CD20靶向Cy7-Rituximab分子探针的制备及在小鼠活体荧光成像中的应用

以B淋巴细胞表面CD20抗原靶向的单克隆抗体Rituximab为载体, 通过共价键偶联荧光基团菁染料Cy7, 获得了新型荧光分子探针Cy7-Rituximab. 利用全光谱紫外-可见分光光度仪、SDS-聚丙烯酰胺凝胶电泳和基质辅助激光解析电离飞行时间质谱等对该探针结构进行表征, 并通过激光共聚焦显微镜观察了其在弥漫大B细胞淋巴瘤(DLBCL)细胞中的摄取情况. 选用BALB/C裸鼠为模型, 尾静脉注射 Cy7-Rituximab, 通过活体荧光成像系统观察了其在小鼠体内的分布情况. 研究结果表明, 修饰后的Cy7-Rituximab保持了原有抗体的免疫活性. 活体荧光成像结果表明, 在CD20高表达的脾脏部位监测到该分子探针的特异性浓集.     

活细胞线粒体中SO2-3/HSO-3的荧光成像

在五甲川菁染料的基础上,合成了一种新型的中位溴取代的五甲川菁染料Cy5-Br,其结构通过质谱和核磁共振波谱表征。性能测试结果表明,该染料作为荧光探针的发射波长可达665 nm,具有较低的细胞毒性,而且对二氧化硫衍生物 SO2-3和 HSO-3具有很好的选择性。该探针在665 nm 处的荧光发射强度随着SO2-3/HSO-3浓度的增加不断降低,具有良好的线性荧光响应。 Cy5-Br可以进入活细胞线粒体,对 SO2-3和HSO-3的浓度进行实时共聚焦荧光成像。     

活细胞线粒体中SO32-/HSO3-的荧光成像

在五甲川菁染料的基础上,合成了一种新型的中位溴取代的五甲川菁染料Cy5-Br,其结构通过质谱和核磁共振波谱表征.性能测试结果表明,该染料作为荧光探针的发射波长可达665 nm,具有较低的细胞毒性,而且对二氧化硫衍生物SO2-3和HSO-3具有很好的选择性.该探针在665 nm处的荧光发射强度随着SO2-3/HSO-3浓度的增加不断降低,具有良好的线性荧光响应.Cy5-Br可以进入活细胞线粒体,对SO2-3和HSO-3的浓度进行实时共聚焦荧光成像.     

一种高效灵敏的DNA荧光探针的合成及应用

设计合成了一种新型噻唑橙二聚体荧光染料Bi-TO3, 采用荧光发射光谱、 圆二色光谱及活细胞荧光成像等方法研究了其与DNA的相互作用. 在10 mmol/L Tris-HCl缓冲液(pH=7.4)中, Bi-TO3的固有荧光极弱, 量子产率小于0.001%; 与小牛胸腺DNA结合后, 其荧光可显著增强约950倍, 但对RNA和蛋白等生物大分子及黏度等环境因素则无明显响应. 紫外吸收光谱及圆二色光谱滴定实验表明, Bi-TO3以小沟结合模式与DNA作用, 且对AT序列有选择性. 实验结果表明, 在缓冲溶液中Bi-TO3的荧光增强信号与低浓度范围的poly(dA-dT)₂仍呈良好的线性关系, 检出限为13.3 ng/mL, 灵敏较度高; 且Bi-TO3可在较低浓度范围(6~12 μmol/L)内应用于活细胞荧光成像.     

TiO_2胶体对菁染料荧光的影响

用紫外 -可见光谱及荧光光谱研究了吸附在TiO2 胶体表面上的两种典型菁染料的光物理性质。吸收光谱表明染料基态分子存在着两种异构体;荧光实验表明TiO2 胶体对菁染料荧光发射强度有增强作用。这是由于染料分子被TiO2 胶体吸附后 ,其分子内的扭转运动受阻 ,从而单重激发态染料分子内扭转运动无辐射跃迁量子效率减少所致。文中还讨论了染料结构对染料 -TiO2 体系荧光发射的影响。     

吲哚类菁染料Cy3嵌入的核壳荧光纳米颗粒的制备及其性质研究

以蛋白质或多肽修饰的吲哚类菁染料Cy3为内核, 采用实验条件简单的油包水反相微乳液方法成核, 通过正硅酸乙酯水解形成的网状二氧化硅包壳的方法制备吲哚类菁染料Cy3嵌入的核壳荧光纳米颗粒. 考察了以不同等电点的蛋白质和多肽修饰的Cy3为内核材料对吲哚类菁染料Cy3嵌入的核壳荧光纳米颗粒制备的影响. 结果表明, 分别采用人免疫球蛋白(IgG)或多聚赖氨酸修饰的Cy3为内核材料, 都能制备荧光强度高、荧光稳定性强和染料泄漏极少的Cy3嵌入的核壳荧光纳米颗粒. 进一步对Cy3嵌入的核壳荧光纳米颗粒进行了表征, 并将基于这一新型的荧光纳米颗粒建立起来的生物标记方法初步应用于流感病毒DNA的检测, 其检测线性范围为3.18×10-10～1.27×10-9 mol/L, 检测下限为3.51×10-10 mol/L, 相关系数r为0.986 5.     

2D-DIGE-HPLC-nESI MS/MS对2,4-二硝基苯磺酸损伤HaCaT细胞差异蛋白质的鉴定

采用Cy2、Cy3和Cy5荧光染料标记蛋白,建立了人角质形成细胞HaCaT受2,4-二硝基苯磺酸(DNBS)刺激前后的双向胶内差异凝胶电泳(2D-DIGE)图谱,每组平行样本数为3.凝胶采用蛋白荧光染料Deep Purple进行后染色(Post-stain).DeCyder定量分析软件在每块凝胶上平均检测到1 200个...     

TiO2胶体对菁染料荧光的影响

用紫外－可见光谱及荧光光谱了吸附在ＴｉＯ２胶体表面上的两种典型菁染料的光物理性质。吸收光谱表明染料基态分子存在着两种异构体;荧光实验表明ＴｉＯ２胶体对菁染料荧光发射强度增强作用。这是出于染料分子被ＴｉＯ２胶体吸附后,其分子内的扭转运动受阻。从而单重激发态染料分子内扭转运动无辐射跃迁量子效率减少所致。文中还讨论了染料结构对染料－ＴｉＯ２胶有附后,其分子内的扭转运动受阻,从而单重激发态染料分子内扭转运     

不同溶剂对方块菁染料(SQ-3)LB膜吸收及荧光光谱的影响

利用荧光光谱、紫外可见吸收光谱研究了方块菁染料在不同极性的溶剂中的光谱性质的变化,并利用LB膜技术、Brewster角显微镜及荧光光谱、紫外可见吸收光谱研究了不同极性的铺展溶剂对染料的铺展形态及制备的LB膜样品的光谱性能的影响,说明了分子组装在形成功能薄膜中的重要性.     

一种含聚乙二醇非离子亲水基团的新型水溶性荧光染料的合成及细胞成像

通过向吲哚环“N”位上引入一个含聚乙二醇(PEG)醚链的非离子亲水基团,合成了一种新型水溶性不对称五甲川吲哚菁染料。 用核磁(¹H NMR)和高分辨质谱(HRMS)表征染料的结构,测试了染料的光谱性能,标记牛血清白蛋白(BSA),并对固定细胞和活细胞分别染色。 结果表明,该染料在水中的最大紫外吸收波长和荧光发射波长分别为648和668 nm,斯托克斯(stokes)位移为20 nm,荧光量子产率(Ф)为0.13,以碘钨灯为光源光照8 h后,染料光降解率为5.8%。 用染料的NHS活性酯标记牛血清白蛋白,标示率(D/P)为1.16。 对固定细胞染色发现,染料可对细胞整体着色,对细胞核染色最明显,能清晰看到核仁。 对活细胞染色发现,有少量染料跨膜进入细胞内部,对细胞质和细胞核有微弱染色,但在活细胞膜上聚集明显。     

Electrochemical Modulation of the Fluorescence of Cyanine Dye Cy5

Organic fluorescent dyes are widely used in single molecule localization microscopy, where their performances are determined by the photophysical properties. Herein, we utilized a sensitive method to modulate the fluorescence of organic dyes by external potentials using a combination of electrochemical cell and super‐resolution fluorescent microscopy. Cy5 (cyanine dye) was chosen as a model molecule considering its wide application and commercial availability. We applied different potentials on the Au electrode to change the Coulombic charge microenvironment of Cy5. When the electrode potential was adjusted negatively, Cy5 displayed a better photostability. This method is proved effective in adjusting the fluorescence of organic dyes.     

Fluorescence properties and photophysics of the sulfoindocyanine Cy3 linked covalently to DNA

The sulfoindocyanine Cy3 is one of the most commonly used fluorescent dyes in the investigation of the structure and dynamics of nucleic acids by means of fluorescence methods. In this work, we report the fluorescence and photophysical properties of Cy3 attached covalently to single-stranded and duplex DNA. Steady-state and time-resolved fluorescence techniques were used to determine fluorescence quantum yields, emission lifetimes, and fluorescence anisotropy decays. The existence of a transient photoisomer was investigated by means of transient absorption techniques. The fluorescence quantum yield of Cy3 is highest when attached to the 5' terminus of single-stranded DNA (Cy3-5' ssDNA), and decreases by a factor of 2.4 when the complementary strand is annealed to form duplex DNA (Cy3-5' dsDNA). Substantial differences were also observed between the 5'-modified strands and strands modified through an internal amino-modified deoxy uridine. The fluorescence decay of Cy3 became multiexponential upon conjugation to DNA. The longest lifetime was observed for Cy3-5' ssDNA, where about 50% of the decay is dominated by a 2.0-ns lifetime. This value is more than 10 times larger than the fluorescence lifetime of the free dye in solution. These observations are interpreted in terms of a model where the molecule undergoes a trans-cis isomerization reaction from the first excited state. We observed that the activation energy for photoisomerization depends strongly on the microenvironment in which the dye is located. The unusually high activation energy measured for Cy3-5' ssDNA is an indication of dye-ssDNA interactions. In fact, the time-resolved fluorescence anisotropy decay of this sample is dominated by a 2.5-ns rotational correlation time, which evidences the lack of rotational freedom of the dye around the linker that separates it from the terminal 5' phosphate. The remarkable variations in the photophysical properties of Cy3-DNA constructs demonstrate that caution should be used when Cy3 is used in studies employing DNA conjugates.     

In vivo Fluorescence Imaging of Tumors using Molecular Aptamers Generated by Cell‐SELEX

Poor sensitivity and low specificity of current molecular imaging probes limit their application in clinical settings. To address these challenges, we used a process known as cell‐SELEX to develop unique molecular probes termed aptamers with the high binding affinity, sensitivity, and specificity needed for in vivo molecular imaging inside living animals. Importantly, aptamers can be selected by cell‐SELEX to recognize target cells, or even surface membrane proteins, without requiring prior molecular signature information. As a result, we are able to present the first report of aptamers molecularly engineered with signaling molecules and optimized for the fluorescence imaging of specific tumor cells inside a mouse. Using a Cy5‐labeled aptamer TD05 (Cy5‐TD05) as the probe, the in vivo efficacy of aptamer‐based molecular imaging in Ramos (B‐cell lymphoma) xenograft nude mice was tested. After intravenous injection of Cy5‐TD05 into mice bearing grafted tumors, noninvasive, whole‐body fluorescence imaging then allowed the spatial and temporal distribution to be directly monitored. Our results demonstrate that the aptamers could effectively recognize tumors with high sensitivity and specificity, thus establishing the efficacy of these fluorescent aptamers for diagnostic applications and in vivo studies requiring real‐time molecular imaging.     

基于双色荧光传感器的癌细胞成像及microRNA定量检测

癌细胞中microRNA(miRNA)的灵敏成像对于疾病的诊断治疗具有重要意义, 其中miRNA-21通常在多种癌细胞中异常表达. 本文将DNA功能化的金纳米颗粒与发射波长分离的荧光染料FAM和Cy5.5修饰的DNA通过含有光控基团PC-linker的DNA4作为桥梁进行自组装, 构建了纳米传感器GDC. 将302 nm紫外光作为启动开关, 用其照射该体系时, Cy5.5修饰的DNA3被释放, 其荧光强度可作为内参比信号, 用于标定进入细胞的组装体含量; 细胞中miRNA-21作为催化分子, 与外加燃料Fuel DNA共同作用下可实现催化放大, FAM修饰的DNA2被释放且被猝灭的荧光信号得以恢复, 并作为检测信号. 通过2种荧光信号强度(FL)的检测及FL_FAM/FL_Cy5.5比值的计算, 达到定量分析细胞中miRNA含量的目的. 该体系可扣除因细胞内组装体含量不同造成的背景信号误差, 不仅能显著提高检测准确度, 还因存在催化循环而大大降低了检出限, 比传统方法至少降低了3个数量级. 该传感器的检出限为23.1 pmol/L, 通过定量计算得出HeLa细胞中miRNA的含量为0.0236 nmol/L.     

Preparation of luminescent Cy5 doped core-shell SFNPs and its application as a near-infrared fluorescent marker

Cy5 dye is widely used as a biomarker in the research fields of life science because of its excitation at wavelengths above 600 nm where autofluorescence of bio-matter is much reduced. However, Cy5 dye could not be encapsulate into silica directly to form stable nanoparticles by using of the traditional methods. In this paper, an improved method had been developed to prepare Cy5 dye doped core-shell silica fluorescent nanoparticles (SFNPs), employing biomolecules conjugated Cy5 as the core material and silica coating produced from the hydrolysis TEOS (tetraethyl orthosilicate) in the water-in-oil microemulsion. To obtain stable Cy5 dye doped SFNPs with core-shell structure, five kinds of biomolecules with different iso-electric point (pI) have been selected to conjugate Cy5 for preparation of core-shell SFNPs. Results demonstrated that very bright and photostable Cy5 doped core-shell SFNPs could be both prepared by use of positive polysine conjugated Cy5 or IgG conjugated Cy5 as the core material, respectively. IgG conjugated Cy5 doped core-shell SFNPs was selected as a demonstration to be characterized and applied as a near-infrared fluorescent marker in cell recognition. The results showed that Cy5 doped core-shell SFNPs prepared by conjugating with a positive biomolecules IgG as the core material were luminescent and stable. About 110 Cy5 dye molecules could be doped in one nanoparticle with size of 42 ± 5 nm. The breast cancer cells had been selectively recognized by use of the near-infrared fluorescent marker based on the Cy5-IgG doped core-shell SFNPs. And the results demonstrated that this Cy5 doped core-shell SFNPs fluorescence marker was superior to the pure Cy5 dye marker for cell recognition in photostability and detection sensitivity.     

Development of fluorescent glucose bioprobes and their application on real-time and quantitative monitoring of glucose uptake in living cells

We developed a novel fluorescent glucose bioprobe, GB2-Cy3, for the real-time and quantitative monitoring of glucose uptake in living cells. We synthesized a series of fluorescent glucose analogues by adding Cy3 fluorophores to the α-anomeric position of D-glucose through various linkers. Systematic and quantitative analysis of these Cy3-labeled glucose analogues revealed that GB2-Cy3 was the ideal fluorescent glucose bioprobe. The cellular uptake of this probe competed with the cellular uptake of D-glucose in the media and was mediated by a glucose-specific transport system, and not by passive diffusion. Flow cytometry and fluorescence microscopy analyses revealed that GB2-Cy3 is ten times more sensitive than 2-NBDG, a leading fluorescent glucose bioprobe. GB2-Cy3 can also be utilized for the quantitative flow cytometry monitoring of glucose uptake in metabolically active C2C12 myocytes under various treatment conditions. As opposed to a glucose uptake assay performed by using radioisotope-labeled deoxy-D-glucose and a scintillation counter, GB2-Cy3 allows the real-time monitoring of glucose uptake in living cells under various experimental conditions by using fluorescence microscopy or confocal laser scanning microscopy (CLSM). Therefore, we believe that GB2-Cy3 can be utilized in high-content screening (HCS) for the discovery of novel therapeutic agents and for making significant advances in biomedical studies and diagnosis of various diseases, especially metabolic diseases.     

Mapping Live Cell Viscosity with an Aggregation‐Induced Emission Fluorogen by Means of Two‐Photon Fluorescence Lifetime Imaging

Intracellular viscosity is a crucial parameter that indicates the functioning of cells. In this work, we demonstrate the utility of TPE‐Cy, a cell‐permeable dye with aggregation‐induced emission (AIE) property, in mapping the viscosity inside live cells. Owing to the AIE characteristics, both the fluorescence intensity and lifetime of this dye are increased along with an increase in viscosity. Fluorescence lifetime imaging of live cells stained with TPE‐Cy reveals that the lifetime in lipid droplets is much shorter than that from the general cytoplasmic region. The loose packing of the lipids in a lipid droplet results in low viscosity and thus shorter lifetime of TPE‐Cy in this region. It demonstrates that the AIE dye could provide good resolution in intracellular viscosity sensing. This is also the first work in which AIE molecules are applied in fluorescence lifetime imaging and intracellular viscosity sensing.     

Carbocyanine dyes as efficient reversible single-molecule optical switch

We demonstrate that commercially available unmodified carbocyanine dyes such as Cy5 (usually excited at 633 nm) can be used as efficient reversible single-molecule optical switch, whose fluorescent state after apparent photobleaching can be restored at room temperature upon irradiation at shorter wavelengths. Ensemble photobleaching and recovery experiments of Cy5 in aqueous solution irradiating first at 633 nm, then at 337, 488, or 532 nm, demonstrate that restoration of absorption and fluorescence strongly depends on efficient oxygen removal and the addition of the triplet quencher beta-mercaptoethylamine. Single-molecule fluorescence experiments show that individual immobilized Cy5 molecules can be switched optically in milliseconds by applying alternating excitation at 633 and 488 nm between a fluorescent and nonfluorescent state up to 100 times with a reliability of >90% at room temperature. Because of their intriguing performance, carbocyanine dyes volunteer as a simple alternative for ultrahigh-density optical data storage. Measurements on single donor/acceptor (tetramethylrhodamine/Cy5) labeled oligonucleotides point out that the described light-driven switching behavior imposes fundamental limitations on the use of carbocyanine dyes as energy transfer acceptors for the study of biological processes.