Miniaturization of devices to study chemical interactions and processes in liquid samples has led to the emergence of microfluidics and construction of lab-on-a-chip systems. Present work was devoted to implementation, development and testing of microfluidic systems with detection by confocal Raman microscopy and surface enhanced Raman scattering under the conditions of training department. Several options of performing standard macroscopic measurements in microscopic scales were explored. A method for measuring thermal stability of biopolymers in microsystems with contactless detection of temperature has been designed and tested. Furthermore, possibilites for studying the SERS effect within microfluidic channels were explored. It was demonstrated that the microfluidic chips provide promising opportunity to study hydrodynamics of liquids at microscopic level and chemical reactions and kinetics.

Circulating tumor cells (CTCs) are known to escape from the primary tumor site and may settle down at the distant organ to grow a second tumor. CTCs are one of causes initiating carcinoma metastasis. Detection of CTCs has been considered to be valuable for cancer management, including diagnosis, prognosis, and clinical treatment management. However, efficient isolation, enumeration, characterization, and genetic analysis of CTCs in whole-blood samples from cancer patients are very challenging due to their extremely low concentration and rare nature (per CTC in blood cells is 1:106–109). With the increasing worldwide death rate associated with cancer, there is a desperate demand for a high-sensitivity, high-throughput, and low-cost detection and separation system. My doctoral research focused on the design and fabrications of the screening system for the detection of CTCs with further analysis of captured CTCs, such as immunofluoresce staining and fluorescence in-situ hybridization (FISH). The distinct significance of this research is that the development of the computer-controlled rotational holder with a series of six inverted microfluidic chips reduced the cost by significantly reducing the consumption of magnetic carriers (25% of the consumed amount used in the commercial CellSearch® system), increasing the capture efficiency by manipulating the blood sedimentation in the microchannel, enhancing the system stability by integrating the micromagnets on the plain glass slide substrate, and achieving high throughput because of the high flow rate (2.5 mL/hr) and large screening volume (screening up to six chips in parallel with each containing 2.5 mL of blood). Immunofluorescence staining and the FISH method have been performed to prove the capability of the system. In addition, the system has been successfully applied for patient samples screening. The incorporation of micromagnets has demonstrated that micromagnets provide localized magnetic forces to scatter the target cancer cells and free nanoparticles throughout the whole channel substrate to increase the channel space usage by 13%. Four cancer cell lines, including COLO 205 (colorectal cancer), SK-BR-3 (breast cancer), MCF-7 (breast cancer), and PC3 (prostate cancer), were spiked in blood samples from healthy donors to verify high capture efficiency of the developed system. On average, over a 97% capture rate was demonstrated for all cell lines. Moreover, the developed screening system has been successfully screened over 40 patient samples, including metastatic lung cancer, breast cancer, prostate cancer, and colorectal cancer. After capture of CTCs, immunofluorescence staining was used to identified the captured cancer cells and the FISH method was performed to characterize the isolated cancer cells by studying the gene expression of CTCs from breast cancer. The proposed automated immunomagnetic microchip-based screening system shows high capture efficiency (average 97% for three spiked cell lines), high throughput (15 mL of blood sample per screening), high sensitivity, high specificity, and low nanoparticle consumption (75% less than CellSearch® system). The screening system provides great promise as a clinical tool for early cancer diagnosis, diagnosis, personalized therapy, and treatment monitoring. / text

Molecular analysis of free circulating (fc)DNA has the potential to change the face of medicine, specifically in cancer diagnostics and in monitoring the efficacy of cancer treatments. In this study, a microfluidic device using AC electrokinetics is developed for rapid concentration and detection of fcDNA from blood. The device concentrates fcDNA using a combination of AC electrothermal flow and dielectrophoresis. The electrothermal fluid motion drives fcDNA towards the center of the electrode where dielectrophoretic trapping occurs. Once fcDNA is collected at the center, the concentration in the sample can be determined by fluorescent analysis using an intercalating dye binding to the double-stranded DNA. Effects of operating parameters are investigated to optimize the device's design. The electrokinetic device isolates high molecular weight DNA and can distinguish from low molecular weight DNA. Quantitative detection of fcDNA in physiologically relevant concentrations is demonstrated toward rapid diagnostics of cancer and monitoring of treatment efficacy.

This dissertation develops technology for microfluidic point-of-care immunoassay devices. This research (2004–2007) improved microfluidic immunoassay performance by reducing reagent consumption, decreasing analysis time, increasing sensitivity, and integrating processes using a lab-on-a-chip. Estimates show that typical hospital laboratories can save $1.0 million per year by using microfluidic chips. Our first objective was to enhance mixing in a microfluidic channel, which had been one of the main barriers to using these devices. Another goal of our studies was to simplify immunoassays by eliminating surfactants. Manufacturers of latex immunoassays add surfactants to prevent non-specific aggregation of microspheres. However, these same surfactants can cause false positives (and negatives) during diagnostic testing. This work, published in Appendix A (© 2006 Elsevier) shows that highly carboxylated polystyrene (HCPS) microspheres can replace surfactants and induce rapid mixing via diffusion in microfluidic devices. Our second objective was to develop a microfluidic device using fiber optics to detect static light scattering (SLS) of microspheres in Appendix B (© 2007 Elsevier). Fiber optics were used to deliver light emitting diode (LED) or laser light. A miniature spectrometer was used to measure 45° forward light scattering collected by optical fiber. Latex microspheres coated with PR3 proteins were used to test for the vasculitis marker, anti-PR3. No false negatives or positives were observed. A limit of detection (LOD) of 50 ng mL⁻¹ was demonstrated. This optical detection system works without fluorescence or chemiluminescence markers. It is cost effective, small, and re-usable with simple rinsing. The final objective in this dissertation, published in Appendix C (© 2007 Elsevier), developed a multiplex immunoassay. A lab-on-a-chip was used to detect multiple antibodies using microsphere light scattering and quantum dot (QD) emission. We conjugated QDs onto microspheres and named this configuration “nano-on-micro” or “NOM”. Upon radiation with UV light, strong light scattering is observed. Since QDs also provide fluorescent emission, we are able to use increased light scattering for detecting antigen-antibody reactions, and decreased QD emission to identify which antibody is present.

Cells in living organisms are constantly experiencing a variety of mechanical cues. From the stiffness of the extra cellular matrix to its topography, not to mention the presence of shear stress and tension, the physical characteristics of the microenvironment shape the cells’ fate. A rapidly growing body of work shows that cellular responses to these stimuli constitute regulatory mechanisms in many fundamental biological functions. Substrate strains were previously shown to be sensed by cells and activate diverse biochemical signaling pathways, leading to major remodeling and reorganization of cellular structures. The majority of studies had focused on the stretching avoidance response in near-uniform strain fields. Prior to this work, the cellular responses to complex planar strain fields were largely unknown. In this thesis, we uncover various aspects of strain sensing and response by first developing a tailored lab-on-a-chip platform that mimics the non-uniformity and complexity of physiological strains. These microfluidic cell stretchers allow independent biaxial control, generate cyclic stretching profiles with biologically relevant strain and strain gradient amplitudes, and enable high resolution imaging of on-chip cell cultures. Using these microdevices, we reveal that strain gradients are potent mechanical cues by uncovering the phenomenon of cell gradient avoidance. This work establishes that the cellular mechanosensing machinery can sense and localize changes in strain amplitude, which orchestrate a coordinated cellular response. Subsequently, we investigate the effect of multiple changes in stretching directions to further explore mechanosensing subtleties. The evolution of the cellular response shed light on the interplay of the strain avoidance and the newly demonstrated strain gradient avoidance, which were found to occur on two different time scales. Finally, we extend our work to study the influence of cyclic strains on the early stages of cancer development in epithelial tissues (using MDCK-RasV12 system), which was previously largely unexplored. This work reveals that external mechanical forces impede the healthy cells’ ability to eliminate newly transformed cells and greatly promote invasive protrusions, as a result of their different mechanoresponsiveness. Overall, not only does our work reveal new insights regarding the long-range organization in population of cells, but it may also contribute to paving the way towards new approaches in cancer prevention treatments.

abstract: Cancer is the second leading cause of death in the United States and novel methods of treating advanced malignancies are of high importance. Of these deaths, prostate cancer and breast cancer are the second most fatal carcinomas in men and women respectively, while pancreatic cancer is the fourth most fatal in both men and women. Developing new drugs for the treatment of cancer is both a slow and expensive process. It is estimated that it takes an average of 15 years and an expense of $800 million to bring a single new drug to the market. However, it is also estimated that nearly 40% of that cost could be avoided by finding alternative uses for drugs that have already been approved by the Food and Drug Administration (FDA). The research presented in this document describes the testing, identification, and mechanistic evaluation of novel methods for treating many human carcinomas using drugs previously approved by the FDA. A tissue culture plate-based screening of FDA approved drugs will identify compounds that can be used in combination with the protein TRAIL to induce apoptosis selectively in cancer cells. Identified leads will next be optimized using high-throughput microfluidic devices to determine the most effective treatment conditions. Finally, a rigorous mechanistic analysis will be conducted to understand how the FDA-approved drug mitoxantrone, sensitizes cancer cells to TRAIL-mediated apoptosis. / Dissertation/Thesis / Ph.D. Chemical Engineering 2013

Depuis quelques décennies, les matériaux composites organique/inorganique font l’objet de nombreux travaux de recherches. En raison de leurs propriétés uniques et intermédiaires entre les deux mondes minérales et organiques, ces matériaux sont d’un grand intérêt pour des nombreuses applications tel que le domaine, d’optique, la microfluidique, la microélectronique…, La synthèse de ce type des matériaux est réalisée à moindre coût en deux étapes : La synthèse du réseau inorganique est effectué par procédé sol-gel , tant qu’à la partie organique des compositions de type résines négatives et positives possède la particularité d’être photo-réticulables sous irradiation (UV et visible). Parallèlement, est apparue la lithographie par écriture laser (spot de quelques microns). Elle s’avère très pertinente pour la mise au point d’un procédé pour lequel des objets de petites dimensions (quelques Microns) et de petites surfaces sont à réaliser car elle permet de s’affranchir de la fabrication de masques. Cette technique associée aux résines négatives, n’est pas idéale pour la fabrication d’objets de grandes surfaces en raison de temps de fabrication induits trop long. Il est, par exemple, extrêmement compliqué et couteux d’utiliser l’écriture Laser pour la réalisation de dispositifs microfluidiques. En effet, la création de canaux de taille micronique nécessite une très grande surface à insoler. Il est donc bien plus pertinent de travailler sur l’association de l’écriture laser avec une résine de type positive. L’objectif principal de ce travail est la synthèse, l’optimisation et mise en œuvre d’un matériau hybride photosensible de type résine positive : Application à la fabrication des capteurs microfluidiques. Notre choix s’est porté sur le poly(amic acid) PAA de masse molaire 2340 g/mol comme partie organique, connu pour ces bonnes propriétés mécaniques et sa grande stabilité thermique. Le travail est centré d’une part, sur la synthèse d’une résine positive photosensible à la longueur d’onde utilisée (365 nm) à base du polymère PAA. En général, les PAA sont très solubles dans une solution alcaline aqueuse, dû à la présence d’acide carboxylique. Afin d’améliorer le contraste entre la partie insolée et non insolée après le développement, un inhibiteur de dissolution 1,3,5-tris[(2-vinyloxy)ethoxy]benzène (TVEB) est greffé au PAA via la fonction vinyl éther. Ce dernier permet la réduction de la teneur en acide carboxylique dans le motif répétitif du polymère et comme conséquence diminuer la dissolution de la partie non insolée. D’autre part, la synthèse du matériau hybride à base de la résine photosensible optimisée, est réalisée par greffage d’un précurseur ORMOSIL le 4-vinyléther-phenyltriéthoxysilane (VEPTES) pré-hydrolysé par procédé sol-gel comme partie inorganique à notre polymère. Afin d'optimiser le matériau, une étude structurale a été réalisée depuis la synthèse du solution jusqu'à l'obtention des dépôts et enfin la création des canaux microfluidiques. Une amélioration significative au niveau des propriétés mécaniques et thermiques est notée au niveau du polymère par ajout d’une partie minérale. / In recent decades, the organic / inorganic composite materials are the subject of many research works. Because of their unique properties and intermediate between inorganic and organic worlds, these materials are of great interest for many applications such as the area, optical, microfluidics, microelectronics ... The synthesis of this type of materials is carried out at a lower cost in two stages: The synthesis of inorganic network is made by sol-gel process, as well as the organic part of the negative and positive resin type compositions has the particularity of being photo-crosslinked under irradiation ( UV and visible).Meanwhile, the lithography by laser writing has appeared (spot a few microns). It is particularly appropriate for the development of a method for which small objects (a few microns) and small surfaces are to achieve because it eliminates the production of masks. This technique associated with negative resins, is not ideal for manufacturing large objects surfaces due to induced production time too long. It is, for example, be extremely complicated and expensive to use writing laser for producing microfluidic devices. Indeed, the creation of micron-sized channels requires a very large surface area to be exposed. It is therefore more appropriate to work on the combination of laser writing with a resin positive type. The main objective of this work is the synthesis, optimization and implementation of a photosensitive hybrid material resin positive type: Application to the fabrication of microfluidic sensors. Our choice fell on the poly(amic acid) PAA with molar mass of 2340 g/mol as an organic part, known for its good mechanical properties and high thermal stability.The work focuses on a part, on the synthesis of a positive photosensitive resin at the wavelength used (365 nm) based on the PAA polymer. In general, PAA are very soluble in an aqueous alkaline solution, due to the presence of carboxylic acid. In order to improve the contrast between the irradiated and unirradiated part after the development, an dissolution inhibitor 1,3,5-tris [(2-vinyloxy) ethoxy] benzene (TVEB) is grafted to the PAA via the vinyl ether function. This allows the reduction of the carboxylic acid content in the repeating unit of the polymer and as a consequence reduces the dissolution of the non-exposed part.On the other part, the synthesis of the hybrid material based on the optimized photosensitive resin is formed by grafting a precursor ORMOSIL 4-vinylether-phenyltriethoxysilane (VEPTES) pre-hydrolyzed by sol-gel method as the inorganic part to our polymer. In order to optimize the material, a structural study was conducted for the synthesis of the solution until the deposits and the creation of microfluidic channels. A significant improvement in mechanical and thermal properties is recorded at the polymer by adding an inorganic portion.

Master of Science / Department of Chemistry / Christopher T. Culbertson / The field of microfluidic devices has being developed quickly. It is aimed at integration of many chemical functions in a single chip, such as sample pretreatment, preconcentration, separation and detection, which provides a number of advantages including simplicity, automation, reduced analysis time, decrease in amount of samples and reduced formation of waste. Its potential applications have been conducted in the fields such as biotechnology, pharmaceuticals, life sciences, public health, agriculture and related areas. Membrane technology has been applied in analytical chemistry for many years and has won substantial growth in microfluidics over the past 10 years. Membrane is used to control transfer of some kinds of species, which can be employed for sample concentration, sample preparation, sample filtration and microreactors and so on. Sol-gel process, which usually involves catalytic hydrolysis of sol-gel precursor(s) and catalytic polycondensation of the hydrolyzed products and other sol-gel-active components present in the reaction medium to form a macromolecular network structure, is one of the most suitable methods for membrane fabrication. In this work, titanium membrane was fabricated inside glass microchips using the precursor of titanium isopropoxide. The resulted membranes demonstrated the excellent preconcentration effect. Followed separation and detection were also achieved. CE has been highly accepted as an efficient separation technique for qualitative and quantitative determination, which is performed using a narrow-bore capillary tube. It offers advantages of simplicity, high resolution separation, and minimal cost in terms of analysis time and sample consumption. In this work, protein separations were carried out by CE. Laser-induced fluorescence was used as detection. On-column fluorescence labeling using a fluorogenic labeling reagent was made. Under suitable experimental conditions, an excellent separation performance with about 1.4 million theoretical plate numbers was achieved.

Blood flow through microcirculation in both simple and complex geometry has been difficult to predict due to the composition and complex behavior of blood at the microscale. Blood is a dense suspension of deformable red blood cells that is comparable in dimensions to the microchannels that it flows through. As a result, rheological properties at the microscale can vastly differ from bulk rheological properties due to non-continuum effects. To further develop our understanding of blood microflow; experimental techniques should be explored. In this work, we explore micro-particle image velocimetry (μPIV) and two-beam fluorescence cross-correlation spectroscopy (2bFCCS) in the application of characterizing blood in microflow conditions. For the development of the μPIV analysis, a polydimethylsiloxane co-flow channel is used to observe blood flow in controlled conditions. Flow conditions (velocity profile and blood layer thickness) are selected based on an analytical model and compared to experimental measurement. The experimental results presented indicate that current flow conditions are inadequate in providing a controlled rate of shear on the blood layer in the co-flow channel and further optimization are required to improve the measurement of the velocity profile. For the development of the 2bFCCS application for blood flow analysis, a wide glass capillary microfluidic device is used to complete the verification of fluorescence fluid admissibility, the effect of laser intensity on inducing photobleaching and the velocity measurement performance. The experimental measurement of the velocity profile is validated against the theoretical profile for a rectangular channel. Results of the velocity profile of high concentration red blood cells show promise in the technique’s ability to measure blood microflows closer to physiological conditions.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23233号 / 工博第4877号 / 新制||工||1761(附属図書館) / 京都大学大学院工学研究科化学工学専攻 / (主査)教授 宮原 稔, 教授 佐野 紀彰, 教授 松坂 修二 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM