Antibody-conjugated Quantum Dots Research Articles

Quantum dots thermal stability improves simultaneous phenotype-specific telomere length measurement by FISH-flow cytometry

Telomere length analysis has been greatly simplified by the quantitative flow cytometry technique FISH-flow. In this method, a fluorescein-labeled synthetic oligonucleotide complementary to the telomere terminal repeat sequence is hybridized to the telomere sequence and the resulting fluorescence measured by flow cytometry. This technique has supplanted the traditional laborious Southern blot telomere length measurement techniques in many laboratories, and allows single cell analysis of telomere length in high-throughput sample formats. Nevertheless, the harsh conditions required for telomere probe annealing (82 °C) has made it difficult to successfully combine this technique with simultaneous immunolabeling. Most traditional organic fluorescent probes (i.e. fluorescein, phycoerythrin, etc.) have limited thermal stability and do not survive the high temperature annealing process, despite efforts to covalently crosslink the antigen–antibody–fluorophore complex. This loss of probe fluorescence has made it difficult to measure FISH-flow in complex lymphocyte populations, and has generally forced investigators to use fluorescent-activated cell sorting to pre-separate their populations, a laborious technique that requires prohibitively large numbers of cells.In this study, we have substituted quantum dots (nanoparticles) for traditional fluorophores in FISH-flow. Quantum dots were demonstrated to possess much greater thermal stability than traditional low molecular weight and phycobiliprotein fluorophores. Quantum dot antibody conjugates directed against monocyte and T cell antigens were found to retain most of their fluorescence following the high temperature annealing step, allowing simultaneous fluorescent immunophenotyping and telomere length measurement. Since quantum dots have very narrow emission bandwidths, we were able to analyze multiple quantum dot antibody conjugates (Qdot 605, 655 and 705) simultaneously with FISH-flow measurement to assess the age-associated decline in telomere length in both human monocytes and T cell subsets. With quantum dot immunolabeling, the mean decrease rate in telomere length for CD4+ cells was calculated at 41.8 bp/year, very close to previously reported values using traditional flow-FISH and Southern blotting. This modification to the traditional flow-FISH technique should therefore allow simultaneous fluorescent immunophenotyping and telomere length measurement, permitting complex cell subset-specific analysis in small numbers of cells without the requirement for prior cell sorting.

Sensitive and multiplexed detection of proteomic antigens via quantum dot aggregation

A rapid, single-step, solution-phase method for quantifying multiple proteomic biomarkers is described. Nanoscale quantum dot–antibody conjugates self-assemble into microscale aggregates in the presence of a specific antigen through antibody-antigen molecular recognition. These aggregates are easily discriminated from the individual components by flow cytometry. Quantum dot (QD) aggregates can be quantified and correlated to the antigen concentration. Two QD populations with distinct emission spectra are used for detecting two proteomics antigens in a single reaction volume. Multiplexed detection of vascular endothelial growth factor A and angiopoietin-2 is demonstrated at the physiologically relevant, picomolar concentration range. Nonmultiplexed detection of the antigens is also demonstrated, with a femtomolar sensitivity limit. This technique may be optimized for low-cost early detection and frequent screening of cancers and other diseases as well as detection of the biological response to therapy. From the Clinical Editor In this paper a rapid, single-step, solution-phase method is described with multiplexed detection at physiological relevant picomolar concentration range and nonmultiplexed detection with a femtomolar sensitivity limit. The technique may enable low-cost early detection of cancers and other diseases as well as detection of the biological response to therapy.

Tracking of Qdot Conjugated Titin Antibodies in Single Myofibril Stretch Experiments Reveals Ig-domain Unfolding at Physiological Sarcomere Lengths

The mechanical characteristics of titin in muscle sarcomeres were previously studied by us in single myofibril stretch experiments, where the extensibility of I-band titin segments was usually measured under static conditions. Here we investigated the behavior of I-band titin during and after stretch of single rabbit psoas myofibrils in real-time. The focus was on titin's proximal Ig-domain region, whose stretch dynamics were analyzed by labeling the myofibrils specifically in the N2A-titin domain using antibody-conjugated quantum dots, which stained the periphery of the myofibril but did not enter the myofilament lattice. Qdot labels were tracked to obtain the stretch-dependent change in epitope distance (across Z-disc) and sarcomere length (SL) over time. In contrast to what was expected from the current titin extensibility model, at sarcomere lengths of 2.5 and 3.8 μm, titin's proximal Ig-domain region elongated continuously, in proportion to the half I-band length. Already at ∼2.6 μm SL the proximal Ig-segment length exceeded the value expected if all Ig-domains remain folded. Our results suggest that Ig-domains unfold in parallel with PEVK-titin extension at physiological sarcomere lengths and under relatively low forces. By reducing the antibody-Qdot concentration, we succeeded in observing titin Ig-domain dynamics in myofibrils at the single-molecule level.

Comparative breast tumor imaging to identify the expression of type I IGF receptor (IGF1R) by anti-IGF1R antibody-conjugated small molecule fluorophore or quantum dots in vivo.

Abstract Abstract #6001 The type I insulin-like growth factor (IGF) receptor (IGF1R) is a transmembrane receptor tyrosine kinase involved in breast cancer proliferation, survival, and metastasis. Several antibodies against IGF1R, including some in clinical trials, downregulate the level of IGF1R both in cell line studies and in in vivo animal experiments. Thus, this downregulation is a biodynamic marker of antibody delivery to the tumor. To date, identification of IGF1R expression and its downregulation in vivo in a non-invasive way is still challenging. In this study, we sort to use two different fluorescent technologies, small molecule fluorophores, or quantum dots (QDs), to detect receptor expression and its downregulation by antibodies in vivo. Alexa 680 is a small molecule fluorophore that can be excited with a peak emission at 705 nm. QDs are nanocrystals that emit fluorescence upon excitation, and QDs with a peak emission at 705 nm was used in this study. Both Alexa 680 and QDs were conjugated with AVE-1642, a humanized anti-IGF1R antibody developed by sanofi-aventis. After conjugation, they both were able to detect the expression of IGF1R and its downregulation in in vitro cell line studies. We used mouse xenograft tumor models to examine their efficacies in vivo. AVE-1642 conjugated Alexa 680 or QDs (705 nm) was intravenously delivered to mice bearing mammary xenograft tumors that express IGF1R. After 24 hours of circulation, Alexa 680 fluorescence was shown to solely localize to the xenograft tumor, with little non-specific targeting to other tissue or organs in mice. In addition, Alexa 680 fluorescence was diminished in tumors that had downregulated IGF1R by antibody pretreatment. In contrast, conjugated QDs fluorescence was mainly localized to liver, spleen, bone marrow and lymph nodes, with very small amount of QDs in the xenograft tumors. The QDs fluorescence in xenograft tumors remained unchanged even when IGF1R was downregulated by antibody pretreatment. Furthermore, we identified that QDs were mainly localized to the hepatic sinusoids, and subsequently engulfed by Kupffer cells. Taken together, our data suggest that small molecule fluorophores, such as Alexa 680, are useful agents to detect the expression and downregulation of IGF1R in vivo. Our data provide valuable insight not only into the clinical development of anti-IGF1R antibodies, but also to other targeted therapy as well. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6001.

Kinetics of molecular recognition mediated nanoparticle self-assembly

A method of multiple protein biomarker detection, comprising providing at quantum dot-antibody conjugates that have an affinity for at least two different protein biomarkers; contacting the conjugates with a sample from a subject; allowing the proteins to bridge the antibodies, forming protein biomarker/quantum dot-antibody conjugate agglomerates; detecting the presence of the biomarkers by excitation of the agglomerates.

Simultaneous Detection of Multifood-Borne Pathogenic Bacteria Based on Functionalized Quantum Dots Coupled with Immunomagnetic Separation in Food Samples

This paper reports a method that simultaneously detects three food-borne pathogenic bacteria, Salmonella typhimurium, Shigella flexneri, and Escherichia coli O157:H7, via an approach that combines magnetic microparticles for the enrichment and antibody-conjugated semiconductor quantum dots (QDs) as fluorescence markers. Using the water-in-oil reverse microemulsions method, the gamma-Fe(2)O(3) magnetic nanoparticles were coated with silica to empower the particles with high dispersibility and broad compatibility to biomacromolecules. The magnetic beads were then modified with amino silane, which could immobilize antibodies by glutaraldehyde treatment. The immunized magnetic beads and pathogenic bacteria formed "bead-cell" complexes in the enrichment procedure. QDs with different emission wavelengths (620, 560, and 520 nm) were immobilized with anti-S. typhimurium antibody, anti-S. flexneri antibody, and anti-E. coli O157:H7 antibody, respectively. Fluorescence microscope images and the fluorescence intensity of QDs labeled "sandwich" complexes (conjungated with antibodies against S. typhimurium, S. flexneri, and E. coli O157:H7, respectively) demonstrated that antibody-conjugated QDs could attach to the surface of bacterial cells selectively and specifically. In our method, we could detect food-borne pathogen bacteria in a food matrix at 10(-3) cfu/mL. We determined that a high concentration of proteins in food matrix would decrease the sensitivity of this method. This method, of which the detection procedures are completed within 2 h, can be applied to the rapid and cost-effective monitoring of bacterial contamination in food samples.

Detection and downregulation of type I IGF receptor expression by antibody-conjugated quantum dots in breast cancer cells

The type I insulin-like growth factor (IGF) receptor (IGF1R) is a transmembrane tyrosine kinase involved in breast cancer proliferation, survival, and metastasis. Several monoclonal antibodies directed against the receptor are in clinical trials. In order to develop a methodology to detect and measure IGF1R levels in breast cancer cells, we covalently conjugated an IGF1R antibody, AVE-1642, with quantum dots (Qdots), which are nanocrystals that emit fluorescence upon excitation. AVE-1642 Qdots only bound to cells that express IGF1R, and measured IGF1R levels by fluorescence emission at 655 nm. After binding to the cell surface, AVE-1642 Qdots underwent receptor mediated endocytosis, localized to endosome, and later translocated into the nucleus. Treating MCF-7 cells with AVE-1642 Qdots, but not unconjugated Qdots alone, downregulated IGF1R levels and rendered cells refractory to IGF-I stimulation. Furthermore, cell proliferation was slightly inhibited by AVE-1642 Qdots, but not the unconjugated Qdots. Our data suggest that AVE-1642 Qdots can be used to detect IGF1R expression and measure changes in cell surface receptor levels. In addition, the inhibitory effect of AVE-1642 Qdots to cell proliferation implies that it may serve as a traceable therapeutic agent.

Quantum Dot Self-Assembly for Protein Detection with Sub-Picomolar Sensitivity

A novel approach to sensitive and rapid antigen detection is described. In the presence of a specific antigen, quantum dot-antibody conjugates rapidly self-assemble into agglomerates that are typically more than 1 order of magnitude larger than their individual components. The size distribution of the agglomerated colloids depends on, among other things, the relative concentration of quantum dot conjugates and antigen molecules. Quantum dot agglomerates mediated by antigen recognition were characterized by measuring their light scattering and fluorescence characteristics in an unmodified flow cytometer. Protein antigens angiopoietin-2 and mouse IgG were detected to sub-picomolar concentrations using this method. This simple technique enables the potential simultaneous detection of multiple antigenic biomarkers directly from physiological media and could be used for early detection and frequent screening of cancers and other diseases.

Imaging biomarkers of inflammation in situ with fun ctionalized quantum dots in the dextran sodium sulfate (DSS) model of mouse colitis

Myeloperoxidase (MPO) and proinflammatory cytokines play an important role in the development of inflammation. These markers are generally measured using tedious ELISA procedures. In this study, a novel technique utilizing antibody conjugated quantum dot nanoparticles was developed to detect Myeloperoxidase, Interleukin-1alpha (IL-1alpha) and Tumor Necrosis Factor-alpha (TNF-alpha) in vivo in the dextran sodium sulfate (DSS) model of experimental colitis. Colitis was induced in animals (n = 8 animals/group) by feeding 4% DSS solution ad libitum for seven to eight days. Quantum Dots (QDs) exhibiting fluorescence at various wavelengths were conjugated to MPO, IL-1alpha and TNF-alpha polyclonal antibodies and tested in vivo at various stages of colitis. Tissue sections obtained were imaged with confocal microscope. The image intensity obtained from the tissue specimen was correlated with clinical activity measured as Disease Activity Index (DAI). Myeloperoxidase, IL-1alpha and TNF-alpha were visualized with quantum dots on various days of disease. The intensity of quantum dots increased with the increase in inflammation. The increase in intensity showed an excellent correlation with the DAI based on the clinical parameters. The study demonstrated that multiple biomarkers can be detected simultaneously and their quantitative expression correlated well with clinical disease severity. This novel technology should facilitate design of a novel optical platform for imaging various biomarkers of inflammation, early detection of acute and chronic disease markers and inflammation-mediated cancer markers. This detection may also facilitate determination of therapeutic success.

Dynamic Basis for One-Dimensional DNA Scanning by the Mismatch Repair Complex Msh2-Msh6

The ability of proteins to locate specific sites or structures among a vast excess of nonspecific DNA is a fundamental theme in biology. Yet the basic principles that govern these mechanisms remain poorly understood. For example, mismatch repair proteins must scan millions of base pairs to find rare biosynthetic errors, and they then must probe the surrounding region to identify the strand discrimination signals necessary to distinguish the parental and daughter strands. To determine how these proteins might function we used single-molecule optical microscopy to answer the following question: how does the mismatch repair complex Msh2-Msh6 interrogate undamaged DNA? Here we show that Msh2-Msh6 slides along DNA via one-dimensional diffusion. These findings indicate that interactions between Msh2-Msh6 and DNA are dominated by lateral movement of the protein along the helical axis and have implications for how MutS family members travel along DNA at different stages of the repair reaction.

Characterization of the Functional Binding Properties of Antibody Conjugated Quantum Dots

Antibody conjugated quantum dots are an emerging technology for high-resolution labeling of biological systems. In this work we determined the number of functional antibodies (i.e., antibodies that are sterically available for functional binding to target proteins) conjugated to semiconductor quantum dots. This is critical for the interpretation of biological data labeled with these methods. We found that the number of available functional antibodies varied significantly for different conjugation methods and are lower than previously estimated. These results may suggest potential strategies for improving quantum dot labeling of biological preparations.

Quantum Dot Probes for Monitoring Dynamic Cellular Response: Reporters of T Cell Activation

Antibody-conjugated quantum dots (QDs) have been used to map the expression dynamics of the cytokine receptor interleukin-2 receptor-alpha (IL-2Ralpha) following Jurkat T cell activation. Maximal receptor expression was observed 48 h after activation, followed by a sharp decrease consistent with IL-2R internalization subsequent to IL-2 engagement. Verification of T cell activation and specificity of QD labeling were demonstrated using fluorescence microscopy, ELISA, and FACS analyses. These antibody conjugates provide a versatile means to rapidly determine cell state and interrogate membrane associated proteins involved in cell signaling pathways. Ultimately, incorporation with a microfluidic platform capable of simultaneously monitoring several cell signaling pathways will aid in toxin detection and discrimination.

Avidin: a natural bridge for quantum dot-antibody conjugates.

We describe the preparation and characterization of bioinorganic conjugates in which luminescent semiconductor CdSe-ZnS core-shell nanocrystal quantum dots (QDs) were coupled to antibodies through the use of an avidin bridge adsorbed to the nanocrystal surface via electrostatic self-assembly. Avidin, a highly positively charged protein, was found to adsorb tightly to QDs modified with dihydrolipoic acid, which gives their surface a homogeneous negative charge. QD conjugation to biotinylated antibodies subsequently is readily achieved. Fluoroimmunoassays utilizing these antibody conjugated QDs were successful in the detection of protein toxins (staphylococcal enterotoxin B, cholera toxin). QD-antibody conjugates formed in such a facile manner permit their use as a common immuno reagent, and in the development of multianalyte detection.