The objective of this LDRD was to develop microdevice strategies for dealing with samples to be examined in biological detection systems. This includes three sub-components: namely, microdevice fabrication, sample delivery to the microdevice, and sample processing within the microdevice. The first component of this work focused on utilizing Sandia's surface micromachining technology to fabricate small volume (nanoliter) fluidic systems for processing small quantities of biological samples. The next component was to develop interfaces for the surface-micromachined silicon devices. We partnered with Micronics, a commercial company, to produce fluidic manifolds for sample delivery to our silicon devices. Pressure testing was completed to examine the strength of the bond between the pressure-sensitive adhesive layer and the silicon chip. We are also pursuing several other methods, both in house and external, to develop polymer-based fluidic manifolds for packaging silicon-based microfluidic devices. The second component, sample processing, is divided into two sub-tasks: cell collection and cell lysis. Cell collection was achieved using dielectrophoresis, which employs AC fields to collect cells at energized microelectrodes, while rejecting non-cellular particles. Both live and dead Staph. aureus bacteria have been collected using RF frequency dielectrophoresis. Bacteria have been separated from polystyrene microspheres using frequency-shifting dielectrophoresis. Computational modeling was performed to optimize device separation performance, and to predict particle response to the dielectrophoretic traps. Cell lysis is continuing to be pursued using microactuators to mechanically disrupt cell membranes. Novel thermal actuators, which can generate larger forces than previously tested electrostatic actuators, have been incorporated with and tested with cell lysis devices. Significant cell membrane distortion has been observed, but more experiments need to be conducted to determine the effects of the observed distortion on membrane integrity and cell viability. Finally, we are using a commercial PCR DNA amplification system to determine the limits of detectable sample size, and to examine the amplification of DNA bound to microspheres. Our objective is to use microspheres as capture-and-carry chaperones for small molecules such as DNA and proteins, enabling the capture and concentration of the small molecules using dielectrophoresis. Current tests demonstrated amplification of DNA bound to micron-sized polystyrene microspheres using 20-50 microliter volume size reactions.
This report contains the summary of the 'Magnetophoretic Bead Trapping in a High-Flowrate Biological Detection System' LDRD project 74795. The objective of this project is to develop a novel biodetection system for high-throughput sample analysis. The chief application of this system is in detection of very low concentrations of target molecules from a complex liquid solution containing many different constituents--some of which may interfere with identification of the target molecule. The system is also designed to handle air sampling by using an aerosol system (for instance a WESP - Wet Electro-Static Precipitator, or an impact spray system) to get air sample constituents into the liquid volume. The system described herein automatically takes the raw liquid sample, whether air converted or initially liquid matrix, and mixes in magnetic detector beads that capture the targets of interest and then performs the sample cleanup function, allowing increased sensitivity and eliminating most false positives and false negatives at a downstream detector. The surfaces of the beads can be functionalized in a variety of ways in order to maximize the number of targets to be captured and concentrated. Bacteria and viruses are captured using antibodies to surface proteins on bacterial cell walls or viral particle coats. In combination with a cell lysis or PCR (Polymerase Chain Reaction), the beads can be used as a DNA or RNA probe to capture nucleic acid patterns of interest. The sample cleanup capability of this system would allow different raw biological samples, such as blood or saliva to be analyzed for the presence of different infectious agents (e.g. smallpox or SARS). For future studies, we envision functionalizing bead surfaces to bind to chemical weapons agents, radio-isotopes, and explosives. The two main objectives of this project were to explore methods for enhancing the mixing of the capture microspheres in the sample, and to develop a novel high-throughput magnetic microsphere trap. We have developed a novel technique using the magnetic capture microspheres as 'stirrer bars' in a fluid sample to enhance target binding to the microsphere surfaces. We have also made progress in developing a polymer-MEMS electromagnet for trapping magnetic spheres in a high-flowrate fluid format.
In order to develop the next generation of high peak intensity lasers, new grating technology providing higher damage thresholds and large apertures is required. The current assumption is that this technical innovation will be multilayer dielectric gratings, wherein the uppermost layer of a thin film mirror is etched to create the desired binary phase grating. A variant of this is explored with the upper grating layer being a lower density gelatin-based volume phase grating in either sol-gel or dichromated gelatin. One key benefit is the elimination of the etching step.
Networked systems of low-cost, small, integrable chemical sensors will enable monitoring of Nonproliferation and Materials Control targets and chemical weapons threats. Sandia-designed prototype chemical sensor systems are undergoing extended field testing supported by DOE and other government agencies. A required surety component will be verification of microanalytical system performance, which can be achieved by providing a programmable source of chemical signature(s) for autonomous calibration of analytical systems. In addition, such a controlled chemical source could be used to dispense microaliquots of derivatization reagents, extending the analysis capability of chemical sensors to a wider range of targets. We have developed a microfabricated system for controlled release of selected compounds (calibrants) into the analytical stream of microsensor systems. To minimize pumping and valve requirements of microfluidic systems, and to avoid degradation issues associated with storage of dilute solutions, we have utilized thermally labile organic salts as solid-phase reservoir materials. Reproducible deposition of tetrapropyl ammonium hydroxide onto arrays of microfabricated heating elements can provide a pair of calibration marker compounds (one fast and one slow-eluting compound) for GC analyses. The use of this microaliquot gas source array for hydrogen generation is currently under further development. The goal of the latter effort will be to provide a source of high-pressure, low viscosity GC carrier gas for Sandia's next-generation microfabricated gas-phase chemical analysis systems.
Thermal property measurements of 18650 cells for the Space Shuttle Orbiter Advanced Hydraulic Power System (AHPS, formerly known as EAPU) have been performed. Cells were measured from three commercial manufacturers: E-One MOLI (12 cells), Panasonic (5 cells) and Sanyo (5 cells). Thermal property measurements of the MOLI 18650 cells included measurements of specific heat, self discharge (microcalorimetry), overcharge response and thermal runaway by accelerating rate calorimetry (ARC). The Panasonic and Sanyo cells were measured only for thermal runaway response in the ARC at increasing states of charge (3.8V, 4.0V, 4.2V, 4.3V).
This study describes the technical and economic benefits derived from adding an energy storage component to an existing building cooling, heating, and power system that uses microturbine generation to augment utility-provided power. Three different types of battery energy storage were evaluated: flooded lead-acid, valve-regulated lead-acid, and zinc/bromine. Additionally, the economic advantages of hybrid generation/storage systems were evaluated for a representative range of utility tariffs. The analysis was done using the Distributed Energy Technology Simulator developed for the Energy Storage Systems Program at Sandia National Laboratories by Energetics, Inc. The study was sponsored by the U.S. DOE Energy Storage Systems Program through Sandia National Laboratories and was performed in coordination with the University of Maryland's Center for Environmental Energy Engineering.
To date, all varistors used in ferroelectric neutron generators have been supplied from a single, proprietary source, General Electric Corporate Research and Development (GE CR&D). To protect against the vulnerability of a single source, Sandia initiated a program in the early 1980's to develop a second source for this material. A chemical preparation process for making homogeneous, high purity ZnO-based varistor powder was generated, scaled to production quantities, and transferred to external suppliers. In 1992, the chem-prep varistor program was suspended when it appeared there was sufficient inventory of GE CR&D material to supply ferroelectric neutron generator production for many years. In 1999, neutron generator production schedules increased substantially, resulting in a predicted exhaustion of the existing supply of varistor material within five years. The chem-prep program was restarted in January, 2000. The goals of the program were to (1) duplicate the chem-prep powder synthesis process that had been qualified for WR production, (2) demonstrate sintered billets from the chem-prep powder met requirements, (3) develop a process for rod fabrication and demonstrate that all component specifications could be met, and (4) optimize the process from powder synthesis through component fabrication for full-scale production. The first three of these goals have been met and are discussed in this report. A facility for the fabrication of production quantities of chem-prep powder has been established. All batches since the restart have met compositional requirements, but differences in sintering behavior between the original process and the restarted process were noted. Investigation into the equipment, precipitant stoichiometry, and powder processing procedures were not able to resolve the discrepancies. It was determined that the restarted process, which incorporated Na doping for electrical stability (a process that was not introduced until the end of the initial program and had not been investigated for processing effects), was responsible for the differences. Rod components fabricated since the restart have met requirements and have performed at a level comparable to chem-prep rods from the original program and GE CR&D rods currently in production.
This reports describes the successful extension of artificial immune systems from the domain of computer security to the domain of real time control systems for robotic vehicles. A biologically-inspired computer immune system was added to the control system of two different mobile robots. As an additional layer in a multi-layered approach, the immune system is complementary to traditional error detection and error handling techniques. This can be thought of as biologically-inspired defense in depth. We demonstrated an immune system can be added with very little application developer effort, resulting in little to no performance impact. The methods described here are extensible to any system that processes a sequence of data through a software interface.
The Unstructured Time-Domain ElectroMagnetics (UTDEM) portion of the EMPHASIS suite solves Maxwell's equations using finite-element techniques on unstructured meshes. This document provides user-specific information to facilitate the use of the code for applications of interest.
Semiconductor bridges (SCBs) are commonly used as initiators for explosive and pyrotechnic devices. Their advantages include reduced voltage and energy requirements and exceptional safety features. Moreover, the design of systems which implement SCBs can be expedited using electrical simulation software. Successful use of this software requires that certain parameters be correctly chosen. In this paper, we explain how these parameters can be identified using optimization. We describe the problem focusing on the application of a direct optimization method for its solution, and present some numerical results.
A commercial plasma enhanced chemical vapor deposition (PECVD) technique with planetary substrate rotation was used to apply a thin (200-400 nm thick) conformal diamond-like carbon (DLC) coating (known as a diamond-like nanocomposite (DLN)) on LIGA fabricated Ni-Mn alloy parts. The PECVD technique is known to overcome the drawbacks associated with the line-of-sight nature of physical vapor deposition (PVD) and substrate heating inherent with traditional chemical vapor deposition (CVD). The purpose of the present study is to characterize the coverage, adhesion, and tribological (friction and wear) behavior of DLN coatings applied to planar and sidewall surfaces of small featured LIGA Ni-Mn fabricated parts, e.g. 280 {micro}m thick sidewalls. Friction and wear tests were performed in dry nitrogen, dry air, and air with 50% RH at Hertzian contact pressures ranging from 0.3 to 0.6 GPa. The friction coefficient of bare Ni-Mn alloy was determined to be 0.9. In contrast, low friction coefficients ({approx}0.02 in dry nitrogen and {approx}0.2 in 50% RH air) and minimal amount of wear were exhibited for the DLN coated LIGA Ni-Mn alloy parts and test coupons. This behavior was due to the ability of the coating to transfer to the rubbing counterface providing low interfacial shear at the sliding contact; resultantly, coating one surface was adequate for low friction and wear. In addition, a 30 nm thick titanium bond layer was determined to be necessary for good adhesion of DLN coating to Ni-Mn alloy substrates. Raman spectroscopy and cross-sectional SEM with energy dispersive x-ray analysis revealed that the DLN coatings deposited by the PECVD with planetary substrate rotation covered both the planar and sidewall surfaces of LIGA fabricated parts, as well as narrow holes of 300 {micro}m (0.012 inch) diameter.
We have found that developing a computational framework for reconstructing error control codes for engineered data and ultimately for deciphering genetic regulatory coding sequences is a challenging and uncharted area that will require advances in computational technology for exact solutions. Although exact solutions are desired, computational approaches that yield plausible solutions would be considered sufficient as a proof of concept to the feasibility of reverse engineering error control codes and the possibility of developing a quantitative model for understanding and engineering genetic regulation. Such evidence would help move the idea of reconstructing error control codes for engineered and biological systems from the high risk high payoff realm into the highly probable high payoff domain. Additionally this work will impact biological sensor development and the ability to model and ultimately develop defense mechanisms against bioagents that can be engineered to cause catastrophic damage. Understanding how biological organisms are able to communicate their genetic message efficiently in the presence of noise can improve our current communication protocols, a continuing research interest. Towards this end, project goals include: (1) Develop parameter estimation methods for n for block codes and for n, k, and m for convolutional codes. Use methods to determine error control (EC) code parameters for gene regulatory sequence. (2) Develop an evolutionary computing computational framework for near-optimal solutions to the algebraic code reconstruction problem. Method will be tested on engineered and biological sequences.
The CABle ANAlysis (CABANA) portion of the EMPHASIS suite is designed specifically for the simulation of cable SGEMP. The code can be used to evaluate the response of a specific cable design to threat or to compare and minimize the relative response of difference designs. This document provides user-specific information to facilitate the application of the code to cables of interest.
The ability to detect Weapons of Mass Destruction biological agents rapidly and sensitively is vital to homeland security, spurring development of compact detection systems at Sandia and elsewhere. One such system is Sandia's microseparations-based pChemLab. Many bio-agents are serious health threats even at extremely low concentrations. Therefore, a universal challenge for detection systems is the efficient collection and selective transport of highly diffuse bio-agents against the enormous background of benign particles and species ever present in the ambient environment. We have investigated development of a ''front end'' system for the collection, preconcentration, and selective transport of aerosolized biological agents from dilute (1-10 active particles per liter of air) atmospheric samples, to ultimate concentrations of {approx}20 active particles per microliter of liquid, for interface with microfluidic-based analyses and detection systems. Our approach employs a Sandia-developed aerosol particle-focusing microseparator array to focus size-selected particles into a mating microimpinger array of open microfluidic transport channels. Upon collection (i.e., impingement, submergence, and liquid suspension), microfluidic dielectrophoretic particle concentrators and sorters can be employed to further concentrate and selectively transport bio-agent particles to the sample preparation stages of microfluidic analyses and detection systems. This report documents results in experimental testing, modeling and analysis, component design, and materials fabrication critical to establishing proof-of-principle for this collection ''front end''. Outstanding results have been achieved for the aerodynamic microseparator, and for the post-collection dielectrophoretic concentrator and sorter. Results have been obtained for the microimpinger, too, but issues of particle-trapping by surface tension in liquid surfaces have proven difficult. Subsequent particle submergence into liquid suspension for microfluidic transport has been demonstrated only inefficiently despite significant and varied effort. Importantly, the separate technologies whose development is described, (inertial microseparator, dielectrophoretic corduroy concentrator/sorter) should each, independently, prove greatly useful in a variety of additional applications.
Earth Power Resources, Inc. recently completed a combined rotary/core hole to a depth of 3,813 feet at it's Hot Sulphur Springs Tuscarora Geothermal Power Project Lease Area located 70-miles north of Elko, Nevada. Previous geothermal exploration data were combined with geologic mapping and newly acquired seismic-reflection data to identify a northerly tending horst-graben structure approximately 2,000 feet wide by at least 6,000 feet long with up to 1,700 feet of vertical offset. The well (HSS-2) was successfully drilled through a shallow thick sequence of altered Tertiary Volcanic where previous exploration wells had severe hole-caving problems. The ''tight-hole'' drilling problems were reduced using drilling fluids consisting of Polymer-based mud mixed with 2% Potassium Chloride (KCl) to reduce Smectite-type clay swelling problems. Core from the 330 F fractured geothermal reservoir system at depths of 2,950 feet indicated 30% Smectite type clays existed in a fault-gouge zone where total loss of circulation occurred during coring. Smectite-type clays are not typically expected at temperatures above 300 F. The fracture zone at 2,950 feet exhibited a skin-damage during injection testing suggesting that the drilling fluids may have caused clay swelling and subsequent geothermal reservoir formation damage. The recent well drilling experiences indicate that drilling problems in the shallow clays at Hot Sulphur Springs can be reduced. In addition, average penetration rates through the caprock system can be on the order of 25 to 35 feet per hour. This information has greatly reduced the original estimated well costs that were based on previous exploration drilling efforts. Successful production formation drilling will depend on finding drilling fluids that will not cause formation damage in the Smectite-rich fractured geothermal reservoir system. Information obtained at Hot Sulphur Springs may apply to other geothermal systems developed in volcanic settings.
The AlGaInN material system is used for virtually all advanced solid state lighting and short wavelength optoelectronic devices. Although metal-organic chemical vapor deposition (MOCVD) has proven to be the workhorse deposition technique, several outstanding scientific and technical challenges remain, which hinder progress and keep RD&A costs high. The three most significant MOCVD challenges are: (1) Accurate temperature measurement; (2) Reliable and reproducible p-doping (Mg); and (3) Low dislocation density GaN material. To address challenge (1) we designed and tested (on reactor mockup) a multiwafer, dual wavelength, emissivity-correcting pyrometer (ECP) for AlGaInN MOCVD. This system simultaneously measures the reflectance (at 405 and 550 nm) and emissivity-corrected temperature for each individual wafer, with the platen signal entirely rejected. To address challenge (2) we measured the MgCp{sub 2} + NH{sub 3} adduct condensation phase diagram from 65-115 C, at typical MOCVD concentrations. Results indicate that it requires temperatures of 80-100 C in order to prevent MgCp{sub 2} + NH{sub 3} adduct condensation. Modification and testing of our research reactor will not be complete until FY2005. A new commercial Veeco reactor was installed in early FY2004, and after qualification growth experiments were conducted to improve the GaN quality using a delayed recovery technique, which addresses challenge (3). Using a delayed recovery technique, the dislocation densities determined from x-ray diffraction were reduced from 2 x 10{sup 9} cm{sup -2} to 4 x 10{sup 8} cm{sup -2}. We have also developed a model to simulate reflectance waveforms for GaN growth on sapphire.
Structured adaptive mesh refinement (SAMR) methods are being widely used for computer simulations of various physical phenomena. Parallel implementations potentially offer realistic simulations of complex, three-dimensional applications. But achieving good scalability for large-scale applications is non-trivial. Performance is limited by the partitioners ability to efficiently use the underlying computer's resources. The goal of our research project is to improve scalability for general SAMR applications executing on general parallel computers. We engineer the dynamically adaptive meta-partitioner, able to select and configure the most appropriate partitioning method at run-time, based on system and application state. This presentation gives an overview of our project, reports on recent achievements, and discusses the project's significance in a wider scientific context.
The Container Analysis Fire Environment (CAFE) computer code has been developed to model all relevant fire physics for predicting the thermal response of massive objects engulfed in large fires. It provides realistic fire thermal boundary conditions for use in design of radioactive material packages and in risk-based transportation studies. The CAFE code can be coupled to commercial finite-element codes such as MSC PATRAN/THERMAL and ANSYS. This coupled system of codes can be used to determine the internal thermal response of finite element models of packages to a range of fire environments. This document is a user manual describing how to use the three-dimensional version of CAFE, as well as a description of CAFE input and output parameters. Since this is a user manual, only a brief theoretical description of the equations and physical models is included.
The formulation, implementation and usage of a numerical solution verification code is described. This code uses the Richardson extrapolation procedure to estimate the order of accuracy and error of a computational program solution. It evaluates multiple solutions performed in numerical grid convergence studies to verify a numerical algorithm implementation. Analyses are performed on both structured and unstructured grid codes. Finite volume and finite element discretization programs are examined. Two and three-dimensional solutions are evaluated. Steady state and transient solution analysis capabilities are present in the verification code. Multiple input data bases are accepted. Benchmark options are included to allow for minimal solution validation capability as well as verification.
This report is a white paper summarizing the literature and different approaches to the problem of calibrating computer model parameters in the face of model uncertainty. Model calibration is often formulated as finding the parameters that minimize the squared difference between the model-computed data (the predicted data) and the actual experimental data. This approach does not allow for explicit treatment of uncertainty or error in the model itself: the model is considered the %22true%22 deterministic representation of reality. While this approach does have utility, it is far from an accurate mathematical treatment of the true model calibration problem in which both the computed data and experimental data have error bars. This year, we examined methods to perform calibration accounting for the error in both the computer model and the data, as well as improving our understanding of its meaning for model predictability. We call this approach Calibration under Uncertainty (CUU). This talk presents our current thinking on CUU. We outline some current approaches in the literature, and discuss the Bayesian approach to CUU in detail.
This report presents a modification of a previous model for the statistical distribution of linear antenna impedance. With this modification a simple formula is determined which yields accurate results for all ratios of modal spectral width to spacing. It is shown that the reactance formula approaches the known unit Lorentzian in the lossless limit.
Sandia National Laboratories performs many expensive tests using inertial measurement units (IMUs)--systems that use accelerometers, gyroscopes, and other sensors to measure flight dynamics in three dimensions. For the purpose of this report, the metrics used to evaluate an IMU are cost, size, performance, resolution, upgradeability and testing. The cost of a precision IMU is very high and can cost hundreds of thousands of dollars. Thus the goals and results of this project are as follows: (1) Examine the data flow in an IMU and determine a generic IMU design. (2) Discuss a high cost IMU implementation and its theoretically achievable results. (3) Discuss design modifications that would save money for suited applications. (4) Design and implement a low cost IMU and discuss its theoretically achievable results. (5) Test the low cost IMU and compare theoretical results with empirical results. (6) Construct a more streamlined printed circuit board design reducing noise, increasing capabilities, and constructing a self-contained unit. Using these results, we can compare a high cost IMU versus a low cost IMU using the metrics from above. Further, we can examine and suggest situations where a low cost IMU could be used instead of a high cost IMU for saving cost, size, or both.
The absence of agreed definitions and metrics for supercomputer RAS obscures meaningful discussion of the issues involved and hinders their solution. This paper seeks to foster a common basis for communication about supercomputer RAS, by proposing a system state model, definitions, and measurements. These are modeled after the SEMI-E10 specification which is widely used in the semiconductor manufacturing industry.
Relatively small motion measurement errors manifest themselves principally as a phase error in Synthetic Aperture Radar (SAR) complex data samples, and if large enough become observable as a smearing, blurring, or other degradation in the image. The phase error function can be measured and then deconvolved from the original data to compensate for the presumed motion error, ultimately resulting in a well-focused image. Techniques that do this are termed 'autofocus' algorithms. A very popular autofocus algorithm is the Phase Gradient Autofocus (PGA) algorithm. The nearly universal, and typically reasonable, assumption is that the motion errors are less than the range resolution of the radar, allowing solely a phase correction to suffice. Very large relative motion measurement errors manifest themselves as an unexpected additional shifting or migration of target locations beyond any deterministic migration during the course of the synthetic aperture. Degradation in images from data exhibiting errors of this magnitude are substantial, often rendering the image completely useless. When residual range migration due to either real or apparent motion errors exceeds the range resolution, conventional autofocus algorithms fail. Excessive residual migration is increasingly encountered as resolutions become finer, less expensive inertial sensors are used, and operating ranges become longer (due to atmospheric phenomena). A new migration-correction autofocus algorithm has been developed that estimates the excessive residual migration and applies phase and frequency corrections to properly focus the image. This overcomes the conventional constraint that motion errors not exceed the SAR range resolution.
This report describes an integrated approach for designing communication, sensing, and control systems for mobile distributed systems. Graph theoretic methods are used to analyze the input/output reachability and structural controllability and observability of a decentralized system. Embedded in each network node, this analysis will automatically reconfigure an ad hoc communication network for the sensing and control task at hand. The graph analysis can also be used to create the optimal communication flow control based upon the spatial distribution of the network nodes. Edge coloring algorithms tell us that the minimum number of time slots in a planar network is equal to either the maximum number of adjacent nodes (or degree) of the undirected graph plus some small number. Therefore, the more spread out that the nodes are, the fewer number of time slots are needed for communication, and the smaller the latency between nodes. In a coupled system, this results in a more responsive sensor network and control system. Network protocols are developed to propagate this information, and distributed algorithms are developed to automatically adjust the number of time slots available for communication. These protocols and algorithms must be extremely efficient and only updated as network nodes move. In addition, queuing theory is used to analyze the delay characteristics of Carrier Sense Multiple Access (CSMA) networks. This report documents the analysis, simulation, and implementation of these algorithms performed under this Laboratory Directed Research and Development (LDRD) effort.
The control volume finite element method (CVFEM) was developed to combine the local numerical conservation property of control volume methods with the unstructured grid and generality of finite element methods (FEMs). Most implementations of CVFEM include mass-lumping and upwinding techniques typical of control volume schemes. In this work we compare, via numerical error analysis, CVFEM and FEM utilizing consistent and lumped mass implementations, and stabilized Petrov-Galerkin streamline upwind schemes in the context of advection-diffusion processes. For this type of problem, we find no apparent advantage to the local numerical conservation aspect of CVFEM as compared to FEM. The stabilized schemes improve accuracy and degree of positivity on coarse grids, and also reduce iteration counts for advection-dominated problems.
Electrical contact resistance testing was performed by hot-switching a simulated gold-platinum metal microelectromechanical systems contact. The experimental objective was to determine the sensitivity of the contact resistance degradation to current level and environment. The contact resistance increased sharply after 100 hot-switched cycles in air. Hot-switching at a reduced current and in nitrogen atmosphere curtailed contact resistance degradation by several orders of magnitude. The mechanism responsible for the resistance degradation was found to be arc-induced decomposition of adsorbed surface contaminants.
Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would be useful in developing structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine selective reagents that can cross-link lysine residues to other lysine residues or the amino terminus. Previous work using these cross-linkers has shown that for several proteins of known structure, the number of cross-links that can be obtained experimentally may be small compared to what would be expected from the known structure, due to the relative reactivity, distribution, and solvent accessibility of the lysines in the protein sequence. To overcome these limitations we have investigated the use of cross-linking reagents that can react with other reactive sidechains in proteins. We used 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the carboxylic acid containing residues, aspartic acid (D), glutamic acid (E), and the carboxy terminus (O), for cross-linking reactions. Once activated, the DEO sidechains can react to form 'zero-length' cross-links with nearby primary amine containing resides, lysines (K) and the amino terminus (X), via the formation of a new amide bond. We also show that the EDC-activated DEO sidechains can be cross-linked to each other using dihydrazides, two hydrazide moieties connected by an alkyl cross-linker ann of variable length. Using these reagents, we have found three new 'zero-length' cross-links in ubiquitin consistent with its known structure (M1-E16, M1-E18, and K63-E64). Using the dihydrazide cross-linkers, we have identified 2 new cross-links (D21-D32 and E24-D32) unambiguously. Using a library of dihydrazide cross-linkers with varying arm length, we have shown that there is a minimum arm length required for the DEO-DEO cross-links of 5.8 angstroms. These results show that additional structural information can be obtained by exploiting new cross-linker chemistry, increasing the probability that the protein target of choice will yield sufficient distance constraints to develop a structural model.
Membrane proteins make up a diverse and important subset of proteins for which structural information is limited. In this study, chemical cross-linking and mass spectrometry were used to explore the structure of the G-protein-coupled photoreceptor bovine rhodopsin in the dark-state conformation. All experiments were performed in rod outer segment membranes using amino acid 'handles' in the native protein sequence and thus minimizing perturbations to the native protein structure. Cysteine and lysine residues were covalently cross-linked using commercially available reagents with a range of linker arm lengths. Following chemical digestion of cross-linked protein, cross-linked peptides were identified by accurate mass measurement using liquid chromatography-fourier transform mass spectrometry and an automated data analysis pipeline. Assignments were confirmed and, if necessary, resolved, by tandem MS. The relative reactivity of lysine residues participating in cross-links was evaluated by labeling with NHS-esters. A distinct pattern of cross-link formation within the C-terminal domain, and between loop I and the C-terminal domain, emerged. Theoretical distances based on cross-linking were compared to inter-atomic distances determined from the energy-minimized X-ray crystal structure and Monte Carlo conformational search procedures. In general, the observed cross-links can be explained by re-positioning participating side-chains without significantly altering backbone structure. One exception, between C3 16 and K325, requires backbone motion to bring the reactive atoms into sufficient proximity for cross-linking. Evidence from other studies suggests that residues around K325 for a region of high backbone mobility. These findings show that cross-linking studies can provide insight into the structural dynamics of membrane proteins in their native environment.
Clostridial neurotoxins, such as botulinum and tetanus, are generally thought to invade neural cells through a process of high affinity binding mediated by gangliosides, internalization via endosome formation, and subsequent membrane penetration of the catalytic domain activated by a pH drop in the endosome. This surface recognition and internalization process is still not well understood with regard to what specific membrane features the toxins target, the intermolecular interactions between bound toxins, and the molecular conformational changes that occur as a result of pH lowering. In an effort to elucidate the mechanism of tetanus toxin binding and permeation through the membrane a simple yet representative model was developed that consisted of the ganglioside G{sub tlb} incorporated in a bilayer of cholesterol and DPPC (dipalmitoylphosphatidyl choline). The bilayers were stable over time yet sensitive towards the binding and activity of whole toxin. A liposome leakage study at constant pH as well as with a pH gradient, to mimic the processes of the endosome, was used to elucidate the effect of pH on the toxin's membrane binding and permeation capability. Topographic imaging of the membrane surface, via in situ tapping mode AFM, provided nanoscale characterization of the toxin's binding location and pore formation activity.
This report describes a system level study on the use of a swarm of sea gliders to detect, confirm and kill littoral submarine threats. The report begins with a description of the problem and derives the probability of detecting a constant speed threat without networking. It was concluded that glider motion does little to improve this probability unless the speed of a glider is greater than the speed of the threat. Therefore, before detection, the optimal character for a swarm of gliders is simply to lie in wait for the detection of a threat. The report proceeds by describing the effect of noise on the localization of a threat once initial detection is achieved. This noise is estimated as a function of threat location relative to the glider and is temporally reduced through the use of an information or Kalman filtering. In the next section, the swarm probability of confirming and killing a threat is formulated. Results are compared to a collection of stationary sensors. These results show that once a glider has the ability to move faster than the threat, the performance of the swarm is equal to the performance of a stationary swarm of gliders with confirmation and kill ranges equal to detection range. Moreover, at glider speeds greater than the speed of the threat, swarm performance becomes a weak function of speed. At these speeds swarm performance is dominated by detection range. Therefore, to future enhance swarm performance or to reduce the number of gliders required for a given performance, detection range must be increased. Communications latency is also examined. It was found that relatively large communication delays did little to change swarm performance. Thus gliders may come to the surface and use SATCOMS to effectively communicate in this application.
