Modular microfluidics for point-of-care protein purifications.

Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured to suit a variety of fluidic operations or biochemical processes. We demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules.

An optimized nanoparticle separator enabled by electron beam induced deposition.

Size-based separations technologies will inevitably benefit from advances in nanotechnology. Direct-write nanofabrication provides a useful mechanism for depositing/etching nanoscale elements in environments otherwise inaccessible to conventional nanofabrication techniques. Here, electron beam induced deposition was used to deposit an array of nanoscale features in a 3D environment with minimal material proximity effects outside the beam-interaction region. Specifically, the membrane component of a nanoparticle separator was fabricated by depositing a linear array of sharply tipped nanopillars, with a singular pitch, designed for sub-50 nm nanoparticle permeability. The nanopillar membrane was used in a dual capacity to control the flow of nanoparticles in the transaxial direction of the array while facilitating the sealing of the cellular-sized compartment in the paraxial direction. An optimized growth recipe resulted which (1) maximized the growth efficiency of the membrane (which minimizes proximity effects) and (2) preserved the fidelity of the spacing between nanopillars (which maximizes the size-based gating quality of the membrane) while (3) maintaining sharp nanopillar apexes for impaling an optically transparent polymeric lid critical for device sealing.

Diverse and conserved nano- and mesoscale structures of diatom silica revealed by atomic force microscopy.

An outstanding example of biological pattern formation at the single cell level is the diversity of biomineral structures in the silica cell walls of the unicellular eukaryotic algae known as diatoms. We present a survey of cell wall silica structures of 16 diatom species, which included all major cell wall components (valves, girdle bands and setae), imaged across the nano-, meso- and microscales using atomic force microscopy. Because of atomic force microscopy's superior ability to image surface topology, this approach enabled visualization of the organization of possible underlying organic molecules involved in mineral structure formation. Diatom nanoscale silica structure varied greatly comparing the same feature in different species and different features within a single species, and frequently on different faces of the same object. These data indicate that there is not a strict relation between nanoscale silica morphology and the type of structure that contains it. On the mesoscale, there was a preponderance of linear structures regardless of the object imaged, suggesting that assembly or organization of linear organic molecules or subcellular assemblies that confine a linear space play an essential and conserved role in structure formation on that scale. Microscale structure imparted an overall influence over nano- and mesoscale structure, indicating that shaping of the silica deposition vesicle plays a key role in structure formation. These results provide insights into the design and assembly principles involved in diatom silica structure formation, facilitating an understanding of the native system and potentially aiding in development of biomimetic approaches.

Size-selectivity and anomalous subdiffusion of nanoparticles through carbon nanofiber-based membranes.

A simulation is presented here that serves the dual functions of generating a nanoporous membrane replica and executing the Brownian motion of nanoparticles through the virtual membrane. Specifically, the concentration profile of a dilute solution of fluorescent particles in a stochastic and SiO(2)-coated carbon nanofiber (oxCNF), nanoporous membrane was simulated. The quality of the simulated profile was determined by comparing the results with experimental concentration profiles. The experimental concentration profiles were collected adjacent to the oxCNF membrane surface from time-lapse fluorescence microscopy images. The simulation proved ideal as an accurate predictor of particle diffusion-the simulated concentration profile merged with the experimental profiles at the inlet/exit surfaces of the oxCNF membrane. In particular, the oxCNF barrier was found to hinder the transport of 50 and 100 nm particles and transmembrane trajectories were indicative of anomalous subdiffusion; the diffusion coefficient was found to be a function of time and space.

Comparison of the indentation and elasticity of E. coli and its spheroplasts by AFM.

Atomic force microscopy (AFM) provides a unique opportunity to study live individual bacteria at the nanometer scale. In addition to providing accurate morphological information, AFM can be exploited to investigate membrane protein localization and molecular interactions on the surface of living cells. A prerequisite for these studies is the development of robust procedures for sample preparation. While such procedures are established for intact bacteria, they are only beginning to emerge for bacterial spheroplasts. Spheroplasts are useful research models for studying mechanosensitive ion channels, membrane transport, lipopolysaccharide translocation, solute uptake, and the effects of antimicrobial agents on membranes. Furthermore, given the similarities between spheroplasts and cell wall-deficient (CWD) forms of pathogenic bacteria, spheroplast research could be relevant in biomedical research. In this paper, a new technique for immobilizing spheroplasts on mica pretreated with aminopropyltriethoxysilane (APTES) and glutaraldehyde is described. Using this mounting technique, the indentation and cell elasticity of glutaraldehyde-fixed and untreated spheroplasts of E. coli in liquid were measured. These values are compared to those of intact E. coli. Untreated spheroplasts were found to be much softer than the intact cells and the silicon nitride cantilevers used in this study.

Cellular secretion studied by force microscopy.

Using the optical microscope, real adventures in cellular research began in earnest in the latter half of the nineteenth century. With the development of the electron microscope, ultramicroscopy, and improved cell staining techniques, significant advances were made in defining intracellular structures at the nanometer level. The invention of force microscopy, the atomic force microscope (AFM) in the mid 1980s, and the photonic force microscope (PFM) in the mid 1990s, finally provided the opportunity to study live cellular structure-function at the nanometer level. Working with the AFM, dynamic cellular and subcellular events at the molecular level were captured in the mid 1990s, and a new cellular structure 'the porosome' in the plasma membrane of all secretory cells has been defined, where specific docking and fusion of secretory vesicles occur. The molecular mechanism of fusion of the secretory vesicle membrane at the base of the porosome membrane in cells, and the regulated release of intravesicular contents through the porosome opening to the extracellular space, has been determined. These seminal discoveries provide for the first time a molecular mechanism of cell secretion, and the possibility to ameliorate secretory defects in disease states.

Measuring cell surface elasticity on enteroaggregative Escherichia coli wild type and dispersin mutant by AFM.

Enteroaggregative Escherichia coli (EAEC) is pathogenic and produces severe diarrhea in humans. A mutant of EAEC that does not produce dispersin, a cell surface protein, is not pathogenic. It has been proposed that dispersin imparts a positive charge to the bacterial cell surface allowing the bacteria to colonize on the negatively charged intestinal mucosa. However, physical properties of the bacterial cell surface, such as rigidity, may be influenced by the presence of dispersin and may contribute to pathogenicity. Using the system developed in our laboratory for mounting and imaging bacterial cells by atomic force microscopy (AFM), in liquid, on gelatin coated mica surfaces, studies were initiated to measure cell surface elasticity. This was carried out in both wild type EAEC, that produces dispersin, and the mutant that does not produce dispersin. This was accomplished using AFM force-distance (FD) spectroscopy on the wild type and mutant grown in liquid or on solid medium. Images in liquid and in air of both the wild-type and mutant grown in liquid and on solid media are presented. This work represents an initial step in efforts to understand the pathogenic role of the dispersin protein in the wild-type bacteria.

Automated image analysis of atomic force microscopy images of rotavirus particles.

A variety of biological samples can be imaged by the atomic force microscope (AFM) under environments that range from vacuum to ambient to liquid. Generally imaging is pursued to evaluate structural features of the sample or perhaps identify some structural changes in the sample that are induced by the investigator. In many cases, AFM images of sample features and induced structural changes are interpreted in general qualitative terms such as markedly smaller or larger, rougher, highly irregular, or smooth. Various manual tools can be used to analyze images and extract more quantitative data, but this is usually a cumbersome process. To facilitate quantitative AFM imaging, automated image analysis routines are being developed. Viral particles imaged in water were used as a test case to develop an algorithm that automatically extracts average dimensional information from a large set of individual particles. The extracted information allows statistical analyses of the dimensional characteristics of the particles and facilitates interpretation related to the binding of the particles to the surface. This algorithm is being extended for analysis of other biological samples and physical objects that are imaged by AFM.

Molecular transport in a crowded volume created from vertically aligned carbon nanofibres: a fluorescence recovery after photobleaching study.

Rapid and selective molecular exchange across a barrier is essential for emulating the properties of biological membranes. Vertically-aligned carbon nanofibre (VACNF) forests have shown great promise as membrane mimics, owing to their mechanical stability, their ease of integration with microfabrication technologies and the ability to tailor their morphology and surface properties. However, quantifying transport through synthetic membranes having micro- and nanoscale features is challenging. Here, fluorescence recovery after photobleaching (FRAP) is coupled with finite difference and Monte Carlo simulations to quantify diffusive transport in microfluidic structures containing VACNF forests. Anomalous subdiffusion was observed for FITC (hydrodynamic radius of 0.54 nm) diffusion through both VACNFs and SiO(2)-coated VACNFS (oxVACNFs). Anomalous subdiffusion can be attributed to multiple FITC-nanofibre interactions for the case of diffusion through the VACNF forest. Volume crowding was identified as the cause of anomalous subdiffusion in the oxVACNF forest. In both cases the diffusion mode changes to a time-independent, Fickian mode of transport that can be defined by a crossover length (R(CR)). By identifying the space-and time-dependent transport characteristics of the VACNF forest, the dimensional features of membranes can be tailored to achieve predictable molecular exchange.

Automated analysis of fluorescence microscopy images to identify protein-protein interactions.

The identification of protein interactions is important for elucidating biological networks. One obstacle in comprehensive interaction studies is the analyses of large datasets, particularly those containing images. Development of an automated system to analyze an image-based protein interaction dataset is needed. Such an analysis system is described here, to automatically extract features from fluorescence microscopy images obtained from a bacterial protein interaction assay. These features are used to relay quantitative values that aid in the automated scoring of positive interactions. Experimental observations indicate that identifying at least 50% positive cells in an image is sufficient to detect a protein interaction. Based on this criterion, the automated system presents 100% accuracy in detecting positive interactions for a dataset of 16 images. Algorithms were implemented using MATLAB and the software developed is available on request from the authors.

Mounting of Escherichia coli spheroplasts for AFM imaging.

The cytoplasmic membrane of Escherichia coli (E. coli) is the location of numerous, chemically specific transporters and recognition elements. Investigation of this membrane in vivo by atomic force microscopy (AFM) requires removal of the cell wall and stable immobilization of the spheroplast. AFM images demonstrate that spheroplasts can be secured with warm gelatin applied to the mica substrate just before the addition of a spheroplast suspension. The resulting preparation can be repeatedly imaged by AFM over the course of several hours. Confocal fluorescence imaging confirms the association of the spheroplasts with the gelatin layer. Gelatin molecules are known to reorder into a network after heating. Entrapment within this gelatin network is believed to be responsible for the immobilization of spheroplasts on mica.

DNA microarrays detect 4-nonylphenol-induced alterations in gene expression during zebrafish early development.

Technological advances in the biological sciences have led to a growing realization of the inherent complexity of the toxic actions of man-made chemicals and industrial compounds. An organism's response to toxic exposure is often a complex summation of the individual responses of various different cell types, tissues, and organs within an individual. Furthermore, within a population, various factors including gender, age, fitness, exposure history, genetic variation, and developmental stage significantly affect how each individual will react following exposure. Because of this complexity, characterizing the responses of organisms to environmental toxin exposure is an area of research well suited to the utilization of the gene-expression profiling capability of DNA microarrays. Microarrays are capable of screening large numbers of genes for response to environmental exposure, with the resulting genesets comprising de facto biomarkers for such exposures. In many cases, the genesets described contain response transcripts anticipated from known mechanistic pathways, but in other cases, equally indicative biomarkers may be found that are unexpected. We investigated the response of zebrafish embryos exposed in vitro to the environmental contaminant 4-nonylphenol (4NP). Nonylphenol is one of several alkylphenol ethoxylate compounds widely used in agricultural and industrial processes that have become ubiquitous environmental contaminants. By combining data from differing levels of exposure, we have identified a group of genes that appear indicative of embryo exposure to 4NP at concentrations ranging from high near-lethal levels to lower, more environmentally relevant levels. These biomarker sets can be further expanded and adapted for use in environmental monitoring as well as in mechanistic studies of complex toxicological mechanisms during both early and adult developmental stages.

AFM imaging of bacteria in liquid media immobilized on gelatin coated mica surfaces.

Immobilization of particulates, especially biomolecules and cells, onto surfaces is critical for imaging with the atomic force microscope (AFM). In this paper, gelatin coated mica surfaces are shown to be suitable for immobilizing and imaging both gram positive, Staphylococcus aureus, and gram negative, Escherichia coli, bacteria in both air and liquid environments. Gelatin coated surfaces are shown to be superior to poly-L-lysine coated surfaces that are commonly used for the immobilization of cells. This cell immobilization technique is being developed primarily for live cell imaging of Rhodopseudomonas palustris. The genome of R. palustris has been sequenced and the organism is the target of intensive studies aimed at understanding genome function. Images of R. palustris grown both aerobically and anaerobically in liquid media are presented. Images in liquid media show the bacteria is rod shaped and smooth while images in air show marked irregularity and folding of the surface. Significant differences in the vertical dimension are also apparent with the height of the bacteria in liquid being substantially greater than images taken in air. In air immobilized bacterial flagella are clearly seen while in liquid this structure is not visible. Additionally, significant morphological differences are observed that depend on the method of bacterial growth.

Automated high-throughput probe production for DNA microarray analysis.

DNA microarrays have become an established tool for gene expression profiling. Construction of these microarrays using immobilized cDNAs is a common experimental strategy. However, this is extremely laborious, requiring the preparation of hundreds or thousands of cDNA probes. To minimize this initial bottleneck, we developed a comprehensive high-throughput robotic system to prepare DNA probes suitable for microarray analysis with minimal user intervention. We describe an automated system using the MultiPROBE Nucleic Acid Purification Workstation to provide the liquid handling and other functions needed to optimize this process. We were able to carry out fully automated plasmid cDNA isolation, PCR assay setup, and PCR purification and also to direct the characterization and tracking of DNA probes during processing. Protocols began with the initial preparation of a plasmid DNA archive of bacterial stocks in parallel 96-well plates (192 samples/run) and continued through to the dilution and reformatting of chip-ready DNA probes in 384-well format. These and other probe production procedures and additional instrument systems were used to process fully a set of mouse cDNA clones that were then validated by differential gene expression analysis.

Towards environmental toxicogenomics -- development of a flow-through, high-density DNA hybridization array and its application to ecotoxicity assessment.

Assessment of the environmental hazard posed by soils/sediments containing low to moderate levels of contaminants using standard analytical chemical methods is uncertain due (in part) to a lack of information on contaminant bioavailability, the unknown interactive effects of contaminant mixtures, our inability to determine the species of a metal in an environmental matrix, and the relative sensitivity of bioassay species. Regulatory agencies compensate for this uncertainty by lowering cleanup goals, but in this process they effectively exclude otherwise attractive cleanup options (i.e. bioremediation). Direct evaluations of soil and sediment toxicity preclude uncertainty from most of these sources. However, the time and cost of chronic toxicity tests limits their general application to higher levels of tiered toxicity assessments. Transcriptional level (mRNA) toxicity assessments offer great advantages in terms of speed, cost and sample throughput. These advantages are currently offset by questions about the environmental relevance of molecular level responses. To this end a flow-through, high-density DNA hybridization array (genosensor) system specifically designed for environmental risk assessment was developed. The genosensor is based on highly regular microchannel glass wafers to which gene probes are covalently bound at discrete (200-microm diameter spot) and addressable (250-microm spot pitch) locations. The flow-through design enables hybridization and washing times to be reduced from approximately 18 h to 20 min. The genosensor was configured so that DNA from 28 environmental samples can be simultaneously hybridized with up to 64 different gene probes. The standard microscopic slide format facilitates data capture with most automated array readers and, thus high sample throughput (> 350 sample/h). In conclusion, hardware development for molecular analysis is enabling very tractable means for analyzing RNA and DNA. These developments have underscored the need for further developmental work in probe design software, and the need to relate transcriptional level data to whole-organism toxicity indicators.

Improving spot homogeneity by using polymer substrates in matrix-assisted laser desorption/ionization mass spectrometry of oligonucleotides.

We describe a method for improving the homogeneity of MALDI samples prepared for analysis of small, single-stranded oligonucleotides using the widely used DNA matrix system, 3-hydroxypicolinic acid/picolinic acid/ ammonium citrate. This matrix system typically produces large crystals around the rim of the dried sample and requires tedious searching of this rim with the laser. However, when a substrate is prepared using both Nafion and a hydrophilic, high-molecular-weight polymer, such as linear polyacrylamide, linear poly(ethylene oxide), or methyl cellulose, oligonucleotide-doped matrix crystals tend to be smaller and more uniformly distributed across the entire spot, thus decreasing the time that is required for locating a usable signal. In addition to MALDI characterization of the spatial distribution of "sweet spots," fluorescence microscopy allows for imaging dye-labeled DNA in dried MALDI spots. The mechanism of enhanced uniformity may involve increased viscosity in the MALDI sample droplet due to partial solubilization of the substrate by the MALDI sample solvent as well as partitioning of the matrix or DNA between the solvent and the undissolved portion of the polymer substrate.

Modification of an automated liquid-handling system for reagent-jet, nanoliter-level dispensing.

Reducing the scale of biochemical reactions is becoming commonplace. Examples include the screening of large libraries of chemical compounds or gene sequences. These applications demand the ability to transfer sub-microliter volumes of fluid. To this end, we have modified a Hamilton MICROLAB 2200 with high-speed solenoids and a liquid pressurization system to modulate volume delivery down to the nanoliter level. Additional modifications include the use of sapphire-tipped dispensing nozzles and a high-resolution substage to assist in the construction of DNA microarrays. Techniques for characterizing the dispensed volume are presented.

Spin-column isolation of DNA-protein interactions from complex protein mixtures for AFM imaging.

Applications of atomic force microscopy (AFM) to investigate structural-functional interactions between DNA and proteins, at the molecular level, should prove valuable for gaining a better understanding of gene expression. Specific genomic DNA-protein interactions occur within a sea of intracellular proteins. Successful AFM imaging requires isolating the specific DNA-protein complex free of background protein contamination. Using spin-column chromatography, we report the successful isolation and AFM imaging of transcription factor DNA complexes from DNA molecules incubated with crude cell lysates. This method should be applicable for the isolation and imaging of other specific DNA-protein complexes pertinent to functional genomic research.

Identifying sequence similarities between DNA molecules.

An atomic force microscope (AFM) imaging technique is described to compare sequences between two different DNA molecules and precisely locate nonhomologies in DNA strands. Sequence comparisons are made by forming heteroduplexes between the two molecules and, by AFM imaging the intact molecules formed, identifying both homologous and nonhomologous regions. By forming heteroduplexes between linearized wildtype pSV-beta-galactosidase plasmid (6821 bp) and a series of deletion mutants we have identified nonhomologies (deletions) as small as 22 bp and as large as 418 bp. Furthermore, by incorporating our technique for AFM-mediated restriction mapping of DNA these mutations can be positioned relative to EcoRI restriction sites. These results suggest AFM can be useful in identifying molecular level similarities and differences in DNA.

Comparative analyses of the secondary structures of synthetic and intracellular yeast MFA2 mRNAs.

The overall folded (global) structure of mRNA may be critical to translation and turnover control mechanisms, but it has received little experimental attention. Presented here is a comparative analysis of the basic features of the global secondary structure of a synthetic mRNA and the same intracellular eukaryotic mRNA by dimethyl sulfate (DMS) structure probing. Synthetic MFA2 mRNA of Saccharomyces cerevisiae first was examined by using both enzymes and chemical reagents to determine single-stranded and hybridized regions; RNAs with and without a poly(A) tail were compared. A folding pattern was obtained with the aid of the MFOLD program package that identified the model that best satisfied the probing data. A long-range structural interaction involving the 5' and 3' untranslated regions and causing a juxtaposition of the ends of the RNA, was examined further by a useful technique involving oligo(dT)-cellulose chromatography and antisense oligonucleotides. DMS chemical probing of A and C nucleotides of intracellular MFA2 mRNA was then done. The modification data support a very similar intracellular structure. When low reactivity of A and C residues is found in the synthetic RNA, approximately 70% of the same sites are relatively more resistant to DMS modification in vivo. A slightly higher sensitivity to DMS is found in vivo for some of the A and C nucleotides predicted to be hybridized from the synthetic structural model. With this small mRNA, the translation process and mRNA-binding proteins do not block DMS modifications, and all A and C nucleotides are modified the same or more strongly than with the synthetic RNA.