Properties of Self‐Assembling Apoferritin and Its Tb3+ Complex: A New Luminescent Biotool for Sensing of Poly(arginine) and Its Protein Conjugates

The apoferritin protein and apoferritin–Tb³⁺ complex were demonstrated to form oligomeric and polymeric self‐assemblies in neutral aqueous solutions, based on characterization by using luminescence and UV/Vis spectroscopy, dynamic light scattering, and transmission electron microscopy. Addition of a 20‐mer or higher poly(arginine) to the solution resulted in coprecipitation through nanoscale interactions, while biological proteins and other poly(amino acids) rarely yielded precipitates under the conditions employed. The apoferritin–Tb³⁺ complex assembly exhibited a particularly long‐lived green luminescence in aqueous solution, and its poly(arginine)‐selective precipitation behavior was followed by monitoring the changes in luminescence. The poly(arginine)‐tagged albumin precipitated selectively and quantitatively, so that the apoferritin–Tb³⁺ complex can function as a new luminescent biotool for the sensing of poly(arginine) and its protein conjugates.     

Optical Temperature-Sensing Performance of Nd3+:MF2 (M=Ba,Ca, Sr) Crystalline Powders Prepared by Combustion Synthesis

There is a large interest in luminescent materials for application as temperature sensors. In this scenario, we investigate the performance of neodymium-doped alkaline-earth fluoride (Nd³⁺:MF₂; M=Ba, Ca, Sr) crystalline powders prepared by combustion synthesis for optical temperature-sensing applications based on the luminescence intensity ratio (LIR) technique. We observe that the near-infrared luminescence spectral profile of Nd³⁺ changes with the temperature in a way that its behavior is suitable for optical thermometry operation within the first biological window. We also observe that the thermometric sensitivities of all studied samples change depending on the spectral integration range used in the LIR analysis. Nd³⁺:CaF₂ presents the largest sensitivity values, with a maximum absolute sensitivity of 6.5×10⁻³/K at 824 K and a relative sensitivity of 1.71 %/K at human-body temperature (310 K). The performance of CaF₂ for optical thermometry is superior to that of β-NaYF₄, a standard material commonly used for optical bioimaging and temperature sensing, and on par with the most efficient oxide nanostructured materials. The use of thermometry data to help understand structural properties via Judd-Ofelt intensity standard parameters is also discussed.     

The inorganic perspective of nerve growth factor: interactions of Cu2+ and Zn2+ with the N-terminus fragment of nerve growth factor encompassing the recognition domain of the TrkA receptor

There is a significant overlap between brain areas with Zn(2+) and Cu(2+) pathological dys-homeostasis and those in which the nerve growth factor (NGF) performs its biological role. The protein NGF is necessary for the development and maintenance of the sympathetic and sensory nervous systems. Its flexible N-terminal region has been shown to be a critical domain for TrkA receptor binding and activation. Computational analyses show that Zn(2+) and Cu(2+) form pentacoordinate complexes involving both the His4 and His8 residues of the N-terminal domain of one monomeric unit and the His84 and Asp105 residues of the other monomeric unit of the NGF active dimer. To date, neither experimental data on the coordination features have been reported, nor has one of the hypotheses according to which Zn(2+) and Cu(2+) may have different binding environments or the Ser1 α-amino group could be involved in coordination been supported. The peptide fragment, encompassing the 1-14 sequence of the human NGF amino-terminal domain (NGF(1-14)), blocked at the C terminus, was synthesised and its Cu(2+) and Zn(2+) complexes characterized by means of potentiometric and spectroscopic (UV/Vis, CD, NMR, and EPR) techniques. The N-terminus-acetylated form of NGF(1-14) was also investigated to evaluate the involvement of the Ser1 α-amino group in metal-ion coordination. Our results demonstrate that the amino group is the first anchoring site for Cu(2+) and is involved in Zn(2+) coordination at physiological pH. Finally, a synergic proliferative activity of both NGF(1-14) and the whole protein on SHSY5Y neuroblastoma cell line was found after treatment in the presence of Cu(2+). This effect was not observed after treatment with the N-acetylated peptide fragment, demonstrating a functional involvement of the N-terminal amino group in metal binding and peptide activity.     

Aptamer‐Based Luminescence Energy Transfer from Near‐Infrared‐to‐Near‐Infrared Upconverting Nanoparticles to Gold Nanorods and Its Application for the Detection of Thrombin

A new luminescence energy transfer (LET) system has been designed for the detection of thrombin in the near‐infrared (NIR) region by utilizing NIR‐to‐NIR upconversion lanthanide nanophosphors (UCNPs) as the donor and gold nanorods (Au NRs) as the acceptor. The use of upconverting NaYF₄:Yb³⁺,Tm³⁺ nanoparticles with sharp NIR emission peaks upon NIR excitation by an inexpensive infrared continuous wave laser diode provided large spectral overlap between the donor and the acceptor. Both the Au NRs and carboxyl‐terminated NaYF₄:Yb³⁺,Tm³⁺ UCNPs were first modified with different thrombin aptamers. When thrombin was added, a LET system was then formed because of the specific recognition between the thrombin aptamers and thrombin. The LET system was used to monitor thrombin concentrations in aqueous buffer and human blood samples. The limits of detection for thrombin are as low as 0.118 nM in buffer solution and 0.129 nM in human serum. The method was also successfully applied to thrombin detection in blood samples.     

Three New Ln-Decavanadates Materials:Synthesis,Structure, and Photoluminescent Sensing for Detection of Zn2+ and Co2+

Three new compounds, [Ln(H₂O)₈]₂[V₁₀O₂₈] · 8H₂O [Ln = Ho ( 1 ), Tb ( 2 )] and [Eu(H₂O)₈]₂[V₁₀O₂₈] · 9H₂O ( 3 ), were successfully synthesized by evaporating the mixture of K₆V₁₀O₂₈ · 10H₂O and LnCl₃ · 6H₂O. Notably, three vanadates are composed of [Ln(H₂O)₈]³⁺ cation, decavanadates ([V₁₀O₂₈]^6–) anion. Meanwhile, free water molecules generate different type water clusters to connect [V₁₀O₂₈]^6– anions and coordination cations to form 3D supramolecular structure. The fluorescence measurements reveal that characteristic photoluminescence of Tb^III and Eu^III is quenched in presence of [V₁₀O₂₈]^6–, then the impacts of variational decavanadates ions concentration on the fluorescence intensities of LnCl₃ (Ln = Tb, Eu) systems and different acetate solution [M(CH₃COO)₂; M = Ni, Cr, Cu, Co, Zn] on fluorescence intensities of Ln-decavanadates (Ln = Eu, Tb) systems are investigated.