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

Miniaturised nucleic acid analysis systems

The design and implementation of miniaturised systems for analysis of nucleic acids from various biological samples has undergone extensive development. Several advances have been made particularly with the integration of nucleic acid amplification and detection, where amplification is most often polymerase chain reaction (PCR). Sample preparation remains a major obstacle for achieving a quantitative analysis employing full miniaturised integration. Miniaturised devices for nucleic acid sample preparation, amplification and detection have to be further developed in order to achieve a fully integrated system, which ultimately can perform single cells genomic analysis with sample-in-answer-out ability. In this thesis, three miniaturised systems have been presented, which can be used for purification and preconcentration of DNA, pre-amplification and long-term storage of DNA, and amplification with real-time detection of DNA, respectively. The first miniaturised system applies isotachophoresis for pretreatment of DNA, where the DNA sample can be purified and concentrated using a discontinuous electrolyte system. Both qualitative and quantitative information can be acquired simultaneously. The second miniaturised system employs simple isothermal multiple displacement amplification, (MDA) for whole genome amplification (WGA) of human genomic DNA. The miniaturised WGA process showed a high efficiency of 95.8%, and the fidelity of the amplified products is extremely high as suggested by single-nucleotide polymorphisms analysis. For the last system, we developed a bidirectional shunting PCR microdevice equipped with real-time fluorescence detection, which allows higher flexibility and fast thermocycling by combining both advantages of stationary PCR and continuous-flow PCR. Real-time monitoring of RNase P PCR amplification from lower concentration human genomic DNA down to ~24 copy numbers or 12 cells was achieved. The three systems described in this thesis can be readily adapted to current reported miniaturised platforms. Such a fully integrated device capable of quantitative nucleic acid analysis remains an enigma, and with further development will represent significant importance for the development of point-of-care device.Das Design und die Implementierung miniaturisierter Systeme für die Analyse von Nukleinsäuren aus verschiedenen biologischen Proben haben eine beträchtliche Entwicklung erlebt. Fortschritte wurden insbesondere bei der Integration der Nukleinsäure-Vervielfältigung und -Detektion gemacht, wobei die Vervielfältigung meistens auf der Polymerase-Kettenreaktion (Polymerase Chain Reaction, PCR) beruht. Die Probenvorbereitung bleibt ein Haupthemmnis bei dem Versuch, eine quantitative Analyse mit vollständig miniaturisierten Systemen zu verwirklichen. Miniaturisierte Geräte für die Probenvorbereitung, Vervielfältigung und Detektion von Nukleinsäuren müssen weiter entwickelt werden, um ein vollständig integriertes System zu verwirklichen, das letztendlich in der Lage ist, die Genomanalyse einzelner Zellen mit "sample-in-answer-out"-Fähigkeit durchzuführen. In dieser Arbeit werden drei miniaturisierte Systeme präsentiert, die jeweils für die DNA-Aufreinigung und -Vorkonzentrierung, deren Vor-Vervielfältigung und Langzeit-Speicherung bzw. der Vervielfältigung mit Echtzeitdetektion von DNA verwendet werden können. Das erste miniaturisierte System nutzt die Isotachophorese zur Vorbehandlung der DNA, bei der die DNA-Probe in einem diskontinuierlichen Elektrolytsystem gereinigt und aufkonzentriert werden kann. Dabei können sowohl qualitative als auch quantitative Informationen simultan aufgenommen werden. Das zweite miniaturisierte System verwendet die simple Methode der isothermischen Multiplen Displacement Amplification (MDA) für die Vervielfältigung des gesamten Genoms (Whole Genome Amplification, WGA) der humanen, genomischen DNA. Der miniaturisierte WGA-Prozess zeigte eine hohe Effizienz von 95,8% und die Wiedergabetreue des vervielfachten Produkts ist extrem hoch, was durch die Ergebnisse einer Single Nucleotide Polymorphism (SNP) Analyse angedeutet wurde. Für des letzte System entwickelten wir ein kleines bidirektionales shunting-PCR-Instrument, in dem die injizierte DNA durch eine temperierte Zone mehrfach hin und her verschoben wird. Ausgestattet mit einem Fluoreszens Detektor, erreicht man eine höhere Flexibilität und schelle Temperaturzyklen, und kombiniert so die Vorteile der stationären PCR und der Durchfluss-PCR. Eine Echtzeitdetektion der RNase-P-PCR-Vervielfältigung von niedrig konzentrierter, humaner genomischer DNA mit einem Minimum von ~24 Kopien oder 12 Zellen wurde erreicht. Die drei in dieser Arbeit beschriebenen Systeme können direkt an aktuelle miniaturisierte Aufbauten angepasst werden. Ein solches vollständig integriertes, zur quantitativen Nukleinsäure-Analyse fähiges Gerät bleibt ein Mysterium, und zusammen mit weiteren Verbesserungen wäre es von großer Bedeutung für die Entwicklung von point-of-care Geräten

Microfluidic PCR with plasmonic imaging for rapid multiplexed characterization of DNA from microbial pathogens

Bloodstream infections (BSIs) are a critical concern in modern medicine due to their continued prevalence in modern hospitals, along with high costs and attributable mortality, particularly among those who are immunocompromised. The current gold standard for detection and characterization of causative pathogens involves cell culture, which can take 24-48 hours to complete, increasing time to adequate treatment and thus mortality. The rise of antimicrobial resistance in hospital acquired infections has reduced the effectiveness of broad spectrum antimicrobial treatments, resulting in a clear need for a rapid, sensitive technique for characterization of resistance markers in microbial pathogens without cell culture. Here we present the development of a microfluidic platform for polymerase chain reaction (PCR) mediated amplification of microbial gene targets in a continuous flow system for potential coupling with sample preparation systems to reduce time to diagnosis from days to within two hours. This culminated in a thermoelectric cooler mediated fluidic thermocycler with a recirculating assay region for real-time hybridization measurements to minimize assay time. We subsequently demonstrated development of a low-cost optical system for localized surface plasmon resonance imaging using a digital micromirror device and tuned nanoprism monolayers for DNA hybridization with a spectral resolution of 2nm. This LSPR imaging system was integrated in-flow into the microfluidic thermocycler, enabling detection of input E. coli DNA samples at a minimum concentration of 5fg/ [microliter]. We further demonstrated multiplex detection of target markers, indicating potential for assaying target panels for characterization of pathogens. Overall, the studies in this dissertation demonstrate a microfluidic PCR system with built-in sensitive LSPR detection of DNA hybridization. It should serve as a starting point for exploration of and expansion with fluidic sample preparation with a focus on rapid characterization of pathogens.Biomedical Engineerin

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

Development of a CMOS-compatible PCR chip : comparison of design and system strategies

Altres ajuts: this work was partly funded by the Consejo Superior de Investigaciones Científicas (CSIC), by grant TIC97-0569 from the Comision Interministerial de Ciencia y Tecnología (CICYT).In the last decade research in chips for DNA amplification through the polymerase chain reaction (PCR) has been relatively abundant, but has taken very diverse approaches, leaving little common ground for a straightforward comparison of results. Here we report the development of a line of PCR chips that is fully compatible with complementary-metal-oxide-semiconductor (CMOS) technology and its revealing use as a general platform to test and compare a wide range of experimental parameters involved in PCR-chip design and operation. Peltier-heated and polysilicon thin-film driven PCR chips have been produced and directly compared in terms of efficiency, speed and power consumption, showing that thin-film systems run faster and more efficiently than Peltier-based ones, but yield inferior PCR products. Serpentine-like chamber designs have also been compared with standard rectangular designs and with the here reported rhomboidal chamber shape, showing that serpentine-like chambers do not have detrimental effects in PCR efficiency when using non-flow-through schemes, and that chamber design has a strong impact on sample insertion/extraction yields. With an accurate temperature control (±0.2 °C) we have optimized reaction kinetics to yield sound PCR amplifications of 25 µl mixtures in 20 min and with 24.4 s cycle times, confirming that a titrated amount of bovine albumin serum (BSA, 2.5 µg µl−1) is essential to counteract polymerase adsorption at chip walls. The reported use of a CMOS-compatible technological process paves the way for an easy adaption to foundry requirements and for a scalable integration of electro-optic detection and control circuitr

Flexible sensors technology for Point-Of-Care diagnostics with integrated micro fluidics on paper

Nowadays the hospitals and the medical centres face a huge challenge finding solutions to improve the efficiency of medical diagnosis. The scope of this project was to develop a “Point-of-Care Diagnostic” (POCD) device, that can give a better alternative for genetic analysis, instead of the usual methods of PCR (polymerase chain reaction). This device is composed by three layers. The first layer which works as a transporter and filter was built on paper. The second layer is the substitute of the regular thermocycling phase in the PCR technique and the third layer incorporates an interdigital capacitor that works as a DNA (deoxyribonucleic acid) sensor with high sensitivity to detect DNA hybridisation. These last two layers were made in kapton film. The devices were produced with microfabrication methods using inkjet printing, lithographic and deposition processes. The device’s characterisation was based on impedance spectroscopy methods. With the purpose of testing the device, the capacitor was functionalised with the YWHAZ gene. However, this process can be performed with any other gene. Due to its characteristics, the device under study was designed to run RT-qPCR (Real time quantitative polymerase chain reaction) and presents itself as an effective way to substitute the traditional PCR techniques. Even more, as the transport of samples to a laboratory and the recruitment of specialised personnel are not necessary, costs and response time are reduced

Design and characterization of convective thermal cyclers for high-speed DNA analysis

An ideal polymerase chain reaction (PCR) system should be capable of rapidly amplifying a wide range of targets in both single and multiplex formats. Unfortunately, the timescales and complexities involved in many existing technologies impose significant limitations on achievable throughput. Buoyancy driven PCR is emerging as a simplified version of thermally driven bio-analysis systems. Here, we demonstrate a simplified convectively driven thermocycler capable of performing single and multiplex PCR for amplicons ranging from 191 bp to 1.3 kb within 10 to 50 minutes using 10 to 25 µL reaction volumes. By positioning two independent thermoelectric heating elements along the perimeter of a flow loop reactor constructed using ordinary plastic tubing, a buoyancy-driven flow is established that continuously circulates reagents through temperature zones associated with the PCR process. Unlike conventional benchtop thermocyclers, this arrangement allows reactions to be performed without the need for dynamic temperature control of inactive hardware components while maintaining comparable product yields and requiring no modifications to standard PCR protocols. We also provide a general correlation that can be applied to design reactor geometries satisfying virtually any combination of reagent volume and cycling time. In addition to offering an attractive combination of cost and performance, this system is readily adaptable for portable battery powered operation, making it feasible to perform PCRbased assays in a broader array of settings