Antibody-conjugated Quantum Dots Research Articles

Recent Research Advances of Antibody-conjugated Quantum Dots

Abstract Quantum Dots (QDs) are water-soluble semiconductor particles on the nanometer scale. Due to their quantum confinement of charge carriers in tiny spaces, QDs show some unique and fascinating optical properties, and are characterized as sharp and symmetrical emission spectra, high quantum yields, broad absorption spectra, good chemical and photo-stability and size dependent emission wavelength tunability. QDs are emerging as a new class of fluorescent labels for biology andmedicine. A major problem in biological applications is how to couple biomolecules to ODs, and remain their activity as well. In recent years, studies in the preparation and application of QDs are reported extensively. However, few reviews have focused on the ODs bioconjugation. Herein, we introduced recent research progress of various coupling reactions which have been used for preparing antibody conjugated ODs, including noncovalent and covalent conjugation. The limitations and the future trend of the biological application of QDs were also discussed.

Highly Specific and Ultrasensitive Graphene-Enhanced Electrochemical Detection of Low-Abundance Tumor Cells Using Silica Nanoparticles Coated with Antibody-Conjugated Quantum Dots

A dual signal amplification immunosensing strategy that offers high sensitivity and specificity for the detection of low-abundance tumor cells was designed. High sensitivity was achieved by using graphene to modify the immunosensor surface to accelerate electron transfer and quantum dot (QD)-coated silica nanoparticles as tracing tags. High specificity was further obtained by the simultaneous measurement of two disease-specific biomarkers on the cell surface using different QD-coated silica nanoparticle tracers. The immunosensor was constructed by covalently immobilized capture antibodies on a chitosan/electrochemically reduced graphene oxide film-modified glass carbon electrode. Cells were captured with a sandwich-type immunoreaction and the different QD-coated silica nanoparticle tracers were captured on the surface of the cells. Each biorecognition event yields a distinct voltammetric peak, which position and size reflects the corresponding identity and amount of the respective antigen. This strategy was vividly demonstrated by the simultaneous immunoassay of EpCAM and GPC3 antigens on the surface of the human liver cancer cell line Hep3B using anti-EpCAM-CdTe- and anti-GPC3-ZnSe-coated silica nanoparticle tracers. The two tracers gave comparable sensitivity, and the immunosensor exhibited high sensitivity and specificity with excellent stability, reproducibility, and accuracy, indicating its wide range of potential applications in clinical and molecular diagnostics.

Detection of Escherichia coli in drugs with antibody conjugated quantum dots as immunofluorescence probes

Based on outstanding optical properties of quantum dots (QDs), a rapid and specific method was established to determine Escherichia coli (E. coli) in drugs. Rabbit anti-goat antibody was covalently conjugated to water-soluble ZnS/CdSe QDs via a cross-linking reaction and the formed conjugate was named as IgG-QDs. The bio-fluorescent probe was verified using agarose gel electrophoresis. Attributing to the affinity between goat anti-E. coli antibody and rabbit anti-goat antibody, E. coli was bio-labeled by IgG-QDs successfully, which was then contributed to detecting E. coli in drugs specifically. The results demonstrated that bioactivity and biorecognition of the antibody was not influenced by the conjugation reaction and IgG-QDs could recognize E. coli specifically. Cell recovery rates for E. coli in clomipramine hydrochloride were 82.90% and 73.64%, respectively. The entire detecting process only spent less than 4h. The method was proved to be rapid and specific, and it could be used to detect stressed and injured cells.

Design of a universal biointerface for sensitive, selective, and multiplex detection of biomarkers using surface plasmon resonance imaging

This paper reports on the sensitive, selective, and simultaneous detection of four protein biomarkers involved in metastasis of various cancers, namely Fas, angiopoietin-2 (Ang-2), human epidermal growth factor receptor 2 (HER2), and matrix metallopeptidase-9 (MMP-9) using an antibody-conjugated quantum dot (QD) chip and surface plasmon resonance imaging (SPRi) biosensors. Initially, a self-assembled monolayer film of l-cysteine, using glutaraldehyde as a linker and QDs as signal enhancement moieties, was employed to immobilize anti-Fas for the detection of Fas as a model protein in buffer. The biointerface was characterized using confocal microscopy, atomic force microscopy, and scanning electron microscopy to provide evidence of uniform surface coverage by the QDs. The SPRi signal was enhanced 100-fold to achieve a detection limit of 25 pg mL(-1) after applying biotinylated detection antibody-conjugate streptavidin-modified QDs. Secondly, this signal amplification strategy was applied to sequentially detect Fas, HER2, MMP-9, and Ang-2 at low concentrations on a protein-microprinted/gold-coated SPRi chip. The results showed the absence of cross-reactivity among these proteins and the feasibility of the approach for multiplex detection of biomarkers as required for the accurate diagnosis of various diseases.

21. The emerging role of nanotechnology in anatomical pathology

Background Interpretation of subcellular localisation by immu-noelectron microscopy is often difficult due to low probe density on target structures. Correlation studies can provide more accurate context and spatial resolution but such approach has hindered by a lack of suitable probes, furthermore, conventional light microscopy will also limit the interpretation due to the resolution limit from diffraction. Nanoparticles such as quantum dots are recently being introduced in research applications. These nanocrystals offer the potential of universal markers and can be visualised by light and electron microscopy. Super-resolution light microscopy overcomes the limit of light diffraction hence it allows a higher image resolution than the conventional light microscopy. Aims To investigate quantum dot for use as immunocytochemi-cal probes for correlation between light and electron microscopy. Furthermore the image quality between super-resolution, confocal and fluorescence light microscopy were also assessed. Methods Quantum dot and colloidal gold probes have been assessed using anti-somatostatin labelling of secretory granules in human somatostatinoma tumour cells. Images of quantum dot labelled tissues were further examined under fluorescence light, confocal and super-resolution light microscopy. Results Antibody conjugated quantum dot immunolabelling was highly specific and can be visualised by light and electron microscopy. Super-resolution microscopy displayed superior details of the target structures compare to fluorescence light and confocal microscopy. Conclusions Quantum dot nanocrystals offer new possibilities for correlation studies incorporating light microscopy, electron microscopy and super resolution, providing a more sensitive and accurate assessment in diagnostic anatomical pathology. Interpretation of subcellular localisation by immu-noelectron microscopy is often difficult due to low probe density on target structures. Correlation studies can provide more accurate context and spatial resolution but such approach has hindered by a lack of suitable probes, furthermore, conventional light microscopy will also limit the interpretation due to the resolution limit from diffraction. Nanoparticles such as quantum dots are recently being introduced in research applications. These nanocrystals offer the potential of universal markers and can be visualised by light and electron microscopy. Super-resolution light microscopy overcomes the limit of light diffraction hence it allows a higher image resolution than the conventional light microscopy. To investigate quantum dot for use as immunocytochemi-cal probes for correlation between light and electron microscopy. Furthermore the image quality between super-resolution, confocal and fluorescence light microscopy were also assessed. Quantum dot and colloidal gold probes have been assessed using anti-somatostatin labelling of secretory granules in human somatostatinoma tumour cells. Images of quantum dot labelled tissues were further examined under fluorescence light, confocal and super-resolution light microscopy. Antibody conjugated quantum dot immunolabelling was highly specific and can be visualised by light and electron microscopy. Super-resolution microscopy displayed superior details of the target structures compare to fluorescence light and confocal microscopy. Quantum dot nanocrystals offer new possibilities for correlation studies incorporating light microscopy, electron microscopy and super resolution, providing a more sensitive and accurate assessment in diagnostic anatomical pathology.

Recent Research Advances of Antibody-conjugated Quantum Dots

Quantum dots(QDs) are water-soluble semiconductor particles on the nanometer scale.QDs are emerging as a new class of fluorescent labels for biology and medicine.A major problem in biological applications is how to couple biomolecules to QDs,and remain their activity as well.In recent years,studies in the preparation and application of QDs were reported extensively.Here,we introduced the research progress of coupling reactions that have been used for preparing antibody conjugated QDs,including noncovalent and covalent.Limitations and the future trend of the biological application of QDs were also demonstrated.

In VitroandIn VivoImaging of Prostate Cancer Angiogenesis Using Anti-Vascular Endothelial Growth Factor Receptor 2 Antibody-Conjugated Quantum Dot

ObjectiveAuthors aimed to determine the targeting ability of vascular endothelial growth factor receptor 2 (VEGFR2)-conjugated quantum dots (QDs) in vitro, and apply it for a xenograft prostate cancer mouse model.Materials and MethodsConjugation reaction of QDs was performed by using the N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and sulfo-(N-hydroxysulfosuccinimide) (Sulfo-NHS). The human umbilical vein cord endothelial cells (HUVECs) were incubated with QDs, conjugated with antiVGFR2, to see a specific binding in vitro. Fluorescent cell images were taken by a confocal microscope. The human prostate cancer cells (PC3) were injected to five nude mice on hind limbs to make the xenograft tumor model. QD-antiVEGFR2 antibody complex was injected into the tumor model and fluorescence measurements were performed at 1, 4, 9, 12, 15, and 24 hours after the injection.ResultsThe specific interaction between HUVECs and QD-antiVEGFR2 antibody was clearly shown in vitro. The in vivo fluorescence image disclosed that there was an increased signal of tumor, 12 hours after the injection of QDs.ConclusionBy showing endothelial cells binding with QDs-antiVEGFR2 antibodyand an experimental application of the antibody for VEGFR2 imaging in the prostate cancer xenograft mouse model, we suggests that the antibody-conjugated QDs can be a potential imaging tool for angiogenesis of the cancer.

Biosensors: Rapid Detection of Hendra Virus Using Magnetic Particles and Quantum Dots (Adv. Healthcare Mater. 5/2012)

Point-of-care methodologies for the rapid detection of emerging viruses are essential for combatting viral outbreaks. On page 631, P. Falcaro, L.-F. Wang, and co-workers report on progress towards the detection of the Hendra virus, based on antibody-conjugated quantum dots and antibody-conjugated magnetic particles.

CSE1L, a Novel Microvesicle Membrane Protein, Mediates Ras-Triggered Microvesicle Generation and Metastasis of Tumor Cells

Tumor-derived microvesicles are rich in metastasis-related proteases and play a role in the interactions between tumor cells and tumor microenvironment in tumor metastasis. Because shed microvesicles may remain in the extracellular environment around tumor cells, the microvesicle membrane protein may be the potential target for cancer therapy. Here we report that chromosome segregation 1-like (CSE1L) protein is a microvesicle membrane protein and is a potential target for cancer therapy. v-H-Ras expression induced extracellular signal-regulated kinase (ERK)-dependent CSE1L phosphorylation and microvesicle biogenesis in various cancer cells. CSE1L overexpression also triggered microvesicle generation, and CSE1L knockdown diminished v-H-Ras-induced microvesicle generation, matrix metalloproteinase (MMP)-2 and MMP-9 secretion and metastasis of B16F10 melanoma cells. CSE1L was preferentially accumulated in microvesicles and was located in the microvesicle membrane. Furthermore, anti-CSE1L antibody-conjugated quantum dots could target tumors in animal models. Our findings highlight a novel role of Ras-ERK signaling in tumor progression and suggest that CSE1L may be involved in the "early" and "late" metastasis of tumor cells in tumorigenesis. Furthermore, the novel microvesicle membrane protein, CSE1L, may have clinical utility in cancer diagnosis and targeted cancer therapy.

Detection of melanoma using antibody-conjugated quantum dots in a coculture model for high-throughput screening system

We proposed an effective strategy for evaluating the targeting specificity of an antibody-conjugated quantum dot (QD) nanoprobe in a coculture system mimicking an in vivo-like tumor microenvironment in which cancer cells grow with normal cells. Analysis of the images was performed with automated confocal microscopy. We have employed a melanoma-melanocyte coculture model to assess the specific binding of QDs conjugated with melanoma antibodies. Conjugation of antibodies to the QD significantly improved the melanoma specificity, while unconjugated antibody alone suffered from non-specific binding to melanocytes. Concentration-dependent binding and competitive inhibition studies with QD-antibody conjugates reproducibly proved the specificity to melanoma cells against melanocytes. The specificity and targeting efficiency of nanoprobes evaluated in a simple coculture model may provide a reasonable assessment for the in vitro diagnosis of early stage melanoma development before in vivo studies. Further, a rapid and sensitive cancer cell detection system demonstrated herein may allow for the development of high-throughput screening platforms for early cancer diagnosis and anti-cancer therapeutics.

Preparation and characterization of human malignant melanoma ganglioside ScFv antibody conjugated quantum dot probe

Melanoma is a highly aggressive and deadly skin cancer. Few treatment options are available for patients with metastatic melanoma, and the global incidence of melanoma is increasing faster than that of other cancers. Therefore, it is vitally important to uncover and use genetic and epigenetic regulatory mechanisms at work during the development and progression of melanoma for better prevention, diagnosis and clinical management. The single-chain antibody (ScFv) has good stability, high antigen affinity, low molecular weight, blood and normal tissue clearance rate, faster and easier penetration into the tumor tissue, which made ScFv to have extremely wide range of application in tumor diagnosis, treatment and prevention. In this study,the prokaryotic expression vector with the anti-human melanoma gaglioside single chain variable fragment antibody gene (GD/ScFvMEL) was constructed, expressed in Escherichia coliand purified by cation-exchange chromatography. Purified GD/ScFvMEL was combined withquantum dots (QD), which was evaluated by fluorescence spectrophotometer and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis. Finally,GD/ScFvMEL coated with QD (QD-GD/ScFvMEL) was transformed into malignant melanoma A375 cell line and human gastric cancer MGC-803 cell line to analyze its function by fluorescence microscopy. It was demonstrated that QD-GD/ScFvMEL could specifically bind with melanoma A375 cells and could not bind with stomach cancer MGC-803 cells, which showed that QD-GD/ScFvMEL could be used as a probe to diagnose melanoma. These results suggested that QD-GD/ScFvMEL has been successfully generated and may serve as a probe to detect malignant melanoma. Key words: Melanoma, preparation, single-chain antibody, quantum dot, probe.

Bead-Based Competitive Fluorescence Immunoassay for Sensitive and Rapid Diagnosis of Cyanotoxin Risk in Drinking Water

Due to the increased occurrence of cyanobacterial blooms and their toxins in drinking water sources, effective management based on a sensitive and rapid analytical method is in high demand for security of safe water sources and environmental human health. Here, a competitive fluorescence immunoassay of microcystin-LR (MCYST-LR) is developed in an attempt to improve the sensitivity, analysis time, and ease-of-manipulation of analysis. To serve this aim, a bead-based suspension assay was introduced based on two major sensing elements: an antibody-conjugated quantum dot (QD) detection probe and an antigen-immobilized magnetic bead (MB) competitor. The assay was composed of three steps: the competitive immunological reaction of QD detection probes against analytes and MB competitors, magnetic separation and washing, and the optical signal generation of QDs. The fluorescence intensity was found to be inversely proportional to the MCYST-LR concentration. Under optimized conditions, the proposed assay performed well for the identification and quantitative analysis of MCYST-LR (within 30 min in the range of 0.42-25 μg/L, with a limit of detection of 0.03 μg/L). It is thus expected that this enhanced assay can contribute both to the sensitive and rapid diagnosis of cyanotoxin risk in drinking water and effective management procedures.

Biofunctional Quantum Dots: Controlled Conjugation for Multiplexed Biosensors

Semiconductor quantum dots possess unique photophysical properties such as bright emission with narrow wavelength bandwidth and extremely broad and strong absorption. In combination with their size-dependent color tunability, quantum dots have been proposed as ideal candidates for multiplexed optical bioanalysis for more than a decade. However, the unavailability of stable, reproducible, biocompatible quantum dots with controlled and functional multiple biolabeling has restricted these nanocrystals to research applications. In this issue of ACS Nano, Jennings et al. demonstrate the versatile use of quantum dot antibody conjugates produced by commercially available kits that allow an easy and fast labeling. This Perspective highlights the potential of novel quantum dot bioconjugation approaches in combination with state-of-the-art detection methods and technologies for successful and widely applicable multiplexed biosensing.

WITHDRAWN: Synthesis and Characterization of Anticancer Drug and Antibody conjugated Quantum Dots as A Novel Theranostic Probe for Pancreatic Cancer Detection and Therapy

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Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis

Cellular microvesicles and nanovesicles (exosomes) are involved in many disease processes and have major potential as biomarkers. However, developments in this area are constrained by limitations in the technology available for their measurement. Here we report on the use of fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles. In this system vesicles are visualized by light scattering using a light microscope. A video is taken, and the NTA software tracks the brownian motion of individual vesicles and calculates their size and total concentration. Using human placental vesicles and plasma, we have demonstrated that NTA can measure cellular vesicles as small as ∼50 nm and is far more sensitive than conventional flow cytometry (lower limit ∼300 nm). By combining NTA with fluorescence measurement we have demonstrated that vesicles can be labeled with specific antibody-conjugated quantum dots, allowing their phenotype to be determined. From the Clinical EditorThe authors of this study utilized fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles, demonstrating that NTA is far more sensitive than conventional flow cytometry.

Abstract 5326: Quantitative molecular profiling of biomarkers for pancreatic cancer with quantum dots

Abstract The detection of cancer biomarkers is important for diagnosis, disease stage forecasting, and clinical management. Since tumor populations are inherently heterogeneous, a key challenge is the quantitative profiling of membrane biomarkers, rather than secreted biomarkers, at the single cell level. The detection of cancer biomarkers is also important for imaging and therapeutics since membrane proteins are commonly selected as targets. Many methods for detection of membrane proteins yield ensemble averages and hence have limited application for analysis of heterogeneous populations or single cells. Fluorescence-based methods allow detection at the single cell level, however, photobleaching presents a major limitation in obtaining quantitative information. Quantum dots overcome the limitations associated with photobleaching, however, the development of quantum dot – antibody (QD-Ab) conjugates that allow quantitative profiling of biomarkers has remained challenging. Here we demonstrate quantitative in vitro profiling and multiplexing of molecular biomarkers associated with precursor lesions of pancreatic adenocarcinoma using QD-Ab conjugates. We demonstrate quantitative spatial profiling of several biomarkers for pancreatic cancer, including mesothelin, prostate stem cell antigen, and claudin-4 in several pancreatic cancer cell lines and a normal pancreas cell line. We also show tumor targeting in a mouse model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5326. doi:10.1158/1538-7445.AM2011-5326

Characterization and Cancer Cell Specific Binding Properties of Anti-EGFR Antibody Conjugated Quantum Dots

Synthesis of biologically active antibody conjugated quantum dots (QDs) has been of great importance in cellular imaging and diagnostics. Cetuximab (or Erbitux) is the first monoclonal antibody drug which targets the epidermal growth factor receptor (EGFR) overexpressed in most cancer cells. In the present work, we investigated three different conjugation strategies to obtain the biologically functional QD-cetuximab conjugates for the tumor-specific imaging. Successful conjugation of cetuximab to QDs was achieved using PEG conjugated polymer-coated QDs and two long-chain heterobifunctional linkers, sulfo-LC-SPDP and sulfo-SMCC. The dissociation constant of the QD-cetuximab conjugates to EGFR was determined to be 0.61 +/- 0.28 nM. The cancer cell-specific binding ability of the QD-cetuximab conjugates was evaluated in vitro, and the cellular internalization of the QD-cetuximab conjugates was clearly demonstrated in live cells by confocal microscopy. The cellular imaging experiments using the QD-cetuximab conjugates showed a clear endocytosis pathway, which was evidenced by the colocalization of the QD-cetuximab conjugates with dye-labeled transferrin. These results suggest that the QD-cetuximab conjugates as an imaging modality for tumor EGFR overexpression can be expected to provide important information on the expression levels of EGFR on the cancer cells.

Quantification of Functional Binding Sites Per Quantum Dot

Antibody conjugated quantum dots have become increasingly useful in the study of proteins both in vitro and in vivo and are particularly valuable in single molecule experiments due to their narrow emission spectra and photostability. Despite their wide range of uses, it has been difficult to determine the number of functional binding sites per quantum dot. Previous studies focused on the characterization the total number of antibodies conjugated to quantum dots rather than the quantification of functional antibodies per quantum dot. Understanding the later is especially important to interpretation of data obtained in single molecule experiments using quantum dot labeled proteins. Additionally, since the number of functional binding sites per quantum dot may vary depending on the antibody conjugation method and also between quantum dot preparations, it is necessary to develop a simple and rapid method to test this experimentally. In this study, we use a direct read-out from the protein itself to determine the average number of proteins bound per quantum dot. By using radio-labeled enhanced green fluorescent protein (EGFP) constructs conjugated to common affinity tags used for protein labeling, we will quantify the number of functional binding sites per quantum dot and the specificity of quantum dot labeling in vitro. This method will be extremely useful in the interpretation of data obtained using quantum dot labeled proteins.

Synthesis and Characterization of Anti-HER2 Antibody Conjugated CdSe/CdZnS Quantum Dots for Fluorescence Imaging of Breast Cancer Cells.

The early detection of HER2 (human epidermal growth factor receptor 2) status in breast cancer patients is very important for the effective implementation of anti-HER2 antibody therapy. Recently, HER2 detections using antibody conjugated quantum dots (QDs) have attracted much attention. QDs are a new class of fluorescent materials that have superior properties such as high brightness, high resistance to photo-bleaching, and multi-colored emission by a single-light source excitation. In this study, we synthesized three types of anti-HER2 antibody conjugated QDs (HER2Ab-QDs) using different coupling agents (EDC/sulfo-NHS, iminothiolane/sulfo-SMCC, and sulfo-SMCC). As water-soluble QDs for the conjugation of antibody, we used glutathione coated CdSe/CdZnS QDs (GSH-QDs) with fluorescence quantum yields of 0.23∼0.39 in aqueous solution. Dispersibility, hydrodynamic size, and apparent molecular weights of the GSH-QDs and HER2Ab-QDs were characterized by using dynamic light scattering, fluorescence correlation spectroscopy, atomic force microscope, and size-exclusion HPLC. Fluorescence imaging of HER2 overexpressing cells (KPL-4 human breast cancer cell line) was performed by using HER2Ab-QDs as fluorescent probes. We found that the HER2Ab-QD prepared by using SMCC coupling with partially reduced antibody is a most effective probe for the detection of HER2 expression in KPL-4 cells. We have also studied the size dependency of HER2Ab-QDs (with green, orange, and red emission) on the fluorescence image of KPL-4 cells.

Detecting Proteins in Highly Autofluorescent Cells Using Quantum Dot Antibody Conjugates

We have applied quantum dot (Qdot) antibody conjugates as a biomolecular probe for cellular proteins important in biogeochemical cycling in the sea. Conventional immunological methods have been hampered by the strong autofluorescence found in cyanobacteria cells. Qdot conjugates provide an ideal alternative for studies that require long-term imaging of cells such as detection of low abundance cellular antigens by fluorescence microscopy. The advantage of Qdot labeled probes over conventional immunological methods is the photostability of the probe. Phycoerythrin bleaches in cyanobacterial cells under prolonged UV or blue light excitation, which means that the semiconducting nanocrystal probe, the Qdot, can yield a strong fluorescent signal without interference from cellular pigments.