Ian M. Kennedy, PhD - US grants

The effect of pressure on the sooting of flames will be studied in a variable pressure gradient wind tunnel. Soot will be monitored accurately through sampling and radiation probes, and properties will yield to advanced optical techniques (extinction and scattering). Soot generation and disappearance will be documented as a function of the pressure field and geometry. The possibility to control soot formation by design-dependent pressure fields could be an outcome of this fundamental work. The effect of shear and radiation losses will be included in this study.

A project has been developed which is concerned with the dispersion of particles in a turbulent shear flow. Single particles of variable size shall be injected onto the center-line of a turbulent jet; laser light scattering will be used to measure the position of the particle across a plane of the jet. It is intended that Lagrangian statistics of particle dispersion be obtained as a function of the relative time scales for particle motion and for turbulence. A second project is being developed to study the effect of mixing rates on turbulent diffusion flames which are confined in a duct. In particular, the formation of soot and its radiation are of interest in confined diffusion flames. As the axial pressure gradient is increased, mixing rates are enhanced and the formation of soot particles may be suppressed. Measurements of soot volume fraction and velocities will be made in these flows. A third area of interest is in particle formation in mixing flows. This work is related to condensation in mixing layers, aerosol nucleation kinetics and possibly material synthesis in flames. A numerical study is being performed on the effect of mixing on a condensational aerosol in a mixing layer. An experiment is planned in which laminar flows containing trace amounts of ammonia and hydrogen chloride form particles via a relatively slow reaction.

Thermal remediation can be used to treat hazardous wastes found at Superfund sites. These sites can incorporate chlorinated hydrocarbons and heavy toxic metals. The thermal treatment of these materials can release hazardous byproducts into the environment. This project seeks to determine the risk to human populations as a result of a release of chlorinated by-products or metals. The chlorinated compounds of most interest are the dioxins. The impact of the rate of mixing of waste and air on the formation of dioxins will be studied in a wind tunnel experiment. Samples of by-products will be collected for later analysis with the toxicology projects for the presence of dioxins. The focus of the metals will be collected for later analysis with the toxicology projects for the presence of dioxins. The focus of the metals research will be on chromium. Chromium is a non volatile metal that tends to form an ultrafine aerosol in high temperature systems. The hexavalent form of chromium is very toxic; the other valence states are not toxic. Hence, it is important to be able to predict the state of chromium emissions and to design systems to minimize the formation of the hexavalent form. This requires knowledge of the kinetics of chromium oxidation in combustion systems. A low pressure burner will be constructed to undertake experiments in a simple, laminar pre-mixed flame that is seeded with chromium. An on-line time of flight mass spectrometer will provide measurement of chromium intermediates. The results will be used to tune a kinetic model of chromium oxidation for application in the design of practical systems. Collaboration with the University of Colorado Boulder will permit laser induced fluorescence measurements of reactive intermediates to be undertaken. Modeling of the dynamics of the chromium aerosol will also be undertaken to predict the size of the aerosol particles. The toxicity of the particles may change as they age in the atmosphere. Artificial aging in a chamber will be used to simulate the reaction of particles in the atmosphere, on their way to human populations. The toxicity of the aged particles will be studied by analytical chemistry as well as by the various bioassays that are available to use throughout the Superfund program. The toxicity of the aged particles will be assessed in vitro, as well as in vivo with animal exposures to artificially condition combustion generated aerosols.

The Superfund program at University of California Davis has produced assays for many environmentally hazardous materials. The application of these bioassays in fast, multi-analyte instruments requires further work that will be undertaken in the Biosensor projects. Initially, engineering of surface microdot systems will be carried out for model assays for atrazine and metabolites. Small dots of antibodies will be printed onto a surface and then exposed to samples. The sensitivity of the surface microdot system will be explored on the model assay. Automation of steps in cell based assays will also be investigated. Novel fluorescence labels will be developed in collaboration with other projects in the program. Three classes of new labels will be tested; they include porphyrins and lanthanides that are amenable to time resolved fluorescence detection. Fluorescence resonance energy transfer (FRET) will be investigated as a promising label. Experiments will be carried out on simple solutions and on a model assay for atrazine to evaluate sensitivity and detection limits. Microdroplets will be investigated a novel format for fluorescence detection. Micron sized droplets of sample with be interrogated with a laser. The possibility of using non linear optical processes, such as droplet lasing, in the microdroplets will be determined. Microfabrication technology will be used to design and fabricate miniaturized systems for immunoassays. Sample handling and processing will be incorporated into the miniaturized device. Ultrasonic agitation will be investigated as a way to improve binding rates. Finally, the miniaturized immunoassay system will be interfaced with novel detection methods, using new fluorophores and new detection formats (such as microdroplets). The sensitivity, detection limits, and turn around time of the packaged system, will be evaluated on atrazine and other compounds of interest, including dioxin and human reproductive hormones.

This award in the Nanoscale Science and Engineering Initiative (Nanoscale: Interdisciplinary Research Teams) will support the application of materials science and engineering to biosystems and environmental biology at the nanoscale. This new approach can open up new and exciting possibilities for researchers in many fields of biology. A multidisciplinary team has been assembled to explore the potential for the application of semiconductor quantum dots to two areas in particular, environmental immunoassays and cell biology. The unique optical properties that are available via the quantum confinement effect inherent to nanoscale clusters of semiconductor material offer unique possibilities in biosystems research. The emission of light from quantum dots is dependent on the size of the cluster. Hence, it is possible to design clusters of various sizes to emit at a desired wavelength. The visible part of the optical spectrum can be covered by a range of quantum dots whose emissions are spectrally distinct, permitting their use in simultaneous bioassays for environmental pollutants and hazards, as well as replacements of conventional fluorophores in studies of peptide chemistry and binding to cell receptors. In addition, the magnetic properties of quantum dots can be used for the manipulation of biological molecules in magnetic fields.
Quantum dots will be synthesized initially by an established method that can produce CdSe clusters capped by ZnS that permits bioconjugation to molecules of interest via a carboxyl group. Reverse micelles will also be studied as an optional scheme; this method may offer a better control on particle size. A major thrust of the effort will be directed to the identification of quantum dot materials that absorb in the visible spectrum and that can avoid the excitation of strong background fluorescence that is typical of complex environmental samples. Metal oxides will be given particular attention by using our established laser ablation method to screen new materials that have a small bulk bandgap, such as VO. Iron oxide will be studied because of its particularly interesting magnetic properties, and the additional benefit that ferromagnetism may confer in terms of manipulation in bioassays and cell migration studies. A new scheme for the improvement of the laser ablation synthesis of quantum dot materials will be investigated by selecting clusters on-the-fly, based on their functional performance i.e., their optical or magnetic properties. The usefulness of the quantum dots in environmental bioassays will be explored by applying them to immunoassays that are designed to detect a class of molecules that are important in agriculture, atrazines. Simultaneous assays will be designed using spectrally distinct quantum dots as labels in assays using a class specific antibody for atrazine, and using compound specific assays for members of the atrazine family. Monodispersed quantum dots will also be evaluated in cell based systems.The quantum dot surface will be coupled to peptide ligands via functional groups, and used to probe intact lymphoma cells. Analysis will include flow cytometry, tissue or cell staining, and peptide trafficking studies. The quantum dot performance will be compared with that of a conventional fluorochrome such as BODIPY. Initial experiments will involve quantum dots with only one defined wavelength. Once the cellular techniques are perfected, and when monodispersed quantum dots with a series of different sizes are available, multiplexed analysis will be performed using a series of peptides that have different binding affinities to the cell.
Research in biology depends increasingly on the development of rapid and sensitive measurement technologies. The use of nanoscale materials in biology for labeling molecules, with the unique properties that arise as a result of the very small scale of these materials, will play a significant role in contributing to progress in this development. The results of this work will lead to the development of improved, miniaturized detection methods for pollutants in the environment and in human populations, as well as providing a valuable new tool for studying fundamental processes at the cellular level.

Miniaturized biosensors can enable the toxicology projects to undertake their assays with high throughput and potentially with greater sensitivity. The Biosensor project aims to implement those bio assays that have been developed in the toxicology projects into usable biosensors. The project will have a fundamental aspect, and an applied aspect in which we intend to implement these assays. The fundamental aspects will investigate new nano scale materials for bio labeling, particularly with application to immunoassays. Long lifetime nano scale phosphors have been found to be particularly useful for labeling haptens, analytes, or antibodies in an immunoassay. We will focus on the use of the lanthanide elements, in particular europium, and also other wavelengths that can be offered by the use of materials such as terbium oxide. We shall also investigate a novel format for carrying out immunoassays in a micro droplet. Samples that contain pico liters can be interrogated for very long times with our photobleaching labels, with the potential for approaching single molecule detection limits in assays. The more practical aspect of the project will be concerned with implementing existing assays in miniaturized biosensors on a chip. We shall make use of micro fabrication techniques to make micro channels on a chip in which we shall carry out the immunoassays. We shall make use of indium tin oxide (ITO) films as waveguides and as electrodes to manipulate nanoparticles labels and antibodies in channels. We shall use evanescent detection of the particle labels within the channel, and use an electrostatic field to enhance binding to antibodies, and potentially to regenerate antibodies within the channel. We shall also attempt to improve the detection of DNA for sampling in soils. This will assist Project 1 in undertaking their measurements of toxin-consuming bacteria within soils. The Biosensor project will implement an in vitro assay for dioxin for use in Dr. Denison's project and will work with Dr. Lasley's project to implement a miniaturized, portable biosensor for markers of reproductive health.

The objective of this research is to develop a bioassay system consisting of a low-cost disposable assay disk and a compact, portable read-out instrument - analogous to a biomolecular disk drive. The approach is to adapt magnetic recording heads from disk and tape-based storage devices for use in detecting magnetically-tagged biomolecules on removable microfluidic assay substrates. Two different types of assay substrates will be constructed using microfabrication techniques, and methods to deposit biological probe molecules onto these substrates will be developed. A DNA assay will be performed as a benchmark to demonstrate the sensitivity and specificity of the new magnetic detection system. The results of this benchmark assay will be compared with the results of a conventional, optically-scanned DNA microarray.

Intellectual merit: This project will explore the use of precision mechanical scanning for the detection of magnetically-tagged biomolecules. Micromagnetic models will be developed and refined using experimental measurements. Precision control and signal processing techniques will be investigated to enhance the sensitivity of the instrument. Surface treatments to suppress non-specific binding of the magnetic labels will be explored.

Broader Impact: This project will address the growing need for compact, sensitive, bioassays that can be used for environmental fieldwork or for point-of-care medical diagnostics. The project will integrate education and research. Engineering students will be trained in interdisciplinary biotechnology research. Outreach to high-school students will be performed through the California State Summer School for Mathematics and Science (COSMOS) program.

Miniaturized biosensors can enable the toxicology projects to undertake their assays with high throughput and potentially with greater sensitivity. The Biosensor project aims to implement those bio assays that have been developed in the toxicology projects into usable biosensors. The project will have a fundamental aspect, and an applied aspect in which we intend to implement these assays. The fundamental aspects will investigate new nano scale materials for bio labeling, particularly with application to immunoassays. Long lifetime nano scale phosphors have been found to be particularly useful for labeling haptens, analytes, or antibodies in an immunoassay. We will focus on the use of the lanthanide elements, in particular europium, and also other wavelengths that can be offered by the use of materials such as terbium oxide. We shall also investigate a novel format for carrying out immunoassays in a micro droplet. Samples that contain pico liters can be interrogated for very long times with our photobleaching labels, with the potential for approaching single molecule detection limits in assays. The more practical aspect of the project will be concerned with implementing existing assays in miniaturized biosensors on a chip. We shall make use of micro fabrication techniques to make micro channels on a chip in which we shall carry out the immunoassays. We shall make use of indium tin oxide (ITO) films as waveguides and as electrodes to manipulate nanoparticles labels and antibodies in channels. We shall use evanescent detection of the particle labels within the channel, and use an electrostatic field to enhance binding to antibodies, and potentially to regenerate antibodies within the channel. We shall also attempt to improve the detection of DNA for sampling in soils. This will assist Project 1 in undertaking their measurements of toxin-consuming bacteria within soils. The Biosensor project will implement an in vitro assay for dioxin for use in Dr. Denison's project and will work with Dr. Lasley's project to implement a miniaturized, portable biosensor for markers of reproductive health.

Rapid, highly sensitive and specific, flexible, simple and cost-effective diagnostic techniques to detect and differentiate the causes of infections are critically needed to distinguish dangerous infections from common ones. We propose developing a simple multiplexed assay to identify the causes of viral fevers. Technologies that derive from recent advances in nanoscale engineering and micro-fluidics present synergies that can be leveraged in the development of multiplexed assays in large-scale screening applications. A simple and robust platform for quantitative multiprotein immunoanalysis has been developed with the use of magnetic core/shell nanoparticles (MLNPs) as a carrier. The magnetic properties of the MLNPs allow their manipulation by an external magnetic field in the separation and washing steps in the immunoassay;enhanced binding kinetics are also provided by the oscillating magnetic field. The luminescent properties arise from the the addition of lanthanide elements to oxide hosts or the addition of lanthanide chelates to porous silica hosts. Silver nano-islands within the latter particles provide significant enhancement of signal [unreadable] leading to the possibility of single particle detection. The optical emission from the particles also provides an internal calibration of the detection system [unreadable] this is important in reducing uncertainties in particle-based assays. Multiplexed sandwich immunoassay involves dual binding events on the surface of the MLNPs functionalized with the capture antibodies. Secondary antibodies labeled with conventional organic dyes (Alexa Fluor) can be used as reporters. Alternatively, the secondary label can be very small porous silica particles with chelate emission enhanced with silver, for a much greater signal and hence sensitivity. The amount of the bound secondary antibody is directly proportional to the concentration of the analyte in the sample. In our approach, the fluorescence intensity of the reporter dye is related to the luminescence signal of the MLNPs. In this way, the intrinsic luminescence of the MLNPs serves as an internal standard in the quantitative immunoassay. This technology will be implemented in a micro-fluidic system that will be automated. We will utilize reagents developed for Rift Valley fever virus (RVFV) detection to develop a prototype assay that will detect RVFV antibody and antigen. We will also develop monoclonal antibodies and recombinant proteins to three other viruses that cause hemorrhagic fever in humans: Dengue, Lassa, and Ebola virus. Using these reagents, we will develop a prototype test that will identify samples from individuals infected with these viruses. Validation with experimental and clinical samples will begin in the final year of the grant.

It is the goal of this project Biosensors, to apply new and emerging technologies to implement bioassays that our colleagues at Davis have developed, with improvements in speed and/or sensitivity compared to conventional methods. Several new schemes for detection will be tested, including a nanowell format for trapping and interrogating our non-bleaching nanoparticle labels for DNA and immune assays; a magnetic/luminescent nanoparticle format for DNA assays; and a nanostructured liquid core waveguide for enhancement of fluorescence detection. The targets will include the pyrethroid metabolite 3-PBA, emerging problem compounds such as TCC, TCS and PBDE, and genes for microbes that are used in bioremediation activities. A sensor for TCDD and related compounds will be developed based on AhR technology.

Remediation of Superfund sites can release nanoscale particles into the environment, along with hazardous vapors. The health effects of these complex mixtures and materials, especially emerging materials produced by the nanotechnology industry, are not sufficiently well understood. It has been hypothesized that the adverse health effects due to exposure to environmental particles (e.g., airborne particulate matter) is, at least in part, due to generation of reactive oxygen species (ROS), but more data are needed to test this mechanism. This project focuses on 3 main areas: (I) using a laboratory combustion system to create complex mixtures of toxic by-products emitted from thermal processing of materials that contain brominated flame retardants; (II) characterizing the generation of reactive oxygen species from nanoscale materials in the environment; and (III) making use of nanotechnology to answer questions that have arisen in earlier studies of nanoparticle toxicity. In the first aim, this project will construct a small, laboratory-scale system to simulate the thermal processing of materials containing brominated flame retardants; the gaseous and particulate by-products of this process will be examined by other projects in this proposal using bioassays for polybrominated diphenyl ethers (PBDEs) and brominated dioxins and furans. In the second aim we will quantitatively measure two of the most important ROS - hydroxyl radical (OH) and hydrogen peroxide (H{2}O{2}) - formed by nanoparticles in a cell-free surrogate lung fluid and, later, in cell cultures. We will also examine how simulated atmospheric reactions alter the ability of nanoparticles to form ROS. In the third aim of this project we will synthesize a range of novel, multi-functional nanoparticles in order to study several questions related to nanomaterial toxicity, in part in conjunction with other projects in the UC Davis Superfund Program.