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Molecular diagnostics is a fast growing field with new technologies being developed constantly. There is a demand for more sophisticated molecular tools able to detect a multitude of molecules on a single molecule level with high specificity, able to distinguish them from other similar molecules. This becomes very important for infectious diagnostics with the increasing antibiotic resistant viruses and bacteria, in gene based diagnostics and for early detection and more targeted treatments of cancer. For increased sensitivity, simplicity, speed and user friendliness, novel readouts are emerging, taking advantage of new technologies being discovered in the field of nanotechnology.  This thesis, based upon four papers, examines two novel electrical readouts for amplified single molecule detection. Target probing is based upon the highly specific amplification technique rolling circle amplification (RCA). RCA enables localized amplification resulting in a long single stranded DNA molecule containing tandem repeats of the probing sequence as product. Paper I demonstrates sensitive detection of bacterial genomic DNA using a magnetic nanoparticles-based substrate-free method where as few as 50 bacteria can be detected. Paper II illustrates a new sensor concept based on the formation of conducting molecular nanowires forming a low resistance circuit. The rolling circle products are stretched to bridge an electrode gap and upon metallization the resistance drops by several orders of magnitude, resulting in an extremely high signal to noise ratio. Paper III explores a novel metallization technique, demonstrating the efficient incorporation of boranephosphonate modified nucleotides during RCA.  In the presence of a silver ion solution, defined metal nanoparticles are formed along the DNA molecule with high spatial specificity. Paper IV demonstrates the ability to manipulate rolling circle products by dielectrophoresis. In the presence of a high AC electric field the rolling circle products stretch to bridge a 10 µm electrode gap.

We have employed a differential detector combined with dielectrophoretic (DEP) translation in a microfluidic channel to monitor dielectric response of single cells and particularly to track phenomenon related to apoptosis. Two different cell lines were studied; Chinese hamster ovary cells (CHO) and MRC5 cells. Dielectric response was quantified by a factor called Force Index. Force Index was studied statistically to identify apoptotic subpopulations. Another direction of this work was to monitor changes in the cytoplasm conductivity following inhibition of mitochondrial ATP production by Oligomycin. To make the DEP response mostly sensitive to the cytoplasm conductivity, medium conductivity and DEP frequency were adjusted such that Clausius Mossotti factor and hence DEP response become less sensitive to cell radius. Chinese hamster ovary cells were used in this work and the impact of different concentrations of Oligomycin has been studied. We show that following exposure to Oligomycin at 8 μg/ml, cytoplasm conductivity drops. The majority of the changes takes place within one hour of exposure to the drug. Furthermore, double shell models has been used to estimate cytoplasm conductivity in a medium with conductivity of 0.42 S/m and the drop in the cytoplasm conductivity following treatment with Oligomycin was estimated to be ≈ 0.16 S/m. The magnitude of the decrease in the cytoplasm conductivity is evidence that Glycolysis is active as an energy production pathway within the cell. This approach can be used to quantify Glycolysis versus mitochondria ATP production which has an application in Warburg effect in cancer cells and monitoring bioprocesses.

The extraordinary properties of the single wall carbon nanotubes (SWCNTs) have stimulated an enormous amount of research towards the realization of SWCNT-based products for different applications ranging form nanocomposites to nanoelectronics. Their high charge mobility, exceedingly good current-carrying capacities and ability to be either semiconducting or metallic render them ideal building blocks for nanoelectronics. For nanoelectronic applications, either individual or parallel aligned SWCNTs are advantageous. Moreover, closely packed arrays of parallel SWCNTs are required in order to sustain the relatively large currents found in high frequency devices. Two key areas still require further development before the realization of large-scale nanoelectronics. They are the reproducible control of the nanotubes spatial position/orientation and chiral management. In terms of nanotube orientation, different techniques have been demonstrated for the fabrication of horizontally aligned SWCNTs with either post synthesis routes (e.g. dielectrophoresis and Langmuir-Blodgett approach) or direct growth (e.g. chemical vapor deposition (CVD)). The low temperature of the production process, allowing the formation of aligned nanotubes on pretty much any substrate, is the main advantage of the post synthesis routes, while the poor levels of reproducibility and spatial control, and the limited quality of the aligned tubes due to the inherently required process steps are limitations. The simplicity, up-scalability, along with the reproducible growth of clean high quality SWCNTs with well-controlled spatial, orientation and length, make CVD the most promising for producing dense horizontally well-aligned SWCNTs. These CVD techniques suffer some drawbacks, namely, that because they are synthesized using catalyst particles (metals or non-metals) the catalyst material can contaminate the tubes and affect their intrinsic properties. Thus, the catalyst-free synthesis of aligned SWNT is very desirable. This thesis comprises detailed and systematic experimental investigations in to the fabrication of horizontally aligned SWCNTs using both post growth (Dielectrophoresis) and direct growth (CVD) methods. Both catalyst-assisted and catalyst-free SWCNTs are synthesized by CVD. While metallic nanoparticles nucleate and grow SWCNTs, opened and activated fullerene structures are used for all carbon catalyst-free growth of single wall and double wall carbon nanotubes. The systematic studies allow for a detailed understanding of the growth mechanisms of catalyst and catalyst-free grown SWCNTs to be elucidated. The data significantly advances our understanding of horizontally aligned carbon nanotubes by both post synthesis alignment as well as directly as-synthesized routes. Indeed, the knowledge enables such tubes to be grown in high yield and with a high degree of special control. It is shown, for the first time, how one can grow horizontally aligned carbon nanotubes in crossbar configurations in a single step and with bespoke crossing angles. In addition, the transport properties of the aligned tubes at room temperature are also investigated through the fabrication of devices based on these tubes. / Die außergewöhnlichen Eigenschaften von einwandigen Kohlenstoffnanoröhren (engl. single wall carbon nanotubes, SWCNTs) haben bemerkenswerte Forschungsaktivitäten zur Verwirklichung von auf SWCNTs basierenden Anwendungen für verschiedene Bereiche, die von Nanokompositen bis hin zur Nanoelektronik reichen, stimuliert. Ihre hohe Ladungsträgermobilität und die außerordentlichen hohen Ladungsdichten, die in SWCNTs erreicht werden können sowie ihre Eigenschaft, entweder halbleitend oder metallisch zu sein, machen sie zu idealen Konstituenten von nanoelektronischen Schaltkreisen. Für Anwendungen in der Nanoelektronik sind entweder einzelne oder parallel angeordnete SWCNTs vorteilhaft. Darüber hinaus sind dicht gepackte Anordnungen von SWCNTs erforderlich, um die relativ hohen Ströme in Hochfrequenzbauelementen zu transportieren. Für eine erfolgreiche Realisierung von großskaligen nanoelektronischen Bauteilen, die auf SWCNTs basieren, sind noch zwei enorm wichtige Kernprobleme zu lösen, die weitere Forschungsanstrengungen erfordern: die reproduzierbare und verlässliche Kontrolle der räumlichen Positionierung und Orientierung der Nanoröhren sowie die Kontrolle der Chiralität der einzelnen SWCNTs. Hinsichtlich der Orientierung der Nanoröhren kann die horizontal parallele Ausrichtung von SWCNTs mit verschiedenen Techniken erreicht werden. Diese setzen entweder nach dem eigentlichen Wachstum der Röhren ein (Post-Synthese-Methoden wie z.B. Dielektrophorese oder Langmuir-Blodgett-Techniken) oder erreichen direkt während des Wachstums (z.B. durch Chemical-Vapor-Deposition-Methoden (CVD)) die parallele Anordnung. Durch die niedrigen Prozesstemperaturen, die während des Herstellungsprozesses erforderlich sind, erlauben die nach der eigentlichen Synthese stattfindenden Ausrichtungsmethoden die parallele Anordnung von Nanoröhren auf nahezu jedem Substrat, jedoch stellen die geringe Reproduzierbarkeit dieser Prozesse, die schwierige Kontrollierbarkeit der räumlichen Anordnung und die limitierte Qualität der ausgerichteten Röhren aufgrund der erforderlichen Prozessschritte natürliche Beschränkungen dieser Techniken dar. Die einfache Durchführung und ihre Skalierbarkeit, zusammen mit dem reproduzierbaren Wachstum qualitativ sehr hochwertiger SWCNTs mit hoher Kontrolle von räumlicher Anordnung, Orientierung und Länge machen die CVD-Methode zur erfolgversprechendsten Technik für die Herstellung von dichtgepackten hochparallelen horizontalen Anordnungen von SWCNTs. Diese CVD-Ansätze weisen jedoch auch einige Nachteile auf, die in den bei der Synthese verwendeten Katalysatorpartikeln (metallisch oder nicht-metallisch) begründet liegen, da das Katalysatormaterial die Röhren kontaminieren und dadurch ihre intrinsischen Eigenschaften beeinflussen kann. Daher ist eine katalysatorfreie Synthesemethode für ausgerichtete SWCNTs ein höchst erstrebenswertes Ziel. Die vorliegende Arbeit beschreibt detaillierte und systematische experimentelle Untersuchungen zur Herstellung von horizontalen, parallel ausgerichteten Anordnungen von SWCNTs unter Verwendung von Methoden, die sowohl nach dem eigentlichen Wachstum der Nanoröhren (Dielektrophorese) als auch während des Wachstums ansetzen (CVD). Bei den CVD-Methoden werden sowohl solche, die auf der Verwendung von Katalysatoren basieren, als auch katalysatorfreie Techniken verwendet. Während metallische Nanopartikel den Ausgangspunkt für das Wachstum von SWCNTs darstellen, werden geöffnete und aktivierte Fullerenstrukturen verwendet, um das katalysatorfreie Wachstum von reinen ein- oder mehrwandigen Nanoröhren zu erreichen. Die systematischen Untersuchungen ermöglichen ein tiefgehendes Verständnis der Wachstumsmechanismen von SWCNTs, die unter Verwendung von Katalysatoren oder katalysatorfrei erzeugt synthetisiert wurden. Die erzielten Ergebnisse erhöhen in einem hohen Maß das Verständnis der Herstellung von horizontal parallel angeordneten Nanoröhren, die durch Post-Synthese-Methoden oder direkt während des Wachstumsprozesses ausgerichtet wurden. Die erzielten Einsichten erlauben die Herstellung solcher Strukturen mit hoher Ausbeute und mit einem hohen Maß an räumlicher Kontrolle der Anordnung. Zum ersten Male kann ein Verfahren präsentiert werden, mit dem horizontal parallel angeordnete Nanoröhren in gekreuzten Strukturen mit wohldefinierten Kreuzungswinkeln hergestellt werden können. Zusätzlich werden die Transporteigenschaften von parallel ausgerichteten Nanoröhren bei Raumtemperatur, durch die Herstellung von auf den dargestellten Strukturen basierenden Bauelementen, untersucht.

Les travaux de cette thèse portent sur l’idée de démontrer que la croissance des nanofils entre deux électrodes prédéfinies et plus particulièrement la croissance horizontale à l’intérieur des tranchées d’oxyde peut être utilisée dans l’optique d’une intégration 3D. Cela permettrait donc à terme de pouvoir directement fabriquer les couches actives semi-conductrices d’un transistor MOS dans les niveaux supérieurs d’une puce CMOS tout en respectant le budget thermique, et sans avoir recours à des étapes de collage de puces. Au cours de ce projet de recherche, nous nous sommes intéressés en premier lieu au développement et à l’optimisation du procédé « nanodamascène » mis en place pour guider des nanofils SiGe dans des tranchées d’oxyde directement sur un substrat SiO2/Si. À part de cette technique d’intégration, nous avons aussi utilisé la technique de diélectrophorèse pour orienter et localiser des nanofils dispersés dans une solution liquide entre des électrodes prédéfinies. Les résultats de ces études ont permis en premier lieu de fabriquer des transistors à canaux nanofils sur l’oxyde, avec un objectif final de montrer la possibilité d’établir un transistor dans le BEOL d’une puce CMOS. / The work of this thesis deals with the idea of demonstrating that the growth of nanowires between two predefined electrodes and more particularly the horizontal growth inside the oxide trenches can be used in the context of a 3D integration. This would help to directly manufacture the active semiconductor layers of a MOS transistor in the upper levels of a CMOS chip while respecting the thermal budget, and without resorting to chip bonding steps. During this project, we focused on the development and optimization of the "nanodamascene" process implemented to guide SiGe nanowires in oxide trenches directly on SiO2/Si substrate. Apart from this integration technique, we have also used the dielectrophoresis technique to orient and localize nanowires dispersed in a liquid solution between predefined electrodes. The results of these studies made it possible in the first place to manufacture nanowire channel transistors on the oxide, with a goal of which will be to demonstrate the possibility of establishing a transistor in the BEOL of a CMOS chip.

L'objectif de ces travaux de thèse a été de démontrer la possibilité, en se basant sur une approche prédictive, de contrôler avec précision la fonctionnalisation d'un matériau composite, d'isotrope à anisotrope, sous l'application d'un champ électrique. Ces derniers matériaux présentent en effet un fort potentiel pour des applications futures telles que des condensateurs intégrés ou bien encore des composites conducteurs thermiques. Une première approche théorique des différentes forces et mécanismes entrant en jeux lors de l'élaboration de composites anisotropes par chaînage a permis d'identifier les paramètres impactant le procédé d'élaboration. A la suite de cette étude théorique, un modèle de formation de chaînes de particules sous champ électrique a été développé afin de prédire la dynamique de chaînage. Le modèle choisi (méthode moment dipolaire effectif) a permis la simulation de plus de 4500 particules. Les paramètres ayant au préalable été identifiés ont ensuite été mesurés. Pour la permittivité des particules, une méthode de mesure diélectrophorétique a été développée, ce qui est une première dans le cas de particules céramiques. L'élaboration des composites anisotropes a été couplé avec un suivi novateur, en temps réel, d'un marqueur (permittivité) de la formation de chaînes, permettant d'obtenir la dynamique de structuration des particules. Afin de valider l'aspect prédictif du modèle numérique, une comparaison a été effectuée entre la dynamique mesurée et simulée. Les résultats obtenus ont démontré une très bonne fiabilité des prédictions du modèle, même si des progrès sont encore réalisables aux faibles taux de chargement. Dans un dernier temps, une preuve de concept a été démontrée, de la réalisation de composites anisotropes dont les particules sont alignées perpendiculairement au champ électrique. / This study was aimed to demonstrate the possibility, based on a predictive approach, to tailor the structure of a composite from isotropic to anisotropic when applying an electric field. This composites have great potential for future applications such as embed capacitors or thermally conductive composites. A theoretical approach of the forces and mechanisms acting in the elaboration of anisotropic composites by chaining allowed identifying the key parameters. Based on this approach a model of particle chaining under electric field was established to predict the structuration dynamics. This model (effective dipole moment) allowed simulating more than 4500 particles. The parameters previously identified were then measured, and for the particle permittivity, a dielectrophoretic measurement method was developed, which was a first for ceramic particles. The elaboration of anisotropic composites was coupled to a novel on-line monitoring of a chaining marker (permittivity), allowing to obtain the structuration dynamics. To validate the predictive aspect of the model, experimental and numerical dynamics were compared showing the robustness and accuracy of the model, even if improvement is still possible at low filler content. In the last part, a proof of concept was demonstrated of the elaboration of anisotropic composites with fillers oriented normally to the direction of the electric field.

abstract: Biological fluids contain information-rich mixtures of biochemicals and particles such as cells, proteins, and viruses. Selective and sensitive analysis of these fluids can enable clinicians to accurately diagnose a wide range of pathologies. Fluid samples such as these present an intriguing challenge to researchers; they are packed with potentially vital information, but notoriously difficult to analyze. Rapid and inexpensive analysis of blood and other bodily fluids is a topic gaining substantial attention in both science and medicine. Current limitations to many analyses include long culture times, expensive reagents, and the need for specialized laboratory facilities and personnel. Improving these tests and overcoming their limitations would allow faster and more widespread testing for disease and pathogens, potentially providing a significant advantage for healthcare in many settings. Both gradient separation techniques and dielectrophoresis can solve some of the difficulties presented by complex biological samples, thanks to selective capture, isolation, and concentration of analytes. By merging dielectrophoresis with a gradient separation-based approach, gradient insulator dielectrophoresis (g-iDEP) promises benefits in the form of rapid and specific separation of extremely similar bioparticles. High-resolution capture can be achieved by exploiting variations in the characteristic physical properties of cells and other bioparticles. Novel implementation and application of the technique has demonstrated the isolation and concentration of blood cells from a complex biological sample, differentiation of bacterial strains within a single species, and separation of antibiotic-resistant and antibiotic-susceptible bacteria. Furthermore, this approach allows simultaneous concentration of analyte, facilitating detection and downstream analysis. A theoretical description of the resolving capabilities of g-iDEP was also developed. This theory explores the relationship between experimental parameters and resolution. Results indicate the possibility of differentiating particles with dielectrophoretic mobilities that differ by as little as one part in 100,000,000, or electrophoretic mobilities differing by as little as one part in 100,000. These results indicate the potential g-iDEP holds in terms of both separatory power and the possibility for diagnostic applications. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2015

abstract: Efficient separation techniques for organelles and bacteria in the micron- and sub-micron range are required for various analytical challenges. Mitochondria have a wide size range resulting from the sub-populations, some of which may be associated with diseases or aging. However, traditional methods can often not resolve within-species size variations. Strategies to separate mitochondrial sub-populations by size are thus needed to study the importance of this organelle in cellular functions. Additionally, challenges also exist in distinguishing the sub-populations of bio-species which differ in the surface charge while possessing similar size, such as Salmonella typhimurium (Salmonella). The surface charge of Salmonella wild-type is altered upon environmental stimulations, influencing the bacterial survival and virulence within the host tissue. Therefore, it is important to explore methods to identify the sub-populations of Salmonella. This work exploits insulator-based dielectrophoresis (iDEP) for the manipulation of mitochondria and Salmonella. The iDEP migration and trapping of mitochondria were investigated under both DC and low-frequency AC conditions, establishing that mitochondria exhibit negative DEP. Also, the first realization of size-based iDEP sorting experiments of mitochondria were demonstrated. As for Salmonella, the preliminary study revealed positive DEP behavior. Distinct trapping potential thresholds were found for the sub-populations with different surface charges. Further, DEP was integrated with a non-intuitive migration mechanism termed absolute negative mobility (ANM), inducing a deterministic trapping component which allows the directed transport of µm- and sub-µm sized (bio)particles in microfluidic devices with a nonlinear post array under the periodic action of electrokinetic and dielectrophoretic forces. Regimes were revealed both numerically and experimentally in which larger particles migrate against the average applied force, whereas smaller particles show normal response. Moreover, this deterministic ANM (dANM) was characterized with polystyrene beads demonstrating improved migration speed at least two orders of magnitude higher compared to previous ANM systems with similar sized colloids. In addition, dANM was induced for mitochondria with an AC-overlaid waveform representing the first demonstration of ANM migration with biological species. Thus, it is envisioned that the efficient size selectivity of this novel migration mechanism can be employed in nanotechnology, organelle sub-population studies or fractionating protein nanocrystals. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2015

abstract: Microfluidics has shown great potential in rapid isolation, sorting, and concentration of bioparticles upon its discovery. Over the past decades, significant improvements have been made in device fabrication techniques and microfluidic methodologies. As a result, considerable microfluidic-based isolation and concentration techniques have been developed, particularly for rapid pathogen detection. Among all microfluidic techniques, dielectrophoresis (DEP) is one of the most effective and efficient techniques to quickly isolate and separate polarizable particles under inhomogeneous electric field. To date, extensive studies have demonstrated that DEP devices are able to precisely manipulate cells ranging from over 10 μm (mammalian cells) down to about 1 μm (small bacteria). However, very limited DEP studies on manipulating submicron bioparticles, such as viruses, have been reported. In this dissertation, rapid capture and concentration of two different and representative types of virus particles (Sindbis virus and bacteriophage M13) with gradient insulator-based DEP (g-iDEP) has been demonstrated. Sindbis virus has a near-spherical shape with a diameter ~68 nm, while bacteriophage M13 has a filamentous shape with a length ~900 nm and a diameter ~6 nm. Under specific g-iDEP experimental conditions, the concentration of Sindbis virus can be increased two to six times within only a few seconds, using easily accessible voltages as low as 70 V. A similar phenomenon is also observed with bacteriophage M13. Meanwhile, their different DEP behavior predicts the potential of separating viruses with carefully designed microchannels and choices of experimental condition. DEP-based microfluidics also shows great potential in manipulating blood samples, specifically rapid separations of blood cells and proteins. To investigate the ability of g-iDEP device in blood sample manipulation, some proofs of principle work was accomplished including separating two cardiac disease-related proteins (myoglobin and heart-type fatty acid binding protein) and red blood cells (RBCs). Consistent separation was observed, showing retention of RBCs and passage of the two spiked protein biomarkers. The numerical concentration of RBCs was reduced (~70 percent after one minute) with the purified proteins available for detection or further processing. This study explores and extends the use of the device from differentiating similar particles to acting as a sample pretreatment step. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2017

Orientadores: Stanislav Alexandrovich Moshkalev, Mario Antonio Bica de Moraes / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-08-11T00:23:59Z (GMT). No. of bitstreams: 1 LeonEras_JorgeAugusto_D.pdf: 28434284 bytes, checksum: 042fbff5bb19c08014235156fdc57ee4 (MD5) Previous issue date: 2007 / Resumo: Não informado / Abstract: Not informed. / Doutorado / Física da Matéria Condensada / Doutor em Ciências