Reaction‐Based Fluorescent Probe for Detection of Endogenous Cyanide in Real Biological Samples

Herein, two compounds ( 1 a and 1 b ) were rationally constructed as novel reaction‐based fluorescent probes for CN^? by making use of the electron‐withdrawing ability of the cyano group that was formed from the sensing reaction. Notably, this design strategy was first employed for the development of fluorescent CN^? probes. The experimental details showed that probe 1 a exhibited a fluorescence turn‐on response to CN^?, whereas other anions, biological thiols, and hydrogen sulfide gave almost no interference. The detection limit of probe 1 a for CN^? was found to be 0.12 μM . The sensing reaction product of 1 a with CN^? was characterized by NMR spectroscopy and mass spectrometry. TD‐DFT calculations demonstrated that the formed cyano group drives the intramolecular charge transfer (ICT) process from coumarin dye to the cyano group and thus the original strong ICT from the coumarin dye to the 3‐position pyridyl vinyl ketone substituent is weakened, which results in recovery of coumarin fluorescence. The practical utility of 1 a was also examined. By fabricating paper strips, probe 1 a can be used as a simple tool to detect CN^? in field measurements. Moreover, probe 1 a has been successfully applied for quantitative detection of endogenous CN^? from cassava root.     

Active‐Site‐Matched Fluorescent Probes for Rapid and Direct Detection of Vicinal‐Sulfydryl‐Containing Peptides/Proteins in Living Cells

Vicinal‐sulfydryl‐containing peptides/proteins (VSPPs) play a crucial role in human pathologies. Fluorescent probes that are capable of detecting intracellular VSPPs in vivo would be useful tools to explore the mechanisms of some diseases. In this study, by regulating the spatial separation of two maleimide groups in a fluorescent dye to match that of two active cysteine residues contained in the conserved amino acid sequence (–CGPC–) of human thioredoxin, two active‐site‐matched fluorescent probes, o‐Dm‐Ac and m‐Dm‐Ac, were developed for real‐time imaging of VSPPs in living cells. As a result, the two probes can rapidly respond to small peptide models and reduced proteins, such as WCGPCK (W‐6), WCGGPCK (W‐7), and WCGGGPCK (W‐8), reduced bovine serum albumin (rBSA), and reduced thioredoxin (rTrx). Moreover, o‐Dm‐Ac displays a higher binding sensitivity with the above‐mentioned peptides and proteins, especially with W‐7 and rTrx. Furthermore, o‐Dm‐Ac was successfully used to rapidly and directly detect VSPPs both in vitro and in living cells. Thus, a novel probe‐design strategy was proposed and the synthesized probe applied successfully in imaging of target proteins in situ.     

A Selective Near‐Infrared Fluorescent Probe for In Vivo Imaging of Thiophenols from a Focused Library

Thiophenols are highly toxic industrial materials that, once released, will accumulate in the environment, and ultimately in human bodies, thereby causing serious health problems. To achieve their selective and sensitive detection, a novel near‐infrared (NIR) fluorescent probe ( CCP‐1 ) from a focused library was developed for thiophenol species. Our studies show that CCP‐1 displays a thiophenol‐triggered 28‐fold fluorescence intensity enhancement at 706 nm, with a detection limit of 34 nm observed. It is also able to differentiate thiophenols from various other thiol‐containing analytes including hydrogen sulfide, hydrogen persulfide, and aliphatic thiols. In total, the desirable properties (e.g., excitation/emission in the NIR region, good cell‐membrane permeability, intracellular stability, and low cytotoxicity) make CCP‐1 a potential candidate for thiophenol detection both in vitro and in vivo. In addition, CCP‐1 , for the first time, successfully visualized thiophenols in mice models of thiophenol inhalation.     

新型香豆素类氟离子荧光探针的合成及细胞成像研究

设计合成了一类基于分子内电荷转移（Intramolecular Charge Transfer,ICT）的香豆素类F^-荧光探针CS1,CS2和CS3,经¹H NMR,¹³C NMR,IR和HRMS表征了相应探针的结构,并解析了探针CS3的晶体结构.通过核磁和质谱实验验证了探针与F^-的作用机理是氟化物脱硅基.光谱分析实验结果显示,CS1,CS2和CS3均具有较好的选择性和灵敏度,且均能成功实现人乳腺癌细胞（MCF^-7）中F^-的检测.     

New Chemodosimetric Reagents as Ratiometric Probes for Cysteine and Homocysteine and Possible Detection in Living Cells and in Blood Plasma

In this work, we have rationally designed and synthesized two new reagents ( L₁ and L₂ ), each bearing a pendant aldehyde functionality. This aldehyde group can take part in cyclization reactions with β‐ or γ‐amino thiols to yield the corresponding thiazolidine and thiazinane derivatives, respectively. The intramolecular charge‐transfer (ICT) bands of these thiazolidine and thiazinane derivatives are distinctly different from those of the molecular probes ( L₁ and L₂ ). Such changes could serve as a potential platform for using L₁ and L₂ as new colorimetric/fluorogenic as well as ratiometric sensors for cysteine (Cys) and homocysteine (Hcy) under physiological conditions. Both reagents proved to be specific towards Cys and Hcy even in the presence of various amino acids, glucose, and DNA. Importantly, these two chemodosimetric reagents could be used for the quantitative detection of Cys present in blood plasma by using a pre‐column HPLC technique. Such examples are not common in contemporary literature. MTT assay studies have revealed that these probes have low cytotoxicity. Confocal laser scanning micrographs of cells demonstrated that these probes could penetrate cell membranes and could be used to detect intracellular Cys/Hcy present within living cells. Thus, the results presented in this article not only demonstrate the efficiency and specificity of two ratiometric chemodosimeter molecules for the quantitative detection of Cys and Hcy, but also provide a strategy for developing reagents for analysis of these vital amino acids in biological samples.     

Phenoxazine‐based Near‐infrared Fluorescent Probes for the Specific Detection of Copper (II) Ions in Living Cells

Abstract : It is well known that copper ions play a critical role in various physiological processes. However, a variety of human diseases are tightly correlated with copper overload. Although there are numerous fluorescent probes capable of detecting copper ions, most of them are “turn‐off” probes owing to copper (II) ions fluorescence quenching effect, resulting in poor sensitivity. Herein, a novel “turn‐on” near‐infrared (NIR) fluorescent probe PZ‐N based on phenoxazine was designed and synthesized for the selective detection of copper (II) ions (Cu²⁺). Upon the addition of Cu²⁺, the probe could quickly react with Cu²⁺ and emit strong fluorescence, along with colour change from colourless to obvious blue. Moreover, the probe PZ‐N showed good water solubility, high selectivity, and excellent sensitivity with low limit of detection (1.93 nM) towards copper (II) ions. More importantly, PZ‐N was capable of effectively detecting Cu²⁺ in living cells.     

Functional Near‐Infrared Fluorescent Probes

Near‐infrared (NIR) fluorescent probes have attracted much attention, but despite the availability of various NIR fluorophores, only a few functional NIR probes, that is, probes whose absorption and/or fluorescence spectra change upon specific reaction with biomolecules, have been developed. However, functional probes operating in the NIR range that can be targeted to protons, metal ions, nitric oxide, β‐galactosidase, and cellular stress markers are expected to be effective for fluorescence imaging in vivo. This Focus Review concentrates on these functional NIR probes themselves, not their applications.     

Versatile Fluorescent Probes for Imaging the Superoxide Anion in Living Cells and In Vivo

The superoxide anion (O₂^.?) is widely engaged in the regulation of cell functions and is thereby intimately associated with the onset and progression of many diseases. To ascertain the pathological roles of O₂^.? in related diseases, developing effective methods for monitoring O₂^.? in biological systems is essential. Fluorescence imaging is a powerful tool for monitoring bioactive molecules in cells and in vivo owing to its high sensitivity and high temporal‐spatial resolution. Therefore, increasing numbers of fluorescent imaging probes have been constructed to monitor O₂^.? inside live cells and small animals. In this minireview, we summarize the methods for design and application of O₂^.?‐responsive fluorescent probes. Moreover, we present the challenges for detecting O₂^.? and suggestions for constructing new fluorescent probes that can indicate the production sites and concentration changes in O₂^.? as well as O₂^.?‐associated active molecules in living cells and in vivo.     

Discerning the Chemistry in Individual Organelles with Small‐Molecule Fluorescent Probes

Principle has it that even the most advanced super‐resolution microscope would be futile in providing biological insight into subcellular matrices without well‐designed fluorescent tags/probes. Developments in biology have increasingly been boosted by advances of chemistry, with one prominent example being small‐molecule fluorescent probes that not only allow cellular‐level imaging, but also subcellular imaging. A majority, if not all, of the chemical/biological events take place inside cellular organelles, and researchers have been shifting their attention towards these substructures with the help of fluorescence techniques. This Review summarizes the existing fluorescent probes that target chemical/biological events within a single organelle. More importantly, organelle‐anchoring strategies are described and emphasized to inspire the design of new generations of fluorescent probes, before concluding with future prospects on the possible further development of chemical biology.     

Versatile Probes for the Selective Detection of Vicinal‐Dithiol‐Containing Proteins: Design,Synthesis, and Application in Living Cells

Endogenous vicinal‐dithiol‐containing proteins (VDPs) that have two thiol groups close to each other in space play a significant importance in maintaining the cellular redox microenvironment. Approaches to identify VDPs mainly rely on monitoring the different concentration of monothiol and total thiol groups or on indirect labeling of vicinal thiols by using p‐aminophenylarsenoxide ( PAO ). Our previous work has reported the direct labeling of VDPs with a highly selective receptor PAO analogue, which could realize fluorescence detection of VDPs directly in living cells. Herein, we developed a conjugated approach to expand detectable tags to nitrobenzoxadiazole (NBD), fluorescein, naphthalimide, and biotin for the synthesis of a series of probes. Different linkers have also been introduced toward conjugation of VTA2 with these functional tags. These synthesized flexible probes with various features will offer new tools for the potential identification and visualization of vicinal dithiols existing in different regions of VDPs in living cells. These probes are convenient tools for proteomics studies of various disease‐related VDPs and for the discovery of new drug targets.     

FRET‐Based Mitochondria‐Targetable Dual‐Excitation Ratiometric Fluorescent Probe for Monitoring Hydrogen Sulfide in Living Cells

Hydrogen sulfide (H₂S) is connected with various physiological and pathological functions. However, understanding the important functions of H₂S remains challenging, in part because of the lack of tools for detecting endogenous H₂S. Herein, compounds Ratio‐H₂S 1/2 are the first FRET‐based mitochondrial‐targetable dual‐excitation ratiometric fluorescent probes for H₂S on the basis of H₂S‐promoted thiolysis of dinitrophenyl ether. With the enhancement of H₂S concentration, the excitation peak at λ≈402 nm of the phenolate form of the hydroxycoumarin unit drastically increases, whereas the excitation band centered at λ≈570 nm from rhodamine stays constant and can serve as a reference signal. Thus, the ratios of fluorescence intensities at λ=402 and 570 nm (I₄₀₂/I₅₇₀) exhibit a drastic change from 0.048 in the absence of H₂S to 0.36 in the presence of 180 μM H₂S; this is a 7.5‐fold variation in the excitation ratios. The favorable properties of the probe include the donor and acceptor excitation bands, which exhibit large excitation separations (up to 168 nm separation) and comparable excitation intensities, high sensitivity and selectivity, and function well at physiological pH. In addition, it is demonstrated that the probe can localize in the mitochondria and determine H₂S in living cells. It is expected that this strategy will lead to the development of a wide range of mitochondria‐targetable dual‐excitation ratiometric probes for other analytes with outstanding spectral features, including large separations between the excitation wavelengths and comparable excitation intensities.     

Two‐Photon Fluorescent Probes for Metal Ions

Two‐photon microscopy (TPM) has become an indispensible tool in biology and medicine owing to the capability of imaging the intact tissue for a long period of time. To make it a versatile tool in biology, a variety of two‐photon probes for specific applications are needed. In this context, many research groups are developing two‐photon probes for various applications. In this Focus Review, we summarize recent results on model studies and selected examples of two‐photon probes that can detect intracellular free metal ions in live cells and tissues to provide a guideline for the design of useful two‐photon probes for various in vivo imaging applications.     

Colorimetric and Fluorescent Bimodal Ratiometric Probes for pH Sensing of Living Cells

pH measurement is widely used in many fields. Ratiometric pH sensing is an important way to improve the detection accuracy. Herein, five water‐soluble cationic porphyrin derivatives were synthesized and their optical property changes with pH value were investigated. Their pH‐dependent assembly/disassembly behaviors caused significant changes in both absorption and fluorescence spectra, thus making them promising bimodal ratiometric probes for both colorimetric and fluorescent pH sensing. Different substituent identity and position confer these probes with different sensitive pH‐sensing ranges, and the substituent position gives a larger effect. By selecting different porphyrins, different signal intensity ratios and different fluorescence excitation wavelengths, sensitive pH sensing can be achieved in the range of 2.1–8.0. Having demonstrated the excellent reversibility, good accuracy and low cytotoxicity of the probes, they were successfully applied in pH sensing inside living cells.     

Design of Ratiometric Fluorescent Probes Based on Arene–Metal‐Ion Interactions and Their Application to CdII and Hydrogen Sulfide Imaging in Living Cells

Non‐coordinative interactions between a metal ion and the aromatic ring of a fluorophore can act as a versatile sensing mechanism for the detection of metal ions with a large emission change of fluorophores. We report the design of fluorescent probes based on arene–metal‐ion interactions and their biological applications. This study found that various probes having different fluorophores and metal binding units displayed significant emission redshift upon complexation with metal ions, such as Ag^I, Cd^II, Hg^II, and Pb^II. X‐ray crystallography of the complexes confirmed that the metal ions were held in close proximity to the fluorophore to form an arene–metal‐ion interaction. Electronic structure calculations based on TDDFT offered a theoretical basis for the sensing mechanism, thus showing that metal ions electrostatically modulate the energy levels of the molecular orbitals of the fluorophore. A fluorescent probe was successfully applied to the ratiometric detection of the uptake of Cd^II ions and hydrogen sulfide (H₂S) in living cells. These results highlight the utility of interactions between arene groups and metal ions in biological analyses.     

Fluorescent Probes Based on Nucleophilic Substitution–Cyclization for Hydrogen Sulfide Detection and Bioimaging

The design, synthesis, properties, and cell imaging applications of a series of 2‐pyridyl disulfide based fluorescent probes (WSP1, WSP2, WSP3, WSP4 and WSP5) for hydrogen sulfide detection are reported. The strategy is based on the dual‐nucleophilicity of hydrogen sulfide. A hydrogen sulfide mediated tandem nucleophilic substitution‐cyclization reaction is used to release the fluorophores and turn on the fluorescence. The probes showed high sensitivity and selectivity for hydrogen sulfide over other reactive sulfur species, including cysteine and glutathione.     

Water‐Soluble Fluorescent Probes Based on Dendronized Polyfluorenes for Cell Imaging

Novel water‐soluble dendronized fluorescent polyfluorenes (DFPFs) are prepared from hydrophilic monomers and hydrophobic comonomers. Incomplete energy transfer is found to result in a two‐color emission of the DFPFs at around 410 and 650 nm. The incomplete energy transfer can be attributed to the poor compatibility between the fluorene and benzothiadiazole units. Polyethylene oxide (PEO) encapsulation of the DFPFs shows over 90% cell viability, indicating good biocompatibility. These DFPFs show differential cellular uptake. P1 with fewer PEO chains exhibits limited cellular membrane uptake and low brightness in cells. By contrast, P3 with more PEO chains is efficiently internalized by cells and accumulated in the cytoplasm. A strong fluorescence from whole cells is also observed.     

Small Molecular Fluorescent Probes for Imaging of Viscosity in Living Biosystems

Viscosity, as a vital microenvironment parameter, is tightly associated with multitudinous cellular processes and diseases. Recently, precise visualization of viscosity has started to arouse more and more interest. However, owing to the complicated character, it is still a huge challenge to directly observe viscosity in living systems. In this regard, mounting fluorescence probes are being increasingly fabricated to map viscosity inside live cells and small animals. In this minireview, the viscosity-sensitive small molecular fluorescent probes used in bioimaging are comprehensively summarized, mainly focusing on the last three years. Moreover, the current challenges and opportunities for the development of viscosity-specific fluorescent probes will be discussed.     

A Naphthalimide-Based Fluorescent Probe for the Detection and Imaging of Mercury Ions in Living Cells

The selective and efficient monitoring of mercury (Hg²⁺) contamination found in the environment and ecosystem has been carried out. Thus, a new 1,8-naphthalimide-based fluorescent probe NADP for the detection of Hg²⁺ based on a fluorescence enhancement strategy has been designed and synthesized. The NADP probe can detect Hg²⁺ with high selectivity and sensitivity and a low detection limit of 13 nm . The detection mechanism was based on a Hg²⁺-triggered deprotection reaction, resulting in a dramatic change in fluorescence from colorless to green at physiological pH. Most importantly, biological investigation has shown that the NADP probe can be successfully applied to the monitoring of Hg²⁺ in living cells and zebrafish with low cytotoxicity.