一种新型快速检测半胱氨酸的荧光探针EI北大核心CSCD

基于三苯胺母体的强供电子能力,设计合成了一种共轭性良好的新型半胱氨酸(Cys)荧光探针。采用荧光光谱法和紫外-可见光谱法研究了目标探针T-Probe对半胱氨酸(Cys)的光谱响应。结果表明:目标探针分子与Cys作用后,荧光发射波长有约20 nm红移,荧光强度发生明显的增强,在365 nm紫外灯下,溶液由青色变为蓝色;探针分子选择性识别Cys的检测限为98.4 nmol/L,且灵敏度较高。     

基于萘酰亚胺的生物硫醇探针的合成及对含硫醇氨基酸的检测

合成了以4-羟基萘酰亚胺为荧光团,2,4-二硝基苯磺酰氧基为特异性识别基团的生物硫醇探针4-(2,4-二硝基苯磺酰氧基)-正丁基-1,8-萘酰亚胺(DNSBN).吸收光谱和荧光光谱结果表明, DNSBN对半胱氨酸(Cys)、同型半胱氨酸(Hcy)和谷胱甘肽(GSH)3种生物硫醇分子具有高效的检测识别能力,不受其它17种天然氨基酸的干扰.同时,通过荧光滴定实验证实了此探针是一种比率型探针,555 nm处的荧光强度与溶液中的生物硫醇分子浓度在0 ~ 20 μmol/L范围内呈良好的线性关系,对Cys、Hcy和GSH的检出限(3σ)分别为25.9、92.0和77.9 nmol/L.而吸收光谱、荧光光谱和质谱表征数据显示,生物硫醇与2,4-二硝基苯磺酸酯发生亲核取代反应并导致磺酸酯的分解.随着识别基团的解离,探针分子的d-PeT (donor-excited photoinduced electron transfer) 效应被解除,并出现非常明显的比色与荧光变化.HeLa细胞成像实验表明,探针DNSBN具有良好的生物相容性,能够对细胞外源性生物硫醇分子进行检测.     

基于分子内电荷转移机制半胱氨酸荧光探针的合成及应用

利用半胱氨酸(Cys)诱导的α,β-不饱和醛酮的加成环化反应来恢复探针的分子内电荷转移过程(ICT),成功合成一种专一性识别半胱氨酸的荧光探针。研究表明,探针分子仅对Cys具有显著的青色荧光增强响应,明显区分于非硫醇氨基酸和含硫醇氨基酸(同型半胱氨酸和谷胱甘肽),荧光可以恢复42倍,具有较好的稳定性。MDA-MB-231细胞内Cys的荧光成像证明了该有机分子具有潜在检测细胞内Cys的能力。     

一种基于查尔酮衍生物的荧光探针对巯基氨基酸的检测及细胞成像

生物硫醇(如半胱氨酸(Cys)、同型半胱氨酸(Hcy)及谷胱甘肽(GSH))与生物体和细胞中的许多生理和病理过程密切相关。荧光探针是对生物硫醇灵敏检测与成像的有力工具。本文合成了一种可检测生物硫醇的基于2′-羟基查尔酮荧光团开启型荧光探针1。探针中的2,4-二硝基苯磺酸酯基团既作为反应识别基团,又作为荧光猝灭基团。在DMSO/Tris(体积比8/2,pH=8.4)中,探针1与生物硫醇反应后释放出前体化合物3,3具有激发态分子内质子转移(ESIPT)和聚集诱导发光(AIE)特性,从而导致长波长荧光发射及较大的斯托克斯位移。探针1具有合成简单、灵敏度高、选择性高、细胞毒性低等优点,可以方便地检测溶液和活细胞中的生物硫醇。     

基于黄酮醇类半胱氨酸荧光探针的设计、合成及性能研究

以丹皮酚和糠醛为原料,黄酮醇为荧光团,丙烯酸酯为识别基团,设计并合成了一种黄酮醇类半胱氨酸荧光探针2-(2-呋喃基)-7-甲氧基色酮-3-丙烯酸酯(HFAC),采用核磁共振(¹H NMR、¹³C NMR)和高分辨质谱(HRMS-ESI)确证其结构。吸收光谱和发射光谱结果表明,探针HFAC对半胱氨酸(Cys)具有高效的检测识别能力。同时,荧光实验证实了探针HFAC是一种"关-开"型荧光探针,525 nm处的荧光强度与Cys浓度在20～400μmol/L范围内呈良好的线性关系,线性方程为y=0.4608x+3.994(R²=0.9987),检出限为37.9 nmol/L,并在365 nm紫外光下发出亮绿色荧光。A549细胞荧光成像实验表明,探针HFAC具有低毒性、良好的细胞膜通透性和生物相容性,具有快速检测细胞内/外源生物硫醇Cys的能力。     

特异性识别半胱氨酸荧光探针的合成及细胞成像研究

基于光诱导电子转移(PET)机制,利用Cys亲核性较强,能够与探针分子发生亲核取代反应,使丙烯酰基离去,使探针分子体系内PET过程失效,合成了一种特异性识别半胱氨酸的荧光探针。当向探针溶液分别加入多种测试物时,除与Cys结构类似的Hcy和GSH会引起探针溶液微弱的荧光变化外,其他氨基酸均不会引起探针溶液荧光强度的变化,该探针对Cys具有良好的选择性和灵敏度,可在生理条件下检测Cys,并且区分Hcy和GSH。同时,该探针成功实现了细胞内Cys的荧光成像,为在生物学及医学中的实际应用建立了一种特异性识别Cys的分析方法。     

荧光探针在半胱氨酸检测的应用

半胱氨酸(Cys)是三种生物硫醇之一,是20种天然氨基酸中唯一一种含还原性巯基的天然氨基酸,是组成细胞内多肽和蛋白质的基本氨基酸之一。其参与体内细胞的氧化还原调控,调节体内氧化还原平衡,维持机体正常代谢,在生理过程中发挥着至关重要的作用。然而体内的Cys浓度水平异常会引起一系列生理疾病,体内的Cys浓度作为几种疾病的生物标志物具有临床意义。因此有效地识别和检测半胱氨酸受到越来越多的研究者们的青睐。相较传统检测方法,荧光探针因其操作简单、灵敏度高、响应迅速和实时检测等优点,已被广泛用于检测生物硫醇。本文基于常见荧光团的结构性能特征,综述了近三年来检测Cys的荧光探针,重点概述了其传感机制,并对其生物应用进行了简要说明,展望了未来Cys探针的研究方向与应用前景。     

荧光素衍生物作为Hg2+比色/荧光双模式检测探针及其荧光成像

合成并表征了一种荧光素衍生物——硫代异氰酸苯酯荧光素(FHBS);采用紫外-可见光谱和荧光光谱,在乙醇/4-羟乙基哌嗪乙磺酸(HEPES)(体积比1∶1,pH=7.0)溶液中,研究了FHBS作为检测探针对Hg2+的识别性能.结果表明,该探针对Hg2+具有高选择性和灵敏性,其它阴、阳离子的存在不干扰其对汞离子的测定.另外,Hg2+的加入使探针溶液从无色变为亮黄色,荧光从无色变为亮绿色,因此该探针为Hg2+的比色/荧光双模式探针,并可用于细胞的荧光成像.     

选择性检测谷胱甘肽的荧光探针

本文设计合成了一种基于BODIPY衍生物选择性检测谷胱甘肽的比率式荧光探针1。荧光探针1中BODIPY的3位连有苯乙炔基团,5位连有咪唑盐离去基团,利用其与谷胱甘肽和半胱氨酸反应机理的不同实现了对谷胱甘肽的选择性检测。紫外可见吸收光谱和荧光光谱实验结果表明探针分子1与谷胱甘肽反应后的光谱发生明显红移,可以实现对谷胱甘肽的比率式检测。探针分子1对谷胱甘肽有极高的选择性,不受其它氨基酸尤其是半胱氨酸的干扰。荧光滴定实验表明探针分子1可实现对谷胱甘肽的定量检测,检测限为3.3×10-8 mol/L。探针分子成功地应用于活体细胞中检测谷胱甘肽。     

基于罗丹明荧光信号报告基团的细胞通透性铜离子荧光探针

分别以罗丹明B和罗丹明6G为荧光信号报告基团,以增强水溶性为目的的羟乙基肼为修饰基团,合成了反应型的Cu_(2+)离子选择性荧光探针分子L1和L2.紫外光谱和荧光光谱等分析结果表明,探针分子L1和L2对Cu_(2+)离子具有高灵敏度、高选择性的光谱识别行为.探针分子对Cu_(2+)离子的识别过程是通过Cu_(2+)离子催化水解控制氧杂蒽荧光信号的螺环酰肼基团实现荧光信号的开启,从而达到识别检测Cu_(2+)离子的目的,对Cu_(2+)离子的检出限均可达到10-8mol/L量级.同时,探针分子对常见金属离子和铵离子均具有较强的抗干扰能力.由于羟乙基肼的引入增强了探针的水溶性,使得探针L1和L2具有良好的细胞通透性和低毒性,实现了其对β-胰岛细胞(INS-1细胞)中Cu_(2+)离子的荧光成像检测.     

A Novel Fluorescent Probe Based on Perylene Derivative for Hg2+ Ions and Biological Thiols and its Application in Live Cell Imaging and Theoretical Calculations

The selective and sensitive detection of biothiols; cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) in aqueous solutions is of considerable importance because of their pivotal roles in maintaining the reducing environment in the cells. This study describes a strategy for the determination of biothiols based on the PDI/Met‐Hg²⁺complex platform. We designed and fabricated methionine modified perylene diimide molecule as a selective sensing probe for Hg²⁺ ions in aqueous solutions ( PDI/Met‐Hg ²⁺). The complex between perylene bisimide derivative ( PDI/Met) and Hg²⁺ was investigated and it demonstrated turn‐on fluorescence response for the detection of the biological thiols. Besides, PDI/Met displayed fluorescence quenching response in the presence of mercury ions and the emission intensity of PDI/Met‐Hg²⁺ was recovered after transferring biothiols (Cys, Hcy, and GSH). Thus, PDI/Met could be utilized as a fluorescent chemosensor for the sequential recognition of mercury ions and biological thiols.     

A sensitive and selective detection method for thiol compounds using novel fluorescence probe

In this work, a sensitive and selective detection method based on fluorescence resonance energy transfer (FRET) was developed for analyzing thiol compounds by using a novel fluorescent probe. The new fluorescent probe contains a disulfide bond which selectively reacts with nucleophilic thiolate through the thiol-disulfide exchange reaction. An obvious fluorescence recovery can be observed upon addition of the thiol compound in the fluorescent probe solution due to the thiol-disulfide exchange reaction and the destruction of FRET. This novel probe was successfully used to determine dithiothreitol (DTT), glutathione (GSH) and cysteine (Cys). The limits of detection (LOD) were 2.0 μM for DTT, 0.6 μM for GSH, and 0.8 μM for Cys. This new detection method was further investigated in the analysis of compound amino acid injection.     

Design of activatable red-emissive assay for cysteine detection in aqueous medium with aggregation induced emission characteristics

A novel water-soluble red-emissive AIE fluorescence probe for cysteine (Cys) in situ was prepared and the performance of selectivity and sensitivity has been carefully investigated in this study. The probe was established on the electrostatic interaction of sulfonate functionalized tetraphenylethene (TPE) and polycation generated by the reaction between a polymer bearing dinitrobenzenesulfonate groups and Cys. From the experimental results, it was easy to distinguish Cys from glutathione (GSH) and homocysteine (Hcy) with a detection limit of 73 nmol/L. The assay system also possessed strong anti-interference ability against multitudinous amino acids. The Stokes shift was 142 nm and the emission ranged from 550 nm to 850 nm. In addition, double responses in fluorescence and ultraviolet-visible spectra also make the red-emissive assay ideal for sensitive detection and quantification of Cys for most purposes, especially in-situ monitoring of Cys in aqueous medium.     

Novel fluorescent probe for the selective detection of organophosphorous nerve agents through a cascade reaction from oxime to nitrile via isoxazole

A novel fluorescent probe based on fluoresceinyloxime was developed for detection of such organophosphorous compounds as nerve agents. The probe underwent an abnormal cascade reaction from oxime to nitrile via isoxazole in aqueous solvent and displayed a highly selective fluorescence response to diethylchlorophosphate (DCP) with the detection limit of 10 nM DCP in HEPES buffer.     

Installation of efficient quenching groups of a fluorescent probe for the specific detection of cysteine and homocysteine over glutathione in solution and imaging of living cells

Herein, we report the synthesis and characterisation of a new fluorescent probe 4-(7-nitro-benzo[1,2,5]oxadiazol-4-yl)-benzaldehyde (NBOB) installed with quenching groups for highly selective and sensitive sensing of biothiols. The probe itself is non-fluorescent due to the presence of quenching groups and photoinduced electron transfer (PET) process. Thus, sensitivity of the probe towards thiols was significantly improved by quenching effects. NBOB has been shown to exhibit selective reactivity towards cysteine (Cys) and homocysteine (Hcy) over glutathione (GSH) under stoichiometric conditions. The response mechanism was proved by ¹H NMR, LCMS and theoretical calculation. The probe NBOB has been shown to react with Cys present in Vero cells by fluorescence microscopy.     

Cysteine‐Mediated Intracellular Building of Luciferin to Enhance Probe Retention and Fluorescence Turn‐On

The development of sensitive and selective small molecular probes that enable real‐time detection of endogenous cysteine (Cys) has become an attractive topic because of the essential roles played by Cys in controlling the cellular nitrogen balance and in maintaining biological redox homeostasis. Herein, we report a Cys‐specific probe, 2‐cyanobenzothiazol‐6‐yl acrylate (CBTOA), that shows not only fluorescence turn‐on for sensitive detection of endogenous Cys but also enhanced probe retention inside cells for real‐time monitoring of Cys levels upon external stimulation. Cys‐mediated intracellular formation of luciferin from CBTOA was the key strategy leading to this new type of fluorogenic probe. CBTOA showed fast response to Cys in living cells and liver tissue slices with high sensitivity and selectivity. By using CBTOA as a real‐time probe, we were able to monitor the change in Cys levels in living HeLa cells under ROS‐induced oxidative stress as well as in human mesenchymal stem cells during adipogenic differentiation.     

A Sensitive and Selective Fluorescent Probe for Cysteine Based on a New Response‐Assisted Electrostatic Attraction Strategy: The Role of Spatial Charge Configuration

A new strategy for fast fluorescent detection of cysteine (Cys), based on a response‐assisted electrostatic attraction, is demonstrated. By utilizing this strategy, we designed and synthesized three fluorescent probes for the specific detection of Cys under actual physiological conditions. The probe m‐ CP , a coumarin fluorophore conjugated with a substituted methyl pyridinium group through an unsaturated ketone unit, showed highly selective and sensitive detection for cysteine (Cys) over homocysteine (Hcy) and glutathione (GSH). The kinetic analysis indicated that the sensing process was highly accelerated (a response time less than 1 min) by the response‐assisted electrostatic attraction. More importantly, control experiments with isomeric probes first demonstrated that the spatial charge configuration of the probe played an important role in Cys‐preferred selectivity and kinetic rate acceleration. Furthermore, the practical utility of the probe m‐ CP in the fluorescent labeling of Cys residues within proteins was demonstrated. Finally, these probes were employed in living cell imaging with HeLa cells, in which it displayed satisfactory cell permeability and enabled us to distinguish active thiols in the cytoplasm, nucleus, and mitochondria.     

A fluorescence turn-on probe for cysteine and homocysteine based on thiol-triggered benzothiazolidine ring formation

We synthesized a new coumarin-based probe TP, containing a disulfide moiety, to detect biothiols in cells. A fluorescence turn-on response is induced by the thiol–disulfide exchange of the probe, with subsequent intramolecular benzothiazolidine ring formation giving rise to a fluorescent product. The probe exhibits an excellent selectivity for cysteine (Cys) and homocysteine (Hcy) over glutathione (GSH) and other amino acids. The fluorescent probe also exhibits a highly sensitive fluorescence turn-on response to Cys and Hcy with detection limits of 0.8 μM for Cys and 0.5 μM for Hcy. In addition, confocal fluorescence microscopy imaging using RAW264.7 macrophages demonstrates that the probe TP could be an efficient fluorescent detector for thiols in living cells.     

用于谷胱甘肽检测的新型罗丹明衍生物荧光探针EI北大核心CSCD

合成了一种反应型近红外荧光探针N-Rh-GSH,该探针以罗丹明衍生物为荧光母体,通过与谷胱甘肽(GSH)作用触发螺环的开关来实现信号的响应,其开环释放的荧光产物具有760 nm的近红外发射波长。细胞实验表明,该探针可实现对活细胞中GSH的成像。