Deirdre R. Meldrum - US grants

The overall objective of this research career award is to develop new methods and tools for automated large-scale DNA sequencing. Initially, the specific aims of the award are to: Train extensively in the field of genomics via lab rotations, courses and seminars in the Department of Molecular Biotechnology. In the lab training the candidate will perform all steps of DNA sequencing via current methods. This experience combined with the candidate's electrical engineering background will be used to develop fast, efficient methods to automate certain steps in large-scale DNA sequencing procedures. Develop a prototype robot that will dispense sub-microliter to sub- nanoliter volumes of reagents for DNA sequencing reactions. A precise robotic positioning system will be designed and built to access individual wells on 384-well or 864-well microtiter plates and alternate denser formats. Investigate and develop a new, automated procedure for loading sequencing reactions onto gels from high density formats. Currently this step in large-scale DNA sequencing is performed manually but with higher density reactions this will be more problematic. Explore alternate electrophoretic formats that require no net migration. Signal processing and identification techniques will be employed to identify DNA molecules in free solution. While performing the steps of large-scale DNA sequencing in lab training during the first year of the five year SERCA program, bottlenecks in large-scale DNA sequencing procedures will be identified. The remainder of the award will be used to focus on solving these problems using new, automated techniques. Ultimately, these technological developments will increase the throughput in DNA sequencing and contribute to the sequencing of the Human Genome. Deirdre Meldrum, the candidate, received her Ph.D. in Electrical Engineering from Stanford University in 1992. At Stanford and the Jet Propulsion Laboratory, Deirdre worked extensively on robotic and controls for space structures, space robots, and computer disk drives. Her strong background in robotics, controls, dynamics, and electrical/mechanical design will be invaluable for fulfilling the goal of automating steps in large-scale DNA sequencing. Throughout the award period and beyond, Deirdre will train and work closely with the faculty in the Department of Molecular Biology while maintaining close ties with the Department of Electrical Engineering. Leroy Hood, the principle faculty advisor, is Professor and Chair of the Department of Molecular Biotechnology at the University of Washington.

The overall objective of this research career award is to develop new methods and tools for automated large-scale DNA sequencing. Initially, the specific aims of the award are to: Train extensively in the field of genomics via lab rotations, courses and seminars in the Department of Molecular Biotechnology. In the lab training the candidate will perform all steps of DNA sequencing via current methods. This experience combined with the candidate's electrical engineering background will be used to develop fast, efficient methods to automate certain steps in large-scale DNA sequencing procedures. Develop a prototype robot that will dispense sub-microliter to sub- nanoliter volumes of reagents for DNA sequencing reactions. A precise robotic positioning system will be designed and built to access individual wells on 384-well or 864-well microtiter plates and alternate denser formats. Investigate and develop a new, automated procedure for loading sequencing reactions onto gels from high density formats. Currently this step in large-scale DNA sequencing is performed manually but with higher density reactions this will be more problematic. Explore alternate electrophoretic formats that require no net migration. Signal processing and identification techniques will be employed to identify DNA molecules in free solution. While performing the steps of large-scale DNA sequencing in lab training during the first year of the five year SERCA program, bottlenecks in large-scale DNA sequencing procedures will be identified. The remainder of the award will be used to focus on solving these problems using new, automated techniques. Ultimately, these technological developments will increase the throughput in DNA sequencing and contribute to the sequencing of the Human Genome. Deirdre Meldrum, the candidate, received her Ph.D. in Electrical Engineering from Stanford University in 1992. At Stanford and the Jet Propulsion Laboratory, Deirdre worked extensively on robotic and controls for space structures, space robots, and computer disk drives. Her strong background in robotics, controls, dynamics, and electrical/mechanical design will be invaluable for fulfilling the goal of automating steps in large-scale DNA sequencing. Throughout the award period and beyond, Deirdre will train and work closely with the faculty in the Department of Molecular Biology while maintaining close ties with the Department of Electrical Engineering. Leroy Hood, the principle faculty advisor, is Professor and Chair of the Department of Molecular Biotechnology at the University of Washington.

The long term objective of this proposal is to automate and improve a key area and current bottleneck of genomic research: the automated handling of submicroliter fluid samples. Specifically, the research proposed in this application is the development of a novel, high-throughput, high-yield, fully automated system capable of performing restriction enzyme digests, PCPs, and sample preparation for DNA sequencing. The immediate goals of this system are to process 5,000 1 mu l samples per day, at a cost per finished sequence base pair of less than 20 cents. This will be accomplished through a 10 fold reduction in reagents and sample volumes, and a 10 fold increase in throughput derived from automation, parallelization, and integration of: (I) prior technology developed by the proposing team, and (2) new technology that will be developed during the course of this project. The system will interface in an automated fashion with microtiter plates, Eppendorf tubes, or capillaries at the front end, and with electrophoretic capillaries, electrophoretic gel loading combs, or microtiter plates at the back end. Other input/output media are also possible. The proposed system uses glass capillary tubes and several novel methods to reduce sample size, automate the handling of small fluid samples, reduce thermal cycling and incubation times, and minimize the amount of disposables used to perform DNA sequencing. All processing steps are performed within these capillaries. This use of capillaries also helps facilitate sample containment and minimize evaporation losses, which are particularly important for the high surface area to volume ratios of small samples. An important feature of the proposed system is that it is consistent with current methods of sample handling. Furthermore, it will reduce costs, increase yield, and increase throughput in deriving DNA sequence. The system described in this proposal will greatly aid the Human Genome Project in meeting its sequencing goals. It will also have a synergistic effect outside of the Human Genome Project, benefiting efforts in clinical testing, medical diagnostics, pharmaceutical development, environmental testing, DNA fingerprinting, and agricultural research.

DESCRIPTION: (Applicant's Abstract) Over the past 3 years the Genomation Laboratory of the University of Washington (15W) and partner/subcontractor Orca Photonic Systems, Inc., havedesigned, developed, and tested ACAPELLA, an automated fluid sample handling system. This system prepares final reaction volumes of 1 to 2 microliters inside glass capillaries for reactions such as PCR, sequencing reactions, and restriction digests. Approximately 44,000 capillaries have been run through the system to test the hardware, software, and biology. Some biology tests were run in parallel with the UW Genome Center to validate the results. The testing results demonstrate that the first generation system, the ACAPELLA-1K, can process samples with high reliability, no contamination, and high quality at a rate of 1,000 samples in 8 hours. The second-generation system, the ACAPELLA-5K, has just come on-line and will process 5,000 samples in 8 hours. The 5K system available at the start of this project will include automated thermal cycling. In this advanced development project, the ACAPELLA-5K system will be enhanced to provide a contiguous, automated process stream, from initial DNA samples to the sequencer. Thermal cycling, purification, sample pooling, sample storage and archiving, will be integrated with ACAPELLA's existing sample preparation capabilities. To validate ACAPELLA technology in the operational sequencing center environment, extensive alpha- and beta-tests will be performed with NIH Genome Centers. Alpha-testing will take place in the UW Genome Center. Beta-testing will take place in the UW Genome Center, the Washington University St. Louis Genome Sequencing Center (subcontract), and the Whitehead Institute/MIT Center for Genome Research (no subcontract, volunteer with existing funds). To ensure ACAPELLA remains viable with the state-of-the-art in DNA sequencing technologies, we will assess and develop methods for direct, automated loading to capillary electrophoresis systems and microchip sequencers. Technology associated with this project will be disseminated and commercialized to bring ACAPELLA processors to the point where they can be deployed to the major genome centers on a commercial basis.

Quantification of Minimal Residual Disease (MRD) is a general concern in oncology since this parameter is likely to give valuable information to clinicians to customize chemotherapeutic treatments and to anticipate possible relapses. An automated system will be developed for the quantification of MRD by using real-time PCR to quantify cancer cells in a background of non pathologic DNA. The system will be derived from an existing high-throughput sample handling system (developed by Meldrum and team) named Acapella. Acapella has a pipelined serial architecture which makes it possible to develop an adaptive PCR control algorithm ensuring a level of sensitivity and accuracy for the quantification results specified by the clinician. In the R21 phase of the project, a critical component of the system is the real-time thermocycler. It will provide DNA quantification results of greater precision than what is currently possible with commercial instruments such as the ABI PRISM 7700 Sequence Detector by taking advantage of a high performance custom fluorescence analyzer and sophisticated data analysis methods. The fluorescence analyzer will have a signal to noise ratio and a dynamic range of at least one order of magnitude larger than the optics used on commercial systems. This unique feature will permit precise measurements of the amplification kinetics during at least five cycles in the exponential phase of the reaction. The amplification yield will he derived from these data using statistical estimators customized to meet the requirements of real-time PCR data analysis. The sample DNA content will be derived from the amplification yield and the calibrated fluorescence measurements of the reaction kinetics. This new approach will make it possible to run series of real-time PCRs with more flexibility than would be possible if the assay was based on standard reactions required to have the same amplification yield as the clinical samples. PCR conditions will be adapted online without concerns about possible differences of amplification rate. The assay control algorithm will adapt the reaction DNA content, the primer selection, and the number of PCRs to meet the clinician requirements for particular patient DNA. During the R21 phase of the project a prototype of the real-time thermocycler will be developed to demonstrate the optic performance and the possibility to estimate the PCR amplification rates with 5% accuracy. A conceptual design of the fully integrated process from patient blood sample to MRD quantification data will be completed to allow assessment of expected performance, cost, and risks associated with the development of the fully engineered system. The development of the various hardware and software components along with their integration into the automated system will take place during the R33 phase of the project. Performance of the system will he evaluated on real biological samples provided by the UW Department of Laboratory Medicine. Results returned by the system will he compared with results of t(14; 18) PCR performed in this department with an ABI PRISM 7700 for the diagnosis of patients suffering from follicular lymphomas.

The availability of genome sequences for both prokaryotes and eukaryotes is laying the foundation for a revolution that will ultimately transform biology from a largely descriptive and reductionist predictive science. The growing ability to analyze whole biological systems based on genomic information is creating snapshots of cells at the transcriptional and translational level, which are providing preliminary insights into cellular complexity. However, to understand complex molecular outcomes such as cell proliferation, differentiation, apoptosis, and pathogenesis, it will be necessary to determine how the parts are integrated in time and space to form complex, dynamic cellular functions, and how cellular interactions create higher-order functions. Such analyses require the simultaneous measurement of many variable sin real-time, and due to heterogeneity in cellular populations, these analyses need to be carried out for individual cells. Therefore, a major barrier to achieving this objective is the lack of available technology for carrying out highly multi-variant, dynamic analyses at the level of individual cells, based on genomic information. A second major barrier is the lack of available technology for processing individual cells, based on genomic information. A second major barrier is the lack of available technology for processing microsamples for genome-based expression analysis, to allow comparative initiative is to meet these challenges with an interdisciplinary team of experts from genomic sciences, microanalytical chemistry, and microsystems engineering who will develop and apply enabling technology for analysis and processing of individual cells. We propose to design and build fully integrated and automated microsystems for the interrogation of individual cells. This core technology will then be converted into modules designed for specific applications, which will push the limits of detection to the minium, in some cases, to single molecule levels This enabling technology will be directed towards specific research problems in two main areas: 1) automated detection of rare cells in cell populations, and 2) real-time analysis of metabolism in individual cells. Both areas are applicable to eukaryotes and prokaryotes, and depend on availability of genome sequence data, but not necessary complete genomes. The integrated biologically-active microsystems we develop will push the limits of detection, tackle the module-to-module interconnect problem that is ubiquitous in all integrated microsystems, emphasize overall systems integration, and enable the production of comprehensive data sets. Ultimately, these microsystems will have far- reaching applications for both basic and applied research in broad areas of biomedical systems biology.

Revised Abstract: The broad, long-term goal of this research is to develop technology for carrying out highly multivariant, dynamic analyses at the level of individual cells, based on genomic information. Microscale instrument systems will be developed that include technologies for highly sensitive detection of metabolites and specific proteins, for combining and analyzing multiple sensor inputs, and for efficient and sensitive separations of cells, metabolites, and internal cell contents. These systems will be developed primarily for two applications: 1) Modular microscale interfaces to a commercially available benchtop mass spectrometer and a microscale mass spectrometer will be developed for highly selective and sensitive metabolomic analysis of prokaryotes. This system will be used to analyze metabolic shifts in single cells and in real time with the ultimate goal of correlating genetic expression with metabolic outcomes. 2) Develop an integrated microscale cell culture instrument system that performs microscopic and chemical processing for eukaryote analysis. The specific application for this system is dense, high-throughput, loss of heterozygosity (LOH) analysis of progeny colonies from single Saccharomyces Cerevisiae cells. This system will be used to study the budding process in yeast and how the frequency of chromosome abnormalities effects aging and cancer development. The goals of this research are complementary to and contribute to the long-term goals of an NIH NHGRI Center of Excellence in Genomic Science, the Microscale Life Sciences Center, 1 P50 HG002360. The microscale instrument systems developed under these programs will comprise a low-cost, flexible, reconfigurable, benchtop toolbox that will push the limits of detection, tackle the module-to-module interconnect problem that is ubiquitous in all integrated microsystems, emphasize overall systems integration, and enable the production of comprehensive data sets. Ultimately, these microsystems will have far-reaching applications for both basic and applied research in broad areas of biomedical systems biology.

DESCRIPTION (provided by applicant): Preparing well-diffracting crystals is the key step in biomacromolecular crystallography and is in particular a challenge for high-throughput structure determination projects of proteins as part of structural genomics and medicinal protein crystallography projects. Another challenge is to freeze the crystals obtained to 100K in such a manner that they remain well-diffracting and with no ice crystals. This is a critical step for X-ray data collection at synchrotron beam lines. The next critical step is to place the crystal in the X-ray beam and center the crystal precisely at the proper position where cryo-stream, X-rays and rotation axes of the goniometer intersect. The aim of our proposal is to obtain a completely automated procedure for all these steps: no manual intervention for crystal growth, cryofreezing and crystal centering. This unique approach can significantly remove all bottlenecks between protein production and the initiation of X-ray data collection for biomacromolecular crystallography. The equipment to be developed would be able to work with very small amounts of protein samples since volumes per experiment are in the low nanoliter range. The aim of the proposed project is to develop, design, and build a complete prototype sample processor that automates all process steps from initial protein sample, through automated detection of crystal growth, though delivery of a completely characterized, cryocooled sample to the synchrotron or other x-ray diffraction facility. Specific performance goals of this prototype system include: 1) A complete pipeline: sample preparation, sample sealing, conditioned storage for crystal growth, automated identification of crystal growth, cryocooling, and characterization of the samples in anticipation of x-ray crystallography. 2) Fully-automated, "hands-free" processing of samples. 3) Throughput of at least 500 samples/hour. 4) In-line image acquisition and processing for automated identification of crystal growth. The proposal has not only potential for a major impact of full automation of all steps between purified protein production and starting the X-ray data collection process in structural genomics projects. In addition, it has the potential to be coupled with combinatorial libraries of chemical compounds, which would allow thousands of compounds to be tested for crystal growth of a drug target protein in the presence of numerous different compounds.

DESCRIPTION (provided by applicant): 3D microscopy represents a powerful new cell analysis tool for early detection and diagnosis of cancer, but its future use may be limited because methods for preparation of samples are cumbersome, inefficient, labor intensive and generally imprecise. Current methods for cytological sample collection are manual and distributed in nature through various physicians' office laboratories and local hospitals, with the actual analysis being centralized at regional clinical laboratories. Among the cytological specimens are sputum, gynecological and colorectal scrapes, fine needle aspirates, urinary tract, and gastrointestinal samples. We propose the development of a new automated system that will transform these difficult and messy clinical specimens into an optimal format for 3D microscopy morphological and molecular analysis. The model and method we propose is comprised of three sequential steps. First, at the distributed site, an automated sample processor dissociates and fixes cells and debris for shipment in an automation compatible canister. Second, after shipment to a centralized clinical laboratory, the specimen canisters are loaded into an automatic processor that performs cleanup (debris removal), specimen/assay specific staining (and counterstaining), and finally embedding of cells of interest in glass microcapillary tubes (about 50 mu m ID), with cells being spaced at regular intervals (about 200 mu m) within a tube. This preparation format is uniquely suited for integration with multiple 3D imaging platforms for true 3D volumetric assessment of cell morphology and molecular probe and/or stain density distribution. The proposed system also enables use of cytometric flow sorting for enrichment of cells of interest at an intermediate stage of the sample preparation process. The potential impact to improved human health through rapid diagnostic screening will be illustrated using a high impact emerging technology, optical tomography. In summary, the aim of the proposed project is to develop, design, and build a complete sample processing system that automates the process of sample cleanup, assay specific staining, and mounting of cells into glass microcapillary tubes, and a tube positioning and rotation scanner mechanism for 3D microscopy analysis of cell morphology for the early detection of cancer.

The Center for Nanotechnology in Society at Arizona State University (CNS-ASU) helps ensure "that advances in nanotechnology bring about improvements in the quality of life for all Americans" (PL 108-153). The Center's vision is that research into the societal aspects of nanoscale science and engineering (NSE), carried out in close collaboration with NSE scientists and combined with public engagement, will improve deliberation and decision making about NSE. CNS-ASU builds the capacity to address the societal implications of NSE by creating a broad institutional network, instituting a coherent research program, promoting innovative educational opportunities, and engaging in meaningful participation and outreach activities, especially with under-represented communities. Its goal is nothing less than charting a path toward new ways of organizing the production of knowledge and developing and testing new processes of anticipatory governance to meet the emerging promises and challenges of NSE.

CNS-ASU joins Arizona State University with the University of Wisconsin - Madison, the Georgia Institute of Technology, North Carolina State University, Rutgers, The State University of New Jersey, and other universities, individuals, and groups in the academic and private sector, as well as the International Nanotechnology and Society Network (www.nanoandsociety.org) that ASU is developing. At ASU, the project's two guiding organizations are the Consortium for Science, Policy, and Outcomes (www.cspo.org), which provides an institutional home for science and technology policy scholarship and engagement, and the Biodesign Institute (www.biodesign.org), which provides a substrate of NSE research and a test bed for interdisciplinary collaboration.

CNS-ASU will implement a program of research and engagement called "real-time technology assessment" (RTTA), which consists of four methods of inquiry: mapping the research dynamics of the NSE enterprise and its anticipated societal outcomes; monitoring the changing values of the public and of researchers regarding NSE; engaging researchers and various publics in deliberative and participatory forums; and reflexively assessing the impact of the information and experiences generated by its activities on the values held and choices made by the NSE researchers in its network. Through RTTA, CNS-ASU will probe the hypothesis that trajectories of NSE innovation can be steered toward socially desirable goals, and away from undesirable ones, by introducing a greater capacity for reflexiveness - that is, social learning that can expand the range of conscious choice - into knowledge-producing institutions. It organizes the research around two broad NSE-in-society themes: freedom, privacy, and security; and human identity, enhancement, and biology.

The Center's educational and training plan includes innovations at the undergraduate, graduate, and postdoctoral level that encourage interdisciplinary opportunities among NSE students and social science and humanities students. Partnerships with proven programs, including the Hispanic Research Center (www.asu.edu/clas/hrc) and the Center for Ubiquitous Computing (http://cubic.asu.edu), ensure recruitment and retention of students from under-represented groups. A collaboration with the Center for Research on Education in Science, Mathematics, Engineering, and Technology (http://cresmet.asu.edu), CNS-ASU generates training modules for high school teachers in NSE-in-society.

Designed as a "boundary organization" at the interface of science and society, CNS-ASU provides an operational model for a new way to organize research through improved contextual awareness, which can signal emerging problems, enable anticipatory governance, and guide trajectories of NSE knowledge and innovation toward socially desirable outcomes, and away from undesirable ones. In pursuit of this broadest impact, CNS-ASU trains a cadre of interdisciplinary researchers to engage the complex societal implications of NSE; catalyzes more diverse, comprehensive, and adventurous interactions among a wide variety of publics potentially interested in and affected by NSE; and creates new levels of awareness about NSE-in-society among decision makers ranging from consumers to scientists to high level policy makers.

DESCRIPTION (provided by applicant): Increasingly, it is becoming apparent that understanding, predicting, and diagnosing disease states is confounded by the inherent heterogeneity of in situ cell populations. This variation in cell fate can be dramatic, for instance, one cell living while an adjacent cell dies. Thus, in order to understand fundamental pathways involved in disease states, it is necessary to link preexisting cell state to cell fate in the disease process at the individual cell level. The Microscale Life Sciences Center (MLSC) at the University of Washington is focused on solving this problem, by developing cutting-edge microscale technology for high throughput genomic-level and multi-parameter single-cell analysis, and applying that technology to fundamental problems of biology and health. Our vision is to address pathways to disease states directly at the individual cell level, at increasing levels of complexity that progressively move to an in vivo understanding of disease. We propose to apply MLSC technological innovations to questions that focus on the balance between cell proliferation and cell death. The top three killers in the US, cancer, heart disease and stroke, all involve an imbalance in this cellular decision-making process. Because of intrinsic cellular heterogeneity in the live/die decision, this fundamental cellular biology problem is an example of one for which analysis of individual cells is essential for developing the link between genomics, cell function, and disease. The specific systems to be studied are proinflammatory cell death (pyroptosis) in a mouse macrophage model, and neoplastic progression in the Barrett's Esophagus (BE) precancerous model. In each case, diagnostic signatures for specific cell states will be determined by measuring both physiological (cell cycle, ploidy, respiration rate, membrane potential) and genomic (gene expression profiles by single-cell proteomics, qRT-PCR and transcriptomics;LOH by LATE-PCR) parameters. These will then be correlated with cell fate via the same sets of measurements after a challenge is administered, for instance, a cell death stimulus for pyroptosis or a predisposing risk factor challenge (acid reflux) for BE. Ultimately, time series will be taken to map out the pathways that underlie the live/die decision. Finally, this information will be used as a platform to define cell-cell interactions at the single-cell level, to move information on disease pathways towards greater in vivo relevance. New technology will be developed and integrated into the existing MLSC Living Cell Analysis cassette system to support these ambitious biological goals including 1) automated systems for cell placement, off-chip device interconnects, and high throughput data analysis with user friendly interfaces;2) new optical and electronic sensors based on a new detection platform, new dyes and nanowires;and 3) new micromodules for single-cell qRT-PCR, LATE-PCR for LOH including single-cell pyrosequencing, on-chip single-cell proteomics, and single-cell transcriptomics using barcoded nanobeads.

This exploratory project will provide effective mechanisms to motivate and engage middle school students in the practice of engineering through hands-on community-based service learning projects by establishing sustainable linkages between higher education, K-12, and community partners, developing professional learning communities (PLCs) comprised of engineering faculty and K-12 educators, and creating appropriately aligned educational pathways that integrates STEM with social studies and language arts. The university- school district- community partnership is at the heart of the proposed project.
The proposed ?Partnership, Pathway and Pipeline for Engineering Education: Engaging Middle School Students with Curricular Integration and Societal Relevance? will be led by the Dean of Engineering at ASU and the superintendent of CUSD. The project will leverage and scale the resources of both ASU and CUSD and allow project outcomes to be incorporated into the strategic plans and organizational missions of both institutions. Central to this project is the identification of engineering service learning projects that facilitate integrated learning of science, math, social studies and language arts. These STEM-based integrated activities are designed to engage students and captivate their interest while promoting problem-solving and entrepreneurial skills by addressing a real world needs in their local communities.

The overall goal of this project is to increase the awareness, motivation and self-perception of Arizona students in their formative middle school years to engineering opportunities and educational pathways that lead to successful engineering careers. In doing so, the State of Arizona will have in place a more socially-embedded comprehensive and effective educational pipeline that will provide the needed Arizona engineering workforce to compete in a global competitive high technology economy. This project will result in a sustainable partnership across institutions and communities to improve and diversify the K-12 pipeline into engineering and technology.

All organisms require chemical elements such as carbon (C), nitrogen (N), and phosphorus (P) for growth and development. P is of particular interest because it is especially important in construction of genetic material such as DNA and RNA and is often present in limiting concentrations in the environment. This project will investigate the biological rules that determine the elemental recipe ("stoichiometry") of microorganisms that grow under severely P deficient conditions in a set of unique desert springs in Mexico.

This study will use both laboratory and field experiments, combined with cutting-edge methods of molecular biology and genomics, to investigate how changes in P supply alter the ecological and evolutionary dynamics of these microorganisms and to reveal how they cope with shortages of this essential chemical element. Furthermore, the study will help us understand how human alterations in P supply, such as those driven by inefficient use of fertilizer or by inputs of P-rich sewage, affect microbial ecosystems. It will also help in discovery of new genes and genetic strategies by which organisms efficiently use P. These discoveries may be useful for agriculture and other settings in light of growing concern about the finite supply of economically recoverable P for fertilizer production. Finally, the project will produce bi-lingual science education products that will enhance science education in Arizona and nationally for both K/12 students and the general public.

DESCRIPTION (provided by applicant): High throughput live-cell microarray screening technology for dynamic, multiparameter sensing of single-cell metabolic phenotypes is proposed. The proposal addresses Common Fund priorities by extending the range of signatures available to the LINCS centers. A sandwich microarray, called the "Cellarium" will be developed and used to analyze individual live cells. The bottom layer of the sandwich supports cells in shallow microwells etched in glass. The top layer of the sandwich seals the cells in the 150-picoliter microwells, and incorporates extracellular fluorescent sensors for multiparameter detection of the metabolic analytes, oxygen, pH and glucose. Chemical isolation is achieved when the two layers are compressed together with a flat metal spring allowing dynamic measurement of transmembrane fluxes without Intracellular probes. Single-cell analysis which directly reveals heterogeneity in metabolic response to perturbations within an isogenic cell population is critical to biological inference. This microarray is an extensible tool for deriving a standardized multiparameter set of data that can be integrated in a coordinated way into LINCS. The specific aims of the project are: 1) develop a disposable microarray ("Cellarium") for dynamic, high throughput, multiparameter metabolic measurements of perturbation-induced signatures of live single cells;2) modify a commercial microarray scanner to read out the Cellarium;3) verify the effectiveness of this technology across a range of cell types by simultaneously monitoring 02, pH, glucose and ATP responses;4) validate the platform by analyzing the distribution of metabolic signatures of single cells in response to perturbations;5) develop written and graphical standard operating procedures that enable reproducible data generation;6) develop active participation with LINCS partners in the instrument development process to ensure efficient device and methods translation. PUBLIC HEALTH RELEVANCE: Determinants of human health and disease depend, ultimately, on the biological state of individual cells. This technology will quantify the distributions of key metabolic parameters, on a cell-by-cell basis, among a cell population. The instrument measures previously inaccessible indicators of cell state and cellular responses to perturbations facilitating new insights into underlying molecular pathways and biological mechanisms.

This EAGER proposal seeks funding to extend the Sensorbot network design, range and coverage by incorporating multi-hop functionality. Sensorbot developed by the PIs with internal ASU funding is a miniaturized, low-cost, underwater, autonomous sensor platform. A single Sensorbot constitutes a network node, capable of measuring and reporting the properties of the fluid in which it is located. With EAGER funding, short-range communicating Sensorbots will be intelligently link together to extend their coverage over long distances in a multi-hop networking approach, thus overcoming the range limitation of standard underwater optical communication.

If successful, once deployed, the network of Sensorbots is expected to not only achieve continuous, autonomous, long term monitoring campaigns but to sustain two-way communication within the network. The goal of this project is to significantly extend the spatial range and coverage of a network of optically-communicating, deep ocean sensors.

Broader Impacts:

The Sensorbots were created to measure a wide range of low-temperature hydrothermal fluid properties. Sensors for pH, dissolved oxygen, trace metals, and temperature are now in operation, and several others are in the early stages of development. Currently under development are fluorescent polymers that respond to methane, sulfate, CO2, and other critical analytes in areas of diffuse flow in hydrothermal regions of the deep ocean. With improved communications, optical multi-hop networked Sensorbots will have broad and immediate application to a diverse array of ocean sensing fields. This potentially transformative proposal is expected to yield solutions to previously intractable questions about the extent of biogeochemical flux on the ocean floor. This project will include one post doc and two undergraduate students.

DESCRIPTION (provided by applicant): Current RT-PCR analysis of single-cells has been strictly limited to analysis of disassociated cells, preventing its compatibility with in-situ analsis of cell states and loses information of initial cell location and morphology within the native tissue. We propose to develop an innovative microfluidic based tool for clinical and scientific users to analyze gene expression heterogeneity, in situ, using single-cell mRNA expression analysis. The device uses a two-photon laser to serially lyse individual cells at known coordinates within a 3D tissue. Differing from conventional single-photon laser lysis, two-photon laser lysis relies on the nonlinear interaction between an ultrafast pulsed light source and the biological material to achieve an energy transfer to the cell precisely within the nanometer-scale focal volume. The lysate is immediately transported to an emulsion-based (oil-droplet) qRT-PCR module to profile mRNA expression. Carryover contamination between sequentially lysed cells is minimized by optimizing laser power and by using hydrodynamic flow focusing with precise flow rate control. Because of the small scale of the microfluidic channels, the total volume flux for sample processing is reduced to microliters, the elapsed time interval between cell lysing and lysate encapsulation is on the order of seconds, and completion of qRT-PCR is on the order of one hour. This technology is well suited to basic biomedical research and clinical applications such as assessing tumor cell population heterogeneity in single-cell gene expression. Additionally, the technology is also amenable to future developments to increase the number of genes that can be quantified. The ultimate implementation would be a highly multiplexed platform capable of detecting dozens of mRNA sequences for each initial droplet eluted from the sample. PUBLIC HEALTH RELEVANCE: We propose to develop an innovative integration of nanometer-resolution laser lysis and microfluidic tool to analyze single-cell heterogeneity in situ using qRT-PCR. In its ultimate implementation, users can simply load a tissue slice in the device, select the target cells based on their location and morphology, and then the system delivers mRNA expression of dozens of genes of individual cells in one hour.

PROJECT SUMMARY (See instructions): The aim of this research is to further develop and apply the novel tools, 3D optical tomography and labs on chips, to better understand the physical attributes of cancer cells. Deeper understanding of cancer cells' physical attributes will allow their quantitative comparison between cancer grades, and before and after genomic and chemotherapeutic interventions, and is an important component of progress toward cures and preventions. Both of these technologies will be applied to single cells. To present a tractable research plan, the cancer cell types studied are colon and esophageal, but the results may be relevant to other cancers. Attributes of immortalized cell lines representing both cancers and cells from patient biopsies will be characterized. Optical Tomography: 3D optical tomography provides two types of information at the 100-nm length scale: The first, receiving primary emphasis, is morphometric (structural: shape, size, chromatin texture and density), relying on absorption imaging analogous to the type (H&E and Feulgen staining) of imagery that cancer cytopathologists have used for decades to diagnose cancer clinically. The second is functional (localized protein concentration), relying on antibody and other fluorescent staining. Among the novel contributions harnessed for this work is the instrumentation and software to perform cell CT imaging for the first time. This provides truly isotropic resolution on cells suspended in their natural state, eliminating ambiguities due to overlapping features in thick sections and smears, and problems with orientation dependence, distortions due to flattening, and the incomplete sampling inherent in all 2D imaging methods. Isotropic resolution confers two salient advantages on this method for understanding cancer: 1) Measurements are robust and repeatable, as they represent the entire 3D cell, not randomlyselected plane(s). 2) The measurements are exquisitely sensitive to the neoplastic progression status of a cell. This research will produce and derive such parameters as nuclear to cytoplasmic ratio and ploidy with unprecedented precision; and texture features like heterochromatin distribution and granularity scale, and metrics related to the nuclear membrane infoldings, invaginations and protrusions, impossible to measure in 2D, which constitute signatures common to many cancer cells. Labs on Chips: Before and after cancer relevant modifications, physiological parameters including respiration rate, pH, ion fluxes and ATP concentrations, and transcriptome levels will be quantified within and between cells and their sealed microenvironments. Correlations will be explored between physiological and transcriptomic variables, and between these and the morphometric, densitometric, and protein expression level and localization measurements from cell CT. Our measurements, and such correlations or the lack thereof, before and after genetic and chemotherapeutic modifications, will provide new insights into the biological physics of cancer.

In this project, a team of engineers and biologists will work together to develop an Ocean Microbial Surveyor (OMS); a miniature, autonomous genetic analyzer that can quantitatively analyze DNA and RNA of the microbial biomass sampled from an aquatic environment such as the ocean. Current technologies are typically expensive, cumbersome, complex, and power-hungry, limiting the number of users as well as the number of systems that can be deployed for scientific measurements. The OMS developed in this project will leverage technology from the biomedical research field to enable new capabilities for ocean monitoring, to make these new technologies easily accessible to oceanographers, and to increase the number of systems that can be deployed for scientific discovery. The OMS will be inexpensive, low power, small and autonomous to enable large networks of OMS systems to be used and to provide new measurement capabilities. Marine microbes account for more than 90% of the ocean biomass, but they are among the most under-studied life forms because of their often-inaccessible habitats. It is important to be able to measure microbes in their natural environment over time and space (spatiotemporal) scales to understand the effects of climate change and other environmental factors on microbial abundance, distribution, function, and production. The long-term goal is to integrate the OMS into other ocean testing platforms and ultimately as a broad network of industry-refined, multi-target microbial analyzers dispersed throughout the globe, in the hands of diverse scientific investigators.

The broader impacts of this project are to build science, technology, engineering, and mathematics (STEM) talent, innovate for the future, improve society, and engage a wide audience in the technology and science. STEM talent will be increased by providing excellent training opportunities for two postdoctoral fellows and numerous undergraduate students in a multidisciplinary research environment. The innovative technology developed in this project will inspire diverse generations of scientists and engineers to explore the oceans and enable access to new data from the genetic instruments that may lead to new discoveries. Monitoring marine microbial activities may help society understand critical issues such as how climate change and other environmental stresses impact the primary production from marine microbes. The new genetic sensors will be displayed and operated in the Biosphere 2 Ocean habitat, near Tucson, Arizona, to provide outreach to groups of school children and adults enabling them to observe science-driven technology development and ocean microbial monitoring experiments.

The OMS to be developed in this project is based on the integration of loop-mediated isothermal DNA and RNA amplification (LAMP) with microfluidics to produce a small footprint, low energy consumption analytical module that can be used on various platforms such as buoys, autonomous underwater vehicles (AUVs), and robotic floats. LAMP is a fast, selective, sensitive isothermal reaction that requires only "one-pot" and produces large quantities of amplification product, enabling simple optical colorimetric detection. The completed OMS prototype, incorporating the LAMP capability, will be able to perform 50 assays per deployment with a user-specified sampling interval. Each assay will measure a user-designed pair of DNA and RNA targets in triplicate. A prototype OMS will be developed that integrates all hardware systems into a single microfluidic chip. The project plan includes development of the hardware subsystems including chassis design, power circuitry, pump and valve automation, illumination and imaging systems, and thermal control design. A microfluidic chip for microbial lysis and nucleic acids extraction (on-chip extraction) will be developed on the benchtop and then integrated into a single microfluidic device with an "on-chip quantification" unit for measurement of DNA and RNA from captured microbes. LAMP reagents will be lyophilized with a company partner, and a chip will be developed to dispense and lyophilize reagents. The target cost of the OMS unit will be less than 1/10th that of existing instruments, supporting experiments requiring multi-node sampling of microbial abundance and/or function.