>Magister Scientiae - MSc / The discovery of cell free foetal DNA (cffDNA) in 1997 allows for the combination of accuracy as well as non-invasiveness for prenatal diagnosis. This non-invasive genetic test requires only a maternal blood sample from which the cffDNA can be isolated and analysed. In this work cffDNA was isolated from a maternal blood sample using a micro-fluidic device which was fabricated using hot embossing and laser ablation techniques. The DNA sample was first pre-concentration by electrokinetic trapping (EKT) and then isotachophoresis (ITP). The concentrated sample was then separated by size using capillary electrophoresis (CE), all in a single device. All parameters and processes concerned with the micro-fluidic device were optimised sequentially. These parameters include both the chemical components as well as the physical processes which occur. The DNA used for the optimisation protocol was analysed using fluorescence spectroscopy, agarose gel electrophoresis as well as an Agilent Bioanalyser. The optimised protocol included a 9% acrylamide/pDMA matrix, 3 M N,N-dimethylurea as a denaturing agent, with tris based buffers for pre-concentration steps and 1X TBE (tris/borate/EDTA) buffer for capillary electrophoresis. The applied voltage of ITP was 300 V and CE was carried out at 180 V. The timing at which DNA was extracted from the device was kept at time = 60 s intervals. The optimised protocol was then used for real sample analysis and these samples were obtained from mothers pregnant with male foetuses. The DNA extracted from the micro-fluidic device was then analysed using real time PCR (RT-PCR) in order to distinguish which was maternal and which was foetal. This was carried out by amplification of male and general (present in male and female) genes respectively. RT-PCR results confirmed that only the male specific gene was amplified in initial samples exiting the device and it was thus successful in isolating cffDNA from a maternal plasma sample.

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: Volumetric cell imaging using 3D optical Computed Tomography (cell CT) is advantageous for identification and characterization of cancer cells. Many diseases arise from genomic changes, some of which are manifest at the cellular level in cytostructural and protein expression (functional) features which can be resolved, captured and quantified in 3D far more sensitively and specifically than in traditional 2D microscopy. Live single cells were rotated about an axis perpendicular to the optical axis to facilitate data acquisition for functional live cell CT imaging. The goal of this thesis research was to optimize and characterize the microvortex rotation chip. Initial efforts concentrated on optimizing the microfabrication process in terms of time (6-8 hours v/s 12-16 hours), yield (100% v/s 40-60%) and ease of repeatability. This was done using a tilted exposure lithography technique, as opposed to the backside diffuser photolithography (BDPL) method used previously (Myers 2012) (Chang and Yoon 2004). The fabrication parameters for the earlier BDPL technique were also optimized so as to improve its reliability. A new, PDMS to PDMS demolding process (soft lithography) was implemented, greatly improving flexibility in terms of demolding and improving the yield to 100%, up from 20-40%. A new pump and flow sensor assembly was specified, tested, procured and set up, allowing for both pressure-control and flow-control (feedback-control) modes; all the while retaining the best features of a previous, purpose-built pump assembly. Pilot experiments were performed to obtain the flow rate regime required for cell rotation. These experiments also allowed for the determination of optimal trapezoidal neck widths (opening to the main flow channel) to be used for cell rotation characterization. The optimal optical trap forces were experimentally estimated in order to minimize the required optical power incident on the cell. Finally, the relationships between (main channel) flow rates and cell rotation rates were quantified for different trapezoidal chamber dimensions, and at predetermined constant values of laser trapping strengths, allowing for parametric characterization of the system. / Dissertation/Thesis / Demonstration of process flow in the microvortex chip / Cell rotation in a 50 microns wide (at the neck) trapezoidal chamber,at a flow rate of 95 microliters/min at approximately 0.25 rev/s / Cell rotation in a 70 microns wide (at the neck) trapezoidal chamber,at a flow rate of 7 microliters/min at approximately 0.125 rev/s / M.S. Bioengineering 2013

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.

Neste trabalho usamos pulsos de femtossegundos na fabricação de estruturas poliméricas em escala microscópica, através da técnica de fotopolimerização via absorção de dois fótons. Graças ao confinamento espacial da polimerização, resultante do processo de absorção de dois fótons, este método permite a fabricação de microestruturas tridimensionais complexas, com alta resolução, visando diversas aplicações tecnológicas, de fotônica até biologia. Inicialmente, desenvolvemos a técnica de fotopolimerização via absorção de dois fótons, desde a implantação da montagem óptica até a confecção dos sistemas de movimentação e controle do posicionamento do feixe laser. Através da fabricação e caracterização de microestruturas, produzidas em resinas acrílicas, o sistema foi aperfeiçoado permitindo a produção de microestruturas da pordem de 30um com razoável resolução espacial. Uma vez que a maior parte as microestruturas reportadas na literatura são elementos passivos, ou seja, suas propriedades ópticas não podem ser controladas por meios externos, numa segunda etapa deste projeto produzimos microestruturas opticamente ativas. Neste caso, a microfabricação foi feita em resinas acrílicas dopadas Rodamina B, exibindo, portanto, fluorescência quando excitadas com luz de comprimento de onda em torno de 540nm. Finalmente, visando a produção eficiente de estruturas em escala milimétrica para aplicações biológicas, implementamos também um sistema de fotopolomerização via absorção de um fóton. / In this work we used femtosecond pulses to fabricate polymeric structures at microscopic scale, by using the two-photon photopolymerization technique. Due to the spatial confinement of the polymerization, provided by the two-photon absorption, this method allows for the fabrication of complex three-dimensional microstructures, with high resolution, aiming to several technological applications, from photonics to biology. Initially, we developed the two-photon polimerization technique, from the optical setup to the mechanical systems to control the movement and the positioning of the laser beam. Through the fabrication and characterization os microestrutures, produced in acrylic resin, the apparatus was improved, allowing the fabriation of 30-um microstructures with reasonable spatial resolution. Since most the report in the literature are passive elements that is, their optical properties cannot be altered by any external means, in a second stage of this project we fabricated optical active microstructures. In this case, the microfabrication was carried out in acrylic resins doped with Rodamine B, exhibiting, consenquently, fluorescence when excited with light at 540nm. Finally, in order to eficiently produce milimetric structures for biological applications, we also implemented a one-photon polimerization setup.

Orientador: Lucila Helena Deliesposte Cescato / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-08-14T05:44:49Z (GMT). No. of bitstreams: 1 GutierrezRivera_LuisEnrique_D.pdf: 4586179 bytes, checksum: dcf2f61b429cf573374569e52bacf96f (MD5) Previous issue date: 2009 / Resumo: Nano-Peneiras são dispositivos de microfiltração que se diferenciam das membranas filtrantes por apresentarem uma distribuição homogênea de poros e baixa rugosidade na sua superfície. Estas características as tornam uma melhor alternativa para processos de separação que requerem alta seletividade na indústria de biotecnologia, fármacos e química. Por outro lado estas peneiras quando atingem poros com dimensões de alguns centos de nanômetros e estão feitas de materiais biocompativeis e biodegradáveis, são utilizadas em Micro-dispositivos Bio Eletro Mecânicos, BIO-MEMS. Entre as aplicações mais relevantes de peneiras poliméricas dentro dos BIO-MEMS podemos mencionar os bio-sensores e as biocapsulas para sistemas de liberação de drogas através de implantes dentro do corpo. Neste trabalho desenvolvemos um processo de fabricação de Peneiras Poliméricas Nanométricas utilizando a associação de técnicas de litografia interferométrica, litografia óptica, e micro-moldagem. As Peneiras foram fabricadas utilizando um sistema de litografia interferométrica UV, que foi construído durante esta tese. Foi estudada a homogeneidade dos poros das peneiras fabricadas através da analises estadísticas de imagens digitais de microscopia eletrônica. Por outro lado fizemos analises de seletividade da filtração utilizando partículas esféricas de poliestireno de diferentes tamanhos. Os resultados da analises estadística da morfologia dos poros mostraram uma alta qualidade na distribuição e na homogeneidade dos poros das peneiras que foram formados através da litografia interferométrica. Por outro lado os resultados das medidas de filtração de partículas apresentaram uma excelente seletividade na separação de partículas por tamanho, que confirmaram a homogeneidade das dimensões dos poros das nanopeneiras poliméricas. / Abstract: Sieves are microfiltration devices that in comparison with conventional membranes present a homogeneous pore size distribution and low roughness on the surface. These characteristics become them very attractive for use in processes of separation of particles by size. When the pores reach sub-micrometric dimensions and the sieves are made in biocompatible and biodegradable materials, they can reach interesting applications in Bio-MEMS. We can mention applications such as Bio-sensor and Bio-Capsules for Drug Delivery Systems. In this work we developed a process for fabrication of polymeric submicrometric sieves using the association of interference lithography, optical lithography and micromolding (soft lithography). The submicrometric pores are recorded using UV interference lithography, while the sustaining structure is recorded by optical lithography. Soft lithography techniques were used in order to transfer the recorded structures to appropriate materials as well as to reduce the costs of fabrication. Measurements of the pore size distribution shown a variation of 7 % in the pore diameter, along the whole sieve area of about 1cm X 1 cm, for submicrometric sieves in SU-8 while this variation was about 15 % for the sieves molded in PLLA (Poly-L-Lactide). This variation is much smaller than that presented for the better homogeneity commercial membranes. / Doutorado / Doutor em Ciências

The miniaturization afforded by the integration of microfluidic technologies within lab-on-a-chip devices has greatly enhanced analytical capabilities in several key applications. Microfluidics has been utilized in a wide range of areas including sample preparation and analysis, DNA microarrays, cell detection, as well as environmental monitoring. The use of microfluidics in these applications offer many unique advantages: reduction in the required sample size, reduction in analysis time, lowered cost through batch fabrication, potentially higher throughput and the vision of having such devices used in portable systems. Nanopore sensors are a relatively new technology capable of detection and analysis with single-molecule sensitivity, and show promise in many applications related to the diagnosis and treatment of many diseases. Recently, some research groups demonstrated the integration of nanopores within microfluidic devices to increase analytical throughput. This thesis describes a methodology for integrating nanopore sensors within microfluidic devices with the aim of enhancing the analytical capabilities required to analyze biomolecular samples. In this work, the first generation of an integrated nanopore-microfluidic device contained multiple independently addressable microfluidic channels to fabricate an array of nanopore sensors using controlled breakdown (CBD). Next, for the second generation, we added pneumatic microvalves to manipulate electrical and fluidic access through connected microfluidic channels. As a proof-of-concept, single molecules (single- and double-stranded DNA, proteins) were successfully detected in the devices. It is also demonstrated that inclusion of the microfluidic via (microvia) limited the exposed area of the embedded silicon nitride membrane to the solution. This helped in localizing nanopore formation by confining the electric field to specific regions of the insulating membrane while significantly reducing high frequency noise in the ionic current signal through the reduction of chip capacitance. The devices highlighted in this thesis were designed and fabricated using soft lithography techniques which are available in most biotechnology laboratories. The core of this thesis is based on two scientific articles (Chapters 3 and 4), which are published in peer-reviewed scientific journals. These chapters are preceded by an introductory chapter and another chapter detailing the experimental setup and the methods used during the course of this study.

Orientador: Cecília Amélia de Carvalho Zavaglia / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecânica / Made available in DSpace on 2018-08-27T18:19:16Z (GMT). No. of bitstreams: 1 Carvalho_ClaudioRobertoCutrim_D.pdf: 4942527 bytes, checksum: c95fc9ea9d486c5f468d6780906cc6c5 (MD5) Previous issue date: 2015 / Resumo: Este trabalho tem como objetivo a fabricação de 3 classes de dispositivos implantáveis para liberação de drogas, produção intra-corpórea de proteínas recombinantes e regeneração de tecidos. Os dispositivos fabricados, a saber são: biorreator implantável para produção de proteínas recombinantes intra-corpóreo, um protótipo construído de titânio (usinado) e outro bioaborvivel construído com poli(ácido láctico-co-glicólico) 50/50% através da litografia macia; sistema anti-infecção composto de termoresistores para produção de calor com o objetivo de inibir o crescimento bacteriano associado com microreservatórios para liberação de antibióticos bactericidas; neurotubos e dispositivos de liberação de drogas implantáveis, o primeiro para fomentar a regeneração de nervos periféricos, e o último com objetivo de liberar drogas, os materiais usados na fabricação destes dispositivos foram o Poli-Dimetil Siloxano(PDMS) e o PSG(Poli-Sebacato de Glicerol).Os 4 dispositivos tem como processo de fabricação principal a tecnologia de litografia macia. Em relação aos resultados, o biorreator implantável produziu as proteínas recombinantes BMP-2 (induzem formação óssea) verificadas através de testes in vitro de detecção das proteínas recombinantes; os resultados do desenvolvimento do neurotubo e dispositivos implantáveis, resultaram em testes de liberação de antibióticos in vivo com sucesso em ratos da raça Winstar; o sistema anti-infecção apresentou resultados animadores quanto a função de seus componentes principais: o termoresistor através da monitoração da produção de corrente, e os reservatórios liberadores de antibióticos, através dos testes in vitro com culturas bacterianas. Palavras-chave: Litografia macia, sistema anti infecção, biorreator, regeneração nervosa / Abstract: The main goal of this work is the manufacturing of four classes of implantable devices for delivering drugs, recombinant proteins for intra-body production and tissue regeneration. The 3 devices are: implantable bioreactor for the production of recombinant proteins intra- corporeal, one prototype built in titanium(machined) and the other one bioabsorbable, constructed with poly (lactic-co-glycolic acid) 50/50% through the soft lithography technology; System anti-infection compounds of resistance thermometers for heat production with the goal inhibiting bacterial growth with the associated microreservoirs to deliver bactericidal antibiotics; Neurotubes and implantable drugs release devices, the first dedicated for peripheral nerve regeneration, and second one for release drugs, the materials used in the manufacture of these devices was Poly-dimethyl siloxane (PDMS) and PSG (poly-glycerol sebacate). The 4 devices has main manufacturing process the soft lithography technology. About results, the implantable bioreactor produced recombinant proteins BMP-2 (induce bone formation) verified through testing in-vitro for detection of the recombinant protein; The development of neurotube and implantable devices results in successfully liberation tests of antibiotics in-vivo in rats Winstars. The anti-infection system showed encouraging results as the function of its main components: the termoresistor through the tests of current production and monitoration, and the releasing reservoirs of antibiotics, through the testes in vitro with bacterial cultures. Key-words: Soft lithography, nerve regeneration, bioreactor, system anti infection / Doutorado / Materiais e Processos de Fabricação / Doutor em Engenharia Mecânica

The development of a polyimide and metals multi-user surface micro-machining process for Micro-electro-mechanical Systems (MEMS) is presented. The process was designed to be as general as possible, and designed to be capable to fabricate different designs on a single silicon wafer. The process was not optimized with the purpose of fabricating any one specific device but can be tweaked to satisfy individual needs depending on the application. The fabrication process uses Polyimide as the structural material and three separated metallization layers that can be interconnected depending on the desired application. The technology allows the development of out-of-plane compliant mechanisms, which can be combined with six variations of different physical principles for actuation and sensing on a single processed silicon wafer. These variations are: electrostatic motion, thermal bimorph actuation, capacitive sensing, magnetic sensing, thermocouple-based sensing and radio frequency transmission and reception.

Micro- and nanofabricated sample environments are useful tools for characterizing diffusion in confined aqueous environments. The steady-state diffusion and adsorption of unilamellar lipid vesicles in arrays of hydrophilic micropillars is investigated. Gradients in the coverage of fluorescently labeled, pillar-supported lipid films, formed from vesicle fusion, are determined from 3D z-stack images using confocal microscopy. The gradients are the result of preferential adsorption of vesicles near the tops of the pillars, which progressively deplete them from solution as they diffuse toward the base of the array. However, the increased propensity for vesicle adsorption near the pillar tops compared to the confined spaces between pillars results in the formation of confluent supported lipid bilayers at the pillar tops that resist the adsorption of additional vesicles while leaving the pillar surfaces below available for binding. This results in a reduction in the numbers of depleted vesicles compared to what one would anticipate based on diffusive fluxes. The resulting inhomogeneous spatial profiles of lipid structures on the pillars are the result of the system being maintained in a dissipative, nonequilibrium steady state during incubation of the pillar arrays in the vesicle solution, which is ultimately quenched by rinsing away the unbound, freely diffusing vesicles.