Tony J. Huang - US grants

This NIRT proposal focuses on developing manufacturable NanoElectroMechanical Systems (NEMS) in which light-driven artificial molecular shuttles are used as the "active nanostructures." The PIs propose to (1) exploit the "bottom-up" based synthetic routes to create novel photoactive bistable rotaxane-based molecular shuttles, (2) develop multiscale computer-aided design methodologies to help design and optimize the molecular structures/properties of the molecular shuttles, (3) experimentally quantify the response time for photoactive bistable rotaxanes switching from one state to another, (4) develop hierarchical nanomanufacturing process for the creation of bistable rotaxane-based polymer micro/nano structures with controllable molecular architecture, and (5) demonstrate a new class of functional MEMS/NEMS devices with photoactive bistable rotaxane-based molecular machines as the key "active nanostructures," including microfluidic platforms, chemical/biological sensors, and energy conversion/storage systems.

Intellectual Merit: The PIs proposed research will lead to the creation of new synthetic routes for artificial molecular machines, and new computer-aided methodologies for the design and optimization of molecular structures/properties. The proposed research will result in breakthrough achievements in real functional molecular-machine-based NEMS. Thus, they aim their effort at the essence of nanotechnologys promise, proving the value of the enormous investment already made and stimulating accelerated activities in these areas.

Broader Impacts: The PI and three co-PIs each has longstanding commitments to education and community outreach activities. With the support from this NIRT grant, their team from four institutes will be able to expand the existing or start new outreach activities for minority groups, high-school students, undergraduates, high-school teachers, and middle-school students. The progress in our proposed endeavors will be included in courses being taught at the four universities. In addition, immersing their students in this interdisciplinary approach to nanotechnology, involving organic chemistry, computational material science, and nanoengineering will better equip students to adjust to the ever-changing scientific world, enabling them to develop into future leaders.

Abstract
ECCS-0801922
T. Huang, PA ST U University Park

Objective: The proposal focuses on developing a new class of ultra-small, all-optical plasmonic switches using photoactive rotaxanes as active components. The proposed photoactive rotaxane-based all-optical plasmonic switches could achieve unprecedented performance (size: molecular level; energy consumption: 1-2 eV; excellent reversibility and flexibility) and be integral components for the future ultra-small, ultra-fast plasmonic circuits and very large scale electronics and photonics integration (VLSEPI).

Intellectual merits: This project introduces light-driven molecular machines into optical device settings. Molecular machines driven by light have several advantages: they can be switched much faster; they do not produce any waste; light can be used for dual purposes¡Vinducing (writing) as well as detecting (reading) molecular motions. Experimental and numerical investigations will shed some light on the fundamental understanding of controlling plasmonics at molecular level. More importantly, with molecular machines¡¦ advantages in their size, energy consumption, speed, and controllability at molecular level, we expect that once established, the proposed rotaxane-based plasmonic switches will be welcomed in many applications such as optical communication.

Broader Impact: The PI will partner with the Penn State Center for Nanoscale Science and develop outreach activities around the theme of ¡¥from molecular shuttles to nanomechanics, nanoelectronics, and nanophotonics¡¦. A suite of demonstrations will imitate molecular machines¡¦ mechanical motions, broadly constructed and idealized in their operation by macroscopic models. The results developed in the past as well as from this proposal will be used to illustrate what molecular machines can achieve, and they will be delivered to museums, high-schools, and summer programs.

Objective
The objective of this research is to develop an opto-fluidic hybrid miniaturized flow cytometry. The approach is to implement an integrated, fully functionalized on-chip system, which is capable of (1) three-dimensional (3D) focusing of cells, (2) flexible manipulation of light in both X-Y and X-Z planes, and (3) simultaneous collection of all three types of light signals (forward scattering, side scattering, and fluorescence).

Intellectual Merit
The proposed research will address two major problems in the existing miniaturized flow cytometry chips: lack of flow focusing in the vertical (Z) direction and lack of on-chip focusing of light. Its functionality and simplicity of fabrication and assembly will make the proposed system attractive for both fundamental biomedical research and clinical applications. Moreover, it will shed light on our understanding in the light and fluid interactions at microscale, providing momentum to the emerging field of optofluidics.

Broader Impact
In addition to its technical contributions, the project will impact society by implementing vigorous education and outreach programs that are closely integrated with the proposed research and designed for groups at all levels (general public, undergraduates, and graduate students). Underrepresented students will participate extensively in the proposed research and be recruited through the Penn State WISER and MURE programs. Results and expertise developed during the course of this project will be incorporated into the PI's teaching activities. To obtain the broadest possible impact, the PI will partner with the Penn State MRSEC to develop a suite of demonstrations in MEMS/NEMS and deliver them to the general public.

DESCRIPTION (Provided by the applicant) Abstract: The Laser Scanning Confocal Microscopy (LSCM) has a unique capability to resolve below- surface tissue structures at a sub-cellular level. With this capability, LSCM has contributed compelling results in early cancer detection. Conventionally, LSCM is based on an assembly of bulky, expensive components[unreadable]motor-controlled scanning mirrors, translation stages, and high numerical aperture (NA) lenses[unreadable]to achieve sub-cellular resolution and optical sectioning. Consequently, applications of LSCM in cancer diagnosis, especially for cancer in hard-to-reach parts of human bodies (such as digestive and respiratory systems), have been limited. In order to maximize the potential of LSCM in early cancer diagnosis, it is necessary to develop miniature LSCM systems that can be fitted on the tip of an endoscope probe or catheter needle to facilitate high-resolution, minimally-invasive, in vivo confocal microscopic imaging. In view of the tremendous potential and significant challenges in developing miniature LSCM, I propose a transformative technical route-Optofluidic Laser Scanning Confocal Microscope (O- LSCM). Specifically, I will (1) address optical and mechanical challenges in the implementation of O-LSCM, (2) characterize and optimize pre-packaged O-LSCM, and (3) construct O-LSCM imaging probe and apply it to tissue imaging for early cancer detection. The proposed O-LSCM realizes all optical and mechanical functions needed for an LSCM in a single embodiment via the control of fluid flows in a microfluidic chamber. O-LSCM has no moving components, and it can be conveniently realized with a simple micro-fabrication technique (i.e., micro mold injection). Therefore, fabrication and assembly process can be dramatically simplified, the assembled system can be miniaturized to such an extent that it is suitable for endoscopic applications, and reliability and optical alignment precision of the system can be significantly improved. With its strong functionalities, simplicity, compactness, and robustness, the proposed O-LSCM will facilitate the design of next-generation endoscopic confocal microscopy systems and will benefit a wide range of biological studies and clinical applications. Public Health Relevance: Laser scanning confocal microscopy (LSCM) is a powerful technique for obtaining high resolution three-dimensional (3D) microscopic images of various biological samples. The long-term objective of this grant is to invent a paradigm-shifting technical route to LSCM-Optofluidic Laser Scanning Confocal Microscope (O-LSCM). With its strong functionalities, simplicity, compactness, and robustness, the proposed O-LSCM will facilitate the design of next-generation high-resolution, minimum-invasive endoscopic confocal microscopy systems and will benefit a wide range of biological studies and clinical applications (such as early cancer detection).

PI: Tony Jun Huang, Pennsylvania State University
Proposal Number: CBET 1160568

The proposed effort seeks NSF funding for student participation grants for the 2011 ASME Society-Wide Micro and Nano Technology Forum to be held during the ASME IMECE 2011 in Denver, Co. The proposed Forum will bring together ASME members and others, with a focus on new developments in the field of micro and nanotechnology. For the past three years the forum has been enthusiastically embraced by ASME IMECE attendees, in particular students; each year, there have been more than 150 poster presentations and about 300 attendees at the forum. The proposed NSF awards will be used to further nurture student development by encouraging participation from a select group of meritorious students working in the general area of micro-/nanoscale engineering by providing them partial travel grants including expenses related to conference registration fees and lodging. The travel awards will be decided by a panel of experts from the Micro and Nano Forum organizing committee, and will be based on the technical quality of the poster abstracts submitted and statement from their research advisors.

In the past, the participating students at the ASME Micro and Nano Forum have greatly valued the opportunity given to them by the Forum to showcase their research, interact with their peers, and meet people outside of their immediate environment. Besides this, the Forum also provides opportunities to increase student exposure to cutting-edge research in the frontiers of micro and nano technologies, and increase student abilities with respect to tools that will make them competitive in a research environment, namely, team work and project management, oral and written technical communication skills, ethics, and overall research acumen. In addition, the students will get an opportunity to attend technical presentations (over 2000 presentations and posters are anticipated to be presented at IMECE 2011) relevant to their current research, and also in other mechanical engineering fields. This will expose them to solutions and challenges that may be relevant to their current projects, while at the same time provide opportunities to discover exciting new research activities to pursue in future. In this regards, the mission of the forum fits well with the mission of NSF (in particular the Engineering Directorate) in attracting young research talents and mentoring them for a career in science and engineering.

Direct exposure of the participating students to leaders in their research fields (technical organizing committee members, judges, etc.) will provide them with a unique opportunity to disseminate their most recent research, and receive first-hand information on available opportunities for postdoctoral positions, as well as faculty positions. ASME traditionally hosts several Grand Challenge sessions where speakers from industry or government identify critical technical challenges facing the nation in various fields. In addition, ASME award lectures by prominent researchers, special sessions on ethics and the next-generation engineering education curriculum, and technical tours to local industry and national laboratories represent other opportunities for student learning and growth. Like in the past, personnel from NSF, DoD agencies, national laboratories, and industry are expected to have a strong presence at the conference. Members from these groups will be involved in providing professional development seminars, workshops, information sessions and recruitment activities, and will provide further new avenues for student development as well. While selecting students for the travel awards, every effort will be made to include and encourage student participation from both minority and traditionally underrepresented student groups in engineering.

Project Summary: The program focuses on developing a new class of high-resolution, high-speed, low-power-consumption, full-color electronic paper displays (E-PADs) using molecular motors as active components. The specific molecular motors to be used are redgreen-blue (RGB) tri-station catenanes. The proposed research will (1) investigate the electrochromic properties of RGB catenanes in polymeric matrix, and (2) construct a prototype color-switchable E-PAD based on RGB catenanes.

Intellectual Merit: Artificial molecular motors and machines such as RGB catenanes represent a promising and challenging field of interdisciplinary research. If successful, this research could lead to a new technology for energy efficient, high speed and full color display technology.

Broader Impact: In addition to its technical contributions, this NSF project will impact society by implementing vigorous education and outreach programs that are closely integrated with the proposed research and designed for groups at all levels. Underrepresented students will participate extensively in the proposed research.

CBET-1248221
PI: Huang

The travel grant request is for partial travel support for graduate students presenting posters in a Poster Symposium at the ASME Micro-Nano Technology Forum at the ASME IMECE 20112 in Houston, TX. The plan is to support nearly 40 meritorious graduate students from a pool of nearly 150-200 posters that will be presented during this forum. Students selected will be on the basis of merit and diversity.

Professional development of talented, diverse group of scientists in critical emerging areas is an important need for the nation and a priority for NSF. The travel grant is focused on doctoral students preparing for careers in academia and industry and in the vital technological area of nano and micro technology that NSF, CBET and the Thermal Transport program have heavily invested in for over a decade. The ASME IMECE is the premier conference for mechanical engineers and in the nano-technology area, and is therefore a good venue for the proposed poster presentations. The students will be exposed to cutting edge emerging research across the country and world, and those presenting will be able to further develop their professional skills in technical presentations. Beyond that, students attending will have the opportunity to learn from a variety of sources, as approximately 2000 presentations are generally presented at this annual meeting, further facilitating idea development for future careers in research. It is anticipated based on previous year attendance that over 150 presentations will be made, and there will be over 300 attendees.

Nano technology science research is now maturing and leading to transformational technology. Supporting this grant will foster interactions between budding researchers and entrepreneurs and will promote the technology and its transformational aspects.

The conference is expected to help in professional development, and students will be supported with an eye on diversity and encouraging under-represented group participation. Student selection will be done by an expert panel organized by the PIs from the posters submitted to the symposium. Opportunities that exist for postdoctoral positions and faculty as well as opportunities facilitated by other governmental agencies (e.g. DoD) will allow for important career development analyses. By attending, students will also have the option to attend professional development workshops and seminars that go beyond the traditional scientific development into areas important for career success, such as public speaking skills and resume/CV preparation.

? DESCRIPTION: The lack of a single-cell manipulation technique that can simultaneously achieve high throughput, high precision, and high cell integrity is a major roadblock for studies of intercellular communication. Recently, our interdisciplinary team has developed a surface acoustic wave (SAW)-based microfluidic platform called acoustic tweezers that possesses significant advantages over existing cell-manipulation techniques for single-cell analysis. Our acoustic tweezers platform is able to modulate the distances between individual cells with sub-micron precision. In addition, it is highly scalable and capable of creating a large array of celluar arrangements for high-throughput studies. Cells do not need to be labelled and can be cultured in their native media. Furthermore, the acoustic power and frequency used to manipulate cells are in the same range as those used in ultrasonic imaging, which has proven to be highly biocompatible. Finally, the components required for SAW generation are small and inexpensive, and the device itself is easy to operate. With these advantages, the acoustic tweezers are groundbreaking in their ability to provide precise spatiotemporal control of intracellular communication at the single-cell level in a high-throughput manner while preserving cell integrity. The transformative potential of acoustic tweezers has already been demonstrated in studies on gap junction-mediated functional intercellular communication in several homotypic and heterotypic cell populations by visualizing the transfer of fluorescent dyes between cells. Our objective in this project is to conduct advanced development of acoustic tweezers and validate them in studies on the effects of intercellular communication on metabolic pathways within the cell. We will, therefore, pursue the following specific aims: (1) advanced development of acoustic tweezers for high-yield, high-throughput characterization of intercellular communication and purinosome assembly at the single-cell level; (2) multi-parametric investigation of purinosome assembly in a primary cell model using acoustic tweezers; and (3) single-cell analyses of purinosome assembly and purine metabolism in a neuronal model using acoustic tweezers. At the completion of the proposed project, we hope to uncover the mechanism for how a genotype affects complex phenotype using Lesch-Nyhan disease (LND) as the disease model and purinosome as an indicator of metabolic state. Due to its unique ability to create multicellular assemblies with prescribed architectures in high throughput, we expect that the acoustic tweezers will become an invaluable tool for single-cell analysis and will fulfill many unmet needs in the bioengineering, biomedical, and pharmaceutical research communities.

This Small Business Technology Transfer (STTR) Phase I project will demonstrate the feasibility of microfluidic-based, bio-compatible, bio-safe, fluorescence-activated cell sorters. Cell sorters are powerful, high-throughput, single-cell characterization and purification tools that are vital for labs in fields such as molecular biology, pathology, plant biology, stem cell biology, and medical diagnostics. Despite their significant impact, current commercial cell sorters have a variety of drawbacks. High instrument costs (average cost: ~$500,000), high maintenance (maintenance cost: ~$30,000 per year; highly trained personnel needed), significant biosafety concerns, and reduction of cell viability and functionality make conventional cell sorters less effective in many applications and inhibit their widespread use. To address limitations of existing cell sorters, an innovative approach is proposed that features two key technologies: 1) a "microfluidic drifting" based cell-focusing technique; and 2) a cell-deflection technique using chirped interdigitated transducers (IDTs). The proposed microfluidic cell sorter eliminates the generation of hazardous aerosols and preserves high cell viability and functions.

The broader impact/commercial potential of this project, if successful, will be the development of the most bio-compatible and bio-safe cell sorters for researchers and scientists. According to a 2011 BCC research report, the instrument market for flow cytometers and cell sorters accounted for $1.4 billion in 2010 and is expected to grow at a CAGR of 9.8% from 2010 to 2015 ($2.2 billion). The served available market (SAM) is estimated to be ~$200 million. Compared with the existing cell sorters, the proposed microfluidic cell sorter will have the following advantages: 1) high bio-compatibility; 2) high bio-safety; and 3) low costs and low maintenance. In addition, the cell sorter will be more accessible to researchers and address existing unmet needs in the market (e.g., sorting fragile or sensitive cells while preserving high viability and functions). It will accelerate research findings and improve diagnostics and therapeutics. It will also create more job opportunities as the company grows.

DESCRIPTION: The ability to perform high-throughput, high-purity, multi-parametric cell sorting is extremely important for many biomedical studies and clinical applications. In the past few decades, fluorescence-activated cell sorters have become the gold standard technique in the field. However, current cell sorters suffer from an inability to maintain cell integrity during the cell- sorting process. Conventional cell-sorting processes are reported to significantly reduce cell viability and function (30-70% reduction) for many fragile or sensitive cells such as neurons, stem cells, liver cells, dendritic cells, sperm cells, and even neutrophils from healthy individual. In addition, our recent preliminary results indicate that gene expression can be significantly altered during the cell-sorting process, even for robust cells (such as HeLa cells). These drawbacks significantly limit the usefulness of cell sorters in many biomedical studies and clinical applications and have created many unmet needs. For example, human induced pluripotent stem (iPS) cells have opened a new field for modeling human diseases using human cells directly. They can be extremely useful for drug screening and personalized medicine. However, today it is still impossible to use cell sorters or any other existing methods to isolate undifferentiated iPS cells in a high-throughput, high-purity, and high-cell-integrity manner. This unmet need has significantly hindered progress in stem cell research and therapy. Our objective is to address these unmet needs by demonstrating standing surface acoustic wave (SSAW) based, high-cell-integrity sorters. When compared to conventional sorters, the proposed SSAW cell sorter is substantially smaller and less expensive, and is expected to significantly improve post-sorting cell viability, function, and gene expression for both fragile and robust cells. In particular, we will (1) develop a SSAW-based flow cytometer that achieves sheathless, multi-color, high-throughput single-cell analysis; (2) demonstrate a high-throughput, single-cell deflecting unit using focused interdigital transducers (f-IDTs); (3) establish a fully integrated, SSAW-based cell sorter system proven with human blood samples to outperform a state-of-the-art cell sorter; and (4) demonstrate sorting of induced pluripotent stem (iPS) cells with maintained cell integrity. With unprecedented capabilities to maintain cell integrity, even for fragile cells, our proposed SSAW-based cell sorter will not only become a more compact, affordable, and easy-to-maintain replacement to the existing cell sorters, but also fill many unmet needs in both fundamental biomedical research and clinical diagnosis and therapeutics.

PI: Costanzo, Francesco
Proposal Number: 1438126

The objective of the proposed work is to study the hydrodynamic interactions between deformable microparticles, and specifically between a micro-bubble and a cell. The idea is to create a micro-bubble using a laser next to the cell, and then use acoustic methods to obtain information about the mechanical properties of the cell. Such information would be used mainly for diagnostic purposes, but also for therapeutic purposes. This idea that a cell's mechanical properties can be used as a biomarker for pathogenic processes is currently being used to diagnose malaria, and there is some evidence that mechanical biomarkers may be used to diagnose cancer. The proposed work could lead directly from fluid dynamics research to applications. The proposed research will lead to more effective, cheaper, and faster cell-based on-chip diagnostic and therapeutic devices. As such, this research can have a major impact on public health world-wide.

Cellular mechanical properties have been found to be valuable indicators for pathogenesis and pathophysiology. This has led to the identification of a new class of biomarkers: mechanical biomarkers that offer some advantages over traditional biochemical biomarkers. While a number of mechanical biomarker-based microfluidic devices have already been proposed in the literature, the full potential of mechanical biomarkers in microfluidic-based diagnostics and therapeutics has yet to be revealed. One reason is the fact that no techniques are currently available for the quantitative assessment of cell deformability in relation to the forces acting on them. Current approaches for estimating the radiation forces on objects in streaming flows are based on classical solutions for idealized geometries (typically spheres) and small deformation of elastic inclusions in the flow. The proposed research will use computational techniques based on the immersed finite element method to advance knowledge in these areas. The goal is to relate cell deformability to the hydrodynamic forces imposed on a cell or on a group of cells in a microfluidic device. The validation of the proposed computational framework will be done against experiments with cancer cells in an opto-thermally-generated and acoustically-activated surface bubbles microfluidic device. The co-PIs propose to involve undergraduate students in research and to leverage already existing initiatives at Penn State in order to reach underrepresented minority students: the Women in Engineering Program and the Multicultural Engineering Program.

This award by the Fluid Dynamics Program of the CBET Division is co-funded by the Instrument Development for Biological Research (IDBR) Program of the Division of Biological Infrastructure.

An award is made to The Pennsylvania State University to develop integrated plasmofluidic microscopy (PFM) that can conduct high-throughput, high-sensitivity single-molecule studies in a simple, compact, cost-effective, user-friendly format. With its unprecedented ability to achieve high sensitivity and high throughput simultaneously while using a relatively simple, low-cost format, the proposed PFM has the potential to transform the field of single-molecule studies and accelerate discoveries in many disciplines in biology, biochemistry, and medicine. This technology would be expected to have numerous diverse applications with immediate, positive implications. Examples include the study of molecular dynamics far from equilibrium, the characterization of molecular properties such as protein conformational changes and folding, or the investigation of protein-protein or protein-DNA interaction kinetics and dynamics. The proposed PFM will be disseminated through 1) recording and distributing videos on experimental procedures, 2) recruiting beta-testing labs, 3) hosting demo sessions at conferences, and 4) collaborating with industry partners. Female and underrepresented minority students will participate extensively in the proposed research projects.

The proposed PFM takes advantage of unique features offered by both plasmonics (i.e., the interaction between light and nanomaterials) and microfluidics. It improves the sensitivity of traditional nanoaperture-based sensors by 20-120 times. In addition to improved sensitivity, the proposed PFM technique provides more precise fluidic control, minimizes consumption of samples and reagents, and reduces equipment costs. This project will improve understanding on the interactions of light, fluid, and molecules at the micro/nano scale, contributing to the emerging field of plasmofluidics. The proposed PFM-based single-molecule studies on the T4 replisome will also improve understanding of the DNA replication process, which will not only elucidate fundamental processes of biology and biochemistry but also shed light on the therapeutics of many diseases, such as cancer and infectious diseases.

? DESCRIPTION (provided by applicant): This is a new application for an R01 grant to establish the intellectual and technical framework to permit the study of viral infection on the single-cell level to be as tractable as the study of viral infection on the population level using the plaque assay. The world is ill equipped to deal with the (re)emergence of diseases caused by RNA viruses. The viral RNA genome is replicated by the virus-encoded RNA-dependent RNA polymerase (RdRp), an enzyme that, in most cases, lacks proofreading activity and a cellular repair mechanism to usurp. As a result, each progeny genome differs from another in the population by one or more nucleotide changes. The Cameron laboratory and colleagues have discovered that the genetic diversity created by replication errors made by the RdRp permits the virus to clear bottlenecks that would otherwise lead to viral extinction. Therefore, it is becoming increasingly clear that attenuated viruses for use as vaccine strains can be created by altering the nucleotide incorporation fidelity of the RdRp. Unexpectedly, the Cameron laboratory has observed that using conventional approaches to study this class of vaccine candidate in cell culture masks the attenuated phenotype. The attenuated phenotype is only observed by evaluating the infection at the level of the single cell instead of the population. This observatio motivated the development of techniques to study viral infection on the single-cell level. Our foray into this area was funded by the PSU Huck Institutes of the Life Sciences. In this proposal, we present a set of experimental objectives that will move single-cell virology from a descriptive art to a quantitative science that can be implemented broadly by the virology community not only to understand viral population dynamics but to reveal between-individual differences that may underlie susceptibility to viral infection. Importantly, this technology is essential to advancing RdRp fidelity as a target and mechanism for viral attenuation and vaccine development, thereby addressing an urgent public-health need. We will, therefore, pursue the following specific aims: (1) Elucidate parameters governing diverse kinetics of viral genome replication at the single-cell level; (2) Establish a data and statistical analysis pipeline for th single-cell virology experiment and develop mechanistic models of infection; and (3) Enhance capabilities of the microfluidic platform for characterization of viral infections at the single-cel level.

The broader/commercial impact of the Small Business Technology Transfer (STTR) Phase II project will be a cell sorter, a new research tool for life science research, animal reproduction, and cell-based therapy. In the past decade, cell sorters have become vital in many fields, such as molecular and cellular biology, immunology, plant biology, animal reproduction, and medical diagnostics and therapeutics. Despite their significant impact, current cell sorters have the following drawbacks: high equipment and maintenance costs, significant bio-safety concerns, and reduced cell viability and function. These drawbacks reduce the effectiveness of cell sorters in many important research studies and clinical applications. Enabled by this innovation, researchers will be able to better understand the causes of diseases, identify new therapies, and test new drugs and vaccines. It also has the potential to improve dairy production efficiency, and aid medical doctors in making better decisions about diagnosis and treatment. In Phase II, the goal is to improve performance of the instrument, and validate the performance with end users.

This STTR Phase II project will demonstrate the feasibility of a microfluidic-based, bio-compatible, bio-safe, fluorescence-activated cell sorter. Cell sorters are powerful, high-throughput, single-cell characterization and purification tools that are vital for labs in fields such as molecular biology, pathology, plant biology, stem cell biology, and medical diagnostics. The technology is based on acoustofluidic (i.e., the fusion of acoustics and microfluidics) cell sorting chips that preserve the integrity and functionality of sorted cells. Current cell sorting systems reduce cell viability, integrity, and cell function due to high shear stress, high impact force, and high driving voltage, which reduces their effectiveness as a research tool, and in clinical applications. Unlike current cell sorters that use electrostatic force to sort cells, which require 12,000 V of driving voltage, the proposed technology uses acoustic tweezers to sort cells, and requires only 10 V, which significantly reduces cell damage. Compared with existing cell sorters, the proposed microfluidic cell sorter will have the following advantages: 1) high bio-compatibility; 2) high bio-safety; and 3) lower costs and lower maintenance. In addition, the cell sorter will be more accessible to researchers and address existing unmet needs in the market (e.g., sorting fragile or sensitive cells while preserving high viability and function). This will accelerate research findings and improve diagnostics and therapeutics.

Abstract B cell non-Hodgkin lymphomas (bNHL) are the most common lymphoma subtype representing >85% of all NHLs. bNHL are typically treated the anti-CD20 antibody (e.g., rituximab) alone or in combination with chemotherapy. There are currently, however, no biological methods or markers to predict the sensitivity or resistance to rituximab (or any other) antibody therapy. A key feature of antibody activity occurs through natural killer (NK) cell-mediated killing of antibody-coated target tumor cells, however, antitumor activity and subsequent resistance, is poorly understood. In this application, we propose to develop and validate a high throughput droplet based microfluidic platform to investigate the key features of NK cells associated with rapid, slow or inactive tumor killing kinetics in NHL. We will first adapt a novel approach and integrate the biocompatible acoustofluidic droplet sorter during the droplet microarray formation to determine the phenotypes of immune-target cell interaction in microfluidic droplets. We will validate a droplet-based microfluidic device to interrogate single-cell dynamic responses and cell-cell interactions within intact droplets. Next, we will demonstrate a high-purity (>95%), high-throughput (>10,000 events/s), four-channel acoustofluidic droplet sorter to integrate with droplet analysis array. The downstream 4-channel sorting will allow, after establishing the kinetic profiles of interactions, to identify and sort droplets containing active lymphocytes into a distinctive pool; separate basal lymphocytes into another pool based on fluorescence. A unique function of selecting sorting criteria based on imaging analysis can be provided by the combination of droplet imaging array and acoustofluidic droplet sorters, which is unachievable for conventional fluorescence activated droplet sorters (FADS) since imaging tracking is inherently tricky in high-speed flow. Thus, our approach serves as a ?bottom-up? method of classification, by first identifying distinct functional categories and then probing the content of the individual cell category to determine the key factors for the molecular classification of heterogeneous immune functions of NK cells related to target cell kill. In addition, we will identify NK cell heterogeneity and bio-functional characteristics to discover novel drug combinations for NK cell dependent immunotherapy via an integrated acoustofluidic droplet sorting platform. We will demonstrate that the accuracy of phenotype identification of our device and its suitability for clinical applications by monitoring and classifying NK/NHL single cell interactions in the presence of monoclonal antibodies and performing biochemical secretome assay from ?hyperactive?, ?basal? and non-responsive pools. By combing these findings with drug screening and identification of phenotype altering drugs, we will demonstrate the applicability of this technology for personalized medicine and rational clinical immunotherapeutic applications. We envision our platform may be leveraged in a variety of single-cell analysis applications in immunotherapy and it will provide high value to the bioengineering, biomedical, and therapeutic research communities.

Cells are fundamental building blocks of life. The ability to manipulate single cells individually in a liquid environment with high precision will enable many fundamental biological studies on cell-to-cell and cell-to-environment interactions that could not be achieved before. Such discoveries will provide key insights into the effect of the environment on cellular structure, function, and signaling, and would have wide applications across multiple disciplines in life sciences, agriculture and medicine. Due to their small size and also soft nature, it is extremely difficult to handle single cells with a physical device, such as mechanical tweezers, without causing damage to the delicate subcellular structures. Alternatively, focused electrical fields, laser beams, and ultrasound waves can be utilized to generate microscale forces at the focal point, serving as virtual tweezers for single cell manipulation. Among them, the 'acoustic tweezers' are the most compatible with the cells due to cell's higher tolerance of sound pressure over electrical field and laser illumination. However, unlike the laser beams that can be easily focused and steered with a glass lens and a rotating mirror, agile focusing and steering of ultrasound waves requires complex and expensive transducer arrays and control electronics. This situation has prevented the wide use of 'acoustic tweezers' in single cell manipulation. The proposed work is to develop an acoustic phased array to enable acoustic steering and focusing of ultrasound beams for their applications in high-resolution acoustic manipulation of single cells.

Among all existing single cell manipulation techniques, the ultrasound phased array has the greatest potential due to its unique ability to achieve electronic beam forming (without physically scanning the transducer(s)). Using this unique beam forming technique to focus ultrasonic radiation at precise locations presents unprecedented manipulation capabilities compared with other methods. However, in current ultrasound phased array systems, multiple channels of ultrasound signals are first converted into electronic ones with an array of transducer elements. The phase shift is then accomplished in the electronic domain. As the operation frequency and the number of the channels increases, the phased array system becomes increasingly complex, bulky, power-consuming and costly. Therefore, current acoustic phased arrays are not suitable for on-chip microfluidic platforms for single cell manipulation. To address this issue, this research aims to develop a new micromachined ultrasound phased array. It will consist of an array of micromachined silicon acoustic delay lines with tunable delay lengths to create the desired phase shift for multiple ultrasound signals without the need for electronic conversion. This enables ultrasound beam forming with a single-element transducer and a single-channel ultrasound transceiver. With advanced micromachining processes, it can be readily fabricated and even integrated together with microfluidic components onto the same substrate for single-chip operation. To achieve the research objective, the following three research tasks will be accomplished: 1) Conduct acoustic and electromechanical design and optimization of the acoustic phase shifter; 2) Develop an on-chip microfabrication process to achieve multi-channel integration; and 3) Demonstrate dexterous acoustic manipulation and positioning of single cells using the ultrasound phased array. This multidisciplinary research is expected to provide unique learning and training opportunities for both graduate/undergraduate students. Students in grades 7-12 will be involved through Engineering Summer Camps and Open House activities. Research results will be incorporated into the PI's course development at all levels. They will also be disseminated through publications, outreach activities, invited talks, and a project website.

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.

Abstract Exosomes are nanosized extracellular vesicles that contain biomolecules (DNA, mRNA, miRNA, and other functional proteins) from their cell of origin. Exosomes are secreted from nearly all cell types, and as a result, they are found in most biological fluids, including blood, saliva, urine, and cerebrospinal fluid. Over the past decade, the transfer of exosomal biomolecules to recipient cells has been implicated in a variety of biological processes. Consequently, exosomes have increasingly been the focus of many studies in biomedical research. Due to their distinct molecular signatures, exosomes have been identified as a potentially transformative circulating biomarker for the diagnosis and prognosis of multiple diseases, including cancer, neurodegenerative diseases (i.e., Parkinson?s and Alzheimer?s), as well as diseases of the kidney, liver, and placenta. In addition to diagnostic applications, exosomes are an ideal drug delivery system in many therapeutic applications. While the versatility of exosomes renders them an excellent candidate for a variety of biomedical applications, difficulties in the consistent, effective isolation of exosomes have greatly limited their utility. Current approaches for exosome isolation involve lengthy procedures, require highly trained personnel, suffer from low repeatability, low yield, low purity, and/or low post-sorting exosome integrity. As a result, there exists a critical need in the research communities for a simple, rapid, efficient, and biocompatible approach for isolating exosomes form biological fluids or in vitro cell culture. In this R01 project, we will address this unmet need by developing an acoustofluidic (i.e., the fusion of acoustics and microfluidics) platform for high-purity, high-yield, high-biocompatibility, automated exosome isolation. The proposed acoustofluidic technology will have the following features: 1) Automated exosome processing which reduces operator-to-operator variability and enables simple, consistent isolation results with improved biohazard containment; 2) Reduces the amount of time necessary to go from biofluid (e.g., 1 mL undiluted blood) to isolated exosomes (<5 min processing time vs ~8 hrs processing time with alternative technologies); 3) Higher exosome recovery rate (>90%) in comparison to benchmark technologies (5?25%); 4) Greater exosome purity (>80%) in comparison to benchmark technologies (~33%); 5) Less contamination from other circulating factors, including non-native serum proteins (e.g., albumin and immunoglobulin) and particles with similar sizes, including various types of lipoproteins; 6) Low-cost and point- of-care design; and 7) ability to handle both large and small sample volumes (maximum sample volume: ~30 mL; minimum sample volume: ~10 µL), which is extremely challenging with existing approaches. With these unique features, the proposed acoustofluidic technology has the potential to greatly simplify and expedite workflows in exosome-related biomedical research and aid in the discovery of new exosomal biomarkers.

Project Summary/Abstract: This UG3/UH3 application is responsive to the NIH Common Fund initiative RFA-RM-18-028 ?Advancing Extracellular RNA (exRNA) Communication Research: Towards Single Extracellular Vesicle (EV) Sorting, Isolation, and Analysis of Cargo?. This proposal is built on a foundation of a 5-year NIH Common Fund ?Extracellular RNA Communication Consortium Stage 1 (ERCC1)? project to develop salivary exRNA biomarkers for gastric cancer detection where a panel of highly discriminatory salivary extracellular RNA (exRNAs) have been developed (discovered and definitively validated) for gastric cancer, scientifically and translationally credentialed salivary exRNA for systemic disease detection. This ERCC Stage 2 project is to develop an innovative technology, AcoustoFluidic Separation (AFS) coupling with Surface Enhanced Raman Spectroscopy (SERS), towards single EV isolation and to characterize exRNA cargos associated with specific EV subpopulations based on the cells of origin, intended target cells and biolfuids. Eight Specific Aims with twelve quantitative milestones in two phases (UG3, UH3) are in place to test the central hypothesis that exosomes are the EV from gastric cancer tissue/cells of origin harboring the nine validated discriminatory salivary exRNA biomarkers, transported through vasculature, homing into salivary glands (target organ) and into saliva. Four Specific Aims in the UG3 Phase are to develop the AFS technology as a standard operating procedure (SOP) for rigor and reproducibility (Aim 1); SERS development for EV fingerprinting and co-localization of exRNA targets to singe EV (Aim 2); two independent ?Rigor and Reproducibility Labs (R&R Labs)? to evaluate the AFS SOP (Aim 3) and approaches to share strategies, protocols, tools with broader scientific community with DMRR (Aim 4). In the UH3 Phase four Specific Aims are in place to optimize, refine and scale up the AFS SOP (Aim 5); perform sorting, tracking of validated salivary exRNA biomarkers for gastric cancer detection from cells of origin, to blood, to salivary glands and to saliva (Aim 6); ?R&R Labs? to optimize AFS to other human biofluids (Aim 7) and approaches to share strategies, protocols, tools with broader scientific community with DMRR (Aim 8). Completing these Aims and goals based on the outcome of the ERCC1 project is logical and highly impactful as the outcomes of the ERCC2 project will deliver a set of novel technologies, AFS in tandem with SERC, for rapid, high yield (6X over current technologies) and single EV level isolation for salivary biomarker development for systemic disease detection. These will constitute the foundation of ?exRNA Saliva Liquid Biopsy (exRNA- SLB)? where the diagnostic and therapeutic functionality of exRNAs can be fully realized when the range of EV subpopulations from a given cell source can be characterized and analyzed for molecular cargos. 1

PROJECT SUMMARY This R01 application is responsive to the NIH initiative PAR-19-253 ?Focused Technology Research and Development?. Automated liquid handling technologies are valuable in many areas of biomedical research. For example, robotic pipetting systems have been extensively utilized to automate assays, thereby eliminating errors associated with manual pipetting and significantly improving reproducibility. However, the majority of automated liquid handling technologies suffer from a fundamental constraint: they rely on physical contact with a solid structure in order to manipulate liquid reagents. Therefore, traces of a reagent inevitably adsorb onto the contact surface and can possibly later dissolve into another liquid sample. Thus, the risk of cross-contamination due to this undesirable ?fouling of the surface? limits the transport surfaces to a single type of working liquid plus reagent combination. Recently, we invented digital acoustofluidics (DAF), an acoustic-based, programmable, contact- free, liquid handling technology, which overcomes the key obstacles associated with the existing liquid handling methods. In this R01 project, we will develop and validate a DAF fluid processing system with the following features: (1) Rewritability, programmability, and ability to perform complex, cascade reactions: We will demonstrate the ability of DAF to transport and mix ?fluidic bits? (i.e., droplets) along prescribed, arbitrary routes without cross-contamination, leading to a 104-fold increase in the number of allowable combinations of reagent inputs on a single device (as compared with conventional platforms); (2) Biocompatibility: Instead of being directly subjected to strong acoustic pressure or high electric fields, the droplets are manipulated in a contactless, gentle manner. Our preliminary results show that the DAF platform has no significant effect on the viability of cells; (3) Versatility: DAF is not restricted to fluids with specific acoustic, electrical, hydrodynamic, or magnetic properties. This versatility makes DAF suitable for handling a wide range of liquids, even for challenging samples such as low-polarity fluids (e.g., organic solvents), sticky or viscous samples (e.g., blood and sputum), and solids (e.g., fecal samples and model organisms); (4) Miniaturization and convenient integration: Our DAF platform provides an unprecedented level of miniaturization and cost-effectiveness compared with existing robotic liquid handling systems. In addition, it is designed to be integrated with a variety of multi-well plates, enabling it to be seamlessly integrated into existing biomedical research laboratories. With the aforementioned advantages, the proposed DAF technology has the potential to exceed current industry standards, address unmet needs in the field, and provide a compelling platform for the development of a robust, rewritable, high- throughput, and digitally-programmable fluidic processor. We will validate its performance across two established biomedical applications: protein crystal chemistry, and high-throughput drug screening. In this regard, we aim to demonstrate the far-reaching potential of DAF to enable improved research in areas ranging from clinical chemistry to fundamental biology.

Recent swift advances in additive manufacturing have demonstrated its great potential in tailoring the local and global properties of produced structures by including micro- or nano-particles into polymer matrix composites. However, current approaches have been limited by the challenge in precision spatial controls of embedded particles, which usually have diverse material properties, sizes, and shapes, making particle manipulation in a viscous polymer fluid difficult. This collaborative research award will conduct fundamental research to transform an additive manufacturing technology that leverages digital light processing for photopolymerization printing and acoustic holography to accurately “tweeze” micro/nano-particles in a polymer resin. The research will greatly impact basic science fields in acoustic tweezers, materials processing, metamaterials, and biomaterials, etc. Moreover, the studied acoustic holography additive manufacturing technology will advance many engineering applications through enabling novel metamaterials containing, e.g., lattice-like patterns for ultrasonic signal processing devices, cellulose-based reinforced architectures for customized repair of aircraft composite structures, or patterned micro-vessels for personalized biomimetic bone tissue regeneration. Through education and outreach activities, this project will also broaden the participation of underrepresented minorities, improve STEM education, and increase public engagements with science and technologies. The multidisciplinary nature of this project will provide unique learning and training opportunities for graduate and undergraduate students.

The overall objective of this research is to understand an acoustic holography enabled additive manufacturing mechanism to fabricate multifunctional composites that contain high-resolution, versatile patterns of diverse micro/nano-particles such as cellulose nanofibrils, carbon-based particles, and cells, etc. First, an acoustic holography-based particle patterning mechanism will be established to construct and reconfigure versatile particle patterns in viscous resins by studying a frequency multiplexing-based method for dynamically controlling multifrequency acoustic fields. Acoustic wave interactions with particles in viscous resins will be uncovered through particle image velocimetry and acoustic field scanning, and a theoretical model for rapid prediction of the particle patterning process will be developed and validated. Next, the knowhow of the acoustic holography-based particle patterning will be fused with the digital light processing-based photopolymerization to create a versatile, high-resolution apparatus for scalable additive manufacturing. Then, the apparatus will be utilized to develop and study novel multifunctional composites such as topological metamaterial composites containing periodic lattice-like patterns of micro-particles. Both theoretical and experimental methodologies will be utilized to further discover the effects of different periodic particle patterns on different properties of additively manufactured composites, including anisotropic elasticity, acoustic band gaps, Dirac cones, and topological states, etc.

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.

Project Summary/Abstract: This U18 application is responsive to the NIH?s RADx-rad Emergency Responses to the COVID- pandemic for new or non-traditional technologies developed for single extracellular vesicle, exosome and extracellular RNA (exRNA) isolation and analysis and reposition them for detection of SARS-CoV-2. The applicant?s group is a grantee in the NIH Common Fund ?Extracellular RNA Communication (ERC)? Program advancing a new and emerging technology of Acoustofluidic Separation (AFS) for label-free, high yield and purity exosomes from biofluids which is coupled to extracellular RNA characterization using Surface Enhanced Raman Spectroscopy (SERS) for single EV identification. This U18 application is to reposition the AFS EV technology and SERS for the non-invasive earliest detection of SARS-CoV-2 in saliva of infected patients. Host immunity to SARS-CoV-2 will also be assessed in the saliva samples, permitting the earliest detection of SARS-CoV-2 infection and host immunity non-invasively in a saliva sample. Five Specific Aims are in place to reposition the saliva-based AFS and SERS technologies, in a 2-year U18 proposal, to test the hypothesis that an integrated multi-parametric non-invasive saliva test for SARS-CoV- 2 infection, viral load and host immunity test demonstrating clinical performance surpassing current saliva-based SARS-CoV-2 EUA tests.

The placenta is essential for fetal development and growth, maternal homeostasis, and broadly, pregnancy health. Yet, our ability to non-invasively probe placental health during human pregnancy is hampered by its deep intrauterine location and its highly vascular composition, rendering the placenta largely inaccessibly for safe and dynamic investigation. Whereas placental research has been advanced by cell culture, ex vivo systems, animal models, and postpartum analyses, these indirect approaches provide ex post facto information about placental health. Placental imaging has revolutionized the field of placental medicine, but resolution at the molecular, cellular, or metabolic level remains limited. To address these challenges, we and others have focused on the release of extracellular vesicles (EVs) from placental trophoblasts, which, in humans, are directly bathed in maternal blood. We focused on exosomes (now termed small EVs or sEVs), microvesicles, and apoptotic blebs, which are continuously and abundantly released from trophoblasts into the maternal circulation and are accessible throughout pregnancy by peripheral blood tests. Among these EVs, we focus mainly on placental sEVs, which harbor messages that are seldom expressed by any other cell types and execute unique placental biological functions, such as an antiviral response. While informative, recent data indicate that sEVs are not a uniform population of vesicles, but comprise several subgroups, defined as large sEVs, small sEVs, and exomeres. In addition to their size, these sEV subtypes are characterized by distinctive cargo. Although the recent discovery of sEV subpopulations has excited researchers due to their potential to revolutionize the field of non-invasive diagnostics, sEV subpopulations have yet to be utilized in clinical settings. This is largely due to the difficulties associated with separation and isolation the nano-sized sEV subpopulations. Our group has now developed advanced acoustofluidic technologies designed to effectively, reproducibly, and rapidly isolate sEVs from blood. We show that we can separate placental sEVs into their specific subpopulations, which has not been previously accomplished. Our proposed investigation therefore focuses on the production of human placental sEV subpopulations, along with their RNA and proteome cargo. We posit that, by profiling these analytes from sEV subpopulations, we can illuminate a unique landscape of bioactive molecules that are relevant to placental health. To reduce data complexity, we propose a machine learning pipeline that will be used to probe the sub-sEV spectra during normal and pathological pregnancies. Further, we will improve our ability to purify sEV subpopulations from lipoproteins, and generate a single, integrated device that can reliably separate vesicles in real time across human gestation. We believe that our automated acoustofluidic approach to separating sEV subpopulations in a high-yield, biocompatible manner is critical to unlocking the clinical utility of sEVs. Insights gained from our investigation will improve non-invasive diagnostics during pregnancy and may uncover new targets for personalized placental therapeutics.