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DOE OSTI2019

Optimization of uranium and plutonium separations using TEVA and UTEVA cartridges for MC-ICP-MS analysis of environmental swipe samples

We report that the analysis of environmental swipe samples for ultra-trace uranium (U) and plutonium (Pu) determinations is essential in the nuclear safeguards community. While mass spectrometry techniques for U and Pu detection continually improve, established separation methods are seldom reevaluated. Currently, actinide separations within the forensics community predominantly employ either Eichrom TEVA® or UTEVA® resins. The direct optimization of U and Pu separations utilizing both resins has not been widely reported. Here, several methods were explored with goals of increasing analyte recovery, acquiring cleaner blanks, and improving the separation efficiency of ultra-trace levels of U and Pu from environmental swipe samples. Finally, the optimized separation methodology of U and Pu was examined using certified reference materials and archived environmental swipe samples.

98 NUCLEAR DISARMAMENT, SAFEGUARDS, AND PHYSICAL P↗

DOE OSTI2018

Automated Separation of Uranium and Plutonium from Environmental Swipe Samples for Multiple Collector Inductively Coupled Plasma Mass Spectrometry

A fully automated method for the separation of low-concentration uranium from plutonium in environmental swipe samples has been developed. The offline chromatography system features renewable 1 mL Eichrom TEVA and UTEVA column generation from bulk resin slurry. Discrete fractions of the separated actinides are delivered into user defined vials for future analysis. Clean room background levels were achieved outside of a cleanroom environment with this method. Purification of uranium and plutonium from various sample matrixes and at various concentrations was successful. Major and minor isotope ratios for both elements were measured via multiple collector inductively coupled plasma mass spectrometry and were in good agreement with certified reference values. Lastly, validation of the separation method was conducted on archived environmental samples and agreed with values previously reported using standard column chemistry.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2016

Oak ridge national laboratory automated clean chemistry for bulk analysis of environmental swipe samples

To shorten the lengthy and costly manual chemical purification procedures, sample preparation methods for mass spectrometry are being automated using commercial-off-the-shelf (COTS) equipment. This addresses a serious need in the nuclear safeguards community to debottleneck the separation of U and Pu in environmental samples—currently performed by overburdened chemists—with a method that allows unattended, overnight operation. In collaboration with Elemental Scientific Inc., the prepFAST-MC2 was designed based on current COTS equipment that was modified for U/Pu separations utilizing Eichrom™ TEVA and UTEVA resins. Initial verification of individual columns yielded small elution volumes with consistent elution profiles and good recovery. Combined column calibration demonstrated ample separation without crosscontamination of the eluent. Automated packing and unpacking of the built-in columns initially showed >15% deviation in resin loading by weight, which can lead to inconsistent separations. Optimization of the packing and unpacking methods led to a reduction in the variability of the packed resin to less than 5% daily. The reproducibility of the automated system was tested with samples containing 30 ng U and 15 pg Pu, which were separated in a series with alternating reagent blanks. These experiments showed very good washout of both the resin and the sample from the columns as evidenced by low blank values. Analysis of the major and minor isotope ratios for U and Pu provided values well within data quality limits for the International Atomic Energy Agency. Additionally, system process blanks spiked with 233U and 244Pu tracers were separated using the automated system after it was moved outside of a clean room and yielded levels equivalent to clean room blanks, confirming that the system can produce high quality results without the need for expensive clean room infrastructure. Comparison of the amount of personnel time necessary for successful manual vs. automated chemical separations showed a significant decrease in hands-on time from 9.8 hours to 35 minutes for seven samples, respectively. This documented time savings and reduced labor translates to a significant cost savings per sample. Overall, the system will enable faster sample reporting times at reduced costs by limiting personnel hours dedicated to the chemical separation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2020

Sampling and Analysis Plan for Investigation of Undeclared Nuclear Activities - 20104

The nuclear-centered energy policy in South Korea is moving towards a nuclear-free era. It also facilitates an action or progress on the denuclearization of the Korean peninsula. The Nuclear Safety and Security Commission (NSSC) launched a new R and D project which attempts to verify nuclear activities via analyzing environmental samples. Environmental samples possibly contain significant radiological information relevant to past undeclared activities. In order to precisely verify them, it is essential to provide 1) the concrete information of target facilities, 2) appropriate methodologies and procedures, and 3) well-suited instrumentation and equipment corresponding to sample signatures. It is also important to provide scientific reliability or validity in any process such as sample collection, handling, transportation and laboratory analysis. Therefore, the information of nuclear facilities (fuel fabrication plant, graphite moderated reactor, reprocessing plant) in North Korea are reviewed in order to determine parameters for appropriate sampling methods representing a facility's characteristics. Secondly, a sampling procedure for radioactive elements is being developed based on international and domestic standards such as ASTM (American Society for Testing and Materials), KS (Korean industrial Standards), and International Atomic Energy Agency (IAEA) manuals. The procedure contains some specific information including sampling process, sampling tools, amount of sample, sample identification, and other related information. Developing the qualified national guidelines is most important since sampling and analyzing results can be used as evidences of nuclear activities. The guideline developed through this work will clearly define various methodologies such as document verification, sampling procedures, collection methods, sample storage criteria, chain of custody, and transportation conditions. The final objective of this work is to establish the infrastructure and regulatory framework for assessment of nuclear activities, and provide a national guideline for sampling and analysis. This will bring confidence in the results and play a critical role in complete denuclearization progress in South Korea. (authors)

98 NUCLEAR DISARMAMENT, SAFEGUARDS, AND PHYSICAL P↗

DOE OSTI2018

Automated Clean Chemistry for Bulk Analysis of Environmental Swipe Samples – Final Report

Sample preparation methods for mass spectrometry are being automated using commercial-off-the-shelf (COTS) equipment to shorten lengthy and costly manual chemical purification procedures. This development addresses a serious need in the International Atomic Energy Agency’s (IAEA) Network of Analytical Laboratories (NWAL) to increase efficiency in the Bulk Analysis of Environmental Samples for Safeguards program with a method that allows unattended, overnight operation. In collaboration with Elemental Scientific Inc. (ESI), the prepFAST-MC2 was designed based on COTS equipment. It was modified for uranium/plutonium separations using renewable columns packed with Eichrom TEVA® and UTEVA® resins, with a chemical separation method based on the Oak Ridge National Laboratory (ORNL) NWAL chemical procedure. Initial verification of individual columns yielded small elution volumes with consistent elution profiles and good recovery. Combined column calibration demonstrated ample separation without crosscontamination of the eluent. Automated packing and unpacking of the built-in columns initially showed >15% deviation in resin loading by weight, which can lead to inconsistent separations. Optimization of the packing and unpacking methods led to a reduction in the variability of the packed resin to less than 5% daily. Based on the initial experience at ORNL with the prepFAST-MC2, a second-generation model, the prepFAST-SR, was built by ESI. The newly designed prepFAST-SR has several upgrades compared with the original prepFAST-MC2. The upgrades largely addressed issues with the reliability of unpacking the Eichrom columns and with ventilation to ensure adequate removal of acid vapors. Both systems are currently installed in the Ultra-Trace Forensics Science Center at ORNL. Separations of mixed uranium and plutonium samples containing certified reference materials were analyzed by multi-collector inductively coupled plasma mass spectrometry and yielded good results. Current efforts have demonstrated a wider applicability of the prepFAST system. Near-quantitative removal of metal interferences was achieved with the system in both uranium and plutonium separated fractions. Successful system validation was completed with several archived samples. Isotopic results from archived samples and certified reference materials were well within data quality limits for the IAEA NWAL. Additional COTS equipment has been evaluated for its potential to aid the prepFAST-SR system in reducing the time allotments and cleanroom infrastructure requirements for accurate separations. Overall, these efforts help ensure continued efficient and effective operation of the NWAL. Comparison of the amount of personnel time necessary for successful manual vs. automated chemical separations showed a significant decrease in hands-on time from 9.8 hours to 35 minutes for seven samples, respectively. This documented time savings and reduced labor translates to a significant cost savings per sample. Overall, the system will enable faster sample reporting times at reduced cost by limiting personnel hours dedicated to the chemical separation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2017

Automated Clean Chemistry for Bulk Analysis of Environmental Swipe Samples - FY17 Year End Report

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

NASA NTRS2002

Lipid Biomarkers for Methanogens in Hypersaline Cyanobacterial Mats for Guerrero Negro, Baja California Sur

Analyses of sediments from the vicinity of active methane seeps have uncovered a particular suite of lipid biomarker patterns that characterize methane consuming archaea and their syntrophic, sulfate reducing partners. These isoprenoid biomarkers, largely identified by their anomalously light carbon isotopic signatures, have been a topic of intense research activity and are recorded in numerous methane-rich environments from Holocene to Cenozoic. This phenomenon has implications for depleted kerogens at 2.7 Ga on early Earth (Hinrichs 2002). In contrast, the lipid biosignatures of methane producing archaea are not readily identified through distinct isotopic labels and have received comparably little attention in analyses of archaea in environmental samples. Indeed, environmental analyses generally detect only free archaeal lipids, not the intact, polar molecules found in the membrane of living organisms. As part of the Ames NAI, the 'Early Microbial Ecosystem Research Group' (EMERG) is working to understand microbial processes in the hypersaline cyanobacterial mats growing in the salt evaporation ponds of the Exportadora de Sal at Guerrero Negro, Baja California Sur, Mexico. The aim of this study was to develop methods by which we could identify the organisms responsible for methane generation in this environment. While the ester-bound fatty acids, hopanoids and wax esters provide a means to identify most of the bacterial components of these mats, the archaea which Ere evidently present through genomic assays and the fact of intense methane production (Hoehler et al. 200l), have not been identified through their corresponding lipid signatures. Archaeal core lipids present a number of analytical challenges. The core lipids of methanogens comprise C20, C40 and sometimes C25 isoprenoid chains, linked through ether bonds to glycerol. As well as archaeal (C20), sn-2- and sn-3-hydroxyarchaeol are associated particularly with methylotrophic methanogens. Recently, we have also identified a dihydroxyarchaeol in a hyperthermophilic methanogen (Summons et al. 2002). Additional structural diversity is encoded into the polar head groups that are attached to the glycerol ether cores. The C20 core lipids are readily analyzed by GC-MS as their volatile trimethylsilyl derivatives while compounds with intact polar head groups can only be detected using LC-MS approaches. Our approach was to utilize the alternative of an ether cleavage reagent (BBr3 vs. HI) and a hydride reducing agent to convert all ether lipids to hydrocarbon in order to provide a vertical profile of quantitative information that might be matched to methane fluxes. We have found that while conventional acid hydrolysis and HI treatment will destroy hydroxyarchaeols, molecular information remains intact through use of BBr3 for ether cleavage. This method revealed the presence of traces of biphytane and various ether alkyls associated with some sulfate reducing bacteria within the mat structure. An interesting, and potentially valuable, byproduct of the method utilizing HI was the identification of abundant homohopanoids after superhydride reduction. Evidently present as sulfur-bound diagenetic products these hopanoids are likely cyanobacterial biomarkers in the early stages of diagenetic preservation.

Jahnke, Linda L.↗

NASA NTRS2012

Rugged, Portable, Real-Time Optical Gaseous Analyzer for Hydrogen Fluoride

Hydrogen fluoride (HF) is a primary evolved combustion product of fluorinated and perfluorinated hydrocarbons. HF is produced during combustion by the presence of impurities and hydrogen- containing polymers including polyimides. This effect is especially dangerous in closed occupied volumes like spacecraft and submarines. In these systems, combinations of perfluorinated hydrocarbons and polyimides are used for insulating wiring. HF is both highly toxic and short-lived in closed environments due to its reactivity. The high reactivity also makes HF sampling problematic. An infrared optical sensor can detect promptly evolving HF with minimal sampling requirements, while providing both high sensitivity and high specificity. A rugged optical path length enhancement architecture enables both high HF sensitivity and rapid environmental sampling with minimal gaseous contact with the low-reactivity sensor surfaces. The inert optical sample cell, combined with infrared semiconductor lasers, is joined with an analog and digital electronic control architecture that allows for ruggedness and compactness. The combination provides both portability and battery operation on a simple camcorder battery for up to eight hours. Optical detection of gaseous HF is confounded by the need for rapid sampling with minimal contact between the sensor and the environmental sample. A sensor is required that must simultaneously provide the required sub-parts-permillion detection limits, but with the high specificity and selectivity expected of optical absorption techniques. It should also be rugged and compact for compatibility with operation onboard spacecraft and submarines. A new optical cell has been developed for which environmental sampling is accomplished by simply traversing the few mm-thick cell walls into an open volume where the measurement is made. A small, low-power fan or vacuum pump may be used to push or pull the gaseous sample into the sample volume for a response time of a few seconds. The optical cell simultaneously provides for an enhanced optical interaction path length between the environmental sample and the infrared laser. Further, the optical cell itself is comprised of inert materials that render it immune to attack by HF. In some cases, the sensor may be configured so that the optoelectronic devices themselves are protected and isolated from HF by the optical cell. The optical sample cell is combined with custom-developed analog and digital control electronics that provide rugged, compact operation on a platform that can run on a camcorder battery. The sensor is inert with respect to acidic gases like HF, while providing the required sensitivity, selectivity, and response time. Certain types of combustion events evolve copious amounts of HF, very little of other gases typically associated with combustion (e.g., carbon monoxide), and very low levels of aerosols and particulates (which confound traditional smoke detectors). The new sensor platform could warn occupants early enough to take the necessary countermeasures.

Pilgrim, Jeffrey↗

DOE OSTI2011

Concepts for Environmental Radioactive Air Sampling and Monitoring

Environmental radioactive air sampling and monitoring is becoming increasingly important as regulatory agencies promulgate requirements for the measurement and quantification of radioactive contaminants. While researchers add to the growing body of knowledge in this area, events such as earthquakes and tsunamis demonstrate how nuclear systems can be compromised. The result is the need for adequate environmental monitoring to assure the public of their safety and to assist emergency workers in their response. Two forms of radioactive air monitoring include direct effluent measurements and environmental surveillance. This chapter presents basic concepts for direct effluent sampling and environmental surveillance of radioactive air emissions, including information on establishing the basis for sampling and/or monitoring, criteria for sampling media and sample analysis, reporting and compliance, and continual improvement.

emissions↗

DOE OSTI2023

Building 151 NNMA Clean Laboratory Project Soil Sampling and Analysis Plan

This Soil Sampling and Analysis Plan (SAP) was prepared by the Environmental Function Area (EFA)/Technical Services Department (TSD) of the Environment, Safety & Health (ES&H) Directorate for the Project Management Office (PMO) for the proposed Building 151 NNMA Clean Laboratory Project (project) located immediately south of Building 154. The purpose of the SAP was to describe the procedures for collection and analysis of environmental samples and evaluation of analytical data (chemical and radiological) to determine management options of excavated soil during project construction in accordance with Lawrence Livermore National Laboratory’s (LLNL) Soils Screening and Management Plan (SSMP) (LLNL, 2022), which is consistent with U.S. Environmental Protection Agency (EPA) guidance for developing Data Quality Objectives for environmental data (EPA, 2006). The SAP was developed in accordance with the SSMP and based on preliminary design information provided to EFA by PMO. In addition to environmental samples, geotechnical samples will be collected by Consolidated Engineering Laboratories (CEL) on behalf of PMO. Geotechnical samples will be collected from a dedicated geotechnical boring location or co-located with environmental samples. Collection of geotechnical samples is determined by CEL and is therefore not discussed in this SAP.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2013

Ultra-trace determination of Strontium-90 in environmental soil samples from Qatar by collision/reaction cell-inductively coupled plasma mass spectrometry (CRC-ICP-MS/MS)

Because of the very low level of {sup 90}Sr in the environmental soil samples and its determination by beta counting may take several weeks, we developed a procedure for ultra-trace determination of {sup 90}Sr using collision reaction cell-inductively coupled plasma tandem mass spectrometry (CRC-ICP-MS/MS, Agilent 8800). Soil samples were dried at 105 deg. C and then heated in a furnace at 550 deg. C to remove any organics present. 500 g of each soil samples were aliquoted into 2000 ml glass beakers. Each Soils samples were soaked in 2 ppm Sr solution carrier to allow determination of chemical yield. The solid to liquid ratio was 1:1. Finally the soil samples were dried at 105 deg. C. Five hundred milliliters concentrated nitric acid and 250 ml hydrochloric acid volumes were added on 500 g soil samples. The samples were digested on hot plate at 80 deg. C to prevent spraying with continuous manual mixing. The leachate solution was separated. The solids were rinsed with 500 ml deionized water, warmed on a hot plate and the leachate plus previous leachate were filtered and the total volume was reduced to 500 ml by evaporation. Final leachate volume was transferred to a centrifuge tubes. The centrifuge tubes were centrifuged at 3,500 rpm for 10 min. The leachate was transferred to a 1 L beaker and heated on a hot plate to evaporate the leachate to dryness. The reside was re-dissolved in 100 ml of 2% HNO{sub 3} and reduced by evaporation to 10 mL. The solution was measured directly by CRC-ICP-MS/MS by setting the first quadruple analyzer to m/z 90 and introducing oxygen gas into the reaction cell for elimination isobar interference from zirconium-90. The method was validated by measurements of standard reference materials and applied on environmental soil samples. The overall time requirement for the measurement of strontium-90 by CRC-ICP-MS/MS is 2 days, significantly shorter than any radioanalytical protocol currently available. (authors)

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

DOE OSTI2023

U328 Electrical Reliability Project Soil Sampling and Analysis Plan

Lawrence Livermore National Laboratory's (LLNL’s) Project Management Office (PMO) is planning to install an underground electrical duct bank at the 3200 block of the Livermore Site (project). The proposed project will include a new electrical substation and feeders, demolition and replacement of the U328C utility building with a new structure to the west, re-routing of utilities, and update of the parking lot. This Sampling and Analysis Plan (SAP) outlines the procedures to collect and evaluate environmental samples for disposition of the excavated soil during the project and geotechnical samples for pavement design. This SAP has been prepared and is organized to be consistent with LLNL's Soils Screening and Management Plan (SSMP) (LLNL, 2022), as well as the U.S. Environmental Protection Agency's (EPA) Data Quality Objectives (DQO) process (EPA, 2006). In addition to the environmental samples, geotechnical samples will be collected by Consolidated Engineering Laboratories (CEL) on behalf of PMO. Geotechnical samples will be collected from a dedicated geotechnical boring location or co-located with environmental samples. Collection and evaluation of the geotechnical samples is determined by CEL and is therefore not discussed in this SAP.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2017

Ligand-Capped CdTe Quantum Dots as a Fluorescent Nanosensor for Detection of Copper Ions in Environmental Water Sample

In this work, as a novel fluorescent nano-sensor, a ligand-capped CdTe QDs (CdTe-L QDs) was designed for the detection and quantification of Cu{sup 2+} ions in environmental water samples. The synthesized QDs were characterized by transmission electron microscopy (TEM), thermo-gravimetric (TG) analysis, Fourier transform infrared (FTIR), UV–Vis spectrophotometry and fluorescence spectroscopy. Optical properties of the produced nanosensor were monitored by UV–Vis and fluorescence spectrophotometry. It was observed that fluorescence intensity of the produced nano-sensor selectively quenched by adding Cu{sup 2+} ions in comparison to other metal ions tested. Using CdTe-L QDs, a rapid and facile analytical method was developed to determine Cu{sup 2+} ions in the concentration range of 5.16 ± 0.07 × 10{sup − 8} mol L{sup − 1}–1.50 ± 0.03 × 10{sup − 5} mol L{sup − 1} with a detection limit of 1.55 ± 0.05 × 10{sup − 8} mol L{sup − 1}. The nanosensor was successfully applied for the determination of Cu{sup 2+} ions in various water samples, and the results were compared with the standard method. .

77 NANOSCIENCE AND NANOTECHNOLOGY↗

NASA NTRS2014

Mobilization Protocols for Hybrid Sensors for Environmental AOP Sampling (HySEAS) Observations

The protocols presented here enable the proper mobilization of the latest-generation instruments for measuring the apparent optical properties (AOPs) of aquatic ecosystems. The protocols are designed for the Hybrid Sensors for Environmental AOP Sampling (HySEAS) class of instruments, but are applicable to the community of practice for AOP measurements. The protocols are organized into eleven sections beyond an introductory overview: a) cables and connectors, b) HySEAS instruments, c) platform preparation, d) instrument installation, e) cable installation, f) test deployment, g) test recovery, h) maintenance, i) shipping, j) storage, and k) smallboat operations. Each section concentrates on documenting how to prevent the most likely faults, remedy them should they occur, and accomplishing both with the proper application of a modest set of useful tools. Within the twelve sections, there are Socratic exercises to stimulate thought, and the answers to these exercises appear in Appendix A. Frequently asked questions (FAQs) are summarized in a separate section after the answers to the exercises in Appendix B. For practitioners unfamiliar with the nautical terms used throughout this document plus others likely encountered at sea, an abbreviated dictionary of nautical terms appears in Appendix C. An abbreviated dictionary of radiotelephone terms is presented in Appendix D. To ensure familiarity with many of the tools that are presented, Appendix E provides a description of the tools alongside a thumbnail picture. Abbreviated deployment checklists and cable diagrams are provided in Appendix F. The document concludes with an acknowledgments section, a glossary of acronyms, a definition of symbols, and a list of references.

Mobilizarion↗

DOE OSTI2021

Merging Fungal and Bacterial Community Profiles via an Internal Control

Integrated measurements of fungi and bacteria are critical to understand how interactions between these taxa drive key processes in ecosystems ranging from soils to animal guts. High-throughput amplicon sequencing is commonly used to census microbiomes, but the genetic markers targeted for fungi and bacteria (typically ribosomal regions) are domain-specific so profiling must be performed separately, obscuring relationships between these groups. To solve this problem, we developed a spike-in method with an internal control (IC) construct containing primer sites commonly used for bacterial and fungal taxonomic profiling. The internal control offers several advantages: estimation of absolute abundances, estimation of fungal to bacterial ratios (F:B), integration of bacterial and fungal profiles for holistic community analysis, and lower costs compared to other quantitation methods. To validate the IC as a scaling method, we compared IC-derived measures of F:B to measures from quantitative PCR (qPCR) using a commercial mock community (the ZymoBiomic Microbial Community DNA Standard II, containing two fungi and eight bacteria) and complex environmental samples. For both the mock community and the environmental samples, the IC produced F:B values that were statistically consistent with qPCR. Merging the environmental fungal and bacterial profiles based on the IC-derived F:B values revealed new relationships among samples in terms of community similarity. As a result, this IC method is the first spike-in method to employ a single construct for cross-domain amplicon sequencing, offering more reliable measurements.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Current and future federal and state sampling guidance for per- and polyfluoroalkyl substances in environmental matrices

Per- and polyfluoroalkyl substances (PFAS) are a class of emerging contaminants composed of an estimated 5000 to 10,000 human-made, fluorinated, organic chemicals. Due to the complexity of PFAS, the need for multiple environmental matrix considerations and the absence of a promulgated federal standard for environmental sampling and analysis, U.S. states have begun developing health-based regulatory and/or guidance values for a limited number of PFAS in environmental matrices. As there is a growing body of science to inform PFAS sampling guidance standard development, it is important to understand which U.S. states are implementing sampling guidelines and how they plan to handle emerging PFAS. This critical review discusses the current and impending federal and state sampling guidelines for PFAS in environmental matrices, the data gaps surrounding PFAS sampling guidance in U.S. states, and the future impacts of impending guidance documents and regulations. Ten federal guidance documents are available for PFAS sampling guidance and analysis. The maximum number of PFAS covered in these guidance documents is 25 analytes spanning across 8 unique media. While the EPA has developed several different sampling and analytical guidelines for PFAS, there is no formal regulation of PFAS or requirements of states to enforce these guidelines. Consequently, only 31 states have informally adopted sampling guidelines, while the other 19 states have no guidance documentation in place for PFAS. The introduction of new PFAS sampling guidelines by the EPA, as well as updated analytical guidelines that target more PFAS or total organofluoride, is expected to continuously shift the landscape of federal and state guidance for PFAS sampling moving forward.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2012

PhiSiGns: an online tool to identify signature genes in phages and design PCR primers for examining phage diversity

Background: Phages (viruses that infect bacteria) have gained significant attention because of their abundance, diversity and important ecological roles. However, the lack of a universal gene shared by all phages presents a challenge for phage identification and characterization, especially in environmental samples where it is difficult to culture phage-host systems. Homologous conserved genes (or “signature genes”) present in groups of closely related phages can be used to explore phage diversity and define evolutionary relationships amongst these phages. Bioinformatic approaches are needed to identify candidate signature genes and design PCR primers to amplify those genes from environmental samples; however, there is currently no existing computational tool that biologists can use for this purpose. Results: Here we present PhiSiGns, a web-based and standalone application that performs a pairwise comparison of each gene present in user-selected phage genomes, identifies signature genes, generates alignments of these genes, and designs potential PCR primer pairs. PhiSiGns is available at (http://www.phantome.org/phisigns/; http:// phisigns.sourceforge.net/) with a link to the source code. Here we describe the specifications of PhiSiGns and demonstrate its application with a case study. Conclusions: PhiSiGns provides phage biologists with a user-friendly tool to identify signature genes and design PCR primers to amplify related genes from uncultured phages in environmental samples. This bioinformatics tool will facilitate the development of novel signature genes for use as molecular markers in studies of phage diversity, phylogeny, and evolution.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2012

Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A-G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the present study, we have developed an ELISA-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotype A, B, C, D, E and F. With engineered high-affinity antibodies, the assays have sensitivities in buffer of 8 fM (1.2 pg/mL) for serotypes A and B, and 32 fM (4.9 pg/mL) for serotypes C, D, E, and F. Using clinical and environmental samples (serum and milk), the microarray is capable of detecting BoNT/A-F to the same levels as in standard buffer. Cross reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical or environmental samples.

Zhang, Yanfeng↗