Binary electrokinetic separation of target DNA from background DNA primers.

This report contains the summary of LDRD project 91312, titled ''Binary Electrokinetic Separation of Target DNA from Background DNA Primers''. This work is the first product of a collaboration with Columbia University and the Northeast BioDefense Center of Excellence. In conjunction with Ian Lipkin's lab, we are developing a technique to reduce false positive events, due to the detection of unhybridized reporter molecules, in a sensitive and multiplexed detection scheme for nucleic acids developed by the Lipkin lab. This is the most significant problem in the operation of their capability. As they are developing the tools for rapidly detecting the entire panel of hemorrhagic fevers this technology will immediately serve an important national need. The goal of this work was to attempt to separate nucleic acid from a preprocessed sample. We demonstrated the preconcentration of kilobase-pair length double-stranded DNA targets, and observed little preconcentration of 60 base-pair length single-stranded DNA probes. These objectives were accomplished in microdevice formats that are compatible with larger detection systems for sample pre-processing. Combined with Columbia's expertise, this technology would enable a unique, fast, and potentially compact method for detecting/identifying genetically-modified organisms and multiplexed rapid nucleic acid identification. Another competing approach is the DARPA funded IRIS Pharmaceutical TIGER platform which requires many hours for operation, and an 800k$ piece of equipment that fills a room. The Columbia/SNL system could provide a result in 30 minutes, at the cost of a few thousand dollars for the platform, and would be the size of a shoebox or smaller

Safety analysis of high pressure 3He-filled micro-channels for thermal neutron detection.

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

Innovative high pressure gas MEM's based neutron detector for ICF and active SNM detection.

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

Quantification of false positive reduction in nucleic acid purification on hemorrhagic fever DNA.

Columbia University has developed a sensitive highly multiplexed system for genetic identification of nucleic acid targets. The primary obstacle to implementing this technology is the high rate of false positives due to high levels of unbound reporters that remain within the system after hybridization. The ability to distinguish between free reporters and reporters bound to targets limits the use of this technology. We previously demonstrated a new electrokinetic method for binary separation of kb pair long DNA molecules and oligonucleotides. The purpose of this project 99864 is to take these previous demonstrations and further develop the technique and hardware for field use. Specifically, our objective was to implement separation in a heterogeneous sample (containing target DNA and background oligo), to perform the separation in a flow-based device, and to develop all of the components necessary for field testing a breadboard prototype system

Novel detection methods for radiation-induced electron-hole pairs.

Most common ionizing radiation detectors typically rely on one of two general methods: collection of charge generated by the radiation, or collection of light produced by recombination of excited species. Substantial efforts have been made to improve the performance of materials used in these types of detectors, e.g. to raise the operating temperature, to improve the energy resolution, timing or tracking ability. However, regardless of the material used, all these detectors are limited in performance by statistical variation in the collection efficiency, for charge or photons. We examine three alternative schemes for detecting ionizing radiation that do not rely on traditional direct collection of the carriers or photons produced by the radiation. The first method detects refractive index changes in a resonator structure. The second looks at alternative means to sense the chemical changes caused by radiation on a scintillator-type material. The final method examines the possibilities of sensing the perturbation caused by radiation on the transmission of a RF transmission line structure. Aspects of the feasibility of each approach are examined and recommendations made for further work

Magnetophoretic bead trapping in a high-flowrate biological detection system.

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

Viral RNA testing and automation on the bead-based CBNE detection microsystem.

We developed prototype chemistry for nucleic acid hybridization on our bead-based diagnostics platform and we established an automatable bead handling protocol capable of 50 part-per-billion (ppb) sensitivity. We are working towards a platform capable of parallel, rapid (10 minute), raw sample testing for orthogonal (in this case nucleic acid and immunoassays) identification of biological (and other) threats in a single sensor microsystem. In this LDRD we developed the nucleic acid chemistry required for nucleic acid hybridization. Our goal is to place a non-cell associated RNA virus (Bovine Viral Diarrhea, BVD) on the beads for raw sample testing. This key pre-requisite to showing orthogonality (nucleic acid measurements can be performed in parallel with immunoassay measurements). Orthogonal detection dramatically reduces false positives. We chose BVD because our collaborators (UC-Davis) can supply samples from persistently infected animals; and because proof-of-concept field testing can be performed with modification of the current technology platform at the UC Davis research station. Since BVD is a cattle-prone disease this research dovetails with earlier immunoassay work on Botulinum toxin simulant testing in raw milk samples. Demonstration of BVD RNA detection expands the repertoire of biological macromolecules that can be adapted to our bead-based detection. The resources of this late start LDRD were adequate to partially demonstrate the conjugation of the beads to the nucleic acids. It was never expected to be adequate for a full live virus test but to motivate that additional investment. In addition, we were able to reduce the LOD (Limit of Detection) for the botulinum toxin stimulant to 50 ppb from the earlier LOD of 1 ppm. A low LOD combined with orthogonal detection provides both low false negatives and low false positives. The logical follow-on steps to this LDRD research are to perform live virus identification as well as concurrent nucleic acid and immunoassay detection

Search for eccentric black hole coalescences during the third observing run of LIGO and Virgo

Despite the growing number of confident binary black hole coalescences observed through gravitational waves so far, the astrophysical origin of these binaries remains uncertain. Orbital eccentricity is one of the clearest tracers of binary formation channels. Identifying binary eccentricity, however, remains challenging due to the limited availability of gravitational waveforms that include effects of eccentricity. Here, we present observational results for a waveform-independent search sensitive to eccentric black hole coalescences, covering the third observing run (O3) of the LIGO and Virgo detectors. We identified no new high-significance candidates beyond those that were already identified with searches focusing on quasi-circular binaries. We determine the sensitivity of our search to high-mass (total mass M>70 M⊙) binaries covering eccentricities up to 0.3 at 15 Hz orbital frequency, and use this to compare model predictions to search results. Assuming all detections are indeed quasi-circular, for our fiducial population model, we place an upper limit for the merger rate density of high-mass binaries with eccentricities 0<e≤0.3 at 0.33 Gpc−3 yr−1 at 90\% confidence level

Ultralight vector dark matter search using data from the KAGRA O3GK run

Among the various candidates for dark matter (DM), ultralight vector DM can be probed by laser interferometric gravitational wave detectors through the measurement of oscillating length changes in the arm cavities. In this context, KAGRA has a unique feature due to differing compositions of its mirrors, enhancing the signal of vector DM in the length change in the auxiliary channels. Here we present the result of a search for U(1)B−L gauge boson DM using the KAGRA data from auxiliary length channels during the first joint observation run together with GEO600. By applying our search pipeline, which takes into account the stochastic nature of ultralight DM, upper bounds on the coupling strength between the U(1)B−L gauge boson and ordinary matter are obtained for a range of DM masses. While our constraints are less stringent than those derived from previous experiments, this study demonstrates the applicability of our method to the lower-mass vector DM search, which is made difficult in this measurement by the short observation time compared to the auto-correlation time scale of DM