Exploring and understanding how the smallest scale features of a cell affect biochemical reactions has always been a challenge. Nanoscale fabrication advancements have allowed scientists to create small volume reaction containers that resemble the physical scale of cell membranes. Engineers seek to use biological design principles to manipulate information and import new functionality to such synthetic devices, which in turn, play a crucial role in allowing them to explore the effects of physical transport and extreme conditions of temperature and pH on reaction systems. Engineered reaction containers can be physically and chemically defined to control the flux of molecules of different sizes and charge. The design and testing of such a container is described here. It has a volume of 19 pL and has defined slits of 10-200 nm. The device successfully contains DNA and protein molecules and has been used to conduct and analyze enzyme reactions under different substrate concentrations and a continuous cell-free protein synthesis. The effect of DNA concentration and slit size on protein yield is also discussed. Glucose oxidase and horseradish peroxidase were loaded in the small volume container and fed with a solution containing glucose and Amplex Red™ to produce Resorufin. Fluorescent microscopy was used to monitor the reaction, which was carried out under microfluidic control. Enzyme kinetics were characterized and compared with conventional scale results. Continuous cell free protein synthesis in arrays of nanoporous, picoliter volume containers has also been achieved. A multiscale fabrication process allows for the monolithic integration of the containers and an addressable microfluidic network. Synthesis of enhanced green fluorescent protein (eGFP) in the nanoporous containers continues beyond 24 hours and yields more than twice the amount of protein, on a per volume basis, than conventional scale batch reactions. These picoliter, nanoporous containers provide new ways for quick determination of enzyme kinetics and continuous protein synthesis in microfluidic systems. They can be used in a wide variety of applications such as drug discovery, clinical diagnostics and high-throughput screening.

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In this thesis, new methods that address some current limitations in nanoelectrospray mass spectrometry (nESI-MS) analysis are presented. One of the major objectives is the potential gain in sensitivity that can be obtained when employing the proposed techniques. In the first part of this thesis, a new emitter, based on the generation of electrospray from a spray orifice with variable size, is presented. Electrospray is generated from an open gap between the edges of two individually mounted, pointed tips. The fabrication and evaluation of two different types of such emitters is presented; an ESI emitter fabricated from polyethylene terephtalate (Paper I), and a high-precision silicon device (Paper II). Both emitters were surface-treated in a selective way for an improved wetting of the gap and to confine the sample solution into the gap. In the second part of this thesis, different methods for improved sensitivity of nESI-MS analysis have been developed. In Paper III, a method for nESI-MS analysis from discrete sample volumes down to 1.5 nL is presented, using commercially available nESI needles. When analyzing attomole amounts of analyte in such a small volume of sample, an increased sensitivity was obtained, compared to when analyzing equal amounts in conventional nESI-MS analysis. To be able to analyze smaller sample volumes, needles with a narrower orifice and a higher flow resistance were needed. This triggered the development of a new method for fabrication of fused silica nESI needles (Paper IV). The fabrication is based on melting of a fused silica capillary by means of a rotating plasma, prior to pulling the capillary into a fine tip. Using the described technique, needles with sub-micrometer orifices could be fabricated. Such needles enabled the analysis of sample volumes down to 275 pL, and a further improvement of the sensitivity was obtained. In a final project (Paper V), nESI-MS was used to study the aggregation behavior of Aβ peptides, related to Alzheimer’s disease. An immunoprecipitation followed by nESI-MS was employed. This technique was also utilized to study the selectivity of the antibodies utilized. / QC 20101112

L’ADN tumoral circulant (ADNtc) porte des altérations spécifiques de la tumeur des patients, qui sont détectables par un acte minimalement invasif. L’ADNtc représente donc un biomarqueur d’intérêt pour le suivi de l’évolution du cancer. Sa détection requière une technique hautement sensible et quantitative. Dans ce contexte, ce travail de thèse a porté sur la quantification et le suivi de l’ADNtc par PCR digitale en gouttelettes (PCRdg). Cet outil permet la détection d’altérations à l’échelle d’un ADN unique, offrant ainsi une sensibilité allant jusqu’à 0.001%. La détection de cet ADNtc a été réalisée par l’évaluation des biomarqueurs tels qu’une mutation spécifique de la tumeur, la fragmentation de l’ADNtc et l’hyperméthylation de séquences cibles. D’une part, nous avons observé que chez les patients atteints de cancer, l’ADN muté circulant est plus fragmenté que l’ADN non muté, et que cet ADN circulant de patients est globalement plus fragmenté que chez les sujets sains. D’autre part, une corrélation entre les pourcentages d’ADN muté et d’ADN hyperméthylé circulants a été observée au cours du suivi de patients. Ceci suggère la possibilité d’un suivi précis et quantitatif de l’ADNtc par l’évaluation de l’hyperméthylation en alternative à la détermination du statut mutationnel. Nous avons ensuite appliqué nos tests de détection de l’ADNtc dans le cadre de deux études cliniques. L’étude PLACOL, incluant 82 patients atteints de cancer colorectal métastatique, a permis de mettre en évidence deux facteurs pronostiques : un seuil de 0.1 ng/mL et la mesure de la pente de décroissance de la concentration en ADN muté ou hyperméthylé circulant. Dans la seconde étude, portant sur le mélanome métastatique dans le contexte d’une thérapie ciblée (vémurafenib), une corrélation inverse entre les concentrations d’ADNtc et de vémurafenib a été observée. Ces résultats suggèrent le potentiel clinique de l’ADNtc pour l’orientation thérapeutique des patients atteints de cancer avancé. / Circulating tumor DNA (ctDNA) carries tumor-specific alterations that are detectable by minimally invasive sampling. It represents a highly pertinent marker for cancer monitoring during patients’ follow-up. CtDNA detection requires a highly sensitive and quantitative technique. In this context, this project focused on ctDNA quantification and monitoring by picoliter-droplet digital PCR. Thanks to the compartmentalization in millions of picoliter droplets, this tool allowed the detection of single DNA molecule with a sensitivity reaching 0.001%. Testing of ctDNA was performed through the evaluation of different potential biomarkers: specific mutations, ctDNA fragmentation, and hypermethylation of target sequences. On one hand, we observed in cancer patients that ctDNA is more fragmented than wild-type DNA, and, globally more fragmented than circulating DNA in healthy individuals. On the other hand, a strong correlation between percentages of hypermethylated and mutated DNA was observed during the follow-up of patients. Such results suggest the feasibility to precisely and quantitatively monitor ctDNA by the evaluation of hypermethylation as an alternative to the determination of mutational status. We have applied such ctDNA detection strategies in the context of two clinical studies. The PLACOL study, enrolling 82 metastatic colorectal cancer patients, allowed to highlight two prognostic factors: a ctDNA concentration threshold of 0.1 ng / mL, and the evaluation of ctDNA decreasing slope. In the second study, ctDNA was monitored in 11 melanoma patients in the context of a targeted therapy (vemurafenib). An inverse correlation between the concentrations of vemurafenib and ctDNA was demonstrated. These results suggest the clinical relevancy of ctDNA in advanced cancer patients, for the optimization of therapeutic management.