Wanda Lyon

Identification of Upper Respiratory Tract Pathogens Using Electrochemical Detection on an Oligonucleotide Microarray

Bacterial and viral upper respiratory infections (URI) produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD) on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae) and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluinza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format.

Use of a multiplexed CMOS microarray to optimize and compare oligonucleotide binding to DNA probes synthesized or immobilized on individual electrodes.

The CombiMatrix microarray with 12,544 electrodes supports in situ electrochemical synthesis of user-defined DNA probes. As an alternative, we immobilized commercially synthesized DNA probes on individual electrodes coated with electropolymerized polypyrrole (Ppy). Hybridization was measured using a biotinylated target oligonucleotide and either Cy5-streptavidin and fluorescence detection or horseradish peroxidase-streptavidin and enzyme-enhanced electrochemical detection. Detection efficiencies were optimized by varying the deposition of the Ppy, the terminal groups on the DNA probes, and other factors that impacted fluorescence quenching and electrical conductivity. Optimized results were compared against those obtained using a microarray with the same DNA sequences synthesized in situ. Immobilized probes produced higher fluorescence signals, possibly by providing a greater stand off between the Cy5 on the target oligonucleotide and the quenching effects of the Ppy and the platinum electrode.

Keratin-based antimicrobial textiles, films, and nanofibers

The combination of appealing structural properties, biocompatibility, and the availability of renewable and inexpensive raw materials, make keratin-based materials attractive for a variety of applications. In this paper, we report on the antimicrobial functionalization of keratin-based materials, including wool cloth and regenerated cellulose/keratin composite films and nanofibers. The functionalization of these materials was accomplished utilizing a facile chlorination reaction that converts the nitrogen-bearing moieties of keratin into halamine compounds. Halamine-charged wool cloth exhibited rapid and potent bactericidal activity against several species of bacteria and induced up to a 5.3 log (i.e., 99.9995%) reduction in the colony forming units of Bacillus thuringiensis spores within 10 min. Keratin-containing composites were prepared by the spin coating and coaxial electrospinning of extracted/oxidized alpha-keratin and cellulose acetate (CA) solubilized in formic acid, followed by CA deacetylation. Regenerated cellulose/keratin materials chlorinated to display halamines were also effective in killing Escherichia coli and Staphylococcus aureus bacteria. Electrospun core/shell nanofibers engineered to maximize keratin-Cl surface area displayed higher activity against S. aureus than films composed of the same materials. The halamine-based antimicrobial functionalization methods demonstrated for keratin-based materials in this paper are anticipated to translate to other protein biopolymers of interest to the biomaterials community.

Sporicidal/Bactericidal Textiles via the Chlorination of Silk

Bacterial spores, such as those of the Bacillus genus, are extremely resilient, being able to germinate into metabolically active cells after withstanding harsh environmental conditions or aggressive chemical treatments. The toughness of the bacterial spore in combination with the use of spores, such as those of Bacillus anthracis, as a biological warfare agent necessitates the development of new antimicrobial textiles. In this work, a route to the production of fabrics that kill bacterial spores and cells within minutes of exposure is described. Utilizing this facile process, unmodified silk cloth is reacted with a diluted bleach solution, rinsed with water, and dried. The chlorination of silk was explored under basic (pH 11) and slightly acidic (pH 5) conditions. Chloramine-silk textiles prepared in acidified bleach solutions were found to have superior breaking strength and higher oxidative Cl contents than those prepared under caustic conditions. Silk cloth chlorinated for ≥1 h at pH 5 was determined to induce >99.99996% reduction in the colony forming units of Escherichia coli, as well as Bacillus thuringiensis Al Hakam (B. anthracis simulant) spores and cells within 10 min of contact. The processing conditions presented for silk fabric in this study are highly expeditionary, allowing for the on-site production of protein-based antimicrobial materials from a variety of agriculturally produced feed-stocks.

Detection of orexin A neuropeptide in biological fluids using a zinc oxide field effect transistor.

Biomarkers which are indicative of acute physiological and emotional states are studied in a number of different areas in cognitive neuroscience. Currently, many cognitive studies are conducted based on programmed tasks followed by timed biofluid sampling, central laboratory processing, and followed by data analysis. In this work, we present a sensor platform capable of rapid biomarker detection specific for detecting neuropeptide orexin A, found in blood and saliva and known as an indicator of fatigue and cognitive performance. A peptide recognition element that selectively binds to orexin A was designed, characterized, and functionalized onto a zinc oxide field effect transistor to enable rapid detection. The detection limit using the sensor platform was sub-picomolar in water, and picomolar to nanomolar levels in saliva and serum. The transistor and recognition element sensor platform can be easily expanded, allowing for multiple biomarkers to be detected simultaneously, lending itself to complex biomarker analysis applicable to rapid feedback for neuroscience research and physiological monitoring.

Microelectrode array biosensor for studying carbohydrate-mediated interactions

Carbohydrate-mediated host-pathogen interactions are essential to bacterial and viral pathogenesis, and represent an attractive target for the development of antiadhesives to prevent infection. We present a versatile microelectrode array-based platform to investigate carbohydrate-mediated protein and bacterial binding, with the objective of developing a generalizable method for screening inhibitors of host-microbe interactions. Microelectrode arrays are well suited for interrogating biological binding events, including proteins and whole-cells, and are amenable to electrochemical derivitization, facilitating rapid deposition of biomolecules. In this study, we achieve microelectrode functionalization with carbohydrates via controlled polymerization of pyrrole to individual microelectrodes, followed by physisorption of neoglycoconjugates to the polypyrrole-coated electrodes. Bioactivity of the immobilized carbohydrates was confirmed with carbohydrate-binding proteins (lectins) detected by both fluorescent and electrochemical means. The platforms ability to analyze whole-cell binding was demonstrated using strains of Escherichia coli and Salmonella enterica, and the dose-dependent inhibition of S. enterica by a soluble carbohydrate antiadhesive.

A Plasmid Containing the Human Metallothionein II Gene Can Function as an Antibody-assisted Electrophoretic Biosensor for Heavy Metals

Abstract Different forms of heavy metals affect biochemical systems in characteristic ways that cannot be detected with typical metal analysis methods like atomic absorption spectrometry. Further, using living systems to analyze interaction of heavy metals with biochemical systems can be laborious and unreliable. To generate a reliable easy-to-use biologically-based biosensor system, the entire human metallothionein-II (MT-II) gene was incorporated into a plasmid (pUC57-MT) easily replicated in Escherichia coli. In this system, a commercial polyclonal antibody raised against human metal-responsive transcription factor-1 protein (MTF-1 protein) could modify the electrophoretic migration patterns (i.e. cause specific decreases in agarose gel electrophoretic mobility) of the plasmid in the presence or absence of heavy metals other than zinc (Zn). In the study here, heavy metals, MTF-1 protein, and polyclonal anti-MTF-1 antibody were used to assess pUC57-MT plasmid antibody-assisted electrophoretic mobility. Anti-MTF-1 antibody bound both MTF-1 protein and pUC57-MT plasmid in a non-competitive fashion such that it could be used to differentiate specific heavy metal binding. The results showed that antibody-inhibited plasmid migration was heavy metal level-dependent. Zinc caused a unique mobility shift pattern opposite to that of other metals tested, i.e. Zn blocked the antibody ability to inhibit plasmid migration, despite a greatly increased affinity for DNA by the antibody when Zn was present. The Zn effect was reversed/modified by adding MTF-1 protein. Additionally, antibody inhibition of plasmid mobility was resistant to heat pre-treatment and trypsinization, indicating absence of residual DNA extraction-resistant bacterial DNA binding proteins. DNA binding by anti-DNA antibodies may be commonly enhanced by xenobiotic heavy metals and elevated levels of Zn, thus making them potentially effective tools for assessment of heavy metal bioavailability in aqueous solutions and fluid obtained from metal implant sites.

Evaluating an Upper Respiratory Disease Panel on the Portable MinION Sequencer

The MinION was used to evaluate upper respiratory disease infections using both whole genome amplification (WGA), targeted sequencing, and was found to have tremendous potential for field use. The MinION nanopore sequencer was been released to community testers for evaluation using a variety of sequencing applications. The MinION was used to evaluate upper respiratory disease infections using both whole genome amplification and targeted sequencing, and was found to have tremendous potential for field use. In this study, we tested the ability of the MinION nanopore sequencer to accurately identify and differentiate clinical bacterial and viral samples via targeted sequencing and whole genome sequencing. The current nanopore technology has limitations with respect to error rate but has steadily improved with development of new flow cells and kits. Upper respiratory disease organisms were successfully identified and differentiated down to the strain level with 87-98% alignment to our reference genome database. The ability to differentiate strains by amplicon and whole genome sequencing on the MinION was accomplished despite the observed average per 100-base error rate averaged 1.2E-01. This study offers evidence of the utility of sequencing to identify and differentiate both viral and bacterial species present within clinical samples.