The objective of this work is to develop a micro/nanofluidic-based single molecule detection (SMD) scheme, which would allow us to inspect individual protein or protein complex study protein-protein interactions and their dynamics. This is a collaboration work with MD Anderson Cancer Center and we applied this scheme to study functions of various proteins related to cancer progression in hope to shed new light on cancer research. State-of-the-art micro/nano-fabrication technology is used to provide fused silica micro/nano-fluidic channel devices as our detection platform. Standard contact photolithography, projection photolithography and advanced electron-beam lithography are used to fabricate micro/nano-fluidic channel with width ranging from 100nm to 2?m. The dimensions of these miniaturized biochips are designed to ensure single molecule resolution during detection and shrinking the detection volume leads to increase in signal-to-noise ratio, which is very critical for SMD. To minimize surface adsorption of protein, a fused silica channel surface coating procedure is also developed and significantly improved the detection efficiency. A fluorescent-labeled protein sample solution is filled in the fluidic channel by capillary force, and proteins are electro-kinetically driven through the fluidic channel with external voltage source. Commercial functionalized Quantum Dots (Qdots) are used as fluorescent labels due to its various advantages over conventional organic dyes for single molecule multi-color detection application. A fluorescence correlation spectrometer system, equipped with a 375nm diode laser, 60x water immersion objective with N.A. of 1.2 and two avalanche photodiodes (APD) is implemented to excite single molecules as well as collect emitted fluorescence signals. A two-dimensional photon burst analysis technique (photon counts vs. burst width) is developed to analyze individual single molecule events. We are able to identify target protein or protein complex directly from cell lysate based on fluorescence photon counts, as well as study the dynamics of protein-protein interactions. More importantly, with this technique we are also able to assess interactions between three proteins, which cannot be done with current ensemble measurement techniques. In summary, the technique described in this work has the advantages of high sensitivity, short processing time (2-3 minutes), very small sample consumption and high resolution quantitative analysis. It could potentially revolutionize the area of protein interaction research and provides us with more clues for the future of cancer diagnostics and treatments.

abstract: Driven by the curiosity for the secret of life, the effort on sequencing of DNAs and other large biopolymers has never been respited. Advanced from recent sequencing techniques, nanotube and nanopore based sequencing has been attracting much attention. This thesis focuses on the study of first and crucial compartment of the third generation sequencing technique, the capture and translocation of biopolymers, and discuss the advantages and obstacles of two different nanofluidic pathways, nanotubes and nanopores for single molecule capturing and translocation. Carbon nanotubes with its constrained structure, the frictionless inner wall and strong electroosmotic flow, are promising materials for linearly threading DNA and other biopolymers for sequencing. Solid state nanopore on the other hand, is a robust chemical, thermal and mechanical stable nanofluidic device, which has a high capturing rate and, to some extent, good controllable threading ability for DNA and other biomolecules. These two different but similar nanofluidic pathways both provide a good preparation of analyte molecules for the sequencing purpose. In addition, more and more research interests have move onto peptide chains and protein sensing. For proteome is better and more direct indicators for human health, peptide chains and protein sensing have a much wider range of applications on bio-medicine, disease early diagnoses, and etc. A universal peptide chain nanopore sensing technique with universal chemical modification of peptides is discussed in this thesis as well, which unifies the nanopore capturing process for vast varieties of peptides. Obstacles of these nanofluidic pathways are also discussed. In the end of this thesis, a proposal of integration of solid state nanopore and fixed-gap recognition tunneling sequencing technique for a more accurate DNA and peptide readout is discussed, together with some early study work, which gives a new direction for nanopore based sequencing. / Dissertation/Thesis / Doctoral Dissertation Physics 2015

Les recherches menées au cours de cette thèse constituent une première étape de développement de méthodes expérimentales de concentration de nanoparticules à l'aide de composants micro-nanofluidiques. L'objectif principal est donc d'explorer différentes architectures de systèmes micro-nanofluidiques où l'étape de concentration est effectuée par effet d'exclusion stérique et/ou ionique sous l'application d'un champ de pression et/ou électrique. Une attention toute particulière a été portée sur les méthodes de caractérisation, comprenant notamment les méthodes de particule Tracking Micro-PIV et de microscopie par fluorescence pour mesurer la répartition en nanoparticules et quantifier les facteurs de concentration. Le premier axe concerne la concentration de nanoparticules dans des architectures de type « Bypass ». Dans le cas de la filtration stérique, une modélisation par méthode de différence finie permet de prédire l'apparition d'une zone localisée où la concentration est d'une centaine à un millier de fois plus élevée que la concentration initiale après une heure d'opération. Des composants micro-nano fluidique en silicium ont été réalisés afin de mener une étude paramétrique. En accord avec le modèle proposé, cette étude montre que le nombre de Peclet est le paramètre déterminent dans le choix du design et des conditions d'expérimentations optimums. Concernant la préconcentration par effet électrocinétique, les expérimentations ont essentiellement consisté à explorer le phénomène d'ICP (Ion Concentration Polarisation) et d'appliquer cette technique pour la concentration de nanoparticules. Enfin le type de géométries « Bypass » a été testé sous différentes conditions. Ainsi, le couplage avantageux de phénomènes électro-hydrodynamiques tel que le « streaming potentiel » permet d'ouvrir la voie à des systèmes de préconcentration à actionnements manuels, rapides et très simples d'utilisation. Le deuxième axe d'étude est quant à lui dédié à la conception et l'utilisation de configuration micro-nanofluidique plus originales. Y sont notamment étudiés des systèmes à configuration radial offrant une meilleure stabilité lors des étapes de préconcentration électrocinétiques. Sur la base des performances et limitations des différents systèmes micro-nanofluidiques réalisés, le dernier chapitre est une mise en perspective des champs d'applications potentiels, notamment pour les laboratoires sur puces. / The researches conducted during this thesis consist in a first step for the development of experimental methods applied to the concentration of nanoparticles using micro-nanofluidic devices. The main aim is to explore different system architectures where the préconcentration step are achieved using steric and/or ion exclusion under the influence of a pressure and/or electric field. A special attention is directed toward the characterization methods including Micro-Particle Image Velocimetry micro-PIV and fluorescent microscopy to measure the nanoparticles repartition and to quantify the concentration folds. The first axis deals with the preconcentration of nanoparticles within « Bypass » like architectures. Concerning the steric filtration, a theoretical model using finite element method allows to predict the rising of a located preconcentration zone where the local concentration is enhanced 1000 fold as compared to the initial concentration after 1h of concentration operation. Silicon Micro-nanofluidic devices were fabricated in order to conduct a parametric study. According to the proposed theoretical model, this study shows that the Peclet number is a key parameter to choose the optimal design and experimental conditions. Concerning the electrokinetic preconcentration, the experiments mainly consisted in exploring the ICP phenomenon (Ion Concentration Polarization) and in using this technic to preconcentrate nanoparticles. Finally the Bypass geometry was tested in many conditions. Thus, the advantageous coupling of electro-hydrodynamic phenomena such as the so called “streaming potential” opens new ways to fast, simple and manual preconcentration systems suitable for LOC applications. The second axis is dedicated to the conception and utilization of original micro-nanofluidic configurations. Will also be studied radial micro-nanofluidic devices offering better stability during electrokinetic preconcentration. On the basis of the performances and limitations inherent to each systems, the last chapter will focus on the potential applications relative to LOC.

Drying and evaporation from nanoscale conduits are two ubiquitous phenomena found in nature. As these two nanoscale liquid-vapor phase change phenomena are significantly “accelerated” compared with the corresponding ones at micro- and macro-scales, various industrial applications, including oil recovery, electronic cooling, membrane desalination, and energy harvesting, have been developed. Despite their important implications, the fundamental mechanisms for these two accelerated phase-change processes have not been completely understood. For drying, it is widely accepted that liquid corner flow and film flow could significantly enhance mass transport in microscale conduits other than the sole contribution by vapor diffusion. However, it is unclear if the same principles apply to smaller scales and if the vapor diffusivity will change at the nanoscale. For evaporation, the evaporation kinetics at the nanoscale interface, rather than liquid/vapor transport toward/from the interface, determine the ultimate transport limit, which can be significantly higher than the classical prediction derived under quasi-equilibrium evaporation conditions. Still, the contributions to such enhanced kinetically limited evaporation remain unclear. This thesis aims to answer these unsolved questions by conducting systematic experimental studies on drying and evaporation from single nanochannels and nanopores. We used state-of-art fabrication to create close-end 2D nanochannels with heights from 29 to 122 nm and measure water drying in such channels using an optical microscope. Combining with the channel confinement study and relative humidity study, we decoupled the individual contributions from vapor and liquid transport to the drying and extracted the water vapor diffusivity in nanochannels. We also developed a hybrid nanochannel-nanopore design to achieve and measure kinetically limited evaporation flux from silicon nitride nanopores and graphene nanopores with pore diameters ranging from 24 to 347 nm. Our results show that the evaporation flux increases with the decreasing diameter for both types of nanopores. Furthermore, graphene nanopores overall exhibit higher evaporation fluxes than silicon nitride nanopores with similar diameters. We attribute the diameter-dependent evaporation flux to the diameter-dependent hydronium ion concentration in silicon nitride nanopores and the edge-facilitated evaporation in graphene nanopores, respectively. We expect this work to advance our understanding of nanoscale fast drying and evaporation and provide design guidance for novel nanoporous membrane evaporators.

85% des patients atteints de cancer du pancréas présentent au diagnostic des formes avancées de la maladie qui empêchent leur prise en charge thérapeutique efficace. Il est donc urgent de mettre en évidence des marqueurs diagnostics permettant de détecter plus tôt ces cancers, mais également leur rechute, afin d'améliorer leur prise en charge. Les miARNs (micro acides ribonucléiques) sont des biomarqueurs du cancer du pancréas, présentant une valeur clinique démontrée pour la détection précoce des tumeurs et le suivi de la réponse au traitement. Cependant, les méthodes actuelles d'extraction et de détection de ces molécules ne sont pas adaptées à une utilisation clinique. Les nouvelles technologies issues des méthodes de micro et nanofabrication ont le potentiel de permettre la mise en place de tests diagnostiques, offrant un haut degré de portabilité et de robustesse, une lecture en temps réel, et à bas coût. Nous proposons ici une plateforme nanofluidique couplée à une détection en fluorescence permettant la mesure en temps réel d'interactions moléculaires en milieu hyper-confiné. Nous décrivons dans un premier temps la plateforme de détection via un modèle théorique à une dimension basé sur la dynamique moléculaire permettant de prédire la capture spécifique des miARNs dans un nanocanal fonctionnalisé. L'originalité du système réside dans une accroche non homogène des miARNs sur la surface du capteur. Ainsi, nous démontrons que l'étude du profil spatial d'hybridation engendré permet de déterminer l'affinité du miARN capturé avec la séquence sonde en une seule étape, sans lavage. Nous démontrons également l'excellente spécificité du biocapteur qui permet la discrimination rapide (moins de 10 minutes) de SND (single nucleotide difference). Les performances du dispositif pour des applications au plus près des problématiques biologiques dans le cadre de la détection du cancer du pancréas sont enfin discutées : les effets de la préparation d'échantillon types biofluides complexes sur l'extraction de miARNs sont étudiés, puis deux approches permettant la détection de miARNs endogènes sont décrites et comparées, conduisant à la détection de miARNs extraits de cultures cellulaires modèles du cancer du pancréas. / 85% of patients affected by pancreatic adenocarcinoma (PDA) are diagnosed at an advanced stage, preventing effective care and curative treatments. Therefore, it is urgent to identify reliable biomarkers for the early detection of disease status, including relapse. MiRNAs (micro ribonucleic acids) are biomarkers of PDA, with demonstrated clinical value for early detection of tumors and monitoring of response to treatment. However, current methods of extraction and detection of miRNA are not compatible with clinical use. New technologies derived from micro and nanofabrication methods have the potential to facilitate the implementation of diagnostic tests, by offering a high degree of portability and robustness, short time to results at low cost. Here, we propose a nanofluidic platform coupled to fluorescence detection for the real time measurement of molecular interactions in a confined environment. We first describe the detection platform via a one-dimension theoretical model based on molecular dynamics to predict the capture of miRNAs into biofunctionalized nanochannels. The originality of the system lies in the non-homogeneous hybridization of miRNA targets onto the sensor. We demonstrate that the analysis of the spatial hybridization profile enables the determination of the affinity of the captured miRNA with the probe sequence in a wash-free single step. We then show the rapid discrimination (less than 10 minutes) of single nucleotide difference (SND) using this strategy. The performance of the device in the context of pancreatic cancer detection is discussed: the effect of sample preparation of complex biofluids is studied and two labeling approaches compatible with the detection of endogenous miRNAs are described and compared, leading to the detection of miRNAs extracted from model cell cultures of pancreatic cancer.

A nanofluidic biosensor using surface-enhanced Raman scattering (SERS) was developed to detect the β-amyloid (Aβ) protein, one of the biomarkers of Alzheimer’s disease (AD). Recent studies have indicated that investigating changes in relative concentrations of structure specific Aβ oligomers in cerebral spinal fluid (CSF) during the progression of AD could be important indicators for diagnosing AD pre-mortem. However, there is no definitive pre-mortem diagnosis of AD thus far because of the lack of technology available for sensitive Aβ detection. Hence, the development of a system for detecting the structure specific Aβ oligomers, along with the concentrations of these oligomers in CSF, would be useful in the investigation of the molecular mechanisms of Aβ cytotoxicity associated with AD. In this thesis, a nanofluidic trapping device trapping system for detecting biomolecules at sub-picomolar concentrations was developed for using SERS. The device, with a microchannel leading to a nanochannel, carries out dual functions: encouraging sizedependent trapping of gold nanoparticles (60nm) at the entrance of the nanochannel as well as restricting the target molecules between the gaps created by the aggregated nanoparticles. Initially, the trapping capability of the nanofluidic device was tested using fluorescent polystyrene and gold nanoparticles. UV-vis absorption spectroscopy was used to characterize the gold nanoparticle clusters at the entrance to the nanochannel. The device established controlled, reproducible, SERS active sites within the interstices of gold nanoparticle clusters and shifted the plasmon resonance to the near infrared, in resonance with incident laser light. Two strongly Raman active molecules, adenine and Congo red, were used to test the feasibility of the SERS nanofluidic device as a platform for the detection of multiple analytes. The results showed that strong SERS signals were obtained from the nanoparticle clusters at the nanochannel entrance. Once the feasibility of the approach was determined with strong Raman molecules, Aβ was detected using this nanofluidic SERS platform. Distinct surface-enhanced Raman spectra of Aβ was observed in different conformational states as a function of concentration and structure (monomer versus oligomer form) due to Aβ refolding from α-helical to a predominantly β-pleated sheet form. The sensor was also shown to potentially distinguish Aβ from insulin and albumin, confounder proteins in cerebral spinal fluid. Thus, a novel platform was developed to detect picomoler levels of Aβ with the ultimate goal of facilitating the diagnosis and understanding of Alzheimer’s disease by means of detecting structure specific oligomers of Aβ.

abstract: This thesis describes several experiments based on carbon nanotube nanofludic devices and field-effect transistors. The first experiment detected ion and molecule translocation through one single-walled carbon nanotube (SWCNT) that spans a barrier between two fluid reservoirs. The electrical ionic current is measured. Translocation of small single stranded DNA oligomers is marked by large transient increases in current through the tube and confirmed by a PCR (polymerase chain reaction) analysis. Carbon nanotubes simplify the construction of nanopores, permit new types of electrical measurement, and open new avenues for control of DNA translocation. The second experiment constructed devices in which the interior of a single-walled carbon nanotube field-effect transistor (CNT-FET) acts as a nanofluidic channel that connects two fluid reservoirs, permitting measurement of the electronic properties of the SWCNT as it is wetted by an analyte. Wetting of the inside of the SWCNT by water turns the transistor on, while wetting of the outside has little effect. This finding may provide a new method to investigate water behavior at nanoscale. This also opens a new avenue for building sensors in which the SWCNT functions as an electronic detector. This thesis also presents some experiments that related to nanofabrication, such as construction of FET with tin sulfide (SnS) quantum ribbon. This work demonstrates the application of solution processed IV-VI semiconductor nanostructures in nanoscale devices. / Dissertation/Thesis / Ph.D. Physics 2011

La filtration de molécules est un enjeu vital dans les domaines biomédicaux tels que la dialyse jusqu’à la production à grande échelle d’eau potable. Dans les dernières décennies, des matériaux nanoporeux ont permis des avancées significatives, mais s’appuient toujours sur une géométrie de type "passoire" où une membrane avec de petits trous permet la sélection des molécules cibles. Ceci entraîne notamment une diminution du transport à travers ces trous, et rend les procédés de séparation coûteux en énergie. Ici je développe plusieurs approches innovantes pour la filtration, inspirées par des filtres biologiques (les reins humains, les aquaporines). Je définis de nouveaux concepts pour la séparation, en m’appuyant sur des modèles simples. J’explore notamment des arrangements topologiques différents, mais aussi l’idée d’une passoire active, où la taille des trous peut par exemple varier dans le temps. Tous ces principes pourraient être implémentés à partir d’éléments existants et fournir des alternatives pour la dialyse ou le recyclage des eaux usées. Ces recherches amènent aussi des questions fondamentales originales en physique. En particulier, des grandeurs définies traditionnellement à l’équilibre comme la pression osmotique ou la perméabilité d’un pore ne sont pas bien définies quand le pore a des propriétés qui dépendent du temps. Pour autant, on imagine aisément qu’il est possible d’étendre ces concepts hors d’équilibre, et que cela aura de nombreuses conséquences pour la filtration, et même le pompage ionique. / Filtering specific molecules is a challenge faced for several vital needs: from biomedical applications like dialysis to the intensive production of clean water. The domain has been boosted over the last decades by the possibilities offered by nanoscale materials. Filtration is however always designed according to a sieving perspective: a membrane with small and properly decorated pores allows for the selection of the targeted molecules. This inevitably impedes the flux and transport, making separation processes costly in terms of energy. Here I investigate several innovative approaches to separation and filtration. I draw inspiration from biological systems (the human kidney, biological channels like aquaporins) and rationalize some new concepts for sieving, based on simple models. These approaches rely on different alternative strategies: either exploring new topologies or the idea of active sieving, with dynamically responsive channels and pores. All these principles could be readily mimicked using existing technologies to build artificial dialysis devices or alternatives for advanced water recycling. In a broader perspective, these approaches open fundamental questions in the fields of statistical physics and fluid dynamics. In particular, traditional in equilibrium quantities like osmotic pressure and permeability are not defined when the pore has an active component, yet one easily imagines that such concepts could be extended to these out-of equilibrium situations. This has numerous consequences on filtration and desalination, but also on ionic pumping and sorting.

This diploma thesis focuses on the preparation of the liposomes, containing lipids with gadolinium, which are used for a contrast magnetic resonance imaging. The liposomes were prepared by the lipid film hydration followed by an extrusion and also by a new nanofluid mixing method on the NanoAssemblr Benchtop. The preparation technology has been optimized for parameters such as the composition of lipids, the flow rate ratio and total flow rate. The method of modification of the liposomes surface by gadolinium complexes has been developed. This method is using a conjugation reaction between the lipids containing cyanuric acid and Gd-DOTA. Prepared Gd-liposomes, which contain gadolinium, were complexly defined by the characterization techniques of DLS and NTA. The morphology of liposomes was observed by TEM and cryo-TEM. Methods for the determination of phospholipid content (Stewart test) and residual water in the lyophylisates of liposomes (Karl-Fischer titration) were used. Gadolinium in liposomal preparations was determined by ICP-OES. Using MR, the concept of gadolinium liposomes was verified and designed for MRI imaging of thrombi. The concept describing the mechanism of liposomes formation based on the experimentally proven existence of a phospholipid bilayer fragment has been developed. This concept is based on the experimentally proven existence of a phospholipid bilayer fragment.

Les enjeux de réduction des consommations des transports amplifie l'intérêt pour de nouveaux stockages d'énergie a forte densité de puissance et d'amortisseurs performants permettant d'alléger les structures. Les systèmes Hétérogènes Lyophobes (SHL) utilisent le mouillage forcé de matrices poreuses pour convertir les énergies mécaniques en énergies interfaciales qui peuvent ensuite être restituée(stockage) ou dissipée(amortisseur) selon le choix de liquide et de matériau. Ces systèmes ont été étudiés depuis les années 80 du fait de leurs propriétés prometteuses pour ces applications en amortissement et stockage d'énergie mécaniques mais aussi comme objet physique.L'application de ces systèmes requière de comprendre leurs intrusion et extrusion du liquide hors du volume poreux sur une plage de température et de durée d'intrusion.Un dispositif d'intrusion à haute pression (100 MPa) a été développé pour étudier l'impact de la température (5-70°C) et de la durée d'intrusion sur trois décades afin de mesurer précisément le comportement dynamique à échelle macroscopique.Ce dispositif a été utilisé pour étudier un matériau de porosité cylindrique (MCM-41) qui a été rendu non mouillant par un greffage hydrophobe en condition anhydre stricte qui a été intégré au laboratoire.Les pressions d'intrusions de l'eau dans le MCM-41 sur une large plage de durée et de température ont confirmé le modèle d'ancrage de la ligne de contact au cours de l'intrusion et à l'extrusion le modèle de nucléation de bulle avec la contribution majeure de la tension de ligne, mesurée sur les autres solutions aqueuses. Des mesures inédites avec de l'eau deutérée, des solutions salines jusqu'à saturation et en pH alcalin ont éclairé la contribution des liaisons hydrogènes, des ions et des silanols sur la pression d'intrusion et la dissipation d'énergie.Le deuxième matériau étudié (ZIF-8) présente une structure cristalline de cages nanométriques connectées par des ouvertures commensurables avec les molécules d'eau dont l'intrusion présente une hystérèse réduite pour le stockage d'énergie. Les mesures dynamiques précises ont montré au-dessus de 35°C un comportement inédit de constance de la pression d'intrusion sur trois décades de durée. Aux températures inférieures une surpression de grande ampleur en puissance -1/2 contraste avec les modèles de réponse linéaire et présente une dépendance affine en température.Des mesures dynamiques inédites avec de l'eau deutérée ont révélé un impact significatif des liaisons hydrogènes. Des particules de différentes durée de synthèse ont montré que la pression d'intrusion est principalement pilotée par des défauts et non la capillarité.Ces résultats inédits ont amené à réaliser des expériences de diffusion de neutrons sous pression qui ont apporté des mesures inédites de déformation des pores cylindriques sous l'effet de l'intrusion et confirmé l'existence de cluster d'eau séparé au sein du ZIF-8. / The stakes of reducing the consumption of transportation drives the interest in high density energy storage as well as shock absorbers to reduce the weight and power of vehicles.Heterogeneous Lyophobic Systems leverage forced wetting to convert mechanical energy into interfacial energy which can be recovered later (storage) or dissipated (damper) depending on the choice of liquid and material.These systems have been studied since the 80s because of their promising properties for damping and storage applications but also as a physics topic.Actual use of these systems require the understanding and control of their intrusion and extrusion pressures in a relevant range of temperatures and over different time scales.A macroscopic high pressure intrusion device (100 MPa) was developed to study the impact of temperature (5-70°C) and the duration of intrusion over three decades to measure precisely the dynamic behavior.This device was used to study a material with cylindrical pores (MCM-41) which was made non-wetting thank to anhydrous hydrophobic grafting protocol that has beenintegrated into the laboratory.Water intrusion pressures in the MCM-41 over a wide rangeof time and temperature confirmed the model of anchored contact line duringintrusion and the bubble nucleation model during extrusion.This confirmed the key contribution of line tension which was quantified and in water and on other aqueous solutions.Original measures with deuterated water, saturated brine andhigh pH have clarified the contributions from hydrogen bonds, ions and silanols to the intrusion pressure and energy dissipation.The second material (ZIF-8) is a crystal composed of nanometric cavities connected by openings as large as water molecules. Water intrusion has a low hysteresis suitable for storage and above 35°C a unprecedented behavior of constant pressure of intrusion spanning three orders of intrusion duration.At lower temperatures, intrusion pressures see ample increase scaling as a power law -1/2 which conflicts with linear response behavior. This descriptive model depends linearly on temperature, dropping to zero at 35°C.First measurements on deuterated water showed similar pattern and a strong impact of hydrogen bonds. Particles from different synthesis durations showed that the intrusion pressure is heavily dependent on inner defects and not capillarity.Those new results driving questions about microscopic mechanisms lead to neutron scattering experiments under pressure. These brought unprecedented measures of cylindrical pores deformations under intrusion and confirmed the division of water in ZIF-8 cavities.