Design and development of a microfluidic platform for use with colorimetric gold nanoprobe assays

Due to the importance and wide applications of the DNA analysis, there is a need to make genetic analysis more available and more affordable. As such, the aim of this PhD thesis is to optimize a colorimetric DNA biosensor based on gold nanoprobes developed in CEMOP by reducing its price and the needed volume of solution without compromising the device sensitivity and reliability, towards the point of care use. Firstly, the price of the biosensor was decreased by replacing the silicon photodetector by a low cost, solution processed TiO2 photodetector. To further reduce the photodetector price, a novel fabrication method was developed: a cost-effective inkjet printing technology that enabled to increase TiO2 surface area. Secondly, the DNA biosensor was optimized by means of microfluidics that offer advantages of miniaturization, much lower sample/reagents consumption, enhanced system performance and functionality by integrating different components. In the developed microfluidic platform, the optical path length was extended by detecting along the channel and the light was transmitted by optical fibres enabling to guide the light very close to the analysed solution. Microfluidic chip of high aspect ratio (~13), smooth and nearly vertical sidewalls was fabricated in PDMS using a SU-8 mould for patterning. The platform coupled to the gold nanoprobe assay enabled detection of Mycobacterium tuberculosis using 3 8l on DNA solution, i.e. 20 times less than in the previous state-of-the-art. Subsequently, the bio-microfluidic platform was optimized in terms of cost, electrical signal processing and sensitivity to colour variation, yielding 160% improvement of colorimetric AuNPs analysis. Planar microlenses were incorporated to converge light into the sample and then to the output fibre core increasing 6 times the signal-to-losses ratio. The optimized platform enabled detection of single nucleotide polymorphism related with obesity risk (FTO) using target DNA concentration below the limit of detection of the conventionally used microplate reader (i.e. 15 ng/μl) with 10 times lower solution volume (3 μl). The combination of the unique optical properties of gold nanoprobes with microfluidic platform resulted in sensitive and accurate sensor for single nucleotide polymorphism detection operating using small volumes of solutions and without the need for substrate functionalization or sophisticated instrumentation. Simultaneously, to enable on chip reagents mixing, a PDMS micromixer was developed and optimized for the highest efficiency, low pressure drop and short mixing length. The optimized device shows 80% of mixing efficiency at Re = 0.1 in 2.5 mm long mixer with the pressure drop of 6 Pa, satisfying requirements for the application in the microfluidic platform for DNA analysis.

Predictive analytical and numerical modeling for orthogonal cutting

In this thesis, a predictive analytical and numerical modeling approach for the orthogonal cutting process is proposed to calculate temperature distributions and subsequently, forces and stress distributions. The models proposed include a constitutive model for the material being cut based on the work of Weber, a model for the shear plane based on Merchants model, a model describing the contribution of friction based on Zorev’s approach, a model for the effect of wear on the tool based on the work of Waldorf, and a thermal model based on the works of Komanduri and Hou, with a fraction heat partition for a non-uniform distribution of the heat in the interfaces, but extended to encompass a set of contributions to the global temperature rise of chip, tool and work piece. The models proposed in this work, try to avoid from experimental based values or expressions, and simplifying assumptions or suppositions, as much as possible. On a thermo-physical point of view, the results were affected not only by the mechanical or cutting parameters chosen, but also by their coupling effects, instead of the simplifying way of modeling which is to contemplate only the direct effect of the variation of a parameter. The implementation of these models was performed using the MATLAB environment. Since it was possible to find in the literature all the parameters for AISI 1045 and AISI O2, these materials were used to run the simulations in order to avoid arbitrary assumption.

Digital microfluidics on paper

This thesis is one of the first reports of digital microfluidics on paper and the first in which the chip’s circuit was screen printed unto the paper. The use of the screen printing technique, being a low cost and fast method for electrodes deposition, makes the all chip processing much more aligned with the low cost choice of paper as a substrate. Functioning chips were developed that were capable of working at as low as 50 V, performing all the digital microfluidics operations: movement, dispensing, merging and splitting of the droplets. Silver ink electrodes were screen printed unto paper substrates, covered by Parylene-C (through vapor deposition) as dielectric and Teflon AF 1600 (through spin coating) as hydrophobic layer. The morphology of different paper substrates, silver inks (with different annealing conditions) and Parylene deposition conditions were studied by optical microscopy, AFM, SEM and 3D profilometry. Resolution tests for the printing process and electrical characterization of the silver electrodes were also made. As a showcase of the applications potential of these chips as a biosensing device, a colorimetric peroxidase detection test was successfully done on chip, using 200 nL to 350 nL droplets dispensed from 1 μL drops.

Non-gyrator type active inductors

Modern CMOS radio frequency (RF) Receivers have enabled efficient and increasing applications. The main requirement is to have system in a single chip, in order to minimize area and cost. For the purpose it is required the development of inductorless circuits for the key blocks of an RF receiver. Examples of this key blocks are RC oscillators, RF band pass filters, and Low Noise Amplifiers. The present dissertation presents an inductorless wideband MOSFET-only RF Non-Gyrator Type of Active Inductors with low area, low cost, and very low power, capable of covering the whole WMTS, and ISM, band and intended for biomedical applications. The proposed circuit is based on a floating capacitor connected between two controlled current sources. The first current source, which is controlled by the circuit input voltage, has two objectives: supply current to the capacitor (

The role of objects in creative collaborations

Objects matter when professionals collaborate to create new products. Chapter 1 explains the intention of this work, to apply theories on objects in the empirical context of fashion design. Chapter 2 addresses the question of how creative professionals learn about and use strategy tools to turn their artistic fame into a commercial success. For Chapter 3 I collected ethnographic data on the development of a seasonal collection from the idea to the presentation at Fashion Week. The result is a deep insight into the collaborative processes and material objects used when a stable team of designers works with several outside experts. Chapter 4 applies the knowledge of the role of objects in fashion design gained during the ethnography in the context of online co-creation and crowd sourced fashion items. The synthesis of the empirical studies allows me to present the conceptual leap in Chapter 5. In the theoretical essay I review the findings on the role of objects in collaborations in relation to practice theory, present the new concept of the comprehensive object and conclude by stating the possibilities for future research.

Appreciating the power of words and imaginative organizing: A triadic model of ethics practiced in everyday conversations

This thesis provides an alternative framework to analyze power and ethics practiced in everyday conversations, which constitute processes of organizing. Drawing upon narrative frameworks, the analyses of messages posted on an online message board demonstrate people’s imaginative capacity to create relevant stories, in respect of their precise grasp of factual understandings, contextual relevance and evaluative/moral appropriateness, by appropriating others’ words. Based on the empirical analyses, the thesis indicates that studies on power and ethics in organizations can be re-oriented towards appreciating irremediable power imbalances by offering alternative ways of member’s denoting experiences of power.

Development of an experimental setup for metal cutting research

Analytical, numerical and experimental models have been developed over time to try to characterize and understand the metal cutting process by chip removal. A true knowledge of the cutting process by chip removal is required by the increasing production, by the quality requirements of the product and by the reduced production time, in the industries in which it is employed. In this thesis an experimental setup is developed to evaluate the forces and the temperature distribution in the tool according to the orthogonal cutting model conditions, in order to evaluate its performance and its possible adoption in future works. The experimental setup is developed in a CNC lathe and uses an orthogonal cutting configuration, in which thin discs fixed onto a mandrel are cut by the cutting insert. In this experimental setup, the forces are measured by a piezoelectric dynamometer while temperatures are measured by thermocouples placed juxtaposed to the side face of the cutting insert. Three different solutions are implemented and evaluated for the thermocouples attachment in the cutting insert: thermocouples embedded in thermal paste, thermocouples embedded in copper plate and thermocouples brazed in the cutting insert. From the tests performed in the experimental setup it is concluded that the adopted forces measurement technique shows a good performance. Regarding to the adopted temperatures measurement techniques, only the thermocouples brazed in the cutting insert solution shows a good performance for temperature measurement. The remaining solutions show contact problems between the thermocouple and the side face of the cutting insert, especially when the vibration phenomenon intensifies during the cut. It is concluded that the experimental setup does not present a sufficiently robust and reliable performance, and that it can only be used in future work after making improvements in the assembly of the thermocouples.

Configurador de sistemas distribuídos de controlo especificados através de redes de Petri IOPT

O objetivo principal deste trabalho é desenvolver um protótipo de ferramenta que permita a geração de ficheiros de configuração de sistemas distribuídos de controlo em plataformas específicas permitindo a integração de um conjunto de componentes previamente definidos. Cada componente é caracterizado como um módulo, identificando-se o conjunto de sinais e eventos de entrada e saída, bem como o seu comportamento, normalmente especificado através de um modelo em redes de Petri IOPT – RdP-IOPT (Input-Output Place-Transitions). O formato PNML (Petri Net Markup Language) será utilizado para a representação de cada componente. Os componentes referidos poderão ser obtidos através de vários métodos, nomeadamente através de ferramentas em desenvolvimento, que se encontram disponíveis em http://gres.uninova.pt/IOPT-Tools/ e também através da sua edição no editor de IOPT, como resultado da partição de um modelo expresso em IOPT, utilizando o editor Snoopy-IOPT em conjugação com a ferramenta SPLIT. Serão considerados várias formas para interligação dos componentes, incluindo-se ligações diretas e wrappers assíncronos num contexto de sistemas Globalmente Assíncronos Localmente Síncronos - GALS bem como diferentes tipos de barramentos e ligações série, incluindo Network-On-Chip específicos. A descrição da interligação entre componentes é gerada automaticamente pela ferramenta desenvolvida, tendo em conta resultados de dissertações de mestrado anteriores. As plataformas especificas de suporte à implementação incluem FPGA’s da serie Xilinx Spartan3,3E e Xilinx Virtex, e várias placas de desenvolvimento.

Regulação epigenética de um gene de resposta a stress de frio em arroz (OsDREB1B)

O arroz (Oryza sativa L.) constitui a principal fonte de alimento para mais de metade da população mundial. O frio tem um grande impacto no desenvolvimento da planta de arroz com efeitos negativos ao nível da produtividade e economia mundial. Os mecanismos epigenéticos associados a alterações estruturais da cromatina estão envolvidos nos mecanismos de resposta das plantas a stresses abióticos. O foco do presente trabalho consistiu na compreensão da influência de fatores epigenéticos, nomeadamente de modificações de histonas, na regulação transcricional de um gene específico (OsDREB1B) envolvido na resposta de arroz ao stress de frio. O estudo da regulação epigenética deste gene envolveu a utilização de plantas de arroz com mutações em enzimas epigenéticas e drogas remodeladoras da cromatina. Neste trabalho mostramos que o gene OsDREB1B é induzido pelo stress de frio (4 °C) em todas os genótipos de arroz analisados, no entanto, a indução do gene em resposta ao stress de frio é maior em Nipponbare que em Dongjin. Nos genótipos que apresentam epimutações, nomeadamente o knockout de osdrm2 (DMT706) e de oshac704 (HAC704), o gene OsDREB1B é mais expresso que no respetivo genótipo selvagem (Dongjin), sendo que a indução da expressão em resposta ao frio foi maior em DMT706 do que em HAC704. A análise do padrão de marcas histónicas ao longo do promotor de OsDREB1B, através da imunoprecipitação da cromatina (ChIP) com anticorpos específicos, revelou que, em resposta ao frio (4ºC), Dongjin apresenta uma menor predominância da marca H3K9ac na região de -519 a -355 pares de bases (antes do ATG), enquanto que Nipponbare possui maior presença de H4K5ac na região de -280 a -121 pares de bases, sugerindo que a regulação epigenética de OsDREB1B seja influenciada pelo genótipo. No caso dos epi-mutantes (HAC704 e DMT706) registou-se um empobrecimento das marcas histónicas testadas sugerindo que a indução da expressão do OsDREB1B em reposta ao stress provocado pelo frio não depende necessariamente da presença de OsDRM2 nem de OsHAC704. Uma visão integrada da paisagem epigenética e do transcriptoma, perspetiva uma melhor compreensão dos mecanismos envolvidos na resposta da planta a condições ambientais adversas, abrindo novas linhas de trabalho no sentido de obter plantas mais tolerantes a fatores de stress.