Molecular diagnostics offer quick access to information for healthcare decision-making towards personalized therapeutics, but complicated procedures requiring extensive labor and infrastructure restrict their use. Droplet-based technologies can expand the accessibility of molecular diagnostics by miniaturizing devices, shortening sample-to-answer times, decreasing costs and increasing throughput. Methods for droplet manipulation are central to the automation of molecular diagnostics protocols. The innovative method, wire-guided droplet manipulation (WDM), is the actuation of liquid droplets in a hydrophobic milieu with a wire, or needle, guide. In this work, WDM is demonstrated for the automation of the polymerase chain reaction (PCR) on reprogrammable platforms for the diagnosis of cardiovascular infections. WDM is used to minimize thermal resistance by convective heat transfer for PCR amplification at a maximum speed of 8.67 s/cycle. The oil-water interfacial boundary is shown to passively partition molecular contaminants from sample matrices, including blood and heart valve tissue. Molecular self-assembly at the oil-water interface is used to increase PCR efficiency with blood in situ and is used as an innovative sensing modality for real-time monitoring of PCR amplification. Temperature feedback controlled droplet actuation is achieved by using a thermocouple loop as a functionalized wire-guide. Our novel methodology for real-time PCR, droplet-on-thermocouple silhouette real-time PCR (DOTS qPCR), utilizes interfacial effects to achieve droplet actuation, relief from PCR inhibitors and amplification sensing, for a sample-to-answer time as short as 3 min 30 s. DOTS qPCR addresses three major issues for rapid PCR—sample preparation, rapid thermocycling and sensitive real-time detection—on an inexpensive, disposable device with smartphone-based detection. In contrast, commercially available real-time PCR systems rely on fluorescence detection, have substantially higher threshold cycles, and require expensive optical components and extensive sample preparation. Due to the advantages of low threshold cycle detection we anticipate extending this technology towards trending biological research applications such as single cell, single nucleus, and single DNA molecule analyses, especially in droplet microfluidic platforms.

Macrophage foam cells are the major culprits in atherosclerotic lesions, having a prominent role in both lesion initiation and progression. With atherosclerosis being the main factor underlying cardiovascular complications, there is a long-standing interest on finding ways to reverse lipid buildup in plaques. Studies have shown that promoting reverse cholesterol transport (RCT) from macrophage foam cells is anti-atherogenic because it alleviates the cholesterol burden of the plaques. The goal of this thesis was to gain insight into the mechanisms that govern cholesterol efflux from macrophage foam cells. The first part of this study looked at the ability of different oxysterols to promote cholesterol efflux in unloaded as compared to lipid-loaded macrophages, and our major finding here is that epoxycholesterol decreases efflux in lipid-loaded macrophages. It appears that epoxycholesterol does so by impairing the release cholesterol from its cellular storage site, the lipid droplet (LD), where it accumulates in the form of cholesteryl esters (CE). These results highlighted the importance of cholesterol release from LDs for efflux; indeed, this process is increasingly being recognized as the rate-limiting step for RCT in vivo. Subsequent experiments aimed at elucidating the mechanisms that govern LD CE hydrolysis in macrophage foam cells lead to the discovery of a novel pathway involved in cholesterol efflux. Macrophage CE hydrolysis is classically defined as being entirely dependent on neutral CE hydrolases. In the second part of this study, we demonstrate that in addition to the canonical CE hydrolases, which mediate neutral lipid hydrolysis, lysosomal acid lipase (LAL) also participates in the hydrolysis of cytoplasmic CE. Autophagy is specifically triggered in macrophages by atherogenic lipoproteins and delivers LD CE to LAL in lysosomes, thus generating free cholesterol for efflux. This autophagy-mediated cholesterol efflux is a process that is primarily dependant on the ABCA1 transporter and, importantly, is important for whole-body RCT. Overall, the studies presented in this thesis support that macrophage LD CE hydrolysis is rate-limiting for cholesterol efflux and shed light on the mechanisms of cholesterol mobilization for efflux in macrophage foam cells.

Droplet evaporation is ubiquitous to multitude of applications such as microfluidics, surface patterning and ink-jet printing. In many of the process like food processing tiny concentrations of suspended particles may alter the behavior of an evaporating droplet remarkably, leading to partially viscous and partially elastic dynamical characteristics. This, in turn, may lead to some striking mechanical instabilities, such as buckling and rupture. In this thesis, we provide a comprehensive physical description of the vaporization, self-assembly, agglomeration and buckling kinetics of sessile nanofluid droplet pinned on a hydrophobic substrate in various configurations. We have deciphered five distinct regimes of droplet lifecycle. Regime I-III consists of evaporation induced preferential agglomeration that leads to the formation of unique dome shaped inhomogeneous shell with stratified varying density liquid core. Regime IV involves capillary pressure initiated shell buckling and stress induced shell rupture. Regime V marks rupture induced cavity inception and growth. We provide a regime map explaining the droplet morphology and buckling characteristics for droplets evaporating on various substrates. Specifically, we find that final droplet volume and radius of curvature at buckling onset are universal functions of particle concentration. Furthermore, flow characteristics inside the heated and unheated droplets are investigated and found to be driven by the buoyancy effects. Velocity magnitudes are observed to increase by an order at higher temperatures with self-similar flow profiles. With an increase in the surface temperature, droplets exhibit buckling from multiple sites over a larger sector in the top half of the droplet. In addition, irrespective of the initial nanoparticle concentration and substrate temperature, hydrophobicity and roughness, growth of daughter cavity (subsequent to buckling) inside the droplet is found to be controlled by the solvent evaporation rate from the droplet periphery. The results are of great significance to a plethora of applications like DNA deposition and nanofabrication. In the next part of the thesis, we deploy the droplet in a rectangular channel. The rich physics governing the universality in the underlying dynamics remains grossly elusive. Here, we bring out hitherto unexplored universal features of the evaporation dynamics of a sessile droplet entrapped in a 3D confined fluidic environment. Increment in channel length delays the completion of the evaporation process and leads to unique spatio-temporal evaporation flux and internal flow. We show, through extensive set of experiments and theoretical formulations, that the evaporationtimescale for such a droplet can be represented by a unique function of the initial conditions. Moreover, using same theoretical considerations, we are able to trace and universally merge the volume evolution history of the droplets along with evaporation lifetimes, irrespective of the extent of confinement. These results are explained in the light of increase in vapor concentration inside the channel due to greater accumulation of water vapor on account of increased channel length. We have formulated a theoretical framework which introduces two key parameters namely an enhanced concentration of the vapor field in the vicinity of the confined droplet and a corresponding accumulation lengthscale over which the accumulated vapor relaxes to the ambient concentration. Lastly, we report the effect of confinement on particle agglomeration and buckling dynamics. Compared to unconfined scenario, we report non-intuitive suppression of rupturing beyond a critical confinement. We attribute this to confinement-induced dramatic alteration in the evaporating flux, leading to distinctive spatio-temporal characteristics of the internal flow leading to preferential particle transport and subsequent morphological transitions. We present a regime map quantifying buckling & non-buckling pathways. These results may turn out to be of profound importance towards achieving desired morphological features of a colloidal droplet, by aptly tuning the confinement space, initial particle concentration, as well as the initial droplet volume. These findings may have implications in designing functionalized droplet evaporation devices for emerging engineering and biomedical applications.

Today’s aerospace industry continues to exploit liquid hydrocarbon fossil fuels. Motivated by operational considerations, continued availability and cost, this is likely to be the case for many years, despite the obvious environmental concerns. The interplay of liquid atomisation, spray vaporisation and the combustion process are intricately linked. However, the physical process of fuel injection and its atomisation into tiny droplets prior to combustion remains poorly understood. Because atomisation governs the size of the fuel droplets, and therefore their subsequent evaporation rate, adjusting the injection sequence is of paramount importance and will have far-reaching repercussions on many aspects of the combustion process, for example pollutant formation. In the context of jet engines, kerosene is usually injected in its liquid form via an airblast-type fuel injector. A coflowing high-speed airstream destabilises the liquid fuel, which is thus sprayed into fine droplets into the combustion chamber. The prediction of this phenomenon for various operating conditions relevant to the aeronautical industry requires a deeper understanding of the mechanisms involved in the interaction of the two fluids. A key element in predicting the complex behaviour of spray formation and evolution in jet engines is accurate modelling of fuel atomisation. Atomisation represents one of the key challenges that remains to be undertaken to make predictive computational simulations possible. However, the inherent multi-physics and multi-scale nature of this process limits numerical investigations. Thanks to the steady progress in computer power and Computational Fluid Dynamics (CFD) methods, computational modelling of injection systems emerges as a promising tool that can drive the design of future devices. This research project sets out to investigate the atomisation process in detail, in particular in providing physical insight into the fundamental physics of the phenomenon, in conjunction with an analysis on wetting behaviours and liquid droplet tracking. High-fidelity numerical simulations are performed using a novel in-house state-of-the-art multiphase flow modelling capability, RCLSFoam. The performance of the numerical scheme is demonstrated on typical two-dimensional and three-dimensional benchmark test cases relevant to both multiphase flow modelling and atomisation, and validated against other computational methods. An informed and systematic qualitative assessment of the topological variations of the phase interface during primary atomisation of a liquid film is made through dynamical analysis, while investigating an extensive domain of operating conditions at ambient and aero-engine injection conditions relevant to industry. This analysis demonstrated the influence of shear-driven instabilities on the atomisation process. The shear stress and difference in inertia between liquid and gas are observed to play a significant role in the atomisation process. In addition, the key physical mechanisms and their competing effects have been mapped out in order to predict the evolution of the process according to the operating conditions of the injection system. The proposed cartography gathers four different atomisation mechanisms. In particular, for sufficiently high liquid injection speeds, three-dimensional wave modes were observed to co-exist (the “3-D wave mode” regime). For very low liquid flow rates, accumulated liquid at the atomising edge undergoes deformation by which droplets are generated (the “accumulation” regime). For an increasing gas injection speed and a fixed liquid velocity, the effects of surface tension were observed to result in the generation of streamwise ligaments only, which tend to pair up (the “ligament-merging” regime). Finally, “vortex action” is another observed mechanism by which the liquid film is fragmented. Overall, this research project culminated in (i) the study of dynamic wetting behaviours, with the implementation and validation against experimental data of the Kistler dynamic contact model; and (ii) the demonstration of an algorithm for droplet capture and subsequent post-processing analysis of the droplet characteristics.

abstract: "Sensor Decade" has been labeled on the first decade of the 21st century. Similar to the revolution of micro-computer in 1980s, sensor R&D; developed rapidly during the past 20 years. Hard workings were mainly made to minimize the size of devices with optimal the performance. Efforts to develop the small size devices are mainly concentrated around Micro-electro-mechanical-system (MEMS) technology. MEMS accelerometers are widely published and used in consumer electronics, such as smart phones, gaming consoles, anti-shake camera and vibration detectors. This study represents liquid-state low frequency micro-accelerometer based on molecular electronic transducer (MET), in which inertial mass is not the only but also the conversion of mechanical movement to electric current signal is the main utilization of the ionic liquid. With silicon-based planar micro-fabrication, the device uses a sub-micron liter electrolyte droplet sealed in oil as the sensing body and a MET electrode arrangement which is the anode-cathode-cathode-anode (ACCA) in parallel as the read-out sensing part. In order to sensing the movement of ionic liquid, an imposed electric potential was applied between the anode and the cathode. The electrode reaction, I_3^-+2e^___3I^-, occurs around the cathode which is reverse at the anodes. Obviously, the current magnitude varies with the concentration of ionic liquid, which will be effected by the movement of liquid droplet as the inertial mass. With such structure, the promising performance of the MET device design is to achieve 10.8 V/G (G=9.81 m/s^2) sensitivity at 20 Hz with the bandwidth from 1 Hz to 50 Hz, and a low noise floor of 100 ug/sqrt(Hz) at 20 Hz. / Dissertation/Thesis / M.S. Electrical Engineering 2013

La microfluidique en gouttes permet l’analyse de systèmes biochimiques à grand débit, en utilisant de faibles volumes réactionnels, à faible coût. Dans l’état de l’art, le volume des gouttes varie entre 2 pL et 4 nL, un millier à million de fois inférieur au volume d’un puit de microplaque. Miniaturiser d’avantage les volumes réactionnels permettrait d’augmenter encore les débits d’analyse, de d’avantage réduire les coûts et ouvre également l’accès à de nouvelles études, telles que les études sur molécule unique ou la délivrance de médicaments. La première partie de ce travail de thèse concerne la miniaturisation des opérations classiques de la microfluidique en gouttes à l’échelle femtolitrique: production, stabilité, biocompatibilité, mélange en gouttes, coalescence, tri, division de gouttes, production à la demande ont été démontrés avec succès sur des gouttelettes femtolitriques. Le tout a été permis en restant dans les limites de la technologie PDMS et des standards classiques de la lithographie. La seconde partie s’intéresse à certaines applications biologiques issues du couplage de gouttelettes picolitriques et femtolitriques. Une plateforme permettant l’encodage in situ de gouttes à l’aide de codes barres d’ADN lisibles par séquençage a été construite. Deux autres applications ont été envisagées: un projet concernant l’émergence des chromosomes dans un monde prébiotique qui nécessitait des facteurs de dilution de l’ordre de 1:1000 et un projet de cartographie génotype-phénotype sur une enzyme qui nécessitait deux dilutions par 10 ont bénéficié de la miniaturisation à l’échelle femtolitrique. / Droplet-based microfluidics has demonstrated its multiple advantage over standard microtiter plates technologies by increasing analysis throughputs, decreasing costs and enabling the encapsulation of single cells into individual reservoirs. In the state-of-the-art, droplet volumes usually range from 2 pL to 4nL, one thousand to one million times smaller than microtiter plate wells. This PhD work focuses on the miniaturization of biological reservoirs down to the femtoliter scale which would enable an increase of throughputs of analysis and open up access to new studies, such as single-molecule studies or drug delivery. The first part of this manuscript concentrates on the miniaturization of elementary operations of droplet-based microfluidics down to the femtoliter scale. Production, mixing, electrocoalescence, DEP sorting, splitting, drop-on-demand, stability, biocompatibility were successfully demonstrated on droplets of a few micrometers diameter. The second part of this manuscript focuses on some biological applications that were developed with the LBC. A platform for the in situ encoding of droplets with DNA barcodes readable per sequencing was developped. Two other applications were envisioned: a project studying the conditions that prevailed the apparition of chromosomes in an early RNA world and a genotype-phenotype mapping project benefited from downscaling to the femtoliter scale.

This work detailed the physical and chemical characterization of oil water mixtures prepared using fresh and weathered Macondo related oils under different conditions of mixing energy/time and in the presence/absence of chemical dispersants. The results indicated that WAFs produced consistent, droplet free solutions for both source and weathered oils with concentration ranges that represented the soluble components of the oil used. Chemically enhanced WAFs prepared with the source oil generated a large amount of micron-size droplets; however the viscosity of the weathered oils were a limiting factor for the preparation of CEWAFs with weathered oils. Droplet size distributions were influenced by the amount of energy in the system and the oil weathering stage, when high energy WAFs were made the increase in weathering of the oil resulted in the formation of smaller droplets that were more stable over time.

Macrophage foam cells are the major culprits in atherosclerotic lesions, having a prominent role in both lesion initiation and progression. With atherosclerosis being the main factor underlying cardiovascular complications, there is a long-standing interest on finding ways to reverse lipid buildup in plaques. Studies have shown that promoting reverse cholesterol transport (RCT) from macrophage foam cells is anti-atherogenic because it alleviates the cholesterol burden of the plaques. The goal of this thesis was to gain insight into the mechanisms that govern cholesterol efflux from macrophage foam cells. The first part of this study looked at the ability of different oxysterols to promote cholesterol efflux in unloaded as compared to lipid-loaded macrophages, and our major finding here is that epoxycholesterol decreases efflux in lipid-loaded macrophages. It appears that epoxycholesterol does so by impairing the release cholesterol from its cellular storage site, the lipid droplet (LD), where it accumulates in the form of cholesteryl esters (CE). These results highlighted the importance of cholesterol release from LDs for efflux; indeed, this process is increasingly being recognized as the rate-limiting step for RCT in vivo. Subsequent experiments aimed at elucidating the mechanisms that govern LD CE hydrolysis in macrophage foam cells lead to the discovery of a novel pathway involved in cholesterol efflux. Macrophage CE hydrolysis is classically defined as being entirely dependent on neutral CE hydrolases. In the second part of this study, we demonstrate that in addition to the canonical CE hydrolases, which mediate neutral lipid hydrolysis, lysosomal acid lipase (LAL) also participates in the hydrolysis of cytoplasmic CE. Autophagy is specifically triggered in macrophages by atherogenic lipoproteins and delivers LD CE to LAL in lysosomes, thus generating free cholesterol for efflux. This autophagy-mediated cholesterol efflux is a process that is primarily dependant on the ABCA1 transporter and, importantly, is important for whole-body RCT. Overall, the studies presented in this thesis support that macrophage LD CE hydrolysis is rate-limiting for cholesterol efflux and shed light on the mechanisms of cholesterol mobilization for efflux in macrophage foam cells.

In vitro studies have shown that the major membrane phospholipid phosphatidylcholine (PC) can positively influence the incorporation of cholesterol in lipid membranes. The influence of PC on the cellular trafficking of LDL-derived free cholesterol was investigated. Sterol regulatory-defective (SRD)-4 cells are Chinese hamster ovary (CHO)-derived fibroblasts that display vastly elevated rates for the synthesis and catabolism of PC. SRD-4 cells harbor two known gene mutations: a mutation in the functional allele for SCAP, resulting in defective feedback suppression of cholesterol biosynthesis; and a loss-of-function mutation in the functional allele for acyl-CoA:cholesterol acyl transferase (ACAT), an endoplasmic reticulum (ER)-localized enzyme that esterifies free cholesterol. Incubation of SRD-4 cells with 50 µg/ml low density lipoprotein (LDL) for 18 h resulted in lysosomal accumulation of free cholesterol as revealed by filipin staining. This accumulation was not evident following LDL treatment of parental CHO7 cells, and was blunted in SRD-2 cells that express a constitutively-active form of SREBP-2 and overproduce cholesterol but have functional ACAT activity. Treatment of SRD-2 cells with LDL in the presence of an ACAT inhibitor 58-035 resulted in robust lysosomal cholesterol accumulation that was reversible upon drug washout, supporting that cholesterol trafficking in cholesterol-loaded cells is dependent on ACAT activity and, more specifically, ER free cholesterol levels. Lysosomal accumulation of LDL-derived cholesterol was prevented in SRD-4 cells supplemented with lyso-PC (50 µM), a substrate for PC synthesis through the reacylation pathway, and also in cells treated with bromoenol lactone (BEL), an inhibitor of phospholipase A2 implicated in bulk PC turnover. In a counter study, lysosomal LDL-derived cholesterol accumulation was induced in parental CHO-7 cells using R-propranolol, which inhibits the conversion of phosphatidic acid to diacylglycerol (DAG), a substrate in the CDP-choline pathway. This blockage was also relieved through co-treatment with lyso-PC. These studies support that PC to free cholesterol ratios in downstream organellar membranes can influence cholesterol trafficking out of lysosomal compartments in cholesterol-loaded cells.

Cell-free transcription and translation systems promise to accelerate and simplify the engineering of synthetic proteins, biological circuits or metabolic pathways. Microfluidic droplet platforms can generate millions of reactions in parallel. This allows cell-free reactions to be miniaturized down to picoliter volumes. Nevertheless, the true potential of microfluidics have not been reached for cell-free bioengineering. Better approaches are needed for reaching sufficient in-drop expression levels while efficiently creating DNA diversity among droplets. This work develops a droplet microfluidic platform for single or multiple protein expression from a single DNA coated bead per droplet. This opens up the possibility to diversify a million droplets for synthetic biology applications.