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Biomedical subjects

David R Walt

Publications and source records attributed to David R Walt.

At least 19 recordsLinked to original sources
PubMed2007

Detecting oxygen consumption in the proximity of Saccharomyces cerevisiae cells using self-assembled fluorescent nanosensors.

We describe a strategy for the preparation and self-assembly of fluorescent nanosensors onto Saccharomyces cerevisiae cell surfaces for dynamically measuring oxygen concentration in the proximity of living cells. Amine functionalized polystyrene nanobeads were impregnated with an oxygen-sensitive ruthenium(II) complex and the beads' surface was coated with polyethylenimine. The resulting nanosensors were assembled on individual S. cerevisiae cells in a controlled manner at physiological pH for continuously monitoring oxygen consumption. This approach exemplifies a general scheme for assembling fluorescent nanosensors on cells for the non-invasive, reversible, and real-time measurement of other physiologically relevant processes, such as the efflux of protons and carbon dioxide, or the influx of glucose.

Fluorescent Dyes↗
PubMed2006

Phenotypic consequences of promoter-mediated transcriptional noise.

A more complete understanding of the causes and effects of cell-cell variability in gene expression is needed to elucidate whether the resulting phenotypes are disadvantageous or confer some adaptive advantage. Here we show that increased variability in gene expression, affected by the sequence of the TATA box, can be beneficial after an acute change in environmental conditions. We rationally introduce mutations within the TATA region of an engineered Saccharomyces cerevisiae GAL1 promoter and measure promoter responses that can be characterized as being either highly variable and rapid or steady and slow. We computationally illustrate how a stable transcription scaffold can result in "bursts" of gene expression, enabling rapid individual cell responses in the transient and increased cell-cell variability at steady state. We experimentally verify computational predictions that the rapid response and increased cell-cell variability enabled by TATA-containing promoters confer a clear benefit in the face of an acute environmental stress.

Base Sequence↗
PubMed2006

Fiber-based single cell analysis of reporter gene expression in yeast two-hybrid systems.

Yeast two-hybrid (Y2H) systems conventionally are used when screening for protein-protein interactions. A recurring problem with Y2H systems is the high prevalence of both false positives and false negatives. Distinguishing between real results and artifacts to characterize a protein-protein interaction properly can often be time-consuming and laborious. In this article, we report a new method for statistically evaluating single cell reporter gene transcriptional activation and expression in yeast two-hybrid systems. The system is based on statistical analysis of many yeast cells contained in a fiber-optic array, allowing for thorough characterization of reporter gene expression at different stringency levels.

Analysis of Variance↗
PubMed2006

Digital readout of target binding with attomole detection limits via enzyme amplification in femtoliter arrays.

In this communication, single molecules of beta-galactosidase were captured on a 1 mm femtoliter array using biotin-streptavidin binding. The femtoliter arrays, containing 24 000 individual reaction chambers, permit digital concentration readout as the percentage of reaction vessels that successfully capture a target molecule is correlated to the bulk target concentration. This capture and readout approach should prove useful for DNA and antibody assays that utilize an enzyme label to catalyze the generation of a fluorescent signal.

Biotin↗
PubMed2006

Microbead chemical switches: an approach to detection of reactive organophosphate chemical warfare agent vapors.

In this article, we describe the preparation and application of microbeads that exhibit a "turn on" fluorescence response within seconds of exposure to diethyl chlorophosphate (DCP) vapor. This sensing approach is modeled after the mechanism for acetylcholinesterase enzyme activity inhibition and uses a specific and irreversible reaction between phosphoryl halides and a fluorescent indicator. The microbeads are fabricated by adsorbing fluoresceinamine (FLA) onto carboxylate-functionalized polymer microbeads coated with poly(2-vinylpyridine) (PVP). When the microbeads are subjected to DCP vapor, the conversion of FLA into a phosphoramide causes a rapid and intense fluorescence increase. The PVP layer provides a high density of proton-accepting pyridine nitrogen sites that neutralize the HCl released during the reaction, thereby maintaining high product fluorescence, even after vapor exposure. No significant response is observed when the microbeads are subjected to other nerve agent simulants, a mustard gas simulant, and volatile organics. The size, sensitivity, and subsecond response of these microbeads make them suitable for nerve agent vapor detection and inclusion into microbead sensor arrays.

Journal Article↗
PubMed2006

Fiber-optic microsphere-based arrays for multiplexed biological warfare agent detection.

We report a multiplexed high-density DNA array capable of rapid, sensitive, and reliable identification of potential biological warfare agents. An optical fiber bundle containing 6000 individual 3.1-mum-diameter fibers was chemically etched to yield microwells and used as the substrate for the array. Eighteen different 50-mer single-stranded DNA probes were covalently attached to 3.1-mum microspheres. Probe sequences were designed for Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella melitensis, Clostridium botulinum, Vaccinia virus, and one biological warfare agent (BWA) simulant, Bacillus thuringiensis kurstaki. The microspheres were distributed into the microwells to form a randomized multiplexed high-density DNA array. A detection limit of 10 fM in a 50-microL sample volume was achieved within 30 min of hybridization for B. anthracis, Y. pestis, Vaccinia virus, and B. thuringiensis kurstaki. We used both specific responses of probes upon hybridization to complementary targets as well as response patterns of the multiplexed array to identify BWAs with high accuracy. We demonstrated the application of this multiplexed high-density DNA array for parallel identification of target BWAs in spiked sewage samples after PCR amplification. The array's miniaturized feature size, fabrication flexibility, reusability, and high reproducibility may enable this array platform to be integrated into a highly sensitive, specific, and reliable portable instrument for in situ BWA detection.

Biological Warfare↗
PubMed2006

Digital concentration readout of single enzyme molecules using femtoliter arrays and Poisson statistics.

Methods for accurately quantifying the concentration of a particular analyte in solution are all based on ensemble responses in which many analyte molecules give rise to the measured signal. In this paper, single molecules of beta-galactosidase were monitored using a 1 mm diameter fiber optic bundle with 2.4 x 10(5) individually sealed, femtoliter microwell reactors. By observation of the buildup of fluorescent products from single enzyme molecule catalysis over the array of reaction vessels and by application of a Poisson statistical analysis, a digital concentration readout was obtained. This approach should prove useful for single molecule enzymology and ultrasensitive bioassays. More generally, the ability to determine concentration by counting individual molecules offers a new approach to analysis of dilute solutions.

Biological Assay↗
PubMed2006

Fiber-optic microarray for simultaneous detection of multiple harmful algal bloom species.

Harmful algal blooms (HABs) are a serious threat to coastal resources, causing a variety of impacts on public health, regional economies, and ecosystems. Plankton analysis is a valuable component of many HAB monitoring and research programs, but the diversity of plankton poses a problem in discriminating toxic from nontoxic species using conventional detection methods. Here we describe a sensitive and specific sandwich hybridization assay that combines fiber-optic microarrays with oligonucleotide probes to detect and enumerate the HAB species Alexandrium fundyense, Alexandrium ostenfeldii, and Pseudo-nitzschia australis. Microarrays were prepared by loading oligonucleotide probe-coupled microspheres (diameter, 3 mum) onto the distal ends of chemically etched imaging fiber bundles. Hybridization of target rRNA from HAB cells to immobilized probes on the microspheres was visualized using Cy3-labeled secondary probes in a sandwich-type assay format. We applied these microarrays to the detection and enumeration of HAB cells in both cultured and field samples. Our study demonstrated a detection limit of approximately 5 cells for all three target organisms within 45 min, without a separate amplification step, in both sample types. We also developed a multiplexed microarray to detect the three HAB species simultaneously, which successfully detected the target organisms, alone and in combination, without cross-reactivity. Our study suggests that fiber-optic microarrays can be used for rapid and sensitive detection and potential enumeration of HAB species in the environment.

Base Sequence↗
PubMed2006

Fiber optic array biosensors.

Optical fiber arrays provide a powerful substrate for creating high-density sensing systems that can address a variety of biological problems. The fiber substrate can be used to create femtoliter wells that can be loaded with individual beads to create high-density arrays for multiplexed screening and analysis. In addition, living cells can be loaded into the arrays, and their individual responses monitored over long time periods, enabling functional screening of biologically active compounds. Adherent cells can be attached to the fiber substrate to provide a rapid method for observing cell migration and for screening anti-migratory compounds. Finally, individual enzyme molecules can be loaded into the array wells enabling single molecule detection via enzyme-catalyzed signal amplification.

Animals↗
PubMed2005

Monitoring "promiscuous" drug effects on single cells of multiple cell types.

Recent advances in genomics and molecular pathogenesis studies have determined that many diseases are caused by a multiplicity of factors. New drug regimens may consist of multiple biologically active agents designed to act synergistically on multiple biochemical targets. Live cell assays are becoming a standard for identifying new drug candidates with an emphasis on "homogeneous" living cell assays in which multiple cell lines are mixed and monitored simultaneously. In this study, we used a high-density single living cell array, based on an optical imaging fiber bundle microwell array, to simultaneously monitor "promiscuous" drug effects on single cells of multiple cell types. Such a capability allows for a more comprehensive understanding of how cells dynamically respond to combinatorial drug libraries or how different cellular pathways and regulation circuits respond cooperatively to drugs in individual cells.

Combinatorial Chemistry Techniques↗
PubMed2005

Extending the longevity of fluorescence-based sensor arrays using adaptive exposure.

Fluorescent microbead sensor arrays were prepared to determine sensor array longevity. Sensor longevity is limited by photobleaching of the dyes attached to the microbeads and presents one of the biggest drawbacks of most fluorescent dye-based arrays. Responses of an array of organic vapor sensors were acquired for 2 weeks to evaluate the sensor performance over time. Photobleaching effects were overcome in two ways: (1) by limiting the excitation light power and gradually increasing the power at a rate comparable to the sensor photobleaching rates and (2) by illuminating subsections of the array through an optical slit. Both approaches extended the longevity of a sensor array. During the longevity study, the sensor arrays were employed to test their ability to correctly distinguish between responses to seven vapors. A high classification accuracy (99.8%) was obtained after 17,700 exposures for vapor responses collected over two weeks using only approximately 8% of the array's surface area.

Journal Article↗
PubMed2005

Development of a microfluidic platform with an optical imaging microarray capable of attomolar target DNA detection.

In this paper, DNA hybridization in a microfluidic manifold is performed using fluorescence detection on a fiber-optic microarray. The microfluidic device integrates optics, sample transport, and fluidic interconnects on a single platform. A high-density optical imaging fiber array containing oligonucleotide-labeled microspheres was developed. DNA hybridization was observed at concentrations as low as 10 aM with response times of less than 15 min at a flow rate of 1 microL/min using 50 microL of target DNA samples. The fast response times coupled with the low sample volumes and the use of a high-density, fiber-optic microarray format make this method highly advantageous. This paper describes the initial development, optimization, and integration of the microfluidic platform with imaging fiber arrays.

Bacillus thuringiensis↗
PubMed2005

Detection of Salmonella spp. using microsphere-based, fiber-optic DNA microarrays.

Salmonella spp. are one of the most problematic food pathogens in public health, as they are responsible for food poisoning associated with contamination of meat, poultry, and eggs. Thus, rapid and sensitive detection of Salmonella spp. is required to ensure food safety. In this study, a fiber-optic DNA microarray using microsphere-immobilized oligonucleotide probes specific for the Salmonella invA and spvB genes was developed for detection of Salmonella spp. Microarrays were prepared by randomly distributing DNA probe-functionalized microspheres (3.1-microm diameter) into microwells created by etching optical fiber bundles. Hybridization of the probe-functionalized microspheres to target DNA from Salmonella was performed and visualized using Cy3-labeled secondary probes in a sandwich-type assay format. In this study, 10(3)-10(4) cfu/mL of the target organism could be detected after 1-h hybridization without any additional amplification. The DNA microarray showed no cross-reactivity with other common food pathogens, including E. coli and Y. enterocolitica, and could even detect Salmonella spp. from cocktails of bacterial strains with only moderate loss of sensitivity due to nonspecific binding. This work suggests that fiber-optic DNA microarrays can be used for rapid and sensitive detection of Salmonella spp. Since fiber-optic microarrays can be prepared with different probes, this approach could also enable the simultaneous detection of multiple food pathogens.

Bacterial Proteins↗
PubMed2005

Individual cell migration analysis using fiber-optic bundles.

In this paper we describe a novel optical fiber-based technology for analyzing cell migration. Cells were labeled with a membrane-bound fluorescent dye and distributed onto a polished optical fiber bundle. When a cell passes over one of the individual fibers in the bundle, the membrane-bound dye causes a large intensity increase, which stays for a given "residence time" until the cell departs from the fiber. Residence time increases significantly upon exposure to an antimigratory drug, indicating a decrease in cell migration. This approach provides a simple migration assay and does not require sophisticated tracking software. By using optical fiber bundles containing smaller individual fibers with higher spatial resolution, this approach was employed to develop a migration assay based on subcellular imaging. The subcellular imaging platform allows for rapid analysis of migratory potential, reducing experimental time from several hours in a standard assay to 5 min using this technology.

Cell Movement↗
PubMed2005

Array-based binary analysis for bacterial typing.

An allele-specific oligonucleotide microarray was developed for rapid typing of pathogens based on analysis of genomic variations. Using a panel of Escherichia coli strains as a model system, selected loci were sequenced to uncover differences, such as single- or multiple-nucleotide polymorphisms as well as insertion/deletions (indels). While typical genomic profiling experiments employ specific sequences targeted to genomic DNA unique to a single strain or virulent gene, the present array is designed to type bacteria based on a patterned signature response across multiple loci. In the signature concept, all strains are interrogated by hybridizing their amplified DNA to an array containing multiple probe sequences. Allele-specific oligonucleotide probe sequences targeting each of these variable regions were synthesized and included in a custom fiber-optic array. For each locus, a set of specific probe sequences is selected, such that hybridization gives a binary signal/no signal response to each of the probes. Using this strategy for multiple loci, many pathogens or microorganisms could be classified using a limited number of probes. Because of the advantages of the fiber-optic array platform over other array formats, including sensitivity and speed, the platform described in this paper is capable of supporting a high-throughput diagnostic strategy.

Bacterial Typing Techniques↗
PubMed2005

Optical fiber-based sensors: application to chemical biology.

Optical fibers have been used to develop sensors based on nucleic acids and cells. Sensors employing DNA probes have been developed for various genomics applications and microbial pathogen detection. Live cell-based sensors have enabled the monitoring of environmental toxins, and have been used for fundamental studies on populations of individual cells. Both single-core optical fiber sensors and optical fiber sensor arrays have been used for sensing based on nucleic acids and live cells.

Biosensing Techniques↗
PubMed2005

Personalized exposure assessment: promising approaches for human environmental health research.

New technologies and methods for assessing human exposure to chemicals, dietary and lifestyle factors, infectious agents, and other stressors provide an opportunity to extend the range of human health investigations and advance our understanding of the relationship between environmental exposure and disease. An ad hoc Committee on Environmental Exposure Technology Development was convened to identify new technologies and methods for deriving personalized exposure measurements for application to environmental health studies. The committee identified a "toolbox" of methods for measuring external (environmental) and internal (biologic) exposure and assessing human behaviors that influence the likelihood of exposure to environmental agents. The methods use environmental sensors, geographic information systems, biologic sensors, toxicogenomics, and body burden (biologic) measurements. We discuss each of the methods in relation to current use in human health research; specific gaps in the development, validation, and application of the methods are highlighted. We also present a conceptual framework for moving these technologies into use and acceptance by the scientific community. The framework focuses on understanding complex human diseases using an integrated approach to exposure assessment to define particular exposure-disease relationships and the interaction of genetic and environmental factors in disease occurrence. Improved methods for exposure assessment will result in better means of monitoring and targeting intervention and prevention programs.

Biomarkers↗