A Fluorescent Probe with Aggregation‐Induced Emission for Detecting Alkaline Phosphatase and Cell Imaging

Alkaline phosphatase (ALP) is associated with many diseases, and its accurate detection is of great significance. Fluorescent compounds with aggregation‐induced emission (AIE) feature show beneficial advantages for serving as fluorescent probes. Herein, an AIE‐active “turn on” probe for ALP detection was synthesized through incorporating a strong electron‐withdrawing group (cyano) in the middle and the recognition moiety phosphate group at the end, thereby rendering a D–A–D structure with a relatively high conjugation degree and good water solubility. It was found that the probe TPE‐CN‐pho is highly sensitive to ALP in aqueous solution. In the presence of ALP, the hydrophilic phosphate group on the probe is rapidly removed, resulting in a decrease in water solubility and subsequent formation of aggregates, thereby achieving aggregation‐induced emission. Moreover, the probe TPE‐CN‐pho has also been successfully applied to imaging ALP in living cells.     

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.     

Structure‐Dependent cis/trans Isomerization of Tetraphenylethene Derivatives: Consequences for Aggregation‐Induced Emission

The isomerization and optical properties of the cis and trans isomers of tetraphenylethene (TPE) derivatives with aggregation‐induced emission (AIEgens) have been sparsely explored. We have now observed the tautomerization‐induced isomerization of a hydroxy‐substituted derivative, TPETH‐OH, under acidic but not under basic conditions. Replacing the proton of the hydroxy group in TPETH‐OH with an alkyl group leads to the formation of TPETH‐MAL, for which the pure cis and trans isomers were obtained and characterized by HPLC analysis and NMR spectroscopy. Importantly, cis‐TPETH‐MAL emits yellow fluorescence in DMSO at ?20 °C whereas trans‐TPETH‐MAL shows red fluorescence under the same conditions. Moreover, the geometry of cis‐ and trans‐TPETH‐MAL remains unchanged when they undergo thiol–ene reactions to form cis‐ and trans‐TPETH‐cRGD, respectively. Collectively, our findings improve our fundamental understanding of the cis/trans isomerization and photophysical properties of TPE derivatives, which will guide further AIEgen design for various applications.     

5‐Formyluracil as a Multifunctional Building Block in Biosensor Designs

In organisms 5‐formyluracil (5fU), which is known as a vital natural nucleobase, is widely present. Despite the recent development of sensor designs for organic fluorescent molecules for selective targeting applications, biocompatible and easily operated probe designs that are based on natural nucleobase modifications have rarely been reported. Here, we introduce the idea of 5fU as a multifunctional building block to facilitate the design and synthetic development of biosensors. The azide group was derived from the sugar of a nucleoside, which can be further used in the selective binding of cells or organelles through click chemistry with alkynyl‐modified targeting groups. The aldehyde group of 5fU can react with different chemicals to generate environmentally sensitive nucleobases that have obvious characteristics, which precious reactants cannot achieve for selective fluorogenic switch‐on detection of a specific target. We first synthesized 5fU analogues that had aggregation‐induced emission properties, and then we used triphenylphosphonium as a mitochondria‐targeting group to selectively image mitochondria in cancer cells and mouse embryonic stem cells. Additionally, the reagents exhibit a high selectivity for reaction with 5fU, which means that the method can also be used for the detection of 5fU. Combining the two characteristics, the idea of 5fU as a multifunctional building block in biosensor designs may potentially be applicable in 5fU site‐specific microenvironment detection in future research.     

An AIE‐Based Probe for Rapid and Ultrasensitive Imaging of Plasma Membranes in Biosystems

The abnormality of the plasma membrane (PM) is an important biomarker for cell status and many diseases. Hence, visualizing the PM, especially in complex systems, is an emerging field in the life sciences, especially in low‐resource settings. Herein, we developed a water‐soluble PM‐specific probe utilizing electrostatic and hydrophobic interaction strategies with aggregation‐induced emission as the signal output. The probe could image the PM with many advanced features (wash‐free, ultrafast staining process, excellent PM specificity, and good biocompatibility), which were demonstrated by the PM imaging of neurons. The probe allowed for the first time the imaging of erythrocytes in the complex brain environment through a fluorescence‐based method. Moreover, the PM of the epidermal and partial view of the eyeball structure of live zebrafish are also revealed.     

A High Contrast Tri‐state Fluorescent Switch: Properties and Applications

A high contrast tri‐state fluorescent switch (FSPTPE) with both emission color change and on/off switching is achieved in a single molecular system by fusing the aggregation‐induced emissive tetraphenylethene (TPE) with a molecular switch of spiropyran (SP). In contrast to most of the reported solid‐state fluorescent switches, FSPTPE only exists in the amorphous phase in the ring‐closed form owing to its highly asymmetric molecular geometry and weak intermolecular interactions, which leads to its grinding‐inert stable cyan emission in the solid state. Such an amorphous phase facilitates the fast response of FSPTPE to acidic gases and induces the structural transition from the ring‐closed form to ring‐open form, accompanied with the “Off” state of the fluorescence. The structural transition leads to a planar molecular conformation and high dipole moment, which further results in strong intermolecular interactions and good crystallinity, so when the acid is added together with a solvent, both the ring‐opening reaction and re‐crystallization can be triggered to result in an orange emissive state. The reversible control between any two of the three states (cyan/orange/dark) can be achieved with acid/base or mechanical force/solvent treatment. Because of the stable initial state and high color contrast (Δλ=120 nm for cyan/orange switch, dark state Φ_F<0.01 %), the fluorescent switch is very promising for applications such as displays, chemical or mechanical sensing, and anti‐counterfeiting.     

Dual‐Emission Channels for Simultaneous Sensing of Cysteine and Homocysteine in Living Cells

The development of a fluorescent probe to distinguish between cysteine (Cys) and homocysteine (Hcy) is always a challenge owing to their structural similarity, and the simultaneous detection of Cys and Hcy by utilizing different emission channels is especially difficult. In this work, we designed and synthesized a new fluorescent probe to differentiate between Cys and Hcy on the basis of a coumarin derivative with a chlorine atom and an α,β‐unsaturated aldehyde. Cys and Hcy induced different cascade reactions with the probe, which led to different products with distinct photophysical properties. The nonfluorescent probe responded to Cys and emitted strong blue fluorescence, whereas it reacted with Hcy and generated yellow fluorescence without interference from glutathione. In addition, the probe was successfully applied to distinguish between Cys and Hcy in living cells.     

In Vitro Light‐Up Visualization of a Subunit‐Specific Enzyme by an AIE Probe via Restriction of Single Molecular Motion

Enzymes contain several subunits to maintain different biological functions. However, it remains a great challenge for specific discrimination of one subunit over another. Toward this end, the fluorescent probe TPEMA is now presented for highly specific detection of the B subunit of cytosolic creatine (CK) kinase isoenzyme (CK‐B). Owing to its aggregation‐induced emission property, TPEMA shows highly boosted emission toward CK‐B with a fast response time and very low interference from other analytes, including the M subunit of CK (CK‐M). With the aid of a Job plot assay, ITC assay and molecular dynamics simulation, it was directly confirmed that the remarkably enhanced fluorescence of TPEMA in the presence of CK‐B results from the restriction of single molecular motion in the cavity. Selective wash‐free fluorescence imaging of CK‐B in macrophages under different treatments was successfully demonstrated.     

DCDHF Fluorophores for Single‐Molecule Imaging in Cells

There is a persistent need for small‐molecule fluorescent labels optimized for single‐molecule imaging in the cellular environment. Application of these labels comes with a set of strict requirements: strong absorption, efficient and stable emission, water solubility and membrane permeability, low background emission, and red‐shifted absorption to avoid cell autofluorescence. We have designed and characterized several fluorophores, termed “DCDHF” fluorophores, for use in live‐cell imaging based on the push–pull design: an amine donor group and a 2‐dicyanomethylene‐3‐cyano‐2,5‐dihydrofuran (DCDHF) acceptor group, separated by a π‐rich conjugated network. In general, the DCDHF fluorophores are comparatively photostable, sensitive to local environment, and their chemistries and photophysics are tunable to optimize absorption wavelength, membrane affinity, and solubility. Especially valuable are fluorophores with sophisticated photophysics for applications requiring additional facets of control, such as photoactivation. For example, we have reengineered a red‐emitting DCDHF fluorophore so that it is dark until photoactivated with a short burst of low‐intensity violet light. This molecule and its relatives provide a new class of bright photoactivatable small‐molecule fluorophores, which are needed for super‐resolution imaging schemes that require active control (here turning‐on) of single‐molecule emission.     

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.     

Assembly of Water-soluble AIE-active Fluorescent Organic Nanoparticles for Ratiometric Detection of Hypochlorite in Living Cells

Hypochlorous acid (HOCl) plays a crucial role in many physiological processes and is widely used as bleach, deodorant and fungicide. In this work, we designed an amphiphilic hydrazone fluorescent molecule THG-1 containing hydrophilic sugar units and hydrophobic tetraphenylethylene unit for ratiometric detection of HOCl with high sensitivity and excellent selectivity based on HOCl-triggered hydrolyzation reaction and aggregation-induced emission (AIE) effect. The detection mechanism was verified by liquid chromatograph mass spectrometry experiments and scanning electron microscope (SEM) tests. Contrast experiments revealed that the numbers of lactose unit and hydrazone linker were essential for assembly of THG-1 and detection of HOCl. In addition, THG-1 was successfully used for imaging of exogenous and endogenous HOCl in living cells.     

Recent Advances in the Z/E Isomers of Tetraphenylethene Derivatives: Stereoselective Synthesis,AIE Mechanism,Photophysical Properties,and Application as Chemical Probes

Focused research on the Z/E isomers of tetraphenylethene (TPE) derivatives is scarce in comparison with the thousands of luminogens with AIE properties (AIEgens) that have been synthesized based on the TPE moiety. The similar chemical and physical properties of the Z/E isomers make them difficult to separate by using conventional chromatographic techniques. However, they can be isolated by introducing polar groups and the pure isomers exhibit very different photophysical properties, mechanochromism, and host–guest coordination, as well as assisting in deciphering the AIE mechanism. In this Minireview, we present an overview of the disagreement regarding the AIE mechanism between the restriction of intramolecular vibration and photoinduced Z/E isomerization. Then, we discuss the development of (Z)‐/(E)‐TPE derivatives, their use in host–guest detection, and their mechanoluminescence properties, with a focus on their photophysical characteristics. Finally, we explore the stereoselective synthesis of pure (Z)‐/(E)‐TPE derivatives.     

A Sequential Dual‐Lock Strategy for Photoactivatable Chemiluminescent Probes Enabling Bright Duplex Optical Imaging

Chemiluminescence (CL)‐based technologies have revolutionized in vivo monitoring of biomolecules. However, significant technical hurdles have limited the achievement of trigger‐controlled, bright, and enriched CL signal. Herein, a dual‐lock strategy uses sequence‐dependent triggers for bright optical imaging with real‐time fluorescent signal and ultra‐sensitive CL signal. These probes can obtain an analyte‐triggered accumulation of stable pre‐chemiluminophore with aggregation‐induced emission (AIE), and then the pre‐chemiluminophore exhibits a rapid photooxidation process (1,2‐dioxetane generation) by TICT‐based free‐radical addition, thereby achieving an enrichment and bright CL signal. The dual‐lock strategy expands the in vivo toolbox for highly accurate analysis and has for the first time allowed access to accurately sense and trace biomolecules with high‐resolution, dual‐mode of chemo‐fluoro‐luminescence, and three‐dimensional (3D) imaging in living animals.     

Water‐Soluble Triarylborane Chromophores for One‐ and Two‐Photon Excited Fluorescence Imaging of Mitochondria in Cells

Three water‐soluble tetracationic quadrupolar chromophores comprising two three‐coordinate boron π‐acceptor groups bridged by thiophene‐containing moieties were synthesised for biological imaging applications. Compound 3 containing the bulkier 5‐(3,5‐Me₂C₆H₂)‐2,2′‐(C₄H₂S)₂‐5′‐(3,5‐Me₂C₆H₂) bridge is stable over a long period of time, exhibits a high fluorescence quantum yield and strong one‐ and two‐photon absorption (TPA), and has a TPA cross section of 268 GM at 800 nm in water. Confocal laser scanning fluorescence microscopy studies in live cells indicated localisation of the chromophore at the mitochondria; moreover, cytotoxicity measurements proved biocompatibility. Thus, chromophore 3 has excellent potential for one‐ and two‐photon‐excited fluorescence imaging of mitochondrial function in cells.     

A “Distorted‐BODIPY”‐Based Fluorescent Probe for Imaging of Cellular Viscosity in Live Cells

Cellular viscosity is a critical factor in governing diffusion‐mediated cellular processes and is linked to a number of diseases and pathologies. Fluorescent molecular rotors (FMRs) have recently been developed to determine viscosity in solutions or biological fluid. Herein, we report a “distorted‐BODIPY”‐based probe BV‐1 for cellular viscosity, which is different from the conventional “pure rotors”. In BV‐1 , the internal steric hindrance between the meso‐CHO group and the 1,7‐dimethyl group forced the boron–dipyrrin framework to be distorted, which mainly caused nonradiative deactivation in low‐viscosity environment. BV‐1 gave high sensitivity (x=0.62) together with stringent selectivity to viscosity, thus enabling viscosity mapping in live cells. Significantly, the increase of cytoplasmic viscosity during apoptosis was observed by BV‐1 in real time.     

Peptide‐Templated Acyl Transfer: A Chemical Method for the Labeling of Membrane Proteins on Live Cells

The development of a method is described for the chemical labeling of proteins which occurs with high target specificity, proceeds within seconds to minutes, and offers a free choice of the reporter group. The method relies upon the use of peptide templates, which align a thioester and an N‐terminal cysteinyl residue such that an acyl transfer reaction is facilitated at nanomolar concentrations. The protein of interest is N‐terminally tagged with a 22 aa long Cys‐E3 peptide (acceptor), which is capable of forming a coiled‐coil with a reporter‐armed K3 peptide (donor). This triggers the transfer of the reporter to the acceptor on the target protein. Because ligation of the two interacting peptides is avoided, the mass increase at the protein of interest is minimal. The method is exemplified by the rapid fluorescent labeling and fluorescence microscopic imaging of the human Y₂ receptor on living cells.