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Materials for the concentration of nitroenergetic and perchlorate targets from groundwater were developed for monitoring applications. Novel molecular imprinting and structure direction techniques were used to obtain the necessary materials. The potential for concentration of targets from ground and surface waters as well as from soil extracts was demonstrated. The functional characteristics of the materials were evaluated in order to determine the constraints on there use in inline preconcentration of targets ahead of IMS and electrochemical sensing techniques. The systems were subsequently developed at the bench level and function was evaluated using commercially available portable sensors. An initial portable prototype was also developed and evaluated under a field scenario.

Reverse micelles (RMs) containing aqueous solutions of Ag+ ions in their core produce fluorescent Ag nanoclusters (NCs), upon exposure to gamma irradiation. The fluorescence spectra of the NCs evolves over days to weeks after the exposure, and usually show large increases in intensity. Responses of as high as 4 x 105 CPS/Gy were reached. A dosage of as low as 0.5 Gys (10% of the lethal dosage for humans) produces NCs having fluorescence intensities significantly higher than background. The RMs can be employed in novel gamma radiation detectors with appearance of fluorescence indicating that radiation was once present. In applications involving detection of fissile materials, the evolution of the fluorescence spectra over time may provide additional information about the radiation source. A two-phase liquid system is used for RM formation in a simple procedure. This synthesis method may be adapted to produce NCs from many other metal ions., The original document contains color images.

A program was initiated in June 2009 to evaluate the potential of porous organosilicate sorbents in the capture or neutralization of light gas threats. Of primary interest were targets identified by the TIC/TIM Task Force as priority threats. A number of approaches were taken for increasing binding capacity and reactivity in the sorbent materials: grafting of metalloporphyrins, incorporation of sites for metal coordination, and incorporation of acidic and basic groups. In addition, the impact of morphological considerations, such as pore size and organization, were evaluated. Target capture by the sorbents was evaluated using microbreakthrough testing completed at the Edgewood Chemical Biological Center. Development of relevant sorbent morphologies was also pursued. Overall, the sorbents show promise, with several offering improvements in target capture and retention over state-of-the-art materials., The original document contains color images. Prepared in collaboration with Edgewood Chemical Biological Center, Aberdeen Proving Grounds, MD. Sponsored in part by DTRA.

The health consequences of sand particle inhalation are incompletely understood. This project evaluated the respiratory toxicity of sand particles collected at military bases near Fort Irwin USA, in Iraq (Camp Victory, Taji and Talil), and Khost Afghanistan. Our primary focus was on assessing the role of soluble metals in the respiratory toxicity of the sand particles using in vitro and in vivo methods. Replicating rat type II alveolar cell cultures (RLE-6TN) were exposed to sand extracts or vehicle control in serum-free media for 24 h. Cytotoxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and assessment of lactate dehydrogenase leakage., Published in Inhalation Toxicology, v26 n7 p405-416, 2013. The original document contains color images. Prepared in collaboration with the The Joint Pathology Center, Division of Biophysical Toxicology/Biophysical Toxicology Laboratory, Malcolm Grow Medical Clinic, Joint Base Andrews Naval Air Facility, MD. Prepared in cooperation with the Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh. Sponsored in part by the Military Operational Medicine Research Program and ONR.

This report summarizes the results of studies directed at the development of organosilicate sorbents for the capture and preconcentration of nitroenergetics targets and perchlorates from natural water sources. Two distinct types of sorbents were developed. The first, directed at nitroenergetics, utilizes new approaches to target templating and a hierarchical structure. The second, directed at perchlorates, utilizes the hierarchical structure with alkylammonium surface modifications. Both materials provide semiselective target capture and offer advantages over commercially available products. Application of the sorbents in single and mixed target solutions is demonstrated. Nitroenergetic capture from natural water sources is also evaluated., The original document contains color images.

The potential for encapsulating RNA within tunable, semi-permeable structures for storage and transportation purposes offers an interesting approach to the reduction of stringent storage requirements that often hamper the field application of genetic analysis methods. In this study, we assessed the potential for application of functionalized, porous silicate sorbents in maintaining nucleic acid integrity. Mesoporous silica nanoparticles (MSNs) with and without incorporated stabilizing reagents were used to encapsulate triosephosphate isomerase mRNA of Arabidopsis thaliana. The absorption, elution, and the long-term stability of the RNA were monitored by using quantitative real-time RT-PCR. The results indicate that adsorbed RNA can be eluted from the sorbents using simple buffers and employed directly for downstream molecular diagnostic assays without any further processing. RNA integrity can be maintained for extended time periods under refrigeration temperatures in the presence of covalently immobilized stabilizing compounds. This study provides initial evidence of the potential for application of MSNs in transportation and storage. They may also have utility in sample collection and processing in restrictive environments., Published in PLOS One, v7 n11, 30 Nov 2012. The original document contains color images.

Observations of enhanced growth of melanized fungi under low-dose ionizing radiation in the laboratory and in the damaged Chernobyl nuclear reactor suggest they have adapted the ability to survive or even benefit from exposure to ionizing radiation. However, the cellular and molecular mechanism of fungal responses to such radiation remains poorly understood. Using the black yeast Wangiella dermatitidis as a model, we confirmed that ionizing radiation enhanced cell growth by increasing cell division and cell size. Using RNA-seq technology, we compared the transcriptomic profiles of the wild type and the melanin-deficient wdpks1 mutant under irradiation and non-irradiation conditions. It was found that more than 3000 genes were differentially expressed when these two strains were constantly exposed to a low dose of ionizing radiation and that half were regulated at least two fold in either direction. Functional analysis indicated that many genes for amino acid and carbohydrate metabolism and cell cycle progression were down-regulated and that a number of antioxidant genes and genes affecting membrane fluidity were up-regulated in both irradiated strains. However, the expression of ribosomal biogenesis genes was significantly up-regulated in the irradiated wild-type strain but not in the irradiated wdpks1 mutant, implying that melanin might help to contribute radiation energy for protein translation. Furthermore, we demonstrated that long-term exposure to low doses of radiation significantly increased survivability of both the wild-type and the wdpks1 mutant, which was correlated with reduced levels of reactive oxygen species (ROS), increased production of carotenoid and induced expression of genes encoding translesion DNA synthesis. Our results represent the first functional genomic study of how melanized fungal cells respond to low dose ionizing radiation and provide clues for the identification of biological processes, molecular pathways and individual genes regular, Published in PLoS ONE, v7 n11, 6 Nov 2012. The original document contains color images.

Geobacter spp. can acquire energy by coupling intracellular oxidation of organic matter with extracellular electron transfer to an anode (an electrode poised at a metabolically oxidizing potential) forming a biofilm extendingmany cell lengths away from the anode surface. It has been proposed that long-range electron transport in such biofilms occurs through a network of bound redox cofactors thought to involve extracellularmatrix c-type cytochromes, as occurs for polymers containing discrete redox moieties. Here, we report measurements of electron transport in actively respiring Geobacter sulfurreducens wild type biofilms using interdigitated microelectrode arrays. Measurements when one electrode is used as an anode and the other electrode is used to monitor redox status of the biofilm 15 micrometers away indicate the presence of an intrabiofilm redox gradient, in which the concentration of electrons residing within the proposed redox cofactor network is higher farther from the anode surface. The magnitude of the redox gradient seems to correlate with current which is consistent with electron transport from cells in the biofilm to the anode, where electrons effectively diffuse from areas of high to low concentration, hopping between redox cofactors., Published online before print September 5, 2012, Proceedings of the National Academy of Sciences. The original document contains color images.

Proteorhodopsins (PRs) are retinal-binding photoproteins that mediate light-driven proton translocation across prokaryotic cell membranes. Despite their abundance, wide distribution and contribution to the bioenergy budget of the marine photic zone, an understanding of PR function and physiological significance in situ has been hampered as the vast majority of PRs studied to date are from unculturable bacteria or culturable species that lack the tools for genetic manipulation. In this study, we describe the presence and function of a horizontally acquired PR and retinal biosynthesis gene cluster in the culturable and genetically tractable bioluminescent marine bacterium Vibrio campbellii. Pigmentation analysis, absorption spectroscopy and photoinduction assays using a heterologous over-expression system established the V. campbellii PR as a functional green light absorbing proton pump. In situ analyses comparing PR expression and function in wild type (WT) V. campbellii with an isogenic DpR deletion mutant revealed a marked absence of PR membrane localization, pigmentation and light-induced proton pumping in the DpR mutant. Comparative photoinduction assays demonstrated the distinct upregulation of pR expression in the presence of light and PR-mediated photophosphorylation in WT cells that resulted in the enhancement of cellular survival during respiratory stress. In addition, we demonstrate that the master regulator of adaptive stress response and stationary phase, RpoS1, positively regulates pR expression and PR holoprotein pigmentation. Taken together, the results demonstrate facultative phototrophy in a classical marine organoheterotrophic Vibrio species and provide a salient example of how this organism has exploited lateral gene transfer to further its adaptation to the photic zone., Pub. in PLoS One, v7 n6, p1-8, Jun 2012.

Vibrio harveyi is a Gram-negative bacterium found in tropical and temperate marine environments as a free-living organism or in association with aquatic animals. We report the first sequenced genome of a Vibrio harveyi strain, CAIM 1792, the etiologic agent of the bright red syndrome of the Pacific white shrimp Litopenaeus vannamei., Journal of Bacteriology, v194 n8 p2104, 2012. Sponsored in part by NRL. The original document contains color images.

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6. Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5., Pub. in Journal of Visualized Experiments, v59, p1-6, January 2012. The original document contains color images.

The abundance of mammalian 18S and 28S ribosomal RNA can decrease the detection sensitivity of bacterial or viral targets in complex host-pathogen mixtures. A method to capture human RNA in a single step was developed and characterized to address this issue. For this purpose, capture probes were covalently attached to magnetic microbeads using a dendrimer linker and the solid phase was tested using rat thymus RNA (mammalian components) with Escherichia coli RNA (bacterial target) as a model system. Our results indicated that random capture probes demonstrated better performance than specific ones presumably by increasing the number of possible binding sites, and the use of a tetrame-thylammonium-chloride (TMA-Cl-) based buffer for the hybridization showed a beneficial effect in the selectivity. The subtraction efficiency determined through real-time RT-PCR revealed capture-efficiencies comparable with commercially available enrichment kits. The performance of the solid phase can be further fine tuned by modifying the annealing time and temperature., Publsihed in the Journal of Biomedicine and Biotechnology, v2011, 2011. Government or Federal Purpose Rights License. The original document contains color images.

Dielectric measurements have been made on three organosiloxane liquid crystal compounds exhibiting a smectic A (SmA) to smectic C* (SmC*) transition, the SmA phase being of the de Vries type. The electroclinic response of the molecules in the de Vries phase of these compounds exhibits a double-peak profile, and is thus different from the conventional chiral SmA phase, a feature explained on the basis of an antiferroelectric (AF) block model. The differential interactions arising from the different molecular ends of these siloxane-based compounds, which are the basis for the AF block model, can also be expected to enhance the layer translational order. We present x-ray integrated intensity data that show a high (~0.9) translational order in the SmA phase. Dielectric relaxation spectra bring out the fact that the magnitude of the soft mode relaxation parameters is dependent on the number of siloxane groups in the terminal part of the molecule. A range-shrinking analysis of the temperature-dependent dielectric relaxation strength has been carried out, using a power-law expression. The characteristic exponent shows a systematic growth with range shrinking and reaches limiting values comparable to that predicted for the 2D Ising universality class., Published in Journal of Physics: Condensed Matter, v23 n105902, 2011. The original document contains color images.

The development of porphyrin-embedded mesoporous organosilicate materials for application to the detection of volatile hydrocarbon solvents is described. Design of the receptor and optical indicator construct begins with parallel selection of the porphyrin indicator and design of the mesoporous sorbent. For the porphyrin indicator, high binding affinity and strong changes in spectrophotometric character upon target interaction are desired. The sorbent should provide high target binding capacity and rapid binding kinetics. A number of porphyrin/metalloporphyrin variants and organosilicate sorbents were evaluated to determine the characteristics of their interaction with the targets, benzene, toluene, and hexane. The selected porphyrin candidates were covalently immobilized within a benzene-bridged sorbent. This construct was applied to the detection of targets using both fluorescence- and reflectance-based protocols. The use of red, green, and blue (RGB) color values from the constructs in a highly simplified detection scheme is described., Published in Sensors, v11 p886-904, 14 Jan 2011. Sponsored in part by DTRA.

Biosensors based on antibody recognition have a wide range of monitoring applications that apply to clinical environmental, homeland security, and food problems. In an effort to improve the limit of detection of the Naval Research Laboratory (NRL) Array Biosensor, magnetic nanoparticles (MNPs) were designed and tested using a fluorescence-based array biosensor. A full optimization study of the antibody-modified MNPs and their use in the biosensor was performed. The results demonstrate the impact of magnetic extraction using the MNPs labeled with fluorescent proteins both for target preconcentration and for subsequent integration into immunoassays performed under flow conditions for enhanced signal generation., Published in Analytical Biochemistry, 2010. Prepared in collaboration with University of Florida, Gainesville, FL; Armstrong Atlantic State University, Savannah, GA; and George Mason University, Manassas, VA.

Background: Spatial epidemiology is useful but difficult to apply in developing countries due to the low availability of digitized maps and address systems, accurate population distributions, and computational tools. A community-based mapping approach was used to demonstrate that participatory geographic information system (PGIS) techniques can provide information helpful for health and community development. Results: The PGIS process allowed for the rapid determination of sectional (neighborhood) boundaries within the city of Bo, Sierra Leone. When combined with data about hospital laboratory visits, a catchment area for one hospital in Bo could be established. A survey of households from within the catchment area determined that the average population per household (about 6 individuals) was similar to that found in the 2004 census. However, we also found that the average house was inhabited by more than one household, for an average of 17.5 inhabitants per residential building, which is critical information to know when estimating population size using remote imagery that can detect and enumerate buildings. Conclusions: The methods developed in this paper serve as a model for the involvement of communities in the generation of municipal maps and their application to community and health concerns., Published in the International Journal of Health Geographics, v9 n56 p1-8, 2010. Prepared in collaboration with Njala University, Sierra Leone; and George Mason University, Fairfax, VA.

Porphyrins are a family of highly conjugated molecules that strongly absorb visible light and fluoresce intensely. These molecules are sensitive to changes in their immediate environment and have been widely described for optical detection applications. Surfactant-templated organosilicate materials have been described for the semi-selective adsorption of small molecule contaminants. These structures offer high surface areas and large pore volumes within an organized framework. The organic bridging groups in the materials can be altered to provide varied binding characteristics. This effort seeks to utilize the tunable binding selectivity, high surface area, and low materials density of these highly ordered pore networks and to combine them with the unique spectrophotometric properties of porphyrins. In the porphyrin-embedded materials (PEMs), the organosilicate scaffold stabilizes the porphyrin and facilitates optimal orientation of porphyrin and target. The materials can be stored under ambient conditions and offer exceptional shelf-life. Here, we report on the design of PEMs with specificity for organophosphates and compounds of similar structure., Published in Sensors, v10 p2315-2331, 2010. Performed in collaboration with NOVA Research Incorporated, Alexandria, VA. Naval Research Laboratory work unit no. 69-8765. Sponsored in part by the Defense Threat Reduction Agency (DTRA; BA08PRO015) and the Department of Defense Strategic Environmental Research and Development Program (SERDP; ER-1604).

The use of peptides to mediate the delivery and uptake of nanoparticle (NP) materials by mammalian cells has grown significantly over the past 10 years. This area of research has important implications for the development of new therapeutic materials and for the emerging field of NP-mediated drug delivery. In this review, we highlight recent advances in the delivery of various NPs by some of the more commonly employed cellular delivery peptides and discuss important related factors such as NP-peptide bioconjugation, uptake efficiency, intracellular fate and toxicity. We also highlight various demonstrations of therapeutic applications of NP-peptide conjugates where appropriate. The paper concludes with a brief forward-looking perspective discussing what can be expected as this field develops in the coming years., Published in Therapeutic Delivery, v1 n3 p411-433, 2010. Sponsored in part by DTRA, ONR, NRL and the NRL-NSI.

The stimulation of subsurface microbial metabolism often associated with engineered bioremediation of groundwater contaminants presents subsurface microorganisms, which are adapted for slow growth and metabolism in the subsurface, with new selective pressures. In order to better understand how Geobacter species might adapt to selective pressure for faster metal reduction in the subsurface, Geobacter sulfurreducens was put under selective pressure for rapid Fe(III) oxide reduction. The genomes of two resultant strains with rates of Fe(III) oxide reduction that were 10-fold higher than those of the parent strain were resequenced. Both strains contain either a single base-pair change or a 1 nucleotide insertion in a GEMM riboswitch upstream of GSU1761, a gene coding for the periplasmic c-type cytochrome designated PgcA. GSU1771, a gene coding for a SARP regulator, was also mutated in both strains. Introduction of either of the GEMM riboswitch mutations upstream of pgcA in the wild-type increased the abundance of pgcA transcripts, consistent with increased expression of pgcA in the adapted strains. One of the mutations doubled the rate of Fe(III) oxide reduction. Interruption of GSU1771 doubled the Fe(III) oxide reduction rate. This was associated with an increased in expression of pilA, the gene encoding the structural protein for the pili thought to function as microbial nanowires. The combination of the GSU1771 interruption with either of the pgcA mutations resulted in a strain that reduced Fe(III) as fast as the comparable adapted strain. These results suggest that the accumulation of a small number of beneficial mutations under selective pressure, similar to that potentially present during bioremediation, can greatly enhance the capacity for Fe(III) oxide reduction in G. sulfurreducens., Published in Environmental Microbiology, 2010.

Metal ion complexes are playing an increasing role in the development of antimicrobials. We review here the antimicrobial properties of cobalt coordination complexes in oxidation state 3+. In addition to reviewing the cobalt complexes containing polydentate donor ligands, we also focus on the antimicrobial activity of the homoleptic [Co(NH3)6]3+ ion., Pub. in Pharmaceuticals v3, p1711-1728, 2010. Sponsored in part by NRL.

We elucidate the polymer dynamics of a liquid crystal elastomer based on the time-dependent response of the pendent liquid crystal mesogens. The molecular tilt and switching time of mesogens are analyzed as a function of temperature and cross-linking density upon application of an electric field. We observe an unexpected maximum in the switching time of the liquid crystal mesogens at intermediate field strength. Analysis of the molecular tilt over multiple time regimes correlates the maximum response time with a transition to entangled polymer dynamics at a critical field strength.

Materials for the concentration of nitroenergetic targets from groundwater are sought for monitoring and remediation applications. For this work, periodic mesoporous organosilicas incorporating diethylbenzene bridges in their pore walls were applied for the adsorption of nitroenegetic targets from aqueous solution. The materials were synthesized by co-condensing precursors to improve structural characteristics. Molecular imprinting of the pore surfaces was employed through the use of a novel target-like surfactant to further enhance selectivity for targets of interest (nitroenergetics and their breakdown products) over targets of similar structure (p-cresol and p-nitrophenol). The impact of variations in precursor ratios and amounts of imprint molecule was evaluated. These results were applied to the development of materials possessing organization on both the meso and macro scales. The incorporation of macropores provides a material with enhanced flow through characteristics, thereby reducing back pressure when used in column formats. Concentration of targets from soil sample extracts using these materials was demonstrated., The original document contains color images. Work unit no. 69-9629-0-8-5

Zoonotic microbes have historically been, and continue to emerge as, threats to human health. The recent outbreaks of highly pathogenic avian influenza virus in bird populations and the appearance of some human infections have increased the concern of a possible new influenza pandemic, which highlights the need for broad-spectrum detection methods for rapidly identifying the spread or outbreak of all variants of avian influenza virus. In this study, we demonstrate that high-density resequencing pathogen microarrays (RPM) can be such a tool. The results from 37 influenza virus isolates show that the RPM platform is an effective means for detecting and subtyping influenza virus, while simultaneously providing sequence information for strain resolution, pathogenicity, and drug resistance without additional analysis. This study establishes that the RPM platform is a broad-spectrum pathogen detection and surveillance tool for monitoring the circulation of prevalent influenza viruses in the poultry industry and in wild birds or incidental exposures and infections in humans., Pub. in Journal of Clinical Microbiology, p988-993, v47 n4, Apr 2009

Fluorescence-based flow cytometry dates back to the 1960s. Essentially, cells or particles are aligned in a flow stream and optically interrogated. Size, density, and fluorescence at multiple wavelengths can be quantified. In many cases, tags, such as fluorescently labeled antibodies, are mixed with the samples prior to analysis so that specific targets or cell functions can be identified. Currently, large, complex, laboratory- based flow cytometers are required to perform medical diagnostics, such as white blood cell counts and immunoassays to detect infection, or for environmental monitoring applications, such as classification of marine algae. In the traditional design, the sample containing the particles or cells is pumped out of a small tube into a much larger, concentric pipe that is carrying filtered water. This hydrodynamic focusing puts all the particles into the center of the wider "sheath" stream, which then is tapered to a smaller diameter. The particles are thus "focused" and pass single-file through the laser beams for analysis. Over the last decade, flow cytometers have become smaller in size and less expensive, but this sheath flow design is not amenable to miniaturization to the point that the systems are portable. NRL's Center for Bio/Molecular Science and Engineering has developed a microfluidic sheath flow system that is robust, simple to fabricate, and very compact. This sheath flow device forms the basis of a microflow cytometer that has demonstrated the capability for 4-color analysis that is competitive with the larger, commercial systems., Published in NRL Review, p152-154, 2009.

Rapid and effective detection and identification of emerging microbiological threats and potential biowarfare agents is very challenging when using traditional culture-based methods. Contemporary molecular techniques, relying upon reverse transcription and/or polymerase chain reaction (RT-PCR/PCR) provide a rapid and effective alternative, however, such assays are generally designed and optimized to detect only a limited number of targets, and seldom are capable of differentiation among variants of detected targets. To meet these challenges, we have designed a broad-range resequencing pathogen microarray (RPM) for detection of tropical and emerging infectious agents (TEI) including biothreat agents: RPM-TEI v 1.0 (RPM-TEI). The scope of the RPM-TEI assay enables detection and differential identification of 84 types of pathogens and 13 toxin genes, including most of the class A, B and C select agents as defined by the Centers for Disease Control and Prevention (CDC, Atlanta, GA). Due to the high risks associated with handling these particular target pathogens, the sensitivity validation of the RPM-TEI has been performed using an innovative approach, in which synthetic DNA fragments are used as templates for testing the assay's limit of detection (LOD). Assay specificity and sensitivity was subsequently confirmed by testing with full-length genomic nucleic acids of selected agents. The LOD for a majority of the agents detected by RPM-TEI was determined to be at least 104 copies per test. Our results also show that the RPM-TEI assay not only detects and identifies agents, but is also able to differentiate near neighbors of the same agent types, such as closely related strains of filoviruses of the Ebola Zaire group, or the Machupo and Lassa arenaviruses., Published in PloS ONE v4 i8 August 2009.

Glass microscope slides are considered by many as the substrate of choice for microarray manufacturing due to their amenability to various surface chemistry modifications. The use of silanes to attach various functional groups onto glass slides has provided a versatile tool for the covalent immobilization of many diverse biomolecules of interest. We recently noted a dramatic reduction in biomolecule immobilization efficiency on standard microscope slides prepared using a well-characterized silanization method. A survey of commercial soda-lime slides yielded the surprising result that slides purchased prior to 2008 had superior immobilization efficiencies when compared to those purchased after 2008. Characterization of the slides by X-ray photoelectron spectroscopy (XPS), contact angle measurements, and atomic force microscopy (AFM), revealed a significant correlation (R > 0.9) between magnesium content, surface roughness, and bioimmobilization efficiency. High performance slides had higher magnesium content and higher root-meansquare (rms) roughness (P < 0.005) than slides with lower bioimmobilization efficiencies. Although the exact mechanism of how magnesium content and surface roughness affect silane deposition has not yet been defined, we show that recent changes in the chemical and physical properties of commercial soda-lime slides affect the ability of these slides to be covalently modified., Published in Analytical Chemistry, v82 n1 p406-412, 1 Jan 2010. Sponsored in by the Defense Threat Reduction Agency and the Office of Naval Research.

Significant challenges exist in constructing and manipulating the building blocks of a nanoscale device. After such a device is assembled, it is an additional challenge to electronically address or measure responses at the molecular level. We demonstrate the usefulness of individual virus particles as scaffolds to enable design, construction, and measurement of nanoscale molecular electronics memory devices. Such devices are of great interest for their potential to enable lightweight, low-cost, and low-power technologies such as handheld electronic sensors and inexpensive disposable molecular memories. Taking advantage of molecular electronics requires developing novel techniques for organizing nanosized materials into usable devices. As biological interactions are better understood, there has been great interest in using the specificity and strong interactions present in biology for this purpose. By combining the structural specificity provided by biological systems with the material properties of synthetic systems it is possible to develop novel devices on the nanoscale. Using viruses as nanoscale scaffolds for devices offers the promise of exquisite control of positioning on the nanoscale, using a particle that can either interface with lithographically defined structures, or undergo further self-assembly into extended structures by itself. We use cowpea mosaic virus (CPMV) as a nanoscale scaffold for constructing molecular electronic circuits for use in memory applications. Nanocircuit Assembly: Using genetically engineered CPMV allows the generation of specific patterns of functional amino acids, which provide a means to assemble complex structures with high spatial specificity on the nanometer scale. Using genetically engineered CPMV allows the generation of specific patterns of functional amino acids, which provide a means to assemble complex structures with high spatial specificity on the nanometer scale., Published in the 2008 NRL Review, p187-189, 2008.

The use of Forster or fluorescence resonance energy transfer (FRET) as a spectroscopic technique has been in practice for over 50 years. A search of ISI Web of Science with just the acronym FRET returns more than 2300 citations from various areas such as structural elucidation of biological molecules and their interactions, in vitro assays, in vivo monitoring in cellular research, nucleic acid analysis, signal transduction, light harvesting and metallic nanomaterials. The advent of new classes of fluorophores including nanocrystals, nanoparticles, polymers, and genetically encoded proteins, in conjunction with ever more sophisticated equipment, has been vital in this development. This review gives a critical overview of the major classes of fluorophore materials that may act as donor, acceptor, or both in a FRET configuration. We focus in particular on the benefits and limitations of these materials and their combinations, as well as the available methods of bioconjugation., Published in Angewandte Chemie Int. Ed., v45 p4562-4588, 2006. The original document contains color images. Prepared in collaboration with George Mason University, Manassas, VA, and the National Research Center on nanoStructures and bioSystems at Surfaces, Modena, Italy.

From October 2004 through March 2005, the Naval Research Laboratory (NRL) transformed a research protocol for gene-based pathogen identification into a demonstration project called Silent Guardian. Silent Guardian executed a bio-surveillance operation in the Washington, DC, region during the weeks surrounding the 2005 Presidential inauguration. To perform this bio-surveillance, nasal wash specimens were collected from patients presenting with flu-like symptoms at six military clinics in the Military District Washington and taken to NRL for testing. Simultaneous analysis for both common respiratory pathogens and bioterrorism agents was performed 24 hours a day for two months. The Silent Guardian Demonstration achieved four major objectives: (1) Pathogens were unequivocally identified from a general urban population within 24 hours; (2) The ability to sequence genes from more than 20 pathogen species was demonstrated in a production mode; (3) The capability of moving cutting edge technology from research to production within a six-week period was demonstrated by combined military/civilian teams; and (4) A clear path was identified that could lead to an automated, portable, user friendly system. This is the first documented application of DNA microarray technology to operational, broad-spectrum strain-level pathogen identification in an urban population., Published in NRL Review, p85-91, 2006.

The presence of trace environmental contamination of soil and ground water is an ongoing concern. Historically, improper practices during the manufacture and storage of explosives, such as 2,4,6-trinitrotoluene (TNT), were the primary causes. Now, the need for trace detection of explosives is further heightened by current homeland security concerns. Required are easy-to-use, sensitive, and fast methods to facilitate the detection and quantification of these contaminants, whether to monitor remediation or provide surveillance. Biosensors have been found effective for solution-phase detection of environmental contaminants, with NRL pioneering the use of flow displacement immunoassays. Our work utilized the Luminex100 (a commercial flow cytometer), which can perform highly multiplexed assays by discriminating between up to 100 different bead sets, each of which can be used to test for a different target. While the Luminex100 has been shown to be a highly effective biosensor for sandwich or competitive immunoassays, the goal of this project was to demonstrate for the first time a displacement immunoassay on this platform, and to test its suitability for TNT detection in the marine environment. We evaluated four different TNT monoclonal antibodies, two recombinant TNT antibodies, and a control antibody simultaneously for the rapid detection of TNT and other nitroaromatics. TNT was detected at 0.1 ppb and could be quantified over the range of 1 ppb to 10 ppm. In addition, the assay was shown to be effective in various matrixes such as lake water, sea water, and soil extracts. This ability to function in the maritime environment is critical for Naval applications, such as trace detection of explosive materials onboard interdicted vessels, anti-mine operations, or detection of unexploded ordinance., Published in Naval Research Laboratory (NRL) Review, p1-3, 2006. Sponsored in part by NSF. Prepared in collaboration with University of Puerto Rico.

Porphyrin-embedded periodic mesoporous organosilica (PMO) materials were developed for sunlight-stimulated degradation of cyclic organics. The binding affinity and selectivity of the PMOs act to bring porphyrin and target into close proximity, provide extensive surface area, and a stable substrate for porphyrin immobilization. Aqueous samples incubated with these PMO materials showed a decrease in analyte concentration greater than that expected based on adsorption and uncatalyzed photoconversion during 5 h of sunlight illumination. Both materials show selective binding of TNT over structurally similar compounds such as p-nitrophenol. Template directed molecular imprinting was used in an attempt to increase the selectivity of the catalyst., Published in Catalysis Communications, v8 p1052-1056, 2007.

Nematic liquid crystal elastomers are thermally actuated to produce macroscopic, anisotropic shape changes. Uniaxial contraction and extension of liquid crystal elastomers can be achieved by cycling the temperature of the material through the nematic to isotropic phase transition. In this report, a new approach is introduced by layering liquid crystal elastomer films to create thermally actuated stacks. A heating element and thermally conductive grease embedded between elastomer films provide a means for rapid internal heat application and distribution when a current is passed through the heating element, thus providing contractile force production in a minimal amount of time. Upon voltage application, stacks composed of two 100 m-thick films and a single heating element produce 18% strain between contracted and relaxed states. In addition, the stacked elastomer films are capable of producing 10% contraction within 1 s and the blocked stress of the thermally actuated stacked films is calculated to be 130 kPa. The stacking approach provides new opportunities to use liquid crystal elastomers in applications requiring forces greater than those capable of being produced by single elastomer films., Published in Sensors and Actuators A, v133 p500-505, 2007.

Hazardous chemical vapors pose a serious challenge to the security and well-being of our nation and to our forces abroad. These can be dangerous chemical warfare agents (CWAs) and explosives or health hazards such as toxic industrial chemicals and materials (TICs and TIMs). A long-standing problem with chemical vapor detectors is their susceptibility to false alarms, which results in disruption of normal activities. In detectors that use a chemically sensitive coating layer, such false alarms are caused by inadequate selectivity toward target molecules. Therefore, there is a critical need to develop novel chemical coatings that can enhance selectivity of detection for specific targets or analytes. Cavitands solve this problem by enhancing the selectivity of detection towards chemical vapors., Published in the 2005 NRL Review, p171-173. Sponsored in part by the Joint Services NMCBD Program.

The RAPTOR is a commercial, portable (5.6 kg) automated fiber optic biosensor able to detect up to four biological threat agents simultaneously. It performs rapid (5 to 13 minute) fluorescent sandwich immunoassays on the surface of four short polystyrene optical probes. The optical waveguide can be reused up to forty times, or until a positive result is obtained, reducing the logistical burden for field operations. The newest version of the RAPTOR uses the proven reliability of peristaltic pumps for its fluidics, which has greatly increased its mean time to failure. On the biochemical side, the use of Alexa Fluor 647 to label tracer antibodies and immobilization of biotinylated capture antibodies on avidincoated waveguides have improved signal generation by a factor of 5. In combination with improved optics, the new RAPTOR has improved limits of detection by a log order. Recently, we demonstrated the capability of the device to test for eight targets by combining multiple antibodies on each waveguide. These improvements make the RAPTOR ready for use wherever rapid on-site biodetection is required., See also ADM001523.

To meet environmental remediation goals, there is a need for rapid, quantitative detection of hazardous pollutants such as explosives. Biosensors provide a rapid, specific, sensitive, portable, and inexpensive means to fulfill those needs. The Naval Research Laboratory has developed two methods for measuring TNT and RDX. These methods employ either the Analyte 2000 or the FAST 2000 optical instruments, both engineered by Research International (Woodinville WA) in collaboration with NRL. These biosensors, based on fluorescence immunoassay techniques, are interfaced to portable computers for instrument control and data analysis. Both biosensors are portable, and easily set-up within 30 minutes on a small table. The Analyte 2000 is a fiber optic biosensor capable of simultaneously monitoring four optical probes. It is based on a competitive fluoroimmunoassay, in which a fluorescent molecule, similar to the analyte, competes with the analyte for binding sites on antibodies immobilized on the surface of an optical probe. In this format, the fluorescence signal is inversely proportional to the amount of analyte in the sample. Results are determined in 12-17 minutes depending on the analyte. Multiple analyses are performed on the same fiber probe to reducing probe to probe variation issues for quantitation. The Fast 2000 is a continuous flow immunosensor based on a displacement immunoassay, with the key components being antibodies specific for the analyte immobilized on a membrane support, fluorescent signal molecules similar to the analyte saturated on the immobilized antibodies, and a fluorescent detector. Upon injection of an explosive contaminated sample, fluorescent signal molecules are released into the flow stream and detected by a detector. The FAST 2000 quantitates samples with minimal sample preparation and reagent addition. Analysis is complete within five minutes, with the fluorescent signal being proportional to the analyte concentration in the sample.

Prepared in collaboration with Shipley Co. Inc. Marlborough, MA.

An NRL continuous flow immunosensor, known as the FAST 2000, has been developed that is adaptable for use in a variety of environmental monitoring platforms. During the past year, the FAST 2000 instrument was tested in initial field trials at numerous military bases identified by the EPA as priority Superfund cleanup sites. The field study objective was to measure the effectiveness and efficiency of the Flow Immunosensor in performing on-site field analysis for two selective explosives, TNT and RDX. Samples containing unknown concentrations of explosives were collected from groundwater and monitoring wells at various locations and analyzed in NRL sensor with no sample pretreatment or concentration. For data validation and in conjunction with the tests performed on-site by the Flow Immunosensor, independent laboratory analyses of lab splits from the field samples and explosives standards were performed by High Performance Liquid Chromatography using the EPA SW846 Method 8330 for explosives. In addition to the contaminated field samples, appropriate controls, blanks and interferents were tested in the lab for certification and validation data requirements. Results were evaluated based on accuracy, precision, rate of false positives/negatives, cost, time, and waste generation.

A new non-rubbing technique for uniform, planar alignment of liquid crystals has been developed. It involves a combination of chemisorption and UV-induced photo-dimerization. By varying chemical structure of the outermost part of the alignment layer, it has been possible to control the pretilt of the liquid crystal molecules at the surface ranging from 0 to 20 degrees. A feature of the process is that it is a room temperature process and applicable to a variety of substrates. Data on LCD cells fabricated using these alignment layers are presented.

The NRL fiber optic biosensor is a device which measures the formation of a fluorescent complex at the surface of an optical fiber. Antibodies provide a mechanism for recognizing an analyte of interest and immobilizing a fluorescent complex onto the fiber surface. The fiber optic biosensor is fast, sensitive, and it permits analysis of hazardous materials remote from the instrumentation. Antibodies coated on the fiber are stable for up to two years of storage prior to use. A portable fiber optic biosensor has been developed which is able to monitor four fiber probes simultaneously. Quadruplicates or 4 separate samples can be analyzed with the multiple fiber system. The biosensor has been used to measure concentration of toxins, proteins, small molecules in the parts per billion (microgram/L) range in under 10 minutes. The fiber optic biosensor is currently being applied for detection of explosives, pathogens, and toxic materials which pollute the environment. On-site TNT analysis of explosive contaminated groundwater and leachate samples has been demonstrated., Presented at Workshop on Tri-Service Environmental Technology held in Hershey, PA on 20-22 May 1996. Pub. in Proceedings of the Tri-Service Environmental Technology Workshop, p287-290, May 1996. This article is from ADA429790 Proceedings of the Tri-Service Environmental Technology Workshop, "Enhancing Readiness Through Environmental Quality Technology" Held in Hershey, PA on 20-22 May 1996

The correlation between molecular structure and electrooptic performance is studied for novel room temperature chiral smectic A liquid crystals. It is observed that slight modifications of the hydrocarbon chain connected to the chiral center result in dramatic differences in their electrooptic performance. If the hydrocarbon chain is shortened or if a double bond is localized at the end of the hydrocarbon chain, the tilt angle, electroclinic coefficient, and switching time are significantly suppressed.

Liposome encapsulated hemoglobin is being developed as hem an artificial resuscitative fluid for in vivo oxygen delivery. in the present report, we examine the effect of accumulation of liposome encapsulated hemoglobin on the structure of reticuloendothelial organs following administration of liposome encapsulated bovine hemoglobin in the normovolemic rat. We have also examined the administration of the liposome vehicle, tetrameric bovine hemoglobin, and liposome encapsulated bovine hemoglobin that had been lyophilized with 300 mM trehalose and rehydrated just before injection. Following injection into the tail vein, rats were sacrificed and liver, spleen, kidney, and lung harvested at 2 h, 24 h, 1 week, and 2 weeks for analysis. Gross pathologic findings of animals injected with liposome en may capsulated hemoglobin showed statistically significant splenomegaly with a waxy parenchymal pallor at early time points. Microscopic findings indicate that the liver and spleen are principally involved with liposome encapsulated hemoglobin removal over the course of 24 h with transient cytoplasmic vacuolization in tissue resident phagocytes as evidenced by both light and electron microscopic examination. Presence of liposome encapsulated hemoglobin in these vacuoles was confirmed by oil red 0 and prussian blue stains. Splenic weight was observed to decline after 24 h but still remained significant above sham-treated controls at 2 weeks and could be correlated with increased hematopoietic activity.

The correlation between molecular structure and electrooptic performance is studied for two series of ferroelectric liquid crystalline materials. The first series (Series 1) consist of siloxy units attached to the hydrocarbon chain at the non-chiral end of the molecule; while the second series (Series 2) contain only hydrocarbon chains at the end of the molecule. Series 1 exhibit chiral smectic A (SmA) and smectic C (SmC). It is observed that slight modifications in either the siloxy or the hydrocarbon chain length has strong effect on the electrooptic properties. Increasing the number of siloxy unit in the chain (Series 1) increases the temperature range of SmA, and reduces the SmA-SmC transition temperature. All materials have melting temperatures below room temperature, and exhibit high values of induced tilt angles. In Series 2, the materials show a broad SmA temperature range, and melting temperatures much higher than in Series 1. However, the SmA phase is found to supercool to subambient temperatures. If the hydrocarbon chain is shortened, tilt angle, electroclinic coefficient and switching time are significantly suppressed. Comparison of the electrooptic performance in SmA between the two series show that Series 1 show a much higher tilt angles than Series 2, while the later exhibit much faster response times.

We have examined three different methods of endotoxin determination utilizing the Limulus Amebocyte Lysate (LAL) assay to accurately determine endotoxin levels in Liposome Encapsulated Hemoglobin (LEH), (1) the gel-clot method, (2) chromogenic spectroscopic-based LAL, and (3) the turbidimetric method which determines endotoxin levels in solutions based on the time needed to reach a specific degree of turbidity. Both the chromogenic and turbidimetric methods require significant dilution of the LEH preparation before accurate measurement can be made. We have tested the levels of endotoxin in LEH solutions using these methods and measured LEH, liposome, and hemoglobin samples spiked with known amounts of endotoxin. A comparison of the three methods shows that the absolute value of endotoxin measured in LEH by the three methods can vary significantly. However, within any one assay the spiked amount of endotoxin in the sample can be accurately measured. The accuracy of these methods may also be complicated by the binding of endotoxin to LEH. This was evident by mixing free endotoxin with LEH followed by centrifugation to separate the LEH. Biological activity of endotoxin bound to LEH was measured by exposure to RAW264.7 followed by the expression of tumor necrosis factor., Prepared in cooperation with Geo-Centers, Inc.,Fort Washington, Md.