Development of a rapid prototyping method for hard polymer microfluidic systems tested through iterative design of a PCR chamber chip

Tese de mestrado integrado, Engenharia Biomédica e Biofísica (Engenharia Clínica e Instrumentação Médica), Universidade de Lisboa, Faculdade de Ciências, 2014One of the challenges of working with polymer microfluidics is the lack of an established prototyping method which allows for easy translation to industrial production. By combining Hot Embossing and Computer Numerically Controlled Milling a microfluidic rapid prototyping method was established for Polycarbonate and Cyclic Olefin Polymer. This method was then tested and optimized through an iterative design process of a microfluidic Polymerase-Chain Reaction chamber. The fabrication method proved to be suitable for microfluidic prototyping, allowing for rapid design changes and fabrication of good quality copies in a simple and straightforward fashion.Uma das dificuldades em trabalhar com microfluídica em polímeros é a falta da existência de um método de prototipagem que permita uma passagem simples para um ambiente de produção industrial. Neste trabalho foi desenvolvido um método de prototipagem rápida para microfluídica em Policarbonato e Cyclic Olefin Polymer utilizando uma Fresadora de Controlo Numérico Computorizado e Hot Embossing. Este método foi testado e optimizado através de um processo de design iterativo de uma câmara microfluídica de Reacção em Cadeia da Polimerase em Policarbonato. O método desenvolvido provou ser adequado para prototipagem microfluídica, permitindo alterações rápidas ao desenho e fabricação de várias cópias com boa qualidade de cada desenho

Design and Optimization Methods for Pin-Limited and Cyberphysical Digital Microfluidic Biochips

<p>Microfluidic biochips have now come of age, with applications to biomolecular recognition for high-throughput DNA sequencing, immunoassays, and point-of-care clinical diagnostics. In particular, digital microfluidic biochips, which use electrowetting-on-dielectric to manipulate discrete droplets (or "packets of biochemical payload") of picoliter volumes under clock control, are especially promising. The potential applications of biochips include real-time analysis for biochemical reagents, clinical diagnostics, flash chemistry, and on-chip DNA sequencing. The ease of reconfigurability and software-based control in digital microfluidics has motivated research on various aspects of automated chip design and optimization.</p><p>This thesis research is focused on facilitating advances in on-chip bioassays, enhancing the automated use of digital microfluidic biochips, and developing an "intelligent" microfluidic system that has the capability of making on-line re-synthesis while a bioassay is being executed. This thesis includes the concept of a "cyberphysical microfluidic biochip" based on the digital microfluidics hardware platform and on-chip sensing technique. In such a biochip, the control software, on-chip sensing, and the microfluidic operations are tightly coupled. The status of the droplets is dynamically monitored by on-chip sensors. If an error is detected, the control software performs dynamic re-synthesis procedure and error recovery.</p><p>In order to minimize the size and cost of the system, a hardware-assisted error-recovery method, which relies on an error dictionary for rapid error recovery, is also presented. The error-recovery procedure is controlled by a finite-state-machine implemented on a field-programmable gate array (FPGA) instead of a software running on a separate computer. Each state of the FSM represents a possible error that may occur on the biochip; for each of these errors, the corresponding sequence of error-recovery signals is stored inside the memory of the FPGA before the bioassay is conducted. When an error occurs, the FSM transitions from one state to another, and the corresponding control signals are updated. Therefore, by using inexpensive FPGA, a portable cyberphysical system can be implemented.</p><p>In addition to errors in fluid-handling operations, bioassay outcomes can also be erroneous due the uncertainty in the completion time for fluidic operations. Due to the inherent randomness of biochemical reactions, the time required to complete each step of the bioassay is a random variable. To address this issue, a new "operation-interdependence-aware" synthesis algorithm is proposed in this thesis. The start and stop time of each operation are dynamically determined based on feedback from the on-chip sensors. Unlike previous synthesis algorithms that execute bioassays based on pre-determined start and end times of each operation, the proposed method facilitates "self-adaptive" bioassays on cyberphysical microfluidic biochips.</p><p>Another design problem addressed in this thesis is the development of a layout-design algorithm that can minimize the interference between devices on a biochip. A probabilistic model for the polymerase chain reaction (PCR) has been developed; based on the model, the control software can make on-line decisions regarding the number of thermal cycles that must be performed during PCR. Therefore, PCR can be controlled more precisely using cyberphysical integration.</p><p>To reduce the fabrication cost of biochips, yet maintain application flexibility, the concept of a "general-purpose pin-limited biochip" is proposed. Using a graph model for pin-assignment, we develop the theoretical basis and a heuristic algorithm to generate optimized pin-assignment configurations. The associated scheduling algorithm for on-chip biochemistry synthesis has also been developed. Based on the theoretical framework, a complete design flow for pin-limited cyberphysical microfluidic biochips is presented.</p><p>In summary, this thesis research has led to an algorithmic infrastructure and optimization tools for cyberphysical system design and technology demonstrations. The results of this thesis research are expected to enable the hardware/software co-design of a new class of digital microfluidic biochips with tight coupling between microfluidics, sensors, and control software.</p>Dissertatio

Electrowetting and Droplet Transport in Digital Microfluidic Chips for Mixing Applications

Microfluidics- based biochips have varies applications like high throughput analyses, DNA sequencing, automated drug discovery, real time bio-molecular recognition, parallel immunoassays, single cell studies and protein RNA interaction and environmental toxicity monitoring. Based upon the fluid flow pattern microfluidic based devices can be categorized in two types. They are continuous flow microfluidic device and discrete flow microfluidic device or digital microfluidic. The continuous flow uses permanently etched microchannels, micro-pumps, and micro-valves for the application such as mixing, splitting, and transportation. In contrast to this discrete type flow uses array of electrodes, voltage to controlled droplet independently for the same application

Development of electrochemical platforms for DNA sensing

[eng] The present doctoral thesis is framed in the research and development (R & D) project between a private biotechnology company of molecular diagnostics Genomica SAU, the Institute for Bioengineering of Catalonia (IBEC), the University of Barcelona, and the Microfluidics ChipShop Company. The main objective of the project is making, implementation and marketing of a diagnostic device for early detection of DNA sequences involved with cancer. The multi device, or lab-on-chip (LOC), consists of a central automation unit (CAU), a system in miniature of DNA amplification or chain reaction polymerase (mini-PCR), and a biosensing platform (DNA chip) that consisting of a matrix or electrochemical array. The three elements are integrated by a microfluidic system in sandwich format cartridge. For this purpose, the aim of this thesis was the creation, characterization and optimization of the biochemical recognition platform between two single strands of DNA of dissimilar lengths but with some complementary sequences for the subsequent electrochemical detection of a hybridization event between them. Then, the integration into the cartridge of above platform was done. For the creation of this platform, we chose to use a self-assembled monolayer (SAM) of thiols as biorecognition interface of the 14 DNA sequences that are part of the project. During optimization of the interface chips individual gold and various molecules were used being chosen the molecule with two arms disulfide of polyethylene glycol (PEG) and a malaimida group at the end of one of them. This linker (or MalPEG linker) reacts with the gold surface due to the dative interaction between the sulfur atoms of the disulfide and the gold atoms from the surface of the chips. At the same time, the malaimida group reacts with the thiol group of the capture probes, joining. The PEG groups function as anti-adhesion molecules. Surface plasmon resonance (SPR) and cyclic voltammetry (CV) were techniques used to characterize the substrate and the hybridization event. For the manufacture of the cartridge, this was divided into two main blocks, the biosensing or electrochemical block and PCR block. The electrochemical block is composed of 4 layers, one of 64 working electrodes and gold paths for contact with the potentiostat, another layer that defines the area of the sensors must be functionalized gold and isolating the gold surface of the tracks. The third layer is a double-sided adhesive that has a hexagonal hole working as hybridization chamber, and the last layer is a screen printing layer with the reference electrode (RE) and counter electrodes. The above layers form an electrochemical cell wherein the hybridization will occurs. Regarding the PCR block, this is a system of two layers with a type microfluidic channel kind loop and its function is to contain the solutions during the process of DNA amplification by the mini-PCR. During the integration of the optimized SAM into an electrochemical cartridge a manual and automated ways were used to immobilize the capture probes. Several tests were performed in order to obtain the best conditions and ratios between the molecules to maximize the hybridization signal during the electrochemical detection.[spa] El presente trabajo de tesis está enmarcado en un proyecto de investigación y desarrollo (I+D) entre la empresa privada Genomica S.A.U., el Instituto de Bioingeniería de Cataluña (IBEC), la Universidad de Barcelona y la empresa alemana ChipShop Microfluidics. El objetivo principal es el desarrollo, puesta a punto y comercialización de un dispositivo electroquímico de diagnóstico médico para etapas tempranas de cáncer. El objetivo de la tesis es la creación, optimización y posterior integración de una interfaz de biosensado de ADN en el dispositivo de diagnóstico, siendo pieza fundamental en el desarrollo de éste. La interfaz escogida fue una monocapa autoensamblada (SAM) que hace las veces de biosensor y que es capaz de anclar secuencias de ADN como sondas de captura y así poder detectar, selectivamente, las secuencias objetivo complementarias. El dispositivo también cuenta con un sistema microfluídico y un sistema de amplificación de ADN de reacción en cadena de la polimerasa en miniatura. La SAM esta inmovilizada en un array electroquímico que consta de 64 electrodos de trabajo que funcionan como elemento transductor de la señal electroquímica redox de los eventos de hibridación que ocurren sobre ellos. La funcionalización y puesta a punto del dispositivo se llevó a cabo inmovilizando múltiples sondas de captura después de una optimización de las concentraciones entre las diferentes partes constituyentes de la monocapa. Técnicas ópticas y electroquímicas fueron utilizadas para la caracterización de cada etapa y técnicas de fotolitografiado y de impresión por pantalla fueron utilizadas para la fabricación de los componentes del dispositivo. Finalmente, y después de algunos cambios surgidos durante el desarrollo del dispositivo, se llega a un diseño final y a las pruebas con muestras reales, proceso que aún está en etapa experimental

Microfabrication and Applications of Opto-Microfluidic Sensors

A review of research activities on opto-microfluidic sensors carried out by the research groups in Canada is presented. After a brief introduction of this exciting research field, detailed discussion is focused on different techniques for the fabrication of opto-microfluidic sensors, and various applications of these devices for bioanalysis, chemical detection, and optical measurement. Our current research on femtosecond laser microfabrication of optofluidic devices is introduced and some experimental results are elaborated. The research on opto-microfluidics provides highly sensitive opto-microfluidic sensors for practical applications with significant advantages of portability, efficiency, sensitivity, versatility, and low cost

Lab-on-a-chip nucleic-acid analysis towards point-of-care applications

Recent infectious disease outbreaks, such as Ebola in 2013, highlight the need for fast and accurate diagnostic tools to combat the global spread of the disease. Detection and identification of the disease-causing viruses and bacteria at the genetic level is required for accurate diagnosis of the disease. Nucleic acid analysis systems have shown promise in identifying diseases such as HIV, anthrax, and Ebola in the past. Conventional nucleic acid analysis systems are still time consuming, and are not suitable for point-ofcare applications. Miniaturized nucleic acid systems has shown great promise for rapid analysis, but they have not been commercialized due to several factors such as footprint, complexity, portability, and power consumption. This dissertation presents the development of technologies and methods for a labon-a-chip nucleic acid analysis towards point-of-care applications. An oscillatory-flow PCR methodology in a thermal gradient is developed which provides real-time analysis of nucleic-acid samples. Oscillating flow PCR was performed in the microfluidic device under thermal gradient in 40 minutes. Reverse transcription PCR (RT-PCR) was achieved in the system without an additional heating element for incubation to perform reverse transcription step. A novel method is developed for the simultaneous pattering and bonding of all-glass microfluidic devices in a microwave oven. Glass microfluidic devices were fabricated in less than 4 minutes. Towards an integrated system for the detection of amplified products, a thermal sensing method is studied for the optimization of the sensor output. Calorimetric sensing method is characterized to identify design considerations and optimal parameters such as placement of the sensor, steady state response, and flow velocity for improved performance. An understanding of these developed technologies and methods will facilitate the development of lab-on-a-chip systems for point-of-care analysis