A novel system for large-scale gene expression analysis: bacterial colonies array

In the present work, we report the use of bacterial colonies to optimize macroarray technique. The devised system is significantly cheaper than other methods available to detect large-scale differential gene expression. Recombinant Escherichia coli clones containing plasmid-encoded copies of 4,608 individual expressed sequence tag (ESTs) were robotically spotted onto nylon membranes that were incubated for 6 and 12 h to allow the bacteria to grow and, consequently, amplify the cloned ESTs. The membranes were then hybridized with a beta-lactamase gene specific probe from the recombinant plasmid and, subsequently, phosphorimaged to quantify the microbial cells. Variance analysis demonstrated that the spot hybridization signal intensity was similar for 3,954 ESTs (85.8%) after 6 h of bacterial growth. Membranes spotted with bacteria colonies grown for 12 h had 4,017 ESTs (87.2%) with comparable signal intensity but the signal to noise ratio was fivefold higher. Taken together, the results of this study indicate that it is possible to investigate large-scale gene expression using macroarrays based on bacterial colonies grown for 6 h onto membranes.     

阵列光镊与微流控芯片技术的结合与应用

在生物医学研究领域中,阵列光镊与微流控芯片的结合已经成为进行细胞操纵、转移以及少量细胞样品分选等方面最有希望的方法之一。光镊技术对样品具有非接触弹性控制、无机械损伤、可无菌操作等优势,以及微流控芯片分析的高效、多功能、微型化、低成本等优势,成为芯片实验室(Lab-on-a-Chip)的重要研究方面。该文概述了阵列光镊技术的形成与研究现状以及微流控芯片技术的发展与应用现状,分析了在不同阵列光镊形成方法下结合微流控芯片可实现的功能与应用,并对其发展趋势进行了展望。     

Rapid identification of ubiquitination and SUMOylation target sites by microfluidic peptide array

SUMOylation and ubiquitination are two essential post translational modifications (PTMs) involved in the regulation of important biological processes in eukaryotic cells. Identification of ubiquitin (Ub) and small ubiquitin-related modifier (SUMO)-conjugated lysine residues in proteins is critical for understanding the role of ubiquitination and SUMOylation, but remains experimentally challenging. We have developed a powerful in vitro Ub/SUMO assay using a novel high density peptide array incorporated within a microfluidic device that allows rapid identification of ubiquitination and SUMOylation sites on target proteins. We performed the assay with a panel of human proteins and a microbial effector with known target sites for Ub or SUMO modifications, and determined that 80% of these proteins were modified by Ub or specific SUMO isoforms at the sites previously determined using conventional methods. Our results confirm the specificity for both SUMO isoform and individual target proteins at the peptide level. In summary, this microfluidic high density peptide array approach is a rapid screening assay to determine sites of Ub and SUMO modification of target substrates, which will provide new insights into the composition, selectivity and specificity of these PTM target sites.     

Miniaturized embryo array for automated trapping, immobilization and microperfusion of zebrafish embryos

Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP). The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale.     

Single-strand conformation polymorphism analysis using capillary array electrophoresis for large-scale mutation detection

This protocol describes capillary array electrophoresis single-strand conformation polymorphism (CAE-SSCP), a screening method for detection of unknown and previously identified mutations. The method detects 98% of mutations in a sample material and can be applied to any organism where the goal is to determine genetic variation. This protocol describes how to screen for mutations in 192 singleplex or up to 768 multiplex samples over 3 days. The protocol is based on the principle of sequence-specific mobility of single-stranded DNA in a native polymer, and covers all stages in the procedure, from initial DNA purification to final CAE-SSCP data analysis, as follows: DNA is purified, followed by PCR amplification using fluorescent primers. After PCR amplification, double-stranded DNA is heat-denatured to separate the strands and subsequently cooled on ice to avoid reannealing. Finally, samples are analyzed by capillary electrophoresis and appropriate analysis software.     

Photoautotrophic culture of Coffea arabusta somatic embryos: development of a bioreactor for large-scale plantlet conversion from cotyledonary embryos

Somatic embryos were developed from in vitro-grown leaf discs of Coffea arabusta in modified Murashige and Skoog medium under 30 micromol m(-2) s(-1) photosynthetic photon flux (PPF). Cotyledonary stage embryos were selected from the 14-week-old cultures and were placed under a high (100 micromol m(-2) s(-1) PPF for 14 d. These pretreated embryos were grown photoautotrophically in three different types of culture systems: Magenta vessel; RITA-bioreactor (modified to improve air exchange); and a specially designed temporary root zone immersion bioreactor system (TRI-bioreactor) with forced ventilation. The aims of the study were to achieve large-scale embryo-to-plantlet conversion, and to optimize growth of plantlets under photoautotrophic conditions. The plantlet conversion percentage was highest (84 %) in the TRI-bioreactor and lowest in the modified RITA-bioreactor (20 %). Growth and survival of converted plantlets following 45 d of photoautotrophic culture in each of the three culture systems were studied. Fresh and dry masses of leaves and roots of plantlets developed in the TRI-bioreactor were significantly greater than those of plantlets developed in the modified RITA-bioreactor or Magenta vessel. The net photosynthetic rate, chlorophyll fluorescence and chlorophyll contents were also highest in plantlets grown in the TRI-bioreactor. Normal stomata were observed in leaves of plantlets grown in the TRI-bioreactor, whereas they could be abnormal in plantlets from the modified RITA-bioreactor. Survival of the plants after transfer from culture followed a similar pattern and was highest in the group grown in the TRI-bioreactor, followed by plants grown in the modified RITA-bioreactor and Magenta vessel. In addition, ex vitro growth of plants transferred from the TRI-bioreactor was faster than that of plants from the other culture systems.     

Telomerase catalyzed fluorescent probes for sensitive protein profiling based on one-dimensional microfluidic beads array

A nucleic acid-based signal amplified method for multiple proteins detection based on one-dimensional beads array using telomerase catalyzed fluorescent probes has been developed in this paper. The biotin labeled fluorescent probes were synthesized by telomerase in homogeneous solution. Approximately 360-480 fluorescein molecules were inserted in each probe. The limit of detection for p53 protein is 1.1 pM (S/N=3) and a 3 orders of linear dynamic range is obtained. The sensitivity is nearly two orders higher than the two-site "sandwich" immunoassay using the same platform. Using this method, cellular p53 protein contents of as few as 85 CNE2 cells per mul sample can be determined specifically. The expression changes of p53, c-myc and beta-actin in CNE2 cells were further examined as a function of anti-cancer drug treatment, and the results are consistent with our previous reports. Compared with immuno-polymerase chain reaction and immuno-rolling circle amplification, this method is simple, fast, cheap and suitable for multi-protein analysis. The multiplexed proteins profiling of cellular samples should facilitate the new opportunities to the fundamental research of tumor development and progression, especially to the low abundant tumor-associated proteins analysis.     

Guidance of engineered tissue collagen orientation by large-scale scaffold microstructures

The tensile strength and stiffness of load-bearing soft tissues are dominated by their collagen fiber orientation. While microgrooved substrates have demonstrated a capacity to orient cells and collagen in monolayer tissue culture, tissue engineering (TE) scaffolds are structurally distinct in that they consist of a three-dimensional (3-D) open pore network. It is thus unclear how the geometry of these open pores might influence cell and collagen orientation. In the current study we developed an in vitro model system for quantifying the capacity of large scale ( approximately 200 microm), geometrically well-defined open pores to guide cell and collagen orientation in engineered tissues. Non-degradable scaffolds exhibiting a grid of 200 microm wide rectangular pores (1:1, 2:1, 5:1, and 10:1 aspect ratios) were fabricated from a transparent epoxy resin via high-resolution stereolithography. The scaffolds (n=6 per aspect ratio) were surface modified to support cell adhesion by covalently grafting GRGDS peptides, sterilized, and seeded with neonatal rat skin fibroblasts. Following 4 weeks of static incubation, the resultant collagen orientation was assessed quantitatively by small angle light scattering (SALS), and cell orientation was evaluated by laser confocal and scanning electron microscopy. Cells adhered to the struts of the pores and proceeded to span the pores in a generally circumferential pattern. While the cell and collagen orientations within 1:1 aspect ratio pores were effectively random, higher aspect ratio rectangular pores exhibited a significant capacity to guide global cell and collagen orientation. Preferential alignment parallel to the long strut axis and decreased spatial variability were observed to occur with increasing pore aspect ratio. Intra-pore variability depended in part on the spatial uniformity of cell attachment around the perimeter of each pore achieved during seeding. Evaluation of diamond-shaped pores [Sacks, M.S. et al., 1997. J. Biomech. Eng. 119(1), 124-127] suggests that they are less sensitive to initial conditions of cell attachment than rectangular pores, and thus more effective in guiding engineered tissue cell and collagen orientation.     

Genetically stable cell lines of cucumber for the large-scale production of diploid somatic embryos

For the initiation of embryogenic cucumber ( Cucumis sativus L.) cell lines, from excised radicles, directly in liquid medium, the culture regime, explant density and type and concentration of hormones were adjusted so that pro-embryogenic masses (PEMs) were formed within about 8 weeks. The established cucumber cell lines were maintained tor several years without loss of embryogenic and genetic stability. The ploidy level of somatic embryos from different cucumber eell lines was either diploid or tetraploid and depended on the ploidy level of Ihe cell line. Cucumber cell lines that produced only diploid embryos were obtained by selecting completely diploid explant material and growing it in the dark during the initiation phase. Mixoploid explains could lead to tetraploid or mixoploid ceil lines. Isolation and additional selection and subculturing of single PEMs resulted in either completely diploid or tetraploid cell lines, indicating that all cells of individual PEMs are either diploid or tetraploid. The ernbryogenic cucumber cell Imes, differing only in ploidy level, were indistinguishable in growth rate and embryogetiic potential and were genetically stable over several years.     

Novel microbial photobioelectrochemical cell using an invasive ultramicroelectrode array and a microfluidic chamber

Objective

To fabricate a novel microbial photobioelectrochemical cell using silicon microfabrication techniques.

Results

High-density photosynthetic cells were immobilized in a microfluidic chamber, and ultra-microelectrodes in a microtip array were inserted into the cytosolic space of the cells to directly harvest photosynthetic electrons. In this way, the microbial photobioelectrochemical cell operated without the aid of electron mediators. Both short circuit current and open circuit voltage of the microbial photobioelectrochemical cell responded to light stimuli, and recorded as high as 250 pA and 45 mV, respectively.

Conclusion

A microbial photobioelectrochemical cell was fabricated with potential use in next-generation photosynthesis-based solar cells and sensors.

     

High throughput flow cytometry based yeast two-hybrid array approach for large-scale analysis of protein-protein interactions

The analysis of protein-protein interactions is a key focus of proteomics efforts. The yeast two-hybrid system (Y2H) has been the most commonly used method in genome-wide searches for protein interaction partners. However, the throughput of the current yeast two-hybrid array approach is hampered by the involvement of the time-consuming LacZ assay and/or the incompatibility of liquid handling automation due to the requirement for selection of colonies/diploids on agar plates. To facilitate large-scale Y2H assays, we report a novel array approach by coupling a GFP reporter based Y2H system with high throughput flow cytometry that enables the processing of a 96-well plate in as little as 3 min. In this approach, the yEGFP reporter has been established in both AH109 (MATa) and Y187 (MATα) reporter cells. It not only allows the generation of two copies of GFP reporter genes in diploid cells, but also allows the convenient determination of self-activators generated from both bait and prey constructs by flow cytometry. We demonstrate a Y2H array assay procedure that is carried out completely in liquid media in 96-well plates by mating bait and prey cells in liquid YPD media, selecting the diploids containing positive interaction pairs in selective media and analyzing the GFP reporter directly by flow cytometry. We have evaluated this flow cytometry based array procedure by showing that the interaction of the positive control pair P53/T is able to be reproducibly detected at 72 hr postmating compared with the negative control pairs. We conclude that our flow cytometry based yeast two-hybrid approach is robust, convenient, quantitative, and is amenable to large-scale analysis using liquid-handling automation.     

A partial ordering approach for functional diversity

Functional diversity is generally regarded as the constituent of biological diversity that considers how the species functional traits affect ecosystem processes. Due to its ecological relevance, a number of indices of functional diversity have been proposed to date based on distinct objectives and motivations. Such proliferation of indices can be at least partially overcome by a more fundamental mathematical approach. In this paper we propose an intrinsic ordering approach for abundance-weighted measures of functional diversity that is similar to the Lorenz curves used by ecologists for ordering evenness measures. We then discuss the relevance of a number of functional diversity indices that have a behavior compatible with the proposed partial ordering.     

On-chip oligonucleotide ligation assay using one-dimensional microfluidic beads array for the detection of low-abundant DNA point mutations

The detection of low-abundant DNA point mutations is very important for the early prediction of cancer, diagnostics of disease and clinical prognosis. In this paper, an on-chip oligonucleotide ligation approach that arrayed a series of functionalized beads in a single microfluidic channel was described for detection of low-abundant point mutations in p53 gene. This gene carried the point mutation with high diagnostic value for assessment of tumor progression and resectional borders. This work extended our prior efforts using one-dimensional (1-D) microfluidic beads array for protein and nucleic acid molecular profiling, and displayed high discrimination sensitivity to mutations detection due to the enhanced mass transport capability caused by microfluidic addressing format of beads array. As a demonstration, it was found that the on-chip beads ligation held high mutation discrimination sensitivity in 1 pM quantities at a SNR (signal-to-noise ratio) >2 using synthesized DNA oligonucleotides in accordance with target fragment. The RT-PCR products of tumor cell line A549, CNE2 and SKBr-3 were further examined to distinguish the point mutation at codon 175 of p53 gene. This approach was capable of detecting a point mutation in a p53 oncogene at a level of 1 mutant in 1000 wild-type sequences using PCR products without the need of LDR amplification. Additionally, this on-chip beads ligation approach also displayed other microfluidic-based advantages of simple handling (one sample injection per test), little reagent quantities, and low potential of contaminations.     

Distinct roles for Galpha and Gbetagamma in regulating spindle position and orientation in Caenorhabditis elegans embryos

Correct placement and orientation of the mitotic spindle is essential for segregation of localized components and positioning of daughter cells. Although these processes are important in many cells, few factors that regulate spindle placement are known. Previous work has shown that GPB-1, the Gbeta subunit of a heterotrimeric G protein, is required for orientation of early cell division axes in C. elegans embryos. Here we show that GOA-1 (a Galphao) and the related GPA-16 are the functionally redundant Galpha subunits and that GPC-2 is the relevant Ggamma subunit that is required for spindle orientation in the early embryo. We show that Galpha and Gbetagamma are involved in controlling distinct microtubule-dependent processes. Gbetagamma is important in regulating migration of the centrosome around the nucleus and hence in orientating the mitotic spindle. Galpha is required for asymmetric spindle positioning in the one-celled embryo.     

Concept, design and implementation of a cardiovascular gene-centric 50 k SNP array for large-scale genomic association studies

A wealth of genetic associations for cardiovascular and metabolic phenotypes in humans has been accumulating over the last decade, in particular a large number of loci derived from recent genome wide association studies (GWAS). True complex disease-associated loci often exert modest effects, so their delineation currently requires integration of diverse phenotypic data from large studies to ensure robust meta-analyses. We have designed a gene-centric 50 K single nucleotide polymorphism (SNP) array to assess potentially relevant loci across a range of cardiovascular, metabolic and inflammatory syndromes. The array utilizes a "cosmopolitan" tagging approach to capture the genetic diversity across approximately 2,000 loci in populations represented in the HapMap and SeattleSNPs projects. The array content is informed by GWAS of vascular and inflammatory disease, expression quantitative trait loci implicated in atherosclerosis, pathway based approaches and comprehensive literature searching. The custom flexibility of the array platform facilitated interrogation of loci at differing stringencies, according to a gene prioritization strategy that allows saturation of high priority loci with a greater density of markers than the existing GWAS tools, particularly in African HapMap samples. We also demonstrate that the IBC array can be used to complement GWAS, increasing coverage in high priority CVD-related loci across all major HapMap populations. DNA from over 200,000 extensively phenotyped individuals will be genotyped with this array with a significant portion of the generated data being released into the academic domain facilitating in silico replication attempts, analyses of rare variants and cross-cohort meta-analyses in diverse populations. These datasets will also facilitate more robust secondary analyses, such as explorations with alternative genetic models, epistasis and gene-environment interactions.     

DNA probe attachment on plastic surfaces and microfluidic hybridization array channel devices with sample oscillation

DNA probe immobilization on plastic surfaces and device assembly are both critical to the fabrication of microfluidic hybridization array channel (MHAC) devices. Three oligonucleotide (oligo) probe immobilization procedures were investigated for attaching oligo probes on four different types of plastic surfaces (polystyrene, polycarbonate, poly(methylmethacrylate), and polypropylene). These procedures are the Surmodics procedure, the cetyltrimethylammonium bromide (CTAB) procedure, and the Reacti-Bind procedure. To determine the optimal plastic substrate and attachment chemistry for array fabrication, we investigated plastic hydrophobicity, intrinsic fluorescence, and oligo attachment efficiency. The Reacti-Bind procedure is least effective for attaching oligo probes in the microarray format. The CTAB procedure performs well enough to use in array fabrication, and the concentration of CTAB has a significant effect on oligo immobilization efficiency. We also found that use of amine-modified oligo probes resulted in better immobilization efficiency than use of unmodified oligos with the CTAB procedure. The oligo probe immobilization on plastic surfaces by the Surmodics procedure is the most effective with regard to probe spot quality and hybridization sensitivity. A DNA hybridization assay on such a device results in a limit of detection of 12pM. Utilizing a CO(2) IR laser machining and adhesive layer approach, we have developed an improved procedure for realizing a DNA microarray inside a microfluidic channel. This device fabrication procedure allows for more feasible spot placement in the channel and reduced sample adsorption by adhesive tapes used in the fabrication procedure. We also demonstrated improved hybridization kinetics and increased detection sensitivity in MHAC devices by implementing sample oscillation inside the channel. A limit of detection of 5pM has been achieved in MHAC devices with sample oscillation.     

Multienzyme-nanoparticles amplification for sensitive virus genotyping in microfluidic microbeads array using Au nanoparticle probes and quantum dots as labels

A novel microfluidic device with microbeads array was developed and sensitive genotyping of human papillomavirus was demonstrated using a multiple-enzyme labeled oligonucleotide-Au nanoparticle bioconjugate as the detection tool. This method utilizes microbeads as sensing platform that was functionalized with the capture probes and modified electron rich proteins, and uses the horseradish peroxidase (HRP)-functionalized gold nanoparticles as label with a secondary DNA probe. The functionalized microbeads were independently introduced into the arrayed chambers using the loading chip slab. A single channel was used to generate weir structures to confine the microbeads and make the beads array accessible by microfluidics. Through "sandwich" hybridization, the enzyme-functionalized Au nanoparticles labels were brought close to the surface of microbeads. The oxidation of biotin-tyramine by hydrogen peroxide resulted in the deposition of multiple biotin moieties onto the surface of beads. This deposition is markedly increased in the presence of immobilized electron rich proteins. Streptavidin-labeled quantum dots were then allowed to bind to the deposited biotin moieties and displayed the signal. Enhanced detection sensitivity was achieved where the large surface area of Au nanoparticle carriers increased the amount HRP bound per sandwiched hybridization. The on-chip genotyping method could discriminate as low as 1fmol/L (10zmol/chip, SNR>3) synthesized HPV oligonucleotides DNA. The chip-based signal enhancement of the amplified assay resulted in 1000 times higher sensitivity than that of off-chip test. In addition, this on-chip format could discriminate and genotype 10copies/μL HPV genomic DNA using the PCR products. These results demonstrated that this on-chip approach can achieve highly sensitive detection and genotyping of target DNA and can be further developed for detection of disease-related biomolecules at the lowest level at their earliest incidence.