Capabilities
Complementary equipment and facilities located throughout PNNL/Battelle provide capabilities used to support the discovery, analysis, validation and implementation of biomarkers. These capabilities help us improve our understanding of intrinsic (e.g., genetic, metabolic) and extrinsic (e.g., environmental) factors associated with acute and chronic exposure to toxicants and their contributions to chronic health effects and disease. Capabilities include:
Data from coordinated experiments are integrated, combining genomic, proteomic, metabonomic, and lipid data, and novel imaging for controlled and stressed conditions to measure and identify biosignatures.
Ecosystem Delivery System
Biological Monitoring and Modeling
While analyses of various excised and intact tissues are performed routinely at the lab, more recently the focus has been on non-invasive biomonitoring (e.g., breath, urine and saliva). Modeling of biological structure and physiological function vary from the simple to the more complex (e.g., virtual respiratory tract). For deployment tools, sensor systems have been developed to address biomonitoring of both non-volatiles and volatiles.
PNNL operates a 3-dimensional cell culture system which incorporated cell lines relevant to human exposures and allows cells to differentiate into 3-dimensional tissues similar that of live host. PNNL also uses cell culture lines with reporter cells to characterize biological activity in response to nanomaterial exposure.
Scientists are working to understand how cell signaling networks integrate with competing environmental stimuli to promote a range of cellular responses that underlie adaptive and pathologic behavior. Understanding the mechanisms will aid in identifying and detecting biomarkers of influence in these signaling pathways.
Computational Bioinformatics and Visualization Tools
PNNL is developing new approaches to handling the large amounts of data generated as part of the biosignature discovery process and the integration of that data into complex models. Researchers are in the midst of developing common databases and interfaces that will be linked into collaborative problem-solving environments. One such tool, OmniViz, has been developed to mine biology information. Another tool used includes ProMat, where the combination of the ELISA microarray platform with the ProMAT data analysis tool provides a powerful approach for the rapid, sensitive and high-throughput measurement of proteins in complex biological samples. Visualization tools are providing expedited interpretation and visualization of integrated data streams; and new methods to identify key biomolecules, integrated biosignatures, and interactions or pathways related to exposure or response of a system.
Biological Response Pathway
PNNL has broad capabilities in dosimetry. In the application to environmental biomarkers, microscopy with single molecule sensitivity is used to study cell membranes to understand local processes, analyze differential protein binding of nanomaterials, and better understand nanomaterial-cell interactions that lead to toxicity as well as the drivers for internalization of these nanomaterials. Additional capabilities include In vitro and in vivo exposure systems to provide integrated experimental results that help identify cellular dose-metrics illustrating nanomaterial dose response and distinct pathways for biosignature identification. Results represent new capabilities for dosimetry, screening for inflammatory response and mode of action indicators.
External Cell/Tissue Dose (Delivery)
PNNL has multiple capabilities to quantify the effects of contaminants or other stressors on ecosystems and communities, investigating the effects across muitiple levels from molecular to microbial communities to higher level communities. Examples include, toxin screening using reporter gene assays, phytoplankton analysis and phytoplankton species identification using MALDI-MS, endocrine disruption studies, micro-algae screening for maximum CO2 fixation and integrated experiments using aqautic mesocosms.
The lab utilizes Affymetrix arrays, Universal Fingerprint arrays, organism-specific microarrays, quantitative RT-PCR in genomic studies.
In conjunction with Battelle Toxicology Northwest, unique capabilities in inhalation toxicology and pulmonary dosimetry exist on the PNNL campus. Generated aerosols are characterized for exposure concentration and particle size distribution and controlled using real-time monitoring, including optical light scattering instrumentation and particle mass monitors. Acute, subchronic, and chronic inhalation toxicity studies of new pharmaceutical and biopharmaceutical candidates are performed, and more recently toxic biological and chemical agents have been studied.
PNNL's High-Performance Mass Spectrometry Facility with its cutting-edge mass spectrometry methods focus on global proteomics research and the Accurate Mass and Time (AMT) Tag process, and is being used to identify protein biomarkers. Capabilities allow visualization and analyses of cell proteins in great detail. State-of-the-art instruments are available for challenging research in proteomics, cell signaling, cellular molecular machines, and high-molecular weight systems. Proteomics (top down & bottoms up approaches). Two approaches to proteomics analysis are supported at PNNL. The top down approach or intact protein analysis uses 2-dimensional gel electrophoresis and utilizes large amounts of sample, has a dynamic range of 10^4 and femtomole sensitivity. This approach is supported by LC-MS/MS, MALDI MS/MS and LC-FTMS, HPLC, MALD TOF/TOF. The bottoms up or peptide analysis (proteolyzed proteins) approach uses LC-FTICR and AMT tag approach, has ultra-high resolution, analyzes minute amounts of sample, has a dynamic range of 10^6 and a zetomole sensitivity. Methods are selected based upon desired outcomes.
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PNNL's approach to nanomaterials characterization and assessing material interaction with biological systems and the environment uses complementary techniques including electron and light microscopy, nuclear magnetic resonance (NMR) and mass spectroscopy methods, For example, scanning force microscopy and time of flight secondary ion mass spectrometry provide distinctive morphological information useful in looking at surface interactions.
Particle Physico-chemical Properties
PNNL has developed methods to identify metabolome components and mass and time tags for highly sensitive, rapid metabolite detection and profiling. The metabolite profiling methods using nuclear magnetic resonance (NMR) imaging and mass spectroscopy (MS) methods allow us to assess shifts in metabolites within a cell or organism providing a direct measure of functional biochemical status; a close link from the molecular components within that cell or organism and its physiology, or active state. NMR methods include non-invasive analysis of the low molecular weight, proton-containing metabolites. NMR is non-sample consuming, thus it may be used to follow metabolic response over time. A capillary liquid chromatography-mass spectrometry (LC-MS)-based method has been developed for the analysis of lipid-soluble metabolites in various biofluids and tissues using minimal sample volumes.
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PNNL has several efforts in microarray development. Examples include a Tree of Life phylogenetic microarray to discern microbial changes in native and perturbed environments; protein ELISA (Enzyme-linked immunosorbent assay) microarray platform that is flexible, highly sensitive and capable of high-throughput protein measurements. The high sensitivity of this assay platform is due to the use of separate antibodies for capture and detection along with the integration of an amplification step for enhanced detection sensitivity. In order to efficiently evaluate the large data outputs from ELISA microarray experiments a bioinformatics program, Protein Microarray Analysis Tool (ProMAT) was developed. Another application of PNNL's microarray technology includes a salmonid-based DNA microarray for testing of exposure to contaminants in salmon populations.
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PNNL staff use nuclear magnetic resonance (NMR) and electronic paramagnetic resonance (EPR) instruments in the High-Field Magnetic Resonance Facility. Ongoing research utilizes high-resolution spectroscopy of biological objects where slow (1 to 100 Hz) magic angle spinning is being used to discover and study biomarkers (e.g., pulmonary phospholipidosis accumulation) in live animals in real time.
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Two approaches to proteomics analysis are supported at PNNL. The top down approach or intact protein analysis utilizes large amounts of sample, and has a dynamic range of 10^4 and femtomole sensitivity. This approach is supported by LC-MS/MS, MALDI MS/MS and LC-FTMS, HPLC, MALD TOF/TOF. The bottom up or peptide analysis (proteolyzed proteins) approach uses LC-FTICR and AMT tag approach, has ultra-high resolution, analyzes minute amounts of sample, has a dynamic range of 10^6 and a zetomole sensitivity. Methods are selected based upon desired outcomes.
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PNNL's integrated biomarker approach, a science-based approach, promotes a more accurate, cost effective method to determine change agents of environmental health. It provides the scientific basis for the use of biomarkers in risk-based management of contaminants, monitors ecosystem health based upon the systems response and solves intractable problems associated with mixtures of contaminant and climate change. We are also developing and using advanced computational models for integration of biosignatures and for predicting health outcomes. In addition, we are demonstrating the unique integrated architecture that facilitates inference of biological relationships and subsequent identification of relevant environmental biomarkers that guides analysts who information risk management.
Various sensor technologies are developed and used at PNNL and vary by type and application. In biomarker identification we have focused on ELISA microarrays, electrochemical-based nanosensors, immunoassay PCR sensors, PCR primer arrays and Tree-of-Life Chips. Developments have include a broad range of applications (e.g., microfluidic/ electro-chemical sensor platforms to detect exposure in humans to toxic chemicals using non-invasive (e.g., saliva, urine, breath) sensor technologies; PCR primer arrays capable of accurately enumerating an unlimited number of microbial (bacterial, fungal, archaeal, and protozoan) species).
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Water Chemistry and Chemical Properties
Routine use of the following PNNL capabilities aids in characterization of the environment:
- Inductively-coupled plasma mass spectrometers (ICP-MS) and atomic emission spectrometer (ICP-AES) for ultra-low-level metals analyses
- Atomic absorption spectrometer (AAS), including graphite furnace atomic absorption (GFAA) for metals speciation
- Cold vapor atomic fluorescence (CVAF) units for mercury and methylmercury analysis
- Ion chromatographs for analysis of nutrients and speciation of arsenic and other metals
- Gas chromatographs for organic analyses of tributyltin, PAHs, PCBs, and other compounds
- High performance liquid chromatographs (HPLC) for specialized sample preparation
- Liquid scintillation analyzer for radionuclides and analysis of C-14 labeled material
- Gamma counter for radionuclide dating of sediment and analysis of isotopes
- Alpha counter for radionuclides and dating of sediments
- Liquid Chlorolab system for determination of photosynthetic oxygen production and dark respiration rates.