Quantum dot bio-conjugate: as a western blot probe for highly sensitive detection of cellular proteins (original) (raw)

Luminescent quantum dots for cellular analysis

SPIE Proceedings, 2006

The paper describes the fabrication, characterization and applications of novel luminescent quantum dots fluorescence resonance energy transfer (FRET) based enzymatic activity probes. The luminescent probes are based on FRET between luminescent quantum dots that serve as donors and rhodamine acceptors that are immobilized to the surface of the quantum dots through peptide linkers that contain selective enzymatic cleavage sites. Upon enzymatic cleavage of the peptide linkers the rhodamine molecules no longer provide an efficient energy transfer channel to the quantum dots, which lightes up the initially quenched the quantum dots. The quantum dots based probes were applied for detecting enzyme activity and screening enzyme inhibitors. They were also used for the measurement of extracellular matrix metallproteinases (MMPs) activity in normal and cancerous breast cells tissues.

Impacts of quantum dots in molecular detection and bioimaging of cancer

Bioimpacts, 2014

Introduction: A number of assays have so far been exploited for detection of cancer biomarkers in various malignancies. However, the expression of cancer biomarker(s) appears to be extremely low, therefore accurate detection demands sensitive optical imaging probes. While optical detection using conventional fluorophores often fail due to photobleaching problems, quantum dots (QDs) offer stable optical imaging in vitro and in vivo. Methods: In this review, we briefly overview the impacts of QDs in biology and its applications in bioimaging of malignancies. We will also delineate the existing obstacles for early detection of cancer and the intensifying use of QDs in advancement of diagnostic devices. Results: Of the QDs, unlike the II-VI type QDs (e.g., cadmium (Cd), selenium (Se) or tellurium (Te)) that possess inherent cytotoxicity, the I-III-VI 2 type QDs (e.g., AgInS 2 , CuInS 2 , ZnS-AgInS 2 ) appear to be less toxic bioimaging agents with better control of band-gap energies. As highly-sensitive bioimaging probes, advanced hybrid QDs (e.g., QD-QD, fluorochrome-QD conjugates used for sensing through fluorescence resonance energy transfer (FRET), quenching, and barcoding techniques) have also been harnessed for the detection of biomarkers and the monitoring of delivery of drugs/genes to the target sites. Antibody-QD (Ab-QD) and aptamer-QD (Ap-QD) bioconjugates, once target the relevant biomarker, can provide highly stable photoluminescence (PL) at the target sites. In addition to their potential as nanobiosensors, the bioconjugates of QDs with homing devices have successfully been used for the development of smart nanosystems (NSs) providing targeted bioimaging and photodynamic therapy (PDT). Conclusion: Having possessed great deal of photonic characteristics, QDs can be used for development of seamless multifunctional nanomedicines, theranostics and nanobiosensors.

Quantum dots as fluorescent bio-labels in cancer diagnostic

2006

In this work we present and discuss some results concerning the application of colloidal semiconductor quantum dots for cancer diagnostic. We have prepared and applied different core-shell semiconductor quantum dots such as Cadmium Teluride-Cadmium Sulfide (CdTe-CdS) and Cadmium Sulfide -Cadmium Hydroxide (CdS/Cd(OH) 2 ). For the purpose of diagnostic with living cells, the CdS/Cd(OH) 2 presented best results, maintaining high levels of luminescence as well high stability in biological media. The quantum dots were obtained in aqueous medium, by reacting Cd 2+ and S 2in the presence of sodium polyphosphate as the stabilizing agent. Subsequent surface passivation with Cd(OH) 2 was carried out to improve luminescence. At a pH of 7,2 the quantum dots were functionalized with a 0.01% glutaraldehyde solution and then, incubated with living healthy and neoplastic cells (glial, glioblastoms and cervical) and tissues (breast) in culture medium. Tissue and cell staining were evaluated by the laser scanning confocal microscopy. Fluorescence Microscopy was used as a primary tool in order to explore the labeling of the samples. The procedure presented in this work, shown to be very efficient as a potential tool for fast and precise cancer diagnostic.

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.

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.

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.

Quantum Dots: Proteomics characterization of the impact on biological systems

Journal of Physics: Conference Series, 2009

Over the past few years, Quantum Dots have been tested in most biotechnological applications that use fluorescence, including DNA array technology, immunofluorescence assays, cell and animal biology. Quantum Dots tend to be brighter than conventional dyes, because of the compounded effects of extinction coefficients that are an order of magnitude larger than those of most dyes. Their main advantage resides in their resistance to bleaching over long periods of time (minutes to hours), allowing the acquisition of images that are crisp and well contrasted. This increased photostability is especially useful for three-dimensional (3D) optical sectioning, where a major issue is bleaching of fluorophores during acquisition of successive z-sections, which compromises the correct reconstruction of 3D structures. The long-term stability and brightness of Quantum Dots make them ideal candidates also for live animal targeting and imaging. The vast majority of the papers published to date have shown no relevant effects on cells viability at the concentration used for imaging applications; higher concentrations, however, caused some issues on embryonic development. Adverse effects are due to be caused by the release of cadmium, as surface PEGylation of the Quantum Dots reduces these issues. A recently published paper shows evidences of an epigenetic effect of Quantum Dots treatment, with general histones hypoacetylation, and a translocation to the nucleus of p53. In this study, mice treated with Quantum Dots for imaging purposes were analyzed to investigate the impact on protein expression and networking. Differential monoand bidimensional electrophoresis assays were performed, with the individuation of differentially expressed proteins after intravenous injection and imaging analysis; further, as several authors indicate an increase in reactive oxygen species as a possible mean of damage due to the Quantum Dots treatment, we investigated the signalling pathway of APE1/Ref1, a protein involved in the response to oxidative stress. Our results, although preliminary, suggest several interesting point of discussion on Quantum Dots imaging for in vivo diagnostic application, but also for a new therapeutic approach.

Fluorescence-based analysis of cellular protein lysate arrays using quantum dots

Methods in molecular biology (Clifton, N.J.), 2007

Reverse-phase protein microarrays (RPPMAs) enable heterogeneous mixtures of proteins from cellular extracts to be directly spotted onto a substrate (such as a protein biochip) in minute volumes (nanoliter-to-picoliter volumes). The protein spots can then be probed with primary antibodies to detect important posttranslational modifications such as phosphorylations that are important for protein activation and the regulation of cellular signaling. Previously, we relied on chromogenic signals for detection. However, quantum dots (QDs) represent a more versatile detection system because the signals can be time averaged and the narrow-emission spectra enable multiple protein targets to be quantified within the same spot. We found that commercially available pegylated, streptavidin-conjugated QDs are effective detection agents, with low-background binding to heterogeneous protein mixtures. This type of test, the RPPMAs, is at the forefront of an exciting, clinically-oriented discipline th...

New Quantum-Dot-Based Fluorescent Immunosensor for Cancer Biomarker Detection

Chemosensors

Cancer antigen 15-3 (CA 15-3) is a biomarker for breast cancer used to monitor response to treatments and disease recurrence. The present work demonstrates the preparation and application of a fluorescent biosensor for ultrasensitive detection of the cancer antigen CA 15-3 protein tumor marker using mercaptopropionic-acid-functionalized cadmium telluride (CdTe@MPA) quantum dots (QDs) conjugated with CA 15-3 antibodies. First, the QDs were synthesized by the hydrothermal route, resulting in spherical nanoparticles up to 3.50 nm in diameter. Subsequently, the QD conjugates were characterized by Fourier transform infrared spectroscopy (FTIR), UV absorption, and fluorescence. The interaction between the conjugates and the protein was studied by fluorescence spectroscopy in buffer and in 10-fold diluted commercial human serum. Calibration in spiked serum samples gave a detection limit of 0.027 U/mL, 1000-fold lower than the clinical limit for CA 15-3 (25 U/mL to 30 U/mL), indicating that...