Investigation of the Doughnut Effect in Cy3-Labeled Protein Microarrays using Scanning Near-Field Optical Microscope

We demonstrate the fluorescence mapping of protein microarrays by the technique of scanning near-field optical microscopy (SNOM) and confocal microscopy. Micron sized spots (300 μm) of human Immunoglobulin G (hIgG) protein with and without a Cy3 dye labeling have been fabricated on glass substrates by an immobilization method which makes use of calixcrown derivatives termed Prolinker. We have also tried to probe into the well-known “doughnut effect” observed in fluorescence images of proteins using the SNOM technique. The topographic and fluorescence SNOM images revealed that the number of proteins at the boundary of the spot were more than at the center in the case of the microarray spot which showed brighter luminescence at the edge than at the center in the confocal image.     

基于Gene Ontology的表达谱与基因功能相似性的相关性研究

聚类是芯片数据分析中被广泛使用的方法。未知基因的功能通常通过其与已知基因在不同生物状态下具有表达相似性来进行预测。然而，还未有人就这种通过表达相似性来进行功能注释的方法的可靠性进行评估。本文利用Gene Ontology对表达相似性和基因功能相似性的相关关系进行了全面的研究。研究表明，尽管表达谱的相似性和基因功能相似性之间有一定的依赖关系，但相关性较弱。在Gene Ontology的三大类中，相对生物过程和分子功能，基因表达谱的相似性更有助于细胞组分的注释。本文的研究结果对于基因功能的预测有一定的指导意义。     

Functionalized Self-Assembled Monolayers on Gold as Binding Matrices for the Screening of Antibody-Antigen Interactions

Two different types of -substituted alkanethiol/disulfide compounds have been used to prepare monolayer architectures on gold serving as platforms for the immobilization of receptor probe molecules – antibodies. These are: (i) carboxylic acid alkanethiols post-reacted with amino biotin to generate streptavidin surfaces, and (ii) N-hydroxysuccinimide-terminated disulfide surfaces. The properties of the monolayers, with and without attached receptor probe molecules, were analysed using infrared spectroscopy, ellipsometry, fluorescence scanning and atomic force microscopy. Several experimental parameters, such as condensation reagents, additives, probe and target concentrations and immobilization time, were systematically varied to determine the dynamic range and to optimize the sensitivity and signal-to-noise ratio of the biochip platforms. Fluorescence screening using Cy5-labelled antigens finally demonstrated that both surfaces could be successfully employed to immobilize the antibodies. The pros and cons of the two approaches are also discussed.Received November 15, 2002; accepted March 25, 2003 Published online July 28, 2003     

DNA microarray: a high throughput approach for methylation detection

We described a DNA microarray-based method combined with bisulphite treatment of DNA and regular PCR to examine hyper-methylation in promoter 1A of APC gene. A set of oligonucleotide probes were designed and immobilized on the aldehyde-coated glass slides for detecting the methylation pattern of 15 selected CpG sites in the region. The methylation status of 30 colorectal tumor samples have been examined by both of methylation-specific PCR (MS-PCR) and the present microarray method. The methylation pattern of the 15 CpG sites for the samples have been obtained with the microarray. A total of 19 samples out of 30 were methylated by microarray, in which five samples cannot be detected by MS-PCR due to the methylated CpG patterns not accordant to the MS-PCR primers. The detecting ratio for methylation of APC gene of colorectal tumor samples increased from 46.7% with MS-PCR to 63.3% with the microarray, which successfully demonstrated that DNA microarray-based method not only can obtained the methylation patterns for the related genes, but also decrease the false-negative results of methylation status by the conventional MS-PCR for the investigated genes.     

纳米荧光小球标记在蛋白质微阵列检测中的应用研究

研究了不经分离、一次性制备氨基化联吡啶钌掺杂的双层二氧化硅纳米小球的方法。实验证明该纳米小球尺寸均匀,光稳定性、水溶性好,分散稳定。通过简单的偶联反应后,它能有效的和蛋白质结合,结合后的蛋白能保持其生物活性。以此纳米荧光小球为标记物,应用于蛋白质微阵列的定量检测,结果发现其效果明显优于相同条件下以异硫氰酸荧光素(FITC)为标记物的定量结果,检出限可以达到3.5 mg/L。     

Electrochemical Detection of Protons Produced in an Electrode Reaction Using Interdigitated Microarray Electrodes

This work describes the use of interdigitated array electrodes (IDAE) for proton detection. Methanol electrooxidation in sulfuric acid solution was exemplified. Reduction currents originating in the reaction product generated by methanol electrooxidation on a Pt generator electrode were observed at the Pt collector electrode, the potential of which was fixed in the hydrogen evolution region. In order to reduce the background current of hydrogen evolution, an Hg‐plated Pt collector electrode was fabricated. Compared to the Pt collector electrode, the reduction current observed at the Hg collector electrode was extremely small. The product detected was found to be a proton from the current responses observed at Pt and Hg collector electrodes.     

Bayesian selection of graphical regulatory models

We define a new class of coloured graphical models, called regulatory graphs. These graphs have their own distinctive formal semantics and can directly represent typical qualitative hypotheses about regulatory processes like those described by various biological mechanisms. They admit an embellishment into classes of probabilistic statistical models and so standard Bayesian methods of model selection can be used to choose promising candidate explanations of regulation. Regulation is modelled by the existence of a deterministic relationship between the longitudinal series of observations labelled by the receiving vertex and the donating one. This class contains longitudinal cluster models as a degenerate graph. Edge colours directly distinguish important features of the mechanism like inhibition and excitation and graphs are often cyclic. With appropriate distributional assumptions, because the regulatory relationships map onto each other through a group structure, it is possible to define a conditional conjugate analysis. This means that even when the model space is huge it is nevertheless feasible, using a Bayesian MAP search, to a discover regulatory network with a high Bayes Factor score. We also show that, like the class of Bayesian Networks, regulatory graphs also admit a formal but distinctive causal algebra. The topology of the graph then represents collections of hypotheses about the predicted effect of controlling the process by tearing out message passers or forcing them to transmit certain signals. We illustrate our methods on a microarray experiment measuring the expression of thousands of genes as a longitudinal series where the scientific interest lies in the circadian regulation of these plants.     

MALDI imaging mass spectrometry reveals multiple clinically relevant masses in colorectal cancer using large‐scale tissue microarrays

For identification of clinically relevant masses to predict status, grade, relapse and prognosis of colorectal cancer, we applied Matrix‐assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) to a tissue micro array containing formalin‐fixed and paraffin‐embedded tissue samples from 349 patients. Analysis of our MALDI‐IMS data revealed 27 different m/z signals associated with epithelial structures. Comparison of these signals showed significant association with status, grade and Ki‐67 labeling index. Fifteen out of 27 IMS signals revealed a significant association with survival. For seven signals (m/z 654, 776, 788, 904, 944, 975 and 1013) the absence and for eight signals (m/z 643, 678, 836, 886, 898, 1095, 1459 and 1477) the presence were associated with decreased life expectancy, including five masses (m/z 788, 836, 904, 944 and 1013) that provided prognostic information independently from the established prognosticators pT and pN. Combination of these five masses resulted in a three‐step classifier that provided prognostic information superior to univariate analysis. In addition, a total of 19 masses were associated with tumor stage, grade, metastasis and cell proliferation. Our data demonstrate the suitability of combining IMS and large‐scale tissue micro arrays to simultaneously identify and validate clinically useful molecular marker. Copyright © 2017 John Wiley & Sons, Ltd.     

Parallel detection and quantitative analysis of specific binding of proteins by oblique-incidence reflectivity difference technique in label-free format

In this work, we parallelly detected the specific binding between microarray targets including 12 different kinds of proteins and the probe solution containing five corresponding antibodies and quantitatively analyzed the interactions between CDH13 and solution phase anti-CDH13 at six different probe concentrations by oblique-incidence reflectivity difference (OIRD) method in label-free format. The detection sensitivity reached 10 ng/mL. The experimental results indicate that the OIRD method is a promising and competing technique not only in research work but also in clinic.     

Application of Polysaccharide Microarray Technology for the Serodiagnosis of Burkholderia pseudomallei Infection (Melioidosis) in Humans

Burkholderia pseudomallei is the causative agent of melioidosis, a bacterial infection endemic in tropical regions including southeast Asia and northern Australia. B. pseudomallei contains structurally unique polysaccharides (capsular polysaccharide and O?antigen saccharides of lipopolysaccharide). A polysaccharide microarray platform was developed by immobilizing these polysaccharides onto glass slides. Employing this microarray, we were able to demonstrate the presence of antibodies to these polysaccharide antigens in the sera of melioidosis patients, but not in serum from nonmelioidosis human subjects. The advantages of this polysaccharide microarray technology over the conventional tests for the serodiagnosis of melioidosis are discussed.