Direct and indirect immunolabelling of HeLa cells with quantum dots

With excellent optical properties, quantum dots (QDs) have been made as attractive molecular probes for labelling cells in biological research. In this study high‐quality CdSe QDs prepared in a paraffin–oleic acid system were used as fluorescent labels in direct and indirect detection of carcinoembryonic antigen (CEA), a cancer marker expressed on the surface of HeLa cells. The primary antibody (Ab) (rabbit anti‐CEA8) and secondary Ab (goat anti‐rabbit IgG) were covalently linked to carboxyl‐functioned CdSe QDs, and both the QDs–antibody and QDs–IgG probes were successfully used to label HeLa cells. The present study demonstrates the practicability of CdSe QDs as an attractive type of fluorescent labels for biological applications such as protein probes and cell imaging. Copyright © 2008 John Wiley & Sons, Ltd.     

Bioconjugated quantum dots for multiplexed and quantitative immunohistochemistry

Bioconjugated quantum dots (QDs) provide a new class of biological labels for evaluating biomolecular signatures (biomarkers) on intact cells and tissue specimens. In particular, the use of multicolor QD probes in immunohistochemistry is considered one of the most important and clinically relevant applications. At present, however, clinical applications of QD-based immunohistochemistry have achieved only limited success. A major bottleneck is the lack of robust protocols to define the key parameters and steps. Here, we describe our recent experience, preliminary results and detailed protocols for QD-antibody conjugation, tissue specimen preparation, multicolor QD staining, image processing and biomarker quantification. The results demonstrate that bioconjugated QDs can be used for multiplexed profiling of molecular biomarkers, and ultimately for correlation with disease progression and response to therapy. In general, QD bioconjugation is completed within 1 day, and multiplexed molecular profiling takes 1-3 days depending on the number of biomarkers and QD probes used.     

Detection of tumor marker CA125 in ovarian carcinoma using quantum dots

The fluorescent labeling of biological materials usingsmall-molecule organic dyes is widely employed in bio-logical imaging and clinical diagnosis. Organic fluoro-phores, however, have certain characteristics that limittheir advantages in some applications. These limitationsinclude narrow excitation bands and broad emissionbands with red spectral tails, which make the simultaneousevaluation of several light-emitting probes difficult due tospectral overlap. Also, many organic dyes exhibit highp…     

In vivo cancer targeting and imaging with semiconductor quantum dots

We describe the development of multifunctional nanoparticle probes based on semiconductor quantum dots (QDs) for cancer targeting and imaging in living animals. The structural design involves encapsulating luminescent QDs with an ABC triblock copolymer and linking this amphiphilic polymer to tumor-targeting ligands and drug-delivery functionalities. In vivo targeting studies of human prostate cancer growing in nude mice indicate that the QD probes accumulate at tumors both by the enhanced permeability and retention of tumor sites and by antibody binding to cancer-specific cell surface biomarkers. Using both subcutaneous injection of QD-tagged cancer cells and systemic injection of multifunctional QD probes, we have achieved sensitive and multicolor fluorescence imaging of cancer cells under in vivo conditions. We have also integrated a whole-body macro-illumination system with wavelength-resolved spectral imaging for efficient background removal and precise delineation of weak spectral signatures. These results raise new possibilities for ultrasensitive and multiplexed imaging of molecular targets in vivo.     

Potentials and pitfalls of fluorescent quantum dots for biological imaging

Fluorescent semiconductor nanocrystals, known as quantum dots (QDs), have several unique optical and chemical features. These features make them desirable fluorescent tags for cell and developmental biological applications that require long-term, multi-target and highly sensitive imaging. The improved synthesis of water-stable QDs, the development of approaches to label cells efficiently with QDs, and improvements in conjugating QDs to specific biomolecules have triggered the recent explosion in their use in biological imaging. Although there have been many successes in using QDs for biological applications, limitations remain that must be overcome before these powerful tools can be used routinely by biologists.     

In vivo molecular and cellular imaging with quantum dots

Quantum dots (QDs), tiny light-emitting particles on the nanometer scale, are emerging as a new class of fluorescent probe for in vivo biomolecular and cellular imaging. In comparison with organic dyes and fluorescent proteins, QDs have unique optical and electronic properties: size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to the development of multifunctional nanoparticle probes that are very bright and stable under complex in vivo conditions. A new structural design involves encapsulating luminescent QDs with amphiphilic block copolymers and linking the polymer coating to tumor-targeting ligands and drug delivery functionalities. Polymer-encapsulated QDs are essentially nontoxic to cells and animals, but their long-term in vivo toxicity and degradation need more careful study. Bioconjugated QDs have raised new possibilities for ultrasensitive and multiplexed imaging of molecular targets in living cells, animal models and possibly in humans.     

量子点在生物学中的研究进展

量子点作为一种新型的荧光标记物近年来已在生物学中获得广泛应用。本文总结了量子点的主要光学特性,其中包括荧光激发和发射光谱特性、量子产额、光漂白特性和荧光寿命等。重点综述了量子点在细胞标记、活体和组织成像、组合标记和光动力学治疗等生物学中的应用及其最新研究进展。同时讨论了量子点在应用中可能存在的细胞毒性等主要问题,最后对量子点在生物学中的应用前景作了展望。     

From analog to digital: exploring cell dynamics with single quantum dots

Semiconductor quantum dots (QDs) have emerged as new fluorescent probes for biology. When combined with ultrasensitive optical techniques, they allow motions of individual biomolecules to be tracked in live cells with high signal-to-noise and over unprecedented durations. Single QD imaging readily offers a powerful tool to investigate the organization in cell membranes. Altogether QDs will contribute to more advanced biological imaging and enable new studies on the dynamics of cellular processes.Robert Feulgen Lecture 2005 presented at the Joint Meeting of the Society for Histochemistry and The Histochemical Society in Noordwijkerhout, The Netherlands     

Application of quantum dots as probes for correlative fluorescence, conventional, and energy-filtered transmission electron microscopy.

Luminescent semiconductor quantum dots (QDs) are a new class of fluorescent label with wide-ranging applications for cell imaging. The electron density and elemental composition of these materials permit the extension of their use as probes in conventional electron microscopy (TEM) and energy-filtered TEM (EFTEM). Here we illustrate the feasibility of using streptavidin-conjugated QDs as TEM tags by labeling a nuclear protein on cell sections and obtaining correlative fluorescence and TEM data. We also show that QD probes can be employed in conjunction with immunogold for co-localization of proteins at the ultrastructural level. Furthermore, by obtaining cadmium elemental maps of CdSe/ZnS QDs distributed on a nuclear structure, we demonstrate the potential of QDs for co-localization of multiple proteins when used in combination with EFTEM.     

Cadmium-containing quantum dots: properties,applications, and toxicity

The marriage of biology with nanomaterials has significantly accelerated advancement of biological techniques, profoundly facilitating practical applications in biomedical fields. With unique optical properties (e.g., tunable broad excitation, narrow emission spectra, robust photostability, and high quantum yield), fluorescent quantum dots (QDs) have been reasonably functionalized with controllable interfaces and extensively used as a new class of optical probe in biological researches. In this review, we summarize the recent progress in synthesis and properties of QDs. Moreover, we provide an overview of the outstanding potential of QDs for biomedical research and innovative methods of drug delivery. Specifically, the applications of QDs as novel fluorescent nanomaterials for biomedical sensing and imaging have been detailedly highlighted and discussed. In addition, recent concerns on potential toxicity of QDs are also introduced, ranging from cell researches to animal models.

     

Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots

Semiconductor quantum dots (QDs) are among the most promising emerging fluorescent labels for cellular imaging. However, it is unclear whether QDs, which are nanoparticles rather than small molecules, can specifically and effectively label molecular targets at a subcellular level. Here we have used QDs linked to immunoglobulin G (IgG) and streptavidin to label the breast cancer marker Her2 on the surface of fixed and live cancer cells, to stain actin and microtubule fibers in the cytoplasm, and to detect nuclear antigens inside the nucleus. All labeling signals are specific for the intended targets and are brighter and considerably more photostable than comparable organic dyes. Using QDs with different emission spectra conjugated to IgG and streptavidin, we simultaneously detected two cellular targets with one excitation wavelength. The results indicate that QD-based probes can be very effective in cellular imaging and offer substantial advantages over organic dyes in multiplex target detection.     

Molecular profiling of single cells and tissue specimens with quantum dots

Quantum dots are tiny light-emitting particles on the nanometer scale. They are emerging as a new class of biological label with properties and applications that are not available with traditional organic dyes and fluorescent proteins. Recent advances, as reported in Science and Nature Biotechnology, have led to quantum dot bioconjugates that are highly luminescent and stable. These bioconjugates raise new possibilities for studying genes, proteins and drug targets in single cells, tissue specimens and even in living animals.     

Immunofluorescent labeling of CD20 tumor marker with quantum dots for rapid and quantitative detection of diffuse large B-cell non-Hodgkin's lymphoma

Fluorescent semiconductor quantum dots (QDs) are newfound nanocrystal probes which have been used in bioimaging filed in recent years. The purpose of this study is to evaluate the diagnostic value of specific QDs coupled to rituximab monoclonal antibody against CD20 tumor markers for patients with diffuse large B-cell lymphoma (DLBCL). In current study rituximab-conjugated quantum dots (QDs-rituximab) were prepared against CD20 tumor markers for detection of CD20-positive cells (human Raji cell line) using flowcytometry. A total of 27 tumor tissue samples were collected from patients with DLBCL and 27 subjects with negative pathological tests as healthy ones, which stained by QD-rituximab. The detection signals were obtained from QDs using fluorescence microscopy. The flowcytometry results demonstrated a remarkable difference in fluorescent intensity and FL2-H + (CD20-positive cells percentage) between two groups. Both factors were significantly higher in Raji in comparison with K562 cell line (P < 0.05). Lot of green fluorescence signals was observed due to the selectively binding of QD-rituximab to CD20 tumor markers which overexpressed in tumor tissues and a few signals observed on the defined healthy ones. Based on these observations the cut-off point was 46.8 dots and the sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 89.5%, 91.3%, and 100%, respectively (LR+, 9.52; LR−, 0). The QD - rituximab could be beneficial as a bioimaging tool with high sensitivity to provide an accurate molecular imaging technique for identifying CD20 tumor markers for early diagnosis of the patients with DLBCL.     

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。     

Improved QD-BRET conjugates for detection and imaging

Self-illuminating quantum dots, also known as QD-BRET conjugates, are a new class of quantum dot bioconjugates which do not need external light for excitation. Instead, light emission relies on the bioluminescence resonance energy transfer from the attached Renilla luciferase enzyme, which emits light upon the oxidation of its substrate. QD-BRET combines the advantages of the QDs (such as superior brightness and photostability, tunable emission, multiplexing) as well as the high sensitivity of bioluminescence imaging, thus holding the promise for improved deep tissue in vivo imaging. Although studies have demonstrated the superior sensitivity and deep tissue imaging potential, the stability of the QD-BRET conjugates in biological environment needs to be improved for long-term imaging studies such as in vivo cell tracking. In this study, we seek to improve the stability of QD-BRET probes through polymeric encapsulation with a polyacrylamide gel. Results show that encapsulation caused some activity loss, but significantly improved both the in vitro serum stability and in vivo stability when subcutaneously injected into the animal. Stable QD-BRET probes should further facilitate their applications for both in vitro testing as well as in vivo cell tracking studies.     

Bioconjugated quantum dots for cancer research: Present status,prospects and remaining issues

Semiconductor quantum dots (QDs) are nanoparticles in which charge carriers are three dimensionally confined or quantum confined. The quantum confinement provides size-tunable absorption bands and emission color to QDs. Also, the photoluminescence (PL) of QDs is exceptionally bright and stable, making them potential candidates for biomedical imaging and therapeutic interventions. Although fluorescence imaging and photodynamic therapy (PDT) of cancer have many advantages over imaging using ionizing radiations and chemo and radiation therapies, advancement of PDT is limited due to the poor availability of photostable and NIR fluorophores and photosensitizing (PS) drugs. With the introduction of biocompatible and NIR QDs, fluorescence imaging and PDT of cancer have received new dimensions and drive. In this review, we summarize the prospects of QDs for imaging and PDT of cancer. Specifically, synthesis of visible and NIR QDs, targeting cancer cells with QDs, in vitro and in vivo cancer imaging, multimodality, preparation of QD-PS conjugates and their energy transfer, photosensitized production of reactive oxygen intermediates (ROI), and the prospects and remaining issues in the advancement of QD probes for imaging and PDT of cancer are summarized.     

Lead sulfide near-infrared quantum dot bioconjugates for targeted molecular imaging

In this paper, we report the use of lead sulfide quantum dot (PbS QD) bioconjugates as near infrared (NIR) contrast agents for targeted molecular imaging with expanded emission wavelengths beyond 1000 nm. The red-shifted emission band, coupled with the small particle size, which will facilitate clearance, both afford PbS QDs unique properties for noninvasive, high resolution in vivo NIR imaging applications. We have performed imaging experiments at the molecular level using surface-modified PbS NIR QDs, together with our lab-built NIR imaging system. This novel instrumentation and fluorescent contrast agent have enabled us to study the relatively unexplored NIR biomedical imaging spectral region of 900-1200 nm. Preliminary experimental results indicate that PbS-QD/antibody bioconjugates are promising candidates for targeted NIR molecular imaging and future in vivo NIR tissue imaging applications.     

Enhancement of cell internalization and photostability of red and green emitter quantum dots upon entrapment in novel cationic nanoliposomes

Two quantum dots (QDs), a green emitter, CdSe and a red emitter, CdSe with ZnS shell are encapsulated into novel liposomes in two different formulations including cationic liposomes. Quantum dots have proven themselves as powerful inorganic fluorescent probes, especially for long‐term, multiplexed imaging and detection. Upon delivery into a cell, in endocytic vesicles such as endosomes, their fluorescence is quenched. We have investigated the potential toxic effects, photophysical properties and cell internalization of QDs in new formulation of liposomes as an in vitro vesicle model. Entrapment of QDs into liposomes is brought about with a decrease in their intrinsic fluorescence and toxicities and an increase in their photostability and lifetime. The biomimetic lipid bilayer of liposomes provides high biocompatibility, thereby enhancing the effectiveness of fluorescent nanoparticles for biological recognition in vitro and in vivo. The prepared lipodots could effectively prevent QDs from photo‐oxidation during storage and when exposed to ultraviolet (UV) light. Moreover, the flow cytometry of HEK 293 T cells showed that the cell internalization of encapsulated QDs in (DSPC/CHO/DOPE/DOAB) liposome is enhanced 10 times compared with non‐encapsulated QD (bare QDs).     

dsDNA-coated quantum dots

Because of their unique spectral properties, quantum dots (QDs) have recently proved useful as fluorescent labels for biosensing probes. We developed a versatile QD label by modifying dsDNA with biotin and thiol groups at opposite ends and attaching it to quantum dots via a metal-thiol bond. These dsDNA-coated QDs fluorescently label their targets through biotin-streptavidin binding and show excellent histological results when used to detect biotin-labeled chromosome probes. The dsDNA coating also circumvented the common problems of aggregation and steric hindrance that occur with other QD probes.     

Characterization and cancer cell targeted imaging properties of human antivascular endothelial growth factor monoclonal antibody conjugated CdTe/ZnS quantum dots

High luminescence quantum yield water‐soluble CdTe/ZnS core/shell quantum dots (QDs) stabilized with thioglycolic acid were synthesized. QDs were chemically coupled to fully humanized antivascular endothelial growth factor₁₆₅ monoclonal antibodies to produce fluorescent probes. These probes can be used to assay the biological affinity of the antibody. The properties of QDs conjugated to an antibody were characterized by ultraviolet and visible spectrophotometry, fluorescent spectrophotometry, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transmission electron microscopy and fluorescence microscopy. Cell‐targeted imaging was performed in human breast cancer cell lines. The cytotoxicity of bare QDs and fluorescent probes was evaluated in the MCF‐7 cells with an MTT viability assay. The results proved that CdTe/ZnS QD–monoclonal antibody nanoprobes had been successfully prepared with excellent spectral properties in target detections. Surface modification by ZnS shell could mitigate the cytotoxicity of cadmium‐based QDs. The therapeutic effects of antivascular endothelial growth factor antibodies towards cultured human cancer cells were confirmed by MTT assay. Copyright © 2014 John Wiley & Sons, Ltd.