Pushing the limits of detection for proteins secreted from single cells using quantum dots (original) (raw)
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Nanobiotechnology: quantum dots in bioimaging
Expert Review of Proteomics, 2007
Many biological systems, including protein complexes, are natural nanostructures. To better understand these structures and to monitor them in real time, it is becoming increasingly important to develop nanometer-scale signaling markers. Single-molecule methods will play a major role in elucidating the role of all proteins and their mutual interactions in a given organism. Fluorescent semiconductor nanocrystals, known as quantum dots, have several advantages of optical and chemical features over the traditional fluorescent labels. These features make them desirable for long-term stability and simultaneous detection of multiple signals. Here, we review current approaches to developing a biological application for quantum dots.
Semiconductor quantum dots for bioimaging and biodiagnostic applications
Annual review of analytical chemistry (Palo Alto, Calif.), 2013
Semiconductor quantum dots (QDs) are light-emitting particles on the nanometer scale that have emerged as a new class of fluorescent labels for chemical analysis, molecular imaging, and biomedical diagnostics. Compared with traditional fluorescent probes, QDs have unique optical and electronic properties such as size-tunable light emission, narrow and symmetric emission spectra, and broad absorption spectra that enable the simultaneous excitation of multiple fluorescence colors. QDs are also considerably brighter and more resistant to photobleaching than are organic dyes and fluorescent proteins. These properties are well suited for dynamic imaging at the single-molecule level and for multiplexed biomedical diagnostics at ultrahigh sensitivity. Here, we discuss the fundamental properties of QDs; the development of next-generation QDs; and their applications in bioanalytical chemistry, dynamic cellular imaging, and medical diagnostics. For in vivo and clinical imaging, the potential ...
Quantum dots for detection, identification and tracking of single biomolecules in tissue and cells
The study of basic cell ultrastructure and intracellular physiological functions has been greatly aided by detection and identifi cation of single macromolecules. Since the current in situ labeling methods for directly correlative (light and electron) microscopy observations have a number of substantial limitations, the semiconductor nanocrystals quantum dots gain distinguished with long-term imaging and high photostability. The quantum dots (Qdots) have quickly fi lled in the role, being found to be superior to traditional organic dyes on several counts. It has been estimated that Qdots are 20 times brighter and 100 times more stable than traditional fl uorescent reporters. Nowadays, a wide variety of quantum dots conjugated to secondary antibodies suitable for multiple labeling have become commercially available. They make possible the study of biological processes, both in the membrane or in the cytoplasm, at a truly molecular scale and with high spatial and temporal resolutions. By applying Qdots with different size and color light we might achieve multiple labeling of proteins. However, the use of particles with different size is problematic for high-resolution imaging, semi-quantitative measurement of epitope numbers, or when epitope density is high. Recently we report new electron microscopy method for immunolabeling, where two approaches are performed to distinguish yet unattainable spatial resolution: i) for fi rst time as small as 1 nm nanoparticles were applied and observed and ii) the scanning transmission electron microscope (STEM) equipped with an energy dispersive X-ray (EDX) detector were used to distinguish equal in size small labels. We prove that various quantum dots in the range between 1–5 nm can be observed and identified at these conditions. Our method is not limited by the necessity of using labels of different sizes and, therefore could open a number of new biological applications requiring small labels. Because the only requirement is that labels have different chemical compositions that can be differentiated by an EDX spectrometer, this method is not restricted to only two labels. The number of different labels depends on the number of species with sufficiently different X-ray spectra that can be produced.
International journal of nanomedicine, 2006
Semiconductor quantum dots (QDs) are a new class of fluorescent labels with broad applications in biomedical imaging, disease diagnostics, and molecular and cell biology. In comparison with organic dyes and fluorescent proteins, quantum dots have unique optical and electronic properties such as size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to multifunctional nanoparticle probes that are highly bright and stable under complex in vitro and in vivo conditions. New designs involve encapsulating luminescent QDs with amphiphilic block copolymers, and linking the polymer coating to tumor-targeting ligands and drug-delivery functionalities. These improved QDs have opened new possibilities for real-time imaging and tracking of molecular targets in living cells, for multiplexed analysis of biomolecular markers in clinical tissue specimens, and for ultrasensitive ...
2005
Abstract: Enhanced peptide-coated quantum dots (with high brightness and high saturation intensity) were developed. Two high-affinity targeting" velcro-pairs" based on avidin-biotin and fluorescine-antibody interactions were demonstrated and used to specifically target single proteins in membranes of live cells. Single molecule spectroscopy and imaging of individual quantum dot-labeled lipid rafts receptors were performed. Software tools were developed to analyze individual diffusion and trafficking trajectories.
Quantum Dots for Live Cells, in Vivo Imaging, and Diagnostics
Science, 2005
Research on fluorescent semiconductor nanocrystals (also known as quantum dots or qdots) has evolved over the past two decades from electronic materials science to biological applications. We review current approaches to the synthesis, solubilization, and functionalization of qdots and their applications to cell and animal biology. Recent examples of their experimental use include the observation of diffusion of individual glycine receptors in living neurons and the identification of lymph nodes in live animals by near-infrared emission during surgery. The new generations of qdots have farreaching potential for the study of intracellular processes at the single-molecule level, high-resolution cellular imaging, long-term in vivo observation of cell trafficking, tumor targeting, and diagnostics.
Immunohistochemical Detection With Quantum Dots
Quantum dot (QD) conjugates have many immunohistochemical applications. The optical, excitation/emission, and photostable properties of QDs offer several advantages over the use of chromogens or organic fluorophores in these applications. Here, we describe the use of QD conjugates to detect primary antibody binding in fixed tissue sections. We also describe the use of QDs in simultaneous and sequential multilabeling procedures and in combination with enzyme-based signal amplification techniques. QD conjugates expand the arsenal of the immunohistochemist and increase experimental flexibility in many applications.
Biofunctional quantum dots as fluorescence probe for cell-specific targeting
Colloids and Surfaces B: Biointerfaces, 2014
We describe here the synthesis, characterization, bioconjugation, and application of water-soluble thioglycolic acid TGA-capped CdTe/CdS quantum dots (TGA-QDs) for targeted cellular imaging. Antihuman epidermal growth factor receptor 2 (HER2) antibodies were conjugated to TGA-QDs to target HER2-overexpressing cancer cells. TGA-QDs and TGA-QDs/anti-HER2 bioconjugates were characterized by fluorescence and UV-Vis spectroscopy, X-ray diffraction (XRD), hydrodynamic sizing, electron microscopy, and gel electrophoresis. TGA-QDs and TGA-QDs/anti-HER2 were incubated with cells to examine cytotoxicity, targeting efficiency, and cellular localization. The cytotoxicity of particles was measured using an MTT assay and the no observable adverse effect concentration (NOAEC), 50% inhibitory concentration (IC 50 ), and total lethal concentration (TLC) were calculated. To evaluate localization and targeting efficiency of TGA-QDs with or without antibodies, fluorescence microscopy and flow cytometry were performed. Our results indicate that antibody-conjugated TGA-QDs are well-suited for targeted cellular imaging studies.
Fluorescent immunolabeling of cancer cells by quantum dots and antibody scFv fragment
Journal of Biomedical Optics, 2009
Semiconductor quantum dots ͑QDs͒ coupled with cancer-specific targeting ligands are new promising agents for fluorescent visualization of cancer cells. Human epidermal growth factor receptor 2/neu ͑HER2/neu͒, overexpressed on the surface of many cancer cells, is an important target for cancer diagnostics. Antibody scFv fragments as a targeting agent for direct delivery of fluorophores offer significant advantages over full-size antibodies due to their small size, lower cross-reactivity, and immunogenicity. We have used quantum dots linked to anti-HER2/neu 4D5 scFv antibody to label HER2/neuoverexpressing live cells. Labeling of target cells was shown to have high brightness, photostability, and specificity. The results indicate that construction based on quantum dots and scFv antibody can be successfully used for cancer cell visualization. © 2009 Society of Photo-Optical Instrumentation Engineers. Keywords: immunolabeling; human epidermal growth factor receptor 2/neu ͑HER2/neu͒; quantum dots ͑QDs͒; 4D5 scFv antibody; human breast cancer SKBR-3 cells.