An Ultrasensitive Cyclization‐Based Fluorescent Probe for Imaging Native HOBr in Live Cells and Zebrafish

Bromine has been reported recently as being the 28^th essential element for human health. HOBr, which is generated in vivo from bromide, is a required factor in the formation of sulfilimine crosslinks in collagen IV. However, to date, no method for the specific detection of native HOBr in vivo has been reported. Herein, we develop a simple small molecular probe for imaging HOBr based on a specific cyclization catalyzed by HOBr. The probe can be easily synthesized in high yield through a Suzuki cross‐coupling reaction. The probe exhibits ultrahigh sensitivity at the picomole level, in addition to specificity for HOBr and real‐time response. Importantly, without Br^? stimulation, this probe reports native HOBr levels in HepG2 cells. Thus, the probe is a promising new tool for imaging endogenous HOBr and may provide a means for finding new physiological functions of HOBr in living organisms.     

A Multisite‐Binding Switchable Fluorescent Probe for Monitoring Mitochondrial ATP Level Fluctuation in Live Cells

Adenosine triphosphate (ATP), commonly produced in mitochondria, is required by almost all the living organisms; thus fluorescent probes for monitoring mitochondrial ATP levels fluctuation are essential and highly desired. Herein, we report a multisite‐binding switchable fluorescent probe, ATP‐Red 1 , which selectively and rapidly responds to intracellular concentrations of ATP. Live‐cell imaging indicated that ATP‐Red 1 mainly localized to mitochondria with good biocompatibility and membrane penetration. In particular, with the help of ATP‐Red 1 , we successfully observed not only the decreased mitochondrial ATP levels in the presence of KCN and starvation state, but also the increased mitochondrial ATP levels in the early stage of cell apoptosis. These results indicate that ATP‐Red 1 is a useful tool for investigating ATP‐relevant biological processes.     

“Bottom‐Up” Fabrication of BODIPY‐Functionalized Fluorescent Hyperbranched Glycopolymers for Hepatoma‐Targeted Imaging

A novel type of multivalent and highly specific fluorescent hyperbranched glycopolymers h‐P(GalEA‐co‐VBPT‐co‐BYMA) (hPGVB) is designed and prepared successfully via a facile “bottom‐up” strategy. The acetylated hPGVB is prepared by one‐pot reversible addition‐fragmentation chain transfer (RAFT) copolymerization of acrylate‐type galactose monomers AcGalEA and methacrylate‐type fluorescent monomers BYMA in presence of an inimer‐type RAFT chain transfer agent. After deacetylation, the resulting amphiphilic hPGVB can self‐assemble into stable nanoparticles in aqueous media, showing strong green fluorescence with relative high quantum yields and good photostability. The cell viability study indicates the excellent biocompatibility of the hPGVB fluorescent nanoparticles (FNPs) against HepG2 and NIH3T3 cells. More importantly, comparing with the galactose‐free fluorescent hyperbranched polymers h‐P(OEGMA‐co‐VBPT‐co‐BYMA), hPEVB FNPs can be selectively internalized by asialoglycoprotein (ASGP) receptor‐rich HepG2 cells, indicating their potential application in the bioimaging fields.     

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.     

A BODIPY‐based Fluorescent Probe for Thiophenol

Thiophenol has been listed as one of the main sources of pollution. Sensitive probes for thiophenol are very significant. Herein, a BODIPY‐based fluorescent probe, named BDP, for poisonous thiophenol detection has been reported. BDP shows rapid response (15 s) and clear fluorescence enhancement (30 folds) to thiophenol in solution. The intensity of fluorescence and concentration of thiophenol have a good linear relationship. The detection limit is as low as 13.6 nmol · L⁻¹. BDP is stable towards pH and light radiation. Cell experiments demonstrate that BDP has good cell membrane penetrability, low cell toxicity and excellent imaging properties in living cells. Therefore, BDP has significant value on the detection of thiophenol in solution and in living cell.     

NeuO: a Fluorescent Chemical Probe for Live Neuron Labeling

To address existing limitations in live neuron imaging, we have developed NeuO , a novel cell‐permeable fluorescent probe with an unprecedented ability to label and image live neurons selectively over other cells in the brain. NeuO enables robust live neuron imaging and isolation in vivo and in vitro across species; its versatility and ease of use sets the basis for its development in a myriad of neuronal targeting applications.     

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.     

Asymmetric Rhodamine‐Based Fluorescent Probe for Multicolour In Vivo Imaging

To achieve rapid and sensitive detection of cancer, activatable fluorescent probes targeting proteases that are overexpressed in various types of cancer have been developed, based on the hydroxymethyl rhodamine green (HMRG) scaffold. However, to visualize altered activities of multiple enzymes in cancer sites, other scaffolds with distinct fluorescence properties from those of HMRG are needed. A novel asymmetrically modified rhodamine with suitable absorption/emission, brightness and equilibrium constant of intramolecular spirocyclization, working in the yellow/orange region, is introduced. As a proof of concept, a probe targeting γ‐glutamyl transpeptidase (gGlu‐HMJCR) was developed on the basis of the new scaffold. Simultaneous visualization and discrimination of tumours expressing γ‐glutamyl transpeptidase (with gGlu‐HMJCR) and cathepsins (with Z‐Phe‐Arg‐HMRG) by colour were achieved in a mouse model in vivo.     

A Naphthalimide‐Based Fluorescence “Turn‐On” Probe for the Detection of Pb2+ in Aqueous Solution and Living Cells

An easily available naphthalimide‐based fluorescent probe NPA for Pb²⁺ detection was successfully developed. NPA exhibited an obvious fluorescence turn‐on response toward Pb²⁺ in aqueous solution and in living cells. Moreover, a series of model compounds were rationally designed and synthesized in order to explore the sensing mechanism and binding mode of NPA with Pb²⁺.     

Mapping Live Cell Viscosity with an Aggregation‐Induced Emission Fluorogen by Means of Two‐Photon Fluorescence Lifetime Imaging

Intracellular viscosity is a crucial parameter that indicates the functioning of cells. In this work, we demonstrate the utility of TPE‐Cy, a cell‐permeable dye with aggregation‐induced emission (AIE) property, in mapping the viscosity inside live cells. Owing to the AIE characteristics, both the fluorescence intensity and lifetime of this dye are increased along with an increase in viscosity. Fluorescence lifetime imaging of live cells stained with TPE‐Cy reveals that the lifetime in lipid droplets is much shorter than that from the general cytoplasmic region. The loose packing of the lipids in a lipid droplet results in low viscosity and thus shorter lifetime of TPE‐Cy in this region. It demonstrates that the AIE dye could provide good resolution in intracellular viscosity sensing. This is also the first work in which AIE molecules are applied in fluorescence lifetime imaging and intracellular viscosity sensing.     

A Perylene Bisimide Cyclophane as a “Turn‐On” and “Turn‐Off” Fluorescence Probe

A rigid, covalently linked perylene‐3,4:9,10‐tetracarboxylic acid bisimide (PBI) cyclophane was synthesized by imidization of a bay‐substituted perylene bisanhydride with p‐xylylenediamine. The interchromophoric distance of approximately 6.5 Å establishes an ideal rigid cavity for the encapsulation of large aromatic compounds such as perylene and anthracene with binding constants up to 4.6×10⁴ M ^?1 (in CHCl₃). For electron‐poor guest molecules, the complexation process is accompanied by a significantly increased fluorescence, whereas the emission intensity is dramatically quenched by more electron‐rich guests because of the formation of charge‐transfer complexes. Furthermore, the influence of the PBI core twist on the binding constant results in a remarkable selectivity towards more flexible aromatic guest molecules.     

Self‐Assembly of Chiral Propeller‐like Supermolecules with Unusual “Sergeants‐and‐Soldiers” and “Majority‐Rules” Effects

Chiral amplification is an interesting phenomenon in supramolecular chemistry mainly observed in complicated systems in which cooperative effect dominate. Herein, chiral, supramolecular, propeller‐like architectures have been constructed through coassembly of an achiral disk‐shaped molecule and chiral amino acid derivatives driven by intermolecular hydrogen bonding. Both the “sergeants‐and‐soldiers” principle and “majority‐rules” effect are applicable in these discrete four‐component supermolecules, which are the simplest supramolecular system ever reported that exhibit chiral amplification.     

Fluorescent Self-Threaded Peptide Probes for Biological Imaging

A general synthetic method creates a new class of covalently connected, self-threaded, fluorescent molecular probes with figure-eight topology, an encapsulated deep-red fluorophore, and two peripheral peptide loops. The globular molecular shape and rigidified peptide loops enhance imaging performance by promoting water solubility, eliminating probe self-aggregation, and increasing probe stability. Moreover, the peptide loops determine the affinity and selectivity for targets within complex biological samples such as cell culture, tissue histology slices, or living subjects. For example, a probe with cell-penetrating peptide loops targets the surface of cell plasma membranes, whereas, a probe with bone-targeting peptide loops selectively stains the skeleton within a living mouse. The unique combination of bright deep-red fluorescence, high stability, and predictable peptide-based targeting is ideal for photon intense fluorescence microscopy and biological imaging.     

A 3,7‐Dihydroxyphenoxazine‐based Fluorescent Probe for Selective Detection of Intracellular Hydrogen Peroxide

A novel N‐borylbenzyloxycarbonyl‐3,7‐dihydroxyphenoxazine fluorescent probe (NBCD) for detecting H₂O₂ in living cells is described. The probe could achieve high selectivity for detecting H₂O₂ over other biological reactive oxygen species (ROS). In addition, upon addition of H₂O₂, NBCD exhibited color change from colorless to pink, which makes it a “naked‐eye” probe for H₂O₂ detection. NBCD could not only be used to detect enzymatically generated H₂O₂ but also to detect H₂O₂ in living systems by using fluorescence spectroscopy, with a detection limit of 2 μm . Importantly, NBCD enabled the visualization of epidermal growth factor (EGF)‐stimulated H₂O₂ generation inside the cells.     

Construction of a Selective Fluorescent Probe for GSH Based on a Chloro‐Functionalized Coumarin‐enone Dye Platform

Glutathione (GSH), the most abundant intracellular biothiol, protects cellular components from damage caused by free radicals and reactive oxygen species (ROS), and plays a crucial role in human pathologies. A fluorescent probe that can selectively sense intracellular GSH would be very valuable for understanding of its biological functions and mechanisms of diseases. In this work, a 3,4‐dimethoxythiophenol‐substituted coumarin‐enone was exploited as a reaction‐type fluorescent probe for GSH based on a chloro‐functionalized coumarin‐enone platform. In the probe, the 3,4‐dimethoxythiophenol group functions not only as a fluorescence quencher through photoinduced electron transfer (PET) to ensure a low background fluorescence, but also as a reactive site for biothiols. The probe displays a dramatic fluorescence turn‐on response toward GSH with the long‐wavelength emission (600 nm) and significant Stokes shift (100 nm). The selectivity of the probe toward GSH over cysteine (Cys), homocysteine (Hcy), and other amino acids was demonstrated. Assisted by laser‐scanning confocal microscopy, we have demonstrated that the probe could specifically sense GSH over Cys/Hcy in human renal cell carcinoma SiHa cells.     

Diketopyrrolopyrrole Bis-Phosphonate Conjugate: A New Fluorescent Probe for In Vitro Bone Imaging

The synthesis of a conjugate molecule between an unusual red-fluorescent diketopyrrolopyrrole (DPP) unit and a bis-phosphonate (BP) precursor by a click-chemistry strategy to target bone tissue and monitor the interaction is reported. After thorough investigation, conjugation through a triazole unit between a γ-azido rather than a β-azido BP and an alkyne-functionalized DPP fluorophore group turned out to be the winning strategy. Visualization of the DPP-BP conjugate on osteoclasts and specific antiresorption activity were successfully demonstrated.