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abstract: The flow of liquid PDMS (10:1 v/v base to cross-linker ratio) in open, rectangular silicon micro channels, with and without a hexa-methyl-di-silazane (HMDS) or poly-tetra-fluoro-ethylene (PTFE) (120 nm) coat, was studied. Photolithographic patterning and etching of silicon wafers was used to create micro channels with a range of widths (5-50 μm) and depths (5-20 μm). The experimental PDMS flow rates were compared to an analytical model based on the work of Lucas and Washburn. The experimental flow rates closely matched the predicted flow rates for channels with an aspect ratio (width to depth), p, between one and two. Flow rates in channels with p less than one were higher than predicted whereas the opposite was true for channels with p greater than two. The divergence between the experimental and predicted flow rates steadily increased with increasing p. These findings are rationalized in terms of the effect of channel dimensions on the front and top meniscus morphology and the possible deviation from the no-slip condition at the channel walls at high shear rates. In addition, a preliminary experimental setup for calibration tests on ultrasensitive PDMS cantilever beams is reported. One loading and unloading cycle is completed on a microcantilever PDMS beam (theoretical stiffness 0.5 pN/ µm). Beam deflections are actuated by adjusting the buoyancy force on the beam, which is submerged in water, by the addition of heat. The expected loading and unloading curve is produced, albeit with significant noise. The experimental results indicate that the beam stiffness is a factor of six larger than predicted theoretically. One probable explanation is that the beam geometry may change when it is removed from the channel after curing, making assumptions about the beam geometry used in the theoretical analysis inaccurate. This theory is bolstered by experimental data discussed in the report. Other sources of error which could partially contribute to the divergent results are discussed. Improvements to the experimental setup for future work are suggested. / Dissertation/Thesis / Masters Thesis Mechanical Engineering 2014

abstract: DNA and DNA nanoassemblies such as DNA origamis have large potential in biosensing, drug delivery, nanoelectronic circuits, and biological computing requiring suitable methods for migration and precise positioning. Insulator-based dielectrophoresis (iDEP) provides an efficient and matrix-free approach for manipulation of micro-and nanometer-sized objects. In order to exploit iDEP for naturally formed DNA and DNA nanoassemblies, a detailed understanding of the underlying polarization and dielectrophoretic migration is essential. The shape and the counterion distribution are considered two essential factors in the polarization mechanism. Here, the dielectrophoretic behavior of 6-helix bundle (6HxB) and triangle DNA origamis with identical sequences but substantial topological differences was explored. The polarizability models were discussed for the two species according to their structural difference. The experimental observations reveal distinct iDEP trapping behavior in low frequency AC electric fields in addition to numerical simulations showing a considerable contribution of the electrophoretic transport of the DNA origami species in the DEP trapping regions. Furthermore, the polarizabilities of the two species were determined by measuring the migration times through a potential landscape exhibiting dielectrophoretic barriers. The resulting migration times correlate to the depth of the dielectrophoretic potential barrier and the escape characteristics of the DNA origamis according to an adapted Kramer’s rate model. The orientations of both species in the escape process were studied. Finally, to study the counterion distribution around the DNA molecules, both λ-DNA and 6HxB DNA were used in a phosphate buffer containing magnesium, revealing distinctive negative dielectrophoretic trapping behavior as opposed to positive trapping in a sodium/potassium phosphate buffer system. / Dissertation/Thesis / Presentation for Lin Gan's thesis defense (orginally in pptx exported in PDF) / Doctoral Dissertation Chemistry 2015

abstract: X-ray crystallography is the most widely used method to determine the structure of proteins, providing an understanding of their functions in all aspects of life to advance applications in fields such as drug development and renewable energy. New techniques, namely serial femtosecond crystallography (SFX), have unlocked the ability to unravel the structures of complex proteins with vital biological functions. A key step and major bottleneck of structure determination is protein crystallization, which is very arduous due to the complexity of proteins and their natural environments. Furthermore, crystal characteristics govern data quality, thus need to be optimized to attain the most accurate reconstruction of the protein structure. Crystal size is one such characteristic in which narrowed distributions with a small modal size can significantly reduce the amount of protein needed for SFX. A novel microfluidic sorting platform was developed to isolate viable ~200 nm – ~600 nm photosystem I (PSI) membrane protein crystals from ~200 nm – ~20 μm crystal samples using dielectrophoresis, as confirmed by fluorescence microscopy, second-order nonlinear imaging of chiral crystals (SONICC), and dynamic light scattering. The platform was scaled-up to rapidly provide 100s of microliters of sorted crystals necessary for SFX, in which similar crystal size distributions were attained. Transmission electron microscopy was used to view the PSI crystal lattice, which remained well-ordered postsorting, and SFX diffraction data was obtained, confirming a high-quality, viable crystal sample. Simulations indicated sorted samples provided accurate, complete SFX datasets with 3500-fold less protein than unsorted samples. Microfluidic devices were also developed for versatile, rapid protein crystallization screening using nanovolumes of sample. Concentration gradients of protein and precipitant were generated to crystallize PSI, phycocyanin, and lysozyme using modified counterdiffusion. Additionally, a passive mixer was created to generate unique solution concentrations within isolated nanowells to crystallize phycocyanin and lysozyme. Crystal imaging with brightfield microscopy, UV fluorescence, and SONICC coupled with numerical modeling allowed quantification of crystal growth conditions for efficient phase diagram development. The developed microfluidic tools demonstrated the capability of improving samples for protein crystallography, offering a foundation for continued development of platforms to aid protein structure determination. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2016

abstract: Over the past fifty years, the development of sensors for biological applications has increased dramatically. This rapid growth can be attributed in part to the reduction in feature size, which the electronics industry has pioneered over the same period. The decrease in feature size has led to the production of microscale sensors that are used for sensing applications, ranging from whole-body monitoring down to molecular sensing. Unfortunately, sensors are often developed without regard to how they will be integrated into biological systems. The complexities of integration are underappreciated. Integration involves more than simply making electrical connections. Interfacing microscale sensors with biological environments requires numerous considerations with respect to the creation of compatible packaging, the management of biological reagents, and the act of combining technologies with different dimensions and material properties. Recent advances in microfluidics, especially the proliferation of soft lithography manufacturing methods, have established the groundwork for creating systems that may solve many of the problems inherent to sensor-fluidic interaction. The adaptation of microelectronics manufacturing methods, such as Complementary Metal-Oxide-Semiconductor (CMOS) and Microelectromechanical Systems (MEMS) processes, allows the creation of a complete biological sensing system with integrated sensors and readout circuits. Combining these technologies is an obstacle to forming complete sensor systems. This dissertation presents new approaches for the design, fabrication, and integration of microscale sensors and microelectronics with microfluidics. The work addresses specific challenges, such as combining commercial manufacturing processes into biological systems and developing microscale sensors in these processes. This work is exemplified through a feedback-controlled microfluidic pH system to demonstrate the integration capabilities of microscale sensors for autonomous microenvironment control. / Dissertation/Thesis / Ph.D. Bioengineering 2012

abstract: This work demonstrated a novel microfluidic device based on direct current (DC) insulator based dielectrophoresis (iDEP) for trapping individual mammalian cells in a microfluidic device. The novel device is also applicable for selective trapping of weakly metastatic mammalian breast cancer cells (MCF-7) from mixtures with mammalian Peripheral Blood Mononuclear Cells (PBMC) and highly metastatic mammalian breast cancer cells, MDA-MB-231. The advantage of this approach is the ease of integration of iDEP structures in microfliudic channels using soft lithography, the use of DC electric fields, the addressability of the single cell traps for downstream analysis and the straightforward multiplexing for single cell trapping. These microfluidic devices are targeted for capturing of single cells based on their DEP behavior. The numerical simulations point out the trapping regions in which single cell DEP trapping occurs. This work also demonstrates the cell conductivity values of different cell types, calculated using the single-shell model. Low conductivity buffers are used for trapping experiments. These low conductivity buffers help reduce the Joule heating. Viability of the cells in the buffer system was studied in detail with a population size of approximately 100 cells for each study. The work also demonstrates the development of the parallelized single cell trap device with optimized traps. This device is also capable of being coupled detection of target protein using MALDI-MS. / Dissertation/Thesis / Ph.D. Chemistry 2013

abstract: Rapid and reliable separation and analysis of proteins require powerful analytical methods. The analysis of proteins becomes especially challenging when only small sample volumes are available, concomitantly with low concentrations of proteins. Time critical situations pose additional challenges. Due to these challenges, conventional macro-scale separation techniques reach their limitations. While microfluidic devices require only pL-nL sample volumes, they offer several advantages such as speed, efficiency, and high throughput. This work elucidates the capability to manipulate proteins in a rapid and reliable manner with a novel migration technique, namely dielectrophoresis (DEP). Since protein analysis can often be achieved through a combination of orthogonal techniques, adding DEP as a gradient technique to the portfolio of protein manipulation methods can extend and improve combinatorial approaches. To this aim, microfluidic devices tailored with integrated insulating obstacles were fabricated to create inhomogeneous electric fields evoking insulator-based DEP (iDEP). A main focus of this work was the development of pre-concentration devices where topological micropost arrays are fabricated using standard photo- and soft lithographic techniques. With these devices, positive DEP-driven streaming of proteins was demonstrated for the first time using immunoglobulin G (IgG) and bovine serum albumin. Experimentally observed iDEP concentrations of both proteins were in excellent agreement with positive DEP concentration profiles obtained by numerical simulations. Moreover, the micropost iDEP devices were improved by introducing nano-constrictions with focused ion beam milling with which numerical simulations suggested enhancement of the DEP effect, leading to a 12-fold increase in concentration of IgG. Additionally, concentration of β-galactosidase was observed, which seems to occur due to an interplay of negative DEP, electroosmosis, electrokinesis, diffusion, and ion concentration polarization. A detailed study was performed to investigate factors influencing protein DEP under DC conditions, including electroosmosis, electrophoresis, and Joule heating. Specifically, temperature rise within the iDEP device due to Joule heating was measured experimentally with spatial and temporal resolution by employing the thermosensitive dye Rhodamine B. Unlike DNA and cells, protein DEP behavior is not well understood to date. Therefore, this detailed study of protein DEP provides novel information to eventually optimize this protein migration method for pre-concentration, separation, and fractionation. / Dissertation/Thesis / Ph.D. Chemistry 2014

Polydimethylsiloxane (PDMS) microfluidic channels, fabricated using low cost and simple soft lithography methods, conventionally have rectangular cross-sections. Despite being often used for organs-on-a-chip and cardiovascular research, these devices do not mimic the circular cross-sections of blood vessels in the human body, creating potential inaccuracies in observed flow conditions and cell behaviours. The purpose of this thesis is to (i) compare and optimize fabrication techniques for microchannels with circular cross-sections, (ii) assess biocompatibility of different surface functionalization approaches for Human Umbilical Vein Endothelial Cell (HUVEC) adhesion and growth, (iii) culture HUVECs within circular microchannels to mimic blood vessel features, and (iv) compare gene expression of HUVECs cultured in 3D circular microchannels to those cultured on 2D surfaces. We show that wire molding is superior to the gas stream technique for producing circular cross-section microchannels with high aspect ratios, circularity, and channel geometry precision. Fibronectin (FN) and polydopamine (PD) surface coatings on PDMS, as well as alternative collagen substrates, were tested for biocompatibility with HUVECs in 2D cultures; fibronectin coated PDMS (PDMS-FN) substrates facilitated cell attachment, spreading and growth. We demonstrate the capability of growing HUVECs on the inner surface of circular PDMS microchannels created using the wire-mold method and treated with fibronectin. A syringe pump was used to induce shear stress on the HUVECs grown in circular microchannels. Relative to static growth conditions, longer cell culture growth periods were more feasible under flow and altered cell morphology was observed. Finally, Microarray analysis revealed significantly different gene expression profiles for HUVECs cultured within PDMS-FN circular cross-section microchannels as compared to HUVECs cultured on PDMS-FN in a 2D environment, thereby highlighting the critical importance of in vitro conditions for mimicking the in vivo reality.

Orientadores: Lucimara Gaziola de La Torre, Reinaldo Gaspar Bastos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-25T04:50:36Z (GMT). No. of bitstreams: 1 Oliveira_AlineFurtado_M.pdf: 2165174 bytes, checksum: d3aa7acdee3edc7f222daf3f7a82f910 (MD5) Previous issue date: 2014 / Resumo: A microfluídica é uma ciência que opera em pequenos volumes de fluídos dentro de canais em dimensões de micrômetros (10-6 m). Estes sistemas permitem controlar moléculas no espaço e no tempo, gerando resultados rápidos e confiáveis num sistema precisamente controlado e capaz de mimetizar ambientes celulares. Os dispositivos microfluídicos apresentam uma diversidade de geometrias aplicáveis para diversas áreas de pesquisas, sendo que a capacidade de formar gradientes permite avaliar as condições e o desempenho celular microbiano. Assim, este trabalho teve como objetivo desenvolver dispositivos microfluídicos capazes de formar gradiente de concentração difusivo e investigar sua aplicabilidade em bioprocessos. Diante disso, foram propostos três modelos de dispositivos usando materiais biocompatíveis: (i) dispositivo em base de vidro, denominado de Vidro-vidro; (ii) em base de vidro e poli dimetilsiloxano (PDMS), chamado de Vidro-PDMS e (iii) vidro e PDMS modificado quimicamente para tornar a superfície hidrofílica, Vidro-mPDMS. Os três dispositivos foram avaliados quanto à capacidade de formação de gradiente de concentração difusivo, os quais apresentaram um perfil linear. Além disso, validou-se o estudo do comportamento de Saccharomyces cerevisiae ATCC 7754 num gradiente de concentração de glicose de 0 a 40 g/L de glicose, sendo usado o dispositivo vidro-vidro. Foi observado que houve crescimento de células ao longo das câmaras microfluídicas, e isso possibilitou na determinação de parâmetros cinéticos, os quais não apresentaram diferença estatisticamente significativa com o cultivo em batelada convencional. As condições da microfluídica possibilitaram também a determinação da cinética de Monod, usando menores intervalos de gradiente. Portanto, este dispositivo microfluídico mostrou-se uma ferramenta com potencial para investigar comportamento celular frente à diferença de concentração e contribuirá para a otimização de bioprocessos através da determinação de parâmetros cinéticos / Abstract: Microfluidic is a science that operates in small amounts of fluids inside channels in dimensions of micrometers (10-6 m). These systems allow the precise control of molecules in space and time, generating fast and reliable results and it can also be used to mimics environment cellular . Microfluidic devices can be produced in diversity of geometries, it can be applied in several scientific areas and especially the formation of concentration gradients can be used to evaluate conditions and performance of microbial cell. Therefore, this work had the objective to develop microfluidic devices that are able to generate diffusive concentration gradients and investigate their applicability in bioprocesses. In this context, we propose three models of microfluidics devices using biocompatible materials: (i) Glass-based device, named glass-glass; (ii) glass and poli dimetilsiloxane (PDMS) based device, Glass-PDMS and (iii) glass and chemically modified PDMS (hydrophilic surface), Glass-mPDMS. The three devices were evaluated by their capacity of generating difusive concentration gradient, demonstrating linear concentration profile. Furthermore, the behavior of Saccharomyces cerevisiae ATCC 7754 inside of glucose concentration gradient ranging from 0 to 40 g/L were validated, using the glass-glass device . It was observed that cell growth along the microfluidic chambers, having determined the kinetic parameters, which was considered statistically similar to conventional batch cultivation. Conditions of microfluidics also allowed determination of the Monod kinetic, using smaller intervals gradient Therefore, the use of concentration gradient in microfluidic device is a potential tool for investigate of microbial cell behavior against the concentration difference and it can contribute to the optimization of bioprocesses through the determination of kinetic parameters / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestra em Engenharia Química

Orientador: José Alberto Fracassi da Silva / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-20T05:22:01Z (GMT). No. of bitstreams: 1 Campos_RichardPifferSoaresde_M.pdf: 3349460 bytes, checksum: 5efdb4dd4409b9d1b6771593c8874edf (MD5) Previous issue date: 2012 / Resumo: Os micro sistemas de análise total consistem de dispositivos da ordem de centímetros que tem como objetivo a integração de várias etapas analíticas em um único chip, tais como etapas de tratamento de amostra, separação por eletroforese capilar, ou mesmo a integração de sensores em canais microfluídicos. O poli(dimetilsiloxano), PDMS, é um dos polímeros mais adotados para a fabricação destes microdispositivos, devido a suas propriedades elastoméricas, transparência óptica, permeabilidade gasosa, biocompatibilidade, fácil moldagem, relativa alta resistência química e baixo custo de fabricação, além de poder ser facilmente moldado e selado, resultando em microcanais com boa resolução. Além disso, é possível a fabricação de canais por ablação a laser sobre o polímero curado. Entretanto, a característica altamente hidrofóbica do PDMS faz com que sua aplicação para soluções aquosas seja problemática e analitos pouco polares possam sofrer forte adsorção nas paredes do canal, tornando pobre a reprodutibilidade do processo. Neste sentido, estratégias para modificar o material nativo ou mesmo a superfície dos canais vêm sendo estudadas. Neste trabalho, foi inicialmente estudada a modificação estrutural do PDMS, que consiste na utilização de um reticulante (contendo função orgânica polar metacrilato ou amina) na formação do substrato. Também foi realizada a modificação da superfície do substrato de PDMS por reação topológica, com a introdução de polietileno glicol, além da modificação do processo convencional de reticulação do PDMS Sylgard 184, pela adição do surfactante Silwet-L77 a este processo. O PDMS modificado foi avaliado quanto a sua hidrofobicidade, por medida do ângulo de contato com a água, em relação às propriedades do fluxo eletrosmótico gerado no microcanal e as modificações foram estudadas por métodos espectroscópicos. A reação de modificação de superfície do PDMS com divinil éter de polietileno glicol apresentou as melhores características hidrofílicas dentre as modificações estudadas e mobilidade do fluxo eletrosmótico com valor de 3,6x10 cm V s. Em adição, as modificações puderam ser caracterizadas por métodos de espectroscopia (IR e Raman), que se mostraram eficientes na avaliação tanto da rota de modificação quanto do produto final / Abstract: The micro total analysis systems consist of devices in the order of centimeters that aim to integrate several analytical steps on a single substrate, such as sample treatment, injection, or even integrated sensors on microfluidic channels. Poly(dimethylsiloxane), PDMS, is one of the most used polymers for microfabrication due to its elastomeric properties, optical transparency, gas permeability, biocompatility, relatively high chemical resistance and low fabrication costs. PDMS can also be easily cast and sealed, resulting in microchannels with good resolution. On top of that, it is possible to fabricate the microchannels using the lase ablation technique on the cured PDMS. However, the highly hydrophobic characteristic of PDMS makes its aqueous applications problematic. Moreover, non-polar analytes can adsorb on the channel walls, leading to poor reproducibility. In this sense, strategies to modify the raw material or channel surface have been proposed. In this work, the structural modification of PDMS, involving the use of a crosslinking agent (containing the methacrylate or amine polar functions) was studied. In addition, the surface modification of PDMS by topologic reaction with polyethylene glycol and the modification of the conventional PDMS Sylgard 184 crosslinking by the addition of Silwet-L77 surfactant were also performed. The hydrophobicity of modified PDMS was evaluated by water contact angle measurements and the modifications were studied by spectroscopic methods. The electroosmotic flow (EOF) generated in the microchannels was also evaluated. The best hydrophilic characteristic among the studied modifications were obtained with the polyethylene glycol divinyl ether PDMS modification. This device presented an EOF of 3,6x10 cm V s. In addition, the modifications could be characterized by spectroscopic methods (Raman and IR) and those techniques were efficient in the evaluation of the reaction routes as well as the final products / Mestrado / Quimica Analitica / Mestre em Química