反应激活型酶荧光探针的研究进展

酶在维持生物体内稳态与生命活动的正常运行方面发挥着举足轻重的作用。某些特定酶含量及活性的异常与人类重大疾病的发生与发展密切相关。因此,生物体内特定酶的实时原位检测及可视化成像具有重要的意义。化学荧光探针具有选择性好、灵敏度高及高时空分辨率可视化成像等优点,近年来研究者设计合成了大量的可用于生物体系内酶识别与可视化成像的荧光探针。目前识别酶的荧光探针主要有两类:(1)基于酶对荧光探针分子中酶抑制剂基团的识别引起探针荧光信号的变化;(2)基于酶对荧光探针特异性催化反应来实现识别前后荧光信号的激活,称为反应激活型酶荧光探针。对反应激活型酶荧光探针的设计策略及4种重大疾病相关的生物标志酶(单胺氧化酶、β-半乳糖苷酶、硝基还原酶、γ-谷氨酰转肽酶)的识别可视化荧光探针研究进展进行了综述,对未来酶识别荧光探针的研究方向进行了展望。     

反应型HClO/ClO-荧光探针的研究进展

次氯酸/次氯酸根是活性氧化物中重要的信号分子，在生物医药和环境安全等方面发挥着重要作用。反应型荧光探针基于出色的灵敏度、实时成像和生物兼容性好等特点，广泛应用于HClO/ClO^-的检测。本文评述了近5年反应型HClO/ClO^-荧光探针的研究进展，包括反应型HClO/ClO^-荧光探针的设计策略和识别机理，并从HClO/ClO^-引发的反应机理角度（碳碳双键的反应、硫族化合物的反应、醛肟基团的反应、腙/席夫碱的反应、酰肼/磺酰肼的反应、N,N-二甲基硫代氨基甲酸酯的反应和苯硼酸/硼酸酯的反应）概述了反应型HClO/ClO^-荧光探针的特点和实际应用，指出反应型HClO/ClO^-荧光探针的发展方向是合成性能优异的识别基团，构建选择性好、水溶性好、低荧光背景、光化学性能稳定和生物毒性低的反应型近红外HClO/ClO^-荧光探针，实现对生物体内外HClO/ClO^-的可视化检测及机理探索。     

检测β-半乳糖苷酶荧光探针的研究进展

β-半乳糖苷酶(β-Gal)是重要的水解酶之一,与多种疾病的发生发展有关,是细胞衰老的重要生物标志物,生物体中β-Gal的荧光成像对于生物学研究具有重要意义。近年来,荧光探针因其光毒性低、生物分子自发荧光干扰小、光散射低、组织穿透能力强、稳定性好,可以实现活体动物肿瘤组织成像等优点成为检测体内小分子物质的新型手段。如今已发展了大量的识别生物体系内β-Gal的荧光探针,这些探针可以可视化的监测体内β-Gal的生物活性。本文对目前检测β-Gal的荧光探针的研究进展进行了综述,对将来β-Gal识别荧光探针的研究方向进行了展望。     

基于醛基取代的氟硼二吡咯类荧光母体探针的合成及其应用

荧光探针具有灵敏度高、可实时检测、精准诊断与成像可视化等优点，被广泛应用于生物医药、信息存储、化学分析等领域。氟硼二吡咯（BODIPY）类荧光探针因其优异的光物理化学特性而被广泛设计与开发使用。该文综述了醛基取代的BODIPY荧光探针的分子设计策略和功能化应用，包括α位醛基BODIPY、β位醛基BODIPY、meso位醛基BODIPY和1,7-位醛基BODIPY的不同位点醛基调控的BODIPY荧光母体探针及其在阴离子检测、生物硫醇识别及细胞成像方面的研究进展。设计新型的醛基取代BODIPY荧光探针将在精准诊疗上具有巨大的发展空间。     

基于二氟化硼-二吡咯甲烷(BODIPY)类染料的次氯酸荧光探针的研究进展

次氯酸(HCl O)作为一种生物体内关键的活性氧物种(ROS),在多种正常的生化功能和异常病理过程中扮演着非常重要的角色。因此,识别与实时准确地监测细胞内次氯酸在活动位点的浓度变化对于生物学研究和临床诊断极其重要。而在所有的检测方法中,荧光探针法由于其灵敏度高、选择性好、易于操作、实时可视化检测、原位检测、无损检测、响应时间快速、所需试剂量小等优点而引起广大科研工作者的兴趣并将其用于生物体内次氯酸的检测及其生理功能的研究。重点综述了近年来基于BODIPY类染料的次氯酸荧光分子探针的设计合成、检测机理及其在生物成像上的应用研究进展。     

用于卵巢癌标志物β-Gal检测的荧光探针研究进展

卵巢癌是最常见的妇科癌症之一，其患者大多数在晚期被诊断出，导致卵巢癌患者的死亡率高。因此，对隐匿症状的早期诊断是患者生存率提高的重要保障。β-半乳糖苷酶(β-Gal)在原发性卵巢癌中的过表达而被认定为卵巢癌生物标志物，所以快速精准的β-Gal检测技术对卵巢癌早期诊断至关重要。近年来，荧光探针技术已成为生物体内监测和可视化的有力工具。目前已经报道了大量的用于检测β-Gal的荧光探针，但是，用于卵巢癌β-Gal检测和可视化成像的荧光探针相对较少。主要综述用于卵巢癌细胞生物标志物β-Gal检测的荧光探针的研究进展，并对探针的发展进行展望。     

基于醛基取代的氟硼二吡咯类荧光母体探针的合成及其应用

荧光探针具有灵敏度高、可实时检测、精准诊断与成像可视化等优点,被广泛应用于生物医药、信息存储、化学分析等领域。氟硼二吡咯(BODIPY)类荧光探针因其优异的光物理化学特性而被广泛设计与开发使用。该文综述了醛基取代BODIPY荧光团的分子设计策略和功能化应用,包括α位醛基-BODIPY、β位醛基-BODIPY、meso位醛基-BODIPY和1,7-位醛基-BODIPY的不同位点醛基调控的BODIPY荧光母体探针及其在阴离子检测、生物硫醇识别及细胞成像等方面的研究进展。设计新型的醛基取代BODIPY探针,未来在精准诊疗上具有发展空间。     

花菁类近红外荧光探针在生物检测中的应用研究进展

随着现代生物学和生物技术的发展,近红外荧光探针作为一种重要的技术手段在许多领域都展现出了重要的应用价值。因具有背景干扰低、细胞损伤小、样品穿透性强、检测灵敏度高等优点,经常用于分子识别、医学诊断、生物分子检测以及生物成像等方面。本综述从金属离子检测、含硫小分子检测、活性氧（ROS）/活性氮（RNS）检测、酶识别、肿瘤细胞识别及治疗以及细胞内pH响应等方面,介绍了近年来花菁类荧光探针在生物检测中的应用研究进展。同时指出了花菁类荧光探针亟待解决的问题,通过花菁母体的结构修饰和改造提高探针的光稳定性、灵敏度、靶向性和水溶性,有望使其在生物检测以及疾病的诊断方面得到进一步应用发展。     

氧杂蒽类荧光探针识别性能研究进展

氧杂蒽结构是一类重要的荧光发色团，作为构建荧光探针的平台，受到了研究者的广泛关注。识别机制基于环的开关来实现荧光信号的有无。氧杂蒽类探针的研究从最初的离子识别到生物小分子识别、医学诊疗等方面，都取得了很大的进展。综述了近年来氧杂蒽类荧光探针的识别性能，对探针的设计思路及识别机制进行了总结。     

有机小分子谷胱甘肽荧光探针研究进展

荧光探针是一种会对pH、温度、特定的物质产生荧光响应的分子。荧光探针由3个基本单元：荧光基团、连接体和识别基团组成。荧光基团具有讯号传递功能,能直接影响到探针对检测对象的敏感性。识别基因作为一种具有特定或识别作用的结构,其特征在于其高度的选择性和特异性。连接体就是在荧光基团和识别基团之间起到衔接的作用。谷胱甘肽(GSH)是由谷氨酸、半胱氨酸和甘氨酸构成的三肽类物质。它在抗HIV、抗过敏、保护巯基酶的活性以及抗氧化和维持体内血红球蛋白表面细胞膜的结构稳定性等方面有着重要作用,并且它还可以用作食品添加剂,广泛应用于环境监测。基于此,开发以谷胱甘肽为被测物质的多功能或多通道的荧光探针分子就成了一项具有挑战性的新任务。主要叙述了基于谷胱甘肽的荧光探针的设计合成和使用方面的最新进展。     

Harnessing Fluorescent Moenomycin A Antibiotics for Bacterial Cell Wall Imaging Studies

The imaging of peptidoglycan (PGN) dynamics in living bacteria facilitates the understanding of PGN biosynthesis and wall-targeting antibiotics. The main tools for imaging bacterial PGN are fluorescent probes, such as the well-known PGN metabolic labeling probes. However, fluorescent small-molecule probes for labeling key PGN-synthesizing enzymes, especially for transglycosylases (TGases), remain to be explored. In this work, the first imaging probe for labeling TGase in bacterial cell wall studies is reported. We synthesized various fluorescent MoeA-based molecules by derivatizing the natural antibiotic moenomycin A (MoeA), and used them to label TGases in living bacteria, monitor bacterial growth and division cycles by time-lapse imaging, and study cell wall growth in the mecA-carrying methicillin-resistant Staphylococcus aureus (MRSA) strains when the β-lactam-based probes were unsuitable.     

Novel fluorescent phosphonic acid esters for discrimination of lipases and esterases

Lipases and esterases are responsible for carboxylester hydrolysis inside and outside cells and are useful biocatalysts for (stereo)selective modification of synthetic substrates. Here we describe novel fluorescent suicide inhibitors that differ in structure and polarity for screening and discrimination of lipolytic enzymes in enzyme preparations. The inhibitors covalently react with the enzymes to form fluorescent lipid-protein complexes that can be resolved by gel electrophoresis. The selectivities of the inhibitors were determined by using different (phospho)lipase, esterase and cholesterol esterase preparations. The results indicate that formation of an inhibitor-enzyme complex is highly dependent on the chemical structure of the inhibitor. We identified inhibitors with very low specificity, and other derivatives that were highly specific for certain subgroups of lipolytic enzymes such as lipases and cholesterol esterases. A combination of these substrate-analogous activity probes represents a useful toolbox for rapid identification and classification of serine hydrolase enzymes.     

One Scaffold,Different Organelle Sensors: pH-Activable Fluorescent Probes for Targeting Live Microglial Cell Organelles**

Targeting live cell organelles is essential for imaging, understanding, and controlling specific biochemical processes. Typically, fluorescent probes with distinct structural scaffolds are used to target specific cell organelles. Here, we have designed a modular one-step synthetic strategy using a common reaction intermediate to develop new lysosomal, mitochondrial, and nucleus-targeting pH-activable fluorescent probes that are all based on a single boron dipyrromethane scaffold. The divergent cell organelle targeting was achieved by synthesizing probes with specific functional group changes to the central scaffold resulting in differential fluorescence and pK_a. Specifically, we show that the functional group transformation of the same scaffold influences cellular localization and specificity of pH-activable fluorescent probes in live primary microglial cells with pK_a values ranging from ∼3.2–6.0. We introduce a structure-organelle-relationship (SOR) framework to target nuclei ( NucShine ), lysosomes ( LysoShine ), and mitochondria ( MitoShine ) in live microglia. This work will result in future applications of SOR beyond imaging to target and control organelle-specific biochemical processes in disease-specific models.     

Biomedical Applications of Fluorescent and Magnetic Resonance Imaging Dual-Modality Probes

Molecular imaging plays a critical role in biomedical research. The combination of different modalities can generate complementary information and provide synergistic advantages over single modality alone. Noninvasive and nonradioactive fluorescent imaging (FI)/magnetic resonance imaging (MRI) dualmodality probes fuse the high sensitivity of FI and the high temporal and spatial resolution and deep-tissue penetration of MRI, and their increasing applications have been reported in biomedical research and clinical practices, including cell labeling, enzyme activity measurement, tumor diagnosis and therapy, and anatomical localization and real-time assessment during surgery.     

Synergistically integrated nanoparticles as multimodal probes for nanobiotechnology

Current biomedical imaging techniques including magnetic resonance imaging (MRI), positron emission tomography (PET), and computed X-ray tomography (CT) are vital in the diagnosis of various diseases. Each imaging modality has its own merits and disadvantages, and a single technique does not possess all the required capabilities for comprehensive imaging. Therefore, multimodal imaging methods are quickly becoming important tools for state-of-the-art biomedical research and clinical diagnostics and therapeutics. In this Account, we will discuss synergistically integrated nanoparticle probes, which will be an essential tool in multimodal imaging technology. When inorganic nanoparticles are introduced into biological systems, their extremely small size and their exceptional physical and chemical properties make them useful probes for biological diagnostics. Nanoparticle probes can endow imaging techniques with enhanced signal sensitivity, better spatial resolution, and the ability to relay information about biological systems at the molecular and cellular levels. Simple magnetic nanoparticles function as MRI contrast enhancement probes. These magnetic nanoparticles can then serve as a core platform for the addition of other functional moieties including fluorescence tags, radionuclides, and other biomolecules for multimodal imaging, gene delivery, and cellular trafficking. For example, MRI-optical dual-modal probes composed of a fluorescent dye-doped silica (DySiO(2)) core surrounded by magnetic nanoparticles can macroscopically detect neuroblastoma cancer cells via MRI along with subcellular information via fluorescence imaging. Magnetic nanoparticles can also be coupled to radionuclides ((124)I) to construct MRI-PET dual-modal probes. Such probes can accurately detect lymph nodes (LNs), which are critical for assessing cancer metastasis. In vivo MRI/PET images can clearly identify small (approximately 3 mm) LNs along with precise anatomical information. Systems using multicomponent nanoparticles modified with biomolecules can also monitor gene expression and other markers in cell therapeutics studies. We have used hybrid stem cell-magnetic nanoparticle probes with MRI to monitor in vivo stem cell trafficking. MRI with hybrid probes of magnetic nanoparticles and adenovirus can detect target cells and can monitor gene delivery and the expression of green fluorescent proteins optically. Each component of such multimodal probes complements the other modalities, and their synergistic materials properties ultimately provide more accurate information in in vitro and in vivo biological systems.     

Improving Colorimetric Assays through Protein Enzyme-Assisted Gold Nanoparticle Amplification

The discovery of the DNA-mediated assembly of gold nanoparticles was a great moment in the history of science; this understanding and chemical control enabled the rational design of functional nanomaterials as novel probes in biodetection. In contrast with conventional probes such as organic dyes, gold nanoparticles exhibit high photostability and unique size-dependent optical properties. Because of their high extinction coefficients and strong distance dependent optical properties, these nanoparticles have emerged over the past decade as a promising platform for rapid, highly sensitive colorimetric assays that allow for the visual detection of low concentrations of metal ions, small molecules, and biomacromolecules. These discoveries have deepened our knowledge of biological phenomena and facilitated the development of many new diagnostic and therapeutic tools. Despite these many advances and continued research efforts, current nanoparticle-based colorimetric detection systems still suffer from several drawbacks, such as limited sensitivity and selectivity. This Account describes the recent development of colorimetric assays based on protein enzyme-assisted gold nanoparticle amplification. The benefits of such detection systems include significantly improved detection sensitivity and selectivity. First, we discuss the general design of enzyme-modified nanoparticle systems in colorimetric assays. We show that a quantitative understanding of the unique properties of different enzymes is paramount for effective biological assays. We then examine the assays for nucleic acid detection based on different types of enzymes, including endonucleases, ligases, and polymerases. For each of these assays, we identify the underlying principles that contribute to the enhanced detection capability of nanoparticle systems and illustrate them with selected examples. Furthermore, we demonstrate that the combination of gold nanoparticles and specific enzymes can probe enzyme dynamics and function with high specificity, offering substantial advantages in both sensitivity and specificity over conventional detection methods. The screening of nuclease, methyltransferase, protease, and kinase activities can be colorimetrically performed in a straightforward manner. Finally, we discuss examples of colorimetric assays for metal ions and small molecules that constitute important advances toward visual monitoring of enzyme catalytic functions and gene expression. Although these enzyme-assisted assay methods hold great promise for myriad applications in biomedicine and bioimaging, the application of the described techniques in vivo faces formidable challenges. In addition, researchers do not fully understand the interactions of gold nanoparticles with enzyme molecules. This understanding will require the development of new techniques to probe enzyme substrate dynamics at the particle interface with higher spatial resolution and chemical specificity.     

An Expanded Set of Fluorogenic Sulfatase Activity Probes

Fluorogenic probes that are activated by an enzymatic transformation are ideally suited for profiling enzyme activities in biological systems. Here, we describe two fluorogenic enzyme probes, 3‐O‐methylfluorescein‐sulfate and resorufin‐sulfate, that can be used to detect sulfatases in mycobacterial lysates. Both probes were validated with a set of commercial sulfatases and used to reveal species‐specific sulfatase banding patterns in a gel‐resolved assay of mycobacterial lysates. The fluorogenic probes described here are suitable for various assays and provide a starting point for creating new sulfatase probes with improved selectivity for mycobacterial sulfatases.     

Fluorescent hydroxyapatite‐loaded biodegradable polymer nanoparticles with folate decoration for targeted imaging

The preparation of the luminescent hydroxyapatite (HAP)‐loaded biocompatible nanoparticles (NPs) for targeted imaging of cancer cells is described. Currently, cellular imaging using fluorescent probes is an important technique for the early diagnosis of cancer. Compared with the quantum dots, luminescent HAP is a new fluorescent material with many advantages such as low toxicity, biocompatibility, thermal stability, resistance to erosion, and low prices. Thus, luminescent HAP has enormous potential to be used as biological fluorescent probes. However, luminescent HAP is water‐insoluble, low sensitivity, which limit its application in the field of cellular imaging. Surface modification of NPs with targeting molecule was carried out to achieve its target function. Thus, novel fluorescent NPs with low toxicity, high sensitivity, and good photostability were prepared to be used for targeted imaging of cancer cells. This study initially explored the applications of luminescent HAP in the field of targeted cellular imaging. This NPs platform will be a promising tool for molecular imaging and medical diagnostics, especially the detection of cancer at its early stage. © 2013 American Institute of Chemical Engineers AIChE J, 59: 4494–4501, 2013     

Target-cancer-cell-specific activatable fluorescence imaging probes: rational design and in vivo applications

Conventional imaging methods, such as angiography, computed tomography (CT), magnetic resonance imaging (MRI), and radionuclide imaging, rely on contrast agents (iodine, gadolinium, and radioisotopes, for example) that are "always on." Although these indicators have proven clinically useful, their sensitivity is lacking because of inadequate target-to-background signal ratio. A unique aspect of optical imaging is that fluorescence probes can be designed to be activatable, that is, only "turned on" under certain conditions. These probes are engineered to emit signal only after binding a target tissue; this design greatly increases sensitivity and specificity in the detection of disease. Current research focuses on two basic types of activatable fluorescence probes. The first developed were conventional enzymatically activatable probes. These fluorescent molecules exist in the quenched state until activated by enzymatic cleavage, which occurs mostly outside of the cells. However, more recently, researchers have begun designing target-cell-specific activatable probes. These fluorophores exist in the quenched state until activated within targeted cells by endolysosomal processing, which results when the probe binds specific receptors on the cell surface and is subsequently internalized. In this Account, we present a review of the rational design and in vivo applications of target-cell-specific activatable probes. In engineering these probes, researchers have asserted control over a variety of factors, including photochemistry, pharmacological profile, and biological properties. Their progress has recently allowed the rational design and synthesis of target-cell-specific activatable fluorescence imaging probes, which can be conjugated to a wide variety of targeting molecules. Several different photochemical mechanisms have been utilized, each of which offers a unique capability for probe design. These include self-quenching, homo- and hetero-fluorescence resonance energy transfer (FRET), H-dimer formation, and photon-induced electron transfer (PeT). In addition, the repertoire is further expanded by the option for reversibility or irreversibility of the signal emitted through these mechanisms. Given the wide range of photochemical mechanisms and properties, target-cell-specific activatable probes have considerable flexibility and can be adapted to specific diagnostic needs. A multitude of cell surface molecules, such as overexpressed growth factor receptors, are directly related to carcinogenesis and thus provide numerous targets highly specific for cancer. This discussion of the chemical, pharmacological, and biological basis of target-cell-specific activatable imaging probes, and methods for successfully designing them, underscores the systematic, rational basis for further developing in vivo cancer imaging.