This work introduces technology for rapid pathogen detection using handheld optical immunoassay and wire-guided droplet PCR systems. There have been a number of cases of foodborne or waterborne illness among humans that are caused by pathogens such as Escherichia coli O157:H7, Salmonella typhimurium, Influenza A H1N1, and the norovirus. The current practices to detect such pathogenic agents are: cell/viral culturing, immunoassays, or polymerase chain reactions (PCRs). These methods are essentially laboratory-based methods that are not at all real-time and thus unavailable for early-monitoring of such pathogens. They are also very difficult to be implemented in field, preventing early detection opportunities. This dissertation is divided into three papers that present methodologies towards the expeditious detections of infectious pathogens and the miniaturization of these detection systems towards field-deployable and point-of-care applications. Specifically, the work presented focuses on two methodologies: (1) light scatter detection using immunoagglutination assays with optimized Mie light scatter parameters in a real biological matrix consisting of plant tissue, and (2) wire-guided droplet manipulations for rapid and improved sample analysis, preparation, and PCR thermocycling. Both of these methods carry a collective objective towards providing high impact technologies for addressing the issues of food-related outbreaks and overall public safety. In the first paper, the direct and sensitive detection of foodborne pathogens from fresh produce samples was accomplished using a handheld lab-on-a-chip device, requiring little to no sample processing and enrichment steps for a near-real-time detection and truly field-deployable device. The detection of Escherichia coli K12 and O157:H7 in iceberg lettuce was achieved utilizing optimized Mie light scatter parameters with a latex particle immunoagglutination assay. The system exhibited good sensitivity, with a limit of detection of 10 CFU mL⁻¹ and an assay time of <6 min. Minimal pretreatment with no detrimental effects on assay sensitivity and reproducibility was accomplished with a simple and cost-effective KimWipes filter and disposable syringe. Mie simulations were used to determine the optimal parameters (particle size d, wavelength λ, and scatter angle θ) for the assay that maximize light scatter intensity of agglutinated latex microparticles and minimize light scatter intensity of the tissue fragments of iceberg lettuce, which were experimentally validated. This introduces a powerful method for detecting foodborne pathogens in fresh produce and other potential sample matrices. The integration of a multi-channel microfluidic chip allowed for differential detection of the agglutinated particles in the presence of the antigen, revealing a true field-deployable detection system with decreased assay time and improved robustness over comparable benchtop systems. In the second paper, we demonstrate a novel method of wire-guided droplet manipulations towards very quick RT-PCR. Because typical RT-PCR assays take about 1–2 h for thermocycling, there is a growing need to further speed up the thermocycling to less than 30 min. Additionally, the PCR assay system should be made portable as a point- of-care detection tool. Rapid movements of droplets (immersed in oil) over three different temperature zones make very quick PCR possible, as heating/cooling will be made by convective heat transfer, whose heat transfer coefficients are much higher than that of conduction, the latter of which is used in most conventional PCR systems. A 30-cycle PCR of a 160 bp gene sequence amplified from 2009 H1N1 influenza A (human origin) was successfully demonstrates in 6 min and 50 sec for a very large 10 μL droplet (with additional 4 min for reverse transcription). The proposed system has a potential to become a rapid, portable, point-of-care tool for detecting influenza A. In the third paper, a wire-guided CNC apparatus was used to perform droplet centrifugation, DNA extraction, and VQ-PCR thermocycling on a single superhydrophobic surface measuring 25 mm by 55 mm and a multi-chambered PCB heater. This methodology exhibited no limitations on the complexity and configuration of procedures that it can perform, making it versatile and far-reaching in its applications. The only modification required for adding or implementing changes for a new protocol is through simple pre-defined programming. The highly adaptive and flexible system was used to execute easily pre-programmed droplet movements and manipulations for the rapid detection of Escherichia coli from PCR detection. Serial dilutions were performed to simulate a diluted field sample with a high level of accuracy. Centrifugation of the diluted sample containing E. coli demonstrated a novel approach to sample pre-treatment. Furthermore, the extraction of DNA from the sample droplet containing E. coli was also performed on the same superhydrophobic surface as the previous 2 steps, requiring less than 10 min. Following extraction, the genetic material was amplified using wire-guided droplet PCR thermocycling, successfully completing 30 cycles of Peptidase D (a long 1500 bp sequence) in 10 min. The droplet centrifugation process was determined to greatly improve the positive band intensity over the non-centrifuged sample. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of accuracy and automation.

<p>Conventional rocket propellants such as monomethyl hydrazine (MMH) and hydrazine have been used for decades due to their high specific impulse and performance. However, interest in greener alternatives, including HAN or HAN-based propellants, has grown due to high levels of toxicity and difficulties in the handling and storage of conventional fuels. Included among potential propellants are monopropellants nitromethane (NM) and isopropyl nitrate (IPN) and their blends. Though large-scale investigations on the ignition and combustion of these fuels have been done, the ignition and combustion processes of these monopropellant fuels are still not well understood. Droplet studies have been traditionally and extensively employed to decipher the influence of ambient conditions and fuel properties on ignition and combustion of different fuels. These fundamental studies allow for the isolation of different factors such as ambient temperature and initial droplet size among others, to provide a deeper understanding of their effects in overall spray combustion.</p> <p> </p> <p>The research described here seeks to add to the knowledge on the ignition and combustion processes of NM and IPN through single droplet ignition and combustion studies. To this end, the first effort has been to establish a suitable method of studying the ignition and combustion of droplets in conditions similar to those in practical systems. Droplet ignition delay measurements for NM and IPN droplets have also been conducted, and the influence of ambient temperature and droplet size has been studied. The double flame structures of NM and IPN, representative of hybrid combustion, have also been observed. In addition, the applicability of the hybrid combustion model, developed to predict mass burning rates for hypergolic fuels exhibiting hybrid burning including MMH, UDMH and hydrazine, has been assessed. Lastly, the ability of the quasi-steady droplet ignition model to predict ignition delays of IPN and NM monopropellant droplets is also discussed.</p>

The effect of superheat on the disruption of liquid droplets in a compressible gas flow was investigated experimentally in a small-scale, supersonic wind tunnel. Aerodynamically generated ethanol droplets of an average diameter of 0.1 mm were injected via a normal sonic jet into a Mach 1.8 freestream. Both nonsuperheated and superheated droplets were injected with initial Weber numbers of approximately 700. The droplets and flow structure were photographed using the shadowgraph method. The relatively high momentum of the liquid droplets typically caused them to pass out of the sonic jet structure. Nonsuperheated droplets showed no signs of disrupting after traveling over 200 mm downstream from the injection point. Only droplets with injection temperatures above the predicted boiling point at tunnel freestream static pressure (48°C) showed signs of disruption, typically after they left the sonic jet structure (30 to 100 mm downstream of the injection point). Droplets in this range of temperatures appeared to begin to boil from the downstream side of the droplet, shedding a vapor cloud before disrupting completely in the chaotic mode. Droplets with temperatures above the boiling point at the exit plane of the sonic jet began to disrupt in the chaotic mode almost instantly (within 1 exit nozzle diameter).

Two new mechanisms of droplet splashing and breakup during impact have been identified and analyzed. One is the internal rupture of spreading droplet film through formation of holes, and the other is the splashing of droplet due to its freezing during spreading. The mechanism of film rupture was investigated by two different methods. In the first method, circular water films were produced by directing a 1 mm diameter water jet onto a flat, horizontal plate for 10 ms. In the second method, films were produced by making 0.6 mm water droplets impact a solid surface mounted on the rim of a rotating flywheel. Substrate wettability was varied over a wide range, including superhydrophobic. In both cases, the tendency to film rupture first increased and then decreased with contact angle. A thermodynamic stability analysis predicted this behavior by showing that films would be stable at very small or very large contact angle, but unstable in between. Film rupture was also found to be promoted by increasing surface roughness or decreasing film thickness. To study the effect of solidification, the impact of molten tin droplets (0.6 mm diameter) on solid surfaces was observed for a range of impact velocities (10 to 30 m/s), substrate temperatures (25 to 200°C) and substrate materials (stainless steel, aluminum and glass) using the rotating flywheel apparatus. Droplets splashed extensively on a cold surface but on a hot surface there was no splashing. Splashing could be completely suppressed by either increasing the substrate temperature or reducing its thermal diffusivity. An analytical model was developed to predict this splashing behavior. The above two theories of freezing-induced splashing and film rupture were combined to predict the morphology of splats typically observed in a thermal spray process. A dimensionless solidification parameter, which takes into account factors such as the droplet diameter and velocity, substrate temperature, splat and substrate thermophysical properties, and thermal contact resistance between the two, was developed. Predictions from the model were compared with a wide range of experimental data and found to agree well.

Two new mechanisms of droplet splashing and breakup during impact have been identified and analyzed. One is the internal rupture of spreading droplet film through formation of holes, and the other is the splashing of droplet due to its freezing during spreading. The mechanism of film rupture was investigated by two different methods. In the first method, circular water films were produced by directing a 1 mm diameter water jet onto a flat, horizontal plate for 10 ms. In the second method, films were produced by making 0.6 mm water droplets impact a solid surface mounted on the rim of a rotating flywheel. Substrate wettability was varied over a wide range, including superhydrophobic. In both cases, the tendency to film rupture first increased and then decreased with contact angle. A thermodynamic stability analysis predicted this behavior by showing that films would be stable at very small or very large contact angle, but unstable in between. Film rupture was also found to be promoted by increasing surface roughness or decreasing film thickness. To study the effect of solidification, the impact of molten tin droplets (0.6 mm diameter) on solid surfaces was observed for a range of impact velocities (10 to 30 m/s), substrate temperatures (25 to 200°C) and substrate materials (stainless steel, aluminum and glass) using the rotating flywheel apparatus. Droplets splashed extensively on a cold surface but on a hot surface there was no splashing. Splashing could be completely suppressed by either increasing the substrate temperature or reducing its thermal diffusivity. An analytical model was developed to predict this splashing behavior. The above two theories of freezing-induced splashing and film rupture were combined to predict the morphology of splats typically observed in a thermal spray process. A dimensionless solidification parameter, which takes into account factors such as the droplet diameter and velocity, substrate temperature, splat and substrate thermophysical properties, and thermal contact resistance between the two, was developed. Predictions from the model were compared with a wide range of experimental data and found to agree well.

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Spray drying is a process, which produces powders from the fluid state. This type of process is mostly used in the industrial sector. In this process, a liquid slurry is atomized, forming droplets, which are dried with hot air. During spray drying these droplets will interact and upon impact can show different types of interactions; droplet-droplet collisions as well as interactions with partially or completely dried particles, leading to agglomeration. The result of collision gives properties of the dried powder. The focus of the thesis is to investigate the droplet-droplet collision outcomes of WPC 80 (Whey Protein Concentrate 80) and Lactose. Then the effects of the absolute droplet diameter and the droplet diameter ratios are to be determined. Existing experimental setup and Image Processing Tool of MATLAB is used to study the collision outcome. The outcomes are shown in a regime map. The present results are compared with different products result and literature study. It is observed that there is an effect on collision outcome for different droplet size ratios and no effect for absolute droplet diameter.

Le carcinome épidermoïde cutané (CEC) représente un enjeu important par sa fréquence et sa gravité potentielle.L’agressivité de ce cancer est liée à l’invasion péri-nerveuse (IPN), mode d’envahissement tumoral reconnu comme un facteur de mauvais pronostic.L’objectif de ce travail est de s’intéresser aux mécanismes favorisant l’IPN, en comparant 2 groupes appariés de CEC humains, avec et sans IPN.Pour cela nous avons réalisé une étude de facteurs et récepteurs neurotrophiques, de marqueurs de la transition épithélio-mésenchymateuse (TEM), et de la molécule NCAM1, par analyse immunohistochimique à partir de pièces chirurgicales de CEC et par analyse moléculaire en droplet digital PCR sur des cellules tumorales microdisséquées.L’analyse immunohistochimique a trouvé une forte expression de BDNF, TrkB, p75NGFR, Snail 1 et NCMA1 dans les cellules tumorales péri-nerveuses, contrastant avec une faible expression de ces marqueurs dans les cellules tumorales à distance du nerf. L’E-cadhérine était diminuée dans les cellules tumorales péri-nerveuses.L’analyse moléculaire en ddPCR montrait une diminution d’expression de l’E-cadhérine et une surexpression de BDNF, TrkB, p75NGFR, Snail1, Slug, Zeb2, Twist1 et NCAM1 dans les cellules tumorales péri-nerveuses par rapport aux cellules tumorales distantes du nerf.Nous avons démontré dans ce travail que l’invasion péri-nerveuse dans les CEC humains est liée aux neurotrophines, à la TEM et implique NCAM1. / Cutaneous squamous cell carcinoma (SCC) is an important issue because of its frequency and potential severity.The aggressiveness of this cancer is related to perineural invasion (PNI), a mode of tumor dissemination recognized as a poor prognosis factor.The aim of this work is to study the mechanisms of PNI, comparing 2 matched- groups of human SCC with and without PNI.For this, we studied neurotrophins, epithelial-mesenchymal transition (EMT) markers, and the NCAM1 molecule, by immunohistochemistry analysis on surgical pieces of SCC and by molecular analysis with digital-droplet PCR on laser-microdissected tumor cells.Immunohistochemistry analysis found strong expression of BDNF, TrkB, p75NGFR, Snail 1 and NCMA1 in perineural tumor cells, contrasting with weak expression of these markers in tumor cells distant from the nerves. E-cadherin was decreased in perineural tumor cells.Molecular analysis in ddPCR showed decreased expression for E-cadherin and overexpression of BDNF, TrkB, p75NGFR, Snail1, Slug, Zeb2, Twist1 and NCAM1 in perineural tumor cells compared to tumor cells distant from the nerves.We have demonstrated in this work that PNI in human SCC is linked to neurotrophins and EMT, and involves NCAM1.