Dissertations / Theses on the topic 'Lab on a chip'

The development of a novel method of droplet levitation to be employed in lab-on-a-chip (LOC) applications relies upon the mechanism of thermocapillary convection (due to the temperature dependence of surface tension) to drive a layer of lubricating gas between droplet and substrate. The fact that most droplets of interest in LOC applications are aqueous in nature, coupled with the fact that success in effecting thermocapillary transport in aqueous solutions has been limited, has led to the development of a technique for the controlled encapsulation of water droplets within a shell of inert silicone oil. These droplets can then be transported, virtually frictionlessly, resulting in ease of transport due to the lack of friction as well as improvements in sample cross-contamination prevention for multiple-use chips. Previous reports suggest that levitation of spherical O(nL)-volume droplets requires squeezing to increase the apparent contact area over which the pressure in the lubricating layer can act allowing sufficient opposition to gravity. This research explores thermocapillary levitation and translation of O(nL)-volume single-phase oil droplets; generation, capture, levitation, and translation of O(nL)-volume oil-encapsulated water droplets to demonstrate the benefits and applicability to LOC operations.

The main target of the LAB on CHIP phD project, funded by Fondazione Cariparo, was to develop a device to allow the handy production of CHO cell clones in order to built recombinant proteins for therapeutic targets. In particular, a major aim is to reduce time and costs associated with clones manufacturing. Cultivated mammalian cells have become the dominant system for the production of recombinant proteins for clinical applications, because of their capacity for proper protein folding, assembly and post-translational modification. The quality and efficacy of a protein can be superior when expressed in mammalian cells versus other hosts such as bacteria, plants and yeast. Today more than 60 % of all recombinant protein pharmaceuticals are produced in mammalian cells. Expression vectors for recombinant cell line generation generally use a strong viral or cellular promoter/enhancer to drive the expression of the recombinant gene. But non-viral gene transfer remains the preferred approach to generate stable cell lines for manufacturing purposes: calcium phosphate transfection, electroporation, lipofection. Transfection is a complex process, and in order to be successful all steps involved must work efficiently. The device developed is based on the physical phenomenon called electroporation, that is the formation of temporary pores in the plasmatic membrane upon application of electric fields. As the biological world is intrinsically variable, common approaches for improving electrotransfection rely on time consuming empirical attempts. It is therefore important to develop new methods enabling a fine control of all critical parameters involved to identify causes of failure and to improve efficiency. On-chip electroporation through capacitive currents can be such a method. To directly assess the formation of pores in the cell membrane, we performed patch-clamp experiments during on-chip electroporation. Thus, most promising protocols were selected and assessed for their electrotransfection efficiency. Moreover, patch clamp experiments allowed to study the dynamics of pores formation and resealing. Regarding the develop of the device, the biocompatibility of titanium dioxide, selected as dielectric material, was tested. The inertness against cellular environment and the state of cell culture were considered. Cell cultures showed healthy state and normal development, good adhesion and normal replication time. No chemical reactions that can damage the culture were observed. The chemical inertness was considered in the reverse direction too. Metabolic products of cell culture did not lead to chemical corrosion of the dielectric surface. So, the patch-clamp on-chip electroporation recordings allowed to select the promising protocol that was tested on CHO cultures. The prototype proposed demonstrated electrotransfection through capacitive coupling between cell and chip. The electroporation efficiency obtained is around 30%. Moreover, the selectivity of the device was demonstrated, and its applicability both in electrotransfection and electroporation for staining application. Collateral results were obtained concerning the formation of pore on attached and free membrane and the possibility of study pore dynamics
Scopo principale del progetto di Dottorato "LAB on CHIP" finanziato dalla Fondazione Cariparo è stato lo sviluppo di un dispositivo che agevoli la creazione di cloni di cellule CHO per la produzione di proteine a scopo terapeutico. In particolare il fine ultimo è quello di ridurne tempi e costi associati alla produzione. Le cellule di mammifero in coltura sono ormai il sistema più diffuso per la produzione di proteine per applicazioni cliniche. La qualità e l'efficacia di una proteina possono essere superiore se essa è espressa in cellule di mammifero rispetto ad altri organismi, quali batteri, piante e lieviti. Ad oggi più del 60 % di tutte le proteine ricombinanti per applicazioni farmaceutiche è prodotto in cellule di mammifero. Vettori di espressione per la creazione di linee cellulari stabili da DNA ricombinante utilizzano vettori virali per indurre l'espressione del gene. Ma la transfezione senza l'ausilio di virus rimane l' approccio prediletto per la generazione di linee stabili per questi scopi. La transfezione è un processo complesso e, affinchè avvenga con successo, tutti i sottoprocessi coinvolti devono svolgersi efficientemente. Il dispositivo proposto si basa sul fenomeno fisico chiamato elettroporazione, che non è altro che la formazione di pori temporanei nella membrana plasmatica a seguito dell'applicazione di opportuni campi elettrici. I comuni approcci utilizzati per migliorare la transfezione tramite elettroporazione richiedono tempi lunghi e possono essere inefficaci. E' importante poter sviluppare metodi nuovi che permettano un controllo di tutti i parametri critici coinvolti in modo da poterne identificare le cause in caso di fallimento e dunque migliorare l'efficienza. L'elettroporazione su chip utilizzzando correnti capacitive può essere un valido approccio. Per poter rilevare la formazione di pori, sono stati fatti esperimenti di patch-clamp su chip durante l'elettroporazione. In tal modo sono stati selezionati i protocolli più promettenti. Per quanto riguarda lo sviluppo del dispositivo, ne è stata verificata la biocompatibilità. Si è valutato lo stato delle colture cellulari che hanno mostrato normali sviluppo, adesione e tempo di replicazione. Non sono state rilevate reazioni chimiche tra il mezzo di coltura e il diossido di titanio. Non si sono inoltre rilevati problemi di corrosione o danneggiamento dell'ossido a causa di prodotti metabolici della cellula. Gli esperimenti di patch-clamp hanno permesso di selezionare un protocollo che è stato poi testato sulle cellule in coltura. Il prototipo sviluppato ha dimostrato l'elettroporazione di cellule CHO in coltura, ottenendo un'efficienza media del 30 %. E' stata inoltre dimostrata la selettività di tale dispositivo e la sua applicabilità sia per la transfezione che per l'introduzione nella cellula di marcatori. Risultati "collaterali" ottenuti riguardano la dimostrazione della formazione di pori temporanei sia sulla membrana adesa che su quella libera e la possibilità di studiare la dinamica dei pori

LTCC -pakkaus Lab-on-a-chip -sovellukseen. Tiivistelmä. Tässä työssä suunniteltiin, valmistettiin ja testattiin uusi pakkaustekniikka ”Lab-on-a-chip” (LOC) -sovellukseen. Pakkaus tehtiin pii-mikrosirulle, jolla voidaan mitata solujen kiinnittymistä sirun pintaan solujen elinkelpoisuuden indikaattorina. Luotettavuustestaukset tehtiin daisy-chain -resistanssimittauksilla solunkasvatusolosuhteissa. Lisäksi työssä selvitettiin LTCC- ja ”Lab-on-a-chip” -teknologioiden perusteet teoreettiselta pohjalta. Mikrosirun pakkauksessa käytettiin joustavaa LTCC-teknologiaa. Sähköisiin kontakteihin ja niiden suojauksiin käytettiin sekä johtavia että eristäviä epoksi-liimoja. LOC-sovelluksiin on tärkeää kehittää uusia pakkausmenetelmiä jotta näiden laitteiden kaikki ominaisuudet saadaan toimimaan luotettavasti. Pakkaus testattiin samoissa olosuhteissa missä sitä tullaan käyttämään ja pakkaus kesti kaikki nämä haasteet. Lisäksi esitetty valmistusprosessi on sellainen, että sitä voidaan käyttää myös muihin ”Lab-on-a-chip” -sovelluksiin.Abstract. This work presents design, manufacturing and testing of new packaging method for Lab-on-a-chip (LOC) application. Packaging was made for silicon microchip which can measure cell adhesion on chips surface as indication of cell viability. Reliability testing was done with daisy-chain resistance measurement in real conditions. Moreover basic theory of LTCC and Lab-on-a-chip technology is presented. Resilient LTCC technology was used for packaging material and conductive/insulating epoxies were applied for electrical contacts and barriers against the environment. It is fundamentally important to develop new packaging methods for LOC applications, so all the properties can be utilized reliably. Packaging was tested under the cell growth conditions and the package showed to withstand all these challenges. Moreover the presented packaging method is possible to use also in other Lab-on-a-chip applications.

Les fonctions magnétiques sont aujourd'hui omniprésentes dans les systèmes Lab-on-Chip. Une découverte surprenante est que tandis que la recherche Lab-on-Chip se concentre sur la miniaturisation, les fonctions magnétiques sur puce sont généralement assurées par des aimants centimétriques. Comparés à ces aimants centimétriques, les champs générés par les micro-aimants bénéficient de lois d'échelle conduisant à des gradients de champ considérablement amplifiés et donc à des forces magnétiques proportionnellement accrues. Le but de cette thèse était de démontrer le potentiel des Lab-on-Chips à base de micro-aimants. Les micro-aimants haute performance ont été intégrés avec succès dans les matériaux Lab-on-Chip les plus pertinents, y compris le polymère, le silicium et le papier. Nous avons étudié des fonctions sur puce basées sur l'interaction de structures mécaniques et de micro-aimants actionnés par des gradients magnétiques, des forces et des couples. Enfin, nous avons simulé, fabriqué et testé une variété de nouvelles puces couvrant un large champ d'applications telles que les études cellulaires-mécaniques, la magnétophorèse, la manipulation de fluides sur puce et le diagnostic auprès du patient. Nous concluons que les micro-aimants intégrés présentent un grand potentiel pour les applications de laboratoire sur puce et devraient être plus largement exploités
Magnetic functions are nowadays ubiquitous in Lab-on-Chip systems. A surprising finding is that while Lab-on-Chip research focalizes on miniaturization, on-chip magnetic functions are usually driven by centimetric magnets. Compared to those centimetric magnets, fields generated by micro-magnets benefit from scaling laws leading to dramatically increased field gradients and thus proportionally improved magnetic forces. The aim of this thesis was to demonstrate the potential of micro-magnet based Lab-on-Chips. High-performance micro-magnets were successfully integrated in the most relevant Lab-on-Chip materials including polymer, silicon and paper. We studied on-chip functions based on the interaction of mechanic structures and micro-magnets actuated by magnetic gradients, forces and torque. Finally, we simulated, fabricated and tested a variety of new chips covering a large field of applications such as cell-mechanics studies, magnetophoresis, on-chip fluid handling and Point-of-Care diagnostics. We conclude that integrated micro-magnets show great potential for lab-on-chip applications and should be more widely exploited

Les travaux de cette thèse s’inscrivent dans un projet de développement d’un Lab-On-Chip destiné à la biodétection. Les plateformes conçues sont basées sur des circuits optiques intégrés sur niobate de lithium. La particularité de ces circuits est qu’ils intègrent le phénomène d’interférence à la fonction de guidage des ondes lumineuses.La fonction d’interférométrie est assurée grâce à une cavité Fabry-Pérot intégrée à un guide d’onde rectiligne et à une structure Mach-Zehnder. Lorsque la surface des supports de ces circuits est bio-fonctionnalisée, ces microsystèmes deviennent sensibles à des molécules cibles. Cette sensibilité se traduit par une variation de l’indice effectifde l’onde en propagation par couplage évanescent modifiant ainsi les conditions de résonance du résonateur Fabry-Pérot. Le vrai challenge de ce travail réside essentiellement dans la bio-fonctionnalisation du niobate de lithium.A notre connaissance, ce matériau favori en optique guidée grâce à ses propriétés physiques exceptionnelles n’a été que rarement sujet à des modifications chimiques de surface. L’implantation réussie de groupements fonctionnels amines à la surface de ce matériau a permis de générer un lien covalent entre ce support et les groupements fonctionnels des molécules sondes. En raison de la grande affinité entre l’avidine et la biotine, ce couple a servi de modèle pour la mise au point de ces bio-capteurs. Un suivi en temps réel des interactions à la surface était rendu possible par une expérimentation sur l’un des bio-capteurs
The work of this thesis is part of a project of a Lab-On-Chip development intended for biosensing. The de-signed platforms are based on integrated optical circuits on lithium niobate. The peculiarity of these circuits isthat they incorporate the phenomenon of interference with the function of guiding light waves. The interferometricfunction is provided by a Fabry-Perot cavity embedded in a straight waveguide and a Mach-Zehnder structure.When the surface of these circuits substrates is biofunctionalized, these microsystems become sensitive to targetmolecules. This sensitivity results in a variation of the effective index of the propagation wave by evanescent cou-pling and modifying the resonance conditions of the Fabry-Perot resonator. The real challenge of this work liesin the biofunctionalization of lithium niobate. To our knowledge, this guided optics favorite material thanks toits exceptional physical properties has been hitherto rarely subject to chemical surface modifications. Successfulimplementation of amino functions on the surface of this material has generating a covalent bond between thissubstrate and the functional groups of the probe molecules. Due to the high affinity between avidin and biotin, thiscouple served as a model for the development of biosensors. A real-time monitoring of surface interactions wasmade possible by experimentation on one of biosensors

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Esta tese apresenta um novo conceito de dispositivo microfluídico flexível, utilizando a borracha natural como material alternativo, de baixo custo, flexível e elástico, para a preparação de plataformas lab-on-a-chip. A preparação do dispositivo é realizada com a dispersão do látex sobre o molde. Este é construído em placas de acrílico (PMMA - Poli-metilmetacrilato) contendo a configuração do dispositivo. O látex deve recobrir toda a estrutura, utilizando o método casting. Posteriormente, o molde contendo látex é submetido a tratamento térmico para a formação das membranas de borracha natural. As membranas retiradas do molde replicam a configuração do dispositivo em sua superfície. Filmes de policloreto de vinil (PVC) são utilizados como recobrimento interno a fim de evitar a absorção de água e a liberação de composição proteica das membranas de borracha natural. Os dispositivos flexíveis de borracha natural foram implementados como sensores óptico e eletroquímicos, utilizando fibras de carbono flexíveis como eletrodos internos. Torna-se importante ressaltar que as membranas de borracha natural são transparentes (quando consideradas a região vísivel do espectro eletromagnético), bem como biocompatíveis, possibilitando a combinação de propriedades mecânicas, ópticas e biológicas num único dispositivo. Por meio das análises eletroquímicas, demonstrou-se que possuem boa estabilidade e resistência quando submetidos a testes prolongados, mantendo a característica da curva de potencial, bem como o curto intervalo de tempo necessário para atingir a corrente estacionária durante os processos eletroquímicos. Em segunda instância, utilizaram-se os microdispositivos de borracha natural como microreatores para a síntese de nanopartículas de magnetita (Fe3O4) decoradas com nanopartículas de ouro (FeO4-AuNPs). A síntese de partículas decoradas (Fe3O4-AuNPs) mostrou-se efetiva, obtendo relativo grau...
This thesis reports a new concept of flexible microfluidic device, using natural rubber as alternative material, flexible and stretchable, for based-plataforms of lab-on-a-chip-devices. The device preparation is carried out dropping the latex over the mold, supported on acrylic dishes, containing the configuration of the device. The latex should cover the entire structure by casting method and, subsequentely subjecting it to thermal treatment to form the natural rubber membranes. The membranes demolded should replicate the configuration of the device on its surface. Poly(vinyl chloride) (PVC) films are implement as covering layer on internal surface, avoiding thw water absorption e protein compounds leaching from natural rubber membranes. The flexible devices of natural rubber were implemented as optical and electrochemical sensors, using flexible carbon fibers as electrodes internal electrodes. Becomes important emphasize, that the natural rubber membranes are transparent when considering the visible region in the electromagnetic sprectrum as well as it is biocompatible, allowing the combination of mechanical, optical and biological properties in a single device. Rely on electrochemical analysis devices demonstrate good stability and resistance for long term stability maintaining the characteristic curve of potential as well as a short interval of time necessary to reach the stationary current for each electrochemical process. In a second instance, we used the natural rubber-based microfluidic as microreactor for the synthesis of magnetic nanoparticles (Fe3O4) decorated with gold nanoparticles (Fe3O4-AuNPs). The synthesis of decorated nanoparticles (Fe3O4-AuNPs) shows effectiveness in order to obtain high degree of homogeneity on gold nanoparticles distribution over the surface of magnetite particles, reaching averages sizes of 6.3 nm
FAPESP: 2011/23362-0

The measurement of chemical concentrations within the oceans is crucial for our understanding of its biogeochemical processes. Such data is vital for; studies of climate and environment change; natural resource management; assessment of pollution, human impact and biodiversity. Current measurement methods (sampling and large in situ instrumentation) cannot provide the quantity and quality of biogeochemical data that is required. The factors limiting the widespread collection of quality data include, sample degradation/contamination, instrument size, cost and insufficient sensitivity. New technologies allow the manufacture of Lab On a Chip (L.O.C.) devices that can be used as small, low-cost and low power sensors. There are numerous demonstrations of these devices in the laboratory where it is possible to use standard bench top equipment to operate them. Within the National Oceanography Centre Southampton and the Nanoscale System Integration Research Group at Southampton University it has been proposed that integrated L.O.C. devices could be used autonomously and remotely in the marine environment. These innovative micro-devices could provide in situ real time synoptic data on processes with temporal and spatial scales smaller than currently sampled. This report details the development, laboratory testing and field trial of the world‟s first deep sea in situ L.O.C. chemical sensor. Preparatory work for the design of the next generation of marine L.O.C. devices including low-cost fabrication in fluoropolymer materials (which are naturally robust and chemically resistant) is also presented. The L.O.C. devices within this study use a reagent based colorimetric protocol to determine the concentration of a chemical within a sea-water sample. The optical absorption when the reagent is mixed with a sample is proportional to the chemical concentration and is measured using a double beam spectrophotometer. This method can be used in the metrology of a number of chemical species including nutrients and pollutants and therefore this technology is generic. The detection of nitrite and nitrate at a wavelength of 540nm is used as a proof of concept within this report. Nitrite samples are combined with α-napthylamine and sulphanilamide to form a coloured dye. The absorption of the dye is proportional to the nitrite concentration. Nitrate is reduced to nitrite using a cadmium column and then measured in same manner. The L.O.C. devices are fabricated using negative photolithography on photosensitive epoxy resin. Micro channels measuring 500 by 500μm are used to create micromixers, optical detection paths and fluid delivery ports on a device with a footprint of 45 by 45mm. The absorption is measured with low powered portable electronics, a modulated light emitting diode source and photodiode detector both coupled to polymer fibres. The mixer uses a three dimensional split and recombine technique to ensure effective mixing of the chemicals and sample. On the laboratory bench the sensor was capable of continuously sampling nitrite concentration levels in sea-water at 60μl/min with a limit of detection of 47.6nM and a precision of 89.3nM at 15μM. Once reconfigured it was capable of detecting nitrate in seawater, at the same flow rates with a limit of detection of 1.75μM and a precision of 9.26μM at 100μM. An in situ version of the sensor, packaged within a pressure compensated housing measuring Ø120mm by 300mm, was deployed in the mid-Atlantic. It provided key functionality and construction methodologies for future generation devices. These trials also identified the developments necessary for the sensor to work as efficiently at depths of 1500m as on the laboratory bench.

This work presents the development of electrowetting on dielectric and liquid dielectrophoresis as a platform for chemistry, biochemistry and biophysics. These techniques, typically performed on a single planar surface offer flexibility for interfacing with liquid handling instruments and performing biological experimentation with easy access for visualisation. Technology for manipulating and mixing small volumes of liquid in microfluidic devices is also crucially important in chemical and biological protocols and Lab on a Chip devices and systems. The electrodynamic techniques developed here have rapid droplet translation speeds and bring small droplets into contact where inertial dynamics achieve rapid mixing upon coalescence. In this work materials and fabrication processes for both electrowetting on dielectric and liquid dielectrophoresis technology have been developed and refined. The frequency, voltage and contact angle dependent behaviour of both techniques have been measured using two parallel coplanar electrodes. The frequency dependencies of electrowetting and dielectrophoretic liquid actuation indicate that these effects are high and low-frequency limits, respectively, of a complex set of forces. An electrowetting based particle mixer was developed using a custom made electrode array and the effect of varying voltage and frequency on droplet mixing was examined, with the highest efficiency mixing being achieved at 1 kHz and 110 V in about 0.55 seconds. A composite electrodynamic technique was used to develop a reliable method for the formation of artificial lipid bilayers within microfluidic platforms for measuring basic biophysical aspects of cell membranes, for biosensing and drug discovery applications. Formation of artificial bilayer lipid membranes (BLMs) was demonstrated at the interface of aqueous droplets submerged in an organic solvent-lipid phase using the liquid dielectrophoresis methods developed in this project to control the droplet movement and bring multiple droplets into contact without coalescence. This technique provides a flexible, reconfigurable method for forming, disassembling and reforming BLMs within a microsystem under simple electronic control. BLM formation was shown to be extremely reliable and the BLMs formed were stable (with lifetimes of up to 20 hours) and therefore were suitable for electrophysiological analysis. This system was used to assess whether nanoparticle-membrane contact leads to perturbation of the membrane structure. The conductance of artificial membranes was monitored following exposure to nanoparticles using this droplet BLM system. It was demonstrated that the presence of nanoparticles with diameters between 50 and 500 nm can damage proteinfree membranes at particle concentrations in the femtomolar range. The effects of particle size and surface chemistry were also investigated. It was shown that a large number of nanoparticles can translocate across a membrane, even when the surface coverage is relatively low, indicating that nanoparticles can exhibit significant cytotoxic effects

Orientador: Aldo Eloizo Job
Banca: Deuber Lincon da Silva Agostini
Banca: Frank Nelson Crespilho
Banca: Ivan Helmuth Bechtold
Banca: Lucas Fugikawa Santos
Resumo: Esta tese apresenta um novo conceito de dispositivo microfluídico flexível, utilizando a borracha natural como material alternativo, de baixo custo, flexível e elástico, para a preparação de plataformas lab-on-a-chip. A preparação do dispositivo é realizada com a dispersão do látex sobre o molde. Este é construído em placas de acrílico (PMMA - Poli-metilmetacrilato) contendo a configuração do dispositivo. O látex deve recobrir toda a estrutura, utilizando o método casting. Posteriormente, o molde contendo látex é submetido a tratamento térmico para a formação das membranas de borracha natural. As membranas retiradas do molde replicam a configuração do dispositivo em sua superfície. Filmes de policloreto de vinil (PVC) são utilizados como recobrimento interno a fim de evitar a absorção de água e a liberação de composição proteica das membranas de borracha natural. Os dispositivos flexíveis de borracha natural foram implementados como sensores óptico e eletroquímicos, utilizando fibras de carbono flexíveis como eletrodos internos. Torna-se importante ressaltar que as membranas de borracha natural são transparentes (quando consideradas a região vísivel do espectro eletromagnético), bem como biocompatíveis, possibilitando a combinação de propriedades mecânicas, ópticas e biológicas num único dispositivo. Por meio das análises eletroquímicas, demonstrou-se que possuem boa estabilidade e resistência quando submetidos a testes prolongados, mantendo a característica da curva de potencial, bem como o curto intervalo de tempo necessário para atingir a corrente estacionária durante os processos eletroquímicos. Em segunda instância, utilizaram-se os microdispositivos de borracha natural como microreatores para a síntese de nanopartículas de magnetita (Fe3O4) decoradas com nanopartículas de ouro (FeO4-AuNPs). A síntese de partículas decoradas (Fe3O4-AuNPs) mostrou-se efetiva, obtendo relativo grau...
Abstract: This thesis reports a new concept of flexible microfluidic device, using natural rubber as alternative material, flexible and stretchable, for based-plataforms of lab-on-a-chip-devices. The device preparation is carried out dropping the latex over the mold, supported on acrylic dishes, containing the configuration of the device. The latex should cover the entire structure by casting method and, subsequentely subjecting it to thermal treatment to form the natural rubber membranes. The membranes demolded should replicate the configuration of the device on its surface. Poly(vinyl chloride) (PVC) films are implement as covering layer on internal surface, avoiding thw water absorption e protein compounds leaching from natural rubber membranes. The flexible devices of natural rubber were implemented as optical and electrochemical sensors, using flexible carbon fibers as electrodes internal electrodes. Becomes important emphasize, that the natural rubber membranes are transparent when considering the visible region in the electromagnetic sprectrum as well as it is biocompatible, allowing the combination of mechanical, optical and biological properties in a single device. Rely on electrochemical analysis devices demonstrate good stability and resistance for long term stability maintaining the characteristic curve of potential as well as a short interval of time necessary to reach the stationary current for each electrochemical process. In a second instance, we used the natural rubber-based microfluidic as microreactor for the synthesis of magnetic nanoparticles (Fe3O4) decorated with gold nanoparticles (Fe3O4-AuNPs). The synthesis of decorated nanoparticles (Fe3O4-AuNPs) shows effectiveness in order to obtain high degree of homogeneity on gold nanoparticles distribution over the surface of magnetite particles, reaching averages sizes of 6.3 nm
Doutor

This dissertation develops technology for microfluidic point-of-care (POC) immunoassay devices, divided into three papers, and explores the use of a quartz crystal microbalance for real time monitoring of blood coagulation in a fourth paper. The concept of POC testing has been well established around the world. With testing conveniently brought to the vicinity of the patient or testing site, results can be obtained in a much shorter time. There has been a global push in recent years to develop POC molecular diagnostics devices for resource-limited regions where well equipped centralized laboratories are not readily accessible. POC testing has applications in medical/veterinary diagnostics, environmental monitoring, as well as defense related testing. In the first paper, we demonstrated the use of latex immunoagglutination assays within a microfluidic chip to be an effective and sensitive method for detecting the bovine viral diarrhea virus. In the second paper the feasibility and general ease of integrating liquid core optical components onto a microfluidic lab-on-a-chip type device, for point-of-care AI diagnosis is demonstrated. In the third paper particle agglutination assays, utilizing light scattering measurements at a fixed angle from incident light delivery, for pathogen detection are explored in both Rayleigh and Mie scatter regimes through scatter intensity simulations and compared to experimental results. In the fourth paper a quartz crystal microbalance was used for real-time monitoring of fibrinogen cross-linking on three model biomaterial surfaces.

Ce travail de thèse s’est porté sur le développement technologique d’un laboratoire sur puce permettant la caractérisation ultrasonore de milieux biologiques en canal microfluidique grâce à des transducteurs de ZnO fonctionnant à une fréquence centrale de 1 GHz. Ce système permet de caractériser, par la transmission d’ondes longitudinales de volume au travers de ce canal, les propriétés mécaniques du milieu sous investigation. Les ondes de volume sont guidées dans une direction parallèle à la surface du substrat grâce à l’introduction de miroirs à 45° obtenus par gravures humides. Cette thèse s’est portée plus particulièrement sur les développements technologiques des briques de base (transducteur ultrasonore en couche mince, miroirs acoustiques, canal microfluidique, lentille cylindrique) ainsi que leur intégration dans un laboratoire sur puce à base de silicium et PDMS. Ces élément ont été étudiés et optimisés afin de réussir le guidage d’ondes dans le substrat de silicium vers le canal microfluidique d’un transducteur émetteur vers un transducteur récepteur. Ce système a permis de réaliser des essais de détection de particules, des mesures de concentration de milieu de référence et également de faire des premières mesures sur des liquides biologiques grâce à l’utilisation de lentilles cylindriques obtenues par gravure plasma
This thesis presents an acoustofluidics platform for elastic characterization of biological samples using ultra high frequency (~1GHz) ultrasonic bulk acoustic waves (BAW). Passive 45° mirror planes obtained by wet chemical etching can be used to control bulk acoustic wave to transmit in the directions parallel to the surface of the silicon wafer. Zinc oxide (ZnO) thin film transducers were deposited by radio frequency sputtering on the other side of the wafer, which act as emitter/receiver after aligned with the mirrors. A microchannel fabricated using ICP technology was inserted between 45° mirror and vertical mirrors to realize the real time biosensing applications. To validate the design and technology of the silicon and PDMS-based platform, the propagation of bulk acoustic waves through the microfluidic channel was studied. This lab-on–a-chip platform was used to characterize different concentrations of chemical solutions in the microfluidic channel and detect latex particles passing through the channel. Moreover, with this design, a confocal cylindrical lens using ICP technology was integrated in the microsystem. The confocal lens controls the phase of acoustic waves for focusing which is used to characterize and detect biosamples (e.g. blood cells), especially on-line to evaluate the concentration of red blood cells

Microfluidics holds great promise to revolutionize various areas of biological engineering, such as single cell analysis, environmental monitoring, regenerative medicine, and point-of-care diagnostics. Despite the fact that intensive efforts have been devoted into the field in the past decades, microfluidics has not yet been adopted widely. It is increasingly realized that an effective system integration strategy that is low cost and broadly applicable to various biological engineering situations is required to fully realize the potential of microfluidics. In this article, we review several promising system integration approaches for microfluidics and discuss their advantages, limitations, and applications. Future advancements of these microfluidic strategies will lead toward translational lab-on-a-chip systems for a wide spectrum of biological engineering applications.

The development of a microfluidic-based process is presented for the production of nanomaterials in continuous-flow microreactors. A flow focusing configuration was used enabling a controllable mixing process to assist the formation of the nanomaterials through precipitation, which was triggered by a solvent exchange process. Initially, Pluronic® tri-block copolymers were used as model polymeric biomaterials, relating to drug delivery applications, to investigate the production of empty polymeric micelles (PMs). Following the production of empty PMs, the production of copolymer stabilized organic β-carotene nanopartilces (NPs) was also investigated. The formation of both PMs and NPs, within microfluidic reactors, was further analysed by computational fluid dynamics (CFD) models in order to gain more insight into the nanoprecipitation process. It has been shown that, besides the important role played by the width of the focused stream, the combined effect of reactor dimension, fluid properties, and flow condition significantly influenced the mixing condition and therefore the nucleation and growth process. When low water soluble molecules were co-precipitated together with polymeric stabilizer, competitive reactions resulted in the formation of two types of NPs, i.e., either with or without loading drug. The obtained results were interpreted by taking into consideration a new parameter representing the mismatching between the aggregations of the two precipitant species (polymer and drug), which played a decisive role in determining the size and polydispersity of the obtained NPs. Finally, the established microfluidic production procedure was examined from a drug delivery point of view, by encapsulating a clinically relevant drug in PMs. PMs containing mithramycin were prepared and tested in vitro as a therapeutic protocol for beta-thalassemia. In conclusion, the results of this study had demonstrated that microfluidics could facilitate the production of nanostructures for drug delivery purposes, and offer a novel method to control their properties including particle size, size distribution and pharmaceutical efficacy.

Recent years have seen considerable interest in the use of microfabricated systems in chemical and biological due to their significant advantages in terms of speed, analytical throughput, yield, unit cost, footprint, reagent requirements and control. Inevitably this has led to a growing interest in transferring chromatographic methods to planar chip formats since techniques using high performance LC play such a prominent role in modern bioanalysis. In addition, the manipulation of multiphase (or segmented) flows within microfluidic channels has been recently investigated as a promising approach for large-scale experimentation in biology and chemistry. Importantly, flow segmentation allows for the compartmentalisation of reagent volumes ranging from a few femtolitres to hundreds of nanolitres within a continuous and immiscible fluid, the production of monodisperse droplets at high frequencies, the accurate control of droplet contents and the ability to perform kinetic analysis with high precision. Accordingly the integration of droplet-based microfluidics with HPLC has the potential to dramatically reduce dispersion and minimise dead volume effects by using droplets to collect fractions of the column effluent. This basic progress preserves the chemical identity of each fraction allowing further analysis downstream. In this work, microfluidic devices were fabricated using thermoset polyester (TPE) to operate under high pressure which is required for LC separation and high frequency droplet generation. The optical characteristics of the fabricated devices were assessed for feasibility of optical detections for droplets. Substrate resistance to pressure also was investigated for droplet generation with high frequency. Lastly, droplets were generated under various conditions by adjusting flow-rates and the oil viscosity. Secondly LC separation columns were formed in TPE channels using two different column materials: particulate and polymer monolithic columns. The packed channels were investigated by SEM. In addition, permeability was calculated from back25 pressures measured as a function of flow-rates and compared with columns. Neurotransmitters were separated by the columns to estimate performance. Thirdly, the both operations, LC separation and droplet-based microfluidics, were combined in a single planar format. Sequential operations of separation, compartmentalisation and concentration gradient generation were integrated on a single chip and characterised using confocal laser-induced fluorescence detection. Finally, a preliminary investigation is reported into the possibility of the indirect electrochemical detection as a universal detection that can monitor electrochemically detectable samples as well as non- or less-electroactive bio samples. Amino acids were separated by a commercial RPHPLC column and detected indirectly.

The development of microlectronic lab-on-a-chip devices (LOACs) can now be pursued thanks to the continous advances in silicon technology. LOACs are miniaturized devices whose aim is to perform in a more efficient way specific chemical or biological analysis protocols which are usually carried out with traditional laboratory equipment. In this application area, CMOS technology has the potential to integrate LOAC functionalities for cell biology applications in single chips, e.g. sensors, actuators, signal conditioning and processing circuits. In this work, after a review of the state of the art, the development of a CMOS prototype chip for individual cell manipulation and detection based on dielectrophoresis will be presented. Issues related to the embedded optical and capacitive detection of cells will be discussed together with the main experimental results obtained in manipulation and detection of living cells and microparticles.

The development of microlectronic lab-on-a-chip devices (LOACs) can now be pursued thanks to the continous advances in silicon technology. LOACs are miniaturized devices whose aim is to perform in a more efficient way specific chemical or biological analysis protocols which are usually carried out with traditional laboratory equipment. In this application area, CMOS technology has the potential to integrate LOAC functionalities for cell biology applications in single chips, e.g. sensors, actuators, signal conditioning and processing circuits. In this work, after a review of the state of the art, the development of a CMOS prototype chip for individual cell manipulation and detection based on dielectrophoresis will be presented. Issues related to the embedded optical and capacitive detection of cells will be discussed together with the main experimental results obtained in manipulation and detection of living cells and microparticles.

Microfluidics is a science and a technology which deals with manipulation and control of small volumes of fluids flowing in channels of micro-scale size. It is currently used for Labs-On-a-Chip (LoCs) applications mainly. In this context, recently fluids have been used in the discrete form of droplets or bubbles dispersed into another immiscible fluid. In this case, droplets or bubbles can be exploited as a means to transport digital information between microfluidic components, with sequences of particles (i.e. droplets or bubbles) representing sequences of binary values. LoCs are today realized through monolithic devices in which samples are processed by passing them through a predetermined sequence of elements connected by fixed and preconfigured microfluidic channels. To increase reusability of LoCs, effectiveness and flexibility, networking functionalities can be introduced so that the sequence of elements involved in the processing can be dynamically selected. Accordingly, in this thesis we introduce the Networked LoC (NLoC) paradigm that brings networking concepts and solutions in microfluidic systems such as LoCs. More specifically, in this paper the need for the introduction of the NLoC paradigm is motivated, its required functions are identified, a system architecture is proposed, and the related physical level design aspects, such as channel characterization, information representation and information capacity are investigated; finally a switching device is proposed and studied.

Background While conventional chemotherapy and radiation therapy have improved the survival of many cancer patients, there are still major disadvantages associated with these treatments such as high toxicity and drug-resistance. The possibility to manipulate the immune system to recognize and kill tumor cells is very attractive despite numerous obstacles remaining to be overcome. In particular, the ability of the immune system to destroy disseminated metastases in a specific way makes immunotherapy an attractive alternative to conventional therapies. Nevertheless, other unconventional technologies emerged in recent years seem to be very promising; in particular the analysis and monitoring of single cell-to-cell interactions and the capability to individually control single cells by Lab-on-a-chip devices have become of great interest in different areas of life sciences. These new technologies, in combination with progresses reached in anti-tumor vaccines, could be useful to improve immune T cell responses against tumor antigen for a more efficient immunotherapy. Aims This thesis focuses on two tumor immunotherapy issues: 1) design, realization and validation of innovative Lab-on-a-chip devices for immune system study, that allows single tumor cell and effector cell interaction, detection and isolation; 2) identification of molecular mechanisms that prevent EBV-associated tumors (e.g. Burkitt’s lymphoma) recognition by T cells and study of their potential correction by specific treatments. The main goal of this study remains indeed the evaluation of an integrated strategy for immunotherapy development enhancing for malignancies treatment. Methods Biocompatibility test, generation of memory CTL cultures, 51Cr release assay, IFN-Elispot, proteasomes purification, western blot assay, enzymatic assay, immunofluorescence, RT-PCR. Main Results As concerns the first part of the thesis, a main achievement was the design of Lab-on-a-chip platform that combined microfluidics and electronics together, consisting in a matrix of up to thousand microwells where living cells can be deposited. Subsequently, different materials have been evaluated to identify the most biocompatible ones for biosensor manufacturing. Once developed Lab-on-a-chip prototype, it has been tested from a functional point of view. In particular, it has been demonstrated that the biosensor is able to isolate and trap single cells inside microwells by dielectrophoresis, that recovered cells are still alive and that their biological functions and gene expression remain unaltered. Furthermore, tumor cell lysis by immune effector cells could be successfully monitored inside device microwells, showing that biosensor could be used for cell to cell interaction studies. Regarding the second aim of this thesis, it has been identified a new epitope-specific T cell response against EBV nuclear antigen 1 (EBNA1). It has also been demonstrated that CTLs specific for another EBNA1-derived epitope (referred as HPV) are detectable in the majority of HLA-B35 individuals, and recognize EBV-transformed B lymphocytes (LCL) but not Burkitt’s lymphoma (BL). Afterwards LCL and BL have been compared for their antigen processing machinery, demonstrating that one of the major differences was at the proteasome level; indeed, proteasomes from BL cells have displayed a far lower chymotryptic and tryptic-like activities. Interestingly, it has also been shown that treatment with proteasome inhibitors partially restored the capacity of BL cells to present the HPV epitope. Conclusions The results achieved in single cell manipulation and cell to cell analysis interaction by Lab-on-a-chip technology, and the findings reached to improve BL immune recognition, represent an implementation of innovative tools that could allow important progresses in cancer diagnosis and immunotherapy.

Esta disertación abarca una investigación en tecnologías Lab-on-a-Chip (LoC) que permiten acoplar luz a capas biológicas celulares como biofilms bacterianos o monocapas de eucariotas, con el objetivo de transformar células en componentes fotónicos vivos adquiriendo un rol óptico dual: transdusor y elemento de medición. El concepto de componentes fotónicos vivos supone múltiples posibilidades en su monitoreo sin contacto y mínima invasión del proceso biológico basado en una respuesta espectral autoreferencial a lo largo del tiempo del ensayo. Sin embargo, la implementación de los ya mencionados elementos fotónicos vivos presenta retos multifacéticos: los aspectos biológicos, las simulaciones numéricas, el diseño óptico, los avances en la microfabricación a bajo costo y la adaptación de nuevos materiales para fabricación PhLOC y cultivo celular en la interfaz óptica y el procesamiento de datos. Particularmente, nos centramos en monitorear biofilms bacterianos y monocapas de células de mamífero debido a su relevancia en la salud pública. Los biofilms bacterianos son un gran riesgo a la salud debido a su ubicuidad, dinamismo y resistencia a los biocidas. Razón por la cual requiere un control intensivo, idealmente con disposición de instrumentación miniaturizada. Por otro lado, las monocapas celulares se han estudiado extensivamente por su relación con las afectaciones crónicas como la diabetes y enfermedades cardiovasculares. Nuestras contribuciones sobre las interfaces ópticas se enfocan en conexiones ópticas robustas y estandarizadas desde y hacia PhLoCs, usando un prototipado rápido y económico basado en procesamiento de un laser de CO2. Una caracterización detallada de Polimetilmetacrilato (PMMA) mecanizada por laser permite crear conexiones ”plug” a conectores de fibra óptica SMA estándar, que se han comparado con sus homólogos comerciales y las cuales son viables para un acoplamiento de luz en guías de onda de capas finas de polímero en una configuración de PhLOC de alta relación Señal-Ruido (SNR). Así mismo, se desarrolló un software modular de interfaces para el control integral del equipo de laboratorio. Este se basó en una el lenguaje de programación libre ”Python”. Además de encargarse del extensivo procesamiento de datos implícito en el monitoreo de un respuesta espectral, la interacción con el kit de desarrollo de software Qt demostró buenos resultados para representaciones gráficas en tiempo real. Nuestra contribución sobre la instrumentación miniaturizada para la monitorización de las capas de bacterias estaba dirigida a la integración de componentes fotónicos en sustratos termoplásticos (particularmente el PMMA). Esto proporciona una plataforma de bajo costo para el estudio de la colonización de superfície en sistemas de distribución de aguas. Al modificar localmente la superficie de la zona de detección, logramos una adhesión preferencial y una detección óptica de bacterias en estadios tempranos de adhesion superficial en condiciones estáticas a través de los segmentos de fibra óptica empotradas en los sustratos modificados. Para la implementación de prototipos que simulen el flujo y las condiciones de presión en los sistemas reales de distribución de agua, también pudimos explorar la integración de guias de onda de polímero con canales de fluido; poniendo en práctica favorablemente nuevas estrategias de fabricación para el encapsulado en PMMA de estructuras SU-8 obtenidas por fotolitografía. Utilizando estos dispositivos y explotando nuestros resultados positivos en términos de interconectores ópticos y interfaz informática el monitoreo de una población circular bacteriana arrojó que, bajo estas condiciones, la colonización de superficie bacteriana podría ser asociada con una respuesta espectral characterística con el tiempo. . Finalmente, se han investigado los ajustes necesarios al paradigma PhLoC para la implementación de monocapas celulares de mamífero como componentes fotónicos vivos. Concretamente, dirigimos nuestros esfuerzos en la evaluación numérica y optimización del confinamiento de luz en capas irregulares de ambientes con bajo índice de refracción y el desarrollo de estrategias adecuadas para el confinamiento de luz en dichas estructuras, tomando en cuenta las restricciones biológicas, mucho más evidentes aquí que en el caso de los biofilms. Con este fin, se estudiaron diferentes materiales tanto en cuestión de compatibilidad con las propiedades previamente establecidas, como las técnicas viables de microfabricación y bio compatibilidad. A fin de cuentas, basados en los resultados de los materiales adecuados, se aplicaron dos opciones de arquitecturas PhLoc a culturas celulares in vitro en diferentes etapas de diferenciación o procesos de inflamación, respectivamente.
This dissertation encompasses our research on Lab-on-a-Chip (LoC) technologies enabling light coupling into biological cell layers like bacterial biofilms or monolayers of eukaryotes, with the aim of making the cells act as living photonic components in the dual role of optical transducer and reporter. The concept of living photonics suggests a host of possibilities in terms of contactless and minimal invasive monitoring of biological processes based on a self-referenced spectral response over time. The implementation of such living photonic elements however presented a very multifaceted challenge, ranging from biological aspects over numerical simulations and optical design, advancements in low-cost micro-fabrication and adaptation of novel materials for PhLoC fabrication and cell culture to optical interfacing and data processing. In particular, we focussed on monitoring bacterial biofilms and mammalian cell monolayers for their relevance in public health. Bacterial biofilms are a major risk due to their ubiquity, resistance to biocides and dynamism and therefore require an intensive control, for which miniaturised and affordable instrumentation would be ideal, very few though is available. Cell monolayers on the other hand are studied extensively in relation with chronic conditions like cardiovascular diseases or diabetes, Our contributions regarding optical interfacing focus on robust and standardised optical connections to and from a PhLoC using a low-cost fast prototyping approach based on CO2-laser processing. In particular, careful characterisation of poly-methylmetacrylate (PMMA) laser machining allowed reliable ‘plug’ connections to standard 𝑆��𝑀��𝐴�� fiber-optics connectors, which were benchmarked against commercial counterparts and applied to light coupling in thin film polymeric waveguides in a high Signal-to-Noise ratio (SNR) PhLoC configuration. Here, optical simulations were mainly employed in the design. In addition, we developed a modular software interface for integral control of laboratory equipment based on the cross platform and open source programming language Python. Besides taking care of the rather extensive data processing implicit in long-term spectral monitoring via efficient number crunching modules like Numpy, interfacing with the Qt software development kit proved apt for real time graphical feedback with fast response times. Our contributions regarding miniaturised monitoring instrumentation of bacterial biofilms focus on integrating photonic components in thermoplastic substrates - in particular commercial grade PMMA - to provide a cheap platform for the study of biofilm colonisation in water distribution systems. By locally modifying the surface in the detection zone, we achieved preferential adhesion and early optical detection of bacteria in static conditions via fiber-optics segments embedded in the modified substrates. For the implementation of prototypes resembling the flux and pressure conditions in real water distribution systems, we also explored the integration of polymeric waveguides with fluidic channels, successfully implementing novel fabrication strategies for the encapsulation of photolithographically obtained SU-8 structures in PMMA PhLoCs . Using these devices, and exploiting our positive results in terms of optical interconnects and software interface, monitoring of a circulating bacterial population suggested that bacterial surface colonisation can in such circumstances indeed be associated with a distinct spectral response over time. Last, we investigated the adjustments to the PhLoC paradigm necessary regarding the implementation of the much thinner mammalian cell monolayers as living photonics. Concretely, we focussed our efforts on the numerical evaluation an optimisation of light confinement in thin irregular layers in low-refractive index environments and the development of suitable strategies to couple light to such structures, taking into account the biological constrains, which were much more pronounced here as compared to biofilms. To that end, different materials were studied in terms of compatibility with the established material parameters, available microfabrication techniques and bio-compatibility. Finally, based on the results regarding suitable materials, we applied two of the resulting PhLoC architectures to in vitro cell cultures in different stages of differentiation or inflammatory processes, respectively.

Las plataformas Micro / Nanofluídicas, simples y miniaturizadas son especialmente interesantes debido a sus ventajas como la reducción de los volúmenes de muestra y reactivos, la disminución del tiempo de análisis, la posibilidad de portabilidad y la integración de técnicas analíticas convencionales. Además, es importante señalar el papel que pueden jugar los nanomateriales en términos de mejora de las propiedades electroquímicas después de ser integrados en plataformas microfluídicas, o incluso modificaciones superficiales de los transductores. Así, la combinación de la nanotecnología, la electroquímica y la microfluícia, podría proporcionar una plataforma de detección muy potente, por lo que, en esta Tesis se estudian diferentes dispositivos microfluídicos con transductores electroquímicos integrados para aplicaciones bioanalíticas. En esta Tesis se exponen también los aspectos generales y los resultados experimentales, a partir de una introducción general, la cual presenta los trabajos más recientes relacionados con el uso de nanomateriales y tecnologías lab-on-a-chip como una sinergia prometedora para una amplia gama de aplicaciones. Después se presenta la detección electroquímica de proteínas mediante el uso de puntos cuánticos como marcadores. En primera instancia, se describe un chip microfluídico híbrido compuesto por una canal de polidimetilsiloxano flexible (PDMS) y policarbonato (PC) como substrato. Este substrato a su vez tiene impreso electrodos serigrafiados integrados de carbono (SCPE). El dispositivo desarrollado combina las ventajas de los chips microfluídicos flexibles, tales como su bajo coste, la posibilidad de ser desechables y la susceptibilidad de ser producidos en masa con las ventajas de la electroquímica por su facilidad de integración y la posibilidad de ser miniaturizables. En la segunda parte, se realizó la detección electroquímica de puntos cuánticos como marcadores en un ensayo para la determinación de un biomarcador de la enfermedad de Alzheimer: Apolipoproteína E (ApoE). El inmunocomplejo se llevó a cabo mediante el uso de partículas magnéticas tosilactivadas, las que fueron a su vez utilizadas como plataforma de preconcentración de muestra dentro de un canal microfluídico. Debido a la necesidad de lograr límites inferiores de detección en inmunoensayos, en esta Tesis se han propuesto diferentes estrategias para mejorar la sensibilidad de los dispositivos. La primera de ellas es el uso de un campo magnético para inmovilizar una mayor cantidad de partículas magnéticas en una disposición controlable dentro de un canal microfluídico con el fin de obtener una zona de precocentración, donde se lleva a cabo el inmunoensayo. La segunda estrategia que se presenta en esta Tesis es el uso de un sistema de reciclaje de fabricación propia. En esta parte, el incremento de la señal de los puntos cuánticos se demuestra mediante el uso de una bomba peristáltica externa conectada a un chip microfluídico que forma un sistema cerrado. Después de esta demostración, se propuso una micro-bomba peristáltica con válvulas integradas. Todas las etapas de fabricación se optimizaron así como también se desarrolló un software para su control. Por último, el bismuto, que es un material bien conocido para aglomerar los metales pesados, fue usado para aglomerar los puntos cuánticos cuyo núcleo está formado por cadmio II, de esta forma se pudo mejorar la señal electroquímica mediante la reducción de los QDs junto con el Bismuto III. Diferentes optimizaciones fueron hechas usando canales microfluídicos. Adicionalmente, se presentan otras nueva plataforma basada en diamante dopado con boro, transductor utilizado para la determinación electroquímica de la atrazina basado en el desarrollo de un magneto-inmunoensayo. Este inmunoensayo se realizó mediante un ensayo competitivo con peroxidasa de rábano silvestre (HRP) como marcador enzimático y micropartículas magnéticas como plataforma de preconcentración. Otra plataforma propuesta es el transistor orgánico de efecto campo de doble puerta, como transductor para biosensores, desarrollado por la tecnología de inyección de tinta sobre un substrato flexible. Este tipo de transistores orgánicos tiene ventajas importantes para biosensores en términos de coste de fabricación y biocompatibilidad, así como su posibilidad de integración en microcanales. Para demostrar la aplicabilidad de este dispositivo en el campo biológico, se ha funcionalizado su capa externa con un anticuerpo de captura que detecta una proteína modelo sin ningún marcador. Se realiza la fabricación del dispositivo, teniendo en cuenta su estructura, los materiales que lo componen, sus características eléctricas y posibles aplicaciones. Por último, se exponen las conclusiones generales y futuras propuestas.
Simple and miniaturized micro / nanofluidic platforms are especially interesting due to their advantages like the reduction of sample and reagent volumes, the decrease of the analysis time, the possibility of portability and the integration of conventional analytical techniques. Furthermore it is important to point out the role that nanomaterials can play in terms of enhancing electrochemical properties after being integrated into the microfluidic platform or even in the electrode, where the detection event will be performed. Combined together, nanotechnology, electrochemistry and microfluidics could provide a really powerful biosensor platform, thus in the present Thesis different microfluidic platforms with integrated electrodes as transducers in biosensing applications were evaluated. General aspects and experimental results are exposed, starting from a General Introduction that describes various aspects related with the use of nanomaterials and lab-on-a-chip technologies as a promising synergy for a wide range of applications. The electrochemical detection of proteins (ex. Apolipoprotein-E, ApoE) by using CdS or CdSe@ZnS Quantum Dots (QDs) as labels has been one of the main objectives of this Thesis. The immunocomplex was performed by using tosylactivated magnetic beads as preconcentration platform into the same microfluidic system. Due to the need to achieve a lower limit of detection of the immunoassays, different strategies for electrochemical signal enhancing are proposed. The first one is the use of a magnetic field to immobilize magnetic beads in a controllable way into a microfluidic channel in order to obtain a stable magnetic plug where the immunoassay is performed. The second strategy is the use of a home-made recycling system. In this part, the increasing signal of QDs is demonstrated by using an external peristaltic pump connected to a microfluidic chip forming a loop system. After this demonstration, a micro-peristaltic pump with integrated valves is also proposed. All the fabrication steps have been optimized and the software for sequential control of the valves also has been developed. Finally, bismuth is used as it is a well-known material that agglomerates with heavy metals. We took advantages of this property for improving the electrochemical signal of QDs, due to the cadmium content that QDs have in their core. Optimization of the bismuth concentration has been done in order to achieve the highest signal. This detection has been performed in batch system as well as in microfluidic mode. In addition, another novel platform for electrochemical determination of a pesticide (atrazine) based on magneto-immunoassay using boron-doped diamond (BDD) electrode is presented. BDD electrode has been modified by electroreduction of potassium tetrachloroplatinate (K2PtCl4) in order to grow platinum nanoparticles (Pt-NPs) onto the electrode surface. The immunoassay was based on a direct competitive assay using horseradish peroxidase (HRP) as enzymatic label and magnetic microparticles as preconcentration platform. A flexible organic double gate Bio-Field Effect Transistor (Bio-FET) developed by inkjet technology onto a flexible substrate is also presented. This kind of organic transducers has important advantages for biosensors in terms of fabrication cost and biocompatibility as well as their integration into microchannels. To demonstrate the applicability of this device in the biological field, its functionalization with a capture antibody, in order to detect a model protein in a label-free mode was performed. The device fabrication, its structure, materials composition optimization, electrical characteristics and other functionalities are also discussed. Finally, the general conclusions are exposed including some opinions / recommendations for further continuation of the research in the field.

In recent times, metallic nanoparticle plasmonics coupled with applications towards biosensing has gathered momentum to the point where commercial R&D are investing large resources in developing the so-called localized surface plasmon resonance (LSPR) biosensors. Conceptually, the main motivation for the research presented within this thesis is achievement of fully-operational LSPR biosensor interfaced with the state-of-the-art microfluidics, allowing for very precise control of sample manipulation and stable read-out. LSPR sensors are specifficaly engineered by electron beam lithography nanofabrication technique, where nanoparticle interactions are optimized to exhibit increased sensitivity and higher signal-to-noise ratio. However, the overall performance of LSPR lab-on-a-chip device depends critically on the biorecognition layer preparation in combination with surface passivation. As an introduction, the principles of plasmonic biosensing are identified encompassing both Surface Plasmon Resonance (SPR) and Localized SPR. Being successfully implemented into commercial product, the governing physics of SPR is compared to LSPR in chapter 1, together with advantages and disadvantages of both. Chapter 2 describes methods necessary for LSPR biosensor development, beginning with nano-fabrication methods, the modelling tool (COMSOL Multiphisics), while the basics of micro-fabrication in microfluidics conclude this chapter, where passive and active microfluidics networks are discerned. Particularly attractive optical properties are exhibited by closely-coupled nanoparticles (dimers), with the dielectric gap of below tens of nm, which were theoretically predicted to be very suitable as LSPR biosensing substrates. Chapter 3 is subjected to optical characterization (dependence on the size of the dielectric gap) of nanofabricated dimer arrays. The acquired data demonstrate the advantages of the nanofabrication methods presented in chapter 2 and the technique for fast and reliable determination of nanoparticle characteristic parameters. The initial biosensing-like experiments presented in chapter 4 (no integration with microfluidics) proved for the first time, the theoretical predictions of higher sensitivity, yielding additionally the specific response as function of analyte size and dielectric gap between nanoparticles. The overall response of different dimer arrays (various gaps) provides information about adopted conformation of analyte protein once immobilized. Broad resonances of dimers feature higher noise when employing them for the real-time LSPR biosensing. As a way to circumvent such problem, the feasibility of employing far-field interaction within the nanoparticle array to spectrally narrow resonance is investigated in chapter 5 by optimizing the array periodicity and introducing thin waveguiding layers. Finally, the concluding chapter 6 is dedicated to a full assembly of a Lab-on-a-chip (LOC) LSPR biosensor, starting with interfacing plasmonic substrates with compatible active microfluidic networks, allowing the precise sample delivery and multiplexing. The prototype device consisting of 8 individual sensors is presented with typical modes of operation. The bulk refractive index determination of various samples demonstrates the working principle of such device. Finally, various strategies of biorecognition layer formation are discussed within the on-going research.

La presente tesis doctoral se centra en la integración del novedoso biosensor nanointerferométrico de guías de onda bimodal (BiMW biosensor) en una plataforma lab-on-chip para la detección temprana de biomarcadores relacionados con el diagnóstico clínico de enfermedades directamente en la muestra del paciente. Nuestro grupo ya ha demostrado a nivel de laboratorio la gran utilidad del biosensor BiMW para la detección de biomarcadores clínicos, pero su integración en una plataforma portátil tipo LOC que pueda ser empleada fuera del laboratorio sigue constituyendo un gran reto científico y tecnológico. Con este objetivo en mente, en esta Tesis hemos llevado a cabo optimizaciones en el diseño de las guías de ondas, sistemas de acoplamiento de la luz, la microfluidica, y los sistemas de lectura que sin duda ayudaran a conseguir su integración en un sistema portátil de análisis. Para ello se ha llevado a cabo un estudio en profundidad del sensor BiMW previamente desarrollado mediante herramientas de modelado ysimulación. Su diseño ha sido optimizado para el sensado con luz visible acoplada en una guía de onda tipo rib con dimensiones nanométricas. Se han realizado estudios para optimizar: las dimensiones del rib para maximizar la sensibilidad del sensor, el efecto de la longitud total del dispositivo y la ubicación del escalón bimodal. Con el propósito de resolver el reto relacionado con el acople de la luz en las guías nanométricas de la BiMW, dos tipos de estructuras con disminuciones graduales han sido diseñadas, simuladas, fabricadas y evaluadas, discutiendo las ventajas e inconvenientes de dicha aproximación. Para conseguir una señal lineal de medida, evitando la lectura interferométrica, se ha diseñado, implementado y evaluado experimentalmente un sistema de modulación. Para conseguir un biosensor de respuesta multiplexada se han diseñado, simulado, fabricado y finalmente caracterizados divisores 1x2, 1x4 y 1x8. Además, se ha implementado un método de lectura multiplexada por software y comparado con los métodos analógicos. Por último, se discuten los pasos que quedan para conseguir el dispositivo POC final. El trabajo de esta Tesis doctoral ha ayudado a conseguir la integración del biosensor nanointerferométrico BiMW en una futura plataforma tipo LOC que pueda ser empleada fuera del entorno del laboratorio.
This doctoral Thesis focuses on the integration of the novel Bimodal Waveguide Nanointerferometric Biosensor (BiMW) into a Lab-On-a-Chip (LOC) platform which can allow the direct detection of biomarkers for diseases diagnosis directly in the patient´s sample. Even if real bioanalytical applications have been widely reported by our group using the BiMW as an optical transducer, its integration into a pre-commercial and portable LOC platform remains a difficult challenge. In this Thesis, we have accomplished optimizations in the waveguide structure, optical subsystems, microfluidic integration and read-out subsystems which greatly will help on its road towards a portable point-of-care device for on-site applications. Firstly, the BiMW previously developed in our group has been in-deep analysed, setting the tools for its modelling, simulation and experimental characterization. Its design has been optimized for sensing with visible light coupled in a rib waveguide with nanometric dimensions. Simulation studies of the BiMW included: rib size optimization for maximizing its sensitivity as biosensor, the effects of the length of the sensor and the location of the step junction. With the aim to solve the challenges related to the light in-coupling in nanometric structures, two kinds of tapers have been designed, simulated, fabricated and finally characterized, demonstrating the advantages and disadvantages of such solution. With the aim to overcome the issues related to the interferometric signal readout, a chirp modulation system has been theoretically designed and experimentally implemented and characterized. Splitter series of 1x2, 1x4 and 1x8 for on-chip multiplexing have been designed, simulated, fabricated and characterized. A multiplexed detection readout method has been implemented via software and compared to analogue methods. Finally, the remaining challenges in terms of integration towards a final POC device are discussed. The work done in this Thesis has greatly helped to push forward the integration of the BiMW nanointerferometric biosensor into a future portable biosensing platform intended for on-site use

Rolled-up nanotechnology based on strain-engineering is a powerful tool to manufacture three-dimensional hollow structures made of virtually any kind of material on a large variety of substrates. The aim of this thesis is to address the key features of different on- and off-chip applications of rolled-up microtubes through modification of their basic framework. The modification of the framework pertains to the tubular structure, in particular the diameter of the microtube, and the material which it is made of, hence achieving different functionalities of the final rolled-up structure. The tuning of the microtube diameter which is adjusted to the individual size of an object allows on-chip studies of single cells in artificial narrow cavities, for example. Another modification of the framework is the addition of a catalytic layer which turns the microtube into a self-propelled catalytic micro-engine. Furthermore, the tuneability of the diameter can have applications ranging from nanotools for drilling into cells, to cargo transporters in microfluidic channels. Especially rolled-up microtubes based on low-cost and easy to deposit materials, such as silicon oxides, can enable the exploration of novel systems for several scientific topics. The main objective of this thesis is to combine microfluidic features of rolled-up structures with optical sensor capabilities of silicon oxide microtubes acting as optical ring resonators, and to integrate these into a Lab-on-a-Chip system. Therefore, a new concept of microfluidic integration is developed in order to establish an inexpensive, reliable and reproducible fabrication process which also sustains the optical capabilities of the microtubes. These integrated microtubes act as optofluidic refractrometric sensors which detect changes in the refractive index of analytes using photoluminescence spectroscopy. The thesis concludes with a demonstration of a functional portable sensor device with several integrated optofluidic sensors
Die auf verspannten Dünnschichten basierende „rolled-up nanotechnologie“ ist eine leistungsfähige Methode um dreidimensionale hohle Strukturen (Mikroröhrchen) aus nahezu jeder Art von Material auf einer großen Vielfalt von Substraten herzustellen. Ausgehend von der Möglichkeit der Skalierung des Röhrchendurchmessers und der Modifikation der Funktionalität des Röhrchens durch Einsatz verschiedener Materialien und Oberflächenfunktionalisierungen kann eine große Anzahl an verschiedenen Anwendungen ermöglicht werden. Eine Anwendung behandelt unter anderem on-chip Studien einzelner Zellen wobei die Mikroröhrchen, an die Größe der Zelle angepasste, Reaktionscontainer darstellen. Eine weitere Modifikation der Funktionalität der Mikroröhrchen kann durch das Aufbringen einer katalytischen Schicht realisiert werden, wodurch das Mikroröhrchen zu einem selbstangetriebenen katalytischen Mikro-Motor wird. Hauptziel dieser Arbeit ist es Mikrometer große optisch aktive Glasröhrchen herzustellen, diese mikrofluidisch zu kontaktieren und als Sensoren in Lab-on-a-Chip Systeme zu integrieren. Die integrierten Glasröhrchen arbeiten als optofluidische Ringresonatoren, welche die Veränderungen des Brechungsindex von Fluiden im inneren des Röhrchens durch Änderungen im Evaneszenzfeld detektieren können. Die Funktionsfähigkeit eines Demonstrators wird mit verschiedenen Flüssigkeiten gezeigt, dabei kommt ein Fotolumineszenz Spektrometer zum Anregen des Evaneszenzfeldes und Auslesen des Signals zum Einsatz. Die entwickelte Integrationsmethode ist eine Basis für ein kostengünstiges, zuverlässiges und reproduzierbares Herstellungsverfahren von optofluidischen Mikrochips basierend auf optisch aktiven Mikroröhrchen

Lab-on-a-chip (LOC) technologies entail the miniaturization of analytical systems, and the reduction of required sample and reagent volumes. LOC devices offer compact alternatives to classical instrumentation while delivering comparable performance and disposable formats. These aspects make disposable LOC a clear candidate to support distributed chemical sensing applications; however, the need of accessory services and readout obstructs the materialization of pervasively distributed LOC solutions. In this thesis methods and devices to solve this problem are investigated. A distinctive aspect of this work is the pursuit of solutions based on disposable LOC elements specifically conceived to exploit ubiquitous infrastructure for readout and evaluation. Consumer electronic devices, such as cell phones are ubiquitous platforms with residual capabilities that can be used for chemical sensing, if properly interfaced. This work investigates elements and tools needed to empower cell phones as readers of disposable LOC devices and commercial disposable tests. Access to flexible fabrication of LOC devices at low cost is an important requisite for testing ideas and implementing customized solutions. A first contribution in this thesis is the development of a platform for mask less fabrication of 3D microstructures, which coexists on a routine fluorescence microscope. This microscope projection lithography system (MPLS) is capable of controlled 3D micro structuring, including cavities and cantilever geometries, and the sealing of monolithic micro cavities to glass substrates as well as the connection to large scale service areas. This fabrication platform and other fabrication methods were used along this thesis to provide disposable optical and fluidic components. Besides custom-made LOC solutions there are well-established commercial disposable devices, which are essentially compatible with decentralized diagnosis, except for the use of specialized readers that confine them to medical centers. The implementation of high dynamic range (HDR) imaging with standard cell phones, using the phone screen to control exposure, shows that sensitivity and resolution can be boosted to permit robust evaluation of this type of disposable tests, in decentralized scenarios. Solutions employing commercial tests, which have not been designed for cell phone evaluation, are typically suboptimal and the investigation of customized LOC components occupies a central role in this thesis. Accordingly, one important aspect to evaluate LOC devices in compact configurations is to be able to image the LOC at a close distance from the phone camera, a condition for which phones cameras are not able to focus. In addition, different phone brands and models have different optical specifications, and a practical refocusing solution should adapt to all of them. In this work an adaptive lens concept, complemented by phone time-lapse acquisition, which can be integrated in disposable LOCs, is demonstrated. The implementation of sensitive detection methods, such as surface plasmon resonance (SPR), which is compatible with label free protocols that simplify sample conditioning, is central to the materialization of ubiquitous LOCs readable with cell phones. In this thesis a disposable optical coupler, conditioning illumination taken from the phone screen, is used to create an angle resolved SPR signal from a LOC, which is read with the phone front camera. Tested performance is comparable with commercial compact SPR modules and detection of β2 microglobulin, which is an established marker for cancer, inflammatory disorders, and kidney disease, is within the diagnostics range for blood and urine. Finally, fluorescence detection within classical LOC devices is tailored to be detectable with consumer cameras. In this case a disposable optical coupler and fluidics is designed to condition laser illumination into total internal reflection excitation, while DSLR and phone cameras capture optically separated fluorescence. The system configuration supports a broad dynamic range and HDR imaging enables localized resolution boost at selected detection ranges. Detection of free fucose, a diagnostic marker for liver cirrhosis and several cancer forms, is shown feasible with a HDR implementation, as one last example of practical LOC detection schemes for decentralized scenarios.

An accurate estimation of physiological buffer capacity and total titratable buffer concentration of blood can give a great deal of insight into the physiological stability of a patient and yet it remains an undervalued diagnostic marker. This thesis highlights the need for a lab-on-chip device to quantify buffer capacity of whole blood samples by estimating the total titratable buffer concentration. Buffer capacity is quantified by titrating the buffer to its end point using monoprotic acids. More sophisticated ways include electrolytic titration, i.e. producing a proton flux using electrodes in a controlled environment. This thesis looks at a novel approach to electrolytic (coulometric) titration by inhibiting the production of OH ions during electrolysis and titrating the sample due to the proton flux from the anode. By definition, is the amount of acid or base added to change the pH of 1 litre of buffer by 1 pH unit. The carbonic acid bicarbonate buffer system is the most important buffer that maintains the body's pH within a stable range. To quantify this buffer's total buffering concentration, it is important to know and indicate its titration end point which signifies the total exhaustion of all buffering constituents. Colorimetric indicators have been used to indicate this end point which can be quantified through cameras or spectrophotometric techniques. Using this novel coulometric titrator and the colorimetric end point detector, this thesis presents a portable lab-on-chip prototype to spectrophotometrically quantify total titratable buffer concentration. Clinically, this device could benefit patients with sickle cell disease, nephritic disease and those admitted in accident and emergency wards. This research work is aimed at presenting a proof-of-concept for a device that can titrate nano-litre samples and be able to detect the end point of a titration in a controlled way.

Abstract There has been considerable interest in developing microsensors integrated within lab-on-a-chip structures for the analysis of single cells; however, substantially less work has focused on developing "active" assays, where the cell‘s metabolic and physiological function is itself controlled on-chip. The heart attack is considered the largest cause of mortality and morbidity in the western world. Dynamic information during metabolism from a single heart cell is difficult to obtain. There is a demand for the development of a robust and sensitive analytical system that will enable us to study dynamic metabolism at single-cell level to provide intracellular information on a single-cell scale in different metabolic conditions (such as healthy or simulated unhealthy conditions). The system would also provide medics and clinicians with a better understanding of heart disease, and even help to find new therapeutic compounds. Towards this objective, we have developed a novel platform based on five individually addressable microelectrodes, fully integrated within a microfluidic system, where the cell is electrically stimulated at pre-determined rates and real-time ionic and metabolic fluxes from active, beating single heart cells are measured. The device is comprised of one pair of pacing microelectrodes, used for field-stimulation of the cell, and three other microelectrodes, configured as an enzyme-modified lactate microbiosensor, used to measure the amounts of lactate produced by the heart cell. The device also enables simultaneous in-situ microscopy, allowing optical measurements of single-cell contractility and fluorescence measurements of extracellular pH and cellular Ca2+ from the single beating heart cell at the same time, providing details of its electrical and metabolic state. Further, we have developed a robust microfluidic array, wherein a sensor array is integrated within an array of polydimethylsiloxane (PDMS) chambers, enabling the efficient manipulation of single heart cells and real-time analysis without the need to regenerate either working electrodes or reference electrodes fouled by any extracellular constituents. This sensor array also enables simultaneous electrochemical and optical measurements of single heart cells by integrating an enzyme-immobilized microsensor. Using this device, the fluorescence measurements of intracellular pH were obtained from a single beating heart cell whose electrical and metabolic states were controlled. The mechanism of released intracellular [H+] was investigated to examine extracellular pH change during contraction. In an attempt to measure lactate released from the electrically stimulated contracting cell, the cause of intracellular pH change is discussed. The preliminary investigation was made on the underlying relationship between intracellular pH and lactate from single heart cells in controlled metabolic states.

Monitoring microorganisms in natural water is central to understanding and managing risks to human health and ecosystems. Some phytoplankton can produce toxic blooms which are harmful to aquatic ecosystems and human health. Kariena brevis is responsible for Harmful Algal Blooms and produces brevetoxin which can lead to gastrointestinal and neurological problems in mammals. Traditional methods for Harmful Algal Bloom monitoring require sample collection and preservation for later study in laboratories where they are generally processed using microscopy which can take many hours or days. Laboratory equipment for this application has been adapted for ship-board use. Portable instrument systems that incorporate sample preparation and detection have been also developed for environmental applications. However, very few are suitable for deployment in the environment (either as a hand-held or in situ system) and often require laboratory infrastructure or personnel to facilitate sample collection and processing. Current in situ systems are large, expensive, and require expert users to operate them. Thus these existing systems do not provide marine science with the high spatial resolution data required to enable a better understanding of the diversity, function and community structure of marine microorganisms. Ideal in situ sensors should provide sample analysis over wide areas and at many depths for long periods of time. This remains a significant challenge. One possible solution is to develop numerous cheap sensors which could be incorporated into autonomous underwater vehicles or an argofloats network. Micro systems are excellent candidates as when mature, they could be mass produced to enable them to meet this particular spatial mapping requirement. The use of fully automatic and accurate micro total analysis systems, also known as lab-on-a-chip, can overcome the challenges of highly integrated in situ systems for incorporation into environmental monitoring vehicles and stations. Lab-on-a-chip technology appears well suited for environmental monitoring with its main advantages being the possibility of miniaturization, portability, reduced reagent consumption and automation. Molecular biology tools combined with microfluidic technology have been seen as a potential technical solution for in situ environmental applications. The purpose of this work has been to develop key functions in independent microchips that perform elements of a complete biological assay for ribonucleic acid phytoplankton metrology from the sample preparation to the detection step. Specifically the system is being developed to analyse the large subunit of the ribulose-bisphosphate carboxylase (rbcL) gene of phytoplankton Kariena brevis, a species responsible for Harmful Algal Blooms. This thesis reports the development of three lab-on-a-chip devices which perform microalga cell lysis, nucleic acid purification and real-time ribonucleic acid detection. The aim was to demonstrate proof-of concept for each device separately in order to obviate the need to tackle the complications of system integration (which remains a challenge), while understanding performance needed and comparing that achieved to the most likely scenarios for real-world applications. Future research should integrate these separate chips into an integrated single chip design to achieve fully automated chips with “sample-in” to “answer-out” capability.

Continuous progress in the nanotechnology field has allowed for the emergence of powerful, nanopore-based detection technology. Solid-state nanopores were developed for next-generation sequencing and single-molecule detection. They are advantageous over their biological counterpart because they offer robustness, stability, tunable pore size and the ability to be integrated within a microfluidic device. With all of these attractive attributes, solid-state nanopores are a top contender for point-of-care diagnostic technologies. However, hindering their performance is an inability to distinguish between small molecules, pore-clogging, and the detection rate's dependence on sample concentration. The concentration-dependent detection rate becomes particularly evident at low sample concentrations (<1 nM), sometimes taking hours for the nanopore to sense a single molecule because of diffusion. The inability to distinguish between small molecules can be addressed using DNA nanostructures; however, pore-clogging and variable detection rates hinder its potential in a clinical setting. This thesis proposes a microfluidic device design and methodology that seeks to mitigate pore-clogging and improve the detection rate for dilute samples. DNA coated microbeads will create a bead column within the microfluidic device and confine the target molecules to an extremely small (20 nL) volume. The sample can be washed, ridding the contaminants, and eluted on-chip, so the sample is purified and concentrated, affording a more reliable sensing performance. First, a magnetic microbead DNA assay was optimized off-chip, and the capture and release efficiencies were monitored using a Biotek™ Epoch™ 2 spectrophotometer (Chapter 2). Next, a novel microfluidic device design was optimized and validated to ensure precise sample manipulation (Chapter 3). Finally, the microbead assay was incorporated into the microfluidic device for sample concentration (Chapter 4). Fluorescence microscopy results suggest successful DNA elution from the microbeads within the microfluidic device, allowing for a 28.5 X concentration increase. This platform shows promise for sample preconcentration by reducing the starting DNA sample volume of 25 µL to 20 nL, which could improve the speed of solid-state nanopore sensing.

Surface acoustic wave (SAW) devices based on the piezoelectric principle have been used extensively in telecommunication applications over the last 20 years, but have recently shown promise in the area of biomedical applications due to their efficient micro-fluidic functions and highly sensitive and label-free detection of pathogens, bacteria, cells, DNA and proteins. There are two types of surface acoustic wave modes: i.e., Rayleigh SAW (R-SAW) and shear horizontal SAW (SH-SAW). R-SAW is widely used for microfluidics and sensing in dry conditions, whereas SH-SAW is mainly used for sensing in liquid conditions. This thesis firstly reviewed the current theoretical and research progress related to these devices and application within the biomedical fields to date, and then the SH-SAW was applied into a novel lab-on-chip combining both bio-sensing and micro-fluidic functions. Simulations of the SH-SAW propagation on 36o Y-cut LiTaO3 were undertaken. Results showed a weak vertical wave component, and at a 90° rotation cut, the crystal was able to support a vertical Rayleigh component showing mixed sensing and streaming possibilities on a single crystal. Experimental investigation of the SH-SAW identified the ability for the shear wave to support mixing, pumping, heating, nebulisation and ejection of sessile droplets on the surface of the crystal with a theoretical explanation for the behaviour presented. A comparison with a standard R-SAW devices made of 128o Y-cut LiNbO3 and sputtered ZnO films was performed. This novel behaviour of digital microfludics, i.e., using sessile droplet with the SH-SAW, demonstrated by this work offers the possibility to manufacture a fully integrated micro-fluidic bio-sensing platform using a single crystal to realise a range of micro-fluidic functions.

Dans cette thèse, nous nous intéressons à la problématique de l’intégration de capteurs plasmoniques performants et bas coût sur des dispositifs de type smartphone, en vue d’applications de diagnostic biomédical. A cette fin, nous proposons deux biocapteurs « smart ». Premièrement, un système de détection colorimétrique à base de nanoparticules d’or est mis en œuvre pour détecter de l’ADN. Le système intègre une détection synchrone logicielle mise en œuvre au sein du smartphone, où les signaux physiques transitent par la voie audio. Le processus de diagnostic prend moins de 15 minutes pour une limite de détection de 0.77 nM, approximativement 6 fois meilleure que la sensibilité usuelle d’un spectromètre UV-Vis conventionnel, à temps de mesure identique. Dans une seconde partie, un capteur à résonance plasmon de surface en configuration de Kretschmann, se distinguant par une sensibilité à la phase optique, est développé. Le design monolithique et compact repose sur un interféromètre à dédoublement latéral et une modulation de phase. Le contrôle et la lecture du prototype s’effectue également par smartphone. La modulation de phase est de type sinusoïdale et une sensibilité importante est obtenue, autour de 2,3 10-6 RIU avec une dynamique de 7 10-3 RIU, chiffres obtenus pour une puce optique standard et un temps d’intégration de 100 ms. Ce second dispositif est ensuite testé pour la détection de protéines (Troponine I cardiaque), en fonctionnalisant la surface par ADN Tro4
In this thesis, we investigate the possibility and potential for integration of portable optical biosensor for diagnostic purposes. To this end, we propose two “smart” biosensor systems. In the first part of this thesis, a DNA biosensor combining single-wavelength colorimetry and digital Lock-in Amplifier within a smartphone is proposed. Utilizing full advantage of audio channel and digital signal processing capacity of a smartphone, we have built a handheld DNA AuNp colorimetry biosensor. Based on the results, the diagnostic process takes only 15 minutes of reaction time while offering a limit of detection around 0.77 nM which is 6 times better than a desktop UV-Vis spectrometer.In second part of the thesis, a Shearing interferometer based Surface Plasmon Resonance (SiSPR) biosensor is proposed. SiSPR allows for phase sensitive detection on conventional Kretschmann configuration. Its monolithic design reduces optical parts, costs and allows portable application. The essence of SiSPR is a reflective layer in addition to plasmonic layer. To extract phase information from SiSPR, a sinusoidal phase modulation is achieved by modulation of the laser injection current. For a 100 ms measurement and a standard optical chip, the sensitivity of the SiSPR is around 2.3x10-6 RIU with a dynamic range of 7.0x10-3 RIU, which is better than amplitude SPR devices. Finally, Tro4 DNA surface modification on the SiSPR chip is demonstrated for future cardiac Troponin I diagnostic

La tecnologia lab-on-chip si sta sviluppando molto velocemente dati i numerosi ambiti di applicazioni nell’area biomedica, della sensoristica, per il controllo ambientale e della sicurezza. Da questo punto di vista, è una scommessa realizzare un laboratorio ottico all’interno di un unico chip. In questi chip dovrebbero essere presenti: la sorgente ottica (laser), gli elementi di controllo (guide d’onda, lenti, polarizzatori, etc.), il canale per l’analisi del campione e il sensore ottico. I vantaggi nello sviluppare questa tecnologia sono legati all’ottenimento di elevata sensibilità, la riduzione dei volumi da analizzare, costi ridotti e la trasportabilità del dispositivo. Al fine di ottenere questi risultati, è importante sviluppare un laser microfluidico appropriato che funge da sorgente luminosa all’interno di questo laboratorio ottico integrato in un unico chip. Questo lavoro è stato compiuto in collaborazione con il gruppo guidato dal Dott. Luigino Criante presso il Centro per le NanoScienze e Tecnologia dell’Istituto Italiano di Tecnologia (CNST-IIT). Durante questo lavoro sono state progettate differenti cavità in collaborazione con il gruppo dell’IIT dove tali dispositivi sono stati realizzati. Tutte le misure e i test delle caratteristiche dei dispositivi sono state effettuate presso il dipartimento di Scienze e Ingegneria della Materia, dell'Ambiente ed Urbanistica (SIMAU) dell’Università Politecnica delle Marche. In questa tesi vengono descritti i processi per la realizzazione e la caratterizzazione di diversi microlaser optofluidici basati su cavità Fabry-Perot e cavità emisferica realizzate attraverso due tecniche di fabbricazione: la microlavorazione con laser a femtosecondi e la tecnologia di stampa inkjet. Con queste tecnologie una cavità standard di tipo Fabry-Perot è stata integrata all’interno di un chip optofluidico. I microlaser sono stati testati con differenti coloranti laser come la Rodamina 6G, il Pirrometene e il DCM. I migliori risultati sono stati una emissione di linea inferiore a ~0.6nm e un fattore di qualità Q~10^3 ottenuto utilizzando come mezzo attivo una miscela di Rodamina 6G disciolta in alcol etilico ad una concentrazione di 5·mMol. L’emissione laser è stata rivelata ad una densità di energia di soglia pari a 1.8 μJ/mm^2, circa un ordine di grandezza inferiore allo stato dell’arte dei laser optofluidici. Tali prestazioni, unite alla stabilità meccanica e alla resistenza chimica di questi dispositivi realizzati completamente all’interno di un supporto in vetro, li rende dei promettenti candidati nello sviluppo di futuri chip microfluidici in applicazioni biosensoristiche.
The lab-on-chip technology is growing very quickly because of the wide range applications in the biomedical area and sensing for environment control and security. In this frame, it is a challenge to realize a complete optical lab in a single chip. This chip should contain the optical source (laser), the optical control elements (waveguides, lenses, polarizers, etc.), the sample analysis channels and the light detector. The advantages of developing of such technology will be to get high sensitivity, use of low testing volumes, low cost and device portability. In order to get these results, it is important to develop a suitable microfluidic laser that acts as a light source in this optical lab in a chip. This work has been carried out in collaboration with the group led by Dott. Luigino Criante at the Center for Nano Science and Technology at Italian Institute of Technology (CNEST-IIT). We have designed different cavities in collaboration with the IIT group where devices were fabricated. All the measurements and tests of the characteristics of the devices were performed at the SIMAU Department of the Polytechnic University of Marche. In this thesis work has been reported the realization and characterization of different optofluidic microlasers based on Fabry-Perot and hemispherical cavity fabricated by exploiting two fabrication techniques: the femtosecond laser micromachining and the inkjet printing technology. In this way a standard Fabry-Perot cavity has been integrated into an optofluidic chip. The microlasers were tested with different laser dyes such as Rhodamine 6G, Pyrromethene and DCM. The best result was an emission bandwidth below ~0.6nm and a quality factor Q~10^3 measured when using Rhodamine 6G dissolved in ethanol at concentration of 5·mMol as active medium. Laser emission was detected at a threshold energy density as low as 1.8 μJ/mm^2 about one order of magnitude lower than state-of-the-art optofluidic lasers. These performances and mechanical and chemical robustness of these devices fully embedded in glass make them promising for future development in optofluidic chips to be exploited in biosensing applications.

Thesis (Ph.D.)--University of Cincinnati, 2006.
Advisor: Dr. Chong H. Ahn. Title from electronic thesis title page (viewed Dec. 22, 2009). Keywords: Blood Separation; Lab-on-a-chip; Point-of-care; On chip pressure actuator. Includes abstract. Includes bibliographical references.

Instrument miniaturization is led by the desire to perform rapid diagnosis in remote areas with high throughput and low cost. In addition, miniaturized instruments hold the promise of consuming small sample volumes and are thus less prone to cross-contamination. Gas chromatography (GC) is the leading analytical instrument for the analysis of volatile organic compounds (VOCs). Due to its wide-ranging applications, it has received great attention both from industrial sectors and scientific communities. Recently, numerous research efforts have benefited from the advancements in micro-electromechanical system (MEMS) and nanotechnology based solutions to miniaturize the key components of GC instrument (pre-concentrator/injector, separation column, valves, pumps, and the detector). The purpose of this dissertation is to address the critical need of developing a micro GC system for various field- applications. The uniqueness of this work is to emphasize on the importance of integrating the basic components of μGC (including sampling/injection, separation and detection) on a single platform. This integration leads to overall improved performance as well as reducing the manufacturing cost of this technology. In this regard, the implementation of micro helium discharge photoionization detector (μDPID) in silicon-glass architecture served as a major accomplishment enabling its monolithic integration with the micro separation column (μSC). For the first time, the operation of a monolithic integrated module under temperature and flow programming conditions has been demonstrated to achieve rapid chromatographic analysis of a complex sample. Furthermore, an innovative sample injection mechanism has been incorporated in the integrated module to present the idea of a chip-scale μGC system. The possibility of using μGC technology in practical applications such as breath analysis and water monitoring is also demonstrated. Moreover, a nanotechnology based scheme for enhancing the adsorption capacity of the microfabricated pre-concentrator is also described.
Ph. D.

In dieser Arbeit wird eine neue Methode und ein neuartiges FET -Sensorelement zum Nachweis von Flüssigkeitsbewegungen vorgestellt, das zudem bei Bedarf auch als Chemo- oder Biosensor fungieren kann. Das Einsatzspektrum von FET-basierten Sensoren in Lab on a Chip-Systemen wird dadurch entscheidend erweitert. Bei dem entwickelten FET-Sensor Bauelement handelt es sich um einen normally-on n-leitenden Dünnschichtfeldeffekttransistor mit Ti-Au-Kontakten, basierend auf Silicon-on-Insulator- Substraten, wobei das natürliche Oxid des Siliziumfilms als Schnittstelle zum Elektrolyten bzw. zur Flüssigkeit verwendet wird. Der mit 10exp16 Bor Atomen pro cm³ p-dotierte Siliziumdünnfilm hat eine Dicke von nur 55 nm und ist durch eine 95 nm dicke Siliziumdioxidschicht vom darunterliegenden Siliziumsubstrat von 600 µm Dicke elektrisch isoliert. Aufgrund der geringen Schichtdicke durchdringt die feldempfindliche Raumladungs- bzw. Verarmungszone die gesamte Dünnschicht, so dass durch Anlegen einer Backgatespannung am Substrat der spezifische Widerstand und die Empfindlichkeit des Bauelements eingestellt werden können. Grundlegende ISFET-Funktionalitäten wie die Empfindlichkeit auf Änderungen der Ionenstärke und des pH-Wertes werden nachgewiesen und ein ENFET-Glukosesensor realisiert. Zudem wird im Hinblick auf die Separation von Emulsionen der Nachweis erbracht, dass die Benetzung mit Hexan und Toluol eine Änderung der spezifischen Leitfähigkeit bewirkt, und die Empfindlichkeit des Bauelements nach Beschichtung mit einem hydrophoben Methacrylatcopolymerfilm erhalten bleibt. Hinsichtlich der Verwendung des FET-Sensor Bauelements zum Nachweis von Flüssigkeitsbewegungen wird zunächst ein theoretisches Modell entwickelt, dessen Kernaussage ist, dass sich in einem rechteckigen Kanal der relative Bedeckungsgrad mit Flüssigkeit direkt proportional zum Drainstrom des FET-Sensors verhält. Basierend auf diesem theoretischen Modell, welches experimentell belegt wird, können mittels eines einzelnen FET-Sensors Füllstand und Füllgeschwindigkeit bzw. bei bekannter Füllgeschwindigkeit Kapillarvolumen und Kapillargeometrie bestimmt werden. Abweichungen von der direkten Proportionalität erlauben zudem, Rückschlüsse auf die Benetzungseigenschaften der Kapillaren und die Dynamik an der Halbleitergrenzfläche zu ziehen. Ist ein Sensorelement vollständig mit Flüssigkeit bedeckt, wird mittels Lösungsmitteltropfen als Markerobjekten die Strömungsgeschwindigkeit bestimmt. Ändert sich die Ionenkonzentration im Elektrolyten als Funktion der Strömungsgeschwindigkeit, so kann die Strömungsgeschwindigkeit durch Messung der Ionenkonzentration mittels FET-Sensor ebenfalls ermittelt werden. Als wichtigster Demonstrator für die Verwendung des FET-Sensors wird ein komplexes Lab on a Chip-System zur Separation von Emulsionen auf chemisch strukturierten Oberflächen entwickelt, bei dem der Separationsvorgang mittels FET-Sensorarray verfolgt werden kann. Zur einfachen Herstellung chemisch modifizierter Oberflächen für die Separationsexperimente werden die Abscheidung von nanoskaligen hydrophoben Methacrylatcopolymerfilmen und die selektive Fluorsilanisierung von Oberflächen sowie deren Lösungsmittelbeständigkeit in Wasser, Toluol und Aceton untersucht. Dabei zeigt sich, dass die Hydrophobie nach Lösungsmittelbehandlung weitestgehend erhalten bleibt, Wasserrückstände im Methacrylatfilm aber zu einer reversiblen Schichtdegradation führen können. Als Modellsystem werden Hexan-Wasser- bzw. Toluol-Wasser-Emulsionen verwendet, die auf Oberflächen getrennt werden, deren eine Seite hydrophil, und deren andere Seite hydrophob ist (Stufengradient). Der Separationsprozess beruht auf der großen Affinität des Wassers hin zu polaren Oberflächen, wobei das wenig selektive Lösungsmittel zur unpolaren Seite gedrängt wird. Zur Erlangung eines tieferen Verständnisses des Prozesses werden die Tropfenkoaleszenz und der Einfluss geometrischer Beschränkungen untersucht. Die Versuche werden sowohl auf offenen Oberflächen als auch im Spalt, unter Verwendung von hydrophilen und hydrophoben Oberflächen, durchgeführt. Es zeigt sich, dass sich die Dynamik der Tropfenkoaleszenz im Spalt umgekehrt zur Dynamik auf offenen Oberflächen verhält. Dies wird mittels eines hierzu entwickelten theoretischen Modells erklärt, welches die Minimierung der Oberflächenenergie und Hystereseeffekte einbezieht. Das Lab on a Chip-System schließlich besteht aus einem mit Siliziumnitrid beschichteten FET-Sensorchip, auf den eine Separationszelle aufgeklebt ist. Neben dem Einlass für die Emulsion ist ein weiterer Einlass vorhanden, durch den Salzsäure für eine pH-Reaktion zugegeben werden kann. Der gesamte Separationsprozess sowie die anschließende pH-Reaktion, lassen sich bequem am PC anhand der Änderung der Stromstärke der einzelnen Sensoren verfolgen und analysieren. Wichtige Ergebnisse hier sind: 1) Mittels eines quasi 1-dimensionalen Sensorarrays kann der Verlauf einer Flüssigkeitsfront in einem 2-dimensionalen Areal überwacht bzw. dargestellt werden. 2) Anhand der Signatur des Signalverlaufs bei pH-Änderung und Flüssigkeitsbewegung, können beide Prozesse unterschieden werden. Der Sensor kann also zum Nachweis von Flüssigkeitsbewegungen und zugleich als Chemosensor eingesetzt werden. Es wurde also nicht nur ein neuartiges, äußerst robustes, chemikalienbeständiges und biokompatibles Multifunktionssensorelement mit Abmessungen im Mikrometer- bis Millimeterbereich entwickelt, sondern auch eine neue Methode entwickelt, mit der es möglich ist, sowohl (bio-)chemische Reaktionen als auch die Bewegung von Flüssigkeiten in Lab on a Chip-Systemen nachzuweisen.

Many scientists and engineers are turning to lab-on-a-chip systems for cheaper and high throughput analysis of chemical reactions and biomolecular interactions. In this work, we developed several lab-on-a-chip modules based on novel manipulations of individual microbeads inside microchannels. The first manipulation method employs arrays of soft ferromagnetic patterns fabricated inside a microfluidic channel and subjected to an external rotating magnetic field. We demonstrated that the system can be used to assemble individual beads (1-3µm) from a flow of suspended beads into a regular array on the chip, hence improving the integrated electrochemical detection of biomolecules bound to the bead surface. In addition, the microbeads can follow the external magnet rotating at very high speeds and simultaneously orbit around individual soft magnets on the chip. We employed this manipulation mode for efficient sample mixing in continuous microflow. Furthermore, we discovered a simple but effective way of transporting the microbeads on-chip in the rotating field. Selective transport of microbeads with different size was also realized, providing a platform for effective sample separation on a chip. The second manipulation method integrates magnetic and dielectrophoretic manipulations of the same microbeads. The device combines tapered conducting wires and fingered electrodes to generate desirable magnetic and electric fields, respectively. By externally programming the magnetic attraction and dielectrophoretic repulsion forces, out-of-plane oscillation of the microbeads across the channel height was realized. Furthermore, we demonstrated the tweezing of microbeads in liquid with high spatial resolutions by fine-tuning the net force from magnetic attraction and dielectrophoretic repulsion of the beads. The high-resolution control of the out-of-plane motion of the microbeads has led to the invention of massively parallel biomolecular tweezers.