This paper presents the results of an ongoing effort to develop a direct solder bumping process for electronics packaging. The proposed process entails delivering molten droplets onto specific locations on electronic devices to form solder bumps. This study is focused on investigating droplet deposition behaviors that affect solder bump characteristics such as final bump volume, shape, and adhesion strength. The occurrence of droplet bouncing has a strong influence on these characteristics. The potential for a droplet to bounce in the absence of solidification was modeled in discrete stages based on energy conservation. Wetting and target surface roughness were identified as the critical parameters affecting bouncing. The experimental results showed that improvements in wetting and decreases in surface roughness retard bouncing. These observations agreed well with the trends predicted by the energy conservation based model. The knowledge acquired in this study is expected to contribute to the development of an efficient solder bumping process. / Singapore-MIT Alliance (SMA)

This article reports the recent progress of re-search made in the Droplet-Based Manufacturing Laboratory at MIT. The study has been focused on obtaining a fundamental understanding of microdroplet deposition and applying the technology to various practical applications. Specific scientific contributions include the development of an analytical model for droplet splashing/recoiling, an in situ droplet size control methodology, and a study of microstructure design for spray forming. The research per-formed in the lab provides both fundamental knowledge base and practical process developments for a range of manufacturing applications, including electronics packaging, spray forming and freeform fabrication. / Singapore-MIT Alliance (SMA)

Ce projet de thèse portait sur le développement d’outils microfluidiques pour la toxicologie et la recherche contre le cancer. En permettant l’analyse simultanée d’un très grand nombre de réactions biologiques ou chimiques réalisés dans des compartiments indépendants (ie. gouttelettes), la microfluidique de gouttes offre une sensibilité de détection et une précision sans précédent pour l’analyse de molécules biologiques, telles que l’ADN ou les Anticorps, en comparaison des expériences réalisées conventionnellement en tubes ou en microplaques (essais en « bulk » ou volume). Ce format permet également de réaliser des expériences à très haut débit et est particulièrement pertinent pour la toxicologie, où des analyses robustes de l’effet des médicaments sont nécessaires. De même, ces procédures sont également très adaptées à l’analyse de cellules uniques pour le séquençage ADN ou ARN et l’épigénomique. Tout cela fait de la microfluidique en goutte un outil puissant pour la toxicologie et la recherche sur le cancer. En premier temps, une méthode du comptage précise des cellules encapsulée dans des microgouttelettes, nommée « hémocytométrie microfluidique », a été développée. Un nouvel algorithme de comptage a été proposé. Des cellules bactériennes (Escherichia Coli) et des cellules de 2 lignées humaines différentes (HL60 and H1975) ont été testées. Le nombre de chaque type de cellules a été déterminé avec une haute corrélation entre la théorie (basée sur la distribution de Poisson) et les résultats expérimentaux. Avec ces résultats robustes, un protocole de microfluidique en goutte a été mis en place pour interroger la viabilité cellulaire et la prolifération des 2 lignées humaines. Ces résultats sont en concordance avec ceux de la littérature. Pour la toxicologie, 3 différents modèles, y compris des microsomes (extrait de cellules d’insectes infectées par un baculovirus exprimant le cytochrome P450 3A4 humain, CYP3A4), HepG2-CYP3A4 (modifiée génétiquement pour exprimer le gène CYP3A4 humain), et HepaRG, une lignée hépatique, ont été évaluées pour l’activité enzymatique du CYP3A4, une enzyme largement utilisée en routine pour le criblage de médicament candidat. Les microsomes ont permis de développer un essai fluorogénique permettant de mesurer l’inhibition du CYP3A4. Cependant, ni l’utilisation des microsomes ni des cellules HepG2 exprimant CYP3A4 n’a donné de résultats satisfaisants en microgouttelettes. L’utilisation des cellules HepaRG, une lignée cellulaire qui conserve la majorité de l’expression des cytochromes P450 et des récepteurs nucléaires nécessaire à leur expression, a montré des résultats encourageant à la fois sur les tests de mesure de l’activité enzymatique et d’analyse de l’induction du CYP3A4. Pour la recherche sur le cancer, 4 essais originaux de PCR digitale en gouttes ont été mis en place pour la détection et la quantification de mutations (NRAS, DNMT3A, SF3B1 and JAK2) importante pour les syndromes myélodysplasiques, un groupe hétérogène de maladies touchant les cellules souches hématopoïétiques caractérisées par une hématopoïèse inefficace et des cytopénies périphériques. Finalement, un essai de PCR sur cellule unique encapsulées au sein de billes agarose a été proposé. / This thesis project consists in developing droplet-based microfluidic tools for toxicology and cancer research. Owing to its large numbers of discretized volumes, sensitivity of detection of droplet-based microfluidics for biological molecules such as DNA and antibody is much higher than bulk assays. This high throughput format is particularly suitable for experiments where a robust dose-response curve is needed, as well as for single cell analysis with applications in genomic or sequencing and epigenetics. All above makes droplet-based microfluidics a powerful tool for toxicology and cancer research. In a first part of the work, an accurate cell counting method, named “microfluidics hemocytometry”, has been developed. A new counting algorithm was proposed to count the cells within each droplet. Escherichia Coli and two different human cell lines (HL60 and H1975) were used to validate our strategy. The number of each type of cells in droplets was determined with a high consistency between theory (Poisson distribution) and experimental results. With these robust results, a droplet-based microfluidic protocol has then been established to inquiry both cell viability and proliferation for the two human cell lines. The results are in good agreement with the one of the literature. For the toxicology, 3 different biological models, including microsomes (extracted from baculovirus-infected insect cell expressing human CYP3A4), HepG2-CYP3A4 (genetically modified to express the human CYP3A4 gene) and HepaRG liver cells lines were evaluated for enzymatic activity of cytochromes P450 (CYP3A4), a routinely used enzyme for drug candidate screening. Microsome-based assays were used to validate a fluorogenic inhibition assay. However neither microsome-based assay nor the assay using CYP3A4 expressing HepG2 gave satisfying results in droplet-based format. However, HepaRG cells, a hepatic function-conserved cell line with most cytochrome and related nuclear receptors, demonstrated high relevance both for enzymatic activity testing and CYP3A4 expression induction study. For cancer research, 4 different picoliter droplet-based PCR assays were developed for the detection and quantification of mutations (NRAS, DNMT3A, SF3B1 and JAK2) present in Myelodysplastic syndromes, a heterogeneous group of clonal bone marrow hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral cytopenias. Furthermore, a single cell multistep PCR assay using encapsulation of target DNA in agarose droplets was proposed.

Diese Doktorarbeit untersucht die verschiedenen dynamischen Prozesse, welche sich an der Tropfenoberfläche abspielen, und der Methoden, die für deren Untersuchung verwendet wurden. Das Ziel dieser Arbeit ist es, die entscheidenden Eigenschaften, die einen Einfluss auf das mechanische Verhalten der Grenzfläche haben, zu identifizieren. Wir verwenden die hydrodynamisch erzwungene Deformation eines Tropfens in einem Mikrokanal, um die mechanischen Eigenschaften der Oberfläche zu untersuchen. Diese Methode wird auf drei verschiedene Fälle angewendet. Als erstes verfolgen wir die zeitliche Entwicklung einer Grenzflächenverformung, um die Dynamik der Tensidadsorption an einer Oberfläche zu untersuchen. Dabei kalibrieren wir die Tropfenverformung als Funktion von Tropfengröße und Oberflächenspannung. Diese Technik wird auf den Fall eines perfluorierten Tensids, welches von industriellem und wissenschaftlichem Interesse ist, angewendet. Wir zeigen die Möglichkeit von Messungen der dynamischen Oberflächenspannung auf Zeitskalen von zehn Millisekunden und gewinnen daraus kinetische Eigenschaften der Moleküle. Wir vergleichen die Dynamik, welche mit der klassischen Pendant-Drop-Methode gemessen werden kann mit denen der Mikrofluidik. Es zeigt sich, dass die Adsorption für den Pendant Drop von der Di usion begrenzt wird, während in der Mikrofluidik die Anbindung an die Oberfläche der langsamere Prozess ist. Der Unterschied entsteht durch das Flussprofil in der Mikrofluidik, welches konvektiven Transport induziert. Danach untersuchen wir die Verformung unter verschiedenen räumlichen Beschränkungen im mikrofluidischen Kanal. Die Tropfenverformung wird mit einer zweidimensionalen numerischen Simulationen und mit einem dreidimensionalen Modell eines Rotationsellipsoids verglichen. In beiden Fällen wird eine qualitative Übereinstimmung festgestellt, jedoch existieren auch spürbare Abweichungen vom Experiment. Die Abweichungen vom zweidimensionalen Modell ist erklärbar mit dem sinkenden Einfluss der viskosen Spannungen mit der Kanalhöhe, hervorgerufen durch Beiträge von Deformationen außerhalb der Beobachtungsebene, welche von dem Modell nicht wiedergegeben werden. Die Abweichungen vom dreidimensionalen Modell kommen von den räumlichen Beschränkungen, welche die Tropfenform von einem Rotationsellipsoid abweichend verformt. Die Untersuchung zeigt die Schwierigkeiten bei der Beschreibung von viskosen Kräfte für Abmessungen, die zu groß sind um als zweidimensional betrachtet zu werden, aber wo die Wechselwirkungen mit den Kanalwänden nicht vernachlässigbar sind. Wir diskutieren ebenfalls den Fall der trägen Relaxation des Tropfens bei Reynoldszahlen von Re 10, für welchen Oszillationen der Tropfenoberfläche beobachtet werden. Wir zeigen, dass die Oszillationen als hydrodynamische Analogie zu einer hookeschen Feder beschrieben werden können, wobei die Oberflächenspannung als Federkonstante fungiert und die Dämpfung durch die Viskosität der Flüssigkeit bestimmt wird. Die Methode liefert korrekte Ergebnisse sowohl für reine Grenzflächen als auch für Grenzflächen mit Tensiden, was zu einer zusätzliche Möglichkeit führt, die Oberflächenspannung aus der Frequenz der Verformungen zu bestimmen. Die viskose Relaxation wurde auch hierbei von den Kanalwänden beeinflusst. Als letztes wenden wir die Methode der mikrofluidischen Tensiometrie auf die Kinetik einer Polymerisationsreaktion auf der Tropfenoberfläche an. Der Einfluss der Reagenzkonzentration auf die Reaktionszeit wird untersucht, ebenso wie der E ekt der Gegenwart von Tensidmolekülen. Erste Ergebnisse dieser Untersuchung zeigen, dass die Deformation einer komplexen Grenzfläche nicht mehr allein durch die Oberflächenspannung beschrieben werden kann. Vielmehr muss die Beschreibung der mechanischen Eigenschaften der Grenzfläche notwendigerweise die Entstehung der Viskoelastizität an der Oberfläche mit in Betracht ziehen. Diese Erkenntnis erö net neue Möglichkeiten, mit Hilfe von Mikrofluidik die mechanischen Eigenschaften von komplexen Grenzflächen, wie zum Beispiel kolloidbesetzte Grenzflächen oder Membranen, zu charakterisieren.

Nous avons pour but de développer un système microfluidique en gouttes, capable, à l’échelle de la cellule/bactérie unique, de détecter et de co-localiser plusieurs marqueurs génétiques, en utilisant une version digitale et multiplexée de la réaction de polymérisation en chaîne (PCR). Les systèmes de PCR digitale actuellement commercialisés ne le permettent toujours pas. Un tel prototype garantira la présence de multiples marqueurs à l’intérieur d’un même génome, ce qui permettra l’identification du pathogène avec précision et un taux de faux-positifs proche de zéro. Comme première application, nous démontrerons la possibilité de co-localiser quatre gènes de virulence de la souche O157:H7 d’Escherichia coli, un pathogène majeur, qui est détecté dans des échantillons alimentaires ou provenant de fèces cliniques pouvant aussi contenir des E. coli non pathogènes porteuses d’une partie des gènes de virulence. Avant de procéder à des tests TaqMan multicolores en point final, E. coli sera d’abord encapsulée dans des gouttes micrométriques et lysée par la chaleur in situ. Notre objectif est de démontrer que ce test peut être appliqué avec succès à un petit ensemble d’échantillons cliniques ou alimentaires / We aim to develop a prototype of droplet-based microfluidic system capable of detecting and colocalizing multiple genetic markers at the single cell/bacteria level, using the Polymerase Chain Reaction (PCR) in a digital multiplexed version. This cannot be achieved using current commercial digital PCR systems, and should increase the sensitivity and reliability of the detection of pathogens. Importantly, the system will guarantee the presence of multiple markers within the same genome and enable accurate identification, and bring the false positive rate close to zero. As a first application, we will demonstrate the possibility to co-localize 3 virulence genes in the E.coli strain O157:H7, a major foodborne pathogen, which has to be detected in clinical feces samples or food samples, which may also contain non pathogenic E. coli carrying only a subset of these virulence genes. E. Coli will be encapsulated in micrometric droplets, lysed by heating in situ prior performing a multicolour end-point Taqman assay. Our objective is to demonstrate that this test can be successfully applied to real clinical or food samples

With the rapid progress of next-generation sequencing (NGS) technologies, new tools and methods have emerged to investigate the transcriptomics of various organisms. RNA sequencing (RNA-seq) employs NGS to evaluate the presence and abundance of RNA transcripts in biological samples. This technique offers a comprehensive snapshot of the RNA dynamics within cells. With the ability to profile the entire transcriptome of organisms rapidly and accurately, RNA-seq has become the state-of-the-art method for transcriptome profiling, surpassing the traditional microarray approach. Single-cell RNA sequencing (scRNA-seq) was introduced in 2009 to profile the single-cell gene expression in highly heterogeneous samples such as brain tissue and tumors. The advancement of scRNA-seq technologies enables the in-depth transcriptomic study in each cell subtype. When selecting an scRNA-seq method, researchers must weigh the trade-off between profiling more single cells versus obtaining more comprehensive transcripts per cell, while considering the overall costs. The throughput of full-length scRNA-seq methods is usually lower, as each single cell needs to be processed separately to produce scRNA-seq libraries. However, full-length methods enable the researchers to investigate the splicing variants and allele-specific expression. Non-full-length methods only capture the 3' or 5' ends of transcripts, which limits their application in isoform detection, but as cells are pooled after barcoding for cDNA synthesis, the throughput is 2–3 orders of magnitude higher than full-length methods. We developed a droplet-based platform for full-length single-cell RNA-seq, which enabled the efficient recovery of full-length mRNA from individual cells in a high-throughput manner. The developed platform can process ~8,000 single cells within 2 days and detect ~20% more genes compared to Drop-seq. Besides scRNA-seq technology development, we also applied a low-input RNA-seq method to study the transcriptomics in different biological samples. When handling precious biological samples, a low-input method is necessary to profile the transcriptome of homogeneous cell populations. We first studied the epigenomic and transcriptomic regulations in colorectal cancer (CRC) using MOWChIP-seq, a low-input high-throughput method, in conjunction with our low-input RNA-seq approach. Fusobacterium nucleatum (Fnn) is closely related to the progression of cancers like CRC and pancreatic cancer. However, the molecular mechanisms of how Fnn adjusts the tumor microenvironment (TME) and leads to poor clinical outcomes are still unclear. In this in-vitro study, we characterized how hypoxia, an important TME ignored by previous research, facilitates Fnn infection of CRC and corresponding alterations of global epigenome and transcriptome. We infer that hypoxia has similar effects as Fnn infection alone on the CRC cells. The Fnn infection under hypoxia further boosts the proliferation and progression of CRC. We then applied our low-input RNA-seq method to study brain neuroscience and immunology. Psychedelics like DOI show promising clinical efficacy in patients with psychiatric conditions. Although psychedelics exhibit rapid antidepression action and long-lasting effectiveness compared to conventional treatment, their acute psychotic symptoms and potential for drug abuse discourage their application in clinical practice. In this case, it is important to comprehend the molecular mechanisms responsible for psychedelics' clinical efficacy. This understanding can pave the way for the development of improved treatments that do not rely on psychedelics. After profiling the transcriptome of mouse brain samples exposed to psychedelics with different post-exposure times, we concluded that the psychedelic-induced transcriptomic variations are more transient than epigenomic changes. In the second brain neuroscience project, we first applied 3-color FACS sorting to differentiate four neuron and non-neuron subtypes in human postmortem prefrontal cortex tissues. Then we profiled the gene expression of the four subtypes and validated the FACS sorting by examining the expression of marker genes. Differentially expressed genes between each subtype and the others were extracted and proceeded to gene ontology analysis. We identified unique altered biological pathways related to each subtype. The immunology research focuses on revealing the difference between low-grade inflammation and monocyte exhaustion, as well as the unique biological pathways they regulate. Therefore, we profiled the transcriptome of bone marrow-derived monocytes stimulated by PBS control, a low- or high-dose LPS. In addition to wild-type mice, we also included TRAM-deficient and IRAK-M-deficient mice. We concluded that low-dose LPS specifically regulates the TRAM-dependent pathway of TLR4 signaling, and high-dose LPS exclusively upregulates exhaustion markers by impacting metabolic and proliferative pathways. / Doctor of Philosophy / Transcriptomics is the comprehensive study of RNA transcripts derived from an organism's genome. RNA plays a vital role in maintaining the fundamental functions of cells and organisms. In eukaryotes, the genetic information stored in the DNA of cells is transferred to messenger RNA (mRNA) molecules through a process called transcription. These mRNA molecules serve as a bridge between DNA and proteins, as they carry the instructions encoded in genes to ribosomes for protein synthesis. Studying mRNA transcripts reveals various cellular mechanisms and their impact on overall organism function, gene regulation, and disease pathways. With the aid of next-generation sequencing, various RNA-seq approaches have been developed to study mRNA transcripts quantitatively in the past decades. To better understand the gene expression regulations in biological samples, we first applied bulk RNA-seq to profile the transcriptome of various samples under different conditions. Our in-house bulk RNA-seq protocol has been proven to be both high-performance and cost-effective compared to commercial kits. To better understand cellular diversity and uncover rare cell types in heterogeneous biological samples, we developed a droplet-based scRNA-seq platform that can recover full-length mRNA transcripts in a high throughput manner. It can profile the transcriptome of thousands of single cells within two days. It combines the advantages of the droplet-based scRNA-seq method (high throughput) and the well plate-based scRNA-seq method (full-length mRNA recovery).

L’efficacité des traitements contre diverses pathologies dépend dans bien des cas de la précocité de la prise en charge des patients. Ce contexte pousse de nos jours les chercheurs à élaborer de nouvelles méthodes de diagnostic, généralement basées sur la détection de biomarqueurs spécifiques, permettant d’établir une corrélation entre un dérèglement moléculaire de l’organisme et la survenue d’une maladie. L’objectif de ces travaux était, par l’utilisation de la microfluidique digitale en gouttelettes, d’établir de nouvelles procédures simples et reproductibles, témoignant d’une sensibilité importante afin de déterminer d’infimes variations de l’état moléculaire de l’organisme à travers la recherche de biomarqueurs spécifiques. Pour cela nous avons élaboré une nouvelle gamme de tensioactifs fluorées adaptés aux applications biologiques en microfluidique digitale, ainsi que différentes stratégies d’étude des variations de l’expression de microARN extrait d’échantillons biologiques, basées respectivement sur les réactions induites par hybridation nucléotidique et sur la réaction de RT-PCR digitale. / Efficiency of treatments for various diseases depends in many cases in precocity of patient management. Nowadays, this context urges researchers to develop new methods of diagnosis, generally based on the detection of specific biomarkers. These new methods allowing to establish correlations between physiological disorders and arisen of diseases states.The aim of this study was, by the use of droplet-based microfluidic, to work out a simple and reproducible procedure, with an increased sensitivity, to determine tiny variations of physiological state through the detection of specific biomarkers. Thus, we developed a new range of fluorinated surfactants fitted to biological applications in droplet-based microfluidics as well as various strategies to study variations of microRNA expressions in a biological sample. These methods, based on DNA-template reaction and digital PCR reaction, allows performing a substantial number of simultaneous reactions in micro-compartments (microdroplets) of picolitre volumes.

Ce travail de thèse rend compte de la mise en oeuvre d’un système de synthèse en continu dupoly(acide acrylique) en milieu CO2 supercritique (15 MPa et 75 °C). Nous avons conçu undispositif microfluidique résistant aux hautes pressions permettant l’écoulement de gouttes desolution aqueuse de monomère dans une phase continue constituée d’un mélangesupercritique d’éthanol dans du CO2 et contenant l’amorceur azobisisobutyronitrile (AIBN).Nous avons déterminé par spectroscopie IR la répartition des différentes espèces chimiquesdu mélange en fonction de la pression et la température, puis caractérisé la décompositionthermique de l’amorceur selon la composition du milieu réactionnel par spectroscopie UVVis.Enfin, nous avons montré que les gouttes sont comparables à des réservoirs demonomère alimentant sans cesse la phase continue. En raison de ce transfert rapide vis-à-visde la conversion de l’AA en chaîne polymère, la réaction de polymérisation s’effectuecontinûment avec un rapport molaire monomère sur amorceur constant durant tout le tempsde séjour dans le microcanal (jusqu’à 41 min). Une gamme large de masses molaires avec desindices de polymolécularité faibles a été obtenue : de 20 000 à 120 000 g.mol-1 pour 1,35 à1,70, en variant simplement les concentrations de monomère de la solution aqueuse initiale.Les paramètres expérimentaux influençant les propriétés du poly(acide acrylique) obtenu,ainsi que le lieu de la polymérisation ont été étudiés. / In this work, a continuous microfluidic device was developed to perform the synthesis ofpoly(acrylic acid) in supercritical CO2 (15 MPa and 75°C). This high pressure resistantdevice allows generating segmented flows in microcanal: an aqueous solution of monomerwas dispersed in a mixture of ethanol in CO2 containing initiator AIBN. The distribution ofdifferent components in this biphasic system has been determined by IR spectroscopyaccording to the pressure and the temperature. The thermal decomposition of AIBN indifferent reaction media has been investigated using UV-Vis spectroscopy. During thereaction, the droplets were used as reservoirs which insure the transfer of monomer to thecontinuous phase. Because of this rapid transfer compared to the reaction conversion, thepolymerization reaction was carried out continuously with a constant molar ratio betweenmonomer and initiator throughout the residence time (up to 41 min). It has been showed thata large range of molecular weights of poly(acrylic acid) (20 000 and 120 000 g.mol-1) withlow polydispersity index (1.35 à 1.70) can be obtained by just changing the initial monomerconcentration in the droplets. The effect of other parameters influencing the properties ofpoly(acrylic acid) as well as the locus of polymerization have been discussed.

Waterborne or foodborne diseases are caused by consuming contaminated fluids or foods. The presence of pathogenic microorganisms can contaminate food or drinking water. These microorganisms can cause sickness even if they are present in minimal concentrations. The World Health Organization (WHO) has defined the standards for clean drinking water as the absence of E. coli in a 100 mL collected volume. Contaminated water or food can cause many diseases, and diarrhea is one of a prominent disease. Early detection of contamination in food or drinking water is critical. Conventional culture-based methods are time-consuming, labour intensive, and not suitable for on-site testing. Nucleic acid-based tests are sensitive and can rapidly detect pathogens. Microfluidic technology can play a significant role to develop low-cost, rapid, integrated, and portable nucleic acid-based detection devices. Microfluidic systems for isothermal amplification assays can be classified into two groups such as droplet-based and chamber-based systems. In this thesis, both droplet-based and chamber-based approaches were used to build the microfluidic methods for isothermal amplification assays. First, a simple electromechanical probe (tweezers) was developed that can manipulate a small aqueous droplet in a bi-layer oil phase. The tweezer consisted of two needles positioned close to each other and used polarization of the aqueous droplet in an applied electrical field to confine the droplet between the needles with minimal solid contact. AC electric potential was applied to the two metal electrodes. Droplet acquired a charge from the high voltage electrode and consequently performed an oscillatory motion with the same electrode. This droplet motion was controlled using two parameters of electric potential and frequency of the applied signal. Initially, electrically actuated droplet (0.3 µL) motion was investigated for a range of applied potential (400-960 Volts) and frequencies (0.1-1000 Hz). The droplet motion with high voltage electrode was characterized into three modes such as detachment, oscillation, and attachment. Mechanical motion of tweezer was used to transport droplet to various positions. Consequently, operations such as transportation, extraction, and merging were demonstrated. First, droplet (5 µL) transportation was characterized under the applied potential of 2000 Volts at various frequencies (5 to 1000 Hz). The droplet was successfully transported to the speed of 15 mm/s at higher frequencies (100 or 1000 Hz). Droplets of various volumes (12-80 µL) were extracted by increasing applied electric potential, from 0 to 6000 Volts at 5 Hz. Then, the operation of droplets merging was demonstrated using operational conditions for electrical tweezer. Finally, electrical tweezer was used to prepare samples for isothermal amplification assays. Two droplets consisted of various reagents of isothermal amplification assays, were transported and merged using the electrical tweezer. Then, a merged droplet (25 µL) was transported and immobilized in the amplification zone. The temperature of the amplification zone (~65°C) was maintained using an in-situ heater. DNA amplification was verified by measuring the off-chip end-point fluorescence intensity of isothermal assays. Second, an integrated microfluidic device has been developed to prepare a sample for isothermal amplification assays. And in-situ real-time amplification assays were performed to detect bacteria. The device consisted of two chambers (lysis and amplification) connected through a microchannel. A low-cost fabrication method was introduced to embed two resistive wire heaters around both chambers. Initially, bacteria cells were thermally lysed in the lysis chamber at 92°C for 5 min. Then, DNA was electrophoretically transported from lysis to the amplification chamber. The electric potential of 10 Volts was applied for 10 min for DNA transportation. Next, transported DNA was amplified at 65°C and DNA amplification was detected by measuring in-situ fluorescence intensity in the real-time format. The operation of the integrated microfluidic device was demonstrated in three steps. 1) Operation of individual components. 2) Operation of two components in a coupled format. 3) Integrated operation of three components with measurement of fluorescent intensity in a real-time format. The bacteria samples with the concentration of 100 CFU/mL were detected in less than one hour. / Thesis / Doctor of Philosophy (PhD)

Droplet-based microfluidics is a powerful tool permitting massive-scale single-cell analysis in pico-/nano-liter water-in-oil droplets. It has been integrated into various library preparation techniques to accomplish high-throughput scRNA-seq, scDNA-seq, scATAC-seq, scChIP-seq, as well as scMulti-omics-seq. These advanced technologies have been providing unique and novel insights into both normal differentiation and disease development at single-cell level. In this thesis, we develop four new droplet-based tools for single-cell omics profiling. First, the developed Drop-BS is the first droplet-based platform to construct single-cell bisulfite sequencing libraries for DNA methylome profiling and allows production of BS library of 2,000-10,000 single cells within 2 d. We applied the technology to separately profile mixed cell lines, mouse brain tissues, and human brain tissues to reveal cell type heterogeneity. Second, the new Drop-ChIP platform only requires two steps of droplet generation to achieve multiple steps of reactions in droplets such as single-cell lysis, chromatin fragmentation, ChIP, and barcoding. Third, we aim to establish a droplet-based platform to accomplish high-throughput full-length RNA-seq (Drop-full-seq), which both current tube-based and droplet-based methods cannot realize. Last, we constructed an in-house droplet-based tool to assist single-cell ATAC-seq library preparation (Drop-ATAC), which provided a low-cost and facile protocol to conduct scATAC-seq in laboratories without the expensive instrument. / Doctor of Philosophy / Microfluidics is a collection of techniques to manipulate fluids in the micrometer scale. One of microfluidic techniques is called "droplet-based microfluidics". It can manipulate (i.e., generate, merge, sort, split, etc) pico-/nano-liter of water-in-oil droplets. First, since the water phase is separated by the continuous oil phase, these droplets are discrete and individual reactors. Second, droplet-based microfluidics can achieve highly parallel manipulation of thousands to millions of droplets. These two advantages make droplet-based microfluidics an ideal tool to perform single-cell assays. Over the past 10 years, various droplet-based platforms have been developed to study single-cell transcriptome, genome, epigenome, as well as multi-ome. To expand droplet-based tools for single-cell analysis, we aim to develop four novel platforms in this thesis. First, Drop-BS, by integrating droplet generation and droplet fusion techniques, can achieve high-throughput single-cell bisulfite sequencing library preparation. It can generate 10,000 single-cell BS libraries within 2 days which is difficult to achieve for conventional library preparation in tubes/microwells. Second, we developed a novel and facile Drop-ChIP platform to prepare single-cell ChIP-seq library. It is easy to operate since it only requires two steps of droplet generation. It also generates higher quality of data compared to previous work. In addition, we are working on the development and characterization of the other two droplet-based tools to achieve full-length single-cell RNA-seq and single-cell ATAC-seq.