Browsing by Subject "Microfluidics"

Detection and quantification of biomarkers directly from complex clinical specimens is desired and often required by healthcare professionals for the effective diagnosis and screening of disease, and for general patient care. Current methodologies to accomplish this task have critical shortcomings. Laboratory immunoassays, most notably enzyme-linked immunosorbent assay (ELISA) require extensive clinical infrastructure and complex user intervention steps to generate results and often are accompanied by a lengthy time-to-result. Conversely, available point-of-care (POC) diagnostic solutions, most notably available lateral flow immunoassays (LFIAs), often struggle with sensitivity and specificity in complex fluids, lack quantitative output and are not easily multiplexed. In this dissertation I will discuss the design, fabrication, testing, and refinement of an all-in-one fluorescence microarray integrated into a passive microfluidic fluid handling system to create a versatile and automated POC platform that can detect biomarkers from complex samples for disease management with the relative ease-of-use of an LFIA and the performance of a laboratory-grade test. The platform is driven by capillary and gravitational forces and automates all intervention steps after the addition of the sample and running buffer at the start of testing. The microfluidic cassette is built on a (poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) polymer brush which imparts two key functionalities, (1) it eliminates cellular and protein binding, and when combined with the vertical orientation of the microfluidic cassette prevents settling of debris during all assay steps. This allows for impressive sensitivities and specificities to be obtained from samples as complex as undiluted whole blood even when relying on gentle capillary and hydrostatic pressures for cassette operation. (2) Paradoxically, printed biorecognition elements can be stably and non-covalently immobilized into the POEGMA allowing for all reagents needed to conduct a sandwich immunoassay in a single step to be easily inkjet printed as spatially discrete spots into the POEGMA brush, which also stabilizes them at room temperature. Additionally, the microfluidic cassette is compatible with the “D4Scope” a handheld fluorescence detector that can quantify the output of the microfluidic cassette in seconds at the POC and is the only piece of auxiliary equipment required to operate the test.

This dissertation discusses early cassette prototypes and characterizes the performance of major device iterations (Chapter 2) before moving into three clinical applications of the cassette. First, a multiplexed serological test to detect antibodies against different proteins of the SARS-CoV-2 virus was developed (Chapter 3). Second, a multiplexed COVID-19 diagnostic test that simultaneously differentiates which variant you are infected with was developed (Chapter 4). Third, a sensitive fungal infection test for the diagnosis of talaromycosis was developed (Chapter 5). Finally, a rapidly iterative yet highly scalable injection molding fabrication process flow was created and characterized to improve performance and translatability of the cassette (Chapter 6).

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.

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.

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.

The quantitative measurement of inorganic ions in the atmosphere is an important aspect in environmental science. The three most important inorganic ions are sulfate, nitrate and ammonium, which are the most abundant components of atmospheric pollutants and have a significant impact on rainfall, atmospheric visibility and human health. To accurately and quickly measure the distribution of inorganic ions in the vertical and horizontal directions of the atmosphere, a compact and automatic real-time detection system is in need.

The research performed in this study is aimed at developing the science and technology for an aerosol detection system that combines digital microfluidics technology, aerosol impaction and chemical detection on the same chip. The system will be smaller and faster with respect to current aerosol analyzing instruments. The chip in this study performs the integrated functions of aerosol collection, extraction, and quantitative detection in real-time, unlike current benchtop methods that require operator handling and laboratory equipment. All functions are realized in dedicated sections on a digital microfluidic platform.

This thesis will present the design and test of individual components of the aforementioned functions. The digital microfluidics chip design includes transparent top and bottom plates for light absorbance measurement. The droplets are dispensed, transported and mixed on chip with other droplets by activating electrodes individually with a 50V AC sine voltage.

In Chapters 3 and 4, the issues involving droplet transportation are addressed, including droplet movement between the air and silicone oil media and droplet transport across the aerosol impaction area. Next, an aerosol impactor and a chip-to-world chamber are demonstrated and tested with lab generated sulfate aerosol. The collected aerosol showed a clear pattern on the impaction plate, and the collection efficiency inside the chip was 96%.

In Chapter 5, the development of colorimetric methods are described as well as experimental testing for inorganic ion detection. Three well-known tests for detecting sulfate, nitrate and ammonium were first adjusted to adapt to on-chip measurement conditions, the adjustments including the choices of solvent, concentration ranges and mixing ratios. The particle measurement results using a conventional spectrometer were compared with on-chip measurements in terms of absorbance range, limit of detection, sensitivity (based on the coefficient of determination and the slope of the linear regression) and signal-to- noise ratio (presented with standard deviation/average of absorbance measurements).

The thin oil film between the droplet and the top/bottom plate, which is naturally formed, plays an important role in lubrication and reduces contact angle hysteresis. However, these oil films are not always uniform in thickness. During the absorbance measurement tests, varied sizes of oil lenses were observed at the oil/top plate interface, and the size and position of the oil lenses randomly changed when a droplet moved between electrodes. The absorbance measured in the normal direction to the chip’s surface was affected by these oil lenses and, thus, not stable for multiple measurements of the same droplet or for different droplets. To solve this problem, optical fibers were introduced horizontally inside the chip, and measurements taken in this direction proved to produce stable results.

Prototypes of the chip have been fabricated, and the impaction and on-chip colorimetric tests for sulfate and ammonium were successful. Although this study was designed to build the fundamentals of a novel detection system of inorganic ions in aerosol, the potential use of the designed system is not limited to atmospheric studies. Applications can extend to testing the quality of drinking water, detection of nitroaromatic explosives or other experiments based on colorimetry.

Every cell in our body contains the same genetic material, but what differentiates one cell type from another is the way in which that material is interpreted. Hidden in the 98% of non-coding DNA sequences, once referred to as “junk DNA,” are the instructions for how to turn genes on and off (i.e., the operating system). As in any other language, decoding the instructions between DNA and gene expression is the key for understanding transcriptional regulation. Without understanding the grammar of transcriptional regulation, we cannot tell which sequence changes affect gene expression and how.

In this study, we have focused on defining the mechanisms mediating gene expression in response to steroid hormones (predominantly 17β-estradiol) as a model system for other non-steroid transcription systems that can be exploited to define general principals governing steroid responsiveness upon target genes. In particular, we have demonstrated that DNA sequence constraints define the functionally active steroid nuclear receptor (sNR) gene regulatory elements in the genome, and this functionality is restricted to elements that vary from the consensus palindromic elements by one or two nucleotides, named nuclear receptor functional enhancers (NRFEs). At NRFEs, the chromatin binding of steroid nuclear receptors is not only correlated with active eRNA production, but also RNAPII occupancy as well as hormone-dependent coregulator and transcription factor (TF) recruitment. Moreover, steroid nuclear receptors with mutated DNA binding domains (DBD), were shown to still interact with chromatin, yet lack hormone-dependent transcriptional activity, highlighting the fact that steroid nuclear receptors can interact with chromatin in a transcriptionally inactive state (i.e., the majority of sNR chromatin interacting events identified in ChIP studies are not linked directly to transcriptional events) (Chapter 2). We further demonstrate that the palindromic architecture of the regulatory element is the underlying mechanism that governs chromatin interaction by steroid nuclear receptors, and the non-functional chromatin interacting sites (non-NRFEs) observed in ChIP-seq studies are subject to the same rules and constraints as NRFEs. Thus, NRFE vs. non-NRFE binding is dictated at the individual nucleotide level, and the residence time (or strength of binding) is determined by the number and locations of variants within the consensus element. The basis of these rules and DNA constraints follow specific algebraic relationships. These findings quantitatively define how steroid nuclear receptors select the ‘appropriate’ regulatory targets out of a very large number of highly similar sequences in the genome, thus eliciting a specific cellular response (Chapter 3).

Estradiol is a potent mitogen in the mouse uterus, a well-characterized tissue used to study the underlying mechanisms of estrogen-mediated transcriptional regulation. Previously shown, estradiol-mediated transcriptional regulation in the mouse uterus is biphasic and can be divided into initial (early) phase and subsequent (late) phase transcriptional events. In this study, we demonstrate that late phase estradiol-mediated transcription requires the early phase transcripts and low-affinity estrogen receptor α (ERα) ligands cannot sustain late phase hormone-mediated transcriptional events. In addition, the interaction of ERα with chromatin is (1) immediate, (2) does not change locations after initial contact, (3) is retained the longest at NRFEs, and (4) is depleted prior to the late phase transcriptional events. Collectively, this indicates that estradiol-mediated late phase transcripts are regulated secondary to early induced transcripts (i.e., the early induced transcripts activate other transcription factors which are responsible for producing the late phase transcripts). Furthermore, the AF2 coactivator surface of ERα is not required for hormone-dependent ERα recruitment to NRFEs, estrogen-independent basal transcription requires ERα binding at NRFEs and the growth factor insulin-like growth factor 1 (IGF-1) activates ERα by recruitment of ERα to NRFEs (Chapter 4).

Our understanding of the physiology and transcriptional regulation of steroid hormones was significantly advanced following the generation of mutant mouse models possessing disruptions (knockouts) of the steroid nuclear receptor genes. In this study, we identified the molecular defects caused by a homozygous missense mutation in ERα identified in an 18 year-old woman with complete estrogen insensitivity syndrome; a clinical presentation similar to those of ERα knockout (αERKO) female mice. From these studies, we identified a potential therapeutic, Diethylstilbestrol (DES), for treating this estrogen insensitivity condition. Treatment of this patient with DES is underway and we remain involved as collaborators in this clinical study. Our studies also characterize the molecular defects caused by a different homozygous mutation in ERα identified in two sisters and a brother that likewise exhibit complete estrogen insensitivity (Chapter 5).

Hormone-dependent transcriptional regulation requires the recruitment of coregulators to the regulatory regions of target genes. This recruitment is determined by the overall surface topography of the sNR. Phage display is a widely-used research technique for screening highly diverse peptide libraries to enrich for sNR-binding clones. It requires two primary components for affinity selection: (1) a phage display cDNA library, and (2) purified recombinant sNR protein. High level expression of soluble biologically-active sNR protein is particularly challenging due to its largely hydrophobic ligand binding domain. In this study, we overcame this challenge by constructing a protein expression system that provides the factors responsible for protein folding of sNRs (Hsp90, Hsp40, Hsp70, Hop and p23) at levels comparable with the amount of over expressed sNR (Chapter 6). Affinity selection in phage display involves panning of a phage library to enrich for sNR-binding clones followed by their amplification. This amplification step enriches for clones that have a growth advantage, introducing bias into the selection that favors faster growing clones regardless of the selection pressure. To eliminate this bias, individual phage must be separated into different growth chambers so they cannot compete for bacterial hosts. To do this, we used microfluidic flow-focusing technology (MFFT) to generate monodisperse droplet based compartments to encapsulate individual phage clones and achieve non-competitive amplification of millions of phage clones having different growth characteristics. The elimination of growth-based competition ensures that selection of binding clones is driven only by the binding strength of each clone for the sNR. The successful development of a MFFT platform and proof of principal demonstration, allowed us to then implement a high throughput MFFT droplet system. This project was the first application to introduce this novel technology to the National Institute of Environmental Health Sciences (NIEHS) (Chapter 6).

Transcriptional regulation takes place at the level of single cells. However, many traditional techniques involve homogenizing tissue samples composed of millions of cells, and thus can only deal with population averages. Single-cell sequencing reveals the inherent properties of a single cell from the large scale of the genome, information critical for understanding cellular heterogeneity in cancer and response/resistance to therapy. Using our new high throughput MFFT droplet system, genome-wide gene expression profiling of individual cells can be done by separating thousands of individual cells into nanoliter-sized aqueous droplets, associating a different barcode with each cell’s RNAs, and sequencing them all together. This results in transcripts from thousands of individual cells that are all identified by their cell of origin. Here, we establish a droplet microfluidic method to sequence genomes of single cells from dissociated, complex tissues using a custom fluorosurfactant (i.e., a triblock copolymer consisting of a polyethylene glycol (PEG) center block covalently bound to two perfluorinated polyether (PFPE) blocks by amide linking groups), to address two of the major challenges in performing biological, drop-based assays: to stabilize aqueous droplets in fluorocarbon oils and to make the droplets compatible with biological molecules and cells (Chapter 6).

Post-translational modification by SUMO is an important mechanism to regulate transcription. Tamoxifen is used in the treatment and prevention of ER positive breast cancer. Tamoxifen is metabolized predominantly by the cytochrome P450 system to several primary and secondary metabolites, some of which exhibit more antiestrogenic effects than tamoxifen itself. In this study, we demonstrate that the more antiestrogenic effect of endoxifen versus tamoxifen is due post-translational modification by SUMO and inhibition of SUMO derepresses endoxifen’s anti-estrogenic activity. This mechanism of transcriptional repression was also demonstrated in other antiestrogens including fulvestrant, raloxifene, bazedoxifene, idoxifene and lasofoxifene (Chapter 7).

Promising applications in the design of various biological systems hold critical implications as heralded in the rising field of synthetic biology. But, to achieve these goals, the ability to synthesize in situ DNA constructs of any size or sequence rapidly, accurately and economically is crucial. Today, the process of DNA oligonucleotide synthesis has been automated but the overall development of gene and genome synthesis technology has far lagged behind that of gene and genome sequencing. This has meant that numerous ideas go unfulfilled due to scale, cost and impediments in the quality of DNA due to synthesis errors.

This thesis presents the development of a multi-tool ensemble platform targeted at gene synthesis. An inkjet oligonucleotide synthesizer is constructed to synthesize DNA microarrays onto silica functionalized cylic olefin copolymer substrates. The arrays are married to microfluidic wells that provide a chamber to for enzymatic amplification and assembly of the DNA from the microarrays into a larger construct. Harvested product is then amplified off-chip and error corrected using a mismatch endonuclease-based reaction. This platform has the potential to be particularly low-cost since it employs standard phosphoramidite reagents and parts that are cheaper than optical and electrochemical systems. Genes sized 160 bp to 993 bp were successfully harvested and, after error correction, achieved up to 94% of intended functionality.

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.

Digital microfluidic (DMF) technology is being utilized for commercial applications such as point-of-care diagnostics, sample processing and genomic library preparation. Advances in this field offer exciting possibilities into immunoassays, enzymatic analysis, and next-generation sequencing. However, typical biomolecular protocols performed in laboratories are far more complex, requiring large reagent volumes, long processing times and provide a low throughput analysis. Using a DMF platform enables overcoming experimental barriers of manual laboratory protocol execution, allowing for scaling the platform geometry, assay times, volumes of reagents used, and minimizing the use of external mechanical equipment. The DMF platform also allows for easy integration with detection systems enabling real-time data analysis and efficient resource allocation. This thesis explores theoretical and experimental approaches for carrying out enhanced biomolecular binding to magnetic beads on a DMF platform as part of a sample preparation protocol. Different DMF prototypes were designed using standard microfabrication procedures to compare passive mixing, active mixing on electrode arrays and local bead actuation on an integrated current-wire electrode, whereby current is sequenced through copper wires fabricated on the electrode to generate magnetic-fields on-chip that cause the magnetic beads in the assay to move relative to the antibody. By making use of an integrated fluorescence detection system, the binding efficiency for each of these approaches is determined. The current-wire device design proves to be a valuable tool in creating an integrated DMF system to carry out intensive bead binding in an assay allowing for lower reagent volumes, shorter assay times and reduced surface area, thus impacting device yield.

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).

Single-cell analysis (SCA) tools have important applications in the analysis of phenotypic heterogeneity, which is difficult or impossible to analyze in bulk cell culture or patient samples. SCA tools thus have a myriad of applications ranging from better credentialing of drug therapies to the analysis of rare latent cells harboring HIV infection or in Cancer. However, existing SCA systems usually lack the required combination of programmability, flexibility, and scalability necessary to enable the study of cell behaviors and cell-cell interactions at the scales sufficient to analyze extremely rare events. To advance the field, I have developed a novel, programmable, and massively-parallel SCA tool which is based on the principles of computer circuits. By integrating these magnetic circuits with microfluidics channels, I developed a platform that can organize a large number of single particles into an array in a controlled manner.

My magnetophoretic circuits use passive elements constructed in patterned magnetic thin films to move cells along programmed tracks with an external rotating magnetic field. Cell motion along these tracks is analogous to the motion of charges in an electrical conductor, following a rule similar to Ohm’s law. I have also developed asymmetric conductors, similar to electrical diodes, and storage sites for cells that behave similarly to electrical capacitors. I have also developed magnetophoretic circuits which use an overlaid pattern of microwires to switch single cells between different tracks. This switching mechanism, analogous to the operation of electronic transistors, is achieved by establishing a semiconducting gap in the magnetic pattern which can be changed from an insulating state to a conducting state by application of electrical current to an overlaid electrode. I performed an extensive study on the operation of transistors to optimize their geometry and minimize the required gate currents.

By combining these elements into integrated circuits, I have built devices which are capable of organizing a precise number of cells into individually addressable array sites, similar to how a random access memory (RAM) stores electronic data. My programmable magnetic circuits allow for the organization of both cells and single-cell pairs into large arrays. Single cells can also potentially be retrieved for downstream high-throughput genomic analysis.

In order to enhance the efficiency of the tool and to increase the delivery speed of the particles, I have also developed microfluidics systems that are combined with the magnetophoretic circuits. This hybrid system, called magnetomicrofluidics, is capable of rapidly organizing an array of particles and cells with the high precision and control. I have also shown that cells can be grown inside these chips for multiple days, enabling the long-term phenotypic analysis of rare cellular events. These types of studies can reveal important insights about the intercellular signaling networks and answer crucial questions in biology and immunology.

Droplet microfluidics is a powerful platform for both fundamental and applied biomedical research. The droplets are small in size with a diameter of 1-300 um. Thus, they could function as a miniaturized environment for quantitative and qualitative analysis. Each droplet composes of water shielded by an immiscible organic shell which enables independent control over different droplets. The large surface to volume ratio of spherical structure allows rapid mass and heat transfer, which could enable more homogeneous chemical reactions. Moreover, since multiple identical droplets could be generated simultaneously, parallel analysis for large amount of samples are possible. The use of microfluidics brings more power to droplet technology. The precise control over the flow allows droplet with preferable size and structure to be generated, which is critical for quantitative analysis, homogeneous chemical reaction as well as some in vivo applications.

Nonetheless, generation of stable, monodispersed and well controlled emulsions to meet specific biological functions are still challenging. First of all, to form more biocompatible W/O/W DE, the microfluidics devices must be patterned with desired surface wettability. W/O emulsion could only form in hydrophobic environment and the O/W emulsions could only form in hydrophilic environment. Differential patterning of the surface wettability to meet the needs are challenging. Second, DE are stabilized by two amphiphilic surfactants, one for the oil phase and the other for the water phase. Selection of appropriate surfactants should hook with specific biological application to ensure stability and biocompatibility. Third, the choice of fluid and contents in the fluid will affect the viscosity and capillary number of interfacial interaction, and eventually influences the droplet formation. The choice of biocompatible medium and buffer must take this into consideration. Fourth, the adoption of emulsions for the specific application requires optimization of the processing techniques in order to meet the needs for final analysis. For instance, control of droplet rupture for content release, modulation of oil phase permeability, quantitative analysis of content with flow cytometry, etc.

In this thesis, we will first demonstrate the design and fabrication of PDMS-based devices for automatic and high-throughput DE formation in Chapter 2. In the following chapters, we will demonstrate the successful adoption of the microfluidics generated DE for different biological applications. In chapter 3, we will illustrate the application of DE as a micro-incubator for cellular studies such genetic circuit behavior and performance in bacterial cells cultured in DE droplets and formation of 3D mammalian cell spheroid. In chapter 4, we will show the successful application of DE as drug carriers for intranasal drug delivery. In chapter 5, we showed the application of microfluidics generated DE as template for microparticle synthesis and the use of these microparticles as therapeutic agents in nucleic acid induced inflammations in autoimmune diseases.

While drug-loaded biodegradable polymer microparticles have found many therapeutic applications, bulk manufacturing methods produce heterogeneous populations of particles. A more highly controlled manufacturing method may provide the ability improve the microparticle characteristics such as the drug release profile. Microfluidic droplet-makers manipulate liquids on the scale of tens of microns and can produce highly regular and controlled emulsions. However, microfluidic droplet manufacturing is not typically designed for clinical translation and the chemicals used are often not biocompatible.

I developed a two-chip PDMS-based microfluidic device that can manufacture PLGA microparticle loaded with hydrophilic or hydrophobic drugs. I characterized protein-loaded microparticles made using this device and compared them with bulk-generated microparticles. The microfluidics-generated microparticles had similar release curves and encapsulation efficiencies as bulk-generated microparticles but a much narrower size distribution. I generated peanut protein-loaded microparticles with this device and tested them in a mouse model of peanut allergy, improving the particles as the project evolved to have a higher loading level and lower burst release. The microparticles improved the safety and efficacy of an immunotherapy protocol. I also encapsulated hydrophilic and hydrophobic chemotherapeutic drugs for a brain cancer model.

High-throughput processing technologies hold critical position in biomedical research. These include screening of cellular response based on phenotypic difference and production of homogeneous chemicals and biologicals for therapeutic applications. The rapid development of microfluidics technology has provided an efficient, controllable, economical and automatable processing platform for various applications. In particular, emulsion droplet gains a lot of attention due to its uniformity and ease of isolation, but the application of water-in-oil (W/O) single emulsion is hampered by the presence of the oil phase which is incompatible with aqueous phase manipulation and the difficulty in modifying the droplet environment.

This thesis presents the development of a double emulsion (DE) droplet platform in microfluidics and two applications: (1) high-throughput screening of synthetic gene and (2) production of multicellular spheroids with adjustable microenvironment for controlling stem cell differentiation and liver tissue engineering. Monodisperse DE droplets with controllable size and selective permeability across the oil shell were generated via two microfluidics devices after optimization of device design and flow rates.

Next, bacterial cells bearing synthetic genes constructed from an inkjet oligonucleotide synthesizer were encapsulated as single cells in DE droplets. Enrichment of fluorescent signals (~100 times) from the cells allowed quantification and selection of functionally-correct genes before and after error correction scheme was employed. Permeation of Isopropyl β-D-1-thiogalactopyranoside (IPTG) molecules from the external phase triggered target gene expression of the pET vector. Fluorescent signals from at least ~100 bacteria per droplet generated clearly distinguishable fluorescent signals that enabled droplets sorting through fluorescence-activated cell sorting (FACS) technique.

In addition, DE droplets promoted rapid aggregation of mammalian cells into single spheroid in 150 min. Size-tunable human mesenchymal stem cells (hMSC) spheroids could be extracted from the droplets and exhibited better differentiation potential than cells cultured in monolayer. The droplet environment could be altered by loading matrix molecules in it to create spheroid-encapsulated microgel. As an example, hMSC spheroid was encapsulated in alginate or alginate-RGD microgel and enhanced osteogenic differentiation was found in the latter case.

Lastly, the capability of forming spheroids in DE droplet was applied in liver tissue engineering, where single or co-culture hepatocyte spheroids were efficiently produced and encapsulated in microgel. The use of alginate-collagen microgel significantly improved the long-term function of the spheroid, in a manner similar to forming co-culture spheroids of hepatocytes and endothelial progenitor cells at a 5 to 1 ratio. The hepatocyte spheroid encapsulated in microgel could be useful for developing bioartificial liver or drug testing platform or applied directly for hepatocyte transplantation.

Cyber-Physical Systems (CPS) feature synergetic integration of multiple subsystems to control physical environments through cycles of sensing and actuation. The correct-by-design paradigm aims to provide guarantees on the performance of CPS by utilizing formally-proven algorithms for synthesis and validation of various system components. This paradigm postulates the ability to both derive adequate abstractions of the system, and mathematically formalize design requirements. Therefore, developing mathematical tools that allow system designers to easily model CPS, and to capture design requirements, is imperative to such paradigm.

This dissertation provides theoretical and experimental contributions towards modeling and development of assured and adaptive CPS. In particular, we propose Delayed Action Game (DAG) to aid with modeling CPS where part of the state is hidden from the controller. The formalism deploys the concept of delaying actions as means to hide them from other players without the usage of private variables, allowing the use of off-the-shelf model checkers for analysis. Based on a DAG model, we design an algorithm that utilizes model checkers to synthesize optimal strategies. In addition, we propose Context-Aware Probabilistic Temporal Logic (CAPTL) to aid with formalizing temporal requirements that can naturally described as a set of objectives that are prioritized based on some probabilistic conditions. Furthermore, we develop the algorithm that allow for synthesizing optimal strategies for a Markov Decision Process (MDP) that satisfy a given CAPTL-based requirement.

We deploy the theoretical frameworks in two application domains: human-robot interaction and digital microfluidics, with the goal of designing systems to be more adaptive to their environments. First, we develop protocols for supervisory systems where a human operator, supervising a number of Unmanned Aerial Vehicles (UAVs), can intermittently perform geolocation tasks to aid in detection of possible attacks. We model the system as a DAG, and further use it to synthesize security-aware human-UAV protocols that both provide UAV path plans, increasing the chances of attack detection, and specify the time instances at which the operator is advised to perform a geolocation task. Second, we propose a stochastic game-based framework for droplet routing in Micro-Electrode-Dot Array (MEDA) biochips. The framework utilizes the ability to sense microelectrode health to synthesize routing plans that adapt to the microelectrode degradation levels in run-time. Using multiple real-life bioassays for evaluation, we show that the framework increases the probability of successful completion of benchmark bioassays. Finally, we adapt the framework to utilize Deep Reinforcement Learning (DRL) algorithms to achieve the same task.

Electrophoresis is the migration of charged particles in a heterogeneous fluid under the influence of an electric field. This project is work toward an electrophoretic separation system on a custom CMOS chip. Modeling, fabrication, and testing of an AMI ABN 1.5 um CMOS chip for this application is discussed. The unique approach is to build the entire system using conventional CMOS integrated circuit technology, such that the separation area is fabricated on the chip with integrated control and detection circuitry. To achieve the desired functionality, a novel configuration of an electrophoresis system is implemented. In this system, instead of using only one electrode at each end of the separation area, a multitude of electrodes beneath the entire separation area are utilized, enabling better control of high electric fields using very small voltages over small areas. Electronic circuits control the position and strength of the electric field to drive the separations and to simultaneously detect the location and concentration of samples within the separation area. Ultimately, the project was successful at showing that implementing an electrophoresis system on standard CMOS is possible.