京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22065号 / 工博第4646号 / 新制||工||1724(附属図書館) / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 井上 康博, 教授 中部 主敬, 教授 横川 隆司 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

<div><div><div><p>The advent of nano and micro class satellites has generated new demand for compact and efficient propulsion systems. Traditional propulsion technologies have been miniaturized for the CubeSat platform and new technology solutions have been proposed to address this demand. However, each of these approaches has disadvantages when applied within the context of a CubeSat. One potential low mass and power alternative is Film-Evaporation MEMS Tunable Array (FEMTA) micropropulsion which is capable of generating 150μN of thrust using 0.65W of electrical power and ultra-pure deionized water as propellant. The FEMTA thruster is etched into a 1cm × 1cm × 0.3mm silicon substrate using standard photolithography and microfabrication techniques. Each thruster consists of a 4 μm wide nozzle and platinum resistive heaters. Capillary pressure prevents the water from leaking through the nozzle and the heaters induce film-evaporation at the fluid interface to generate thrust. FEMTA has been in development at Purdue University since 2015 under the NASA SmallSat Technology Partnership Program and is currently on its 5th generation design. While these generations of FEMTA have successfully demonstrated the viability of the propulsion technique under ideal conditions, multiple reliability and performance related issues have been identified. More specifically, high vacuum tests have shown that the current FEMTA design is susceptible to quiescent propellant mass loss due to ice generation and leaking at the nozzle. These mass ejections can limit the lifespan and performance of the thruster and can induce undesired attitude perturbations on the host spacecraft. The purpose of this researchidentify the root causes of the quiescent mass loss mechanims hrough simulation and direct experimentation. Based on the results of these investigations, a next generation design is proposed, fabricated, and tested. Microfabrication was performed at Purdue’s Birck Nanotechnology Center and vacuum and thrust stand tests were performed at the High Vacuum Lab in the Aerospace Sciences Laboratory at Purdue.</p></div></div></div>

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Understanding the dynamics of micro- and nanometer-sized objects like molecules, particles, and living cells in biological systems and biomaterials has become a key component in biomedical research. Consequently, significant progress has been made for the development of imaging platforms, fluorescent probes, and computational tools to visualize and quantify biological processes at different length and time scales. However, despite such advances, achieving a reliable measurement accuracy on the dynamic behavior of these microscopic vehicles in diverse biological contexts is challenging. Subsequently, the motivation behind this dissertation is to develop new robust microfluidic velocimetry techniques to investigate molecular transport and cell migration within an in-vitro microfluidic platform.

This diploma thesis deals with cavitation flow in the microscale, which remains an area with a lack of sufficient description of this phenomenon. At the same time, microfluidics is a field experiencing a dramatic rise in numerous biochemical applications, which underlines the relevance of researches of this type. In theoretical part of the thesis, cavitation was described in detail. In the practical part, a microfluidic device with a cavitation orifice was designed and manufactured. The ANSYS program was used for this design. An experiment was performed with the designed microchip, the aim of which was to observe a cavitating flow on the orifice. This measurement took place at the microfluidic laboratory at Victor Kaplan Department of Fluid Engineering. Due to the failure of the experiment, a CFD model of two-phase cavitation flow was built. The conclusions of the thesis were compiled from the findings of measurement and the results of modeling.

Modern drug development is an astronomically expensive and time consuming undertaking. Because of this, studying the pharmacokinetic properties of drugs in vitro has become an integral step early in the process of drug development, with the goal of preventing costly failures late in the process, and dangerous side effects. Artificial phospholipid bilayers known as droplet interface bilayers (DIBs) have the potential to be used for these pharmacokinetics assays, combining the low cost of cell-free assays with the ability to more closely mimic structures found in life than current cell-free in vitro techniques. Combined with the reproducibility, ease of use, and low reagent consumption found with microfluidic methods, disruptive new low cost techniques for assessing pharmacokinetics in drug development may be possible using DIBs as an artificial cell membrane model. In this work, I establish the potential of DIBs to be used as a pharmacokinetics modelling platform, and advance the use of microfluidic methods for carrying out pharmacokinetics assays in drug discovery. I first developed a new microfluidic platform for the formation of DIBs, which sought to solve some of the shortcomings of current microfluidic methods for DIB formation (Chapter 2). This device is the first that can be used to form DIB networks from dissimilar droplets in parallel, without use of active controls, and with droplet contact gentle enough to enable use of biomimetic lipid mixtures. I examine for the first time the behaviour of phospholipids on microfluidic devices, and characterise the interaction that they have with a common material used to construct microfluidic devices (Chapter 3). Not only has this interaction never been studied before, but my unexpected findings indicate a new area requiring further study in order to advance the adoption of DIBs on microfluidic devices. In collaboration with my colleague Jaime Korner, I use my newly developed microfluidic platform to carry out an on-chip permeation assay for the first time using biomimetic lipid formulations and bespoke compartments modelled after the human intestine. We demonstrate that this on-chip assay has predictive accuracy greater than that of a current widely used cell-free technique (Chapter 4). Finally, I demonstrate that a DIB based microfluidic platform enables, and is critical for, characterising the effect of structural features such as membrane asymmetry on drug permeation. With this, I find measurable, previously unknown effects of membrane asymmetry on the absorption of the chemotherapy drug doxorubicin, highlighting a possible contributing factor to chemoresistance in some cancers (Chapter 5). I find, and demonstrate throughout the body of this work that microfluidic methods and DIBs can not only provide alternatives to current cell-free in vitro pharmacokinetics assays, but that they can exceed the performance of existing assays, and be used for entirely new ways of examining pharmacokinetics. Through building bespoke artificial cell membranes from the ground up, I hope to demonstrate herein the great potential of these powerful new cell-free methods. / Graduate / 2022-09-12

Graphene electronics represent a developing field where many material properties and devices characteristics are still unknown. Researching several possible fabrication processes creates a fabrication process using resources found at Cal Poly a local industry sponsor. The project attempts to produce a graphene network in the shape of a fractal Sierpinski carpet. The fractal geometry proves that PDMS microfluidic channels produce the fine feature dimensions desired during graphene oxide deposit. Thermal reduction then reduces the graphene oxide into a purified state of graphene. Issues arise during thermal reduction because of excessive oxygen content in the furnace. The excess oxygen results in devices burning and additional oxidation of the gate contacts that prevents good electrical contact to the gates. Zero bias testing shows that the graphene oxide resistance decreases after thermal reduction, proving that thermal reduction of the devices occurs. Testing confirms a fabrication process producing graphene electronics; however, revision of processing steps, especially thermal reduction, should greatly improve the yield and functionality of the devices.

La qualité de la prise en charge d’un patient repose sur la disponibilité d’outils diagnostiques performants. Le développement de biocapteurs s’appuyant sur le phénomène de transfert d’énergie par résonance de type Förster permet la détection de biomarqueurs spécifiques avec une grande précision et une bonne sensibilité. Le FRET (Förster Resonance Energy Transfer) est un processus de transfert d’énergie dépendant de la distance fréquemment utilisé dans les applications de biodétection, dans lesquelles les reconnaissances, fonctions et structures biologiques sont de l’ordre de 1 à 20 nm. Cette thèse de doctorat présente la mise en œuvre de procédés de fabrication et de fonctionnalisation pour la détection optique de molécules d’intérêt biologique par FRET, en particulier pour des applications en diagnostic clinique et en séquençage d’ADN.Ce travail présente une étude du phénomène de FRET sur des surfaces d’aluminium fonctionnalisées, première étape du développement d’une plateforme de séquençage par FRET exalté par effet plasmonique. La détection quantitative du phénomène de FRET sur des surfaces d’aluminium fonctionnalisées par silanisation est développée, et les résultats de caractérisation de la fonctionnalisation par différentes méthodes (angle de contact, spectroscopie FTIR, imagerie de fluorescence) sont discutés en détails.Ce manuscrit expose également le développement d’un dispositif microfluidique fonctionnalisé pour la réalisation d’un immunodosage par FRET multiplexé. Dans l’optique de concevoir un dispositif intégré fonctionnalisé pour la détection par FRET en conditions microfluidiques, j’ai développé une stratégie pour la réalisation d’un biocapteur optique microfluidique par FRET multiplexé. Les résultats préliminaires de FRET entre deux anticorps fluorescents dans un canal microfluidique démontrent la faisabilité d’une telle plateforme de biodétection. / Patient care quality relies on the availability of efficient diagnostics tools. Development of biosensors based on Förster resonance energy transfer (FRET) allows for the detection of specific biomarkers with high precision and sensitivity. FRET is a distance dependent energy transfer process that is frequently used in biosensing applications, in which biological recognition, functions or structures are within the 1 to 20 nm length scale. This PhD thesis presents the establishment of fabrication and functionalization processes for the optical FRET detection of molecules of biological interest, toward an application in clinical diagnostics and DNA sequencing.This work presents a FRET study on functionalized aluminum surfaces, which is the first step towards the development of a sequencing platform using plasmonics enhanced FRET. Quantitative FRET detection on silanized aluminum surfaces was extensively investigated and the results of different characterization methods (contact angle, FTIR spectroscopy, fluorescence imaging) are discussed in details.This manuscript also describes the development of a functionalized microfluidic device for the realization of a multiplexed FRET immunoassay. With the aim of designing a functionalized integrated device for FRET detection in microfluidic conditions, I developed a strategy for the realization of a microfluidic optical multiplexed FRET biosensor. Preliminary FRET results between two labeled antibodies in a microfluidic channel demonstrate the feasibility of such a biosensing platform.

abstract: Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has enabled the determination of damage-free protein structures at ambient temperatures and of reaction intermediate species with time resolution on the order of hundreds of femtoseconds. However, currently available XFEL facility X-ray pulse structures waste the majority of continuously injected crystal sample, requiring a large quantity (up to grams) of crystal sample to solve a protein structure. Furthermore, mix-and-inject serial crystallography (MISC) at XFEL facilities requires fast mixing for short (millisecond) reaction time points (𝑡"), and current sample delivery methods have complex fabrication and assembly requirements. To reduce sample consumption during SFX, a 3D printed T-junction for generating segmented aqueous-in-oil droplets was developed. The device surface properties were characterized both with and without a surface coating for improved droplet generation stability. Additionally, the droplet generation frequency was characterized. The 3D printed device interfaced with gas dynamic virtual nozzles (GDVNs) at the Linac Coherent Light Source (LCLS), and a relationship between the aqueous phase volume and the resulting crystal hit rate was developed. Furthermore, at the European XFEL (EuXFEL) a similar quantity and quality of diffraction data was collected for segmented sample delivery using ~60% less sample volume than continuous injection, and a structure of 3-deoxy-D-manno- octulosonate 8-phosphate synthase (KDO8PS) delivered by segmented injection was solved that revealed new structural details to a resolution of 2.8 Å. For MISC, a 3D printed hydrodynamic focusing mixer for fast mixing by diffusion was developed to automate device fabrication and simplify device assembly. The mixer was characterized with numerical models and fluorescence microscopy. A variety of devices were developed to reach reaction intermediate time points, 𝑡", on the order of 100 – 103 ms. These devices include 3D printed mixers coupled to glass or 3D printed GDVNs and two designs of mixers with GDVNs integrated into the one device. A 3D printed mixer coupled to a glass GDVN was utilized at LCLS to study the oxidation of cytochrome c oxidase (CcO), and a structure of the CcO Pr intermediate was determined at 𝑡" = 8 s. / Dissertation/Thesis / Supplementary Video D.1 - Droplet formation in a 3D printed droplet generator / Doctoral Dissertation Chemistry 2019