Browsing by Subject "Acoustofluidics"

Cancer comprises a collection of diseases, all of which begin with abnormal tissue growth from various stimuli, including (but not limited to): heredity, genetic mutation, exposure to harmful substances, radiation as well as poor dieting and lack of exercise. The early detection of cancer is vital to providing life-saving, therapeutic intervention. However, current methods for detection (e.g., tissue biopsy, endoscopy and medical imaging) often suffer from low patient compliance and an elevated risk of complications in elderly patients. As such, many are looking to “liquid biopsies” for clues into presence and status of cancer due to its minimal invasiveness and ability to provide rich information about the native tumor. In such liquid biopsies, peripheral blood is drawn from patients and is screened for key biomarkers, chiefly circulating tumor cells (CTCs). Capturing, enumerating and analyzing the genetic and metabolomic characteristics of these CTCs may hold the key for guiding doctors to better understand the source of cancer at an earlier stage for more efficacious disease management.

The isolation of CTCs from whole blood, however, remains a significant challenge due to their (i) low abundance, (ii) lack of a universal surface marker and (iii) epithelial-mesenchymal transition that down-regulates common surface markers (e.g., EpCAM), reducing their likelihood of detection via positive selection assays. These factors potentiate the need for an improved cell isolation strategy that can collect CTCs via both positive and negative selection modalities as to avoid the reliance on a single marker, or set of markers, for more accurate enumeration and diagnosis.

The technologies proposed herein offer a unique set of strategies to focus, sort and template cells in three independent microfluidic modules. The first module exploits ultrasonic standing waves and a class of elastomeric particles for the rapid and discriminate sequestration of cells. This type of cell handling holds promise not only in sorting, but also in the isolation of soluble markers from biofluids. The second module contains components to focus (i.e., arrange) cells via forces from acoustic standing waves and separate cells in a high throughput fashion via free-flow magnetophoresis. The third module uses a printed array of micromagnets to capture magnetically labeled cells into well-defined compartments, enabling on-chip staining and single cell analysis. These technologies can operate in standalone formats, or can be adapted to operate with established analytical technologies, such as flow cytometry. A key advantage of these innovations is their ability to process erythrocyte-lysed blood in a rapid (and thus high throughput) fashion. They can process fluids at a variety of concentrations and flow rates, target cells with various immunophenotypes and sort cells via positive (and potentially negative) selection. These technologies are chip-based, fabricated using standard clean room equipment, towards a disposable clinical tool. With further optimization in design and performance, these technologies might aid in the early detection, and potentially treatment, of cancer and various other physical ailments.

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.

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.

Droplet microfluidics technology holds immense potential for single-cell level biomedical engineering studies, motivating the study of cellular immunotherapy, cell interactions, drug screening, and single-cell dynamics. However, existing droplet microfluidics have limited pairing efficiency, complex manual operation procedures, and low in-droplet cell manipulation capacity, which hinder their potential application in single-cell analyses with high sample purity or uniformity requirements. Several efforts, such as electrophoretic, magnetic, and optical droplet sorters, have been used to overcome some limitations. Still, they need the all-powerful capability of acoustics to solve all the problems. As a dynamic, precise, contact-free, and biocompatible force, acoustics can improve pairing efficiency in droplets and provide an ideal tool for active droplet manipulation. In this dissertation, I demonstrate the reinvention of droplet microfluidics, reporting the multifunctionality of acoustics for high throughput, high-precision droplet sorting, and active droplet manipulation. I propose a modular acoustofluidics platform designed for the streamlined sorting and collection of effector-target (i.e., NK92-K562) cell pairs, facilitating efficient monitoring of the real-time dynamics of immunological response formation. Coupled with transcriptional and protein expression analyses, I evaluated the synergistic effect of doxorubicin on the cellular immune response. In addition, because of the non-contact nature of acoustics, I can actively and simultaneously manipulate multiple cell-encapsulated droplets, which other methods cannot achieve. The proposed acoustofluidic platform can provide promising building blocks for high-throughput quantitative single-cell level coculture to understand intercellular communication while empowering immunotherapy through precision manipulation and analysis of immunological synapses.

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.

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.