Fluorescent Probes for Selective Recognition of Hypobromous Acid: Achievements and Future Perspectives

Reactive oxygen species (ROS) have been implicated in numerous pathological processes and their homeostasis facilitates the dynamic balance of intracellular redox states. Among ROS, hypobromous acid (HOBr) has a high similarity to hypochlorous acid (HOCl) in both chemical and physical properties, whereas it has received relatively little attention. Meanwhile, selective recognition of endogenous HOBr suffers great challenges due to the fact that the concentration of this molecule is much lower than that of HOCl. Fluorescence-based detection systems have emerged as very important tools to monitor biomolecules in living cells and organisms owing to distinct advantages, particularly the temporal and spatial sampling for in vivo imaging applications. To date, the development of HOBr-specific fluorescent probes is still proceeding quite slowly, and the research related to this area has not been systematically summarized. In this review, we are the first to review the progress made so far in fluorescent probes for selective recognition and detection of HOBr. The molecular structures, sensing mechanisms, and their successful applications of these probes as bioimaging agents are discussed here in detail. Importantly, we hope this review will call for more attention to this rising field, and that this could stimulate new future achievements.     

Acidic‐pH‐Activatable Fluorescence Probes for Visualizing Exocytosis Dynamics

Live imaging of exocytosis dynamics is crucial for a precise spatiotemporal understanding of secretion phenomena, but current approaches have serious limitations. We designed and synthesized small‐molecular fluorescent probes that were chemically optimized for sensing acidic intravesicular pH values, and established that they can be used to sensitively and reliably visualize vesicular dynamics following stimulation. This straightforward technique for the visualization of exocytosis as well as endocytosis/reacidification processes with high spatiotemporal precision is expected to be a powerful tool for investigating dynamic cellular phenomena involving changes in the pH value.     

A Sensitive and Selective Fluorescent Probe for Cysteine Based on a New Response‐Assisted Electrostatic Attraction Strategy: The Role of Spatial Charge Configuration

A new strategy for fast fluorescent detection of cysteine (Cys), based on a response‐assisted electrostatic attraction, is demonstrated. By utilizing this strategy, we designed and synthesized three fluorescent probes for the specific detection of Cys under actual physiological conditions. The probe m‐ CP , a coumarin fluorophore conjugated with a substituted methyl pyridinium group through an unsaturated ketone unit, showed highly selective and sensitive detection for cysteine (Cys) over homocysteine (Hcy) and glutathione (GSH). The kinetic analysis indicated that the sensing process was highly accelerated (a response time less than 1 min) by the response‐assisted electrostatic attraction. More importantly, control experiments with isomeric probes first demonstrated that the spatial charge configuration of the probe played an important role in Cys‐preferred selectivity and kinetic rate acceleration. Furthermore, the practical utility of the probe m‐ CP in the fluorescent labeling of Cys residues within proteins was demonstrated. Finally, these probes were employed in living cell imaging with HeLa cells, in which it displayed satisfactory cell permeability and enabled us to distinguish active thiols in the cytoplasm, nucleus, and mitochondria.     

Imaging molecular events in single living cells

Fluorescence imaging could be the most powerful technique available for observing spatial and temporal dynamics of biomolecules in living cells, if fluorescent indicators for the relevant biomolecules become available. We have recently developed fluorescent indicators for a variety of second messengers or protein phosphorylations. Using the indicators, we have visualized spatial and temporal dynamics of these molecular events in single living cells. These fluorescent indicators are becoming an indispensable tool for understanding the complex mechanism of signal transduction in living cells.     

荧光纳米探针用于细胞器pH检测的研究进展

pH稳态对于维持活细胞细胞器的正常功能具有重要作用. 细胞器内pH稳态被打破会导致细胞器功能的紊乱, 进而引发癌症、 神经退行性疾病等相关疾病. 因此, 在活细胞水平上定量测定pH并对其波动进行实时监测对于理解相关疾病的发生机制非常重要. 基于非侵入、 高时空分辨率成像的优势, 荧光探针非常适合用于活细胞内pH的检测. 本综述总结了近些年利用不同种类荧光纳米探针对不同细胞器进行pH成像的研究工作, 并对荧光纳米探针应用面临的机遇与挑战进行了展望.     

ECHO probes: a concept of fluorescence control for practical nucleic acid sensing

An excitonic interaction caused by the H-aggregation of fluorescent dyes is a new type of useful photophysical process for fluorescence-controlled nucleic acid sensing. This critical review points out the recent advances in exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes, which have a fluorescence-labeled nucleotide in which two molecules of thiazole orange or its derivatives are linked covalently. ECHO probes show absorption shift and emission switching depending on hybridization with the target nucleic acid. The hybridization-sensitive fluorescence emission of ECHO probes and the further modification of probes have made possible a variety of practical applications, such as multicolor RNA imaging in living cells and facile detection of gene polymorphism (144 references).     

Photochemical control of biological processes

Photochemical regulation of biological processes offers a high level of control to study intracellular mechanisms with unprecedented spatial and temporal resolution. This report summarizes the advances made in recent years, focusing predominantly on the in vivo regulation of gene function using irradiation with UV light. The majority of the described applications entail the utilization of photocaging groups installed either on a small molecule modulator of biomolecular function or directly on a biological macromolecule itself.     

Micrometer-scale transient ion transport for real-time pH assay in living rat brains

Ion transport has been widely used for various applications such as sensing, desalination and energy conversion; however, nearly all applications are based on steady-state ion transport. Herein, we for the first time demonstrate the capability of transient ion transport for in vivo sensing with both high spatial (∼μm) and temporal (∼ms) resolution by using pH as the model target. Transient ion transport behavior (i.e., time-dependent ion current change) was observed by applying high-frequency pulse potential. Importantly, we proposed the ion distribution transient model for this time-dependent ion transport behavior. With this model, the temporal resolution of the as-developed pH microsensor based on ion current was improved to the ms level, thus satisfying the requirement of neurochemical recording. Moreover, our microsensor features good reproducibility, selectivity, and reversibility, and can thus real-time monitor the pH change in living rat brains. This study demonstrates the first example of in vivo sensing based on ion transport, opening a new way to neurochemical monitoring with ultrahigh spatiotemporal resolution. This study is also helpful to understand the transient process of asymmetric ion transport.

Micrometer-scale transient ion transport has been successfully used for constructing a high spatiotemporal resolution and performance microsensor, which could be used for real-time monitoring the change of pH in rat brains.     

Enhanced fluorescence cyanide detection at physiologically lethal levels: reduced ICT-based signal transduction

Three water-soluble fluorescent probes have been specifically designed to determine free cyanide concentrations up to physiologically lethal levels, >20 microM. The probes have been designed in such a way as to afford many notable sensing features, which render them unique with regard to signal transduction, photophysical characteristics, and their application to physiological cyanide determination and safeguard. The probes are readily able to reversibly bind free aqueous cyanide with dissociation constants around 4 microM3. Subsequent cyanide binding modulates the intramolecular charge transfer within the probes, a change in the electronic properties within the probes, resulting in enhanced fluorescence optical signals as a function of increased solution cyanide concentration. The ground-state chelation with cyanide produces wavelength shifts, which also enable the probes to sense cyanide in both an excitation and emission ratiometric manner, in addition to enhanced fluorescence signaling. This has enabled a generic cyanide sensing platform to be realized that is not dependent on fluorescent probe concentration, probe photodegradation, or fluctuations in the intensity of any employed excitation sources, ideal for remote cyanide sensing applications. Further, the >600 nm fluorescence emission of the probes potentially allows for enhanced fluorescence ratiometric cyanide sensing in the optical window of tissues and blood, facilitating their use for the transdermal monitoring of cyanide for mammalian safeguard or postmortem in fire victims, both areas of active research.     

Synthesis and Supramolecular Functional Assemblies of Ratiometric pH Probes

Tracking pH with spatiotemporal resolution is a critical challenge for synthetic chemistry, chemical biology and beyond. Over the last decade, different small probes and supramolecular systems have emerged for in cellulo or in vivo pH tracking. However, pH reporting still presents critical limitations, such as background reduction, improved sensor stability, cell targeting, endosomal escape, near- and far-infrared ratiometric pH tracking and adaption to new imaging techniques (i.e., super-resolution). These challenges will require the combined efforts of synthetic and supramolecular chemistry working together to develop the next generation of smart materials that will resolve current limitations. Herein, recent advances in the synthesis of small fluorescent probes, together with new supramolecular functional systems employed for pH tracking, are described with an emphasis on ratiometric probes. The combination of organic synthesis and stimuli-responsive supramolecular functional materials will be essential to solve future challenges of pH tracking, such as improved signal to noise ratio, on target activation and microenvironment reporting.     

基于功能核酸的细胞荧光成像

细胞是生物体基本的结构和功能单元,对活细胞中特定生物组分进行动态分析,将为相关生命活动过程的研究提供重要信息。荧光成像为细胞分析提供了一种操作简单、灵敏度高、可实时监测细胞微观动态分子过程的光学生物成像技术。发展高性能的荧光探针用于活细胞成像已成为研究热点。功能核酸是一类具有特殊化学和生物学功能的寡核苷酸分子,除了天然存在的核酶(Ribozyme)和核糖开关(Riboswitch)之外,还包括通过指数富集的配体系统进化技术(SELEX)筛选获得的核酸适体和脱氧核酶(DNAzyme)。功能核酸由于具有合成简单、免疫原性低、相对分子质量小、化学稳定性高、易于修饰等优点,在生物成像领域受到广泛关注。本文主要综述了基于功能核酸的荧光探针在细胞成像领域中的应用研究,总结了该领域面临的挑战,并对其未来发展方向进行了展望。     

Photocaged morpholino oligomers for the light-regulation of gene function in zebrafish and Xenopus embryos

Morpholino oligonucleotides, or morpholinos, have emerged as powerful antisense reagents for evaluating gene function in both in vitro and in vivo contexts. However, the constitutive activity of these reagents limits their utility for applications that require spatiotemporal control, such as tissue-specific gene disruptions in embryos. Here we report a novel and efficient synthetic route for incorporating photocaged monomeric building blocks directly into morpholino oligomers and demonstrate the utility of these caged morpholinos in the light-activated control of gene function in both cell culture and living embryos. We demonstrate that a caged morpholino that targets enhanced green fluorescent protein (EGFP) disrupts EGFP production only after exposure to UV light in both transfected cells and living zebrafish (Danio rerio) and Xenopus frog embryos. Finally, we show that a caged morpholino targeting chordin, a zebrafish gene that yields a distinct phenotype when functionally disrupted by conventional morpholinos, elicits a chordin phenotype in a UV-dependent manner. Our results suggest that photocaged morpholinos are readily synthesized and highly efficacious tools for light-activated spatiotemporal control of gene expression in multiple contexts.     

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.     

Recent advances in formaldehyde-responsive fluorescent probes

Formaldehyde is one of the simplest reactive carbonyl species. In view of the harmfulness of formaldehyde in nature and humans, it is of great signifi cance to further elucidate roles and functions of formaldehyde by a noninvasive detection approach. Fluorescent probes have become a popular tool to track and detect formaldehyde in vitro and in vivo, which have attracted more and more interest recently. This review focuses on various reaction mechanisms to design the fluorescent probes for detecting formaldehyde.     

Sensitive and rapid detection of endogenous hydrogen sulfide distributing in different mouse viscera via a two-photon fluorescent probe

Development of efficient methods for detection of endogenous H₂S in living cells and tissues is of considerable signi?cance for better understanding the biological and pathological functions of H₂S. Two-photon (TP) fluorescent probes are favorable as powerful molecular tools for studying physiological process due to its non-invasiveness, high spatiotemporal resolution and deep-tissues imaging. Up to date, several TP probes for intracellular H₂S imaging have been designed, but real-time imaging of endogenous H₂S-related biological processes in tissues is hampered due to low sensitivity, long response time and interference from other biothiols. To address this issue, we herein report a novel two-photon fluorescent probe (TPP-H₂S) for highly sensitive and fast monitoring and imaging H₂S levels in living cells and tissues. In the presence of H₂S, it exhibits obviously improved sensitivity (LOD: 0.12 μM) and fast response time (about 2 min) compared with the reported two-photon H₂S probes. With two-photon excitation, TPP-H₂S displays high signal-to-noise ratio and sensitivity even no interference in cell growth media. As further application, TPP-H₂S is applied for fast imaging of H₂S in living cells and different fresh tissues by two-photon confocal microscope. Most importantly we first measured the endogenous H₂S level in different viscera by vivisection and found that the distribution of endogenous H₂S mostly in brain, liver and lung. The excellent sensing properties of TPP-H₂S make it a practically useful tool for further studying biological roles of H₂S.     

High efficient probes with Schiff base functional receptors for hypochlorite sensing under physiological conditions

A series of novel and convenient fluorescent probes with Schiff base functionality were presented for direct detection of OCI via the irreversible OCl-promoted oxidation and hydrolyzation reaction in formation of the ring-opened product, fluorescein. Prominent high sensitivity, selectivity and antiinterference OCl-induced fluorescence and color change over a wide range of tested metal ions performance were observed for each probe under physiological conditions, thus making the probes well suitable for sensing of OCl in living cells.     

Recent progress in Michael addition-based fluorescent probes for sulfur dioxide and its derivatives

In this review, we have summarized the recent progress of Michael addition-based fluorescent probes for SO₂ and its derivatives in the design strategies, sensing performances, detection mechanisms and applications.     

硝基取代苯腙类荧光受体的阴离子识别与传感性能

合成了含萘荧光基团的硝基取代苯腙类受体,利用紫外-可见分光光度法、荧光发射光谱法和核磁等方法研究了受体的阴离子识别与光化学传感性能. 结果表明,在DMSO有机溶剂体系中,单硝基取代受体选择性比色和荧光识别氟离子,而双硝基取代受体可以比色和荧光识别氟离子和醋酸根离子. 归因于腙=N-NH基团质子酸性的进一步增强,双硝基取代受体能够在DMSO-H2O体系中实现对氟离子的比色和荧光识别. 此类受体是有效的“off-on”型阴离子荧光传感分子.