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.     

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.     

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.     

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.     

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.     

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.     

Quinolinium‐Based Fluorescent Probes for the Detection of Thiophenols in Environmental Samples and Living Cells

A new type of fluorescent probes for thiophenols, 6HQM‐DNP and 7HQM‐DNP, containing 6‐ or 7‐hydroxy quinonlinium as fluorophore and 2,4‐dinitrophenoxy (DNP) as nucleophilic recognition unit were constructed. As ethers, these non‐fluorescent probe molecules can release the corresponding fluorescent quinolinium (6HQM and 7HQM) through aromatic nucleophilic substitution (S_NAr) by thiolate anions from thiophenols. The sensing reaction is highly sensitive (detection limit of 8 nM for 7HQM‐DNP) and highly selective to thiophenols over aliphatic thiols and other nucleophiles under neutral conditions (pH 7.3). The probes respond rapidly to thiophenols, with second‐order rate constants k=45 M ^?1 s^?1 for 7HQM‐DNP and 24 M ^?1 s^?1 for 6HQM‐DNP. Furthermore, the selective detection of thiophenols in living cells by 7HQM‐DNP was demonstrated by confocal fluorescence imaging. In addition, these quinolinium salts show excellent chemical and thermal stability. In conclusion, this type of probes may find use in the detection of thiophenols in environmental samples and biosystems.     

Highly Selective and Sensitive Near‐Infrared‐Fluorescent Probes for the Detection of Cellular Hydrogen Sulfide and the Imaging of H2S in Mice

Herein, we report the development of two fluorescent probes for the highly selective and sensitive detection of H₂S. The probes take advantage of a Cu^II? cyclen complex, which acts as a reaction center for H₂S and as a quencher of BODIPY (boron‐dipyrromethene)‐based fluorophores with emissions at 765 and 680 nm, respectively. These non‐fluorescent probes could only be turned on by the addition of H₂S, and not by other potentially interfering biomolecules, including reactive oxygen species, cysteine, and glutathione. In a chemical system, both probes detected H₂S with a detection limit of 80 nM . The probes were successfully used for the endogenous detection of H₂S in HEK 293 cells, for measuring the H₂S‐release activity of dietary organosulfides in MCF‐7 cells, and for the in vivo imaging of H₂S in mice.     

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.     

A Selective,Nontoxic, OFF–ON Fluorescent Molecular Sensor Based on 8‐Hydroxyquinoline for Probing Cd2+ in Living Cells

In spite of the fact that cadmium(II) has been recognized as a highly toxic element and that excessive exposure to this metal ion has been reported to have many adverse effects on human health, very few selective and specific fluorescent probes are available for imaging Cd²⁺ in living cells. Herein, we report the spectroscopic and photochemical characterization of 5‐(5‐chloro‐8‐hydroxyquinolinylmethyl)‐2,8‐dithia‐5‐aza‐2,6‐pyridinophane ( L ) as a fluorescent sensor for the selective imaging of Cd²⁺ in living cells. In particular, the response of L to Cd²⁺ was first assessed in aqueous solutions, sodium dodecyl sulfate micelles, and liposomes, and subsequently in living cells by fluorescence microscopy techniques. Cytofluorimetric analyses of leukemic HL‐60 cells loaded with L also allowed evaluation of the toxicity of the probe and the selective analysis of its intracellular fluorescence in the presence of Cd²⁺. Furthermore, the 1:1 complex species [Cd( L )H₂O]²⁺ responsible for the OFF–ON chelation enhancement of fluorescence (CHEF) effect on L was structurally characterized; time‐dependent DFT calculations allowed the prediction of theoretical excitations, which were comparable with the experimental ones.     

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.     

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.