近红外荧光BSA-CdSeTe探针的制备及在生物学中的应用

本文用半胱氨酸作表面修饰剂,水相合成水溶性的近红外量子点CdSeTe,并偶联标记BSA,形成近红外BSA-CdSeTe探针。对CdSeTe QDs和BSA-CdSeTe探针进行荧光光谱分析,探讨了pH值、CdSeTe用量和反应时间这三个因素对近红外BSA-CdSeTe探针荧光强度的影响,对近红外BSA-CdSeTe探针进行了体外细胞成像实验和细胞毒性实验。结果表明,制得的CdSeTe粒径约5 nm,BSA-CdSeTe粒径约19 nm。当λex≈470 nm时,二者的λem均在750 nm左右。在pH=8、1m L CdSeTe和反应120 min时体系的荧光强度最大。当BSACdSeTe探针浓度在4~200μg·mL~(-1)范围之间,L929细胞的存活率均在85%之上。该探针在近红外荧光显微镜中可视,与L929细胞共孵育后可实现实时成像。因此,近红外荧光探针BSA-CdSeTe是一种可以进行实时细胞成像且生物相容性良好的纳米探针。     

水相合成近红外CdTe量子点用于体液中硫醇的检测

通过降低反应物中L-半胱氨酸(L-Cys)的比例,在水相快速合成了近红外CdTe量子点,使之对巯基化合物产生荧光响应. 并以此构建了一种基于表面配体缺失的CdTe近红外荧光量子点的巯基探针,为生物样品中的硫醇检测提供简便经济、高灵敏度和高选择性的新方法. 在其它多种氨基酸和生物液体中主要离子、分子共存的情况下,我们所制备的近红外量子点对L-Cys、同型半胱氨酸(Hcy)和谷胱甘肽(GSH)的荧光检测中显示了良好的选择性和灵敏度. 在血清和细胞提取液中,加标5.0 μmol·L^-1硫醇的回收率均在90%～109%范围内. 该方法对L-Cys,Hcy和GSH的检出限(3s)分别为43,46和63 nmol·L^-1.     

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.     

一种基于香豆素-苯并噻唑荧光探针的合成及性能研究

以香豆素为母体,丙烯酸酯为识别基团,设计、合成了荧光探针3-(2-苯并噻唑基)香豆素-7-丙烯酸酯(探针1),用于高灵敏、选择性检测活细胞中的还原型谷胱甘肽(GSH)。探针1与GSH反应后,溶液由无色变为亮绿色,荧光强度与GSH浓度在0～10μmol/L范围内呈现良好的线性关系,检出限为74 nmol/L;此外,探针还具有选择性高、抗干扰能力强和稳定性好等优点。荧光成像实验表明,探针1具有良好的细胞膜通透性、低毒性和生物相容性,可用于细胞内GSH浓度的快速检测。     

基于聚集诱导发光的荧光多肽探针检测谷胱甘肽EI北大核心CSCD

采用固相合成法将聚集诱导发光分子NI-COOH与GSH响应的多肽序列共价连接,得到兼具NI探针的聚集诱导发光性能和GSH响应性的多肽探针(NI-GFFKESSRGD),可用于GSH的体外检测。在50~600μmol/L的GSH浓度范围内,多肽探针的荧光强度和GSH浓度呈良好的线性关系,在该范围内可对未知浓度的GSH样品进行准确定量,误差小于0.5%。当多肽的浓度进一步增加至2.0%(w/w)时,可形成GSH响应的超分子水凝胶,其微观形貌上呈现纳米纤维交联的网状结构,流变性能上具有良好的粘弹特性和剪切稀化特点,是一类优秀的可注射性材料。多肽探针对成纤维细胞和肿瘤细胞具有良好的细胞相容性,表明该探针在非入侵性的活体生物分子检测上具有良好的应用前景。     

一种新型一氧化氮比率型近红外荧光探针的制备及应用

该文以邻苯二胺修饰的［c］［1, 2, 5］噻二唑-5, 6-二胺作为一氧化氮（NO）识别基团和电子受体,芴衍生物作为荧光基团和电子供体,合成了一种新型检测NO的近红外荧光探针。通过紫外可见吸收光谱和荧光光谱研究探针分子的光谱学性质及检测NO的可行性。该探针与NO反应后生成苯并三氮唑结构,分子内电荷转移（ICT）效应加强,在近红外区的荧光明显增强。相较于传统的增强型或猝灭型NO荧光探针,该文制备的荧光探针通过比率计量荧光检测信号,实现了背景荧光低、抗干扰能力强的NO近红外荧光检测。该荧光探针受外界干扰小,且不与其他活性氧、活性氮反应,能够对不同浓度的NO产生快速、灵敏的荧光响应,对NO的检测线性范围为0～10 μmol/L,检出限为28.88 nmol/L。选择性实验表明,该探针对NO的响应具有专一性和抗干扰性。该文制备的比率型荧光探针能实现NO近红外荧光分析和检测,具有背景荧光低、抗干扰能力强的优点,可用于生物样品中NO的检测。     

γ-谷氨酰转移酶近红外比率型荧光探针的设计与成像

γ-谷氨酰转肽酶(GGT)在诸多肿瘤组织中高表达,是一种重要的肿瘤标志物,对其活性的准确检测有助于相关疾病的早期诊断与治疗.本文将谷胱甘肽(GSH)共价修饰于七甲川花菁染料上作为GGT的酶切位点,研制了一例可用于GGT活性检测的近红外比率型荧光探针(Cy-GGT).该探针具有良好的水溶性,向其溶液中加入GGT,荧光发射波长由808 nm蓝移至616 nm (蓝移192 nm).较大的发射位移可避免两个信号之间的串扰,提高检测的准确性.实验表明,探针Cy-GGT可特异性检测GGT活性,检测限为0.02 mU/mL,并可用于活细胞和动物体中GGT的活性检测和荧光成像,能够区分肿瘤细胞与正常细胞,有望在相关疾病的早期诊断与治疗中发挥积极作用.     

一种用于活细胞中检测还原性谷胱甘肽的试卤灵类荧光探针

设计并合成了一种试卤灵类的荧光探针R1,通过质谱,1HNM R谱及13C NM R谱等手段对探针R1进行表征。并采用荧光光谱法和紫外-可见光谱法研究了探针R1对小分子生物硫醇包括还原型谷胱甘肽(GSH)和L-半胱氨酸(Cys)的光谱响应。实验结果表明,探针R1在p H 7.4磷酸缓冲溶液中对小分子生物硫醇均表现出快速、灵敏的显色和荧光响应,以及较好的选择性。与GSH作用后,探针R1的溶液颜色由无色变为红色。在5~100μmol/L范围内,探针R1的荧光强度与GSH浓度呈线性关系,对GSH的检出限为0.3μmol/L。此外,探针R1可跨过细胞膜,可应用于He La细胞内源性的GSH的激光共聚焦成像,对研究各类病理状态下细胞内氧化还原状态异常有一定的辅助作用。     

基于聚集诱导发光的荧光多肽探针检测谷胱甘肽

采用固相合成法将聚集诱导发光分子NI-COOH与GSH响应的多肽序列共价连接,得到兼具NI探针的聚集诱导发光性能和GSH响应性的多肽探针(NI-GFFKESSRGD),可用于GSH的体外检测。在50～600μmol/L的GSH浓度范围内,多肽探针的荧光强度和GSH浓度呈良好的线性关系,在该范围内可对未知浓度的GSH样品进行准确定量,误差小于0.5%。当多肽的浓度进一步增加至2.0%(w/w)时,可形成GSH响应的超分子水凝胶,其微观形貌上呈现纳米纤维交联的网状结构,流变性能上具有良好的粘弹特性和剪切稀化特点,是一类优秀的可注射性材料。多肽探针对成纤维细胞和肿瘤细胞具有良好的细胞相容性,表明该探针在非入侵性的活体生物分子检测上具有良好的应用前景。     

基于新型萘环稠合硼氟二吡咯化合物(BODIPY)的氟离子荧光探针及其细胞成像研究

设计、合成了一种基于新型萘环稠合硼氟二吡咯化合物(BODIPY)5的氟离子比率计量型和荧光猝灭型分子探针.通过紫外-可见光谱实验发现,该探针在氟离子存在时光谱红移100nm,进入近红外区域,可用于肉眼比色检测.荧光光谱分析表明,氟离子可促使荧光猝灭.细胞成像研究表明探针分子5可在活体细胞中专一性地识别氟离子.     

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.     

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.     

Development of a Two-photon Ratiometric Fluorescent Probe for Glutathione and Its Applications in Living Cells

Glutathione(GSH), as the most abundant intracellular biothiol, plays an important role in the redox homeostasis of the organism. Abnormal concentrations of GSH in cells may lead to many malignant diseases, such as cancer, liver damage and neurodegenerative diseases. It is urgent to develop effective methods to detect GSH in living organisms. In this work, a new two-photon ratiometric fluorescent probe Co-GSH based on the coumarin-chalcone dye platform was judiciously developed. Based on the Michael-addition reaction, Co-GSH was able to identify GSH with high selectivity and sensitivity. Furthermore, assisted by laser-scanning confocal microscopy, Co-GSH could specifically response GSH over the other biothiols, including Cys and Hcy, in living HeLa cells by using one-and two-photon modes.     

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.     

Simultaneous Visualization of Endogenous Homocysteine,Cysteine, Glutathione,and their Transformation through Different Fluorescence Channels

Studying numerous biologically important species simultaneously is crucial to understanding cellular functions and the root causes of related diseases. Direct visualization of endogenous biothiols in biological systems is of great value to understanding their biological roles. Herein, a novel multi‐signal fluorescent probe was rationally designed and exploited for the simultaneous sensing of homocysteine (Hcy), cysteine (Cys), and glutathione (GSH) using different emission channels. This probe was successfully applied to the simultaneous discrimination between and visualization of endogenous Hcy, Cys, GSH, and their transformation in living cells.     

A Multi‐signal Fluorescent Probe with Multiple Binding Sites for Simultaneous Sensing of Cysteine,Homocysteine, and Glutathione

A novel fluorescent probe was developed by integrating chlorinated coumarin and benzothiazolylacetonitrile and exploited for simultaneous detection of cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). Featuring four binding sites and different reaction mechanisms for different biothiols, this probe exhibited rapid fluorescence turn‐on for distinguishing Cys, Hcy, and GSH with 108‐, 128‐, 30‐fold fluorescence increases at 457, 559, 529 nm, respectively, across different excitation wavelengths. Furthermore, the probe was successfully applied to the fluorescence imaging of endogenous Cys and GSH and exogenous Cys, Hcy, and GSH in living cells.     

A Multi‐signal Fluorescent Probe with Multiple Binding Sites for Simultaneous Sensing of Cysteine,Homocysteine, and Glutathione

A novel fluorescent probe was developed by integrating chlorinated coumarin and benzothiazolylacetonitrile and exploited for simultaneous detection of cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). Featuring four binding sites and different reaction mechanisms for different biothiols, this probe exhibited rapid fluorescence turn‐on for distinguishing Cys, Hcy, and GSH with 108‐, 128‐, 30‐fold fluorescence increases at 457, 559, 529 nm, respectively, across different excitation wavelengths. Furthermore, the probe was successfully applied to the fluorescence imaging of endogenous Cys and GSH and exogenous Cys, Hcy, and GSH in living cells.     

Simultaneous Visualization of Endogenous Homocysteine,Cysteine, Glutathione,and their Transformation through Different Fluorescence Channels

Studying numerous biologically important species simultaneously is crucial to understanding cellular functions and the root causes of related diseases. Direct visualization of endogenous biothiols in biological systems is of great value to understanding their biological roles. Herein, a novel multi‐signal fluorescent probe was rationally designed and exploited for the simultaneous sensing of homocysteine (Hcy), cysteine (Cys), and glutathione (GSH) using different emission channels. This probe was successfully applied to the simultaneous discrimination between and visualization of endogenous Hcy, Cys, GSH, and their transformation in living cells.     

A Reversible Fluorescent Probe for Real-Time Quantitative Monitoring of Cellular Glutathione

The ability to monitor and quantify glutathione (GSH) in live cells is essential in order to gain a detailed understanding of GSH-related pathological events. However, owing to their irreversible response mechanisms, most existing fluorescent GSH probes are not suitable for this purpose. We have developed a ratiometric fluorescent probe (QG- 1 ) for quantitatively monitoring cellular GSH. The probe responds specifically and reversibility to GSH with an ideal dissociation constant (K_d) of 2.59 mm and a fast response time (t_1/2=5.82 s). We also demonstrate that QG- 1 detection of GSH is feasible in a model protein system. QG- 1 was found to have extremely low cytotoxicity and was applied to determine the GSH concentration in live HeLa cells (5.40±0.87 mm ).     

A colorimetric,ratiometric and water-soluble fluorescent probe for simultaneously sensing glutathione and cysteine/homocysteine

A chlorinated coumarin-aldehyde was developed as a colorimetric and ratiometric fluorescent probe for distinguishing glutathione (GSH), cystenine (Cys) and homocysteine (Hcy). The GSH-induced substitution-cyclization and Cys/Hcy-induced substitution-rearrangement cascades lead to the corresponding thiol-coumarin-iminium cation and amino-coumarin-aldehyde with distinct photophysical properties. The probe can be used to simultaneously detect GSH and Cys/Hcy by visual determination based on distinct different colors – red and pale-yellow in PBS buffer solution by two reaction sites. From the linear relationship of fluorescence intensity and biothiols concentrations, it was determined that the limits of detection for GSH, Hcy and Cys are 0.08, 0.09 and 0.18 μM, respectively. Furthermore, the probe was successfully used in living cell imaging with low cell toxicity.