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.     

荧光探针在氰化物分析中的研究进展

氰化物极易与细胞色素氧化酶键合,抑制电子转移、导致组织缺氧,从而显示较强的毒性。荧光化学传感器作为简易、灵敏且可视化的方法广泛应用于氰化物的检测。本文对荧光探针在氰化物检测中的应用进行综述,概述了氰化物的毒性机制和荧光探针对氰化物的响应机理。同时,本文总结了荧光探针在水体、食品和生物组织中对氰化物的检测及生物成像中的应用,并对荧光探针在结构设计中如何提高生物兼容性和靶向性等进行了展望,以期为光化学探针分子的设计及应用提供理论与研究依据。     

Optical Properties and Response Mechanism Analysis of Multi-branched Fluorescent Probes Based on Intramolecular Charge Transfer

In this work, the optical properties of fluorescent probes used for detection of biothiol were studied by employing time-dependent density functional theory. By calculating the single photon absorption and emission properties of probe Mol.1, Mol.2 and Mol.3 before and after reaction with cysteine and homocysteine, we have investigated the effect of carboncarbon triple bond and benzene ring on the properties of fluorescent probes. It is found that the oscillator strength of probe molecules increases gradually with the improvement of the structure of the electron donor triphenylamine and the addition of carbon-carbon triple bonds, and better properties of fluorescence probes have also been demonstrated. At the same time, the effect of different number of side branches on the molecular properties of the probe was also studied. The results showed that compared with single-branched molecule Z1 and tribranched probe Mol.3, two side probe molecules Z2 had higher oscillator strength andbetter detection effect. In addition, the new single-branched probe Mol.4 with the addition of carbon-carbon triple bonds and benzene rings has better probe properties and simpler structure than the tribranched probe Mol.3.     

A chloroacetate based ratiometric fluorescent probe for cysteine detection in biosystems

The specific detection of cysteine (Cys) over homocysteine (Hcy), glutathione (GSH) and other amino acids is of great significance for studying its biological functions as well as for the diagnosis of related diseases. Chloroacetyl group was often used as a reaction site for cysteine fluorescent probes for its sensitivity and selectivity. However, high background fluorescence and low stability are common problems encountered by such probes. Here, four chloroacetyl group based fluorescent probes (C1, C2, C3, and H4) was synthesized for a comparative study. We found that the inefficient quenching ability of chloroacetyl group turned into an advantage when connected with a ratiometric fluorophore. With the modification of chloroacetyl group, probe H4 displayed excellent ratiometric property and great selectivity for Cys, the stability was also improved. Additionally, the probe was successfully applied for quantitative detection of Cys in fetal bovine serum and real-time imaging in living HeLa cells with low toxicity.     

Small molecule fluorescent probes of protein vicinal dithiols

The vicinal dithiol motif is widely present in proteins, and is critical for proteins' structures and functions.In recent years, a variety of fluorescent probes with high specificity and outstanding optical properties for sensing protein vicinal dithiols have been developed. In this review, we summarized the fluorescent probes of protein vicinal dithiols in literature. These probes are classified into four types based on their acceptor sites, i.e., biarsenical probes, monoarsenical probes, dimaleimide probes and diacrylate probes.Through analyzing the properties of different probes, we expect that this review would help readers further understand the structural factors of these probes and provide the design strategy for novel fluorescent probes with improved properties.     

官能化罗丹明基金属阳离子荧光探针

重金属离子对环境和生物体具有极强的生理毒性,故高选择性和高灵敏度的离子检测荧光探针的研究有着重要意义。荧光分子探针在表达分子间识别行为以及复杂的环境、生命体系的内状态信息方面具有优异的性能,已广泛深入的用于构建新型功能性探针分子。本文综述了近年来基于罗丹明的金属阳离子如Hg₂₊、Cu₂₊、Fe₃₊等荧光分子探针的研究进展,包括探针的结构特征、检测机理、检测水平,更重要的是其在环境检测、生物成像、分子器件等方面的新应用,并对荧光探针所面临的问题和发展前景做了分析。     

A carbazole-hemicyanine dye based ratiometric fluorescent probe for selective detection of bisulfite (HSO3−) in cells and C. elegans

A colorimetric and ratiometric fluorescent probe for selective detection of HSO₃^- based on the 1,4-nucleophilic addition reaction was successfully synthesized and applied to the detection of exogenous HSO₃^- in Hela cells and C. elegans.     

多肽荧光探针在蛋白质检测中的应用

荧光探针法是痕量蛋白质检测的重要方法,其中多肽荧光探针得到了广泛的应用.本文综述了3种主要类型多肽荧光探针,即单荧光标记探针、双荧光标记探针和与其他材料形成复合物的探针的结构特点、检测原理以及不同类型多肽荧光探针在蛋白质定性、定量检测和酶活性测定等方面的应用,并对多肽荧光探针的未来发展方向进行了展望.     

Fluorescent probes for detection of biothiols based on “aromatic nucleophilic substitution-rearrangement” mechanism

“Aromatic nucleophilic substitution-rearrangement (SNAr-rearrangement)” mechanism provided a powerful tool to design fluorescent probes for the discrimination between biothiols.     

缺氧响应荧光探针的成像及治疗应用

由于肿瘤内部细胞远离血管, 其氧气消耗量远远超出血液供应量, 因此容易导致肿瘤缺氧. 肿瘤缺氧会引发肿瘤扩散加速、 诱导某些基因过表达及产生药物抗药性等问题. 基于此, 发展性能优异的缺氧响应荧光探针对肿瘤的诊断和治疗具有重要意义. 本文对缺氧响应荧光探针在成像及治疗方面的应用进展进行了综合评述, 介绍了硝基、 偶氮键和醌3种常用的缺氧响应基团, 并探讨了它们在缺氧微环境下的识别机理; 介绍了缺氧响应荧光探针的构建及其在生物成像方面的最新研究成果; 总结了缺氧响应荧光探针在基因治疗、 光动力学治疗、 化学治疗及协同治疗方面的研究进展; 展望了缺氧响应荧光探针在临床诊断和治疗方面的应用前景.     

Molecular engineering of ultra-sensitive fluorescent probe with large Stokes shift for imaging of basal HOCl in tumor cells and tissues

Fluorescent probes have been widely employed in biological imaging and sensing. However, it is always a challenge to design probes with high sensitivity. In this work, based on rhodamine skeleton, we developed a general strategy to construct sensitivity-enhanced fluorescent probe with the help of theoretical calculation for the first time. As a proof of concept, we synthesized a series of HOCl probes. Experiment results showed that with the C-9 of pyronin moiety of rhodamine stabilized by an electron donor group, probe DQF-S exhibited an importantly enhanced sensitivity (LOD: 0.2 nmol/L) towards HOCl together with fast response time (<10 s). Moreover, due to the breaking symmetrical electron distribution by another electron donor group, the novel rhodamine probe DQF-S displayed a far red to near-infrared emission (>650 nm) and large Stokes shift. Bioimaging studies indicated that DQF-S can not only effectively detect basal HOCl in various types of cells, but also be successfully applied to image tumor tissue in vivo. These results demonstrate the potential of our design as a useful strategy to develop excellent fluorescent probes for bioimaging.     

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.     

Fluorescent probes for detection of biothiols based on “aromatic nucleophilic substitution-rearrangement” mechanism

Biothiols, including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) play important roles in physiological processes, and the detection of thiol using fluorescent probes has attracted attention due to their high sensitivity and selectively and invasive on-time imaging. However, the similar structures and reactivity of these biothiols present great challenges for selective detection. This review focused on the the “aromatic nucleophilic substitution-rearrangement (S_NAr-rearrangement) mechanism”, which provided a powerful tool to design fluorescent probes for the discrimination between biothiols. We classify the fluorescent probes according to types of fluorophores, such as difluoroboron dipyrromethene (BODIPY), nitrobenzoxadiazole (NBD), cyanine, pyronin, naphthalimide, coumarin, and so on. We hope this review will inspire exploration of new fluorescent probes for biothiols and other relevant analytes.     

A Quencher-Based Blood-Autofluorescence-Suppression Strategy Enables the Quantification of Trace Analytes in Whole Blood

Precise quantification of trace components in whole blood via fluorescence is of great significance. However, the applicability of current fluorescent probes in whole blood is largely hindered by the strong blood autofluorescence. Here, we proposed a blood autofluorescence-suppressed sensing strategy to develop an activable fluorescent probe for quantification of trace analyte in whole blood. Based on inner filter effect, by screening fluorophores whose absorption overlapped with the emission of blood, a redshift BODIPY quencher with an absorption wavelength ranging from 600–700 nm was selected for its superior quenching efficiency and high brightness. Two 7-nitrobenzo[c] [1,2,5] oxadiazole ether groups were introduced onto the BODIPY skeleton for quenching its fluorescence and the response of H₂S, a gas signal molecule that can hardly be quantified because of its low concentration in whole blood. Such detection system shows a pretty low background signal and high signal-to-back ratio, the probe thus achieved the accurate quantification of endogenous H₂S in 20-fold dilution of whole blood samples, which is the first attempt of quantifying endogenous H₂S in whole blood. Moreover, this autofluorescence-suppressed sensing strategy could be expanded to other trace analytes detection in whole blood, which may accelerate the application of fluorescent probes in clinical blood test.     

Molecular engineering of ultra-sensitive fluorescent probe with large Stokes shift for imaging of basal HOCl in tumor cells and tissues

Fluorescent probes have been widely employed in biological imaging and sensing. However, it is always a challenge to design probes with high sensitivity. In this work, based on rhodamine skeleton, we developed a general strategy to construct sensitivity-enhanced fluorescent probe with the help of theoretical calculation for the first time. As a proof of concept, we synthesized a series of HOCl probes. Experiment results showed that with the C-9 of pyronin moiety of rhodamine stabilized by an electron donor group, probe DQF-S exhibited an importantly enhanced sensitivity (LOD: 0.2 nmol/L) towards HOCl together with fast response time (<10 s). Moreover, due to the breaking symmetrical electron distribution by another electron donor group, the novel rhodamine probe DQF-S displayed a far red to near-infrared emission (>650 nm) and large Stokes shift. Bioimaging studies indicated that DQF-S can not only effectively detect basal HOCl in various types of cells, but also be successfully applied to image tumor tissue in vivo. These results demonstrate the potential of our design as a useful strategy to develop excellent fluorescent probes for bioimaging.     

谷胱甘肽和凋亡酶-3响应的环肽分子荧光探针

设计、合成了一类新型谷胱甘肽(glutathione,GSH)和凋亡酶-3(Caspase-3)响应的环肽分子荧光探针.该类探针主要由能量共振转移(FRET)分子荧光对、Caspase-3特异性识别多肽序列和GSH响应双硫键组成,分为不含穿膜肽序列(CP)和包含穿膜肽序列(cp CP)的两种不同环肽分子荧光探针.2种环肽分子荧光探针均能实现在GSH和Caspase-3同时存在情况下的精确成像,同时具有良好的响应性、特异性和高信噪比.该类环肽分子荧光探针在细胞培养环境中具有良好的稳定性和生物相容性.利用该探针,可以实现对星形孢菌素(STS)诱发的细胞凋亡进行实时、原位的成像监测,并对抗肿瘤药物阿霉素(DOX)和顺铂(cisplatin)诱导的细胞凋亡进行成像.这种具有多重响应并能用于精确成像的分子荧光探针将极大地促进疾病的精确诊断.     

Syntheses and characterizations of novel pyrrolocoumarin probes for SNAP-tag labeling technology

SNAP-tag technology is a revolutionary protein labeling technology employing in various biological studies. Since low signal/noise ratio and severe overlap between the FRET donors/acceptors often occurred in applying present fluorescent probes and thus limited the further applications, development of new fluorescent probes with excellent fluorescent properties is still of request by today’s SNAP-tag technology. In this paper, a number of SNAP-tag protein probes have been developed by incorporating a novel pyrrolocoumarin fluorophore recently developed by our group. Examination of these novel synthetic compounds shows all these materials possess satisfactory fluorescent properties. Among these, probe 7 exhibits the most excellent characters, and its quantum yield, maximum emission wavelength and Stocks shift reach to 0.44, 534 nm and 112 nm, respectively. Further analysis of structure-property relationship indicates that the probes with a longer C3-substituted alkyl (such as pentyl) give stronger fluorescence.     

近红外荧光探针检测金属离子、小分子和生物大分子

荧光探针技术是近年来发展迅速的一种荧光分析方法,具有灵敏度高、选择性好、操作简便和响应迅速等特点,受到环境及生命科学领域的青睐.随着荧光探针技术的发展,近红外一区荧光探针由于具有发射波长长(600～ 900 nm)、对细胞损伤小、组织穿透性强和自发荧光背景低等优点,被广泛应用于细胞、组织等复杂生物体系中生物分子的检测、...     

A near-infrared fluorescent probe for monitoring fluvastatin-stimulated endogenous H2S production

Most reported fluorescent probes have limitations in practical applications in living systems due to the strong autofluorescence background,construction of probes with near-infrared(NIR) fluorescence emission is an accessible approach for addressing this challenge.We here designed a NIR fluorescent probe for monitoring the endogenous production of H_2S in living cells.The designed probe showed significant NIR fluorescence turn-on response to H_2S with high selectivity,enabling the sensitive detection H_2S.Importantly,the probe could be applied in monitoring the endogenous production of H_2S in raw 264.7 macrophages.This study showed that fluvastatin can promote the activity of cystathionineγ-lyase(CSE) for generation H_2S.     

Development of Small-Molecule Fluorescent Probes Targeting Enzymes

As biological catalysts, enzymes are vital in controlling numerous metabolic reactions. The regulation of enzymes in living cells and the amount present are indicators of the metabolic status of cell, whether in normal condition or disease. The small-molecule fluorescent probes are of interest because of their high sensitivity and selectivity, as well as their potential for automated detection. Fluorescent probes have been useful in targeting particular enzymes of interest such as proteases and caspases. However, it is difficult to develop an ideal fluorescent probe for versatile purposes. In the future, the design and synthesis of enzyme-targeting fluorescent probes will focus more on improving the selectivity, sensitivity, penetration ability and to couple the fluorescent probes with other available imaging molecules/technologies.