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.     

偶氮苯衍生物探针在乏氧细胞成像中的应用

细胞不受控制的生长增殖和异常的血管系统导致肿瘤部位氧气供应不足,氧气浓度低于正常组织.细胞乏氧是大多数实体瘤的共同特征,可用作恶性组织和癌症进展的指标.准确的乏氧检测和成像对癌症患者的诊断和临床治疗至关重要.荧光成像具有高灵敏度、无创、实时等优点,常被用于癌症检查.偶氮基团由于其对荧光基团的荧光猝灭作用和还原断裂荧光恢...     

An Unsymmetrical Squaraine-Based Activatable Probe for Imaging Lymphatic Metastasis by Responding to Tumor Hypoxia with MSOT and Aggregation-Enhanced Fluorescent Imaging

Optoacoustic imaging has great potential for preclinical research and clinical practice, and designing robust activatable optoacoustic probes for specific diseases is beneficial for its further development. Herein, an activatable probe has been developed for tumor hypoxia imaging. For this probe, indole and quinoline were linked on each side of an oxocyclobutenolate core to form an unsymmetrical squaraine. A triarylamine group was incorporated to endow the molecule with the aggregation enhanced emission (AEE) properties. In aqueous media, the squaraine chromophore aggregates into the nanoprobe, which specifically responds to nitroreductase and produces strong optoacoustic signals due to its high extinction coefficient, as well as prominent fluorescence emission as a result of its AEE feature. The nanoprobe was used to image tumor metastasis via the lymphatic system both optoacoustically and fluorescently. Moreover, both the fluorescence signals and three-dimensional multispectral optoacoustic tomography signals from the activated nanoprobe allow us to locate the tumor site and to map the metastatic route.     

Developing Novel Fabrication and Optimisation Strategies on Aggregation-Induced Emission Nanoprobe/Polyvinyl Alcohol Hydrogels for Bio-Applications

The current study describes a new technology, effective for readily preparing a fluorescent (FL) nanoprobe-based on hyperbranched polymer (HB) and aggregation-induced emission (AIE) fluorogen with high brightness to ultimately develop FL hydrogels. We prepared the AIE nanoprobe using a microfluidic platform to mix hyperbranched polymers (HB, generations 2, 3, and 4) with AIE (TPE-2BA) under shear stress and different rotation speeds (0–5 K RPM) and explored the FL properties of the AIE nanoprobe. Our results reveal that the use of HB generation 4 exhibits 30-times higher FL intensity compared to the AIE alone and is significantly brighter and more stable compared to those that are prepared using HB generations 3 and 2. In contrast to traditional methods, which are expensive and time-consuming and involve polymerization and post-functionalization to develop FL hyperbranched molecules, our proposed method offers a one-step method to prepare an AIE-HB nanoprobe with excellent FL characteristics. We employed the nanoprobe to fabricate fluorescent injectable bioadhesive gel and a hydrogel microchip based on polyvinyl alcohol (PVA). The addition of borax (50 mM) to the PVA + AIE nanoprobe results in the development of an injectable bioadhesive fluorescent gel with the ability to control AIEgen release for 300 min. When borax concentration increases two times (100 mM), the adhesion stress is more than two times bigger (7.1 mN/mm²) compared to that of gel alone (3.4 mN/mm²). Excellent dimensional stability and cell viability of the fluorescent microchip, along with its enhanced mechanical properties, proposes its potential applications in mechanobiology and understanding the impact of microstructure in cell studies.     

Fluorescent Bioprobes: Structural Matching in the Docking Processes of Aggregation‐Induced Emission Fluorogens on DNA Surfaces

Whereas most conventional DNA probes are flat disklike aromatic molecules, we explored the possibility of developing quadruplex sensors with nonplanar conformations, in particular, the propeller‐shaped tetraphenylethene (TPE) salts with aggregation‐induced emission (AIE) characteristics. 1,1,2,2‐Tetrakis[4‐(2‐triethylammonioethoxy)phenyl]ethene tetrabromide (TPE‐ 1 ) was found to show a specific affinity to a particular quadruplex structure formed by a human telomeric DNA strand in the presence of K⁺ ions, as indicated by the enhanced and bathochromically shifted emission of the AIE fluorogen. Steady‐state and time‐resolved spectral analyses revealed that the specific binding stems from a structural matching between the AIE fluorogen and the DNA strand in the folding process. Computational modeling suggests that the AIE molecule docks on the grooves of the quadruplex surface with the aid of electrostatic attraction. The binding preference of TPE‐ 1 enables it to serve as a bioprobe for direct monitoring of cation‐driven conformational transitions between the quadruplexes of various conformations, a job unachievable by the traditional G‐quadruplex biosensors. Methyl thiazolyl tetrazolium (MTT) assays reveal that TPE‐ 1 is cytocompatible, posing no toxicity to living cells.     

Tricyano-Methylene-Pyridine Based High-Performance Aggregation-Induced Emission Photosensitizer for Imaging and Photodynamic Therapy

Photosensitizers equipped with high reactive oxygen species (ROS) generation capability and bright emission are essential for accurate tumor imaging and precise photodynamic therapy (PDT). However, traditional aggregation-caused quenching (ACQ) photosensitizers cannot simultaneously produce desirable ROS and bright fluorescence, resulting in poor image-guided therapy effect. Herein, we report an aggregation-induced emission (AIE) photosensitizer TCM-Ph with a strong donor–π–acceptor (D–π–A) structure, which greatly separates the HOMO–LUMO distribution and reduces the ΔE_ST, thereby increasing the number of triplet excitons and producing more ROS. The AIE photosensitizer TCM-Ph has bright near-infrared emission, as well as a higher ROS generation capacity than the commercial photosensitizers Bengal Rose (RB) and Chlorine e6 (Ce6), and can effectively eliminate cancer cells under image guidance. Therefore, the AIE photosensitizer TCM-Ph has great potential to replace the commercial photosensitizers.     

Design of Dual‐Emission Chemosensors for Ratiometric Detection of ATP Derivatives

Nucleoside pyrophosphate (nucleoside PP) derivatives are widespread in living cells and play pivotal roles in various biological events. We report novel fluorescence chemosensors for nucleoside PPs that make use of coordination chemistry. The chemosensors, which contain two Zn^II–dipicolylamine units, bind strongly to nucleoside PPs (K_app>10⁶ M ^?1) in aqueous solution and sense them by a dual‐emission change. Detailed fluorescence and UV/Vis spectral studies revealed that the emission changes of the chemosensors upon binding to nucleoside PPs can be ascribed to the loss of coordination between Zn^II and the acridine fluorophore. This is a unique sensing system based on the anion‐induced rearrangement of the coordination. Furthermore, we demonstrated the utility of these chemosensors in real‐time monitoring of two important biological processes involving nucleoside PP conversion: the apyrase‐catalyzed hydrolysis of nucleoside PPs and the glycosyl transfer catalyzed by β‐1,4‐galactosyltransferase.     

A FRET Probe for Cell‐Based Imaging of Ganglioside‐Processing Enzyme Activity and High‐Throughput Screening

Gangliosides are important signaling molecules in the cell membrane and are processed by several enzymes. Deficiencies in these enzymes can cause human lysosomal storage diseases. Building an understanding of the pathways of glycosphingolipid catabolism requires methods for the analysis of these enzymatic activities A GM3‐derived FRET probe was synthesized chemoenzymatically for the detection and quantitation of a range of ganglioside‐degrading enzymes, both in cell lysates and in living cells. This is the first substrate that enables the ratiometric fluorogenic assay of sphingolipid ceramide N‐deacylase and endoglycoceramidase and can detect and localize neuraminidase activity in living cells. It is therefore a valuable tool for building a better understanding of membrane‐confined enzymology. It also enables the robust and reliable assay of ganglioside‐degrading enzymes in a microtiter plate, thus opening the door to screening for novel or engineered biocatalysts or for new inhibitors.     

Fluorogenic Protein Probes with Red and Near-Infrared Emission for Genetically Targeted Imaging**

Fluorogenic probes are important tools to image proteins with high contrast and no wash protocols. In this work, we rationally designed and synthesized a small set of four protein fluorogens with red or near-infrared emission. The fluorophores were characterized in the presence of albumin as a model protein environment and exhibited good fluorogenicity and brightness (fluorescence quantum yield up to 36 %). Once conjugated to a haloalkane ligand, the probes reacted with the protein self-labeling tag HaloTag with a high fluorescence enhancement (up to 156-fold). The spectroscopic properties of the fluorogens and their reaction with HaloTag were investigated experimentally in vitro and with the help of molecular dynamics. The two most promising probes, one in the red and one in the near-infrared range, were finally applied to image the nucleus or actin in live-cell and in wash-free conditions using fluorogenic and chemogenetic targeting of HaloTag fusion proteins.     

Fluorination Enhances NIR-II Fluorescence of Polymer Dots for Quantitative Brain Tumor Imaging

Here, we describe a fluorination strategy for semiconducting polymers for the development of highly bright second near-infrared region (NIR-II) probes. Tetrafluorination yielded a fluorescence QY of 3.2 % for the polymer dots (Pdots), over a 3-fold enhancement compared to non-fluorinated counterparts. The fluorescence enhancement was attributable to a nanoscale fluorous effect in the Pdots that maintained the molecular planarity and minimized the structure distortion between the excited state and ground state, thus reducing the nonradiative relaxations. By performing through-skull and through-scalp imaging of the brain vasculature of live mice, we quantitatively analyzed the vascular morphology of transgenic brain tumors in terms of the vessel lengths, vessel branches, and vessel symmetry, which showed statistically significant differences from the wild type animals. The bright NIR-II Pdots obtained through fluorination chemistry provide insightful information for precise diagnosis of the malignancy of the brain tumor.     

Bio‐/Chemosensors and Imaging with Aggregation‐Induced Emission Luminogens

Aggregation‐induced emission (AIE) luminogens show abnormal fluorescent behavior; they are non‐emissive in solution, but they become strongly emissive after aggregation. Sensing and imaging are the major applications of AIE luminogens. By properly manipulating the aggregation and deaggregation of AIE molecules, various bio‐/chemosensors have been developed. Moreover, AIE molecules with targeting groups have been devised for imaging of organelles and cancer cells. In this account, we report our recent work on the application of AIE luminogens for the construction of bio‐/chemosensors and imaging.

     

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.     

Ultra‐fast Cycling for Multiplexed Cellular Fluorescence Imaging

Rapid analysis of single and scant cell populations is essential in modern diagnostics, yet existing methods are often limited and slow. Herein, we describe an ultra‐fast, highly efficient cycling method for the analysis of single cells based on unique linkers for tetrazine (Tz)/trans‐cyclooctene (TCO)‐mediated quenching. Surprisingly, the quenching reaction rates were more than 3 orders of magnitude faster (t_1/2 <1 s) than predicted. This allowed multi‐cycle staining and immune cell profiling within an hour, leveraging the accelerated kinetics to open new diagnostic possibilities for rapid cellular analyses.     

用于生物标记的半导体量子点研究

半导体量子点的独特光学性质使之成为理想的荧光探针材料,在生物医学领域具有广阔的应用前景.本文评述了目前量子点合成、表面修饰、结合生物分子的方法,以及半导体量子点在生物标记应用中相对于传统有机染料的优点.     

Multistimuli-Responsive Luminescence of Naphthalazine Based on Aggregation-Induced Emission

Stimuli-responsive luminescent materials, which are dependent on changes in physical molecular packing modes, have attracted more and more interest over the past ten years. In this study, 2,2-dihydroxy-1,1-naphthalazine was synthesized and shown to exhibit different fluorescence emission in solution and solid states with characteristic aggregation-induced emission (AIE) properties. A remarkable change in the fluorescence of 2,2-dihydroxy-1,1-naphthalazine occurred upon mechanical grinding, heating, or exposure to solvents. According to the characterization by solid-state fluorescence spectroscopy, X-ray crystallography, differential scanning calorimetry, and X-ray powder diffraction, the fluorescence change could be attributed to transitions between two structurally different polymorphs. These significant properties could also give 2,2-dihydroxy-1,1-naphthalazine more potential applications as a multifunctional material.     

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.     

Intracellular Self-assembly of TPE-biotin Nanoparticles Enables Aggregation-Induced Emission Fluorescence for Cancer-Targeted Imaging

Fluorogens with aggregation-induced emission (AIE) characteristics have recently been widely applied for studying biological events, and fluorogens with "smart" properties are especially desirable. Herein, we rationally designed and synthesized a biotinylated and reduction-activatable probe (Cys(StBu)-Lys(biotin)-Lys(TPE)-CBT (begin{document}$textbf{1}$end{document})) with AIE properties for cancer-targeted imaging. The biotinylated probe begin{document}$textbf{1}$end{document} can be actively uptaken by the biotin receptor-overexpressing cancer cells, and then "smartly" self-assemble into nanoparticles inside cells and turn the fluorescence "On". Employing this "smart" strategy, we successfully applied probe begin{document}$textbf{1}$end{document} for cancer-targeted imaging. We envision that this biotinylated intelligent probe begin{document}$textbf{1}$end{document} might be further developed for cancer-targeted imaging in routine clinical studies in the near future.