Published as part of Chemical Reviews special issue “Fluorescent Probes in Biology”. Seeing is believing! Chemistry makes the invisible visible through the use of imaging agents that can monitor the elements and molecules of life at a variety of length scales with both spatial and temporal resolution. In this context, fluorescence and luminescence provide visual readouts that can be widely adopted by specialists and nonspecialists alike, from laboratory researchers to medical clinicians and field technicians to children playing with glowsticks and fireflies during the long nights of summer. This thematic issue, Fluorescent Probes in Biology, delves into the latest research achievements in this storied and highly active field in the design and development of chemical reagents to decipher new fundamental biology and to translate this knowledge to advanced diagnostic and/or therapeutic platforms. Each paper in this issue focuses on molecular principles of probe design, applied to a particular biological question for analyte detection, chemical platform consisting of a small molecule, macromolecule, nanomaterial, or hybrid scaffold, or disease biomarker. State-of-the art research and future prospects in unmet needs pervade all these informative reviews. A key emerging theme in this collection is the broad use of activity-based sensing, a termed coined by our laboratory and advanced by our team and many others across the world, which is defined as using molecular reactivity rather than molecular recognition for analyte detection. (1) We organize this discussion across fluorescent probes for specific bioanalytes, fluorescent probes derived from a specific type of scaffold (e.g., small molecule, protein, nanomaterial, or hybrid), or fluorescent probes for biomedical applications. A foundational use of fluorescent probes in biology is their application in detecting the chemistry of elements and molecules of life to decipher their physiological and/or pathological contributions to living systems. New and our laboratory have collaborated to write a review on small-molecule sensors for transition metal ions in biological specimens (10.1021/acs.chemrev.3c00819). A focus is on the use of binding-based sensing and activity-based sensing approaches, where the former strategy exploits traditional lock-and-key metal–ligand coordination bonding for selective metal chelation and detection, whereas the latter concept leverages the diverse reactivity of metal ions for their sensing. These approaches are applied to image bioavailable metal pools, termed the labile metal pool, over a variety of biological length scales and in a variety of cell and animal models with metal and oxidation state selectivity, revealing new biological concepts such as transition metal signaling and metalloallostery in health and disease. Activity-based sensing offers a powerful approach to use reaction chemistry as a strategy for analyte detection. Lippert and Domaille and their teams review activity-based sensing strategies for reactive oxygen species (ROS) and reactive nitrogen species (RNS) across a diverse array of reaction scaffolds. These families of transient small molecules are toxic and lead to oxidative stress and oxidative damage when overproduced in aberrant quantities, but privileged members such as nitric oxide and hydrogen peroxide are also potent signaling agents that modify proteins, nucleic acids, and other biological targets with high specificity when produced in the right time and space. A key design feature is to develop reactions with selective one- and two-electron chemistry, as many of these ROS and RNS operate by free radical chemistry. Pluth and his colleagues write an excellent piece on activity-based sensing approaches on hydrogen sulfide and related reactive sulfur species (10.1021/acs.chemrev.3c00683). Hydrogen sulfide (H₂S) is an important representative of the ever-growing class of gasotransmitters along with nitric oxide, ammonia, carbon monoxide, carbon dioxide, and ethylene, as well as the primary reactive sulfur species. This review scholarly outlines reaction types for use in selective reactive sulfur species detection, from reduction of oxidized nitrogen motifs, electrophilic reactions, metal precipitation and metal coordination, and disulfide exchange. These tools have revealed a central role for H₂S and reductant-labile and sulfane sulfur species, including persulfides and polysulfides, as signaling agents that act by protein post-translational modifications. Zhang and Liu showcase design and application of fluorescent probes for physical microenvironments within cellular contexts (10.1021/acs.chemrev.3c00573). Foundational parameters such as pH, temperature, voltage, mechanical force, polarity, and viscosity offer physical markers that pervade all cells across all kingdoms of life. Fluorescent probes can be used to visualize dynamic changes in the cellular environment and be leveraged to expand fundamental knowledge and enrich diagnostic tools to better human health. Fluorescent probes can be constructed from a variety of chemical components that span synthetic small molecules to biological macromolecules to materials. Moreover, combinations of these basic building blocks can give rise to unique sensor platforms. Beyond traditional small-molecule organic fluorophores, Lo and colleagues review transition metal complexes that possess requisite optical properties and biocompatibility for bioimaging. These imaging agents often exhibit high photostability derived from complexes with inert metal–ligand bonds and can be used for activity-based sensing applications for bioanalyte detection along with protein and organelle imaging. Demirer and Beyene and their laboratories provide a comprehensive overview of fluorescent carbon nanomaterials and their applications for biomedical and environmental imaging, sensing, and cargo delivery applications (https://pubs.acs.org/doi/10.1021/acs.chemrev.3c00581). Graphene and graphene oxide, carbon nanotubes and related carbon nanohoops, along with carbon dots showcase the range of materials that exhibit interesting photophysics for such applications. Zhang and his team write on nanocrystals for bioimaging (10.1021/acs.chemrev.3c00506), focusing on nanocrystal designs that absorb and emit in the near-infrared region (700–1700 nm). This optical profile enables deep tissue penetration that is necessary for in vivo imaging applications. Nanocrystals can be tailored for fluorescence, bioluminescence, and chemiluminescence imaging in intensity and time-resolved modes. Moreover, advanced sensor design by controlling energy transfer pathways gives rise to Cerenkov luminescent, X-ray excited luminescent, and persistent luminescent imaging. Kikuchi and colleagues review the exciting field of hybrid small-molecule/protein fluorescent probes (https://pubs.acs.org/doi/10.1021/acs.chemrev.3c00549), where these systems combine the molecular tunability of small-molecule reagents via chemical synthesis with biological scaffolds that enable site-specific delivery and augmented compatibility with living systems. This work is built upon the myriad of site-selective protein labeling reactions using exogenous peptide/small protein tags, enzymatic post-translational modifications, bioorthogonal reactions for genetically incorporated unnatural amino acids, and ligand-directed chemistry. Indeed, imaging of protein trafficking and conformational changes and bioanalytes with high signal-to-noise responses can be achieved using these hybrid platforms. In addition to fluorescent probes as reagents for learning about fundamental biology, they can also be applied in advanced diagnostic applications for medicine. Urano and Fujita write on activity-based fluorescence detection in cancer (10.1021/acs.chemrev.3c00612). These diverse classes of probes take advantage of biomarkers that are intrinsically elevated in various cancers. By administering an activatable probe to a disease specimen, from whole blood and serum to tumor tissue, a fluorogenic response can enable rapid cancer imaging with high contrast. A distinguished international team of Gunnlaugsson, James, Tang, Li, and Lewis and their colleagues provides a broad overview of fluorescent probes for disease diagnostics (10.1021/acs.chemrev.3c00776). They emphasize the design strategies behind these reagents, which open the door to applications in detecting bioactive molecules associated with diseases spanning organ damage, inflammation, cancers, cardiovascular diseases, and brain disorders. They also highlight future unmet needs to achieve accurate detection and identification of biomarkers for biomedical research with activity-based sensing reagents. Another large international collaboration between Kim, Sessler, Peng, James, Zeng, He, and Sharma and their laboratories focuses on theranostic fluorescent probes (10.1021/acs.chemrev.3c00778). This large field leverages the ability of activity-based sensing triggers that are activated by disease biomarkers to achieve spatiotemporal control in the context of drug delivery. When combined with a concomitant imaging response, these dual theranostic agents can achieve both targeted therapeutic delivery of a medicine and imaging readout. Indeed, photodynamic, photothermal, and sonodynamic therapeutic applications are but some of the numerous possibilities afforded by this theranostic approach using small-molecule to nanomaterial scaffolds. I hope that this collection of reviews will be a useful resource to introduce the exciting world of fluorescent probes to a broad range of newcomers as well as experts in the field, where chemistry can light the way to new knowledge and societal benefit. C.J.C. thanks the NIH (GM 79465, GM 139245, ES 28096) for support. Christopher J. Chang is the Edward and Virginia Taylor Professor of Bioorganic Chemistry at Princeton University. He completed his B.S. and M.S. degrees from Caltech in 1997 with Harry Gray, a Fulbright scholarship with Jean-Pierre Sauvage, a Ph.D. from MIT in 2002 with Dan Nocera, and a postdoc at MIT with Steve Lippard. Chris started his independent career at UC Berkeley in 2004 before moving to Princeton in 2024. The Chang laboratory focuses on metals in biology and energy. His group has pioneered the concept of activity-based sensing, showing that selectivity in sensor design is achievable by reaction-based methods that go beyond traditional receptors that operate by lock-and-key binding. His laboratory’s work has changed dogma in the inorganic and chemical biology communities by showing that transition metals are not merely active site cofactors in proteins but also serve as dynamic, allosteric regulators of protein function through metalloallostery, launching a field of transition metal signaling. This article references 1 other publications. This article has not yet been cited by other publications.

中文翻译：

---

简介：生物学中的荧光探针


作为 Chemical Reviews 特刊“Fluorescent Probes in Biology”的一部分发表。眼见为实！化学通过使用成像剂使不可见的事物变得可见，成像剂可以在各种长度尺度上以空间和时间分辨率监测生命的元素和分子。在这种情况下，荧光和发光提供了视觉读数，可以被专家和非专业人士广泛采用，从实验室研究人员到医学临床医生和现场技术人员，再到在夏季漫长的夜晚玩荧光棒和萤火虫的儿童。本期主题为 Fluorescent Probes in Biology，深入探讨了这一传奇且高度活跃的领域在化学试剂的设计和开发方面的最新研究成果，以破译新的基础生物学并将这些知识转化为先进的诊断和/或治疗平台。本期的每篇论文都侧重于探针设计的分子原理，应用于分析物检测的特定生物学问题，由小分子、大分子、纳米材料或混合支架或疾病生物标志物组成的化学平台。未满足需求的最新研究和未来前景贯穿所有这些内容丰富的评论。该系列中一个关键的新兴主题是基于活动的传感的广泛使用，该术语由我们的实验室创造，并由我们的团队和世界各地的许多其他人提出，其定义为使用分子反应性而不是分子识别进行分析物检测。（1） 我们通过特定生物分析物的荧光探针、来自特定类型支架的荧光探针（例如、小分子、蛋白质、纳米材料或杂交）或用于生物医学应用的荧光探针。荧光探针在生物学中的基本用途是它们用于检测生命元素和分子的化学性质，以破译它们对生命系统的生理和/或病理贡献。New 和我们的实验室合作撰写了一篇关于生物样本中过渡金属离子的小分子传感器的综述 （10.1021/acs.chemrev.3c00819）。重点是使用基于结合的传感和基于活性的传感方法，其中前者策略利用传统的锁和钥匙金属-配体配位键进行选择性金属螯合和检测，而后一种概念利用金属离子的多样化反应性进行传感。这些方法应用于在各种生物长度尺度上以及在各种具有金属和氧化态选择性的细胞和动物模型中对生物可利用的金属池（称为不稳定金属池）进行成像，揭示了新的生物学概念，例如健康和疾病中的过渡金属信号传导和金属流失。基于活性的传感提供了一种强大的方法，可以将反应化学用作分析物检测策略。Lippert 和 Domaille 及其团队回顾了各种反应支架中基于活性氧 （ROS） 和活性氮 （RNS） 的传感策略。 这些瞬时小分子家族是有毒的，当过量产生时会导致氧化应激和氧化损伤，但一氧化氮和过氧化氢等特权成员也是有效的信号转导剂，当在正确的时间和空间产生时，它们会以高度特异性修饰蛋白质、核酸和其他生物靶标。一个关键的设计特征是利用选择性单电子和双电子化学反应来开发反应，因为许多 ROS 和 RNS 都是通过自由基化学作用的。Pluth 和他的同事写了一篇关于硫化氢和相关活性硫物质的基于活性的传感方法的优秀文章 （10.1021/acs.chemrev.3c00683）。硫化氢 （H₂S） 是与一氧化氮、氨、一氧化碳、二氧化碳和乙烯以及主要活性硫物质一起不断增长的气体递质类别的重要代表。本综述从学术上概述了用于选择性活性硫物质检测的反应类型，包括氧化氮基序的还原、亲电反应、金属沉淀和金属配位以及二硫键交换。这些工具揭示了 H₂S 和还原剂不稳定和硫烷硫物质（包括过硫化物和多硫化物）作为通过蛋白质翻译后修饰起作用的信号转导剂的核心作用。Zhang 和 Liu 展示了荧光探针在细胞环境中物理微环境中的设计和应用 （10.1021/acs.chemrev.3c00573）。pH 值、温度、电压、机械力、极性和粘度等基本参数提供了遍布所有生命王国中所有细胞的物理标志物。 荧光探针可用于可视化细胞环境中的动态变化，并用于扩展基础知识和丰富诊断工具，以改善人类健康。荧光探针可以由各种化学成分构成，这些成分涵盖合成小分子、生物大分子和材料。此外，这些基本构建模块的组合可以产生独特的传感器平台。除了传统的小分子有机荧光团之外，Lo 及其同事还回顾了具有生物成像所需光学特性和生物相容性的过渡金属络合物。这些显像剂通常表现出来自具有惰性金属-配体键的复合物的高光稳定性，可用于生物分析物检测以及蛋白质和细胞器成像的基于活性的传感应用。Demirer 和 Beyene 及其实验室全面概述了荧光碳纳米材料及其在生物医学和环境成像、传感和货物运输应用 （https://pubs.acs.org/doi/10.1021/acs.chemrev.3c00581） 中的应用。石墨烯和氧化石墨烯、碳纳米管和相关的碳纳米箍以及碳点展示了为此类应用展示有趣光物理学的材料范围。Zhang 和他的团队撰写了关于用于生物成像的纳米晶体 （10.1021/acs.chemrev.3c00506） 的文章，重点介绍了在近红外区域 （700–1700 nm） 吸收和发射的纳米晶体设计。这种光学轮廓可实现体内成像应用所需的深层组织穿透。纳米晶体可以针对强度和时间分辨模式下的荧光、生物发光和化学发光成像进行定制。 此外，通过控制能量传递途径的先进传感器设计产生了 Cerenkov 发光、X 射线激发发光和持续发光成像。Kikuchi 及其同事回顾了令人兴奋的小分子/蛋白质混合荧光探针 （https://pubs.acs.org/doi/10.1021/acs.chemrev.3c00549） 领域，在这些领域中，这些系统通过化学合成将小分子试剂的分子可调性与生物支架相结合，从而实现位点特异性递送并增强与生命系统的兼容性。这项工作建立在使用外源性肽/小蛋白标签、酶促翻译后修饰、基因掺入非天然氨基酸的生物正交反应和配体定向化学的无数位点选择性蛋白质标记反应之上。事实上，使用这些混合平台可以实现蛋白质运输和构象变化以及具有高信噪比响应的生物分析物的成像。除了作为学习基础生物学的试剂的荧光探针外，它们还可以应用于医学的高级诊断应用。Urano 和 Fujita 撰写了关于癌症中基于活性的荧光检测的文章 （10.1021/acs.chemrev.3c00612）。这些不同类别的探针利用了在各种癌症中固有升高的生物标志物。通过对疾病标本（从全血和血清到肿瘤组织）进行可激活探针，荧光反应可以实现高对比度的快速癌症成像。由 Gunnlaugsson、James、Tang、Li 和 Lewis 及其同事组成的杰出国际团队提供了用于疾病诊断的荧光探针的广泛概述 （10.1021/acs.chemrev.3c00776）。 他们强调了这些试剂背后的设计策略，这些策略为检测与器官损伤、炎症、癌症、心血管疾病和脑部疾病相关的生物活性分子打开了大门。它们还强调了未来未满足的需求，即使用基于活性的传感试剂实现生物医学研究中生物标志物的准确检测和鉴定。Kim、Sessler、Peng、James、Zeng、He 和 Sharma 及其实验室之间的另一项大型国际合作侧重于治疗诊断荧光探针 （10.1021/acs.chemrev.3c00778）。这个大领域利用了由疾病生物标志物激活的基于活动的传感触发器的能力，在药物输送的背景下实现了时空控制。当与伴随的成像反应相结合时，这些双重治疗诊断药物可以实现药物的靶向治疗递送和影像学读数。事实上，光动力学、光热学和声动力学治疗应用只是这种使用小分子到纳米材料支架的治疗诊断学方法提供的众多可能性中的一部分。我希望这组评论将成为有用的资源，向该领域的广大新人和专家介绍令人兴奋的荧光探针世界，化学可以照亮通往新知识和社会效益的道路。CJC 感谢 NIH（GM 79465、GM 139245、ES 28096）的支持。Christopher J. Chang 是普林斯顿大学 Edward 和 Virginia Taylor 生物有机化学教授。他于 1997 年师从 Harry Gray 获得加州理工学院的学士和硕士学位，与 Jean-Pierre Sauvage 一起获得富布赖特奖学金，2002 年与 Dan Nocera 一起获得麻省理工学院博士学位，并在麻省理工学院与 Steve Lippard 一起获得博士后。 Chris 于 2004 年在加州大学伯克利分校开始了他的独立职业生涯，然后于 2024 年搬到普林斯顿。Chang 实验室专注于生物学和能源中的金属。他的团队开创了基于活动的传感概念，表明传感器设计中的选择性可以通过基于反应的方法实现，这些方法超越了通过锁键结合运行的传统受体。他的实验室的工作改变了无机和化学生物学界的教条，表明过渡金属不仅是蛋白质中的活性位点辅因子，而且还通过金属变构作为蛋白质功能的动态变构调节剂，开创了过渡金属信号传导领域。本文参考了 1 其他出版物。本文尚未被其他出版物引用。