Plasmon-Enhanced Photoluminescence of SiC Quantum Dots for Cell Imaging Applications

Strong photoluminescence enhancement of chemically inert and biocompatible SiC quantum dots (QDs) ensured by their near-field coupling with multipolar localized plasmons is experimentally demonstrated. The main physical mechanisms responsible for this phenomenon are described with the use of three-dimensional FDTD simulations. Nano-Ag/SiN_X/glass plasmonic substrates were shown to be efficiently used for significant luminescence enhancement of fibroblast cells labeled with the SiC QDs. The proposed approach allows a plasmon-induced enhancement of fluorescent cell imaging.     

单克隆抗体ND-1-量子点荧光探针对大肠癌细胞的特异成像研究

目的制备抗人大肠癌单克隆抗体ND-1的量子点荧光探针,实现对大肠癌细胞的靶向成像。方法采用共价偶联方法,以1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC)和N-羟基硫代琥珀酰亚胺(NHS)为缩合剂,通过在反应体系中加入不同摩尔比例的单克隆抗体ND-1和游离量子点QD605进行条件优化,制备偶联产物ND-1-QD605荧光探针;利用荧光光谱扫描技术对ND-1-QD605进行光学特性表征,并检测其抗光漂白能力;利用免疫荧光方法检测ND-1-QD605对大肠癌细胞的靶向结合能力。结果在量子点QD605与单克隆抗体ND-1摩尔比1:40条件下,可实现二者的高效偶联;荧光光谱分析显示ND-1-QD605保留了游离量子点QD605优良的荧光特性;在激发光照射1h内,ND-1-QD605荧光强度未发生明显改变;荧光显微镜观察可见该探针能够与表达有相应抗原LEA的人大肠癌CCL187细胞特异性结合,呈现高灵敏度、特异性荧光成像。结论制备的单克隆抗体ND-1的量子点荧光探针具有大肠癌细胞靶向成像能力,有望为大肠癌的体内靶向成像研究和临床诊断提供新方法。     

Compact Quantum Dots for Single-molecule Imaging

Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology ^1-4. To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study.Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation ⁵. Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large ^4,6,7.Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past ^8,9. The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed Hg_xCd_1-xSe core coated with an insulating Cd_yZn_1-yS shell, further coated with a multidentate polymer ligand modified with short polyethylene glycol (PEG) chains (Figure 1). Compared with conventional CdSe nanocrystals, Hg_xCd_1-xSe alloys offer greater quantum yields of fluorescence, fluorescence at red and near-infrared wavelengths for enhanced signal-to-noise in cells, and excitation at non-cytotoxic visible wavelengths. Multidentate polymer coatings bind to the nanocrystal surface in a closed and flat conformation to minimize hydrodynamic size, and PEG neutralizes the surface charge to minimize nonspecific binding to cells and biomolecules. The end result is a brightly fluorescent nanocrystal with emission between 550-800 nm and a total hydrodynamic size near 12 nm. This is in the same size range as many soluble globular proteins in cells, and substantially smaller than conventional PEGylated QDs (25-35 nm).     

Quantum Dots in Cell Biology

Quantum dots are semiconductor nanocrystals that have broad excitation spectra, narrow emission spectra, tunable emission peaks, long fluorescence lifetimes, negligible photobleaching, and ability to be conjugated to proteins, making them excellent probes for bioimaging applications. Here the author reviews the advantages and disadvantages of using quantum dots in bioimaging applications, such as single-particle tracking and fluorescence resonance energy transfer, to study receptor-mediated transport.     

Targeting of Embryonic Stem Cells by Peptide-Conjugated Quantum Dots

Background

Targeting stem cells holds great potential for studying the embryonic stem cell and development of stem cell-based regenerative medicine. Previous studies demonstrated that nanoparticles can serve as a robust platform for gene delivery, non-invasive cell imaging, and manipulation of stem cell differentiation. However specific targeting of embryonic stem cells by peptide-linked nanoparticles has not been reported.

Methodology/Principal Findings

Here, we developed a method for screening peptides that specifically recognize rhesus macaque embryonic stem cells by phage display and used the peptides to facilitate quantum dot targeting of embryonic stem cells. Through a phage display screen, we found phages that displayed an APWHLSSQYSRT peptide showed high affinity and specificity to undifferentiated primate embryonic stem cells in an enzyme-linked immunoabsorbent assay. These results were subsequently confirmed by immunofluoresence microscopy. Additionally, this binding could be completed by the chemically synthesized APWHLSSQYSRT peptide, indicating that the binding capability was specific and conferred by the peptide sequence. Through the ligation of the peptide to CdSe-ZnS core-shell nanocrystals, we were able to, for the first time, target embryonic stem cells through peptide-conjugated quantum dots.

Conclusions/Significance

These data demonstrate that our established method of screening for embryonic stem cell specific binding peptides by phage display is feasible. Moreover, the peptide-conjugated quantum dots may be applicable for embryonic stem cell study and utilization.     

量子点在癌症研究中的应用

半导体量子点具有长时间、多目标和灵敏度高等独特的光化学性质,这些特性使量子点成为细胞标记和生物应用中得到了广泛的应用。利用量子点目标定位癌细胞,对于寻找癌变部位具有指导的作用。近年来,利用量子点作为光动力学治疗癌症的能量供体也得到了一定的研究。简单地介绍了量子点独特的光学性质,并从量子点标记癌细胞、可视化癌细胞表面功能和在光动力学治疗癌症等方面综述了量子点在癌症诊断和治疗中的应用。     

量子点光学成像技术在肿瘤诊断和治疗的应用

纳米技术在生物医学的进展使其在肿瘤的诊治中应用日益广泛。荧光纳米粒子中的量子点(Quantum Dots),具备光学成像特性在肿瘤中应用中显示出独特的优势。其作为一种荧光半导体纳米粒子,具有荧光强度高、稳定性强、激发波谱宽、发射波谱窄等光学特性。同时,它可以结合其他功能基团,包括靶向模式、治疗因素和成像探针,为临床肿瘤诊断和治疗提供了新的潜力。本文就量子点的类型和特点及量子点的肿瘤体外和体内成像进行综述。     

体内光量子点可视化图像在脑肿瘤中的应用

恶性胶质瘤年发病率约为5/100,000。美国每年有超过14,000例的新发恶性脑胶质瘤患者。治疗主要以手术治疗为主,手术肿瘤的切除程度影响患者的预后。外科手术治疗脑肿瘤需要精确定位脑肿瘤组织在正常脑组织中的位置以便能够获得精确的组织活检和肿瘤的完全切除。量子点是稳定存在的,产生荧光的可视化半导体纳米晶体。静脉注射量子点伴随着网状内皮系统和巨噬细胞的隔离。巨噬细胞可渗入到肿瘤组织并且能够吞噬通过静脉注射的光量子来产生可视化的肿瘤标记。通过巨噬细胞介导,将光量子运输至肿瘤组织展现了一种新兴技术来标记术前肿瘤组织。由于肿瘤组织中的光量子可以被光学成像和光谱学工具来探测,因此在脑肿瘤组织活检和切除中可以为外科医生提供可视化得实时反馈。     

Imaging Depths of Near-infrared Quantum Dots in First and Second Optical Windows

AbstractPotential advantages of quantum dot (QD) imaging in the second optical window (SOW) at 1,000 to 1,400 nm over the first optical window (FOW) at 700 to 900 nm have attracted much interest. QDs that emit at 800 nm (800QDs) and QDs that emit at 1,300 nm (1,300QDs) are used to investigate the imaging depths at the FOW and SOW. QD images in biologic tissues are processed binarized via global thresholding method, and the imaging depths are determined using the criteria of contrast to noise ratio and relative apparent size. Owing to the reduced scattering in the SOW, imaging depth in skin can be extended by approximately three times for 1,300QD/SOW over 800QD/FOW. In liver, excitation of 1,300QD/SOW can be shifted to longer wavelengths; thus, the imaging depth can be extended by 1.4 times. Effects of quantum yield (QY), concentration, incidence angle, polarization, and fluence rate F on imaging depth are comprehensively studied. Under F approved by the Food and Drug Administration, 1,300QDs with 50% QY can reach imaging depths of 29.7 mm in liver and 17.5 mm in skin. A time-gated excitation using 1,000 times higher F pulses can obtain the imaging depth of ≈ 5 cm. To validate our estimates, in vivo whole-body imaging experiments are performed using small-animal models.     

Characterization of VCAM-1-Binding Peptide-Functionalized Quantum Dots for Molecular Imaging of Inflamed Endothelium

Inflammation-induced activation of endothelium constitutes one of the earliest changes during atherogenesis. New imaging techniques that allow detecting activated endothelial cells can improve the identification of persons at high cardiovascular risk in early stages. Quantum dots (QDs) have attractive optical properties such as bright fluorescence and high photostability, and have been increasingly studied and developed for bio-imaging and bio-targeting applications. We report here the development of vascular cell adhesion molecule-1 binding peptide (VCAM-1 binding peptide) functionalized QDs (VQDs) from amino QDs. It was found that the QD fluorescence signal in tumor necrosis factor (TNF-) treated endothelial cells in vitro was significantly higher when these cells were labeled with VQDs than amino QDs. The VQD labeling of TNF--treated endothelial cells was VCAM-1 specific since pre-incubation with recombinant VCAM-1 blocked cells'' uptake of VQDs. Our ex vivo and in vivo experiments showed that in the inflamed endothelium, QD fluorescence signal from VQDs was also much stronger than that of amino QDs. Moreover, we observed that the QD fluorescence peak was significantly blue-shifted after VQDs interacted with aortic endothelial cells in vivo and in vitro. A similar blue-shift was observed after VQDs were incubated with recombinant VCAM-1 in tube. We anticipate that the specific interaction between VQDs and VCAM-1 and the blue-shift of the QD fluorescence peak can be very useful for VCAM-1 detection in vivo.     

鼠源性人乳腺癌单链抗体库的构建与乳腺癌相关抗体的筛选

目的:获得乳腺癌的噬菌体呈现型单链抗体(scFv)库,筛选与乳腺癌细胞特异结合的抗体,为乳腺癌的诊断和治疗奠定基础。方法:用乳腺癌细胞系MCF-7、T47D、MDA-MB-435免疫BALB/c小鼠,取脾脏提取总RNA,用RT-PCR分别扩增抗体重、轻链可变区(VH和VL)基因,经Linker连接形成scFv基因片段。将scFv基因片段与噬菌粒载体pCANTAB5E的连接产物转化大肠杆菌TG1。用辅助噬菌体M13KO7进行超感染,获得重组噬菌体抗体。选用乳腺癌细胞系MCF-7和人正常肝细胞系HL02做正负差异的筛选细胞,通过5轮筛选,随机挑取克隆,经phage-ELISA筛选特异性结合MCF-7细胞的scFv。结果:构建了1个库容为1.3×106的单链抗体库。筛选到2株与MCF-7细胞有较高结合活性的噬菌体-单链抗体scFv-873和scFv-874。数据库搜索表明这2株单链抗体基因是与以往抗体序列不同的新基因。用Westernblot检测了这2株单链抗体在琥珀密码子非抑制型菌株TOP10中的表达情况。结论:筛选到2个与乳腺癌细胞结合特异性较好的单链抗体,为乳腺癌的诊断和治疗研究奠定了基础。     

Complexes of Streptavidin-Fused Antigens with Biotinylated Antibodies Targeting Receptors on Dendritic Cell Surface: A Novel Tool for Induction of Specific T-Cell Immune Responses

The choice of tools that enable efficient targeting of exogenous antigens (Ag) for processing and presentation by professional Ag-presenting cells (APC) remains limited. This represents, indeed, a bottleneck in development of vaccines inducing specific T-cell responses. Here, we describe a novel strategy of Ag delivery into APCs. The Ag of choice is fused to the N- or C-terminus of streptavidin (SA) and tetrameric Ag-SA or SA-Ag fusion proteins are produced in E. coli and purified by 2-Iminobiotin-Agarose affinity chromatography. Alternatively, Ag-SA proteins are purified from urea extracts of E. coli inclusion bodies and refolded in vitro into functional tetramers. Complexes with biotinylated antibodies targeting cell surface receptors are formed and used to deliver the Ags of choice for processing and presentation by APCs and induction of Ag-specific CD4+ and CD8+ T-cell responses in vitro and in vivo.     

近场光学显微镜结合量子点标记人胃腺癌SGC-7901细胞靶点的观察

扫描近场光学显微镜突破衍射极限,具有纳米量级的空间分辨率,量子点(QD s)标记有荧光强度高且抗光漂白能力强等优点。结合上述两种技术,对人胃腺癌SGC-7901细胞膜表面特异性结合的叶酸受体(FR)进行成像探测,获得了叶酸受体在SGC-7901细胞膜表面上的分布,以及细胞内化外源性叶酸过程中叶酸受体在细胞膜表面的分布变化,成像的光学分辨率达到120 nm。实验结果表明:特异性结合的叶酸受体在SGC-7901细胞膜表面的分布,绝大部分是以聚集体的形式存在。随着SGC-7901细胞内化叶酸量的增加,叶酸受体在细胞膜表面的分布密度逐渐降低,并在经过120 m in左右趋于稳定。上述方法和手段为实现单细胞水平上靶点分布和变化的长期监测,肿瘤细胞内化受体的机制研究提供了新的技术途径。     

以双歧杆菌为载体实现量子点在体靶向肿瘤的可行性研究

应用双歧双歧杆菌作为量子点输送载体,为小动物在体生物肿瘤成像提供依据. 采用电穿孔方法,将
二棕榈酰磷脂酰胆碱（DPPC）包被的硫硒镉水溶性量子点转入细菌内,得到“量子点-细菌”复合探针,在
“量子点-细菌”表面通过1-(3-二甲氨基丙基)-3-乙基碳二亚胺 (EDC)活化法进一步偶联叶酸分子,制得
“量子点-细菌-叶酸”复合纳米生物探针. 将纳米生物探针经尾静脉注入Lewis肺癌小鼠体内,采用冰冻组
织切片,考察探针在小鼠体内脏器及肿瘤的分布情况. 结果表明,在肿瘤部位检测到较强的量子点光致发光
信号,而在肺、肝、脾等脏器中只检测到微弱的量子点发光信号. “量子点-细菌-叶酸”复合纳米生物探
针小动物在体肿瘤靶向成像是可行的.     

基于单个B细胞抗体基因扩增技术筛选马IgG1单克隆抗体*

在马的免疫学研究领域中,由于目前市场上缺乏商业化的马IgG单克隆抗体,使得对马的B细胞研究受到很大阻碍,IgG是B细胞受体(BCR)的重要构成成分,与B细胞分化成熟相关。为了获得马IgG特异性单克隆抗体,利用单个B细胞扩增技术进行抗体筛选。首先,将马IgG蛋白(EqIgG1-C)密码子优化后合成到真核表达载体pcDNA3.4上,纯化出抗原蛋白。随后,使用蛋白质免疫小鼠,分离脾细胞后利用流式细胞术分离特异性单个B细胞,扩增出抗体重链和轻链的可变区基因,用overlapping PCR方法扩增出线性化的完整抗体,并进行鉴定。结果从80个B细胞中获得了27株特异性重组单克隆抗体,并挑选出3株线性结合活性最强的抗体基因构建到表达载体上,共转染Expi293F^TM细胞后表达纯化,经过ELISA和Western blot验证,显示获得的抗体可以和EqIgG1-C蛋白有良好的结合作用。使用该方法可以省时高效的获得特异性抗体,为马的免疫学研究提供了重要研究工具,为鼠单克隆抗体筛选提供了技术拓展。     

Selective Targeting of Tumorigenic Cancer Cell Lines by Microtubule Inhibitors

For anticancer drug therapy, it is critical to kill those cells with highest tumorigenic potential, even when they comprise a relatively small fraction of the overall tumor cell population. We have used the established NCI/DTP 60 cell line growth inhibition assay as a platform for exploring the relationship between chemical structure and growth inhibition in both tumorigenic and non-tumorigenic cancer cell lines. Using experimental measurements of “take rate” in ectopic implants as a proxy for tumorigenic potential, we identified eight chemical agents that appear to strongly and selectively inhibit the growth of the most tumorigenic cell lines. Biochemical assay data and structure-activity relationships indicate that these compounds act by inhibiting tubulin polymerization. Yet, their activity against tumorigenic cell lines is more selective than that of the other microtubule inhibitors in clinical use. Biochemical differences in the tubulin subunits that make up microtubules, or differences in the function of microtubules in mitotic spindle assembly or cell division may be associated with the selectivity of these compounds.     

Synthesis of Quantum Dots Labeled Short Peptides and Imaging the T cell Surface Receptors with QDs-Labeled Peptides

Semiconductor quantum dots have been used for labeling many biomacromolecules and small molecules, but it remains a challenge to couple it with short active peptides that play critical roles in many physiological processes. Several binding methods for QDs and short peptides have been reported, but all with some limitations in amino acid sequence. In this paper, we report a method for synthesis of quantum dots labeled short peptides that is appropriate to any short peptide. The quantum dots (CdTe)-labeled short peptides were verified and characterized by RP-HPLC. The QDs-labeled peptides were applied to monitor the specific binding between two immune peptides and T cell surface receptors. The quantum dots-labeled immune peptides provide a powerful method for studying immunological functions of these peptides, and an effective strategy for monitoring their complex modulating processes in vivo.     

量子点在生物学中的应用

量子点是一种无机荧光材料,它具有独特的光物理特性,如其激发光谱宽且连续分布,而发射光谱呈对称分布且宽度窄,而且可通过改变量子点内核的尺寸对其发射光波长进行精细调节等。量子点的这些特性正引起人们日益广泛的关注,并在很多领域得到了应用。本文介绍了量子点的组成以及它的光学特性,同时介绍并讨论了近年来量子点在生物学领域应用的进展以及存在的问题。