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Quantitative microarray analysis of intact glycolipid - CD1d interaction and correlation with cell-based cytokine production

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JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 2008年第37期130卷 12348-12354页

作者： Liang, Pi-Hui Imamura, Masakazu Li, Xiangming Wu, Douglass Fujio, Masakazu Guy, Richard T. Wu, Bing-Ching Tsuji, Moriya Wong, Chi-Huey Rockefeller Univ Aaron Diamond AIDS Res Ctr New York NY 10016 USA Acad Sinica Genom Res Ctr Taipei 11529 Taiwan Scripps Res Inst Dept Chem La Jolla CA 92037 USA Natl Taiwan Univ Sch Pharm Taipei 100 Taiwan

The protein CD1d binds self and foreign glycolipids for presentation to CD1-restricted T cells by means of TCR recognition and activates T(H)1 and T(H)2 chemokine release. In this study, a variety of glycolipid ligands were attached to a microarray surface and their binding with dimeric CD1d was investigated. An alpha-galactosyl ceramide (alpha-GalCer) bearing a carbamate group at the 6'-OH position was tethered to the surface, and the dissociation constant on surface with CD1d was determined to reflect the multivalent interaction. Competition assays were then used to determine the dissociation constants (K-i) of new and intact glycolipids in solution. The 4-fluorophenyloctanoyl-modified alpha-GalCer (18) was found to bind most strongly with CD1d (K-i 0.21 mu M), 2 orders of magnitude stronger than alpha-GalCer and more than three times more selective than alpha-GalCer for IFN-gamma release from NKT cells. Various alpha-GalCer analogues were analyzed, and the results showed that the binding affinity of glycolipids to CD1d correlates well with IFN-gamma production but poorly with IL-4 secretion by NKT cells, suggesting that tighter binding ligands could bias cytokine release through the T(H)1 pathway.

关键词： Antigen Presentation Antigens, CD1/chemistry Antigens, CD1/immunology Antigens, CD1/metabolism Antigens, CD1d Binding, Competitive Galactosylceramides/chemistry Galactosylceramides/metabolism Humans Interferon-gamma/biosynthesis Interferon-gamma/immunology Interleukin-4/biosynthesis Interleukin-4/immunology Killer Cells, Natural/immunology Kinetics microarray analysis/methods Spectrometry, Fluorescence Th1 Cells/immunology Th1 Cells/immunology T-Lymphocytes/immunology

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Polydiacetylene liposome arrays for selective potassium detection

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JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 2008年第15期130卷 5010-+页

作者： Lee, Jiseok Kim, Hyong-Jun Kim, Jinsang Univ Michigan Dept Mat Sci & Engn Ann Arbor MI 48109 USA Univ Michigan Dept Chem Engn Ann Arbor MI 48109 USA Univ Michigan Dept Macromol Sci & Engn Ann Arbor MI 48109 USA Univ Michigan Dept Biomed Engn Ann Arbor MI 48109 USA

Potassium is an important cation in biology, and quantitative detection of the extracellular potassium level is important. However, selective detection of extracellular physiological potassium is a challenging task due to the presence of sodium in a much higher concentration. In this contribution, we describe the development of practical polydiacetylene (PDA) liposome-based microarrays to selectively detect potassium even in the presence of sodium. We utilize the fact that the G-rich ssDNA can fold into a G-quadruplex via intramolecular hydrogen bonding by wrapping around a potassium ion exclusively. We rationally design the PDA liposome in such a way that the G-rich ssDNA probes are presented densely at the liposome surface and form bulky quadruplexes upon binding with K+. The resulting bulky quadruplexes are sterically hindered and repulse each other and impose mechanical stress on the PDA backbone, resulting in the conformational change of the ene-yne backbone of the PDA. As a result, polydiacetylene liposomes turn into the emissive red phase from the nonfluorescent blue phase.

关键词： Acetylation Circular Dichroism Liposomes/chemistry microarray analysis/methods Molecular Structure Polyacetylenes/chemistry Polymers/chemistry Potassium/analysis

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Fluidic and air-stable supported lipid bilayer and cell-mimicking microarrays

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JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 2008年第19期130卷 6267-6271页

作者： Deng, Yang Wang, Yini Holtz, Bryan Li, Jingyi Traaseth, Nathan Veglia, Gianluigi Stottrup, Benjamin J. Elde, Robert Pei, Duanqing Guo, Athena Zhu, X. -Y. MicroSurfaces Inc Minneapolis MN 55421 USA Univ Minnesota Dept Chem Minneapolis MN 55455 USA Augsburg Coll Dept Phys Minneapolis MN 55454 USA Univ Minnesota Dept Neurosci Minneapolis MN 55455 USA Univ Minnesota Dept Pharmacol Minneapolis MN 55455 USA

As drug delivery, therapy, and medical imaging are becoming increasingly cell-specific, there is a critical need for high fidelity and high-throughput screening methods for cell surface interactions. Cell membrane-mimicking surfaces, i.e., supported lipid bilayers (SLBs), are currently not sufficiently robust to meet this need. Here we describe a method of forming fluidic and air-stable SLBs through tethered and dispersed cholesterol groups incorporated into the bottom leaflet. Achieving air stability allows us to easily fabricate SLB microarrays from direct robotic spotting of vesicle solutions. We demonstrate their application as cell membrane-mimicking microarrays by reconstituting peripheral as well as integral membrane components that can be recognized by their respective targets. These demonstrations establish the viability of the fluidic and air-stable SLB platform for generating content microarrays in high throughput studies, e.g., the screening of drugs and nanomedicine targeting cell surface receptors.

关键词： Biomimetic Materials/chemistry Calcium-Binding Proteins/chemistry Calcium-Binding Proteins/metabolism Cell Membrane/chemistry Cell Membrane/metabolism Cholesterol Esters/chemistry Cholesterol Esters/metabolism Fatty Acids, Monounsaturated/chemistry G(M1) Ganglioside/chemistry Glass/chemistry Lipid Bilayers/chemistry Membrane Fluidity Membrane Proteins/chemistry Membrane Proteins/metabolism microarray analysis/methods Microscopy, Fluorescence Phosphatidylcholines/chemistry Phosphatidylethanolamines/chemistry Polyethylene Glycols/chemistry Quaternary Ammonium Compounds/chemistry Quaternary Ammonium Compounds/chemistry Sarcoplasmic Reticulum Calcium-Transporting ATPases/chemistry Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism

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Affinity as a tool in life science

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BIOTECHNIQUES 2008年第5期44卷 649-654页

作者： Uhlen, Mathias AlbaNova Univ Ctr Sch Biotechnol Royal Inst Technol SE-10691 Stockholm Sweden

The use of affinity-based tools has become invaluable as a platform for basic research and in the development of drugs and diagnostics. Applications include affinity chromatography and affinity tag fusions for efficient purification of proteins as well as methods to probe the protein network interactions on a whole-proteome level. A variety of selection systems has been described for in vitro evolution of affinity reagents using combinatorial libraries, which make it possible to create high-affinity reagents to virtually all biomolecules, as exemplified by generation of therapeutic antibodies and new protein scaffold binders. The strategies for high-throughput generation of affinity reagents have also opened up the possibility of generating specific protein probes on a whole-proteome level. Recently, such affinity proteomics have allowed the detailed analysis of human protein expression in a comprehensive manner both in normal and disease tissue using tissue microarrays and confocal microscopy.

关键词： Biological Science Disciplines/methods Biological Science Disciplines/trends Chromatography, Affinity/methods Chromatography, Affinity/trends microarray analysis/methods microarray analysis/trends Molecular Probe Techniques/trends

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Fluorometric assay for quantitation of biotin covalently attached to proteins and nucleic acids

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BIOTECHNIQUES 2007年第4期43卷 503-507页

作者： Batchelor, Robert H. Sarkez, Adam Cox, W. Gregory Johnson, Iain Invitrogen Mol Probes Detect Technol Eugene OR 97402 USA

As a component of the (strept)avidin affinity system, biotin is often covalently linked to Proteins or nucleic acids. We describe here a microplate-based high-throughput fluorometric assay for biotin linked to either Proteins or nucleic acids based (on fluorescence resonance energy transfer (FRET). This assail utilizes a complex of Alexa Fluor (R) 488 dye-labeled avidin with a quencher dye, 2-(4'-hydroxyazobenzene) benzoic acid (HABA), occupying the biotin binding sites of the avidin. In the absence of biotin, HABA quenches the fluorescence emission of the Alexa Fluor 488 dyes via FRET HABA is displaced when biotin binds to the Alexa Fluor 488 dye-labeled avidin, resulting in decreased FRET e). This mechanism results in an increase in fluorescence intensity directly related to the amount of biotin present in. the sample. The assay is able to detect as little as 4 pmol biotin in a 0.1 mL volume within 15 min of adding sample to the reagent, with a Z-factor >0.9.

关键词： Biotin/metabolism Fluorescence Resonance Energy Transfer/methods Immunoassay/methods Immunoassay/methods microarray analysis/methods Nucleic Acids/metabolism Protein Binding Proteins/metabolism

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Bioactive microarrays immobilized on low-fouling surfaces to study specific endothelial cell adhesion

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BIOMACROMOLECULES 2007年第11期8卷 3668-3673页

作者： Monchaux, Emmanuelle Vermette, Patrick Univ Sherbrooke Dept Chem Engn Lab Bioingn Biophys Sherbrooke PQ J1K 2R1 Canada Univ Sherbrooke Inst Geriatr Sherbrooke PQ J1H 4C4 Canada

With the aim to study how to modulate the specific endothelial cell patterning and responses on biomaterials surfaces, bioactive microarrays were developed and validated for specific cell patterning. These microarrays were made of low-fouling surfaces, that prevent nonspecific cell adhesion, bearing bioactive molecules at given known locations by presenting specific ligands to cell receptors. Arrays of bioactive molecules (RGD, REDV, and SVVYGLR sequences and vascular endothelial growth factor (VEGF)) were immobilized on a carboxy-methyl-dextran low-fouling surface and were exposed to human endothelial cells and fibroblasts to screen for the effect of bioactive spot molecular composition on cell adhesion. Endothelial cells only were sensitive to RGD peptide co-immobilized with REDV or SVVYGLR sequences: they induced a reduction in cell spreading and a loss of actin stress fibers. RGD co-immobilized with VEGF also resulted in the reorganization of actin filaments and focal points in endothelial cells. Combination of RGD with these endothelial cell-selective biomolecules did not elicit a strong adhesion phenotype but rather one characteristic of migrating cells.

关键词： Actins/metabolism Amino Acid Sequence Cell Adhesion/drug effects Cells, Cultured Endothelial Cells/cytology Endothelial Cells/metabolism Focal Adhesions/drug effects Focal Adhesions/metabolism Humans microarray analysis/instrumentation microarray analysis/methods Molecular Structure Peptides/pharmacology Phalloidine/metabolism Vascular Endothelial Growth Factor A/pharmacology

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Isolation of rare circulating tumour cells in cancer patients by microchip technology

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NATURE 2007年第7173期450卷 1235-U10页

作者： Nagrath, Sunitha Sequist, Lecia V. Maheswaran, Shyamala Bell, Daphne W. Irimia, Daniel Ulkus, Lindsey Smith, Matthew R. Kwak, Eunice L. Digumarthy, Subba Muzikansky, Alona Ryan, Paula Balis, Ulysses J. Tompkins, Ronald G. Haber, Daniel A. Toner, Mehmet Harvard Univ Sch Med Surg Serv Massachusetts Gen Hosp Boston MA 02114 USA Harvard Univ Sch Med BioMEMS Resource Ctr Massachusetts Gen Hosp Boston MA 02114 USA Shriners Hosp Children Boston MA 02114 USA Harvard Univ Sch Med Massachusetts Gen Hosp Canc Ctr Boston MA 02114 USA

Viable tumour- derived epithelial cells ( circulating tumour cells or CTCs) have been identified in peripheral blood from cancer patients and are probably the origin of intractable metastatic disease(1-4). Although extremely rare, CTCs represent a potential alternative to invasive biopsies as a source of tumour tissue for the detection, characterization and monitoring of non-haematologic cancers(5-8). The ability to identify, isolate, propagate and molecularly characterize CTC subpopulations could further the discovery of cancer stem cell biomarkers and expand the understanding of the biology of metastasis. Current strategies for isolating CTCs are limited to complex analytic approaches that generate very low yield and purity(9). Here we describe the development of a unique microfluidic platform ( the 'CTC-chip') capable of efficient and selective separation of viable CTCs from peripheral whole blood samples, mediated by the interaction of target CTCs with antibody ( EpCAM)- coated microposts under precisely controlled laminar flow conditions, and without requisite pre- labelling or processing of samples. The CTC- chip successfully identified CTCs in the peripheral blood of patients with metastatic lung, prostate, pancreatic, breast and colon cancer in 115 of 116 ( 99%) samples, with a range of 5-1,281 CTCs per ml and approximately 50% purity. In addition, CTCs were isolated in 7/7 patients with early- stage prostate cancer. Given the high sensitivity and specificity of the CTC- chip, we tested its potential utility in monitoring response to anti- cancer therapy. In a small cohort of patients with metastatic cancer undergoing systemic treatment, temporal changes in CTC numbers correlated reasonably well with the clinical course of disease as measured by standard radiographic methods. Thus, the CTC- chip provides a new and effective tool for accurate identification and measurement of CTCs in patients with cancer. It has broad implications in advancing both cancer bio

关键词： Cell Separation/methods Cell Separation/methods Female Humans Male microarray analysis/instrumentation microarray analysis/methods Microfluidics/instrumentation Microfluidics/methods Neoplasm Metastasis Neoplasms/blood Neoplasms/diagnosis Neoplasms/drug therapy Neoplasms/pathology Neoplastic Cells, Circulating/pathology Neoplastic Cells, Circulating/pathology Sensitivity and Specificity

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Surface free energy and microarray deposition technology

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ANALYST 2007年第3期132卷 192-195页

作者： McHale, Glen Nottingham Trent Univ Sch Biomed & Nat Sci Nottingham NG11 8NS England

microarray techniques use a combinatorial approach to assess complex biochemical interactions. The fundamental goal is simultaneous, large-scale experimentation analogous to the automation achieved in the semiconductor industry. However, microarray deposition inherently involves liquids contacting solid substrates. Liquid droplet shapes are determined by surface and interfacial tension forces, and flows during drying. This article looks at how surface free energy and wetting considerations may influence the accuracy and reliability of spotted microarray experiments.

关键词： Chemistry, Physical Dust microarray analysis/methods Oligonucleotide Array Sequence analysis Physicochemical Phenomena Pressure Surface Properties Water/chemistry Wettability Wettability

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Regression analysis and modelling of data acquisition for SELDI-TOF mass spectrometry

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BIOINFORMATICS 2007年第11期23卷 1401-1409页

作者： Skold, Martin Ryden, Tobias Samuelsson, Viktoria Bratt, Charlotte Ekblad, Lars Olsson, Håkan Baldetorp, Bo Lund Univ Ctr Math Sci SE-22100 Lund Sweden Univ Hosp Ctr Oncol SE-22185 Lund Sweden Lund Univ Dept Oncol S-22185 Lund Sweden

Motivation: Pre-processing of SELDI-TOF mass spectrometry data is currently performed on a largel y ad hoc basis. This makes comparison of results from independent analyses troublesome and does not provide a framework for distinguishing different sources of variation in data. Results: In this article, we consider the task of pooling a large number of single-shot spectra, a task commonly performed automatically by the instrument software. By viewing the underlying statistical problem as one of heteroscedastic linear regression, we provide a framework for introducing robust methods and for dealing with missing data resulting from a limited span of recordable intensity values provided by the instrument. Our framework provides an interpretation of currently used methods as a maximum-likelihood estimator and allows theoretical derivation of its variance. We observe that this variance depends crucially on the total number of ionic species, which can vary considerably between different pooled spectra. This variation in variance can potentially invalidate the results from naive methods of discrimination/classification and we outline appropriate data transformations. Introducing methods from robust statistics did not improve the standard errors of the pooled samples. Imputing missing values however-using the EM algorithm-had a notable effect on the result;for our data, the pooled height of peaks which were frequently truncated increased by up to 30%.

关键词： Algorithms Computer Simulation Data Interpretation, Statistical microarray analysis/methods Models, Statistical Regression analysis Reproducibility of Results Sensitivity and Specificity Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods

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Fluorescent microsphere fiber optic microsensor array for direct iodide detection at low picomolar concentrations

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ANALYST 2007年第3期132卷 268-272页

作者： Wygladacz, Katarzyna Bakker, Eric Purdue Univ Dept Chem W Lafayette IN 47907 USA

Fluorescent optode microspheres doped with the halide-selective receptor [ 9] mercuracarborand-3 and a lipophilic pH fluoroionophore were found to exhibit picomolar limits of detection to iodide at pH 3.5, and were used to monitor the precipitation of iodide by silver ions at subnanomolar concentrations, just above their calculated solubility.

关键词： Chemical Precipitation Fiber Optic Technology Fluorescence Fluorescence Hydrogen-Ion Concentration Iodides/analysis Ionophores/chemistry microarray analysis/methods Microspheres Optical Fibers Organomercury Compounds/chemistry Precipitation

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