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We present a microfluidic immunoassay platform based on the use of linear microretroreflectors embedded in a transparent polymer layer as an optical sensing surface, and micron-sized magnetic particles as light-blocking labels. Retroreflectors return light directly to its source and are highly detectable using inexpensive optics. The analyte is immuno-magnetically pre-concentrated from a sample and then captured on an antibody-modified microfluidic substrate comprised of embedded microretroreflectors, thereby blocking reflected light. Fluidic force discrimination is used to increase specificity of the assay, following which a difference imaging algorithm that can see single 3 μm magnetic particles without optical calibration is used to detect and quantify signal intensity from each sub-array of retroreflectors. We demonstrate the utility of embedded microretroreflectors as a new sensing modality through a proof-of-concept immunoassay for a small, obligate intracellular bacterial pathogen, Rickettsia conorii, the causative agent of Mediterranean Spotted Fever. The combination of large sensing area, optimized surface chemistry and microfluidic protocols, automated image capture and analysis, and high sensitivity of the difference imaging results in a sensitive immunoassay with a limit of detection of roughly 4000 R. conorii per mL.

Check valves are often essential components in microfluidic devices, enabling automated sample processing for diagnostics at the point of care. However, there is an unmet need for a check valve design that is compatible with rigid thermoplastic devices during all stages of development—from initial prototyping with a laser cutter to final production with injection molding. Here, we present simple designs for a passive, normally closed check valve that is manufactured from commonly available materials with a CO2 laser and readily integrated into prototype and production thermoplastic devices. The check valve consists of a thermoplastic planar spring and a soft elastomeric pad that act together to seal against fluid backflow. The valve’s cracking pressure can be tuned by modifying the spring’s planar geometry and thickness. Seal integrity is improved with the addition of a raised annular boss beneath the elastomeric pad. To demonstrate the valve’s usefulness, we employ these valves to create a finger-operated on-chip reagent reservoir and a finger-actuated pneumatic pump. We also apply this check valve to passively seal a device to enable portable detection of RNA from West Nile virus in a laser-cut device.

Digital microfluidics (DMF) is a powerful technique for sample preparation and analysis for a broad range of biological and chemical applications. In many cases, it is desirable to carry out DMF on an open surface, such that the matrix surrounding the droplets is ambient air. However, the utility of the air-matrix DMF format has been severely limited by problems with droplet evaporation, especially when the droplet-based biochemical reactions require high temperatures for long periods of time. We present a simple solution for managing evaporation in air-matrix DMF: just-in-time replenishment of the reaction volume using droplets of solvent. We demonstrate that this solution enables DMF-mediated execution of several different biochemical reactions (RNA fragmentation, first-strand cDNA synthesis, and PCR) over a range of temperatures (4-95 °C) and incubation times (up to 1 h or more) without use of oil, humidifying chambers, or off-chip heating modules. Reaction volumes and temperatures were maintained roughly constant over the course of each experiment, such that the reaction kinetics and products generated by the air-matrix DMF device were comparable to those of conventional benchscale reactions. This simple yet effective solution for evaporation management is an important advance in developing air-matrix DMF for a wide variety of new, high-impact applications, particularly in the biomedical sciences.

Digital microfluidics (DMF) is a powerful technique for simple and precise manipulation of microscale droplets of fluid. This technique enables processing and analysis of a wide variety of samples and reagents and has proven useful in a broad range of chemical, biological, and medical applications. Handling of "real-world" samples has been a challenge, however, because typically their volumes are greater than those easily accommodated by DMF devices and contain analytes of interest at low concentration. To address this challenge, we have developed a novel "world-to-DMF" interface in which an integrated companion module drives the large-volume sample through a 10 μL droplet region on the DMF device, enabling magnet-mediated recovery of bead-bound analytes onto the device as they pass through the region. To demonstrate its utility, we use this system for extraction of RNA from human whole blood lysates (110-380 μL) and further purification in microscale volumes (5-15 μL) on the DMF device itself. Processing by the system was2-fold faster and consumed 12-fold less reagents, yet produced RNA yields and quality fully comparable to conventional preparations and supporting qRT-PCR and RNA-Seq analyses. The world-to-DMF system is designed for flexibility in accommodating different sample types and volumes, as well as for facile integration of additional modules to enable execution of more complex protocols for sample processing and analysis. As the first technology of its kind, this innovation represents an important step forward for DMF, further enhancing its utility for a wide range of applications.

Fission barrier height is one of the least known nuclear parameters, with experimental data, acquired decades ago, existing only close to beta-stability line. Availability of heavy radioactive beams offers possibility to investigate fission of more exotic nuclei and using the state of the art detection technique such as the active target we can even probe their fission barriers heights with precision has not been reached so far. The present status of fission barrier measurement is going to be explained in this paper. We are going to discuss the possibilities to stage experimental studies of fission barrier heights at new generation of ISOL facilities such as HIE-ISOLDE and active target ACTAR TPC. As an example we select the experiment IS581, being prepared for execution at the HIE-ISOLDE facility (CERN).

We have developed an automated quality control (QC) platform for next-generation sequencing (NGS) library characterization by integrating a droplet-based digital microfluidic (DMF) system with a capillary-based reagent delivery unit and a quantitative CE module. Using an in-plane capillary-DMF interface, a prepared sample droplet was actuated into position between the ground electrode and the inlet of the separation capillary to complete the circuit for an electrokinetic injection. Using a DNA ladder as an internal standard, the CE module with a compact LIF detector was capable of detecting dsDNA in the range of 5-100 pg/μL, suitable for the amount of DNA required by the Illumina Genome Analyzer sequencing platform. This DMF-CE platform consumes tenfold less sample volume than the current Agilent BioAnalyzer QC technique, preserving precious sample while providing necessary sensitivity and accuracy for optimal sequencing performance. The ability of this microfluidic system to validate NGS library preparation was demonstrated by examining the effects of limited-cycle PCR amplification on the size distribution and the yield of Illumina-compatible libraries, demonstrating that as few as ten cycles of PCR bias the size distribution of the library toward undesirable larger fragments.

Field-deployable detection technologies in the nation's water supplies have become a high priority in recent years. The unattended water sensor is presented which employs microfluidic chip-based gel electrophoresis for monitoring proteinaceous analytes in a small integrated sensor platform. The instrument collects samples directly from a domestic water flow. The sample is then processed in an automated microfluidic module using in-house designed fittings, microfluidic pumps and valves prior to analysis via Sandia's microChemLab module, which couples chip-based electrophoresis separations with sensitive LIF detection. The system is controlled using LabVIEW software to analyze water samples about every 12 min. The sample preparation, detection and data analysis has all been fully automated. Pressure transducers and a positive control verify correct operation of the system, remotely. A two-color LIF detector with internal standards allows corrections to migration time to account for ambient temperature changes. The initial unattended water sensor prototype is configured to detect protein biotoxins such as ricin as a first step toward a total bioanalysis capability based on protein profiling. The system has undergone significant testing at two water utilities. The design and optimization of the sample preparation train is presented with results from both laboratory and field testing.

Formaggio di Fossa is an Italian traditional cheese of the Montefeltro area (Emilia Romagna and Marche regions) characterized by a particular step of ripening that is carried out into pits (infossamento) borne in the sandstone. Since the XIV century, the inhabitants were used to set food, especially cereals and cheese, into pits to preserve them during winter and to protect them from invaders. The aim of the present work is to study physical and chemical features of this product with particular reference to the presence of the most important biogenic amines ( -Phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermine and spermidine), compared with a control cheese fully ripened in factory. Formaggio di Fossa showed higher values of Aw, pH, humidity, proteins, pH 4,6-soluble nitrogen (NCN) and water soluble nitrogen (NPN) and much lower amounts of fat. Much higher amounts of total biogenic amines were detected in Formaggio di Fossa than in control cheese, where their concentration was very low. Cadaverine, putrescine and tyramine were the most concentrated biogenic amines. Nevertheless, thyramine was present at levels suggested as compatible with GMPs. Histamine was detected at low amounts, far from potentially toxic levels.

The ability to monitor cell signaling events is crucial to the understanding of immune defense against invading pathogens. Conventional analytical techniques such as flow cytometry, microscopy, and Western blot are powerful tools for signaling studies. Nevertheless, each approach is currently stand-alone and limited by multiple time-consuming and labor-intensive steps. In addition, these techniques do not provide correlated signaling information on total intracellular protein abundance and subcellular protein localization. We report on a novel phosphoFlow Chip (pFC) that relies on monolithic microfluidic technology to rapidly conduct signaling studies. The pFC platform integrates cell stimulation and preparation, microscopy, and subsequent flow cytometry. pFC allows host-pathogen phosphoprofiling in 30 min with an order of magnitude reduction in the consumption of reagents. For pFC validation, we monitor the mitogen-activated protein kinases ERK1/2 and p38 in response to Escherichia coli lipopolysaccharide (LPS) stimulation of murine macrophage cells (RAW 264.7). pFC permits ERK1/2 phosphorylation monitoring starting at 5 s after LPS stimulation, with phosphorylation observed at 5 min. In addition, ERK1/2 phosphorylation is correlated with subsequent recruitment into the nucleus, as observed from fluorescence microscopy performed on cells upstream of flow cytometric analysis. The fully integrated cell handling has the added advantage of reduced cell aggregation and cell loss, with no detectable cell activation. The pFC approach is a step toward unified, automated infrastructure for high-throughput systems biology.

We are developing an automated system for the simultaneous, rapid detection of a group of select agents and toxins in the environment. To detect toxins, we modified and automated an antibody-based approach previously developed for manual medical diagnostics that uses fluorescent eTag reporter molecules and is suitable for highly multiplexed assays. Detection is based on two antibodies binding simultaneously to a single antigen, one of which is labeled with biotin while the other is conjugated to a fluorescent eTag through a cleavable linkage. Aqueous samples are incubated with the mixture of antibodies along with streptavidin-coated magnetic beads and a photoactive porphyrin complex. In the presence of antigen, a molecular complex is formed where the cleavable linkage is held in proximity to the photoactive group. Upon excitation at 680 nm, free radicals are generated, which diffuse and cleave the linkage, releasing the eTags. Released eTags are analyzed using capillary gel electrophoresis with laser-induced fluorescence detection. Limits of detection for ovalbumin and botulinum toxoid individually were 4 (or 80 pg) and 16 ng/mL (or 320 pg), respectively, using the manual assay. In addition, we demonstrated the use of pairs of antibodies from different sources in a single assay to decrease the rate of false positives. Automation of the assay was demonstrated in a flow-through format with higher LODs of 32 ng/mL (or 640 ng) each of a mixture of ovalbumin and botulinum toxoid. This versatile assay can be easily modified with the appropriate antibodies to detect a wide range of toxins and other proteins.

For domestic and military security, an autonomous system capable of continuously monitoring for airborne biothreat agents is necessary. At present, no system meets the requirements for size, speed, sensitivity, and selectivity to warn against and lead to the prevention of infection in field settings. We present a fully automated system for the detection of aerosolized bacterial biothreat agents such as Bacillus subtilis (surrogate for Bacillus anthracis) based on protein profiling by chip gel electrophoresis coupled with a microfluidic sample preparation system. Protein profiling has previously been demonstrated to differentiate between bacterial organisms. With the goal of reducing response time, multiple microfluidic component modules, including aerosol collection via a commercially available collector, concentration, thermochemical lysis, size exclusion chromatography, fluorescent labeling, and chip gel electrophoresis were integrated together to create an autonomous collection/sample preparation/analysis system. The cycle time for sample preparation was approximately 5 min, while total cycle time, including chip gel electrophoresis, was approximately 10 min. Sensitivity of the coupled system for the detection of B. subtilis spores was 16 agent-containing particles per liter of air, based on samples that were prepared to simulate those collected by wetted cyclone aerosol collector of approximately 80% efficiency operating for 7 min.

Advances in molecular biology, microfluidics, and laboratory automation continue to expand the accessibility and applicability of these methods beyond the confines of conventional, centralized laboratory facilities and into point of use roles in clinical, military, forensic, and field-deployed applications. As a result, there is a growing need to adapt the unit operations of molecular biology (e.g., aliquoting, centrifuging, mixing, and thermal cycling) to compact, portable, low-power, and automation-ready formats. Here we present one such adaptation, the rotary zone thermal cycler (RZTC), a novel wheel-based device capable of cycling up to four different fixed-temperature blocks into contact with a stationary 4-microliter capillary-bound sample to realize 1-3 second transitions with steady state heater power of less than 10 W. We demonstrate the utility of the RZTC for DNA amplification as part of a highly integrated rotary zone PCR (rzPCR) system that uses low-volume valves and syringe-based fluid handling to automate sample loading and unloading, thermal cycling, and between-run cleaning functionalities in a compact, modular form factor. In addition to characterizing the performance of the RZTC and the efficacy of different online cleaning protocols, we present preliminary results for rapid single-plex PCR, multiplex short tandem repeat (STR) amplification, and second strand cDNA synthesis.

We report the development of a hand-held instrument capable of performing two simultaneous microchip separations (gel and zone electrophoresis), and demonstrate this instrument for the detection of protein biotoxins. Two orthogonal analysis methods are chosen over a single method in order to improve the probability of positive identification of the biotoxin in an unknown mixture. Separations are performed on a single fused-silica wafer containing two separation channels. The chip is housed in a microfluidic manifold that utilizes o-ring sealed fittings to enable facile and reproducible fluidic connection to the chip. Sample is introduced by syringe injection into a septum-sealed port on the device exterior that connects to a sample loop etched onto the chip. Detection of low nanomolar concentrations of fluorescamine-labeled proteins is achieved using a miniaturized laser-induced fluorescence detection module employing two diode lasers, one per separation channel. Independently controlled miniature high-voltage power supplies enable fully programmable electrokinetic sample injection and analysis. As a demonstration of the portability of this instrument, we evaluated its performance in a laboratory field test at the Defence Science and Technology Laboratory with a series of biotoxin variants. The two separation methods cleanly distinguish between members of a biotoxin test set. Analysis of naturally occurring variants of ricin and two closely related staphylococcal enterotoxins indicates the two methods can be used to readily identify ricin in its different forms and can discriminate between two enterotoxin isoforms.

The design, fabrication, and demonstration of a hand-held microchip-based analytical instrument for detection and identification of proteins and other biomolecules are reported. The overall system, referred to as muChemLab, has a modular design that provides for reliability and flexibility and that facilitates rapid assembly, fluid and microchip replacement, troubleshooting, and sample analysis. Components include two independent separation modules that incorporate interchangeable fluid cartridges, a 2-cm-square fused-silica microfluidic chip, and a miniature laser-induced fluorescence detection module. A custom O-ring sealed manifold plate connects chip access ports to a fluids cartridge and a syringe injection port and provides sample introduction and world-to-chip interface. Other novel microfluidic connectors include capillary needle fittings for fluidic connection between septum-sealed fluid reservoirs and the manifold housing the chip, enabling rapid chip priming and fluids replacement. Programmable high-voltage power supplies provide bidirectional currents up to 100 microAlpha at 5000 V, enabling real-time current and voltage monitoring and facilitating troubleshooting and methods development. Laser-induced fluorescence detection allows picomolar (10(-11) M) detection sensitivity of fluorescent dyes and nanomolar sensitivity (10(-9) M) for fluorescamine-labeled proteins. Migration time reproducibility was significantly improved when separations were performed under constant current control (0.5-1%) as compared to constant voltage control (2-8%).

The document is a progress report on a first generation rapid testing microfluidic device with integrated sidewall mirrors being conducted by the Sandia National Laboratories team..

Purpose :The purpose of this study is to examine the relationship between the occurrence of a difficult intubation and (1) the use of neuromuscular blocking agents INMB) and (2) the presence of airway injuries. It is a retrospective analysis of data from a trauma registry. Materials and Methods :Registry records of patients (n = 160) who required emergent endotracheal intubation or establishment of a surgical airway over a 3.5-year period in the emergency department were reviewed. Risk factors for difficult intubations were identified and analyzed using multivariate logistic regression analysis. Results :NMB were used in 75% of patients requiring intubation. Fifteen percent of the intubations were considered difficult. No association was found between the presence of airway injuries and difficult intubations; however, the use of succinylcholine was associated with a lower risk of difficult intubations compared with intubations where a nondepolarizing NMB was used. Conclusions :The use of succinylcholine may result in fewer difficult intubations in the trauma patient than when a nondepolarizing NMB is used. The presence of airway injuries did not appear to predispose to difficult intubations.

Next-generation sequencing (NGS) is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM). The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories.

Optical measurements of particle size and concentration were made at the chromium plating tank and exhaust system at a commercial hexavalent chromium plating facility. Particles were examined at three locations in the exhaust system: 1) directly at the hexavalent chromium plating bath surface, 2) at the exit of a cyclone separator located in the exhaust system approximately three to four meters downstream of the bath, and 3) in the exhaust stack, downstream of the induced draft fan and all abatement devices. Particle diameters at the bath surface ranged from 0.3 to 25 μm. Downstream of the cyclone exit and mesh pad filters, particle top sizes were approximately 5 and 0.7 mm, respectively. On a mass basis, the collection efficiency of all abatement devices was 99.997%. Assuming that droplets in the flow consist primarily of water and chromium, correcting the total particle mass flow against water content gives a chromium emission rate of 64,000 μg/hr, which compares favorably with a value of 77,00...

We have developed an automated quality control (QC) platform for next-generation sequencing (NGS) library characterization by integrating a droplet-based digital microfluidic (DMF) system with a capillary-based reagent delivery unit and a quantitative CE module. Using an in-plane capillary-DMF interface, a prepared sample droplet was actuated into position between the ground electrode and the inlet of the separation capillary to complete the circuit for an electrokinetic injection. Using a DNA ladder as an internal standard, the CE module with a compact LIF detector was capable of detecting dsDNA in the range of 5-100 pg/μL, suitable for the amount of DNA required by the Illumina Genome Analyzer sequencing platform. This DMF-CE platform consumes tenfold less sample volume than the current Agilent BioAnalyzer QC technique, preserving precious sample while providing necessary sensitivity and accuracy for optimal sequencing performance. The ability of this microfluidic system to validate NGS library preparation was demonstrated by examining the effects of limited-cycle PCR amplification on the size distribution and the yield of Illumina-compatible libraries, demonstrating that as few as ten cycles of PCR bias the size distribution of the library toward undesirable larger fragments.

We are developing an instrument to continuously monitor metal aerosol emissions in applications including industrial process vents (e.g., exhaust stacks from electroplating baths), waste treatment processes (incinerators), and boilers and industrial furnaces (coal-fired power plants). The monitoring technique is based on laser spark spectroscopy (LASS; also known as laser-induced breakdown spectroscopy - LIBS), in which a pulsed laser is used to rapidly heat a particle and produce a plasma (or laser ‘spark’). The light emission from the spark is spectrally resolved and analyzed to identify the elemental constituents of the particle and quantify the abundance of the measured species. One feature of LASS is that it can measure atomic species embedded in either solid particles or fine liquid droplets, which account for a large percentage of metal emissions from applications of interest. In the initial work described here, we have focused on the application of the LASS technique for measuring chromium emissions from electroplating baths. This paper describes the approach used for measuring the total chromium concentration in laboratory simulations of electroplating aerosols. Chromium concentrations less than 1 mg/scm can be measured. This work forms the basis for future applications to incineration and fossil power plants.

Next-generation sequencing (NGS) technology is a promising tool for identifying and characterizing unknown pathogens, but its usefulness in time-critical biodefense and public health applications is currently limited by the lack of fast, efficient, and reliable automated DNA sample preparation methods. To address this limitation, we are developing a digital microfluidic (DMF) platform to function as a fluid distribution hub, enabling the integration of multiple subsystem modules into an automated NGS library sample preparation system. A novel capillary interface enables highly repeatable transfer of liquid between the DMF device and the external fluidic modules, allowing both continuous-flow and droplet-based sample manipulations to be performed in one integrated system. Here, we highlight the utility of the DMF hub platform and capillary interface for automating two key operations in the NGS sample preparation workflow. Using an in-line contactless conductivity detector in conjunction with the capillary interface, we demonstrate closed-loop automated fraction collection of target analytes from a continuous-flow sample stream into droplets on the DMF device. Buffer exchange and sample cleanup, the most repeated steps in NGS library preparation, are also demonstrated on the DMF platform using a magnetic bead assay and achieving an average DNA recovery efficiency of 80% ± 4.8%.

Rapid and specific characterization of bacterial endospores is dependent on the ability to rupture the cell wall to enable analysis of the intracellular components. In particular, bacterial spores from the bacillus genus are inherently robust and very difficult to lyze or solubilize. Standard protocols for spore inactivation include chemical treatment, sonication, pressure, and thermal lysis. Although these protocols are effective for the inactivation of these agents, they are less well suited for sample preparation for analysis using proteomic and genomic approaches. To overcome this difficulty, we have designed a simple capillary device to perform thermal lysis of bacterial spores. Using this device, we were able to super heat (195 degrees C) an ethylene glycol lysis buffer to perform rapid flow-through rupture and solubilization of bacterial endospores. We demonstrated that the lysates from this preparation method are compatible with CGE as well as DNA amplification analysis. We further demonstrated the flow-through lysing device could be directly coupled to a miniaturized electrophoresis instrument for integrated sample preparation and analysis. In this arrangement, we were enabled to perform sample lysis, fluorescent dye labeling, and protein electrophoresis analysis of bacterial spores in less than 10 min. The described sample preparation device is rapid, simple, inexpensive, and easily integratable with various microfluidic devices.

In a multiyear research agreement with Tenix Investments Pty. Ltd., Sandia has been developing field deployable technologies for detection of biotoxins in water supply systems. The unattended water sensor or UWS employs microfluidic chip based gel electrophoresis for monitoring biological analytes in a small integrated sensor platform. This instrument collects, prepares, and analyzes water samples in an automated manner. Sample analysis is done using the {mu}ChemLab{trademark} analysis module. This report uses analysis results of two datasets collected using the UWS to estimate performance of the device. The first dataset is made up of samples containing ricin at varying concentrations and is used for assessing instrument response and detection probability. The second dataset is comprised of analyses of water samples collected at a water utility which are used to assess the false positive probability. The analyses of the two sets are used to estimate the Receiver Operating Characteristic or ROC curves for the device at one set of operational and detection algorithm parameters. For these parameters and based on a statistical estimate, the ricin probability of detection is about 0.9 at a concentration of 5 nM for a false positive probability of 1 x 10{sup -6}.

Rapid identification of aerosolized biological agents following an alarm by particle triggering systems is needed to enable response actions that save lives and protect assets. Rapid identifiers must achieve species level specificity, as this is required to distinguish disease-causing organisms (e.g., Bacillus anthracis) from benign neighbors (e.g., Bacillus subtilis). We have developed a rapid (1-5 minute), novel identification methodology that sorts intact organisms from each other and particulates using capillary electrophoresis (CE), and detects using near-infrared (NIR) absorbance and scattering. We have successfully demonstrated CE resolution of Bacillus spores and vegetative bacteria at the species level. To achieve sufficient sensitivity for detection needs ({approx}10{sup 4} cfu/mL for bacteria), we have developed fiber-coupled cavity-enhanced absorbance techniques. Using this method, we have demonstrated {approx}two orders of magnitude greater sensitivity than published results for absorbing dyes, and single particle (spore) detection through primarily scattering effects. Results of the integrated CE-NIR system for spore detection are presented.

We present a rapid method for the identification of viruses using microfluidic chip gel electrophoresis (CGE) of high-copy number proteins to generate unique protein profiles. Viral proteins are solubilized by heating at 95 degrees C in borate buffer containing detergent (5 min), then labeled with fluorescamine dye (10 s), and analyzed using the microChemLab CGE system (5 min). Analyses of closely related T2 and T4 bacteriophage demonstrate sufficient assay sensitivity and peak resolution to distinguish the two phage. CGE analyses of four additional viruses--MS2 bacteriophage, Epstein-Barr, respiratory syncytial, and vaccinia viruses--demonstrate reproducible and visually distinct protein profiles. To evaluate the suitability of the method for unique identification of viruses, we employed a Bayesian classification approach. Using a subset of 126 replicate electropherograms of the six viruses and phage for training purposes, successful classification with non-training data was 66/69 or 95% with no false positives. The classification method is based on a single attribute (elution time), although other attributes such as peak width, peak amplitude, or peak shape could be incorporated and may improve performance further. The encouraging results suggest a rapid and simple way to identify viruses without requiring specialty reagents such as PCR probes and antibodies.

The overarching goal is to develop novel technologies to elucidate molecular mechanisms of the innate immune response in host cells to pathogens such as bacteria and viruses including the mechanisms used by pathogens to subvert/suppress/obfuscate the immune response to cause their harmful effects. Innate immunity is our first line of defense against a pathogenic bacteria or virus. A comprehensive 'system-level' understanding of innate immunity pathways such as toll-like receptor (TLR) pathways is the key to deciphering mechanisms of pathogenesis and can lead to improvements in early diagnosis or developing improved therapeutics. Current methods for studying signaling focus on measurements of a limited number of components in a pathway and hence, fail to provide a systems-level understanding. We have developed a systems biology approach to decipher TLR4 pathways in macrophage cell lines in response to exposure to pathogenic bacteria and their lipopolysaccharide (LPS). Our approach integrates biological reagents, a microfluidic cell handling and analysis platform, high-resolution imaging and computational modeling to provide spatially- and temporally-resolved measurement of TLR-network components. The Integrated microfluidic platform is capable of imaging single cells to obtain dynamic translocation data as well as high-throughput acquisition of quantitative protein expression and phosphorylation information of selected cell populations. The more » platform consists of multiple modules such as single-cell array, cell sorter, and phosphoflow chip to provide confocal imaging, cell sorting, flow cytomtery and phosphorylation assays. The single-cell array module contains fluidic constrictions designed to trap and hold single host cells. Up to 100 single cells can be trapped and monitored for hours, enabling detailed statistically-significant measurements. The module was used to analyze translocation behavior of transcription factor NF-kB in macrophages upon activation by E. coli and Y. pestis LPS. The chip revealed an oscillation pattern in translocation of NF-kB indicating the presence of a negative feedback loop involving IKK. Activation of NF-kB is preceded by phosphorylation of many kinases and to correlate the kinase activity with translocation, we performed flow cytometric assays in the PhosphoChip module. Phopshorylated forms of p38. ERK and RelA were measured in macrophage cells challenged with LPS and showed a dynamic response where phosphorylation increases with time reaching a maximum at {approx}30-60min. To allow further downstream analysis on selected cells, we also implemented an optical-trapping based sorting of cells. This has allowed us to sort macrophages infected with bacteria from uninfected cells with the goal of obtaining data only on the infected (the desired) population. The various microfluidic chip modules and the accessories required to operate them such as pumps, heaters, electronic control and optical detectors are being assembled in a bench-top, semi-automated device. The data generated is being utilized to refine existing TLR pathway model by adding kinetic rate constants and concentration information. The microfluidic platform allows high-resolution imaging as well as quantitative proteomic measurements with high sensitivity (

Towards designing a portable diagnostic device for detecting biological toxins in bodily fluids, we have developed microfluidic chip-based immunoassays that are rapid (20 minutes), require minimal sample volume (10 microL) and have appreciable sensitivity and dynamic range (microM-pM). The microfluidic chip is being integrated with miniaturized electronics, optical elements, fluid-handling components, and data acquisition software to develop a portable, self-contained device. The device is intended for rapid, point-of-care (and, in future, point-of-incident) testing in case of an accidental or intentional exposure/intoxication to biotoxins. Detection of toxins and potential host-response markers is performed using microfluidic electrophoretic immunoassays integrated with sample preconcentration and mixing of analytes with fluorescently labeled antibodies. Preconcentration is enabled by photopolymerizing a thin, nanoporous membrane with a MW cut-off of approximately 10 kDa in the sample loading region of the chip. Polymeric gels with larger pores are located adjacent to the size exclusion membrane to perform electrophoretic separation of antibody-analyte complex and excess antibody. Measurement of the ratio of bound and unbound immune-complex using sensitive laser-induced fluorescence detection provides quantitation of analyte in the sample. We have demonstrated electrophoretic immunoassays for the biotoxins ricin, Shiga toxin I, and Staphylococcal enterotoxin B (SEB). With off-chip mixing and no sample preconcentration, the limits of detection (LOD) were 300 pM for SEB, 500 pM for Shiga toxin I, and 20 nM for ricin. With a 10 min on-chip preconcentration, the LOD for SEB is10 pM. The portable device being developed is readily applicable to detection of proteinaceous biomarkers of many other diseases and is intended to represent the next-generation diagnostic devices capable of rapid and quantitative measurements of multiple analytes simultaneously.

This document is a safety analysis of a novel neutron detection technology developed by Sandia National Laboratories. This technology is comprised of devices with tiny channels containing high pressure {sup 3}He. These devices are further integrated into large scale neutron sensors. Modeling and preliminary device testing indicates that the time required to detect the presence of special nuclear materials may be reduced under optimal conditions by several orders of magnitude using this approach. Also, these devices make efficient use of our {sup 3}He supply by making individual devices more efficient and/or extending the our limited {sup 3}He supply. The safety of these high pressure devices has been a primary concern. We address these safety concerns for a flat panel configuration intended for thermal neutron detection. Ballistic impact tests using 3 g projectiles were performed on devices made from FR4, Silicon, and Parmax materials. In addition to impact testing, operational limits were determined by pressurizing the devices either to failure or until they unacceptably leaked. We found that (1) sympathetic or parasitic failure does not occur in pressurized FR4 devices (2) the Si devices exhibited benign brittle failure (sympathetic failure under pressure was not tested) and (3) the Parmax devices failed unacceptably. FR4 devices were filled to pressures up to 4000 + 100 psig, and the impacts were captured using a high speed camera. The brittle Si devices shattered, but were completely contained when wrapped in thin tape, while the ductile FR4 devices deformed only. Even at 4000 psi the energy density of the compressed gas appears to be insignificant compared to the impact caused by the incoming projectile. In conclusion, the current FR4 device design pressurized up to 4000 psi does not show evidence of sympathetic failure, and these devices are intrinsically safe.

An innovative helium3 high pressure gas detection system, made possible by utilizing Sandia's expertise in Micro-electrical Mechanical fluidic systems, is proposed which appears to have many beneficial performance characteristics with regards to making these neutron measurements in the high bremsstrahlung and electrical noise environments found in High Energy Density Physics experiments and especially on the very high noise environment generated on the fast pulsed power experiments performed here at Sandia. This same system may dramatically improve active WMD and contraband detection as well when employed with ultrafast (10-50 ns) pulsed neutron sources.

A single substrate intended for a 3-dimensional (3D) edge-cooled multichip module (MCM) has been built and thermally tested. The substrate, with dimensions 1.9 in. by 2 in., is mounted in a fluid cooled block at one end. To test this cooling architecture and verify the accuracy of thermal models, we constructed thermal test modules using alumina (Al/sub 2/O/sub 3/), aluminum nitride (AIN), and CVD diamond substrate materials. Each module was populated with an array of 16 Sandia ATC03 test chips with resistive heaters and temperature sensing diode thermometers. Comparative measurements of the 3 substrates were made in which the top row of 4 die were heated at 5 W each for a total of 20 W. The maximum temperature differences between the heated die and the interface with the cold chuck, /spl Delta/T/sub JS/, were 24, 126, and 265/spl deg/C for diamond, AIN and Al/sub 2/O/sub 3/, respectively. Measurements on the diamond thermal test module, uniformly heated at a total power of 45 W, gave a measured substrate-to-sink temperature of /spl Delta/T /spl AP/ 20/spl deg/C. An extrapolation of our experimental data indicates that the diamond edge-cooled substrate could dissipate a total power /spl AP/ 192 W for a maximum junction-to-ambient temperature of /spl Delta/T/sub JA/ /spl AP/ 124/spl deg/C. If multiple substrates were 3 mounted in the fluid cooled block, spaced 0.075 in. apart, the volumetric power density would be about 850 W/in/sup 3/.

This paper presents the first systematic engineering study of the impact of chemical formulation and surface functionalization on the performace of free-standing microfluidic polymer elements used for high-pressure fluid control in glass microsystems. System design, chemical wet-etch processes, and laser-induced polymerization techniques are described, and parametric studies illustrate the effects of polymer formulation, glass surface modification, and geometric constraints on system performance parameters. In particular, this study shows that highly crosslinked and fluorinated polymers can overcome deficiencies in previously-reported microvalve architectures, particularly limited solvent compatibility. Substrate surface modification is shown effective in reducing the friction of the polymer-glass interface and thereby facilitating valve actuation. A microchip one-way valve constructed using this architecture shows a 2 x 10(8) ratio of forward and backward flow rates at 7 MPa. This valve architecture is integrated on chip with minimal dead volumes (70 pl), and should be applicable to systems (including chromatography and chemical synthesis devices) requiring high pressures and solvents of varying polarity.

Rapid, automated sample preparation of bacterial cells and spores is required for threat analysis by remotely deployed chemical and biological warning systems. Sandia is designing, building, and testing an automated front-end sample preparation system based on miniature and microfluidic components, with the goal of concentrating bacterial species collected from the air, harvesting and solubilizing proteins from them, and delivering them to Sandia’s MicroChemLab capillary gel electrophoresis system1,2 for analysis (Fig. 1). Miniature, motorized valves and pumps control flow between system components connected by fused silica capillaries (Fig. 4). Sample processing modules include concentration by dielectrophoresis in an array of insulating posts or by mechanical filtration; heat-activated chemical lysis; mechanical filtration; removal of chemical lysis agents by size exclusion chromatography (SEC); and in-capillary fluorescent labeling.

Rapid identification of viral and bacterial species is dependent of the ability to manipulate biological agents into a form where they are directly analyzed. Many of these species of interest, such as bacterial spores, are inherently hearty and very difficult to lyse or solubilize. Standard protocols for spore inactivation include, chemical treatment, sonication, pressure and thermal lysis. While these protocols are effective for the inactivation of these agents they are less well suited for sample preparation for analysis using capillary electrophoresis techniques. In order to overcome this difficulty we fabricated a simple capillary device to perform thermal lysis of vegatative bacterial cells and bacterial spores. Using an ethylene glycol buffer to super heat bacterial spores we were able to perform rapid flow through lysis and solubilzation of these agents. This device was then coupled to a sample preparation station for on-line fluorescamine dye lableling and buffer exchange for direct analysis using a miniaturized capillary electrophoresis instrument. Using this integrated device were we enabled to perform sample lysis, labeling and protein fingerprint analysis of vegatative bacterial cells, bacterial spores and viruses in less than 10 minutes. The described device is simple, inexpensive and easily integratable with various microfluidic devices.

Photorefractive screening-photovoltaic solitons are observed in lithium niobate. Two-dimensional bright circular solitons are formed thanks to a strong static bias field, externally applied, opposite to the photovoltaic internal field. The dynamics of the soliton formation is monitored and compared to a time-dependent numerical model allowing determination of the photovoltaic field. Efficient single mode waveguides are shown to be memorized by the soliton beam for a long time.

The μChemLab™ program is developing hand-portable systems for detecting a broad range of chemical, biological, and viral agents in both gas and liquid samples. The μChem Lab liquid sample analyzer employs electrokinetic sample injection, chip-based electrophoretic microseparations and laser-induced florescence detection to analyze liquid samples. A second-generation liquid phase prototype is described. The device incorporates improvements from technological advances and applied research experience. New features include a modular design that readily accommodates on-chip preconcentration and additional separation techniques. The redesign reduces hardware failures, minimizes downtime during component replacement, improves usability, and provides increased sensitivity. Improvements have been made without compromising previous system performance.

We recently tested µChemLab™ /CB, a hand-portable detection system developed for protein biotoxins, with biotoxin isoforms at Defense Science and Technology Laboratory (Dstl). We found the device is capable of detecting and discriminating various toxins and toxin isoforms.