Near-infrared fluorescent probes: a next-generation tool for protein-labeling applications

The development of near-infrared (NIR) fluorescent probes over the past few decades has changed the way that biomolecules are imaged, and thus represents one of the most rapidly progressing areas of research. Presently, NIR fluorescent probes are routinely used to visualize and understand intracellular activities. The ability to penetrate tissues deeply, reduced photodamage to living organisms, and a high signal-to-noise ratio characterize NIR fluorescent probes as efficient next-generation tools for elucidating various biological events. The coupling of self-labeling protein tags with synthetic fluorescent probes is one of the most promising research areas in chemical biology. Indeed, at present, protein-labeling techniques are not only used to monitor the dynamics and localization of proteins but also play a more diverse role in imaging applications. For instance, one of the dominant technologies employed in the visualization of protein activity and regulation is based on protein tags and their associated NIR fluorescent probes. In this mini-review, we will discuss the development of several NIR fluorescent probes used for various protein-tag systems.

This minireview describes the development of NIR chemical probes for various protein-tag systems.     

荧光素类荧光分子探针

荧光素衍生物是重要的荧光探针,在检测和生物成像等领域中显示出巨大的前景。因此,急需对功能性荧光素结构探针的设计策略进行深入研究。通常通过引入醛基或酯化到荧光素呫吨环和苯部分来构建探针,由于其高活性,这些衍生物可以与分析物复合以发生颜色和荧光强度的变化。本文总结了荧光素的修饰位点及方法,介绍了荧光素探针的合成、性质及应用,并对近五年荧光素探针对不同分析物(包括金属阳离子、阴离子、小分子和生物大分子)的检测进行分类说明,旨在为高灵敏度荧光素探针的筛选和生物检测提供参考,并推动其在分析物传感和检测中的进一步应用。     

Zebrafish as a good vertebrate model for molecular imaging using fluorescent probes

Fluorescent probes have been used extensively to monitor biomolecules and biologically relevant species in vitro and in vivo. A new trend in this area that has been stimulated by the desire to obtain more detailed information about the biological effects of analytes is the change from live cell to whole animal fluorescent imaging. Zebrafish has received great attention for live vertebrate imaging due to several noticeable advantages. In this tutorial review, recent advances in live zebrafish imaging using fluorescent probes, such as fluorescent proteins, synthetic fluorescent dyes and quantum dots, are highlighted.     

Near-infrared small molecular fluorescent dyes for photothermal therapy

This review aims to provide a summary of the progress in fluorescent dyes for photothermal therapy in recent years and it is classified according to the structure of organic molecules including cyanines, phthalocyanines, rhodamine analogues and BODIPYs.     

Biomarker-targeted fluorescent probes for breast cancer imaging

This review summarized fluorescent probes for breast cancer imaging according to different biomarkers probes recognized.     

Boronate-based fluorescent probes for imaging cellular hydrogen peroxide

The syntheses, properties, and biological applications of the Peroxysensor family, a new class of fluorescent probes for hydrogen peroxide, are presented. These reagents utilize a boronate deprotection mechanism to provide high selectivity and optical dynamic range for detecting H2O2 in aqueous solution over similar reactive oxygen species (ROS) including superoxide, nitric oxide, tert-butyl hydroperoxide, hypochlorite, singlet oxygen, ozone, and hydroxyl radical. Peroxyresorufin-1 (PR1), Peroxyfluor-1 (PF1), and Peroxyxanthone-1 (PX1) are first-generation probes that respond to H2O2 by an increase in red, green, and blue fluorescence, respectively. The boronate dyes are cell-permeable and can detect micromolar changes in H2O2 concentrations in living cells, including hippocampal neurons, using confocal microscopy and two-photon microscopy. The unique combination of ROS selectivity, membrane permeability, and a range of available excitation/emission colors establishes the potential value of PR1, PF1, PX1, and related probes for interrogating the physiology and pathology of cellular H2O2.     

萘酰亚胺类荧光分子探针的研究进展

荧光分子探针作为一种有效的金属离子检测手段,不仅使用方便,而且具有高灵敏度,高选择性等突出的优点.作者综述了萘酰亚胺类荧光分子探针的最新研究进展;指出萘酰亚胺化合物具有独特的荧光化学性质(如荧光量子产率高、荧光发射波长适中、斯托克斯位移大、光稳定性好、结构易于修饰等),因此被广泛应用于荧光探针研究领域,并且在合成、离子识别、检测及细胞成像等方面不断取得新的应用.     

Activatable imaging probes with amplified fluorescent signals

Current optical imaging probe applications are hampered by poor sensitivity and specificity to the target, but molecular-level fluorescent signal activation strategies can efficiently overcome these limitations. Recent interdisciplinary research that couples the imaging sciences to fluorophore, peptide, polymer, and inorganic-based chemistry has generated novel imaging probes that exhibit high sensitivity and low background noise in both in vitro and in vivo applications. This feature article introduces and discusses the various approaches described by the term "fluorescent signal activation methods" with respect to their unique imaging probe design strategies and applications.     

Near-infrared fluorescent probes: synthesis and spectroscopic investigations of a few amphiphilic squaraine dyes

With the objective of developing near-infrared fluorescence probes for biological applications, a few squaraine dyes 3a-d, containing amphiphilic substituents, were synthesized and their photophysical properties have been investigated in the presence and absence of the organized media. These dyes exhibited absorption in the range 630-650 nm, with significant absorption coefficients (epsilon = 1-3 x 10(5) M(-1) cm(-1)) in the aqueous medium. The fluorescence spectra of these dyes showed emission maximum from 660 to 675 nm, depending on the nature of substituents. The fluorescence quantum yields were in the range from 0.15 to 0.21 in ethanol, but 10 times lower values were observed (phi(f) = 0.01-0.02) in the aqueous medium. In the presence of micelles such as cetyltrimethylammonium bromide, sodium dodecyl sulfate, and Triton X-100, these dyes showed negligible changes in their absorption properties, whereas a significant enhancement (5-10-folds) in their fluorescence yields was observed. Picosecond time-resolved studies indicated that these dyes show single-exponential decay in ethanol and ethanol-water mixtures; however, they exhibit biexponential decay with longer lifetimes in the presence of the micellar media. The results indicate that these novel amphiphilic squaraine dyes 3a-d, which exhibit favorable photophysical properties, good solubility in the aqueous medium, and interact efficiently with micelles, can have potential biological applications as near-infrared fluorescence sensors.     

Seminaphthofluorescein-based fluorescent probes for imaging nitric oxide in live cells

Fluorescent turn-on probes for nitric oxide based on seminaphthofluorescein scaffolds were prepared and spectroscopically characterized. The Cu(II) complexes of these fluorescent probes react with NO under anaerobic conditions to yield a 20-45-fold increase in integrated emission. The seminaphthofluorescein-based probes emit at longer wavelengths than the parent FL1 and FL2 fluorescein-based generations of NO probes, maintaining emission maxima between 550 and 625 nm. The emission profiles depend on the excitation wavelength; maximum fluorescence turn-on is achieved at excitations between 535 and 575 nm. The probes are highly selective for NO over other biologically relevant reactive nitrogen and oxygen species including NO(3)(-), NO(2)(-), HNO, ONOO(-), NO(2), OCl(-), and H(2)O(2). The seminaphthofluorescein-based probes can be used to visualize endogenously produced NO in live cells, as demonstrated using Raw 264.7 macrophages.     

Sulfonate-based fluorescent probes for imaging hydrogen peroxide in living cells

Based on the mechanism of H₂O₂-mediated hydrolysis of sulfonates, two fluorescein disulfonates compounds (FS-1 and FS-2) were designed and synthesized as the highly selective and sensitive fluorescent probes for imaging H₂O₂ in living cells. The probes were detected with elemental analysis, IR, ¹H NMR and ¹³C NMR. Upon reaction with H₂O₂, the probes exhibit strong fluorescence responses and high selectivity for H₂O₂ over other reactive oxygen species and some biological compounds. Furthermore, the sulfonate-based probes, as novel fluorescent reagents, are cell-permeable and can detect micromolar changes in H₂O₂ concentrations in living cells by using confocal microscopy. Supported by the National Basic Research Program of China (Grant No. 2007CB936000), the National Natural Science Funds for Distinguished Young Scholar (Grant No. 20725518), Major Program of the National Natural Science Foundation of China (Grant No. 90713019), the National Natural Science Foundation of China (Grant No. 20875057), the Natural Science Foundation of Shandong Province, China (Grant No. Y2007B02), and the Science and Technology Development Programs of Shandong Province, China (Grant No. 2008GG30003012)     

基于磺酸酯的荧光探针用于活细胞内过氧化氢的成像检测

基于过氧化氢（H2O2）特异性催化水解磺酸酯,设计合成了新型绿色荧光探针：荧光素二磺酸酯（FS—1）和二氯荧光素二磺酸酯（FS-2）两种螺环内酯型化合物,用于活细胞内过氧化氢的检测．探针结构由元素分析、IR、^1H NMR及^13C NMR表征．实验表明：探针FS-1和FS-2在模拟生物体系中检测过氧化氢具有良好的选择性和灵敏度,且线性范围较宽．细胞成像显示：探针FS-1和FS-2用于PMA刺激或外加不同浓度H2O2孵育的小鼠腹膜巨噬细胞均呈现明亮的绿色荧光,且能对细胞内H2O2微摩尔级浓度变化产生响应,证明两探针均具有良好的膜渗透性、高的选择性及良好的灵敏度．该方法的建立对研究生物体内H2O2的产生,H2O2导致的各种疾病机制及H2O2介导的信号转导途径具有重要的理论及实际意义．     

Near-infrared fluorescence probes for surgical navigation

Optical imaging is a promising tool for visualizing fundamental biological processes including disease progression, detection of tumors, and therapeutic monitoring non-invasively. Unlike visible light, near-infrared fluorescence (NIRF) imaging (beyond 700–1,700 nm) offers a competitive advantage to yield high-resolution images within a certain penetration depth (few millimeters to centimeters depending on NIR window). The last few years have witnessed rapid development of new NIRF probes within the span of whole NIR window, including small-molecule dyes, inorganic nanoparticles, and organic macromolecules. Benefitted by this, we observe a continual surge in the number of preclinical and clinical studies of NIRF imaging in surgery and related applications. At present, NIRF-guided imaging has emerged as a quintessential procedure to assist surgeons for intraoperative delineation and resection of tumors. Moreover, NIRF imaging is also used to improve the intraoperative staging, identify the hidden lesion in diseased organs, map lymph node metastases, detect tumor margins, and highlight vital organs intraoperatively. Considering rapid advancement of this field, we review recent progress in the development of NIRF probes, cancer-targeting strategies and their application for surgical navigation, particularly for the sentinel lymph node mapping, detection of tumors, and angiography. Moreover, we spotlight surgical navigation instrumentation that is currently used for intraoperative tumor detection.     

Nile-red and Nile-blue-based near-infrared fluorescent probes for in-cellulo imaging of hydrogen sulfide

Hydrogen sulfide has recently been identified as a biologically responsive species. The design and synthesis of fluorescence probes, which are constructed with Nile-red or Nile-blue fluorophores and a fluorescence-controllable dinitrophenyl group, for hydrogen sulfide are reported in this paper. The Nile-red–dinitrophenyl-ether-group-based probe (1a) is essentially non-fluorescent because of the inhibition of the photo-induced electron-transfer process; when the dinitrobenzene moiety is removed by nucleophilic substitution with the hydrosulfide anion, probe 1a is converted into hydroxy Nile red, eliciting a H₂S-induced fluorescence turn-on signal. Furthermore, probe 1a has high selectivity and sensitivity for the hydrosulfide anion, and its potential for biological applications was confirmed by using it for real-time fluorescence imaging of hydrogen sulfide in live HeLa cells. The Nile-blue–dinitrobenzene-based probe (1b) has gradually diminishing brightness in the red-emission channel with increased hydrogen-sulfide concentration. Thus, this paper reports a comparative study of Nile-red and Nile-blue-based hydrogen-sulfide probes. Graphical Abstract

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The chronological evolution of small organic molecular fluorescent probes for thiols

Abnormal concentrations of biothiols such as cysteine, homocysteine and glutathione are associated with various major diseases. In biological systems, the structural similarity and functional distinction of these three small molecular thiols has not only required rigorous molecular design of the fluorescent probes used to detect each thiol specifically, but it has also inspired scientists to uncover the ambiguous biological relationships between these bio-thiols. In this minireview, we will discuss the evolution of small organic molecular fluorescent probes for the detection of thiols over the past 60 years, highlighting the potent methodologies used in the design of thiol probes and their particular applications in the semi-quantification of cellular thiols and real-time labelling. At the same time, the present challenges that limit their further application will be discussed. We hope that this minireview will promote future research to enable deeper insight into the crucial role of thiols in biological systems.

The chronological evolution of small organic molecular fluorescent probes for thiols: from separation dependency analysis to cellular specific analysis, what''s next?     

Recent advances in molecular fluorescent probes for organic phosphate biomolecules recognition

Organic phosphate biomolecules (OPBs) are indispensable components of eukaryotes and prokaryotes, such as acting as the fundamental components of cell membranes and important substrates for nucleic acids. They play pivotal roles in various biological processes, such as energy conservation, metabolism, and signal modulation. Due to the difficulty of detection caused by variety OPBs, investigation of their respective physiological effects in organisms has been restrained by the lack of efficient tools. Many small fluorescent probes have been employed for selective detection and monitoring of OPBs in vitro or in vivo due to the advantages of tailored properties, biodegradability and in situ high temporal and spatial resolution imaging. In this review, we summarize the recent advances in fluorescent probes for OPBs, such as nucleotides, NAD(P)H, FAD/FMN and PS. Importantly, we describe their identification mechanisms in detail and discuss the general strategies for these OPBs probe designs, which provide new insights and ideas for the future probe designs.     

Selective turn-on fluorescent probes for imaging hydrogen sulfide in living cells

Hydrogen sulfide (H(2)S) is an important biological messenger but few biologically-compatible methods are available for its detection. Here we report two bright fluorescent probes that are selective for H(2)S over cysteine, glutathione and other reactive sulfur, nitrogen, and oxygen species. Both probes are demonstrated to detect H(2)S in live cells.