Browsing by Author "Huang, Tony Jun"

Acoustic-based techniques have emerged as a promising avenue for the precise manipulation of particles, combining the disciplines of acoustics, physics, and biotechnology. Utilizing sound waves, this method allows for the gentle, non-invasive movement and positioning of particles, from minute biological entities to larger synthetic materials. Such automated manipulation harnesses the intricacies of acoustic radiation forces and streaming, offering advantages in terms of scalability, precision, and integration into various systems. As biotechnological demands grow, the potential of acoustic-based platforms to influence fields like drug delivery, diagnostics, and cellular research becomes increasingly evident. This defense delves into the development of two platforms utilizing automated acoustic technologies for particle manipulation aimed at advancing biological applications. The first part showcases a digital piezoelectric-based platform, adept at dynamic particle manipulation through the modulation of acoustic streaming, enhanced with surrounding barrier structures. We built a programmable droplet-handling platform to demonstrate the basic functions of planar-omnidirectional droplet transport, merging droplets, and in situ mixing via a sequential cascade of biochemical reactions. The ensuing part unveils a novel platform tailored for the meticulous long-term observation of single cell physical attributes, founded on 2D acoustic patterning of single cell array and automatic phase modulation. By adaptively segmenting and fitting the movement, we are able to monitor the density, compressibility and size fluctuation of the sample at the same time. These innovations have the potential to revolutionize biological endeavors, notably in large-scale drug screening and the proactive surveillance of cellular responses to distinct environmental stimulations over extended periods.

Acoustic waves, as a form of mechanical vibration, not only induces the force directly on the object, but also induces the motion of the medium that propagates throughout the system. The study of acoustofluidic mainly focuses on the exploration of the underlying mechanism of the acoustic waves and fluid motion and the methodology of applying this technique to practical applications. Featuring its contactless, versatile, and biocompatible capabilities, the acoustofluidic method makes itself an ideal tool for biosample handling. As the majority of the bio-related samples (e.g., cell, small organism, exosome) possess their native environment within liquids, there is an urgent need to study the acoustic induced fluid motion in order to cooperate with the development of the acoustic tweezing technique. While both the theoretical study and application exploration have been established for the combination of acoustics and microfluidics, the fluid motion on a larger scale is still under-developed. One reason is that, although the acoustofluidic methods hold great potential in various biomedical applications, there is a limited way to form an organized motion in a larger fluid domain, which may lead to the imprecise manipulation of the target. On the other hand, the theoretical study for the microfluidic domain is on the basis of a simplified model with certain assumptions, when applying to the larger fluid area, and significantly influences both the accuracy and computation cost. In this dissertation, we have first developed a series of theoretical and numerical methods in order to provide insights into the acoustofluidic phenomenon in different domain scales. Specifically, we explored the non-linear acoustic dynamics in fluids with the perturbation theory and Reynolds’ stress theory. Then we presented that the vortex streaming can be predicted and designed with our theoretical and numerical study, which can be utilized for various fluid systems and expanded to practical biomedical applications. The boundary-driven streaming and Reynolds’ stress-induced streaming are studied and applied to the digital acoustofluidic droplet handling platform and droplet spinning system, respectively. We demonstrated that within the digital acoustofluidic platform, the droplet can be manipulated on the oil layer in a dynamic and biocompatible manner. Meanwhile, in the droplet spinning system, we can predict and guide the periodic liquid-air interface deformation, as well as the particle motion inside the droplet. We demonstrated that with the theoretical and experimental study, this platform can be utilized for the nanoscale particle (e.g., DNA molecule and exosome) concentration, separation, and transport. Next, based on our study of the acoustically induced fluid motion, we developed an integrated acoustofluidic rotational tweezing platform that can be utilized for zebrafish larvae rapid rotation (~1s/rotation), multi-spectral imaging, and phenotyping. In this study, we have conducted a systematic study including theory development, acoustofluidic device design/fabrication, and flow system implementation. Moreover, we have explored the multidisciplinary expansion combining the acoustofluidic zebrafish phenotyping device with the computer-vision-based 3D model reconstruction and characterization. With this method, we can obtain substantial information from a single zebrafish sample, including the 3D model, volume, surface area, and deformation ratio. Moreover, with the design of the continuous flow system, a flow-cytometry-like system was developed for zebrafish larvae morphological phenotyping. In this study, a standard workflow is established which can directly transfer the groups of samples to a statistical digital readout and provide a new guideline for applying acoustofluidic techniques to biomedical applications. This work represents a complete fusion of acoustofluidic theory, experimental function, and practical application implementation.

Showing increased application in biological and medical fields, acoustofluidics is a combined technology between acoustics and microfluidics. The core function of acoustofluidics is a label-free and contact-free manipulation of particles in the fluid, which can be applied as active separation, active mixing, and active concentration. Since in therapeutic and diagnostic applications, contamination in the samples can significantly interference analysis results and treatment outcome, proper per-screening of the sample can significantly decrease the target detection threshold and avoiding interferences come from noise and misreading. The acoustofluidic technology derive a particle manipulation based on physical properties of the particles and fluids, specifically, the size of the particle, densities for the particles and fluid, and the viscosity of the fluid, which generate a screening system that can separate particles with different sizes and densities. By utilizing this property, acoustofluidics has been applied on separating multiple biological particles and objects including circulating cancer cells, red blood cells, and multiple populations of vesicles. These reagent-free and contact-free separations have been demonstrated biocompatible for cells and vesicles and can conserve the cell viabilities and vesicle cargoes including DNA, miRNA, and proteins. However, current achievements on acoustofluidic manipulation focus on general analysis of the separated components, which are not disease specific biomarkers, and the body fluid using for separation are limited to blood and artificial isotonic solutions including phosphate-buffered saline. Although these works demonstrated acoustofluidic technology is eligible for separating bio-particles that have diagnosis and therapeutic functions, lack of real cases related applications and diseases specific investigations still make the technology’s application abilities being restricted to possibilities but not promised functions. To deeply investigate and demonstrate the acoustofluidic technology’s potential on diagnostic application, the technology was evaluated by using samples related with multiple specific diseases. Since the acoustofluidic technology has been demonstrated eligible for isolating exosomes, which are 50-200 nm vesicles secreted from cells, pathology related exosomes were selected for diagnostic application investigation. Exosomes’ vesicle structures make them ideal candidate for diagnosis, since vesicles formed by lipid bilayer membrane contain both proteins or nucleic acids as cargoes inside and transmembrane or membrane proteins and polysaccharides on the surface. Furthermore, the forming and secreting pathologies of exosomes are highly dependent on endocytosis and exocytosis pathologies, which are influenced by cellular metabolism. Exosomes’ cargoes have been found specifically correlated with secreting cells populations, indicates depending on types of cells, like tumor cells or stem cells, the secreted exosomes will contain different molecules that can be used as biomarkers for reversed identifying secreting cells. Except high values on biological and medical research and applications, exosomes’ small size makes the vesicles difficult for isolation and increase the cost on both equipment and time aspects. Since acoustofluidics provides an active approach for separating nanometer sized particles and the isolation is a continuous procedure, the simple and rapid exosome isolation the acoustofluidics can provide makes the technology high valuable. Considering these improvements, the acoustofluidics can provide on exosome related fields, demonstrating acoustofluidic devices separated exosomes containing disease biomarkers and could be used for diagnostic applications become a necessary step for validating the technology’s ability. In this dissertation, the first attempt for validating acoustofluidic exosome separation’s diagnostic potential was made for isolating salivary exosomes aimed at human papillomavirus (HPV) induced oropharyngeal cancer diagnosis. Different with previous research that worked on blood exosome separation, a unique property of this study is achieving exosome separation from saliva, which is a more unstable system on components and physical properties than blood. By isolating salivary exosome using the acoustofluidic technology and processing down-stream digital droplet polymerase chain reaction (PCR) analysis, HPV-16 virus, which has been found can induce oropharyngeal cancer, was found majorly distributed in isolated exosome fractions. Since saliva has complex components that cause inaccuracy analysis result, the application of acoustofluidic technology can increase the diagnostic sensitive and enable saliva based liquid biopsy for early screening of oropharyngeal cancer. In the next work, we further demonstrate the acoustofluidic technology’s advantage on rapid isolation of exosomes benefits the time sensitive diagnosis. The acoustofluidic devices were applied for isolating exosomes from mice models that were induced to traumatic brain injury (TBI), which can develop to chronic diseases or deteriorate in short term. Since these outcomes induced by improper or untimely treatments, fast screening of TBI becomes critical for achieving ideal therapeutic outcomes. By collecting plasma from mice and deriving exosome isolation through the acoustofluidics devices, isolated exosome samples with less contamination were found compared with original plasma. Protein analysis further indicates isolated exosomes keeps several exosome specific and neuron damage specific proteins, indicates the acoustofluidic technology is biocompatible and low harmful for exosome structures and components. High isolation purity achieved by the acoustofluidic technology also benefits downstream analysis by decreasing detection noise. In flow cytometer analysis, the acoustofluidic devices isolated exosomes demonstrated TBI disease biomarker increasing in 24 h after the mice were induced to TBI, while the plasma sample cannot demonstrate this tendency. The success of revealing early stage TBI biomarker changes indicates the acoustofluidic technology not only can benefit diagnosis, but also eligible for achieving diagnosis in a very early stage of the pathology. Since the acoustofluidic technology had demonstrated a promising performance on biocompatibility and rapid separation, other time-sensitive samples, including live virus was applied for evaluating the device’s performance. To achieve better control and eliminate irrelevant variable, we use cultured reverse transcription virus that is used for mammal cells transfection as target for isolation. The acoustofluidic technology showed reliable isolation of the murine leukemia virus and majority of the virus particles were separated out from the original sample. Virus viability was further validated robust based on the transfection experiments that using acoustofluidic separated virus and original virus samples demonstrated similar level transfection rates. This work indicates except vesicles like exosomes, the acoustofluidic technology is also eligible for isolating virus and keeping its viability, which significantly expands the application of the technology. Next, to expend the acoustofluidic technology’s functions, we utilized the concentration and manipulation ability of the device for deriving high efficiency membrane degradation. By generating strong microstreaming and microstreaming derived shear stress, the acoustofluidic devices can generate strong vertex flow fields in channel that can capture and lyse mammal cells. Since the acoustofluidic cell lysis is totally a physical process without participation of any chemical reagent and demonstrates a high lysis efficiency, this acoustofluidics application has potential for achieving high efficiency cell analysis. Since the acoustofluidic technology has demonstrated potential for concentration and lysis effect by generating high flow rate microstreaming vertex, we further investigated whether similar effect can derive exosome concentration and lysis. By generating acoustofluidic vertex in droplet containing exosome, nanoparticles, and small molecule drugs, exosome concentration and lysis effects were utilized for high efficiency drug loading and carrier encapsulation. Derived by the acoustofluidic concentration effect, the porous nanoparticles and drug molecules are concentrated in small area of the fluid system and this active concentration increasing induces a high drug loading rate. Simultaneously, the acoustofluidic vertex disrupts exosome membrane and concentrates exosomes with the nanoparticles, which induces exosome encapsulation. These exosome encapsulated drug-loaded nanoparticles demonstrate high intake rate of cells and derive more efficient drug delivery rate. Since the drug loading and exosome encapsulation are physical processes, the acoustofluidic technology derived particle manipulation has potential for deriving loading and encapsulation for large varieties of drugs, particles, and vesicles, which significantly expand the technology’s application.

In this study, we present a novel on-chip 2D hologram device of an on-chip hologram device capable of generating both arbitrary 3D nodes and 2D microscale nodes. As the first true 3D hologram folded on chip, our approach combines a refined fabrication process with a novel theoretical framework, which employs Fresnel transformation to describe the patterns, enabling the shaping of patterns in 3D, thus broadening the potential applications of lab-on-chip devices and establishing a new generation of acoustic tweezers. We also reduced the complexity of setup and manufacturing thus achieving high resolution and low tolerance with lithographic techniques. Our work holds promise for a wide range of applications, including biomedical, material handling, and sensing technologies, marking a noteworthy advancement in the pursuit of efficient and precise acoustic manipulation.

The largest obstacle in nanoscale microscopy is the diffraction limit. Although several means of achieving sub-diffraction resolution exist, they all have shortcomings such as cost, complexity, and processing time, which make them impractical for widespread use. Additionally, these technologies struggle to find a balance between a high resolution and a large field of view. In this introduction of dissertation, we provide an overview of various microsphere based super resolution techniques that address the shortcomings of existing platforms and consistently achieve sub-diffraction resolutions. Initially, the theoretical basis of photonic nanojets, which make microsphere based super resolution imaging possible, are discussed. In the following sections, different type of acoustofluidic scanning techniques and intelligent nanoscope are explored. The introduction concludes with an emphasis on the limitless potential of this technology, and the wide range of possible biomedical applications.First, we have documented the development of an acoutofluidic scanning nanoscope that can achieve both high resolution and large field of view at the same time, which alleviates a long-existing shortcoming of conventional microscopes. The acoutofluidic scanning nanoscope developed here can serve as either an add-on component to expand the capability of a conventional microscope, or could be paired with low-cost imaging platforms to develop a stand-alone microscope for portable imaging. The acoutofluidic scanning nanoscope achieves high-resolution imaging without the need for conventional high-cost and bulky objectives with high numerical apertures. The field of view of the acoutofluidic scanning nanoscope is much larger than that from a conventional high numerical aperture objective lens, and it is able to achieve the same resolving power. The acoutofluidic scanning nanoscope automatically focuses and maintains a constant working distance during the scanning process thanks to the interaction of the microparticles with the liquid domain. The resolving power of the acoutofluidic scanning nanoscope can easily be adjusted by using microparticles of different sizes and refractive indices. Additionally, it may be possible to further improve the performance of this platform by exploring additional microparticle sizes and materials, in combination with various objectives. Altogether, we believe this acoutofluidic scanning nanoscope has potential to be integrated into a wide range of applications from portable nano-detection to biomedicine and microfluidics. Next, we developed a dual-camera acoustofluidic nanoscope with a seamless image merging algorithm (alpha blending process). This design allows us to precisely image both the sample and the microspheres simultaneously and accurately track the particle path and location. Therefore, the number of images required to capture the entire field of view (200 × 200 μm) by using our acoustofluidic scanning nanoscope is reduced by 55-fold compared with previous designs. Moreover, the image quality is also greatly improved by applying an alpha blending imaging technique, which is critical for accurately depicting and identifying nanoscale objects or processes. This dual-camera acoustofluidic nanoscope paves the way for enhanced nanoimaging with high resolution and a large field of view. Next, we developed an acoustofluidic scanning nanoscope via fluorescence amplification technique. Nanoscale fluorescence signal amplification is a significant feature for many biomedical and cell biology research area. Different types of fluorescence amplification techniques were studied; however, those technologies still need a complex process and rely on an elaborate optical system. To conquer these limitations, we developed an acoustofluidic scanning nanoscope via fluorescence amplification with hard PDMS membrane technique. The microsphere magnification by photonic nanojets effect with the hard PDMS could deliver certain focal distance to maximize the amplification. Moreover, a bidirectional acoustofluidic scanning device with an image processing also developed to perform 2D scanning of large field of view area. In the image processing procedure, we applied a correction of lens distortion to provide a restored distortion image. This fluorescence amplification via acoustofluidic nanoscope allow us to afford a nanoscale fluorescence imaging. Next, we developed an intelligent nanoscope that combines machine learning and microsphere array-based imaging to: (1) surpass the diffraction limit of the microscope objective with microsphere imaging to provide high-resolution images; (2) provide large field-of-view imaging without the sacrifice of resolution by utilizing a microsphere array; and (3) rapidly classify nanomaterials using a deep convolution neural network. The intelligent nanoscope delivers more than 46 magnified images from a single image frame so that we collected more than 1,000 images within 2 seconds. Moreover, the intelligent nanoscope achieves a 95% nanomaterial classification accuracy using 1,000 images of training sets, which is 45% more accurate than without the microsphere array. The intelligent nanoscope also achieves a 92% bacteria classification accuracy using 50,000 images of training sets, which is 35% more accurate than without the microsphere array. This platform accomplished rapid, accurate detection and classification of nanomaterials with miniscule size differences. The capabilities of this device wield the potential to further detect and classify smaller biological nanomaterial, such as viruses or extracellular vesicles. Lastly, this chapter serves a conclusion. Here, I discuss current issues regarding the acoustofluidic scanning nanoscope across review the current limitations of the technology and give suggestions for different direction of microsphere imaging. Moreover, I provide my perspective on the future development of acoustofluidic scanning nanoscope and potential new applications. I discuss how the technologies developed in this dissertation can be improved and applied to new applications in nanoimaging.

Handling of fluids is essential for a majority of applications involving liquid phase reactions in chemistry, biology, and biomedicine. In contrast to manual pipetting in conventional small workshops, automated liquid handling techniques have brought unrivaled accuracy, precision, speed, and repeatability to modern biomedical researches and pharmaceutical industries. Despite their benefits, most advanced liquid handling techniques (e.g., microfluidics and micro-plates) lack fluidic rewritability due to surface-adsorption-induced contaminations on solid-liquid interfaces, limiting their capability of performing complex cascade reactions or high-content combinatorial screening on reusable fluid carriers. To date, the lack of fluidic rewritability still remains as a challenge for engineering scientists to achieve the automated processing of ‘fluidic bits’ in a manner similar to ‘electronic bits’ within a miniature chip. In this work, we approach the fluidic rewritability by contactlessly manipulating aqueous droplets floating on a dense, immiscible carrier fluid layer using acoustic-streaming-induced hydrodynamic gradients. The presented acoustic streaming-based liquid handling (i.e., digital acoustofluidics) devices can be categorized into three versions. (1) The first version of digital acoustofluidic devices actuate floating droplets and small objects by actively propelling them along a straight path following the horizontal direction of acoustic wave propagation. (2) In contrast, the second version employs acousto-hydrodynamic potential traps on the surface of the carrier fluid layer to attract and capture the floating droplets at the equilibrium position of the triggered butterfly-shaped streaming pattern. By selectively exciting the immersed interdigital transducers and sequentially triggering the localized acousto-hydrodynamic traps, the floating droplets can be transported, merged, mixed, split, and generated in a contact-free and programmable manner. (3) The third version of digital acoustofluidic devices is built upon the second version by integrating additional channel-shaped acoustic streaming vortices under high-amplitude excitations, enabling dual-mode manipulation using a single unit transducer. Furthermore, based on the scalable feature of the channel-shaped acoustic streaming vortices, fundamental droplet logic control can be achieved without solid-liquid interactions.

Altogether, this article summarizes the trials-and-errors, working mechanism, design principle, controlling strategy, and potential improvement directions of our digital acoustofluidics platform to facilitate the future development of compact liquid handling workstation with fluidic rewritability. Furthermore, it is our hope that our results and efforts can benefit the explorations in acoustic streaming and associated meso-/micro-manipulation techniques. Lastly, we hope the concept of fluidic rewritability in digitized liquid handling may motivate future microfluidic engineers to develop real Lab-on-a-Chip devices to enable high-speed automation of reactions with dynamic reconfigurability and controllability.

Acoustofluidic technologies, the integration of acoustics into microfluidics, offer rich possibilities for particle manipulation in life sciences. One promising aspect of these technologies is acoustic micro-vortex steaming, resulting from the energy dissipation of acoustics into fluids. There are two opposing directions for the development of micro-vortex steaming: the first one is increasing rotational flow to enhance microscale fluid motion for laminar fluid mixing and capture of biological particles; the other is suppressing rotational flow to create stable acoustic pressure fields for particle patterning, deflection and separation. Although these developments have demonstrated success in microfluidic mixing and cell separation, their ability to realize nanoparticle separation and precisely control fluid mixing, particularly for viscous samples, multi-fluids, and sequential fluids, remains limited. In this dissertation, we target at expanding micro-vortex streaming based acoustofluidic technologies by addressing the existed technological hurdles and introducing new physical concept of topological insulator. To this end, we first developed a sharp edge based acoustofluidic micromixer that enables robust and strong mixing. Robust, efficient, and strong mixing in microfluidics is essential to viscous biological sample preparation. Inspired by the concept that the energy band of phononic crystals depends strongly on their structure height and substrate thickness, we maximized micro-vortex streaming via rational design of the microchamber and glass substrate thickness. The device is able to not only mix fluids across a wide range of flow rates up to 150 µL min-1, but also process fluids with viscosities to 95.9 mPa.s. Using this strong micro-vortex streaming, we were able to realize on-chip liquefaction for human stool samples. This device provides a promising platform to be integrated with portable stool diagnostics. Next, we developed a sharp edge based acoustofluidic micromixer capable of achieving rapid, multi-fluid (≥2) and multi-step (≥2) mixing, which is difficult to realize in hydrodynamic fluid focusing method. Rapid, multi-step and multi-fluid mixing is critical to nanomaterial synthesis for drug delivery. These novel capabilities are realized simply by varying the strength and sites of micro-vortex streaming. With this platform, we synthesized homogeneous poly(lactide-co-glycolide)-block-poly (ethylene glycol) (PLGA-PEG) nanoparticles by rapid mixing, high-molecular PLGA-PEG nanoparticles by strong mixing, PLGA-lipid core-shell nanoparticles by two-step mixing and chitosan nanoparticles by three-fluid mixing. When combined with varying flow rates and reagent concentrations, the acoustofluidic platform allows for nanoparticle synthesis with unprecedented control of nanoparticle size and structure. We have also developed a surface acoustic wave (SAW)-based, disposable acoustofluidic platform for bacteria separation by suppressing micro-vortex streaming and enhancing acoustic pressure field. SAW induced micro-vortex streaming is generated by the viscous attenuation of SAW propagation. In SAW-based devices, the acoustic steaming competes with acoustic radiation force. To overcome SAW induced steaming, we generated standing SAW to form time-averaged momentum flux in opposite direction and then cancelling it out. To increase acoustic radiation force, we designed unidirectional transducers that enable SAW to propagate primarily in one direction, thus tremendously increasing acoustic energy intensity. Using this device, we were able to pattern 400 nm polystyrene particles within the disposable microchannel, as well as separate 600 nm silicon dioxide and 200 nm silver nanoparticles from 1 µm polystyrene particles. Additionally, our disposable device achieved high-purity separation of bacteria from human red blood cells (RBCs). This method of unidirectional transducer design provides a way of enhancing acoustic radiation force to suppress acoustic streaming. Finally, we developed a valley hall based topological acoustofluidic device with the characteristic of chiral micro-vortex streaming by introducing the concept of topological insulator. Topological insulators, which originate from condensed matter physics, have recently been exploited for unconventional wave propagation. One of the prominent features of valley-hall based topological insulators is chiral vortex feature of energy flux. By electroplating copper micropillars on a lithium niobate substrate, we established hexagonal latticed copper pillars with valley hall effect in microfluidics, where SAW was utilized as excitation source. We numerically and experimentally demonstrated clockwise and anticlockwise of vortex streaming by tracing 200 nm fluorescent polystyrene particles. To our knowledge, this is the first visualization of chiral vortex feature in topological insulators. Within the microfluidic community, this allows for novel functionalities including unidirectional particle rotation and back-movement immune particle transport. In the topological physics space, the liquid domain within microfluidic devices enables a new technology for characterizing topological spin textures, which is difficult to be realized in solid or air domain. Furthermore, multiphysics nature of the system enriches the physics of topological insulators. Therefore, the topological acoustofluidics developed here expands not only the field of microfluidics but also the field of topological insulators. In summary, this dissertation serves to further the knowledge of micro-vortex streaming along three planes: enhancing micro-vortex streaming, suppressing micro-vortex streaming, and introducing topological physics to generate chiral micro-vortex streaming. Finally, I will provide my perspective for the next-generation development of micro-vortex streaming based technologies and possible emerging applications. I hope that eventually the microfluidic and physical communities can benefit from each other.

Acoustofluidics is an emerging research field that combines both acoustics and fluid dynamics. With acoustic tweezers technique being developed for years, it is featured for its contactless, noninvasive, and biocompatibility which makes the method suitable for various applications in the field of biology, material sciences, and chemistry. Especially when handling small objects, e.g., cells, nanoparticles, C. elegans, and zebrafish larvae, the native environment involved is mainly liquid. During the acoustic propagation inside the liquid, fluid motion will also be initiated and will influence the object movement in addition to the acoustic radiation forces. This brings up the multidisciplinary study combining the acoustic wave and fluid motion for object manipulation within liquids. This technical development has revealed huge potential for applying acoustofluidic studies into different applications. However, there are still several technical bottlenecks that must be overcome for acoustofluidic technology to provide maximum impact. For example, cell patterning using standing acoustic waves commonly has the regular grid-like shape and sees the fluid motion as an unwanted side effect without an effective way to minimize it. The current target particle size that can be controlled using acoustics is between ~mm to µm, thus hindering the exploration of nanoscale objects. In this dissertation, I explored the combined effect of acoustics and fluid dynamics, and validated that the interplay of both effects can derive new research insights and can be applied to particles with a smaller size range (i.e., nanometer). Specifically, I studied the synergetic effect of acoustics and flow in three classic fluid systems: bulk fluids, droplets, and continuous flow. For bulk fluids, we designed an acoustofluidic holography platform that can initiate and utilize fluid motion with arbitrary designed acoustic fields. With the design and implementation of the holographic acoustic lens, our method can pattern cells into an arbitrary shape that can potentially benefit tissue engineering or cell mechanics studies. Besides patterning, we also demonstrated that, with the same experimental configuration, we can utilize vortex acoustic streaming to achieve different functions, e.g., particle rotation, concentration, and separation. For droplets, we observed a new physics phenomenon which can drive the spin of a liquid droplet using surface acoustic wave. With this external angular momentum and Stokes drift effect, we found the nanoparticles can be rapidly concentrated or differentially concentrated in one spinning droplet. Furthermore, we demonstrated that the single spinning droplet can serve as one unit that possesses a specific function and we can assemble the units for a more flexible manipulation function. We built a dual droplet acoustofluidic centrifuge system that can achieve nanoparticle separation and transport and utilized the platform for exosome subgroup separation. For continuous flow, as acoustic separation technique has been developed for years, we have explored two directions that may be utilized for small animal blood apheresis study. One direction is the high-throughput platelet separation using a plastic device. This method significantly increased the throughput and moved one step towards clinical usage. Another direction is building the integrated system for plasma separation. Built around the surface acoustic wave separator, we assembled the fluid driving unit, temperature control unit, and separation unit into a prototype-like system. We then performed the proof-of-concept experiment to identify the feasibility of applying the acoustofluidic separation method to small animal models (i.e., mice).

Microfluidic technologies, and the subset of devices that integrate acoustics into their designs (known as acoustofluidic devices), present great potential for solving the challenges of the future. One specific subset of these technologies, termed sharp-edge based acoustofluidics, has shown promise in a variety applications; specifically, previous work has explored the use of this technology in applications such as fluid pumping and mixing, cell stimulation, and bio-sample preparation. However, even though there are a vast number of applications that sharp-edge based acoustofluidics have been applied to, there are several shortcomings that need to be addressed.

First, and perhaps most critically, very little is known about the fundamental mechanism of this platform’s operation. The search for novel applications has left a gap in the knowledge base for understanding how these devices work on a fundamental level; gaining a better understanding of how the technology works may open the door to finding new and previously unimagined applications. Second, although not a problem that is specifically limited to sharp-edge based acoustofluidic devices, the technology suffers from serious limitations in real world applicability. That is, even though these devices have advantages over traditional techniques, including speed, cost, and ease of use, they are unable to be taken advantage of. For this reason, there is a critical need to demonstrate a viable pathway to real-world usage.

In an attempt to tackle these shortcomings, we begin our research by investigating the vibrational profile generated within a sharp-edge mixer. Throughout this exploration we uncover that the mechanism behind the technology’s success is relatively low frequency flexural waves which have wavelengths commensurate with the overall dimensions of the technology. This is in contrast to the previous belief that waves with lengths many times larger than the device itself were dominating; as a result, we developed and explored a novel platform for particle manipulation based on wave interference (not unlike high frequency based acoustofluidic platforms). This technology offers a new technique for interacting with micro particles and cells in an open fluid chamber. In order to improve the technology’s adoptability, we also developed and characterized two unique and portable control platforms towards eventual point-of-care (POC) use.

Altogether, this work serves to further the knowledge and relevance of sharp-edge based technology. It is our hope that this work can serve as a starting point for future explorations into novel platforms which make use of the small wavelength vibrations achievable with this low cost setup. Additionally, we hope that this work may motivate the broader field to transition their technology into equally accessible platforms, such that the microfluidics community as a whole can bring their useful technology to practical applications.

Technologies that can manipulate single particles and cells in a high precision, high throughput and contact-free manner have long been motivated by applications in materials science, physics, medicine, and the life sciences. However, current single cell manipulation tools, such as atomic force microscopy, optical tweezers, and micropipette aspiration, suffer from low throughputs, insufficient repeatability over the single cell assay, and physical contacts that can negatively influence testing results. On the other hand, conventional acoustic tweezers can manipulate cells in a high biocompatible and contact-free manner but lack the precision to selectively manipulate single cells for biophysical studies. In this dissertation, I demonstrate the reinvention of the acoustic tweezers for high throughput, high precision, high repeatable and selective single cell manipulation. With the analytical simulation of acoustic waves, I propose both the spatial and temporal modulation methods to generate and control surface acoustic waves to dynamically manipulate single cells. And further biophysical analysis studies, such as the assay of differences in intercellular adhesion among cancer cell lines with different malignancies and metastatic potentials are demonstrated.

As rapid developments in technology merge acoustics and microfluidics, acoustofluidic technology has been increasingly employed in biophysical and biomedical research to address various challenges, especially in the fields of tissue engineering, liquid biopsy, clinical diagnostics and therapeutics. Acoustofluidic technologies offer highly biocompatible, label-free and contact-less manipulation of objects based on differential effects including acoustic streaming and acoustic radiation force. However, acoustofluidic technologies have not been fully implemented into model animal studies to simplify the manipulation, lower the cost, and increase the throughput. In addition, despite the expansion of the scope of acoustic-based particle manipulation technologies from the micro to the nanoscale over the past decade, limitations continue to pose challenges in manipulating sub-100 nm particles using acoustic waves. As a result, developing an acoustic technique capable of manipulating sub-100 nm particles would strengthen the capabilities of acoustic manipulation and fulfill needs in areas such as biomedicine, biophysics, optics, electronics, and materials science.

First, with the nematode Caenorhabditis elegans (C. elegans) employed as a model animal in the field of developmental biology, neuroscience, human diseases, aging and drug screening for more than 50 years, we sought to extend acoustofluidic technologies into C. elegans research to address the key drawbacks of current C. elegans analysis procedures. An acoustofluidic chip capable of rotating C. elegans in both static and continuous flow in a controllable, precise, high-throughput and stable manner was then developed. Rotational manipulation was achieved by exposing C. elegans to a surface acoustic wave (SAW) field that generated a vortex inside a microchannel. By controlling the propagation of the SAW, we achieved bidirectional and stepwise rotation of C. elegans. Using this chip, we have clearly imaged the dopaminergic neurons, as well as the vulval muscles and muscle fibers of the C. elegans in different orientations. These achievements are difficult to realize through conventional microscopy. After that, another tool for effectively isolating and categorizing large quantities of C. elegans based on different phenotypes was developed as an integrated acoustofluidic chip. This chip was capable of identifying worms of interest based on expression of a fluorescent protein in a continuous flow and then separating them accordingly in a high-throughput manner. For example, L3 worms can be processed at a throughput of around 70 worms/min with a sample purity over 99%, which remains over 90% when the throughput is increased to around 115 worms/min. In our acoustofluidic chip, the time period to complete the detection and sorting of one worm is only 50 ms, which outperforms nearly all existing microfluidics-based worm sorting devices and may be further reduced to achieve higher throughput.

Moving forward with the experience we gained through manipulating C. elegans via acoustofluidics, we are aiming to solve a critical issue in current acoustofluidic technologies. Although acoustic fields have been increasingly used to pattern, focus, and separate micro- and nanometer-sized particles for biomedical applications, the acoustic-based separation of nanoscale bioparticles in sub-100 nm range remains a significant challenge. To address this problem, we present Acoustic Nanoscale Separation via Wave-pillar Excitation Resonance (ANSWER), allowing for the high-resolution, tunable separation of nanoscale bioparticles ranging from 50 nm to 1000 nm. We created virtual acoustic pillars that enable iterative deflection of particles for precision separation via an excitation resonance. Controlling the cut-off diameter is optimized by acoustic frequency, power, and microchannel dimensions in separating sub-100 nm particles. To demonstrate the potential of our ANSWER platform in biomedical applications, we have shown its ability to fractionate small extracellular vesicle (sEV) subpopulations. For the first time, sEV subpopulations can be rapidly separated (<10 minutes) directly from human plasma without sample preprocessing or complex nanofabrication. Due to its high separation purity (>96% small exosomes, >80% exomeres), ANSWER shows promise as a powerful tool that will enable more in-depth studies into the complexity, heterogeneity, and functionality of sEV subpopulations. To simplify the operation and keep the biological components in their native environment, separation without sheathflow was then discussed with the ANSWER platform. The same sheathless separation concept was then extended to the microscale for the isolation of plasma directly from human whole blood.

The work in this dissertation presents a comprehensive investigation and exploration of both the mechanism of specialized acoustic field generation and modulation, as well as the application of highly controllable manipulation of model animals and nanoscale (< 100 nm) biological particles. We hope our work can benefit and enable new possibilities in the relevant research fields.