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At least 199 records · Page 11

Serrating Nozzle Surfaces for Complete Transfer of Droplets

A method of ensuring the complete transfer of liquid droplets from nozzles in microfluidic devices to nearby surfaces involves relatively simple geometric modification of the nozzle surfaces. The method is especially applicable to nozzles in print heads and similar devices required to dispense liquid droplets having precise volumes. Examples of such devices include heads for soft printing of ink on paper and heads for depositing droplets of deoxyribonucleic acid (DNA) or protein solutions on glass plates to form microarrays of spots for analysis. The main purpose served by the present method is to ensure that droplets transferred from a nozzle have consistent volume, as needed to ensure accuracy in microarray analysis or consistent appearance of printed text and images. In soft printing, droplets having consistent volume are generated inside a print head, but in the absence of the present method, the consistency is lost in printing because after each printing action (in which a drop is ejected from a nozzle), a small residual volume of liquid remains attached to the nozzle. By providing for complete transfer of droplets (and thus eliminating residual liquid attached to the nozzle) the method ensures consistency of volume of transferred droplets. An additional benefit of elimination of residue is prevention of cross-contamination among different liquids printed through the same nozzle a major consideration in DNA microarray analysis. The method also accelerates the printing process by minimizing the need to clean a printing head to prevent cross-contamination. Soft printing involves a hydrophobic nozzle surface and a hydrophilic print surface. When the two surfaces are brought into proximity such that a droplet in the nozzle makes contact with the print surface, a substantial portion of the droplet becomes transferred to the print surface. Then as the nozzle and the print surface are pulled apart, the droplet is pulled apart and most of the droplet remains on the print surface. The basic principle of the present method is to reduce the liquid-solid surface energy of the nozzle to a level sufficiently below the intrinsic solid-liquid surface energy of the nozzle material so that the droplet is not pulled apart and, instead, the entire droplet volume becomes transferred to the print surface. In this method, the liquid-solid surface energy is reduced by introducing artificial surface roughness in the form of micromachined serrations on the inner nozzle surface (see figure). The method was tested in experiments on soft printing of DNA solutions and of deionized water through 0.5-mm-diameter nozzles, of which some were not serrated, some were partially serrated, and some were fully serrated. In the nozzles without serrations, transfer was incomplete; that is, residual liquids remained in the nozzles after printing. However, in every nozzle in which at least half the inner surface was serrated, complete transfer of droplets to the print surface was achieved.

Kim, Chang-Jin "CJ"↗

Salt-Induced Polymorphs Observed in Colloidal Single Crystals

Polymorphs are solid materials with the same chemical composition but different crystallographic structures. A unique aspect of polymorphs is that they exhibit different physical properties, such as solubility, melting point, density, color, hardness, and bioavailability. Here, we synthesized polymorphs of colloidal crystals engineered with DNA by slow-cooling gold nanoparticle-core programmable atom equivalents (PAEs, particles with DNA sequences that control their bonding characteristics) under salt concentrations ranging from 0.5 to 4 M NaCl. This approach yielded a diverse set of single-crystalline phases with cubic, tetragonal, and hexagonal lattice symmetries. The structural transitions observed here arise solely from the modulation of interparticle repulsion via ionic strength and thermal processing. Notably, in certain cases, we observed diffusionless phase transformations, wherein the superlattices evolve from cubic to lower-symmetry tetragonal lattices. By tuning the thermal stability and salt concentration, we captured intermediate, metastable body-centered tetragonal structures during the slow-cool process, indicating that subtle changes in free energy can direct crystallization to low-symmetry phases. In conclusion, this study demonstrates that thermal and ionic parameters can be tuned to access and stabilize colloidal crystal polymorphs with emergent structures and interesting functional properties.

Colloidal crystallization↗

Rational Design of Nanoplasmonic Array Geometries for Biosensing

Background: Molecular diagnostics provide early and accurate diagnosis, which is essential for the prevention and treatment of infectious as well as chronic diseases. These tests are designed to detect disease-specific bioanalytes such as nucleic acid (DNA or RNA) or protein (antigens, antibodies) biomarkers. In the context of infectious disease diagnosis, nucleic acid-based detection methods are known to provide more specific and sensitive results. Here, the presence of a unique sequence belonging to the pathogenic genomic material is targeted to identify species, organism, genera and/or antimicrobial resistant gene markers. The majority of the common nucleic acid based diagnostic techniques require amplification (polymerase chain reaction, isothermal amplification etc.) of the pathogenic genetic material prior to detection impacting diagnostic speed, complexity, and cost thereby limiting ease of use. Thus, the development of simplified nucleic acid-based diagnostics that can be even used in resource-poor settings may hugely benefit patients across the globe. Nanopath is a molecular diagnostics company utilizing a solid-state nanosensor to enable sequence-specific detection of target nucleic acids without the need of amplification. These nanostructures enable ultra-sensitive biomarker detection using geometric, feature-dependent properties highly dependent on the local dielectric environment, allowing them to be sensitive to low concentration binding events. This paper describes an application of this approach to provide highly relevant clinical information within a single doctor’s office visit. Intro: The Nanopath team is in collaboration with NASA (National Aeronautics and Space Administration) and NIST (National Institute of Standards and Technology) to push the bounds of the fundamental physics associated with their biosensing platform. The ability of metals to support electromagnetic surface waves gives rise to surface plasmons when optically illuminated. This property, and its strong sensitivity to changes in the local refractive index, allows for the use of metal nanoparticles as ultra-sensitive transducers. In prior work by members of this team, ensembles of randomly oriented nanoparticles (i.e., colloidal nanorods dispersed on chip) were employed for sequence-specific nucleic acid sensing (1-3). While these particle sensors have the advantage of rapid fabrication, they suffer from low sensitivity and quality factor due to the random particle dispersity. In contrast, in this study we employ ordered array nanoparticle ensembles which can be used to improve sensor sensitivity and figure-of-merit. Study Methods Overview: In this talk, we detail the results of sensing experiments and computational simulations to outline a rational design of the structure of these plasmonic nanoparticle arrays for biomolecular sensing. Through simulation and experiment, we iteratively tailor nanostructure dimension to provide high quality signal and large resonance shifts upon modeled nucleic acid binding. In particular, full-wave electromagnetic simulations were conducted using Lumerical photonic simulation software in which periodic boundary conditions were applied in the x- and y- dimensions for each of the nanoplasmonic sensor geometries. To simulate the resonance response to changes in the bulk solution in contact with the sensor surface, the refractive index of the surrounding media was changed appropriately. Nucleic acid hybridization events were modeled using either using spherical structures approximating the relevant radius of genomic material as estimated by polymer models, or as conformal layers with the known refractive indices for nucleic acids. On the basis of initial simulations, nanosensors were fabricated using traditional electron-beam lithography protocols at NIST. To evaluate consensus between simulations and experiments, bulk sensing experiments were carried out in which the resonance peaks were obtained by submerging the sensors in refractive index standards. Key nanosensor characteristics including resonance peak locations, resonance peak shifts as a function of refractive index, and figure of merit (FOM) of extinction curves were examined between the experimental and simulation results prior to proceeding with simulations on additional geometries and more complex solution conditions, and further device fabrication. This iterative process is repeated toward a rational design of nanoplasmonic array geometries for biosensing optimizing response for targeted disease detection. In summary, this study puts forth a methodology for rational design and characterization of regularly spaced nanoparticle arrays for optics-based biosensing. The results of this study will allow for more informed design of nanostructure geometries towards sequence-specific nucleic acid detection. These improved designs have the potential to improve clinical sensitivity and limit-of-detection across disease indication.

sensor↗

Microbiological Sampling Methods and Sanitation of Edible Plants Grown on ISS

Pathogenic microbes on the surfaces of salad crops and growth chambers pose a threat to the health of crew on International Space Station. For astronauts to safely consume spacegrown vegetables produced in NASA's new vegetable production unit, VEGGIE, three technical challenges must be overcome: real-time sampling, microbiological analysis, and sanitation. Raphanus sativus cultivar Cherry Bomb II and Latuca sativa cultivar Outredgeous, two saled crops to be grown in VEGGIE, were inoculated with Salmonella enterica serovar Typhimurium (S. Typhimurium), a bacterium known to cause food-borne illness~ Tape- and swab-based sampling techniques were optimized for use in microgravity and assessed for effectiveness in recovery of bacteria from crop surfaces: Rapid pathogen detection and molecular analyses were performed via quantitative real-time polymerase chain reactiop using LightCycler® 480 and RAZOR® EX, a scaled-down instrument that is undergoing evaluation and testing for future flight hardware. These methods were compared with conventional, culture-based methods for the recovery of S. Typhimurium colonies. A sterile wipe saturated with a citric acid-based, food-grade sanitizer was applied to two different surface materials used in VEGGIE flight hardware that had been contaminated with the bacterium Pseudomonas aeruginosa,. another known human pathogen. To sanitize surfaces, wipes were saturated with either the sanitizer or sterile deionized water and applied to each surface. Colony forming units of P. aeruginosa grown on tryptic soy agar plates were enumerated from surface samples after sanitization treatments. Depending on the VEGGIE hardware material, 2- to 4.5-log10 reductions in colony-forming units were observed after sanitization. The difference in recovery of S. Typhimurium between tape- and swab- based sampling techniques was insignificant. RAZOR® EX rapidly detected S. Typhimurium present in both raw culture and extracted DNA samples.

Parrish, Charles H. II↗

Clear as mud redefined: Tunable transparent mineral scaffolds for visualizing microbial processes below ground

Microbes inhabiting complex porous microenvironments in sediments and aquifers catalyze reactions that are critical to global biogeochemical cycles and ecosystem health. However, the opacity and complexity of porous sediment and rock matrices have considerably hindered the study of microbial processes occurring within these habitats. Here, we generated microbially compatible, optically transparent mineral scaffolds to visualize and investigate microbial colonization and activities occurring in these environments, in laboratory settings and in situ. Using inexpensive synthetic cryolite mineral, we produced optically transparent scaffolds mimicking the complex 3D structure of sediments and rocks by adapting a suspension-based, freeze-casting technique commonly used in materials science. Fine-tuning of parameters, such as freezing rate and choice of solvent, provided full control of pore size and architecture. The combined effects of scaffold porosity and structure on the movement of microbe-sized particles, tested using velocity tracking of fluorescent beads, showed diverse yet reproducible behaviors. The scaffolds we produced are compatible with epifluorescence microscopy, allowing the fluorescence-based identification of colonizing microbes by DNA-based staining and fluorescence in situ hybridization (FISH) to depths of 100 µm. Additionally, Raman spectroscopy analysis indicates minimal background signal in regions used for measuring deuterium and 13 C enrichment in microorganisms, highlighting the potential to directly couple D 2 O or 13 C stable isotope probing and Raman-FISH for quantifying microbial activity at the single-cell level. To demonstrate the relevance of cryolite scaffolds for environmental field studies, we visualized their colonization by diverse microorganisms within rhizosphere sediments of a coastal seagrass plant using epifluorescence microscopy. The tool presented here enables highly resolved, spatially explicit, and multimodal investigations into the distribution, activities, and interactions of underground microbes typically obscured within opaque geological materials until now.

36 MATERIALS SCIENCE↗

Molecular Identification of Microbial Contaminants

Microorganisms can have significant impacts on the success of NASA’s missions, including the integrity of materials, the reliability of scientific results, and maintenance of crew health. Robust cleaning and sterilization protocols are currently in place in NASA facilities, but agency experts agree that microbial contamination is unavoidable and its impact on NASA’s missions and science must be minimized. Therefore, it is critical to understand: 1) what specific microorganisms are present, 2) how they may impact scientific objectives, and 3) how to select appropriate mitigation strategies. The Marshall Space Flight Center (MSFC) Planetary Protection (PP) microbiology lab historically relied solely upon enumeration of culturable microbial contamination associated with spacecraft materials or cleanrooms. However, this process is time consuming, many microbes cannot be cultured, and very few can be identified with any fidelity using NASA standard microbiological methods. The work described in this white paper includes the establishment of molecular identification capabilities at MSFC, including DNA isolation, amplification, purification, and Sanger sequencing. This capability will not only improve planetary protection efforts at MSFC (i.e. by identifying contaminating microorganisms in cleanrooms or on spacecraft) but also offers a service center-wide for the identification of contaminants that arise in other projects, processing locations, or during set up and roll out of spacecraft. This work also lays the foundation for higher throughput efforts to identify large populations of microbes across the lifetime of a project and serves as the starting point for future work into whole genome sequencing, non-culture based methods, or additional characterization studies. Ultimately, accurate identification informs appropriate mitigation strategies, increasing the chances of success for NASA’s missions and objectives.

C. D. Cassilly↗

Megafans and Trumpeter Bird Biodiversity-Psophia Phylogeography and Landscape Evolution in Amazonia

Based on geomorphic character and mapped geology, geologists have interpreted the landscape surrounding the Andes Mountains as becoming progressively younger to the East. These sedimentary materials filled the late Miocene swampland that formerly occupied central and western Amazonia. Apart from the ancient landscapes of the Guiana Highlands (top right, figure 1a), Zone Ac is the oldest, followed by Zone Aw, within which megafan Jw is older than megafan Je (figure 1a). DNA-based paleogeography of the trumpeters shows that younger clades diverge from parent lineages with increasing distance from the Andes chain. Thus, Psophia napensis diverges from the P. crepitans parent, and P. ochroptera diverges from P. napensis. The P. ochroptera population is confined solely to the Je megafan (figure 1a). The same trend is seen on the south side of the Amazon depression. Since the timing of the events seems to be of exactly the same order [post-Miocene for the land surfaces and trumpeter divergence within the last 3 million years (figure 1d)], it seems reasonable to think that the megafans provided the substrate on which new bird lineages could speciate. Such physical controls of evolution are becoming more important in the understanding of biodiversity.

Wilkinson, Justin↗

De Novo Synthesis and Functional Study of Primitive Polypeptides in the Prebiotic Protein World

DNA, RNA and proteins within a lipid-bound membrane are the core components of life, but the order of their appearance during the origin of life is still under debate. The widely accepted RNA World hypothesis states that RNA likely emerged prior to proteins and DNA since RNA can serve both replicative and catalytic roles. While biochemists have reproduced the synthesis, polymerization, and replication of nucleotides and RNA under controlled prebiotic conditions, it is clear that such complex organic molecules were are not present in significant amounts in the the starting prebiotic material on Earth either from endogenous production or meteoritic input. In contrast, amino acids are naturally abundant in various prebiotic contexts such as carbonaceous chondrites and Urey-Miller type experiments, and many studies have demonstrated that under plausible prebiotic conditions amino acids could condense or polymerize to give rise to short peptides. These findings support the basis of a Protein World hypothesis for life, however little has been done to study the functions of such primitive peptides. Here we present our novel synthetic biology-based approach to the de novo synthesis of billions of primitive peptidesproteins derived from a limited set of naturally abundant proteinogenic amino acids such as glycine, alanine, aspartic acid, glutamic acid, valine and serine. Of these peptides, the ones with divalent metal-binding capability are of particular interest and will be screened and identified. Certain divalent metals were likely present in prebiotic environments. Not only do they coordinate well with amino acids, but they also catalyze reactions, which are difficult to achieve in organic chemistry. Since D-chiral and non-proteinogenic amino acids are also abundant in the universe and may provide insight into the pathway by which life developed, we will also discuss methods to analyze primitive peptides consisting of these amino acids and D-chiral and non-proteinogenic amino acids. By understanding this naturalistic pathway, we will be able to better understand how life developed here on Earth. Since these amino acids are abundant in universe, this work provide insight into pathways by which life developed on Earth and, by extension, the probability of life arising elsewhere.

Fujishima, Kosuke↗

Making the most of your electrons: Challenges and opportunities in characterizing hybrid interfaces with STEM

Inspired by the unique architectures composed of hard and soft materials in natural and biological systems, synthetic hybrid structures and associated hard-soft interfaces have recently evoked significant interest. Soft matter is typically dominated by structural fluctuations even at room temperature, while hard matter is governed by rigid mechanical behavior. This dichotomy offers considerable opportunities to leverage the disparate properties offered by these components across a wide spectrum spanning from basic science to engineering insights with significant technological overtones. Such hybrid structures, which include polymer nanocomposites, DNA functionalized nanoparticle superlattices, and metal organic frameworks to name a few, have delivered promising insights into the technologically relevant applications such as catalysis, environmental remediation, optoelectronics, and medicine. The interfacial structure between the hard and soft phases demonstrates features across a variety of length scales and often strongly influence the functionality of hybrid systems. While scanning/transmission electron microscopy (S/TEM) has proven to be a valuable tool for acquiring intricate molecular and nanoscale details of these interfaces, the unusual nature of hybrid composites presents a suite of challenges that make assessing or establishing structure–property relationships especially difficult. There are additional considerations at all stages of sample analysis from preparing electron-transparent samples to obtaining sufficient contrast to resolve the interface between dissimilar materials given the dose sensitivity of soft materials. Here, we discuss each of these challenges and supplement a review of recent developments in the field with additional experimental investigations and simulations to present solutions for attaining a nano or molecular-level understanding of these interfaces. These solutions present a host of opportunities for investigating the role interfaces play in this unique class of functional materials.

36 MATERIALS SCIENCE↗

Predictions for Radiation Shielding Materials

Radiation from galactic cosmic rays (GCR) and solar particle events (SPE) is a serious hazard to humans and electronic instruments during space travel, particularly on prolonged missions outside the Earth s magnetic fields. Galactic cosmic radiation (GCR) is composed of approx. 98% nucleons and approx. 2% electrons and positrons. Although cosmic ray heavy ions are 1-2% of the fluence, these energetic heavy nuclei (HZE) contribute 50% of the long-term dose. These unusually high specific ionizations pose a significant health hazard acting as carcinogens and also causing microelectronics damage inside spacecraft and high-flying aircraft. These HZE ions are of concern for radiation protection and radiation shielding technology, because gross rearrangements and mutations and deletions in DNA are expected. Calculations have shown that HZE particles have a strong preference for interaction with light nuclei. The best shield for this radiation would be liquid hydrogen, which is totally impractical. For this reason, hydrogen-containing polymers make the most effective practical shields. Shielding is required during missions in Earth orbit and possibly for frequent flying at high altitude because of the broad GCR spectrum and during a passage into deep space and LunarMars habitation because of the protracted exposure encountered on a long space mission. An additional hazard comes from solar particle events (SPEs) which are mostly energetic protons that can produce heavy ion secondaries as well as neutrons in materials. These events occur at unpredictable times and can deliver a potentially lethal dose within several hours to an unshielded human. Radiation protection for humans requires safety in short-term missions and maintaining career exposure limits within acceptable levels on future long-term exploration missions. The selection of shield materials can alter the protection of humans by an order of magnitude. If improperly selected, shielding materials can actually increase radiation damage due to penetration properties and nuclear fragmentation. Protecting space-borne microelectronics from single event upsets (SEUs) by transmitted radiation will benefit system reliability and system design cost by using optimal shield materials. Long-term missions on the surface of the Moon or Mars will require the construction of habitats to protect humans during their stay. One approach to the construction is to make structural materials from lunar or Martian regolith using a polymeric material as a binder. The hydrogen-containing polymers are considerably more effective for radiation protection than the regolith, but the combination minimizes the amount of polymer to be transported. We have made composites of simulated lunar regolith with two different polymers, LaRC-SI, a high-performance polyimide thermoset, and polyethylene, a thermoplastic.

Kiefer, Richard L.↗

Restoration and Modification of Magnetosome Biosynthesis by Internal Gene Acquisition in a Magnetotactic Bacterium

We report that the Integration of a large-sized DNA fragment into a chromosome is an important strategy for characterization of cellular functions in microorganisms. Magnetotactic bacteria synthesize intracellular organelles comprising membrane-bound single crystalline magnetite, also referred to as magnetosomes. Magnetosomes have gained interest in both scientific and engineering sectors as they can be utilized as a material for biomedical and nanotechnological applications. Although genetic engineering of magnetosome biosynthesis mechanism has been investigated, the current method requires cumbersome gene preparation processes. Here, the chromosomal integration of a plasmid containing ≈27 magnetosome genes (≈26 kbp region) in a non-magnetic mutant of Magnetospirillum magneticum AMB-1 using a broad-host-range plasmid is shown. The genome sequencing of gene-complemented strains reveals the chromosomal integration of the plasmid with magnetosome genes at a specific site, most likely by catalysis of an endogenous transposase. Magnetosome production is successfully enhanced by integrating a variation of magnetosome gene operons in the chromosome. This chromosomal integration mechanism will allow the design of functional magnetosomes de novo and M. magneticum AMB-1 may be used as a chassis for the designed magnetosome production.

59 BASIC BIOLOGICAL SCIENCES↗

Martian Neutron Energy Spectrometer (MANES)

High energy charged particles of extragalactic, galactic, and solar origin collide with spacecraft structures and planetary atmospheres. These primaries create a number of secondary particles inside the structures or on the surfaces of planets to produce a significant radiation environment. This radiation is a threat to long term inhabitants and travelers for interplanetary missions and produces an increased risk of carcinogenesis, central nervous system (CNS) and DNA damage. Charged particles are readily detected; but, neutrons, being electrically neutral, are much more difficult to monitor. These secondary neutrons are reported to contribute 30-60% of the dose equivalent in the Shuttle and MIR station. The Martian atmosphere has an areal density of 37 g/sq cm primarily of carbon dioxide molecules. This shallow atmosphere presents fewer mean free paths to the bombarding cosmic rays and solar particles. The secondary neutrons present at the surface of Mars will have undergone fewer generations of collisions and have higher energies than at sea level on Earth. Albedo neutrons produced by collisions with the Martian surface material will also contribute to the radiation environment. The increased threat of radiation damage to humans on Mars occurs when neutrons of higher mean energy traverse the thin, dry Martian atmosphere and encounter water in the astronaut's body. Water, being hydrogeneous, efficiently moderates the high energy neutrons thereby slowing them as they penetrate deeply into the body. Consequently, greater radiation doses can be deposited in or near critical organs such as the liver or spleen than is the case on Earth. A second significant threat is the possibility of a high energy heavy ion or neutron causing a DNA double strand break in a single strike.

Maurer, R. H.↗

Dynamic self-assembly of compartmentalized DNA nanotubes

Abstract Bottom-up synthetic biology aims to engineer artificial cells capable of responsive behaviors by using a minimal set of molecular components. An important challenge toward this goal is the development of programmable biomaterials that can provide active spatial organization in cell-sized compartments. Here, we demonstrate the dynamic self-assembly of nucleic acid (NA) nanotubes inside water-in-oil droplets. We develop methods to encapsulate and assemble different types of DNA nanotubes from programmable DNA monomers, and demonstrate temporal control of assembly via designed pathways of RNA production and degradation. We examine the dynamic response of encapsulated nanotube assembly and disassembly with the support of statistical analysis of droplet images. Our study provides a toolkit of methods and components to build increasingly complex and functional NA materials to mimic life-like functions in synthetic cells.

59 BASIC BIOLOGICAL SCIENCES↗

Continuous precipitation‐filtration process for initial capture of a monoclonal antibody product using a four‐stage countercurrent hollow fiber membrane washing step

The significant increase in product titers, coupled with the growing focus on continuous bioprocessing, has renewed interest in using precipitation as a low‐cost alternative to Protein A chromatography for the primary capture of monoclonal antibody (mAb) products. In this work, a commercially relevant mAb was purified from clarified cell culture fluid using a tubular flow precipitation reactor with dewatering and washing provided by tangential flow microfiltration. The particle morphology was evaluated using an inline high‐resolution optical probe, providing quantitative data on the particle size distribution throughout the precipitation process. Data were obtained in both a lab‐built 2‐stage countercurrent washing system and a commercial countercurrent contacting skid that provided 4 stages of continuous washing. The processes were operated continuously for 2 h with overall mAb yield of 92 ± 3% and DNA removal of nearly 3 logs in the 4‐stage system. The high DNA clearance was achieved by selective redissolution of the mAb using a low pH acetate buffer. Host cell protein clearance was 0.59 ± 0.08 logs, comparable to that based on model predictions. The process mass intensity was slightly better than typical Protein A processes and could be significantly improved by preconcentration of the antibody feed material.

59 BASIC BIOLOGICAL SCIENCES↗