A unique paradigm for a Turn-ON near-infrared cyanine-based probe: noninvasive intravital optical imaging of hydrogen peroxide

The development of highly sensitive fluorescent probes in combination with innovative optical techniques is a promising strategy for intravital noninvasive quantitative imaging. Cyanine fluorochromes belong to a superfamily of dyes that have attracted substantial attention in probe design for molecular imaging. We have developed a novel paradigm to introduce a Turn-ON mechanism in cyanine molecules, based on a distinctive change in their π-electrons system. Our new cyanine fluorochrome is synthesized through a simple two-step procedure and has an unprecedented high fluorescence quantum yield of 16% and large extinction coefficient of 52,000 M(-1)cm(-1). The synthetic strategy allows one to prepare probes for various analytes by introducing a specific triggering group on the probe molecule. The probe was equipped with a corresponding trigger and demonstrated efficient imaging of endogenous hydrogen peroxide, produced in an acute lipopolysaccharide-induced inflammation model in mice. This approach provides, for the first time, an available methodology to prepare modular molecular Turn-ON probes that can release an active cyanine fluorophore upon reaction with specific analyte.     

Spectroscopic probes with changeable π-conjugated systems

Spectroscopic probes have been extensively investigated and used widely in many fields because of their powerful ability to improve analytical sensitivity, and to offer greater temporal and spatial resolution (in some cases a molecule event may be visualized by the naked eye). So far, different photophysical mechanisms, such as charge transfer, photo-induced electron transfer and fluorescent resonance energy transfer, have been employed to develop various spectroscopic probes with superior properties. However, these photophysical mechanisms depend on the energy levels of molecular orbitals, which are usually difficult to accurately determine. This would lead to the poor prediction of analytical performance of the designed probe. Instead, the change of π-conjugated systems induced by chemical reactions is often accompanied by a distinct alteration in spectroscopic signal, which is more predictable and is of high signal/background ratio. This mechanism can serve as an effective measure for developing excellent spectroscopic probes, but to our knowledge, has not been systematically summarized. In this feature article, we review the development of spectroscopic probes with changeable π-conjugated systems, which is catalogued according to the fluorochromes: fluorescein, rhodamine, spiropyran, squaraine, coumarin, cyanine, etc. Two main strategies for constructing these spectroscopic probes, including ring-closing reaction and nucleophilic addition reaction, are summarized, and the merits and limitations of the probes are discussed.     

Systematic investigation of the aza-Cope reaction for fluorescence imaging of formaldehyde in vitro and in vivo

Increasing evidence has highlighted the endogenous production of formaldehyde (FA) in a variety of fundamental biological processes and its involvement in many disease conditions ranging from cancer to neurodegeneration. To examine the physiological and pathological relevance and functions of FA, fluorescent probes for FA imaging in live biological samples are of great significance. Herein we report a systematic investigation of 2-aza-Cope reactions between homoallylamines and FA for identification of a highly efficient 2-aza-Cope reaction moiety and development of fluorescent probes for imaging FA in living systems. By screening a set of N-substituted homoallylamines and comparing them to previously reported homoallylamine structures for reaction with FA, we found that N-p-methoxybenzyl homoallylamine exhibited an optimal 2-aza-Cope reactivity to FA. Theoretical calculations were then performed to demonstrate that the N-substituent on homoallylamine greatly affects the condensation with FA, which is more likely the rate-determining step. Moreover, the newly identified optimal N-p-methoxybenzyl homoallylamine moiety with a self-immolative β-elimination linker was generally utilized to construct a series of fluorescent probes with varying excitation/emission wavelengths for sensitive and selective detection of FA in aqueous solutions and live cells. Among these probes, the near-infrared probe FFP706 has been well demonstrated to enable direct fluorescence visualization of steady-state endogenous FA in live mouse brain tissues and elevated FA levels in a mouse model of breast cancer. This study provides the optimal aza-Cope reaction moiety for FA probe development and new chemical tools for fluorescence imaging and biological investigation of FA in living systems.

Systematic investigation of various homoallylamines reveals N-p-methoxybenzyl homoallylamine as the optimal 2-aza-Cope reaction moiety for development of highly efficient formaldehyde fluorescent probes for in vitro and in vivo imaging.     

生物硫醇荧光探针的研究进展

生物硫醇(包含半胱氨酸、高半胱氨酸和谷胱甘肽)在生命活动中扮演了重要的角色,其浓度的异常变化与某些疾病息息相关,因此对硫醇的检测具有重要意义.荧光探针因具有灵敏度高、时空分辨率好、无损伤、可视化等优势,在生物硫醇的检测方面得到了高度重视.利用硫醇在分子结构上的共同点(含巯基的氨基酸)和差异(分子大小、亲核性、空间位阻、细胞内含量),可通过迈克尔加成、亲核芳基取代、加成环化等反应实现对硫醇的选择性检测.综述了近3年来硫醇荧光探针领域的研究进展.首先介绍了对硫醇有选择性识别的荧光探针,随后分类讨论了对半胱氨酸、高半胱氨酸和谷胱甘肽各具有特异性检测的荧光探针,并重点介绍了分子设计、识别机理、荧光性质和成像应用,初步探讨了部分探针在监测细胞生命活动中的作用,同时还对本领域的发展提出了展望.     

Aggregation-Induced Emission: Recent Advances in Materials and Biomedical Applications

The concept of aggregation-induced emission (AIE) has opened new opportunities in many research fields. Motivated by the unique feature of AIE fluorogens (AIEgens), during the past decade, many AIE molecular probes and AIE nanoparticle (NP) probes have been developed for sensing, imaging and theranostic applications with excellent performance outperforming conventional fluorescent probes. This Review summarizes the latest advancement of AIE molecular probes and AIE NP probes and their emerging biomedical applications. Special focus is to reveal how the AIE probes are evolved with the development of new multifunctional AIEgens, and how new strategies have been developed to overcome the limitations of traditional AIE probes for more translational applications via fluorescence imaging, photoacoustic imaging and image-guided photodynamic/photothermal therapy. The outlook discusses the challenges and future opportunities for AIEgens to advance the biomedical field.     

分子影像探针

分子影像是近年出现并迅速发展的一个生物医学领域,在疾病的治疗与诊断中发挥着重要作用。同时它又是一门交叉学科,涉及化学、医学、生物、计算机科学、放射科学、材料科学等。分子影像的发展除了需要先进的成像设备外,最关键的是合成新型而高效的成像探针。目前,分子影像探针广泛应用于科学研究和临床,并且也取得了巨大进步。本文主要综述了5种常见的分子影像探针:超声成像探针、X-射线计算机断层成像探针、光学成像探针、核磁共振成像探针、正电子发射计算机断层扫描成像探针,并对分子影像探针的应用进行了概述,最后对分子影像探针的发展进行了展望。     

Aggregation‐Induced Emission: Recent Advances in Materials and Biomedical Applications

The concept of aggregation‐induced emission (AIE) has opened new opportunities in many research fields. Motivated by the unique feature of AIE fluorogens (AIEgens), during the past decade, many AIE molecular probes and AIE nanoparticle (NP) probes have been developed for sensing, imaging and theranostic applications with excellent performance outperforming conventional fluorescent probes. This Review summarizes the latest advancement of AIE molecular probes and AIE NP probes and their emerging biomedical applications. Special focus is to reveal how the AIE probes are evolved with the development of new multifunctional AIEgens, and how new strategies have been developed to overcome the limitations of traditional AIE probes for more translational applications via fluorescence imaging, photoacoustic imaging and image‐guided photodynamic/photothermal therapy. The outlook discusses the challenges and future opportunities for AIEgens to advance the biomedical field.     

Optical imaging techniques in microfluidics and their applications

Microfluidic devices have undergone rapid development in recent years and provide a lab-on-a-chip solution for many biomedical and chemical applications. Optical imaging techniques are essential in microfluidics for observing and extracting information from biological or chemical samples. Traditionally, imaging in microfluidics is achieved by bench-top conventional microscopes or other bulky imaging systems. More recently, many novel compact microscopic techniques have been developed to provide a low-cost and portable solution. In this review, we provide an overview of optical imaging techniques used in microfluidics followed with their applications. We first discuss bulky imaging systems including microscopes and interferometer-based techniques, then we focus on compact imaging systems that can be better integrated with microfluidic devices, including digital in-line holography and scanning-based imaging techniques. The applications in biomedicine or chemistry are also discussed along with the specific imaging techniques.     

Optimized Fluorescent Probe for Specific Imaging of Glutathione S‐Transferases in Living Cells and Mice

GSTP1 has been considered to be a marker for malignancy in many tissues. However, the existing GST fluorescent probes are unfavorable for in vivo imaging because of the limited emission wavelength or insufficient fluorescence enhancement (six‐fold). The limited fluorescence enhancement of GST fluorescent probes is mainly ascribed to the high background signals resulting from the spontaneous reaction between GSH and the probes. In this work, a highly specific GST probe with NIR emission has been successfully developed through optimization of the essential unit of the probe to repress the spontaneous reaction. The novel GST probe exhibits over 100‐fold fluorescence enhancement upon incubation with GSTP1/GSH and high selectivity over other potential interference. In addition, the probe has been proved to be capable of tracking endogenous GST in A549 cells. Finally, the in vivo imaging results demonstrate that the probe can be used for effective imaging of endogenous GST activity in subcutaneous tumor mouse with high contrast.     

Strategies for in vivo imaging of enzyme activity: an overview and recent advances

Imaging of enzyme activity in living subjects promises many applications in both basic and translational researches from helping elucidate the enzyme function and mechanism in biology to better disease detection and monitoring, but the complexity and dynamics of enzymatic reactions in living systems present unique challenges for probe design. This critical review examines the approaches in recent literature to in vivo imaging of the activity of a variety of enzyme targets with an emphasis on the chemical perspective of probe design, structure and function. Strategies for designing enzyme-activated probes based on a variety of molecular scaffolds including small molecules, organic and inorganic nanoparticles, and genetically encoded proteins for commonly used molecular imaging modalities--whole body optical (fluorescence, bioluminescence) imaging, magnetic resonance imaging, and radionuclide-based tomographic imaging, are critically evaluated. Recent advances in combining multiple modalities to imaging enzyme activity in living subjects are also highlighted (255 references).     

Near-infrared fluorescent molecular probes for imaging and diagnosis of nephro-urological diseases

Near-infrared (NIR) fluorescence imaging has improved imaging depth relative to conventional fluorescence imaging in the visible region, demonstrating great potential in both fundamental biomedical research and clinical practice. To improve the detection specificity, NIR fluorescence imaging probes have been under extensive development. This review summarizes the particular application of optical imaging probes with the NIR-I window (700–900 nm) or the NIR-II window (1000–1700 nm) emission for diagnosis of nephron-urological diseases. These molecular probes have enabled contrast-enhanced imaging of anatomical structures and physiological function as well as molecular imaging and early diagnosis of acute kidney injury, iatrogenic ureteral injury and bladder cancer. The design strategies of molecular probes are specifically elaborated along with representative imaging applications. The potential challenges and perspectives in this field are also discussed.

Near-infrared fluorescent molecular probes with improved imaging depth and optimized biodistribution have been reviewed, showing great potential for diagnosis of nephro-urological diseases.     

A melatonin-based targetable fluorescent probe for screening of tumor cells and real-time imaging of glutathione fluctuations in tumor cells

Glutathione(GSH) is a key maintainer of cellular redox balance and plays an important role in many physiological effects. For example, GSH has been widely implicated in cancer initiation, progression and metastasis. Moreover, the concentrations of GSH in tumor cells can influence drug resistance. Given the serious harmfulness of cancer and the important roles of GSH in cancer, it has great significance to development probes for screening of tumor cells and real-time monitoring of GSH fluctuation...     

用于一氧化氮检测的小分子荧光探针研究进展

生物小分子NO以其重要的生理学和病理学作用受到科学家们的广泛关注。高选择性、高灵敏度、低毒性NO分子荧光探针的设计和开发,在环境检测、食品安全及人体内NO检测等领域具有重要意义。本文以小分子荧光探针对NO的识别机制为主线,从唑环的形成、螺内酰胺开环、还原脱氨、二氢吡啶的芳构化、NO与金属络合物的反应、与非金属Se的反应和亚硝胺的形成出发,综述了近年来NO小分子荧光探针的研究进展。对NO探针设计及其识别性能研究方面的工作进行了总结,并讨论了NO荧光探针今后的设计思路和重点研究方向。     

香豆素类荧光探针对硫化氢检测的研究进展

硫化氢(H2S)是目前人们发现的第三类生物内源性“气体信使分子”。其及时检测对人类的健康有着非常大的意义。随着荧光探针技术的发展,有机小分子荧光探针受到广大学者的关注。其中,香豆素因其结构简单,荧光量子产率高以及易于功能化而备受青睐。本文根据探针的识别机理综述近三年来报道的香豆素类H2S荧光探针代表性研究成果,并对其进行了展望,为后续设计开发更具实用价值的H2S荧光探针提供一点有益的参考。     

Recent progress in two-photon small molecule fluorescent probes for enzymes

This review aims to provide a summary of the progress in TP small molecule fluorescent probes for enzymes in recent years and displays the main fluorescent mechanisms that have been applied to design probes.     

荧光/化学发光探针成像检测超氧阴离子自由基的研究进展

超氧阴离子自由基(O·-2)是细胞内氧气单电子还原后最先产生的一类含氧的高活性物种(活性氧,ROS),与生命过程息息相关.正常稳态浓度的O·-2起重要的信号调控作用,包括细胞的增殖、分化、自噬等.但O·-2浓度的异常,又与癌症、神经退行性疾病、糖尿病等多种疾病的发生发展密切相关.因此,监测O·-2浓度的变化对揭示相关疾病的机理具有至关重要作用.由于荧光成像检测方法具有诸多优势,发展高灵敏、高选择性检测O·-2的荧光探针成为揭示相关疾病发生发展分子机制的关键切入点.近年来,随着荧光显微技术的发展,研究者开发了多种荧光/化学发光探针,实现了对细胞及活体内O·-2水平的可视化监测.本文综述了近五年用于检测O·-2的分子探针、纳米探针、蛋白探针以及化学发光探针的研究进展,并对其发展前景进行了展望.     

Marked small molecule libraries: a truncated approach to molecular probe design

A truncated approach to the design of molecular probes from small molecule libraries is outlined, based upon the incorporation of a bioorthogonal marker. The applicability of this strategy to small molecule chemical genetics screens has been demonstrated using analogues of the known stress activated protein kinase (SAPK) pathway activator, anisomycin. Compounds marked with a propargyl group have shown activation of the SAPK pathways comparable to that induced by their parent structures, as demonstrated by immunoblot assays against the downstream target JNK1/2. The considerable advantages of this new approach to molecular probe design have been illustrated through the rapid development of a functionally active fluorescent molecular probe, through coupling of the marked analogues to fluorescent azides using the copper(i)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction. Active molecular probes generated in this study were used to investigate cellular uptake through FACS analysis and confocal microscopy.     

新型荧光/EPR双功能次氯酸探针的构建及性能

构建了一种新型香豆素-萘酰亚胺荧光/电子顺磁共振双功能探针CNNOH,并结合荧光光谱、电子顺磁共振(EPR)波谱和紫外-可见吸收光谱对其性能进行了研究.结果表明,该探针可结合荧光光谱的灵敏性和EPR波谱的特异性进行次氯酸的检测;由于香豆素与萘酰亚胺之间存在荧光共振能量转移(FRET)效应,探针分子具有较大的Stokes位移(135 nm),可有效避免由激发光导致的杂散光对检测的干扰.该双功能探针具有检出限低(0.214μmol/L)、反应速度快(~10 s)、检测范围宽(0~5 mmol/L)、选择性好及在生理条件下稳定的特点,预期在活体细胞检测方面有良好的应用前景.     

Recent progress in two-photon small molecule fluorescent probes for enzymes

Enzymes are macromolecular biological catalysts which can accelerate chemical reactions in living organisms. Almost all the physiological metabolism activities in the cell need enzymes to sustain life via rapid catalysis. Currently, medical research has proved that abnormal enzyme activity is associated with numerous diseases, such as Parkinson’s disease (PD), Alzheimer's disease (AD) and cancers. On the other hand, early diagnosis of those diseases is of great significance to improve the survival rate and cure rate. In the current diagnostic tools, two-photon fluorescent probes (TPFPs) are developing rapidly due to their unique advantages, such as higher spatial resolution, deeper imaging depth, and lower biotoxicity. Therefore, the design and synthesis of two-photon (TP) small molecule enzymatic probes have broad prospects for early diagnosis and treatment of diseases. As of now, scientists have developed many TP small molecule enzymatic probes. This review aims to summarize the TP small molecule enzymatic probes and expound the reaction mechanism.     

Target‐Triggered NIR Emission with a Large Stokes Shift for the Detection and Imaging of Cysteine in Living Cells

Background autofluorescence from biological systems generally reduces the sensitivity of a fluorescent probe for imaging biological targets. Addressing this challenge requires the development of fluorescent probes that produce emission in the near‐infrared region. Herein, we report the design and synthesis of a fluorescent probe that generates an NIR emission with a large Stokes shift upon the selective response to Cys over Hcy and GSH. The probe is designed to consist of two Cys‐sensing sites, an acrylate ester and an aldehyde installed ortho to each other. The reaction of the probe with Cys triggers an excited state intramolecular proton transfer process upon photo‐excitation, thereby producing an NIR emission with a large Stokes shift. Accordingly, this probe hold great promise for the selective detection of Cys in biological systems. We further demonstrate the capacity of this probe for Cys imaging in living cells.