<p>Microfluidic devices using electroosmotic flows (EOFs) in microchannels have been developed and widely applied in chemistry, biology and medicine. Advantages of using these devices include the reduction of reagent consumption and duration for analysis. Moreover the velocity profile of EOFs, in contrast to the parabolic profile found in pressure-driven flows, has a plug-like profile which contributes significantly less to solute dispersion. It also requires no valve to control the flow, which is done with the appropriate application of electrical potentials, thus becomes one of the favourite techniques for sample separation. However, high potentials of several hundred volts are usually required to generate sufficient EOF. These high potentials are not practical for general usage and could cause electrical hazard in some applications. One of the possible solutions is the introduction of zeta potential modulation. The EOF in a microchannel can be controlled by the zeta potential at the liquid/solid interface upon the application of external gate potentials across the channel walls. Combined with AC EOF, it can rectify the oscillating flows and generate pressure that can be used for microfluidic pumping applications. Since the flow induced by the alternating electric field is unsteady and periodic, it is critical to visualize the flow with high spatial and temporal resolutions in order to understand fluid dynamics. A novel method to obtain high temporal resolution for high frequency periodic electrokinetic flows using phase sampling technique in micro particle image velocimetry (PIV) measurements are first developed in order to characterize the AC electroosmotic flow. After that, the principle of zeta potential modulation is demonstrated to transport particles, cells, and other micro organisms using rectified AC EOF in open microchannels. The rectified flow is obtained by synchronous zeta-potential modulation with the driving potential in the microchannel. Subsequently, we found that PDMS might not be the best material for some pumping and biomedical applications as its hydrophobic surface property makes the priming process more difficult in small microchannels and also causes significant protein adsorption from biological samples. A more hydrophilic and biocompatible material, polyurethane (PU), was chosen to replace PDMS. A polyurethane-based soft-lithography microfabrication including its bonding, interconnect integration and in-situ surface modification was developed providing better biocompatibility and pumping performance. Finally, an electroosmotic pumping device driven by zeta potential modulation and fabricated by PU soft lithography was presented. The problem of channel priming is solved by the capillary force induced by the hydrophilic surface. Its flow rate and pressure output were found to be controllable through several parameters such as driving potential, gate potential, applied frequency, and phase lag between the driving and gate potentials.</p> / Doctor of Philosophy (PhD)

<p>Microdroplets find use in a variety of applications ranging from chemical synthesis to biological analysis. However, commercial use of microdroplets has been stymied in many applications, as current devices lack one or more of the critical features such as precise and dynamic control of the droplet size, high throughput and easy fabrication. This work involves design, fabrication and characterization of a microdroplet generating device that uses low cost fabrication, allows dynamic control of the droplet size and achieves parallelized droplet generation for high throughput.</p> <p>Dynamic droplet size control by DC electric field has been demonstrated with the device. By varying the potential from 300 V to 1000 V, the droplet size can change from 140 microns to around 40 microns . The transition of the droplet size just takes few seconds. Parallelized droplet generation has also been demonstrated. The standard deviation of the droplet size is lower than 4% for the three-capillary device and lower than 6% for the five-capillary device under different operating conditions. Highest throughput of 0.75 mL/hour is achieved on the five-capillary device. It has been show that this proposed device has a better performance than the existing PDMS based parallel droplet generating devices. A theoretical model of the droplet generating process has also been developed which is able to predict the droplet size at various potentials. The theoretical results are in good agreement with experimental ones.</p> / Master of Applied Science (MASc)

<p>Sample preparation is a crucial processing step required for molecular biological analysis of environmental samples like water that has a variety of constituents in it. Furthermore, large volumes of sample need to be processed as the prescribed limits of pathogens in water are extremely low. However, microfluidic biosensing devices that can perform rapid molecular biological analysis in the field are designed to handle small sample volumes. In such cases, there is a need for a sample processing device that can reduce (concentrate) a large sample volume into a small one while retaining the biological species present in it. Hollow fibers are appropriate for this purpose of sample reduction and serve as a macro to micro interface for the microfluidic device. The received concentrate from the hollow fiber device requires be further concentrated to several microliters and separated and sorted to various modular components within the microfluidic device. This requires a second stage microfiltration where an integrated membrane can sort based on particulate size. In this thesis, a two stage filtration was designed. A first stage hollow based fiber pre-concentration device is developed that is portable, low cost, has high retention efficiency, low elution volume and is rapid. The hollow fiber device has low elution volume of ~1-3 ml. Controlled experiments were performed to validate the recovery of the hollow fiber device. Simulated 250 ml E.coli contaminated samples were filtered to <5 ml from an original sample volume of 250 ml. No bacteria were present in the filtrate and nearly 100% was recovered at high bacterial concentrations. At low concentrations (~200 cells in the sample) the recovery was less (~50%). A second stage microfiltration device that can be integrated with the microfluidic device and that can reduce the sample still further from ~ 5 ml to 5 μl was designed. Plasma bonding of ultrafiltration and microfiltration membranes using fluorine ions was investigated for fabrication of this device. The bonding of PDMS channels with polysulfone membranes via SF6 plasma was tested via tensile pull tests, burst pressure tests, and analyzed through scanning electron microscopy and electron dispersive x-ray spectroscopy. Quantitative tests on 10kDa and 70kDa polyethersulfone membranes demonstrated increased operational bonding strength of 86.6 and 146.9 kPa increases with three hour plasma application. Microfiltration membranes (0.2 micrometer pore size polyethersulfone) bonded in such a way that was easier to permeate as compared to ultrafiltration membranes. This bonding technique is generic in nature and can be used for integration of other commercially available polyethersulfone membranes with microfluidic devices for applications such as bio separations. No filtration testing was performed with E.coli samples.</p> / Master of Applied Science (MASc)

<p>The production of a novel micropump based on the synthetic jet principle is investigated both numerically and experimentally. The proposed micropump consists of a synthetic jet actuator driven by a vibrating diaphragm issuing into an inverted T- shaped channel structure forming the inlet/outlet channels of the pump.</p> <p>The software package Ansys is used to perform numerical investigations of the operation of the proposed micropump. Simulations were performed to study the effect of changing the inlet/outlet channel dimensions as well as the operating frequency, amplitude and duty cycle of the excitation signal. Inlet/outlet channel widths ranging from 200 to 800 μm and operating amplitude and frequency of excitation of the 5 mm square membrane driving the synthetic jet actuator ranging from 20 to 60 μm and from 20 to 60 Hz respectively were investigated.</p> <p>Based on the findings of the numerical simulations, a prototype design was chosen and produced. Prototype production using microfabrication techniques as well as micromachining was investigated. The final prototype was micromachined using plexiglass as the working material. An experimental setup was constructed to test the performance of the produced prototype, which allowed for measuring the produced flow rate, pressure head, actuation amplitude and frequency.</p> <p>The findings of the numerical simulations verified the possibility to produce a working micropump with flow rates of up to 1.3 ml/min. Simulation results also showed the dependence of the produced flow rate on both the inlet and outlet channel widths. An increase in the inlet channel width resulted in a gain in the average flow rate through the pump while an increase in the outlet channel width results in a reduction in the flow rate. Increases in either the actuation amplitude or frequency of excitation both resulted in an improvement in the produced flow rate. Changes in the ejection duty cycle, or the ejection time relative to the suction time during an actuation cycle, were found to influence the flow rate produced by the pump. A shorter ejection time produced a higher flow rate from the pump as compared to a longer ejection time. It was also found that changes in dimensions or operating parameters affected the fluctuations in the flow rate through the pump associated with the pulsating nature of the synthetic jet. Experimental investigations confirmed the findings of the numerical simulations in terms of the flow rate and the trends in the dependence of the flow rate on operating parameters. Values of maximum back pressure of up to 500 Pa were also reported experimentally and membrane driving powers of up to 122 μW were calculated numerically.</p> / Doctor of Philosophy (PhD)

The metabolism of amino acids produces important chemical signaling molecules called neurotransmitters, which are responsible for carrying out important actions within the human body. There are approximately one hundred identified neurotransmitters. Neurotransmitter study is important due to their involvement in biological, physiological, pharmacological, and pathological functions. Commonly employed methods for neurotransmitter detection are mainly based upon microdialysis. However, the methods suffer from disadvantages. Microdialysis fails to determine the absolute concentration of analytes and therefore requires it to be tied in with an analytical technique such as high performance liquid chromatography or capillary electrophoresis. Although high performance liquid chromatography is the most powerful analytical technique to date, it necessitates high maintenance and suffers from poor temporal resolution. While capillary electrophoresis affords more rapid separations than high performance liquid chromatography, it suffers from poor concentration limits of detection and requires large sample dilutions of highly conductive samples, such as biological fluids. Consequently, research is focused on detection of various amino acids and neurotransmitters employing novel analytical techniques along with traditional capillary electrophoresis. First, a method was developed using traditional capillary electrophoresis with laser induced fluorescence detection to detect two major excitatory neurotransmitters, glutamate and aspartate in planaria. The method was later applied to detect several biogenic amines using micellar electrokinetic chromatography with laser induced fluorescence detection in planaria to study the effect of feeding on the levels of biogenic amines within individual planaria homogenates. The concentration sensitivity issue of capillary electrophoresis led to the use of a new method for sensitive neurotransmitter measurements, gradient elution isotachophoresis. Gradient elution isotachophoresis is an efficient capillary-based enrichment and separation technique based on balancing hydrodynamic counter-flow against electrophoresis. Enrichment is achieved with the aid of high concentrations of leading electrolyte in the counter-flow solution that creates an ionic interface near the capillary inlet. Discrete electrolyte spacers or carrier ampholyte mixtures are used to separate analyte zones. The method was applied to the enrichment and separation of physiologically relevant concentrations of aspartate and glutamate labeled with dansyl chloride, phenyl isothiocyanate, or carboxyfluorescein, succinimidyl ester in artificial cerebrospinal fluid using ultraviolet absorbance detection. Finally, gradient elution isotachophoresis was combined with capillary zone electrophoresis to eliminate the use of spacers and provide rapid separations and enrichment. The technique was applied for the detection of biogenic amines in a glass microfluidic device. / Chemistry

Design and development / Respiratory distress syndrome (RDS) is a major cause of mortality and long-term morbidity annually affecting 14% preterm infants worldwide. Therapies have been developed to overcome this common disorder; however, limitations exist with these treatments that often lead to complications including bronchopulmonary dysplasia (BPD). One approach to address RDS is to implement a microfluidic oxygenator that serves as a respiratory support system for preterm neonates while the lungs fully develop, extra-uterine. This artificial lung assist device (LAD) is characterised by its non-invasiveness (given that it is connected via umbilical vessels), pumpless configuration, ambient air operation, portability and low priming volume. Furthermore, the LAD is formed by single oxygenator units (SOU) that are stacked in a parallel array which allows for usage on different body weights. The objective of this thesis is to design an electrochemical system to provide an in-situ enriched O2 environment able to supply 1.9 ml O2/min for use in the SOU while maintaining the simplicity of operation of the oxygenator. An inexpensive, electrically powered and compact device was envisioned allowing for a higher permeation flux to fully oxygenate the blood. Moreover, the system would be easy to manufacture, low maintenance and avoid the risk of gas contamination. In the initial work, different designs of electrolytic cells were developed and tested. The two- chamber design connected by a gel membrane showed an O2 production 10 times higher than with previous designs with 42 mg O2/L. Subsequently, different supporting electrolytes were tested. NaOH demonstrated a better performance and no degradation of the electrode in contrast to NaCl and Na2SO4. Stainless steel mesh (SSM) and graphite sheet electrodes were then tested; it was observed that stainless steel produced 3.4 times more dissolved oxygen (DO) than graphite with 28.3 mg O2/L. Experimentation with electrolysis of water showed that the DO in water reached stability 3 min after the electrolysis process was initiated measuring a change of DO of 29 mg/L at 3 A. Furthermore, an active oxygenation (AO) system was developed for in-vitro experiments via electrolysis of water and compared to a passive oxygenation (PO) system exposing blood to enriched O2 air and ambient air, respectively. It was demonstrated that AO provided 300% greater oxygenation to blood than PO. The electrolysis chamber designed for the microfluidic oxygenator allows the oxygenator to maintain its essential characteristics of simplicity and low cost while increasing the rate of oxygenation of blood. Preterm neonates suffering from RDS need an artificial lung that can partially support the oxygenation of their blood. Thus, combining the oxygenator with the O2 generation in-situ system enables a greater blood O2 uptake of 300% making possible the development of an efficient artificial lung. / Thesis / Master of Applied Science (MASc)

One of the major technical challenges with creating 3D artificial tissue constructs is the lack of simple and effective methods to integrate vascular networks within them. Without these vascular-like networks, the cells embedded within the constructs quickly become necrotic. This thesis details the use of a commercially available, low-cost, 3D printer modified with a microfluidic printhead in order to generate instantly perfusable vascular-like networks integrated within gel scaffolds seeded with cells. The printhead featured a coaxial nozzle that allowed the fabrication of hollow, gel tubes (500µm–2mm) that can be easily patterned to create single or multi-layered constructs. Media perfusion of the channels caused a significant increase in cell viability. This microfluidic nozzle design was further modified to allow for multi-axial extrusion in order to 3D print and pattern bi- and tri-layered hollow channel structures. Most available methodologies lack the ability to create multi-layered concentric conduits inside natural extracellular matrices, which would more accurately replicate the hierarchal architecture of biological blood vessels. The nozzle used in this work allowed, for the first time, for these hierarchal structures to be embedded within layers of gels in a fast, simple and low cost manner. This scalable design allowed for versatility in material incorporation, thereby creating heterogeneous structures that contained distinct concentric layers of different cell types and biomaterials. This thesis also demonstrates the use of non-extrusion based 3D biofabrication involving planar processing by means of hydrogel adhesion. There remains a lack of effective adhesives capable of composite layer fusion without affecting the integrity of patterned features. Here, silicon carbide was found for the first time to be an effective and cytocompatible adhesive to achieve strong bonding (0.39±0.03kPa) between hybrid hydrogel films. Multi-layered, heterogeneous constructs with embedded high-resolution microchannels (150µm-1mm) were fabricated in this way. With the new 3D fabrication technology developed in this thesis, gel constructs with embedded arrays of hollow channels can be created and used as potential substitutes for blood vessel networks as well as in applications such as drug discovery models and biological studies. / Thesis / Doctor of Philosophy (PhD) / Additive manufacturing (AM) involves any three-dimensional (3D) fabrication technologies that is used to produce a solid model of a predetermined design. AM techniques have recently been used in tissue engineering applications for fabrication of 3D artificial tissues that resemble architectures and material properties similar to that of the native tissue. Utilizing AM for this purpose presents the advantage of increased control in feature patterning, which leads to the realization of more complex geometries. However, there still remains a lack of simple and effective methods to integrate vascular networks within these 3D artificially engineered scaffolds and tissue constructs. Without these vascular-like networks, the cells embedded within the constructs would quickly die due to a lack of nutrient delivery and waste transport. This remains one of the biggest challenges in true 3D tissue engineering. This thesis presents a number of fast, effective and low-cost AM biofabrication techniques to address this challenge.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第24610号 / 工博第5116号 / 新制||工||1978(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 土屋 智由, 教授 横川 隆司, 教授 安達 泰治, 教授 田畑 修(京都先端科学大学) / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Bioinspiration and biomimetics are two increasingly important fields in applied science and mechanics that seek to imitate systems or processes in nature to design improved engineering devices. Here, we are inspired and motivated by microscale internal flow transport phenomena in insect tracheal networks, which are observed to be induced by the rhythmic tracheal wall contractions. These networks have been shown to mange fluid very efficiently compared to current state-of-the-art microfluidic devises. This dissertation presents two versions of a novel bioinspired pumping mechanism that is neither peristaltic nor belongs to impedance mismatch class of pumping mechanisms. The insect-inspired pumping models presented here are expected to function efficiently in the microscale flow regime in a simple channel/tube geometries or a complex network of channels. The first pumping approach shows the ability of inducing a unidirectional net flow by using an inelastic tube or channel with at least two moving contractions. The second pumping approach presents a new concept for directional pumping, namely ``selective pumping in a network.". The results presented here might help in mimicking features of physiological systems in insects and guide efforts to fabricate novel microfluidic devices with improved efficiency. In this study, both theoretical analysis and Stokeslets-meshfree computational methods are used to solve for the 2D and 3D viscous flow transport in several micro-geometries (tubes, channels and networks) with prescribed moving wall contractions. The derived theoretical analysis is based on both lubrication theory and quasi-steady approximations at low Reynolds numbers. The meshfree numerical method is based on the method of fundamental solutions (MFS) that uses a set of singularized force elements ``Stokeslets'' to induce the flow motions. Moreover, the passive particle tracking simulation approach in the Lagrangian frame of reference is also used to strengthen and support our pumping paradigm developed in this dissertation. / Ph. D.

Previous studies have shown that genes can be switched on or off by age, environmental factors, diseases, and lifestyles. The open or compact structures of chromatin is a crucial factor that affects gene expression. Epigenetics refers to hereditary mechanisms that change gene expression and regulations without changing DNA sequences. Epigenetic modifications, such as DNA methylation, histone modification, and non-coding RNA interaction, play critical roles in cell differentiation and disease processes. The conventional approach requires the use of a few million or more cells as starting material. However, such quantity is not available when samples from patients and small lab animals are examined. Microfluidic technology offers advantages to utilize low-input starting material and for high-throughput. In this thesis, I developed novel microfluidic technologies to study epigenomic regulations, including 1) profiling epigenomic changes associated with LPS-induced murine monocytes for immunotherapy, 2) examining cell-type-specific epigenomic changes associated with BRCA1 mutation in breast tissues for breast cancer treatment, and 3) developing a novel microfluidic oscillatory hybridized ChIRP-seq assay to profile genome-wide lncRNA binding for numerous human diseases. We used 20,000 and 50,000 primary cells to study histone modifications in inflammation and breast cancer of BRCA1 mutation, respectively. In the project of whole-genome lncRNA bindings, our microfluidic ChIRP-seq assay, for the first time, allowed us to probe native lncRNA bindings in mouse tissue samples successfully. The technology is a promising approach for scientists to study lncRNA bindings in primary patients. Our works pave the way for low-input and high-throughput epigenomic profiling for precision medicine development. / Doctor of Philosophy / Traditionally, physicians treat patients with a one-size-fits-all approach, in which disease prevention and treatment are designed for the average person. The one-size-fits-all approach fits many patients, but does not work on some. Precision medicine is launched to improve the low efficiency and diminish side effects, and all of these drawbacks are happening in the traditional approaches. The genomic, transcriptomic, and epigenomic data from patients is a valuable resource for developing precision medicine. Conventional approaches in profiling functional epigenomic regulation use tens to hundreds of millions cells per assay, that is why applications in clinical samples are restricted for several decades. Due to the small volume manipulated in microfluidic devices, microfluidic technology exhibits high efficiency in easy operation, reducing the required number of cells, and improving the sensitivity of assays. In order to examine functional epigenomic regulations, we developed novel microfluidic technologies for applications with the small number of cells. We used 20,000 cells from mice to study the epigenomic changes in monocytes. We also used 50,000 cells from patients and mice to study epigenomic changes associated with BRCA1 mutation in different cell types. We developed a novel microfluidic technology for studying lncRNA bindings. We used 100,000-500,000 cells from cell lines and primary tissues to test several lncRNAs. Traditional approaches require 20-100 million cells per assay, and these cells are infected by virus for over-producing specific lncRNA. However, our technology just needs 100,000 cells (non-over-producing state) to study lncRNA bindings. To the best of our knowledge, this is the first allowed us to study native lncRNA bindings in mouse samples successfully. Our efforts in developing microfluidic technologies and studying epigenomic regulations pave the way for precision medicine development.