Mark Hayes, Ph.D. - US grants

ABSTRACT
CTS-0102680
A. Garcia/Arizona State University

This is a NER Grant. Surface phenomena become prominent when fluidic systems and devices are miniaturized since the surface area to volume ration increases. Biological systems capitalize on nanoscale surface phenomena by assembling unique elements such as membranes and vesicles to shuttle materials into and out of cells or to process wastes in organs such as the kidney. Much research is currently underway to find alternatives to moving parts in order to move ultra-small volumes of liquid (i.e. micorfluidics) since mechanical pumps and valves are currently difficult to manufacture at the nanoscale and require very careful choice of materials.

This NER research project is an exploration of the nanoscale engineering of the surface of capillaries to control movement of ultra-small volumes of liquid. It explores the hypothesis that molecular mixtures including photochromic molecules can be attached to the surface to form various types of nanoscale monolayer films in which the solvation and free volume of the active element (i.e. the photochormic molecule) are controlled. This in turn will allow the design of light controlled micorfluidic pumping, switching, and valving systems with biotechnological applications. This project also supports the establishment of collaboration with the NSF-CREST Computational Center for Molecular Structure and Interactions at Jackson State University in order to visualize and interpret our experimental results as well as to guide further experimentation.

ABSTRACT

Broadband biological agent detection is developed whereby chains of labeled paramagnetic nanoparticles are caused to rotate through a magnetic field and signal is enhanced through a lock-in amplification technique. The project, if successful, will allow improving signal to noise ratio for biosensors. Three students are involved and many aspects of the research from nanotechnology, nanomagnetics, sensing, and modeling are all involved. Good outreach activities and teaching are incorporated into the research.

This project is being funded by the CTS/ENG (C Aidun, PD) and ECS/ENG (R Khosla, PD).

Chemistry (12) This project aims to introduce mass spectrometry (MS) into all levels of the undergraduate chemistry and biochemistry curriculum at Arizona State University. A major objective of the project is to use MS as a means for developing "molecular thinking" in students. Acquisition of Gas Chromatography/Mass Spectrometry (GC/MS) and Matrix-Assisted Laser Desorption Ionization - Time of Flight (MALDI-TOF) mass spectrometers are enabling students to perform experiments using mass spectrometry in course-related laboratory work and independent student research. A number of experiments, with increasing complexity, are being adapted from the current scientific literature for instructional purposes at various levels of the curriculum including general chemistry, organic chemistry, instrumental analysis and biochemistry. Assessment of the outcomes of the project is being accomplished through a variety of methods including ongoing evaluation of students, TAs and faculty, as well as exit interviews and tests designed to measure how well the methods help students learn key mass spectrometry and chemistry concepts.

DESCRIPTION (provided by applicant): New molecular information from blood is being used to improve patient outcomes. Assays for established biomarkers have recently advanced (so-called 'high sensitivity', 'variants'and 'ultra', for examples), allowing physicians to use this additional information to provide better care. The enhanced patient outcomes result from more detailed, accurate and precise diagnostic and risk stratification, resulting in more timely and useful care (such as pharmaceuticals, treatments options, and timing of treatments) and avoiding unnecessary and costly actions. The objective of this proposal is to enable a new capability to isolate and concentrate biomarkers from blood in small volumes (200 microliters), at high sensitivity, over short periods of time, and to monitor multiple markers. We initially will focus on biomarkers for myocardial infarction and stroke. Upon completion of the entire project-which this proposal enables-enhanced information will be provided to physicians, allowing for accurate and fast diagnostics and treatments providing better patient outcomes-saving lives and money. To accomplish the proposal objective, a pair of microfluidic techniques will allow processing of small samples of blood to remove unwanted materials and isolate and concentrate the target biomarkers. These two techniques enable the objective because they keep the sample volume minimal and remove unwanted materials that could degrade detection, while quickly isolating and concentrating target species. Physically, this is made possible by exploit a unique combination of dielectrophoretic, flow and electrophoretic forces combined into the two techniques (gradient dielectrophoresis and electrophoretic capture) pioneered in the PI's laboratory. Gradient dielectrophoresis will remove cells and debris (and perhaps concentrate the targets) and electrophoretic capture will isolate and concentrate individual biomarkers away from possible interfering species. The four target biomarkers identified for proof of principle to enable the larger project are: two cardiac markers- myoglobin and cardiac troponin I, (cTnI), a stroke marker-neuron specific enolase (NSE), and an inflammatory marker-tumor necrosis factor-alpha (TNF1). These targets will provide a reasonable test for the success of the strategy and techniques.. PUBLIC HEALTH RELEVANCE: Developing an ability to isolate and concentrate biomarkers from blood using gradient dielectrophoresis and electrophoretic capture improving detection limits by at least two orders of magnitude while keeping the sample volume very modest (200 microliters).

DESCRIPTION (provided by applicant): Diagnostic approaches providing identification of viruses directly from bio-fluids with improved figures of merit (fast, accurate (selective, sensitive), simple, low power, and cost-effective) are needed. A new, innovative micro-fluidic strategy that can contribute to this goal is presented here. The system can rapidly and selectively separate, isolate and concentrate viruses from bio-fluids for direct identification or further assessments (immuno- or geno-recognition). The strategy is based on DC insulator gradient dielectrophoresis (DC-iGDEP) which provides not only the advantage of truly unique and non-linear separation of bioparticles, but also can remove unwanted components that are often present in complex biological samples and interfere with subsequent assays. The approach can fuse location to identification via electric field manipulation of bio-particles, thus avoiding a number of issues with current methods that require prior molecular recognition elements and commonly cold-chain reagents. The basis for the approach is a combination of dielectrophoretic and electro-kinetic forces in a single channel. The long-term objective is to integrate DC-iGDEP into a simple, cost effective, reliable biosensor that will be a component of a diagnostic platform that can be used in the clinical laboratory and ideally, amenable for surveillance and diagnosis in developing countries. As poof-of-concept, we will use dengue viruses as the target model for a blood borne virus and two respiratory viruses, human coronavirus OC43 and influenza that are typically only present in respiratory secretions and not in blood during acute infections. Dengue viruses are one of the most significant emerging infectious pathogens today and newly emerged corona viruses and influenza strains remain as important health concerns, thus the test samples will have real world relevance. Standard virological assays, including virus titer, hemagglutination (HA) and inhibition (HAI), RT-PCR, and other well established serological diagnostic methods will be used in parallel with the platform development to monitor sensitivity, accuracy and overall feasibility of DC-iGDEP. The goal is to isolate/concentrate/separate virus particles from typical venipuncture and respiratory sample volumes. Once developed, the approach can be modified for a broad range of medically important viruses. PUBLIC HEALTH RELEVANCE: Developing an ability to isolate and concentrate viruses from biofluids (saliva, CSF, blood) using gradient dielectrophoresis. This can be developed into devices which can detect virus at earlier phases of infection providing for better care and reduced spread of infectious agents.

DESCRIPTION (provided by applicant): Diagnostic approaches providing identification of phenotypes of pathogens directly from biofluids with improved figures of merit (fast, accurate (selective, sensitive), simple, low power, and cost-effective) is needed. A new, innovative microfluidic strategy that can contribute to this goal is presented here. The system can rapidly and selectively separate, isolate and concentrate pathogens from biofluids for direct identification or further assessments (immuno- or geno-recognition). The strategy is based on DC insulator gradient dielectrophoresis (DC-iGDEP) which provides not only the advantage of truly unique and non-linear separation of bioparticles, but also can remove unwanted components that are often present in complex biological samples and interfere with subsequent assays. The approach can fuse location to identification via electric field manipulation of bioparticles, thus avoiding a number of issues with current methods that require prior molecular recognition elements and commonly cold-chain reagents. The basis for the approach is a combination of dielectrophoretic and electrokinetic forces in a single channel. The system will be demonstrated by isolating and concentrating enterohemorrhagic strain designated E. coli serotype O157 in the presence of background flora and matrix. The long-term objective is to integrate DC-iGDEP into a simple, cost effective, reliable sensor that will be a component of a diagnostic platform that can be used in the clinical laboratory and ideally, amenable for surveillance and diagnosis in developing countries. The goal is to isolate/concentrate/separate pathogen particles from typical venipuncture and respiratory sample volumes. Once developed, the approach can be modified for a broad range of medically important pathogens.

? DESCRIPTION (provided by applicant): Diagnostic approaches providing identification of phenotypes of pathogens directly from biofluids with improved figures of merit (fast, accurate (selective, sensitive), simple, low power, and cost-effective) is needed. As innovative microfluidic strategy that can contribute to this goal is presented here. The system promises to rapidly and selectively separate, isolate and concentrate pathogens from biofluids for direct identification or further assessments (immuno- or geno-recognition). The strategy is based on DC insulator gradient dielectrophoresis (DC-iGDEP) which provides not only the advantage of truly unique and non-linear separation of bioparticles, but also can remove unwanted components that are often present in complex biological samples and interfere with subsequent assays. The approach can fuse location to identification via electric field manipulation of bioparticles, thus avoiding a number of issues with current methods that require prior molecular recognition elements and commonly cold- chain reagents. The basis for the approach is a combination of dielectrophoretic and electrokinetic forces in a single channel. To understand if this is a general approach, isolation and concentration of pathogenic versus non-pathogenic Listeria in the presence of background flora and matrix will be attempted. The long-term objective is to integrate DC-iGDEP into a simple, cost effective, reliable sensor that will be a component of a diagnostic platform that can be used in the clinical laboratory and ideally, amenable for surveillance and diagnosis in developing countries. The goal is to isolate/concentrate/separate pathogen particles from typical biofluids. Once developed, the approach can be modified for a broad range of medically important pathogens.

Microbes are everywhere, and they play major roles in ecosystem function and human health. However, studying microbes is challenging because they are so small and because they live in complex communities. Most microbial species cannot be cultivated in the lab. Therefore, to understand the diversity, evolution, and ecology of microbes, scientists typically extract bulk DNA from the environment. This can reveal which types of microbes are present, but it does not provide information about what each species looks like and which species are carrying out which processes. In this project, researchers will develop a device to separate complex microbial communities into their individual species. The researchers will share this separation technology broadly with other scientists in the microbial systematics, diversity, and ecology communities to help accelerate research in these areas. An interactive outreach module will be developed and brought to K-12 students to help spark appreciation for science, technology, and engineering research.

This project will develop a method using dielectrophoresis to separate microbial communities based on morphotypes. Dielectrophoresis is the movement of uncharged particles in an electric field. Particles of similar size, shape, and surface characteristics move in similar ways and are expected to accumulate in the same compartment of a gated microscopic channel once an electrical field is applied. This approach will advance the fields of microbial systematics and biodiversity science by enabling scientists to link morphological and molecular biodiversity in understudied microbial communities. The separation device will be used to answer two main questions about the evolution of symbiotic, wood-digesting protozoa that live in termite hindguts. First, how did the ancestor of termite hindgut protozoa acquire the ability to digest wood? For this project, tens of thousands of cells from key protozoan species will be separated from the rest of their communities for transcriptome sequencing. Genes for wood digesting enzymes will be identified and phylogenetically analyzed along with related genes from other organisms. Second, have protozoan symbionts transferred from one termite species to another in the genus Zootermopsis? And if so, can this explain the greater diversity of protozoa relative to their hosts? For this project, the hindgut communities of four Zootermopsis species will be separated into their constituent species in order to determine how many protozoan species are present and how closely they are related to one another. This information will be compared to the phylogeny and biogeography of Zootermopsis termites to determine whether and when such transfers may have occurred.

PROJECT SUMMARY We will perform an initial test of viability for a new technique that promises to rapidly and accurately serotype Salmonella isolates. Over 400 deaths, 20k+ hospitalizations and 1.2M illnesses in the United States each year are caused by Salmonella according to the Centers for Disease Control and Prevention (CDC Salmonella Atlas 2013). This pathogen causes the most gastroenteritis and can cause invasive, life threatening infections. Containment of outbreaks requires additional strain characterization to the serotype level. Serotypes are established according to the Kauffman-White scheme, which enables the ability to track and identify outbreaks and a method to determine the success of control efforts. The current serotyping methods are laborious, resulting in high cost and delayed delivery of information. The cost (including capital, facilities, and training) limits its wide spread use. The burden of serotyping falls to state and local public health labs, national reference labs, and government labs. The delays occur from shipping and confirmation of species using conventional techniques and can take up to ten days to obtain results. These delays provide more time for contaminated food vectors to remain in consumer hands and create further infections. We have begun to develop a microdevice technology based on dielectrophoresis that differentiates specific pathogens based on their biophysical properties. This approach is in distinct contrast to genotyping, expression profiles, phenotyping, or metabolic tests, all of which require the laborious procedures and resources noted. Pathogenic and non-pathogenic E. coli have been differentiated using this strategy and gentamicin resistant S. epidermidis and S. aureus have been isolated from their susceptible strains. Data and theory suggests each serotype can be selectively isolated and concentrated thus providing its identity. This proposal is limited to producing a proof-of-principle data set by evaluating the CDC Salmonella validation kit to understand if this strategy is worth further pursuit. An optimized device will be developed and sample property requirements established. The proposed work will directly assess the sensitivity and specificity and provide an estimate for the speed of identification and a rough estimate of the cost. If the data suggest this is a viable strategy, future studies can include characterization of further isolates, more accurate estimates of cost, precise determination of speed, and the development of user-friendly and handheld interfaces. The upside for biophysical serotyping is excellent: results in minutes, minimal cost, wide distribution, and no cold-chain reagents.

PROJECT SUMMARY To better investigate, identify and sense (detect) viruses, high-resolution separation, isolation and concentration is of significant importance. Our team is developing a bioparticle separation scheme based on dielectrophoresis using a microfluidic device that promises, and has demonstrated, extremely high-resolution separations of bioparticles. The separation is based on specific biophysical makeup of the viruses that in turn result in electrodynamic properties that vary for each specific target. If a separation scheme is of high enough resolution, distinct populations of homogeneous particles can be purified. Our preliminary data and recent theoretical modeling suggest that strain- or serotype-specific resolution of virus particles is achievable, however, this has not been explicitly shown. Unlabeled viruses must ultimately be used for the platform to be integrated in various virus assessment workflows. To enable detection of the separation of unlabeled virions, on-chip detection capabilities are developed, addressing this limiting issue in the current level of development. To demonstrate and develop the microfluidic platform we will use gene altered Sindbis virus (Venus and mCherry) which are fluorescent. Once parameters have been established for optimal dielectrophoresis in our system, the wild-type Sindbis virus (SINV) will be compared with mutants that harbor changes in the E2 protein to establish the ability to separate the viruses. Standard molecular biology genotyping and phenotyping will be used to confirm separation of virion populations. Figures of merit (detection limit, dynamic range, sensitivity and specificity) will be determined for the separated viruses along with defining appropriate properties of the incoming sample (ionic strength, pH, additives, etc.) to ensure proper operation of the technique.