Robotic Printing Research Articles

An antibody microarray, in multiwell plate format, for multiplex screening of foodborne pathogenic bacteria and biomolecules

Intoxication and infection caused by foodborne pathogens are important problems worldwide, and screening tests for multiple pathogens are needed because foods may be contaminated with multiple pathogens and/or toxic metabolites. We developed a 96-well microplate, multiplex antibody microarray method to simultaneously capture and detect Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium (S. typhimurium), as well as a biomolecule (chicken immunoglobulin G or IgG employed as a proteinaceous toxin analog) in a single sample. Microarrayed spots of capture antibodies against the targeted analytes were printed within individual wells of streptavidin-coated polystyrene 96-multiwell microtiter plates and a sandwich assay with fluorescein- or Cy3-labeled reporter antibodies was used for detection. (Printing was achieved with a conventional microarray printing robot that was operated with custom-developed microplate arraying software.) Detection of the IgG was realized from ca. 5 to 25ng/mL, and detection of E. coli O157:H7 and S. typhimurium was realized from ca. 10(6) to 10(9) and ca. 10(7) to 10(9)cells/mL, respectively. Multiplex detection of the two bacteria and the IgG in buffer and in culture-enriched ground beef filtrate was established with a total assay (including detection) time of ca. 2.5h. Detection of S. typhimurium was largely unaffected by high concentrations of the other bacteria and IgG as well as the ground beef filtrate, whereas a small decrease in response was observed for E. coli O157:H7. The multiwell plate, multiplex antibody microarray platform developed here demonstrates a powerful approach for high-throughput screening of large numbers of food samples for multiple pathogens and toxins.

Preparation and Use of Microarrays Containing Synthetic Heparin Oligosaccharides for the Rapid Analysis of Heparin–Protein Interactions

Heparin is a highly sulfated, linear polymer that participates in a plethora of biological processes by interaction with many proteins. The chemical complexity and heterogeneity of this polysaccharide can explain the fact that, despite its widespread medical use as an anticoagulant drug, the structure-function relationship of defined heparin sequences is still poorly understood. Here, we present the chemical synthesis of a library containing heparin oligosaccharides ranging from di- to hexamers of different sequences and sulfation patterns. An amine-terminated linker was placed at the reducing end of the synthetic structures to allow for immobilization onto N-hydroxysuccinimide activated glass slides and creation of heparin microarrays. Key features of this modular synthesis, such as the influence of the amine linker on the glycosidation efficiency, the use of 2-azidoglucose as glycosylating agents for oligosaccharide assembly, and the compatibility of the protecting group strategy with the sulfation-deprotection steps, are discussed. Heparin microarrays containing this oligosaccharide library were constructed using a robotic printer and employed to characterize the carbohydrate binding affinities of three heparin-binding growth factors. FGF-1, FGF-2 and FGF-4 that are implicated in angiogenesis, cell growth and differentiation were studied. These heparin chips aided in the discovery of novel, sulfated sequences that bind FGF, and in the determination of the structural requirements needed for recognition by using picomoles of protein on a single slide. The results presented here highlight the potential of combining oligosaccharide synthesis and carbohydrate microarray technology to establish a structure-activity relationship in biological processes.

Identification of Differentially Expressed Genes During Anther Abortion of Taigu Genic Male Sterile Wheat by Combining Suppression Subtractive Hybridization and cDNA Array

Taigu Genic Male Sterile Wheat (TGMSW; Triticum aestivum L.), a dominant genic male sterile germplasm, is of considerable value in the genetic improvement of wheat because of its stable inherence, complete male abortion, and high cross-fertilization rate. To identify specially transcribed genes in sterile anther, a suppression subtractive hybridization (SSH) library was constructed with sterile anther as the tester and fertile anther as the driver. A total of 2 304 SSH inserts amplified by polymerase chain reaction were arrayed using robotic printing. The cDNA arrays were hybridized with 32P-labeled probes prepared from the RNA of forward- and reverse-subtracted anthers. Ninety-six clones were scored as upregulated in sterile anthers compared with the corresponding fertile anthers and some clones were selected for sequencing and analysis in GenBank. Based on their putative functions, 87 non-redundant clones were classified into the following groups: (i) eight genes involved in metabolic processes; (ii) four material transportation genes; (iii) three signal transduction-associated genes; (iv) four stress response and senescence-associated protein genes; (v) seven other functional protein genes; (vi) five genes with no known function; and (vii) another 56 genes with no match to the databases. To test the hybridization efficiency, eight genes were selected and analyzed by Northern blot. The results of the present study provide a comprehensive overview of the genes and gene products involved in anther abortion in TGMSW. (Managing editor: Li-Hui Zhao)

Identification of differently expressed genes in human colorectal adenocarcinoma

To investigate the differently expressed genes in human colorectal adenocarcinoma. The integrated approach for gene expression profiling that couples suppression subtractive hybridization, high-throughput cDNA array, sequencing, bioinformatics analysis, and reverse transcriptase real-time quantitative polymerase chain reaction (PCR) was carried out. A set of cDNA clones including 1260 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent-labeled probes prepared from RNA of human colorectal adenocarcinoma (HCRAC) and normal colorectal tissues. A total of 86 genes were identified, 16 unknown genes and 70 known genes. The transcription factor Somult9 influencing cell differentiation was downregulated. At the same time, Heat shock protein 10 KDis downregulated and Calmoulin is up-regulated. Downregulation of heat shock protein 10 KD lost its inhibition of Ras, and then attenuated the Ras GTPase signaling pathway, increased cell proliferation and inhibited cell apoptosis. Down-regulated transcription factor Somult9 influences cell differentiation and cell-specific gene expression. Down-regulated Somult9 also decreases its binding to calmodulin, accumulates calmodulin as receptor-activated kinase and phosphorylase kinase due to the activation of PhK.

Nanodroplet profiling of enzymatic activities in a microarray

We describe a generic method for the large-scale functional characterization of enzymes in a microarray. Poly- l-lysine and amine reactive slides were coated with fluorogenic substrates sensitive to proteases and phosphatases. Patterning enzymes on the slides by robotic printing produced spatially addressable, segregated droplets that were simultaneously exposed to the on-chip sensors. Multiple enzymes were profiled using this system that provided fluorescence readouts across temporal and stoichiometric dimensions concurrently on a single microarray substrate. This integrated microarray platform is applicable not only for the functional annotation of proteins, but also for the rapid agonist and antagonist discovery and in performing on-chip kinetics.

Encoded fiber-optic microsphere arrays for probing protein-carbohydrate interactions.

Carbohydrate-binding proteins and their glycoconjugate ligands play significant roles in a number of critical biological processes. Immune response, viral membrane fusion, glycoprotein homeostasis, and signaling all involve carbohydratebinding protein mediation at key steps. A more detailed understanding of the exact nature of carbohydrate–protein interactions is expected to render them attractive therapeutic targets. To facilitate these biochemical investigations, glycobiologists require tools that will enable the simultaneous identification of carbohydrate-binding proteins and the oligosaccharide structures they bind. Carbohydrate microarrays were introduced as a promising tool to aid in the discovery of oligosaccharide moieties necessary for carbohydrate-binding protein recognition. Microarrays require small quantities of material, enable simultaneous screening of a defined set of structures against a host of potential binding partners, and, in some cases, are fully amenable to present high-throughput screening technologies, such as robotic printing and fluorescence scanning devices. In the past year, several systems were described that present both natural and synthetically derived carbohydrate structures in array formats. These systems differ in the mode of immobilization (covalent versus noncovalent), the surface upon which the immobilization was performed, and the

Identification of tissue-specific genes in nasopharyngeal epithelial tissue and differentially expressed genes in nasopharyngeal carcinoma by suppression subtractive hybridization and cDNA microarray.

Suppression subtractive hybridization (SSH) was performed for isolation of tissue-specific genes in nasopharyngeal epithelial tissue, by use of cDNAs from human adult nasopharyngeal epithelial tissue as tester and mixed cDNAs from esophagus, lung, liver, heart, stomach, spleen, skeletal muscle, kidney, and skin as drivers. Fourteen differentially expressed genes in nasopharyngeal epithelial tissue were obtained. Among these genes, LPLUNC1 and SPLUNC1 were confirmed to be specifically expressed in nasopharyngeal epithelial tissue and the trachea. A novel transcript of SPLUNC1, which we designate NASG, was found. We also combined SSH and cDNA microarray hybridization to identify genes whose expressions were altered in nasopharyngeal carcinoma (NPC). We used NPC cell line HNE1 and primary human embryo nasopharyngeal epithelial cells in one SSH experiment, and NPC biopsies and normal adult nasopharyngeal epithelial tissue in another. Some 1,200 SSH inserts from four subtractive cDNA libraries were arrayed onto nylon membranes by use of robotic printing. Differential gene expression was verified by hybridizing of the membranes with radioactively labeled first-strand cDNA from NPC cell line HNE1, primary human embryo nasopharyngeal epithelial cells, NPC biopsies, and normal adult nasopharyngeal epithelial tissue. Seventeen differentially expressed genes in NPC were obtained. Among these genes, we identified SPLUNC1 and LPLUNC1 to be down-expressed in NPC biopsies (34/48, 33/48).

Local mean normalization of microarray element signal intensities across an array surface: quality control and correction of spatially systematic artifacts.

Here we present a methodology for the normalization of element signal intensities to a mean intensity calculated locally across the surface of a DNA microarray. These methods allow the detection and/or correction of spatially systematic artifacts in microarray data. These include artifacts that can be introduced during the robotic printing, hybridization, washing, or imaging of microarrays. Using array element signal intensities alone, this local mean normalization process can correct for such artifacts because they vary across the surface of the array. The local mean normalization can be usedfor quality control and data correction purposes in the analysis of microarray data. These algorithms assume that array elements are not spatially ordered with regard to sequence or biological function and require that this spatial mapping is identical between the two sets of intensities to be compared. The tool described in this report was developed in the R statistical language and is freely available on the Internet as part of a larger gene expression analysis package. This Web implementation is interactive and user-friendly and allows the easy use of the local mean normalization tool described here, without programming expertise or downloading of additional software.

Antigen Microarrays for Serodiagnosis of Infectious Diseases

Progress in robotic printing technology has allowed the development of high-density nucleic acid and protein arrays that have increased the throughput of a variety of assays. We generated protein microarrays by printing microbial antigens to simultaneously determine in human sera antibodies directed against Toxoplasma gondii, rubella virus, cytomegalovirus (CMV), and herpes simplex virus (HSV) types 1 and 2 (ToRCH antigens). The antigens were printed on activated glass slides with high-speed robotics. The slides were incubated first with serum samples and subsequently with fluorescently labeled secondary antibodies. Human IgG and IgM bound to the printed antigens were detected by confocal scanning microscopy and quantified with internal calibration curves. Both microarrays and commercial ELISAs were utilized to detect serum antibodies against the ToRCH antigens in a panel of characterized human sera. The detection limit (mean + 2 SD) of the microarray assay was 0.5 pg of IgG or IgM bound to the slides. Within-slide, between-slide, and between-batch precision profiles showed CVs of 1.7-18% for all antigens. Overall, >80% concordance was obtained between microarray assays and ELISAs in the classification of sera; for T. gondii, CMV, and HSV1, concordance exceeded 90%. The microarray is a suitable assay format for the serodiagnosis of infectious diseases and can be easily optimized for clinical use. The ToRCH assay performs equivalently to ELISA and may have potentially important advantages in throughput, convenience, and cost.

Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes.

Comparing patterns of gene expression in cell lines and tissues has important applications in a variety of biological systems. In this study we have examined whether the emerging technology of cDNA microarrays will allow a high throughput analysis of expression of cDNA clones generated by suppression subtractive hybridization (SSH). A set of cDNA clones including 332 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent labeled probes prepared from RNA from ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231 and HBL-100) breast cancer cell lines. Ten clones were identified that were over-expressed by at least a factor of five in the ER-positive cell lines. Northern blot analysis confirmed over-expression of these 10 cDNAs. Sequence analysis identified four of these clones as cytokeratin 19, GATA-3, CD24 and glutathione-S-transferase mu-3. Of the remaining six cDNA clones, four clones matched EST sequences from two different genes and two clones were novel sequences. Flow cytometry and immunofluorescence confirmed that CD24 protein was over-expressed in the ER-positive cell lines. We conclude that SSH and microarray technology can be successfully applied to identify differentially expressed genes. This approach allowed the identification of differentially expressed genes without the need to obtain previously cloned cDNAs.

Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray

A high-capacity system was developed to monitor the expression of many genes in parallel. Microarrays prepared by high-speed robotic printing of complementary DNAs on glass were used for quantitative expression measurements of the corresponding genes. Because of the small format and high density of the arrays, hybridization volumes of 2 microliters could be used that enabled detection of rare transcripts in probe mixtures derived from 2 micrograms of total cellular messenger RNA. Differential expression measurements of 45 Arabidopsis genes were made by means of simultaneous, two-color fluorescence hybridization.