Rashid Bashir - US grants

DESCRIPTION (Adapted from the applicant's abstract): The nucleotide sequence of DNA and mRNA and its expression in various cells is of utmost importance to life scientists because every disease state of biological function could be traced back to a single or a group of genes (DNA sequences). Thus the knowledge of the sequence of these genes and their subsequent regulation should provide powerful therapeutic approaches for disease control at the source of the problem (i.e. genetic level). The objective of the investigators work is to design and implement a silicon- based nano-electro-mechanical-system for the study, characterization and sequencing of individual polynucleotide molecules of DNA and RNA using conductance techniques. Such a system has never been demonstrated in silicon. The proposal takes a new step in fabricating the system in a silicon wafer. The project is high risk and can have significant impact to the area of single molecule electronic detection. The device will consist of two chambers in a silicon wafer with integrated electrodes. These chambers will be separated by a thin membrane with nano-pores defined in it. The DNA molecule will be introduced in chamber 1 and a voltage will be applied across the silicon membrane between electrodes 1 and 2. Since DNA is negatively charged, it will be pulled through the pore under the electric field at a controllable speed. The chambers will be filled with a conducting solution and the conductance measured across the pore will change when the DNA strand passes through the pore. The device described in the proposal will use silicon micro-machining techniques for the fabrication. Silicon processing and micro-machining techniques have been used to develop various micro-electro-mechanical structures for bio-medical application over the past few years. The processing dimensions are approaching regimes where these devices can be interfaced with biological molecules. For instance, lateral features down to 200A (e.g. using electron beam or X-ray lithography) and vertical features down to a 20A (e.g. using growth of oxide or controlled deposition) could be defined. For the device described the thin membrane will be made of silicon using epitaxial lateral overgrowth (ELO) and or made of silicon nitride deposited using low pressure chemical vapor deposition (LPCVD). "Nanapores" (diameter < 50A) will be formed in the membrane (thickness < 500A) which will separate the two chambers. The initial size of the pores will be controlled using direct write electron beam lithography and dry gas phase etching and the final dimension will be achieved using formation of sidewall. The ability to build the system described in this application will further enable the fabrication of other bio-electronic interface devices such as protein separation and purification of charged molecules. In addition, once the basic challenge of moving a single DNA molecule through a silicon pore under and electric field has been demonstrated in a silicon based device, further enhancements can be made by adding fluorescence related components, the next step of the long term goal of the research.

The goal of our research is to develop a new and highly sensitive technique for the characterization and investigation of stress-induced breakdown in the advanced, ultra-thin gate dielectrics which constitute one of the key components for next state-of-the-art and next generation VLSI technology.
Our technique of Noise Spectroscopy is based on the realization that dielectric breakdown must involve the breakage and rearrangement of atomic bonds arising from the impact of energetic carriers (electrons or holes) which invariably leads to the formation of CARRIER TRAP STATES. EXCESS CURRENT NOISE in the tunneling, leakage current through gate dielectrics represents an extremely sensitive probe of both the presence and the nature of these TRAPS which serve as stepping stones in the tunneling process and therefore the detailed and systematic investigation of excess noise via Noise Spectroscopy has the potential of providing direct information on the QUALITY OF DIELECTRIC FILMS immediately after growth as well as the PATHWAY TO DIELECTRIC BREAKDOWN.

The potential impact of this research in the shorter term is the development of a more sensitive and informative technique to ascertain the quality and robustness of ultra thin gate dielectrics which will be useful to the silicon VLSI industry complementary to conventional charge to breakdown and CV techniques. In the longer term, when a clear understanding of the physical mechanism of dielectric breakdown is achieved it should lead to the ability to design and manufacture better and more robust gate dielectrics e.g. via addition of trace impurities to pin atomic motion and reduce trap formation. In terms of education, we intend to involve three graduate students, one for the Noise Spectroscopy technique and the others for dielectric growth. Both aspects will involve state-of-the-art methods in the respective areas. In addition we plan to foster industrial ties to the fullest extent.

0216518
Feinerman

This grant will allow UIC to purchase a high resolution e-beam lithography equipment (Raith 150) with a minimum of 50nm patterning capability guaranteed and sub-10nm patterning has been demonstrated. UIC will set it up in a class 100 clean room bay devoted exclusively to lithography. The reasons for the selection of this particular machine will be elaborated on in section 3c "Description of Research Instrumentation and Needs," and are the patterning performance, metrology capability, ease of use by a diverse population, purchase price, and annual maintenance cost. This machine will benefit 6 other members of the UIC consortium (Notre Dame, NWU, Purdue, UC, IIT, and NIU) as well as a large corporate base around UIC and a national laboratory. Basic science and engineering research are entering a new era where fundamental knowledge of nanoscale materials including magnetic, semiconductors, superconductors, electronic devices, bio-materials, fluids, and related interface phenomena will be essential for the design of advanced devices and engineered systems. A great deal of basic research and innovation will be needed as devices shrink below the natural length scales like the size of the minimum ferromagnetic domain (100-1000 nm), the superconducting coherence length (~ 1-100 nm), or the characteristic size of bio molecules. The equipment will enable these programs and many more that can not be described here due to space limitations:

UIC, Nanomagentism studies, and nanopatterning
Note Dame, Nanoelectronics and high-speed circuits and devices
lIT, Nanoelectronics
NIU, Nanoelectronics
Purdue, NEMS for the Direct DNA Characterization and Sequencing at the Molecular Level
NWU, Two dimensional Photonoic band gap materials for biomolecular
detection
UC, Electronically controlled substrates for mechanistic studies of cell adhesion and migration.

The additional applications mentioned are the 23 letters of support received to support e--beam lithography development at UIC that are in the supplementary documents section. These are letters of interest and intent from 10 companies, and from 9 non-profit institutions. The nano-lithography system will be a part of University of Illinois at Chicago's Micro/Nanofabrication Applications Laboratory (MAL). The MAL has become a regional user facility for the Chicago area and in the last 3.5 years it has increased its user base 7 fold with over 50% of the users coming from outside of UIC. This rapid growth is due to the MAL offering open and inexpensive access, training, and guidance on a versatile set of fabrication and characterization equipment. The addition of e-beam lithography to the PIs facilities will allow them to greatly increase the participation of UIC and the surrounding community in the national nano initiative.

DESCRIPTION (provided by applicant): The recent technological advances in nanotechnology and micromachining of semi-conductor materials present themselves with new opportunities for cheap, small, and sensitive diagnostic devices capable of rapid and highly accurate detection of infectious agents. Surface derivitized cantilever structures have successfully been applied to the detection of DNA, proteins and cells, yet still fully realized at the levels of sensitivity required for practical applications. In addition, detection of viruses has not been fully explored with these technologies. The application of this technique in to detection of aerosolized virus particles is the main goal of the current proposal. This proposal brings together a group of truly interdisciplinary researchers from the fields of micro/nano-systems technology, molecular biology and virology, and bio-separations engineering to develop micro-cantilever-based virus detection techniques and systems which promises performance characteristics exceeding the sensitivity and specificity of PCR amplification assays and ELISAs. Calculated limits of detection of our approach are 10-17 to 10 -18 gm of mass change on the cantilever surface. This translates to the mass of single virus particles. When this method is coupled to currently available monoclonal antibodies against viruses, its specificity could surpass ELISAs since our technique doesn't rely on enzymatic reaction kinetics as does the former. The ability to detect and monitor-in real-time and continual basis- of viruses and their subtypes, particularly the most contagious viruses and bioterrorism agents, can have drastic implications in the confinement and management of the viral epidemics. The long-term objective of this application is to develop a micro-scale, robust, real-time monitoring device, based on micro-machined ultrathin cantilever arrays for the rapid and sensitive detection of infectious agents, particularly bioterrorism agents in field setting and in primary-patient care facilities. The array will be specific for specific pathogens and will have the sensitivity to detect a single virus or toxin molecule. During Phase I, the proposed effort aims to develop dielectrophoresis-based infectious agent trapping, separation and concentration device and a proof-of-principle demonstration for the detection of an air-borne virus on functionalized micro-scale cantilever. The performance value of the devices for trapping, separation, concentration and detection of aerosolized coronavirus particles will be assessed. During Phase II, this sensor design and manufacturing capabilities will be extended and scaled-up to other infectious agents in the form of integrated sensor arrays with capability for on-board signal processing.

DESCRIPTION (provided by applicant): Micro- and nano-systems technology has found increasing use in a wide variety of biomedical applications, including detection and characterization of biological entities. The devices used for such applications are broadly referred to as 'bio-chips' and for example in the case of DNA detection, have even been commercialized. One area of research that has become increasingly important but not very well studied is the handling, manipulation, and characterization of very few cells and micro-organisms using biomedical micro-electro-mechanical-systems technology (BioMEMS). The detection of very low cell concentrations from samples of bodily fluids, tissue samples, soil, water, and food is a challenge that has not been fully realized. The goal of such an effort should be to handle, detect, and characterize a single cell or microorganism, and micro-devices are ideally suited for such studies. In addition, reducing the time-to-result to be able to perform "point-of-use" analysis is also very important. Such endeavors can not only yield very important scientific results but can also be used immediately in practical diagnostic applications in the health and food industry and in biological warfare and hazard prevention systems. This project brings together interdisciplinary researchers from the fields of micro/nano-systems technology and microbiology with the knowledge of Bacillus anthracis (anthrax) to further the state-of-the-art in micro-scale detection and identification of Bacillus anthracis. The knowledge developed herein will also apply to other microorganisms. The PIs have developed novel technologies that serve as the basis and starting point for continued state-of-the-art research. Micro-devices will be developed to rapidly detect the viability of the spores upon germination within one doubling cycle of the organisms, providing an electronic output. The devices will also have built-in electronic filters to concentrate the spores and cells inside the biochips at the detection sites. In parallel, biological analysis will be performed to identify the spore coat proteins, especially those novel to B. anthracis spores. These proteins are surface localized and thus useful for spore detection. The eventual system is envisioned to capture only the spores or pathogens of interested inside the bio-chips using these surface protein receptors, concentrate these microorganisms inside this chip, and electronically detect their viability and germination, while reducing the total time to result to less than possible by any other technology.

Integrated biochips are promising devices for the rapid and accurate detection of biological entities such as cells, bacteria, spores, and viruses. These integrated biochips need to concentrate, trap, lyse, and perform genetic identification, all on the same device. Figure 1 shows an overall integrated biochip device that is being developed in our group. The accurate identification of these cells and microorganisms require the lysing of the cells to extract the DNA and proteins for molecular identification. DNA extraction using electrical means that are low power and integrated on to a microchip format can be very useful for realizing fully integrated bioanalytical devices. The objective of this project is to design and develop a method of extracting the genetic material from viruses within a handheld device which are low power, simple to use and compact. We will work with the Western Reserve Strain of Vaccinia Virus as the model virus as the main goal. As a stretch goal, we will perform the experiments with the Human Coronavirus

The objective of this research is to increase the sensitivity of label-free biomolecular detectors to levels achieved by label-based detectors (e.g. fluorescence) that are sensitive but time-consuming and costly. A method that improves sensitivity without modification of sensor structure or operation principle is highly desirable. The approach is to incorporate activated receptor molecules into biosensors. Single-stranded DNA or RNA-based molecules, namely aptazymes that perform enzyme activity upon binding to small targets will be used as receptor molecules. Accordingly, biosensors will detect the activity of relatively large aptazymes as opposed to directly detecting the binding of small target molecules. Since the activity will be triggered by small target molecules, they will be indirectly detected with greatly amplified sensitivity. The efficacy of the technique will be demonstrated on the surfaces of a few model label-free detection systems (both commercial and nanomechanical sensors built in-house) whereby small molecules whose direct binding would reveal signals too small to detect will be indirectly detected via aptazyme activity. All experimental work will be undertaken by Purdue. The aptazyme synthesis will be performed at the University of Texas at Austin.

This research project intends to make a broad impact on high-throughput detection of biomolecules such as DNA, RNA, proteins and small molecules whose sensitive detection is of top importance for early diagnosis of many cancers that take 500,000 American lives every year. The findings of this project will be a significant step towards the development of fast, low-cost and high-resolution biomolecular detectors and towards the understanding of biomolecular interactions on solid surfaces. This project will also have significant educational contributions by setting an interdisciplinary stage where engineering students and chemists can interact and learn from each other. A part of the project will also be linked to a graduate level laboratory course.

The objective of this research is to develop and integrate the building blocks of a nanowire sensor system using a well-coordinated and interdisciplinary effort in design, fabrication and functionalization to form a nanowire sensor array for the label free electrical detection of target biomolecules. The approach is to use top town silicon nano-fabrication to form the nanowires and use a novel thermally mediate exchange reaction as attachment scheme for site specific functionalization of capture bimolecular at different sensor sites. The proposal focuses on the detection of microRNAs (miRNA), which are usually 21-23 nt in length and occur in abundance in cells. These molecules have recently been found to be critical cellular components whose levels of expression correlate with disease states.

Intellectual Merit: The detection of miRNA sequences in itself has significant scientific and commercial implications. In addition, the proposed work will develop the framework by which individual sensor components, perhaps developed in different laboratories, may be efficiently integrated into sensor arrays. Also, the design platform, fabrication techniques, functionalization schemes, and integration approaches developed in the project have many generic features that can easily be adapted not only to other nano-bio systems, but also to nano-electronics, DNA-computing, nano-mechanics and nano-composites.

Broader Impact: Finally, this integrated design approach will involve non-traditional interdisciplinary interactions, with important implications for graduate and undergraduate education, research, and the practice of engineering. We will develop a senior/graduate level course entitled: Introduction to Bio-Nanotechnology. It will be taught to students from many departments. Our program in collaboration with NCN has graduate and undergraduate components as well as outreach to the research community and the public.

[unreadable] DESCRIPTION (provided by applicant): The recent advances in nanotechnology and nanofabrication of semiconductor materials present themselves with new opportunities for miniaturized, ultra-sensitive, label-free, and parallel sensors capable of rapid and highly accurate analysis of biological and chemical entities. Silicon nanowire sensors, based on field effect modulation of current upon binding of molecules on the nanowire surface, have been successfully demonstrated for the detection of DNA and proteins in fluids on individual devices. However, these devices are still to be realized in an array format, in a robust manner using top-down fabrication techniques, with detection of multiple molecules. In this R21 proposal, we have assembled an interdisciplinary team with the goal of designing, producing, functionalizing, and testing the silicon nanowire sensor array. Our work will be motivated by, (a) the need to detect single stranded DNA molecules for sensing and diagnostic applications, and (b) detect microRNA (miRNA) from cell lysates, which have been recently shown to be indicative of diseases such as cancer. These goals will be accomplished by our team of researchers with expertise in nano-scale computation (Ashraf Alam), novel linker design and surface chemistry (Donald Bergstrom), and micro/nano-fabrication (Rashid Bashir). The following are the specific aims of the project: (i) Use novel nanoscale computation approaches to guide the design and fabrication of the nano-wire sensor elements, (ii) Develop novel top-down silicon fabrication techniques to produce nano-wire arrays within microfluidic devices, (iii) Develop novel techniques to functionalize the individual sensors in an array using laser or electrically mediated thermal exchange reactions, and (iv) Perform the detection experiments in an array of nanowires within a microfluidic biochip and explore the use of nano-particle (dendrimer) based charge amplification schemes. Each specific aim includes novel and innovative state-of-the-art nanoscale research with broad applications in the area of Nanobiotechnology. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Nanotechnology is truly revolutionizing our ability to manufacture and design active devices and systems that are small, cheap, and ultra-sensitive. The need to produce small medical devices for rapid and parallel detection or health monitoring is intensifying. Moreover, as the micro-fabricated devices get smaller and smaller with each new development in materials science and engineering, the need to design and fabricate biologically inspired nanoscale devices and sensors and ultra-compact power sources to drive these devices and sensors are emerging. Nature can provide the tools to address the above needs. Molecular motors, such as ATPase [Noji et al., 1997], bacterial flagellar [Sowa et al., 2005], or viral DNA packaging motors [Guo, 2002;Shu et al., 2003] can be utilized to synthesize power at the nano-scale. Some of these motors can generate force up to tens or hundreds of pico Newtons. Some of them can use energy and generate force, via ATP hydrolysis, or use the force and generate energy in the form of ATP, via electrolocomotive force or pH gradients, with efficiencies in the range of 80-100% [Yasuda et al., 2001;Aksimentiev et al., 2004]. Some of these motors can have rotational speeds of 100-1000 rpm [Sowa et al., 2005]. In the recent years, nanofabrication capabilities have progressed to a point where sub 20nm nanopores, nanowires, and nanotubes can be grown at specific locations on a silicon wafer such that these structures can possibly be interfaced with biological motors for applications such as nanomechanics, filtration, locomotion, and energy generation and harvesting. In this project, we propose to develop active nanostructures and systems based on biological nanomotors. Our focus here would be the use of the bacteriophage phi29 DNA packaging nanomotor that is driven and geared by small RNA molecules termed packaging RNA or "pRNA". This nanomotor has been shown to play a novel and essential role in transporting phi29 genomic DNA into procapsids. As more progress is made in understanding the structure and mechanisms of these novel systems, it is time to evaluate these structures using bionanotechnology-based approaches and to explore the interface between these nanomotors and synthetic structures using top down and bottoms up fabrication technology to form active nanostructures and nanosystems. Our core platform will consist of the pRNA-driving motors anchored on nanoporous membranes on micromachined silicon or Alumina based membrane via a 2-dimensional self- assembled DNA crystal. The use of the DNA self assembled layer will ensure the integrity and functionality of the nanomotor. The development and characterization of this basic platform is a significant challenge in itself and requires a cohesive interdisciplinary approach. We will integrate the nanomotor in a hybrid silicon based device and demonstrate its operation, and then integrate the nanomotor without the capsid and demonstrate the translocation of dsDNA through the motor. Once these tasks are accomplished, it will be possible to investigate various technology modules such as active pumping surfaces within microfluidic channels, active sieving and filtration, and many other applications directly relevant to biology and medicine.

DESCRIPTION (provided by applicant): The recent technological advances in top-down silicon nanotechnology and micromachining of semiconductor materials present themselves with new opportunities for small, sensitive, one time use, point of care diagnostic devices capable of rapid and highly accurate analysis of samples of body fluid. Nano- sensors and microfluidic biochips have been successfully demonstrated for the detection of proteins in fluids, yet still to be realized in a robust array format, with detection of multiple proteins, which can be used in preclinical studies or clinical samples. In this proposal, we have assembled a truly comprehensive team of interdisciplinary researchers with the goal of applied and translational multidisciplinary research for designing and producing robust top-down silicon-based field-effect nano-sensor platform technologies integrated in one-time-use point-of-care diagnostic biochips, functionalized with multiple antibodies, for the ex-vivo detection of cancer proteins from cell lysate from breast aspirates. These tasks will be accomplished by our team of researchers with expertise in clinical translation (Susan Clare at ILJSOM), micro/nano-fabrication of silicon based field effect devices and on-chip sample concentration and separation (Rashid Bashir at Purdue and Luke Lee at UC Berkeley), Simulation and computation of nanosensors (Ashraf Alam at Purdue), and Cancer bio-chemistry (Don Bergstrom at Purdue). The following are the specific aims of the project: (i) Develop on chip cell lysing approaches from breast aspirates, (ii) Develop novel techniques to functionalize the sensor array surfaces with antibodies, while minimizing non-specific adsorption and bio-fouling, (iii) Develop novel computational and simulation strategies for modeling the field effect sensor response upon protein binding in fluids, (iv) Develop robust top-down silicon-based field-effect nano-plate arrays for multiplexed detection of cancer proteins &markers, and (v) Develop integrated biochip sensors and perform extensive tests and preclinical studies. We will keep the focus on the issues and requirements towards assay development to perform ex-vivo preclinical studies with human samples.

CBET-0820887
Cunningham

The proposal was submitted by 5 Investigators and 4 Senior Personnel from the departments of Electrical and Computer Engineering, Mechanical Science and Engineering and Bioengineering. The PI, Brian Cunningham, PhD in Electrical Engineering, UIUC, 1990, Associate Professor of Electrical and Computer Engineering, joined the UIUC faculty after nearly 10 years in industry. His work is supported by SRU Biosystems, NSF, DOD, NIH and the Beckman Institute and he has 23 patents awarded for optical sensors and components and more than 20 patents pending. . Rashid Bashir, PhD from Purdue, 1992, just moved to UICU in the fall of 2007 as the Bliss Professor of Electrical and Computer Engineering % Bioengineering and the Director of MNTL. Dr. Bashir has impressive credentials and support from NSF, NIH and NASA. Ken-Yung Cheng, PhD in Electrical Engineering from Stanford, 1975, Professor of Electrical and Computer Engineering, is the Director of the DARPA University Photonics Research Center for Hyper-Uniform Nanophotonic Technology. Kent Choquette, PhD in Materials Science from the University of Wisconsin-Madison, 1990, joined the UIUC Department of Electrical and Computer Engineering in 2000 after nearly 10 years of experience at AT&T and Sandia National Labs. His photonics research is supported by NSF and DARPA. James Coleman, PhD in EE from UIUC, 1975, Professor of Electrical and Computer Engineering and Materials Science and Engineering, has published extensively and holds 6 patents. The Senior Investigators, Nicholas Fang, Milton Feng, Placid Ferreira and William King (NSF CAREER and DOE PECASE awardee), are likewise well funded. In general, the team leading this effort is outstanding. Ten research projects, with all but one currently receiving support from NSF, NIH, NASA, ONR, or DARPA, were described in some detail including information about how access to the proposed lithography equipment, which would enable imprinting more quickly and of larger areas, would benefit research efforts.

This goal of this Integrative Graduate Education and Research Training (IGERT) award is to create a graduate training program that will produce the next generation of intellectual leaders in Cellular & Molecular Mechanics and Bio-Nanotechnology. This program represents a highly coordinated and interdisciplinary effort to educate Ph.D. students across the University of Illinois at Urbana-Champaign, University of California at Merced, North Carolina Central University, and partner institutions to tackle the important problems in bionanotechnology spanning the molecular-cellular-tissue scale.

How living cells transduce mechanical signals to functionalities at different length scales, from inside cells to their communication with the extra cellular matrix, presents a scientific grand challenge of our times. Recent advancements in micro/nanotechnology, molecular scale imaging, and computational methodologies will catalyze this quantitative biological revolution at a cellular and molecular scale. Students who have been trained at the intersection of these domains have the potential to revolutionize tissue and regenerative engineering, biological energy harvesting, sensing and actuation, cells-as-a-machine, and synthetic biology, to name a few. Unique training efforts in this program include a two-track educational program to educate engineering and biology students to develop depth and breadth in their area of research, new experimental modules and a summer workshop introducing the IGERT trainees to state-of-the-art equipment and laboratory procedures, an exciting iWORLD program with collaborators around the world that will provide IGERT trainees with international research experiences, and a student leader council that participates in the leadership and drives the management of the IGERT.

IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries.

DESCRIPTION (provided by applicant): We propose to establish a Midwest Cancer Nanotechnology Training Center (M-CNTC) at UIUC in collaboration with regional clinical collaborators. The goal of this M-CNTC will be to produce the next generation of intellectual leaders who will define the new frontiers and applications of nanotechnology in cancer research. This goal would be accomplished by creating a highly interdisciplinary environment which educates and empowers the students and post-doctoral fellows in the training center to take leadership roles and address the challenges at the cross-roads of cancer biology, physical sciences, and nanotechnology. It is known that more than a 1.5 million people were diagnosed with cancer and half a million died of cancer in US alone during 2007. In spite of a considerable effort, there has been limited success in reducing per capita deaths from cancer since 1950. This calls for a paradigm shift in the understanding, detection and intervention of the evolution of cancer from a single cell to tumor scale. In this M-CNTC proposal, we have responded to this very timely need and assembled a preeminent interdisciplinary team of researchers and educators across UIUC and clinical collaborators in the region from University of Illinois at Chicago, Mayo Clinic, Indiana University School of Medicine, and Washington University at St. Louis, to train the next generation of engineers, physical sciences, and biologists to address the challenge of understanding, managing, diagnosing, and treatment of cancer using the most recent advancements in nanotechnology. The proposed program represents a highly coordinated and interdisciplinary effort to educate scientists and engineers across UIUC and clinical partner institutions to tackle the important problems in applications of nanotechnology to cancer research. Taking advantage of the expertise in nanofabrication, imaging, nano-materials, and cancer biology in our team, the program is expected to significantly impact quantitative methods used in cancer research. Our unique educational training components will not only transform graduate education on our campus, but also to be an empirically validated model for other programs in this area. PUBLIC HEALTH RELEVANCE: We propose to establish a Midwest Cancer Nanotechnology Training Center (M-CNTC) at UIUC in collaboration with regional clinical collaborators. The goal of this M-CNTC will be to produce the next generation of intellectual leaders who will define the new frontiers and applications of nanotechnology in cancer research.

The crisis in the management of infectious disease for the developed world and in the developing world (global health arena) requires rapid, easy to use, integrated, and inexpensive diagnostic devices for the detection of agents of infectious diseases, i.e. bacteria, viruses. In this application, the development of a ?Lab-on-a-Transistor? is proposed for cell capture and thermal lysing, and ultra rapid techniques for performing nucleic acid amplification on silicon transistors with a direct, rapid electrical detection of the amplified products. The grand challenge of making nucleic acid amplification truly a point-of-care test will be addressed, where the results can be obtained with high accuracy and reliability in less than 5 minutes on a silicon sensor array. To address these challenges, the bacteria Listeria monocytogenes will be used as the model system within the three years of this proposed grant but the technology platform can be applied to other pathogenic microorganisms as well.

Intellectual Merit: The proposed concept of developing a ?Lab-on-a-Transistor? has the following intellectual merit; (a) concentrating single bacteria on individual field effect transistors with in a linear array using dielectrophoresis within a microfluidic channel. Then ultra-localized heating on the surface of the field effect sensor using an ac voltage will be explored. Using this method, thermal lysing of bacteria that are attached on the surface of the transistors will be performed by achieving temperatures of 95oC or higher, (b) the same sensor would be used as a heater to perform nucleic acid amplification reactions using either a polymerase chain reaction or a rolling circuit amplification method, and (c) exploring the use of the transistors themselves for label free electrical detection of the PCR products.

Broad Impact: The proposed work will have a broad impact in the area of silicon based biosensors and the proposed technology could provide significant advances in developing point of care sensors. Graduate students will be involved in summer lecture and hands-on workshops to be held at the Micro and Nanotechnology Laboratory at the University of Illinois. REU summer support will be requested for hiring additional undergraduates to work with the graduate students on the research project during the summer, which will also help to develop a pipeline of graduate student researchers for the future.

The Center for Agricultural Biomedical and Pharmaceutical Nanotechnology (CABPN) will focus on developing nanotechnology platforms that can be applied to three substantially important topics requiring strong industry/academic partnerships: Agriculture, Pharmaceutical Research, and Biomedical Applications. The proposed center will be a single-university center located at the University of Illinois at Urbana-Champaign (UIUC).

The proposal touts advances in health care and agriculture at the confluence of biotechnology and nanotechnology in a "convergence of frontiers." The emphasis will be on taking bio-nanomedical developments from the bench to benefit agriculture and healthcare. Advances in this center are anticipated to enhance the development of vaccines for food animals, and safety of the food supply, monitoring patients in intensive care and tools to make pharmaceutical research more effective. The proposed center will enable assembly of a cohesive University0Industry alliance that will enable Industry participants to communicate their research needs to academia, to facilitate collaboration between companies in different market spaces, and to train graduate students as effective future leaders.

Fostering cooperation between the hard core nanotechnology practitioners (mostly coming out of electronics and materials departments) and those in agriculture, biomedicine, and pharma could provide a cross-discipline platform on which to stir the intellectual melting pot and generate innovative solutions. Should CABPN succeed in transitioning the tools of nanotechnology to commercial use in these unrelated fields while gaining synergies from their interaction at the center, it could be a real driver for commercial innovation. Success could produce jobs and improve the life of the nation. CABPN has planned an active role in mentoring students and has incorporated them into its overall plan. The proposed Center has a system for outreach to minorities to foster training in nanotechnology to the widest group of individuals possible, and plans to have meetings within the I/UCRC framework to widely disseminate the technologies within the staff of the I/UCRC as well as partnering organizations. Publication of works is also planned and the PI has a path to publication that takes into account the commercial interests of the partner organizations.

DESCRIPTION (provided by applicant): Cancer is a leading cause of death worldwide accounting for approximately 13% of all deaths in 2004. It is becoming more and more apparent that cancer is as much a disease of misdirected epigenetics as it is a disease of genetic mutations. Epigenetic alterations occur in the form of DNA methylation changes, an early and frequently observed event in carcinogenesis. Interestingly, cancer-specific methylated DNA from most tumor types is readily available in bodily fluids and biopsy specimens and also exists in the form of free-floating DNA shed by dead cancer cells. A technology capable of detecting aberrant methylation patterns in specific genes extracted from the serum of cancer patients would be of immense clinical value. We propose using solid-state nanopore sensors for the detection of robust cancer biomarkers (specifically DNA methylation patterns) at ultra low concentrations in human serum samples. Nanopore sensors use the principle of electrical current spectroscopy to interrogate individual DNA molecules, with the sensitivity to discern subtle structural motifs in single molecules. Nanopore technology is also well suited for gene based methylation analysis, capable of screening small panels of hypermethylation markers specific to a variety of cancers. Nanopore sensors could potentially play an important role in early cancer detection, risk assessment, disease monitoring, chemoprediction and patient prognosis. In working towards our goal of nanopore based methylation analysis, in this R21 we propose the following specific aims: (i) We will first explore the methylation detection capabilities of nanopore sensors using commercially available, fully methylated DNA fragments, and (ii) We will perform Methylation detection and quantification of methylated plasmid DNA. Depending on the outcomes of Aims 1 and 2, this technique may be extended in a follow on R01 to the analysis of pre-clinical and clinical samples, specifically the detection of aberrant methylation patterns in DNA isolated from the serum of prostate cancer patients. PUBLIC HEALTH RELEVANCE: Direct detection of DNA methylation patterns in cancer genes can be very important in early cancer detection, risk assessment, disease monitoring, and patient prognosis. We propose the use of solid-state nanopore sensors for the detection of these methylation patterns.

Measurements of BPDE-DNA adducts by solid state nanopore and deep sequencing (PQ3) Potent carcinogen Benzo[a]pyrene (BaP) is found in heavily polluted air, smoked food, and tobacco smoke. Once inside cells, BaP can form stable benzo[a]pyrene dihydrodiol epoxide (BPDE) DNA adducts leading to the insertion of incorrect bases during replication. BPDE-DNA adducts are considered biomarkers of exposure to BaP and increased concentrations of BPDE-DNA adducts in white blood cells and in tissues such as lung and esophageal are associated with increased risk of cancer. Current methods for BPDE adduct measurements either involve highly radioactive reagents and are non-specific, or require highly specialized equipment and prohibitively large amounts of DNA for most epidemiological studies. Novel sensors for specific biomonitoring of genomic BPDE adduct which do not require hazardous reagents, or cumbersome procedures will speed the links between BaP exposure and development of various cancers. Furthermore, it is difficult to assess the associations of adduct at specific genomic loci and various cancers. The binding of BPDE on the genomic DNA is not random. Recent reports have demonstrated a preferential distribution of the BPDE-DNA complexes on the methylated CpG dinucleotides and on the mutational hotspots in tumor suppressor p53 and ras oncogene. However, with the existing methodologies BPDE adducts can only be examined at a very limited number of genomic loci. Such investigations would require laborious techniques involving highly radioactive reagents and sequencing gels. Novel non-intensive methodologies for estimating BPDE concentrations on the genome could provide new tools for identifying potential links between BaP exposure and specific mutations and epigenetic alterations that cause cancer. The objective of this proposal is to develop new solid state sensors and novel omic -style methodologies for the measurement of DNA adducts. Both approaches will be specifically tuned to BPDE-DNA measurements and will have the sensitivity needed for subsequent studies in human cancer using surgical tissues without requiring prohibitively large amounts of DNA. Aim 1 will develop a solid state nanopore measurement system for single molecule detection of BPDE-DNA adducts. Aim 2 will develop a protocol for genome mapping BPDE adducts in DNA from cell lines exposed to BaP or BPDE. This research will provide the proof of concept and scientific evidence upon which subsequent experiments can be designed for assessing BaP exposures in specific populations. Achieving these goals will open new areas of research and provide valuable new tools for fast and detailed measurements of long and short term exposure to the BaP. The approaches introduced here can be extended to most other toxins and carcinogens that form DNA adducts. These findings will benefit diverse areas of cancer and scientific research, such as toxicology, where adducts are studied.

The objective of the research is to investigate and promote the use of pico-liter scale droplets for encapsulation and monitoring of bacterial growth, mammalian cell growth, and PCR reactions. The approach is to ascertain both experimentally and numerically, the device design, mechanisms of encapsulation, and biological reactions inside the droplets. The research is expected to result in increased understanding of the fundamental mechanisms as well as realization of practical devices for point-of-care diagnostics and screening applications.
The intellectual merits of the proposed work include the development of novel numerical techniques for simulation of the cell encapsulation process, obtaining fundamental insight into the process of cell encapsulation in the droplets of cell media or buffer, examining the effect of ionic liquid medium on the cells, a systematic experiment and simulation approach to design the devices for optical capture, and whether single cell reactions can be interrogated inside these droplets.
The broader impact of the proposed work is that a further understanding of the mechanism of cell encapsulation process in micro-channels will directly guide the use of droplets as reaction vessels for cells and molecules in biochemical applications. Thus, the proposed study can serves as generic platform that allows the biological activities to be monitored in real time using electrical means resulting in compact devices. The devices would be used to demonstrate fundamentals of microfluidics and bioassays to high school students in science fairs at OSU and as part of a microfluidic module in a 2 week long summer school at UIUC.

Measurements of BPDE-DNA adducts by solid state nanopore and deep sequencing (PQ3) Potent carcinogen Benzo[a]pyrene (BaP) is found in heavily polluted air, smoked food, and tobacco smoke. Once inside cells, BaP can form stable benzo[a]pyrene dihydrodiol epoxide (BPDE) DNA adducts leading to the insertion of incorrect bases during replication. BPDE-DNA adducts are considered biomarkers of exposure to BaP and increased concentrations of BPDE-DNA adducts in white blood cells and in tissues such as lung and esophageal are associated with increased risk of cancer. Current methods for BPDE adduct measurements either involve highly radioactive reagents and are non-specific, or require highly specialized equipment and prohibitively large amounts of DNA for most epidemiological studies. Novel sensors for specific biomonitoring of genomic BPDE adduct which do not require hazardous reagents, or cumbersome procedures will speed the links between BaP exposure and development of various cancers. Furthermore, it is difficult to assess the associations of adduct at specific genomic loci and various cancers. The binding of BPDE on the genomic DNA is not random. Recent reports have demonstrated a preferential distribution of the BPDE-DNA complexes on the methylated CpG dinucleotides and on the mutational hotspots in tumor suppressor p53 and ras oncogene. However, with the existing methodologies BPDE adducts can only be examined at a very limited number of genomic loci. Such investigations would require laborious techniques involving highly radioactive reagents and sequencing gels. Novel non-intensive methodologies for estimating BPDE concentrations on the genome could provide new tools for identifying potential links between BaP exposure and specific mutations and epigenetic alterations that cause cancer. The objective of this proposal is to develop new solid state sensors and novel omic -style methodologies for the measurement of DNA adducts. Both approaches will be specifically tuned to BPDE-DNA measurements and will have the sensitivity needed for subsequent studies in human cancer using surgical tissues without requiring prohibitively large amounts of DNA. Aim 1 will develop a solid state nanopore measurement system for single molecule detection of BPDE-DNA adducts. Aim 2 will develop a protocol for genome mapping BPDE adducts in DNA from cell lines exposed to BaP or BPDE. This research will provide the proof of concept and scientific evidence upon which subsequent experiments can be designed for assessing BaP exposures in specific populations. Achieving these goals will open new areas of research and provide valuable new tools for fast and detailed measurements of long and short term exposure to the BaP. The approaches introduced here can be extended to most other toxins and carcinogens that form DNA adducts. These findings will benefit diverse areas of cancer and scientific research, such as toxicology, where adducts are studied.

DESCRIPTION (provided by applicant): HIV/AIDS affects more than 33 million people throughout the world, and is especially a critical problem in resource-poor regions in sub-Saharan Africa, where 67% of all HIV/AIDS patients live. Antiretroviral therapy (ART) increases the longevity and quality of life for HIV patients, and global efforts have increased the accessibility of such treatment by 30-fold in sub-Saharan Africa between 2003 and 2008. However, the lack of objective diagnostic tests to determine when to start ART and to monitor its success hinders the effective use of treatment. In addition to the counting of CD4+ T Lymphocytes, it is also highly desirable to perform viral load counts at the point-of-care, as both these parameters are needed for the development of the appropriate treatment strategy. HIV viruses could occur at levels ranging from 10pfu/¿l to thousands of pfu/¿l of whole blood, making it challenging to detect these minute quantities of particles. Current tests include both antibody- based and PCR-based and are not available at point-of-care, especially for resource-limited settings. It should be also pointed out that such devices would of course be extremely valuable also for the developed world, for remote settings, at bedside, or at the doctor's office for a range of applications in detection of viruses. As a solution to these problems presented above, micro-fabricated point-of-care (POC) biochips for HIV/AIDS analysis hold tremendous promise. We propose to develop an integrated device for the electrical detection of HIV viral load at point-of-care. We propose to build on our extensive preliminary work to develop electrically- based sensing methods within micro-fluidic biochips to greatly reduce the operating costs, increase portability, and provide simple-to-use diagnostic kits that can be operated by healthcare workers in remote facilities or during home visits. Our integrated approach is innovative as we will; (i) capture the specific viruses from whole blood, (ii) label these viruses using liposomes tagged with antibodies for the viral particles, (iii) lyse the liposomes by lowerin the electrical conductivity of the medium, and detect the changes in the electrical impedance of the medium in the microfluidic capture chamber. The change in impedance is expected to correlate to the number of virus particles captured. We plan to use VSV-G virus spiked in whole human blood as a model for HIV during this R21 to demonstrate the proof of concept of the novel detection scheme. The interdisciplinary collaboration brings together the expertise of the Co-PIs. We will build on the extensive work in Bashir group (UIUC) on development of microfluidic point-of-care tests that are electrically based, and in Lee Lab (UCI) in microfluidics and generation of liposomes of controlled size and properties. With the integration of these two sets of expertise, we expect to address the grand challenge of developing point-of-care viral load assays.

DESCRIPTION (provided by applicant): HIV/AIDS affects more than 33 million people throughout the world, and is especially a critical problem in resource-poor regions in sub-Saharan Africa, where 67% of all HIV/AIDS patients live. Antiretroviral therapy (ART) increases the longevity and quality of life for HIV patients, and global efforts have increased the accessibility of such treatment by 30-fold in sub-Saharan Africa between 2003 and 2008. However, the lack of objective diagnostic tests to determine when to start ART and to monitor its success hinders the effective use of treatment. In addition to the counting of CD4+ T Lymphocytes, it is also highly desirable to perform viral load counts at the point-of-care, as both these parameters are needed for the development of the appropriate treatment strategy. HIV viruses could occur at levels ranging from 10pfu/¿l to thousands of pfu/¿l of whole blood, making it challenging to detect these minute quantities of particles. Current tests include both antibody- based and PCR-based and are not available at point-of-care, especially for resource-limited settings. It should be also pointed out that such devices would of course be extremely valuable also for the developed world, for remote settings, at bedside, or at the doctor's office for a range of applications in detection of viruses. As a solution to these problems presented above, micro-fabricated point-of-care (POC) biochips for HIV/AIDS analysis hold tremendous promise. We propose to develop an integrated device for the electrical detection of HIV viral load at point-of-care. We propose to build on our extensive preliminary work to develop electrically- based sensing methods within micro-fluidic biochips to greatly reduce the operating costs, increase portability, and provide simple-to-use diagnostic kits that can be operated by healthcare workers in remote facilities or during home visits. Our integrated approach is innovative as we will; (i) capture the specific viruses from whole blood, (ii) label these viruses using liposomes tagged with antibodies for the viral particles, (iii) lyse the liposomes by lowerin the electrical conductivity of the medium, and detect the changes in the electrical impedance of the medium in the microfluidic capture chamber. The change in impedance is expected to correlate to the number of virus particles captured. We plan to use VSV-G virus spiked in whole human blood as a model for HIV during this R21 to demonstrate the proof of concept of the novel detection scheme. The interdisciplinary collaboration brings together the expertise of the Co-PIs. We will build on the extensive work in Bashir group (UIUC) on development of microfluidic point-of-care tests that are electrically based, and in Lee Lab (UCI) in microfluidics and generation of liposomes of controlled size and properties. With the integration of these two sets of expertise, we expect to address the grand challenge of developing point-of-care viral load assays.

Nanotechnology has tremendous promise in improving the human health through better imaging, diagnostics or drug delivery. AME2014 International Congress on NanoEngineering for Medicine and Biology (NEMB 2013) will help catalyze and spur on this field by bringing together leading academics with interests in experimental and theoretical aspects of nanotechnology. These experts will discuss topics related to improved prediction of nanoparticle delivery in the body, to novel strategies for using nanomaterials for disease detection and for construction of diagnostic platforms. The exchange of ideas and interactions afforded by this conference will help identify problems and possibilities and will lead to new collaborations and new concepts in the field of nanotechnology and nanomedicine.

This Partnerships for Innovation: Building Innovation Capacity (PFI:BIC) project will develop a mobile sensor technology for performing detection and identification of viral and bacterial pathogens. By means of a smartphone-based detection instrument, the results are shared with a cloud-based data management service that will enable physicians to rapidly visualize the geographical and temporal spread of infectious disease. When deployed by a community of medical users (such as veterinarians or point-of-care clinicians), the PathTracker system will enable rapid determination and reporting of instances of infectious disease that can inform treatment and quarantine responses that are currently not possible with tests performed at central laboratory facilities.

Polymerase Chain Reaction (PCR) and Loop-Mediated Isothermal Amplification (LAMP) currently represent the most sensitive and specific approaches for identification of viral or bacterial pathogens, with intense research focus directed towards miniaturization, acceleration, and automation of the protocol for amplifying disease-specific DNA sequences to easily-measured concentration. The plan is to apply the results of previously NSF-funded advances in photonic crystal enhanced fluorescence (PCEF) and smartphone fluorescence spectroscopy to implement PCR or LAMP assays within sub-µl liquid volumes for reduction in the assay amplification time to register a measurable fluorescent signal. Importantly, the detection approach enables >10x multiplexing of PCR (or LAMP) reactions within a chip that can be "swiped" through a custom handheld detection instrument that interfaces with the back-facing camera of a conventional smartphone in a manner that is similar to reading a credit card. A mobile device software application will guide the user through the assay process, interpret the results of the detection (including correlation of assay measurements with on-chip experimental controls), and communicate results to a cloud-based data management system along with other relevant information provided by the user. Importantly, the app will enable the user to view the results of tests performed by other users, with a mobile device interface that enables simple visualization of the locations, times, and circumstances surrounding positive/negative tests. The system will enable users to request customizable alerts when positive tests occur within the network of users, and to highlight confirmed positive cases when conventional laboratory tests can confirm results of positive field tests. The app will track outcomes and report statistics on system performance, including Receiver Operating Characteristic of assays.

While the system will initially be deployed in the context of equine infectious disease representing an opportunity to mitigate enormous economic losses associated with infectious disease in the horse industry, the developed technology will be equally applicable to humans, food animals, and companion animals. Considering the economic and health impact of ebola, HIV, tuberculosis, and malaria, when PathTracker is fully deployed within developing nations, the potential of the system to save lives by rapid delivery effective treatment, quarantine of infectious patients, and rapid identification/reporting of new cases is enormous.

At the inception of the project, the primary partners are the lead institution: University of Illinois at Urbana-Champaign (Department of Electrical and Computer Engineering, Department of Bioengineering, and National Center for Supercomputing Applications); University of Washington at Seattle (academic institution); Perkin Elmer, Diagnostics R&D Division, (Waltham, MA) (Large business); Motorola Mobility (Chicago, IL) (Large business);and Dr. David Nash, D.VM.(Lexington, KY) (Individual practitioner veterinarian).

In this proposed departmental revolution, the Bioengineering Department at the University of Illinois is aligning its undergraduate curriculum with medical practice and education by designing the curriculum around a simple message, "no solution without a need." The rising cost of healthcare, the increased role of technology in medicine, and emerging ethical dilemmas created by an increasing and aging population require that all bioengineers and healthcare providers understand the social and ethical context of their work to derive solutions that can meet these complex needs. However, students are typically isolated from these social contexts during their technical training, limiting their ability to identify and understand the needs of society and healthcare providers. Consequently, these students are limited in their ability to derive optimal engineering or technological solutions. The Bioengineering curriculum is being changed to achieve four objectives: 1) redesign the curriculum so that societal needs for healthcare and medicine drive the technical content, 2) integrate out-of-class experiences so that students receive hands-on practice with identifying and understanding societal and healthcare provider needs, 3) translate medical assessment practices to align clinical experiences with the curriculum, and 4) develop faculty teaching skills to meet these new challenges by engaging their intrinsic motivations to revolutionize the department. This revolution is driven locally by the creation and launch of the first Engineering-Based College of Medicine in the nation in fall 2018 that will integrate instruction in engineering with clinical and biological sciences.

Catalyzed by the Grinter Report, engineering education was previously revolutionized by aligning its practice and education with science. This alignment created a social-technical duality in engineering where the technical skills were elevated and social skills were relegated. In response, calls have risen for holistic training of engineering students who understand the societal needs and the societal implications of their practice. This change can be accomplished by aligning sub-disciplines of engineering with other holistic disciplines. The next revolution in bioengineering education can be brokered by realigning with healthcare and medicine - areas of impact and practice that holistically integrate social and technical aspects. This revised curriculum integrates clinical experiences that provide a context for students to learn ethnographic methods for user-oriented needs identification and problem scoping. While traditional curricula organize courses by their technical content, the new curriculum organizes courses by the needs that the curriculum will empower students to solve. Needs such as age-related disease, global health, and cancer provide the starting point for students who are navigating their curriculum. The use of new assessment tools such as competency-based models and e-portfolios integrate these new curricular tracks with the clinical experiences. Finally, to help faculty learn how to execute this new curriculum, the Bioengineering faculty are organized into Communities of Practice to execute course and assessment revisions. This process is driven by departmental administration who are guided and supported by organizational change researchers. This revolution is being spread beyond the Illinois campus through accreditation agencies and technical societies.

This National Science Foundation Research Traineeship award to the University of Illinois at Urbana-Champaign will address the next frontier in biotechnology: to engineer, and then decipher and harness, the living three-dimensional brain. The program will provide doctoral students with the skills and knowledge base to develop and utilize miniature brain machinery in an effort to understand and regulate brain activities. To achieve the goals of developing cross-disciplinary researchers, trainees will learn diverse fundamentals in biology, mathematics, engineering, and cognitive science, relevant to miniature brain machinery. The training grant anticipates providing a unique and comprehensive training opportunity for sixty (60) PhD students, including thirty four (34) funded trainees. Trainees will be recruited from neuroscience, cell and developmental biology, molecular and integrative physiology, chemistry, chemical and biomolecular engineering, bioengineering, electrical and computer engineering, and psychology. The training program will foster a culture of innovation and translational research, and will produce a new generation of scientists and engineers prepared to tackle major problems in brain studies that can improve the quality of human life.

The research and training program will bridge two dominant, non-overlapping brain research paradigms: i) cognitive and behavioral studies, focused principally on understanding of adaptation, decision-making, psychology, and learning of an individual using bioimaging and computational tools vs. ii) cell and tissue studies, focused on activities of multiple neuronal cells by altering their internal and external microenvironments comprised of biomolecules, extracellular matrix, and external stimuli. The goal of this NRT training program is to unite these two dominant paradigms in brain science studies and bridge the expertise of cell and molecular biologists, physiologists, chemists, nano/micro technologists, and cognitive neuroscientists. This training program prepares students for studies that enable control over the networks producing behavior and thus to study causal relations. The overarching goal of the program is to provide students with an interdisciplinary curriculum grounded in problem-based learning and an immersive research experience that blends techniques from multiple disciplines. A second goal is to increase the participation of women, underrepresented minorities, and students with disabilities in neuroscience, life sciences, chemical sciences, and engineering fields. A third goal is to train students in communication skills with the public. Evaluative studies conducted throughout this research traineeship project will explore the dynamics and efficacy of interdisciplinary collaboration by students in this program. Project outcomes will be a demonstrated, evaluated model for transformative graduate training that is effective in developing broadly trained professionals.

The NSF Research Traineeship (NRT) Program is designed to encourage the development and implementation of bold, new potentially transformative models for STEM graduate education training. The Traineeship Track is dedicated to effective training of STEM graduate students in high priority interdisciplinary research areas, through comprehensive traineeship models that are innovative, evidence-based, and aligned with changing workforce and research needs.

The goal of this project is to model, design, fabricate and study micrometer to centimeter size soft bio-hybrid robots that bring together artificial elements and living biological cells. Living components promise to provide these robots with a natural organism's abilities to self-assemble, heal, grow, and to adapt to varying environments. This project is made possible by recent advances in fabrication techniques that allow artificial elements to be combined with an array of living cell types such as muscles and neurons. The proposed mini bio-hybrid robots are thus compliant, configurable, biocompatible, and can generate power from local nutrients. Bio-hybrid robots can also exhibit adaptive behaviors in response to uncertain environments, and thus offer the promise of a host of high impact applications including localized drug delivery, environmental exploration and chemical sensing, evaluation of potential surgical sites within the body, and precision manipulation and fabrication. This is in line with the national need to reduce healthcare costs by enhancing medical effectiveness as well as stimulating the development of advanced manufacturing techniques to increase competitiveness. This effort speaks to a broad audience: what can spark the imagination of potential young scientists better than bringing tiny robots to life. Excitement in this area will be leveraged via activities ranging from museum exhibitions to the deployment of high school and K-12 intuitive learning modules, in order to foster interest in advanced computing, mechanics, math, manufacturing, and biology.

Soft robotics is currently constrained by a lack of rigorous engineering methods. Intuition-driven approaches are further strained in the case of bio-hybrid systems due to the inclusion of the living component, which is even less well understood. This project seeks to create a systematic approach based on modeling, simulation, and fabrication to overcome these limitations and enable deployment and scalability. Central to this project is the concept of functional units, hierarchical bio-hybrid assemblies that can be combined into integrated robotic systems capable of higher level functions, much like the organization of living organisms and object-oriented software. By embracing this analogy, five fundamental building blocks (muscle and neuron cells, elastomers, multi-stable buckling structures, wireless conformable electronics) are considered, so as to: (1) introduce a novel mathematical formalism based on Cosserat rod assemblies to model their mechanical response, actuation and interaction with the environment; (2) develop software to simulate the dynamics of composite functional units and to optimize them according to a desired target behavior; (3) fabricate and test obtained solutions, and update models based on experiments; (4) showcase new understanding by engineering integrated bots able to perform complex tasks. Thus, this effort overall deals with enabling the vision of autonomous, adaptive mini-bots and laying the analytical, computational and experimental foundations of a bio-hybrid soft engineering science.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Principal Investigator/Program Director (Last, First, Middle): Bashir, Rashid Summary: Sequencing the human genome has helped to improve our understanding of disease, inheritance and individuality. The growing need for cheaper and faster genome sequencing has prompted the development of new technologies that surpass conventional Sanger chain-termination methods in terms of speed and cost. These next-generation sequencing technologies ? inspired by the $1,000 genome challenge proposed by the National Institutes of Health in 2004 ? are beginning to revolutionize personalized medicine. Nanopore sensors are one of a number of DNA sequencing technologies that are currently poised to meet this challenge. Biological nanopores such as a-hemolysin and MspA, which consist of molecular motors anchored at the pore, have shown very promising results for ionic current based sequencing of ssDNA molecules, and systems using these pores are now being commercialized by Oxford Nanopores Technologies. However, biological nanopores do not provide the potential of direct single nucleotide read since the pore length spans 5-6 bases long. Solid-state nanopores using two-dimensional materials such as graphene, MoS2, and others could address this challenge regarding spatial resolution of sensing and controlling the DNA motion are addressed. As well as robustness and durability, the solid-state approach offers the ability to potentially fabricate high- density arrays of nanopores, attractive mechanical and chemical characteristics, and the possibility of integrating with novel electronic detection mechanisms. Despite the potential promise, to-date solid state nanopores have yet to demonstrate DNA sequencing, and resolving the challenges require discovering new mechanisms of sensing and translocation control. In this proposal, we introduce a completely new type of sensor which has the desired spatial resolution of sub nanometer and can potentially control the translocation of the DNA molecule. This high risk, high reward approach consists of engineering a nanometer scale out of plane diode using a 2D heterostructure consisting of crossed junction of monolayer MoS2 on monolayer WSe2. Unlike the nanopores in single monolayers, the new sensor allows multi-terminal measurements to probe different physical phenomena within the heterostructure simultaneously, enabling correlated measurements and control of the DNA translocation through the nanopore. The out of plane electric fields at the reverse bias junction will allow for sub nanometer spatial probing of the DNA molecule, and can also reduce the stringent requirement of measuring the change in in-plane conductivity of nanoribbons in which nanopores are formed. The applied biases and local electric field can also be used to control the translocation speed of the molecule. Understanding the relative contributions, interaction, and crosstalk of these different signals is the key scientific goal of this proposal. The key technological goal is to use the new readout schema to achieve single base pair resolution in sensing within a solid state nanopore.

Abstract Infectious disease remains the world?s top contributor to death and disability, and, due to their ability to spread rapidly through insect vectors or contact with bodily fluids, there is an urgent need for simple, sensitive and easily translatable point-of-care tests, particularly for infections that are difficult to differentiate based upon clinical symptoms alone. This project develops a novel point-of-care platform to quantitatively diagnose viral infectious diseases (Zika, Dengue (types 1 and 3), and Chikungunya) from whole blood samples using a microfluidic platform that performs sample pre-processing in a microfluidic cartridge followed by real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) in the same cartridge with pre-dried primers specific to pathogen targets. Our handheld, inexpensive (~$500), point-of-care platform communicates its measurements to a smartphone to process a sequence of fluorescence images of the amplification reaction. Image analysis of the dynamic amplification process is used to estimate the pathogen count from amplification initiation points using a novel approach called ?spatial? LAMP (S-LAMP). Our preliminary data shows that the approach provides selectivity and detection limits that are equivalent to conventional Polymerase Chain Reaction (PCR) or LAMP reactions performed with conventional laboratory instruments, and the ability to detect the targets with high specificity from complex media. The S-LAMP approach demonstrates potential to detect 1-5 pathogen copies per reaction. The system will be evaluated using a rigorous tiered approach using plasmids containing the target nucleic acid sequence for initial characterization of detection limits, followed by nucleic acid pathogen extracts, culminating of detection of the pathogens in whole blood using chemical lysis to release target nucleic acids. Partnering with collaborators at the Institute for Infectious Diseases in Brazil, and Carle Clinic in Urbana, the system will be utilized on patient sample repositories and measurements compared to gold standard analysis. Importantly, as detection, image processing, and quantitation are performed with the smartphone microprocessor, detection is easily integrated with a cloud-based database reporting system that facilitates communication with remotely-located physicians and tracking of epidemiological data by health services. The resulting platform will be broadly applicable to a wide variety of multiplexed panels of pathogens that are of interest for human health, animal health, food safety, and environmental monitoring.

Multiplexed Pathogen Detection from Whole Blood for Rapid Detection of Sepsis Abstract Sepsis, an adverse auto-immune response to an infection often causing life-threatening complications, results in the highest mortality and treatment cost of any illness in US hospitals. In this proposal we propose a novel microchip to bridge the existing gaps in the pathogen detection (poor sensitivity and long processing time). Our strategy in the proposed work is performing picolitre volume RT-LAMP reactions on dried blood using our in-house developed bi-phasic reaction to achieve a highly sensitive pathogen count (1-3 CFU/mL). The realization of this microwell array platform potentially overcomes the limitations in sensitivity and time of response of the current blood cultures. We expect that this device will greatly contribute to the faster rapid diagnosis of sepsis. As a final goal, we anticipate that this platform will replace the use of blood culture for pathogen detection.

Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, the virus that causes the coronavirus-associated acute respiratory disease COVID-19) jumped from an animal reservoir to humans in December 2019, it has rapidly spread across the world, bringing death, illness, disruption to daily life, and economic losses to businesses and individuals. The rapid development of the COVID-19 pandemic highlights the shortcomings in the existing laboratory-based testing paradigm for viral diagnostics that features a widespread lack of test kits, extended time delays to obtain test results, and a high rate of false negative tests. The shortcomings of the existing laboratory-based infrastructure are contributing to uncertainty surrounding quarantine failure, confusion among health authorities, and also to a general public anxiety. To address this critical and timely need, in this work, development of point-of-care device is proposed for detecting the presence of SARS-CoV-2 from nasal fluid samples and in 10 minutes.

The fundamental limitations of current assays for viral pathogens stem from their reliance upon polymerase chain reaction (PCR) analysis, which requires complex, labor-intensive, laboratory-based protocols for viral isolation, lysis, and removal of inhibiting materials. Importantly, PCR requires a large number of time-consuming and precise thermal cycles to enzymatically amplify specific RNA sequences. In this work development of an electrical, label-free and surface modification-free point-of-care device for detecting the presence of SARS-CoV-2 (1-3 copy/mL) from nasal fluid samples and in < 10 min. The proposed work will combine Bst polymerase in an isothermal RT-LAMP (reverse transcription loop-mediated isothermal amplification) reaction, with target-specific primers and crumpled graphene field-effect transistors (gFET) to electrically detect the amplification event by sensing the consumption of primers. These reactions offer the possibility of rapidly detecting SARS-CoV-2 using a simple, inexpensive and portable potentiostat and avoiding the necessity of RNA extraction. In order to develop the proposed detection technology, this proposal will first validate twelve sets of RT-LAMP primers specific to SARS-CoV-2. Importantly, No RT-LAMP primers specific to SARS-CoV-2 are available commercially, neither in the literature. Then, the proposal will demonstrate the feasibility of the RT-LAMP-based electrical approach to detect SARS-CoV-2. Likewise, the proposal will demonstrate the approach as a global health technology to contribute to providing low-cost diagnostics around the world using portable and inexpensive heating block and potentiostat for virus detection and quantification.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Principal Investigator/Program Director (Last, first, middle): Bashir, Rashid Project Summary: Sepsis, a life-threatening organ dysfunction caused by a dysregulated host response to infection (Sepsis- 3 definition), is the leading cause of death and most expensive condition in hospitals. Annually, > 30 million people affected worldwide, with at least 1.7 million adults developing sepsis (nearly 270K die) at a cost of $24 billion per year in the U.S. Patients diagnosed with sepsis and no ongoing sign of organ failure have about a 15-30% chance of death. However, the mortality rate can increase up to 40-60% for severe sepsis or septic shock patients. One in three patients who die in a hospital have sepsis. One major factor in these rising mortality rates is the inability to accurately and quickly diagnose potentially septic patients. Likewise, sepsis is a leading cause of hospital readmission (higher proportion than hospitalizations for heart attack, heart failure, COPD, and pneumonia in the U.S.). EDs and ICUs rely on monitoring extremely non-specific parameters (e.g. fever, low blood pressure, increased heart rate) to initiate a clinical diagnosis and begin treatment. These crude indicators cause doctors to mistake early stage sepsis with several other diseases. A positive diagnose of early onset sepsis is critical because mortality increases with delays in treatment. Survival rates have been reported to drop by 7.6% every hour that the proper antibiotics are not administered, and these delays compound unnecessary hospital costs. Over the last 30 years, clinics have used different criteria such as SIRS, LODS and SOFA or qSOFA as screening tools to assess the severity of organ dysfunction in a potentially septic patient. Common factors among these criteria are non-specificity and very high false positive rates. For patients with positive criteria, the final diagnostic test is a blood culture that may take up to 5 day for a negative result. Likewise, blood culture has a very high false negative rate (> 60%) and does not work for fastidious pathogens such as Chlamydia pneumoniae. More importantly, blood culture cannot be a gold standard method for sepsis diagnosis. This technique only detects the presence of bacteria in the bloodstream (bacteremia), which does not necessarily indicate illness. Many non-bacteremic infections can also cause life-threatening sepsis. In order to improve the accuracy and sensitivity of sepsis diagnosis, the Sepsis-3 definition underscores the requirements for both pathogen detection and information about the personalized state of the immune system of the patient. Therefore, we propose to focus our efforts on monitoring selective biomarkers of this immune response. However, no single, or even a combination of biomarkers has been validated for the diagnosis of sepsis. Because no single biomarker is specific enough to predict sepsis, we propose to develop a point-of-care microfluidic biochip for measuring cell-surface and plasma-proteins biomarkers that will be used for contributing in early sepsis diagnosis. The microfluidic biochip will provide a complete white blood cell count (WBC), as well as quantification of CD64 expression on neutrophil (nCD64), procalcitonin (PCT), C-Reactive Protein (CRP) and Interleukin 6 (IL-6). Multiple studies have demonstrated the high sensitivity of these biomarkers to sepsis. The proposed device will combine for the first time the analysis of cell-surface proteins and plasma proteins biomarkers from the same sample of blood. Such a device, combined with the routinely test performed in the hospitals, could significantly accelerate the diagnosis of sepsis and as consequence the clinical decision, to provide the correct treatment to the patients.